Document pmzv2x7zEGVJrMOYdw0ZNZrjB

A Pharmacokinetic Study of Potassium Perfluorooctanoate in the Cynomolgus Monkey Southern Research Institute Study ID: 9921.4 January 14, 2003 Final Report on A Pharmacokinetic Study of Potassium Perfluorooctanoate 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 of perfluorooctanoate 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 perfluorooctanoate, potassium salt. At various times after dosing, serum and urine (24-hour collections) samples were obtained and analyzed by HPLC/MS/MS for levels of intact perfluorooctanoate. The lower limit of quantitation o f the analytical method was 20 ng/mL for serum samples and 10 ng/mL for urine samples. At 0.5 hours after dosing, serum concentrations of perfluorooctanoate were similar in male and female monkeys and ranged from 91,130 to 96,660 ng/mL in male monkeys and from 88,940 to 96,400 ng/mL in female monkeys. Serum concentrations of perfluorooctanoate subsequently declined but, beyond the first few days after dosing, the rate o f decrease appeared to be faster in male monkeys than in female monkeys. On Day 28, the concentrations o f perfluorooctanoate in serum ranged from 1863 to 27,140 ng/mL in the male monkeys and from 7,145 to 33,680 ng/mL in the female monkeys. Due to the relatively high serum concentrations o f the compound on this day, the length of serum collection was extended through Day 123. On Day 123, the serum concentration o f perfluorooctanoate was at or only slightly above the limit o f quantitation (20 ng/mL) in male monkeys and between 885 and 4701 ng/mL in female monkeys. The serum concentration versus time data were subjected to non-compartmental pharmacokinetic analysis. The terminal half-life o f perfluorooctanoate in serum was 13.6,13.7, and 35.3 days in the three male monkeys and 26.8,29.3, and 41.7 days in the three female monkeys. The total body clearance was 4.0, 15.8, and 17.5 mL/day/kg for male monkeys and 3.1, 3.9, and 9.1 mL/day/kg for female monkeys. These estimated values for half-life and clearance indicated that two o f the three male monkeys eliminated perfluorooctanoate at a faster rate than did the female monkeys. The volume o f distribution o f the compound at steady state (Vdss) was similar for both sexes and ranged from 168 to 192 mL/kg for male monkeys and 133 to 270 mL/kg for female monkeys. Perfluorooctanoate was slowly excreted in urine by both male and female monkeys; levels o f perfluorooctanoate, representing as much as 1% o f the administered dose, were present in urine 28 days after dosing. The results o f this study suggested that the pharmacokinetics of perfluorooctanoate may be different in male and female monkeys. 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 3 Serum Levels o f Perfluorooctanoate 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 o f Perfluorooctanoate 4 4 5 5 5 4.0 DISCUSSION 7 5.0 CONCLUSIONS 8 6.0 RECORD ARCHIVES 7.0 REFERENCES 8 8 11 Table 1: Table 2: Table 3: Table 4: Figure 1: Appendix A: Appendix B: Appendix C: TABLE OF CONTENTS (Continued) LIST OF TABLES Page Individual Body Weights 9 Serum Concentrations of Perfluorooctanoate 10 Pharmacokinetic Parameters Calculated from Serum Concentrations of Perfluorooctanoate 11 Urinary Excretion of Perfluorooctanoate 12 LIST OF FIGURES Serum Concentration Profiles of Perfluorooctanoate in Monkeys 14 LIST OF APPENDICES Study Protocol and Amendments A-l Method Validation Report: Validation of Analytical Methods for B-l Determination of Perfluorooctanoate in Monkey Serum and Urine Using H PL C /M S/M S Analytical Method for Determination of Perfluorooctanoate in Monkey Serum and Urine C-l Ill Signature Page A Pharmacokinetic Study of Potassium Perfluorooctanoate in the Cynomolgus Monkey _________________________________ / / / * / os Patricia E. Noker, Ph.D., D.A.B.T. Date Study Director Supervisor, ADME & Pharmacokinetics 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. /V ^ AS / v_ Gregory S. Gorman, Ph.D. Manager, Bioanalytical Chemistry Group Date IV Good Laboratory Practices Disclaimer This study described in this final report was not conducted in strict compliance with the U.S. Food and Dmg 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 o f 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 of Sample Collection: Study Completion: October 4, 2000 October 9, 2000 February 9, 2001 January 14, 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.S. Associate Director, Safety Assessment Gregory S. Gorman, Ph.D. Manager, Bioanalytical Chemistry Group Tsu-Han Lin, Ph.D. Pharmacokineticist Darrell E. Hoskins, D.V.M., Ph.D., A.C.L.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 o f potassium perfluorooctanoate in serum and to estimate urinary clearance at various times following administration of 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 3 male and 3 female monkeys designated for use in this study were selected from an in house colony o f 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 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 o f the daily ration was sufficient to meet 2 nutritional requirements. In addition, the diet was supplemented with fresh fruit/treats several times each week. Tap water (Birmingham public water supply) was available to the monkeys ad libitum during the quarantine and study periods. The monkeys were housed individually in stainless steel cages during the quarantine and the study periods. From Day 0 to the end o f the study, the monkeys were housed in a room that was maintained at a temperature o f 68.0-70.3 F and a relative humidity o f 22.2-65.7%. 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 of short duration. 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 (IACUC). Southern Research is fully accredited by the American Associate for Accreditation of Laboratory Animal Care International (AAALAC). The monkeys used on this study were previously given a single IV bolus dose of perfluorobutanesulfonate (10 mg/kg) on April 10, 2000 (Southern Research Study No. 9921.1); a single IV bolus dose o f potassium perfluorobutanoate (10 mg/kg) on June 13, 2000 (Southern Research Study No. 9921.2); and a single IV bolus dose o f potassium perfluorohexanoate (10 mg/kg) on July 31, 2000 (Southern Research Study No. 9921.3). 2.2 Test Article and Vehicle Test Article: One bottle containing 5.2 grams o f potassium perfluorooctanoate (T-7507; expiration date not supplied; Southern Research Lot No. E09/L-1) was supplied by 3M (St. Paul, MN) and received on May 15, 2000. The test article was stored at room temperature until used. Stability of the test article was the responsibility o f the Sponsor. 3 Vehicle: The vehicle used for the preparation o f the dose formulation of potassium perfluorooctanoate was sterile saline, USP (Phoenix Pharmaceutical Company; St. Joseph, MO; Lot 8070655, expiration date July 2001). The vehicle was stored at room temperature and was considered to be stable when stored according to these conditions. Dose Formulation Preparation: For the single dose formulation o f potassium perfluorooctanoate prepared at 5 mg/mL, the required amount of test article was weighed out in a volumetric flask. Sterile saline was added and the formulation was stirred until in solution. The formulation was stored refrigerated and used for dosing within 4 days 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 perfluorooctanoate at 10 mg/kg by injection into a superficial arm or leg vein. Doses were based upon the Day -1 individual body weights. Doses were administered at a volume of 2 mL/kg. Clinical Observations: All animals were observed twice daily for signs of mortality/moribundity. Each primate was examined shortly after dose administration for clinical signs of toxicity. Additional clinical observations were performed on days of blood collection. Body Weights: Each primate was weighed on Days -1, 4, 7 ,14, 21, and 28. Urine and Feces Collection: Urine and feces were collected for 24-hour intervals on the following days: prior to dose administration (Day -3; baseline), on Day 1 (0-24 hours 4 postdose), on Day 2 (24-48 hours postdose), and on Days 7, 14,21, and 28. The volume of each urine sample was measured upon collection. Urine and feces samples were stored frozen (approximately -20 C or below). Fecal samples will not be analyzed unless specifically requested by the Sponsor. Serum Levels of Perfluorooctanoate: Blood samples (approximately 3 mL) were collected from each primate at approximately 0 (predose) minutes; 0.5, 2, 4, 8, and 24 hours; and on Days 2,4 , 7,11, 14,21,28, 57, 79, 87, and 123 postdose. Samples were collected into tubes without anticoagulant and were allowed to clot at room temperature. The blood samples were then centrifuged, and the serum separated and stored frozen (approximately -20 C or below) until analyzed. Bioanalytical Method Development and Sample Analysis: A bioanalytical method was developed for the analysis o f perfluorooctanoate in serum and urine matrices. The method was validated for accuracy. This method was used for the analysis o f serum and urine samples collected during the study (see Appendices B and C). Data Analyses: The serum concentration data for unchanged perfluorooctanoate were subjected to non-compartment pharmacokinetic analysis using WinNonlin (Standard Edition; Version 1.1; Scientific Consulting Inc.; Cary, NC). Mean values and standard deviations for each parameter were calculated using Microsoft Excel software (Microsoft Corporation; Irvine, CA). The urinary excretion o f perfluorooctanoate at each collection interval was calculated and expressed as a percent o f the administered dose. No other statistical analyses of the data were performed. 3.0 Results 3.1 Mortality No test article-related deaths occurred during the study. One o f the three male monkeys (2052) was euthanized on Day 79 because o f repeated episodes o f self-mutilation; there was no indication that the self-mutilation was related to administration o f perfluorooctanoate. 5 3.2 Clinical Observations No adverse drug-related clinical signs of toxicity were observed for any monkey. Male monkey 2052 was observed on Day 57 o f the study to have several wounds on the right leg. The attending veterinarian diagnosed these wounds to be self-inflicted lacerations. The wounds were cleaned and subsequently flushed with a weak betadine solution every 3-4 days for 4 courses o f treatment. The social enrichment of the monkey was also augmented in order to provide the monkey additional stimulation and to change his focus o f attention. On Day 79, the monkey was examined and several severe lacerations were observed on the leg and foot o f the animal. These lacerations were diagnosed as self-inflicted wounds. Due to the repeated instances o f self-mutilation and the likelihood of continued episodes of self mutilation, the monkey was euthanized on Day 79 for humane reasons. 3.3 Body Weights Body weights for individual monkeys are presented in Table 1. The body weight o f each monkey remained the same between Days -1 and 28. 3.4 Serum and Urine Concentrations of Perfluorooctanoate During the study, an HPLC/MS/MS method was developed and validated for the analysis o f perfluorooctanoate in monkey serum and urine. Details o f the method validation procedure are provided in Appendix B. Details o f the analytical method are provided in Appendix C. The lower limit of quantitation o f the method was 20 ng/mL for serum samples and 10 ng/mL for urine samples. Serum concentrations o f perfluorooctanoate in three male and three female monkeys at various times through Day 123 after administration o f a single iv dose o f 10 mg/kg are presented in Table 2 and plotted in Figure 1. At 0.5 hours after dosing, serum concentrations o f perfluorooctanoate were similar in male and female monkeys and ranged from 91,130 to 96,660 ng/mL in the male monkeys and from 88,940 to 96,400 ng/mL in the female monkeys. Serum concentrations o f perfluorooctanoate subsequently declined at a slow rate; the rate o f decline appeared to be similar in male and female monkeys through at least the 6 first two days after dosing. At 48 hours, serum concentrations o f perfluorooctanoate were between 48,530 and 65,810 ng/mL in the male monkeys and 39,820 and 61,600 ng/mL in the female monkeys. Beyond this time, serum concentrations o f perfluorooctanoate in two o f the three male monkeys decreased at a faster rate than that observed for the third male monkey and for the three female monkeys. On Day 28, the concentrations of perfluorooctanoate in serum ranged from 1863 to 27,140 ng/mL in the male monkeys and from 7,145 to 33,680 ng/mL in the female monkeys; due to these relatively high serum concentrations of the compound on this day, the length o f serum collection was extended through Day 123. On Day 123, the serum concentration o f perfluorooctanoate was at or only slightly above the limit of quantitation (20 ng/mL) in the two surviving male monkeys and between 885 and 4701 ng/mL in the three female monkeys. Pharmacokinetic parameters calculated from serum concentrations o f perfluorooctanoate in individual monkeys are presented in Table 3. The values were derived from noncompartmental pharmacokinetic analysis o f the data. A U C ^ ^ values ranged from 571 to 2501 /ig*day/mL (mean: 1235 /g.day/mL) for male monkeys and from 1094 to 3224 yUgday/mL (mean: 2293 yUg*day/mL) for female monkeys. The terminal half-life o f perfluorooctanoate in serum was 13.6, 13.7, and 35.3 days in the three male monkeys and 26.8, 29.3, and 41.7 days in the three female monkeys. The total body clearance was 4.0, 15.8, and 17.5 mL/day/kg for male monkeys and 3.1, 3.9, and 9.1 mL/day/kg for female monkeys. These estimated values for half-life and clearance indicated that two of the three male monkeys eliminated perfluorooctanoate at a faster rate than did the female monkeys. The volume o f distribution o f the compound at steady state (Vdss) was similar for both sexes and ranged from 168 to 192 mL/kg for the male monkeys and 133 to 270 mL/kg for the female monkeys. 7 The amount o f perfluorooctanoate eliminated in urine by individual monkeys at various times after dosing is presented in Table 4. Sex differences in the urinary excretion o f perfluorooctanoate were not readily apparent prior to Day 21; on Day 21 and Day 28, the female monkeys appeared to eliminate a higher percentage o f the dose in urine than did the male monkeys. The urinary elimination o f perfluorooctanoate was prolonged for both the male and female monkeys; on Day 28 from 0.1 to 1% o f the administered dose was recovered in urine collected from the individual monkeys. 4.0 Discussion Perfluorooctanoate was slowly eliminated by male and female monkeys given a single iv dose o f 10 mg/kg. The results o f this study indicated that beginning on around Day 7 and continuing throughout the remaining period o f sample collection, serum concentrations of perfluorooctanoate were lower in two o f the three male monkeys than in the three female monkeys. On the final day of serum collection (Day 123), serum concentrations o f perfluorooctanoate were below or only slightly above the quantitation limit (20 ng/mL) in the two surviving male monkeys, whereas, on the same day, serum concentrations o f perfluorooctanoate ranged from 885 to 4701 ng/mL in the three female monkeys. These differences in the serum concentrations of perfluorooctanoate were reflected in a shorter serum half-life o f perfluorooctanoate in these two male monkeys (13.6 or 13.7 days) than observed for the three female monkeys (range: 26.8 to 41.7 days). The clearance of perfluorooctanoate also appeared to be higher in two o f the male monkeys than in the three female monkeys. Although the number o f monkeys was limited, these results suggested that the kinetics of perfluorooctanoate may have been different in male and female monkeys. It was observed that the serum concentration data for one of the male monkeys (2052) more closely paralleled the serum concentration profile observed for the female monkeys than that observed for the other two male monkeys. It is possible that the study procedures (e.g., handling, short-term restraint in a chair) or other unknown factors may have produced stress in this monkey resulting in the release o f high levels o f cortisol. Cortisol is known to affect various physiological changes including changes in carbohydrate, protein, and/or lipid metabolism, shifts in electrolyte and water balance, and increases in plasma proteins. Such changes could have possibly led to changes in the 8 disposition of perfluorooctanoate in this male monkey. It is also possible that the self-mutilation activities o f this monkey, which lead to the eventual euthanasia o f the animal, were a response to stress. Although the serum levels o f perfluorooctanoate indicated that at least two o f the three male monkeys may have eliminated perfluorooctanoate at a faster rate than did female monkeys, it was difficult to discern a difference in the urinary excretion o f the compound by male and female monkeys. This may have been related to the fact that the rate o f urinary excretion o f the compound by all the monkeys on study was slow. Less than 20% of the administered dose was excreted in urine by either male or female monkeys within the first 48 hours after dosing. Subsequently, on Day 28, perfluorooctanoate was present in urine collected from male or female monkeys at levels that represented as much as 1.0% o f the dose. 5.0 Conclusions The mean terminal serum half-life o f perfluorooctanoate was 22.0 days in male monkeys and 32.6 days in female monkeys. Unchanged perfluorooctanoate was slowly excreted in urine at a slow rate by male and female monkeys. 6.0 Record Archives Data, specimens, and a copy 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 of Potassium Perfluorooctanoate in the Cynomolgus Monkey Individual Body Weights Animal ID 2052 2054 2211 2058 2059 2061 Dose Level 10 mg/kg 10 mg/kg 10 mg/kg 10 mg/kg 10 mg/kg 10 mg/kg 0 6.6 6.4 5.1 3.4 3.5 4.1 Body Weight (kg) on Study Day 4 7 11 14 Males 6.5 6.4 6.4 6.4 6.3 6.3 6.4 6.3 5.1 5.1 5.1 5.1 Females 3.4 3.5 3.5 3.5 3.5 3.5 3.5 3.4 4.0 4.0 4.1 4.1 21 6.7 6.6 5.4 3.4 3.6 4.1 28 6.6 6.2 5.4 3.5 3.6 4.1 10 Table 2 A Pharmacokinetic Study of Potassium Perfluorooctanoate in the Cynomolgus Monkey Serum Concentrations of Perfluorooctanoate Timepoint 2052 0 115 0.5 hrs 91,130 2 hrs 67,900 4 hrs 74,000 8 hrs 71,200 24 hrs 50,210 48 hrs 65,810 Day 4 43,370 Day 7 45,840 Day 11 39,450 Day 14 37,360 Day 21 33,600 Day 28 27,140 Day 57 15,010 Day 79 9915a Day 87 -- Day 123 -- Serum Concentration (ng/mL) Males Females 2054 2211 2058 2059 BQL 20 57 123 2061 33 96,660 91,370 96,400 88,940 94,080 91,630 90,680 74,280 77,870 71,600 89,160 71,960 70,120 81,620 69,380 60,860 73,650 79,570 61,520 64,170 50,930 64,160 62,890 60,760 41,940 48,530 48,930 61,600 49,670 39,820 42,940 30,490 70,520 41,640 44,370 27,910 29,100 59,820 44,600 36,760 19,320 23,640 38,980 53,980 37,500 11,220 13,430 40,450 45,790 21,910 3,985 5,939 38,750 37,850 10,460 1,863 3,241 33,680 24,300 7,145 467 780 31,690 16,870 4,992 ------ -- -- 95 I l l 8,585 5,453 2,123 BQL 35 3,651 4,701 885 aSample taken prior to humane sacrifice o f animal. BQL = Below the quantitation limit (<20 ng/mL) Page 1 of 1 Table 3 A Pharmacokinetic Study of Potassium Perfluorooctanoate in the Cynomolgus Monkey Pharmacokinetic Parameters Calculated from Serum Concentrations of Perfluorooctanoate Male Parameter 2052 2054 2211 Average SD 2058 Cmax (jUg/mL)a 101 98.4 91.6 97.0 4.9 105 ti/2 (day)b 35.3 13.7 13.6 20.9 12.5 26.8 AUCo-iast Ogday/mL)c 1999 569 633 1067 808 3083 AUCo-infmity Ogday/mL)d 2501 571 634 1235 1097 3224 Cl (mL/day/kg)e 4.0 17.5 15.8 12.4 7.4 3.1 Vdss (mL/kg)r 192 168 184 181 12 133 aMaximum concentration in serum Half-life of the terminal elimination phase cArea under the serum concentration versus time curve calculated from 0 to the last time point dArea under the serum concentration versus time curve calculated from 0 to infinity eTotal body clearance .p Volume o f distribution at steady state 2059 93.0 41.7 2314 2560 3.9 190 Female 2061 103 29.3 1056 1094 9.1 270 Average 100 32.6 2151 2293 5.4 198 SD 6.0 8.0 1023 1090 3.3 69 Page 1 of 1 12 Table 4 A Pharmacokinetic Study of Potassium Perfluorooctanoate in the Cynomolgus Monkey Animal ID 2052 2054 2211 2058 2059 2061 2052 2054 2211" 2058 2059 2061 2052 2054 2211 2058 2059 2061 2052 2054" 2211" 2058 2059 2061a Urinary Excretion of Perfluorooctanoate Urine Concentration Sex (ng/mL) M BQL M BQL M BQL F BQL F BQL F BQL M 6,150 M 9,283 M 27,150 F 5,527 F 1,924 Fb M 3047 M 1174 M 2766 F 3783 F 2085 F 3486 M 3906 M 10350 M 10770 F 4347 F 4253 F 18190 Urine Volume (mL) Total (/g) Baseline 960 - 840 - 490 - 205 - 700 - 170 - Day 1 200 1,230 420 3,899 350 9,503 190 1,050 860 1,655 80 - Day 2 250 762 170 200 120 332 150 567 660 1,376 60 209 Day 7 350 1,367 870 9,005 300 3,231 200 869 110 468 80 1,455 Dose (Mg) 66,000 64,000 51,000 34,000 35,000 41,000 66,000 64,000 51,000 34,000 35,000 41,000 66,000 64,000 51,000 34,000 35,000 41,000 66,000 64,000 51,000 34,000 35,000 41,000 Percent of Dose in Urine (%) -- -- -- 1.9 6.1 18.6 3.1 4.7 -- 1.2 0.3 0.7 1.7 3.9 0.5 2.1 14.1 6.3 2.6 1.3 3.5 BQL = Below the quantitation limit (<10 ng/mL) aConcentration of perfluorooctanoate in the sample greatly exceeded the highest concentration in the standard curve; therefore, urine concentration value may contain significant error bDue to technician error, urine was discarded after the volume was obtained. cConcentration of perfluorooctanoate in the sample was slightly above the highest concentration in the standard curve; urine concentration value should have no significant error. Page 1 o f2 13 Table 4 (Continued) A Pharmacokinetic Study of Potassium Perfluorooctanoate in the Cynomolgus Monkey Urinary Excretion of Perfluorooctanoate Animal ID 2052 2054 2211 2058 2059 2061 2052 2054 2211 2058 2059 2061 2052 2054 2211 2058 2059 2061 Urine Concentration Sex (ng/mL) M 1066 M 776 M 819 F 1673 F 5550 F 6151 M 389 M 575 M 298 F 2,278 F 2,347 F 6,052 M 417 M 79 M 185 F 665 F 2286 F 3126 Urine Volume (mL) 1010 1060 390 170 130 160 1000 710 880 140 150 100 680 660 520 410 150 105 Total (Mg) Day 14 1,077 823 319 284 722 984 Day 21 389 408 262 319 352 605 Day 28 284 52 96 273 343 328 Dose (M) 66,000 64,000 51,000 34,000 35,000 41,000 66,000 64,000 51,000 34,000 35,000 41,000 66,000 64,000 51,000 34,000 35,000 41,000 Percent of Dose in Urine (%) 1.6 1.3 0.6 0.8 2.1 2.4 0.6 0.6 0.5 0.9 1.0 1.5 0.4 0.1 0.2 0.8 1.0 0.8 BQL = Below the quantitation limit (<10 ng/mL) aConcentration of perfluorooctanoate in the sample greatly exceeded the highest concentration in the standard curve; therefore, urine concentration value may contain significant error bDue to technician error, urine was discarded after the volume was obtained. cConcentration of perfluorooctanoate in the sample was slightly above the highest concentration in the standard curve; urine concentration value should have no significant error. Page 2 of 2 Serum concentration (jjg/nnL) 14 M2052 -O- Observed -- Predicted Serum Concentration (ug/mL) M2054 -e- Observed -- Predicted Figure 1 A Pharmacokinetic Study of Potassium Perfluorooctanoate in the Cynomolgus Monkey Serum Concentration Profiles of Perfluorooctanoate in Monkeys 15 M2211 Observed -- Predicted Serum concentration (ug/mL) Serum concentration (|jg/ml_) F2058 -o- Observed -- Predicted Figure 1 (Continued) A Pharmacokinetic Study o f Potassium Perfluorooctanoate in the Cynomolgus Monkey Serum Concentration Profiles o f Perfluorooctanoate in Monkeys 16 F2059 Observed Predicted Serum concentration ((jg/mL) Serum concentration (|jg/mL) F2061 Observed Predicted Figure 1 (Continued) A Pharmacokinetic Study of Potassium Perfluorooctanoate in the Cynomolgus Monkey Serum Concentration Profiles of Perfluorooctanoate in Monkeys Appendix A Study Protocol and Amendments A-l Study Protocol: A Pharmacokinetic Study of Potassium Perfluorooctanoate in the Cynomolgus Monkey Southern Research Study ID: 9921.4 October 4, 2000 Southern Research INSTITUTE STUDY NO.: 9921.4 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 55144-322Q (651) 733-1962; FAX: (651) 733-1773 Protocol Approval: (Initial last page also) October 4, 2000 2P age o f 13 Test Article: Ship Unused Test Article to: John L. Butenhoff Perfluorooctanoate, potassium salt D. Hakes Building B236 3M P.O. Box 33327 55133-3327 3M Center, 224-IN-04 St. Paul, Minnesota 55144-1000 Date A -3 STUDY NO.: 9921.4 2.0 TITLE: 4October , 2000 Page 3 o f 13 A Pharmacokinetic Study of Potassium Perfluorooctanoate in the Cynomolgus Monkey 3.0 OBJECTIVE: The objectives o f this study are to determine the concentration o f perfluorooctanoate in serum and urine at various times following administration o f a single intravenous dose of potassium perfluorooctanoate 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 -3 1 2 7 14 21 28 0: 0 (predose), 0.5, 2, 4, 8 and 24 hours postdose 2 4 7 11 14 21 28 60 Calendar days after completion of the in-life phase 15 days after final Sponsor comments received Date(s) - Year 2000 10/9/00 10/6/00 10/10/00 10/11/00 10/16/00 10/23/00 10/30/00 11/6/00 10/9 - 10/00 10/11/00 10/13/00 10/16/00 10/20/00 10/23/00 10/30/00 11/6/00 1/5/01 A -4 STUDY NO.: 9921.4 6.0 STUDY PERSONNEL: October 4, 2000 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 o f the study, residual bulk test article will be returned to the Sponsor. 7.1 Identity o f the Test Article: Name: Identification: Supplier: Lot Number(s): Special Handling: Perfluorooctanoate, potassium salt T-7507 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 o f the Sponsor. A -5 STUDY NO.: 9921.4 7.2 Identity of the Vehicle : October 4, 2000 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) of expiration provided by the manufacturer when stored appropriately. The bulk vehicle will be stored in accordance with the manufacturer's instructions. 7.3 Formulation: Preparation: The test article will be formulated in sterile saline at a concentration of 5 mg/mL for intravenous administration; briefly, the required amount of test article will be mixed with the required amount o f 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 (Macacafascicularis) Charles River BRF, Inc. (Houston, TX) 3-4 years o f 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, and potassium perfluorohexanoate in study 9921.3. A -6 STUDY NO.: 9921.4 8.1 Justification: October 4, 2000 Page 6 o f 13 Primates are commonly used in preclinical pharmacological and toxicological evaluations o f compounds used or intended for use in humans, or to which humans might be exposed. 8.2 H o u s in g : 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 of 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 B e d d in g : 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 of 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 of the animals. 8.5 Water: Water (Birmingham public water supply) will be supplied ad libitum during the quarantine and study periods via an automatic watering system. Samples of water from the animal facility will be periodically analyzed, and the analyses will be reviewed by the DVMB o f Southern Research to assure that no known contaminants are present that could affect the health of the animals. A -7 STUDY NO.: 9921.4 8.6 Quarantine: October 4, 2000 Page 7 o f 13 All primates were selected from stock animals that will have been quarantined for a minimum o f 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 o f B virus titer (primates found to have a positive B vims 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 staff veterinarian, will determine the course o f 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. 8.7 Psychological Well-Being and Socialization: Nonhuman primates will be provided a psychological well-being program for social enrichment as directed by a veterinarian and approved by the IACUC and in accordance with the appropriate SOP. Nonhuman primates will be provided cage and feeding regimen modifications daily for their psychological well-being. The modifications include, but are not limited to: swings, perches, Kong toys, clean 2liter soft drink bottles, puzzle feeders, nutritionally sound primate treats, unshelled peanuts, and raw fruit. Where possible, primates will be housed proximate to one another for visual and vocal contact. 8.8 Animal Identification: During quarantine, the primates will be individually identified by chest tattoo number or letter combination. Positive identification will be required after every cage change and prior to blood sampling, dose administration, and observation. A-8 STUDY NO.: 9921.4 9.0 EXPERIMENTAL DESIGN: October 4, 2000 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 perfluorooctanoate by injection into a superficial arm or leg vein. Blood samples for serum drug level determinations will be collected from each primate at selected time points during the study. Urine and feces will also be collected at pre determined intervals. A synopsis o f the study design is presented in the following table. Day of Study Study Procedures - la 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 21 28 Health Check X Dose Preparation X Dosing X Clinical Observations XXX X X X X X 28 Body Weights X XX XXX Urine & Feces X XX X XXX Serum Drug Levels XXX X X X XXX 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 (IV) dose o f potassium perfluorooctanoate (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 of the study. 9.3 Clinical Observations: 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 o f mortality/moribundity and overt toxicity. Animals found in extremis will A -9 STUDY NO.: 9921.4 October 4, 2000 Page 9 o f 13 be humanely sacrificed by an overdose of barbiturate followed by exsanguination with appropriate approval. Detailed O bservations: Each primate will be examined shortly after dose administration for detailed clinical signs of toxicity. All findings will be recorded. Additional clinical observations will be performed and recorded on days of blood collection. 9.4 B o d y W e ig h t s : Each primate will be weighed on Days -1 , 4, 7, 14, 21, and 28. 9.5 U r in e a n d Feces C o lle c t io n s : Urine and feces will be collected on Days -3 ,1 (0-24 hours postdose), 2 (24-48 hours post dose), 7, 14, 21, and 28. 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 Se r u m D rug L evels : 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 1 ,1 4 ,2 1 , and 28 days after dosing. Samples will be collected into tubes without anticoagulant and will be allowed to clot at room temperature. The blood samples will then be centrifuged, and the serum will be separated and stored frozen at -2 0 C or below until analyzed. 9.7 B io a n a l y t ic a l M eth o d D e v e lo p m e n t : Bioanalytical method(s) are to be developed for the determination of perfluorooctanoate in serum and urine matrices. The method will be validated for accuracy and should be sensitive to the 1 ppm or less level. If feces and other tissue methods need to be developed, such a decision would result in additional cost. 9.8 B io a n a l y t ic a l Sa m p le A n a ly s is : The serum and urine samples from all monkeys will be analyzed for the concentrations of perfluorooctanoate using the previously validated method. The data will be expressed as equivalents o f potassium perfluorooctanoate. The analytical data on samples collected through and including Day 14 should be available by Day 21 postdose. The Sponsor will use this information to make A -10 STUDY NO.: 9921.4 October 4, 2000 Page 1 0 o f 13 decisions concerning possible blood sampling o f animals beyond Day 28 postdose. Feces will be analyzed only if requested by the Sponsor. 9.9 Animal Disposition: At the end o f 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 o f unchanged perfluorooctanoate, as appropriate and feasible, using a standard pharmacokinetic program. The total amount o f perfluorooctanoate in urine will be calculated and expressed in terms of percent o f 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 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 o f the final report will be retained in the central archives at Southern Research. 12.0 FINAL REPORT: A brief letter report summarizing the serum drug level results will be issued as soon as the information is available. A draft final report will be issued within 60 calendar days after completion 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: Dose formulation preparation Clinical observations Body weight data Serum drug level data Pharmacokinetic parameters Urine excretion data A - 11 STUDY NO.: 9921.4 13.0 R EG U LA TORY REFEREN CES: October 4, 2000 Page 1 1 o f 13 This study will be conducted in accordance with the protocol and the Standard Operating Procedures (SOPs) o f Southern Research, and in accordance with the applicable regulatory requirements, as addressed below. 13.1 protocol Amendments and deviations: A m endm ents: 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 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 GFR Part 58). Data from this study may be submitted to the FDA in support o f an IND/NDA application. Q uality A ssurance 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. 13.3 Facilities Management and Animal Husbandry: Animal care will be in compliance with the SOPs o f Southern Research, the Guidelines fo r the Care and Use o f Laboratory Animals, 7th Edition (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). A -12 STUDY NO.: 9921.4 13.4 A n im a l W elfar e A ct Co m p lia n c e : October 4, 2000 Page 1 2 o f 13 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 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.: 9921.4 14.0 PROTOCOL APPROVALS: This protocol has been reviewed and approved. October 4, 2000 Page 13 o f 13 Study Director: _.y T ___________________ fl/ J 4 /o o Patricia E. Noker, Ph.D., D.A.B.T. Date Study Director Sponsor's Monitor: INITIALS ONLY (See page 2) Date Management Approval: Ward R. Richter, M.S., D.V.M., D.A.C.V.P. / / yAp 7 Date Director, Safety Assessment Department, Southern Research Institute Appendix B Method Validation Report: Validation of Analytical Method for Determination of Perfluorooctanoate in Monkey Serum and Urine Using HPLC/MS/MS  B-l METHOD VALIDATION REPORT VALIDATION OF AN ANALYTICAL METHOD FOR DETERMINATION OF PERFLUOROOCTANECARBOXALATE IN MONKEY SERUM AND URINE USING HPLC/MS/MS STUDY ID: 9921.4.2 Southern Research Institute 2000 Ninth Avenue South P.O. Box 55537 Birmingham, AL 35255-5537 i B -2 SUMMARY Southern Research Institute has successfully validated for 3M an analytical method (BACG 3548) entitled "Determination o f Perfluorooctanecarboxylate in Monkey Serum and Urine: Sample Preparation and Analysis by HPLC Mass Spectrometry/Mass Spectrometry (HPLC/MS/MS)". Calibration standards were prepared by spiking samples of either blank monkey serum or human urine with known amounts o f test article, perfluorooctanecarboxalate (PFOC) and internal standard, perfluorohexanesulfonate (PFHS). The concentration of test article in serum covered a combined range from 10 to 100,000 ng/mL while the urine extended from 5 to 500 ng/mL. Quantitation limits were set at 20 ng/mL for serum and 10 ng/mL for urine. Composite standard curves generated using internal standard quantitation were used to determine the correlation coefficients. For the serum, three composite curves composed of a total o f 44 individual calibration standards extending over concentration ranges from 5000 100,000 ng/mL, 100 - 10,000 ng/mL and 1 0 -5 0 0 ng/mL produced correlation coefficient of 0.9874, 0.9750 and 0.9977 respectively. A single composite curve prepared from urine over a concentration range of 5 - 500 ng/mL and containing 20 individual calibration standards produced a correlation coefficient of 0.9960. B-3 KEY PERSONNEL James D. Johnson, M.S., MBA Manager Bioanalytical Chemistry Group Gregory S. Gorman, Ph.D. Staff Chemist Bioanalytical Chemistry Group Lester Williams, B.S. Associate Chemist II Bioanalytical Chemistry Group George Dollar Associate Biologist I Bioanalytical Chemistry Group t B-4 1. OBJECTIVE The objective of this study was to provide a validated analytical method for the determination of perfluorooctanecarboxalate in monkey serum and urine. 2. SAFETY All necessary procedures to ensure safety o f 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 o f zoonoses, all unpreserved tissues, including blood, plasm a and serum, are to be considered as biohazards and handled with universal precautions. Refer to SOP number SRI 2-5-5 for a description o f 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 Perfluorooctanecarboxalate in Monkey Serum and Urine: 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 blank serum or urine (e.g., 500 pL) was spiked with a known amount o f test article (PFOC) and internal standard (PFHS). To each standard was added 500 pL o f TBA ion-pairing solution, 1 mL o f carbonate/bicarbonate buffer, and 1 mL of deionized water. This mixture was vortexed for approximately 5 seconds. Then 2.5 mL o f ethyl acetate was added and the mixture was placed on a horizontal platform shaker 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 50C under a stream o f dry nitrogen. The residue was reconstituted in 5% 5mM ammonium acetate in deionized water : 95% methanol containing 1.5% formic acid and filtered through a 0.2 pm syringe filter. 3.2 Method Validation Validation for BACG 3548 "Determination o f Perfluorooctanecarboxalate in Monkey Serum and Urine: Sample Preparation and Analysis by HPLC Mass Spectrometry/Mass Spectrometry (HPLC/MS/MS)" consisted of analyzing standard curves prepared in monkey serum or human B-5 urine over various concentration ranges: 5,000 - 100,000 ng/mL, 100 - 10,000 ng/mL and 10 500 ng/mL for serum and 5 - 5 0 0 ng/mL for urine. 5.000 -100.000 ng/mL (Seruml One composite calibration curve comprising 3 sets o f calibration standards in a single analytical run in the 5,000 - 100,000 ng/mL concentration range generated from a total of 22 calibration standards produced a correlation coefficient o f 0.9874. A statistical summary for the composite curve is shown below: Cone. (ng/mL) # o f Stds Mean % Accur. Std. Deviation STANDARD COMPOSITE CURVE 1 (ng/mL) 5000 3 93.9 385 10,000 2 102 31.9 20,000 25,000 23 98.7 107 1193 3917 50,000 75,000 54 101 95.3 2016 3048 100,000 3 102 13580 100 -10.000 ng/mL fSerumi Two composite curves consisting o f a total o f 12 calibration standards encompassing a concentration range o f 100 - 10,00 ng/mL produced correlation coefficient o f 0.9750 The 5,000 ng/mL standards were dropped due to preparation error. A statistical summary for the composite curve is given below: STANDARD COMPOSITE CURVE 2 ing/mLI Cone. (ng/mL) # o f Stds Mean % Accur. Std. Deviation 100 200 500 1000 2000 10,000 2 22222 93.5 109 114 93.8 86.1 102 14.5 32.1 106 14.7 307 852 10 - 500 ng/mL iSeriiml A single composite curve consisting o f 11 standards taken from an analytical run encompassing a concentration range o f 10 - 500 ng/mL produced a correlation coefficient o f 0.9977. No calibration standards were dropped from the run. A statistical summary for the composite curve is given below # B -6 Cone. (ng/mL) # of Stds Mean % Accur. Std. Deviation STANDARD COMPOSITE CURVE 3 ing/m D 10 20 50 100 200 500 2 222 12 96.6 100 101 106 90.6 101 0.51 0.97 1.04 2.08 -- 38.1 5 - 500 ng/mL (Urinel , A composite curve prepared in urine consisting of 20 standards over a range o f 5 - 500 ng/mL produced a correlation coefficient of 0.9960. Only one calibration standard, 5 ng/mL was dropped due to low internal standard response. A statistical summary for the composite curve is given below: STANDARD COMPOSITE CURVE 4 (ng/mL) Cone. (ng/mL) # o f Stds Mean % Accur. Std. Deviation 5 10 20 50 100 200 500 2 333333 94.7 107 107 82.7 107 101 99.6 0.07 0.27 3.41 4.20 8.2 9.0 14.6 3.3 Calculations Calculations were performed using TurboQuan (Version 1.0). The amount o f 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 o f weighting. A quadratic fit with 1/X weighting was determined to be the best f i t : y = ax2 + bx + c where: y - Peak height response of PFOC divided by peak height response of the IS (PFHS) in standards. x = Concentration of the PFOC in standards. a, b, c = Constants derived from the regression analysis. i B -7 4.0 Conclusion A quantitative method (BAGC 3548) has been developed and validated for the determination of perfluorooctanecarboxalate in monkey serum utilizing LC/MS/MS. Quantitation for this method is based on an internal standard (PFHS) using a 1/x weighted quadratic equation. The total concentration range for a serum matrix is 20 - 100,000 ng/mL and 5 - 5 0 0 ng/mL for urine with limits o f quantitation o f 20 and 10 ng/mL respectively. Appendix C Analytical Method for Determination of Perfluorooctanoate in Monkey Serum and Urine C -l Page 1 of 13 ANALYTICAL METHOD Method No.: BACG-3548 Title: Determination of Perfluorooctanecarboxalate in Monkey Serum and Urine: Sample Preparation and Analysis by HPLC Mass Spectrometry/Mass Spectrometry (H PL C /M S /M S ) 1.0 PRINCIPLE Serum or urine samples are obtained from cynomolgus monkeys treated with Perfluorooctanecarboxalate (PFOC). The serum or urine (e.g., 0.5 mL) containing (PFOC) is fortified with an internal standard (IS), perfluorohexanesulfonate (PFHS). 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 of reliable results extends from about 20 to 100,000 ng/mL of PFOC in serum and from 10 to 500 ng/mL in urine. Samples containing PFOC at concentrations greater than 100,000 ng/m L may be diluted with control blank matrix so that the concentration of PFOC will be within the range of reliable results prior to analysis. The mass spectrometry of PFOC and PFHS 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 matrix. In order to maximize sensitivity, this method is based on mixed reaction monitoring of the negative fragment ion (M-COOH)' for PFOC. CAUTION: Since primates may carry a number of zoonoses, all unpreserved tissues, including blood, plasma and serum, are to be considered as biohazards and handled with universal precautions. Refer to SOP number SRI 2-5-5 for a description of safety procedures to be used when handling unpreserved primate tissue. 2.0 REAGENTS AND SOLUTIONS The listed reagents or their equivalents may be used. 2.1 Neat Reagents C-2 Page 2 of 13 ANALYTICAL METHOD Method No.: BACG-3548 Title: Determination of Perfluorooctanecarboxalate in Monkey Serum and Urine: Sample Preparation and Analysis by HPLC Mass Spectrometry/Mass Spectrometry (HPLC/MS/MS) 2.1.1 W ater, deionized and organic free (from in-house purification system; e.g., Ingalls 2 ION) 2.1.2 Methanol, HPLC grade 2.1.3 Perfluorooctanecarboxalate (analyte), as provided by the client 2.1.4 Perfluorohexanesulfonate (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 2.1.11 Sodium Hydroxide 50% solution, Certified grade or equivalent 2.1.12 Blank control monkey urine 2.1.13 Formic Acid 2.2 Prepared Solutions Appropriate changes in the solutions may be made at the discretion of the analyst 2.2.1 5 mM Ammonium acetate in organic free water C-3 Page 3 of 13 ANALYTICAL METHOD Method No.: BACG-3548 Title: Determination of Perfluorooctanecarboxalate in Monkey Serum and Urine: Sample Preparation and Analysis by HPLC Mass Spectrometry/Mass Spectrometry (HPLC/MS/MS) 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. Note: a more dilute solution of NaOH in water may be used to effect smaller pH adjustments. 2.2.3 Carbonate/Bicarbonate Buffer Solution 2.2.3.1 For example, to prepare 100 mL, dissolve approximately 2.65 g o f 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, 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 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 3.6 Filters for HPLC mobile phase filtration apparatus (e.g., Nylon-66, 0.20 /m) C-4 Page 4 of 13 ANALYTICAL METHOD Method No.: BACG-3548 Title: Determination of Perfluorooctanecarboxalate in Monkey Serum and Urine: Sample Preparation and Analysis by HPLC Mass Spectrometry/Mass Spectrometry (HPLC/MS/MS) 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 pipets 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 W ORKING STOCKS Appropriate changes in the concentrations of the solutions may be made at the discretion of the analyst. Actual dilutions will be documented on the preparation sheets. M ain Stock Solution of PFOC ~ 1000 pg/m L , Prepare an ~ 1000 pg/m L solution of PFOC in deionized organic-free water (e.g., accurately weigh about 10 mg PFOC into a 10-mL volumetric flask). Add deionized organic-free water to dissolve. Dilute to the mark. Alternatively, weigh the compound C-5 Page 5 of 13 ANALYTICAL METHOD Method No.: BACG-3548 Title: Determination of Perfluorooctanecarboxalate in Monkey Serum and Urine: Sample Preparation and Analysis by HPLC Mass Spectrometry/Mass Spectrometry (HPLC/MS/MS) 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 In te rn a l S ta n d a rd (PFH S), --200 pg/m L Prepare an --200 pg/m L solution of PFHS 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. Spiking solution of Internal Standard (PFHS), -- 50pg/mL Prepare an -- 50 pg/mL solution of PFHS in deionized organic- free water by pipetting 2 mL of the 200 pg/mL solution into a culture tube and add 6 mL o f deionized water. Mix well. W orking Stock Solutions of PFOC 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. Working Stock Level (WSL) Approximate Concentration (ng/mL) 500,000 250,000 Volume of PFOC solution 5 mL of 1000 pg/mL 5 mL of 500,000 ng/mL Final Volume in deionized organic free water (mL) 10 10 C-6 Page 6 of 13 ANALYTICAL METHOD Method No.: BACG-3548 Title: Determination of Perfluorooctanecarboxalate in Monkey Serum and Urine: Sample Preparation and Analysis by HPLC Mass Spectrometry/Mass Spectrometry (HPLC/MS/MS) 62,500 31,250 15,625 5,000 2,500 1,000 500 250 2.5 mL of 250,000 ng/mL 5 mL of 62,500 ng/mL 5 mL of 31,250 ng/ mL 50 fiL of 1000 /xg/mL stock 5 mL of 5000 ng/mL stock 4 mL of 2500 ng/mL stock 5 mL of 1000 ng/mL stock 5 mL of 500 ng/mL stock 10 10 10 10 10 10 10 10 4.4.2 Summary of concentrations of serum standards: Standard Level Volume and Spike Cone. Approximate Concentration of PFOC in serum (ng/mL) A 50 jlL of 1000 /xg/mL 100,000 B 37.5 /xL of 1000 /xg/mL 75,000 C 25 /xL of 1000 /xg/mL 50,000 D 12.5 /xL of 1000 /xg/mL 25,000 E 10 /xL of 1000 /xg/mL 20,000 C-7 Page 7 of 13 ANALYTICAL METHOD Method No.: BACG-3548 Title: Determination of Perfluorooctanecarboxalate in Monkey Serum and Urine: Sample Preparation and Analysis by HPLC Mass Spectrometry/Mass Spectrometry (HPLC/MS/MS) F 10 pL of 500,000 ng/mL G 10 pL of 250,000 ng/mL H 16 pL of 62,500 ng/mL I 16 pL of 31,250 ng/mL J 16 pL of 15,625 ng/mL K 20 pL of 5,000 ng/mL L 10 pL of 5,000 ng/mL M 10 pL of 2,500 ng/mL N 10 pL of 1,000 ng/mL O 10 pL of 500 ng/mL P 10 pL of 250 ng/mL 10,000 5,000 2,000 1,000 500 200 100 50 20 10 5 5.0 PREPARATION OF SPIKED STANDARDS AND BLANKS Appropriate changes in the concentrations of the solutions may be made at the discretion of the analyst. 5.1 Multiple (e.g., about three) sets of matrix standards and a matrix blank (blank + IS) are analyzed with each set of unknown samples. A matrix double blank (blank-IS) may also be analyzed if desired. 5.2 Into individual --20-mL culture tubes, pipet blank matrix (e.g., 0.5 mL ). Pipet in the appropriate volume as described in the table above for each standard. For the blanks, pipet 10 pL of organic-free water instead of the working stock solution. Add the 10 pL C-8 Page 8 of 13 ANALYTICAL METHOD Method No.: BACG-3548 Title: Determination of Perfluorooctanecarboxalate in Monkey Serum and Urine: Sample Preparation and Analysis by HPLC Mass Spectrometry/Mass Spectrometry (HPLC/MS/MS) o f internal standard stock ( --50 pg/mL) to each tube except the blank-IS (pipet 10 pL o f organic-free water instead) and vortex for --5 seconds. 5.3 To each tube add 500 pL of the TBA ion-pairing solution, 1 mL of carbonate/bicarbonate buffer, and 1 mL of deionized organic free water. Vortex each tube for about 5 seconds. 5.4 Add 2.5 mL of ethyl acetate and extract on horizontal mixer for 1 hour at a low speed setting. 5.5 Remove the tube from the shaker and place in a centrifuge (e.g., 2500 rpm) for about 5 minutes. 5.6 Take off the top ethyl acetate layer and put it into a clean tube and evaporate to dryness (e.g., -- 50 minutes in the Turbo-Vap ) with a gentle stream o f nitrogen and moderate heat (e.g., 50 C). 5.7 Reconstitute the residue in 500 pL of 5% 5 mM ammonium acetate: 95% methanol containing 1.5% formic acid and vortex briefly to mix. Filter the samples through 0.2 pm PVDF or Nylon syringe filters into autosampler vials. 6.0 PREPARATION OF SAMPLES 6.1 Allow each serum sample to thaw to room temperature. Vortex each sample briefly, but vigorously. Pipet an aliquot of each sample (e.g., 0.5 mL) into individual --20 mL culture tubes. If necessary, dilute an aliquot of any sample with blank matrix so that the expected concentration of the test article being analyzed will fall within the concentration range of the standard curve. Add 10 pL o f internal standard stock ( -- 50 pg/mL) to each tube and vortex for a couple of seconds. 6.2 To each tube add 500 pL of the TBA ion-pairing solution, 1 mL of carbonate/bicarbonate buffer, and 1 mL of deionized organic free water. Vortex each tube for about 5 seconds. C-9 Page 9 of 13 ANALYTICAL METHOD Method No.: BACG-3548 Title: Determination of Perfluorooctanecarboxalate in Monkey Serum and Urine: Sample Preparation and Analysis by HPLC Mass Spectrometry/Mass Spectrometry (HPLC/MS/MS) 6.3 6.4 6.5 6.6 7.0 7.1 7.1.1 Add 2.5 mL of ethyl acetate and extract on horizontal mixer for 1 hour at a low speed setting. Remove the tube from the shaker and place in a centrifuge (e.g ., 2500 rpm) for about 5 minutes. Take off the top ethyl acetate layer and put it into a clean tube and evaporate to dryness (e.g., ~ 50 minutes in the Turbo-Vap ) with a gentle stream o f nitrogen and moderate heat (e.g., 50 C). Reconstitute the residue in 500 fiL of 5% 5 mM ammonium acetate: 95% methanol containing 1.5% formic acid and vortex briefly to mix. Filter the samples through 0.2 /xm PV D F or Nylon syringe filters into autosampler vials. Cap vials for analysis. 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 C18, 150 mm x 2 mm ID, or equivalent Guard Column: Elution Flow rate: Injection volume: Mobile phase: Keystone Aquasil C l 8 10 mm x 2 mm 400 L/min. 3 JLL A : 5mM ammonium acetate buffer B: 1.5% formic acid in methanol Gradient Profile: 0 - 0 . 5 min. 50%A : 50%B 0.5 - 7.5 min. 10% A : 90% B linear gradient 7.5-8 min. 10%A : 90% B step gradient C-10 Page 10 of 13 ANALYTICAL METHOD Method No.: BACG-3548 Title: Determination of Perfluorooctanecarboxalate in Monkey Serum and Urine: Sample Preparation and Analysis by HPLC Mass Spectrometry/Mass Spectrometry (HPLC/MS/MS) 7.1.2 ^ Temperature: 8-11 min. Ambient 5 0 % A : 5 0 % B\ PE Sciex API 3000 Triple Quadrupole Mass Spectrometer Conditions Software: PE Sciex TurboQuan Turboion vSprav 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 NC TEM OR RNG QO IQ1 ST ROl IQ2 R02 ST3 R03 DF CEM Value -2000 0 400 -20 -120 5 6 10 6 10 30 40 32 250 1800 C -ll Page 11 of 13 ANALYTICAL METHOD Method No.: BACG-3548 Title: Determination of Perfluorooctanecarboxalate in Monkey Serum and Urine: Sample Preparation and Analysis by HPLC Mass Spectrometry/Mass Spectrometry (HPLC/MS/MS) Parameter NEB CUR CAD QPE POL VCM IPE Value 15 6 5 0 1 0 0 MS/MS Acquisition Conditions Scan type: MRM Polarity: Negative Acquisition mode: Profile Pause time: 5 milliseconds Masses requested: PFOC: Q1 Mass (amu) 412.9 Q3_Mass (amu) 368.9 Dwell Time (ms) 200 PFHS (IS) Q1 Mass (amu) 398.9 Q3 Mass (amu) 398.9 Parameter R02 ST3 R03 Start 50 60 52 Stop 50 60 52 Dwell Time (ms) 200 C-12 Page 12 of 13 ANALYTICAL METHOD Method No.: BACG-3548 Title: Determination of Perfluorooctanecarboxalate in Monkey Serum and Urine: Sample Preparation and Analysis by HPLC Mass Spectrometry/Mass Spectrometry (HPLC/MS/MS) 8.0 CALCULATIONS 8.1 At the end of the analytical run, review each chromatogram to ensure the retention time, peak shape, and peak height and peak area determination of the test article and 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 PFOC PFHS Ton Profile 412.9 to 368.9 398.9 to 398.9 8.2 Plot the peak area response of PFOC divided by the peak area response of the IS (PFHS) from all standards versus the concentration of the test article in the standards. Alternatively, the peak heights may be used instead of peak areas. Obtain the best curve fit of 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 PFOC divided by peak height response of the IS (PFHS) in standards, Concentration of the PFOC in standards, = Constants derived from the regression analysis. 8.3 Using the standard curve, calculate the level of PFHC in each unknown sample. Correct the results of samples for any dilutions. C-13 Page 13 of 13 ANALYTICAL METHOD Method No.: BACG-3548 Title: Determination of Perfluorooctanecarboxalate in Monkey Serum and Urine: Sample Preparation and Analysis by HPLC Mass Spectrometry/Mass Spectrometry (HPLC/MS/MS) 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. 9.0 9.1 10.0 10.1 ACCEPTANCE AND REJECTION CRITERIA Refer to SOP SRI 91-3 for acceptance/rejection criteria except acceptable accuracy for standards is 80-120% of theoretical. REPORTING Results of all analyses are tabulated, and the raw data, original chromatograms, and reports are to be filed in the appropriate study file. Authors: G regory's. Gorman, Ph.D ., Staff Chemist Bioanalytical Chemistry Group Approved by: Uly arns D. John; nnviS, MBA, Manager B/oanalytical mistry Group Date Is. - 3 7-et Date