Document g7dn2ZgwX7anYJ3JML7DEkMG

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. BOX33327 55133-3327 3M Center, 224-IN-04 St. Paul, Minnesota 55144-1000 By: P.E. Noker and G.S. Goman 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 of 10 mg/kg of 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 of 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 fi-om 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 of decrease appeared to be faster in male monkeys than in female monkeys. On Day 28, the concentrations of perfluorooctanoate in serum ranged fi-om 1863 to 27,140 ng/& in the male monkeys and fi-om 7,145 to 33,680 n g / d in the female monkeys. Due to the relatively high serum concentrations of the compound on this day, the length of serum collection was extended through Day 123. On Day 123,the serum concentrationof perfluorooctanoatewas at or only slightly above the limit of quantitation (20 ng/mL) in male monkeys and between 885 and 4701 ng/& in female monkeys. The serum concentration versus time data were subjected to non-compartmental pharmacokinetic analysis. The terminal half-life of 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 of distribution of the compound at steady state (Vd,,) 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. Perfluorooctanoatewas slowly excreted in urine by both male and female monkeys; levels of perfluorooctanoate, representing as much as 1% of the administered dose, were present in urine 28 days after dosing. The results of this study suggested that the pharmacokinetics of perfluorooctanoate may be different in male and female monkeys. i SIGNATURE PAGE TABLE OF CONTENTS PaEe ... 111 GOOD LABORATORY PRACTICES DISCLAIMER iv STUDY SCHEDULE AND PERSONNEL V 1.0 INTRODUCTION 1 2.0 MATERIALS AND METHODS 1 2.1 Test System 1 2.2 Test Article and Vehicle 2 Test Article 2 Vehicle 3 Dose Formulation Preparation 3 Dose Formulation Analyses 3 2.3 Experimental Design 3 Group Assignment and Dose Procedure 3 Clinical Observations 3 Body Weights 3 Urine and Feces Collection 3 Serum Levels of Perfluorooctanoate 4 Bioanalytical Method Development and Sample Analysis 4 Data Analyses 4 3.0 RESULTS 4 3.1 Mortality 4 3.2 Clinical Observations 5 3.3 Body Weights 5 3.4 Serum and Urine Concentrations of Perfluorooctanoate 5 4.0 DISCUSSION 7 5.0 CONCLUSIONS 8 6.0 RECORD ARCHIVES 8 7.0 REFERENCES 8 ii Table 1: Table 2: Table 3: Table 4: Figure 1: Appendix A: Appendix B: Appendix C: TABLE OF CONTENTS (Continued) LIST OF TABLES PaEe Individual Body Weights 9 Serum Concentrations of Perfluorooctanoate 10 Pharmacokinetic Parameters Calculated from Serum Concentrations 11 of Perfluorooctanoate Urinary Excretion of Perfluorooctan0ate 12 LIST OF FIGURES Serum Concentration Profiles of Perfluorooctanoate in Monkeys 14 LIST OF APPENDICES Study Protocol and Amendments A- 1 Method Validation Report: Validation of Analytical Methods for B-1 Determination of Perfluorooctanoate in Monkey Serum and Urine Using HPLC/MS/MS Analytical Method for Determination of Perfluorooctanoate in Monkey Serum and Urine c-1 ... 111 Signature Page A Pharmacokinetic Study of Potassium Perfluorooctanoatein the Cynomolgus Monkey y&>+;>27 3 4 Patricia E. Noker, Ph.D., D.A.B.T. Study Director Supervisor, ADME & Pharmacokinetics Reviewed by: j / / y1/ 0 3 Date Charles D. Hkbert, Ph.D., D.A.B.T. Date Director, Safety Assessment We, the undersigned, were responsible for the conduct of the work and reporting of the results in the listed sections. We concur with the views relative to our body of work as expressed in the discussion and conclusions. Manager, Bioanalytical Chemistry Group iv Good Laboratory Practices Disclaimer T h s study described in this final report was not conducted in strict compliancewith the U.S. Food and Drug Administration (FDA) Good Laboratory Practice (GLP) Regulations (21 CFR Part 58), and neither t h s report nor the raw data were reviewed by the Southern Research Quality Assurance Unit. However, the study was conducted according to the protocol and amendments and the applicable standard operating procedures, and all study procedures, data recording, and reporting were performed in a manner consistent with the standard of GLPs. The final report accurately reflects the raw data obtained during the performance of the study. There were no adverse circumstances that affected the quality or integrity of the study. -7F&-&r ...., 2-L 7 4 Patricia E. Noker, Ph.D., D.A.B.T. Study Director i / / q L/ o 3 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. Hkbert, Ph.D., D.A.B.T. Norman D. Jefferson, B.S. Director, Safety Assessment 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 of this study were to determine the concentration of potassium perfluorooctanoate 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 fiom 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 River BRF, Inc. (Houston, TX) and were an estimated 3-4 years of age when placed on study. Individual animal identification was by chest tattoo. The cynomolgus monkey is an accepted species to support clinical studies of drugs used or intended for use in humans. During the quarantine period, a complete physical examination including a fecal examination for internal parasites, complete blood count (CBC), body weight, and rectal temperature was performed on each of the monkeys. The following procedures were performed on the monkeys during quarantine: (1) Three tuberculin tests were administered to each animal at 2-week intervals. All tuberculin tests were administered intrapalpebrally. The three tuberculin tests were negative for all monkeys. (2) Blood was drawn for CBC and B virus titer. (3) Fecal cultures (screening for Salmonella and Shigella) were obtained, and fecal flotation tests were performed. (4) In general, primates were examined at least once weekly by an approved veterinarian and were observed (cage-side observations recorded by exception only) twice daily for abnormal clinical signs and mortality/moribundity. Housing, feed, water, and socializationprocedures remained the sarne 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 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 of the study, the monkeys were housed in a room that was maintained at a temperature of 68.0-70.3 O 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 1800hours, CST) and 12hours of dark per day. Cage size and animal care conformed to the guidelines of the Guidefor the Care and Use of LaboratoryAnimals, 7th edition'') and the U.S. Department of Agriculture through the Animal Welfare Act (Public Law 99-198) and to the applicable Standard Operating Procedures (SOPS) of Southern Research. The study design was approved by Southern Research's Institute Animal Care and Use Committee (IACUC). Southern Research is filly accredited by the American Associate for Accreditation of Laboratory Animal Care International ( M A C ) . The monkeys used on this study were previously given a single IV bolus dose of perfluorobutanesulfonate (10 mgkg) on April 10, 2000 (Southern Research Study No. 9921.1); a single IV bolus dose of potassium perfluorobutanoate (10 mgkg) on June 13, 2000 (Southern Research Study No. 9921.2); and a single IV bolus dose of potassium perfluorohexanoate (10 m a g ) on July 31,2000 (Southern Research Study No. 9921.3). 2.2 Test Article and Vehicle Test Article: One bottle containing 5.2 grams of potassium perfluorooctanoate (T-7507; expiration date not supplied; Southem 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 of the Sponsor. 3 Vehicle: The vehicle used for the preparation of 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 of 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 of the three male and three female monkeys received a single intravenous (IV) dose of perfluorooctanoate at 10mgkg 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 specificallyrequested 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 123postdose. Sampleswere 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 of perfluorooctanoate 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: The serum concentration data for unchanged perfluorooctanoate were subjected to non-compartmentpharmacokmetic analysis using WinNonlin (Standard Edition; Version 1.1;ScientificConsulting Inc.; C q , NC). Mean values and standard deviations for each parameter were calculated using Microsoft Excel@ software (Microsoft Corporation; Irvine, CA). The urinary excretion of perfluorooctanoate at each collection interval was calculated and expressed as a percent of 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 of the three male monkeys (2052) was euthanized on Day 79 because of repeated episodes of self-mutilation;there was no indication that the self-mutilation was related to administration of 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 of 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 of treatment. The social enrichment of the monkey was also augmented in order to provide the monkey additional stimulationand to change h s focus of attention. On Day 79, the monkey was examined and several severe lacerations were observed on the leg and foot of the animal. These lacerations were diagnosed as self-inflicted wounds. Due to the repeated instances of self-mutilation and the likelihood of continued episodes of selfmutilation, 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 of 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 of perfluorooctanoate in monkey serum and urine. Details of the method validation procedure are provided in Appendix B. Details of the analytical method are provided in Appendix C. The lower limit of quantitation of the method was 20 ng/mL for serum samples and 10 ng/mL for urine samples. Serum concentrations of perfluorooctanoate in three male and three female monkeys at various times through Day 123 after administration of a single iv dose of 10 mgkg are presented in Table 2 and plotted in Figure 1. At 0.5 hours after dosing, serum concentrations of perfluorooctanoatewere similar in male and female monkeys and ranged from 91,130to 96,660 ng/mL in the male monkeys and from 88,940 to 96,400 ng/mL in the female monkeys. Serum concentrations of perfluorooctanoate subsequently declined at a slow rate; the rate of 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 of 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 of perfluorooctanoate in two of 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 of serum collection was extended through Day 123. On Day 123,the serum concentrationof 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. Pharmacokineticparameters calculated fiom serum concentrations of perfluorooctanoatein individual monkeys are presented in Table 3. The values were derived from noncompartmentalpharmacokineticanalysis of the data. AUC,-mw values ranged fiom 571 to 2501 ,ug.day/mL (mean: 1235 ,ugmday/mL) for male monkeys and from 1094 to 3224 ,ueday/mL (mean: 2293 ,ug.day/mL) for female monkeys. The terminal half-life of 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 of distributionof 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 of perfluorooctanoate eliminated in urine by individual monkeys at various times after dosing is presented in Table 4. Sex differences in the urinary excretion of perfluorooctanoate were not readily apparent prior to Day 21; on Day 21 and Day 28, the female monkeys appeared to eliminate a higher percentage of the dose in urine than did the male monkeys. The urinary elimination of perfluorooctanoate was prolonged for both the male and female monkeys; on Day 28 from 0.1 to1% of 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 of 10 mg/kg. The results of this study indicated that beginning on around Day 7 and continuing throughout the remaining period of sample collection, serum concentrations of perfluorooctanoate were lower in two of the three male monkeys than in the three female monkeys. On the final day of serum collection (Day 123), serum concentrations of 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 of perfluorooctanoate ranged fiom 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 of 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 of the male monkeys than in the three female monkeys. Although the number of 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 of high levels of 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 of this monkey, which lead to the eventual euthanasia of the animal, were a response to stress. Although the serum levels of perfluorooctanoate indicated that at least two of 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 of the compound by male and female monkeys. This may have been related to the fact that the rate of urinary excretion of 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% of the dose. 5.0 Conclusions The mean terminal serum half-life of 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 of the final report from t h s study will be stored in the Archives at Southern Research for up to 1year after acceptance of the final report by the Sponsor. After 1year 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 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 Dose Level 0 I 2052 10 m g k g 6.6 2054 10m2Y-k 6.4 221 1 10 m g k g 5.1 Body Weight (kg) on Study Day 4 7 11 14 21 Males 6.5 6.4 6.4 6.4 6.7 6.3 6.3 6.4 6.3 6.6 5.1 5.1 5.1 5.1 5.4 2058 2059 206 1 10 m g k g 3.4 3.4 3.5 3.5 3.5 3.4 10 m g k g 3.5 3.5 3.5 3.5 3.4 3.6 10 m&g 4.1 4.0 4.0 4.1 4.1 4.1 28 I 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 0.5 hrs 4 hrs 48 hrs Day 4 Day 7 Day 11 Day 14 Day 21 Day 28 Day 57 Day 79 Day 87 Day 123 Serum Concentration (ng/mL) Males I Females 2052 I 2054 I 2211 I 2058 2059 I 2061 115 I BQL I 20 I 57 I I I 91,130 96,660 91,370 96,400 I I 67,900 91,630 9 0 , 6 8 0 E 1 I I 74,000 89,160 71,960 70,120 I 1 I 71,200 60,860 73,650 79,570 I I I 50,210 50,930 64,160 62,890 I I I 65,810 48,530 48,930 61,600 I I 43,370 I 42,940 I 30,490 I 70,520 45,840 27,910 %=159,820 39,450 19,320 23,640 38,980 37,360 11,220 13,430 40,450 33,600 I 3,985 I 5,939 I 38,750 27,140 I 1,863 I 3,241 I 33,680 15,010 I 467 I 780 I 31,690 9915" I -- I -- I -- -- I 95 I 111 1 8,585 -- I BQL I 35 I 3,651 81,620 I 69,380 I 61,520 64,170 I 60,760 41,940 I 49,670 I 41,640 I 44,600 I 53,980 39,820 44,370 36,760 ~~~ 37,500 I 45,790 I 37,850 I 24,300 21,910 10,460 7,145 16,870 I 4,992 I 5,453 I 4,701 2,123 885 " Sample taken prior to humane sacrifice of 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 aximum concentration in serum bHalf-lifeof the terminal elimination phase `Area 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 eTotalbody clearance fVolume of distribution at steady state Page 1 of 1 12 Table 4 A Pharmacokinetic Study of Potassium Perfluorooctanoate in the Cynomolgus Monkey Animal ID Urine Concentration Sex (ng/mL) Urine Volume (mu Total (Pg) 2052 M BQL 960 -- 2054 M BQL 840 -- 2211 M BQL 490 -- 2058 F BQ- L 205 -- 2059 F BQL 700 -- 2061 F BQL 170 -- -d- 2052 I M I 6,150 I 200 I 1,230 2054 M 9,283 420 3,899 2211" M 27,150 350 9,503 2058 F 5,527 190 1,050 2059 F 1,924 860 2061 F b 80 1,655 -- Dose (Pug) 66,000 64,000 5 1,000 34,000 35,000 4 1,000 Percent of Dose in Urine (%) ---- ---- I 66,000 I 1.9 64,000 6.1 5 1,000 18.6 34,000 3.1 35,000 4.7 4 1,000 -- 2052 M 3047 250 762 66,000 1.2 2054 M 1174 170 200 64,000 0.3 2211 M 2766 120 332 5 1,000 0.7 2058 F 3783 150 567 34,000 1.7 2059 F 2085 660 1,376 35,000 3.9 2061 F 3486 60 209 4 1,000 0.5 2052 M 3906 350 1,367 66,000 2.1 2054" M 10350 870 9,005 64,000 14.1 221 1" M 10770 300 3,23 1 5 1,000 6.3 2058 F 4347 200 869 34,000 2.6 2059 F 4253 110 468 35,000 1.3 2061 F 18190 80 1,455 4 1,000 3.5 BQL = Below the quantitation limit ( 4 0 ng/mL) a Concentration of perfluorooctanoate in the sample greatly exceeded the highest concentration in the standard curve; therefore, urine concentration value may contain significant error bDueto technician error, urine was discarded after the volume was obtained. 'Concentration 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 of 2 13 Table 4 (Continued) A Pharmacokinetic Study of Potassium Perfluorooctanoate in the Cynomolgus Monkey Animal ID Urine Concentration Sex (ng/mL) Urine Volume 2052 M 2054 M 2211 M 2058 F 2059 F 2061 F 1066 776 819 1673 5550 6151 1010 1060 390 170 130 160 2052 M 2054 M 2211 M 2058 F 2059 F 2061 F 389 575 298 2,278 2,347 6,052 1000 710 880 140 150 100 2052 M 417 680 2054 M 79 660 2211 M 185 520 2058 F 665 410 2059 F 2286 150 2061 F 3126 105 Total (lug) 1,077 823 319 284 722 984 389 408 262 319 352 605 284 52 96 273 343 328 Dose (lug) 66,000 64,000 5 1,000 34,000 35,000 4 1,000 66,000 64,000 5 1,000 34,000 35,000 4 1,000 66,000 64,000 5 1,000 34,000 3 5,000 4 1,000 Percent of Dose in Urine ("/.I 1.6 1.3 0.6 0.8 2.1 2.4 0.6 0.6 0.5 0.9 1.o 1.5 0.4 0.1 0.2 0.8 1.o 0.8 BQL = Below the quantitation limit ( 4 0 ng/mL) a Concentration of perfluorooctanoate in the sample greatly exceeded the highest concentration in the standard curve; therefore, urine concentration value may contain significant error bDueto technician error, urine was discarded after the volume was obtained. 'Concentration 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 1000 J h -I E \ 0) 1 W 100 S 0 .-c, ec.c, I 0 a 0 c 0 0 E 3 L a 1 v) 1 0.1 I 0 14 I I I I 1 I I I I I I I Observed Predicted I I 1 I I I I I I I I 25 50 75 100 125 Time (day) U h I I I I 10 : 0 n i+--- =--=I= I I 8- Observed 0.01 I 0 I I I I 25 50 75 100 125 Time (day) . .. Figure 1 A Pharmacohetic Study of Potassium Perfluorooctanoate in the Cynomolgus Monkey t, Serum Concentration Profiles of Perfluorooctanoate in Monkeys I1000 1 100 10 1 11 I f.: 0.1 1 i 0.01 0 I I I , I 0 n -, I I I I I I I 25 15 M221I I I I I I I I I I I I I I I I I I I I I I I I I I 50 75 Time (day) I I I I I I I / I I I I I 100 I ~~~ 8 Observed - Predicted (3 \ 125 1000 1 ! ! I I 100 b I I I I n I I I I I n I 10 8 Observed \ - Predicted 1 I I 0.1 0 25 < -> 50 75 Time (day) I 100 125 Figure 1 (Continued) A Pharmacolunetic Study of Potassium Perfluorooctanoate in the Cynomolgus Monkey Serum Concentration Profiles of Perfluorooctanoate in Monkeys 1000 J I I . __. I r- + I ._. n E 100 \ 0) 1 v fz .0- 4d ec.cI 10 a, 0 8 c E 3 L 1 a, v> 1 I 0.1 0 25 -. 16 F2059 I I I I I I I I I . - I I 50 75 Time (day) 8- Observed - Predicted I 100 125 1000.0 F2061 100.0 10.0 n n- - E-- n 1.o 8- Observed - Predicted 0.1 0 20 40 60 80 100 120 140 Time (day) Figure 1 (Continued) A Pharmacokinetic Study of Potassium Perfluorooctanoatein the CynomolgusMonkey Serum ConcentrationProfiles of Perfluorooctanoatein Monkeys Appendix A Study Protocol and Amendments A- 1 Study Protocol: A Pharmacokinetic Study of Potassium Perfluorooctanoate in the Cynomolgus Monkey Southern Research Study ID: 9921.4 October 4,2000 SOUTHERNRESEARCH INSTITUTE A-2 STUDY NO.: 9921.4 (. 1.0 SPONSOR REPRESENTATIVE AND CONTACTS: October 4,2000 Page 2 of 13 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 55144-3226 I m (651) 733-1962; FAX:(651) 733-1773 Protocol Approval: (Initial last page also) Test Article: John L. Butenhoff Date Perfluorooctanoate, potassium salt I I Ship Unused Test Article to: D. Hakes Building B236 3M P.O. BOX33327 55133-3327 3M Center, 224-IN-04 St. Paul, Minnesota 55144-1000 I ' A-3 ._ ; F i STUDY NO.: 9921.4 2.0 TITLE: October 4,2000 page 3 of 13 A Pharmacokinetic Study of Potassium Perfluorooctanoate in the Cynomolgus Monkey 3.0 OBJECTIVE: The objectives of this study are to determine the concentration of perfluorooctanoate in serum and urine at various times following administration of 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@)- Year 2000 10/9/00 10/6/00 1011o/oo 10/11/00 1O/ 16/00 10/23/00 10/3O/OO 11/6/00 10/9 - 10/00 10/11/00 10/13/00 10/16/00 10/20/00 10/23/00 10/3O/OO 11/6/00 1/5/01 I ' A-4 STUDY NO.: 9921.4 6.0 STUDY PERSONNEL: October 4,2000 page 4 of 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 Laboratones: Veterinarian: Patricia E. Noker James D. Johnson Ward R. Richter Norman D. Jefferson James D. Johnson LaJuana A. Durbin Darrell E. Hoskins Ph.D., D.A.B.T. M.S., M.B.A. D.V.M., M.S., D.A.C.V.P. B.A. M.S., M.B.A. A.A.S. D.V.M., A.C.L.A.M. (Dipl.) 7.0 TEST & CONTROL ARTICLES: The test article will be supplied by the Sponsor, who will be responsible for documentation of stability, as well as methods of synthesis, fabrication, or derivation. Upon completion of , the study, residual bulk test article will be returned to the Sponsor. 7.1 IDENTITY OF THE TESTARTICLE: Name: Identification: Supplier: Lot Nwnber(s): Special Handling: Perfluorooctanoate, potassium salt T-7507 3M To be documented in the study data. None Characterization: Documentation of the characterization of the test article, including identity, purity, strength, and composition, as well as methods of synthesis, fabrication, or derivation, is the responsibility of the Sponsor. Copies of characterizationdata have been provided to the testing laboratory. Stability & Storage: The bulk test article will be stored at room temperature. Stability of the bulk test article is the responsibility of the Sponsor. A-5 STUDY NO.: 9921.4 7.2 IDENTITY OF THE VEHICLE: October 4,2000 Parre 5 of 13 Name: Supplier: Lot Number(s): Special Handling: Sterile Saline Commercial supplier To be documented in the study data. None Characterization: Documentation of the characterization of the vehicle may be attained by recording all pertinent information fiom 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: i Preparation: The test article will be formulated in sterile saline at a concentration of 5 m g / d 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 refiigerated until used for dosing; formulations of the test article in sterile saline are expected to be stable for weeks when so stored. Dose Formulation Concentration and Homogeneity Analyses: No analysis of dose formulation concentration and homogeneity will be conducted. 8.0 TEST SYSTEM: Species & Strain: Supplier: Age on Day 1: Weight at randomization: Number on Study: Cynomolgus monkeys (Macacafascicularis) Charles River BRF, Inc. (Houston, TX) 3-4 years of age (estimated) 3-7 kg Males -3 Females -3 Animals were previously dosed with potassium perfluorobutanesulfonatein study 9921.1, potassium perfluorobutanoate in study 9921.2, and potassium perfluorohexanoatein study 9921.3. A-6 I STUDY NO.: 9921.4 I \ 8.1 JUSTIFICATION: October 4,2000 Page 6 of 13 Primates are commonly used in preclinical pharmacological and toxicological evaluations of compounds used or intended for use in humans, or to which humans might be exposed. 8.2 HOUSING: During quarantine/acclimation and study, animals will be individually housed in stainless steel, slat-bottom cages. All animals will be housed in a room that provides a minimum of 10 air exchanges per hour. Controls will be set to maintain the animal room at a temperature of 64-84 OF and a relative humidity of 30-70%. A 12-hour lighUl2-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 t' used for excrement absorption. Analyses of the bedding, supplied by the vendor, will be reviewed by the Department of Veterinary Medicine and Bioresources (DVMB) of Southern Research to assure that no known contaminants are present that could interfere with or affect the outcome of the study. 8.4 DIET: Diet will be commercial Certified Primate Chow #5048 (PMI Feeds, Inc.; St. Louis, MO). The primates will be offered feed twice daily, with approximately the recommended daily ration available at each feeding interval. In addition, the diet will be supplemented with 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 of the feed, supplied by the vendor, will be reviewed by the DVMB of Southern Research to assure that no known contaminants are present that could affect the health of the animals. 8.5 WATER: Water (Birmingham public water supply) will be supplied ad libitum during the quarantine and study periods via an automatic watering system. Samples of water from the animal facility will be periodically analyzed, and the analyses will be reviewed by the DVMB of Southern Research to assure that no known contaminants are present that could affect the health of the animals. A-7 STUDY NO.: 9921.4 8.6 QUARANTINE: October 4,2000 Pace 7 of 13 All primates were 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 (prirnates must test negative to all three tests prior to release fkom 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, ShigelZa, or parasites, the Study Director, in conjunction with the staff veterinarian, 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. 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. 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 ~ a e 8e of 13 As only one treatment group will be used in this study, no formal randomization will be required. Doses will be administered by intravenous in.ection to determine the pharmacokinetics of the test article. Each primate (three males, three females) will receive a single dose of potassium perfluorooctanoateby injection into a superficial arm or leg vein. Blood samples for serum drug level determinations will be collected from each primate at selected time points during the study. Urine and feces will also be collected at predetermined intervals. A synopsis of the study design is presented in the following table. i a Denotes week 9.1 RANDOMIZATI&ONGROUPASSIGNMENT: As only one treatment group will be used in this study, no formal randomization will be required. 9.2 DOSEPROCEDURE: Each primate (three males, three females) will receive a single intravenous (IV) dose of 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 of dosing will be Day 0 of the study. 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/moribundityand overt toxicity. Animals found in extremis will A-9 STUDY NO.: 9921.4 October 4,2000 page 9 of 13 be humanely sacrificed by an overdose of barbiturate followed by exsanguination with appropriate approval. Detailed Observations: Each primate will be examined shortly after dose administration for detailed clinical signs of toxicity. All findings will be recorded. Additional clinical observations will be performed and recorded on days of blood collection. 9.4 BODYWEIGHTS: Each primate will be weighed on Days -1,4,7, 14,21, and 28. 9.5 URINE AND FECESCOLLECTIONS: 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 of each urine sample will be measured upon collection. All samples collected will be stored frozen at -20 "C or below prior to analysis (urine) or until further notice by the Sponsor (feces). 9.6 SERUMDRUGLEVELS: 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, 11, 14,21, 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 -20 O C or below until analyzed. 9.7 BIOANALYTICAMLETHODDEVELOPMENT: 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 BIOANALYTICASALMPLEANALYSIS: 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 of 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 10 of 13 decisions concerning possible blood sampling of animals beyond Day 28 postdose. Feces will be analyzed only if requested by the Sponsor. 9.9 ANIMALDISPOSITION: At the end of the study, monkeys will be maintained in the stock colony. 10.0 DATA ANALYSIS: Pharmacokinetic parameters (e.g., AUC, half-life, clearance) will be estimated from serum concentrations of unchanged perfluorooctanoate, as appropriate and feasible, using a standard pharmacokinetic program. The total amount of perfluorooctanoatein urine will be calculated and expressed in terns of percent of dose. .- Mean values and standard deviations will be calculated for each time point and sample type, as appropriate. No other statistical analyses of the data will be performed. 11.0 RECORDS: All raw data pertaining to the conduct of this study, and all samples/specimens collected in this study, will be stored in the Archives at Southern Research Institute for up to 1year 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 brief letter report summarizing the serum drug level results will be issued as soon as the information is available. A draft final report will be issued within 60 calendar days after completion of the in-life aspects of the study. The final report (electronic and hard copies) will be issued within 15 working days after receipt of the Sponsor's final review comments on the draft report. The final report for the present study will include, but not necessarily be limited to the following: Dose formulationpreparation Clinical observations Body weight data Serum drug level data Phannacokinetic parameters Urine excretion data A-1 1 i STUDY NO.: 9921.4 13.0 REGULATORY REFERENCES: October 4,2000 Pace 11 of 13 This study will be conducted in accordance with the protocol and the Standard Operating Procedures (SOPs) of Southern Research, and in accordance with the applicable regulatory requirements, as addressed below. 13.1 PROTOCOL AMENDMENTS AND DEVIATIONS: Amendments: All changes in or revisions 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 operationspertaining to this study, unless specifically defined inthis protocol, will be performed according to the Standard Operating Procedures (SOPs) of Southern Research andor the protocol, and any deviations fiom protocol or SOP will be documented. 13.2 REGULATORCYOMPLIANCE: Good Laboratory Practices: This nonclinical laboratory study will be conducted in the spirit of, but will not require strict compliance with, the U.S. Food and Drug Administration's (FDA) Good Laboratory Practice (GLP) regulations (21 GFR Part 58). Data fiom 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. 13.3 FACILITIEMSANAGEMENATND ANIMALHUSBANDRY: Animal care will be in compliance with the SOPs of Southern Research, the Guidelinesfor the Care and Use of Laboratory Animals, 7thEdition (Institute of Animal Resources, Commission on Life Sciences, National Research Council; National Academy Press; Washington, DC; 1996), and the U.S. Department of Agriculture through the Animal Welfare Act (Public Law 99-198). Southern Research Institute is fully accredited by the American Association for Accreditation of Laboratory Animal Care (AAALAC). A-12 _ . STUDY NO.: 9921.4 13.4 ANIMAW L ELFARAECTCOMPLIANCE: October 4,2000 pane 12 of 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 of study. This study design was reviewed by the IACUC at Southern Research Institute and was approved on 07/26/2000; it was assigned IACUC tracking number 00-07-034. \ A-13 , I STUDY NO.: 9921.4 14.0 PROTOCOL APPROVALS: This protocol has been reviewed and approved. October 4,2000 Page 13 of 13 Study Director: W6A7d Patricia E. Noker, Ph.D., D.A.B.T. Study Director /I/ / + . A D Date Sponsor's Monitor: PE INITIALS ONLY (See page 2) Date &h//k[//&y Management Approval: , Ward R. Richter, M.S., D.V.M., D.A.C.V.P. /fq./yho Date Director, Safety Assessment Department, Southern Research Institute Appendix B Method Validation Report: Validation of Analytical Method for Determination of Perfluorooctanoatein Monkey Serum and Urine Using HPLC/MS/MS f B- 1 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 B-2 SUMMARY Southern Research Institute has successfully validated for 3M an analytical method (BACG 3548) entitled ``Determination of Perfluorooctanecarboxylate in Monkey Serum and Urine: Sample Preparation and Analysis by HPLC Mass Spectrometryh4ass Spectrometry (HPLC/MS/MS)". Calibration standards were prepared by spiking samples of either blank monkey serum or human urine with known amounts of test article, perfluorooctanecarboxalate (PFOC) and internal standard, perfluorohexanesulfonate (PFHS). The concentration of test article in serum covered a combined range fiom 10 to 100,000 ng/rnL 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 of 44 individual calibration standards extending over concentration ranges from 5000 100,000ng/mL, 100 - 10,000ng/mL and 10- 500 ng/mL produced correlation coefficient of 0.9874,0.9750 and 0.9977 respectively. A single composite curve prepared fiom urine over a concentration range of 5 - 500 ng/mL and containing 20 individual calibration standards produced a correlation coefficient of 0.9960. James D. Johnson, M.S., MBA Manager Bioanalytical Chemistry Group Gregory S. Goman, Ph.D. Staff Chemist Bioanalytical Chemistry Group Lester Williams, B.S. Associate,Chemist I1 Bioanalytica1 Chemistry Group George Dollar Associate Biologist I 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 perfluorooctanecarboxalate in monkey serum and urine. 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 unpresewed 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 unpresewed primate tissue. 3. EXPERIMENTAL 3.1 Analytical Procedures The sample preparation and analysis procedures as described in the analytical method entitled ``Determinationof Perfluorooctanecarboxalate in Monkey Serum and Urine: Sample Preparation and Analysis by HPLC Mass SpectrometryMass Spectrometry (HPLCMSMS)" 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 of test article (PFOC) and internal standard (PFHS). To each standard was added 500 pL of TBA ion-pairing solution, 1 mL of carbonatehicarbonate 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 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 of dry nitrogen. The residue was reconstituted in 5% 5 m M 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 "Determinationof Perfluorooctanecarboxalatein Monkey Serum and Urine: Sample Preparation and Analysis by HPLC Mass Spectrometry/Mass Spectrometry (HPLCMS/MS)" consisted of analyzing standard curves prepared in monkey serum or human I 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 - 500 ng/mL for urine. 5.000 - 100,000 ng/mL (Serum) One composite calibration curve comprising 3 sets of calibration standards in a single analytical run in the 5,000 - 100,000ng/mL concentrationrange generated from a total of 22 calibration standards produced a correlation coefficient of 0.9874. A statistical summary for the composite curve is shown below: STANDARD COMPOSITE CURVE 1 (ng/mL) # of Stds Mean % Accur. Std. Deviation 3 2 2 3 5 4 3 93.9 102 98.7 107 101 95.3 102 385 31.9 1193 3917 2016 3048 13580 100 - 10.000 nP/mL (Serum) Two composite curves consisting of a total of 12 calibration standards encompassing a concentrationrange of 100- 10,OOng/mL produced correlation coefficient of 0.9750 The 5,000 n g / d standards were dropped due to preparation error. A statistical summary for the composite curve is given below: # of Stds Mean % Accur. Std. Deviation 2 2 2 2 93.5 109 114 93.8 86.1 14.5 32.1 106 14.7 307 852 10 - 500 ng/mL (Serum) A single composite curve consisting of 11 standards taken from an analytical run encompassing a concentration range of 10 - 500 ng/mL produced a correlation coefficient of 0.9977. No calibration standards were dropped from the run. A statistical summary for the composite curve is given below B-6 STANDARD COMPOSITE CURVE 3 (ndmL) # of Stds Mean % Accur. Std. Deviation 2 2 2 2 1 2 96.6 100 101 106 90.6 101 0.5 1 0.97 1.04 2.08 ---- 38.1 5 - 500 nE/mL (Urine) / A composite curve prepared in urine consisting of 20 standards over a range of 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 (ndmL) # of Stds Mean % Accur. Std. Deviation 2 3 3 3 3 3 3 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 of analyte in the serum extracts (ng/mL) was back calculated using a calibration curve genbrated 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 = a x 2 + 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 3-7 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 l/x weighted quadratic equation. The total concentrationrange for a serum matrix is 20 - 100,000ng/mL and 5 - 500 ng/mL for urine with limits of quantitation of 20 and 10 ng/mL respectively. Appendix C Analytical Method for Determination of Perfluorooctanoate in Monkey Serum and Urine I c-1 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 (HPLC/MS/MS) 1 .o 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/MassSpectrometry (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/mL 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 Perfluorooctanecarboxalatein Monkey Serum and Urine: Sample Preparation and Analysis by HPLC Mass Spectrometry/Mass Spectrometry (HPLC/MS/MS) 2.1.1 2.1.2 2.1.3 2.1.4 2.1.5 2.1.6 2.1.7 2.1.8 2.1.9 2.1.10 2.1.11 2.1.12 2.1.13 2.2 2.2.1 Water, deionized and organic free (from in-house purification system; e.g., Ingalls 210N) Methanol, HPLC grade Perfluorooctanecarboxalate (analyte),as provided by the client Perfluorohexanesulfonate (internal standard), 97 % Ammonium acetate, HPLC grade Blank control monkey serum Sodium Carbonate, Certified ACS Grade or equivalent Sodium Bicarbonate, Certified ACS Grade or equivalent Ethyl Acetate, HPLC grade TetrabutylammoniumHydrogen Sulfate Sodium Hydroxide 50% solution, Certified grade or equivalent Blank control monkey urine Formic Acid Prepared Solutions Appropriate changes in the solutions may be made at the discretion of the analyst 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 2.2.2 2.2.2.1 2.2.3 2.2.3.1 3 .O 3.1 3.2 3.3 3.4 3.5 3.6 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. TBA Ion-Pairing Solution (0.5 M tetrabutylammonium hydroxide) 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. 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. INSTRUMENTS, MATERIALS, AND APPARATUS The following or their equivalents may be used. HPLC pump(s) , autosampler, and triple quadrupole mass spectrometer Autosampler vials with inserts Vortex mixers (e.g., touch mixer and IKA-Vibrax @ platform mixer) Solvent-concentrationapparatus (e.g., Zymark Turbo-Vap @ with source of nitrogen) HPLC mobile phase filtration apparatus Filters for HPLC mobile phase filtration apparatus (e.g., Nylon-66, 0.20 ,urn) 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 WORKING 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. Main Stock Solution of PFOC - 1000 pg/mL Prepare an - 1000 pg/mL 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. I into an appropriate vessel (e.g., culture tube) and add 10 mL of deionized organic-free water. Mix well. Transfer the solution to a clean vessel if desired. Stock Solution of Internal Standard (PFHS), -200 pg/mL Prepare an -200 pg/mL 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), - SOpg/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 of deionized water. Mix well. Working 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) I 62,500 1 3 1,250 I 5,000 2,500 1,000 500 250 I 2.5 mL of 250,000 ng/mL I 10 I 5mLof62,500ng/mL I 10 1 50 pL of 1000pg/mL stock 1 10 5 mL of 5000 ng/rnL stock 10 I I 4 mL of 2500 ng/mL stock 10 5 mL of 1000 ng/mL stock 10 5 rnL of 500 ng/mL stock 10 4.4.2 Summary of concentrations of serum standards: Volume and Spike Conc. II 1I Standard IA 50 pL of 1000 pg/mL 37.5 pL of 1000 pg/mL Ic 25 pL of 1000 pg/mL 12.5 pL of 1000 pg/mL 10 pL of 1000 pg/mL Approximate Concentration of PFOC in serum 100,000 75,000 ~ 50,000 25,000 20,000 c-7 Page 7 of 13 ANALYTICAL METHOD Method No. : BACG-3548 Title: Determination of Perfluorooctanecarboxalatein Monkey Serum and Urine: Sample Preparation and Analysis by HPLC Mass Spectrometry/Mass Spectrometry (HPLC/MS/MS) r I I F 10 p L 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 15,625ng/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 i 10,000 5,000 2,000 1,000 500 200 100 50 0 10 pL of 500 ng/mL 10 P 10 pL of 250 ng/mL 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) of internal standard stock ( - 50 pg/mL) to each tube except the blank-IS (pipet 10 pL of organic-free water instead) and vortex for -5 seconds. 5.3 To each tube add 500 pL of the TBA ion-pairing solution, 1 mL of carbonate/bicarbonatebuffer, and 1mL, 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 of nitrogen and moderate heat (e.g., 50 "C). Y 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 of 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/bicarbonatebuffer, 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 Perfluorooctanecarboxalatein 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 1hour 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 of nitrogen and moderate heat (e.g., 50 "C). Reconstitute the residue in 500 pL of 5 % 5 mM ammonium acetate: 95% methanol containing 1.5% formic acid and vortex briefly to mix. Filter the samples through 0.2 pm PVDF or Nylon syringe filters into autosampler vials. Cap vials for analysis. ANALYSIS BY HIGH PERFORMANCE LIQUID CHROMATOGRAPHY MASS SPECTROMETRY/MASSSPECTROMETRY (HPLCIMSPMS) Conditions are to be optimized if necessary. PLC 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 C18 10 m x 2 m 400 pL/min. 3 PL A : 5mM ammoilium 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% 3 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 f Temperature: 8-11 min. Ambient 50%A : 50%B PE Sciex API 3000 Triple Quadrupole Mass Spectrometer Conditions Software: PE Sciex TurboQuan Turboion Spray Source Note: Values listed under "MS/MS Acquisition Conditions" override parameters in this table. Auxiliary Gas : Air (e.g., Grade 0.1) at 85 pounds per square inch Parameter IS NC TEM OR RNG QO IQ 1 ST RO 1 IQ2 R02 ST3 R03 DF CEM Value -2000 0 400 -20 - 120 5 6 10 6 10 30 40 32 250 1800 c-11 Page 11 of 13 ANALYTICAL METHOD Method No. : BACG-3548 Title: Determination of Perfluorooctanecarboxalatein 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 : Masses requested: PFOC: -412.9 -PFHS (IS) 5 milliseconds -Q=k&md 368.9 398.9 398.9 w stap R02 50 50 ST3 60 60 R03 52 52 Dwell Tuxx&us) 200 Dwell Thc@& 200 c-12 Page 12 of 13 ANALYTICAL METHOD Method No. : BACG-3548 Title: Determination of Perfluorooctanecarboxalatein 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 followiiig mass-to-charge ratios: Analvte PFOC 412.9 to 368.9 PFHS 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 l/concentration of the test article or a quadratic fit). Note: The best curve fit may be dependent on the range of the standard curve and it may be necessary to have more than one standard curve for various concentration ranges using the following: y = ax2+ bx + c where y = x = a, b, c Peak height response of PFOC divided by peak height response of the IS (PFHS) in standards. Concentration of the PFOC in standards. = Coiistants 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/rejectioncriteria 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: Bioanalytical Chemistry Group /a-J?-cm Date