Document 710mD3ROE89rejGEdB4g0gLnj

q Final Report on A Pharmaeokinetic Study of Potassium Perfluorooetanoate in the Cynomolgus Monkey -r--7_-o7.1 \ 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 of 10 mg_g ofperfluorooctanoate, 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 perfluorocctanoate 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 rateof decrease appeared to be faster in male monkeys than in female monkeys. On Day 28, the concentrations ofperfluorooctanoate in serum ranged fi-om1863 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 of the compound on this day, the length of serum collection was extended through Day 123. On Day 123, the serum concentration ofperfluorooctanoate was at or only slightly above the limit of 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 ofperfluorooctanoate 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 sieady 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 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 OFCONTENTS e_ag 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 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 OF CONTENTS (Continued) LIST OF TABLES Table 1: Table 2: Table 3: Table 4: Figure 1: Appendix A: Appendix B: Appendix C: Individual Body Weights 9 Serum Concentrations of Perfluorooctanoate 10 Pharrnacokinetic Parameters Calculated from serum Concentrations 11 of Perfluorooctanoate Urinary Excretion of Perfluorooctanoate 12 LIST OFFIGURES 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 iii Signature Page A Pharmaeokinetic Study of Potassium Perfluorooctanoate in the Cynomolgus Monkey Patricia E. Noker, Ph.D., D.A.B.T. Date Study Director Supervisor, ADME & Pharmacokinetics Reviewed by: Charles D. H_bert, 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. "_reg_"(3._G_an, Ph.D. Manager, Bioanalytical Chemistry Group /-/k- Date iv Good Laboratory Practices Disclaimer This study described in this final reportwas not conducted in strictcompliance with the U.S. Food and Drug Administration (FDA) Good Laboratory Practice (GLP) Regulations (21 CFR Part 58), and neither this reportnor 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. Patricia E. Noker, Ph.D., D.A.B.T. Date Study Director V Study Dates: 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. H6bert, 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 AppendixA. 2.0 Materials and Methods 2.1 Test System The 3 male and 3 female monkeys designated for use in this study were selected from an inhouse colony of monkeys that were housed at Southern Research Institute (Southern Research) prior to use on this study. These monkeys were purchased from Charles 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 intemal parasites, complete blood count (CBC), body weight, and rectal temperature was performed on each of the monkeys. The following procedures were performed on the monkeys during quarantine: (1) Three tuberculin tests were administered to each animal at 2-week intervals. All tuberculin tests were administered intrapalpebrally. The three tuberculin tests were negative for all monkeys. (2) Blood was drawn for CBC and B virus titer. (3) Fecal cultures (screening for Salmonella and Shigella) were obtained, and fecal flotation tests were performed. (4) In general, primates were examined at least once weekly by an approved veterinarian and were observed (cage-side observations recorded by exception only) twice daily for abnormal clinical signs and mortality/moribundity. Housing, feed, water, and socialization procedures remained the same during the quarantine, holding, and study periods. Certified, commercial, dry monkey chow #5048 (PMI Feeds, Inc., St. Louis, MO) was fed to the monkeys 2-3 times each day. The quantity of the daily ration was sufficient to meet 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 F and a relative humidity of 22.2-65.7%. The humidity was within the required range (30-70%) over 90% of the time during the study; excursions below the recommended humidity range were of short duration. Room lights were controlled by an automatic timer set to provide 12 hours of light (0600 to 1800 hours, CST) and 12 hours of dark per day. Cage size and animal care conformed to the guidelines of the Guide for the Care and Use of Laboratory Animals, 7th edition ) and the U.S. Department of Agriculture through the Animal Welfare Act (Public Law 99-198) and to the applicable Standard Operating Procedures (SOPs) of Southem 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 perfluorobutanesuifonate (10 rng/kg) on April 10, 2000 (Southern Research Study No. 9921.1); a single IV bolus dose of potassium perfluorobutanoate (10 mg/kg) on June 13, 2000 (Southern Research Study No. 9921.2); and a single IV bolus dose of 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 of 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 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 perfluomoctanoate 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: required to be performed. Dose concentration and homogeneity analyses were not 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 ofperfluorooctanoate 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: Ufine 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 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 Perfluorooetanoate: 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. Bioanalytieal Method Development and Sample Analysis: A bioanalytical method was developed for the analysis ofperfluorooctanoate 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-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 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 ofperfluorooctanoate. 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 stimulation and to change his 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 ofquantitation 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 timesthrough Day 123 after administration of a single iv dose of 10 mg/kg are presented in Table 2 and plotted in Figure 1. 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 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 ofperfluorooctanoate 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 ofperfluorooctanoate 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 concentration ofperfluorooctanoate 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 ofperfluorooctanoate in individual monkeys are presented in Table 3. The values were derived from noncompartmental pharmacokinetic analysis of the data. AUC0.i_ values ranged from 571 to 2501 /_g.day/mL (mean: 1235 _zg.day/mL) for male monkeys and from 1094 to 3224 /.zgoday/mL (mean: 2293 _zgday/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 distribution of the compound at steady state (Vd_) 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 al_er 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 ofperfluorooctanoate was prolonged for both the male and female monkeys; on Day 28 from 0.1 tol % 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 ofperfluorooctanoate 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 from 885 to 4701 ng/mL in the three female monkeys. These differences in the serum concentrations ofperfluorooctanoate 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 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 ofperfluorooctanoate 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 this study will be stored in the Archives at Southem Research for up to 1 year after acceptance of the final report by the Sponsor. After 1 year and with the permission of the Sponsor's Monitor, the data and any samples/specimens will be shipped to the Sponsor or to the Sponsor's designated archival facility. If materials are to be retained in the archives beyond this date, such continued storage will be for a specific fee determined with the Sponsor. A copy of the final report will be retained in the central archives at Southem 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 Pharmacokinetie Study of Potassium Perfluorooctanoate in the Cynomolgus Monkey Individual Body Weights Animal ID Dose Level 0 2052 10 mg/kg 6.6 2054 10 mg/kg 6.4 2211 10 mg/kg 5.1 2058 10 mg/kg 3.4 2059 10 mg/kg 3.5 2061 10 mg/kg 4.1 Body Weight (kg) on Study Day 4 7 Males 11 14 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 r 3.5 4.0 4.0 3.5 3.5 3.5 3.4 4.1 4.1 21 28 6.7 6.6 6.6 6.2 5.4 5.4 3.4 I 3.5 3.6 3.6 4.1 4.1 . ,_ 10 Table 2 A Pharmacokinetic Study of Potassium Perfluorooctanoate in the Cynomolgus Monkey Serum Concentrations of Perfluorooctanoate Timepoint 0 0.5 hrs 2hrs 4hrs 8hrs 24hrs 48hrs Day4 Day7 Day 11 Day 14 Day 21 Day28 Day 57 Day 79 Day 87 Day 123 2052 115 91,130 67,900 74,000 71,200 _50,210 65,810 43,370 45,840 39,450 37,360 33,600 27,140 15,010 9915 a --- Serum Concentration (ng/mL) Males Females 2054 2211 2058 2059 BQL 20 57 123 96,660 91,370 96,400 88,940 91,630 90,680 74,280 77,870 89,160 71,960 70,120 81,620 60,860 73,650 79,570 61,520 50,930 64,160 62,890 60,760 48,530 48,930 61,600 49,670 42,940 30,490 70,520 41,640 27,910 29,100 59,820 44,600 19,320 23,640 38,980 53,980 11,220 13,430 40,450 45,790 3,985 5,939 38,750 37,850 1,863 3,241 33,680 24,300 467 780 31,690 16,870 .......... 95 111 8,585 5,453 BQL 35 3,651 4,701 2061 33 94,080 71,600 69,380 64,170 41,940 39,820 44,370 36,760 37,500 21,910 10,460 7,145 4,992 2,123 885 o. a Sample taken prior to humane sacrifice of animal. BQL = Below the quantitation limit (<20 ng/mL) Page 1 of 1 t J 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 (_zg/mL)a 101 98.4 91.6 97.0 4.9 105 " tit2(day) b 35.3 13.7 13.6 20.9 12.5 26.8 AUC0.1ast (/zgoday/mL) _ 1999 569 633 1067 808 3083 AUC0-infinity (/.zgoday/mL)a. 2501 571 634 1235 1097 3224 C1(mL/day/kg) _ 4.0 17.5 15.8 12.4 7.4 3.1 Vd,s (mL/kg) f 192 168 184 181 12 133 aMaximum concentration m serum bHalf-life of the terminal elimination phase CArea under the serum concentration versus time curve calculated from 0 to the last time point aArea under the serum concentration versus time curve calculated from 0 to infinity eTotal body clearance fVolume of distribution at steady state 2059 93.0 41.7 2314 2560 3.9 190 Female 2061 Average SD 103 100 6.0 29.3 32.6 8.0 1056 2151 1023 1094 2293 1090 9.1 5.4 3.3 270 198 69 . Page 1 of l 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 2061 u 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 F b 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) 960 840 490 205 700 170 200 420 350 190 860 80 250 170 120 150 660 60 350 870 300 200 110 80 Total (_zg) Baseline ------Day 1 1,230 3,899 9,503 1,050 1,655 -- Day 2 762 200 332 567 1,376 209 Day 7 1,367 9,005 3,231 869 468 1,455 Dose (_g) 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) i Concentration of perfluorooctanoate in the sample greatly exceeded the highest concentration standard curve; therefore, urine concentration value may contain significant error _Due to technician error, urine was discarded after the volume was obtained. in the CConcentration of perfluorooctanoate in the sample was slightly above the highest concentration in the standard curve; urine concentration value should have no significan t error. Page 1 of 2 13 Table 4 (Continued) A Pharmacokinetic Study of Potassium Perfluorooctanoate in the Cynomolgus Monkey Animal ID [ 2052 2054 2211 2058 2059 2061 I 2052 2054 2211 2058 2059 2061 I 2052 2054 2211 2058 2059 2061 Urinary Excretion of Perfluorooctanoate 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 (gg) 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 (_g) 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 standard curve; therefore, urine concentration value may contain significant error _Due to technician error, urine was discarded after the volume was obtained. in the 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 1000 ._1 E 100 L i.._o _ 10 oo_ c 8 E E1 _v o _'--'-_ 0.1 0 25 14 M2052 50 75 Time (day) -e- Observed -- Predicted 100 125 1000 M2054 [ '" ._i 100 i-..... E _o _ 0 -- .o 10 o i o f- 0" Obse_e-_-...-......_.I._.___ _ 1 -:.. ':i.-:_':::-'-::'-'-_:.---. :----_T_-_:"-'_---:..:---..-.---:_.:.E..T...:.._L-....-.:.-..-.:...E..-..:.:.:.-.:....:.-.-.'-:;:-"_---- (,_ . - :::-:-:-- - - : -.:-:-::: : -- : -:-:"-" ::-:::_="'-_E ............. . -- E CO 0.1 0.01 0 25 50 75 100 Time (day) -- Predicted 125 Figure 1 A Pharmacokinetic Study of Potassium Perfluorooctanoate in the Cynomolgus Monkey ,. Serum Concentration Profiles of Perfluorooctanoate in Monkeys 1000 100 --J wv _ r 113 o t- o a.. ,- _ 1 E _ - 0.1 0.01 13 25 15 M2211 _ _--.,,. -_ _ .... ......... " ---:_ 50 75 100 Time (day) -e- Observed -- Predicted 125 1ooo :_-L-_-_?::..... ----_=::.-:---. _ F2058 _J 'ooI CP) :-,--_0 vw _ 0_ 0 "_ r" _ .... i: .-._._._: ::::.__.7__:_ ......... E 2I Q) m, (,r) r_ _ ---....._ --:.._ .......... ............... ................. ..2._r2_.- --_ _ -.- -e,- Observed -- Predicted 0.1 0 .......... 25 .......... ! 513 75 Time (day) 100 125 Figure 1 (Continued) A Pharmacokinetic Study of Potassium Perfluorooctanoate in the Cynomolgus Monkey Serum Concentration Profiles of Perfluorooctanoate in Monkeys 1000 I ........................ _J E lOO v _ O' U C .__0 'C-' 10 r- E 21 0 ....... 16 F2059 _o _ " ,, _ _ -e- Observed _ _ Predicted 0.1 0 25 50 75 100 125 Time (day) 1000.0 F2061 , ...I E 100.0 J, v "_,0 c O *_ 10.0 -- f_ 0 o o -_-- _ o_ e 1.o =- ............... O0 _' _ ---- __ _ .,, ., 0.1 .......................... 0 20 40 60 80 100 Time (day) .: _ -,..... .- ---- *_- Observed _ Predicted 120 140 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-1 ( Study Protocol: A Pharmacokinetic Study of Potassium Perfluorooetanoate in the Cynomolgus Monkey \ Southern Research Study ID: 9921.4 October 4, 2000 SOUTHERN RESEARCH INSTITUTE A-2 STUDY NO.: 9921.4 1.0 SPONSOR REPRESENTATIVE AND CONTACTS: Sponsor: Sponsor's Representative & Study Monitor: 3M Center, 220-2E-02 P.O. Box 33220 St. Paul, Minnesota 55133-3220 John L. Butenhoff, Ph.D., D.A.B.T. 3M Center Building 220-2E-02 St. Paul, Minnesota 55144-322G [b*l'I) (651) 733-1962; FAX: (651) 733-1773 Protocol Approval: (Initiallastpage also) October 4, 2000 p_ge 2 of I3 " John L. Butenhoff Date Test Article: Perfluorooctanoate, potassium salt Ship Unused Test Article to: D. Hakes Building B236 3M P.O. Box 33327 55133-3327 3M Center, 224-IN-04 St. Paul, Minnesota 55144-1000 A-3 " STUDY NO.: 9921.4 2.0 TITLE: October 4, 2000 p_ge3 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 2000 Ninth Avenue P.O. Box 55305 Institute South 35205 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/Q0 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 Pa_e 4 of 1,3 The following are the primary contributors and supervisory personnel participating in this study. Study Direetor: 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 La.luana 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 IDENTITYOFTHE TEST ARTICLE: Name: Identification: Supplier: Lot Number(s): Special Handling: Characterization: Perfluorooetanoate, potassium salt T-7507 3M To be documented in the study data. None 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 characterization data 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 Piige5 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 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 of sterile saline, and the mixture will be stirred until the test article is visibly in solution. Formulations will be stored refi-igerated 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 perfluorobutanesulfonate in study 992 I. I, 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 of 1"4 Primates are commonly evaluations of compounds might be exposed. used in preclinical pharmacological used or intended for use in humans, and toxicological or to which humans 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 F and a relative humidity of 30-70%. A 12-hour light/12-hour dark cycle will be routinely maintained. Animals will be acclimated in the same room used for study. 8.3 BEDDING: None required for caging equipped with flushable pans. For cages equipped with excrement absorption pans, commercial heat-treated hardwood chip bedding will be , used for excrement absorption. Analyses 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 Page7 of 13 All primates were selected from stock animals that will have been quarantined for a minimum of 35 days prior to study start. No prophylactic or therapeutic treatments will be administered during the quarantine period. Standard procedures to be conducted during the quarantine period are as follows: a) A complete physical examination including a fecal examination for internal parasites, complete blood count (CBC), body weight, and body (rectal) temperature will be performed; b) three tuberculin tests at 2-week intervals (administered intrapalpebrally, using alternate eyelids for each test) will be performed on each primate (primates must test negative to all three tests prior to release from quarantine; primates that respond positively to a tuberculin test will be euthanized immediately, while non-responding primates housed in the same room will be held in quarantine for an additional 90 days); c) the blood sample drawn for CBC will also be used for measuring of B virus titer (primates found to have a positive B virus titer will be euthanized immediately); d) a fecal sample for culture (screening for Salmonella and Shigella) will be obtained and submitted to an independent laboratory for analysis (if any primates are found to be positive for Salmonella, Shigella, or parasites, the Study Director, in conjunction with the 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-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 Page8 of 13 As only one treatment group will be usedin required. this study, no formal randomization will be 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 predetermined intervals. A synopsis of the study design is presented in the following table. Study Procedures Health Check Dose Preparation -1" 0 x x 1234 Day of Study 5 6 7 8 9 10 11 12 13 14 21 28 Clinical Observations xxx x x Body Weights x x x Urine & Feces x xx x Serum Drug Levels xxx x x a Denotes week x x x 28 xxx xxx x xxx 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 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 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 of mortality/moribundity and overt toxicity. Animals found in extremis will A-9 STUDY NO.: 9921.4 October 4, 2000 Page9 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 BODY WEIGHTS: Each primate will be weighed on Days -1, 4, 7, 14, 21, and 28. 9.5 URINE AND FECES COLLECTIONS: 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 SERUM DRUG LEVELS: Blood samples (approximately 3 mL) will be collected from each primate at approximately 0 (predose) minutes; 0.5, 2, 4, 8, and 24 hours; and 2, 4, 7, 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 C or below until analyzed. 9.7 BIOANALYTICAL METHOD DEVELOPMENT: 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 BIOANALYTICAL SAMPLE ANALYSIS: 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-IO STUDY NO.: 9921.4 October 4, 2000 PageI0 of13 decisioncsoncemingpossiblbeloodsamplinogfanimalbseyondDay 28 postdose. Feceswillbe analyzeodnlyifrequestebdy theSponsor. 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 ofperfluorooctanoate in urine will be calculated and expressed in terms 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 1 year after acceptance of the final report by the Sponsor. After i 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 aRer 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 draR 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 REGULATORY REFERENCES: October 4, 2000 Pitge11 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 PROTOCOLAMENDMENTSANDDEVIATIONS: 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 operations pertaining to this study, unless sp_ifically defined in this protocol, will be performed according to the Standard Operating Procedures (SOPs) of Southern Research and/or the protocol, and any deviations from protocol or SOP will be documented. 13.2 REGULATORYCOMPLIANCE: 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 CFR Part 58). Data from this study may be submitted to the FDA in support of an IND/NDA application. Quality Assurance Review: As this study will not be conducted in strict compliance with FDA's GLP regulations, neither the in-life activities nor the final report will be audited by the Quality Assurance Unit at Southern Research. 13.3 FACILITIESMANAGEMENTANDANIMALHUSBANDRY: Animal care will be in compliance with the SOPs of Southern Research, the Guidelines for the Care and Use of Laboratory Animals, 7_hEdition (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). Southem Research Institute is fully accredited by the American Association for Accreditation of Laboratory Animal Care (A.AALAC). A-12 STUDY NO.: 9921.4 13.4 ANIMAL WELFARE ACT COMPLIANCE: October 4, 2000 Page12 of 13 By signing this protocol, the Sponsor signifies that there are no generally accepted altematives 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 STUDY NO.: 9921.4 14.0 PROTOCOL APPROVALS: This protocol has been reviewed and approved. October 4, 2000 Pace13 of 1 Study Director: .- 7-_ --_._ Patricia E. Noker, Ph.D., D.A.B.T. Study Director 1__1 /e/,C/oa Date Sponsor's Monitor: _TZ--L_" INITIALS ONLY (See page 2) /'//_/_ Date I '_ Management Approval: k_4/,,/_. '/_Z;/7_/'_//f Ward R. Richter, M.S., D.V.M., D.A.C.V.P. /_#/_'o 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 I 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 Southem 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 Spectrometry/Mass 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 fi'om 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 of 44 individual calibration standards extending over concentration ranges from 5000 100,000 ng/mL, 100 - 10,000 ng/mL and 10 - 500 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. 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 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 procedure._ to ensure safety of the analysts were based on information contained in the Material Safety and Data Sheets (MSDS), provided by 3M for the test article used in this study. CAUTION: Since primates may carry a number of zoonoses, all unpreserved tissues, including blood, plasma and serum, are to be considered as biohazards and handled with universal precautions. Refer to S.OP number SRI 2-5-5 for a description of safety procedures to be used when handling unpreserved primate tissue. 3. EXPERIMENTAL 3.1 Analytical Procedures The sample preparation and analysis procedures as described in the analytical method entitled "Determination of 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 gL) was spiked with a known amount of test article (PFOC) and internal standard (PFHS). To each standard was added 500 gL of TBA ion-pairing solution, 1 mL of carbonate/bicarbonate buffer, and 1 mL of deionized water. This mixture was vortexed for approximately 5 seconds. Then 2.5 mL of ethyl acetate was added and the mixture was placed on a horizontal 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% 5mM ammonium acetate in deionized water : 95% methanol containing 1.5% formic acid and filtered through a 0.2 _tm syringe filter. 3.2 Method Validation Validation for BACG 3548 "Determination of 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 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 - 100,000 ng/mL concentration range 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: Cone. (ng/mL) # of Stds Mean % Accur. Std. Deviation STANDARD COMPOSITE CURVE 1 (ng/mL) 5000 10,000 20,000 25,000 50,000 75,000 100,000 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 ng/mL (Serum) Two composite curves consisting of a total of 12 calibration standards encompassing a concentration range of 100 - 10,00 ng/mL produced correlation coefficient of 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 CQMpOSITE CURVE 2 (n_mL) Cone. (ng/mL) # of Stds Mean % Accur. Std. Deviation 100 200 500 1000 2000 10,000 2 2 2 2 2 2 93.5 109 114 93.8 86.1 102 14.5 32.1 106 14.7 307 852 I0 - 500 n_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 .j B-6 Cone. (ng/mL) # of Stds Mean % Accur. Std. Deviation STANDARD COMPOSITE CURVE 3 (ng/mL) 10 20 50 100 200 500 2 2 2 2 1 2 96.6 100 101 106 90.6 101 0.51 0.97 1.04 2.08 .... 38.1 5 - 500 n_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 (n_mL) Cone. (ng/mL) # of Stds Mean % Accur. Std. Deviation 5 10 20 50 100 200 500 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 generated from a set of calibration standards. The calibration curve was generated by a regression analysis to determine the best fit curve (e.g., linear, quadratic etc.) and amount of weighting. A quadratic fit with 1/X weighting was determined to be the best fit : where: y=ax2+ bx + c 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.  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 - 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 C-1 Method No.: BACG-3548 ANALYTICAL METHOD Page 1 of 13 Title: Determination of Perfluorooctanecarboxalate in Monkey Serum and Urine: Sample Preparation and Analysis by HPLC Mass Spectrometry/Mass Spectrometry (HPLC/MS/MS) 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/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 Method No.: BACG-3548 ANALYTICAL METHOD Page 2 of 13 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 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 Tetrabutylammonium Hydrogen 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 Method No." BACG-3548 ANALYTICAL METHOD . . Page 3 of 13 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 3.0 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), aut0sampler, and triple quadrupole mass spectrometer Autosampler vials with inserts Vortex mixers (e.g., touch mixer and IKA-Vibrax platform mixer) Solvent-concentration apparatus (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 _zm) C-4 ANALYTICAL METHOD Page 4 of 13 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 Analytical balance 3.8 Volumetric flasks (e.g., 10 and 25 mL) 3.9 Disposable Pasteur pipets 3.10 Micropipettor(s) with tips 3.11 Culture tubes with teflon-lined caps 3.12 Centrifuge 3.13 Assorted glassware and syringes 3.14 Culture tubes (vials) for use with solvent-concentration apparatus 3.15 1 mL Plastic syringes with 0.2 _tm PVDF syringe filters 3.16 Variable speed horizontal platform shaker 3.17 pH meter 4.0 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. 4.1 Main Stock Solution of PFOC - 1000 lag/mL 4.1.1 Prepare an -1000 p.tg/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 IC-5 Method No.: BACG-3548 ANALYTICAL METHOD Page 5 of 13 Title: Determination of Perfiuorooctanecarboxalate 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 of deionized organic-free water. Mix well. Transfer the solution to a clean vessel if desired. Stock Solution of Internal Standard (PFHS), -200 l.tg/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), --- 50#g/mL Prepare an - 50/_g/mL solution of PFHS in deionized organic- free water by pipetting 2 mL of the 200 #g/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 #g/mL 5 mL of 500,000 ng/mL Final Volume in deionized organic free water (mL) 10 10 C-6 Method No." BACG-3548 ANALYTICAL METHOD Page 6 of 13 Title: Determination of Perfluorooctanecarboxalate in Monkey Serum and Urine: Sample Preparation and Analysis by HPLC Mass Spectrometry/Mass Spectrometry (HPLC/MS/MS) 62,500 I 31 ,250 15,625 5,000 2,500 1,000 500 250 2.5 mL of 250,000 ng/mL 10 5 mL of 62,500 rig/mE 10 5 mL of 31,250 ng/mL 10 50 gL of 1000 gg/mL stock 10 5 mL of 5000 ng/mL stock 10 4 mL of 2500 ng/mL stock 10 5 mL of 1000 ng/mL stock 10 5 mL of 500 ng/mL stock 10 4.4.2 Summary of concentrations of serum standards: Standard Level A B C D E Volume and Spike Cone. 50 gL of 1000 gg/mL 37.5 #L of 1000/zgimL 25/_L of 1000 gg/mL 12.5 _L of 1000/xg/mL 10 gL of 1000 gg/mL Approximate Concentration of PFOC in serum (ng/mL) 100,000 [ I 75,000 [ 50,000 25,000 20,000 (_-7 ANALYTICAL METHOD Page 7 of 13 Method No.: BACG-3548 Title: Determination of Perfiuorooctanecarboxalate in Monkey Serum and Urine: Sample Preparation and Analysis by HPLC Mass Spectrometry/Mass Spectrometry (HPLC/MS/MS) F 10/zL of 500,000 ng/mL G 10 _tL of 250,000 ng/mL H 16/zL of 62,500 ng/mL I 16 _L of 31,250 ng/mL J 16 _L of 15,625 ng/mL K 20 t-tL of 5,000 ng/mL L 10/_L of 5,000 ng/mL M 10/_L of 2,500 ng/mL N 10 _L of 1,000 ng/mL O 10 _L of 500 ng/mL P 10/zL 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 _tL of organic-free water instead of the working stock solution. Add the 10 #L C-8 Method No.: BACG-3548 ANALYTICAL METHOD Page 8 of 13 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 p.g/mL) to each tube except the blank-IS (pipet 10 _tL of organic-free water instead) and vortex for -5 seconds. .5.3 To each tube add 500 /xL 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 of nitrogen and moderate heat (e.g., 50 *C). 1 5.7 Reconstitute the residue in 500 _L of 5 % 5 mM ammonium acetate: 95 % methanol containing 1.5% formic acid and vortex briefly to mix. Filter the samples through 0.2 #m 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/_L of internal standard stock (- 50 #g/mL) to each tube and vortex for a couple of seconds. 6.2 To each tube add 500 /_L 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 Method No.: BACG-3548 ANALYTICAL METHOD Page 9 of 13 Title: Determination of Perfluorooctanecarboxalate in Monkey Serum and Urine: Sample Preparation and Analysis by HPLC Mass Spectrometry/Mass Spectrometry (HPLC/MS/MS) 6.3 Add 2.5 mL of ethyl acetate and extract on horizontal mixer for 1 hour at a low speed setting. 6.4 .Remove the tube from the shaker and place in a centrifuge (e.g., 2500 rpm) for about 5 minutes. 6.5 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). 6.6 Reconstitute the residue in 500 _L of 5 % 5 mM ammonium acetate: 95% methanol containing 1.5 % formic acid and vortex briefly to mix. Filter the samples through 0.2 t_m PVDF or Nylon syringe filters into autosampler vials. Cap vials for analysis. 7.0 ANALYSIS BY HIGH PERFORMANCE LIQUID CHROMATOGRAPHY MASS SPECTROMETRY/MASS SPECTROMETRY (HPLC/MS/MS) 7.1 Conditions are to be optimized if necessary. 7.1.1 HPLC Conditions Analytical Column: Guard Column: Elution Flow rate: Injection volume: Mobile phase: Gradient Profile" Keystone Scientific Aquasil C18, 150 mm x 2 mm ID, or equivalent Keystone Aquasil C18 10 mmx 2 mm 400 #L/min. 3 _L A: 5mM ammonium acetate buffer B: 1.5 % formic acid in methanol 0 - 0.5 min. 50%A 50%B 0.5 - 7.5 rain. 10% A 90% B linear gradient 7.5-8 min. 10%A 90% B step gradient C-IO W Method No.: BACG-3548 ANALYTICAL METHOD Page 10 of 13 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 50%A 50%B i 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 IQI ST RO1 IQ2 RO2 ST3 RO3 DF CEM Value -2000 0 400 -20 - 120 5 6 I0 6 10 30 40 32 250 1800 C-II ANALYTICAL METHOD Page 11 of 13 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 l 0 0 MS/MS Acquisition Scan type: Polarity: Acquisition mode: Pause time: Conditions MRM Negative Profile 5 milliseconds Masses requested: PFOC: Ol Mass (ainu) 412.9 Q3 Mass (ainu) 368.9 Dwell Time (ms) 200 PFHS (IS) QI Mahdi(ainu) 398.9 _.ar.ltmeter RO2 ST3 RO3 Q3 Mass fan]u) 398.9 SlaLt Stop 50 50 60 60 52 52 Dwell Time (ms) 200 C-12 Method No." BACG-3548 ANALYTICAL METHOD Page 12 of 13 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: AnaLy_ PFOC PFHS Ion 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. Altematively, 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 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. = 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 i Method No." BACG-3548 ANALYTICAL METHOD Page 13 of 13 Title: Determination of Perfluorooctanecarboxalate in Monkey Serum and Urine: Sample Preparation and Analysis by HPLC Mass Spectrometry/Mass Spectrometry (HPLC/MS/MS) 9.0 9.1 10.0 10.1 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. ',. 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. "GregOffryt.-Gorman, Ph.D., Staff Chemist Date " Bioanalytical Chemistry Group Approved by: 0 t_q_/_ / B,fo_asnalDyt.icJaolhnff_/lo_/mn-i/s4t(ryISM,GBroAu,p Manager I .- /,.2 - ,,77"-_ _ Date