Document QMvEvvV5GgX6NXZp5dyRzY845

flR A2.6 - !3>G A Pharmacokinetic Study of Potassium Perfluorooctanesulfonate in the Cynomolgus Monkey Southern Research Institute Study ED: 9921.6 April 22,2003 000375 Final Report on A Pharmacokinetic Study of Potassium Perfluorooctanesulfonate in the Cynomolgus Monkey To: 3M Corporation P.O. Box 33327 55133-3327 3M Center, 224-IN-04 St. Paul, Minnesota 55144-1000 By: P.E. Noker and G.S. Gorman Southern Research Institute 2000 Ninth Avenue South 35205 P.O. Box 55305 Birmingham, Alabama 35255-5305 000376 ABSTRACT The pharmacokinetics and urinary excretion of perfluorooctanesulfonate were investigated in male and female cynomolgus monkeys. Three male and three female monkeys were administered a single iv bolus dose of 2 mg/kg o f perfluorooctanesulfonate, potassium salt (T-6295). At various times after dosing, serum and urine (24-hour collections) samples were obtained and analyzed by HPLC/MS/MS for levels of intact perfluorooctanesulfonate. The lower limit of quantitation of the analytical method was 10 ng/mL for serum samples and 5 ng/mL for urine samples. At 0.5 hours after dosing (earliest time point), serum concentrations of perfluorooctanesulfonate appeared to be slightly higher in male monkeys than in female monkeys and ranged from 13,790 to 16,250 ng/mL in male monkeys and from 7,871 to 11,790 ng/mL in female monkeys. Serum concentrations of perfluorooctanesulfonate then decreased relatively rapidly in each monkey through the first 24 hours after dosing and, at 24 hours, ranged from 7,039 to 9,538 ng/mL in male monkeys and 5,042 to 7,294 ng/mL in female monkeys. With the exception of one male and one female monkey, serum concentrations of perfluorooctanesulfonate remained essentially constant between Days 7 and 70. Thereafter, serum concentrations of perfluorooctanesulfonate decreased in all animals and ranged from 3,803 to 3,840 ng/mL in the male monkeys and from 2,019 to 2,781 ng/mL in the female monkeys on Day 161. The serum concentration versus time data were subjected to non-compartmental pharmacokinetic analysis. The serum terminal half-life o f perfluorooctanesulfonate ranged from 122 to 146 days (mean: 132 days) in male monkeys and from 88 to 138 days (mean: 110 days) in female monkeys. The total body clearance of perfluorooctanesulfonate was 1.0 to 1.2 mL/day/kg in male monkeys and 1.6 to 2.1 mL/day/kg in female monkeys. The volume of distribution o f perfluorooctanesulfonate ranged from 178 to 220 mL/kg and from 231 to 327 mL/kg in male and female monkeys, respectively. Only very low levels (<0.1% of the administered dose) of perfluorooctanesulfonate were measured in urine during any given 24-hour period of sample collection between Day 1 and Day 91 after dosing. The results of this study provided no clear indication that the pharmacokinetics of perfluorooctanesulfonate were different in male and female monkeys. Perfluorooctanesulfonate was eliminated in urine by both male and female monkeys at low levels for a prolonged period of time (>91 days) after iv administration. 000377 1 TABLE OF CONTENTS ag SIGNATURE PAGE iii GOOD LABORATORY PRACTICES DISCLAIMER iv STUDY SCHEDULE AND PERSONNEL v 1.0 INTRODUCTION 1 2.0 MATERIALS AND METHODS 1 2.1 Test System 1 2.2 Test Article and Vehicle 3 Test Article 3 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 4 Urine and Feces Collection 4 Serum Levels of Perfluorooctanesulfonate 4 Bioanalytical Method Development and Sample Analysis 4 Data Analyses 4 3.0 RESULTS 3.1 Mortality 3.2 Clinical Observations 3.3 Body Weights 3.4 Serum and Urine Concentrations o f Perfluorooctanesulfonate 5 5 5 5 5 4.0 DISCUSSION 7 5.0 CONCLUSIONS 7 6.0 RECORD ARCHIVES 7 7.0 REFERENCES 8 000378 Table 1: Table 2: Table 3: Table 4: Table 5: Figure 1: Appendix A: Appendix B: Appendix C: TABLE OF CONTENTS (Continued) LIST OF TABLES Page Individual Body Weights 9 Serum Concentrations o f Perfluorooctanesulfonate 10 Replicate Analysis Comparisons for Potassium Perfluorooctanesulfonate in Monkey Serum 11 Pharmacokinetic Parameters Calculated from Serum Concentrations o f Potassium Perfluorooctanesulfonate 12 Urinary Excretion o f Perfluorooctanesulfonate 13 LIST OF FIGURES Serum Concentration Profile o f Perfluorooctanesulfonate 16 LIST OF APPENDICES Study Protocol and Comments on Study Data A-l Analytical Method for Determination o f Perfluorooctanesulfonate in Monkey Serum and Urine B-l Summary of Pharmacokinetic Parameters Calculated from Serum Concentrations of PFOs and Urinary Excretion Data for PFOs C-l 000379 IV Good Laboratory Practices Disclaimer This study described in this final report was not conducted in strict compliance with the U.S. Food and Drug Administration (FDA) Good Laboratory Practice (GLP) Regulations (21 CFR Part 58), and neither this report nor the raw data were reviewed by the Southern Research Quality Assurance U nit However, the study was conducted according to the protocol and amendments and the applicable standard operating procedures, and all study procedures, data recording, and reporting were performed in a manner consistent with the standard o f GLPs. The final report accurately reflects the raw data obtained during the performance o f the study. There were no adverse circumstances that affected the quality or integrity o f the study. Patricia E. Noker, PhD ., D.A.B.T. Study Director Date 000380 Ill Signature Page A Pharmacokinetic Study o f Potassium Perfluorooctanesulfonate in the Cynomolgus Monkey Patricia E. Noker, Ph.D., D.A.B.T. Study Director Supervisor, ADME & Pharmacokinetics Reviewed by: Date Charles D. Hbert, Ph.D., D.A.B.T. Director, Safety Assessment Date We, the undersigned, were responsible for the conduct o f the work and reporting o f the results in the listed sections. We concur with the views relative to our body o f work as expressed in the discussion and conclusions. Grejgfty ^Gorman, Ph.D. Manager, Bioanalytical Chemistry Group Date 0003S1 Study Dates: Study Schedule and Personnel Study Initiation: Day o f Dosing: Last Day o f Sample Collection: Study Completion: July 26,2001 July 30,2001 January 7,2002 April 22,2003 Study Personnel: Patricia E. Noker, Ph.D., D.A.B.T. Charles D. Hbert, Ph.D., D.A.B.T. Norman D. Jefferson, B.A. Gregory S. Gorman, Ph.D. DaiTell E. Hoskins, D.V.M., Ph.D., A.C.L.A.M. LaJuana A. Durbin, B.S. D. Wayne May, LATG Carolyn R. Oliver, B.S. Study Director Director, Safety Assessment Associate Director, Safety Assessment Manager, Bioanalytical Chemistry Group Veterinarian Supervisor, Large Animal Laboratory Supervisor, Animal Care Supervisor, Study Coordination 000382 1 1.0 Introduction The objectives o f this study were to determine the concentration o f potassium perfluorooctanesulfonate in serum and to estimate urinary clearance at various times following administration o f a single intravenous dose to monkeys. A copy o f the protocol can be found in Appendix A. 2.0 Materials and Methods 2.1 T est System The three male and three female cynomolgus monkeys designated for use in this study were selected from an in-house colony o f monkeys that were housed at Southern Research Institute (Southern Research) prior to use on this study. These monkeys were purchased from Charles River BRF, Inc. (Houston, TX) and were an estimated 3.5-4.5 years o f age when placed on study. Individual animal identification was by chest tattoo. The cynomolgus monkey is an accepted species to support clinical studies o f drugs used or intended for use in humans. During the quarantine period, a complete physical examination including a fecal examination for internal parasites, complete blood count (CBC), body weight, and rectal temperature was performed on each o f the monkeys. The following procedures were performed on the monkeys during quarantine: (1) Three tuberculin tests were administered to each animal at 2-week intervals. All tuberculin tests were administered intrapalpebrally. The three tuberculin tests were negative for all monkeys. (2) Blood was drawn for CBC and B virus titer. (3) Fecal cultures (screening for Salmonella and Shigella) were obtained, and fecal flotation tests were performed. (4) In general, primates were examined at least once weekly by an approved veterinarian and were observed (cage-side observations recorded by exception only) twice daily for abnormal clinical signs and mortality/moribundity. Housing, feed, water, and socialization procedures remained the same during the quarantine, holding, and study periods. 000383 2 Certified, commercial, dry monkey chow #5048 (PMI Feeds, Inc., S t Louis, MO) was fed to the monkeys 2-3 times each day. The quantity o f the daily ration was sufficient to meet nutritional requirements. In addition, the diet was supplemented with fresh 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 o f 66.4-71.5 F and a relative humidity of 25-74%. The humidity was within the required range (30-70%) over 90% o f the time during the study; excursions below the recommended humidity range were o f short duration and had no impact on animal health or the outcome o f the study. Room lights were controlled by an automatic timer set to provide 12 hours o f light (0600 to 1800 hours, CST) and 12 hours o f dark per day. Cage size and animal care conformed to the guidelines o f the Guidefo r the Care and Use o fLaboratory Animals, 7th edition01 and the U.S. Department o f Agriculture through the Animal Welfare Act (Public Law 99-198) and to the applicable Standard Operating Procedures (SOPs) of Southern Research. The study design was approved by Southern Research's Institute Animal Care and Use Committee. Southern Research is frilly accredited by the American Association for Accreditation o f Laboratory Animal Care International. With the exception o f animal 2053, all o f the monkeys used on this study were previously given a single iv bolus dose o f perfluorobutanesulfonate (10 mg/kg) on 4/10/00 (Southern Research Study No. 9921.1); a single iv bolus dose o f potassium perfluorobutanoate (10 mg/kg) on 6/13/00 (Southern Research Study No. 9921.2); a single iv bolus dose of potassium perfluorohexanoate (10 mg/kg) on 7/31/00 (Southern Research Study No. 9921.3); and a single iv bolus dose o f potassium perfluorooctanoate (10 mg/kg) on 10/9/00 (Southern Research Study No. 9921.4). In addition, all monkeys used on this study were given a single iv bolus dose o f potassium perfluorohexanesulfonate (10 mg/kg) on 2/9/01 (Southern Research Study No. 9921.5). 000384 3 2.2 Test A rticle and Vehicle Test A rticle: One bottle containing 6.65 grams o f potassium perfluorooctanesulfonate (T6295; expiration date not supplied; SRI E05/L-1) was supplied by 3M (S t Paul, MN) and received on May 15, 2000. The test article was stored at room temperature until used. Stability o f the test article was the responsibility of the Sponsor. Vehicle: The vehicle used for the preparation o f the dose formulation o f potassium perfluorooctanesulfonate was sterile saline, USP (Phoenix Pharmaceutical Company; St. Joseph, MO; Lot 102049F, expiration date February 2004). The vehicle was stored at room temperature and was considered to be stable when stored according to these conditions. Dose Form ulation Preparation: For the single dose formulation o f potassium perfluorooctanesulfonate prepared at 1 mg/mL, the required amount o f test article was weighed out in a volumetric flask. Sterile saline was added and the formulation was stirred until in solution. The formulation was stored refrigerated and used for dosing on the same day o f preparation; it was considered stable during this period. Dose Form ulation Analyses: Dose concentration and homogeneity analyses were not required to be performed. 2.3 Experim ental Design G roup Assignment and Dose Procedure: As only one treatment group was used in this study, no formal randomization was required. On Day 0, each o f the three male and three female monkeys received a single intravenous (iv) dose o f perfluorooctanesulfonate at 2 mg/kg by injection into a superficial arm or leg vein. Doses were based upon the individual body weights obtained on the day o f dosing. Doses were administered at a volume o f 2 mL/kg. Clinical O bservations: All animals were observed twice daily for signs of mortality/moribundity. Each primate was examined shortly after dose administration for 000385 4 clinical signs o f toxicity. Additional clinical observations were performed on days o f blood collection, with the exception o f Day 21 when clinical observations were not performed due to technician error. Body Weights: Each primate was weighed on Days 0 ,4 ,7 ,1 4 ,2 1 ,2 8 ,4 2 ,5 6 ,7 0 ,8 5 ,1 0 5 , 126, and 151. 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 postdose), on Day 2 (24-48 hours postdose), and on Days 7, 1 5 ,2 1 ,2 8 ,4 2 ,5 6 ,7 0 , and 91. The volume o f each urine sample was measured upon collection. Urine and feces samples were stored frozen (approximately -20 C or below). Fecal samples will not be analyzed unless specifically requested by the Sponsor. Serum Levels of Perfluorooctanesulfonate: Blood samples (approximately 3 mL) were collected from each primate at approximately 0 (predose) minutes; 0 .5 ,2 ,4 , 8 ,2 4 and 48 hours; and on Days 4, 7, 14, 21, 28, 42, 56, 70, 105, and 161 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. s j Bioanalytical M ethod Development and Sample Analysis: Serum and urine samples were analyzed for perfluorooctanesulfonate using a previously validated HPLC/MS/MS method (Appendix B). The lower limit o f quantitation o f the method was 10 ng/mL for serum samples and 5 ng/mL for urine samples. D ata Analyses: The serum concentration data for unchanged perfluorooctanesulfonate were subjected to noncompartmental pharmacokinetic analysis psing 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 00386 5 Corporation; Irvine, CA). The urinary excretion o f perfluorooctanesulfonate at each collection interval was calculated and expressed as a percent o f the administered dose. No other statistical analyses o f the data were performed. 3.0 Results 3.1 M ortality All o f the monkeys in this study survived to the end o f the study. 3.2 Clinical Observations No adverse drug-related clinical signs were noted for any monkey during the course o f this study. 3.3 Body W eights Body weights are presented in Table 1. Each monkey either gained weight or maintained essentially a constant weight between Day 0 and Day 151 (last day during the study that body weights were obtained). 3.4 Serum and Urine Concentrations o f Perfluorooctanesulfonate Serum concentrations o f perfluorooctanesulfonate in three male and three female monkeys at various times through Day 161 after administration o f a single iv dose o f 2 mg/kg are presented in Table 2 and in Figure 1. At 0.5 hours after dosing (earliest time point), serum concentrations o f perfluorooctanesulfonate appeared to be slightly higher in male monkeys than in female monkeys and ranged from 13,790 to 16,250 ng/mL in male monkeys and from 7,871 to 11,790 ng/mL in female monkeys. Serum concentrations of perfluorooctanesulfonate then decreased relatively rapidly in each monkey through the first 24 hours after dosing and ranged from 7,039 to 9,538 ng/mL in the three male monkeys and from 5,042 to 7,294 ng/mL in two o f the female monkeys at 24 hours; data for the other female monkey was not obtained at this time point. A trough in serum concentrations of perfluorooctanesulfonate was observed in individual monkeys between Days 2 and 7. This trough was followed by an increase and subsequent plateau in serum concentrations of 000387 6 perfluorooctanesulfonate; for all except one male and one female monkey (M2054 and F20S8), serum concentrations o f perfluorooctanesulfonate remained essentially constant between Days 7 and 70. Thereafter, serum concentrations o f perfluorooctanesulfonate decreased in all monkeys and ranged from 3,803 to 3,840 ng/mL in the male monkeys and from 2,019 to 2,781 ng/mL in the female monkeys on Day 161. To confirm the reproducibility o f the analytical method, selected serum samples collected from individual monkeys at various times during the study were re-analyzed approximately 3 months after their initial analysis. The results o f these determinations are presented in Table 3. The results o f the first and second analyses o f each serum sample were in good agreement (within 20% o f the initially determined concentration) with each other. Pharmacokinetic parameters calculated from serum concentrations of perfluorooctanesulfonate in individual monkeys are presented in Table 4. AUQ.-^ . values ranged from 1666 to 1877 pgday/mL in male monkeys and from 932 to 1259 figday/mL in female monkeys. The terminal half-life o f perfluorooctanesulfonate in serum ranged from 122 to 146 days (mean: 132 days) in the three male monkeys and from 88 to 138 days (mean: 110 days) in the three female monkeys. The total body clearance o f perfluorooctanesulfonate was 1.0 to 1.2 mL/day/kg in male monkeys and 1.6 to 2.1 mL/day/kg in female monkeys. The volume o f distribution o f perfluorooctanesulfonate ranged from 178 to 220 mL/kg in male monkeys and from 231 to 327 mL/kg in female monkeys. The amount o f perfluorooctanesulfonate eliminated in urine by individual monkeys at various times after dosing is presented in Table 5. Only very low levels (0.1% o f the administered dose) o f perfluorooctanesulfonate were measured in urine during any given 24hour period of sample collection between Day 1 and Day 91 after dosing. There was no clear indication o f a sex-related difference in the urinary excretion o f perfluorooctanesulfonate. The urinary excretion o f perfluorooctanesulfonate was prolonged; detectable levels o f the compound were present in urine on Day 91 (last day o f urine collection) after dosing. 000388 7 4.0 Discussion The results o f this study indicated that perfluorooctanesulfonate was detectable in male and female monkeys for a prolonged period o f time after administration o f a single iv dose o f 2 mg/kg. Among individual male and female monkeys, the apparent terminal elimination half-life o f perfluorooctanesulfonate was estimated to be 88-146 days. In addition, it was found that only low levels o f perfluorooctanesulfonate were excreted in urine by either male or female monkeys during any given 24-hour period o f sample collection. For either sex, the urinary excretion o f perfluorooctanesulfonate was prolonged; the compound was still present in urine at 91 days after dosing (longest time point that urine samples were collected). From the serum concentration versus time data and die urinary excretion data, there was no clear indication the pharmacokinetics o f perfluorooctanesulfonate were different in male and female monkeys. A summary o f estimated pharmacokinetic parameters and urinary excretion data obtained during this and previous investigations with various perfluoro-compounds is presented in Appendix C. For the C6 and C8 carboxylated derivatives, and possibly the C4 carboxylated compound, the serum pharmacokinetics appeared to be different in male and female monkeys. Sex differences in pharmacokinetic parameters were less apparent for the C4, C6, and C8 sulfonated derivatives. 5.0 Conclusions There was no clear indication that the serum kinetics o f perfluorooctanesulfonate were different in male and female monkeys. Perfluorooctanesulfonate was eliminated in urine by both male and female monkeys at low levels for a prolonged period o f time (>91 days) after iv administration. 6.0 Record Archives Data, specimens, and a copy o f the final report from this study will be stored in the Archives at Southern Research for up to 1 year after acceptance of tire final report by the Sponsor. After 1 year and with the permission o f the Sponsor's Monitor, the data and any samples/specimens will be shipped to the Sponsor or to the Sponsor's designated archival facility. If materials are to be retained 000389 8 in the archives beyond this date, such continued storage will be for a specific fee determined with the Sponsor. A copy o f the final report will be retained in the central archives at Southern Research. 7.0 References 1. Institute o f Laboratory Animal Resources, Commission on Life Sciences, National Research Council; National Academy Press; Washington D.C.; 1996. 000390 Table 1 A Pharmacokinetic Study of Potassium Perfluorooctanesulfonate in the Cynomolgus Monkey Individual Body Weights Animal ID 2053 2054 2211 2058 2059 2061 Dose Level 2 mg/kg 2 mg/kg 2 mg/kg 2 mg/kg 2 mg/kg 2 mg/kg 0 6.5 6.4 6.6 3.6 3.5 4.3 Body Weight (kg) on Study Day 47 Males 14 21 6.8 6.6 6.7 6.7 6.7 6.5 6.5 6.5 6.6 6.6 6.6 6.6 Females 3.5 3.6 3.7 3.5 3.5 3.4 3.5 3.4 4.3 4.3 4.3 4.2 28 6.7 6.4 6.5 3.4 3.5 4.1 42 6.6 6.5 6.6 1 3.5 3.5 4.3 j Animal ID | I 2053 2054 1 2211 | I 2058 2059 2061 Dose Level 2 mg/kg 2 mg/kg 2 mg/kg 2 mg/kg 2 mg/kg 2 mg/kg Body Weight (kg) on Study Day 56 70 85 105 126 Males 6.7 6.6 6.7 6.7 6.9 6.4 6.4 6.4 6.5 6.6 6.6 6.6 6.8 6.7 6.9 Females 3.5 3.5 3.7 3.6 3.7 3.5 3.5 3.6 3.6 3.7 4.3 4.3 4.5 4.6 4.6 151 7.0 6.7 7.2 3.8 3.8 4.8 000391 10 Table 2 A Pharmacokinetic Study o f Potassium Perfluorooctanesulfonate in the Cynomolgus Monkey Serum Concentrations o f Perfluorooctanesulfonate Timepoint 2053 0 BQL 0.5 hrs 16,250 2 hrs 11,390 4 hrs 10,273 8 hrs 9,578 24 hrs 8,345 48 hrs 7,998 Day 4 7,354 Day 7 7,589 Day 14 8,630 Day 21 8,547 Day 28 9186b Day 42 9,485 Day 56 7,880 Day 70 9,038 Day 105 4,596 Day 161 3,803 Serum Concentration (ng/mL) Males Females 2054 2211 2058 2059 2061 BQL BQL 5.8 BQL BQL 15,120 13,790 7,871 10,755 11,790 12,480 11,280 7,991 9,500 9,207 11,308 9,585 6,080 9,490 9,200 11,473 10,015 7,358 10,525 8,717 9,538 7,039 5,042 a 7,294 7,636 7,501 5,165 7,860 7,436 9,157 7,277 4,994 6,620 7,825 10,090 7,592 5,163 5,485 6,873 10,040 9,515 5,586 5,995 7,136 7205b 8,380 6,972 5,675 8,023 11,746 9089b 4,990 5,680 8,208 10,580 8,358 6,403 5,730 6,134 8,695 7,406 4,652 6,265 6,204 9,885 6,624 4,457 5,735 6,925 4,423 3,750 3,079 2,197 3,332 3,805 3,840 2,584 2,019 2,781 BQL = Below the quantitation limit (<10 ng/mL) a Initial analysis o f the sample was problematic; there was an insufficient volume o f sample for a reanalysis. b Data represent an average of two analyses o f the sample. Page 1 of 1 000392 11 Table 3 A Pharmacokinetic Study o f Potassium Perfluorooctanesulfonate in the Cynomolgus Monkey Replicate Analysis Comparisons for Potassium Perfluorooctanesulfonate in Monkey Serum Sample" F20614 hr F2058 Day 14 F2061 24 hr F2061Day 7 F2058 Day 42 Results from 10/15/01 (ng/mL) 9,200 5,586 7,294 6,873 6,403 Results from 1/9/02 (ng/mL) 7,512 5,674 7,399 6,737 5,216 Percent Difference -18.3 1.6 1.4 -2.0 -18.5 "These samples were randomly selected from a group o f samples containing a sufficient volume o f serum for analysis. Page 1 of 1 000393 Table 4 A Pharmacokinetic Study o f Potassium Perfluorooctanesulfonate in the Cynomolgus Monkey Pharmacokinetic Parameters Calculated from Serum Concentrations o f Potassium Perfluorooctanesulfonate Parameter 2053 2054 Male 2211 Average SD Cmax (pg/mL)a AUC0.liit (pg*day/mL)b 18.3 14.7 16.1 16.4 1.8 1,078 962 1,165 1,068 102 A U C o.infinity (pg*day/mL)c 1,877 1,666 1,833 1,792 111 ti/2e (day)d 146 127 122 132 13 Clearance (mL/day/kg)e Vdss (mL/kg)f 1.0 1.2 1.1 1.1 0.1 209 220 178 202 22 aMaximum concentration in serum bArea under the serum concentration time curve from 0 to the last time point 'Area under the serum concentration time curve from 0 to infinity dHalf-life of the terminal elimination phase 'Total body clearance Volume o f distribution at steady state g^Value for 0.5 hour time point was excluded from the pharmacokinetic analyses 20588 10.5 672 1,188 138 1.7 327 2059 11.2 677 932 88 2.1 264 Female 2061 Average 12.8 11.5 841 730 1,259 1,126 104 110 1.6 1.8 231 274 SD 1.2 96 172 26 0.3 49 Page 1 of 1 13 Table 5 A Pharmacokinetic Study o f Potassium Perfluorooctanesulfonate in the Cynomolgus Monkey Animal ID 2053 2054 2211 2058 2059 2061 2053 2054 2211 2058 2059 2061 2053 2054 2211 2058 2059 2061 2053 2054 2211 2058 2059 2061 Urinary Excretion o f Perfluorooctanesulfonate Urine Concentration Sex (ng/mL) M BQL M BQL M BQL F BQL F BQL F BQL M 11 M 33 M 22 F 29 F 14 F 10 M 15 M 41 M 24 F 20 F 22 F 16 M 31 M 33 M 27 F 41 F 46 F 84 Urine Volume Total (mL) <M) Baseline 740 -- 300 -- 350 -- 280 -- 150 - 420 -- Day 1 (0-24 hours) 540 5.9 210 6.9 330 7.3 170 4.9 130 1.8 350 3.5 Day 2 '24-48 hours) 450 6.8 160 6.6 180 4.3 210 4.2 160 3.5 200 3.2 Day 7 330 10.2 340 11.2 220 5.9 180 7.4 140 6.4 110 9.2 Dose (m s ) 26,000 25,600 26,400 14,400 14,000 17,200 26,000 25,600 26,400 14,400 14,000 17,200 26,000 25,600 26,400 14,400 14,000 17,200 26,000 25,600 26,400 14,400 14,000 17,200 Percent of Dose inUrine (%) --- -- -- - -- 0.02 0.03 0.03 0.03 0.01 0.02 0.03 0.03 0.02 0.03 0.03 0.02 0.04 0.04 0.02 0.05 0.05 0.05 BQL = Below the quantitation limit (<5 ng/mL) a One o f these samples was mislabeled during collection; due to uncertainty regarding correct identification, the results are not reported. Page 1 of 3 000395 14 Table 5 (Continued) A Pharmacokinetic Study o f Potassium Perfluorooctanesulfonate in the Cynomolgus Monkey Animal ID 2053 2054 2211 2058 2059 2061 2053 2054 2211 2058 2059 2061 2053 2054 2211 2058 2059 2061 2053 2054 2211 2058 2059 2061 Urinary Excretion o f Perfluorooctanesulfonate Urine Concentration Sex (ng/mL) M 32 M 40 M 31 F 37 F 40 F 60 M 14 M 20 M 21 F 37 F 75 F 72 M 31 M 20 M 28 F 31 F 38 F 41 M6 M 11 M 11 F 28 F 34 F 62 Urine Volume (mL) Total (m s ) )ay 15 400 12.8 230 9.2 320 9.9 180 6.7 90 3.6 140 8.4 Day 21 450 6.3 400 8.0 560 11.8 130 4.8 180 13.5 200 14.4 DZS 410 12.7 510 10.2 300 8.4 230 7.1 180 6.8 110 4.5 Day 42 810 4.9 930 10.2 730 8.0 120 3.4 130 4.4 190 11.8 Dose (m s ) 26,000 25,600 26,400 14,400 14,000 17,200 26,000 25,600 26,400 14,400 14,000 17,200 26,000 25,600 26,400 14,400 14,000 17,200 26,000 25,600 26,400 14,400 14,000 17,200 Percent of Dose in Urine (%) 0.05 0.04 0.04 0.05 0.03 0.05 0.02 0.03 0.04 0.03 0.10 0.08 0.05 0.04 0.03 0.05 0.05 0.03 0.02 0.04 0.03 0.02 0.03 0.07 BQL = Below the quantitation limit (<5 ng/mL) a One o f these samples was m islabeled during collection; due to uncertainty regarding correct identification, the results are not reported. Page 2 of 3 000396 15 Table 5 (Continued) A Pharmacokinetic Study o f Potassium Perfluorooctanesulfonate in the Cynomolgus Monkey Urinary Excretion o f Perfluorooctanesulfonate Animal ID 2053 2054 2211 2058 2059 2061 2053 2054 2211 2058 2059 2061 2053 2054 2211 2058 2059 2061 Urine Concentration Sex (ng/mL) Ma M 12 M 12 Fa F 20 F 42 M 13 M 19 M 11 F 27 F 23 F 33 M8 M 10 M9 F 16 F 20 F 41 Urine Volume (mL) 620 1,010 560 340 90 200 600 390 510 220 190 170 660 900 400 300 90 150 Total (Mg) Day 56 -- 12.1 6.7 - 1.8 8.4 Day 70 7.8 7.4 5.6 5.9 4.4 5.6 Day 91 5.3 9.0 3.6 4.8 1.8 6.2 Dose (MS) 26,000 25,600 26,400 14,400 14,000 17,200 26,000 25,600 26,400 14,400 14,000 17,200 26,000 25,600 26,400 14,400 14,000 17,200 Percent of Dose in Urine (%) - 0.05 0.03 -- 0.01 0.05 0.03 0.03 0.02 0.04 0.03 0.03 0.02 0.04 0.01 0.03 0.01 0.04 BQL = Below the quantitation limit (<5 ng/mL) a One o f these samples was mislabeled during collection; due to uncertainty regarding correct identification, the results are not reported. Page 3 of 3 000397 16 M2053 Serum Concentration (ug/mL) Serum Concentration (ug/mL) -- Observe -- Predicted M2054 1 1 r~\____Q____ r __~o----- 0 Observe -- Predicted J 0 20 40 60 BO 100 120 140 160 180 Tim e (Days) Figure 1 A Pharmacokinetic Study of Potassium Perfluorooctanesulfonate in the Cynomolgus Monkey Serum Concentration Profile of Perfluorooctanesulfonate 000398 17 M2211 F2058 Observe -- Predicted -s- Observe -- Predicted Figure 1 (Continued) A Pharmacokinetic Study o f Potassium Perfluorooctanesulfonate in the Cynomolgus Monkey Serum Concentration Profile of Perfluorooctanesulfonate 000399 18 F2059 Serum Concentration (ug/mL) Serum Concentration (ug/mL) -9 - Observe -- Predicted F2061 i t rui ----- e ----- -- Observe -- Predicted o 3 0 20 40 60 80 100 120 140 160 180 Tim e (Days) Figure 1 (Continued) A Pharmacokinetic Study o f Potassium Perfluorooctanesulfonate in the Cynomolgus Monkey Serum Concentration Profile o f Perfluorooctanesulfonate O0400 Appendix A Study Protocol and Comments on Study Data 000401 A -l Study Protocol: A Pharmacokinetic Study of Potassium Perfluorooctanesulfonate in the Cynomolgus Monkey Southern Research Study ID: 9921.6 July 26,2001 Southern Research INSTITUTE 000402 STUDY NO.: 9921.6 A-2 1.0 SPONSOR REPRESENTATIVE AND CONTACTS: Sponsor: 3M Center, 220-2E-02 P.O. Box 33220 St. Paul, Minnesota 55133-3220 Sponsor's R epresentative & Study Monitor: John L. Butenhoff, Ph.D., D.A.B.T. 3M Center Building 220-2E-02 St. Paul, Minnesota 55144-3220 (651) 733-1962; FAX: (651) 733-1773 Protocol Approval: (Initial last page also) July 26,2001 Page 2 of 13 Test Article: Ship Unused Test Article to: John L. Butenhoff * Date Perfluorooctanesulfonate, potassium salt D. Hakes Building B236 3M Center P.O. Box 33327 55133-3327 St. Paul, Minnesota 55144-1000 000403 STUDY NO.: 9921.6 2.0 TITLE: July 26,2001 Page 3 of 13 A Pharmacokinetic Study o f Potassium Perfluorooctanesulfonate in the Cynomolgus Monkey 3.0 OBJECTIVE: The objectives o f this study are to determine the concentration o f perfluorooctanesulfonate in serum and urine at various times following administration o f a single intravenous dose o f potassium perfluorooctanesulfonate 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: i Event Day of Treatment Urine and Feces Collections Serum Drug Levels Draft Report Due Final Report Due Sequence (Day) Date(s) - Year 2001 0 7/30/01 Baseline (predose) 1 (0-24 hour) 2 (24-48 hour) 7 14 21 7/27/01 7/30/01 7/31/01 8/6/01 8/13/01 8/20/01 28 8/27/01 42 9/10/01 56 9/24/01 70 10/8/01 91 10/29/01 0: 0 (predose), 0.5,2,4, and 8 hours postdose 7/36/01 1: 24 hours postdose 7/31/01 2 8/1/01 4 8/3/01 7 8/6/01 14 8/13/01 21 8/20/01 28 8/27/01 42 9/10/01 56 9/24/01 70 10/8/01 105 11/12/01 161 12/31/01 60 Calendar days after completion of the in-life m m phase 15 days after final Sponsor comments received t ________________________ 000404 STUDY NO.: 9921.6 6.0 STUDY PERSONNEL: July 26,2001 P age4of 13 The following are the primary contributors and supervisory personnel participating in this study. Study Director: Alternate Study Director: Director, Safety Assessment: Associate Director: Manager, Bioanalytical Chemistry: Supervisor, In-Life Laboratories: Veterinarian: Patricia E. Noker James D. Johnson Ward R. Richter Norman D. Jefferson James D. Johnson LaJuana A. Durbin Darrell E. Hoskins PhJX, DJLB.T. M.S., MBA. D.V.M., M.S., D.A.C.VP. B.A. M.S., M.BA. AAS. D.V.M., A.C.LAJvi. (DipL) 7.0 TEST & CONTROL ARTICLES: The test article will be supplied by the Sponsor, who will be responsible for documentation o f stability, as well as methods o f synthesis, fabrication, or derivation. Upon completion of the study, residual bulk test article will be returned to the Sponsor. 7.1 Identity of the Test Article: Name: Identification: Supplier: Lot Number(s): Special Handling: Perfluorooctanesulfonate, potassium salt T-6295 3M To be documented in the study data. None Characterization: Documentation o f the characterization o f the test article, including identity, purity, strength, and composition, as well as methods o f synthesis, fabrication, or derivation, is the responsibility o f the Sponsor. Copies o f characterization data have been provided to the testing laboratory. Stability & Storage: The bulk test article will be stored at room temperature. Stability of the bulk test article is the responsibility of the Sponsor. Q00405 A-5 STUDY NO.: 9921.6 7.2 Identity of the Vehicle: July 26,2001 Page 5 of 13 Name: Supplier: Lot Numbers): Special Handling: Sterile Saline Commercial supplier To be documented in the study data. None Characterization : Documentation o f the characterization o f the vehicle may be attained by recording all pertinent information from the container labels, or by retaining the container labels, or copies thereof, in the study data. The vehicle is a commercially available product. Stability & Storage: Sterile saline is considered stable through the date(s) of expiration provided by the manufacturer when stored appropriately. The bulk vehicle will be stored in accordance with the manufacturer's instructions. 7.3 F o r m u l a t io n : Preparation: The test article will be formulated in sterile saline at a concentration o f 1 mg/mL for intravenous administration; briefly, the required amount o f test article will be mixed with the required amount of sterile saline, and the mixture will be stirred until the test article is visibly in solution. Formulations will be stored refrigerated until used for dosing; formulations of the test article in sterile saline are expected to be stable for weeks when So stored. Dose Form ulation Concentration and Homogeneity Analyses: No analysis of dose formulation concentration and homogeneity will be conducted. 8.0 TEST SYSTEM: Species & Strain: Supplier: Age on Day 1: Weight at randomization: Number on Study: Cynomolgus monkeys (Macacafascicularis) Charles River BRF, Inc. (Houston, TX) 3-4 years o f age (estimated) 3-7 kg Males -3 Females -3 Animals were previously dosed with potassium perfluorobutanesulfonate in study 9921.1, potassium perfluorobutanoate in study 9921.2, potassium perfluorohexanoate in study 000406 STUDY NO.: 9921.6 9921.3, potassium perfluorooctanoate in study 9921.4, and potassium perflourohexanesulfonate in 9921.5. July 26,2001 Page 6 of 13 8.1 Justification: Primates are commonly used in preclinical pharmacological and toxicological evaluations o f compounds used or intended for use in humans, or to which humans might be exposed. 8.2 Housing: During quarantine/acclimation and study, animals will be individually housed in stainless steel, slat-bottom cages. All animals will be housed in a room that provides a minimum o f 10 air exchanges per hour. Controls will be set to maintain the animal room at a temperature o f 64-84 F and a relative humidity o f 30-70%. A 12-hour light/12-hour dark cycle will be routinely maintained. Animals will be acclimated in the same room used for study. 8.3 Bedding: None required for caging equipped with flushable pans. For cages equipped with excrement absorption pans, commercial heat-treated hardwood chip bedding will be used for excrement absorption. Analyses o f the bedding, supplied by the vendor, will be reviewed by the Department o f Veterinary Medicine and Bioresources (DVMB) o f Southern Research to assure that no known contaminants are present that could affect the health o f the animals. 8.4 Diet: Diet will be commercial Certified Primate Chow #5048 (PMI Feeds, Inc.; S t Louis, MO). The primates will be offered feed twice daily, with approximately the recommended daily ration available at each feeding interval. In addition, the diet will be supplemented with fresh fruit offered daily and treats offered several times each week. The quantity o f the daily ration will be sufficient to meet nutritional requirements. Analyses o f the feed, supplied by the vendor, will be reviewed by the DVMB o f Southern Research to assure that no known contaminants are present that could affect the health o f the animals. 000407 A-7 STUDY NO.: 9921.6 8.5 W a t e r : July 26,2001 Page 7 of 13 Water (Birmingham public water supply) will be supplied ad libitum during the quarantine and study periods via an automatic watering system. Samples of water from the animal facility will be periodically analyzed, and the analyses will be reviewed by the DVMB o f Southern Research to assure that no known contaminants are present that could affect the health o f the animals. 8.6 Q u a r a n t in e : All primates were selected from stock animals that were quarantined for a minimum o f 35 days upon receipt at Southern Research. No prophylactic or therapeutic treatments were administered during the quarantine period. Standard procedures conducted during the quarantine period were as follows: a) A complete physical examination including a fecal examination for internal parasites, complete blood count (CBC), body weight, and body (rectal) temperature was performed; b) three tuberculin tests at 2-week intervals (administered intrapalpebrally, using alternate eyelids for each test) were performed on each primate. Primates tested negative to all three tests prior to release from quarantine. ; c) the blood sample drawn for CBC was also used for measurement o f B virus titer; d) a fecal sample for culture (screening for Salmonella and Shigella) was obtained and submitted to an independent laboratory for analysis; and e) all primates were examined at least once weekly by a veterinarian and observed (cage-side observations) twice daily for abnormal clinical observations and mortality/moribundity. Throughout the subsequent holding and study periods, monkeys will be maintained under conditions similar to those for quarantine. In addition, quarterly evaluations to be performed on each monkey will include tuberculin testing, body weight determination, and body temperature measurement. Primates that respond positively to a tuberculin test will be euthanized immediately. 8.7 PSYCHOLOGICAL W ELL-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 000408 STUDY NO.: 9921.6 July 26,2001 .Page 8 of 13 peanuts, and raw fruit. Where possible, primates will be housed proximate to one another for visual and vocal contact. 8 .8 A n i m a l I d e n t i f i c a t i o n : The primates will be individually identified by chest tattoo number or letter combination. Positive identification will be required after every cage change and prior to blood sampling, dose administration, and observation. 9.0 EXPERIM ENTAL DESIGN: As only one treatment group will be used in this study, no formal randomization will be required. Doses will be administered by intravenous injection to determine the pharmacokinetics of the test article. Each primate (three males, three females) will receive a single dose of potassium perfluorooctanesulfonate by injection into a superficial aim or leg vein. Blood samples for serum drug level determinations will be collected from each primate at selected time points during the study. Urine and feces will also be collected at pre determined intervals. A synopsis o f the study design is presented in the following table. [" Study Procecures -1 0 Health Check X Dose Preparation X M l Hi 1 lllilllllllWMmfSaffi W Clinical Observations X Body Weights Urine & Feces X X Serum Drug Levels X a Denotes week Day of Study | r1 2 4 7 L i l 21 28 35 42 56 70 84 105 126 147 1 WWHMkpP ! m srawuTM.B I b H w HMI p m ___ ___ .U J X XX XXX X X XX X XXXXXXXXXX X XX XX XXXX X XX X X X X XXX X XXX XX X 9.1 R a n d o m iz a t io n & G r o u p A s s ig n m e n t : As only one treatment group will be used in this study, no formal randomization will be required. 000409 STUDY NO.: 9921.6 9.2 Dose Procedure: July 26,2001 P aee9 o f 13 Each primate (three males, three females) will receive a single intravenous (TV) dose o f potassium perfluorooctanesulfonate (2 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 o f the study. 9.3 Clinical Observations: Daily O bservations: All monkeys will be observed once daily during the holding period and twice daily, morning and afternoon, at least 4 hours apart, during the study for signs o f mortality/moribundity and overt toxicity. Animals found in extremis will be humanely sacrificed by an overdose o f barbiturate followed by exsanguination with appropriate approval. Detailed Observations: Each primate will be examined shortly after dose administration for detailed clinical signs o f toxicity. All findings will be recorded. Additional clinical observations will be performed and recorded on days o f blood collection. 9.4 Body Weights: Each primate will be weighed on Days -1 , 4, 7, 14, 21, and 28 and at bi-weekly (Days 42, 56,70, and 84) or tri-weekly (105,126,147) intervals thereafter through the end o f the study. 9.5 Urine and Feces Collections: Urine and feces will be collected for approximate 24 hour intervals prior to dosing and on Days 1 (0-24 hours postdose), 2 (24-48 hours post dose), 7 ,1 4 ,2 1 ,2 8 ,4 2 ,5 6 , 70 and 91. The volume o f each urine sample will be measured upon collection. All samples collected will be stored frozen at -2 0 C or below prior to analysis (urine) or until further notice by the Sponsor (feces). 9.6 S er u m D r u g L e v e l s: Blood samples (approximately 3 mL) will be collected from each primate at approximately 0 (predose) minutes; 0 .5 ,2 ,4 ,8 , and 24 hours; and 2 ,4 ,7 ,1 4 ,2 1 ,2 8 , 42 ,5 6 ,7 0 ,1 0 5 , and 161 days after dosing. Additional blood samples may be taken at subsequent times if requested by the Sponsor. Samples will be collected into tubes 000410 STUDY NO.: 9921.6 July 26,2001 Page 10 of 13 without anticoagulant and will be allowed to clot at room temperature. The blood samples will then be centrifuged, and the serum will be separated and stored frozen at -2 0 G or below until analyzed. 9.7 B io a n a ly t ic a l M et h o d D e v e lo p m e n t : Bioanalytical method(s) will be developed for the determination o f perfluorooctanesulfonate in serum and urine matrices. The method(s) will be validated for accuracy and ideally will be sensitive to the 1 ppm or less level. If feces and/or other tissues require analyses, these analyses will be negotiated with the Sponsor. 9.8 B i o a n a l y t i c a l S a m p l e A n a l y s i s : The serum and urine samples from all monkeys will be analyzed for concentrations o f perfluorooctanesulfonate using die previously validated method. The data will be expressed as equivalents of potassium perfluorooctanesulfonate. Feces will be analyzed only if requested by the Sponsor. 9.9 A n i m a l D i s p o s i t i o n : At the end o f the study, monkeys will be maintained in the stock colony. In the event untoward reactions or other conditions warrant the euthanasia of a monkey at any time during the sample collection period, a serum sample (5-10 mL) will be obtained from the animal prior to euthanasia. After euthanasia, the animal will be necropsied and the liver and bile (as much as possible) will be removed and stored at approximately -70 C. 10.0 DATA ANALYSIS: Pharmacokinetic parameters (e.g., AUC, half-life, clearance) will be estimated from serum concentrations o f unchanged perfluorooctanesulfonate, as appropriate and feasible, using a standard pharmacokinetic program. The total amount o f perfluorooctanesulfonate in urine will be calculated and expressed in terms of percent o f dose. Mean values and standard deviations will be calculated for each time point and sample type, as appropriate. No other statistical analyses o f the data will be performed. 000411 STUDY NO.: 9921.6 11.0 RECORDS: July 26,2001 Page 11 of 13 All raw data pertaining to the conduct o f this study, and all samples/specimens collected in this study, will be stored in the Archives at Southern Research Institute for up to 1 year after acceptance o f the final report by the Sponsor. After 1 year and with the permission o f the Sponsor's Monitor, the data and any samples/specimens will be shipped to the Sponsor or to the Sponsor's designated archival facility. If materials are to be retained in the archives beyond this date, such continued storage will be for a specific fee determined with the Sponsor. A copy o f the final report will be retained in the central archives at Southern Research. 12.0 FINAL REPORT: A brief letter report summarizing the serum drug level results will be issued as soon as the information is available. A draft final report will be issued within 60 calendar days after completion o f the in-life aspects o f the study. The final report (electronic and hard copies) will be issued within 15 working days after receipt o f the Sponsor's final review comments on the draft report. The final report for the present study will include, but not necessarily be limited to the following: Dose formulation preparation Clinical observations Body weight data Serum drug level data Pharmacokinetic parameters Urine excretion data 13.0 REGULATORY REFERENCES: This study will be conducted in accordance with the protocol and the Standard Operating Procedures (SOPs) o f Southern Research, and in accordance with the applicable regulatory requirements, as addressed below. 13.1 p r o t o c o l A m e n d m e n t s a nd d e v ia t io n s : Amendments: All changes in or revisions o f the approved protocol and the reasons thereof will be documented, signed, and dated by the Study Director, and the Sponsor's Monitor. Amendments will be maintained with the protocol. Written approval (a fax signature or electronic communication, such as email) for changes in the protocol may be granted by the Sponsor's Monitor, but a written amendment will follow. 000412 STUDY NO.: 9921.6 July 26,2001 Pace 12 of 13 Deviations: All operations pertaining to this study, unless specifically defined in this protocol, will be performed according to the Standard Operating Procedures (SOPs) o f Southern Research and/or the protocol, and any deviations from protocol or SOP will be documented. 13.2 Regulatory Compliance: Good L aboratory Practices: This nonclinical laboratory study will be conducted in the spirit of, but will not require strict compliance with, the U.S. Food and Drug Administration's (FDA) Good Laboratory Practice (GLP) regulations (21 CFR Part 58). Data from this study may be submitted to the FDA in support o f an IND/NDA application. Quality Assurance Review: As this study will not be conducted in strict compliance with FDA's GLP regulations, neither the in-life activities nor the final report will be audited by the Quality Assurance Unit at Southern Research. 13.3 Facilities Management and Animal Husbandry: Animal care will be in compliance with the SOPs o f Southern Research, the Guidelines fo r the Care and Use o f Laboratory Animals, 7* Edition (Institute o f Animal Resources, Commission on Life Sciences, National Research Council; National Academy Press; Washington, DC; 1996), and the U.S. Department o f Agriculture through the Animal Welfare Act (Public Law 99-198). Southern Research Institute is fully accredited by the American Association for Accreditation o f Laboratory Animal Care (AAALAC). 13.4 Animal W elfare Act Compliance: By signing this protocol, the Sponsor signifies that there are no generally accepted alternatives to the use o f animals, and that the study described by this protocol does not unnecessarily duplicate previously conducted or reported experiments. Procedures used in this protocol are designed to conform to accepted practices and to minimize or avoid causing pain, distress, or discomfort in the animals. In those circumstances in which required study procedures are likely to cause more than momentary or slight pain or distress, the animals will receive appropriate analgesics or anesthetics unless the withholding of these agents has been justified in writing by the Study Director and/or Sponsor and approved by the IACUC. ooo4ia A-13 STUDY NO.: 9921.6 July 26,2001 Page 13 of 13 The number o f animals selected for use in this study is considered to be the m inim um number necessary to meet scientific and regulatory guidelines for this type o f study. This study design was reviewed by the IACUC at Southern Research Institute and was approved on 07/26/2000; it was assigned IACUC tracking number 00-07-034. 14.0 PROTOCOL APPROVALS: This protocol has been reviewed and approved. Study Director: a> Patricia E. Noker, Ph.D., D.A.B.T. Study Director Sponsor's M onitor: INITIALS ONLY (See page 2) Date /}p r o f 7 Date Management Approval: Ward R. Richter, M.S., D.V.M., D.A.C.V.P. Date Director, Safety Assessment Department, Southern Research Institute (D me 5 M y Motnb* c U m l reh^v^ f>n>brof. A ropy/ o f l h e orymo.1 uja 5iynei amn ty+ke tudy + foY V tft -b Ihe rnombr -fbr 000414 A-14 Comments on Study Data The following is a list o f protocol and/or SOP deviations that occurred during this study. Unless otherwise noted, the Study Director determined these deviations to have no adverse impact on the outcome o f the study. The protocol required that mine collections be conducted on Day 14. In order to accommodate the laboratory schedule, the collections specified for Day 14 were made on Day 15. Due to an oversight at protocol development, the protocol lists Day 161 as 12/31/01 in the "Serum Drug Levels" section o f Section 5.0. Day 161 was actually on 1/7/02, and serum was collected on that day instead o f 12/31/01. Due to technician error, a clinical observation was not recorded on Day 21 (8/20/01). In the data for this study, recording errors occurred but were corrected in accordance with the applicable SOPs. 000415 Appendix B Analytical Method for Determination of Perfluorooctanesulfonate in Monkey Serum and Urine 000416 B -l Page 1 o f 13 ANALYTICAL M ETHOD M ethod No.: BACG-3607 Title: Detennination o f Perfluorooctanesulfonate in Monkey Serum and Urine: Sample Preparation and Analysis by HPLC Mass Spectrometry/Mass Spectrometry (HPLC/MS/MS) 1.0 2.0 2.1 2.1.1 2.1.2 PRINCIPLE Serum or urine samples are obtained from cynomolgus monkeys treated with Perfluorooctanesulfonate (PFOS). The serum or urine (e.g., 0.5 mL) containing (PFOS) is fortified with an internal standard (IS), Perfluorooctanecarboxalate (PFOC). The samples are then mixed with an ion-pairing reagent, buffer and water, followed by extraction with ethyl acetate. The ethyl acetate layer is removed, evaporated to dryness, reconstituted in 95% methanol containing 1.5 % formic acid, 5 % 5mM ammonium acetate, filtered, and transferred to autosampler vials, and analyzed by HPLC Mass Spectrometry/Mass Spectrometry (HPLC/MS/MS). The range o f reliable results extends from about 20 to 10,000 ng/mL o f PFOS in serum and from 10 to 500 ng/mL in urine. Samples containing PFOS at concentrations greater than 10,000 ng/mL may be diluted with control blank matrix so that the concentration o f PFOS will be within the range of reliable results prior to analysis. The mass spectrometry o f PFOS and PFOC is accomplished in the negative ion mode. The ion spray source voltage is set at - 2000 volts which is low enough to greatly reduce the formation of other potentially interfering ions extracted from the matrix. CAUTION: Since primates may carry a number o f zoonoses, all unpreserved tissues, including blood, plasma and serum, are to be considered as biohazards and handled with universal precautions. Refer to SOP number SRI 2-5-5 for a description of safety procedures to be used when handling unpreserved primate tissue. REAGENTS AND SOLUTIONS The listed reagents or their equivalents may be used. Neat Reagents Water, deionized and organic free (from in-house purification system; e.g., Ingalls 21ON) Methanol, HPLC grade 000417 B-2 Page 2 o f 13 ANALYTICAL M ETHOD M ethod No.: BACG-3607 Title: Determination o f Perfluorooctanesulfonate in Monkey Serum and Urine: Sample Preparation and Analysis by HPLC Mass Spectrometry/Mass Spectrometry (HPLC/MS/MS) 2.1.3 Perfluorooctanesulfonate (analyte), as provided by the client 2.1.4 Perfluorooctanecarboxalate (internal standard), 97% 2.1.5 Ammonium acetate, HPLC grade 2.1.6 Blank control monkey serum 2.1.7 Sodium Carbonate, Certified ACS Grade or equivalent 2.1.8 Sodium Bicarbonate, Certified ACS Grade or equivalent 2.1.9 Ethyl Acetate, HPLC grade 2.1.10 Tetrabutylammonium Hydrogen Sulfate, 97% 2.1.11 Sodium Hydroxide 50% solution, Certified grade or equivalent 2.1.12 Blank control monkey urine 2.1.13 Formic Acid, 88% 2.2 P repared Solutions Appropriate changes in the solutions may be made at the discretion o f the analyst 2.2.1 5 mM Ammonium acetate in organic free water 2.2.1.1 For example, to prepare 4 liters, measure out ammonium acetate (e.g., 1.542 g) and add in organic-free water (e.g., 4 L). Mix well and filter through HPL] mobile phase filtration apparatus. 2.2.2 TBA Ion-Pairing Solution (0.5 M tetrabutylammonium hydroxide) B-3 Page 3 o f 13 ANALYTICAL M ETHOD M ethod No.: BACG-3607 Title: Determination o f Perfluorooctanesulfonate in Monkey Serum and Urine: Sample Preparation and Analysis by HPLC Mass Spectrometry/Mass Spectrometry (HPLC/MS/MS) 2.2.2.1 For example, to prepare 25 mL, dissolve 4.24 g o f tetrabutylammonium hydrogen sulfate in deionized water and adjust the pH to 10 with 50% NaOH solution. Note: a more dilute solution of NaOH in w ater may be used to effect smaller pH adjustm ents. 2.2.3 Carbonate/Bicarbonate Buffer Solution for Serum (0.25M/0.25M) 2.2.3.1 For example, to prepare 100 mL, dissolve approximately 2.65 g o f sodium carbonate and approximately 2.10 g o f sodium bicarbonate in 100 mL o f deionized water. Mix well to ensure complete dissolution. 2.2.4 Carbonate/Bicarbonate Buffer Solution for Urine (1.0M/1.0M) 2.2.4.1 For example, to prepare 100 mL, dissolve approximately 10.6 g o f sodium carbonate and approximately 8.4 g o f sodium bicarbonate in 100 mL o f deionized water. Mix well to ensure complete dissolution. 3.0 INSTRUMENTS, MATERIALS, AND APPARATUS The following or their equivalents may be used. 3.1 HPLC pump(s), autosampler, and triple quadrupole mass spectrometer 3.2 Autosampler vials with inserts 3.3 Vortex mixers (e.g., touch mixer and IKA-Vibrax platform mixer) 3.4 Solvent-concentration apparatus (e.g., Zymark Turbo-Vap with source o f nitrogen) 3.5 HPLC mobile phase filtration apparatus 3.6 Filters for HPLC mobile phase filtration apparatus (e.g., Nylon-66, 0.20 pm) 000419 B-4 Page 4 o f 13 ANALYTICAL M ETHOD M ethod No.: BACG-3607 Title: Detennination o f Perfluorooctanesulfonate in Monkey Serum and Urine: Sample Preparation and Analysis by HPLC Mass Spectrometry/Mass Spectrometry (HPLC/MS/MS) 3.7 3.8 3.9 3.10 3.11 3.12 3.13 3.14 3.15 3.16 3.17 4.0 4.1 4.1.1 Analytical balance Volumetric flasks (e.g., 10 and 25 mL) Disposable Pasteur pipettes Micropipettor(s) with tips Culture tubes with teflon-lined caps Centrifuge Assorted glassware and syringes Culture tubes (vials) for use with solvent-concentration apparatus 1 mL Plastic syringes with 0.2 pm PVDF syringe filters Variable speed horizontal platform shaker pH meter PREPARATION OF STOCKS AND WORKING STOCKS Appropriate changes in the concentrations o f the solutions may be made at the discretion o f the analyst. Actual dilutions will be documented on the preparation sheets. M ain Stock Solution of PFOS ~1000 pg/mL Prepare an -1000 pg/mL solution o f PFOS in deionized organic-free water (e.g., accurately weigh about 10 mg PFOS into a 10-mL volumetric flask). Add deionized organic-free water to dissolve. Dilute to the mark. Alternatively, weigh the compound 000420 B-5 Page 5 o f 13 ANALYTICAL M ETHOD M ethod No.: BACG-3607 Title: Determination o f Perfluorooctanesulfonate in Monkey Serum and Urine: Sample Preparation and Analysis by HPLC Mass Spectrometry/Mass Spectrometry (HPLC/MS/MS) 4.2 4.2.1 4.3 4.3.1 4.4 4.4.1 into an appropriate vessel (e.g., culture tube) and add 10 mL o f deionized organic-free water. Mix well. Transfer the solution to a clean vessel if desired. Stock Solution of Internal Standard (PFO Q , ~200 pg/mL Prepare an ~200pg/mL solution o f PFOC in deionized organic-free water (e.g., accurately weigh about 10 mg into a 50-mL volumetric flask). Add deionized organic-free water to dissolve and dilute to the mark with deionized organic-free water. Alternatively, weigh the compound into a an appropriate vessel (e.g., culture tube) and add 50 mL of deionized organic-free water. Mix well. Transfer the solution to a clean vessel if desired. Spiking solution of Internal Standard (P FO Q , ~ 50pg/mL Prepare an ~ 50 pg/mL solution of PFOC in deionized organic- free water by pipetting 2 mL o f the 200 pg/mL solution into a culture tube and add 6 mL o f deionized water. Mix well. W orking Stock Solutions of PFOS 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) Volume o f PFOS solution 500,000 250,000 5 mL o f 1000 pg/mL 5 mL of 500,000 ng/mL Final Volume in deionized organic free water (mL) 10 10 000421 B-6 Page 6 o f 13 ANALYTICAL M ETHOD M ethod No.: BACG-3607 Title: Determination o f Perfluorooctanesulfonate in Monkey Serum and Urine: Sample Preparation and Analysis by HPLC Mass Spectrometry/Mass Spectrometry (HPLC/MS/MS) 62,500 2.5 mL o f 250,000 ng/mL 31,250 5 mL o f 62,500 ng/mL 15,625 5 mL o f 31,250 ng/ mL 5,000 50 pL o f 1000 pg/mL stock 2,500 5 mL o f 5000 ng/mL stock 1,000 4 mL o f 2500 ng/mL stock 500 5 mL o f 1000 ng/mL stock 250 5 mL of 500 ng/mL stock 10 10 10 10 10 10 10 10 4.4.2 Summary of concentrations o f serum standards: Standard Level Volume and Spike Cone. Approximate Concentration of PFOS in serum (ng/mL) A 10 pL o f 500,000 ng/mL 10,000 B 10 pL o f 250,000 ng/mL 5,000 C 16 pL o f 62,500 ng/mL 2,000 D 16 pL o f 31,250 ng/mL 1,000 000422 B-7 Page 7 o f 13 ANALYTICAL M ETHOD M ethod No.: BACG-3607 Title: Determination o f Perfluorooctanesulfonate in Monkey Serum and Urine: Sample Preparation and Analysis by HPLC Mass Spectrometry/Mass Spectrometry (HPLC/MS/MS) E 16 pL o f 15,625 ng/mL F 20 pL o f 5,000 ng/mL G 10 pL o f 5,000 ng/mL H 10 pL o f 2,500 ng/mL I 10 pL o f 1,000 ng/mL J 10 pL o f 500 ng/mL K 10 pL o f 250 ng/mL 500 200 100 50 20 10 5 5.0 PREPARATION OF SPIKED STANDARDS AND BLANKS Appropriate changes in the concentrations o f the solutions may be made at the discretion o f the analyst. 5.1 Multiple (e.g., about three) sets o f matrix standards and a matrix blank (blank + IS) are analyzed with each set o f unknown samples. A matrix double blank (blank-IS) may also be analyzed if desired. 5.2 Into individual ~20-mL culture tubes, pipet blank matrix (e.g., 0.5 m L ). Pipet in the appropriate volume as described in the table above for each standard. For the blanks, pipet 10 pL o f organic-free water instead o f the working stock solution. Add the 10 pL o f internal standard stock (~50 pg/mL) to each tube except the blank-IS (pipet 10 pL o f organic-free water instead) and vortex for ~5 seconds. 5.3 For serum samples add the following to each tube: 500 pL o f the TBA ion-pairing solution, 1 mL o f 0.25M/0.25M carbonate/bicarbonate buffer, and 1 mL of deionized organic free water. Vortex each tube for about 5 seconds. For urine samples add the following to each tube 1 mL o f TBA ion-pairing solution, 1 mL o f 1.0M/1.0M carbonate/bicarbonate buffer and 1 mL o f deionized water. Vortex each tube for about 5 seconds. 00Q423 B-8 Page 8 o f 13 ANALYTICAL M ETHOD M ethod No.: BACG-3607 Title: Determination o f Perfluorooctanesulfonate in Monkey Serum and Urine: Sample Preparation and Analysis, by HPLC Mass Spectrometry/Mass Spectrometry (HPLC/MS/MS) 5.4 Add 2.5 mL o f ethyl acetate and extract on horizontal mixer for 1 hour at a low speed setting. 5.5 Remove the tube from the shaker and place in a centrifuge (e.g., 2500 rpm) for about 5 minutes. 5.6 Take off the top ethyl acetate layer and put it into a clean tube and evaporate to dryness (e.g., ~ 50 minutes in the Turbo-Vap ) with a gentle stream o f nitrogen and moderate heat (e.g., 50 C). 5.7 Reconstitute tire residue in 500 pL o f 5% 5 mM ammonium acetate: 95% methanol containing 1.5% formic acid and vortex briefly to mix. Filter the samples through 0.2 pm PVDF or Nylon syringe filters into autosampler vials. 6.0 PREPARATION OF SAMPLES 6.1 Allow each serum sample to thaw to room temperature. Vortex each sample briefly, but vigorously. Pipet an aliquot o f each sample (e.g., 0.5 mL) into individual ~20-mL culture tubes. If necessary, dilute an aliquot o f any sample with blank matrix so that the expected concentration of the test article being analyzed will fall within the concentration range of the standard curve. Add 10 pL o f internal standard stock (~ 50 pg/mL) to each tube and vortex for a couple of seconds. 6.2 For serum samples add the following to each tube: 500 pL o f the TBA ion-pairing solution, 1 mL o f 0.25M/0.25M carbonate/bicarbonate buffer, and 1 mL o f deionized organic free water. Vortex each tube for about 5 seconds. For urine samples add the following to each tube 1 mL o f TBA ion-pairing solution, 1 mL o f 1.0M/1.0M carbonate/bicarbonate buffer and 1 mL o f deionized water. Vortex each tube for about 5 seconds. 6.3 Add 2.5 mL o f ethyl acetate and extract on horizontal mixer for 1 hour at a low speed setting. 000424 B-9 Page 9 o f 13 ANALYTICAL M ETHOD M ethod No.: BACG-3607 Title: Determination o f Perfluorooctanesulfonate in Monkey Serum and Urine: Sample Preparation and Analysis by HPLC Mass Spectrometry/Mass Spectrometry (HPLC/MS/MS) 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 o f nitrogen and moderate heat (e.g., 50 C). 6.6 Reconstitute the residue in 500 pL o f 5% 5 mM ammonium acetate: 95% methanol containing 1.5% formic acid and vortex briefly to mix. Filter the samples through 0.2 . pm PVDF or Nylon syringe filters into autosampler vials. Cap vials for analysis. 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: Keystone Scientific Aquasil C 1 8 ,150 mm x 2 mm ID, or equivalent Guard Column: Elution Flow rate: Injection volume: Mobile phase: Keystone Aquasil C l8 10 mm x 2 mm 400 pL/min. 5 pL A: 5mM ammonium acetate buffer B: 1.5% formic acid in methanol Gradient Profile: Temperature: 0 - 3 min. 3 - 5min. 5 - 8 min. 8-10 min. Ambient 50%A : 50%B 20% A : 80% B linear gradient 10%A : 90% B step gradient 50%A : 50%B step gradient 7.1.2 PE Sciex API 3000 Triple Quadrupole Mass Spectrometer Conditions Software: PE Sciex TurboQuan 00042S B-10 Page 10 o f 13 ANALYTICAL M ETHOD Method No.: BACG-3607 Title: Determination o f Perfluorooctanesulfonate in Monkey Serum and Urine: Sample Preparation and Analysis by HPLC Mass Spectrometry/Mass Spectrometry (HPLC/MS/MS) Turboion Sorav Source Note: Values listed under "MS/MS Acquisition Conditions" override parameters in this table. Auxiliary Gas: Air (e.g., Grade 0.1) at 85 pounds per square inch Parameter IS NC TEM OR RNG QO IQ1 ST ROl IQ2 R02 ST3 R03 DF CEM Value -2000 0 450 -20 -120 10 11 15 11 20 50 60 52 250 1800 000426 B -ll Page 11 o f 13 ANALYTICAL M ETHOD M ethod No.: BACG-3607 Title: Determination o f Perfluorooctanesulfonate in Monkey Serum and Urine: Sample Preparation and Analysis by HPLC Mass Spectrometry/Mass Spectrometry (HPLC/MS/MS) Parameter NEB CUR CAD QPE POL VCM IPE Value 15 6 5 0 1 0 0 MS/MS Acquisition Conditions Scan type: MRM Polarity: Negative Acquisition mode: Profile Pause time: 5 milliseconds Masses requested: PFOS: 01 Mass faim) 498.9 03 Mass tamul 498.9 PFOC (IS) Ol Mass (amu> 412.9 03 Mass famu) 368.9 Parameter R02 ST3 R03 QO IQ1 ST Start 35 45 37 18 19 23 Stop 35 45 37 18 19 23 Dwell Time (msl 200 Dwell Time (ms~) 200 000427 B-12 Page 12 o f 13 ANALYTICAL M ETHOD M ethod No.: BACG-3607 Title: Determination o f Perfluoiooctanesulfonate in Monkey Serum and Urine: Sample Preparation and Analysis by HPLC Mass Spectrometry/Mass Spectrometry (HPLC/MS/MS) ROl 19 IQ2 20 19 20 8.0 CALCULATIONS 8.1 At the end o f the analytical run, review each chromatogram to ensure the retention time, peak shape, and peak height and peak area determination o f the test article and the IS are acceptable. The data may be smoothed as appropriate. For quantitation, use the ion profiles at the following mass-to-charge ratios: Analyte PFOS PFOC Ion Profile 498.9 to 498.9 412.9 to 368.9 8.2 Plot the peak area response o f PFOS divided by the peak area response o f the IS (PFOC) from all standards versus the concentration of the test article in the standards. Alternatively, the peak heights may be used instead o f peak areas. Obtain the best curve fit o f the data (e.g., quadratic fit weighted with 1/concentration o f the test article or a quadratic fit). Note: The best curve fit may be dependent on the range o f die 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 o f PFOS divided by peak height response of the IS (PFOC) in standards. Concentration o f the PFOS in standards, = Constants derived from the regression analysis. 000428 B-13 Page 13 o f 13 ANALYTICAL M ETHOD M ethod No.: BACG-3607 Title: Determination o f Perfluorooctanesulfonate in Monkey Serum and Urine: Sample Preparation and Analysis by HPLC Mass Spectrometry/Mass Spectrometry (HPLC/MS/MS) 8.3 Using the standard curve, calculate the level o f PFHC in each unknown sample. Correct the results o f samples for any dilutions. NOTE: Due to unresolvable interferences with the test article and/or internal standard from the matrix, external standard quantitation may be used at the discretion o f the supervising mass spectrometrist. 9.0 ACCEPTANCE AND REJECTION CRITERIA 9.1 Refer to SOP SRI 91-3 for acceptance/rejection criteria except acceptable accuracy for standards is 80-120% o f theoretical. 10.0 R EPO R TIN G 10.1 Results o f all analyses are tabulated, and the raw data, original chromatograms, and reports are to be filed in the appropriate study file. Author(s): Lori Coward, BS Sr. Research Associate Bioanalytical Chemistry Group Date Approved by: Greg Gorman, Ph.D. Manager Bioanalytical Chemistry Group 9/ 1/0 ) Date 000429 Appendix C Summary o f Pharmacokinetic Parameters Calculated from Serum Concentrations o f PFOs and Urinary Excretion Data for PFOs 000430 A Pharmacokinetic Study o f Potassium Perfluorooctanesulfonate in the Cynomolgus Monkey Summary of Pharmacokinetic Parameters Calculated from Serum Concentrations o f PFOs j PARAMETER | Study Number | PFO Identification Dose (mg/kg) AUC o-inf(pgday/mL) tvie(days) Clearance (mL/day/kg) Vdss (mL/kg) C4CARBOXYLATE Male Female 9921.2 9921.2 T-7505 T-7505 10 10 4.7 6.6 1.7 1.7 2154 1515 526 443 C4 SULFONATE Male Female 9921.1 9921.1 T-7485 T-7485 10 10 24.3 35.4 4.0 3.5 511 368 254 255 C6CARBOXYLATE Male Female 9921.3 T-7506 10 1.6 9921.3 T-7506 10 4.0 1.5 0.8 6923 2494 953 235 j PARAMETER Study Number PFO Identification Dose (mg/kg) AUC o-taf(ngday/mL) t*. (days) Clearance (mL/day/kg) Vdss (mL/kg) C6 SULFONATE Male Female 9921.5 9921.5 T-7504 T-7504 10 10 7462 5794 141 87 1.3 1.9 287 213 C8 CARBOXYLATE Male Female 9921.4 9921.4 T-7507 10 T-7507 10 1235 2293 20.9 32.6 12.4 5.4 181 198 C8 SULFONATE Male Female 9921.6 9921.6 T-6295 2 1792 T-6295 2 1126 132 110 1.1 1.8 202 274 000431 000432 A Pharmacokinetic Study o f Potassium Perfluorooctanesulfonate in the Cynomolgus Monkey Summary o f the Urinary Excretion o f PFOs Study Number PFO Identification Dose (mg/kg) Day 1 (0-24 hours) Day 2 (24-48 hours) Day 7 Day 14 Day 21 | Day 28 URINARY EXCRETION RATE (MEAN % OF DOSE/DAY) C4CARBOXYLATE C4 SULFONATE C6 CARBOXYLATE Male Female Male Female Male Female 9921.2 T-7505 10 41.6 2.3 0.02 - 9921.2 T-7505 10 46.2 3.1 - 0.02 - 9921.1 T-7485 10 48.9 m1 0.2 0.01 - 9921.1 T-7485 10 66.4 -- 0.2 0.02 - 9921.3 T-7506 10 69.9 - 0.03 -- <0.01 9921.3 T-7506 10 85.1 -- 0.03 <0.01 -- -- -- -- <0.01 <0.01 URINARY EXCRETION RATE ( MEAN % OF DOSE/DAY) C6 SULFONATE C8 CARBOXYLATE C8 SULFONATE Male Female Male Female Male Female Study Number PFO Identification Dose (mg/kg) Day 1 (0-24 hours) Day 2 (24-48 hours) Day 7 Day 14 Day 21 Day 28 Day 42 Day 56 || Day 70 | Day 91 9921.5 T-7504 10 0.09 0.03 0.03 <0.01 <0.01 0.01 0.04 0.02 <0.02 " 9921.5 T-7504 10 0.05 0.05 0.03 0.05 0.03 0.01 0.02 <0.02 0.03 -- 9921.4 T-7507 10 8.9 0.7 7.5 1.2 0.6 0.2 -- - - -- 9921.4 T-7507 10 3.9 2.0 2.5 1.8 1.1 0.9 - -- - -- 9921.6 T-6295 2 0.03 0.03 0.03 48 0.03 0.04 0.03 0.04 0.03 0.02 9921.6 T-6295 2 0.02 0.03 0.05 48 0.07 0.04 0.04 0.03 0.04 0.03 'Urine was not collected at the indicated time bSample was collected on Day IS 1 1 9Ni 1 1