Document 3QK89gmzjBY5jBJbR1JVV410E

3M Medical Department Study: T-6889.3 Analytical Report: FACT TOX-026 LRN-U2782 Study Title 26-Week Capsule Toxicity Study with Ammonium Perfluorooctanoate (APFO/POAA) in Cynomolgus Monkeys Amended Analytical Laboratory Report Title Determination of the Presence and Concentration of Perfluorooctanoate Fluorochemical in Liver, Serum, Urine and Feces Samples Data Requirement Not Applicable Author 3M Environmental Laboratory Analytical Phase Completion Date June 11, 2001 Performing Laboratories Liver, Serum and Urine Analyses Feces Analyses 3M Environmental Laboratory Centre Analytical Laboratories, Inc. Building 2-3E-09, 935 Bush Avenue St. Paul, MN 55106 3048 Research Drive State College, PA 16801 Project Identification 3M Medical Department Study: T-6889.3 Covance In-Life Study: #6329-231 Analytical Report: FACT TOX-026 3M Laboratory Request No. U2782 Total Number of Pages 408 3M Environmental Laboratory Page 1 3M Medical Department Study: T-6889.3 Analytical Report: FACT TOX-026 LRN-U2782 This page has been reserved for specific country requirements. 3M Environmental Laboratory Page 2 3M Medical Departm ent Study: T-6889.3 Analytical Report: FACT TO X-026 LRN-U2782 GLP Compliance Statement Analytical Laboratory Report Title: Determination of the Presence and Concentration of Perfluorooctanoate Fluorochemical in Liver, Serum, Urine and Feces Samples Study Identification Numbers:T-6889.3, FACTTOX-026, LRN U2782 This study was conducted in compliance with United States Environmental Protection Agency (EPA) Good Laboratory Practice (GLP) Standards 40 CFR Part 792, with the exceptions in the bulleted list below. Exceptions to GLP compliance: There was a point during the course of this study in which there were two study directors assigned to the study. This was resolved by amending the protocol. Labels of reagents and solutions did not contain all of GLP requirements. It is unknown if an in-phase inspection was conducted. The audit report could not be located. Paul Lieder, Ph.D., Study Director / / , 7,00i v Date k L -------------- Kris'J. Hansen, Ph.D., Principal Analytical Investigator 05/z y /Oi Date -------- William Reagen, Ph.D., Laboratory Manager ><? /a rj/o y Date John L. Butenhoff, Ph.D., Sponsor Representative Date 3M Environmental Laboratory Page 3 3M Medical Department Study: T-6889.3 Analytical Report: FACT TOX-026 LRN-U2782 GLP Study-Q uality Assurance Statement Analytical Laboratory Report Title: Determination of the Presence and Concentration of Perfluorooctanoate Fluorochemical in Liver, Serum, Urine and Feces Samples Study Identification Numbers:T-6889.3, FACT TOX-026, LRN U2782 This analytical study performed at 3M Environmental Laboratory has been inspected by the 3M Environmental Laboratory Quality Assurance Unit (QAU) as indicated in the following table. The findings were reported to the study director and laboratory management. Inspection Dates Phase Date Reported to Management Study Director 10/26/98 Protocol 10/27/98 9/25/00--9/29/00 10/2/00- 10/6/00 10/9/00, 10/30/00 12/14/00-12/15/00 12/18/00-12/20/00, 5/3/01, 5/4/01,5/7/01,5/8/01,5/9/01 Data Draft Report 10/9/00, 10/30/00 12/21/00, 5/10/01 See Appendix H for individual contractor quality assurance statements. 10/27/98 10/9/00, 10/30/00 12/21/00, 5/10/01 QAUI Repre,sentative L>l Ig/ol Date 3M Environmental Laboratory Page 4 3M Medical Department Study: T-6889.3 Analytical Report: FACT TOX-026 LRN-U2782 Table of Contents GLP Compliance Statement.............................................................................................. 3 GLP Study--Quality Assurance Statement.......................................................................4 Study Personnel and Contributors..................................................................................... 8 Summary............................................................................................................................ 9 Introduction and Purpose................................................................................................... 9 Test System...................................................................................................................9 Specimen Collection..................................................................................................... 10 Specimen Receipt and Maintenance.................................................................................10 Chemical Characterization of Test Substance.................................................................. 11 Procurement.................................................................................................................. 11 Stability Studies............................................................................................................. 11 Dose Confirmation Analyses......................................................................................... 11 Method Summaries............................................................................................................ 12 Centre Analytical Laboratories......................................................................................12 3M Environmental Laboratory.......................................................................................12 Preparatory Methods................................................................................................12 Analytical Methods................................................................................................... 13 Analytical Equipment................................................................................................ 14 Deviations..................................................................................................................... 15 Data Quality Objectives and Data Integrity....................................................................... 15 Data Summary, Analyses, and Results............................................................................. 15 Summary of Quality Control Analyses Results............................................................. 15 Statement of Data Quality.................................................... .........................................16 Summary of Sample Results.........................................................................................16 Statistical Methods and Calculations................................................................................ 16 Statement of Conclusion....................................................................................................17 Appendix A: Chemical Characterization and Control Matrices..........................................18 Appendix B: Protocol, Protocol Amendments and Deviation Summary........................... 20 Appendix C: Extraction and Analytical Methods...............................................................66 FACT-M-1.1, Extraction of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds from Liver for Analysis Using HPLC-Electrospray/Mass Spectrometry, (15 pages)................................................................................................................................ 7 FACT-M-2.1, Analysis of Fluorochemicals in Liver Extracts Using HPLCElectrospray/Mass Spectrometry, (9 pages).....................................................................82 ETS-8-6.0, "Extraction of Potassium Perfluorooctanesulfonate or other Fluorochemical Compounds from Liver for Analysis using HPLC-Electrospray/Mass Spectrometry", (14 pages)................................................................................................................................ 91 ETS-8-7.0, "Analysis of Potassium Perfluorooctane-sulfonate or other Fluorochemicals in Liver Extracts Using HPLC-Electrospray/Mass Spectrometry", (10 pages)................. 105 3M Environmental Laboratory Page 5 3M Medical Department Study: T-6889.3 Analytical Report: FACT TOX-026 LRN-U2782 ETS-8-4.1, Extraction of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds from Serum for Analysis Using HPLC-Electrospray/Mass Spectrometry, (14 pages)..................................................................................................................................i115 FACT-M-3.1, Extraction of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds from Serum or Other Fluid for Analysis Using HPLC-Eiectrospray/Mass Spectrometry, (17 pages)...................................................................................................J29 ETS-8-5.1, Analysis of Potassium Perfluorooctanesulfonate or Other Fluorochemicals in Serum Extracts Using HPLC-Electrospray/Mass Spectrometry, (9 pages)....................... 146 FACT-M-4.1, Analysis of Potassium Perfluorooctanesulfonate or Other Fluorochemicals in Serum or Other Fluid Extracts Using HPLC-Electrospray/Mass Spectrometry, (9 pages)................................................................................................................................. ETS-8-96.0, Extraction of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds from Urine for Analysis Using HPLC-Electrospray/Mass Spectrometry, (14 pages)..................................................................................................................................164 ETS-8-97.0, Analysis of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds in Urine Extracts using HPLC-Electrospray/Mass Spectrometry/Mass Spectrometry, (10 pages)...................................................................................................178 Appendix D: Method Validation Summary (TOX134)......................................................... l188 Appendix E: Data Summary Tables....................................................................................i189 Appendix F: Data Spreadsheets......................................................................................... c203 Appendix G: Example Calculations.....................................................................................i295 Appendix H: Contract Lab Report.......................................................................................<297 Centre Analytical Laboratories, 26-Week Capsule Toxicity Study with Ammonium Perfluorooctanoate (APFO/POAA) in Cynomolgus Monkeys, (110 pages)....................... '208 Appendix I: Report Signature Page................................................................................... 408 Appendix J: Amendment 1 to FACT TOX-026 Final Report...............................................42 3M Environmental Laboratory Page 6 3M Medical Department Study: T-6889.3 Analytical Report: FACT TOX-026 LRN-U2782 List of Tables Table 1. Cynomolgus Monkey Population Demographics for Study (#6329-231)........... 9 Table 2. Target Ions Monitored in 3M Environmental Laboratory Analyses.....................14 Table 3. Characterization of the Control Matrices Used for Urine, Liver and Sera Analyses in Study FACT TOX-026........................................................................18 Table 4. Characterization of Test and Reference Substances in Study FACT TOX-026.. 19 Table 5. TOX134 Method Validation Results Summary.................................................... <188 Table 6. LOQ Values Used in FACT TOX-026 Analyses by Method and Usage Dates....189 Table 7. Recovery Summaries of Fortified Samples in FACT TOX-026.......................... 189 Table 8. Recovery Summaries of Fortified Samples in FACT TOX-026.......................... 190 Table 9. Recovery Summaries of Fortified Samples in FACT TOX-026.......................... 191 Table 10. TOX026 Data Summary of POAA Concentration--Serum (pg/mL)................. 192 Table 11. TOX 026 Data Summary of POAA Concentration--Urine (pg/mL)................... 194 Table 12. TOX026 Data Summary of POAA Concentration--Liver (pg/g)....................... 196 Table 13. TOX 026 Data Summary of POAA Concentration from Centre Analytical Laboratory--Feces (pg/g)...................................................................................... 196 Table 14. FACT TOX-026 Individual POAA Results per Sample--Serum (pg/mL)..........198 Table 14 (continued). FACT TOX-026 Individual POAA Results per Sample--Serum (pg/mL).................................................................................................................. 199 Table 15. FACT TOX-026 Individual POAA Results per Sample--Urine (pg/mL)............200 Table 15 (continued). FACT TOX-026 Individual POAA Results per Sample--Urine (pg/mL).......................................................................... 201 Table 16. FACT TOX-026 Individual POAA Results per Sample--Liver (pg/g)............... 203 3M Environmental Laboratory Page 7 3M Medical Department Study: T-6889.3 Analytical Report: FACT TOX-026 LRN-U2782 Study Personnel and Contributors Study Director Sponsor Paul Lieder, Ph.D., DABT 3M Corporate Toxicology 3M Center, Building 220-2E-02 St. Paul, MN, 55144-1000 651-737-2678 3M Toxicology Services Medical Department 3M Center, Building 220-2E-02 St. Paul, MN, 55133-3220 John L. Butenhoff, Ph.D., Sponsor Representative Analytical Chemistry Laboratories Liver, Serum and Urine Analyses Feces Analyses 3M Environmental Laboratory Centre Analytical Laboratories, Inc Kristen J. Hansen, Ph.D., Analytical Investigator Emily Stauffer, Analytical Investigator 3M Environmental Laboratory Contributing Personnel David R. Barnidge, Ph.D.* Lisa A. Clemen Rhonda S. Dick* Kelly J. Dorweiler* Mark E. Ellefson Sara E. Estes* Barb A. Gramenz* Sarah A. Heimdal* Cari S. Hewitt* Marlene M. Heying* 'Contract lab professional service employees Megan C. Holloway* Harold 0. Johnson Kelly J. Kuehlwein* Glenn Langenburg* Sally A. Linda Joseph C. Pilon* Scott R. Post* Ian A. Smith* Bob W. Wynne* Location of Archives All original raw data, protocol, and analytical report have been archived at the 3M Environmental Laboratory. The test substance and reference substance reserve samples, as well as the specimens pertaining to the analytical phase of this study are archived at the 3M Environmental Laboratory. Feces specimens will be returned from Centre Analytical Laboratories for archiving at the 3M Environmental Laboratory and will be maintained at 3M Environmental Laboratory according to GLP requirements. 3M Environmental Laboratory Page 8 3M Medical Department Study: T-6889.3 Analytical Report: FACT TOX-026 LRN-U2782 Summary Under the conditions of the present studies, the fluorochemical perfluorooctanoate (POAA) was observed in the serum, urine, and liver of most samples collected from cynomolgus monkeys dosed with the test substance during the in-life phase of the study. Introduction and Purpose The purpose of the analytical phase of this study is to determine the presence and concentration of perfluorooctanoate (POAA) in liver, serum, urine and feces samples taken at specified intervals from the 26-week capsule toxicity study in cynomolgus monkeys. The analytical phase of this study was initiated on November 12, 1998. The text and tables in this report cover ONLY the analyses conducted at 3M, unless otherwise specified, and the analogous information on feces analysis can be found in the documents from Centre Analytical Laboratory (Appendix H). Test System A total of 22 cynomolgus male monkeys were obtained from Covance Research Products, Inc. and acclimated for 35 days. The monkeys were 3 to 7 years old and weighed 3-5 kg at the initiation of the treatment. All monkeys were identified with a collar tag. Animals were assigned to the control or treatment group using a computerized blocking procedure designed to achieve body weight balance with respect to treatment groups. The in-life laboratory staff gave each study animal the specified dose orally once daily for at least 183 days, with the exceptions noted in the Covance in-life final report #6329-231. A select number of monkeys were then chosen from the main study to continue on in a recovery group study. The doses administered for each control and subtreatment group are listed in Table 1. Doses were given in the form of Size No. 2 gelatin capsules. Table 1. Cynomolgus Monkey Population Demographics for Study (#6329-231) P o p u la tio n T o ta l S e le c te d fo r S tu d y Group S e le c te d fo r Recovery Group Group 1 Control 66 Group 2 Low-dose (3 mg/kg/day) 4 4 Group 3 M id -do se (10 mg/kg/day) 6 6 Group 4 High-dose (30/20 mg/kg/day)a 6 6 a30 mg/kg/day was suspended on Day 12 and reinitiated on Day 22 at 20 mg/kg/day. N O TE: This in-life information came from Covance in-life final report for study #6329-231. 2 0 2 0 3M Environmental Laboratory Page 9 3M Medical Department Study: T-6889.3 Analytical Report: FACT TOX-026 LRN-U2782 Specim en Collection A total of 947 liver, serum, urine and feces specimens were collected from cynomolgus monkeys (beginning 7 October 1998) and sent to the 3M Environmental Laboratory to be analyzed for perfluorooctanoate (POAA). Serum, urine and feces specimens were collected approximately every two weeks and liver samples were collected at time of sacrifice. An additional 101 specimens of whole blood, plasma, red blood cells, kidneys and testes were collected by Covance (study #6329-231), but were not part of the scope of analysis determined by the study director. The number and type of specimens collected for analyses in the analytical phase of this study are presented below. Specimens Collected from Study Groups 1 through 4 (through 3/31/99): Serum Specimens--292 specimens Liver Specimens-- 19 specimens Urine Specimens--272 specimens Feces Specimens--272 specimens Specimens Collected from the Recovery Group from 4/08/99 to 7/02/99: Serum Specimens--32 specimens Liver Specimens--4 specimens Urine Specimens--28 specimens Feces Specimens--28 specimens Sera, liver and urine samples were extracted beginning on 12 November 1998 using an ion-pairing reagent and methyl-tert-butyl ether (MtBE) or ethyl acetate. Sample extracts were analyzed using high-performance liquid chromatography-electrospray/tandem mass spectrometry (HPLC-ES/MS/MS) in the multiple response monitoring mode. POAA levels in sera and liver were quantitated by external calibration (without an internal standard reference), while the urine analysis was performed by internal calibration (with an internal standard reference). Analytical details are included in this report. Specimen Receipt and Maintenance The 3M Environmental Laboratory received liver, serum, urine and feces specimens collected at predetermined time points of the in-life phase of this study (#6329-231) beginning 11/3/98 to 8/17/99 from Covance. All specimens were received frozen on dry ice and were immediately transferred to storage at -55C 20C and -20C 10C. Feces specimens were shipped to Centre Analytical Laboratories frozen on dry ice for analysis. Control matrices used in the liver, sera, and urine analyses were obtained from various sources and are presented in Appendix A. The remaining portions of the samples analyzed at the 3M Environmental Laboratory will be maintained at the laboratory for a period of time as specified by the regulations and will be kept at -50C 20C or -20C 10C. 3M Environmental Laboratory Page 10 3M Medical Department Study: T-6889.3 Analytical Report: FACT TOX-026 LRN-U2782 Chemical Characterization of Test Substance Ammonium Perfluorooctanoate CAS Number: 3825-26-1 Chemical Formula: C7F15CO2N H / Molecular Weight: 431.1 Chemical characterization information on the test substance and reference substances used in this study is presented in tabular form in Appendix A. Purity determinations are on-going. P ro curem en t One lot of ammonium perfluorooctanoate (Lot number 332) was obtained from 3M Specialty Chemicals. Stability Studies No stability samples were sent for analysis. Dose Confirmation Analyses Dose confirmation analyses were not performed on the test substance dose samples. Since the test material was not mixed with a carrier, dose analyses were not required. 3M Environmental Laboratory Page 11 3M Medical Department Study: T-6889.3 Analytical Report: FACT TOX-026 LRN-U2782 Method Summaries Centre A nalytical Laboratories The methods and analytical equipment settings used by Centre Analytical Laboratories for feces analysis are presented in the contract lab report (see Appendix H). 3M Environmental Laboratory Following is a brief description of the methods used during this analytical phase by the 3M Environmental Laboratory. Detailed descriptions of the methods used in this phase are located in Appendix C. As the present analytical phase progressed, more advanced methods evolved and earlier methods were used with deviations until amendments to the protocol were written. A summary of protocol and method deviations is presented in Appendix B of this report. Preparatory Methods ETS-8-6.0, "Extraction of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds from Liver for Analysis using HPLC-Electrospray/Mass Spectrometry" Liver samples were homogenized in water. An aliquot of each homogenate was spiked with surrogate and extracted using an ion-pairing extraction procedure. An ion-pairing reagent was added to the sample and the analyte ion pair was partitioned into MtBE. The extract was transferred to a centrifuge tube and put onto a nitrogen evaporator until dry. Each extract was reconstituted in 1.0 mL of methanol and passed through a 0.2 pm nylon filter using a 3 cc disposable plastic syringe into glass autosampler vials. ETS-8-4.1, "Extraction of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds from Serum for Analysis using HPLC-Electrospray/Mass Spectrometry" Serum samples were extracted using an ion-pairing extraction procedure. An ion pairing reagent was added to the sample and the analyte ion pair was partitioned into MtBE. The MtBE extract was transferred to a centrifuge tube and put onto a nitrogen evaporator until dry. Each extract was reconstituted in 1.0 mL of methanol, then filtered through a 0.2 pm nylon filter using a 3 cc plastic syringe into glass autovials. ETS-8-96.0, "Extraction of Potassium Perfluorooctanesulfonate or other Fluorochemical compounds from Urine for Analysis Using HPLC-Electrospray/Mass Spectrometry" Urine samples were extracted using an ion-pairing extraction procedure. An ion pairing reagent is added to 2 mL of the sample, and the analyte-ion pair is partitioned into MtBE. The MtBE extract is removed and put onto a nitrogen evaporator until dry. Each extract is reconstituted in 0.5 mL of methanol, then filtered through a 0.2 pm nylon filter using a 3 cc plastic syringe into glass autovials. 3M Environmental Laboratory Page 12 3M Medical Department Study: T-6889.3 Analytical Report: FACT TOX-026 LRN-U2782 Analytical Methods ETS-8-7.0, "Analysis of Potassium Perfluorooctanesulfonate or other Fluorochemicals in Liver Extracts Using HPLC-Electrospray/Mass Spectrometry" ETS-8-5.1, "Analysis of Potassium Perfluorooctanesulfonate or other Fluorochemicals in Serum Extracts Using HPLC-Electrospray/Mass Spectrometry" ETS-8-97.0, "Analysis of Potassium Perflurooctanesulfonate or Other Fluorochemical Compounds in Urine Extracts Using HPLC-Electrospray/Mass Spectrometry" The analyses were performed by monitoring one or more product ions selected from a single primary ion characteristic of a particular fluorochemical using HPLC/ES/MS/MS. For example, molecular ion 413, selected as the primary ion for POAA (C7F15CO2') analysis, was fragmented to produce ion 169,119, and 219. The characteristic ion 169 was monitored for quantitative analysis. In liver and sera analyses, each curve is plotted using linear regression with 1/x weighting. In urine analyses, each curve is plotted with an internal standard using a quadratic fit and 1/x weighting. The method validation study for the extraction and analysis of POAA in sera was not complete at the start of data collection. For method parameters characterized during validation, see Appendix D. 3M Environmental Laboratory Page 13 3M Medical Department Study: T-6889.3 Analytical Report: FACT TOX-026 LRN-U2782 Analytical Equipment The actual analytical equipment settings used in the analysis of liver, sera and urine varied slightly during actual data collection. The following is representative of the settings used during the analytical phase of this study. (Please see Appendix H for the instrument settings used during feces analysis. Liquid Chromatograph: Hewlett-Packard Series 1100 Liquid Chromatograph system Analytical column: Keystone BetasilTM C182x50 mm (5 pm) Column temperature: Ambient Mobile phase components: Component A: 2mM ammonium acetate Component B: methanol Flow rate: 300 pL/min Injection volume: 10 pL Solvent Gradient: Time (minutes) %B 0.0 40% 1.0 40% 4.5 95% 6.5 95% 7.0 40% Mass Spectrometer: Micromass API/Mass Spectrometer Quattro IITMTriple Quadrupole system Software: Mass LynxTM 3.2-3.4 Cone Voltage: 15-60 V Collision Gas Energy: 20-40 eV Mode: Electrospray Negative Source Block Temperature: 150C 10C Electrode: Z-spray Analysis Type: Multiple Reaction Monitoring (MRM) Table 2. Target Ions Monitored in 3M Environmental Laboratory Analyses Target Analyte Primary Ion (a m u ) Product Ion (a m u ) POAA THPFOS 413.0 427.0 119.0, 169.0, 219.0 80.0 T H P F O S was used as a surrogate standard. 3M Environmental Laboratory Page 14 3M Medical Department Study: T-6889.3 Analytical Report: FACT TOX-026 LRN-U2782 Deviations It should be noted that as the analytical phase of this study progressed, method parameters were evaluated to improve analyses. Earlier methods were used with deviations until amendments to the protocol were written. Deviations from the original protocol and methods are documented in Appendix B. Data Quality Objectives and Data Integrity The following data quality objectives (DQOs) were indicated in the protocol for this study: Linearity: The coefficient of determination (r2) equal to or greater than 0.980 Limits of Quantitation (LOQ): The Limit of Quantitation (LOQ) is equal to the lowest acceptable standard in the calibration curve Acceptable Precision: Inter-assay, intra-assay and system are within 30% for the method Acceptable Spike Recoveries: 70-130% for sera, 60-140% for liver and urine Demonstration of Specificity: Specificity to be demonstrated by chromatographic retention time and mass spectral daughter ion characterization. Data Summary, Analyses, and Results Data quality objectives for the analytical phase of this study outlined in the 3M Environmental Laboratory protocol for FACT TOX-026 (see Appendix B) were met with the exceptions noted in this report. Summary of Quality Control Analyses Results Linearity: The coefficient of determination (r2) of the standard curve was >0.980. Calibration Standards: For sera and liver analyses, quantitation of the target a n a ly te u sed lin e a r re g re ssion a na lysis, w e ig h te d 1/x, o f tw o e xtra cte d m atrix cu rve s bracketing each group of samples, or of a single curve preceding the samples. Urine data was determined using a 1/x weighted quadratic fit calibration curve with an internal standard. For sera, liver and urine analyses, high or low points on the curve may have been deactivated to provide a better linear fit over the curve range most appropriate to the data. Low curve points with peak areas less than two times that of the extraction blanks were deactivated to disqualify a data range that may have been significantly affected by background levels of the analyte. Occasionally, a single mid-range curve point that was an obvious outlier was deactivated. Quantitation of the analyte was based on the response of three specific product ions using the multiple response monitoring mode of the instrument (see Appendix C, Analytical Methods). Limits of Quantitation (LOQ): The LOQ is equal to the lowest acceptable standard in the calibration curve (defined as a standard within 30% of the theoretical value), and is at least two times the analyte peak area detected in the matrix blanks. Unless otherwise noted, the LOQ for liver is approximately 75 ng/g, for sera the LOQ is approximately 14 ng/mL and for urine the LOQ is approximately 20 ng/mL. 3M Environmental Laboratory Page 15 3M Medical Department Study: T-6889.3 Analytical Report: FACT TOX-026 LRN-U2782 Blanks: All blanks in liver assays were below the lower limit of quantitation for the compound of interest. In some instances for sera and urine data, the blanks were above the LOQ (refer to Appendix F for clarification). To simplify analyses that were complicated by endogenous levels of fluorochemicals in unexposed monkey sera and monkey liver, rabbit sera and liver were selected as a suitable surrogate matrix. Monkey urine was used for standardization and QC samples in urine analysis. Precision: Not specifically determined within the course of this phase of the study. Matrix Spikes: Sera--Matrix spike samples were prepared from control monkey and/or rabbit sera along with each batch of sera samples. Samples were spiked with a final concentration of approximately 250-500 ng/ml_, levels that approximate the background levels detected in the Group 1 samples. Liver--Matrix spike samples were prepared from control monkey liver along with each batch of liver samples. Samples were spiked at approximately 250 ng/g, levels that approximate the background levels detected in the Group 1 samples. Urine--Matrix spike samples were prepared from control monkey urine along with each batch of urine samples. Samples were spiked at approximately 60 ng/mL, levels that approximate the background levels detected in the Group 1 samples. Surrogates: The surrogate (THPFOS) was added to all samples and standards. THPFOS was not used for quantitation for liver and sera analysis, but was used to monitor for gross instrument failure. THPFOS was used as an internal standard for quantitation of POAA in the urine samples. Statem ent of Data Quality It is not possible to verify true recovery of endogenous analyte from tissues without radio-labeled reference material. The only measurement of accuracy available at this time, matrix spike studies, indicate that the sera, liver, urine, and feces data can be considered to be accurate to within one standard deviation of the average fortified sample recovery. The average fortified sample recovery for sera was 94% with a standard deviation of 11%. The average fortified sample recovery for liver was 90% with a standard deviation of 26%. The average fortified sample recovery for urine was 88% with a standard deviation of 17%. The average fortified sample recovery for feces was 117% with a standard deviation of 22%. Summary of Sam ple Results Samples from Control Animals: Low levels of POAA were often detected in the sera, liver, and urine of the control animals. These levels were significantly lower than those found in the low dose test animals. Samples from Dosed Animals: In general, POAA levels found in the sera, liver, and urine of the test animals increased with dose groups. Detailed sample data tables are presented in Appendices E and F. Statistical Methods and Calculations Statistical methods were limited to the calculation of means and standard deviations. See Appendix G for example calculations used to generate the serum, urine, and liver sample data in FACT TOX-026. Data calculations used for feces sample data are included In the Centre Analytical Laboratories report. 3M Environmental Laboratory Page 16 3M Medical Department Study: T-6889.3 Analytical Report: FACT TOX-026 LRN-U2782 Blanks: All blanks in liver assays were below the lower limit of quantitation for the compound of interest. In some instances for sera and urine data, the blanks were above the LOQ (refer to Appendix F for clarification). To simplify analyses that were complicated by endogenous levels of fluorochemicals in unexposed monkey sera and monkey liver, rabbit sera and liver were selected as a suitable surrogate matrix. Monkey urine was used for standardization and QC samples in urine analysis. Precision: Not specifically determined within the course of this phase of the study. Matrix Spikes: Sera--Matrix spike samples were prepared from control monkey and/or rabbit sera along with each batch of sera samples. Samples were spiked with a final concentration of approximately 250-500 ng/mL, levels that approximate the background levels detected in the Group 1 samples. Liver--Matrix spike samples were prepared from control monkey liver along with each batch of liver samples. Samples were spiked at approximately 250 ng/g, levels that approximate the background levels detected in the Group 1 samples. Urine--Matrix spike samples were prepared from control monkey urine along with each batch of urine samples. Samples were spiked at approximately 60 ng/mL, levels that approximate the background levels detected in the Group 1 samples. Surrogates: The surrogate (THPFOS) was added to all samples and standards. THPFOS was not used for quantitation for liver and sera analysis, but was used to monitor for gross instrument failure. THPFOS was used as an internal standard for quantitation of POAA in the urine samples. Statem ent of Data Quality It is not possible to verify true recovery of endogenous analyte from tissues without radio-labeled reference material. The only measurement of accuracy available at this time, matrix spike studies, indicate that the sera, liver, urine, and feces data can be considered to be accurate to within one standard deviation of the average fortified sample recovery. The average fortified sample recovery for sera was 93% with a standard deviation of 11%. The average fortified sample recovery for liver was 90% with a standard deviation of 26%. The average fortified sample recovery for urine was 88% w ith a sta n d a rd d e via tio n o f 17% . T he a verag e fo rtifie d sa m p le re co ve ry fo r fe c e s w a s 117% with a standard deviation of 22%. Summary of Sample Results Samples from Control Animals: Low levels of POAA were often detected in the sera, liver, and urine of the control animals. These levels were significantly lower than those found in the low dose test animals. Samples from Dosed Animals: In general, POAA levels found in the sera, liver, and urine of the test animals increased with dose groups. Detailed sample data tables are presented in Appendices E and F. Statistical Methods and Calculations Statistical methods were limited to the calculation of means and standard deviations. See Appendix G for example calculations used to generate the serum, urine, and liver sample data in FACT TOX-026. Data calculations used for feces sample data are included in the Centre Analytical Laboratories report. 3M Environmental Laboratory Page 16 3M Medical Department Study: T-6889.3 Analytical Report: FACT TOX-026 LRN-U2782 Statement of Conclusion Under the conditions of the present studies, the fluorochemical perfluorooctanoate was observed in the serum, urine, and liver of most samples collected from cynomolgus monkeys dosed with the test substance during the in-life phase of the study. 3M Environmental Laboratory Page 17 3M Medical Department Study: T-6889.3 Analytical Report: FACT TOX-026 LRN-U2782 Appendix A: Chemical Characterization and Control Matrices Table 3. Characterization of the Control Matrices Used for Urine, Liver and Sera Analyses in Study FACT TOX-026 C o n tro l M a trix R a b b it S e ru m T N -A -2052 R a b b it S e ru m T N -A -2307 R a b b it L iv e r T N -A -0808 R a b b it S e ru m TN -A -2391 R a b b it S e ru m T N -A -2573 S o u rce Expiration D ate Storage C onditions C hem ical Lot # Physical D escription S igm a C hem icals 0 1 /0 1 /2 0 1 0 -20 C 1 0 C 107H 8409 R abbit Serum S igm a C hem icals 01/01/2010 -2 0 C 10C 118H8418 R abbit Serum C om ing Hazelton 01/01/2010 -2 0 C 1 0 C F00011 R abbit Liver S igm a C hem icals 0 1 /0 1 /2 0 1 0 -2 0 C 10C 118H8418 R abbit S erum S ig m a C hem icals 0 1 /0 1 /2 0 1 0 -2 0 C 10C 118H8418 R abbit Serum C o n tro l M a trix S o u rce Expiration D ate S torage C onditions C hem ical Lot # Physical D escription R a b b it L iv e r T N -A -0806 C orning Hazelton 0 1 /0 1 /2 0 1 0 -20C 10C F00009 R abbit Liver R a b b it L iv e r T N -A -0802 C o m in g Hazelton 01/01/2010 -20C 10C F00005 R abbit Liver R a b b it S e ru m 9 9 0 6 2 -0 5 5 S igm a C hem icals 01/01/2010 -2 0 C 1 0 C 118H8418 R abbit Serum M o n k e y U rin e 9 9 0 6 2 -0 6 7 N /R 0 1 /0 1 /2 0 1 0 -2 0 C 10C 106015 M onkey U rine M o n k e y U rin e 9 9 0 6 2 -0 7 6 Covance 0 1 /0 1 /2 0 1 0 -2 0 C 1 0 C 6329-262 M onkey U rine C o n tro l M a trix S o u rce Expiration D ate S torage C onditions C hem ical Lot # Physical Description N/R--not recorded M o n k e y U rin e 9 9 0 6 2 -0 7 8 Covance 0 1 /0 1 /2 0 1 0 -2 0 C 10C 6329-262 M onkey Urine 3M Environmental Laboratory Page 18 3M Medical Department Study: T-6889.3 Analytical Report: FACT TOX-026 LRN-U2782 Table 4. Characterization of Test and Reference Substances in Study FACT TOX-026 Test Substance Reference Substances Laboratory C o v a n c e L a b o ra to rie s , In c . 3M E n v iro n m e n ta l L ab 3M E n viro n m e n ta l Lab C e n tre A n a ly tic a l L a b o ra to rie s Chemical Name A P FO (A m m onium P e rflu o ro o c ta n o a te ) Source Expiration Date Storage Conditions Chemical Lot # Physical Description Purity 3M S pecialty C hem ical N /R A m bient tem perature 332 W hite powder 9 5 .0 -9 5 .2 % :0 (A m m o n iu m luorooctanoate) T N -A -1 4 7 9 S E -0 3 0 A P FO (A m m onium P e rflu o ro o c ta n o a te ) T N -A -0 4 9 7 SD022 ecialty C hem icals 3M S pecialty C hem icals 2010 1/1/2010 A P F O (A m m onium P e rflu o ro o c ta n o a te ) TC R -99030-30 3M S pecialty C hem icals N /R Am bient tem perature A m bient tem perature A m bient tem perature 245 N/R 332 W hite powder Light-colored pow der W hite powder TBD" TBD" 95.0% Analytical Reference Substances Laboratory C e n tre A n a ly tic a l L a b o ra to rie s 3M E n v iro n m e n ta l Lab Chemical Name Source THPFOS SE037 ICN B iom edicals THPFOS SD028 ICN B iom edicals Expiration Date Storage Conditions 1/01/2010 A m bient tem perature 1/1/2010 Am bient tem perature Chemical Lot # 53406 59909 Physical Description Brow n w axy solid Brown pow der Purity T B D ** NA* N/R-- Not recorded 'N one remaining; purity assumed to be 100%. 'Unless otherwise indicated, at the time of quantitation, the purity for all analytes was assumed to be 100%. 3M Environmental Laboratory Page 19 3M Medical Department Study: T-6889.3 Analytical Report: FACT TOX-026 LRN-U2782 Appendix B: Protocol, Protocol Amendments and Deviation Summary 3M Environmental Laboratory Page 20 Protocol #FACT-TOX-026 Study Tide 6-Month Capsule Toxicity Study with Ammonium Perfluorooctanoate (APFO/POAA) in Cynomolgus Monkeys PROTOCOL Author Lisa Clemen Date: October 12, 1998 Performing Laboratory 3M Environmental Technology & Safety Services 3M Environm ental Laboratory 935 Bush Avenue St. Paul, MN 55106 Laboratory Project identification FACT-TOX-026 3M Environmental Laboratory Page 1 of 9 Protocol #FACT-TOX-026 Study Identification 6-Month Capsule Toxicity Study with Ammonium Perfluorooctanoate (APFO/POAA) in Cynomolgus Monkeys Test Material Ammonium perfluorooctanoate (APFO/POAA) Sponsor 3M Toxicology Services - Medical Department 3M Center, Building 220-2E-02 P.O. Box 33220 St. Paul, MN 55133-3220 Sponsor Representative Paul Lieder, Ph.D., DABT 3M Toxicology Services Building 220-2E-02 651-737-2678 Study Director Kristen Hansen, Ph.D. 3M Environmental Technology and Safety Services Building 2-3E-09 651-778-6018 Study Location(s) In vivo Testing Facility Analytical Testing Laboratory Covance Laboratories, Inc. 3301 Kinsman Boulevard Madison, Wisconsin 53704 3M Environmental Laboratory Building 2-3E-09 935 Bush Avenue St. Paul, MN 55106 Proposed Study Timetable Analytical Start Date Analytical Termination Date November 12, 1998 May 12, 1999 3M Environmental Laboratory Page 2 of 9 Protocol #FACT-TOX-026 1. S tudy Six month capsule toxicity study with ammonium perfluorooctanoate (APFO/POAA) in cynomolgus monkeys. 2. Purpose The analytical portion of this study is designed to determine levels of (APFO/POAA) in the liver and serum of cynomolgus monkeys. Based on these results additional tissues or fluids may be analyzed. The in-life portion of this study was conducted at Covance Laboratories, study #6329231. 3. Regulatory Compliance This study will be conducted in accordance with the United States Environmental Protection Agency Good Laboratory Practices Standards, 40 CFR 792. However, analysis of the test material mixture for concentration, solubility, homogeneity, and stability will not be conducted, and is the responsibility of the Sponsor. 4. Quality A ssurance The 3M Environmental Laboratory Quality Assurance Unit will audit the protocol, study conduct, and final report in accordance with the Good Laboratory Practice Standards and 3M Environmental Laboratory Standard Operating Procedures. 5. Test Material 5.1 Identification Ammonium perfluorooctanoate (APFO/POAA) 5.2 Source 3M Specialty Chemical Division 5.3 Physical Description Gelatin capsules 5.4 Purity and Stability Determined by the Sponsor 5.5 Storage Conditions Room temperature _ 5.6 Reserve Samples Responsibility of the Sponsor 5.7 Disposition Specimens will be retained per GLP regulation 5.8 Safety Precautions Refer to MSDS for chemicals used. Wear appropriate laboratory attire, and follow adequate precautions for handling biological materials and preparing samples for analysis. __ 3M Environmental Laboratory Page 3 of 9 Protocol #FACT-TOX-026 6. Control Material 6.1 Identification Monkey liver and serum 6.2 Source Covance Laboratories, Inc. 6.3 Physical Description Liver and serum 6.4 Purity and Stability Not applicable 6.5 Storage Conditions Frozen at -2 0 C 10 C 6.6 Reserve Samples Not applicable 6.7 Disposition Biological tissues and fluids are retained per GLP regulation 6.8 Safety Precautions Refer to MSDS for chemicals used. Wear appropriate laboratory attire, and follow adequate precautions for handling biological materials and preparing samples for analysis. 7. Reference Material 7.1 Identification Ammonium perfluorooctanoate (APFO/POAA), lot #377 or #245 7.2 Source 3M Specialty Chemicals 7.3 Physical Description White powder 7.4 Purity and Stability Responsibility of the Sponsor 7.5 Storage Conditions Room temperature 7.6 Reserve Samples Not applicable 7.7 Disposition Retained as per GLP regulation and 3M Environmental Laboratory policy 7.8 Safety Precautions Refer to MSDS for chemicals used. Wear appropriate laboratory attire, and follow adequate precautions for handling bio lo g ical materials and preparing samples for analysis. 8. Test S ystem Cynomolgus monkeys were used as the test system, and were maintained and dosed as described in Covance protocol #6329-231. Group 1 control animals did not receive the test substance. Groups 2, 3, and 4 received the test substance daily for 26 weeks, in increasing concentration per group. Two animals each from Groups 1, 3, and 4 were designated as recovery animals and were allowed a 13 week recovery period after cessation of treatment. - 3M Environmental Laboratory Page 4 of 9 Protocol #FACT-TOX-026 9. Specimen Receipt The 3M Environmental Laboratory will receive samples of the following body tissues and fluids from the indicated points in the study: Body tissue/fluid Collected Shipped Expected # of samples Serum - all animals 7 days post treatment, every two weeks thereafter Packed on dry ice for shipping Urine, feces - recovery animals Liver - all animals After 6, 30, and 90 days recovery At termination of the study Total number of test animals: 16 Total number of control animals: 6 Packed on dry ice for shipping Shipped with final serum samples on dry ice 264 from main study 78 additional from recovery 18 urine, 18 feces 22 Samples sent to 3M Environmental Laboratories will be received and tracked according to applicable Standard Operating Procedures. 10. Preparatory Methods 10.1 FACT-M-1.0, Extraction of Potassium Perfluorooctanesulfonate or Other Anionic Fluorochemical Surfactant from Liver for Analysis Using HPLC-Electrospray/Mass Spectrometry 10.2 FACT-M-3.1, Extraction of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds from Serum or Other Fluid for Analysis Using HPLCElectrospray/Mass Spectrometry . 10.3 If preparatory m ethods other than th o se listed above are used, an a m en d m en t to this protocol will be written. 11. A nal ytical Methods 11.1 FACT-M-2.0, Analysis of Fluorochemicals in Liver Extracts Using HPLCElectrospray/Mass Spectrometry 11.2 FACT-M-4.1, Analysis of Potassium Perfluorooctanesulfonate or Other Fluorochemicals in Serum or Other Fluid Extracts Using HPLC-Electrospray/Mass Spectrometry ' 11.3 If analytical methods other than those listed above are used, an amendment to this protocol will be written. 3M Environmental Laboratory Page 5 of 9 Protocol #FACT-TOX-026 12. Data Quality Objectives The number of spikes/duplicates, use of surrogates, and information on other data quality indicators is included in the analytical methods. In addition, the following criteria will be met: 12.1 Linearity r2 > 0.980 12.2 Limits of detection / quantitation 12.2.1 Method Detection Limit (MDL) for APFO/POAA a. Serum: 5 ppb b. Liver: 24 ppb 12.2.2 Practical Quantitation Limit (PQL) - Equal to the lowest standard in the calibration curve 12.3 Duplicate acceptable precision < 30% for the method 12.4 Spike acceptable recoveries 70% -130% 12.5 Use of confirmatory methods Indeterminate samples will be re-analyzed 12.6 Demonstration of specificity Chromatographic retention time, mass spectral daughter ion characterization 13. Sub-Contracted A nalysis All analyses as detailed in this protocol will be performed at 3M Environmental Laboratories, Building 2-3E-09, 935 Bush Avenue, St. Paul, MN 55106. 14. S tatistical A nalysis Averages and standard deviations will be calculated. The statistical methods that will be used are described below: ' 14.1 Data transformations and analysis Data will be reported as the concentration (weight/weight or weight/vol) of APFO/POAA or metabolite per tissue or fluid, or of APFO/POAA or metabolite per unit of tissue or fluid. * 14.2 Statistical analysis Statistics used may include regression analysis of concentrations over time, and standard deviations calculated for the concentrations within each dose group. If necessary, simple statistical tests, such as Student's t test, may be applied to evaluate statistical difference. 15. Report A report of the results of the study will be prepared by 3M Environmental Laboratory. The report will include, but not be limited to, the following, when applicable: 15.1 Name and address of the facility performing the study 15.2 Dates upon which the study was initiated and completed 3M Environmental Laboratory Page 6 of 9 Protocol #FACT-TOX-026 15.3 A statement of compliance by the Study Director addressing any exceptions to Good Laboratory Practice Standards 15.4 Objectives and procedures as stated in the approved protocol, including any changes in the original protocol 15.5 The test substance identification by name, chemical abstracts number or code number, strength, purity, and composition or other appropriate characteristics, if provided by the Sponsor 15.6 Stability and the solubility of the test substances under the conditions of administration, if provided by the Sponsor 15.7 A description of the methods used to conduct the test(s) 15.8 A description of the test system 15.9 A description of any circumstances that may have affected the quality or the integrity of the data 15.10 The name of the Study Director and the names of other scientists, professionals, and supervisory personnel involved in the study 15.11 A description of the transformations, calculations, or operations performed on the data, a summary and analysis of the analytical chemistry data, and a statement of the conclusions drawn from the analyses 15.12 Statistical methods used to evaluate the data, if applicable 15.13 The signed and dated reports of each of the individual scientists or other professionals involved in the study, if applicable 15.14 The location where raw data and the final report are to be stored 15.15 A statement prepared by the Quality Assurance Unit listing the dates that study inspections and audits were made, and the dates of any findings reported to the Study Director and M anagement If it is necessary to make corrections or additions to a final report after it has been accepted, the changes will be made in the form of an amendment issued by the Study Director. The amendment will clearly identify the part of the final report that is being amended, the reasons for the amendment, and will be signed by the Study Director. 16. Location of Ra w Data, Records, and Final Report Original data, or copies thereof, will be available at 3M Environmental Laboratory to facilitate audits of the study during its progress and before acceptance of the final report. When the final report is completed, all original paper data, including those items listed below, will be retained in the archives of 3M Environmental Laboratory for at least a period of time as specified by regulation, and as established by 3M Environmental Laboratory Standard Operating Procedures. 3M Environmental Laboratory Page 7 of 9 Protocol #FACT-TOX-026 16.1 The following raw data and records will be retained in the study folder in the study/project archives according to 3M Environmental Laboratory Standard Operating Procedures: 16.1.1 Approved protocol and amendments 16.1.2 Study correspondence 16.1.3 Shipping records 16.1.4 Raw data 16.1.5 Approved final report (original signed copy) 16.1.6 Electronic copies of data 16.2 The following supporting records will be retained separately from the study folder in the archives according to 3M Environmental Laboratory Standard Operating Procedures: 16.2.1 Training records 16.2.2 Calibration records 16.2.3 Instrument maintenance logs 16.2.4 Standard Operating Procedures, Equipment Procedures, and Methods 17. Sample Retention Specimens will be maintained in the laboratory specimen archives for at least a period of time as specified by regulation, and as established by 3M Environmental Laboratory Standard Operating Procedures. 18. Protocol A mendments and deviations Planned changes to the protocol will be in the form of written am endm ents signed by the Study Director and the Sponsor's Representative. Amendments will be considered as part of the protocol and will be attached to the final protocol. All changes to the protocol will be indicated in the final report. Any other changes will be in the form of written deviations, signed by the Study Director and filed with the raw data. 19. A ttachments 19.1 Attachment A Preparatory and analytical methods 3M Environmental Laboratory Page 8 of 9 20. Signatures Protocol #FACT-TOX-026 Paul Lieder, Ph.D, DABT, Sponsor Representative ifoWiW_- Kristen Hansen, Ph.D., 3M Environmental Laboratory Study Director Date Date 3M Environmental Laboratory Page 9 of 9 Study Title 26-Week Capsule Toxicity Study with Ammonium Perfluorooctanoate (APFO) in Cynomolgus Monkeys PROTOCOL AMENDMENT NO. 1 Amendment Date: July 23,1999 Performing Laboratory 3M Environm ental Technology & Safety Services 3M Environmental Laboratory 935 Bush Avenue St. Paul, MN 55106 Laboratory Project Identification ET&SS FACT-TOX026 LIRNU2782 Environmental Laboratory Protocol FACT-TOX026 Amendment No. 1 This amendment modifies the following portion(s) of the protocol: 1. PROTOCOL reads: Section 10.0 and 11.0 list the following methods to use for extraction and analysis: FACT-M-1.0 "Extraction of Potassium Perfluorooctanesulfonate or Other Anionic Fluorochemical Surfactant from Liver for Analysis Using HPLCrElectrospray/Mass Spectrometry" FACT-M-3.1 "Extraction of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds from Serum or Other Fluid for Analysis Using HPLC-Electrospray/Mass Spectrometry" FACT-M-2.0 "Analysis of Fluorochemicals in Liver Extracts Using HPLC-Electrospray/Mass Spectrometry" _ FACT-M-4.1 "Analysis of Potassium Perfluorooctanesulfonate or Other Fluorochemicals in Serum or Other Fluid Extracts Using HPLC-Electrospray/Mass Spectrometry" AMEND to read: The extraction and analytical methods FACT-M-3.1 and FACT-M-4.1, respectively, were updated on 04/27/99 to: ETS-8-4.1 "Extraction of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds from Serum for Analysis Using HPLC-Electrospray/Mass Spectrometry" ETS-8-5.1 "Analysis of Potassium Perfluorooctanesulfonate or Other Fluorochemicals in Serum Extracts Using HPLC-Electrospray/Mass Spectrometry" REASON: The methods were updated to replace the extraction solvent ethyl acetate with a different extraction solvent MTBE (methyl tert butyl ether), POAA and Monoester were removed from the standard mix, and M556 was added to the standard mix. The analytical method was updated to include linear regression with 1/x weighting and a few minor changes in the HPLC 1100 instrument parameters. 2. PROTOCOL reads: Section 10.0 and 11.0 list the following methods to use for extraction and analysis: FACT-M-1.0 "Extraction of Potassium Perfluorooctanesulfonate or Other Anionic Fluorochemical Surfactant from Liver for Analysis Using HPLC-Electrospray/Mass Spectrometry" ETS-8-4.1 "Extraction of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds from Serum or Other Fluid for Analysis Using HPLC-Electrospray/Mass Spectrometry" FACT-M-2.0 "Analysis of Fluorochemicals in Liver Extracts Using HPLC-Electrospray/Mass Spectrometry" ETS-8-5.1 "Analysis of Potassium Perfluorooctanesulfonate or Other Fluorochemicals in Serum or Other Fluid Extracts Using HPLC-Electrospray/Mass Spectrometry" 3M Environmental Laboratory Protocol FACT-TOX026 Amendment No. 1 AMEND to read: The extraction and analytical methods FACT-M-1.0 and FACT-M-2.0, respectively, were updated on 07722/99 to: ETS-8-6.0 "Extraction of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds from Liver for Analysis Using HPLC-Electrospray/Mass Spectrometry" ETS-8-7.0 "Analysis-of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds in Liver Extracts Using HPLC-Electrospray/Mass Spectrometry" REASON: The methods were updated to replace the extraction solvent ethyl acetate with a different extraction solvent MTBE, POAA and Monester were removed from the standard mix, and M556 was added to the standard mix. The analytical method.was updated to include linear regression with 1/x weighting and a few minor changes in the HPLC 1100 instrument parameters. 3. PROTOCOL reads: Section 10.0 and 11.0 list the following methods to use for extraction and analysis: ETS-8-6.0 "Extraction of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds from Liver for Analysis Using HPLC-Electrospray/Mass Spectrometry"ETS-8-4.1 "Extraction of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds from Serum or Other Fluid for Analysis Using HPLC-Electrospray/Mass Spectrometry" ETS-8-7.0 "Analysis of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds in Liver Extracts Using HPLC-Electrospray/Mass Spectrometry" ETS-8-5.1 "Analysis of Potassium Perfluorooctanesulfonate or Other Fluorochemicals in Serum or Other Fluid Extracts Using HPLC-Electrospray/Mass Spectrometry" AMEND TO read: Additional extraction and analytical methods, listed below, were added to the protocol: ETS-8-96.0 "Extraction of Potassium Perfluorooctanesulfonate or other fluorochemical compounds from Urine for Analysis Using HPLC-Electrospray/Mass Spectrometry" ETS-8-97.0 "Analysis of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds in Urine Extracts Using HPLC-Electrospray/Mass Spectrometry" REASON: These methods were developed and validated for urine extraction and analysis after the original protocol was written and approved. QAi# C ni/iV A nm onlo/ / Protocol FACT-TOX026 Amendment No. 1 4. PROTOCOL reads: Section 2.0 lists serum and liver as the matrices of interest for determination of POAA. AMEND to read: Urine will be included for determination of POAA. REASON: The methods were developed and validated for extraction and analysis of urine after the original protocol was written and approved. - 5. PROTOCOL reads: Section 6.0 lists monkey serum and liver as the control matrices received from Covance Laboratories. AMEND TO read: Control matrix monkey urine was obtained from Covance Laboratories and is maintained at a temperature of -20 C 10 C. All traceability information for this matrix will be included in the final report. REASON: Addition of control urine matrix to the analytical protocol. 6. PROTOCOL reads: Section 12.2 lists monkey serum and liver method detection limits. AMEND TO read: Method detection limit for urine is 6 ppb. Reason: Addition of method detection limit for urine matrix. Amendment Approval Paul Lieder, Ph.D., DABT, Sponsor Representative tm d ^ J 9 9 9 v Date Kris J. Hansen, Ph.D., Study Director 3M Environmental Laboratory Date Study Title 6-Month Capsule Toxicity Study with Ammonium Perfluorooctanoate (APFO/POAA) Cynomolgus Monkeys PROTOCOL AMENDMENT NO. 2 Amendment Date: 20 January 2000 Performing Laboratory 3M Environmental Technology & Safety Services 3M Environmental Laboratory 935 Bush Avenue St. Paul, MN 55106 Laboratory Project Identification ET&SS LRN-U2782 FACT TOX-026 Covance Study. 6329-231 3M Medical Department Study: T-6889.3 3M Environmental Laboratory Protocol LRN-U2782 Amendment Number 2 This amendment modifies the following portion(s) of the protocol: 1. Protocol reads: The study director for the present study was identified in the protocol as Kristen J. Hansen, Ph.D. A mend to readi The role of study director for the present study was reassigned to Paul Lieder, Ph.D., as of 20 January 2000. The previous study director, Kristen Hansen, has been reassigned to the role of Principle Analytical Investigator. Reason: The role o f study director w as reassigned in an effort to ensure com pliance with Good Laboratory Practice Standards that outline study personnel requirements (refer to 40 CFR "Part 792). 2. Protocol reads: The sponsor for the present study was identified as Paul Lieder. A mend to read: The role o f sponsor fo r the present study was reassigned to John L. Butenhoff, Ph.D., as of 20 January 2000. Reason: To ensure that the study director does not also carry the duties of study sponsor, the sponsor role w as reassigned. In this manner, personnel responsibilities and workload are more evenly balanced. 3. Protocol reads: 17. Sample Retention: Specim ens will be m aintained in the laboratory specim en archives fo r at least a period of tim e as specified by regulation, and as established by 3M Environmental Laboratory Standard Operating Procedures. A mend to read: 17. Specimen Retention: S pecim ens will be m aintained in the 3M Environm ental Laboratory specim en archives. Any specimens sent to sub-contract laboratories will be returned to the 3M Environmental Laboratory upon completion of analysis and submission of the sub contract laboratory(s) final report. Specimens analyzed at sub-contract laboratories will be returned with the following documentation: the signed original chain of custody and records of storage conditions while at the sub-contract facility. Reason: To define in detail the appropriate disposition o f specim ens analyzed at subcontract laboratories. 3M Environmental Laboratory Protocol LRN-U2782 Amendment Number 2 4. Protocol reads: Section 16 states that the following raw data and records will be retained in the study fold er in the archives according to AM D T-S-8: Approved protocol and amendments; study correspondence; shipping records; raw data; approved final report (original signed copy); and electronic copies o f data. A dditionally, Section 16 states tha t supporting records to be retained separately from the study folder in the archives according to AMDT-S-8 will include at least the following: Training records; calibration records; instrument maintenance logs; Standard Operating Procedures, Equipm ent Procedures, and Methods; and appropriate specimens. A mend to read: Section 16 states: "The original data, or copies thereof, will be available at the 3M Environm ental Laboratory to facilitate audits of the study during its progress and before acceptance of the final report. W hen the final report is com pleted, all original paper data, including: approved protocol and amendments, study correspondence, shipping records, raw data, approved final report, and electronic copies of data will be retained in the archives o f the 3M E nvironm ental Laboratory. All corresponding training records, calibration records, instrument maintenance logs, standard operating procedures, equipm ent procedures, and m ethods will be retained in the archives o f the facility performing each analysis. Reason: To direct subcontract laboratories in the disposition o f the items listed above. 3M Environmental Laboratory Amendment Approval Protocol LRN-U2782 Amendment Number 2 John Butenhoff, P h D ., Sponsor Representative /O , Date Kristen J. Hansen, P h D ., Outgoing Study Director / / - F-c b -' lOOO Date Paul Lieder, PhD ., Incoming Study Director /O t 2 ,0 0 0 Date ll\rf Fn\/irnnm&ntal I ahnrntnrv Study Title 6-Month Capsule Toxicity Study with Ammonium Perfluorooctanoate (APFO/POAA) in Cynomolgus Monkeys PROTOCOL AMENDMENT NO. 3 Amendment Date: 20 April 2000 Performing Laboratories Liver, Serum, and Urine A nalyses 3M Environmental Technology and Safety Services Fluorine Analytical Chemistry Team Building 2-3E-09, 935 Bush Avenue St. Paul, MN 55106 Feces A nalyses Centre Analytical Laboratories, Inc. 3 0 4 8 R esearch D rive State College, PA 16801 Laboratory Project Identification ET&SS LRN-U2782 FACT TOX-026 Covance Study: 6329-231 3M Medical Department Study: T-6889.3 3M Environmental Laboratory Protocol LRN-U2782 Amendment Number 3 This amendment modifies the following portion(s) of the protocol: 1. Protocol reads: The amended section 2.0 text states that this study is designed to determine levels of APFO/POAA in the liver, serum, and urine of cynomolgus monkeys. A mend to read: This study is designed to determine levels of APFO/POAA in the liver, serum, feces, and urine of cynomolgus monkeys. Reason: The analysis of fecal tissue for the target chemical and/or its analytes was added to the scope of the study following the issuance of the protocol. Feces extraction and analytical methods were not validated"and approved prior to protocol approval. 2. Protocol reads: The amended section 6.0 lists monkey liver, serum, and urine. A mend to read: Add: monkey feces with a physical description of feces. Reason: Analysis of fecal tissue for the target chemical and/or its analytes was added to the scope of the study following the issuance of the original protocol. 3. Protocol Reads: The amended Section 12.2.1 items a., b., and c., list the Method Detection Limits for matrices analyzed in this study. A mend to read: The method detection limits for all compounds and matrices will be taken from the methods used for extraction and analysis. Reason: The method detection limits are specific to the 3M Environmental Laboratory. This statement was added to allow for sub-contracted analyses, revised methods, and added matrices. 4. Protocol R eads: Section 13. lists the laboratories that will be conducting analyses for this study. A mend to read: Add: Centre Analytical Laboratories, Inc., 3048 Research Drive, State College, PA 16801 Reason: Feces analyses were added to the scope of this study. The sub-contract laboratory performing analyses was not in the original protocol. 3M Environmental Laboratory Protocol LRN-U2782 Amendment Number 3 5. Protocol Reads: Sections 10.3 and 11.3 state that if methods other than those listed are used in this study, an amendment will be written to include the new methods. A mend to read: The feces extraction and analytical method used by Centre Analytical Laboratories will be; 00M -023-003 (Revision 2), "Determination of Fluorochemical Residues in Monkey/Rat Feces by LC/MS7MS." ' Reason: The sub-contract laboratory performing feces analyses was added to the scope of this study; this method was not validated and approved prior to protocol approval. Amendment Approval John L. Butenhoff, Ph.D., Sponsor Representative P au l Lieder, Ph.D., Study Director Date Date 3M Environmental Laboratory m Study Title 26-Week Capsule Toxicity Study with Ammonium Perfluorooctanoate (APFO) in Cynomolgus Monkeys PROTOCOL AMENDMENT NO. 4 Amendment Date: August 2, 2000 Performing Laboratories Covance Laboratories Inc. 3301 Kinsman Boulevard Madison, WI 53704 3M Environmental Technology & Safety Services 3M Environmental Laboratory 935 Bush Avenue St. Paul, MN 55106 Laboratory Project Identification FACT-TOX-026 LIMS U2782 Covance 6329-231 3M Medical Department Study: T-6889.3 Protocol TOX-026 Amendment 4 This amendment modifies the following portion(s) of the protocol: 1. Protocol reads: S tudy Title: "6-Month Capsule Toxicity protocol for the analytical phase of the study). (on the title page and page 2 of the Amend to read: S tudy Title: "26-Week Capsule Toxicity protocol for the analytical phase of the study). (on the title page and page 2 of the Reason: To correct the title for the analytical phase of the study. 2. Protocol reads: D a ta Q u a lity Objectiv es (Section 12., page 6 ) Amend to read: Add to this section: LOQ in feces of 10 ng/g Reason: To specify the analytical limits for feces analyses. 3. Protocol reads: (page 2) S tudyDirector: Peter J. Thomford and Paul Lieder Testing Facility: Covance Laboratories Sponsor: APME AdHoc APFO Toxicology Working Group and 3M Toxicology Services - M edical Departm ent Sponsor Representative: David Farrar and John Butenhoff Amend to read: S tudy Director: Paul Lieder Testing Facility: 3M Toxicology Services - Medical Department Sponsor: APME AdHoc APFO Toxicology Working Group (including 3M Toxicology Services - Medical Department) Sponsor Representative: David Farrar (in-life) and John Butenhoff (analytical) Reason: To reassign the testing facility, in order to abide by the GLP requirement for one study director. To clarify the responsibilities of all parties included in this study. Protocol TOX-026 Amendment 4 4. Protocol reads: P rincipal Analytical Investigator: Kristen Hansen, Ph.D. (Amendment No 2 to TOX 026, Section 1.) Am end to read: Add: Principal Analytical Investigators: Kristen Hansen, Ph.D. an d Enaksha Wickremesinhe, Ph.D. Re a s o n : To specify the PAI at Centre. 5. Protocol reads: Analytical Termination Date: May 12, 1999 (under Proposed Study Timetable, page 2) Am end to read: Analytical Termination Date: September 3 0 ,2 0 0 0 Reason: To allow for the analyses of all samples. 6. Protocol reads: Location of Raw Data, Records, and Final Report (Section 16., page 7) Am end to read: Add: After issuing their final report, Centre will forward all original study-specific raw data to 3M Environmental Laboratories, together with copies of appropriate facilityspecific raw data applicable to this study. Centre will maintain a copy of the applicable study-specific raw data, protocol and analytical report in the Centre archives. Reason: To specify the archival requirement for the portion of the data developed by Centre. 7. P rotocol reads: Sample Retention (Section 17., page 8) Amend to read: After the analytical report on feces is signed by the PAI, all feces specimens of this study will be returned to 3M Environmental Laboratory. These specimens may then be discarded by written direction of the study director. Specimens of blood, plasma, red blood cells, serum, bile, and urine may also be discarded by written direction of the study director after Protocol TOX-026 Amendment 4 the analytical report for the 3M Environmental Laboratory analyses is signed by the study director. Reason: To specify the handling o f the above biological specimens, and to define when the quality assurance verification is considered complete. Amendment Approval Protocol TOX-026 Amendment 4 John Butenhoff, Ph.D. Sponsor Representative, Analytical Phase / ^ f' P / Date 2 x 5 o - _______ ( y j _________________ __________ / 9 Paul Lieder, Ph.D., D A B T Date 3M Study Director S F fr 2 (h x r Study Title 26-Week Capsule Toxicity Study with Ammonium Perfluorooctanoate (APFO) in Cynomolgus Monkeys PROTOCOL AMENDMENT NO. 5 Amendment Date: October 12, 2000 Performing Laboratory 3M Environmental Technology & Safety Services 3M Environmental Laboratory 935 Bush Avenue St. Paul, MN 55106 Laboratory Project Identification FACT-TOX-026 ET&SS LRN U2782 Covance 6329-231 3M Medical Department Study: T-6889.3 3M Environmental Laboratory Protocol FA C T TOX-026 Amendment No. 5 This amendment modifies the following portion(s) of the protocol: 1. Protocol reads: Principal Analytical Investigators: Kristen Hansen, Ph.D. and Enaksha Wickremesinhe, Ph.D. 2. A mend to read: Principal Analytical Investigators: Kristen Hansen, Ph.D. and Emily Stauffer. Reason: To change role of the Principal Analytical Investigator at Centre Analytical Laboratories. 3M Environmental Laboratory Amendment Approval Protocol FA C T TO X-026 Amendment No. 5 John L. Butenhoff, Ph.D. Sponsor Representative, Analytical Phase Date (? oa Paul Lieder, Ph.D., D A B T 3M Study Director f h b h o Date OO -mdy Mauffer ' Date Centre Analytical Laboratories, Principal Analytical Investigator 3M Environmental Laboratory Record of Deviation Identification Study / Project No. F A C T 'T b X - b 2L Deviation Type SOP ( Check one) O Protocol hvanoL Method Equipment Procedure Other: ETJ-3-5.1 E T 5-*-q?.n li/'ol'l, silh SliltW' s/aei, T/ar/ii, -S/a/Vf, iiD3^ blii l 1, a/nist aiijq1) aJairs, sI jh'i^ `/ii/V1,i II. Description: Required Procedure/process: __ .Ijkie.....m^lkoAj jjg lc'-__Aaq!qu, .iW .. e^lm d-ed ma In * .. Jlfin Aod-clr... prior Jk>.. fiori....... I..Pp lloLinn eac-K dei Cx:1rac.Tj ,__Xh& ._Qj/G5U|jl._C>_Al&q_iifladUrd._Curut-l..Loill. JaLr plo B ti. V. ___ __________ _-....... ....... ............ ..............-... ...... .... .......... ......... -............. -............. Actual Procedure/process: J h k ....&xIra g l e d __ P ia lrix ji a A c d r .... Lutee...fio o lite c i... prior t> Orti .l t \ ..of. p.sl V u c l-f....Dnl^ i k e . U ...4 ondarci...Curvte.... locj... plowed, 2 n Curve...Lucy__ noi_u ie ' '* ' ' U` ' " * J " 1 rX-u-ru~C. OLOlAc t c k . .... Xh t rTkiJ....deviCelici III. Actions Taken: (such as amendment issued, SOP revision, etc.) U)4... Lori lit e Recorded By Date IV. Impact on Study/Project ^ I ihI oo C l U c/ke^.QC- j v r ..A z .von#........................... V < z / / \ j^<haM s.. l^c^c b v * esfaeJ-ec* f\J /2tt4/&LsLC SlJiUsCs. .... ..... , ............. ........ w k h n in '/M ...... ................... . Authorized By (Study Director / Project Lead) Date Spctuvr 3"oLr> tJulioJiot(- -Jiwla tre.c"lcr, Pcul I'ltitf 3M Environmental Laboratorv Deviation No. . 1 Form ETS-4-8.0 (assigned by Study Director or Project Lead at the end of study or project] Record of Deviation I. identification Study / Project No. Deviation Type (Check one) Fa c t -.t d y. - o a i SOP Protocol C o \j ncj> - 131 0 Method Equipment Procedure Other: Document Number _______ Fa c t - m- H- l Date(s) of occurrence il/u-iw, nhnlk U/oj/w I otIm Iw oi/ai/q^ o i/a iiw _____________ II. Description: Required Procedure/process: _____ __ ...... St_M F o A F a ct- tn- 4.1 s fc .. iftit'ai Curvi, s k u)V...-.he...p l v i i d ...u s a k ... lii&L, .I.0 r / JJi.oin .LO.,'4k oui1 lW..i.n.^........ 1 iI.tAj . , ' <J Actual Procedure/process: 1k . V, L l Cu r n . ic tu jtlo-lW .... .....W r rt^ctiiiso , w-eic^ikd \Jx III. Actions Taken: (such as amendment issued, SOP revision, etc.) 'ftw Aeviukton_U)j UOri Its Recorded By C=ty-<- ^ 0- /a m/M IV. Impact on Study / Project r jAAJSrC...U>... <24a.................................... .O.a <?Uh&v<lX^ ...................... ......... ............. .............................. ............................................................. k , b n /n -ic z ) Authorized By (Study Director / Project Lead) Date JpwlJDr! Tolin GiuWVioFP 3M Environmental Laboratory Form ETS-4-8.0 DirecW ! Ptiul Litj-er ^ Deviation No. (assigned by Study Director or Project Lead at the end o f study or project) Record of Deviation I. Identification Study/ProjectNo. T O o ^ tovocnu. 1*321-331 Deviation Type (Check one) SOP Method Equipment Procedure Protocol Other: Document Number fflCT- ft\-3A 4- Date(s) of occurrence _____________________________ FAtr-W-H.) 1_________ H l & l W ________________ II. Description: Required Procedure/process:................................................................................................. ...TV exiracjftn..a n d ... Q oaU lita)....rn&iKpic....k v k d ... ore...FftCT-ft-.?.-!.... nd F f i c r - m - H . L ........ ............ ...................................................................................... Actual Procedure/process: ............................................................................................... ..T/ie-...exWtkon...cimi....QnaUWrtl.... nn-tAK-pdj......u k lk ...Wvl)...kt-...rpjlovvtdl.... CirC ..n s - 8 H I Qnd T3 - J ......................................................................................... III. Actions Taken: (such as amendment issued, SOP revision, etc.) An GihendmenV ko jprvWcl.................. tiiiied........................... .............................. ........................... ...... Recorded By Date C $ b t\ A C^yrrvA\ IV. Impact on Study / Project TVi (W jctW toiU ..ClfCtel"..&L,....OuictPjh.... i?i... dKiJ...-i4ud^: .: : ..;..... Authorized By ((Study D irectop/ Project Lead) -/J n --------- Date 3M Environmental Laboratory Form ETS-4-8.0 D eviatio n N o. _____^ ________ (assigned by Study Director or Project Lead at the end of study or project) Record of Deviation I. Identification Study/ProjectNo. T a m l, C o r n e t (,,331-331 Deviation Type ( Check one) SOP jS^Method Equipment Procedure Protocol Other: Document Number ETJ-8 H. I v hps-j'-S.I Date(s) of occurrence ___________________________ ____________I II. Description: Required Procedure/process: .................................................... T I ' i ' 4-1 PinA Bz' 5....1 Tht- A * Ira c ito n Qod fto nod 1\ _ perPluorpo ciA?G.U, 5 Ci __dar.^tL. fto Oliata.. .................... do Actual Procedure/process: ............................................................................ ... PerP lpuro t>cd Ano cvW-..CPPA/0..U>`ll....b e...pkfc,.................... Grtal^pL .... t x W c W ............Qaal^Jif..... o P...vierunv................................................. III. Actions Taken: (such as amendment issued, SOP revision, etc.) "Thi...the-dcclj..lot I) kL r t v .it A Pd.... irvd'udt..... Phvi.....Co/nJi-DAinp. Recorded By Date (^ x A CJIm k iv\ IV. Impact on Study / Project 4 /21/ ^ T k i cUv-fcd&n. Ui)l hcw&....no....aPPLed..... On..A t ....C>uPcc.inA...>P...4j)U' .......... Authorized By (Study D irector}/Project Lead) jt^ - /o c f c l------------- ' Date 3M Environmental Laboratory Form ETS-4-8.0 D eviatio n No. _____J________ (assigned by Study Director or Project Lead at the end of study or project) Record of Deviation I. Identification Study / Project No. FCT- T b X- Deviation Type SOP (Check one) Protocol C doOCL...... .339- 231 J l Method Equipment Procedure Other: Document Number __ F A C T ' m - I. D Date(s) of occurrence p s io fth , 0 5 113k Ou Ul l P i l l i l i 1 II. Description: Required Procedure/process: JHkkkbA___Ffl LT- tf\ - l . b jhalfU ...i k i _t l h ^ l .................... loili ki- JA.lt,A,_CU...~iLc ..^xkekao__ J&lt^/th_____________ Actual Procedure/process: .................. -t-u.L:i).uLi].............................. b>as...uj&A... sxs..IL l....t-x-uacJaon.... oL caI. III. Actions Taken: (such as amendment issued, SOP revision, etc.) Thu .tv.aiton_U&J_U)nJitn. .r .-Wotrv Jolxrfyrl.................... ifl.t were voiiciaW ... Uiirv ^ M.TC_Ax. Recorded By Oi - Of/'k A i b m e ^ IV. Impact on Study / Project t ..M TTE. Jx1 7^ 1... ^iry r^> iJAA/yrn / kJ ....f je ..n d MTAXLc.... Date u/ivL AuthorizedBy (Study Director / Project Lead) j)k J J M 4 ltiL y Date ipvfliw. Tohn "'C.ultnK.it 3M Environmental Laboratory Form ETS-4-8.0 ^irttVoi-' Pciv.) Li^clti Deviation No. (assigned by Study Director or Project Lead at the end of study or project) Record of Deviation I. Identification Study / Project No. Deviation Type (Check one) F A CT - T bX - O L SOP Protocol Co\JL...io329- P'31 S3 Method Equipment Procedure Other: Document Number fa c t - m - a .o Date(s) of occurrence 5)(0/*, =/"/, sin*, 5Ih lK , C./zc./H, It/nlH , *llHi , II. Description: Required Procedure/process: M eJL ri... FACT- J c j ..A L . ir.lsQ1_Ciu rli W ar f^rU d io irv U)i)l iO pioj-icid... Actual Procedure/process: AFu-... in itial L u rv ^ ..................p lo lW ,.. _lin e a r... redrew bn, lo&i^hltd....l/x . III. Actions Taken: (such as amendment issued, SOP revision, etc.) This..txjQ iW ....Wj..Ltriiltn. Recorded By A CAuuk IV. Impact on Study / Project J ASU. <tf <n tAJfsi#}\sfs d A .A * /T u /s ...04 CMry - /OT^Ft/VC^ri^1^ - o J L U lJ It - Authorized By (Study Director / Project Lead) Date il I h IDo Jc Date Spooler Tolw CxdtrilofC- ' Slud-j ^'iltlVor. Pftu^ 3M Environmental Laboratory Form ETS-4-8.0 Deviation No. (assigned by Study Director or Project Lead at the end of study or project) Record of Deviation Record of Deviation I. Identification Study / Project No. Deviation Type (Check one) F A C T -T oX -.O I L SOP 0 Protocol C o van LL.... L l M ' 2 2 1 Method Equipment Procedure Other: Document Number Fa Lr- m - 2 0 !j (D./alitlAe(s, ) of occulorUrseInMc.e f/Jthn,ijufhh+ilH, LltCh'i II. Description: Required Procedure/process: _____ TC l (u L ITt i i kpA F/tC T- Pv A. D For ___ .............ex.Var. i i. Actual Procedure/process: HeiKoIF....E r - - 5 .1 ..(a i pr&..Qokjkc l... rnilkaiciy.- P'icl.FACI-TA- 3 .1................ iy Qi..-.Hu,.....melbodj....lu ed ... iJ ho...OneLytin^... Tkwe. livtr..CxWeii................................ III. Actions Taken: (such as amendment issued, SOP revision, etc.) ..% \J..wifcyv...W i... LCikn. Recorded By ______ C J\ LCtrCu2-e- Ubn^K IV. Impact on Study / Project j F A c T ' M 'A i M gyr\ e ftw ' p A ^P M -1 0- Authorized By (Study Director / Project Lead) M iliti) Date & ^ // o /a r f Speller Tcliri RultAoif -cSi,lu)ju. rOv._r.e.tlW/- oP.au1l ILie.Ajer 3M Environmental Laboratory Deviation No. a Form ETS-4-8.0 (assigned by Study Director or Project Lead at the end of study or project) Record of Deviation 1. Identification Study / Project No. FACT- TbX- DZL> . . . . . . . . . . L^y...CA?.. . . {$333....i331 . . . . . . . . Deviation Type SOP Method Equipment Procedure (Check one) JJ Protocol O Other: Document Number Date(s) of occurrence II. Description: Required Procedure/process: TJul Dr^-hor^ol lijlf "f k . iiv'ir (L-yTrt\-k\yi Vhkknl J FALT'lh-l.D Actual Procedure/process: Followed.... mkkocl.... FA C T -f il - M III. Actions Taken: (such as amendment issued, SOP revision, etc.) T k i dem';!o/...Ldclj....U)ri'tkjfl. Recorded By Ji IV. Impact on Study / Project f A t T- M ' / /.yu>.........../ ... A i? . Date I I d Od (jh (IJl? (4 ) Authorized By (Study Director / Project Lead) L//JU m F /3 Date "ToKn Suit/ihoiP -SluiC Di^cl' . P[.u\ Li^fr 3M Environmental Laboratory Deviation ITM. ______ _______ Form ETS-4-8.0 (assigned by Study Director or Project Lead at the end of study or project) Record of Deviation I. Identification Study / Project No. ........................_............ FACT- T 0 X-02L>..................... Covcnoi....y .A i: .3 3 i.................. Deviation Type SOP jj Method Equipment Procedure (Check one) Protocol Other: Document Number ______________ E T S -j j- H lo I Date(s) of occurrence _______I o z lo is ln i- oslio lw II. Description: Required Procedure/process: ___ ____ __ ___ HcAkod.._____Adi. Alfcrn.1 sl/MiJurA._jUii.L. UuA..5 r .. Cuiifyj lalug.. CMnCribaJinnS Gad i i t , jxnrt- -fir ?Of)A u t ili lit , p ln irk d __ USia..0- qU.qdrfti'C Actual Procedure/process: _____ ..Cpftt^W iorn..lAitr^ CalcJaH...'dM^.,,&xlcraci\..-Siord... a a d __P.OAA....Curvi, .W.03..ploAleA..____ h/)tar...restosiion............. .......................... ........ ............. III. Actions Taken: (such as amendment issued, SOP revision, etc.) _ 5 j 2l Eooi...kCtrC. Aukm S/iVJL .nm d ftf .w m L. & r .. s e rn n h j>urpoSW - 4o. ei t r /nlKa.__ ................... Qi'd JtiiJjr^....no.i . . . ref t?.r.ltd. & Recorded By IV. Impact on Study / Project Tk>j tW icioA.. Uili..noi...aAvtj-sei-jy............ .ike,..QiALome,...oJc. Authorized By (Study Director } / Project Lead) (h -- Date 8/nff 3M Environmental Laboratory Form ETS-4-8.0 D eviation No. ______^0______ (assigned by Study Director or Project Lead at the end of study or project) Record of Deviation I. Identification Study / Project No. FAtt-Tt>X- C>3iL Deviation Type SOP (Check one) Protocol Couttca.... L 3-1 - 2 31 0 Method Equipment Procedure Other: Document Number ________ ETi-3-qi.i> Date(s) of occurrence s jw lw II. Description: Required Procedure/process: T/yf,..miAKorl..slaks - noly * ... &.m iliM g i,... .................Leiilra-W ..uerjk.ak>o... & U & m L 10_JQmpk j, Luik _ mioltnurt)--o l.o<u....pficjbt^jdL------------------------------------------------- A \ eoaI one C o n tW i^ Calibra W verikc.bo/\ per Id .T6m ptl4 __ CWAJ...... pio CX percml r^Cvuxr ^ __i ^ iik iW.. */- I v i. q Ik ..jp i k d ....,,.(k>Ca.WWi...... .... .......... ............-.................................. Actual Procedure/process: Ont , .Cof\h(M^AQ..ca lik &W \jej-;k c.eAvt>ri per...IS..-Scmple-S....hai ..0-..pferak.. re,a>uer^..uoiUviw- */- 3c Z ..0 [ ...fU , jp .jiid ...CoAd/vk-ktan.............. ............ .... ......................................................................... III. Actions Taken: (such as amendment issued, SOP revision, etc.) D ak..uU ....k ................in -Ik Exa l ... ipre&ckki-k Recorded By 0^ A T V , Aa up AX CA*m^ IV. Impact on Study / Project -b d^km djL _ojLrAuX Date 11*100 Authorized By (Study Director 7 Project Lead) <1 / / / ( / o r '/ 7- J jk .U jJll'b.- Date Y^ 3M Environmental Laboratory Form ETS-4-8.0 Deviation No. II (assigned by Study Director or Project Lead at the end o f study or project) Record of Deviation I. Identification Study / Project No. ....................FP\ CT- Dviation Type (Check one) M - OC SOP |J Protocol CoonUL L>32 ^ - 2 1 1 Method Equipment Procedure Other: Document Number ______ E T i-jM l. D 1Date(s) of occurrence _ _ _ i llofln. qIoIm . ^ 09) 99. p i l o t a II. Description: Required Procedure/process: -Tta...poskc-ol_Liti__ imeiiDcl...,, J I - ' 3 ?..&................................. Uri/n ....fex iari j Actual Procedure/process: a d . ..._.Ti.-5:S.|..( f t ............. M aiolici..mekksci).... n i ......E T .i-< 5'7-5(fftt.....lllrtr... W LjLcl im ik s d i... Mere,..I .kii._ a i.. meihods........... .M e l...when../iI^x.a 4U Uritu.....tX La ck. ____ III. Actions Taken: (such as amendment issued, SOP revision, etc.) -Thd....ddiAftitoa... U?Ctf--.Lftntkn..... ......... ......... .. .................................... Recorded By IV. Impact on Study / Project Date v wc. " I n I,<o ii I # m I Do js p im ^ h ...'k t jA&VYTZcf rn ^ts fh c tb ..M iti. )lo cto rrrce *cfjc(,..thd.. ...................... ( TS- j r - 7 o ) _UJtoo............................p io ..ad-M uic " ' ' ' h lU ir/M .. Authorized By (Study Director / Project Lead) Date Sponsor' Tolvi Bulcilioft jfw E Dihtckr Pcul UeAtr 3M Environmental Laboratory Deviation No. ii Form ETS-4-8.0 (assigned by Study Director or Project Lead at the end of study or project) Record of Deviation I. Identification Study / Project No. ...... .....................J M j t .!M : Q 2 . k ..... ...... .. .Cqmoll.... ^32%: 231.................... Deviation Type SOP 0 Method Equipment Procedure (Check one) Protocol Other: Document Number ETS--a.n ! Date(s) of occurrence _______ i q i z s l w ________ II. Description: Required Procedure/process: M fclM ....kTS -S'K-D jU 4 t J U&i^hkd.. 1/6l~+ 4 U W k a l .XiAQrfL... LOill... .Uj.fe^__ in k rflo.1.S la n ia rd. JdL.. p lo lk l....Lui "- j Gl . Actual Procedure/process: J h i , ...... M M , . ..... L O A i.....file .. tx .le m a l.... A m I tcL ,,UJAvc^__ M ir ili achtel. U-StV t III. Actions Taken: (such as amendment issued, SOP revision, etc.) Thil....................... U)Aj ......LOd I E y, IV. Impact on Study / Project 2: VA/o-hXi. , o j pcS\ v)r> ..c A ^ ^ J h i 'M* msu. *1 -fk* ..C i fh c u ^ . .fh t c&fJtL yM .Le -- ^ ----------------- ,_,______________ kjh .illB M Authorized By (Study Director / Project Lead) Date 7 / ttic/o*'/'JvbTM' 7- y peer*? SfvncrTToiv\ BujtnhoPP SjutL Di.xcW . ?Ciu\ L<tcltr 3M Environmental Laboratory Deviation No. l Form ETS-4-8.0 (assigned by Study Director or Project Lead at the end of study or project) Record of Deviation 1. Identification Study/Project No. Deviation Type ( Check one) SOP 13 M ethodic Equipment Procedure Protocol Other: Document Number P A J,0 Date(s) of occurrence II. Description: Required Procedure/process: ........ K~ th t <2- a / A h C m M & ' A w A .../m A S S .A e /S A x u n .......... h # _ / A A h & A A y S c .:... l ^ xSm l i n ' J h L . /Ti& ^dy &f/baS V s ^ c /- /S. W?S/AstfA ^ zz^ / / ^l/ / pz& M xj/ . ........................................ .C^rh>i .................. Actual Procedure/process: ........... PAL..OsetL....n^zA... g ~ ../y?/c#. dJ</9-f/3OUX-// '&..Qym&r ... 2?.....p f e - U & -. & d s i/Z r A h i. w j - r? /M _ M gO / I T h>^n> / x z / A A h z S -^ A A h i z P P , A hA P z/ III. Actions Taken: (such as amendment issued, SOP revision, etc.) P Z ~ ......................................................................... Recorded By Date iV. Impact on Study / Project 3%jl sni ayimn &(maq U.t-'vk effivu rc\a h>htwc h-C/fy /OaVu^<i/ 1/WtffuA fheu^ a A '^ Ajj- <t Xm^ usr*ct*^tY*4) *-' y /V M - ../?S 7 U ...... pu^r\c~'aAaJ ' LMlh * IakaX axccpfa [&(. / th*o asrevn&^oi. is ridi' inditeti id dp Kr j & f p t i + r \ A s f l C J A A - v y ..... A /c?....K i t - t d i S U . .. f h * /L A A k r . Authorized By (Study Director / Project Lead) k h (P & O to A z - ' rDZ-/&d ^Dat"e Sportior (U-p- John Bu.lt/vHoif 3M Environmental Laboratory Form ETS-4-8.0 iluci^ Director- Paul L'fcAer Deviation No. JH (assigned by Study Director or Project Lead at the end of study or project) Record of Deviation I. Identification Study/Project No. FACT-TOX-026 Covance 6329-231 Deviation Type (Check one) SOP X Method Equipment Procedure Protocol Other: Document Number(s): ETS-8-5.1, ETS-8-7.0, Date(s) of occurrence: and ETS-8-97.0 Entire study II. Description: Required Procedure/process: The method describes the calculations that should be used to convert extract concentration to matrix concentration. Actual Procedure/process: The actual calculation used varied somewhat from that written in the method. One additional factor was added for salt correction. This accommodates the mass difference between the analytical standard (C7F15COONH4) and the target analyte (C7F15COO-) determined after the study was completed. III. Actions Taken: (such as amendment issued, SOP revision, etc.) This deviation was written. Recorded By Date e d it* J \ IV. Impact on Study / Project o i j a i oi The updated calculations accommodate new information and are an improvement. No adverse affect on the study. fc }, o'Z-//~z-/0 / | Authorized By (Study Director / Project Lead) . Date / ? Feb 0 / XrtcW '. ?0u\ tUr 3M Environmental Laboratory Form ETS-4-8.0 D eviatio n No. ______ ljg______ (assigned by Study Director or Project Lead at the end of study or project) Record of Deviation 1. Identification Study / Project No. FACT-TOX-026 Covance 6329-231 Deviation Type ( Check one) SOP X Method Equipment Procedure Protocol Other: Document Number(s): ETS-8-7.0, ETS-8-97.0 Date(s) of occurrence: Entire Study II. Description: Required Procedure/process: These methods state that matrix spike percent recoveries must be within 30% of the spiked concentration. Actual Procedure/process: The matrix spike percent recoveries for liver and urine data were 40% of the spiked concentration. III. Actions Taken: (such as amendment issued, SOPrevision, etc.) This deviation was written. Recorded By Date I A C ltn ^ O i l 1 oi 1 I IV. Impact on Study / Project Data quality stated in the final report will reflect actual recovery of the matrix spikes. No adverse impact on the study. Fh l/li/ol -------- --- ----------------- Authorized By (Study Director / Project L e a d )^ ^> Date ) Pf F e i> 0 ) Sportier EeprtK/ila-iiirt,'. John Buit/iWC DirtcW'. Pftul Otdxr 3M Environmental Laboratory Form ETS-4-8.0 Deviation No. liT_______ (assigned by Study Director or Project Lead at the end of study or project) Record of Deviation I. Identification Study / Project No. FACT-TOX-026 Covance 6329-231 Deviation Type (Check one) SOP Method Equipment Procedure X Protocol Other: Document Number(s): Protocol FACT-TOX-026 Date(s) of occurrence: Entire Study II. Description: Required Procedure/process: Methods ETS-8-7.0 and ETS-8-97.0 state that matrix spike percent recoveries must be within 30% of the spiked concentration. Actual Procedure/process: The matrix spike percent recoveries for liver and urine data were 40% of the spiked concentration. III. Actions Taken: (such as amendment issued, SOP revision, etc.) This deviation was written. Recorded By Date DS M o I IV. Impact on Study / Project Data quality stated in the final report will reflect actual recovery of the matrix spikes. No adverse impact on the study. ............................................................................................ f f t ....0 $ S ' ) o \ ............ Authorized By (Study Director / Project Lead) S1u4l^ Director Sponsor eprtje/ilftlu/f, 7chn &uk/tho 3M Environmental Laboratory Form ETS-4-8.0 Deviation No. _TL (assigned by Study Director or Project Lead at the end of study or project) 3M Medical Departm ent Study: T-6889.3 Analytical Report: FACT TO X-026 LRN-U2782 Appendix C: Extraction and Analytical Methods This appendix includes the following 3M Environmental Laboratory methods: Liver FACT-M-1.1, Extraction of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds from Liver for Analysis Using HPLC-Electrospray/Mass Spectrometry, (15 pages) FACT-M-2.1, Analysis of Fluorochemicals in Liver Extracts Using HPLCElectrospray/Mass Spectrometry, (9 pages) ETS-8-6.0, "Extraction of Potassium Perfluorooctanesulfonate or other Fluorochemical Compounds from Liver for Analysis using HPLC-Electrospray/Mass Spectrometry", (14 pages) ETS-8-7.0, "Analysis of Potassium Perfluorooctane-sulfonate or other Fluorochemicals in Liver Extracts Using HPLC-Electrospray/Mass Spectrometry", (10 pages) Serum ETS-8-4.1, Extraction of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds from Serum for Analysis Using HPLC-Electrospray/Mass Spectrometry, (14 pages) FACT-M-3.1, Extraction of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds from Serum or Other Fluid for Analysis Using HPLCElectrospray/Mass Spectrometry, (17 pages) ETS-8-5.1, Analysis of Potassium Perfluorooctanesulfonate or Other Fluorochemicals in Serum Extracts Using HPLC-Electrospray/Mass Spectrometry, (9 pages) FACT-M-4.1, Analysis of Potassium Perfluorooctanesulfonate or Other F lu o ro ch e m ica ls in S e rum o r O th e r Fluid E xtracts U sing H P L C -E le ctro sp ra y/M a ss Spectrometry, (9 pages) Urine ETS-8-96.0, Extraction of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds from Urine for Analysis Using HPLC-Electrospray/Mass Spectrometry, (14 pages) ETS-8-97.0, Analysis of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds in Urine Extracts using HPLC-Electrospray/Mass Spectrometry/Mass Spectrometry, (10 pages) 3M Environmental Laboratory Page 21 3M Environmental Laboratory M ethod Extraction of Potassium Perfluorooctanesulfonate or Other Fluorochemical compounds from Liver for Analysis U sing HPLC-Electrospray/Mass Spectrometry S'- Method Number: FACT-M-1.1 Author: Lisa Clemen, Glenn Langenburg Approved By: Laboratory Manager Group Leader (Ot/A* A itA ^ Technical Reviewer Adoption Date: 05/26/98 Revision Date: O c Date 6/1 / M Date __________________ i l h i l 'i *} Date 0 Scope and Application ^ 1 Scope: This method is for the extraction of potassium perfluorooctanesulfonate (PFOS) or O other fluorochemical compounds from liver. Applicable Compounds: Fluorochemical surfactants or other fluorinated compounds. -- Matrices: Rabbit, rat, bovine, and monkey liver or other liver as designated in the validation 3 ' rePrt. PL Microsoft 6.0/95 FACT-M-1.1 Extraction of PFOS from Liver Page 1 of 15 2.0 Summary of M ethod 2.1 This method describes the procedure for extracting potassium perfluorooctanesulfonate (PFOS) or other fluorochemicals from liver homogenate using an ion pairing reagent and 5.0 ml of ethyl acetate. In this method, seven fluorochemicals were extracted: PFOS, PFOSA, PFOSAA, EtFOSE-OH, POAA, PFOSEA, and FC-807 monoester (see 3.0 Definitions). An ion pairing reagent is added to the sample and the analyte ion pair is partitioned into ethyl acetate. Four ml of extract are removed and put onto a nitrogen evaporator until dry. Each extract is reconstituted in 1.0 ml of methanol, then filtered through a 3 cc plastic syringe attached to a 0.2 pm nylon filter into glass autovials. 3.0 Definitions______________________________________________________________ 3.1 PFOS: perfluorooctanesulfonate (anion of potassium salt) C8F17S 0 3' 3.2 PFOSA: perfluorooctane sulfonylamide C8F17S 0 2NH2 3.3 PFOSAA: perfluorooctane sulfonylamido (ethyl)acetate C8F17S 0 2N(CH2CH3)CH2C 0 2' 3.4 EtFOSE-OH: 2(N-ethylperfluorooctane sulfonamido)-ethyl alcohol C8F17S 0 2N(CH2CH3)CH2CH20H 3.5 POAA: perfluorooctanoate (anion of ammonium salt) C7F]5COO' 3.6 PFOSEA: perfluorooctane sulfonyl ethylamide C8F17S 0 2N(CH2CH3)H 3.7 FC-807 monoester C8F17S 0 2N(CH2CH3)CH2CH20 - P 0 3H) . 3.8 Surrogate standard 1H,1H,2H,2H perfluorooctane sulfonic acid 4.0 Warnings and Cautions__________________________________________________ 4.1 Health and safety warnings: 4.1.1 Use universal precautions, especially laboratory coats, goggles, and gloves when handling animal tissue, it may contain pathogens. 5.0 Interferences____________________________________________________________ 5.1 There are no known interferences at this time. 6.0 Equipment_______________________________________________________________ 6.1 The following equipment is used while carrying out this method. Equivalent equipment is acceptable. 6.1.1 6.1.2 6.1.3 6.1.4 6.1.5 6.1.6 Ultra-Turrax with T25 grinder attachment for grinding/dispersing/emulsifying Vortex mixer, VWR, Vortex Genie 2 Centrifuge, Mistral 1000 or IEC Shaker, Eberbach or VWR Nitrogen evaporator, Organomation Balance, ( 0.100 g) FACT-M-l.l FvtrarHnn of PFOS from T.ivor Page 2 of 15 7.0 Supplies and M aterials 7.1 Gloves . 7.2 Eppendorf or disposable pipettes * 7.3 Nalgene bottles, capable of holding 250 ml and 1 L 7.4 Wheaton 6 ml Plastic Sampule Vials 7.5 Glass, type A, volumetric flasks 7.6 40 ml glass I-CHEM vials 7.7 Polypropylene centrifuge tubes, 15 ml 7.8 Labels 7.9 Syringes, capable of measuring 5 pL to 50 pL 7.10 Glass, type A, volumetric pipettes 7.11 Graduated pipettes 7.12 Electronic pipettor, Eppendorf or equivalent 7.13 Timer 7.14 Disposable plastic 3 cc syringes 7.15 Filters, nylon syringe filters, 0.2 pm, 25 mm 7.16 Crimp cap autovials Note: Prior to using glassware and bottles, rinse 3 times with methanol and 3 times with Milli- QTMwater. Rinse syringes a minimum of 9 times with methanol, 3 rinses from 3 separate vials. 8.0 Reagents and Standards__________________________________________________ 8.1 ASTM Type I reagent grade water, Milli-QTM or equivalent; all water used in this method should be Milli-QTM water and may be provided by a Milli-Q TOC PlusTM system. 8.2 Sodium hydroxide (NaOH), J.T Baker or equivalent 8.3 Tetrabutylam m onium hydrogen sulfate (TB A ), Kodak or equivalent 8.4 Sodium carbonate (Na2C03), J.T. Baker or equivalent 8.5 Sodium bicarbonate (NaHC03), J.T. Baker or equivalent 8.6 Ethyl acetate, Omnisolv, glass distilled or HPLC grade 8.7 Methanol, Omnisolv, glass distilled or HPLC grade 8.8 Liver tissue, frozen from supplier 8.9 Control matrix or blank matrix for standards, QC checks, blanks, etc. 8.10 Fluorochemical standards 8.10.1 PFOS (3M Specialty Chemical Division), molecular weight = 538 8.10.2 PFOSA (3M Specialty Chemical Division), molecular weight = 499 8.10.3 PFOSAA (3M Specialty Chemical Division), molecular weight = 585 F A C T -M -l.l Extraction of PFOS from Liver Page 3 of 15 8.10.4 EtFOSE-OH (3M Specialty Chemical Division), molecular weight = 571 8.10.5 POAA (3M Specialty Chemical Division), molecular weight = 431 8.10.6 PFOSEA (3M Specialty Chemical Division), molecular weight = 527 8.10.7 FC-807 monoester (3M Specialty Chemical Division). FC-807 is a mixture of triester, diester, and monoester fluorochemical components. The monoester molecular weight = 650 8.10.8 Surrogate Standard: 4-H, perfluorooctane sulfonic acid (1-H,1-H, 2-H,2-H C8F,3S 0 3H) molecular weight = 428 8.10.9 Other fluorochemicals, as appropriate 8.11 Reagent preparation 8.11.1 10 N sodium hydroxide (NaOH): Weigh approximately 200g NaOH. Pour into a 1000 ml beaker containing 500 ml Milli-QTM water, mix until all solids are dissolved. Store in a 1 L Nalgene bottle. 8.11.2 1 N sodium hydroxide (NaOH): Dilute 10N NaOH 1:10. Measure 10 ml of 10N NaOH solution into a 100 ml volumetric flask and dilute to volume using Milli-QTM water. Store in a 125 ml Nalgene bottle. 8.11.3 0.5 M tetrabutylammonium hydrogen sulfate (TBA): Weigh approximately 169 grams of TBA into a 1 L volumetric containing 500 ml Milli-QTM water. Adjust to pH 10 using approximately 44 to 54 ml of 10N NaOH and dilute to volume with Milli-QTM water. While adding the last few ml's of NaOH, add slowly because the pH changes abruptly. Store in a 1 L Nalgene bottle. 8.11.3.1 TBA requires a check prior to each use to ensure pH = 10. Adjust as needed using IN NaOH solution. 8.11.4 0.25M Sodium carbonate/sodium bicarbonate buffer (Na2C0 3/NaHC0 3 ): Weigh approximately 26.5 g of sodium carbonate (NajCOj) and 21.0 g of sodium b icarb o n ate (N a H C 0 3) into a 1 L volum etric flask and b rin g to v o lu m e w ith M illi- QTM water. Store in a 1 L nalgene bottle. 8.12 Standards 8.12.1 Prepare PFOS standards for the standard curve. 8.12.2 Prepare other fluorochemical standards, as appropriate. Multicomponent fluorochemical standards are acceptable (e.g. one working standard solution containing 1.00 ppm PFOS, 1.02 ppm PFOSA, 0.987 ppm PFOSAA, and 1.10 ppm EtFOSE-OH.) 8.12.3 Weigh approximately 100 mg of PFOS into a 100 ml volumetric flask and record the actual weight. 8.12.4 Bring to volume with methanol for a stock standard of approximately 1000 ppm (pg/ml). 8.12.5 Dilute the stock solution with methanol for a working standard 1 solution of approximately 50 ppm. FACT-M-1.1 Extraction of PFOS from Liver Page 4 of 15 8.12.6 Dilute the stock solution with methanol for a working standard 2 solution of approx. 5.0 ppm. 8.12.7 Dilute the stock solution with methanol for a working standard 3 solution of approx. 0.50 ppm. 8.13 Surrogate stock standard preparation 8.13.1 Prepare a surrogate stock standard. Weigh approximately 50-60 mg of surrogate standard 1-H,1-H, 2-H,2-H, C8F13S 0 3H into a 50 ml volumetric flask and record the actual weight. 8.13.2 Bring to volume with methanol for a surrogate stock of approximately 1000-1200 ppm. 8.13.3 Prepare a surrogate working standard. Transfer approximately 0.5 ml of surrogate stock to a 50 ml volumetric flask and bring to volume with methanol for a working standard of 10-20 ppm. Record the actual volume transferred. 8.14 Liver homogenate preparation Note: Thefollowing procedure w ill be much easier to perform with frozen liver tissue. Prevent tissuefrom thawing; keep stored on ice until excising a portion o f 8.14.1 it. Weigh 40 g of blank or control liver into a 250 ml Nalgene bottle containing 100 mis Milli-QTM water. Record the actual weight of liver and total volume of water used. Grind the liver into a finely dispersed homogenate with an Ultra-Turrax T25 grinder (high speed for approximately 3 minutes or until sufficiently homogenized). Rinse grinder with an additional 100 ml of MilliQTM water, to bring the total volume of water added to 200 ml. 8.14.2 To determine the concentration of the blank liver homogenate, transfer ten 1.0 ml aliquots of the homogenate to tared polypropylene tubes, and weigh each aliquot on a balance. The average density of these aliquots is determined and then the concentration (g o f liver/ml o f hom ogenate) can be calculated as follows: 8.14.3 fgrams (g) of liver! x lave, weight of 1.0 ml of homogenate (density) (g/mPl {[grams (g) of liver] + [grams (g) of water]} 8.14.3 Prepare sample livers as described in 8.3.1, but weigh out 1 g of liver, homogenize with 2.5 ml of MilliQTM water, and rinse with another 2.5 ml of MilliQTM water. Use Wheaton 6 ml plastic sampule vials or appropriate receptacle. Rinse grinder unit after every sample with water and then with methanol. Label vials appropriately including study number, sample ID, liver weight, date, and analyst. Record all weights and volumes used. (Do not perform 8.3.2 fo r the sample liver homogenates). FACT-M-1.1 Extraction of PFOS from Liver Page 5 of 15 9.0 Sample Handling_______________________________________________________________ 9.1 All livers are received frozen and must be kept frozen until the extraction is performed. ` 10.0 Quality Control______________________________________________________________ 10.1 Matrix blanks and method blanks 10.1.1 Extract two 1.0 ml aliquots of the liver homogenate (prepared in 8.14.1-2) following this procedure and use as matrix blanks. See Section 11.1.2. 10.1.2 Extract two 1.0 ml aliquots of Milli-QTM water following this procedure and use as method blanks. 10.2 Matrix spikes 10.2.1 Prepare and analyze matrix spike and matrix spike duplicate samples to determine the accuracy of the extraction. 10.2.2 Prepare each spike using liver chosen by the analyst, usually the control liver received with each sample set. 10.2.3 Expected concentrations fall in the mid-range of the initial calibration curve. Additional spikes may be included and may fall in the low-range of the initial calibration curve. . 10.2.4 Prepare one matrix spike and one matrix spike duplicate per 40 samples, with a minimum of 2 matrix spikes per batch. 10.3 Continuing calibration checks 10.3.1 Prepare and analyze continuing calibration check samples to ensure the accuracy of the initial calibration curve. If the percent difference between the initial curve and the continuing check differ by >30%, reanalyze samples analyzed after the last acceptable check. 10.3.2 Prepare one check per group of ten samples. For example, if a sample set = 34, prepare and extract four checks. 10.3.3 Prepare each continuing calibration check from the same blank liver homogenate used to prepare the initial curve. 10.3.4 The expected concentrations fall within the mid-range of the initial calibration curve. Additional spikes may be included that fall in the low-range of the initial calibration curve. This is necessary if the analyst must quantitate using only the low end of the calibration curve (e.g. 10 ppb - 100 ppb, rather than 10 ppb - 1000 ppb). 11.0 Calibration and Standardization_____________________________________________ 11.1 Prepare liver homogenate standards 11.1.1 Transfer 1 ml aliquots of blank/control liver homogenate prepared in 8.14.1-2 to 15 ml centrifuge tubes. FACT-M-1.1 Extraction of PFOS from Liver Page 6 of 15 11.1.2 If the volumes of sample liver homogenates are limited, extract standards with liver homogenate volumes equal to the sample volumes. Do not extract below 0.50 ml of liver homogenate. Record the sample volume on the extraction sheet. 11.1.3 While preparing a total of twenty aliquots of liver homogenate in 15 ml centrifuge tubes, mix or shake between aliquots. 11.1.4 Two 1 ml, or appropriate aliquots, serve as matrix blanks. Typically use the standard concentrations and spiking amounts listed in Table 1 (at the end of this section) to spike, in duplicate, two standard curves, for a total of eighteen standards and two matrix blanks. 11.1.5 Refer to the validation reports FACT-M-l.l-V-1 and FACT-M-2.1-V-1 which list the working ranges and Linear Calibration Range (LCR) for calibration curves. 11.1.6 Use Attachment D as an aid in calculating the concentrations of the working standards. See Section 13.0 to calculate actual concentrations of PFOS in calibration standards. 11.2 To each standard, blank, or QC check, add appropriate amount of surrogate working standard for the concentration to fall within the calibration curve range 10 ppb - 1000 ppb. 11.3 Extract spiked liver homogenate standards following 12.6-12.16 of this method. Use these standards to establish each initial curve on the mass spectrometer. Table 1 Approximate Spiking Amounts for Standards and Spikes Using 1.0 ml of Liver Working Standard pL Approx, final cone, of (Approx. Cone.) PFOS in liver - - Blank 0.500 ppm 4 0.012 ppm 0.500 ppm 10 0.030 ppm 0.500 ppm 20 0.060 ppm 0.500 ppm 40 0.120 ppm 5.00 ppm 10 0.300 ppm 5.00 ppm 20 0.600 ppm 5.00 ppm 30 0.900 ppm 50.0 ppm 4 1.20 ppm 50.0 ppm 6 1.80 ppm 12.0 Procedures____________________________________________________________________ 12.1 Obtain frozen liver samples and homogenize as described in 8.14.3. 12.2 Vortex mix homogenate for 15 seconds, then transfer 1.0 ml or other appropriate volume to a 15 ml polypropylene centrifuge tube. 12.3 Return liver homogenate samples to freezer after extraction amount has been removed. FACT-M-l.l P Ytrartirvn n f P P O f m m T ivfr Page 7 of 15 12.4 Record the liver homogenate volume on the extraction worksheet. The final methanol volume will equal the initial homogenate volume. For example, if 1 ml of homogenate is transferred for extraction, then the final reconstitution methanol volume equals 1 ml. 12.5 Label the tube with the study number, liver ID, date, and analyst initials. See attached worksheet for documenting the remaining steps. 12.6 Spike blank liver homogenate aliquots with the appropriate amount of standard as described in Section 11.1 or Table I in that section for the calibration curve standards. Also prepare matrix spikes and continuing calibration standards. 12.7 Spike all samples, including blanks and standards, ready for extraction with surrogate standard as described in Section 11.2. 12.8 Vortex mix the standard curve samples, matrix spike samples, and continuing calibration samples for 15 seconds. 12.9 To each sample, add 1 ml 0.5 M TBA and 2 ml of the 0.25 M sodium carbonate/sodium bicarbonate buffer. 12.10 Using a volumetric pipette, add 5 ml ethyl acetate. 12.11 Cap each sample and put on the shaker for 20 minutes. 12.12 Centrifuge for 20 to 25 minutes at approximately 3500 rpm, until layers are well separated. 12.13 Transfer 4 ml of organic layer, using a 5 ml graduated glass pipette, to a clean 15 ml centrifuge tube. Label this fresh tube with the same information as in 12.5. 12.14 Put each sample on the analytical nitrogen evaporator until dry, approximately 2 to 3 hours. 12.15 Add 1.0 ml or appropriate volume of methanol to each centrifuge tube using a graduated pipette. Methanol volume equals the initial volume of liver homogenate used for the extraction. 12.16 Vortex mix for 30 seconds. 12.17 Attach a 0.2 pm nylon mesh filter to a 3 cc syringe and transfer the sample to this syringe. Filter into a 1.5 ml glass autovial (or low-volume autovial when necessary). 12.18 Label the autovial with the study number, animal number and gender, sample timepoint, matrix, final solvent, extraction date, and analyst(s) who performed the extraction. 12.19 Cap and store extracts at approximately 4 C until analysis. 12.20 Complete the extraction worksheet, attached to this document, and tape to page of study notebook or include in study binder, as appropriate. 13.0 Data Analysis and Calculations__________________________________________ 13.1 Calculations: 13.1.1 Calculate actual concentrations of PFOS, or other appropriate fluorochemical, in calibration standards using the following equation: FACT-M-1.1 p Y tr a r tin n rvf P F O S frrvm T iv p r Page 8 of 15 ml of Standard x Concentration of Standard ( ug/ml)_____ _ Concentration of Blank Liver Homogenate (g/ml) (see 8.14.2) Final Concentration (fo.g/g) of PFOS in Liver See Attachment D for a sample form to calculate the concentrations of standards. 14.0 M ethod Performance_________________________________________________________ 14.1 The method detection limit (MDL) is analyte and matrix specific. Refer to MDL report for specific MDL and limit of quantitation (LOQ) values (see Attachments B and C). 14.2 The following quality control samples are extracted with each batch of samples to evaluate the quality of the extraction and analysis. 14.2.1 Method blanks and matrix blanks 14.2.2 Matrix spike and matrix spike duplicate samples to determine accuracy and precision of the extraction 14.2.3 Continuing calibration check samples to determine the continued accuracy of the initial calibration curve. 15.0 Pollution Prevention and W aste M anagement________________________________ 15.1 Sample waste is disposed in biohazard containers, flammable solvent waste is disposed in high BTU containers, and used glass pipette waste is disposed in broken glass containers located in the laboratory. 16.0 Records_________________________________________________________________ 16.1 Complete the extraction worksheet attached to this method, and tape into the study notebook or include into study binder, as appropriate. 17.0 Attachments__________________________________________________________________ 17.1 Attachment A, Extraction worksheet 17.2 Attachment B, MDL/LOQ values 17.3 Attachment C, LOQ summary 17.4 Attachment D, Calibration standard concentration worksheet 18.0 References______________________________________________________________ 18.1 The validation reports associated with this method are FACT-M-1.1 and 2.1-V-l. 19.0 Affected Documents__________________________________________________________ 19.1 FACT-M-2.1, "Analysis of Liver Extracts for Fluorochemicals using HPLC-Electrospray Mass Spectrometry" FACT-M-1.1 Fvfrartinn o f PFOS from Tiver Page 9 of 15 20.0 Revisions Revision Number. 1 Reason For Revision Validation o f method to include 7fluo rochemicals, new API/MS(MS) systems, monkey liver cross validation, improvements to ion pairing extraction, M D L study, updates in record keeping and storing policies, etc. Revision Date 08/01/98 FACT-M-1.1 pYtrartirvn r\fP F H 5 frrvm T iv#r Page 10 of 15 Study# Matrix Box# Analyst/Date H,0 Blank Blank Surrogate Std. approx, ppm actual ppm #W FC Mix Std approx. 0.5 ppm actual ppm #W FC Mix Std approx. 5 ppm actual ppm #W FC Mix Std approx. 50 ppm actual ppm #W Comments Blank____________ Std #________________ amount =______________________________________________ Extraction Method/Revision:________________________________________________________Pate & Initials Add Surrogate, Vortex 15 sec.____________________________________________________________________ Pipette Sample_______________________________ Volume_____________ ml___________________________ Pipette 1ml of 0.5 M TBA, pH 10. PH= Std. # Pipette 2 ml of 0.25 Na2CO^/Q.25MNaHCO^ buffer Std. # Pipette 5 ml of ethyl acetate_________________________TN-A- _________________________ Shake 20 min._________________________________ Shaker Speed____________________________________ Centrifuge 20-25 min.____________________________ Centrifuge speed:__________________________________ Remove a4 ml aliquot of organic layer______________________________________________________________ Put onNitrogen Evaporator to dryness________________ Temperature:______________________________________ Add methanol__________ Volume ml________ TN-A- _____________________ Vortex 30 sec._______________________________________________________________________________ Filter using a 3cc B-D syringe with a 0.2um SRI filter into a 1.5 ml autosample vial_________________________________ MS/MSD/__ Cont. Checks: Spiked_____uL of a _____ppm std (____________ ) for a final concentration of _______ ppm. MS/MSD used sample_____________ . Cont. Checks used same matrix as for std curve. Attachment A: Extraction worksheet FACT-M-1.1 E xtraction o f PFO S from T.ivcr Page 11 o f 15 MDL/LOQ values for Rabbit Liver: Compound MDL LOQ Linear Calibration Range (LCR) (PPb) (PPb) Approximate Concentrations to be used for preparing the Standard Calibration Curve PFOS 11.8 37.4 38ppb- 1000 ppb PFOSA 6.06 19.3 20 ppb - 1200 ppb PFOSAA 55.7 177 180 ppb - 1000 ppb EtFOSE-OH 58.7 187 190 ppb - 1800 ppb POAA 23.7 75.5 76 ppb - 1800 ppb PFOSEA Monoester 16.2 51.7 62 ppb - 1200 ppb n/v n/v Monoester was not detectable/quantifiable at the spiked concentrations. MDL/LOQ values for Rat Liver: Compound MDL LOQ Linear Calibration Range (LCR) (PPb) (PPb) Approximate Concentrations to be used for preparing the Standard Calibration Curve PFOS 24.7 78.7 62 ppb - 1200 ppb PFOSA 20.7 65.8 20 ppb - 1200 ppb PFOSAA n/v n/v 62 ppb - 1200 ppb EtFOSE-OH n/v n/v 120 ppb - 1200 ppb POAA n/v n/v 62 ppb - 1200 ppb PFOSEA n/v n/v 120 ppb - 1200 ppb Monoester n/v n/v Monoester was not detectable/quantifiable at the spiked concentrations. MDL/LOQ values for Monkey Liver: Compound MDL LOQ Linear Calibration Range (LCR) (PPb) (PPb) Approximate Concentrations to be used for preparing the Standard Calibration Curve PFOS n/v n/v 59 ppb - 1200 ppb PFOSA 27.4 87.1 28 ppb - 1200 ppb PFOSAA n/v n/v 120 ppb - 1200 ppb EtFOSE-OH n/v n/v 58 ppb - 1200 ppb POAA n/v n/v 120 ppb - 1200 ppb PFOSEA n/v n/v 120 ppb - 1200 ppb Monoester n/v n/v Monoester was not detectable/quantifiable at the spiked concentrations. n/v = Not valid. Upon analyzing the data, value did not pass the criteria set for this characterization. Until further analysis is completed, use the LCR to determine the range of standard concentrations for calibration curve preparation. Attachment B: MDL/LOQ Values FACT-M-1.1 Extraction of PFOS from Liver Page 12 o f 15 Summary Table: Limits of Quantitation Compound Matrix MDL LOQ PFOS Rabbit Bovine 11.8 ppb 37.4 ppb n/d = not determined2 Approximate Linear Range1 Low Standard 38 ppb 60 ppb High Standard 1000 ppb 1200 ppb Rat Monkey 24.7 ppb 78.7 ppb n/v = not valid3 62 ppb 59 ppb 1200 ppb 1200 ppb PFOSA Rabbit 6.06 ppb 19.3 ppb 20 ppb 1200 ppb Bovine n/d n/d 30 ppb 1200 ppb Rat 20.7 ppb 65.8 ppb 6 ppb 1200 ppb Monkey 27.4 ppb 87.1 ppb 6 ppb 1200 ppb PFOSAA Rabbit 55.7 ppb 177 ppb 180 ppb 1900 ppb Bovine n/d n/d 120 ppb 1200 ppb Rat n/v n/v 62 ppb 1200 ppb Monkey n/v n/v 120 ppb 1200 ppb EtFOSE-OH Rabbit 58.7 ppb 187 ppb 190 ppb 1800 ppb Bovine n/d n/d 120 ppb 1200 ppb Rat n/v n/v 120 ppb 1200 ppb Monkey n/v n/v 58 ppb 1200 ppb POAA Rabbit 23.7 ppb 75.5 ppb 76 ppb 1800 ppb Bovine n/d n/d 120 ppb 1200 ppb Rat n/v n/v 62 ppb 1200 ppb Monkey n/v n/v 120 ppb 1200 ppb PFOSEA Rabbit 16.2 ppb 51.7 ppb 62 ppb 1200 ppb Bovine n/d n/d 30 ppb 1200 ppb Rat n/v n/v 120 ppb 1200 ppb Monkey n/v n/v 120 ppb 1200 ppb Monoester Rabbit n/d n/d n/a n/a B o v in e n/d n/d n/a n/a Rat n/d n/d n/a n/a Monkey n/d n/d n/a n/a 1 - Upper Limit chosen where the value was within the Linear Calibration Range (LCR) but did not excessivelyweight the standard curve or affect Repeatability & Reproducibility values. 2 - Not determined refers to no sample was analyzed for this data. 3 - Not valid refers to data fromthe analysis failed to meet specific criteria for a valid MDL/LOQ determination. _____ ________________________ Compound Liver Matrix Rabbit Bovine Rat Monkey Prepared Range of Standards (PPb)(ng/g) 5.95 -1790 6.00- 1200 6.22- 1240 5.93 - 1190 PFOS Range of Average Curve (ppb)(ng/g) 5.95 -1790 6.00- 1200 6.22- 1240 5.93 - 1190 l.('R from \\eragc Curve /, (ppb)(ng/g) 29 5-1190 60 0- 1200 62 2- 1240 59 3 - 1190 Range of Low Std. Curve (ppb)(ng/g) 5.95 -298 n/a n/a n/a LCRfrom Range of LCR rrom 1on Stdl!r High Std. High Std. . Cun'e-iv Curve = ^ , (pib)(ng/g) (ppb)(ng/g) (ppb)(ng'g) 11.9-295 119-1790 119-1790 n/a n/a a a n/a- . n/a * {*n/1a4.y* ._ n/a --- >---ii->k*i * Attachment C: LOQ summary FACT-M-1.1 Extraction cvf PFO S from L iver Page 13 of 15 Compound Liver Matrix Rabbit Bovine Rat Monkey Prepared Range of Standards (ppb)(ng/g) 6.04-1810 5.95- 1190 6.17-1240 5.88-1180 PFOSA Range of Average Curve (ppb)(ng/g) 6.04- 1210 5.95- 1190 6.17-1240 5.88-1180 LCR from Average Curve (PPb)(ng/g) (.1)4- 1210 29 S- 1190 6.17-1240 5.88-1180 Range of Low Std. Curve (PPb)(ng/g) 6.04 - 302 n/a n/a n/a LCR from Range of LCR from LowStd. High Std. High Std. Curve Curve Curve (ppb)(iig'g) (PPb)(ng/g) (ppb)(ng/g) 12 1 312 121 - 1210 302- 1210 n/a n a n .i 1 n/a . n/a n^a n/a n/a Compound Liver Matrix Rabbit Bovine Rat Monkey Prepared Range of Standards (ppb)(ng/g) 6.33 - 1900 5.99 - 1200 6.21 -1240 5.92-1180 PFOSAA Range of KRIiom Average ' Average Curve Curve Range of Low Std. Curve (PPb)(ng/g) (ppli>(ng/g) (ppb)(ng/g) 127- 1900 ! 127- 19001 n/a 120- 1200 122- 1200 n/a 62.1 - 1240 ! 62.1 - 1240 n/a 59.2- 1180 | 118-1180' n/a LI RIrani 1 Range of LCR tram . LowStd. :| High Std. High Std. Curve j Curve 1 urve (pph)(ng.g) (PPb)(ng/g) (ppb)(iig R) n/a --1 n/a IJM n/a;;";! n/a ' n/a r- :n/a - n/a ' n/a n'a ! n/a n'/a Compound Liver Matrix Rabbit Bovine Rat Monkey Prepared Range of Standards (ppb)(ng/g) 5.96- 1790 5.87-1170 6.09- 1220 5.80-1160 EtFOSE-OH Range of Average Curve (PPb)(ng/g) LCR from Range of \Vlldgt. Low Std. Curve ' Curve (Ppb)(ng/g). (PPb)(ng/g) 119-1790 119-1790 n/a 58.7- 1170 117-1170 n/a 122- 1220 122 - 1220 n/a 29.4-1160 58 0- 1160 n/a LCR fioin Range of LCR from I ow Std. High Std. High Std. . Curve' , Curve urve;f (ppb)(ng/g). (PPb)(ng/g) (PPh)(HR/R) n/a - n/a n.j n/a'j'v* n/a l l l l i i i l l l l n/a \ ^ n/a na n/a ' n/a Compound Liver Matrix Rabbit Bovine Rat Monkey Prepared Range of Standards (ppb)(ng/g) 6.06- 1820 5.93-1190 6.15-1230 5.86-1170 POAA Range of Average Curve (ppb)(ng/g) 30.3 - 1820 59.3 - 1190 61.5- 1230 117-1170 1CKfrom Average Curve ' IPpl'Xng'g; 121 - 1821 119-119! 61.5 -:. 1-23!ts 117-1170 Range of I.CK tram Range of LCR from LowStd. Low.Stdi ^ High Std. Jtigli Std. Curve '-Curve v;' Curve Curve-'- (ppb)(ng/g) (ppb)(ng>g) (PPb)(ng/g) (ppfi^ng/g)' 30.3 - 606 . 30.3^ 606;;' 303 - 1820 303- 1S20 n/a n/a n/a liliil n/a .ir/a n/a n/a* ' n/a ,n/a n/a iiS liM iip Compound Liver Matrix Rabbit Bovine Rat Monkey Prepared Range of Standards (ppb)(ng/g) 6.20- 1860 5.92-1180 6.14-1230 5.85-1170 PFOSEA Range of Average Curve (PPb)(ng/g) 62.0- 1240 29.6- 1180 123 - 1230 58.5 - 1170 l.CRfroin Range of Average?.-. Low Std. .. Curve'l l Curve (ppb)(ng/g) (ppb)(ng/g) 62- 1240 n/a 29.6-1180- n/a 123 1230 n/a 117-1170 n/a LCRfrom Range of LowStd.-,-; High Std. ' CiirveH Curve (ppb)(ng/g) (ppb)(ng/gi (ppl n/a , n/a Bi|J| n/a n/a n/a , n/a n/a n/a n/a n/a 1 'h/a ; Monoester was not detectable/quantifiable in liver matrix for the concentration range of4.94 - 1450 ppb. Attachment C: LOQ summary FACT-M-1.1 Extraction o f PFOS from l iver Page 14 of 15 Prep Date(s): 11/2/98 Analyst(s): RWW-IAS Sample Matrix: Monkey Liver Method/Revision FACT-M-1.0 target FC-Mix Analyte(s): Ion Pair Standard Curves--Tissues Study Number: Cross Validation Equipment Number: 1 Final Solvent & TN Number: MeOH TN-A-2076 Blank Tissue/Identifier: Liver FC Mix Std Approx. 0.500 ppm: W398-1004 FC Mix Std Approx. 5.00 ppm: W398-1003 FC Mix Std Approx. 50.0 ppm: W398-1002 Surrogate Std Approx. 16.5 ppm: W398-989 Actual Concentrations of Standards in the FC Mix PFOS PFOSA PFOSAA Std Cone. Std Cone. Std Cone. ug/mL 0.501 0.501 0.501 0.501 0.501 5.01 5.01 5.01 50.1 ug/mL 0.497 0.497 0.497 0.497 0.497 4.97 4.97 4.97 49.7 ug/mL 0.500 0.500 0.500 0.500 0.500 5.00 5.00 5.00 50.0 EtFOSE Std Cone. ug/mL 0.490 0.490 0.490 0.490 0.490 4.90 4.90 4.90 49.0 POAA Std Cone. ug/mL 0.495 0.495 0.495 0.495 0.495 4.95 4.95 4.95 49.5 PFOSEA Std Cone. ug/mL 0.494 0.494 0.494 0.494 0.494 4.94 4.94 4.94 49.4 All Am't Spiked mL 0.002 0.004 0.010 0.020 0.040 0.010 0.020 0.030 0.004 All Liver cone. g/ml 0.169 0.169 0.169 0.169 0.169 0.169 0.169 0.169 0.169 Calculated Concentrations of Standards in the Sample Matrix. PFOS PFOSA PFOSAA EtFOSE Final Cone. Final Cone. Final Cone. Final Cone. ng/g ng/g ng/g ng/g 5.93 5.88 5.92 5.80 11.9 11.8 11.8 11.6 29.6 29.4 29.6 29.0 59.3 58.8 59.2 58.0 119 118 118 116 296 294 296 290 593 588 592 580 889 882 888 870 1186 1176 1183 1160 POAA Final Cone. ng/g 5.86 11.7 29.3 58.6 117 293 586 879 1172 PFOSEA Final Cone. ng/g 5.85 11.7 29.2 58.5 117 292 585 877 1169 Surrogate All Std Cone. Am't Spiked ug/mL mL 16.50 0.005 Surrogate Final Cone. ng/g 488.2 Validated Ranges --Approximate Concentrations Liver PFOS PFOSA PFOSAA EtFOSE-OH POAA PFOSEA Rabbit 40- lOOOppb 20- 1200 ppb 180- 1900 ppb 190- 1800 ppb 80- 1800 ppb 60- 1200 ppb Bovine 60- 1200ppb 30- 1200 ppb 120- 1200 ppb 120- 1200 ppb 80- 1200 ppb 30- 1200 ppb Rat 60- 1200ppb 70- 1200 ppb 60- 1200 ppb 120- 1200 ppb 60- 1200 ppb 120- 1200 ppb Monkey 60- 1200 ppb 90- 1200 ppb 120- 1200 ppb 60- 1200 ppb 120- 1200 ppb 120- 1200ppb Attachment D: Calibration standard concentration worksheet FACT-M-1.1 Extraction of PFOS from Liver Page 15 of 15 3M Environmental Laboratory M ethod Analysis of Fluorochemicals in Liver Extracts U sing H PLC -Electrospray/Mass Spectrometry Method Number: FACT-M-2.1 Author: Lisa Clemen Approved By: Adoption Date: 05/26/98 Revision Date : O b \ o i Laboratory Manager Date 7^2 Group Leader h/l f) Date (h _________________________ Technical Reviewer l I o iI k Date Dm C7Er S ? 0 Scope and Application Scope: This method is for the analysis of extracts of liver or other tissues for fluorochemical surfactants using HPLC-electrospray/mass spectrometry. % Applicable Compounds: Potassium perfluorooctanesulfonate, anionic fluorochemical surfactants, or other ionizable compounds. oS Matrices: Rabbit, rat, bovine, and monkey livers or other livers as designated i.n the *9- validation report. fa)5 j o ]___ Date Word 7.0.1/95 FACT-M-2.1 Analysis of Liver Extract Using ES/MS Page 1 o f 9 2.0 Summary of M ethod 2.1 This method describes the analysis of fluorochemical surfactants extracted from liver using HPLC-electrospray/mass spectrometry. The analysis is performed by monitoring a single ion characteristic of a particular fluorochemical, such as the potassium perfluorooctanesulfonate (PFOS) anion, M/Z= 499. Samples may also be screened to verify compound identification. 3.0 Definitions______________________________________________ _____________________ _ 3.1 Atmospheric Pressure Ionization (API): The Micromass platform systems allow for various methods of ionization by utilizing various sources, probes, and interfaces. These include but are not limited to: Electrospray Ionization (ESI), Atmospheric Pressure chemical Ionization (APcI), Thermospray, etc. The ionization process in these techniques occurs at atmospheric pressure (i.e. not under a vacuum). 3.2 Electrospray Ionization (ES, ESI): a method of ionization performed at atmospheric pressure, whereby ionization occurs through the production of tiny charged droplets in a strong electrical field. 3.3 Mass Spectrometry, Mass Spectrometer (MS), Tandem Mass Spectrometer (MS/MS): The API platforms are equipped with quadrupole mass selective detectors. Ions are selectively discriminated by mass to charge ratio (m/z) and subsequently detected. A single MS may be employed for ion detection or a series (MS/MS) for more specific fragmentation information. 3.4 Conventional vs. Z-spray probe interface: The latest models of Micromass platform systems (post 1998) utilize a "Z-spray" conformation. The spray emitted from a probe is orthogonal to the cone aperture. In the conventional conformation it is aimed directly at the cone aperture, after passing through a tortuous pathway in the counter electrode. Though the configuration is different, the methods of operation, cleaning, and maintenance are the same. However, Z-spray components and conventional components are not compatible with one another, but only w ith sim ilar systems (i.e. Z-spray components are com patible w ith other Z- spray systems, etc.) 3.5 Mass Lynx Software: System software designed for the specific operation of these platform systems. Currently MassLynx has Windows 95 and WindowsNT 3.1 versions. All versions are similar. For more details see the manual specific to the instrument (Micromass Platform II or Quattro II MassLynx or MassLynx NT USER'S GUIDE). 4.0 W arnings and Cautions_________________________________________________________ 4.1 Health and Safety Warnings: 4.1.1 Use caution with the voltage cables for the probe. The probe employs a voltage of approximately 5000 Volts. 4.1.2 When handling samples or solvents wear appropriate protective gloves, eyewear, and clothing. Word 7.0.1/95 FACT-M-2.1 Analysis of Liver Extract Usine ES/MS page 2 o f 9 4.2 Cautions: 4.2.1 Do not run solvent pumps above capacity of 400 bar (5800 psi). If pressure goes over 400 bar, the HP 1100 will initiate automatic shutdown. 4.2.2 Do not run solvent pumps to dryness. 5.0 Interferences__________________________________________________________ _ 5.1 To minimize interferences when analyzing samples for perfluorooctanoate(POAA), teflon should not be used for sample storage or any part of instrumentation that comes in contact with the sample or extract. 6.0 Equipment_____________________________________________________________________ 6.1 Equipment listed below may be modified in order to optimize the system. 6.1.1 Micromass Electrospray Mass Spectrometer 6.1.2 HP 1100 low pulse solvent pumping system and autosampler. 7.0 Supplies and Materials___________________________________________ ;_______ 7.1 Supplies 7.1.1 High purity grade nitrogen gas regulated to approximately 100 psi 7.1.2 HPLC column, specifics to be determined by the analyst. 7.1.3 Capped autovials or capped 15 mL centrifuge tubes. 8.0 Reagents and Standards__________________________________________________ 8.1 Reagents 8.1.1 Methanol, HPLC grade or equivalent. 8.1.2 ASTM , Type I w ater, M illi-QTM water, all w ater used in this m ethod should be M illi-Q TM water and may be provided by a M illi-Q TOC Plus system. 8.1.3 Ammonium acetate, reagent grade or equivalent. 8.2 Standards 8.2.1 Typically one method blank, one matrix blank, and ten matrix standards are prepared during the extraction procedure. See FACT-M-1.1. 9.0 Sample Handling______________________________________________________________ 9.1 Fresh matrix standards are prepared with each analysis. Extracted standards and samples are stored in capped autovials or capped 15 mL centrifuge tubes until analysis. 9.2 If analysis will be delayed, extracted standards and samples may be stored at room temperature or refrigerated at 4 C until analysis can be performed. FACT-M -2.1 Analvsis o f Liver E xtract U sine F.S/MS Page 3 of 9 10.0 Quality Control 10.1 Matrix Blanks and Method Blanks 10.1.1 Analyze a method blank and matrix blank prior to each calibration curve. 10.2 Matrix Spikes 10.2.1 Analyze a matrix spike and matrix spike duplicate with each analysis. With a minimum of 2 spikes per batch. 10.2.2 Expected concentrations will fall in the mid-range of the initial calibration curve. Additional spike concentrations may fall in the low-range of the initial calibration curve. 10.2.3 See section 13 to calculate percent recovery. 10.3 Continuing Calibration Checks 10.3.1 Analyze a mid-range calibration standard after every tenth sample. If a significant change ( 30%) in peak area occurs, relative to the initial standard curve, stop the run. Only those samples analyzed before the last acceptable calibration standard will be used. The remaining samples must be reanalyzed. 10.3.2 See section 13 to calculate percent difference. 11.0 Calibration and Standardization__________________________________________ 11.1 Analyze the extracted matrix standards prior to and following each set of extracts. The average of two standard curves will be plotted by linear regression (y = my + b), not forced through zero, using MassLynx or other suitable software. 11.2 If the curve does not meet requirements, perform routine maintenance or reextract the standard curve (if necessary) and reanalyze. 11.3 For purposes of accuracy when quantitating low levels of analyte, it may be necessary to use the low end o f the calibration curve rather than the full range o f the standard curve. Example: when attempting to quantitate approximately 10 ppb of analyte, generate a calibration curve consisting of the standards from 5 ppb to 100 ppb rather than the full range of the curve (5 ppb to 1000 ppb). This will reduce inaccuracy attributed to linear regression weighting of high concentration standards. 12.0 Procedures______________________________________________________________ 12.1 Acquisition Set up 12.1.1 Click on start button in the Acquisition Control Panel. Set up a sample list. Assign a filename using MO-DAY-last digit of year-sample number, assign a method (MS) for acquiring, and type in sample descriptions. 12.1.2 To create a method click on scan button in the Acquisition control panel and select SIR (Single Ion Recording) or MRM. Set Ionization Mode as appropriate and mass to 499 or other appropriate masses. A full scan is usually collected along with the SIRs. Save acquisition method. If MS/MS instruments are employed, additional product ion fragmentation information may be collected. See Micromass FACT-M-2.1 A n n lv sis n f T .iver Tvvtrar.t I ls in v F.S/M S Page 4 of 9 MassLynx GUIDE TO DATA ACQUISITION for additional information and MRM (Multiple Reaction Monitoring). 12.1.3 Typically the sample list begins with the first set of liver standards and ends with the second set of standards. 12.1.4 Samples are analyzed with a continuing calibration check injected after every tenth sample. Solvent blanks should be analyzed periodically to monitor possible analyte carryover and are not considered samples but may be included as such. 12.2 Using the Autosampler 12.2.1 Set up sample tray according to the sample list prepared in section 12.1.1, 12.2.2 Set-up the HP1100/autosampler at the following conditions or at conditions the analyst considers appropriate for optimal response. Record actual conditions in the instrument logbook: 12.2.2.1 Sample size = 10 pL injection with a sample wash 12.2.2.2 Inject/sample = 1 12.2.2.3 Cycle time =13.5 minutes 12.2.2.4 Solvent ramp = Time 0.00 min. 8.0 min. 11.0 min. 12.0 min. MeOH 40% 90% 90% 40% 2.0 mM Ammonium acetate 60% 10% 10% 60% 12.2.2.5 Press the "Start" button. 12.3 Instrument Sep-up 12.3.1 Refer to ETS-9-24.0 for more details . 12.3.2 Check the solvent level in reservoirs and refill if necessary. 12.3.3 Check the stainless steel capillary at the end of the probe. Use an eye piece to check the tip. The tip should be flat with no jagged edges. If the tip is found to be unsatisfactory, disassemble the probe and replace the stainless steel capillary. 12.3.4 Set HPLC pump to "On". Set the flow to 10 - 500 uL/min or as appropriate. Observe droplets coming out of the tip of the probe. Allow to equilibrate for approximately 10 minutes. 12.3.5 Turn on the nitrogen. A fine mist should be expelled with no nitrogen leaking around the tip of the probe. Readjust the tip of the probe if no mist is observed. 12.3.6 The instrument uses these parameters at the following settings. These settings may change in order to optimize the response: FACT-M-2.1 Analysis of Liver Extract Usine ES/MS Page 5 of 9 12.3.6.1 Drying gas 250-400 liters/hour 12.3.6.2 ESI nebulizing gas 10-15 liters/hour 12.3.6.3 LC constant flow mode flow rate 10 - 500 uL/min 12.3.6.4 Pressure <400 bar (This parameter is not set, it is a guide to ensure the instrument is operating correctly.) 12.3.7 Carefully guide the probe into the opening. Insert probe until it will not go any further. Connect the voltage cables to the probe. 12.3.8 Print the tune page, with its parameters, and store it in the study binder with a copy taped into the instrument log. 12.3.9 Using the cross-flow counter electrode in the ES/MS source is recommended for the analysis of biological matrices. 12.3.10 Click on start button in the Acquisition Control Panel. Press the start button at top of sample list. Ensure start and end sample number includes all samples to be analyzed. 13.0 Data Analysis and Calculations___________________;_______________________ 13.1 Calculations: 13.1.1 Calculate matrix spike percent recoveries using the following equation: % Recovery = Observed Result - Background Result x 100 Expected Result 13.1.2 Calculate percent difference using the following equation: % Difference = Expected Cone. - Calculated Cone, x 100 Expected Cone. 13.1.3 Calculate actual concentration of PFOS anion in total liver (mg): . r ug PFOS anion calc, from std curve' v g of liver used for analysis ) m , , ---------------------------------------------- x Total mass of liver (g) 1000ug/l mg 14.0 Method Performance____________________________________________________ 14.1 Method Detection Limit (MDL) and Limit of Quantitation (LOQ) are method, analyte, and matrix specific. Please see ETS-8-1.1, Attachment B, for a listing of current validated MDL and LOQ values. 14.1 14.2 Solvent Blanks, Method Blanks, and Matrix Blanks 14.1.1 Solvent blanks, method blanks, and matrix blanks values are must be below the lowest standard in the calibration curve. 14.2 Calibration Curves 14.2.1 The r2value for the calibration curve must be 0.980 or better. . FACT-M-2.1 Analvsis o f Liver Extract U sine ES/MS Page 6 of 9 14.3 Matrix Spikes 14.3.1 Matrix spike percent recoveries are must be within 30% of the spiked concentration. 14.4 Continuing Calibration Verifications 14.4.1 Continuing calibration verification percent recoveries must be 30% of the spiked concentration. 14.5 If criteria listed in this method performance section isn't met, maintenance may be performed on the system and samples reanalyzed or other actions as determined by the analyst. Document all actions in the appropriate logbook. 14.6 If data are to be reported when performance criteria have not been met, the data must be footnoted on tables and discussed in the text of the report. 15.0 Pollution Prevention and Waste Management_____________________________ 15.1 Sample waste is disposed in biohazard containers, flammable solvent waste is disposed in high BTU containers, and glass pipette waste is disposed in broken glass containers. All containers are located in the laboratory. 16.0 Records_________________________________________________________________ 16.1 Each page generated for a study must have the following information included either in the header or hand written on the page: study or project number, acquisition method, integration method, sample name, extraction date, dilution factor (if applicable), and analyst. 16.2 Print the tune page, sample list, and acquisition method from MassLynx to include in the appropriate study folder. Copy these pages and tape into the instrument runlog. 16.3 Plot the calibration curve by linear regression, weighted 1/x, then print these graphs and store in the study folder. 16.4 Print data integration summary, integration method, and chromatograms, from MassLynx, and store in the study folder. 16.5 Summarize data using suitable software (Excel 5.0) and store in the study folder, see Attachment A for an example of a summary spreadsheet. 16.6 Back up electronic data to appropriate medium. Record in study notebook the file name and location of backup electronic data. 17.0 Tables. Diagrams. Flowcharts, and Validation Data_______________________ 17.1 Attachment A: FACT-M-2.1 Data reporting spreadsheet FACT-M-2.1 Analysis of Liver Extract Using ES/MS Page 7 of 9 18.0 References 18.1 FACT-M-1.1, "Extraction of Potassium Perfluorooctanesulfonate or Other Fluorochemical compounds from Serum for Analysis Using HPLC-Electrospray/Mass Spectrometry 18.2 ETS-9-24.0, "Operation and Maintenance of the Micromass Atmospheric Pressure Ionization/Mass Spectrometer Quattro II triple quadrupole Systems" 18.3 The validation report associated with this method is FACT-M-l.l-V & 2.1-V-l.. 19.0 Affected Documents_____________________________________________________ 19.1 FACT-M-1.1, "Extraction of Potassium Perfluorooctanesulfonate from Liver for Analysis Using HPLC-Electrospray/Mass Spectrometry" 20.0 Revisions Revision Number. 1 Reason For Revision Section 6.1.2 Clarification of HP1100 system components. Section 11.1 Average of two curves, not standard values, are used for plotting linear regression. Section 12.2.2.4 Clarification of solvent ramp. Section 17.1 Changed from attachment B to A. Revision Date 05/04/99 FACT-M-2.1 Analysis of Liver Extract Using ES/MS Page 8 of 9 Laboratory Study # Study: Test Material: Matrix/Final Solvent: Method/Revision: Analytical Equipment System Number: Instrument Software/Version: Filename: R-Squared Value: Slope: Y Intercept: Date of Extraction/Analyst: Date of Analysis/Analyst: Group Dose Sample# Concentration ug/mL Initial Vol. mL Dilution Factor Final Cone. ug/mL Slope: Taken from linear regression equation. Group/Dose: Taken from the study folder. Sample#: Taken from the study folder. Concentration (ug/mL): Taken from the MassLynx integration summary. Initial Volume (mL): Taken from the study folder. Dilution Factor: Taken from the study folder. Final Cone. (ug/mL): Calculated by dividing the initial volume from the concentration Attachment A: Data Sheet FACT-M-2.0 A nalvsis n f T.iver E xtract U sino F.S/MS Page 9 of 9 3M Environmental Laboratory M ethod E xtraction of P otassium P erfluorooctanesulfonate or other F lu o ro ch em ica l C ompounds from Liv er fo r A nalysis using H P L C - E l e c ir o s p r a y /M ass Spe c t r o m e t r y Method Number: ETS-8-6.0 Author: Lisa Clemen, Robert Wynne Approved By: 0 /) 7 s -- Laboratory Manager u ________________ Adoption Date: T L 1'l Revision Date: " V 2 . - ^ h <7 Date Group Leader Date A _______ :____________________ Technical Reviewer O l/w h 'l Date Exact Copy of Grioinal 7^ - - - - - - - - - - - - - - - - Initial Date 1.0 Scope and Application 1.1 Scope: This method is for the extraction of potassium perfluorooctanesulfonate (PFOS) or other fluorochemical compounds from liver. 1.2 Applicable Compounds: Fluorochemical surfactants or other fluorinated compounds. 1.3 Matrices: Rabbit, rat, bovine, and monkey livers or other tissues as designated in the validation report. Word 6.0/95 ETS-8-6.0 Extraction of PFOS from Liver Page 1 of 14 2.0 Summary of Method 2.1 This method describes the procedure for extracting potassium perfluorooctanesulfonate (PFOS) or other fluorochemical surfactants from liver, or other tissues, using an ion pairing reagent and methyl-terf-butyl ether (MtBE). In this method, seven fluorochemicals can be extracted: PFOS, PFOSA, PFOSAA, EtFOSE-OH,.PFOSEA, M556, and surrogate standard. An ion pairing reagent is added to the sample and the analyte ion pair is partitioned into MtBE. The MtBE extract is transferred to a centrifuge tube and put onto a nitrogen evaporator until dry. Each extract is reconstituted in 1.0 mL methanol then filtered through a 3 cc plastic syringe attached to a 0.2 pm nylon filter into glass autovials. 2.2 These sample extracts are analyzed following method ETS-8-7.0 or other appropriate methods. 3.0 Definitions________________;______________________________ _________ ___________ 3.1 PFOS:perfluorooctanesulfonate (anion of potassium salt) C8F17S 03 3.2 PFOSA: perfluorooctane sulfonylamide C8F17S 02NH2 3.3 PFOSAA: perfluorooctane sulfonylamido (ethyl)acetate C8FI7S 02N(CH2CH3)CH2C 0 2 3.4 EtFOSE-OH: 2(N-ethylperfluorooctane sulfonamido)-ethyl alcohol C,F17S 0 2N(CH2CH3)CH2CH20H 3.5 PFOSEA: perfluorooctane sulfonyl ethylamide C8F17S 02N(CH2CH3)H 3.6 M556: C8F17S 02N(H)(CH2C 00H ) 3.7 Surrogate standard: 1H-1H-2H-2H perfluorooctane sulfonic acid 4.0 Warnings and Cautions__________________________________ :___________ ' 4.1 H ealth and Safety 'Warnings: 4.1.1 Use universal precautions, especially laboratory coats, goggles, and gloves when handling animal tissue, which may contain pathogens. . 5.0 Interferences_______________________________________________________________ 5.1 There are no interferences known at this time. ' 6.0 Equipment______________________ _________________ !__________________ ;________ 6.1 The following equipment is used while performing this method. Equivalent equipment is acceptable. 6.1.1 Ultra-Turrax T25 Grinder for grinding liver samples 6.1.2 Vortex mixer, VWR, Vortex Genie 2 6.1.3 Centrifuge, Mistral 1000 or IEC 6.1.4 Shaker, Eberbach or VWR ETS-8-6.0 Extraction of PFOS from Liver Page 2 of 14 6.1.5 Nitrogen Evaporator, Organomation 6.1.6 Balance (sensitivity to 0.100 g) 7.0 Supplies and Materials___________________________________ _________ ___________ 7.1 Gloves 7.2 Dissecting scalpels 7.3 Eppendorf or disposable pipettes 7.4 Nalgene bottles, capable of holding 250 mL and 1 L 7.5 Volumetric flasks, glass, type A . 7.6 I-CHEM vials, 40 mL glass 7.7 Plastic sampule vials, Wheaton, 6 mL (or appropriate size) 7.8 Centrifuge tubes, polypropylene, 15 mL 7.9 Labels 7.10 Oxford Dispensor--3.0 to 10.0 ml 7.11 Syringes, capable of measuring 5 pL to 50 pL 7.12 Graduated pipettes 7.13 Syringes, disposable plastic, 3 cc 7.14 Syringe filters, nylon, 0.2 pm, 25 mm 7.15 Timer 7.16 Crimp cap autovials and caps 7.17 Crimpers Note: Prior to using glassware and bottles, rinse 3 times with methanol and 3 times with MilliQTM water. Rinse syringes a minimum of 9 times with methanol, 3 rinses from 3 separate vials. 8.0 Reagents and Standards_____________________________________________ _ 8.1 Type I reagent grade water, Milli-QTM or equivalent; all water used in this method should be Milli-QTM water and be provided by a Milli-Q TOC PlusTM system 8.2 Sodium hydroxide (NaOH), J.T Baker or equivalent 8.3 Tetrabutylammonium hydrogen sulfate(TBA), Kodak or equivalent 8.4 Sodium carbonate (N^COj), J.T. Baker or equivalent 8.5 Sodium bicarbonate (NaHC03), J.T. Baker or equivalent 8.6 Methyl-iert-butyl ether, Omnisolv, glass distilled or HPLC grade 8.7 Methanol, Omnisolv, glass distilled or HPLC grade 8.8 Liver, frozen from supplier 8.9 Dry ice from supplier 8.10 Fluorochemical standards 8.10.1 PFOS (3M Specialty Chemical Division), molecular weight = 538 ETS-8-6.0 Extraction of PFOS from Liver Page 3 of 14 8.10.2 PFOSA (3M Specialty Chemical Division), molecular weight = 499 8.10.3 PFOSAA (3M Specialty Chemical Division), molecular weight = 585 8.10.4 EtFOSE-OH(3M Specialty Chemical Division), molecular weight = 570 8.10.5 PFOSEA (3M Specialty Chemical Division), molecular weight = 527 8.10.6 M556 (3M Specialty Chemical Division), molecular weight = 557 8.10.7 Surrogate standard: 4-H, perfluorooctane sulfonic acid (1-H,1-H, 2-H, 2-H CgF,3S03H) molecular weight = 428 8.10.8 Other fluorochemicals, as appropriate 8.11 Reagent preparation NOTE: When preparing larger volumes than listed in reagent, standard, or surrogate preparation, adjust accordingly. 8.11.1 10 N sodium hydroxide (NaOH): Weigh approximately 200 g NaOH. Pour into a 1000 mL beaker containing 500 mL Milli-QTM water, mix until all solids are . dissolved. Store in a 1 L Nalgene bottle. 8.11.2 1 N sodium hydroxide (NaOH): Dilute 10 N NaOH 1:10. Measure 10 mL of ION NaOH solution into a 100 mL volumetric flask and dilute to volume using Milli-QTM water. Store in a 125 mL Nalgene bottle. 8.11.3 0.5 M tetrabutylammonium hydrogen sulfate (TBA): Weigh approximately 169 g of TBA into a 1 L volumetric containing 500 mL Milli-QTM water. Adjust to pH 10 using approximately 44 to 54 mL of 10 N NaOH (While adding the last mL of NaOH, add slowly because the pH changes abruptly). Dilute to volume with Milli-QTM water. Store in a 1 L Nalgene bottle. 8.11.3.1 TBA requires a check prior to each use to ensure pH = 10. Adjust as needed using 1 N NaOH solution. 8.11.4 0.25 M sodium carbonate/sodium bicarbonate buffer (NajCOj/NaHCOj): Weigh approximately 26.5 g of sodium carbonate (NajCOj) and 21.0 g of sodium bicarbonate (N aH C O s) into a l L volum etric flask and bring to volum e w ith M illi- QTM water. Store in a 1 L Nalgene bottle. 8.12 Standards preparation 8.12.1 Prepare PFOS standards for the standard curve. 8.12.2 Prepare other fluorochemical standards, as appropriate. Multicomponent fluorochemical standards are acceptable (for example, one working standard solution containing 1.00 ppm PFOS, 1.02 ppm PFOSA, 0.987 ppm PFOSAA, and 1.10 ppm EtFOSE-OH.) . 8.12.3 Weigh approximately 100 mg of PFOS into a 100 mL volumetric flask and record the actual weight. 8.12.4 Bring to volume with methanol for a stock standard of approximately 1000 ppm (pg/mL). 8.12.5 Dilute the stock solution with methanol for a working standard 1 solution of approximately 50 ppm. ETS-8-6.0 Extraction of PFOS from Liver Page 4 of 14 8.12.6 Dilute the stock solution with methanol for a working standard 2 solution of approx. 5.0 ppm. 8.12.7 Dilute the stock solution with methanol for a working standard 3 solution of approx. 0.50 ppm. 8.13 Surrogate stock standard preparation 8.13.1 Weigh approximately 50-60 mg of surrogate standard l-H.l-H, 2-H, 2-H, C8F13S 03H into a 50 ml volumetric flask and record the actual weight. 8.13.2 Bring to volume with methanol for a surrogate stock of approximately 1000-1200 ppm. 8.13.3 Prepare a surrogate working standard. Transfer approximately 1.0 ml of surrogate stock to a 10 ml volumetric flask and bring to volume with methanol for a working standard of 10-20 ppm. Record the actual volume transferred. 9.0 Sample Handling____________________ ;____________________ _________ 9.1 All samples are received frozen and must be kept frozen until the extraction is performed. 10.0 Quality Control_______________________________________________________ 10.1 Matrix blanks and method blanks 10.1.1 An aliquot of 1.0 mL methanol is used as a solvent blank. 10.1.2 Extract two 1.0 mL aliquots of Milli-QTM water following this procedure and use as method blanks. 10.1.3 Extract two 1.0 mL aliquots of liver homogenate following this procedure and use as matrix blanks. Refer to 11.1.6. - 10.2 Matrix spikes 10.2.1 Prepare and analyze matrix spike and matrix spike duplicate samples to determine the accuracy o f the extraction. 10.2.2 Prepare each spike using a sample chosen by the analyst, usually a control liver received with each sample set. 10.2.3 Expected concentrations will fall in the mid-range of the initial calibration curve. Additional spikes may be included and may fall in the low-range of the initial calibration curve. 10.2.4 Prepare one matrix spike and matrix spike duplicate per 40 samples, with a minimum of 2 matrix spikes per batch. 10.3 Continuing calibration verifications 10.3.1 Prepare continuing calibration verification samples to ensure the accuracy of the initial calibration curve. 10.3.2 Prepare, at a minimum, one continuing calibration verification sample per group of 10 samples. For example, if a sample set - 34, four verifications are prepared and extracted. ' ETS-8-6.0 Extraction of PFOS from Liver Page 5 of 14 10.3.3 Prepare each continuing calibration verification from the same matrix used to prepare the initial curve. 10.3.4 The expected concentrations will fall within the mid-range of the initial calibration curve. Additional spikes may be included that fall in the low-range of the initial calibration curve. This is necessary if the analyst must quantitate using only the low end of the calibration curve (for example, 5 ppb - 100 ppb, rather than 5 ppb - 1000 ppb). 11.0 Calibration and Standardization______________________ ,____________ 11.1 Prepare matrix calibration standards 11.1.1 Weigh approximately 40 gofliver into a 250 mLNalgene bottle containing 200 mLs Milli-QTM water. Grind to a homogeneous solution. 11.1.2 If 40 g is not available, use appropriate amounts of liver and water to ensure a 1:5 ratio. 11.1.3 Refer to 13.0 to calculate the actual density of liver homogenate and the concentration of solid liver tissue dispersed in 1.0 mL of homogenate solution. 11.1.5 Add 1 mL of homogenate to a 15 mL centrifuge tube. Re-suspend solution by shaking between aliquots while preparing a total of eighteen 1 mL aliquots of homogeneous solution in 15 mL centrifuge tubes. 11.1.6 Two 1 mL aliquots, or other appropriate volume, serve as matrix blanks. 11.1.7 Typically use the standard concentrations and spiking amounts listed in Table 1, at the end of this section, to spike, in duplicate, two standard curves, for a total of eighteen samples, two matrix blanks, and two method blanks. 11.1.8 Refer to validation reports ETS-8-6.0 and ETS-8-7.0-V-1 or Attachment B, which lists the working ranges and the Linear Calibration Range (LCR) for calibration curves. 11.1.9 U se Attachm ent C as an aid in calculating the concentrations o f the working standards. Refer to 13.0 to calculate actual concentrations of PFOS in calibration standards. 11.2 To each working standard, blank, or continuing verification, add appropriate amount of surrogate working standard for the concentration to fall within the calibration curve range 5 ppb - lOOOppb. ETS-8-6.0 Extraction of PFOS from Liver Page 6 of 14 11.3 Extract spiked liver homogenates following 12.14-12.25 ofthis method. Use these standards to establish each initial curve on the mass spectrometer. Table 1 Approximate Spiking Amounts for Calibration Standards Working Standard (Approx. Cone.) 0.50 ppm 0.50 ppm 0.50 ppm 0.50 ppm 0.50 ppm 5.0 ppm 5.0 ppm 5.0 ppm 50 ppm pi '2 4 10 20 40 10 20 30 4 Approx, final cone, of PFOS in liver Blank 0.005 ppm 0.010 ppm 0.025 ppm 0.050 ppm 0.100 ppm 0.250 ppm 0.500 ppm 0.750 ppm 1.00 ppm 12.0 Procedure ________________________________ ___________________________ 12.1 Obtain frozen liver samples. 12.2 Cut approximately 1 g of liver using a dissecting scalpel. This part of the procedure is best performed quickly, not allowing the liver to thaw. 12.3 Weigh the sample directly into a tared plastic sampule vial. 12.4 Record the liver weight in'the study notebook. 12.5 Return unused liver portions to freezer. 12.6 Add 2.5 mLs of water to sampule vial. 12.7 Grind the sample. Put the grinderprobe in the sample and grind for about 2 minutes, or until the sample is homogeneous. 12.8 Rinse the probe into the sample with 2.5 mLs water using a pipette. 12.9 Take the grinder apart and clean it with methanol after each sample. Refer to AMDT-EP22. 12.10 Cap the sample and vortex for 15 seconds. Label the sampule vial with the study number, weight, liver ID, date and analyst initials. ETS-8-6.0 Extraction of PFOS from Liver Page 7 of 14 12.11 Pipette 1.0 mL, or other appropriate volume, of homogenate into a 15 mL polypropylene centrifuge tube. Label the centrifuge tube with the identical information as the sampule vial. Refer to attached worksheet for documenting the remaining steps. 12.12 Pipette two 1 mL aliquots of Milli-QTM water to centrifuge tubes. These will serve as method blanks. 12.13 Spike all samples, including blanks and standards ready for extraction with surrogate standard as described in section 11.2. 12.14 Spike each matirx with the appropriate amount of standard as described in 11.1, or Table 1 ofthat section, for the calibration curve standards. Also prepare matrix spikes and continuing calibration standards. . 12.15 Vortex mix the standard curve samples, matrix spike samples, and continuing calibration samples for 15 seconds. 12.16 Check to ensure 0.5 M TBA reagent is at pH 10. Ifnot, adjust accordingly. 12.17 To each sample, add 1 mL 0.5 M TBA and 2 mL of the 0.25 M sodium carbonate/sodium bicarbonate buffer. 12.18 Using an Oxford Dispenser, add 5 mL methyl-terf-butyl ether. 12.19 Cap each sample andput on the shaker at a setting of 300 ipm, for 20 minutes. 12.20 Centrifuge for 20 to 25 minutes at a setting of 3500 rpm, or until layers are well separated. 12.21 Label a fresh 15 mL centrifuge tube with the same information as in 12.10. 12.22 Remove 4.0 mL of the organic layer to the fresh 15 mL centrifuge tube. 12.23 Put each sample on the analytical nitrogen evaporator until dry, approximately 1 to 2 hours. 12.24 Add 1.0 mL to each centrifuge tube using a graduated pipette. 12.25 Vortex m ix for 30 seconds. 12.26 Attach a 0.2 pm nylon mesh filter to a 3 cc syringe and transfer the sample to this syringe. Filter into a 1.5 mL glass autovial or low-volume autovial when necessary. 12.27 Label the autovial with the study number, animal number and gender, sample timepoint, matrix, final solvent, extraction date, and analyst(s) performing the extraction. 12.28 Cap and store extracts at room temperature or at approximately 4 C until analysis. 12.29 Complete the extraction worksheet, attached to this document, and tape in study notebook or include in study binder, as appropriate. ETS-8-6.0 Extraction of PFOS from Liver Page 8 of 14 13.0 Data Analysis and Calculations______________________________________ 13.1 Calculations: 13.1.1 Calculate the average density of the liver homogenate by recording each mass of ten separate 1.0 mL aliquots of homogenate. Average density (mg/mL) = Average mass (mel of the aliquots 1.0 mL aliquot 13.1.2 Calculate the amount of liver (mg) per 1.0 mL homogenate (or concentration of dispersed solid tissue per mL of homogenate suspension) using the following equation: g of Liver x Average density* ofhomogenate (ms/mL'l (g of Liver + g of Water) * refer to 13.1.1 for details. 13.1.3 Calculate actual concentrations ofPFOS and other fluorochemicals in calibration standards using the following equation: uL of Standard x Concentration tug /mL) = Final Concentration (pg/g or mg/kg) mg Liver/1 mL homogenate* of PFOS in Liver *refer to 13.1.2 for details. 14.0 Method Performance__________________________________________________ 14.1 The method detection limit (MDL) is analyte and matrix specific. Refer to MDL report for specific MDL and limit of quantitation (LOQ) values (refer to Attachments B and C). 14.2 The following quality control samples are extracted with each batch o f samples to evaluate the quality of the extraction and analysis. 14.2.1 Method blanks and matrix blanks. 14.2.2 Matrix spike and matrix spike duplicate samples to determine accuracy and . precision of the extraction. 14.2.3 Continuing calibration verification samples to determine the continued accuracy of the initial calibration curve. 14.3 Refer to section 14 of ETS-8-7.0 for method performance criteria. 15.0 Pollution Prevention and Waste Management___________________________ _ 15.1 Sample waste is disposed in biohazard containers, flammable solvent waste is disposed in high BTU containers, and used glass pipette waste is disposed in broken glass containers located in the laboratory. ETS-8-6.0 Extraction of PFOS from Liver Page 9 of 14 16.0 R ecords 16.1 Complete the extraction worksheet attached to this method, and tape in the study notebook or include in the 3-ring study binder, as appropriate. 17.0 Tables, Diagrams. Flowcharts, and Validation Data 17.1 Attachment A, Extraction worksheet _________________ 17.2 Attachment B, MDL/LOQ values and summary 17.3 Attachment C, Calibration standard calculation and concentrationworksheet 18.0 References___________________________________________ _______________ . 18.1 The validation report associated with this method is ETS-8-6.0 & 7.0-V-l. 18.2 AMDT-EP-22, "Routine Maintenance of Ultra-Turrax T-25" 18.3 FACT-M-1.1, "Extraction of PFOS or Other Anionic Fluorochemical Surfactants from Liver for Analysis Using HPLC-Electrospray/Mass Spectrometry" 19.0 Affected Documents__________________________________ _ 19.1 ETS-8-7.0, "Analysis of Liver Extracts for Fluorochemicals using HPLC-Electrospray Mass Spectrometry" 20.0 Revisions Revision N um ber. _________ ;___________________ Reason For Revision Revision Date ETS-8-6.0 Extraction of PFOS from Liver Page 10 of 14 Studv # Matrix Box # Wk/Dav Date Spiked/Analyst ccv MS MSD Surrogate Std approx, ppm actual ppm # FC Mix Std approx. 0.5 ppm actual ppm # FCMix Std approx. 5 ppm actual ppm # FC Mix Std approx. 50 ppm actual ppm # Comments .-- -. - - - - - - - - - - Blank____________LiverHomogenate: Std#_____ ;_______ Liveramount- ___________ g Liver Extraction Method :____________________ '_______________________ Date & Initials Spike surrogate andStandard mix. Vortex IS sec. _ Pipette 1mLof LiverSolution Pipette 1mLoftO-5 M TBA, pH 10. pH= Std. # Pipette2 mLof0.25 Na?COy0.25M NaHCCh Buffer Std. # ~ Dispense5ml of Methyl-t-Butyl Ether_________________ TN-A- ______________________ Shake20 min. Shaker Speed Centrifuge 20-25 min. Centrifuge Speed ! Removea4 mL aliquot oforganic layer Put on Nitrogen Evaporator to dryness Evaporator Temperature _ Add 1.0mLofMethanol_____________ Vortex30sec. ........ Filterusing a 3ccB-Dsyringe with a0.2um SRI filter intoautosamplevial Cont. Cal. Verifications used the same matrix as for the standard curve. ...... ...--....... ........... - . _ Attachment B: MDL/LOQ Values ETS-8-6.0 Extraction of PFOS from Liver Page 11 of 16 MDL/LOQ values for rabbit liver Compound MDL LOQ Linear Calibration Range (LCR) (PPb) (PPb) Approximate concentrations to be used for preparing the Standard Calibration Curve PFOS 8.45 26.9 30 ppb - 1200 ppb PFOSA , 3.50 11.1 12 ppb - 1200 ppb PFOSAA 24.6 78.3 30 p p b -1200 ppb EtFOSE-OH 108 345 60 ppb - 900 ppb* M556 82.3 262 60 ppb -1 200 ppb PFOSEA 33.9 108 30 ppb- 1200 ppb MDL/LOQ values in rat, bovine, and monkey liver were not statistically determined. Two curves in each of these matrices were extracted and analyzed with the rabbit liver curves to determine equivalence. Responses in the rat, bovine, and monkey liver curves were equivalent to the rabbit responses, therefore, their MDL and LOQ will be assumed to be equivalent to those values as determined for the rabbit liver. Refer to LOQ Summary and MDL study in ETS-8-6.0 & 7.0-V-l for further information * EtFOSE-OH estimates only for MDL and LOQ. Did not meet criteria for validation. Compound: PFOS________________________ ._______ _______________ Prepared Range of . LCR'fioxfcu,,- Range of LCRfrdm f Range of Liver range of average 'ave'ui's'?; low std i. high std matrix standards curve curve 'Vvuw/ni curve (ppb) (ng/mL) (ppb) (ng/mL) (ppb)" (ng/mL)' (ppb) (ng/mL) r(ppb)--(iig/mL)i (ppb) (ng/mL) Rabbit 6.1 9 -1 23 7 12 -1200 6-300 12 - $00 60 - 1200 LOR frpmr , highstdj Curve '(ppb) (ng/nib) ; -1200/:. Compound: PFOSA Prepared Liver matrix range of standards (ppb) (ng/mL) Range of average curve (ppb) (ng/mL) Rabbit 6.19 -1237 12 - 1200 LGRfrom.:-; Range of ayecureeif low std i.--: L-.r . curve <ppb),'(n^/mL)- (ppb) (ng/mL) lL R ^n*:1. Range of : ;ddwitdj'. i high std *. * 'curva.' /. curve (ppb) (ng/mL) (ppb) (ng/mL) : LtlRfrom.' high std ' ' - curved V*, ..(ppb) .(ng/m L):: IL.'nOQi/^ 12 - 300 12-300- 60 - 1200 . '60 - 1200 : Compound: PFOSAA Prepared Range of Liver range of average matrix standards curve (ppb) (ng/mL) (ppb) (ng/mL) Rabbit 6 .1 6-1232 12 - 1200 LGR,fi;Qm. Range of avecurye low std , curve (ppb) (ng/mL) (ppb) (ng/mL) 30-1200 30-900 LGRfm Range of low stdi. ; high std curve.; curve (ppb) (ng/mL) (ppb) (ng/mL) 60-900 . N/A LCR from, high std curve (ppb) (ng/mL) N/A Attachment B: MDL/LOQ Values ETS-8-6.0 Extraction of PFOS from Liver Page 12 of 16 Compound: EtFOSE-OH Prepared Range of Liver range of average matrix standards curve (ppb) (ng/mL) (ppb) (ng/mL) Rabbit 6.17 - 1235 31 -9 0 0 LCR from ve curve (ppb) (ng/mL) 31 -9 0 0 Range of low std curve (ppb) (ng/mL) N/A LCR from low std curve (ppb) (ng/mL) N/A Range of high std curve (ppb) (ng/mL) N/A LCR from high std curve (ppb) (ng/mL) N/A Compound: PFOSEA Prepared Range o f Liver range o f average m atrix standards curve (ppb) (ng/mL) (ppb) (ng/mL) Rabbit 6.17 -1235 3 1-12 00 LCR fr o m ave curve . (ppb); (ng/mL) Range o f lo w std curve (ppb) (ng/mL) 3 1 - 1200 . N/A LCR fr o m lo w std curve (ppb). (ng/mL) Range o f high std curve (ppb) (ng/mL) N/A - ' N/A LCR fr o m high std curve (ppb) (ng/mL) N/A. ' Compound: M556 Prepared Liver range of matrix standards (P P b ) (ng/mL) Rabbit 6 .1 7 -1 2 3 5 Range of average curve (ppb) (ng/mL) 31 - 1200 LCRifrom- Range of aye,cutye::.; low std :/ "A *- curve (ppb);v(ng/n|)v. (P P b ) (ng/mL) XGRfiem- Range of LCR from low std : high std high std " r curve; : , curve V curve : .... :(ppb) ;(ng/mL) ; (ppb) (ng/mL) (ppb) (ng/toL) '60^:1200 N/A `1 ' ] N/A . ` N/A \ Attachment C: Standard Calculations ETS-8-6.0 Extraction of PFOS from Liver Page 13 of 14 Ion Pair Standard Curves - Tissue Prep date(s): Analyte(s): Sample matrix: Method/revision: Target analyte(s): FC mix std approx. 0.500 ppm: FC mix std approx. 5.00 ppm: FC mix std approx. 50.0 ppm: Surrogate std approx. 100 ppm: Standard number: Equipment number: Final solvent and TN: Blank liver/identifier: Actual concentrations of standards in the FC mix PFOS PFOSA PFOSAA EtFOSE PFOSEA Std cone Std cone Std cone Std cone Std cone ug/mL ug/mL ug/mL ug/mL ug/mL 0.500 0.500 0.500 0.500 0.500 0.500 0.500 0.500 0.500 0.500 0.500 0.500 0.500 0.500 0.500 0.500 0.500 0.500 0.500 0.500 0.500 0.500 0.500 0.500 0.500 5.00 5.00 5.00 5.00 5.00 5.00 5.00 5.00 5.00 5.00 5.00 5.00 5.00 5.00 5.00 50.0 50.0 50.0 50.0 50.0 M556 Std cone ug/mL 0.500 0.500 0.500 0.500 0.500 5.00 5.00 5.00 50.0 Std cone ug/mL All Ain't spiked mL 0.002 0.004 0.010 0.020 0.040 0.010 0.020 0.030 0.004 AU Density g 0.167 0.167 0.167 0.167 0.167 0.167 0.167 0.167 0.167 Calcnlated concentrations of standards in the sample matrix PFOS PFOSA PFOSAA EtFOSE PFOSEA M556 Final Final Final cone Final Final Final Std cone cone cone ng/g cone cone cone ng/g ng/g ng/g ng/g ng/g ng/g 5.99 5.99 5.99 5.99 5.99 5.99 12.0 12.0 12.0 12.0 12.0 12.0 29.9 29.9 29.9 29.9 29.9 29.9 59.9 59.9 59.9 59.9 59.9 59.9 120 120 120 120 120 120 299 299 299 299 299 299 599 599 599 599 599 599 898 898 898 898 898 898 1198 1198 1198 1198 1198 1198 Surrogate Std cone ng/mL 100 Surrogate Final cone ng/mL 0.500 All Am't spiked mL 0:005 Validated ranges - approximate concentrations Liver PFOS PFOSA PFOSAA Rabbit . 5-1000 ppb 5-1000 ppb 5-1000 ppb Bovine ' Estimates only, use rabbit values. Rat Estimates only, userabbit values. Monkey Estimates only, userabbit values. EtFOSE-OH 5-1000 ppb POAA 5-1000 ppb PFOSEA 5-1000 ppb Attachment C: Standard Calculations ETS-8-6.0 Extraction of PFOS from Liver Page 14 of 14 3M Environmentat, Laboratory M ethod Analysis o f P otassium P erfluorooctanesulfonate or Oth er F luorochem icals in L iver E xtracts Using H PL C -E lectrospray/Mass Spectrom etry Method Number: ETS-8-7.0 Adoption Date: O 'f/z l I n Author: Lisa Clemen, Glenn Langenburg Revision Date: f\lf\ Approved By: fM .7 V -- Laboratory Manager Group Leader --- -- ? A v/ j Date 9/W /& Date A dt/snl-i. Technical Reviewer O llrth Date 3 7^ m V/i >< 53' nv oM ) j 1.0 Scope and Application 1.1 Scope: This method is for the analysis of liver extracts for fluorochemical surfactants using HPLC-electrospray/mass spectrometry. 1.2 Applicable Compounds: Fluorochemical surfactants or other fluorinated compounds, or Tor >* other ionizable compounds. 1.3 Matrices: Rabbit, rat, bovine, monkey liver, or other tissues as designated in the validation cT '* report. -- .J ) Word 6/95 ETS-8-7.0 Page 1 of 10 Analysis of Liver Extract Using ES/MS 2.0 Summary of Method 2.1 This method describes the analysis of fluorochemical surfactants extracted from liver using HPLC-electrospray/mass spectrometry, or similar system as appropriate. The analysis is performed by monitoring a single ion characteristic of a particular fluorochemical, such as the perfluorooctanesulfonate (PFOS) anion, m/z = 499. Additionally, samples may be analyzed using a tandem mass spectrometer to further verify the identity of a compound by detecting daughter ions of the selected parent ion. 3.0 Definitions___________________________ ^________________________________________ 3.1 Atmospheric Pressure Ionization (API): The Micromass Quattro II triple quadrupole systems allow for various methods of ionization by utilizing various sources, probes, and interfaces. These include but are not limited to: Electrospray Ionization (ESI), Atmospheric Pressure chemical Ionization (APcI), Thermospray, etc. The ionization process in these techniques occurs at atmospheric pressure (i.e. not under a vacuum). 3.2 Electrospray Ionization (ES, ESI): a method of ionization performed at atmospheric pressure, whereby ions in solution are transferred to the gas phase via tiny charged droplets. These charged droplets are produced by the application of a strong electrical field. 3.3 Mass Spectrometry, Mass Spectrometer (MS), Tandem Mass Spectrometer (MS/MS): The API Quattro II triple quadrupole mass spectrometer is equipped with two quadrupole mass selective detectors and a collision cell. Ions are selectively discriminated by mass to charge ratio (m/z) and subsequently detected. A single MS may be employed for ion detection or an ion may be selected in the first quadrupole, fragmented in the collision cell, and these fragments may be analyzed in the second quadrupole. 3.4 Conventional vs. Z-spray probe interface: The latest models of Micromass Quattro II triple quadrupole (post 1998) utilize a "Z-spray" conformation. The spray emitted from a probe is orthogonal to the cone aperture. In the conventional conformation it is aimed directly at the cone aperture, after passing through a tortuous pathway in the counter electrode. Though the configuration is different, the methods of operation, cleaning, and maintenance are the same. However, Z-spray components and conventional components are not compatible with one another, but only with similar systems (i.e. Z-spray components are compatible with other Z-spray systems, etc.) 3.5 Mass Lynx Software: System software designed for the specific operation of these Quattro II triple quadrupole systems. Currently MassLynx has Windows 95 and WindowsNT 4.0 versions. All versions are similar. For more details refer to the manual specific to the instrument (Micromass Quattro 13triple quadrupole MassLynx or MassLynx NT User's Guide). 4.0 W arnings and Cautions______________________ 4.1 Health and Safety Warnings: . ______________ 4.1.1 Use caution with the voltage cables for the probe. When engaged, the probe employs a voltage of approximately 5000 Volts. ETS-8-7.0 Analysis of Liver Extract Using ES/MS Page 2 of 10 4.1.2 When handling samples or solvents wear appropriate protective gloves, eyewear, and clothing. ' 4.2 Cautions: 4.2.1 Operate the solvent pumps below a back pressure o f400 bar (5800 psi). If the back pressure exceeds 400 bar, the HP1100 will initiate automatic shutdown. 4.2.2 Do not run solvent pumps to dryness. 5.0 Interferences_________________ _____________________ _________________________ 5.1 To minimize interferences when analyzing samples, Teflon shall not be used for sample storage or any part of instrumentation that comes in contact with the sample or extract. 6.0 Equipment__________________;___________________ _____________________________ 6.1 Equipment listed below may be modified in order to optimize the system. Document any modifications in the raw data as method deviations. 6.1.1 6.1.2 Micromass Quattro E triple quadrupole Mass Spectrometer equipped with an electrospray ionization source. HP 1100 low pulse solvent pumping system, solvent degasser, column compartment, and autosampler 7.0 Supplies and Materials_______________ ;_________________ ;______________________ 7.1 Supplies 7.1.1 High purity grade air regulated to approximately 100 psi (house air system) 7.1.2 HPLC analytical column, specifics to be determined by the analyst and documented in the raw data 7.1.3 Capped autovials or capped 15 m l centrifuge tubes 8.0 Reagents and Standards 8.1 Reagents __________________________________________________ 8.1.1 Methanol, HPLC grade or equivalent . 8.1.2 Milli-QTM water (ASTM type I), all water used in this method should be ATSM type I, or equivalent, and be provided by a Milli-Q TOC Plus system or other vendor 8.1.3 Ammonium acetate, reagent grade or equivalent 8.1.3.1 When preparing different amounts than those listed, adjust accordingly. 8.1.3.2 2.0 mM ammonium acetate solution: Weigh approximately 0.300 g ammonium acetate. Pour into a 2000 mL volumetric container containing 2000 mL Milli-QTM water, mix until all solids are dissolved. Store at room temperature. ETS-8-7.0 Analysis of Liver Extract Using ES/MS Page 3 of 10 8.2 Standards 8.2.1 Typically two method blanks, two matrix blanks, and eighteen matrix standards are prepared during the extraction procedure. Refer to ETS-8-6.0. 9.0 Sample Handling________ ;_________ ___ ______________ ________________________ 9.1 Fresh matrix standards are prepared with each analysis. Extracted standards and samples are stored in capped autovials or capped 15 ml centrifuge tubes until analysis. 9.2 If analysis will be delayed, extracted standards and samples may be stored at room temperature, or refrigerated at approximately 4 C, until analysis can be performed. 10.0 Quality Control________ ;_______________ ;_______________ ;_______ ;________ 10.1 Method Blanks and Matrix Blanks 10.1.1 Solvent blanks, method blanks, and matrix blanks are prepared and analyzed with each batch to determine contamination or carryover. 10.1.2 Analyze a method blank and a matrix blank prior to each calibration curve. 10.2 Matrix Spikes 10.2.1 Matrix spikes are prepared and analyzed to determine the matrix effect on the recovery efficiency. 10.2.2 Matrix spike duplicates are prepared and analyzed to measure the precision and the recovery for each analyte. 10.2.3 Analyze a matrix spike and matrix spike duplicate per forty sample^. With a minimum of 2 spikes per batch. 10.2.4 Matrix spike and matrix spike duplicate concentrations will fall in the mid-range of the initial calibration curve. Additional spike concentrations may fall in the lowrange of the initial calibration curve. 10.3 Continuing Calibration Checks 10.3.1 Continuing calibration verifications are analyzed to verify the continued accuracy of the calibration curve. 10.3.2 Analyze a mid-range calibration standard every tenth sample, with a minimum of one per batch. 11.0 Calibration and Standardization__________________________ '___________ 11.1 Analyze the extracted matrix standards prior to and following each set of sample extracts. The average of two standard curves will be plotted by linear regression (y = mx + b), weighted 1/x, not forced through the origin, using MassLynx or other suitable software. 11.2 If the curve does not meet requirements perform routine maintenance or reextract the standard curve (if necessary) and reanalyze. ETS-8-7.0 Analysis of Liver Extract Using ES/MS Page 4 of 10 11.3 For purposes of accuracy when quantitating low levels of analyte, it may be necessary to use the low end ofthe calibration curve rather than the full range of the standard curve. Example: when attempting to quantitate approximately 10 ppb of analyte, generate a calibration curve consisting of the standards from 5 ppb to 100 ppb rather than the full range of the curve (5 ppb to 1000 ppb). This will reduce inaccuracy attributed to linear regression weighting of high concentration standards. 12.0 Procedures______________________ ______________ ___________________________ 12.1 Acquisition Set up 12.1.1 Set up the sample list. 12.1.1.1 Assign a sample list filename using MO-DAY-last digit of year-increasing letter of the alphabet starting with a 12.1.1.2 Assign a method (MS file) for acquiring 12.1.1.3 Assign an HPLC program (Inlet file) 12.1.1.4 Type in sample descriptions and vial position numbers 12.1.2 To create a method click on method in the Acquisition control panel then mass Spectrometer headings and select SIR (Single Ion Recording) or MRM (Multiple Reaction Monitoring). Set Ionization Mode as appropriate and mass to 499 or other appropriate masses. A frill scan is usually collected along with the SIRs. Save acquisition method. IfMS/MS instruments are employed, additional product ion fragmentation information may be collected. Refer to Micromass MassLynx GUIDE TO DATA ACQUISITION for additional information and MRM. 12.1.3 Typically the analytical batch run sequence begins and ends with a set of extracted matrix standards. . 12.1.4 Samples are analyzed with a continuing calibration verification injected standard after every tenth sample. Solvent blanks should be analyzed periodically to monitor possible analyte carryover and are not considered samples but may be included as such. 12.2 Using the Autosampler 12.2.1 Set up sample tray according to the sample list prepared in Section 12.1.1. 12.2.2 Set-up the HP1100/autosampler at the following conditions or at conditions the analyst considers appropriate for optimal response. Record actual conditions in the instrument logbook: 12.2.2.1 Sample size = 10 pL injection 12.2.2.2 Inject/sample = 1 ' 12.2.2.3 Cycle time = 9 minutes . ETS-8-7.0 Analysis of Liver Extract Using ES/MS Page 5 of 10 12.2.2.4 Solvent ramp conditions Time MeOH 0.00 min. 1.0 min. 4.5 min. 6.5 min. 7.0 min. 9.0 mi. 40% 40% 95% 95% 40% 40% 2.0 mM Ammonium acetate 60% 60% 5% 5% 60% 60% 12.2.2.5 Press the "Start" button. 12.3 Instrument Set-up 12.3.1 Refer to ETS-9-24.0, "Operation and Maintenance of the Micromass Quattro II Triple Quadrupole Mass Spectrometer Fitted with an Atmospheric Pressure Ionization Source," for more details. 12.3.2 Check the solvent level in reservoirs and refill if necessary. 12.3.3 Check the stainless steel capillary at the end of the probe. Use an eyepiece to check the tip. The tip should be flat with no jagged edges. If the tip is found to be unsatisfactory, disassemble the probe and replace the stainless steel capillary. 12.3.4 Turn on the nitrogen. 12.3.5 Open the tune page. Clicks on operate to initiate source block and desolvation heaters. 12.3.6 Open the Inlet Editor. 12.3.6.1 Set HPLC pump to "On" 12.3.6.2 Set th e flow to 10 - 500 uL/min or as appropriate 12.3.6.3 Observe droplets coming out of the tip of the probe. A fine mist should be expelled with no nitrogen leaking around the tip of the probe. Readjust the tip of the probe if no mist is observed 12.3.6.4 Allow to equilibrate for approximately 10 minutes. 12.3.7 The instrument uses these parameters at the following settings. These settings may change in order to optimize the response: 12.3.7.1 Drying gas 250-400 liters/hour 12.3.7.2 ESI nebulizing gas 10-15 liters/hour 12.3.7.3 HPLC constant flow mode flow rate 10 - 500 pL/min 12.3.7.4 Pressure <400 bar (This parameter is not set, it is a guide to ensure the HPLC is operating correctly.) 12.3.7.5 Source block temperature 150 12.3.7.6 Desolvation temperature 250 ETS-8-7.0 Analysis of Liver Extract Using ES/MS Page 6 of 10 12.3.8 Print the time page, with its parameters, and store it in the study binder with a copy taped into the instrument log. 12.3.9 Click on start button in the Acquisition Control Panel (this may vary among MassLynx versions, refer to appropriate MassLynx User's Guide). Ensure start and end sample number includes all samples to be analyzed. 13.0 Data Analysis and Calculations_____________________ ____________________ 13.1 Calculations: 13.1.4 Calculate matrix spike percent recoveries using the following equation: % Recovery = Observed Result - Background Result x 100 Expected Result ' 13.1.5 Calculate percent difference using the following equation: % Difference = Expected Cone. - Calculated Cone, x 100 Expected Cone. 13.1.6 Calculate actual concentrations in matrix (pg/g): fne of PFOS calc, from std. Curve x Dilution Factor! x 1 pg (Initial Weight of Liver (g'l 1000 ng Final Volume (mL) 14.0 Method Performance _________________________________ _____________ 14.1 Method Detection Limit (MDL) and Limit of Quantitation (LOQ) are method, analyte, and . matrix specific. Refer to ETS-8-6.0, Attachment B for a listing of current validated MDL and LOQ values. 14.2 Solvent Blanks, Method Blanks and Matrix Blanks 14.2.1 Solvent blanks, method blanks, and matrix blanks must be below the lowest standard in the calibration curve. . 14.3 Calibration Curves 14.3.1 The r2 value for the calibration must be 0.980 or better. 14.4 Matrix Spikes 14.4.1 Matrix spike percent recoveries must be within 30% of the spiked concentration. 14.5 Continuing Calibration Verification 14.5.1 Continuing calibration verification percent recoveries must be within 30% of the . spiked concentration. 14.6 If criteria listed in the method performance section are not met, maintenance may be performed on the system and samples reanalyzed or other actions as determined by the analyst. Document all actions in the appropriate logbook. ETS-8-7.0 Analysis o f Liver Extract Using ES/MS Page 7 o f 10 14.7 If data are to be reported when performance criteria have not been met, the data must be footnoted on tables and discussed in the text of the report. 15.0 Pollution Prevention and Waste Management______ ______________________ 15.1 Sample extract waste and flammable solvent is disposed in high BTU containers, and glass pipette waste is disposed in broken glass containers located in the laboratory. 16.0 Records _______________ ;_____________________' _________ 16.1 Each page generated for a study must have the following information included either in the header or hand written on the page: study or project number, acquisition method, integration method, sample name, extraction date, dilution factor (if applicable), and analyst. 16.2 Print the tune page, sample list, and acquisition method from MassLynx to include in the appropriate study folder. Copy these pages and tape into the instrument runlog. 16.3 Plot the calibration curve by linear regression, weighted 1/x, then print these graphs and store in the study folder. 16.4 Print data integration summary, integration method, and chromatograms from MassLynx and store in the study folder. 16.5 Summarize data using suitable software (Excel 5.0+) and store in the study folder, refer to Attachment A for an example of a summary spreadsheet. 16.6 Back up electronic data to appropriate medium. Record in study notebook the file name and location ofbackup electronic data. 17.0 Tables. Diagrams. Flowcharts, and Validation Data______________________ 17.1 Attachment A: ETS-8-7.0 Data summary spreadsheet 18.0 References____________________________ _______________________________ 18.1 FACT-M-2.1, "Extraction of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds from Liver for Analysis Using HPLC-Electrospray/Mass Spectrometry" 18.2 ETS-9-24.0, "Operation and Maintenance of the Micromass Atmospheric Pressure Ionization/Mass Spectrometer Quattro II triple quadrupole Systems" 18.3 The validation report associated with this method is ETS-8-6.0 & 7.0-V-l 19.0 Affected Documents___________________________________________________ 19.1 ETS-8-6.0, "Extraction ofPotassium Perfluorooctanesulfonate or Other Fluorochemical Compounds from Liver or Fluid for Analysis Using HPLC-Electrospray/Mass Spectrometry" ETS-8-7.0 , Analysis of LiverExtract Using ES/MS Page 8 of 10 20.0 Revisions Revision Number Reason For Revision Revision Date ETS-8-7.0 Analysis of Liver Extract Using ES/MS Page 9 of 10 Laboratory Study # Study: Test Material: Matrix/Final Solvent: Method/Revision: Analytical Equipment SystemNumber: Instrument Software/Version: Filename: R-Squared Value: Slope: Y Intercept: Date of Extraction/Analyst: Date ofAnalysis/Analyst G roup D ose Sam ple# C oncentration ng/g Initial W t. g D ilution Factor F inal C one, ug/g Group/Dose: Taken from the study folder. Sample#: Taken fromthe study folder. Concentration (ng/g): Taken fromthe MassLynx integration summary. Initial Wt. (g): Taken from the study folder. Dilution Factor: Taken fromthe study folder. Final Cone, (ng/g): Calculated by dividing the initial volume fromthe concentration Attachment A: Summary Spreadsheet ETS-8-7.0 Analysis of Liver Extract Using ES/MS Page 10 of 10 3M Environmental Laboratory M ethod E x t r a c t io n .o f P o ta ssiu m Per flu o r o o c ta n e su lfo n a te o r O t h e r Fluorochem ical com pounds from Serum for Analysis U sing H PLC- E lectro spray /M a ss Spectro m etry Method Number: ETS-8-4.1 Adoption Date: 03/01/99 Author: Lisp. Clemen, Glenn Langenburg * Revision Date: ^ ^ Approved By: Laboratory Manager U l ---------Group Leader Date Date Technical Reviewer Date 2 '5? to' t ? ofl) AO Scope and Application - g .l Scope: This method is for the extraction of potassium perfluorooctanesulfonate (PFOS) or other fluorochemical compounds from serum. O Applicable compounds: Fluorochemical surfactants or other fluorinated compounds. (D .0 .3 ^ S' z>" 0) Matrices: Rabbit, rat, bovine, monkey, and human serum or other fluids as designated in the validation report. Word 6/95 ETS-8-4.1 Extraction of PFOS from Serum Page 1 o f 14 2.0 Summary of Method 2.1 This method describes the procedure for extracting potassium perfluorooctanesulfonate (PFOS) or other fluorochemical surfactants from serum, or other fluids, using an ion pairing reagent and methyl-teri-butyl ether (MtBE). In this method, seven fluorochemicals were extracted: PFOS, PFOSA, PFOSAA, EtFOSE-OH, PFOSEA, M556, and surrogate standard (see 3.0 Definitions). An ion pairing reagent is added to the sample and the analyte ion pair is partitioned into MtBE. The MtBE extract is removed and put onto a nitrogen evaporator until dry. Each extract is reconstituted in 1.0 mL of methanol, then filtered through a 3 cc plastic syringe attached to a 0.2 pm nylon filter into glass autovials. 2.2 These sample extracts are analyzed following method ETS-8-5.1 or other appropriate methods. 3.0 D efinitions_____________________________________________________________________ 3.1 PFOS: perfluorooctanesulfonate (anion of potassium salt) CgF17S 0 3' 3.2 PFOSA: perfluorooctane sulfonylamide C8F17S 0 2NH2 3.3 PFOSAA: perfluorooctane sulfonylamido (ethyl)acetate C8F17S 0 2N(CH2CH3)CH2C 0 2' 3.4 EtFOSE-OH: 2(N-ethylperfluorooctane sulfonamido)-ethyl alcohol C8F 17S 0 2N(CH2CH3)CH2CH20H 3.5 PFOSEA: perfluorooctane sulfonyl ethylamide C8F17S 0 2N(CH2CH3)H 3.6 M556: C8F17SO2N(H)(CH2C 00H ) 3.7 Surrogate standard: 1H-1H-2H-2H perfluorooctane sulfonic acid 4.0 W arnings and Cautions____________________________________________________ 4.1 Health and safety warnings 4.1.1 Use universal precautions, especially laboratory coats, goggles, and gloves when handling animal tissue, which may contain pathogens. 5.0 Interferences__________________________________________________________________ 5.1 There are no interferences known at this time. 6.0 Equipment______________________________________________________________________ 6.1 The following equipment is used while performing this method. Equivalent equipment is acceptable. 6.1.1 Vortex mixer, VWR, Vortex Genie 2 6.1.2 Centrifuge, Mistral 1000 or IEC 6.1.3 Shaker, Eberbach or VWR ETS-8-4.1 Extraction of PFOS from Seram Page 2 o f 14 6.1.4 Nitrogen evaporator, Organomation 6.1.5 Balance ( 0.100 g) 7.0 Supplies and Materials____________________________________________________ 7.1 Gloves 7.2 Eppendorf or disposable pipettes . 7.3 Nalgene bottles, capable of holding 250 mL and 1 L 7.4 Volumetric flasks, glass, type A 7.5 I-CHEM vials, glass, 40 mL glass 7.6 Centrifuge tubes, polypropylene, 15 mL 7.7 Labels -. 7.8 Oxford Dispenser - 3.0 to 10.0 mL 7.9 Syringes, capable of measuring 5 pL to 50 pL 7.10 Graduated pipettes 7.11 Syringes, disposable plastic, 3 cc 7.12 Syringe filters, nylon, 0.2 pm, 25 mm 7.13 Timer 7.14 Crimp cap autovials and caps 7.15 Crimpers Note: Prior to using glassware and bottles, rinse 3 times with methanol and 3 times with Milli-QTM water. Rinse syringes a minimum of 9 times with methanol, 3 rinses from 3 separate vials. 8.0 Reagents and Standards___________________________________________________ 8.1 Type I reagent grade water, Milli-QTM or equivalent; all water used in this method should be Milli-QTM water and may be provided by a Milli-Q TOC PlusTM system 8.2 Sodium hydroxide (NaOH), J.T Baker or equivalent 8.3 Tetrabutylammonium hydrogen sulfate(TBA), Kodak or equivalent 8.4 Sodium carbonate (Na2C03), J.T. Baker or equivalent 8.5 Sodium bicarbonate (NaHC03), J.T. Baker or equivalent 8.6 Methyl-T-Butyl Ether, Omnisolv, glass distilled or HPLC grade 8.7 Methanol, Omnisolv, glass distilled or HPLC grade 8.8 Serum or blood, frozen from supplier 8.9 Fluorochemical standards 8.9.1 PFOS (3M Specialty Chemical Division), molecular weight = 538 8.9.2 PFOSA (3M Specialty Chemical Division), molecular weight = 499 ETS-8-4.1 Extraction of PFOS from Serum Page 3 of 14 8.9.3 PFOSAA (3M Specialty Chemical Division), molecular weight = 585 8.9.4 EtFOSE-OH (3M Specialty Chemical Division), molecular weight = 570 8.9.5 PFOSEA (3M Specialty Chemical Division), molecular weight = 527 8.9.6 M556 (3M Specialty Chemical Division), molecular weight = 557 8.9.7 Surrogate standard: 4-H, perfluorooctane sulfonic acid (1-H,1-H, 2-H, 2-H C8F 13S 0 3H) molecular weight = 428 . 8.9.8 Other fluorochemicals, as appropriate 8.10 Reagent preparation NOTE: When preparing larger volumes than listed in reagent, standard, or surrogate preparation, adjust accordingly. 8.10.1 ION so_dium hydroxide (NaOH): Weigh approximately 200 g NaOH. Pour into a 1000 mL beaker containing 500 mL Milli-QTM water, mix until all solids are dissolved. Store in a 1 L Nalgene bottle. 8.10.2 1 N sodium hydroxide (NaOH): Dilute 10N NaOH 1:10. Measure 10 mL of 10 . N NaOH solution into a 100 mL volumetric flask and dilute to volume using Milli-QTM water. Store in a 125 mL Nalgene bottle. 8.10.3 0.5 M tetrabutylammonium hydrogen sulfate (TBA): Weigh approximately 169 g of TBA into a 1 L volumetric containing 500 mL Milli-QTM water. Adjust to pH 10 using approximately 44 to 54 mL of 10 N NaOH (While adding the last mL of NaOH, add slowly because the pH changes abruptly). Dilute to volume with Milli-QTM water. Store in a 1 L Nalgene bottle. 8.10.3.1 TBA requires a check prior to each use to ensure pH = 10. Adjust as needed using 1 N NaOH solution. 8.10.4 0.25 M sodium carbonate/sodium bicarbonate buffer (Na^CO/NaHCOJ: Weigh approximately 26.5 g of sodium carbonate (Na^O-,) and 21.0 g of sodium bicarbonate (NaHC03) into a 1 L volumetric flask and bring to volume with MilliQTM water. Store in a 1 L Nalgene bottle. 8.11 Standards preparation 8.11.1 Prepare PFOS standards for the standard curve. 8.11.2 Prepare other fluorochemical standards, as appropriate. Multicomponent fluorochemical standards are acceptable (for example, one working standard solution containing 1.00 ppm PFOS, 1.02 ppm PFOSA, 0.987 ppm PFOSAA, and 1.10 ppm EtFOSE-OH.) 8.11.3 Weigh approximately 100 mg of PFOS into a 100 mL volumetric flask and record the actual weight. 8.11.4 Bring to volume with methanol for a stock standard of approximately 1000 ppm (pg/mL). 8.11.5 Dilute the stock solution with methanol for a working standard 1 solution of approximately 50 ppm. 8.11.6 Dilute working standard 1 with methanol for a working standard 2 solution of approx. 5.0 ppm. ETS-8-4.1 Extraction of PFOS from Serum Page 4 o f 14 8.11.7 Dilute working standard 1 with methanol for a working standard 3 solution of approx. 0.50 ppm. 8.12 Surrogate stock standard preparation 8.12.1 Weigh approximately 50-60 mg of surrogate standard l-H.l-H, 2-H, 2-H, C8F 13S 0 3H into a 50 mL volumetric flask and record the actual weight. 8.12.2 Bring to volume with methanol for a surrogate stock of approximately 1000-1200 ppm. * ' 8.12.3 Prepare a surrogate working standard. Transfer approximately 1 mL of surrogate stock to a 10 mL volumetric flask and bring to volume with methanol for a working standard of 100 ppm. Record the actual volume transferred. 9.0 Sample Handling_______________________________________________________________ 9.1 All samples are received frozen and must be kept frozen until the extraction is performed. 9.2 Allow samples to thaw to room temperature prior to extraction. 10.0 OualIty Control______________________________________________________________ 10.1 Solvent Blanks, Method blanks and matrix blanks 10.1.1 An aliquot of 1.0 mL methanol is used as a solvent blank. 10.1.2 Extract two 1.0 mL aliquots of Milli-QTM water following this procedure and use as method blanks. 10.1.3 Extract two 1.0 mL aliquots of the serum following this procedure and use as matrix blanks. See 11.1.4. 10.2 Matrix spikes 10.2.1 Prepare and analyze matrix spike and matrix spike duplicate samples to determine the accuracy of the extraction. 10.2.2 Prepare each spike using a sample chosen by the analyst, usually the control m atrix received w ith each sample set. 10.2.3 Expected concentrations will fall in the mid-range of the initial calibration curve. Additional spikes may be included and may fall in the low-range of the initial calibration curve. 10.2.4 Prepare one matrix spike and matrix spike duplicate per 40 samples, with a minimum of 2 matrix spikes per batch. 10.3 Continuing calibration checks 10.3.1 Prepare continuing calibration check samples to ensure the accuracy of the initial calibration curve. 10.3.2 Prepare, at a minimum, one continuing check per group of 10 samples. For example, if a sample set = 34, four checks are prepared and extracted. 10.3.3 Prepare each continuing calibration check from the same matrix used to prepare the initial curve. ETS-8-4.1 Extraction of PFOS from Serum Page 5 of 14 10.3.4 The expected concentrations will fall within the mid-range of the initial calibration curve. Additional spikes may be included that fall in the low-range of the initial calibration curve. This is necessary if the analyst must quantitate using only the low end of the calibration curve (for example, 5 ppb - 100 ppb, rather than 5 ppb - 1000 ppb). 11.0 Calibration and Standardization_________________;____________________________ 11.1 Prepare matrix calibration standards 11.1.1 Transfer 1 mL of serum to a 15 mL centrifuge tube. 11.1.2 If most sample volumes are less than 1.0 mL, extract standards with matrix volumes equal to the sample volumes. Do not extract less than 0.50 mL of matrix. Record each sample volume on the extraction sheet. 11.1.3 While preparing a total of twenty aliquots in 15 mL centrifuge tubes, mix or shake between aliquots. 11.1.4 Two 1 mL aliquots, or other appropriate volume, serve as matrix blanks. ' Typically use the standard concentrations and spiking amounts listed in Table 1, at the end of this section, to spike, in duplicate, two standard curves, for a total of eighteen standards, two matrix blanks, and two method blanks. 11.1.5 Refer to validation report ETS-8-4.0 & ETS-8-5.0-V-1, which lists the working ranges and the Linear Calibration Range (LCR) for calibration curves. 11.1.6 Use Attachment D as an aid in calculating the concentrations of the working standards. See Section 13.0 to calculate actual concentrations of PFOS in calibration standards. 11.2 To each standard, blank, or continuing check, add appropriate amount of surrogate working standard for the concentration to fall within the calibration curve range 5 ppb 1000 ppb. 11.3 Extract spiked matrix standards following 12.6-12.16 of this method. Use these standards to establish each initial curve on the m ass spectrometer. ETS-8-4.1 Extraction of PFOS from Serum Page 6 o f 14 Table 1 Approximate spiking amounts for standards and spikes Using 1.0 mL of matrix Working standard pL Approx, final cone, of (approx, cone.) analyte in matrix * - - -Blank 0.500 ppm 10 0.005 ppm 0.500 ppm 20 0.010 ppm 5.00 ppm 5 0.025 ppm 5.00 ppm 10 0.050 ppm 5.00 ppm 20 0.100 ppm 50:0 ppm 5 0.250 ppm 50.0 ppm 10 0.500 ppm 50.0 ppm 15 0.750 ppm 50.0 ppm 20 . 1.00 ppm 12.0 Procedure_____________________________________________________________________ 12.1 Obtain frozen samples and allow to thaw at room temperature or in a lukewarm waterbath. 12.2 Vortex mix for 15 seconds, then transfer 1.0 mL or other appropriate volume to a 15 mT, polypropylene centrifuge tube. 12.3 Return unused samples to freezer after extraction amounts have been removed. 12.4 Record the initial volume on the extraction worksheet. 12.5 Label the tube with the study number, sample ID, date and analyst initials. See attached worksheet for documenting the remaining steps. 12.6 Spike all samples, including blanks and standards, ready for extraction with surrogate standard as described in 11.2. 12.7 Spike each matrix with the appropriate amount of standard as described in 11.1, or Table 1 in that section, for the calibration curve standards. Also prepare matrix spikes and continuing calibration standards. 12.8 Vortex mix the standard curve samples, matrix spike samples, and continuing calibration samples for 15 seconds. 12.9 Check to ensure the 0.5 M TBA reagent is at pH 10. If not, adjust accordingly. 12.10 To each sample, add 1 mL 0.5 M TBA and 2 mL of 0.25M sodium carbonate/sodium bicarbonate buffer. 12.11 Using an Oxford Dispenser, add 5 mL methyl-teri-butyl ether. 12.12 Cap each sample and put on the shaker at a setting of 300 rpm, for 20 minutes. 12.13 Centrifuge for 20 to 25 minutes at a setting of 3500 rpm, or until layers are well separated. ETS-8-4.1 Extraction of PFOS from Serum Page 7 o f 14 12.14 Label a fresh 15 mL centrifuge tube with the same information as in 12.5. 12.15 Remove 4.0 mL of the organic layer to this clean 15 mL centrifuge tube. 12.16 Put each sample on the analytical nitrogen evaporator until dry, approximately 1 to 2 hours. 12.17 Add 1.0 mL of methanol to each centrifuge tube using a graduated pipette. 12.18 Vortex mix for 30 seconds. . 12.19 Attach a 0.2 pm nylon mesh filter to a 3 cc syringe and transfer the sample to this syringe. Filter into a 1.5 mL glass autovial or low-volume autovial when necessary. 12.20 Label the autovial with the study number, animal number and gender, sample timepoint, matrix, final solvent, extraction date, and analyst(s) performing the extraction. 12.21 Cap and store extracts at room temperature or at approximately 4 C until analysis. 12.22 Complete the extraction worksheet, attached to this document, and tape in the study notebook or include in study binder, as appropriate. 13.0 Data Analysis and Calculations_____ ;_____________________________________ 13.1 Calculations 13.1.1 Calculate actual concentrations of PFOS, or other applicable fluorochemical, in calibration standards using the following equation: mL of standard x concentration of standard (ug /mLl_________________ mL of standard + mL of surrogate standard + initial matrix volume (mL) Final Concentration (jig/mL) of PFOS in matrix 14.0 Method Performance__________________________________________________ __ 14.1 The method detection limit (MDL) is analyte and matrix specific. Refer to MDL report for specific M D L and limit o f quantitation (L O Q ) values (se e Attachments B and C). 14.2 The following quality control samples are extracted with each batch of samples to evaluate the quality of the extraction and analysis. 14.2.1 Method blanks and matrix blanks. 14.2.2 Matrix spike and matrix spike duplicate samples to determine accuracy and precision of the extraction. 14.2.3 Continuing calibration check samples to determine the continued accuracy of the initial calibration curve. 14.3 Refer to section 14 of ETS-8-5.1 for method performance criteria. 15.0 Pollution Prevention and Waste Management_______________________ 15.1 Sample waste is disposed in biohazard containers, flammable solvent waste is disposed in high BTU containers, and used glass pipette waste is disposed in broken glass containers located in the laboratory. ETS-8-4.1 Extraction of PFOS from Serum Page 8 o f 14 16.0 Records 16.1 Complete the extraction worksheet attached to this method, and tape in the study notebook or include in the 3-ring study binder, as appropriate. 17.0 Attachments___________________________________________________________________ 17.1 Attachment A, Extraction worksheet 17.2 Attachment B, MDL/LOQ values and summary 17.3 Attachment C, Calibration standard concentration worksheet 18.0 R eferences ___________________________________________________________________ 18.1 The validation report associated with this method is ETS-8-4.0 & 5.0-V -l. 18.2 FACT-M-3.1, "Analysis of Serum or Other Fluid Extracts for Fluorochemicals using HPLC-Electrospray Mass Spectrometry" 19.0 A ffected Documents___________________________________________________________ 19.1 ETS-8-5.1, "Analysis of Serum or Other Fluid Extracts for Fluorochemicals using HPLC-Electrospray Mass Spectrometry" 20.0 R evisions____________________ ;_________________________________________________ Revision Number 1 Reason For Revision Section 12.21 Changed to include sample storage at room temperature. Section 12.13 Added the shaker speed. Section 12.17 Final volume is 1.0 mL; not adjusted for initial volumes less than 1.0 mL. Revision Date 04/02/99 ETS-8-4.1 Extraction of PFOS from Serum Page 9 of 14 Extraction Worksheet ETS-8-4.1 Study # Matrix Box # Wk/Day DateSpiked/Analyst CCV MS MSD Surrogate Std approx, ppm actual ppm # FC-Mix approx. 0.5 pm actual ppm # FC-Mix approx. 5 ppm actual ppm n FC-Mix approx. 50 ppm actual ppm # Comments ' - -- -' -- -- -- -- Blank Std # Serum Extraction Method Vortex 15 sec. Pipette Matrix Pipette 1 mL of 0.5 M TBA, pH 10. pH= - - - - - : Volume - amount = mL Std. # Pipette 2 mL of 0.25 Na2COy0.25M NaHC03 buffer Std. # Dispense 5 mL ofmethyl-t-butyl ether TN-A- Shake 20 min. Centrifuge 20-25 min. Remove a 4 mL aliauot of organic layer Put on Nitrogen Evaporator to drvness Add methanol Volume Shaker speed: Centrifuge speed: Temperature: mL TN-A- Vortex 30 sec. Filter using a 3cc B-D syringe with a 0.2um filter into a 1.5 mL autosample vial Cont. Cal. Verifications used same matrix as for std curve. - - - - - - mL Date & Initials Attachment A ETS-8-4.1 Page 10 of 14 MDL/LOQ values for rabbit serum Compound MDL LOQ Linear Calibration Range (LCR) (PPb) (ppb) Approximate concentrations to be used for preparing the Standard Calibration Curve PFOS 1.74 5.55 5 ppb -1000 ppb PFOSA 1.51 4.79 5 ppb - 1000 ppb PFOSAA 3.46 20.5 5 ppb - 1000 ppb . EtFOSE-OH 11.4 36.2 5 ppb - 1000 ppb M 556 6.03 19.2 5 ppb - 1000 ppb PFOSEA 5.71 18.2 5 ppb - 1000 ppb MDL/LOQ values in rat, bovine, monkey, and human serum, and monkey plasma were not statistically determined. Two curves in each of these matrices were extracted and analyzed with the rabbit serum curves to determine equivalence. Responses in the rat, bovine, monkey, and human were equivalent to the rabbit responses, therefore, their MDL and LOQ will be the same values as determined in rabbit serum. Please see LOQ Summary and MDL study in ETS-8-4.0 & 5.0-V-l for further information. Attachment B: MDL/LOQ Summary ETS-8-4.1 Extraction of PFOS from Serum Page 11 o f 14 Compound: PFOS Prepared range Rabbit Serum of standards (ppb) (ng/mL) Full Range Low Curve High curve 1/X 0.995 - 978 4.94 - 248 97.8 - 978 0.995 - 978 Compound: PFOSA Prepared range' Rabbit Serum of standards (ppb) (ng/mL) Full Range 0.993 - 976 Low Curve 4.93 - 97.6 High curve 24.8 - 976 1/X \ 0.993 - 976 Compound: PFOSAA Prepared range Rabbit Serum of standards (ppb) (ng/mL) Full Range Low Curve High curve 1/X 0.991 -974 4.92 - 247 49.2 - 974 0.991 -974 LCR from curve (Ppb) (ng/mL) 24.8 - 978 4.94 - 248 97.8 - 978 4.94 - 978 LCR from curve (ppb) (ng/mL) 4.93 - 976 4.93 - 97.6 24.8 - 978 4.93 - 976 LCR from curve (Ppb) (ng/mL) 24.7 - 974 9.74 - 247 97.4 - 974 9.74 - 974 % Recovery Range 83-108 85-104 85-106 94-111 % Recovery Range 88-103 87-105 93-102 94-103 % Recovery Range 81-111 97-107 85-108 95-115 RSD Range 4.67-11.0 '5.34-12.0 4.84-9.80 4.60-10.5 RSD Range 5.10-14.7 9.85-14.7 5.08-13.9 5.10-14.5 RSD Range 4.18-10.6 6.38-21.8 4.33-12.5 4.11-23.2 Attachment B: MDL/LOQ Summary ETS-8-4.1 Extraction of PFOS from Serum Page 12 o f 14 Compound: EtFOSE-OH Prepared range Rabbit Serum of standards (ppb) (ng/mL) Full Range Low Curve High curve 1IX 0.993 - 976 4.93 - 97.6 49.3 - 976 0.993 - 493 Compound: PFOSEA - Prepared range Rabbit Serum o f standards (ppb) (ng/mL) i Full Range 0.993 - 976 Low Curve 4.93 - 248 High curve 49.3 - 976 1/X 0.993 - 976 Compound: M556 Prepared range Rabbit Serum o f standards (ppb) (ng/mL) Full Range Low Curve High curve 1/X 0.993 - 976 4.93 - 97.6 97.6 - 976 0.993 - 976 LCR from curve (PPb) (ng/mL) 49.3 - 976 9.76-97.6 97.6-976 9.76 - 976 LCR from curve (ppb) (ng/mL) 24.8 - 976 9.76 - 248 49.3 - 976 9.76 - 976 LCR from curve (ppb) (ng/mL) 24.8 - 976 9.76-97.6 97.6 - 976 9.76 - 976 % Recovery Range 77-110 97-107 90-109 86-111 % Recovery Range 96-106 91-110 86-106 95-117 % Recovery Range 88-106 100-105 81-111 97-110 RSD Range 11.2-25.5 14.1-21.3 11.5-19.6 11.1-21.2 RSD Range 10.1-16.2 11.8-19.5 10.2-18.2 10.1-19.1 RSD Range 4.82-17.9 5.95-18.2 5.11-9.74 4.77-19.5 Attachment B: MDL/LOQ Summary ETS-8-4.1 Extraction of PFOS from Serum Page 13 o f 14 0 Ion Pair Standard Curves - Fluids Prep date(s): Standard number: Analyte(s): Equipment number: Sample matrix: Final solvent and TN: Blank fluid/identifier: Method/revision: Target analyte(s): FC mix std approx. 0.500 ppm: FC mix std approx. 5.00 ppm: FC mix std approx. 50.0 ppm: Surrogate std approx. 100 ppm: Actual concentrations of standards in the FC mix PFOS PFOSA PFOSAA EtFOSE PFOSEA Std cone Std cone Std cone : Std cone Std cone ug/mL ug/mL ug/mL ug/mL ug/mL 0.500 0.507 0.532 0.501 0.521 0.500 0.507 0.532 0.501 0.521 5.00 5.07 5.32 5.01 5.21 ' 5.00 5.07 5.32 5.01 5.21 5.00 5.07 5.32 5.01 5.21 50.0 50.1 53.2 50.1 52.1 50.0 50.1 53.2 50.1 52.1 50.0 50.1 53.2 50.1 52.1 50.0 50.1 53.2 50.1 52.1 M556 Std cone ug/mL 0.501 0.501 5.01 5.01 5.01 50.1 50.1 50.1 50.1 All Ain't spiked mL 0.010 0.020 0.005 0.010 0.020 0.005 0.010 0.015 0.020 All Final vol mL 1.015 1.025 1.010 1.015 1.025 1.010 1.015 1.020 1.025 Calculated concentrations of standards in the sample matrix PFOS PFOSA PFOSAA EtFOSE PFOSEA M556 Surrogate Final cone Final cone Final cone Final cone Final cone Final cone Std cone ng/mL ng/mL ng/mL ng/mL ng/mL ng/mL ng/mL 4.93 5.00 5.24 4.94 5.01 5.13 100 9.76 9.89 10.4 9.78 9.93 10.2 24.8 25.1 49.3 5 0 .0 26.3 24.8 25.2 25.8 Surrogate 5 2 .4 49.4 50.1 51.3 Final cone 97.6 98.9 104 97.8 99.3 102 ng/mL 248 251 263 248 252 258 500 493 500 524 494 501 513 735 746 782 737 749 766 976 989 1038 978 993 1017 All Am't spiked mL 0.005 Validated ranges - approximate concentrations Serum PFOS PFOSA PFOSAA Rabbit 5.00-1000 5.00-1000 5.00-1000 Bovine Estimates only. Use values for rabbit. Rat Estimates only. Use values for rabbit. Monkey & Plasma Estimates only. Use values for rabbit. . Human Estimates only. Use values for rabbit. EtFOSE-OH 5.00-1000 PFOSEA 5.00-1000 M556 5.00-1000 Attachment C: Ion Pair Standard Curves ETS-8-4.1 Extraction of PFOS from Serum Page 14 o f 14 V. 3M Environmental Laboratory Method Extraction of P otassium Perfluorooctanesulfonate or Other Fluorochemical compounds from Serum or Other Fluid for Analysis U sing HPLC-Electrospray/Mass Spectrometry Method Number: FACT-M-3.1 Adoption Date: 04/22/98 Author: Lisa Clemen, Glenn Langenburg _ Revision Date: \o Approved By: | y // l / f J o 1^ ' -------- Laboratory Manager ,a) f n Date c1 Group Leader 4 /-z -? / i Date d \ \ ^ A CLyt^K_________________ Technical Reviewer . Date m X u 1J5 Scope and Application 1.1 Scope: This method is for the extraction of potassium perfluorooctanesulfonate (PFOS) ' or other fluorochemical compounds from serum or other fluid. 1.^ Applicable compounds: Fluorochemical surfactants or other fluorinated compounds. I . 3 Matrices: Rabbit, rat, bovine, and monkey serum, rat whole blood, and rat milk curd. S' Word 6/95 FACT-M-3.1 Extraction of PFOS from Serum and Other Fluids Page 1 of 17 ........................... - Jj-Jj5l-L 2.0 Summary of Method 2.1 This method describes the procedure for extracting potassium perfluorooctanesulfonate (PFOS) or other fluorochemicals from serum, blood, or milk curd using an ion pairing reagent and 5.0 ml of ethyl acetate. In this method, seven fluorochemicals were extracted: PFOS, PFOSA, PFOSAA, EtFOSE-OH, POAA, PFOSEA, and FC-807 monoester (see 3.0 Definitions). An ion pairing reagent is added to the sample and the analyte ion pair is partitioned into ethyl acetate. Four ml of extract are removed and put onto a nitrogen evaporator until dry. Each extract is reconstituted in 1.0 ml of methanol, then filtered through a 3 cc plastic syringe attached to a 0.2 pm nylon filter into glass autovials. 3.0 Definitions_______________________________________________________________ 3.1 PFOS: perfluorooctanesulfonate (anion of potassium salt) C8F17S 0 3' 3.2 PFOSA: perfluorooctane sulfonylamide C8F17S 0 2NH2 3.3 PFOSAA: perfluorooctane sulfonylamido (ethyl)acetate CgFnS 0 2N(CH2CH3)CH2C 0 2' 3.4 EtFOSE-OH: 2(N-ethylperfluorooctane sulfonamido)-ethyl alcohol c 8f ;7so 2n (ch2c h 3)c h 2ch2oh ' 3.5 POAA: perfluorooctanoate (anion of ammonium salt) C7F15COO' 3.6 PFOSEA: perfluorooctane sulfonyl ethylamide C8F17S 02N(CH2CH3)H 3.7 FC-807 monoester C8F17S 0 2N(CH2CH3)CH2CH20 - P 0 3H) 3.8 Surrogate standard: 1H-1H-2H-2H perfluorooctane sulfonic acid 4.0 Warnings and Cautions___________________________________________________ 4.1 Health and safety warnings 4.1.1 Use universal precautions, especially laboratory coats, goggles, and gloves when handling animal tissue, which may contain pathogens. 5.0 Interferences____________________________________________________________ 5.1 There are no known interferences at this time. 6.0 Equipment________________________________________________________________ 6.1 The following equipment is used while performing this method. Equivalent equipment is acceptable. 6.1.1 Vortex mixer, VWR, Vortex Genie 2 6.1.2 Centrifuge, Mistral 1000 or IEC 6.1.3 Shaker, Eberbach or VWR 6.1.4 Nitrogen evaporator, Organomation 6.1.5 Balance ( 0.100 g) FACT-M-3.1 Extraction of PFOS from Serum or Other Fluid Page 2 o f 17 7.0 Supplies and Materials 7.1 Gloves 7.2 Eppendorf or disposable pipettes 7.3 Electronic pipettor, Eppendorf or equivalent 7.4 Graduated pipettes ' 7.5 Nalgene bottles, capable of holding 250 mL and 1 L 7.6 Volumetric flasks, glass, type A 7.7 Volumetric pipets, glass, type A 7.8 I-CHEM vials, glass, 40 mL glass 7.9 Crimp cap autovials 7.10 Centrifuge tubes, polypropylene, 15 mL 7.11 Labels . 7.12 Syringes, capable of measuring 5 pL to 50 pL 7.13 Syringes, disposable plastic, 3 cc 7.14 Syringe filters, nylon, 0.2 pm, 25 mm 7.15 Timer Note: Prior to using glassware and bottles, rinse 3 times with methanol and 3 times with Milli-QTM water. Rinse syringes a minimum of 9 times with methanol, 3 rinses from 3 separate vials. 8.0 Reagents and Standards_____________________ ;___________________________ __ 8.1 Type I reagent grade water, Milli-QTM or equivalent; all water used in this method should be Milli-QTM water and may be provided b y a Milli-Q TOC PlusTM system 8.2 Sodium hydroxide (NaOH), J.T Baker or equivalent 8.3 Tetrabutylammonium hydrogen sulfate(TBA), Kodak or equivalent 8.4 Sodium carbonate (Na2C03), J.T. Baker or equivalent 8.5 Sodium bicarbonate (NaHC03), J.T. Baker or equivalent 8.6 Ethyl acetate, Omnisolv, glass distilled or HPLC grade 8.7 Methanol, Omnisolv, glass distilled or HPLC grade 8.8 Serum or blood, frozen from supplier 8.9 Control matrix or blank matrix for purpose of standards, QC checks, blanks, etc. 8.10 Fluorochemical standards 8.10.1 PFOS (3M Specialty Chemical Division), molecular weight = 538 8.10.2 PFOSA (3M Specialty Chemical Division), molecular weight = 499 FACT-M-3.1 Extraction of PFOS from Serum or Other Fluid Page 3 of 17 8.10.3 PFOSAA (3M Specialty Chemical Division), molecular weight = 585 8.10.4 EtFOSE-OH (3M Specialty Chemical Division), molecular weight = 571 8.10.5 POAA (3M Specialty Chemical Division), molecular weight = 431 8.10.6 PFOSEA (3M Specialty Chemical Division), molecular weight = 527 8.10.7 FC-807 monoester (3M Specialty Chemical Division). FC-807 is a mixture of triester, diester, and monoester fluorochemical components. The monoester molecular weight = 650 ' 8.10.8 Surrogate standard: 4-H, perfluorooctane sulfonic acid (1-H,1-H, 2-H, 2-H C8F13S 0 3H) molecular weight = 428 8.10.9 Other fluorochemicals, as appropriate 8.11 Reagent preparation 8.11.1 10 N sodium hydroxide (NaOH): Weigh approximately 200 g NaOH. Pom into a 1000 mL beaker containing 500 mL Milli-QTM water, mix until all solids are dissolved. Store in a 1 L Nalgene bottle. 8.11.2 1 N sodium hydroxide (NaOH): Dilute 10 N NaOH 1:10. Measure 10 mL of 10N NaOH solution into a 100 mL volumetric flask and dilute to volume using Milli-QTM water. Store in a 125 mL Nalgene bottle. 8.11.3 0.5 M tetrabutylammonium hydrogen sulfate (TBA): Weigh approximately 169 g of TBA into a 1 L volumetric containing 500 mL Milli-QTM water. Adjust to pH 10 using approximately 44 to 54 mL of 10 N NaOH and dilute to volume with Milli-QTM water. While adding the last mL of NaOH, add slowly because the pH changes abruptly. Store in a 1 L Nalgene bottle. 8.11.3.1 TBA requires a check prior to each use to ensure pH = 10. Adjust as needed using 1 N NaOH solution. 8.11.4 0.25 M sodium carbonate/sodium bicarbonate buffer (NajCOj/NaHCO-,): Weigh approximately 26.5 g of sodium carbonate (NajCOj) and 21.0 g of sodium bicarbonate (NaHC03) into a 1 L volumetric flask and bring to volume with Milli- QTM water. Store in a 1 L Nalgene bottle. 8.12 Standards preparation 8.12.1 Prepare PFOS standards for the standard curve. 8.12.2 Prepare other fluorochemical standards, as appropriate. Multicomponent fluorochemical standards are acceptable (for example, one working standard solution containing 1.00 ppm PFOS, 1.02 ppm PFOSA, 0.987 ppm PFOSAA, and 1.10 ppm EtFOSE-OH.) 8.12.3 Weigh approximately 100 mg of PFOS into a 100 ml volumetric flask and record the actual weight. 8.12.4 Bring to volume with methanol for a stock standard of approximately 1000 ppm - (pg/ml). 8.12.5 Dilute the stock solution with methanol for a working standard 1 solution of approximately 50 ppm. FACT-M-3.1 Extraction of PFOS from Serum or Other Fluid Page 4 of 17 8.12.6 Dilute the stock solution with methanol for a working standard 2 solution of approx. 5.0 ppm. 8.12.7 Dilute the stock solution with methanol for a working standard 3 solution of approx. 0.50 ppm. 8.13 Surrogate stock standard preparation 8.13.1 Weigh approximately 50-60 mg of surrogate standard 1-H,1-H, 2-H, 2-H, C8FnS 0 3H into a 50 ml volumetric flask and record the actual weight. 8.13.2 Bring to volume with methanol for a surrogate stock of approximately 1000-1200 ppm. 8.13.3 Prepare a surrogate working standard. Transfer approximately 0.5 ml of surrogate stock to a 50 ml volumetric flask and bring to volume with methanol for a working standard of 10-20 ppm. Record the actual volume transferred. 9.0 Sample Handling__________________________________________________________ 9.1 All samples are received frozen and must be kept frozen until the extraction is performed. \* 10.0 Quality Control_____________________________________________________ 10.1 Matrix blanks and method blanks 10.1.1 Extract two 1.0 mL aliquots of the appropriate matrix (serum or blood, with blood samples diluted 1:1 with Milli-QTM water) following this procedure and use as matrix blanks. See 11.1.4. 10.1.2 Extract two 1.0 ml aliquots of Milli-QTM water following this procedure and use as method blanks. 10.2 Matrix spikes 10.2.1 Prepare and analyze matrix spike and matrix spike duplicate samples to determine the accuracy of the extraction. 10.2.2 Prepare each spike using a sample chosen by the analyst, usually the control matrix received with each sample set. 10.2.3 Expected concentrations will fall in the mid-range of the initial calibration curve. Additional spikes may be included and may fall in the low-range of the initial calibration curve. 10.2.4 Prepare one matrix spike and matrix spike duplicate per 40 samples, with a minimum of 2 matrix spikes per batch. 10.3 Continuing calibration checks 10.3.1 Prepare and analyze continuing calibration check samples to ensure the accuracy of the initial calibration curve. If the percent difference between the initial curve and the continuing check differ by >30%, re-analyze samples analyzed after the last acceptable check. 10.3.2 Prepare one check per group of ten samples. For example, if a sample set = 34, prepare and extract four checks. FACT-M-3.1 Extraction of PFOS from Serum or Other Fluid Page 5 of 17 10.3.3 Prepare each continuing calibration check from the same matrix used to prepare the initial curve. 10.3.4 The expected concentration will fall within the mid-range of the initial calibration curve. Additional spikes may be included that fall in the low-range of the initial calibration curve. This is necessary if the analyst must quantitate using only the low end of the calibration curve (for example, 5 ppb - 100 ppb, rather than 5 ppb - 1000 ppb). 11.0 Calibration and Standardization__________________________________________ 11.1 Prepare matrix calibration standards Note: Blood coagulates in air; therefore, minimize air contact until dilution. At this point, add TBA and buffer to each centrifuge tube as in step 12.9, then add 1.0 mL of the diluted matrix sample to each tube. 11.1.1 Transfer 1 mL of serum or 1 mL of blood (blood is diluted 1:1 with Milli-QTM water) to a 15 mL centrifuge tube. The blood is similar in composition to milk curd and can be used in place of milk curd for standard curves when extracting ~ that matrix. 11.1.2 If most sample volumes axe less than 1.0 mL, extract standards with matrix volumes equal to the sample volumes. Do not extract below 0.50 mL of matrix. Record the sample volume on the extraction sheet. 11.1.3 While preparing a total of twenty aliquots in 15 ml centrifuge tubes, mix or shake between aliquots. 11.1.4 Two 1 mL aliquots, or other appropriate volume, serve as matrix blanks. Typically use the standard concentrations and spiking amounts listed in Table 1, at the end of this section, to spike, in duplicate, two standard curves, for a total of eighteen standards and two matrix blanks. 11.1.5 Refer to validation reports FACT-M-3.1-V-1 and FACT-M-4.1-V-1, which list the working ranges and the Linear Calibration Range (LCR) for calibration curves. 11.1.6 Use Attachment D as an aid in calculating the concentrations of the working standards. See Section 13.0 to calculate actual concentrations of PFOS in calibration standards. 11.2 To each standard, blank, or QC check, add appropriate amount of surrogate working standard for the concentration to fall within the calibration curve range 5 ppb -1000 ppb. 11.3 Extract spiked matrix standards following 12.6-12.16 of this method. Use these standards to establish each initial curve on the mass spectrometer. FACT-M-3.1 Extraction of PFOS from Serum or Other Fluid Page 6 of 17 Table 1 Approximate spiking amounts for standards and spikes using 1.0 ml of matrix Working standard pL Approx, final cone, of (approx, cone.) analyte in matrix - - . Blank 0.500 ppm 10 0.005 ppm 0.500 ppm 20 . 0.010 ppm 5.00 ppm 5 0.025 ppm 5.00 ppm 10 0.050 ppm 5.00 ppm 20 0.100 ppm 50.0 ppm 5 0.250 ppm 50.0 ppm 10 0.500 ppm 50.0 ppm 15 0.750 ppm 50.0 ppm 20 1.00 ppm Table 2 Approximate spiking amounts for standards and spikes using 0.5 ml of matrix Working standard p.L Approx, final cone, of (approx, cone.) analyte in matrix - - Blank 0.500 ppm 5 0.005 ppm 0.500 ppm 10 0.010 ppm 5.00 ppm 2.5 0.025 ppm 5.00 ppm 5 0.050 ppm 5.00 ppm 10 0.100 ppm 50.0 ppm 2.5 0.250 ppm 50.0 ppm 5 0.500 ppm 50.0 ppm 7.5 0.750 ppm 50.0 ppm 10 1.00 ppm 12.0 Procedure___________________ ;__________________________________________ 12.1 Obtain frozen samples and allow to thaw. 12.2 Vortex mix for 15 seconds, then transfer 1.0 mL or other appropriate volume to a 15 mL polypropylene centrifuge tube. For blood samples, remove 0.5 mL and dilute to 1.0 mL with Milli-QTM water. As soon after diluting as possible, pipet diluted blood into TBA . buffer mixture shown in step 12.9 and mix well. 12.3 Return samples to freezer after extraction amount has been removed. FACT-M-3.1 Extraction of PFOS from Serum or Other Fluid Page 7 o f 17 12.4 Record the volume on the extraction worksheet. The final methanol volume equals the volume transferred from the sample. For example, if 0.5 mL is removed for a blood sample, the final methanol volume will equal 0.5 mL. 12.5 Label the tube with the study number, sample ID, date and analyst initials. See attached worksheet for documenting the remaining steps. 12.6 Spike each matrix with the appropriate amount of standard as described in 11.1 or Table 1 or 2 in that section for the calibration curve standards. Also prepare matrix spikes and continuing calibration standards. 12.7 Spike all samples, including blanks and standards, ready for extraction with surrogate standard as described in 11.2. 12.8 Vortex mix the standard curve samples, matrix spike samples, and continuing calibration samples for 15 seconds. 12.9 To each sample, add 1 mL 0.5 M TBA and 2 mL of 0.25 M sodium carbonate/sodium bicarbonate buffer. 12.10 Using a volumetric pipette, add 5 mL ethyl acetate. 12.11 Cap each sample and put on the shaker for 20 minutes. 12.12 Centrifuge for 20 to 25 minutes at approximately 3500 rpm, until layers are well separated. 12.13 Transfer 4 mL of organic layer, using a 5 mL graduated glass pipette, to a clean 15 mL centrifuge tube. Label this fresh tube with the same information as in 12.5. 12.14 Put each sample on the analytical nitrogen evaporator until dry, approximately 2 to 3 hours. 12.15 Add 1.0 mL or other appropriate volume of methanol to each centrifuge tube using a graduated pipette. Methanol volume to add equals the initial volume of sample used for the extraction. 12.16 Vortex mix for 30 seconds. 12.17 Attach a 0 .2 p m nylon mesh filter to a 3 cc syringe and transfer the sam ple to this syringe. Filter into a 1.5 mL glass autovial or low-volume autovial when necessary. 12.18 Label the autovial with the study number, animal number and gender, sample timepoint, matrix, final solvent, extraction date, and analyst(s) performing the extraction. 12.19 Cap and store extracts at approximately 4 C until analysis. 12.20 Complete the extraction worksheet, attached to this document, and tape in the study notebook or include in study binder, as appropriate. FACT-M-3.1 Extraction of PFOS from Serum or Other Fluid Page 8 o f 17 13.0 Data A nalysis and Calculations 13.1 Calculations 13.1.1 Calculate actual concentrations of PFOS, or other applicable fluorochemical, in calibration standards using the following equation: mL of standard x concentration of standard fug /mLl_________________ = mL of standard + mL of surrogate standard + initial matrix volume (mL) Final Concentration (pg/mL) of PFOS in matrix 14.0 Method Performance_____________________________________________________ 14.1 The method detection limit (MDL) is analyte and matrix specific. Refer to MDL report for specific MDL and limit of quantitation (LOQ) values (see Attachments B and C). 14.2 The following quality control samples are extracted with each batch of samples to ensure - the quality of the extraction and analysis. 14.2'.l Method blanks and matrix blanks 14.2.2 Matrix spike and matrix spike duplicate samples to determine accuracy and precision of the extraction 14.2.3 Continuing calibration check samples to determine the continued accuracy o f the initial calibration curve 15.0 Pollution Prevention and Waste Management_______________________ 15.1 Sample waste is disposed in biohazard containers, flammable solvent waste is disposed in high BTU containers, and used glass pipette waste is disposed in broken glass containers located in the laboratory. 16.0 Records________________________________________________________________________ 16.1 Complete the extraction worksheet attached to this method, and tape in the study notebook or include in study 3-ring binder, as appropriate. 17.0 Attachments_____________________________________________________________ 17.1 Attachment A, Extraction worksheet 17.2 Attachment B, MDL/LOQ values 17.3 Attachment C, LOQ Summary 17.4 Attachment D, Calibration standard concentration worksheet 18.0 References_______________________________________________________________ 18.1 The validation reports associated with this method are FACT-M-3.1 & 4.1-V-l. FACT-M-3.1 Extraction of PFOS from Serum or Other Fluid Page 9 of 17 19.0 Affected Documents 19.1 FA C T-M -4.1, " Analysis o f Serum or Other Fluid Extracts for Fluorochemicals using HPLC-Electrospray Mass Spectrometry" 20.0 R e v isio n s__________________________________________________________________________ Revision Number 1 - Reason For Revision Validation o f method to include 7 fluorochemicals, an additional matrix, new API/M S(M S) systems, monkey serum cross validation, improvements to ion pairing extraction, M D L study, updates in record keeping and storing policies, etc. Revision Date 07/01/98 FACT-M-3.1 Extraction of PFOS from Serum or Other Fluid Page 10 of 17 Study # Sam ple N um ber 1 set # H ,0 Blank Blank ' FC-M ix approx. 0.5 ppm actual ppm #W - FC-M ix approx. 5 ppm actual ppm #W - FC-M ix approx. 50 ppm actual ppm #W - - Date and Initials for Std. or Comments -- -- - - -- -- -- -- -'- -- -- -- -- -- -- -- -- -- -- -- - - 1Study number where the original work:sheet is located. Blank Std# amount = mL Serum Extraction Method : Date & Initials Vortex 15 sec. Pipette Matrix Volume ml Pipette 1 ml of 0.5 M TBA, pH 10. Std. # Pipette 2 ml of 0.25 Na2COy0.25M NaHCOt buffer Std. # Pipette 5 ml of ethyl acetate TN-A- Shake 20 min. Centrifuge 20-25 min. Centrifuge speed: Remove a 4 ml aliauot of organic layer Put on Nitrogen Evaporator to drvness Evaporator #: Temperature: Add methanol Volume ml TN-A- Vortex 30 sec. Filter using a 3cc B-D syringe with a 0.2um SRI filter into a 1.5 ml autosample vial MS/MSD/__ Cont. Checks: Spiked____ uL of a _____ppm std (____________ ) for a final concentration of _______ ppm. MS/MSD used sample_____________. Cont. Checks used same matrix as for std curve. Surrogate Standard: Spiked____ uL of a _____ppm std (____________ ) to all samples, standards, and blanks Attachment A: Extraction worksheet FACT-M-3.1 Extraction of PFOS from Seram or Other Fluid Page 11 o f 17 MDL/LOQ values for Rabbit Serum: Compound M D L LO Q Linear C alibration Range (LCR ) (ppb) (ppb) Approxim ate concentrations to be used fo r preparing the Standard C alibration Curve PFOS 1.38 4.39 5 ppb - 1000 ppb PFOSA 2.23 7.09 10 ppb - 1000 ppb PFOSAA 2.84 9.04 10 ppb - 1000 ppb EtFOSE-OH 3.90 12.4 15 ppb - 1000 ppb POAA 4.31 13.7 15 ppb-7 5 0 ppb PFOSEA 1.09 3.48 25 ppb - 1000 ppb Monoester 149 248 MDL and LOQ are estimates only. No valid MDL was determinable from MDL study. Any quantitation performed for monoester will be an estimate only. Please refer to FACT-M-3.1 & 4.1-V-l for specifics. M D L/LO Q values fo r R at Serum: Compound M D L LO Q Linear C alibration Range (LC R ) (PPb) (PPb) Approxim ate concentrations to be used fo r preparing the Standard C alibration Curve PFOS 1.27 4.04 10 ppb - 1000 ppb PFOSA 2.14 6.81 25 p p b - 1000ppb PFOSAA 2.32 7.38 10 ppb - 1000 ppb EtFOSE-OH 3.25 10.3 50 ppb - 1000 ppb POAA 1.20 3.81 5 ppb - 1000 ppb PFOSEA 1.84 5.86 10 ppb - 1000 ppb Monoester 149 248 MDL and LOQ are estimates only. No valid MDL was determinable from MDL study. Any quantitation performed for monoester will be an estimate only. Please refer to FACT-M-3.1 & 4.1-V-l for specifics. M D L/LO Q values fo r Bovine Serum: Compound M D L LO Q Linear C alibration Range (LC R ) (PPb) (PPb) Approxim ate concentrations to be used fo r preparing the Standard C alibration Curve PFOS 2.11 6.70 25 ppb - 1000 ppb PFOSA 5.04 16.0 25 ppb - 1000 ppb PFOSAA 2.34 7.45 260 ppb - 1000 ppb EtFOSE-OH 11.3 35.8 50 ppb - 1000 ppb POAA 4.64 14.8 15 ppb - 1000 ppb PFOSEA 3.71 11.8 15 ppb - 1000 ppb Monoester 149 248 MDL and LOQ are estimates only. No valid MDL was determinable from MDL study. Any quantitation performed for monoester will be an estimate only. Please refer to FACT-M-3.1 & 4.1-V-l for specifics. No data is available for MDL or LOQ in Monkey Serum. Use validated Linear Calibration Range instead. Please see Attachment C (LOQ Summary) and MDL study in FACT-M-3.1 & 4.1-V-l for specifics. Attachment A: Extraction worksheet FACT-M-3.1 Extraction of PFOS from Serum or Other Fluid Page 12 o f 17 MDL/LOQ values for Monkey Serum: Compound M D L LO Q Linear C alibration Range (LC R ) (PPb) (ppb) Approxim ate concentrations to be used fo r preparing the Standard C alibration Curve PFOS 1.38 4.39 MDL and LOQ are estimates only. No valid MDL was determinable from MDL study. Any quantitation performed for PFOS will be an estimate only. Please refer to FACT-M-3.1 & 4.1-V-l for specifics. PFOSA 2.23 - 7.09 MDL and LOQ are estimates only. .No valid MDL was determinable from MDL study. Any quantitation performed for PFOSA will be an estimate only. Please refer to FACT-M-3.1 & 4.1-V-l for specifics. PFOSAA 2.84 9.04 MDL and LOQ are estimates only. No valid MDL was determinable from MDL study. Any quantitation performed for PFOSAA will be an estimate only. Please refer to FACT-M-3.1 & 4.1-V-l for specifics. EtFOSE-OH 3.90 12.4 MDL and LOQ are estimates only. No valid MDL was determinable from MDL study. Any quantitation performed for EtFOSE-OH will be an estimate only. Please refer to FACT-M-3.1 & 4.1-V-l for specifics. POAA 4.31 13.7 MDL and LOQ are estimates only. No valid MDL was determinable from MDL study. Any quantitation performed for POAA will be an estimate only. Please refer to FACT-M-3.1 & 4.1 -V-1 for specifics. PFOSEA > 1.09 3.48 MDL and LOQ are'estimates only. No valid MDL was determinable from MDL study. Any quantitation performed for PFOSEA-OH will be an Monoester 149 estimate only. Please refer to FACT-M-3.1 & 4.1-V-l for specifics. 248 MDL and LOQ are estimates only. No valid MDL was determinable from MDL study. Any quantitation performed for EtFOSE-OH will be an estimate only. Please refer to FACT-M-3.1 & 4.1-V-l for specifics. M D L/LO Q values fo r R at W hole Blood: Compound M D L LO Q Linear C alibration Range (LC R ) (ppb) (ppb) Approxim ate Concentrations to be used fo r preparing the Standard C alibration Curve PFOS 1.25 3.96 5 ppb - 1000 ppb PFOSA 1.77 5.65 lO p p b - lOOOppb PFOSAA 17.3 55.0 55 ppb --1000 ppb EtFOSE-OH 7.89 25.1 MDL and LOQ are estimates only. No valid MDL was determinable from MDL study. Any quantitation performed for EtFOSE-OH will be an estimate only. Please refer to FACT-M-3.1 & 4.1-V-l for specifics. POAA 4.73 15.1 15 ppb - 1000 ppb PFOSEA 24.2 77.1 80 ppb - 1000 ppb Monoester 58.0 185 MDL and LOQ are estimates only. No valid MDL was determinable from MDL study. Any quantitation performed for monoester will be an estimate only. Please refer to FACT-M-3.1 & 4.1-V-l for specifics. Please see Attachment C (LOQ Summary) and MDL study in FACT-M-3.1 & 4.1-V-l for specifics. Attachment A: Extraction worksheet FACT-M-3.1 Extraction o f PFOS from Serum or Other Fluid Page 13 o f 17 Ion Pairing Extraction o f Fluorochemicals from Serum and Analysis by API/MS(MS) Summary Table: Lim its o f Quantitation Approximate linear range2 Compound Matrix MDL LOQ Low std High std PFOS Rabbit 1.38 ppb 4.39 ppb 5 ppb 1000 ppb Bovine 2.11 ppb 6.70 ppb 25 ppb 1000 ppb _ Rat 1.27 ppb 4.04 ppb 10 ppb 1000 ppb Monkey n/d n/d 25 ppb 1000 ppb PFOSA Rabbit 2.23 ppb 7.09 ppb 10 ppb 1000 ppb Bovine 5.04 ppb 16.0 ppb 25 ppb 1000 ppb Rat 2.14 ppb 6.81 ppb 25 ppb 1000 ppb Monkey n/d n/d 25 ppb 1000 ppb PFOSAA Rabbit 2.84 ppb 9.04 ppb 10 ppb 1000 ppb Bovine 2.34 ppb 7.45 ppb 263 ppb 1000 ppb Rat - 2.32 ppb 7.38 ppb 10 ppb 1000 ppb Monkey n/d n/d 25 ppb 1000 ppb EtFOSE-OH Rabbit 3.90 ppb 12.4 ppb 15 ppb 1000 ppb - Bovine 11.3 ppb 35.8 ppb 50 ppb 1000 ppb * Rat 3.25 ppb 10.3 ppb 50 ppb 1000 ppb Monkey n/d n/d 10 ppb 1000 ppb POAA Rabbit 4.31 ppb 113.7 ppb 15 ppb 750 ppb Bovine 4.64 ppb 14.8 ppb 5 ppb 1000 ppb Rat 1.20 ppb 3.81 ppb 5 ppb 1000 ppb Monkey n/d n/d 5 ppb 1000 ppb PFOSEA Rabbit 1.03 ppb 3.48 ppb 25 ppb 1000 ppb Bovine 3.71 ppb 11.8 ppb 5 ppb 1000 ppb Rat 1.84 ppb 5.86 ppb 10 ppb 1000 ppb Monkey n/d n/d 5 ppb 1000 ppb Monoester1 Rabbit 149 ppb 474.0 ppb 250 ppb 1000 ppb Bovine 149 ppb 474.0 ppb 250 ppb 1000 ppb Rat 149 ppb 474.0 ppb 250 ppb 1000 ppb Monkey n/d n/d 100 ppb 1000 ppb 1. Values for monoester are estimates only. 2. H ighest standard (approx. 1 5 0 0 ppb) was excluded from final LCR and upper LOQ values due to poor R & R values and excessive weighting of the calibration curve. Compound: PFOS_________________________ Serum Prepared Range of LCR from Range of LCR from Range of LCR from matrix range of average ave curve low std low std high std high std standards curve curve curve curve curve (PPb)(ng/mL) (ppb)(ng/mL) (ppbXng/mL) (PPb)(ng/mL) (ppb)(ng/mL) (ppb)(ng/mL) (ppbXng/mL) Rabbit 4.93 - 1450 4.93 - 1450 49.3- 1000 49.3 - 97.6 4.93- 97.6 97.6 - 1450 97.6 - 1000 Bovine 4.93 - 1450 4.93 - 1450 97.6 - 1000 4.93 - 248 24.8-248 97.6 - 1450 97.6 - 1000 Rat Monkey 4.93 - 1450 4.93 - 976 24.8-976 4.93 - 1450 4.93 - 1450 24.8 - 1000 4.93 -248 24.8 -493 9.76 - 248 24.8 - 493 97.6 - 1450 248 - 1000 97.6 - 1450 97.6 1000 Attachment C: LOQ Summary FACT-M-3.1 Extraction of PFOS from Serum or Other Fluid Page 14 of 17 Compound: PFOSA Serum Prepared matrix range of standards (ppb)(ng/m L) Rabbit 5.00 - 1470 Range of average curve (ppb)(ng/m L) 5.00 - 1470 LCR from ave curve (ppb)(ng/m L) 9.89-1000 Range of low std curve (PPb)(ng/m L) 5.00-251 Bovine 5.00 - 1470 __ 5.00 - 1470 25.1 - 1000 5 .0 0 -9 8 .9 Rat 5.00 - 1470 5.00 - 1470 5 0 .0 -1 0 0 0 9.89 - 500 Monkey 5.00 - 1470 5.00 - 1470 98.9 - 1000 25.1 -5 0 0 LCR from low std curve (PPb)(ng/m L) n/a Range of high std curve (PPb)(ng/m L) 98.9 - 1470 LCR from high std curve (PPb)(ng/m L) 98.9 - 1000 n/a 9 8 .9 - 1470 9 8 .9 - 1 0 0 0 2 5 .1 -5 0 0 98.9 - 1470 98.9 - 1000 25.1-500 98.9- 1470 n/a Compound: PFOSAA Serum Prepared - Range of : matrix range of average standards curve (PPb)(ng/mL) (ppbXng/mL) Rabbit 5,20-1540 5.20-1540 LCR from ! ave curve (ppbXng/mL) 104-1000 Range of low std curve (ppbXng/mL) 5.20-263 LCR from low std : curve (ppbXng/mL) 10.4-263 Bovine 5 .2 0 - 1 5 4 0 5 .2 0 -1 5 4 0 263 -1 0 0 0 1 0 .4 -5 2 1 n/a (1) Rat 5 .2 0 - 1 5 4 0 5 .2 0 - 1540 1 0 4 -1 0 0 0 5.20 - 263 1 0 .4 -2 6 3 Monkey 5 .2 0 - 1540 5 .2 0 - 1540 52.4 - 1000 5 .2 0 -2 6 3 26.3 - 263 Range of high std curve (PPb)(ng/mL) 104-1540 1 0 4 - 1540 1 0 4 - 1540 104-1540 LCR from high std curve (ppbXng/mL) 263 -1 0 0 0 2 6 3 - 1000 2 6 3 - 1000 2 6 3 - 1000 Compound: EtFOSE-OH Serum Prepared Range of matrix range of average standards curve (ppb)(ng/mL) (ppb)(ng/mL) Rabbit 4.94 - 1450 4.94 - 1450 Bovine 4.94 - 1450 4.94 - 1450 Rat 4.94 - 1450 4.94 - 1450 Monkey 4.94 - 1450 4.94 -1450 LCR from ave curve (PPbXng/mL) 49.4-1000 97.8 - 1000 494-1000 97.8 - 1000 Range of low std curve (ppb)(ng/mL) 4.94 - 248 LCR from low std... curve (ppbXng/mL) 9.78 - 248 Range of high std curve (ppb)(ng/mL) LCR from high std curve (ppbXng/mL) 97.8 - 1450 n/a 4.94 - 248 4.94 - 248 4.94 - 248 : : n/a 4.94 - 248 9.78 - 248 97.8 - 1450 97.8 - 1450 97.8 - 1450 248 - 1000 97.8 1000 n/a Attachment C: LOQ Summary FACT-M-3.1 Extraction of PFOS from Serum or Other Fluid Page 15 of 17 Compound: POAA Serum Prepared matrix range of standards (ppb)(ng/m L) Rabbit 5.01 - 1480 Range of average curve (PPb)(ng/m L) 5.13-1510 LCR from ave curve (ppbXng/m L) 25.8 -1 0 0 0 Bovine 5.01 - 1480. 5 .1 3 -1 5 1 0 1 0 2 - 1000 Rat 5.01 - 1480 5 .1 3 -1 5 1 0 5 1 .3 -1 0 0 0 Monkey 5.01 - 1480 5 .1 3 -1 5 1 0 102 - 1000 Range of low std curve (PPb)(ng/m L) 5 .1 3 -2 5 8 5 .1 3 -2 5 8 5 .1 3 -1 0 2 5.13-102 LCR from low std curve (ppb)(ng/m L) n/a 5 .1 3 -2 5 8 5 .1 3 -1 0 2 5.13-102 Range of high std curve (PPb)(ng/m L) 102-1510 102-1510 102-1510 102-1510 LCR from high std curve (ppb)(ng/m L) n/a 258 - 1000 102-1000 258 -1 0 0 0 Compound: PFOSEA Serum Prepared " Range of matrix range of average standards curve (ppbXng/mL) (ppbXng/mL) Rabbit 5vl3- 1510 5.13-1510 LCR from ave curve (ppbXng/mL) 25.8 - 1000 Range of low std curve (ppbXng/mL) 5/13-258 LCR from low std curve (ppbXng/mL) n/a . Range of high std curve (ppbXng/mL) 102 - 1510 LCR from high std curve (ppbXng/mL) n/a Bovine 5 .1 3 -1 5 1 0 5 .1 3 -1 5 1 0 102 - 1000 5.13 - 2 5 8 5.13 - 2 5 8 102 - 1510 258 - 1000 Rat 5 .1 3 -1 5 1 0 5 .1 3 -1 5 1 0 5 1 .3 -1 0 0 0 5.13 - 102 5.13 - 1 0 2 102 - 1510 102 - 1000 Monkey 5.13 - 1510 5.13 - 1510 102 - 1000 5.13 - 1 0 2 5.13 - 1 0 2 102 - .1510 258 - 1000 Compound: Monoester Serum Prepared Range of matrix range of average standards curve (ppbXng/mL) (ppbXng/mL) Rabbit 4.94 - 1450 9.78 - 978 LCR from ave curve (ppbXng/mL) n/a Bovine 4.94 - 1450 97.8 - 1450 n/a Rat 4.94 - 1450 248 - 1450 2 4 8 -1 0 0 0 Monkey 4.94 - 1450 49.4 - 1450 97.8 - 1000 In general, the chromatography for the monoester was very poor (broad peaks, high baseline). Curves for monoester in rabbit and bovine were unacceptable. Any quantitation performed with the monoester is only an estimate and should not be used for reliable, accurate data reporting. Attachment C: LOQ Summary FACT-M-3.1 Extraction of PFOS from Serum or Other Fluid Page 16 of 17 Ion Pair Standard Curves - Fluids Prep date(s): Standard number: Analyte(s): Equipment number: Sample matrix: Final solvent and TN: Blank fluid/identifier: Method/revision: Target analyte(s): FC mix std approx. 0:500 ppm: W398-641 FC mix std approx. 5.00 ppm: W398-640 FC mix std approx. 50.0 ppm: W398-639 Surrogate std approx. 17.71 ppm: W398-605 Actual concentrations of standards in the FC mix PFOS PFOSA PFOSAA EtFOSE- POAA - OH Std cone Std cone Std cone Std cone Std cone ug/mL ug/mL ug/mL ug/mL ug/mL PFOSEA Std cone ug/mL Monoeste r Std cone ug/rnL 0.500 0.500 0.507 .507 0.532 0.532 0.501 0.501 0.509 0.509 0.521 0.521 0.501 0.501 5.00 5.07 5.32 5.01 5.09 5.21 5.01 5.00 5.07 5.32 5.01 5.09 5.21 5.01 5.00 5.07 5.32 5.01 5.09 5.21 5.01 50.0 50.1 53.2 50.1 50.9 52.1 50.1 50.0 50.1 53.2 50.1 50.9 52.1 50.1 50.0 50.1 53.2 50.1 50.9 52.1 50.1 50.0 50.1 53.2 50.1 50.9 52.1 50.1 Calculaited concentrations o f standards in the sample m atrix PFOS Final cone ng/mL PFOSA PFOSAA EtFOSE POAA PFOSEA Monoester Final cone Final cone Final cone Final cone Final cone Std cone ng/mL ng/mL ng/mL ng/mL ng/mL ng/mL 4.93 5.00 9 .7 6 9 .8 9 24.8 25.1 49.3 50.0 97.6 98.9 248 251 493 500 735 746 976 989 5.24 4.94 5.01 5.13 10.4 9.78 9.93 10.2 26.3 24.8 25.2 25.8 52.4 49.4 50.1 51.3 104 97.8 99.3 102 263 248 252 258 524 494 501 513 782 737 749 766 1038 978 993 1017 4.94 9 .7 8 24.8 49.4 97.8 248 494 737 978 All All Am't spiked mL 0.010 0.020 0.005 0.010 0.020 0.005 0.010 0.015 0.020 Final Volume mL 1.015 1.025 1.010 1.015 1.025 1.010 1.015 1.020 1.025 Surrogate All Std cone Am't spiked ng/mL (mL) 2.64 0.005 Surrogate Final cone ng/mL 81.0 Validated ranges - approximate concentrations Sera PFOS PFOSA PFOSAA Rabbit 5-1000 ppb 10-1000 ppb 10-1000 ppb Bovine 25-1000 ppb 25-1000 ppb 263-1000 ppb Rat 10-1000 ppb 25-1000 ppb 10-1000 ppb Monkey Estimates only. Use values for Rabbit EtFOSE-OH 10-1000 ppb 5-1000 ppb 50-500 ppb POAA 10-750 ppb 5-1000 ppb 5-1000 ppb PFOSEA 25-1000 ppb 5-1000 ppb 5-1000 ppb Attachment D: Ion Pair Standard Curves FACT-M-3.1 Extraction of PFOS from Serum or Other Fluid Page 17 o f 17 3M Environmental Laboratory Method A nalysis o f P otassium Perfluorooctanesulfonate o r O th er F luorochem icals in Serum E xtracts Using H P L C -E lectrospray/M ass Spectrom etry Method Number: ETS-8-5.1 \ Author: Lisa Clemen, Robert Wynne Approved By: Laboratory Manager -- Adoption Date: 03/01/99 Revision Date: Hl&fil *1 C , Date Group Leader Date 3 nA ______________________________ Vn ^clin ical Reviewer 55' 7 O dh/ i l / m Date O o " 1^1 Scope and Application____________________________________________________ D lTf Scope: This method describes the analysis of serum extracts for fluorochemical surfactants &<D Q using HPLC-electrospray/mass spectrometry. 143.Applicable Compounds: Fluorochemical surfactants or other fluorinated compounds, or -- ^ other ionizable compounds. 1.3 Matrices: Rabbit, rat, bovine, monkey, and human serum, or other fluids as designated in the validation report. Word 6/95 ETS-8-5.1 Analysis of Serum Extract Using ES/MS Page 1 o f 9 2.0 Summary of M ethod 2.1 This method describes the analysis of fluorochemical surfactants extracted from serum or other fluids, using HPLC-electrospray/mass spectrometry, or similar system as appropriate. The analysis is performed by monitoring a single ion characteristic of a particular fluorochemical, such as the perfluorooctanesulfonate (PFOS) anion, m/z= 499. Additionally, samples may be analyzed using a tandem mass spectrometer to further verify the identity of a compound by detecting daughter ions of the parent ion. 3.0 D e fin it io n s_____________________________________________________________________________ 3.1 Atmospheric Pressure Ionization (API): The Micromass Quattro II triple quadrupole systems allow for various methods of ionization by utilizing various sources, probes, and interfaces. These include but are not limited to: Electrospray Ionization (ESI), Atmospheric Pressure chemicalTonization (APcI), Thermospray, etc. The ionization process in these techniques occurs at atmospheric pressure (i.e., not under a vacuum). 3.2 Electrospray Ionization (ES, ESI): a method of ionization performed at atmospheric pressures whereby ions in solution are transferred to the gas phase via tiny charged droplets. These charged droplets are produced by the application of a strong electrical field. 3.3 Mass Spectrometry, Mass Spectrometer (MS), Tandem Mass Spectrometer (MS/MS): The API Quattro II triple quadrupole systems are equipped with quadrupole mass selective detectors. Ions are selectively discriminated by mass to charge ratio (m/z) and subsequently detected. A single MS may be employed for ion detection or a series (MS/MS) for more specific fragmentation information. 3.4 Conventional vs. Z-spray probe interface: The latest models of Micromass Quattro II triple quadrupole systems (post 1998) utilize a "Z-spray" conformation. The spray emitted from a probe is orthogonal to the cone aperture. In the conventional conformation it is aimed directly at the cone aperture, after passing through a tortuous pathway in the counter electrode. Though the configuration is different, the methods of operation, cleaning, and maintenance are the same. However, Z-spray components and conventional components are not compatible with one another, but only with similar systems (i.e., Z-spray components are compatible with some other Z-spray systems, etc.) 3.5 Mass Lynx Software: System software designed for the specific operation of these Quattro II triple quadrupole systems. Currently MassLynx has Windows 95 and WindowsNT 4.0 versions. All versions are similar. For more details see the manual specific to the instrument (Micromass Quattro II triple quadrupole MassLynx or MassLynx NT User's Guide). 4.0 W a r n in g s a n d C a u t io n s____________________________________________________________ 4.1 Health and Safety Warnings: - 4.1.1 Use caution with the voltage cables for the probe. When engaged, the probe employs a voltage of approximately 5000 Volts. 4.1.2 When handling samples or solvents wear appropriate protective gloves, eyewear, and clothing. ETS-8-5.1 Analysis of Serum Extract Using ES/MS Page 2 of 9 4.2 Cautions: 4.2.1 Do not operate solvent pumps above capacity of 400 bar (5800 psi) back pressure. If the back pressure exceeds 400 bar, the HP 1100 will initiate automatic shutdown. 4.2.2 Do not run solvent pumps to dryness. 5.0 I n t e r fe r e n c e s._____________________________________________________________________ 5.1 To minimize interferences when analyzing samples, teflon should not be used for sample storage or any part of instrumentation that comes in contact with the sample or extract. 6.0 E q u ip m e n t __________________________________________________________ ___________________ 6.1 Equipment listed below may be modified in order to optimize the system. Document any modifications in the raw data as method deviations. 6.1.1 Micromass Quattro II triple quadrupole Mass Spectrometer equipped with an electrospray ionization source 6.1.2 , HP1100 low pulse solvent pumping system, solvent degasser, column compartment, and autosampler 7.0 S u p p l ie s a n d M a t e r ia l s______________________________________________________ _ 7.1 Supplies 7.1.1 High purity grade nitrogen gas regulated to approximately 100 psi (House air system) 7.1.2 HPLC analytical column, specifics to be determined by the analyst and documented in the raw data. 7.1.3 Capped autovials or capped 15 mL centrifuge tubes 8.0 R e a g e n t s a n d S t a n d a r d s______________________________________ ;_______________________ 8.1 Reagents 8.1.1 Methanol, HPLC grade or equivalent 8.1.2 Milli-QTM water, all water used in this method should be Milli-QTM water or equivalent, and may be provided by a Milli-Q TOC Plus system or other vendor 8.1.3 Ammonium acetate, reagent grade or equivalent 8.2 Standards 8.2.1 Typically two method blanks, two matrix blanks, and eighteen matrix standards are prepared during the extraction procedure. See ETS-8-4.1. 9.0 S a m pl e H a n d lin g ______________________________________________________________________ 9.1 Fresh matrix standards are prepared with each analysis. Extracted standards and samples are stored in capped autovials or capped 15 mL centrifuge tubes until analysis. ETS-8-5.1 Analysis of Serum Extract Using ES/MS Page 3 of 9 9.2 If analysis will be delayed, extracted standards and samples can be refrigerated at approximately 4 C, or at room temperature, until analysis can be performed. 10.0 Q u a l it y C o n t r o l _____________________________________________________________________ 10.1 Solvent Blanks, Method Blanks and Matrix Blanks 10.1.1 SolvenUblanks, method blanks and matrix blanks are prepared and analyzed with each batch to determine contamination or carryover. 10.1.2 Analyze a method blank and a matrix blank prior to each calibration curve. 10.2 Matrix Spikes 10.2.1 Matrix spikes are prepared and analyzed to determine the matrix effect on the recovery efficiency. 10.2.2 Matrix spike duplicates are prepared and analyzed to measure the precision and the recovery for each analyte. 10.2.3 Analyze a matrix spike and matrix spike duplicate per forty samples, with a ' minimum of 2 spikes per batch. 10.2.4 Matrix spike and matrix spike duplicate concentrations will fall in the mid-range of the initial calibration curve. Additional spike concentrations may fall in the lowrange of the initial calibration curve. 10.3 Continuing Calibration Verifications 10.3.1 Continuing calibration verifications are analyzed to verily the continued accuracy of the calibration curve. 10.3.2 Analyze a mid-range calibration standard after every tenth sample, with a minimum of one per batch. 11.0 C a l i b r a t i o n a n d S t a n d a r d i z a t i o n ________________________________________________________________________ 11.1 Analyze the extracted matrix standards prior to and following each set of extracts. The average of two standard curves will be plotted by linear regression (y = my + b), weighted 1/x, not forced through zero, using MassLynx or other suitable software. 11.2 If the curve does not meet requirements, perform routine maintenance or reextract the standard curve (if necessary) and reanalyze. 11.3 For purposes of accuracy when quantitating low levels of analyte, it may be necessary to use the low end of the calibration curve rather than the full range of the standard curve. Example: when attempting to quantitate approximately 10 ppb of analyte, generate a calibration curve consisting of the standards from 5 ppb to 100 ppb rather than the full range of the curve (5 ppb to 1000 ppb). This will reduce inaccuracy attributed to linear ' regression weighting of high concentration standards. ETS-8-5.1 Analysis of Serum Extract Usine ES/MS Page 4 of 9 12.0 Procedures 12.1 Acquisition Set up 12.1.1 Click on start button in the Acquisition Control Panel. Set up a sample list. Assign a filename using MO-DAY-last digit of year-sample number, assign a method (MS) for acquiring, and type in sample descriptions. 12.1.2 To create a method click on scan button in the Acquisition control panel and select SIR (Single Ion Recording) or MRM. Set Ionization Mode as appropriate and mass to 499 or other appropriate masses. A full scan is usually collected along with the SIRs. Save acquisition method. If MS/MS instruments are employed, additional product ion fragmentation information may be collected. See Micromass MassLynx GUIDE TO DATA ACQUISITION for additional information and MRM (Multiple Reaction Monitoring). 12.1.3 Typically the analytical batch run sequence begins with a set of extracted matrix standards and ends with a set of extracted matrix standards. 12.1.4 Samples are analyzed with a continuing calibration check injected after every tenth sample. Solvent blanks should be analyzed periodically to monitor possible analyte ' carryover and are not considered samples but may be included as such. 12.2 Using the Autosampler 12.2.1 Set up sample tray according to the sample list prepared in Section 12.1.1. 12.2.2 Set-up the HP1100/autosampler at the following conditions or at conditions the analyst considers appropriate for optimal response. Record actual conditions in the instrument logbook: 12.2.2.1 Sample size = 10 pL injection 12.2.2.2 Inject/sample = 1 12.2.2.3 Cycle time = 13.5 minutes 12.2.2.4 Solvent ramp = Time 0.00 min. 8.50 min. 11.0 min. 12.0 min. MeOH 40% 90% 90% 40% 2.0 mM Ammonium acetate 60% 10% 10% 60% 12.2.2.5 Press the "Start" button. 12.3 Instrument Set-up 12.3.1 Refer to ETS-9-24.0 for more details. 12.3.2 Check the solvent level in reservoirs and refill if necessary. ETS-8-5.1 Analysis of Serum Extract Using ES/MS Page 5 of 9 12.3.3 Check the stainless steel capillary at the end of the probe. Use an eyepiece to check the tip. The tip should be flat with no jagged edges. If the tip is found to be unsatisfactory, disassemble the probe and replace the stainless steel capillary. 12.3.4 Set HPLC pump to "On". Set the flow to 10 - 500 uL/min or as appropriate. Observe droplets coming out of the tip of the probe. Allow to equilibrate for approximately 10 minutes. 12.3.5 Turn oirthe nitrogen. A fine mist should be expelled with no nitrogen leaking around the tip of the probe. Readjust the tip of the probe if no mist is observed. 12.3.6 The instrument uses these parameters at the following settings. These settings may change in order to optimize the response: 12.3.6.1 Drying gas 250-400 liters/hour 12.3.6.2 ESI nebulizing gas 10-15 liters/hour 12.3.6.3 HPLC constant flow mode, flow rate 1 0 - 5 0 0 pL/min 12.3.6.4 Pressure <400 bar (This parameter is not set, it is a guide to ensure the HPLC is operating correctly.) 12.3.7 Carefully guide the probe into the opening. Insert probe until it will not go any further. Connect the voltage cables to the probe. 12.3.8 Print the tune page, with its parameters, and store it in the study binder with a copy taped into the instrument log. 12.3.9 Using the cross-flow counter electrode in the ES/MS source is recommended for the analysis of biological matrices. 12.3.10Click on start button in the Acquisition Control Panel (this may vary among MassLynx versions, see appropriate MassLynx USER'S GUIDE). Press the start button. Ensure start and end sample number includes all samples to be analyzed. 13.0 Data Analysis and Calculations___________________________________________ 13.1 Calculations: 13.1.4 Calculate matrix spike percent recoveries using the following equation: % Recovery = Observed Result - Background Result x 100 Expected Result 13.1.5 Calculate percent difference using the following equation: % Difference = Expected Cone. - Calculated Cone, x 100 Expected Cone. 13.1.6 Calculate actual concentration of PFOS, or other fluorochemical, in matrix - (pg/mL): (ng of PFOS calc, from std. Curve x Dilution Factor) x 1 pg (Initial Volume of matrix (mL) + mL of Surrogate Standard) 1000 ng Final Volume (mL) ETS-8-5.1 Analysis of Serum Extract Using ES/MS Page 6 of 9 14.0 Method Performance 14.1 Method Detection Limit (MDL) and Limit of Quantitation (LOQ) are method, analyte, and matrix specific. Please see ETS-8-4.1, Attachment B, for a listing of current validated MDL and LOQ values. 14.2 Solvent Blanks, Method Blanks, and Matrix Blanks 14.2.1 Solvent blanks, method blanks, and matrix blanks values are must be below the lowest standard in the calibration curve 14.3 Calibration Curves 14.3.1 The r2 value for the calibration curve must be 0.980 or better. 14.4 Matrix Spikes 14.4.1 Matrix spike percent recoveries are must be within 30% of the spiked concentration. 14.5 Continuing Calibration Verifications . t 14.5.1 Continuing calibration verification percent recoveries must be 30% of the spiked concentration. 14.6 If criteria listed in this method performance section isn't met, maintenance may be performed on the system and samples reanalyzed or other actions as determined by the analyst. Document all actions in the appropriate logbook. 14.7 If data are to be reported when performance criteria have not been met, the data must be footnoted on tables and discussed in the text of the report. 15.0 P o l l u t io n P r e v e n t io n and W a ste M a n a g e m e n t ________________________________ __ 15.1 Sample extract waste and flammable solvent is disposed in high BTU containers, and glass pipette waste is disposed in broken glass containers located in the laboratory. 16.0 R ec o r d s_______________________________________________________________________________ 16.1 Each page generated for a study must have the following information included either in the header or hand written on the page: study or project number, acquisition method, integration method, sample name, extraction date, dilution factor (if applicable), and analyst. 16.2 Print the tune page, sample list, and acquisition method from MassLynx to include in the appropriate study folder. Copy these pages and tape into the instrument runlog. 16.3 Plot the calibration curve by linear regression, weighted 1/x, then print these graphs and store in the study folder. 16.4 Print data integration summary, integration method, and chromatograms, from MassLynx, and store in the study folder. ETS-8-5.1 Analysis of Serum Extract Using ES/MS Page 7 of 9 16.5 Summarize data using suitable software (Excel 5.0) and store in the study folder, see Attachment A for an example of a summary spreadsheet. 16.6 Back up electronic data to appropriate medium. Record in study notebook the file name and location of backup electronic data. 17.0 T a b l e s . D ia g r a m s . F lo w c h a r t s, a n d V a l id a t io n D ata____________________________ 17.1 Attachment A: ETS-8-5.1 Data summary spreadsheet. 18.0 R e f e r e n c e s ___________________________________________________________________________ 18.1 FACT-M-4.1, "Extraction of Potassium Perfluorooctanesulfonate or Other Fluorochemical compounds from Serum for Analysis Using HPLC-Electrospray/Mass Spectrometry 18.2 ETS-9-24.0, "Operation and Maintenance of the Micromass Atmospheric Pressure Ionization/Mass Spectrometer Quattro II triple quadrupole Systems" 18.3 The validation report associated with this method is ETS-8-4.0 & 5.0-V-l. 19.0 A f f e c t e d D o c u m e n t s_________________________________________________________________ 19.1 ETS-8-4.1, "Extraction of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds from Serum for Analysis Using HPLC-Electrospray/Mass Spectrometry" 20.0 R e v isio n s_____________________________________________________________________________ Revision Number. 1 Reason For Revision Section 6.1.2 Clarification of HP 1100 system components. Section 11.1 Average of two curves, not standard values, are used for plotting linear regression and added the 1/x weighting of the curve. Section 12.2.2.4 Clarification of solvent ramp. Section 17.1 Changed from attachment B to A. Revision Date 04/02/99 ETS-8-5.1 Analysis of Serum Extract Using ES/MS Page 8 of 9 Laboratory Study # Study: Test Material: Matrix/Final Solvent: Method/Revision: - Analytical Equipment System Number: Instrument Software/Version: Filename: R-Squared Value: Slope: Y Intercept: Date of Extraction/Analyst* Date of Analysis/Analyst: Group Dose Sample# Concentration ug/mL Initial Vol. mL Dilution Factor Final Cone. ug/mL Slope: Taken from linear regression equation. Group/Dose: Taken from the study folder. Sample#: Taken from the study folder. Concentration (ug/mL): Taken from the MassLynx integration summary. Initial Volume (mL): Taken from the study folder. Dilution Factor: Taken from the study folder. Final Cone. (ug/mL): Calculated by dividing the initial volume from the concentration Attachment A: Summary Spreadsheet ETS-8-5.1 Analysis of Serum Extract Using ES/MS Page 9 of 9 3M Environmental Laboratory M ethod Analysis o f P otassium Perfluorooctanesulfonate o r O th er F luo roch em ica ls in Serum or O th er Fluid E xtracts U sing H P L C -E lectrospray/M ass Spectrom etry Method Number: FACT-M-4.1 Adoption Date: 4/22/98 Author: Lisa Clemen, Glenn Langenburg Revision Date: \o - \ -< % Approved By: Laboratory Manager IL a >- ja . Group Leader ---------- / < V / // Date V z -llW Date ! IJ^chnical Reviewer p _____ __ Date 1 > 3 f.O Scope and Application___________________________________________________ ill Scope: This method is for the analysis of extracts from serum or blood for fluorochemical surfactants using HPLC-electrospray/mass spectrometry. %:2 Applicable Compounds: Fluorochemical surfactants or other fluorinated compounds, or _j' other ionizable compounds, w T 3 Matrices: Rabbit, rat, bovine, or monkey serum and rat whole blood or milk curd. Word 6.0.1/95 FACT-M-4.1 Analysis of Serum or Fluid Extract Using ES/MS Page 1 o f 9 initial (i.|s|ni Date 2.0 Summary of Method__________________________________________ ____________ 2.1 This method describes the analysis of fluorochemical surfactants extracted from serum, whole blood, or milk curd using HPLC-electrospray/mass spectrometry, or similar system as appropriate. The analysis is performed by monitoring a single ion characteristic of a particular fluorochemical, such as the potassium perfluorooctanesulfonate (PFOS) anion, M/Z= 499. Samples may also be analyzed using an API/MS/MS system to further verify compound identification. . 3.0 Definitions__________________________________________________ _____________ 3.1 Atmospheric Pressure Ionization (API): The Micromass platform systems allow for various methods of ionization by utilizing various sources, probes, and interfaces. These include but are not limited to: Electrospray Ionization (ESI), Atmospheric Pressure chemical Ionization (APcI), Thermospray, etc. The ionization process in these techniques occurs at atmospheric pressure (i.e. not under a vacuum). 3.2 Electrospray Ionization (ES, ESI): a method of ionization performed at atmospheric pressure, whereby ionization occurs through the production of tiny charged droplets in a strong electrical field. 3.3 Mass Spectrometry, Mass Spectrometer (MS), Tandem Mass Spectrometer (MS/MS): The API platforms are equipped with quadrupole mass selective detectors. Ions are selectively discriminated by mass to charge ratio (m/z) and subsequently detected. A single MS may be employed for ion detection or a series (MS/MS) for more specific fragmentation information. 3.4 Conventional vs. Z-spray probe interface: The latest models of Micromass platform systems (post 1998) utilize a "Z-spray" conformation. The spray emitted from a probe is orthogonal to the cone aperture. In the conventional conformation it is aimed directly at the cone aperture, after passing through a tortuous pathway in the counter electrode. Though the configuration is different, the methods of operation, cleaning, and maintenance are the same. However, Z-spray components and conventional components are not compatible with one another, but only w ith sim ilar systems (i.e. Z-spray com ponents are com patible w ith other Z- spray systems, etc.) 3.5 Mass Lynx Software: System software designed for the specific operation of these platform systems. Currently MassLynx has Windows 95 and WindowsNT 3.1 versions. All versions are similar. For more details see the manual specific to the instrument (Micromass Platform II or Quattro II MassLynx or MassLynx NT USER'S GUIDE). 4.0 Warnings and Cautions____________________________________________________ 4.1 Health and Safety Warnings: - 4.1.1 Use caution with the voltage cables for the probe. The probe employs a voltage of approximately 5000 Volts. 4.1.2 When handling samples or solvents wear appropriate protective gloves, eyewear, and clothing. Word 6.0.1/95 FACT-M-4.1 Analysis of Serum or Fluid Extract Using ES/MS Page 2 of 9 4.2 Cautions: 4.2.1 Do not operate solvent pumps above capacity of 400 bar (5800 psi) back pressure. If the back pressure exceeds 400 bar, the HP 1100 will initiate automatic shutdown. 4.2.2 Do not run solvent pumps to dryness. 5.0 Interferences____________________________________________________________ 5.1 To minimize interferences when analyzing samples for perfluorooctanoate(POAA), teflon should not be used for sample storage or any part of instrumentation that comes in contact with the sample or extract. 6.0 Equipment - _________________________________________________ 6.1 Equipment listed below may be modified in order to optimize the system. - 6.1.1 Micromass Electrospray Mass Spectrometer 6.1.2 ' HP 1100 low pulse solvent pumping system and autosampler 7.0 Supplies and Materials____________________________________________________ 7.1 Supplies 7.1.1 High purity grade nitrogen gas regulated to approximately 100 psi 7.1.2 HPLC analytical column, specifics to be determined by the analyst 7.1.3 Capped autovials or capped 15 ml centrifuge tubes 8.0 Reagents and Standards___________________________________________________ 8.1 Reagents 8.1.1 Methanol, HPLC grade or equivalent 8.1.2 Milli-QTM water, all water used in this method should be Milli-QTM water and may be provided by a Milli-Q TOC Plus system 8.1.3 Ammonium acetate, reagent grade or equivalent 8.2 Standards 8.2.1 Typically one method blank, one matrix blank, and ten matrix standards are prepared during the extraction procedure. See FACT-M-3.1. 9.0 Sample Handling_________________________________________________________ 9.1 Fresh matrix standards are prepared with each analysis. Extracted standards and samples are stored in capped autovials or capped 15 ml centrifuge tubes until analysis. 9.2 If analysis will be delayed, extracted standards and samples can be refrigerated at approximately 4 C until analysis can be performed. FACT-M-4.1 Analysis of Serum or Fluid Extract Using ES/MS Page 3 of 9 10.0 Q u a l it y C o n t r o l 10.1 Method Blanks and Matrix Blanks 10.1.1 Analyze a method blank and a matrix blank prior to each calibration curve. 10.2 Matrix Spikes' - 10.2.1 Analyze a matrix spike and matrix spike duplicate per forty samples. With a minimum of 2 spikes per batch. 10.2.2 Expected spike concentrations will fall in the mid-range of the initial calibration curve. Additional spike concentrations may fall in the low-range of the initial calibration curve. 10.2.3 See Section 13 to calculate percent recovery. 10.3 Continuing Calibration Checks 10.3.1 Analyze a mid-range calibration standard after every tenth sample. If a significant change ( 30%) in peak area occurs, relative to the initial standard curve, stop the run. Only those samples analyzed before the last acceptable calibration standard will be used. The remaining samples must be reanalyzed. 10.3.2 See Section 13 to calculate percent difference. 11.0 Calibration and Standardization__________________________________________ 11.1 Analyze the extracted matrix standards prior to and following each set of extracts. The mean of two standard values, at each standard concentration, will be plotted by linear regression (r2)for the calibration curve using MassLynx or other suitable software. 11.2 The r2 value for the data should be 0.980 or greater. Lower values may be acceptable at the discretion o f the analyst and documented approval o f the Project Lead. 11.3 If the curve does not meet requirements, perform routine maintenance or reextract the standard curve (if necessary) and reanalyze. 11.4 For purposes of accuracy when quantitating low levels of analyte, it may be necessary to use the low end of the calibration curve rather than the full range of the standard curve. Example: when attempting to quantitate approximately 10 ppb of analyte, generate a calibration curve consisting of the standards from 5 ppb to 100 ppb rather than the full range of the curve (5 ppb to 1000 ppb). This will reduce inaccuracy attributed to linear regression weighting of high concentration standards. 12.0 Procedures______________________________________________________________ 12.1 Acquisition Set up 12.1.1 Click on start button in the Acquisition Control Panel. Set up a sample list. Assign a filename using letter-MO-DAY-last digit of year-sample number, assign a method (MS) for acquiring, and type in sample descriptions. FACT-M-4.1 Analysis of Serum or Fluid Extract Using ES/MS Page 4 of 9 12.1.2 To create a method click on scan button in the Acquisition control panel and select SIR (Single Ion Recording). Set Ionization Mode as appropriate and mass to 499 or other appropriate masses. A full scan is usually collected along with the SIRs. Save acquisition method. If MS/MS instruments are employed, additional product ion fragmentation information may be collected. See Micromass MassLynx GUIDE TO DATA ACQUISITION for additional information and MRM (Multiple Reaction Monitoring). 12.1.3 Typically the analytical batch run sequence begins with a set of extracted matrix standards and ends with a set of extracted matrix standards. 12.1.4 Samples are analyzed with a continuing calibration check injected after every tenth sample. Solvent blanks should be analyzed periodically to monitor possible analyte carryover and are not considered samples but may be included as such. 12.2 Using the Autosampler 12.2.1 Set up sample tray according to the sample list prepared in Section 12.1.1. _ 12.2.2 Set-up the HP1100/autosampler at the following conditions or at conditions the analyst considers appropriate for optimal response. Record actual conditions in the instrument logbook: 12.2.2.1 Sample size = 10 pL injection with a sample wash 12.2.2.2 Inject/sample = 1 12.2.2.3 Cycle time = 15 minutes 12.2.2.4 Solvent ramp = Time 0.00 min. 7.5 min. 11.0 min. 11.5 min. MeOH 45% 90% 90% 45% 2.0 mM Ammonium acetate 55% 10% 10% 55% Note: In this instrument configuration, the run must be set up on the electrospray software with a "Waiting for inlet start" message before the "Start:" button is pressed on the HP Workstation. 12.2.2.5 Press the "Start" button. 12.3 Instrument Set-up 12.3.1 Refer to FACT-EP-3.0 for more details. 12.3.2 Check the solvent level in reservoirs and refill if necessary. 12.3.3 Check the stainless steel capillary at the end of the probe. Use an eyepiece to check the tip. The tip should be flat with no jagged edges. If the tip is found to be unsatisfactory, disassemble the probe and replace the stainless steel capillary. FACT-M-4.1 Analysis of Serum or Fluid Extract Using ES/MS Page 5 of 9 12.3.4 Set HPLC pump to "On". Set the flow to 10 - 500 uL/min or as appropriate. Observe droplets coming out of the tip of the probe. Allow to equilibrate for approximately 10 minutes. 12.3.5 Turn on the nitrogen. A fine mist should be expelled with no nitrogen leaking around the tip of the probe. 12.3.6 The instrument uses these parameters at the following settings. These settings may change in order to optimize the response: . 12.3.6.1 Drying gas 250-400 liters/hour 12.3.6.2 ESI nebulizing gas 10-15 liters/hour 12.3.6.3 HPLC constant flow mode flow rate 10 - 500 p.L/min 12.3.6.4 Pressure <400 bar (This parameter is not set, it is a guide to ensure the HPLC is operating correctly.) 12.3.7 Carefully guide the probe into the opening. Insert probe until it will not go any further. Connect the voltage cables to the probe. 12.3.8 Record tune parameters in the instrument log. t 12.3.9 Using the cross-flow counter electrode in the ES/MS source is recommended for the analysis of biological matrices. 12.3.10Click on start button in the Acquisition Control Panel (this may vary among MassLynx versions, see appropriate MassLynx USER'S GUIDE). Press the start button at top of sample list. Ensure start and end sample number includes all samples to be analyzed. 13.0 Data Analysis and Calculations___________________________________________ 13.1 Calculations: 13.1.4 Calculate matrix spike percent recoveries using the following equation: % Recovery = Observed Result - Background Result x 100 Expected Result 13.1.5 Calculate percent difference using the following equation: % Difference = Expected Cone. - Calculated Cone, x 100 Expected Cone. 13.1.6 Calculate actual concentration of PFOS, or other fluorochemical, in matrix (pg/ml): fag of PFOS calc, from std. Curve x Dilution Factor) x 1 ug (Initial Volume of matrix (ml) + ml of Surrogate Standard! 1000 ng Final Volume (mL) 14.0 M e t h o d P er fo r m a n c e _______________________________________________________________ 14.1 Method Detection Limit (MDL) and Limit of Quantitation (LOQ) are method, analyte, and matrix specific. Please see FACT-M-3.1, Attachment A for a listing of current validated MDL and LOQ values. FACT-M-4.1 Analysis of Serum or Fluid Extract Using ES/MS Page 6 of 9 14.2 Method Blanks and Matrix Blanks 14.2.1 Method blanks and matrix blanks will be analyzed with each sample set for possible contamination or carryover. Values are expected to fall below the lowest standard in the calibration curve. 14.3 Matrix Spikes 14.3.1 Matrix spikes are analyzed with each sample set and the percent recoveries are expected to fall within 30% of the spiked concentration. 14.4 Continuing Calibration Checks 14.4.1 Continuing calibration checks are analyzed at a minimum of after every 10 samples with each sample set. The percent recoveries are expected to fall within 30% of the spiked concentration. 14.5 If any criteria listed in the method performance section isn't met, maintenance may be - performed on the system and samples reanalyzed or other actions as determined by the analyst. All actions will be documented in the instrument runlog, the maintenance log, or on the summary sheet with the sample results. 15.0 Pollution Prevention and Waste Management_____________________________ 15.1 Sample extract waste and flammable solvent is disposed in high BTU containers, and glass pipette waste is disposed in broken glass containers located in the laboratory. 16.0 R e c o r d s__________________________________________________________________ 16.1 Store chromatograms in the study or project folder. Each chromatogram must have the following information included either in the header or hand written on the chromatogram: study or project number, acquisition method, integration method, sample name, extraction date, dilution factor (if applicable), and analyst. 16.2 Plot calibration curve by linear regression and store in the study folder. 16.3 Print sample list from MassLynx and tape into the instrument runlog. 16.4 Print data integration summary from MassLynx and tape into the instrument runlog. 16.5 Copy instrument runlog pages, including instrument parameters and sample results, and store in appropriate study folder. 16.6 Summarize data using suitable software and store in the study folder. 16.7 Back up electronic data to appropriate medium. Record in study notebook the file name and location of backup electronic data. 17.0 Tables, Diagrams, Flowcharts, and Validation Data_______________________ 17.1 Attachment A: FACT-M-4.1 Data reporting spreadsheet 17.2 The validation report associated with this method is FACT-M-3.1 & 4.1-V-l. FACT-M-4.1 Analysis of Serum or Fluid Extract Using ES/MS Page 7 of 9 18.0 References 18.1 FACT-EP-3.0, "Operation and Maintenance of the Micromass Atmospheric Pressure Ionization/Mass Spectrometer Platform Systems" 19.0 A f fe c t e d D o c u m e n t s_________________________________________________________________ 19.1 FACT-M-3.1, "Extraction of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds from Serum or Fluid for Analysis Using HPLC-Electrospray/Mass Spectrometry" 20.0 R e v isio n s______________________________________________________________________________ Revision Number. 1 - _ . Reason For Revision Validation o f method to include 7fluo rochemicals addition o f whole blood matrix, surrogate standard, new API/MS(MS) systems, monkey sera cross validation, M D L study, updates in record keeping and storing policies, etc. . Revision Date 07/01/98 FACT-M-4.1 Analysis of Seram or Fluid Extract Using ES/MS Page 8 of 9 Attachment A Laboratory Study # Study: Test Material: Matrix/Final Solvent: Method/Revision: _ Analytical Equipment System Number: Instrument Software/Version: Filename: R-Squared Value: Slope: Y Intercept: Date of Extraction/Analyst; . Date of Analysis/Analyst: Group Dose Sample# Concentration ug/mL Initial Voi. mL . Dilution Factor Final Cone. ug/mL Slope: Taken from linear regression equation. Group/Dose: Taken from the study folder. Sample#: Taken from the study folder. Concentration (ug/mL): Taken from the MassLynx integration summary. Initial Volume (mL): Taken from the study folder. Dilution Factor: Taken from the study folder. Final Cone. (ug/mL): Calculated by dividing the initial volume from the concentration FACT-M-4.0 Analysis of Serum or Fluid Extract Using ES/MS Page 9 of 9 3M Environmental Laboratory Method E x t r a c t io n -o f P o tassium Per flu o r o o c ta n e su lfo n a te o r o th er FLUOROCHEMICAL COMPOUNDS FROM URINE FOR ANALYSIS USING HPLC- Electrospray/Mass Spectrometry/Mass S pectrometry Method Number: ETS-8-96.0 Adoption Date: Author: Lisa Clemen, Glenn Langenburg Revision Date: f \ j ( \ Approved By: )^yj Laboratory Manager ~)Hh? Date 1--------Group Leader Date ( A CluYvfi^v Technical Reviewer Date m 2oX**0! ~.<jP--S--c-o--p--e--a--n d A--p-p--l--ic--a--t-i-o--nScope: This method is for the extraction of potassium perfluorooctanesulfonate (PFOS) o or other fluorochemical compounds from urine. .2l .T Applicable compounds: Fluorochemicals or other fluorinated compounds. Matrices: Human, rat, and monkey urine or other fluids as designated in the validation .o report. co Word 6/95 ETS-8-96.0 Extraction of PFOS from Urine Page 1 of 14 ^^ Initial Is / s ) 0 \ Date 2.0 S u m m a r y o f M e t h o d 2.1 This method describes the procedure for extracting potassium perfluorooctanesulfonate (PFOS) or other fluorochemicals from urine, or other fluids, using an ion pairing reagent and methyl-terl-butyl ether (MtBE). In this method, eight fluorochemicals are extracted: PFOS, PFOSA, PFOSAA, EtFOSE-OH, M556, M570, perfluorooctanoate (POAA), and surrogate standard (see 3.0 Definitions). An ion pairing reagent is added to two ml of sample and the analyte ion pair is partitioned into MtBE. The MtBE extract is removed and put onto a nitrogen evaporator until dry. Each extract is reconstituted in 0.5 ml of methanol, then filtered through a 0.2 pm nylon filter attached to 3 cc plastic syringe into glass autovials. 2.2 These sample extracts are analyzed following method ETS-8-97.0 or other appropriate method. _ 3.0 D e fin it io n s_____________________________________________________________________________ 3.1 PFOS: perfluorooctanesulfonate (anion of potassium salt) C8F17S 0 3' 3.2 PFOSA: perfluorooctane sulfonylamide C8F17S 0 2NH2 3.3 PFOSAA: perfluorooctane sulfonylamido (ethyl)acetate C8F 17S 0 2N(CH2CH3)CH2C 0 2' 3.4 EtFOSE-OH: 2(N-ethylperfluorooctane sulfonamido)-ethyl alcohol C8F17S 0 2N(CH2CH3)CH2CH20H 3.5 M556: C8F17S 0 2N(H)(CH2C 00H ) 3.6 M570: C8F17S 0 2N(CH3)CH2C 00 H 3.7 POAA: perfluorooctanoate C7F15C 0 0 ` 3.8 Surrogate standard THPFOS: 1H-1H-2H-2H perfluorooctane sulfonic acid, used as an internal standard in this method. 4.0 W a r n in g s a nd C a utio ns_______________________________________________________________ 4.1 H ealth and safety warnings 4.1.1 Use universal precautions, especially laboratory coats, goggles, and gloves when handling animal tissue, which may contain pathogens. 5.0 I n te r fe r e n c e s______________________ __________________________________________________ 5.1 At this time, it is unknown how the extraction method is affected by potential interferences that may be present such as conjugated fluorochemicals (eg. Glucuronides). Conjugates may become deconjugated during extraction or analysis resulting in a high bias for reported results of target analytes. ETS-8-96.0 Extraction of PFOS from Urine Page 2 o f 14 6.0 Equipment 6.1 The following equipment is used while performing this method. Equivalent equipment is acceptable. 6.1.1 Vortex mixer, VWR, Vortex Genie 2 6.1.2 Centrifuge, Mistral 1000 or IEC 6.1.3 Shaker, Eberbach or VWR . 6.1.4 Nitrogen evaporator, Organomation 6.1.5 Balance ( 0.100 g) 7.0 Supplies and Materials________________________________________________ _ 7.1 Gloves _ 7.2 Eppendorf or disposable pipettes 7.3 Nalgene bottles, capable of holding 250 ml and 1 L 7.4 Volumetric flasks, glass, type A 7.5 I-CHEM vials, glass, 40 ml glass 7.6 Centrifuge tubes, polypropylene, 15 ml 7.7 Labels 7.8 Oxford Dispenser - 3.0 to 10.0 ml 7.9 Syringes, capable of measuring 2.5 pL to 50 pL 7.10 Graduated pipettes 7.11 Syringes, disposable plastic, 3 cc 7.12 Syringe filters, nylon, 0.2 pm, 25 mm 7.13 Timer 7.14 Crimp cap autovials and caps 7.15 Crimpers Note: Prior to using glassware and bottles, rinse 3 times with methanol and 3 times with Milli-QTM water. Rinse glass syringes a minimum of 9 times with methanol, 3 rinses from 3 separate vials. 8.0 Reagents and Standards________________________________________________________ 8.1 Type I reagent grade water, Milli-QTM or equivalent; all water used in this method should be Milli-QTM water and may be provided by a Milli-Q TOC PlusTM system 8.2 Sodium hydroxide (NaOH), J.T Baker or equivalent 8.3 Tetrabutylammonium hydrogen sulfate(TBA), Kodak or equivalent 8.4 Sodium carbonate (NajCOj), J.T. Baker or equivalent 8.5 Sodium bicarbonate (NaHC03), J.T. Baker or equivalent ETS-8-96.0 Extraction of PFOS from Urine Page 3 o f 14 8.6 Methyl-T-Butyl Ether, Omnisolv, glass distilled or HPLC grade 8.7 Methanol, Omnisolv, glass distilled or HPLC grade 8.8 Urine frozen from supplier 8.9 Fluorochemical standards 8.9.1 PFOS (3M Specialty Chemical Division), molecular weight = 538 8.9.2 PFOSA (3M Specialty Chemical Division), molecular weight = 499 8.9.3 PFOSAA (3M Specialty Chemical Division), molecular weight = 585 8.9.4 EtFOSE-OH (3M Specialty Chemical Division), molecular weight = 570 8.9.5 M556 (3M Specialty Chemical Division), molecular weight = 557 8.9.6 M570 (3M Specialty Chemical Division), molecular weight = 571 8.9.7 POAA (3M Specialty Chemical Division), molecular weight = 452 8.9.8 THPFOS (1-H,1-H, 2-H, 2-H C8F13S 0 3H) molecular weight = 428 8.9.9 Other fluorochemicals, as appropriate 8.10 Reagent preparation NOTE: When preparing larger volumes than listed in reagent, standard, or surrogate preparation, adjust accordingly. 8.10.1 ION sodium hydroxide (NaOH): Weigh approximately 200 g NaOH. Pour into a 1000 ml beaker containing 500 ml Milli-QTM water, mix until all solids are dissolved. Store in a 1 L Nalgene bottle. 8.10.2 1 N sodium hydroxide (NaOH): Dilute 10N NaOH 1:10. Measure 10 ml of 10 N NaOH solution into a 100 ml volumetric flask and dilute to volume using MilliQTM water. Store in a 125 ml Nalgene bottle. 8.10.3 0.5 M tetrabutylammonium hydrogen sulfate (TBA): Weigh approximately 169 g of TBA into a 1 L volumetric containing 500 ml Milli-QTM water. Adjust to pH 10 u sin g ap p ro x im ately 4 4 to 5 4 m l o f 10 N N aO H (W h ile adding the last m l o f NaOH, add slowly because the pH changes abruptly). Dilute to volume with Milli-QTM water. Store in a 1 L Nalgene bottle. 8.10.3.1 TBA requires a check prior to each use to ensure pH = 10.0. Adjust as needed using 1 N NaOH solution. 8.10.4 0.25 M sodium carbonate/sodium bicarbonate buffer (NajCO/NaHCO.,): Weigh approximately 26.5 g of sodium carbonate (Na^O-,) and 21.0 g of sodium bicarbonate (NaHC03) into a 1 L volumetric flask and bring to volume with MilliQTM water. Store in a 1 L Nalgene bottle. 8.11 Standards preparation 8.11.1 Prepare PFOS standards for the standard curve. 8.11.2 Prepare other fluorochemical standards, as appropriate. Multicomponent fluorochemical standards are acceptable (for example, one working standard solution containing 1.00 ppm PFOS, 1.02 ppm PFOSA, 0.987 ppm PFOSAA, and 1.10 ppm EtFOSE-OH.) ETS-8-96.0 Extraction of PFOS from Urine Page 4 of 14 8.11.3 Weigh approximately 100 mg of PFOS into a 100 ml volumetric flask and record the actual weight in the Standard Logbook. 8.11.4 Bring to volume with methanol for a stock standard of approximately 1000 ppm (pg/ml). 8.11.5 Dilute the stock solution with methanol for a working standard 1 solution of approximately 50 ppm. 8.11.6 Dilute'working standard 1 with methanol for a working standard 2 solution of approx. 5.0 ppm. 8.11.7 Dilute working standard 1 with methanol for a working standard 3 solution of approx. 0.50 ppm. 8.12 Surrogate stock standard preparation 8.12.1 Weigh approximately 50-60 mg of surrogate standard 1-H,1-H, 2-H, 2-H, C8FI3S 0 3H into a 50 ml volumetric flask and record the actual weight. 8.12.2 Bring to volume with methanol for a surrogate stock of approximately 1000-1200 ppm. 8.12.3 Prepare a surrogate working standard. Transfer approximately 1 ml of surrogate stock to a 10 ml volumetric flask and bring to volume with methanol for a working standard of 100-120 ppm. Record the actual volume transferred in the Standard Logbook. 9.0 Sample Handling__________________________________________________________ 9.1 All samples are received frozen and must be kept frozen until the extraction is performed. 9.2 Allow samples to thaw to room temperature prior to extraction. 10.0 Quality Control_________________________________________________________ 10.1 Solvent Blanks, Method blanks and matrix blanks 10.1.1 A n aliquot o f 2 .0 m l m ethanol is used as a solvent blank. 10.1.2 Extract two 2.0 ml aliquots of Milli-QTM water following this procedure and use as method blanks. 10.1.3 Extract two 2.0 ml aliquots of the urine following this procedure and use as matrix blanks. See 11.1.4. 10.2 Matrix spikes 10.2.1 Prepare and analyze matrix spike and matrix spike duplicate samples to determine the accuracy of the extraction. 10.2.2 Prepare each spike using a sample chosen by the analyst, usually the control matrix received with each sample set. 10.2.3 Expected concentrations should fall in the mid-range of the initial calibration curve. Additional spikes may be included and may fall in the low-range of the initial calibration curve. ETS-8-96.0 Extraction of PFOS from Urine Page 5 of 14 10.2.4 Prepare one matrix spike and matrix spike duplicate per 40 samples, with a minimum of 2 matrix spikes per batch. 10.3 Continuing calibration verifications 10.3.1 Prepare continuing calibration verification samples to ensure the accuracy of the initial calibration curve. 10.3.2 Prepare, at a minimum, one continuing calibration verification per group of 10 samples. For example, if a sample set = 34, four checks are prepared and extracted. 10.3.3 Prepare each continuing calibration verification from the same matrix used to prepare the initial curve. 10.3.4 The expected concentrations will fall within the mid-range of the initial calibration curve. Additional spikes may be included that fall in the low-range of the initial calibration curve. This is necessary if the analyst must quantitate using only the low end of the calibration curve (for example, 5 ppb - 100 ppb, rather than 5 ppb - 1000 ppb). 11.0 Calibration and Standardization *____________________________________ 11.1 Prepare matrix calibration standards 11.1.1 Transfer 2.0 ml of urine to a 15 ml centrifuge tube. 11.1.2 Record each sample volume on the extraction sheet. 11.1.3 While preparing a total of twenty-two aliquots in 15 ml centrifuge tubes, mix or shake between aliquots. 11.1.4 Two 2.0 ml aliquots serve as matrix blanks. 11.1.5 Typically use the standard concentrations and spiking amounts listed in Table 1, at the end of this section, to spike, in duplicate, two standard curves, for a total of twenty standards, two matrix blanks, and two method blanks. 11.1.6 Refer to validation report FACT-TOX-131, W 2067, which lists the working ranges and the L inear C alibration R ange (L C R ) for calibration curves. 11.1.7 Use Attachment D as an aid in calculating the concentrations of the working standards. See Section 13.0 to calculate actual concentrations of PFOS in calibration standards. 11.2 To each standard, blank, or continuing check, add appropriate amount of surrogate working standard for the concentration to fall within the calibration curve range 10 ppb - 1500 ppb. 11.3 Extract spiked matrix standards following 12.6-12.16 of this method. Use these standards to establish each initial curve on the mass spectrometer. ETS-8-96.0 Extraction of PFOS from Urine Page 6 o f 14 Table 1 Approximate spiking amounts for standards and spikes Using 2.0 ml of matrix Working standard p.L Approx, final cone, Approx, final cone. (approx, cone.) of analyte in matrix Of analyte in solvent -* - Blank - Blank 0.500 ppm 5 1.25 ppb 5.00 ppb 0.500 ppm 10 2.50 ppb 10.0 ppb 0.500 ppm 25 6.00 ppb 25.0 ppb 5.00 ppm 5 12.5 ppb 50.0 ppb 5.00 ppm 10 25.0 ppb 100 ppb 5.00 ppm 25 62.5 ppb 250 ppb 50.0 ppm 5 125 ppb 500 ppb 50.0 ppm 7.5 200 ppb 750 ppb 50.0 ppm 10 v 250 ppb 1000 ppb 50.0 ppm 15 375 ppb 1500 ppb 12.0 Procedure_______________________________________________________________ 12.1 Obtain frozen samples and allow to thaw at room temperature or in a lukewarm waterbath. 12.2 Vortex mix for 15 seconds, then transfer 2.0 ml or other appropriate volume to a 15 ml polypropylene centrifuge tube. 12.3 Return unused samples to freezer after extraction amounts have been removed. 12.4 Record the initial volume on the sample weight/volume worksheet.. See Attachment D. The original weight/volume worksheet is included in the study binder. 12.5 Label the tube with the study number, sample ID, date and analyst initials. See attached w orksheet for docum enting the rem aining steps. 12.6 Spike all samples, including blanks and standards, ready for extraction with surrogate standard as described in 11.2. 12.7 Spike each matrix with the appropriate amount of standard is described in 11.1, or Table 1 in that section, for the calibration curve standards. Also prepare matrix spikes and continuing calibration standards. 12.8 Vortex mix the standard curve samples, matrix spike samples, and continuing calibration samples for 15 seconds. 12.9 Check to ensure the 0.5 M TBA reagent is at pH 10. If not, adjust accordingly. 12.10 To each sample, add 1 ml 0.5 M TBA and 2 ml of 0.25M sodium carbonate/sodium bicarbonate buffer. 12.11 Using an Oxford Dispenser, add 5 ml methyl-tert-butyl ether. 12.12 Cap each sample and put on the shaker at a setting of 300 rpm, for 20 minutes. ETS-8-96.0 Extraction of PFOS from Urine Page 7 of 14 12.13 Centrifuge for 20 to 25 minutes at a setting of 3500 rpm, or until layers are separated. 12.14 Label a fresh 15 ml centrifuge tube with the same information as in 12.5. 12.15 Remove 4.0 ml of the organic layer to this clean 15 ml centrifuge tube. 12.16 Put each sample on the analytical nitrogen evaporator until dry, approximately 30 to 60 minutes. 12.11 Add 0.5 ml of methanol to each centrifuge tube using a graduated pipette. If excessive residue is present, add the methanol and allow the extract to sit for 30 minutes prior to vortexing. 12.17 Vortex mix for 30 seconds. 12.18 Label the autovial with the study number, animal number and gender, sample timepoint, matrix, final solvent, extraction date, fluorochemical components, extraction type, vial file archive number, and analyst(s) performing the extraction. 12.19 Attach a 0.2 pm nylon mesh filter to a 3 cc syringe and transfer the sample to this syringe. Filter into a 1.5 ml glass autovial or low-volume autovial when necessary. 12.20 Cap and store extracts at room temperature or refrigerated at approximately 4 C until analysis. 12.21 Complete the extraction worksheet, attached to this document, and tape in the study notebook or include in study binder, as appropriate. 13.0 Data Analysis and Calculations__________________________________________ 13.1 Calculations 13.1.1 Calculate actual concentrations of PFOS, or other applicable fluorochemical, in calibration standards using the following equation: ml of standard x concentration of standard (ug /ml)__________________ = ml of standard + ml of surrogate standard + initial matrix volume (ml) Final Concentration (pg/ml) of PFOS in matrix 14.0 Method Performance____________________________________________________ 14.1 The method detection limit (MDL) is analyte and matrix specific. Refer to MDL report for specific MDL and limit of quantitation (LOQ) values (see Attachment B). 14.2 The following quality control samples are extracted with each batch of samples to evaluate the quality of the extraction and analysis. 14.2.1 Method blanks and matrix blanks. 14.2.2 Matrix spike and matrix spike duplicate samples to determine accuracy and ... precision of the extraction. 14.2.3 Continuing calibration check samples to determine the continued accuracy of the initial calibration curve. 14.3 Refer to section 14 of ETS-8-97.0 for method performance criteria. ETS-8-96.0 Extraction of PFOS from Urine Page 8 o f 14 15.0 Pollution Prevention and Waste Management 15.1 Human and monkey sample waste is disposed in infectious biohazard waste containers, all other sample waste is disposed in noninfectious biohazard waste containers. Flammable solvent waste is disposed in high BTU containers. Used glass pipette waste is disposed in broken glass containers located in the laboratory. 16.0 Records_________________________________________________________________ 16.1 Complete the extraction worksheet attached to this method, and tape in the study notebook or include in the 3-ring study binder, as appropriate. 17.0 Tables. Diagrams. Flowcharts, and Validation Date_______________________ 17.1 Attachment A, Extraction worksheet 17.2 Attachment B, MDL/LOQ values and summary 17.3 Attachment C, Calibration standard concentration worksheet 17.4 Attachment D, Sample weight/volume worksheet 18.0 References_______________________________________ :______________________ 18.1 The validation report associated with this method is FACT-TOX-131, W2067. 19.0 Affected Documents_____________________________________________________ 19.1 ETS-8-97.0, "Analysis of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds in Urine Extracts Using HPLC-Electrospray Mass Spectrometry/Mass Spectrometry" 20.0 Revisions_______________________________________________________________ Revision Number Reason For Revision Revision Date ETS-8-96.0 Extraction of PFOS from Urine Page 9 o f 14 Extraction Worksheet ETS-8-96.0 Study # Matrix Box # Wk/Dav DateSpiked/Analyst CCV MS MSD Surrogate Std Approx, ppm Actual ppm # FC-Mix approx. 0.5 ppm actual ppm # - FC-Mix approx. 5 ppm actual ppm # - FC-Mix approx. 50 ppm actual ppm # Comments - -- - --' - - - -- - -- -- - -- -- - -- - -- - -- - -- - -- - -- - -- Blank______________________Std #_____________________ amount =_____________ ml Urine Extraction Method_____________________:_____________________________________ Date & Initials Vortex 15 sec._____________________________________________________________________________ Pipette M atrix . spike with appropriate surrogate or FC-M ix Volume__________________ m]___________________________________ Pipette 1 ml of 0.5 M TBA, pH 10. pH= Std. # _________________ Pipette 2 ml of0.25 Na2COy0.25M NaHCO^ buffer________________ Std. # _________________ Dispense 5 ml ofmethyl-t-butyl ether_________________________ TN-A- _____________________ Shake 20 min.__________________________________ Shaker speed:__________________________________ Centrifuge 20-25 min.__________________________ Centrifuge speed:__________________________________ Remove a 4 ml aliquot oforganic layer_____________________________________________________________ Put on Nitrogen Evaporator to dryness__________________ Temperature:__________________________________ Add methanol__________ Volume ml__________ TN-A- ________________________ Vortex 30 sec. __________________________________________________________________________ Filter using a 3cc B-D syringe with a0.2um filter into an autosample vial______________________________________ Cont. Cal. Verifications used same matrix as for std curve. Attachment A ETS-8-96.0 -i D P O Q frrttr T T rm o Page 10 of 14 MDL/LOQ values for human urine Compound MDL LOQ Linear Calibration Range (LCR) PFOS POAA (PPb) 4.6 22.9 (PPb) 14.7 15 ppb - 1500 ppb (in final MeOH extract) 72.9 50 ppb - 1500 ppb (in final MeOH extract) PFOSA n/d PFOSAA n/d. EtFOSE-OH n/d M556 n/d M570 n/d n/d = not determined n/d 25 ppb -1000 ppb (in final MeOH extract) n/d 25 ppb - 1000 ppb (in final MeOH extract) n/d 25 ppb - 1000 ppb (in final MeOH extract) n/d 25 ppb - 1000 ppb (in final MeOH extract) n/d 25 ppb - 1000 ppb (in final MeOH extract) NOTE: to calculate MDL, LOQ, and LCR values in ug/ml of urine divide the above values by 4. MDL/LOQ values in rat and monkey urine were not statistically determined. Two curves in each of these matrices were extracted and analyzed with the human urine curves to determine equivalence. Responses in the rat and monkey were similar to the human responses, therefore, their MDL and LOQ are assumed to be similar to the values determined for human urine. If a suitable amount of clean, control matrix is available, samples will be evaluated versus a curve extracted from urine originating from the same species as the specimens. Please see LOQ Summary and MDL study in FACT-TOX-131, W2067 for further information. Attachment B: MDL/LOQ Summary ETS-8-96.0 Extraction of PFOS from Urine Page 11 o f 14  Compound: PFOS Prepared range of Human Urine standards (ppb) (ng/ml) Full Range n/d LCR from curve (ppb) (ng/ml) n/d % Recovery Range n/d Low Curve n/d n/d n/d High curve n/d n/d n/d 1/X 2.5ppb -15 00 ppb 15 ppb - 1500 ppb 75-116 Compound: POAA Prepared range of Human Urine standards (ppb) (ng/ml) Full Range n/d LCR from curve (ppb) (ng/ml) n/d % Recovery Range n/d Low Curve n/d n/d n/d High curve 1/X, quadratic n/d n/d 2.5ppb - 1500 ppb 50 ppb - 1500 ppb n/d 86-105 RSD Range n/d n/d n/d +/- 30% RSD Range n/d n/d n/d +/- 30% Attachment B: MDL/LOQ Summary ETS-8-96.0 Extraction of PFOS from Urine Page 12 of 14 Ion Pair Standard Curves - Urine Prep date(s): Standard number: Analyte(s): Equipment number: Sample matrix: Final solvent and TN: Blank fluid/identifier: Method/revision: Box Number: Target analyte(s): FC mix std approx. 0.500 ppm: FC mix std approx. 5.00 ppm: FC mix std approx. 50.0 ppm: Surrogate std approx. 100 ppm: Actual concentrations of standards in the FC mix PFOS PFOSA PFOSAA EtFOSE POAA M556 M570 Std cone Std cone Std cone Std cone Std cone Std cone Std cone ug/ml ug/ml ug/ml ug/ml ug/ml ug/ml ug/ml 0.50 0.501 0.500 0.501 0.499 0.500 0.501 0.500 0.501 0.500 0.501 0.499 0.500 0.501 0.500 0.501 0.500 0.501 0.499 0.500 0.501 5.00 5.01 5.00 5.01 4.99 5.00 5.01 5.00 5.01 5.00 5.01 4.99 5.00 5.01 5.00 5.01 5.00 5.01 4.99 5.00 5.01 50.0 50.1 50.0 50.1 49.9 50.0 50.1 50.0 50.1 50.0 50.1 49.9 50.0 50.1 50.0 50.1 50.0 50.1 49.9 50.0 50.1 50.0 50.1 50.0 50.1 49.9 50.0 50.1 All Am't spiked ml 0.005 0.010 0.025 0.005 0.010 0.025 0.005 0.0075 0.010 0.015 All Final vol ml 2.0075 2.0125 2.0275 2.0075 2.0125 2.0275 2.0075 2.0100 2.0125 2.0130 Calculated concentrations of standards in the sample matrix PFOS PFOSA PFOSAA EtFOSE POAA M556 Final cone Final cone Final cone Final cone Final cone Final cone ng/ml ng/ml ng/ml ng/ml ng/ml ng/ml 1.25 1.25 1.25 1.25 1.24 1.25 2.48 2.49 2.48 2.49 2.48 2.48 6.17 6.18 6.17 6.18 6.15 6.17 12.5 12.5 12.5 12.5 12.4 12.5 24.8 24.9 24.8 24.9 24.8 24.8 61.7 61.8 61.7 61.8 61.5 61.7 125 125 125 125 124 125 187 187 187 187 186 187 248 249 248 249 248 248 372 372 372 372 371 372 M570 Final cone ng/ml 1.25 2.49 6.18 12.5 24.9 61.8 125 187 249 372 Surrogate Std cone ng/ml 100 Surrogate Final cone ng/ml 125 All Am't spiked ml 0.0025 Calculated concentrations of standards in methanol extract (0.5 ml final volume) PFOS PFOSA PFOSAA EtFOSE POAA M556 M570 Final cone Final cone Final cone Final cone Final cone Final cone Final cone ng/ml ng/ml ng/ml ng/ml ng/ml ng/ml ng/ml 5.00 5.01 5.00 5.01 4.99 5.00 5.01 10.0 10.0 10.0 10.0 10.0 10.0 10.0 25.0 25.1 25.0 25.1 25.0 25.0 25.1 50.0 50.1 50.0 50.1 49.9 50.0 50.1 100 100 100 100 100 100 100 250 251 250 251 250 250 251 500 501 500 501 499 500 501 750 752 750 752 749 750 752 1000 1002 1000 1002 998 1000 1002 1500 1503 1500 1503 1497 1500 1503 Surrogate Std cone ng/ml 100 Surrogate Final cone ng/ml 500 All Am't spiked ml 0.0025 Attachment C: Standard Calculation Sheet ETS-8-96.0 Extraction of PFOS from Urine Page 13 o f 14 Prep Date(s): Analyst(s): Sample Matrix: Method/Revision: Target Analyte(s): Sample ID Initial Wt/VoL g/mL/L Study Number: Equipment Number: Final Solvent & TN Number: Matrix Blank/Identifier: Box: Comments Summary of method: Notes: Attachment D: Weight/Volume Sheet ETS-8-96.0 Extraction of PFOS from Urine Page 14 of 14 3M Environm ental Laboratory M ethod Analysis o f P otassium Perfluorooctanesulfonate o r O th er F lu o r o c h em ic a l C ompounds in Urine E xtracts U sing H P L C -E lectrospray/M ass Spectrom etry/M ass Spectrom etry Method Number: ETS-8-97.0 '-.vAq,Adoption Date: Author: Lisa Clemen, Robert Wynne, Glenn Langenburg Revision Date: Approved By: Laboratory Manager ----- 7^----------- * Group Leader 7 /^/? 5 Date f/z-S133 ' Date 5 r-- t- la t7 A Ctl/mav tech n ical Reviewer X So3) ' TXW Date Oo :q.O S c o pe and A pplic a tio n D & A O Cj.l Scope: This method describes the analysis of urine extracts for fluorochemicals using -K HPLC-electrospray/mass spectrometry. 2 Applicable Compounds: Fluorochemicals or other ionizable compounds. C3O*3 Matrices: Human, rat, and monkey urine, or other flui.ds as d. esign.ated in. the v.alidation L report. Word 6/95 ETS-8-97.0 Analysis o f Urine Extract Using ES/MS Page 1 of 10 2.0 Summary of M ethod 2.1 This method describes the analysis of fluorochemicals extracted from urine or other fluids, using HPLC-electrospray/mass spectrometry, or similar system as appropriate. The analysis is performed by monitoring a single ion characteristic of a particular fluorochemical, such as the perfluorooctanesulfonate (PFOS) anion, m/z= 499. Additionally, samples may be analyzed using a tandem mass spectrometer to further verify the identity of a'compound by detecting daughter ions of the parent ion. 3.0 D efin itio n s___________ ,________________________________________________________________ 3.1 Atmospheric Pressure Ionization (API): The Micromass Quattro II triple quadrupole systems allow for various methods of ionization by utilizing various sources, probes, and interfaces. These include but are not limited to: Electrospray Ionization (ESI), Atmospheric Pressure chemical Ionization (APcI), Thermospray, etc. The ionization process in these techniques occurs at atmospheric pressure (i.e., not under a vacuum). 3.2 Electrospray Ionization (ES, ESI): a method of ionization performed at atmospheric pressure, whereby ions in solution are transferred to the gas phase via tiny charged droplets. These charged droplets are produced by the application of a strong electrical field. 3.3 Mass Spectrometry, Mass Spectrometer (MS), Tandem Mass Spectrometer (MS/MS): The API Quattro II triple quadrupole mass spectrometer is equipped with two quadrupole mass selective detectors and a collision cell. Ions are selectively discriminated by mass to charge ratio (m/z) and subsequently detected. A single MS may be employed for ion detection or an ion may be selected in the first quadrupole, fragmented in the collision cell, and these fragments may be analyzed in the second quadrupole. 3.4 Conventional vs. Z-spray probe interface: The latest models of Micromass Quattro II triple quadrupole systems (post 1998) utilize a "Z-spray" conformation. The spray emitted from a probe is orthogonal to the cone aperture. In the conventional conformation it is aimed directly at the cone aperture, after passing through a tortuous pathway in the counter electrode. Though the configuration is different, the methods of operation, cleaning, and maintenance are the same. However, Z-spray components and conventional components are not compatible with one another, but only with similar systems (i.e., Z-spray components are compatible with some other Z-spray systems, etc.) 3.5 Mass Lynx Software: System software designed for the specific operation of these Quattro II triple quadrupole systems. Currently MassLynx has WindowsNT 4.0 versions. For more details see the manual specific to the instrument (Micromass Quattro II triple quadrupole MassLynx NT User's Guide). 4.0 W a r n in g s and C autio ns_______________________________________________________ 4.1 Health and Safety Warnings: 4.1.1 Use caution with the voltage cables for the probe. When engaged, the probe employs a voltage of approximately 5000 Volts. ETS-8-97.0 Analysis of Urine Extract Using ES/MS Page 2 of 10 4.1.2 When handling samples or solvents wear appropriate protective gloves, eyewear, and clothing. 4.2 Cautions: 4.2.1 Operate the solvent pumps below a back pressure of 400 bar (5800 psi). If the back pressure exceeds 400 bar, the HP 1100 will initiate automatic shutdown. 4.2.2 Do not run solvent pumps to dryness. . 5.0 In t e r fe r e n c e s_________________________________________________________________________ 5.1 To minimize interferences when analyzing samples, teflon should not be used for sample storage or any part of instrumentation that comes in contact with the sample or extract. 6.0 E quipm ent ~_______________________________________________________________________ 6.1 Equipment listed below may be modified in order to optimize the system. Document any modifications in the raw data as method deviations. 6.1.1 6.1.2 Micromass Quattro II triple quadrupoie Mass Spectrometer equipped with an electrospray ionization source HP1100 low pulse solvent pumping system, solvent degasser, column compartment, and autosampler 7.0 Su pp l ie s a n d M a ter ia l s____________________________ :__________________________________ 7.1 Supplies 7.1.1 High purity grade nitrogen regulated to approximately 100 psi. (House air system) 7.1.2 High purity grade argon regulated to approximately 6 psi. 7.1.3 HPLC analytical column, specifics to be determined by the analyst and documented in the raw data. 7.1.4 Capped autovials or capped 15 mL centrifuge tubes 8.0 R e a g e n t s a n d St a n d a r d s______________________________________________________________ 8.1 Reagents 8.1.1 Methanol, HPLC grade or equivalent 8.1.2 Milli-QTM water (ASTM type I), all water used in this method should be ASTM type I, or equivalent, and may be provided by a Milli-Q TOC Plus system or other vendor 8.1.3 Ammonium acetate, reagent grade or equivalent 8.1.3.1 When preparing different amounts than those listed, adjust accordingly. 8.1.3.2 2.0 mM ammonium acetate solution: Weigh approximately 0.300 g ammonium acetate. Pour into a 2000 mL volumetric container containing 2000 mL Milli-QTM water, mix until all solids are dissolved. Store at room temperature. ETS-8-97.0 Analysis of Urine Extract Using ES/MS Page 3 of 10 8.2 Standards 8.2.1 Typically two method blanks, two matrix blanks, and twenty matrix standards are prepared during the extraction procedure. See ETS-8-96.0. 9.0 Sample Handling__________________________________________________________ 9.1 Fresh matrix standards are prepared with each analysis. Extracted standards and samples are stored in capped autovials or capped 15 mL centrifuge tubes until analysis. 9.2 If analysis will be delayed, extracted standards and samples can be refrigerated at approximately 4 C, or at room temperature, until analysis can be performed. 10.0 Quality Control_________________________________________________________ 10.1 Solvent Blanks,IMethod Blanks and Matrix Blanks 10.1.1 Solvent blanks, method blanks and matrix blanks are prepared and analyzed with each batch to determine contamination or carryover. 10.1.2 Analyze a solvent blank, method blank, and matrix blank prior to each calibration curve. 10.2 Matrix Spikes 10.2.1 Matrix spikes are prepared and analyzed to determine the matrix effect on the recovery efficiency. 10.2.2 Matrix spike duplicates are prepared and analyzed to measure the precision and the recovery for each analyte. 10.2.3 Analyze a matrix spike and matrix spike duplicate per forty samples, with a minimum of 2 spikes per batch. 10.2.4 Matrix spike and matrix spike duplicate concentrations will fall in the mid-range of the initial calibration curve. Additional spike concentrations may fall in the lowrange of the initial calibration curve. 10.3 Continuing Calibration Verifications 10.3.1 Continuing calibration verifications are analyzed to verify the continued accuracy of the calibration curve. 10.3.2 Analyze a mid-range calibration standard after every tenth sample, with a minimum of one per batch. 11.0 Calibration and Standardization__________________________________________ 11.1 Analyze the extracted matrix standards prior to and following each set of extracts. The " average of two standard curves will be plotted by regression (linear or otherwise, see 11.2), weighted 1/x, not forced through zero, with IS reference (surrogate is used as an internal standard) using MassLynx or other suitable software. ETS-8-97.0 Analysis of Urine Extract Using ES/MS Page 4 o f 10 11.2 Use the following parameters for determining a calibration curve for each compound^ Compound Weighting Regression Fit Response type PFOS 1/X Linear IS reference POAA 1/X Quadratic IS reference Suitability of curve Itting parameters for other fluorochemical compounds will addressed by the analyst. 11.3 If the curve does not meet requirements, perform routine maintenance or reextract the standard curve (if necessary) and reanalyze. 11.4 For purposes of accuracy when quantitating low levels of analyte, it may be necessary to use the low end of the calibration curve rather than the full range of the standard curve. Example: whenattempting to quantitate approximately 50 ppb of analyte, generate a calibration curve consisting of the standards from 25 ppb to 250 ppb rather than the full range of the curve (25 ppb to 1000 ppb). This will reduce inaccuracy attributed to linear regression weighting of high concentration standards. 12.0 P r o c e d u r e s_______________________________________________________________________ 12.1 Acquisition Set up 12.1.1 Set up the sample list. 12.1.1.1 Assign a sample list filename using MO-DAY-last two digits of yearincreasing letter of the alphabet starting with a 12.1.1.2 Assign a method (MS file) for acquiring 12.1.1.3 Assign an HPLC program (Inlet file) 12.1.1.4 Type in sample descriptions and vial position numbers 12.1.2 To create a method click on method in the Acquisition control panel then mass spectrometer headings and select SIR (Single Ion Recording) or MRM (Multiple Reaction M onitoring). Set Ionization M ode as appropriate and mass to 499 or other appropriate masses. A full scan is usually collected along with the SIRs. Save acquisition method. If MS/MS instruments are employed, additional product ion fragmentation information may be collected. Refer to Micromass MassLynx GUIDE TO DATA ACQUISITION for additional information and MRM. 12.1.3 Typically the analytical batch run sequence begins and ends with a set of extracted matrix standards. 12.1.4 Samples are analyzed with a continuing calibration verification injected after every tenth sample. Solvent blanks should be analyzed periodically to monitor possible analyte carryover and are not considered samples but may be included as such. 12.2 Using the Autosampler 12.2.1 Set up sample tray according to the sample list prepared in Section 12.1.1. ETS-8-97.0 Analysis of Urine Extract Using ES/MS Page 5 of 10 12.2.2 Set-up the HP1100/autosampler at the following conditions or at conditions the analyst considers appropriate for optimal response. Record actual conditions in the instrument logbook: 12.2.2.1 Sample size = 10 pL injection 12.2.2.2 Inject/sample = 1 12.2.2.3 Cycle time = 9.0 minutes . 12.2.2.4 Solvent ramp = Time 0.00 min 1.0 min. 4.5 min. 6.5 min. 7.0 min. 9.0 min. MeOH 40% 40% 95% 95% 40% 40% 2.0 mM Ammonium acetate 60% 60% 5% 5% 60% 60% 12.2.2.5 Press the "Start" button. 12.3 Instrument Set-up 12.3.1 Refer to ETS-9-24.0, "Operation and Maintenance of the Micromass Quattro II Triple Quadrupole Mass Spectrometer Fitted with an Atmospheric Pressure Ionization Source," for more details. 12.3.2 Check the solvent level in reservoirs and refill if necessary. 12.3.3 Check the stainless steel capillary at the end of the probe. Use an eyepiece to check the tip. The tip should be flat with no jagged edges. If the tip is found to be unsatisfactory, disassemble the probe and replace the stainless steel capillary. 12.3 .4 Turn on the nitrogen. 12.3.5 Open the tune page. Click on operate to initiate source block and desolvation heaters. 12.3.6 Open the Inlet Editor. 12.3.6.1 Set HPLC pump to "On" 12.3.6.2 Set the flow to 10 - 500 uL/min or as appropriate 12.3.6.3 Observe droplets coming out of the tip of the probe. A fine mist should be expelled with no nitrogen leaking around the tip of the probe. Readjust the tip of the probe if no mist is observed - 12.3.6.4 Allow to equilibrate for approximately 10 minutes. 12.3.7 The instrument uses these parameters at the following settings. These settings may change in order to optimize the response: 12.3.7.1 Drying gas 250-400 liters/hour ETS-8-97.0 Analysis of Urine Extract Using ES/MS Page 6 of 10 12.3.7.2 ESI nebulizing gas 10-15 liters/hour 12.3.7.3 HPLC constant flow mode, flow rate 10 - 500 pL/min 12.3.7.4 Pressure <400 bar (This parameter is not set, it is a guide to ensure the HPLC is operating correctly.) 12.3.7.5 Source block temperature 150 12.3.7.6L Desolvation temperature 250 . 12.3.8 Print the tune page, with its parameters, and store it in the study binder with a copy taped into the instrument log. 12.3.9 Click on start button in the Acquisition Control Panel (this may vary among MassLynx versions, refer to appropriate MassLynx User's Guide). Ensure start and end sample number includes all samples to be analyzed. 13.0 D a ta A n a ly sis and C a l c u l a t i o n s ____________________________________________ 13.1 Calculations: 13.1.4 Calculate matrix spike percent recoveries using the following equation: % Recovery = Observed Result - Background Result x 100 Expected Result 13.1.5 Calculate percent difference using the following equation: % Difference = Expected Cone. - Calculated Cone, x 100 Expected Cone. 13.1.6 Calculate actual concentration of PFOS, or other fluorochemical, in matrix (pg/mL): (ng/mL of PFOS calc, from std. Curve x Dilution Factor) x 1 pg_____ Initial Volume of matrix ('mLl 1000 ng Final Volume (m L) 14.0 M e t h o d P er fo r m a n c e ____________________________________________________________ 14.1 Method Detection Limit (MDL) and Limit of Quantitation (LOQ) are method, analyte, and matrix specific. Please see ETS-8-96.0, Attachment B, for a listing of current validated MDL and LOQ values. 14.2 Solvent Blanks, Method Blanks, and Matrix Blanks 14.2.1 Solvent blanks, method blanks, and matrix blanks values must be below the lowest standard in the calibration curve 14.3 Calibration Curves 14.3.1 The r2 value for the calibration curve must be 0.980 or better. 14.4 Matrix Spikes 14.4.1 Matrix spike percent recoveries must be within 30% of the spiked concentration. ETS-8-97.0 Analysis of Urine Extract Using ES/MS Page 7 of 10 14.5 Continuing Calibration Verifications 14.5.1 Continuing calibration verification percent recoveries must be within 30% of the spiked concentration. 14.6 If criteria listed in this method performance section are not met, maintenance may be performed on the system and samples reanalyzed or other actions as determined by the analyst. Document all actions in the appropriate logbook. 14.7 If data are to be reported when performance criteria have not been met, the data must be footnoted on tables and discussed in the text of the report. 15.0 Pollution Prevention and Waste Management_____________________________ 15.1 Sample extract waste and flammable solvent is disposed in high BTU containers, and glass pipette waste is disposed in broken glass containers located in the laboratory. 16.0 Records__________________________________________________________________ 16.1 Each page generated for a study must have the following information included either in the header or hand written on the page: study or project number, acquisition method, integration method, sample name, extraction date, dilution factor (if applicable), and analyst. 16.2 Print the time page, sample list, and acquisition method from MassLynx to include in the appropriate study folder. Copy these pages and tape into the instrument runlog. 16.3 Plot the calibration curve by a linear or quadratic fit, referenced to the internal standard (surrogate), weighted 1/x, then print these graphs and store in the study folder. 16.4 Print data integration summary, integration method, and chromatograms, from MassLynx, and store in the study folder. 16.5 Summarize data using suitable software (Excel 7.0) and store in the study folder, see Attachment A for an example of a summary spreadsheet. 16.6 Back up electronic data to appropriate medium. Record in study notebook the file name and location of backup electronic data. 17.0 Tables. Diagrams. Flowcharts, and Validation Data_______________________ 17.1 Attachment A: ETS-8-97.0 Data summary spreadsheet. 18.0 References______________________________________________________________ 18.1 ETS-9-24.0, "Operation and Maintenance of the Micromass Atmospheric Pressure Ionization/Mass Spectrometer Quattro II triple quadrupole Systems" 18.2 The validation report associated with this method is FACT-TOX-131, W 2067 ETS-8-97.0 Analysis of Urine Extract Using ES/MS Page 8 of 10 19.0 A f f e c t e d D o c u m e n t s 19.1 ETS-8-96.0, "Extraction of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds from Urine for Analysis Using HPLC-Electrospray/Mass Spectrometry" 20.0 R ev isio n s Revision Number. Reason For Revision . Revision Date ETS-8-97.0 Analysis o f Urine Extract Using ES/MS Page 9 o f 10 Laboratory Study # Study: Test Material: Matrix/Final Solvent: Method/Revision: Analytical Equipment System Number: Instrument Software/Version: Filename: R-Squared Value: Slope: Y Intercept: Date of Extraction/Analyst: Date of Analysis/Analyst: Group Dose Sample# Concentration ug/mL Initial Vol. mL Dilution Factor Final Cone. ug/mL Slope: Taken from linear regression equation. Group/Dose: Taken from the study folder. Sample#: Taken from the study folder. Concentration (ug/mL): Taken from the MassLynx integration summary. Initial Volume (mL): Taken from the study folder. Dilution Factor: Taken from the study folder. Final Cone. (ug/mL): Calculated by dividing the initial volume from the concentration Attachment A: Summary Spreadsheet ETS-8-97.0 Analysis o f Urine Extract Using ES/MS Page 10 o f 10 3M Medical Department Study: T-6889.3 Analytical Report: FACT TOX-026 LRN-U2782 Appendix D: Method Validation Summary (TOX134) Table 5. TOX134 Method Validation Results Summary Parameters Linear Range: LOQ: Precision: In te r-a s s a y In tra-assay S ystem P recisio n Accuracy (Monkey sera): Reproducibility: R e s u lts ~ 5-1500 pg/m L (based on m ethod, som e run-to-run variability can be expected) - 10 (jg /m L (based on m ethod, som e run-to-run variability can be expected) <5.4% <21% <3% 88-94% <8.5% 3M Environmental Laboratory Page 22 3M Medical Department Study: T-6889.3 Appendix E: Data Summary Tables Analytical Report: FACT TOX-026 LRN-U2782 Table 6. LOQ Values Used in FACT TOX-026 Analyses by Method and Usage Dates M ethod 1 Effective Date LOQ Usage Dates Sera FACT-M -4.1 E T S -8 -5 .1 E T S -8 -5 .1 10/11/98 4/26/99 4/26/99 pg/m L 0.0137 0.0137 0.0137 11/16/98 to 1/27/99 (Day 9, Wk4, Wk 12, Wk 14) 4/30/99 to 6/23/99 (Wk 6-Wk 10, Wk 16-Wk 34) 7/14/99 to 7/21/99 (Wk 36-40) U rine E T S -8 -9 7 .0 7/28/99 pg/m L 0.0182 8/19/99 to 9/28/99 (Wk 2-Wk 40) L iv e r FACT-M -2.1 6/03/99 pg/g 0.0755 6/23/99 to 10/5/99 (Wk 20, Wk 27, Wk 40) Table 7. Recovery Summaries of Fortified Samples in FACT TOX-026 S erum S am ple N am e MKS05149-MS-3 MKS05149-MSD-3 I05709M-MS 105714M-MS MKS06119-MS-1 MKS06119-MSD-1 MKS07139-MS-1 MKS07139-MSD-1 A v erag e S tandard D eviation % R eco very 84 91 79 108 106 104 92 84 94 11 3M Environmental Laboratory Page 23 3M Medical Department Study: T-6889.3 Analytical Report: FACT TOX-026 LRN-U2782 Table 8. Recovery Summaries of Fortified Samples in FACT TOX-026 U rin e S am ple Nam e % R eco very M K U 08049 MS-1 M K U 08049 MSD-1 M KU 08049 MS-2 M KU08049 MSD-2 MKU08059 MS MKU08059 MSD M K U 0 8 1 7 9 M S 1-1 M K U 0 8 1 7 9 M S D 1-1 M K U 08179 M S 1-2 M K U 08179 M S D 1-2 M KU 08179 M S 1-3 M K U 08179 M SD 1-3 M KU 08179 M S 1-4 M K U 08179 M SD 1-4 M K U 0 8 2 0 9 M S 1-1 M K U 0 8 2 0 9 M S D 1-1 M K U 08209 M S 1-2 M K U 08209 M SD 1-2 M K U 09219 MS-1 M K U 09219 MSD-1 A verage S tandard D eviation 133 92 95 70 91 87 92 104 105 101 74 74 73 73 66 63 90 89 92 95 88 17 3M Environmental Laboratory Page 24 3M Medical Department Study: T-6889.3 Analytical Report: FACT TOX-026 LRN-U2782 Table 9. Recovery Summaries of Fortified Samples in FACT TOX-026 L iv e r S am ple N am e I05709M-MS-3 I05709-MSD-3 105718M-MS-11 I05718M-MSD-12 Average S tandard D eviation % R eco very 74 62 113 111 90 26 3M Environmental Laboratory Page 25 3M Medical Department Study: T-6889.3 Analytical Report: FACT TOX-026 LRN-U2782 Table 10. TOX 026 Data Summary of POAA Concentration--Serum (|jg/mL) G roup 1 0 .0 m g /k g /d a y A verage SD G roup 2 3 m g /k g /d a y A verage SD G roup 3 10 m g /k g /d a y A verage SD T im e p o in t Day 9 W eek 4 W eek 6 W eek 8 W eek 10 W eek 12 W eek 14 W e e k 16 W eek 18 W eek 20 W eek 22 W eek 24 W eek 26 W eek 26/27 W eek 28 W eek 30 W eek 32 W eek 34 W eek 36 0.0613 0 .0 4 7 2 (n=6) 0.206 0 .1 0 5 (n=6) 0.103 0 .0 1 1 3 (n=6) <LOQ (n=6) 0.126 0.0348 (n=6) 0.123 0 .0 5 0 7 (n=6) 0.162 0 .0 6 4 3 (n=6) 0.128 0.0721 (n=6) 0.183 0 .0 6 3 7 (n=6) 0.224 0.0730 (n=6) 0.232 0.131 (n=6) <LOQ (n=4) 0.209 0 .1 5 6 (n=6) 0.223 0 .1 0 5 (n=6) 0.181 0 .0 3 9 1 (o=2) 0.144 0.0238 (n=2) 0.110 0 .0 2 1 6 (n=2) 0.0861 0.0256 (n=2) 0.111 0 .0 4 4 5 (n=2) 126 36.1 (n=5) 98.4 42.7a (n=3) 102 3 3 .6 (n=4) 94.7 2 7 .3 (n=4) 105 3 7 .7 (n = 4 ) 79.6 2 5 .9 (n=4) 90.0 2 8 .9 (n=4) 68.6 2 6 .0 (n=4) 29.4 25.4 (n=4) 33.0 26.0 (n=4) 77.6 24.9 (n=3) 72 .2 68.8 (n=3) 118 2 7 .5 (n=3) 52.5 9 .1 4 (n=5) NS NS NS NS NS 189 4 8 .9 (n=6) 172 7 1 .9 (n=5) 95.8 20.6 (n=6) 97.6 23.5 (n=6) 93.6 1 3 .7 (n=6) 90.6 24.5 (n=6) 92.2 27.6 (o=6) 98.5 4 2 .3 (n=6) 17.4 1 4 .3 (n=6) 96.6 2 6 .6 (n=6) 105 3 6 .3 (n = 6 ) 90.9 2 1 .2 (n=6) 77.4 1 6 .9 (n=4) 74.1 3 3 .1 (n=6) 25.9 7.07 (n=2) 11.3 5 .11 (n=2) 7.60 3.90 (n=2) 3.97 2 .0 9 (n=2) 2.75 1 .8 8 (n=2) G roup 4 3 0 m g /k g /d a y A verage SD 1597 2392 (n=6) 1084 1839 (o=6) 145 2 1 .6 (n=5) 166 9 8 .9 (n=5) 237 158 (n=5) 140 7 7 .5 (0=5) 79.4 28.3 (0=5) 83.9 58.5 (n=5) 36.2 2 1 .2 (o=5) 97.7 1 2 9 (n=5) 58.6 45.6 (n=5) 6 2 .7 74.3 (n=5) 77.8 1 2 6 (o=5) 51.5 7 7 .6 (n=6) NS NS NS NS NS 3M Environmental Laboratory Page 26 3M Medical Department Study: T-6889.3 Analytical Report: FACT TOX-026 LRN-U2782 Table 10. TOX 026 Data Summary of POAA Concentration--Serum (|jg/mL) G roup 1 0 .0 m g /k g /d a y A verage SD G roup 2 3 m g /k g /d a y A verage SD G roup 3 10 m g /k g /d a y A verage SD G roup 4 3 0 m g /k g /d a y A verage SD W eek 38 W eek 40 0.0941 0.0324 (n=2) 0.0738 0.00256 (n=2) NS NS 1.84 1 .4 0 (n=2) 1.18 0 .8 2 7 (n=2) NS NS NS-- No sample <LOQ-- Below limit of quantitation "Group 2, W eek 4, Animal I05706M result was an anomaly and not included in the mean value. See individual results in Table 14. NOTE: Results are expressed as group/gender averages the standard deviation associated with that group/gender. NOTE: It is not possible to verify true recovery of endogenous analyte from tissues without radio-labeled reference material. The only measurement of accuracy available at this time, matrix spike studies, indicate that the sera data are accurate to within one standard deviation of the average fortified sample recovery. The average fortified sample recovery was 94% with a standard deviation of 11%. 3M Environmental Laboratory Page 27 3M Medical Department Study: T-6889.3 Analytical Report: FACT TOX-026 LRN-U2782 Table 11. TOX 026 Data Summary of POAA Concentration--Urine (|jg/mL) G roup 1 0 .0 m g /k g /d a y A verage SD G roup 2 3 m g /k g /d a y A verage SD G roup 3 10 m g /k g /d a y A verage SD T im e p o in t W eek 2 W eek 4 W eek 6 W eek 8 W eek 10 W e e k 12 W eek 14 W eek 16 W eek 18 W eek 20 W eek 22 W eek 24 <LOQ (n=6) 0.152 0 .3 3 7 (n=6) 0.0587 0 .0 7 1 6 (n=6) 0.0161 0 .0 0 9 4 0 (n=6) 0.0177 0 .0 1 1 4 (n=6) 0.0141 0 .0 0 6 4 8 (n=6) 7.96 1 9 .5 (n=6) 0.0299 0.0339 (n=6) 0.0256 0.0248 (n=6) 0.0211 0 .0 1 3 0 (n=6) 0.0231 0.00688 (n=6) 0.0125 0.00749 (n=6) 73.5 38.1 (n=4) 54.9 4.62 (n=4) 65.7 46.9 (n=4) 47.6 2 0 .6 (n=4) 39.9 1 8 .7 (n=4) 48.4 1 8 .8 (n=4) 63.1 4 7 .2 (n=4) 50.2 2 1 .0 (n=4) 37.7 1 9 .3 (n=4) 52.1 9 .6 3 (n=4) 95.8 80.8 (1=3) 46.3 8.52 (n=3) 2 2 1 124 (n=6) 190 9 1 .6 (n=6) 128 5 0 .0 (n=6) 206 73.1 (n=6) 175 9 2 .3 (n=6) 201 92.7 (n=6) 139 5 2 .2 (n=6) 139 5 7 .0 (n=6) 186 6 3 .9 (n=6) 144 1 3 5 (n=6) 158 7 8 .4 (n=6) 157 6 3 .3 (n=6) W eek 26 W eek 28 0.0268 0.0265 (n=6) 0.118 0 .1 4 2 (n=2) 51.6 1 3 .7 (n=3) NS 109 7 5 .2 (n=6) 0.327 0 .0 1 8 2 (n=2) W eek 30 <LOQ (n=2) 0.361 0 .1 1 8 NS (n=2) W eek 32 <LOQ (n=2) 0.114 0.0608 NS (n=2) W eek 34 W eek 36 <LOQ (n=2) 0.0117 0.00841 (n=2) NS NS 0.121 0 .0 3 0 5 (n=2) 0.0502 0.0166 (n=2) W eek 38 <LOQ (n=2) 0.0284 0 .0 1 0 2 NS (n=2) G roup 4 3 0 m g /k g /d a y A verage SD 909 269 (n=6) 240 161 (n=6) 272 140 (n=5) 180 1 0 9 (n=5) 359 449 (n=5) 118 111 (n=5) 72.9 84.0 (n=5) 50.2 67.9 (n=5) 43.1 8 4 .6 (n=5) 44.0 59.9 (n=5) 98.5 1 3 4 (n=5) 56.0 77.2 (n=5) 19.2 2 7 .0 (n=5) NS NS NS NS NS NS 3M Environmental Laboratory Page 28 3M Medical Department Study: T-6889.3 Analytical Report: FACT TOX-026 LRN-U2782 Table 11. TOX 026 Data Summary of POAA Concentration--Urine (pg/mL) G roup 1 0 .0 m g /k g /d a y A verage SD G roup 2 3 m g /k g /d a y A verage SD G roup 3 10 m g /k g /d a y A verage SD G roup 4 3 0 m g /k g /d a y A verage SD W eek 40 <LOQ (n=2) 0.0254 0.0101 NS NS (n=2) <LOQ-- Below limit of quantitation NS-- No sample NOTE: Results are expressed as group/gender averages the standard deviation associated with that group/gender. NOTE: It is not possible to verify true recovery of endogenous analyte from tissues without radio-labeled reference material. The only measurement of accuracy available at this time, matrix spike studies, indicate that the urine data are accurate to within one standard deviation of the average fortified sample recovery. The average fortified sample recovery was 88% with a standard deviation of 17%. 3M Environmental Laboratory Page 29 3M Medical Department Study: T-6889.3 Analytical Report: FACT TOX-026 LRN-U2782 Table 12. TOX 026 Data Summary of POAA Concentration--Liver (fig/g) T im epoint G roup 1 0 .0 m g /k g /d a y A verage SD G roup 2 3 m g /k g /d a y A verage SD G roup 3 10 m g /k g /d a y A verage SD G roup 4 3 0 m g /k g /d a y A verage SD W eek 20 NS 18.3 (n=1) NS NS W eek 27 0.117 0 .0 7 3 0 (9=4) 15.3 3 .0 2 (n=4) 14.0 7 .5 5 (n=4) 42.8 6 3 .3 (n=6) W eek 40 <LOQ (n=2) NS 0.114 0.0441 (n=2) NS NS-- No sample <LOQ-- Below limit of quantitation NOTE: Results are expressed as group/gender averages the standard deviation associated with that group/gender. NOTE: It is not possible to verify true recovery of endogenous analyte from tissues without radio-labeled reference material. The only measurement of accuracy available at this time, matrix spike studies, indicate that the liver data are accurate to within one standard deviation of the average fortified sample recovery. The average fortified sample recovery was 90% with a standard deviation of 26%. Table 13. TOX 026 Data Summary of POAA Concentration from Centre Analytical Laboratory--Feces (fjg/g) G roup 1 0 .0 m g /k g /d a y A verage SD G roup 2 3 m g /k g /d a y A verage SD G roup 3 10 m g /k g /d a y A verage SD G roup 4 3 0 m g /k g /d a y A verage SD T im e p o in t W eek 2 W eek 4 W eek 6 W eek 8 W eek 10 W eek 12 W eek 14 W eek 16 W eek 18 W eek 20 <LOQ (n=6) 0.0214 0 .0 1 7 8 (n=6) 0.0108 0 .0 0 1 9 2 (n=6) 0.0782 0 .1 0 3 (n=6) <LOQ (n=6) 0.0498 0.0894 (n=6) 0.139 0 .3 0 8 (9=6) 0.0572 0.0762 (9=6) 0.258 0.604 (9=6) 0.450 1 .0 8 (9=6) 7.43 6.54 (n=4) 10.4 1 2 .0 (n=4) 12.1 1 4 .1 (9=4) 9.4 6 9.21 (n=4) 3.96 3.68 (n=4) 7.15 5 .6 5 (9=4) 7.50 2 .4 3 (n=4) 6.88 2 .6 2 (n=4) 5.72 7 .1 5 (n=4) 6.81 4.89 (n=4) 15.4 1 0 .2 (9=6) 23.4 1 0 .6 (9=6) 23.3 8.46 (9=6) 41.0 2 5 .0 (n=6) 26.0 1 7 .4 (n=6) 10.3 6.07 (9=6) 27.2 29.4 (9=6) 31.4 2 3 .3 (9=6) 17.3 1 3 .3 (9=6) 52.4 39.5 (n=6) 56.6 73.7 (9=6) 22.0 6.23 (n=6) 101 8 6 .7 (9=5) 36.7 34.2 (9=5) 48.0 34.0 (n=5) 32.0 4 2 .9 (9=5) 19.9 2 5 .2 (n=5) 18.2 2 8 .8 (n=5) 22.1 3 1 .7 (9=5) 37.8 58.1 (9=5) 3M Environmental Laboratory Page 30 3M Medical Department Study: T-6889.3 Analytical Report: FACT TOX-026 LRN-U2782 Table 13. TOX 026 Data Summary of POAA Concentration from Centre Analytical Laboratory--Feces (pg/g) G roup 1 0 .0 m g /k g /d a y A verage SD G roup 2 3 m g /k g /d a y A verage SD G roup 3 10 m g /k g /d a y A verage SD G roup 4 3 0 m g /k g /d a y A verage SD W eek 22 W eek 24 W eek 26 W eek 28-34 W eek 36-40 15.5 3 6 .9 (n=6) 0 .5 1 7 1.13 (n=6) 0.0172 0 .0 0 8 9 2 (n=6) 0.279 0.732 (n=8) 0.0103 0 .0 0 6 8 4 (n=6) 13.8 5 .2 2 (n=3) 6.22 5.45 (n=3) 2.92 1 .3 5 (n=3) NS NS 39.5 2 1 .0 (n=6) 40.5 2 1 .8 (n=6) 43.0 36.9 (n=6) 0.387 0.372 (n=8) 0.0336 0.0313 (n=6) 25.2 36.0 (n=5) 34.6 47.7 (n=5) 10.3 2 0 .8 (n=5) NS NS NS-- No sample <LOQ-- Below limit of quantitation NOTE: Results are expressed as group/gender averages the standard deviation associated with that group/gender. NOTE: It is not possible to verify true recovery of endogenous analyte from tissues without radio-labeled reference material. The only measurement of accuracy available at this time, matrix spike studies, indicate that the feces data are accurate to within one standard deviation of the average fortified sample recovery. The average fortified sample recovery was 117% with a standard deviation of 22%. 3M Environmental Laboratory Page 31 3M Medical Department Study: T-6889.3 Analytical Report: FACT TOX-026 LRN-U2782 Table 14. FACT TOX-026 Individual POAA Results per Sample--Serum (pg/mL) A n im a l ID D ay 9 W eek 4 W eek 6 W eek 8 W eek 10 W eek 12 W eek 14 W eek 16 W eek 18 W eek 20 W eek 22 G roup 1 0.0 m g/kg/day I05709M 105714M 0 .0 6 4 5 0.137 0.299 0.230 <LOQ 0.126 <LOQ <LOQ 0.191 0.105 0.207 0.0994 0.275 0.156 0.275 <LOQ 0.263 0.244 0.342 0.212 0.393 0.157 C ontrol 105715M <LOQ 0 .0 7 8 5 <LOQ <LOQ 0.0919 0.064 0.0981 <LOQ 0.0950 0.115 0.106 105718M 0 .0 8 8 9 0 .3 4 7 <LOQ <LOQ 0.129 0.139 0.153 <LOQ 0.165 0.224 0.269 I05720M I05725M <LOQ nnQd 0 .1 6 7 0.114 <LOQ <LOQ <LOQ <LOQ 0.128 0.110 0.140 nU inVpCcwo 0.182 nV. m1UeJ <LOQ <LOQ 0.193 0.138 0.242 a nm 0.372 <LOQ G roup 2 3.0 m g/kg/day Low Dose 105721M replacement I05702M I05706M 105717M 105721M I05723M 91.6 177 116 119 96.8 3611a 142 56.5 67.5 115 8 3 .7 143 76.4 101 70.5 130 75.9 114 74.8 154 64.0 85.3 55.5 114 72.7 112 58.4 117 55.3 72.6 43.4 103 11.1 16.0 66.8 23.8 17.8 11.4 33.3 69.6 69.8 105 57.5 G roup 3 10 m g/kg/day I05707M I05708M 230 103 197 84.6 84.4 78.8 97.2 67.8 97.4 72.8 94.0 56.9 68.0 103 99.5 60.2 13.4 9.67 107 61.9 122 63.7 M id-D ose 105710M 105712M 173 178 268 E 103 87.9 88.5 82.3 87.9 103 79.0 131 61.3 79.2 73.2 180 46.4 13.7 75.2 137 87.7 168 105716M 228 119 86.2 102 102 84.4 134 91.3 10.1 91.4 106 105719M 220 194 135 133 108 98.4 108 86.5 11.3 107 85.2 G roup 4 I05703M 6338 286 162 175 313 235 84.4 68.2 68.8 38.9 16.4 30 m g/kg/day I05704M 313 251 111 117 128 103 109 113 20.0 92.5 92.7 H igh Dose 105711M 1676 4802 161 102 67.4 32.2 68.2 13.3 19.0 2.39 1.83 105713M 229 66.7 154 100 208 185 36.9 167 27.3 322 96.3 I05722M 151* 273 137 334 467 143 98.8 57.7 45.9 32.9 86.0 I05724M 876 822 E-- No extract remaining ' Sam ple evaporated to dryness, tentative value Shaded areas-- No sample aAnimal I05706M , Group 2, W e e k 4 result is an anomaly. NOTE: It is not possible to verify true recovery of endogenous analyte from tissues without radio-labeled reference material. The only measurement of accuracy available at this time, matrix spike studies, indicate that the sera data are accurate to within one standard deviation of the average fortified sample recovery. The average fortified sample recovery was 94% with a standard deviation of 11 %. W eek 24 E E <LOQ <LOQ <LOQ <LOQ 10.2 146 60.2 89.1 61.4 81.8 116 82.3 115 20.4 90.4 4.29 181 17.0 3M Environmental Laboratory Page 32 3M Medical Department Study: T-6889.3 Analytical Report: FACT TOX-026 LRN-U2782 Table 14 (continued). FACT TOX-026 Individual POAA Results per Sample--Serum (pg/mL) W eek A n im a l ID W eek 26 26& 27 W e e k 28 W e e k 30 W e e k 3 2 W e e k 34 W e e k 36 W e e k 38 W eek 40 G roup 1 0.0 m g/kg/day C ontrol I05709M 105714M 105715M 105718M I05720M I05725M 0.471 0.108 0.101 0.206 0.308 0 .0 6 2 3 0.377 0 .2 1 9 0 .0 9 4 5 0.206 0.308 0.136 0.153 0.209 0.127 0.161 0.0943 0.125 0.0680 0.104 0.0795 0.142 0.0711 0 .1 1 7 0 .0 7 1 9 0.0756 G roup 2 3.0 m g/kg/day Low Dose 105721M replacement G roup 3 10 m g/kg/day M id-D ose I05702M I05706M 105717M 105721M I05723M I05707M I05708M 105710M 105712 M 105716M 105719M 125 141 87.6 77.9 55.4 96.5 79.6 47.9 64.5 44.1 46.0 60.0 121 32.1 47.6 101 78.1 65.2 30.9 21.0 14.9 7.70 10.4 4.85 5.45 2.49 4.08 1.42 - 2.83 0.842 1.77 0 .6 0 0 G roup 4 I05703M 11.1 7.48 30 m g/kg/day I05704M 61.7 58.6 H igh D ose 105711M 1.50 0.877 105713M 299 184 I0 5 7 2 2 M 15.8 6.65 I05724M 489 Shaded areas-- No sample NOTE: It is not possible to verify true recovery of endogenous analyte from tissues without radio-labeled reference material. The only measurement of accuracy available at this time, matrix spike studies, indicate that the sera data are accurate to within one standard deviation of the average fortified sample recovery. The average fortified sample recovery was 94% with a standard deviation of 11%. 3M Environmental Laboratory Page 33 3M Medical Department Study: T-6889.3 Analytical Report: FACT TOX-026 LRN-U2782 Table 15. FACT TOX-026 Individual POAA Results per Sample--Urine (pg/mL) A n im a l ID W e e k 2 W e e k 4 W e e k 6 W e e k 8 W e e k 10 W e e k 12 W e e k 14 W e e k 16 W e e k 18 W e e k 2 0 W e e k 22 W e e k 2 4 G roup 1 0.0 m g/kg/day I05709M 105714M <LOQ <LOQ <LOQ <LOQ 0.172 <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ 0.0344 0.0287 <LOQ <LOQ C ontrol 105715M 105718M I05720M I05725M <LOQ <LOQ <LOQ <LQQ <LOQ 0.0270 <LOQ 0.839 <LOQ 0.0254 <LOQ 0.126 <LOQ 0.0307 <LOQ <LOQ <LOQ 0.0225 <LOQ 0.0359 <LOQ 0.0181 0 .0 1 8 4 <LOQ <LOQ 0 .0 2 2 4 4 7 .7 <LOQ <LOQ 0.0952 0 .0 3 6 6 a r\r\ ^ .L U U <LOQ <LOQ 0.0752 <LOQ <LOQ 0.0213 0.0451 <LOQ <LOQ <LOQ 0.0208 <LOQ <LOQ <LOQ 0.0213 <LOQ G roup 2 3.0 m g/kg/day Mid D ose I05702M I05706M 105717M 105721M 54.7 60.1 48.9 130 55.3 55.5 60.0 48.7 132 55.6 53.2 21.8 49.2 75.4 38.3 27.5 33.3 63.7 43.6 19.2 65.3 62.8 38.8 26.6 58.7 131 40.4 22.9 37.4 62.4 73.1 28.1 36.7 58.3 43.7 12.0 48.9 66.1 44.3 48.9 44.7 53.7 189 36.8 53.3 48.7 G roup 3 I05707M 412 195 157 230 272 170 127 165 157 1648* 58.0 184 10 m g/kg/day M id-high Dose I05708M 105710M 105712M 105716M 199 123 60.9 242 271 264 35.8 133 195 150 54.4 117 221 298 75.2 192 252 233 45.1 94.3 144 365 259 142 171 185 50.3 115 187 162 42.7 98.1 228 194 75.6 204 362 177 59.3 104 168 235 86.9 144 216 212 54.0 112 105719M 288 242 94.3 220 154 130 183 181 258 18.0 255 163 G roup 4 30 m g/kg/day H igh D ose I05703M I05704M 105711M 525 811 941 383 47.6 186 354 193 427 167 282 3.62 1131 328 1.41 175 169 0.725 0.181 156 <LOQ 0.179 136 0.380 <LOQ 170 0.201 0.363 124 0.134 0.352 255 0.334 0.306 155 0.107 105713M 846 444 72.3 257 109 243 134 112 0.406 93.3 237 124 I05722M 983 295 314 193 225 1.35 1.11 2.34 1.67 2.33 0.868 0.248 I05724M 1350 83.0 < LO Q -- Below limit of quantitation Shaded area-- No sample 'T h is sample was not diluted 1/5000; a 2pL injection of D 100 0 was analyzed instead. All other injections were 10pL. This provides a 1/5 dilution of D 1000 for a total of D 5000. A 1 /4 000 dilution of I05 707 M was also analyzed, but was just out of calibration range. NOTE: It is not possible to verify true recovery of endogenous analyte from tissues without radio-labeled reference material. T h e only measurement of accuracy available at this time, matrix spike studies, indicate that the urine data are accurate to within one standard deviation of the average fortified sample recovery. The average fortified sample recovery was 88% with a standard deviation of 17%. W eek 26 0.0208 <LOQ <LOQ <LOQ 0 .0 7 9 7 <LOQ 37.0 53.6 64.2 256 66.6 50.8 72.3 103 107 0.223 57.7 0.0682 37.8 0.215 3M Environmental Laboratory Page 34 3M Medical Department Study: T-6889.3 Analytical Report: FACT TOX-026 LRN-U2782 Table 15 (continued). FACT TOX-026 Individual POAA Results per Sample--Urine (pg/mL) A n im a l ID W e e k 28 W e e k 3 0 W e e k 32 W e e k 3 4 W e e k 3 6 W e e k 3 8 W eek 40 G roup 1 I05709M 0.0 m g/kg/day 105714M C o n tro l 105715M 105718M I0 5 7 2 0 M I05725M 0.218 <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ 0.0176 <LOQ <LOQ <L0Q <LOQ G roup 2 I05702M 3.0 m g/kg/day I05706M M id Dose 105717M 105721M G roup 3 I05707M 10 m g/kg/day I05708M M id-high D ose 105710M 105712M 105716M 105719M 0 .3 1 4 0 .3 4 0 0.278 0.445 0 .1 5 7 0.0712 0.0993 0.142 0 .0 3 8 4 0.0619 0.0357 0.0212 0.0325 <L0Q G roup 4 I05703M 30 m g/kg/day I05704M H igh D ose 105711M 105713M I05722M I05724M <LO Q -- Below limit of quantitation Shaded area-- No sample NOTE: It is not possible to verify true recovery of endogenous analyte from tissues without radio-labeled reference material. The only measurement of accuracy available at this time, matrix spike studies, indicate that the urine data are accurate to within one standard deviation of the average fortified sample recovery. The average fortified sample recovery was 88% with a standard deviation of 17%. 3M Environmental Laboratory Page 35 3M Medical Department Study: T-6889.3 Analytical Report: FACT TOX-026 LRN-U2782 Table 16. FACT TOX-026 Individual POAA Results per Sample--Liver (pg/g) G roup 1 0.0 m g/kg/day C o n tro l G roup 2 3.0 m g/kg/day M id D ose G roup 3 10 m g/kg/day M id-high Dose A n im a l ID I05709M 105714M 105715M I0 5 7 2 5 M 105718M I05720M I0 5 7 0 2 M I0 5 7 0 6 M 105717 M I0 5 7 2 3 M 105721M I0 5 7 0 7 M I0 5 7 0 8 M 105710M 105719 M 105712M 105716M W eek 20 18.3 W eek 27 0 .0 9 1 3 <LOQ 0.226 <LOQ 15.2 18.5 11.3 16.3 21.9 6.29 8.86 18.8 W eek 40 <LOQ <LOQ 0.146 0.0833 G roup 4 I05703M 1.31 30 m g/kg/day I0 5 7 0 4 M 16.0 H igh D ose 105711M 0.216 105713M 83.3 I0 5 7 2 2 M 1.41 I0 5 7 2 4 M 154 'S h a d e d areas-- N o sample. Sam ples w ere taken at sacrifice or time of death, therefore there is only one sam ple per NOTE'. It is not possible to verify true recovery of endogenous analyte from tissues without radio-labeled reference material. Th e only m easurement of accuracy available at this time, matrix spike studies, indicate that the liver data are accurate to within one standard deviation of the average fortified sample recovery. T h e average fortified sample recovery was 90% with a standard deviation of 26% . 3M Environmental Laboratory Page 36 3M Medical Department Study: T-6889.3 Appendix F: Data Spreadsheets Analytical Report: FACT TOX-026 LRN-U2782 3M Environmental Laboratory Page 37 FACT-TOX-026 Covance# 6329-231 Study: Product Number(Test Substance): Matrix: Method/Revision: Analytical Equipment System Number: Instrument Software/Version: Filename: R-Squared Value: Slope: Y-Intercept: Date of Extraction/Analyst: Date of Analysis/Analyst: Date of Data Reduction/Analyst: 6 Month Capsule Toxicity Study with APFO in Cynomolgus Monkeys T-6889.2 (POAA) Monkey Sera FACT-M-3.1 & FACT-M-4.1 Amelia 062498 MassLynx 3.1 See Attachments See Attachments See Attachments See Attachments 11/13/98 IAS 11/16/98, 11/24/98, 12/03/98 HOJ 11/17/98, 11/30/98, 12/07/98,09/29/00 HOJ/KJH Filenames Blks Grp 1 Grp 2 Grp 3 Grp 4 MS, MSD Sam ple Data Day 9 MONKEY SERA Group Dose Sample # Extraction Voi. Ratio POAA Std Correction Factor POAA Dilution Factor POAA Cone. ng/mL Concentration of POAA ug/mL or % Ree. Mean POAA ug/mL Method Blk Matrix Blk QC - 500 ppb H20 Blk-1 H20 Blk-2 Rabbit Sera Blk-1 Rabbit Sera Blk-2 RBS11138-MS RBSl 1138-MSD NA NA 1.00 1.00 1.00 1.00 0.9582 0.9582 0.9582 0.9582 NA NA 1 <LOQ 1 <LOQ 1 <LOQ 1 14.4 1 530 1 489 <LOQ <LOQ <LOQ <LOQ 0.0138 <LOQ 109% ** 100% ** 104% Group 1 Control 0.0 mg/kg/day Group 2 Low Dose 3.0 mg/kg/day Group 3 Mid-High Dose 10 mg/kg/day I05709M I05714M I05715M 105718M I05720M I05725M I05702M I05706M I05717M I05721M I05723M 105707M I05708M I05710M 105712M I05716M I05719M 0.50 0.9582 1 33.7 0.50 0.9582 1 71.4 0.60 0.9582 1 4.68 0.50 0.9582 1 46.4 0.30 0.9582 1 8.23 0.50 0.9582 1 26.0 0.70 0.9582 750 89.2 0.50 0.9582 750 123 0.60 0.9582 750 96.8 - 0.9582 NR NR 0.30 0.9582 75 497 0.50 0.9582 1000 120 0.60 0.9582 1000 64.5 0.50 0.9582 1000 90.1 0.60 0.9582 1000 112 0.50 0.9582 1000 119 0.50 0.9582 1000 115 0.0645 0.137 <LOQ 0.0889 <LOQ 0.0499 91.6 177 116 NR 119 230 103 173 178 228 220 0.0613 126 189 Group 4 High Dose 30 mg/kg/day I05703M I05704M I05711M I05713M I05722M I05724M 0.40 0.9582 5000 529 0.50 0.9582 2000 81.8 0.40 0.9582 2000 - 350 0.60 0.9582 2000 71.8 0.30 0.9582 1 47257 0.40 0.9582 2000 183 6338 313 1676 229 151 876 * 1597 Limit of Quantitation (LOQ): 0.0137 ug/mL *Tentative value, sample evaporated to dryness. Correction factors not applicable for MS/MSD QC data . ++Bracketed by a CCV not within method criteria (31%). POAA = Perfluorooctanoate Date Entered/By: Date Verified/By: 11/21/98, 12/07/98, 10/10/00 LAC 06/21/99 EAD, 10/11/00 KJH, 11/09/00 HOJ POAA A ll 1698003, 4 & 79, 80 111698015-20 112498018, 12039845-47 12039851-56 111698041, 12039859-63 111698075-76 Dilutions 1/1 1/1 1/75, 1/750 1/1000 1/1, 1/5000, 1/2000 1/1 RSD Std. Dev. MS/MSD RPD NA NA 8% 77.1 0.0472 28.7 36.1 25.9 48.9 150 2392 FACT-M-4.1 Sera Day9 FACT-M-4.1 FACT-TOX-026 Covance# 6329-231 Study: Product Number(Test Substance): Matrix: Method/Revision: Analytical Equipment System Number: Instrument Software/Version: Filename: R-Squared Value: Slope: Y-Intercept: Date of Extraction/Analyst: Date of Analysis/Analyst: Date of Data Reduction/Analyst: 6 Month Capsule Toxicity Study with APFO in Cynomolgus Monkeys T-6889.2 (POAA) Monkey Sera FACT-M-3.1 & FACT-M-4.1 Amelia 062498 MassLynx 3.1 See Attachments See Attachments See Attachments See Attachments 11/13/98 IAS 11/16/98,11/24/98,12/03/98 HOJ 11/17/98, 11/30/98, 12/07/98,09/29/00 HOJ/KJH Sam ple Data Day 9 MONKEY SERA Group Dose Sample # Method Blk Matrix Blk QC - 500 ppb H20 Blk-1 H20 Blk-2 Rabbit Sera Blk-1 Rabbit Sera Blk-2 RBSl 1138-MS RBS11138-MSD Group 1 Control 0.0 mg/kg/day Group 2 Low Dose Group 3 Mid-High Dose 10 mg/kg/day I05709M I05714M I05715M 105718M I05720M I05725M I05702M I05706M I05717M I05721M I05723M I05707M I05708M I05710M I05712M I05716M I05719M Group 4 High Dose 30 mg/kg/day I05703M I05704M 105711M I05713M I05722M I05724M Limit of Quantitation (LOQ): 0.0137 ug/mL Correction factors not applicable for MS/MSD QC data Concentration of POAA ug/mL or % Ree. <LOQ <LOQ <LOQ 0.0138 109% 100% 0.0645 0.137 <LOQ 0.0889 <LOQ 0.0499 91.6 177 116 NR 119 230 102.9 173 178 228 220 6338 313 1676 229 151 876 Mean POAA ug/mL <LOQ <LOQ ** ** 104% RSD Std. Dev. MS/MSD RPD NA NA 8% 0.0613 126 77.1 0.0472 28.7 36.1 25.9 189 48.9 * 150 1597 2392 Tentative value, sample evaporated to dryness. Bracketed by a CCV not.within method criteria (31%). POAA = Perfluorooctanoate Date Entered/By: Date Verified/By: 11/21/98, 12/07/98, 10/10/00 LAC 06/21/99 EAD, 10/11/00 KJH, 11/09/00 HOJ Sera Dav9 FACT-TOX-026 Covance# 6329-231 Study: Product Number(Test Substance): Matrix: Method/Revision: Analytical Equipment System Number: Instrument Software/Version: Filename: R-Squared Value: Slope: Y-Intercept: Date of Extraction/Analyst: Date of Analysis/Analyst: Date of Data Reduction/Analyst: 6 Month Capsule Toxicity Study with APFO in Cynomolgus Monkeys T-6889.2 (POAA) Monkey Sera FACT-M-3.1 &FACT-M-4.1 Amelia 062498 MassLynx 3.1 See Attachments See Attachments See Attachments See Attachments 11/13/98 IAS 11/16/98, 11/24/98, 12/03/98,01/04/99 HOJ 11/17/98, 11/30/98, 12/07/98,01/07/99,09/29/00 HOJ/KJH Sam ple Data W eek 4 M ONKEY SERA Group Dose Sample # Extraction Voi. Ratio Method Blk H20 Blk-1 Matrix Blk QC - 500 ppb H20 Blk-2 Rabbit Sera Blk-1 Rabbit Sera Blk-2 RBS11138-MS RBS11138-MSD Group 1 I05709M Control I05714M 0.0 mg/kg/day I05715M I05718M I05720M I05725M Group 2 Low Dose 3.0 mg/kg/day I05702M I05706M 105717M I05721M I05723M Group 3 Mid-High Dose 10 mg/kg/day I05707M I05708M I05710M I05712M I05716M I05719M Group 4 High Dose 30 mg/kg/day I05703M I05704M 105711M I05713M I05722M I05724M Limit of Quantitation (LOQ): 0.0137 ug/mL Correction factors not applicable for MS/MSD QC data NA NA 1.00 1.00 1.00 1.00 0.50 0.50 0.60 0.30 0.30 0.50 0.60 0.40 0.50 0.40 0.50 0.60 0.30 0.40 0.60 0.50 0.40 0.40 0.30 0.30 0.50 0.50 POAA Std Correction Factor POAA Dilution Factor POAA Cone. ng/mL Concentration of POAA ug/mL or % Ree. 0.9582 1 <LOQ <LOQ 0.9582 1 <LOQ <LOQ 0.9582 1 <LOQ <LOQ 0.9582 1 14.4 0.0138 NA 1 530 NA 1 489 109% 100% 0.9582 1 156 0.299 0.9582 1 120 0.230 0.9582 1 49.1 0.0785 0.9582 1 109 0.347 0.9582 1 52.2 0.167 0.9582 1 59.5 0.114 0.9582 750 80.8 96.8 0.9582 1875 804 3611 0.9582 750 98.7 142 0.9582 0.9582 250 23576 NR NR 56.5 NR 0.9582 150 684 197 0.9582 150 353 84.6 0.9582 150 558 268 0.9582 EE E 0.9582 0.9582 150 498 150 676 119 194 0.9582 200 598 286 0.9582 200 524 251 0.9582 5000 301 4802 0.9582 60 20893 66.7 0.9582 200 713 273 0.9582 2000 214 822 *Anomaly, was not included in these values. ^Bracketed by a CCV not within method criteria (31%). POAA = Perfluorooctanoate Date Entered/By: Date Verified/By: NR = Sample not received nor reported. E = Sample evaporated, not analyzed. 11/21/98, 12/07/98, 01/12/99, 10/10/00 LAC 06/21/99 EAD, 10/11/00 KJH, 11/09/00 OJ Filenames BIks Grp 1 Grp 2 Grp 3 Grp 4 Grp 4 MS, MSD Mean POAA ug/mL <LOQ <LOQ ** ** 104% 0.206 * 98.4 98.4* 977 172 1084 POAA A 111698003, 4 & 79, 80 111698045-50 12039866-68,010499021 112498041-46 112498049-50,52 12039881,83 010499020 111698075-76 RSD Std. Dev. MS/MSD RPD NA NA 8% 51.1 0.105 43.4* 42.7* 180 1757 41.7 71.9 170 1839 -M-4.1 Sera Week4 Dilutions 1/1 1/1 1/250, 750, 1875 1/150 1/200 1/60, 2000, 5000 1/1 FACT-M-4.1 FACT-TOX-026 Covance# 6329-231 Study: Product Number(Test Substance): Matrix: Method/Revision: Analytical Equipment System Number: Instrument Software/Version: Filename: R-Squared Value: Slope: Y-Intercept: Date of Extraction/Analyst: Date of Analysis/Analyst: Date of Data Reduction/Analyst: 6 Month Capsule Toxicity Study with APFO in Cynomolgus Monkeys T-6889.2 (POAA) Monkey Sera FACT-M-3.1 & FACT-M-4.1 Amelia 062498 MassLynx 3.1 See Attachments See Attachments See Attachments See Attachments 11/13/98 IAS 11/16/98, 11/24/98, 12/03/98, 01/04/99 HOJ 11/17/98, 11/30/98, 12/07/98, 01/07/99, 09/29/00 HOJ/KJH Sam ple Data W eek 4 MONKEY SERA Group Dose Sample # Method Blk H20 Blk-1 Matrix Blk H20 Blk-2 Rabbit Sera Blk-1 QC - 500 ppb Rabbit Sera Blk-2 RBS11138-MS RBS11138-MSD Group 1 Control I05709M I05714M 0.0 mg/kg/day I05715M I05718M I05720M I05725M Group 2 Low Dose I05702M I05706M 105717M 105721M I05723M Group 3 Mid-High Dose 10 mg/kg/day I05707M I05708M I05710M I05712M 105716M I05719M Group 4 High Dose 30 mg/kg/day I05703M I05704M I05711M I05713M I05722M I05724M Limit of Quantitation (LOQ): 0.0137 ug/mL Correction factors not applicable for MS/MSD QC data Concentration Mean of POAA POAA ug/mL or % Ree. ug/mL <LOQ <LOQ <LOQ <LOQ 0.0138 <LOQ 109% ** 100% ** 104% 0.299 0.230 0.0785 0.347 0.167 0.114 0.206 96.8 3611 * 142 98.4* 56.5 NR 977 197 84.6 268 E 119 194 172 286 251 4802 66.7 273 822 1084 *Anomaly, was not included in these values. **Bracketedby a CCV not within method criteria (31%). RSD Std. Dev. MS/MSD RPD NA NA 8% 51.1 0.105 43.4* 42.7* 180 1757 41.7 71.9 170 1839 POAA = Perfluorooctanoate Date Entered/By: Date Verified/By: NR = Sample not received nor reported. E = Sample evaporated, not analyzed. 11/21/98, 12/07/98,01/12/99, 10/10/00 LAC 06/21/99 EAD, 10/11/00 KJH, 11/09/00 OJ Sera Week4 FACT-TOX-026 Covance# 6329-231 Study: Product Number(Test Substance): Matrix: Method/Revision: Analytical Equipment System Number: Instrument Software/Version: Filename: R-Squared Value: Slope: Y-Intercept: Date of Extraction/Analyst: Date of Analysis/Analyst: Date of Data Reduction/Analyst: 6 Month Capsule Toxicity Study with APFO in Cynomolgus Monkeys T-6889.2 (POAA) Monkey Sera ETS-8-4.1 and ETS-8-5.1 Amelia 062498, Soup 020199 MassLynx 3.2 See Attachments See Attachments See Attachments See Attachments 05/12/99 SAH 06/03/99, 06/07/99, 06/10/99, 06/16/99 HOJ/SAH/MEE/LAC 06/04/99, 06/08/99, 06/11/99, 06/17/99, 11/06/00 HOJ/LAC/KJH Blks Grp 1 Grp 2 Grp 3 Grp 4 MS, MSD Sam ple Data W eek 6 M ONKEY SERA Group Sample # Extraction POAA Std Dose Voi. Ratio Correction Factor Method Blk H20 Blk-3 1.00 0.9582 H20 Blk-4 1.00 0.9582 Matrix Blk Rabbit Sera Blk-3 1.00 0.9582 Rabbit Sera Blk-4 1.00 0.9582 QC - 250 ppb RBS05129-MS-3 1.00 NA RBS05129-MSD-3 1.00 NA RBS05129-MS-4 1.00 NA RBS05129-MSD-4 1.00 NA Group 1 I05709M 0.54 0.9582 Control I05714M 0.74 0.9582 0.0 mg/kg/day I05715M 0.70 0.9582 105718M 0.55 0.9582 I05720M 0.56 0.9582 I05725M 0.67 0.9582 Group 2 I05702M 0.70 0.9582 Low Dose I05706M 0.55 0.9582 3.0 mg/kg/day I05717M 0.56 0.9582 I05721M 0.65 0.9582 I05723M NR 0.9582 Group 3 I05707M 0.72 0.9582 Mid-High Dose I05708M 0.55 0.9582 10 mg/kg/day 105710M 0.53 0.9582 I05712M 0.82 0.9582 105716M 0.67 0.9582 I05719M 0.40 0:9582 Group 4 I05703M 0.49 0.9582 High Dose 30 mg/kg/day I05704M 105711M 0.39 0.9582 0.61 0.9582 I05713M I05722M 0.59 0.9582 0.49 0.9582 Limit of Quantitation (LOQ): 0.0137 ug/mL . Correction factors not applicable for MS/MSD QC data NR = Sample not received nor reported. POAA = Perfluorooctanoate Date Entered/By: 06/08/99, 06/14/99, 06/17/99, 11/06/00 LAC Date Verified/By: 06/21/99 EAD, 11/09/00 HOJ POAA Dilution Factor 1 1 1 1 1 1 1 1 1 1 1 1 1 1 100 100 100 1000 1 1000 100 100 1000 100 100 200 100 1000 1000 1000 POAA Cone. ng/mL 0.00 0.00 0.00 0.00 199 199 229 214 56.3 100 22.5 33.0 3.15 21.0 493 662 489 97.1 NR 63.4 452 569 75.3 602 562 414 450 102 95.1 70.0 Concentration of POAA ug/mL or % Ree. <LOQ (0.0983 ug/mL) <LOQ (0.0983 ug/mL) <LOQ (0.0983 ug/mL) <LOQ (0.0983 ug/mL) 80% 79% 92% 85% <LOQ (0.0983 ug/mL) 0.126 <LOQ (0.0983 ug/mL) <LOQ (0.0983 ug/mL) <LOQ (0.0983 ug/tnL) <LOQ (0.0983 ug/mL) 67.5 115 83.7 143 NR 84.4 78.8 103 87.9 86.2 135 162 111 161 154 137 Mean POAA ug/mL <LOQ <LOQ 80% 89% 0.103 102 95.8 145 Filenames POAA A061099031, 32 & 99, 100 061099043-50 060799015-18,64 060799022-23,27-28,67-68 060799031-32,71-73, 0616990115 061099051-54 RSD Std. Dev. MS/MSD RPD NA NA 0% 7% 11.0 0.0113 32.8 33.6 21.5 20.6 14.9 21.6 ETS-8-5.1 Sera Week6 Dilutions 1/1 1/1 1/100&1000 1/100&1000 1/100,200,&1000 1/1 ETS-8-5.1 FACT-TOX-026 Covance# 6329-231 Study: Product Number(Test Substance): Matrix: Method/Revision: Analytical Equipment System Number: Instrument Software/Version: Filename: R-Squared Value: Slope: Y-Intercept: Date of Extraction/Analyst: Date of Analysis/Analyst: Date of Data Reduction/Analyst: 6 Month Capsule Toxicity Study with APFO in Cynomolgus Monkeys T-6889.2 (POAA) Monkey Sera ETS-8-4.1 and ETS-8-5.1 Amelia 062498, Soup 020199 MassLynx 3.2 See Attachments See Attachments See Attachments See Attachments 05/12/99 SAH 06/03/99, 06/07/99, 06/10/99, 06/16/99 HOJ/SAH/MEE/LAC 06/04/99, 06/08/99, 06/11/99, 06/17/99, 11/06/00 HOJ/LAC/KJH Sam ple Data W eek 6 MONKEY SERA Group Sample # Concentration Mean Dose of POAA ug/mL or % Ree. POAA ug/mL Method Blk H20 Blk-3 <LOQ (0.0983 ug/mL) Matrix Blk H20 Blk-4 <LOQ (0.0983 ug/mL) Rabbit Sera Blk-3 <LOQ (0.0983 ug/mL) <LOQ QC - 250 ppb Rabbit Sera Blk-4 <LOQ (0.0983 ug/mL) RBS05129-MS-3 80% <LOQ RBS05129-MSD-3 79% 80% RBS05129-MS-4 92% RBS05129-MSD-4 85% 89% Group 1 Control I05709M 105714M <LOQ (0.0983 ug/mL) 0.126 0.0 mg/kg/day 105715M <LOQ (0.0983 ug/mL) 105718M <LOQ (0.0983 ug/mL) I05720M <LOQ (0.0983 ug/mL) I05725M <LOQ (0.0983 ug/mL) 0.103 Group 2 I05702M 67.5 Low Dose I05706M 115 I05717M 83.7 I05721M I05723M 143 NR 102 Group 3 I05707M 84.4 Mid-High Dose I05708M 78.8 10 mg/kg/day I05710M 103 I05712M 87.9 I05716M I05719M 86.2 135 95.8 Group 4 I05703M 162 High Dose I05704M 111 30 mg/kg/day 105711M 161 105713M 154 I05722M 137 145 Limit of Quantitation (LOQ): 0.0137 ug/mL . Correction factors not applicable for MS/MSD QC dataNR = Sample not received nor reported. RSD Std. Dev. MS/MSD RPD NA NA 0% 7% 11.0 0.0113 32.8 33.6 21.5 20.6 14.9 21.6 POAA = Perfluorooctanoate Date Entered/By: Date Verified/By: 06/08/99, 06/14/99, 06/17/99, 11/06/00 LAC 06/21/99 EAD, 11/09/00 HOJ Sera Week6 FACT-TOX-026 Covance# 6329-231 Study: Product Number(Test Substance): Matrix: Method/Revision: Analytical Equipment System Number: Instrument Software/Version: Filename: R-Squared Value: Slope: Y-Intercept: Date of Extraction/Analyst: Date of Analysis/Analyst: Date of Data Reduction/Analyst: Sam ple Data W eek 8 M ONKEY SERA Group Sample # Dose Method Blk H20 Blk-3 H20 Blk-4 Matrix Blk Rabbit Sera Blk-3 Rabbit Sera Blk-4 QC - 250 ppb RBS05129-MS-3 RBS05129-MSD-3 RBS05129-MS-4 RBS05129-MSD-4 Group 1 Control I05709M I05714M 0.0 mg/kg/day I05715M I05718M I05720M I05725M Group 2 Low Dose 3.0 mg/kg/day I05702M I05706M I05717M I05721M I05723M Group 3 I05707M Mid-High Dose I05708M 10 mg/kg/day I05710M I05712M 105716M I05719M Group 4 I05703M High Dose I05704M 30 mg/kg/day 105711M I05713M I05722M Limit of Quantitation (LOQ): 0.0137 ug/mL . Correction factors not applicable for MS/MSD QC data 6 Month Capsule Toxicity Study with APFO in Cynomolgus Monkeys T-6889.2 (POAA) Monkey Sera ETS-8-4.1 and ETS-8-5.1 Amelia 062498 MassLynx 3.2 See Attachments See Attachments See Attachments See Attachments 05/12/99 SAH 06/03/99, 06/07/99, 06/10/99 HOJ 06/04/99, 06/08/99, 06/11/99, 11/06/00 HOJ/KJH Blks Grp 1 Grp 2 Grp 3 Grp 4 MS, MSD Extraction Voi. Ratio 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 0.30 0.67 0.34 0.26 0.60 0.57 0.54 0.50 0.57 0.52 NR 0.58 0.63 0.41 0.73 0.56 0.41 0.36 0.42 0.64 0.59 0.61 POAA Std Correction Factor 0.9582 0.9582 0.9582 0.9582 NA NA NA NA 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 POAA Dilution Factor 1 1 1 1 1 1 1 1 1 1 1 1 1 1 100 100 100 100 1 100 100 100 1000 100 100 100 100 100 1000 1000 POAA Cone. ng/mL 0.00 0.00 0.00 0.00 199 199 229 214 32.5 86.2 0.00 0.00 39.3 4.97 431 529 419 707 NR 588 446 379 62.7 597 567 659 513 684 61.8 213 Concentration of POAA ug/mL or % Ree. <LOQ (0.0983 ug/mL) <LOQ (0.0983 ug/mL) <LOQ (0.0983 ug/mL) <LOQ (0.0983 ug/mL) 80% 79% 92% 85% <LOQ (0.0983 ug/mL) <LOQ (0.0983 ug/mL) <LOQ (0.0983 ug/mL) <LOQ (0.0983 ug/mL) <LOQ (0.0983 ug/mL) <LOQ (0.0983 ug/mL) 76.4 101 70.5 130 NR 97.2 67.8 88.5 82.3 102 133 175 117 102 100 334 NR = Sample not received nor reported. Mean POAA ug/mL <LOQ <LOQ 80% 89% <LOQ 94.7 97.6 166 Filenames POAA A061099031, 32 & 99, 100 061099057-62 060799038-41 060799044-46,50-51,80 060799054-56,84-85 061099051-54 RSD Std. Dev. MS/MSD RPD NA NA 0% 7% NA NA 28.8 27.3 24.1 23.5 59.6 98.9 Dilutions 1/1 1/1 1/100 1/100&1000 1/100&1000 1/1 POAA = Perfluorooctanoate Date Entered/By: Date V erified/By: 06/08/99, 06/14/99, 11/06/00 LAC 06/21/99 EAD, 11/09/00 HOJ ETS-8-5.1 Sera Week8 ETS-8-5.1 FACT-TOX-026 Covance# 6329-231 Study: Product Number(Test Substance): Matrix: Method/Revision: Analytical Equipment System Number: Instrument Software/Version: Filename: R-Squared Value: Slope: Y-Intercept: Date of Extraction/Analyst: Date of Analysis/Analyst: Date of Data Reduction/Analyst: Sam ple Data W eek 8 MONKEY SERA Group Dose Sample # Method Blk H20 Blk-3 H20 Blk-4 Matrix Blk Rabbit Sera Blk-3 Rabbit Sera Blk-4 QC - 250 ppb RBS05129-MS-3 RBS05129-MSD-3 RBS05129-MS-4 RBS05129-MSD-4 Group 1 I05709M Control I05714M 0.0 mg/kg/day I05715M I05718M I05720M I05725M Group 2 I057O2M Low Dose I05706M I05717M I05721M I05723M Group 3 I05707M Mid-High Dose I05708M 10 mg/kg/day I057I0M I05712M I05716M I05719M Group 4 I05703M High Dose I05704M 30 mg/kg/day 105711M I05713M I05722M Limit of Quantitation (LOQ): 0.0137 ug/mL Correction factors not applicable for MS/MSD QC data 6 Month Capsule Toxicity Study with APFO in Cynomolgus Monkeys T-6889.2 (POAA) Monkey Sera ETS-8-4.1 and ETS-8-5.1 Amelia 062498 MassLynx 3.2 See Attachments See Attachments See Attachments See Attachments 05/12/99 SAH 06/03/99,06/07/99,06/10/99 HOJ 06/04/99,06/08/99,06/11/99, 11/06/00 HOJ/KJH Concentration Mean of POAA POAA ug/mL or % Ree. ug/mL <LOQ (0.0983 ug/mL) <LOQ (0.0983 ug/mL) <LOQ <LOQ (0.0983 ug/mL) <LOQ (0.0983 ug/mL) <LOQ 80% 79% 80% 92% 85% 89% <LOQ (0.0983 ug/mL) <LOQ (0.0983 ug/mL) <LOQ (0.0983 ug/mL) <LOQ (0.0983 ug/mL) <LOQ (0.0983 ug/mL) <LOQ (0.0983 ug/mL) <LOQ 76.4 101 70.5 130 NR 94.7 97.2 67.8 88.5 82.3 102 133 97.6 175 117 102 100 334 166 . NR = Sample not received nor reported. RSD Std. Dev. MS/MSD RPD NA NA 0% 7% NA NA 28.8 27.3 24.1 23.5 59.6 98.9 POAA = Perfluorooctanoate Date Entered/By: Date Verified/By: 06/08/99,06/14/99,11/06/00 LAC 06/21/99 BAD, 11/09/00 HOJ Sera Week8 FACT-TOX-026 Covance# 6329-231 Study: Product Number(Test Substance): Matrix: Method/Revision: Analytical Equipment System Number: Instrument Software/Version: Filename: R-Squared Value: Slope: Y-Intercept: Date of Extraction/Analyst: Date of Analysis/Analyst: Date of Data Reduction/Analyst: 6 Month Capsule Toxicity Study with APFO in Cynomolgus Monkeys T-6889.2 (POAA) Monkey Sera ETS-8-4.1 and ETS-8-5.1 Madeline 041098 MassLynx 3.2 See Attachments See Attachments See Attachments See Attachments 05/13/99 SRP/JCP 05/17/99, 05/19/99, 06/10/99 MEE/KJH/HOJ 05/18/99,05/20/99,06/11/99, 11/06/00 KJH/MEE/HOJ Blks Grp 1 Grp 2 Grp 3 Grp 4 MS, MSD Sam ple Data W eek 10 M ONKEY SERA CCV outside criteria Group Dose Sample # Method Blk H20 Blk-7 Matrix Blk Matrix Blk QC - 250 ppb H20 Blk-8 Rabbit Sera Blk-7 Rabbit Sera Blk-8 Monkey Sera Blk-7 Monkey Sera Blk-8 RBS05129-MS-7 RBS05129-MSD-7 RBS05129-MS-8 RBS05129-MSD-8 Group 1 Control 0.0 mg/kg/day I05709M I05714M 105715M I05718M I05720M I05725M Group 2 Low Dose 3.0 mg/kg/day I05702M I05706M 105717M I05721M I05723M Group 3 Mid-High Dose I05707M I05708M 10 mg/kg/day I05710M I05712M I05716M I05719M Group 4 I05703M High Dose I05704M 30 mg/kg/day 105711M .I05713M I05722M Limit of Quantitation (LOQ): 0.0137 ug/mL Correction factors not applicable for MS/MSD QC data Extraction Voi. Ratio 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 0.43 0.83 0.79 0.57 0.53 0.45 0.52 0.54 0.42 0.33 NR 0.55 0.77 0.61 0.69 0.66 0.63 0.50 0.73 0.68 0.55 0.53 POAA Std Correction Factor 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 NA NA NA NA 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 POAA = Perfluorooctanoate Date Entered/By: Date Verified/ By: ETS-8-5.1 05/20/99, 05/25/99, 06/14/99, 11/06/00, 11/09/00 LAC 06/21/99 EAD, 06/22/99 LAC, 11/09/00 HOJ POAA POAA Concentration Dilution Cone. of POAA Factor ng/mL ug/mL or % Ree. 1 7.56 1 0.00 <LOQ <LOQ * 1 30.0 1 32.8 0.0288 0.0315 * 1 85.4 1 47.4 1 257 0.0819 0.0454 103% * * * 1 265 106% * 1 258 1 317 103% 127% * * 1 85.5 0.191 1 90.7 0.105 1 75.7 0.0919 1 76.6 0.129 1 70.9 1 51.5 0.128 0.110 100 412 75.9 100 641 114 100 328 74.8 100 532 154 NR NR NR 100 559 97.4 100 585 72.8 100 560 87.9 100 740 103 100 703 102 100 708 108 250 654 313 250 392 128 250 191 67.4 250 477 208 500 517 467 * CCV bracketing these data was outside criteria. NR = Sample not received nor reported. E = Sample evaporated, not analyzed. Sera WeeklO Mean POAA ug/mL <LOQ 0.0301 0.0636 104% 115% 0.126 105 93.6 237 Filenames POAA M051799028, 29, 98-99, & 109, 110 051799040-44, 47 051999016-19 051999022-27 051999030-34, 052499058 051799102-105, 061099073 Dilutions 1/1 1/1 1/100 1/100 1/250&500 1/1 RSD Std. Dev. MS/MSD RPD NA 0.00192 0.0258 3% 20% 27.7 ' 0.0348 36.0 37.7 14.6 13.7 66.8 158 ETS-8-5.1 FACT-TOX-026 Covance# 6329-231 Study: Product Number(Test Substance): Matrix: Method/Revision: Analytical Equipment System Number: Instrument Software/Version: Filename: R-Squared Value: Slope: Y-Intercept: Date of Extraction/Analyst: Date of Analysis/Analyst: Date of Data Reduction/Analyst: 6 Month Capsule Toxicity Study with APFO in Cynomolgus Monkeys T-6889.2 (POAA) Monkey Sera ETS-8-4.1 and ETS-8-5.1 Madeline 041098 MassLynx 3.2 See Attachments See Attachments See Attachments See Attachments 05/13/99 SRP/JCP 05/17/99, 05/19/99, 06/10/99 MEE/KJH/HOJ 05/18/99,05/20/99,06/11/99, 11/06/00 KJH/MEE/HOJ Sam ple Data W eek 10 M ONKEY SERA Group Sample # Concentration Mean RSD Dose of POAA POAA Std. Dev. ug/mL or % Ree. ug/mL MS/MSD RPD Method Blk H20 Blk-7 H20 Blk-8 <LOQ <LOQ * <LOQ NA Matrix Blk Rabbit Sera Blk-7 0.0288 Rabbit Sera Blk-8 0.0315 0.0301 0.00192 Matrix Blk Monkey Sera Blk-7 0.0819 * QC - 250 ppb Monkey Sera Blk-8 RBS05129-MS-7 RBS05129-MSD-7 RBS05129-MS-8 RBS05129-MSD-8 0.0454 103% 106% 103% 127% * 0.0636 * * 104% * * 115% 0.0258 3% 20% Group 1 I05709M 0.191 Control I05714M 0.105 0.0 mg/kg/day 105715M 0.0919 I05718M 0.129 I05720M I05725M 0.128 0.110 0.126 27.7 0.0348 Group 2 I05702M 75.9 Low Dose I05706M 114 I05717M 74.8 I05721M I05723M 154 36.0 NR 105 37.7 Group 3 I05707M 97.4 Mid-High Dose I05708M 72.8 10 mg/kg/day I05710M 87.9 I05712M 103 I05716M I05719M 102 14.6 108 93.6 13.7 Group 4 I05703M 313 High Dose I05704M 128 30 mg/kg/day 105711M 67.4 I05713M 208 66.8 I05722M 467 237 158 Limit of Quantitation (LOQ): 0.0137 ug/mL * CCV bracketing these data was outside criteria. Correction factors not applicable for MS/MSD QC data NR = Sample not received nor reported. E = Sample evaporated, not analyzed. POAA = Perfluorooctanoate Date Entered/By: 05/20/99,05/25/99,06/14/99,11/06/00,11/09/00 LAC Date Verified/ By: 06/21/99 EAD, 06/22/99 LAC, 11/09/00 HOJ Sera WeeklO FACT-TOX-026 Covance# 6329-231 Study: Product Number(Test Substance): Matrix: Method/Revision: Analytical Equipment System Number: Instrument SoftwareAversion: Filename: R-Squared Value: Slope: Y-Intercept: Date of Extraction/Analyst: Date of Analysis/Analyst: Date of Data Reduction/Analyst: 6 Month Capsule Toxicity Study with APFO in Cynomolgus Monkeys T-6889.2 (POAA) Monkey Sera FACT-M-3.1 & FACT-M-4.1, ETS-8-5.1 Madeline 041098 MassLynx 3.1 See Attachments See Attachments See Attachments See Attachments 01/08/99 SAH/JCP 01/21/99,01/27/99 HOJ/MEE 01/25/99, 01/28/99,10/02/00, 10/11/00 KJH/MEE Sam ple Data W eek 12 M ONKEY SERA Group Dose Sample # Extraction Voi. Ratio POAA Std Correction Factor POAA Dilution Factor POAA Cone. ng/mL Concentration of POAA ug/mL or % Ree. Method Blk H20 Blk-1 H20 Blk-2 Matrix Blk Rabbit Sera Blk-1 Rabbit Sera Blk-2 QC - 250 ppb RBS01089-MS RBS01089-MSD Group 1 Control 0.0 mg/kg/day I05709M I05714M I05715M 105718M I05720M I05725M Group 2 I05702M Low Dose I05706M 3.0 mg/kg/day I05717M 105721M I05723M Group 3 I05707M Mid-High Dose I05708M 10 mg/kg/day I05710M 105712M 105716M 105719M Group 4 I05703M High Dose I05704M 30 mg/kg/day 105711M 105713M I05722M I05724M Limit of Quantitation (LOQ): 0.0137 ug/mL Correction factors not applicable for MS/MSD QC data NA NA 1.00 1.00 1.00 1.00 0.50 0.70 0.60 0.40 0.60 0.50 0.60 0.60 0.70 0.50 1.00 0.50 0.60 0.50 0.70 0.60 0.50 0.70 0.60 0.60 0.50 0.70 1.00 0.9582 0.9582 0.9582 0.9582 NA NA 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 1 <LOQ 1 <LOQ 1 <LOQ 1 <LOQ 1 247 1 243 1 108 1 73.0 1 40.2 1 58.2 1 87.9 1 45.6 150 268 150 358 150 272 150 397 NR NR 150 329 150 239 150 276 400 240 150 354 150 344 500 345 150 433 50 405 400 243 400 263 NR NR <LOQ <LOQ <LOQ <LOQ 100% 98% 0.207 0.0994 0.064 0.139 0.140 0.0869 64.0 85.3 55.5 114 NR 94.0 56.9 79.0 131 84.4 98.4 235 103 32.2 185 143 NR Filenames Blks Grp 1 Grp 2 Grp 3 Grp 4 MS, MSD Mean POAA ug/mL <LOQ <LOQ 99% 0.123 79.6 90.6 140 POAA M012199003, 4 & 79, 80 012199016-21 012199024-27 012199030-35, 012799026 012199039-40, 012799027-29 012199074-75 Dilutions 1/1 1/1 1/150 1/150, 1/400 1/50, 1/150, 1/250, 1/400, 1/500 1/1 RSD Std. Dev. MS/MSD RPD NA NA 2% 41.4 0.0507 32.6 25.9 27.1 24.5 55.5 77.5 POAA = Perfluorooctanoate Date Entered/By: Date Verified/ By: 01/27/99, 01/28/99, 10/10/00, 10/13/00 LAC 06/21/99 EAD, 10/11/00, 10/19/00 KJH, 11/09/00 HOJ FACT-M-4.1 Sera W eekl2 FACT-M-4.1 FACT-TOX-026 Covance# 6329-231 Study: Product Number(Test Substance): Matrix: Method/Revision: Analytical Equipment System Number: Instrument Software/Version: Filename: R-Squared Value: Slope: Y-Intercept: Date of Extraction/Analyst: Date of Analysis/Analyst: Date of Data Reduction/Analyst: 6 Month Capsule Toxicity Study with APFO in Cynomolgus Monkeys T-6889.2 (POAA) Monkey Sera FACT-M-3.1 & FACT-M-4.1, ETS-8-5.1 Madeline 041098 MassLynx 3.1 See Attachments See Attachments See Attachments See Attachments 01/08/99 SAH/JCP 01/21/99,01/27/99 HOJ/MEE 01/25/99, 01/28/99, 10/02/00, 10/11/00 KJH/MEE Sam ple Data W eek 12 M ONKEY SERA Group Dose Sample # Concentration of POAA ug/mL or % Ree. Mean POAA ug/mL RSD Std. Dev. MS/MSD RPD Method Blk H20 Blk-1 H20 Blk-2 Matrix Blk Rabbit Sera Blk-1 Rabbit Sera Blk-2 QC - 250 ppb RBS01089-MS RBS01089-MSD Group 1 I05709M Control I05714M 0.0 mg/kg/day I05715M I05718M I05720M I05725M Group 2 I05702M Low Dose I05706M 105717M I05721M I05723M Group 3 I05707M Mid-High Dose I05708M 10 mg/kg/day I05710M I05712M I05716M I05719M Group 4 I05703M High Dose I05704M 30 mg/kg/day 105711M I05713M I05722M I05724M Limit of Quantitation (LOQ): 0.0137 ug/mL <LOQ <LOQ <LOQ <LOQ 100% 98% 0.207 0.0994 0.0639 0.139 0.140 0.0869 64.0 85.3 55.5 114 NR 94.0 56.9 79.0 131 84.4 98 235 103 32.2 185 143 NR <LOQ <LOQ 99% NA NA 2% 0.123 79.6 41.4 0.0507 32.6 25.9 27.1 90.6 24.5 55.5 140 77.5 Correction factors not applicable for MS/MSD QC data POAA = Perfluorooctanoate Date Entered/By: Date Verified/By: 01/27/99, 01/28/99, 10/10/00, 10/13/00 LAC 06/21/99 EAD, 10/11/00, 10/19/00 KJH, 11/09/00 HOJ Sera W eekl2 FACT-TOX-026 Covance# 6329-231 Study: Product Number(Test Substance): Matrix: Method/Revision: Analytical Equipment System Number: Instrument Software/Version: Filename: R-Squared Value: Slope: Y-Intercept: Date of Extraction/Analyst: Date of Analysis/Analyst: Date of Data Reduction/Analyst: 6 Month Capsule Toxicity Study with APFO in Cynomolgus Monkeys T-6889.2 (POAA) Monkey Sera FACT-M-3.1 & FACT-M-4.1, ETS-8-5.1 Madeline 041098 MassLynx 3.1 See Attachments See Attachments See Attachments See Attachments 01/08/99 SAH/JCP 01/21/99,01/27/99 HOJ/MEE 01/25/99, 01/28/99, 10/02/00, 10/11/00 KJH/MEE Sam ple Data W eek 14 M ONKEY SERA Group Dose Sample # Extraction Voi. Ratio Method Blk H20 Blk-1 H20 Blk-2 Matrix Blk Rabbit Sera Blk-1 Rabbit Sera BIk-2 QC - 250 ppb RBS01089-MS RBS01089-MSD Group 1 I05709M Control I05714M 0.0 mg/kg/day I05715M I05718M I05720M I05725M Group 2 I05702M Low Dose I05706M 3.0 mg/kg/day I05717M I05721M I05723M Group 3 I05707M Mid-High Dose I05708M 10 mg/kg/day I05710M I05712M I05716M I05719M Group 4 I05703M High Dose I05704M 30 mg/kg/day 105711M I05713M I05722M I05724M Limit of Quantitation (LOQ): 0.0137 ug/mL Correction factors not applicable for MS/MSD QC data NA NA 1.00 1.00 1.00 1.00 0.50 0.50 0.50 0.50 0.30 0.50 0.50 0.50 0.50 0.35 1.00 0.50 0.50 0.50 0.50 0.45 0.40 0.50 0.50 0.45 0.50 0.50 1.00 POAA Std Correction Factor 0.9582 0.9582 0.9582 0.9582 NA NA 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 POAA Dilution Factor 1 1 1 1 1 i 1 1 1 1 1 1 100 200 100 100 NR 200 200 100 100 200 100 150 150 150 150 150 NR POAA Cone. ng/mL <LOQ <LOQ <LOQ <LOQ 247 243 144 81.6 51.4 80.3 57.3 55.0 381 294 306 428 NR 178 270 322 415 316 451 295 380 215 129 346 NR Concentration of POAA ug/mL or % Ree. <LOQ <LOQ <LOQ <LOQ 100% 98% 0.275 0.156 0.0981 0.153 0.182 0.105 72.7 112 58.4 117 NR 68.0 103 61.3 79.2 134 108 84.4 109 68.2 36.9 98.8 NR Filenames Blks Grp 1 Grp 2 Grp 3 Grp 4 MS, MSD Mean POAA ug/mL <LOQ <LOQ 99% 0.162 90.0 92.2 79.4 POAA M012199003, 4 & 79, 80 012199045-50 012199053-56, 012799033 012199061-62, 64 012799034-36 012199067-71 012199074-75 Dilutions 1/1 1/1 1/100, 1/200 1/100, 1/200 1/150 1/1 RSD Std. Dev. MS/MSD RPD NA NA 2% 39.8 0.0643 32.1 28.9 30.0 27.6 35.6 28.3 POAA = Perfluorooctanoate Date Entered/By: Date Verified/By: 01/27/99,01/28/99,10/10/00,10/13/00 LAC 06/21/99 EAD, 10/11/00, 10/19/00 KJH, 11/09/00 HOJ FACT-M-4.1 Sera W eekl4 FACT-M-4.1 FACT-TOX-026 Covance# 6329-231 Study: Product Number(Test Substance): Matrix: Method/Revision: Analytical Equipment System Number: Instrument Software/Version: Filename: R-Squared Value: Slope: Y-Intercept: Date of Extraction/Analyst: Date of Analysis/Analyst: Date of Data Reduction/Analyst: 6 Month Capsule Toxicity Study with APFO in Cynomolgus Monkeys T-6889.2 (POAA) Monkey Sera FACT-M-3.1 &FACT-M-4.1, ETS-8-5.1 Madeline 041098 MassLynx 3.1 See Attachments See Attachments See Attachments See Attachments 01/08/99 SAH/JCP 01/21/99, 01/27/99 HOJ/MEE 01/25/99,01/28/99, 10/02/00, 10/11/00 KJH/MEE Sam ple Data W eek 14 M ONKEY SERA Group Dose Sample # Concentration of POAA ug/mL or % Ree. Mean POAA ug/mL RSD Std. Dev. MS/MSD RPD Method Blk Matrix Blk QC - 250 ppb H20 Blk-1 H20 Blk-2 Rabbit Sera Blk-1 Rabbit Sera Blk-2 RBS01089-MS RBS01089-MSD <LOQ <LOQ <LOQ <LOQ 100% 98% <LOQ <LOQ 99% NA NA 2% Group 1 Control 0.0 mg/kg/day I05709M I05714M I05715M I05718M I05720M 105725M 0.275 0.156 0.0981 0.153 0.182 0.105 0.162 39.8 0.0643 Group 2 Low Dose I05702M 72.7 I05706M 112 105717M 58.4 105721M 117 32.1 I05723M NR 90.0 28.9 Group 3 I05707M 68.0 Mid-High Dose I05708M 103 10 mg/kg/day I05710M 61.3 I05712M 79.2 I05716M 134 30.0 I05719M 108 92.2 27.6 Group 4 I05703M 84.4 High Dose I05704M 109 30 mg/kg/day 105711M 68.2 I05713M 36.9 I05722M 98.8 35.6 I05724M NR 79.4 28.3 Limit of Quantitation (LOQ): 0.0137 ug/mL Correction factors not applicable for MS/MSD QC data POAA = Perfluorooctanoate Date Entered/By: Date Verified/By: 01/27/99, 01/28/99, 10/10/00, 10/13/00 LAC 06/21/99 EAD, 10/11/00, 10/19/00 KJH, 11/09/00 HOJ Sera W eekl4 FACT-TOX-026 Covance# 6329-231 Study: Product Number(Test Substance): Matrix: Method/Revision: Analytical Equipment System Number: Instrument Software/Version: Filename: R-Squared Value: Slope: Y-Intercept: Date of Extraction/Analyst: Date of Analysis/Analyst: Date of Data Reduction/Analyst: 6 Month Capsule Toxicity Study with APFO in Cynomolgus Monkeys T-6889.2 (POAA) Monkey Sera ETS-8-4.1 and ETS-8-5.1 Madeline 041098 MassLynx 3.1 See Attachments See Attachments See Attachments See Attachments 05/13/99 SRP/JCP 05/17/99, 05/19/99, 06/10/99 MEE 05/18/99,05/20/99,06/11/99, 11/06/00 KJH/MEE/HOJ Blks Grp 1 Grp 2 Grp 3 Grp 4 MS, MSD Sam ple Data W eek 16 M ONKEY SERA* Group Dose Sample # Extraction Voi. Ratio Method Blk Matrix Blk Matrix Blk QC - 250 ppb H20 Blk-7 H20 Blk-8 Rabbit Sera Blk-7 Rabbit Sera Blk-8 Monkey Sera Blk-7 Monkey Sera Blk-8 RBS05129-MS-7 RBS05129-MSD-7 RBS05129-MS-8 RBS05129-MSD-8 Group 1 Control 0.0 mg/kg/day I05709M I05714M I05715M 105718M I05720M I05725M Group 2 Low Dose 3.0 mg/kg/day I05702M I05706M 105717M 105721M I05723M Group 3 Mid-High Dose I05707M I05708M 10 mg/kg/day 105710M I05712M I05716M 105719M Group 4 High Dose I05703M I05704M 30 mg/kg/day 105711M I05713M I05722M I05724M Limit of Quantitation (LOQ): 0.0137 ug/mL Correction factors not applicable for MS/MSD QC data 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 0.54 0.66 0.64 0.51 0.50 0.59 0.60 0.55 0.59 0.52 NR 0.57 0.68 0.56 0.59 0.57 0.64 0.57 0.75 0.60 0.74 0.54 NR POAA Std POAA POAA Concentration Correction Dilution Cone. of POAA Factor Factor ng/mL ug/mL or % Ree. 0.9582 1 7.56 <LOQ 0.9582 1 0.00 <LOQ 0.9582 1 30.0 0.0288 0.9582 1 32.8 0.0315 0.9582 1 85.4 0.0819 0.9582 1 47.4 0.0454 NA 1 257 103% NA 1 265 106% NA 1 258 103% NA 1 317 127% 0.9582 1 155 0.275 0.9582 1 70.7 <LOQ (0.0983 ug/mL) 0.9582 1 6.40 <LOQ (0.0983 ug/mL) 0.9582 1 50.6 <LOQ (0.0983 ug/mL) 0.9582 0.9582 1 48.3 <LOQ (0.0983 ug/mL) 1 3.07 <LOQ (0.0983 ug/mL) 0.9582 100 346 55.3 0.9582 100 417 72.6 0.9582 100 267 43.4 0.9582 100 560 103 0.9582 NR NR NR 0.9582 100 592 99.5 0.9582 100 427 60.2 0.9582 100 428 73.2 0.9582 500 222 180 0.9582 0.9582 100 543 100 578 91.3 86.5 0.9582 250 162 68.2 0.9582 250 355 113 0.9582 10 835 13.3 0.9582 250 516 167 0.9582 250 130 57.7 0.9582 NR NR NR NR = Sample not received nor reported. E = Sample evaporated, not analyzed. * * # * * * * * Mean POAA ug/mL <LOQ 0.0301 0.0636 104% 115% 0.128 68.6 98.5 83.9 . POAA = Perfluorooctanoate Date EnteredTBy: 05/20/99,06/14/99,11/06/00 LAC * CCV bracketing these data was not within criteria. . ETS-8-5.1 Date Verified/By: 06/21/99 EAD, 06/22/99 LAC, 11/09/00 HOJ SeraWeekl6 Filenames POAA Dilutions M051799028, 29, 98-99, & 109, 110 1/1 061099065-69, 72 1/1 051999037-40 1/100 051999043-45, 47-48, 052499059 1/100 051999051-52, 54-55, 061099074 1/10&250 051799102-105, 061099073 1/1 RSD Std. Dev. MS/MSD RPD NA 0.00192 0.0258 3% 20% 56.4 0.0721 37.9 26.0 43.0 42.3 69.7 58.5 ETS-8-5.1 FACT-TOX-026 Covante# 6329-231 Study: Product Number(Test Substance): 6 Month Capsule Toxicity Study with APFO in Cynomolgus Monkeys T-6889.2 (POAA) Matrix: Method/Revision: Monkey Sera ETS-8-4.1 and ETS-8-5.1 Analytical Equipment System Number: Madeline 041098 Instrument Software/Version: MassLynx 3.1 Filename: R-Squared Value: Slope: Y-Intercept: Date of Extraction/Analyst: Date of Analysis/Analyst: Date of Data Reduction/Analyst: See Attachments See Attachments See Attachments See Attachments 05/13/99 SRP/JCP 05/17/99, 05/19/99, 06/10/99 MEE 05/18/99,05/20/99,06/11/99, 11/06/00 KJH/MEE/HOJ* Sam ple Data W eek 16 M ONKEY SERA Group Dose Sample # Method Blk H20 Blk-7 H20 Blk-8 Matrix Blk Rabbit Sera Blk-7 Rabbit Sera Blk-8 Matrix Blk Monkey Sera Blk-7 QC - 250 ppb Monkey Sera Blk-8 RBS05129-MS-7 RBS05129-MSD-7 RBS05129-MS-8 RBS05129-MSD-8 Group 1 Control 0.0 mg/kg/day I05709M I05714M I05715M 105718M I05720M I05725M Group 2 I05702M Low Dose I05706M 105717M I05721M I05723M Group 3 Mid-High Dose I05707M I05708M 10 mg/kg/day I05710M 105712M 105716M I05719M Group 4 High Dose 30 mg/kg/day I05703M I05704M 105711M I05713M I05722M I05724M . Limit of Quantitation (LOQ): 0.0137 ug/mL Correction factors not applicable for MS/MSD QC data Concentration of POAA ug/mL or % Ree. <LOQ <LOQ 0.0288 0.0315 0.0819 0.0454 103% 106% 103% 127% 0.275 <LOQ (0.0983 ug/mL) <LOQ (0.0983 ug/mL) <LOQ (0.0983 ug/mL) <LOQ (0.0983 ug/mL) <LOQ (0.0983 ug/mL) 55.3 72.6 43.4 103 NR 100 60.2 73.2 180 91.3 86.5 68.2 113 13.3 167 57.7 NR Mean POAA ug/mL * <LOQ 0.0301 * * 0.0636 * * 104% * * 115% RSD Std. Dev. MS/MSD RPD NA 0.00192 0.02578 3% 20% 0.128 68.6 56.4 0.0721 37.9 26.0 43.0 98.5 42.3 69.7 83.9 58.5 NR = Sample not received nor reported. E = Sample evaporated, not analyzed. POAA = Perfluorooctanoate Date Entered/By: Date Verified/ By: 05/20/99,06/14/99, 11/06/00 LAC 06/21/99 EAD, 06/22/99 LAC. 11/09/00 HOJ * CCV bracketing these data was not within criteria. Sera Week 16 FACT-TOX-026 Covance# 6329-231 Study: Product Number(Test Substance): Matrix: Method/Revision: Analytical Equipment System Number: Instrument Software/Version: Filename: R-Squared Value: Slope: Y-Intercept: Date of Extraction/Analyst: Date of Analysis/Analyst: Date of Data Reduction/Analyst: 6 Month Capsule Toxicity Study with APFO in Cynomolgus Monkeys T-6889.2 (POAA) Monkey Sera ETS-8-4.1 and ETS-8-5.1 Madeline 041098, Amelia 062498 MassLynx 3.2 See Attachments See Attachments See Attachments See Attachments 05/14/99 SEE 05/18/99, 05/24/99, 05/25/99, 06/07/99 MEE/HOJ/SAH 05/19/99, 05/25/99, 05/27/99, 06/08/99 MEE/HOJ Sam ple Data W eek 18 MONKEY SERA Group Dose Sample # Method Blk H20 Blk-1 H20 BIk-2 Matrix Blk Rabbit Sera Blk-1 Rabbit Sera Blk-2 QC - 250 ppb RBS05149-MS-11 RBS05149-MSD-11 RBS05149-MS-12 RBS05149-MSD-12 Group 1 I05709M Control I05714M 0.0 mg/kg/day I05715M I05718M I05720M I05725M Group 2 I05702M Low Dose I05706M 3.0 mg/kg/day I05717M I05721M Group 3 Mid-High Dose I05707M I05708M 10 mg/kg/day I05710M I05712M I05716M I05719M Group 4 I05703M High Dose I05704M 30 mg/kg/day 105711M I05713M I05722M Limit of Quantitation (LOQ): 0.0137 ug/mL Correction factors not applicable for MS/MSD QC data Extraction Voi. Ratio NA NA 1.00 1.00 1.00 1.00 1.00 1.00 0.63 0.57 0.57 0.48 0.43 0.52 0.69 0.63 0.45 0.47 0.52 0.68 0.48 0.60 0.70 0.75 0.46 0.66 0.32 0.70 0.60 POAA Std Correction Factor 0.9582 0.9582 0.9582 0.9582 NA NA NA ' - NA 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 POAA Dilution Factor 1 1 1 1 1 1 1 1 1 1 1 1 1 1 50 50 250 50 50 50 50 50 50 50 50 50 50 50 50 POAA Cone. ng/mL 0.00 0.00 8.69 1.76 290 236 247 201 173 145 56.5 82.7 86.7 75.1 160 210 125 234 145 137 465 171 147 178 660 276 127 399 575 Concentration of POAA ug/mL or % Ree. <LOQ <LOQ <LOQ <LOQ 116% 95% 99% 80% 0.263 0.244 0.0950 0.165 0.193 0.138 11.1 16.0 66.8 23.8 13.4 9.67 46.4 13.7 10.1 11.3 68.8 20.0 19.0 27.3 45.9 Filenames Blks Grp 1 Grp 2 Grp 3 Grp 4 MS, MSD Mean POAA ug/mL <LOQ <LOQ 105% 90% 0.183 29.4 17.4 36.2 POAA POAA M0518990I3, 14 & 89, 90 051899026-33 052599086-87, 89 60799061 052599092-97 052499030, 32, 34, 052599101, 103 051899084-87 RSD Std. Dev. MS/MSD RPD NA NA 20% 21% 34.8 0.0637 86.5 25.4 82.1 14.3 58.5 21.2 POAA = Perfluorooctanoate Date Entered/By: Date Verified/By: 05/19/99, 05/25/99, 05/27/99, 06/08/99 LAC 06/21/99 EAD Dilutions 1/1 1/1 1/50&250 1/50 1/50 1/1 ETS-8-5.1 Sera Week 18 ETS-8-5.1 FACT-TOX-026 Covance# 6329-231 Study: Product Number(Test Substance): Matrix: Method/Revision: Analytical Equipment System Number Instrument Software/Version: Filename: R-Squared Value: Slope: Y-Intercept: Date of Extraction/Analyst: Date of Analysis/Analyst: Date of Data Reduction/Analyst: Sam ple D ata W eek 18 M ONKEY SERA Group Dose Sample # Method Blk H20Blk-l H20 Blk-2 Matrix Blk Rabbit Sera Blk-1 Rabbit Sera Blk-2 QC - 250 ppb RBS05149-MS-11 RBS05149-MSD-11 RBS05149-MS-12 RBS05149-MSD-12 Group 1 I05709M Control I05714M 0.0 mg/kg/day I05715M 105718M I05720M I05725M Group 2 I05702M Low Dose I05706M 105717M I05721M Group 3 I05707M Mid-High Dose I05708M 10 mg/kg/day I05710M I05712M 105716M I05719M Group 4 I05703M High Dose - I05704M 30 mg/kg/day 105711M I05713M I05722M Limit of Quantitation (LOQ): 0.0137 ug/mL Correction factors not applicable for MS/MSD QC data 6 Month Capsule Toxicity Study with APFO in Cynomolgus Monkeys T-6889.2 (POAA) Monkey Sera ETS-8-4.1 and ETS-8-5.1 Madeline 041098, Amelia 062498 MassLynx 3.2 See Attachments See Attachments See Attachments See Attachments 05/14/99 SEE 05/18/99, 05/24/99, 05/25/99, 06/07/99 MEE/HOJ/SAH 05/19/99, 05/25/99, 05/27/99, 06/08/99 MEE/HOJ Concentration of POAA ug/mL or % Ree. <LOQ <LOQ <LOQ <LOQ 116% 95% . 99% 80% 0.263 0.244 0.0950 0.165 0.193 0.138 11.1 16.0 66.8 23.8 13.4 9.67 46.4 13.7 10.1 11.3 68.8 20.0 19.0 27.3 45.9 Mean POAA ug/mL <LOQ <LOQ 105% 90% 0.183 29.4 17.4 36.2 RSD Std. Dev. MS/MSD RPD NA NA 20% 21% 34.8 0.0637 86.5 25.4 82.1 14.3 58.5 21.2 POAA = Perfluorooctanoate Date Entered/By: Date Verified/By: 05/19/99, 05/25/99, 05/27/99, 06/08/99 LAC 06/21/99 EAD Sera W eekl8 FACT-TOX-026 Covance# 6329-231 0.250746269 Study: Product Number(Test Substance): Matrix: Method/Revision: Analytical Equipment System Number: Instrument Software/Version: Filename: R-Squared Value: Slope: Y-Intercept: Date of Extraction/Analyst: Date of Analysis/Analyst: Date of Data Reduction/Analyst: 6 Month Capsule Toxicity Study with APFO in Cynomolgus Monkeys T-6889.2 (POAA) Monkey Sera ETS-8-4.1 and ETS-8-5.1 Madeline 041098, Amelia 062498, Soup 020199 MassLynx 3.2 See Attachments See Attachments See Attachments See Attachments 05/14/99 SEE 05/18/99, 05/24/99, 05/25/99, 06/10/99, 06/16/99 MEE/HOJ/SAH/LAC 05/19/99, 05/25/99, 05/27/99, 06/11/99, 06/17/99, 11/06/00 MEE/HOJ/LAC/KJH Sam ple Data W eek 20 MONKEY SERA Group Dose Sample # Extraction Voi. Ratio Method Blk H20 Blk-1 H20 Blk-2 Matrix Blk Rabbit Sera Blk-1 Rabbit Sera Blk-2 QC - 250 ppb RBS05149-MS-11 RBS05149-MSD-l 1 RBS05149-MS-12 RBS05149-MSD-12 Group 1 I05709M Control I05714M 0.0 mg/kg/day 105715M I05718M I05720M I05725M Group 2 I05702M Low Dose I05706M 3.0 mg/kg/day I05717M I05721M Group 3 I05707M Mid-High Dose I05708M 10 mg/kg/day I05710M I05712M 105716M I05719M Group 4 I05703M High Dose I05704M 30 mg/kg/day 105711M I05713M I05722M Limit of Quantitation (LOQ): 0.0137 ug/mL Correction factors not applicable for MS/MSD QC data NA NA 1.00 1.00 1.00 1.00 1.00 1.00 0.59 0.66 0.62 0.33 0.53 0.58 0.73 0.65 0.63 0.24 0.43 0.70 0.42 0.65 0.55 0.62 0.49 0.53 0.38 0.68 0.36 POAA Std Correction Factor 0.9582 0.9582 0.9582 0.9582 NA NA NA NA 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 POAA Dilution Factor 1 1 1 1 1 1 1 1 1 1 1 1 1 1 50 50 50 50 50 50 50 500 500 500 50 500 50 500 50 POAA Cone. ng/mL 0.00 0.00 8.69 1.76 290 236 247 201 211 146 74.3 77.2 134 126 271 155 438 348 964 905 660 186 105 138 398 102 18.9 456 248 Concentration of POAA ug/mL or % Ree. <LOQ <LOQ <LOQ <LOQ 116% 95% 99% 80% 0.342 0.212 0.115 0.224 0.242 0.207 17.8 11.4 33.3 69.6 107 61.9 75.2 137 91.4 107 38.9 92.5 2.39 322 32.9 Filenames BIks Grp 1 Grp 2 Grp 3 Grp 4 MS, MSD Mean POAA ug/mL <LOQ <LOQ 105% 90% 0.224 33.0 96.6 97.7 POAA M051899013, 14 & 89, 90 051899055-62 052599107-110 052499045, 061099075-76, 061699018-20 052499051, 53, 55, 061699021-22 051899084-87 RSD Std. Dev. MS/MSD RPD NA NA 20% 21% 32.6 0.0730 78.8 26.0 27.5 26.6 132 129 POAA = Perfluorooctanoate Date Entered/By: Date Verified/By: 05/20/99, 05/25/99, 05/27/99, 06/14/99,06/17/99, 11/06/00 LAC 06/21/99 EAD, 06/22/99 LAC, 11/09/00 HOJ Dilutions 1/1 1/1 1/50 1/50&500 1/50&500 1/1 ETS-8-5.1 Sera Week20 ETS-8-5.1 FACT-TOX-026 Covance# 6329-231 Study: Product Number(Test Substance): Matrix: Method/Revision: Analytical Equipment System Number: Instrument Sofltware/Version: Filename: R-Squared Value: Slope: Y-Intercept: Date of Extraction/Analyst: Date of Analysis/Analyst: Date of Data Reduction/Analyst: Sam ple Data W eek 20 M ONKEY SERA Group Dose Sample # Method Blk H20 Blk-1 H20 Blk-2 Matrix Blk Rabbit Sera Blk-1 Rabbit Sera Blk-2 QC - 250 ppb RBS05149-MS-11 RBS05149-MSD-11 RBS05149-MS-12 RBS05149-MSD-12 Group 1 I05709M Control 105714M 0.0 mg/kg/day I05715M 105718M I05720M I05725M Group 2 I05702M Low Dose I05706M 105717M I05721M Group 3 I05707M Mid-High Dose I05708M 10 mg/kg/day I05710M I05712M 105716M I05719M Group 4 I05703M High Dose I05704M 30 mg/kg/day 105711M I05713M I05722M Limit of Quantitation (LOQ): 0.0137 ug/mL . Correction factors not applicable for MS/MSD QC data 6 Month Capsule Toxicity Study with APFO in Cynomolgus Monkeys T-6889.2 (POAA) Monkey Sera ETS-8-4.1 and ETS-8-5.1 Madeline 041098, Amelia 062498, Soup 020199 MassLynx 3.2 See Attachments See Attachments See Attachments See Attachments 05/14/99 SEE 05/18/99, 05/24/99, 05/25/99, 06/10/99, 06/16/99 MEE/HOJ/SAH/LAC 05/19/99, 05/25/99, 05/27/99, 06/11/99, 06/17/99, 11/06/00 MEE/HOJ/LAC/KJH Concentration of POAA ug/mL or % Ree. <LOQ <LOQ <LOQ <LOQ 116% 95% 99% 80% 0.342 0.212 0.115 0.224 0.242 0.207 17.8 11.4 33.3 69.6 107 61.9 75.2 137 91.4 107 38.9 92.5 2.39 322 32.9 Mean POAA ug/mL <LOQ <LOQ 105% 90% 0.224 33.0 96.6 97.7 RSD Std. Dev. MS/MSD RPD NA NA 20% 21% 32.6 0.0730 78.8 26.0 27.5 26.6 132 129 POAA = Perfluorooctanoate Date Entered/By: Date Verified/By: 05/20/99, 05/25/99, 05/27/99, 06/14/99, 06/17/99, 11/06/00 LAC 06/21/99 EAD, 06/22/99 LAC, 11/09/00 HOJ Sera Week20 FACT-TOX-026 Covance# 6329-231 Study: Product Number(Test Substance): Matrix: Method/Revision: Analytical Equipment System Number: Instrument Software/Version: Filename: R-Squared Value: Slope: Y-Intercept: Date of Extraction/Analyst: Date of Analysis/Analyst: Date of Data Reduction/Analyst: Sam ple Data 6 Month Capsule Toxicity Study with APFO in Cynomolgus Monkeys T-6889.2 (POAA) Monkey Sera ETS-8-4.1 and ETS-8-5.1 Amelia 062498, Madeline 041098 MassLynx 3.2 See Attachments See Attachments See Attachments See Attachments 05/14/99 MCH 05/20/99, 05/26/99, 05/28/99, 06/10/99 HOJ/MEE 05/24/99, 05/27/99, 06/01/99, 06/11/99, 11/06/00 HOJ/MEE/KJH Filenames Blks Grp 1 Grp 2 Grp 3 Grp 4 MS, MSD W eek 22 MONKEY SERA Group Dose Sample # Extraction Voi. Ratio Method Blk H20 Blk-9 Matrix Blk H20 Blk-10 Rabbit Sera Blk-9 QC - 250 ppb Rabbit Sera Blk-10 RBS05149-MS-9 RBS05149-MSD-9 Group 1 I05709M Control I05714M 0.0 mg/kg/day I05715M I05718M I05720M I05725M Group 2 I05702M Low Dose I05706M 3.0 mg/kg/day 105717M Group 3 Mid-High Dose 10 mg/kg/day I05707M I05708M I05710M I05712M 105716M I05719M Group 4 I05703M High Dose I05704M 30 mg/kg/day 105711M I05713M I05722M Limit of Quantitation (LOQ): 0.0137 ug/mL Correction factors not applicable for MS/MSD QC data 1.0 1.0 1.0 1.0 1.0 1.0 0.62 0.68 0.53 0.49 0.31 0.28 0.75 0.32 0.68 0.42 0.55 0.65 0.54 0.46 0.60 0.35 0.28 0.33 0.56 0.50 POAA Std Correction Factor 0.9582 0.9582 0.9582 0.9582 NA NA 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 POAA Dilution Factor 1 1 1 1 1 1 1 1 1 1 1 1 100 100 100 100 100 100 200 100 100 10 100 10 100 100 POAA Cone. ng/mL 0.00 42.5 0.00 32.9 261 273 254 111 58.8 137 120 0.00 547 352 408 533 366 595 472 510 533 601 271 63.2 563 449 Concentration of POAA ug/mL or % Ree. <LOQ 0.0407 <LOQ 0.0315 104% 109% 0.393 0.157 0.106 0.269 0.372 <LOQ (0.0983 ug/mL) 69.8 105 57.5 122 63.7 87.7 168 106 85.2 16.4 92.7 1.83 96.3 86.0 Mean POAA ug/mL NA NA 107% 0.232 77.6 105 58.6 POAA = Perfluorooctanoate Date Entered/By: Date Verified/By: 05/25/99, 05/27/99, 06/02/99, 06/1.4/99, 11/06/00 LAC 06/21/99 EAD, 06/22/99 LAC, 11/09/00 HOJ POAA A052099035, 36 & 98, 99 052099047-51, 061099086 052699025-27 052699028-32, 34-35, 052899104 052699036-42 061099095-96 RSD Std. Dev. MS/MSD RPD NA NA 4% 56.5 0.131 32.1 24.9 34.5 36.3 77.8 45.6 Dilutions 1/1 1/1 1/100 1/100&200 1/10&100 1/1 ETS-8-5.1 Sera Week22 ETS-8-5.1 FACT-TOX-026 Covance# 6329-231 Study: Product Number(Test Substance): Matrix: Method/Revision: Analytical Equipment System Number: Instrument Software/Version: Filename: R-Squared Value: Slope: Y-Intercept: Date of Extraction/Analyst: Date of Analysis/Analyst: Date of Data Reduction/Analyst: Sam ple Data W eek 22 M ONKEY SERA Group Dose Sample # Method Blk H20 Blk-9 H20 Blk-10 Matrix Blk Rabbit Sera Blk-9 Rabbit Sera Blk-10 QC - 250 ppb RBS05149-MS-9 RBS05149-MSD-9 Group 1 I05709M Control I05714M 0.0 mg/kg/day 105715M 105718M I05720M I05725M Group 2 I05702M Low Dose I05706M I05717M Group 3 I05707M Mid-High Dose I05708M 10 mg/kg/day I05710M I05712M 105716M 105719M Group 4 I05703M High Dose I05704M 30 mg/kg/day 105711M I05713M I05722M Limit of Quantitation (LOQ): 0.0137 ug/mL Correction factors not applicable for MS/MSD QC data 6 Month Capsule Toxicity Study with APFO in Cynomolgus Monkeys T-6889.2 (POAA) Monkey Sera ETS-8-4.1 and ETS-8-5.1 Amelia 062498, Madeline 041098 MassLynx 3.2 See Attachments See Attachments See Attachments See Attachments 05/14/99 MCH 05/20/99, 05/26/99, 05/28/99, 06/10/99 HOJ/MEE 05/24/99, 05/27/99, 06/01/99, 06/11/99, 11/06/00 HOJ/MEE/KJH Concentration of POAA ug/mL or % Ree. <LOQ 0.0407 <LOQ 0.0315 104% 109% 0.393 0.157 0.106 0.269 0.372 <LOQ (0.0983 ug/mL) 69.8 105 57.5 122 63.7 87.7 168 106 85.2 16.4 92.7 1.83 96.3 86.0 Mean POAA ug/mL NA NA 107% RSD Std. Dev. MS/MSD RPD NA NA 4% 0.232 77.6 56.5 0.131 32.1 24.9 34.5 105 36.3 ' 77.8 58.6 45.6 POAA = Perfluorooctanoate Date Entered/By: Date Verified/By: 05/25/99, 05/27/99, 06/02/99, 06/14/99, 11/06/00 LAC 06/21/99 EAD, 06/22/99 LAC, 11/09/00 HOJ Sera Week22 FACT-TOX-026 Covance# 6329-231 Study: Product Number(Test Substance): Matrix: Method/Revision: Analytical Equipment System Number: Instrument Software/Version: Filename: R-Squared Value: Slope: Y-Intercept: Date of Extraction/Analyst: Date of Analysis/Analyst: Date of Data Reduction/Analyst: Sam ple Data 6 Month Capsule Toxicity Study with APFO in Cynomolgus Monkeys T-6889.2 (POAA) Monkey Sera ETS-8-4.1 and ETS-8-5.1 Filenames Amelia 062498 MassLynx 3.2 Blks See Attachments Grp 1 See Attachments Grp 2 See Attachments Grp 3 See Attachments Grp 4 05/14/99 MCH MS, MSD 05/20/99,05/26/99,05/28/99 HOJ/SAH/MEE 05/24/99, 05/27/99, 06/01/99, 11/06/00 HOJ/MEE/KJH POAA A052099035, 36 & 98, 99 061099087-90 052699045, 47, 052899105 052699052-59 052699051, 63-64, 052899106-107 061099095-96 W eek 24 M ONKEY SERA Group Dose Sample # Extraction Voi. Ratio Method Blk H20 Blk-9 H20 Blk-10 Matrix Blk Rabbit Sera Blk-9 Rabbit Sera Blk-10 QC - 250 ppb RBS05149-MS-9 RBS05149-MSD-9 Group 1 Control I05709M I05714M 0.0 mg/kg/day I05715M I05718M I05720M I05725M Group 2 I05702M Low Dose 3.0 mg/kg/day I05706M I05717M Group 3 I05707M Mid-High Dose I05708M 10 mg/kg/day I05710M I05712M I05716M I05719M Group 4 I05703M High Dose 30 mg/kg/day I05704M 105711M I05713M I05722M Limit of Quantitation (LOQ): 0.0137 ug/mL Correction factors not applicable for MS/MSD QC data 1.0 1.0 1.0 1.0 1.0 1.0 0.40 0.58 0.31 0.47 0.29 0.38 0.61 0.69 0.43 0.71 0.64 0.61 0.54 0.59 0.54 0.57 0.72 0.52 0.63 0.43 POAA Std Correction Factor 0.9582 0.9582 0.9582 0.9582 NA NA 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 POAA POAA Concentration Dilution Cone. of POAA Factor ng/mL ug/mL or % Ree. 1 0.00 <LOQ 1 42.5 0.0407 1 0.00 <LOQ 1 32.9 0.0315 1 261 1 273 104% 109% 1 Ext Ext 1 Ext Ext 1 5.55 <LOQ (0.0983 ug/mL) 1 55.5 <LOQ (0.0983 ug/mL) 1 77.5 <LOQ (0.0983 ug/mL) 1 5.08 <LOQ (0.0983 ug/mL) 100 64.9 10.2 200 527 100 270 146 60.2 100 660 100 410 89.1 61.4 100 521 81.8 100 656 116 100 507 100 646 82.3 115 20 606 20.4 100 679 90.4 10 233 4.29 200 596 181 10 765 17.0 Ext: Samples lost during sample extraction. Mean POAA ug/mL NA NA 107% <LOQ 72.2 90.9 62.7 RSD Std. Dev. MS/MSD RPD NA NA 4% NA NA 95.3 68.8 23.3 21.2 119 74.3 Dilutions 1/1 1/1 1/100&200 1/100 1/1,10,20,&200 1/1 POAA = Perfluorooctanoate Date Entered/By: Date Verified/By: 05/25/99, 05/27/99, 06/02/99, 11/06/00 LAC 06/21/99 EAD, 11/09/00 HOJ ETS-8-5.1 Sera Week24 FACT-TOX-026 Covance# 6329-231 Study: Product Number(Test Substance): Matrix: Method/Revision: Analytical Equipment System Number Instrument Software/Version: Filename: R-Squared Value: Slope: Y-Intercept: Date of Extraction/Analyst: Date of Analysis/Analyst: Date of Data Reduction/Analyst: Sam ple Data W eek 24 M ONKEY SERA Group Dose Sample # Method Blk H20 Blk-9 H2OBlk-10 Matrix Blk Rabbit Sera Blk-9 Rabbit Sera Blk-10 QC - 250 ppb RBS05149-MS-9 RB S05149-MSD-9 Group 1 I05709M Control I05714M 0.0 mg/kg/day I05715M 105718M I05720M I05725M Group 2 I05702M Low Dose I05706M 105717M Group 3 I05707M Mid-High Dose I05708M 10 mg/kg/day I05710M I05712M I05716M 105719M Group 4 I05703M High Dose I05704M 30 mg/kg/day 105711M 105713M I05722M Limit of Quantitation (LOQ): 0.0137 ug/mL Correction factors not applicable for MS/MSD QC data 6 Month Capsule Toxicity Study with APFO in Cynomolgus Monkeys T-6889.2 (POAA) Monkey Sera ETS-8-4.1 and ETS-8-5.1 Amelia 062498 MassLynx 3.2 See Attachments See Attachments See Attachments See Attachments 05/14/99 MCH 05/20/99, 05/26/99, 05/28/99 HOJ/SAH/MEE 05/24/99, 05/27/99, 06/01/99, 11/06/00 HOJ/MEE/KJH Concentration Mean of POAA POAA ug/mL or % Ree. ug/mL <LOQ 0.0407 NA <LOQ 0.0315 NA 104% 109% 107% Ext Ext <LOQ (0.0983 ug/mL) <LOQ (0.0983 ug/mL) <LOQ (0.0983 ug/mL) <LOQ (0.0983 ug/mL) <LOQ 10.2 146 60.2 72.2 89.1 61.4 81.8 116 82.3 115 90.9 20.4 90.4 4.29 181 17.0 62.7 Ext: Samples lost during sample extraction. RSD Std. Dev. MS/MSD RPD NA NA 4% NA NA 95.3 68.8 23.3 21.2 119 74.3 POAA = Perfluorooctanoate Date Entered/By: Date Verified/By: 05/25/99, 05/27/99, 06/02/99, 11/06/00 LAC 06/21/99 EAD, 11/09/00 HOJ Sera Week24 FACT-TOX-026 Covance# 6329-231 Study: Product Number(Test Substance): Matrix: Method/Revision: Analytical Equipment System Number: Instrument Software/Version: Filename: R-Squared Value: Slope: Y-Intercept: Date of Extraction/Analyst: Date of Analysis/Analyst: Date of Data Reduction/Analyst: Sam ple Data 6 Month Capsule Toxicity Study with APFO in Cynomolgus Monkeys T-6889.2 (POAA) Monkey Sera ETS-8-4.1 & ETS-8-5.1 Amelia 062498 MassLynx 3.2 See Attachments See Attachments See Attachments See Attachments 04/29/99, 05/14/99 SAH 05/17/99, 05/20/99,05/26/99, 06/10/99 HOJ/SAH/MEE 05/19/99, 05/24/99,05/27/99, 06/11/99, 11/06/00 HOJ/MEE/KJH Filenames Blks Grp 1 Grp 2 Grp 3 Grp 4 MS, MSD W eek 26 M ONKEY SERA Group Dose Sample # Extraction Voi. Ratio Method Blk H20 Blk-3 Method Blk Matrix Blk Matrix Blk QC - 250 ppb H20 Blk-4 Rabbit Sera Blk-3 Rabbit Sera Blk-4 MKS05149-MS-3 MKS05149-MSD-3 Group 1 I05709M Control I05714M 0.0 mg/kg/day I05715M I05718M I05720M I05725M Group 2 I05702M Mid Dose I05706M 3.0 mg/kg/day 105717M Group 3 Mid-High Dose 10 mg/kg/day I05707M I05708M I05710M I05719M Group 4 I05703M High Dose I05704M 30 mg/kg/day 105711M I05713M I05722M Limit of Quantitation (LOQ): 0.0137 ug/mL Correction factors not applicable for MS/MSD QC data NA NA 1.0 1.0 1.0 1.0 0.58 0.66 0.57 0.50 0.50 0.52 0.62 0.61 0.61 0.49 0.58 0.65 0.38 0.37 0.70 0.36 0.63 0.51 POAA Std Correction Factor 0.9582 0.9582 0.9582 0.9582 NA NA 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 POAA Dilution Factor 1 1 1 1 1 1 1 1 1 1 1 200 200 200 100 100 200 100 10 100 1 500 20 POAA Cone. ng/mL 0.00 0.00 0.00 0.00 208 226 285 74.1 60.3 108 161 33.8 403 450 279 399 335 327 316 429 451 565 393 422 Concentration of POAA ug/mL or % Ree. <LOQ (0.0250 ug/mL) <LOQ (0.0250 ug/mL) <LOQ (0.0250 ug/mL) <LOQ (0.0250 ug/mL) 83% 91% 0.471 0.108 0.101 0.206 0.308 0.0623 125 141 87.6 77.9 55.4 96.5 79.6 11.1 61.7 1.50 299 15.8 Mean POAA ug/mL <LOQ <LOQ 87% 0.209 118 77.4 77.8 POAA A051799055, 56 & 95, 96 051799067-70 052699015-17 052099120-121, 123 & 052699020 051799086, 052099111, 126 & 052699021-22 061099093-94 Dilutions 1/1 1/1 1/200 1/100&200 1/1,10,20,100,&500 1/1 RSD Std. Dev. MS/MSD RPD NA NA 9% 74.5 0.156 23.3 27.5 21.8 16.9 162 126 POAA = Perfluorooctanoate Date Entered/By: Date Verified/ By: 05/19/98,05/27/99,06/14/99, 11/06/00 LAC 06/21/99 EAD, 06/22/99 LAC ETS-8-5.1 Sera Week26 FACT-TOX-026 Covance# 6329-231 Study: Product NumberfTest Substance): Matrix: Method/Revision: Analytical Equipment System Number: Instrument Software/Version: Filename: R-Squared Value: Slope: Y-Intercept: Date of Extraction/Analyst: Date of Analysis/Analyst: Date of Data Reduction/Analyst: 6 Month Capsule Toxicity Study with APFO in Cynomolgus Monkeys T-6889.2 (POAA) Monkey Sera ETS-8-4.1 & ETS-8-5.1 Amelia 062498 MassLynx 3.2 See Attachments See Attachments See Attachments See Attachments 04/29/99,05/14/99 SAH 05/17/99, 05/20/99, 05/26/99, 06/10/99 HOJ/SAH/MEE 05/19/99, 05/24/99, 05/27/99, 06/11/99, 11/06/00 HOJ/MEE/KJH Sam ple Data W eek 26 MONKEY SERA Group Sample # Concentration Dose of POAA ug/mL or % Ree. Method BIk H20 Blk-3 <LOQ (0.0250 ug/mL) Method BIk H20 Blk-4 <LOQ (0.0250 ug/mL) Matrix BIk Rabbit Sera Blk-3 <LOQ (0.0250 ug/mL) Matrix BIk Rabbit Sera Blk-4 <LOQ (0.0250 ug/mL) QC - 250 ppb MKS05149-MS-3 83% MKS05149-MSD-3 91% Group 1 I05709M 0.471 Control I05714M 0.108 0.0 mg/kg/day I05715M 0.101 105718M 0.206 I05720M 0.308 I05725M 0.0623 Group 2 I05702M 125 Mid Dose I05706M 141 105717M 87.6 Group 3 I05707M 77.9 Mid-High Dose I05708M 55.4 10 mg/kg/day I05710M 96.5 105719M 79.6 Group 4 I05703M 11.1 High Dose I05704M 61.7 30 mg/kg/day 105711M 1.50 105713M 299 I05722M 15.8 Limit of Quantitation (LOQ): 0.0137 ug/mL Correction factors not applicable for MS/MSD QC data Mean POAA ug/mL <LOQ <LOQ 87% 0.209 118 77.4 77.8 RSD Std. Dev. MS/MSD RPD NA NA 9% 74.5 0.156 23.3 27.5 21.8 16.9 162 126 POAA = Perfluorooctanoate Date Entered/By: Date Verified/By: 05/19/98, 05/27/99, 06/14/99, 11/06/00 LAC 06/21/99 EAD, 06/22/99 LAC ETS-8-5.1 Sera Week26 FACT-TOX-026 Covance# 6329-231 Study: Product Numbei^Test Substance): Matrix: Method/Revision: Analytical Equipment System Number: Instrument Software/Version: Filename: R-Squared Value: Slope: Y-Intercept: Date of Extraction/Analyst: Date of Analysis/Analyst: Date of Data Reduction/Analyst: 6 Month Capsule Toxicity Study with APFO in Cynomolgus Monkeys T-6889.2 (POAA) Monkey Sera ETS-8-4.1 & ETS-8-5.1 Filenames Amelia 062498 MassLynx 3.2 Blks See Attachments Grp 1 See Attachments Grp 2 See Attachments Grp 3 See Attachments Grp 4 04/29/99 SAH MS, MSD 04/30/99,05/03/99, 05/04/99, 05/05/99 KJH/MEE/HOJ 05/03/99,05/04/99, 05/05/99, 05/25/99, 11/02/00, 11/06/00 HOJ/KJH POAA A050399003, 4 & 42, 43 043099065-70 050399016-18,36-37 050399021-28, 050599116 050399029-32, 050499016, 050599117 043099073-74 Sam ple Data W eek 26, 27 M ONKEY SERA Group Dose Sample # Extraction Voi. Ratio Method Blk H20 Blk-1 H20 Blk-2 Matrix Blk Rabbit Sera Blk-1 Rabbit Sera Blk-2 QC - 250 ppb I05709M-MS I05714M-MS Group 1 I05709M Wk27 Control I05714M Wk27 0.0 mg/kg/day I05715M Wk27 I05718M Wk26 I05720M Wk26 I05725M Wk27 Group 2 I05702M Wk27 Mid Dose I05706M Wk27 3.0 mg/kg/day I05717M Wk27 105721M Moribund 5721M replacement I05723M Group 3 I05707M Wk27 Mid-High Dose I05708M Wk27 10 mg/kg/day I05710M Wk27 I05712M Wk26 05716M Wk26 I05719M Wk27 Group 4 I05703M Wk27 High Dose I05704M Wk27 30 mg/kg/day 10571 lMWk27 I05713M Wk27 I05722M Wk27 I05724M Moribund Limit of Quantitation (LOQ): 0.0137 ug/mL Correction factors not applicable for.MS/MSD QC data NA NA 1.0 1.0 1.0 1.0 1.0 1.0 1.0 0.50 0.50 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 0.50 0.60 1.0 1.0 1.0 1.0 1.0 1.0 1.0 POAA Std Correction Factor 0.9582 0.9582 0.9582 0.9582 NA NA 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 POAA Dilution Factor 1 1 1 1 1 1 1 1 1 1 1 1 100 100 100 100 100 1000 100 100 100 100 100 100 100 100 1000 100 1000 POAA Cone. ng/mL 0.00 0.00 0.00 0.00 592 500 393 229 98.7 108 161 142 500 673 460 480 626 126 335 497 525 489 680 78.1 612 9.15 192 69.4 511 Concentration of POAA ug/mL or % Ree. <LOQ (0.00983 ug/mL) <LOQ (0.00983 ug/mL) <LOQ (0.00983 ug/mL) <LOQ (0.00983 ug/mL) 79% 108% 0.377 0.219 0.0945 0.206 0.308 0.136 47.9 64.5 44.1 46.0 60.0 121 32.1 47.6 101 78.1 65.2 7.48 58.6 0.877 184 6.65 489 Mean POAA ug/mL <LOQ <LOQ 94% 0.223 52.5 74.1 51.5 RSD Std, Dev, MS/MSD RPD NA NA 17% 47.1 0.105 17.4 9.14 44.7 33.1 151 77.6 POAA = Perfluorooctanoate Date Entered/By: Date Verified/By: 05/04/99,05/05/99,05/25/99, 11/03/00, 11/06/00 LAC 06/21/99 EAD, 11/09/00 HOJ Dilutions 1/1 1/1 1/100 1/100&1000 1/100&1000 1/1 ETS-8-5.1 Sera Week26.27 ETS-8-5.1 FACT-TOX-026 Covance# 6329-231 Study: Product Number(Test Substance): Matrix: Method/Revision: Analytical Equipment System Number: Instrument Software/Version: Filename: R-Squared Value: Slope: Y-Intercept: Date of Extraction/Analyst: Date of Analysis/Analyst: Date of Data Reduction/Analyst: Sam ple Data W eek 26, 27 MONKEY SERA Group Dose Sample # Method Blk H20 Blk-1 H20 Blk-2 Matrix Blk Rabbit Sera Blk-1 Rabbit Sera Blk-2 QC - 250 ppb I05709M-MS I05714M-MS Group 1 I05709M Wk27 Control 0.0 mg/kg/day I05714M Wk27 105715M Wk27 105718M Wk26 I05720M Wk26 I05725M Wk27 Group 2 I05702M Wk27 Mid Dose I05706M Wk27 I05717M Wk27 105721M Moribund I05723M Group 3 I05707M Wk27 Mid-High Dose I05708M Wk27 10 mg/kg/day 105710M Wk27 105712M Wk26 105716M Wk26 I05719M Wk27 Group 4 I05703M Wk27 High Dose I05704M Wk27 30 mg/kg/day 105711M Wk27 I05713M Wk27 I05722M Wk27 I05724M Moribund Limit of Quantitation (LOQ): 0.0137 ug/mL Correction factors not applicable for MS/MSD QC data 6 Month Capsule Toxicity Study with APFO in Cynomolgus Monkeys T-6889.2 (POAA) Monkey Sera ETS-8-4.1 & ETS-8-5.1 Amelia 062498 MassLynx 3.2 See Attachments See Attachments See Attachments See Attachments 04/29/99 SAH 04/30/99, 05/03/99, 05/04/99, 05/05/99 KJH/MEE/HOJ 05/03/99,05/04/99,05/05/99,05/25/99, 11/02/00, 11/06/00 HOJ/KJH Concentration of POAA ug/mL or % Ree. <LOQ (0.00983 ug/mL) <LOQ (0.00983 ug/mL) <LOQ (0.00983 ug/mL) <LOQ (0.00983 ug/mL) 79% 108% 0.377 0.219 0.0945 0.206 0.308 0.136 47.9 64.5 44.1 46.0 60.0 121 32.1 47.6 101 78.1 65.2 7.48 58.6 0.877 184 6.65 489 Mean POAA ug/mL <LOQ <LOQ 94% 0.223 52.5 74.1 51.5 RSD Std. Dev. MS/MSD RPD NA NA 17% 47.1 0.105 17.4 9.14 44.7 33.1 151 77.6 POAA = Perfluorooctanoate Date Entered/By: Date Verified/ By: 05/04/99, 05/05/99, 05/25/99, 11/03/00, 11/06/00 LAC 06/21/99 EAD, 11/09/00 HOJ Sera Week26.27 FACT-TOX-026 Covance# 6329-231 Study: Product Number(Test Substance): Matrix: Method/Revision: Analytical Equipment System Number: Instrument Software/Version: Filename: R-Squared Value: Slope: Y-Intercept: Date of Extraction/Analyst: Date of Analysis/Analyst: Date of Data Reduction/Analyst: 6 Month Capsule Toxicity Study with APFO in Cynomolgus Monkeys T-6889.2 (POAA) Monkey Sera ETS-8-4.1 & ETS-8-5.1 Filenames Soup 020199, Amelia 062498 MassLynx 3.2 Blks See Attachments Grp 1 See Attachments Grp 3 See Attachments MS, MSD See Attachments 06/11/99 RWW 06/22/99, 06/23/99 DRB/MEE 06/23/99, 06/24/99, 11/06/00 DRB/MEE/KJH Sam ple Data W eek 28 MONKEY SERA Group Dose Sample # Extraction Voi. Ratio POAA Std Correction Factor POAA Dilution Factor POAA Cone. ng/mL Concentration of POAA ug/mL or % Ree. Method Blk Matrix Blk Matrix Blk QC - 250 ppb Group 1 Control, 0.0 mg/kg/day Group 3 10 mg/kg/day H20 Blk-1 H20 Blk-2 Rabbit Sera Blk-1 Rabbit Sera Blk-2 Monkey Sera Blk-1 Monkey Sera Blk-2 MKS06119-MS-l MKS06119-MSD-1 105718M I05720M I05712M 105716M 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 0.53 0.56 0.61 0.60 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 NA . NA 0.9582 0.9582 0.9582 0.9582 1 0.00 1 0.00 1 0.00 1 0.00 1 0.150 1 0.00 1 264 1 261 1 84.8 1 122 50 394 50 262 <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ 106% 104% 0.153 0.209 30.9 21.0 Limit of Quantitation (LOQ): 0.0137 ug/mL Correction factors not applicable for MS/MSD QC data POAA S062299003-5 & 44-46 062299016-17 062399016-17 062299040-41 Mean POAA ug/mL <LOQ <LOQ <LOQ 105% 0.181 25.9 RSD Std. Dev. MS/MSD RPD NA NA NA i% 21.6 0.0391 27.2 7.07 Dilutions 1/1 1/1 1/50 1/1 POAA = Perfluorooctanoate Date Entered/By: Date Verified/ By: 06/23/99, 06/25/99, 11/06/00 LAC 08/04/99 GML, 11/09/00 HOJ F.TS-8-5.1 Sera Week28 FACT-TOX-026 Covance# 6329-231 Study: Product Number(Test Substance): Matrix: Method/Revision: Analytical Equipment System Number Instrument Software/Version: Filename: R-Squared Value: Slope: Y-Intercept: Date of Extraction/Analyst: Date of Analysis/Analyst: Date of Data Reduction/Analyst: Sam ple Data W eek 28 M ONKEY SERA Group Dose Sample # Method Blk H20 Blk-1 H20 Blk-2 Matrix Blk Rabbit Sera Blk-1 Rabbit Sera Blk-2 Matrix Blk Monkey Sera Blk-1 Monkey Sera Blk-2 QC - 250 ppb MKS06119-MS-1 MKS06119-MSD-1 Group 1 105718M Control, 0.0 mg/kg/day I05720M Group 3 105712M 10 mg/kg/day 105716M Limit of Quantitation (LOQ): 0.0137 ug/mL Correction factors not applicable for MS/MSD QC data 6 Month Capsule Toxicity Study with APFO in Cynomolgus Monkeys T-6889.2 (POAA) Monkey Sera ETS-8-4.1 & ETS-8-5.1 Soup 020199, Amelia 062498 MassLynx 3.2 See Attachments See Attachments See Attachments See Attachments 06/11/99 RWW 06/22/99, 06/23/99 DRB/MEE 06/23/99,06/24/99, 11/06/00 DRB/MEE/KJH rA rA OO oo Concentration of POAA ug/mL or % Ree. <LOQ <LOQ <LOQ <LOQ 106% 104% 0.153 0.209 30.9 21.0 Mean POAA ug/mL <LOQ <LOQ <LOQ 105% 0.181 25.9 RSD Std. Dev. MS/MSD RPD NA NA NA i% 21.6 0.0391 27.2 7.07 POAA = Perfluorooctanoate Date Entered/By: Date Verified/ By: 06/23/99, 06/25/99,11/06/00 LAC 08/04/99 GML, 11/09/00 HOJ ETS-8-5.1 Sera Week28 FACT-TOX-026 Covance# 6329-231 Study: Product Number(Test Substance): Matrix: Method/Revision: Analytical Equipment System Number: Instrument Software/Version: Filename: R-Squared Value: Slope: Y-Intercept: Date of Extraction/Analyst: Date of Analysis/Analyst: Date of Data Reduction/Analyst: 6 Month Capsule Toxicity Study with APFO in Cynomolgus Monkeys T-6889.2 (POAA) Monkey Sera ETS-8-4.1 & ETS-8-5.1 Soup 020199, Amelia 062498 MassLynx 3.2 See Attachments See Attachments See Attachments See Attachments 06/11/99 RWW 06/22/99, 06/23/99 DRB/MEE 06/23/99,06/24/99, 11/06/00 DRB/MEE/KJH Sam ple Data W eek 30 M ONKEY SERA Group Dose Sample # Extraction Voi. Ratio Method Blk H20 Blk-1 H20 Blk-2 Matrix Blk Rabbit Sera Blk-1 Rabbit Sera BIk-2 Matrix Blk Monkey Sera Blk-1 Monkey Sera Blk-2 QC - 250 ppb MKS06119-MS-1 MKS06119-MSD-l Group 1 I05718M Control, 0.0 mg/kg/day I05720M Group 3 I05712M 10 mg/kg/day 105716M Limit of Quantitation (LOQ): 0.0137 ug/mL Correction factors not applicable for MS/MSD QC data 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 0.65 0.62 0.75 0.55 POAA Std Correction Factor 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 NA NA 0.9582 0.9582 0.9582 0.9582 POAA Dilution Factor 1 1 1 1 1 1 1 1 1 i 50 50 POAA Cone. ng/mL 0.00 0.00 0.00 0.00 0.150 0.00 264 261 86.1 104 234 88.4 Concentration of POAA ug/mL or % Ree. <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ 106% 104% 0.127 0.161 14.9 7.70 Filenames Blks Grp 1 Grp 3 MS, MSD Mean POAA ug/mL <LOQ <LOQ <LOQ 105% 0.144 11.3 POAA S062299003-5 & 44-46 062299018-19 062399020-21 062299040-41 Dilutions 1/1 1/1 1/50 1/1 RSD Std. Dev. MS/MSD RPD NA NA NA i% 16.6 0.0238 45.2 5.11 POAA = Perfluorooctanoate Date Entered/By: Date Verified/ By: 06/23/99, 06/25/99, 11/06/00 LAC 08/04/99 GML, 11/09/00 HOJ ETS-8-5.1 Sera Week30 FACT-TOX-026 Covance# 6329-231 Study: Product Number(Test Substance): Matrix: Method/Revision: Analytical Equipment System Number Instrument Software/Version: Filename: R-Squared Value: Slope: Y-Intercept: Date of Extraction/Analyst: Date of Analysis/Analyst: Date of Data Reduction/Analyst: Sam ple Data W eek 30 M ONKEY SERA Group Dose Sample # Method Blk H20 Blk-1 H20 Blk-2 Matrix Blk Rabbit Sera Blk-1 Rabbit Sera BIk-2 Matrix Blk Monkey Sera Blk-1 Monkey Sera Blk-2 QC - 250 ppb MKS06119-MS-l MKS06119-MSD-l Group 1 105718M Control, 0.0 mg/kg/day I05720M Group 3 I05712M 10 mg/kg/day I05716M Limit of Quantitation (LOQ): 0.0137 ug/mL Correction factors not applicable for MS/MSD QC data 6 Month Capsule Toxicity Study with APFO in Cynomolgus Monkeys T-6889.2 (POAA) Monkey Sera ETS-8-4.1 & ETS-8-5.1 Soup 020199, Amelia 062498 MassLynx 3.2 See Attachments See Attachments See Attachments See Attachments 06/11/99 RWW 06/22/99, 06/23/99 DRB/MEE 06/23/99,06/24/99, 11/06/00 DRB/MEE/KJH rA rA OO oo O' O oo VV Concentration of POAA ug/mL or % Ree. <LOQ <LOQ 106% 104% 0.127 0.161 14.9 7.70 Mean POAA ug/mL <LOQ <LOQ <LOQ 105% 0.144 11.3 RSD Std. Dev. MS/MSD RPD NA NA NA i% 16.6 0.0238 45.2 5.11 POAA = Perfluorooctanoate Date Entered/By: Date Verified/ By: 06/23/99, 06/25/99, 11/06/00 LAC 08/04/99 GML, 11/09/00 HOJ ETS-8-5.1 Sera Week30 FACT-TOX-026 Covance# 6329-231 Study: Product Number(Test Substance): Matrix: Method/Revision: Analytical Equipment System Number: Instrument Software/Version: Filename: R-Squared Value: Slope: Y-Intercept: Date of Extraction/Analyst: Date of Analysis/Analyst: Date of Data Reduction/Analyst: 6 Month Capsule Toxicity Study with APFO in Cynomolgus Monkeys T-6889.2 (POAA) Monkey Sera ETS-8-4.1 & ETS-8-5.1 Soup 020199, Amelia 062498 MassLynx 3.2 See Attachments See Attachments See Attachments See Attachments 06/11/99 RWW 06/22/99,06/23/99 DRB/MEE 06/23/99,06/24/99, 11/06/00 DRB/MEE/KJH Sam ple Data W eek 32 M ONKEY SERA Group Dose Sample # Extraction Voi. Ratio Method Blk H20 Blk-1 H20 Blk-2 Matrix Blk Rabbit Sera Blk-1 Rabbit Sera Blk-2 Matrix Blk Monkey Sera Blk-1 Monkey Sera Blk-2 QC - 250 ppb MKS06119-MS-1 MKS06119-MSD-l Group 1 105718M Control, 0.0 mg/kg/day I05720M Group 3 I05712M 10 mg/kg/day I05716M Limit of Quantitation (LOQ): 0.0137 ug/mL Correction factors not applicable for MS/MSD QC data 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 0.38 0.48 0.72 0.80 POAA Std Correction Factor 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 NA NA 0.9582 0.9582 0.9582 0.9582 POAA Dilution Factor 1 1 1 1 1 1 1 i 1 i 50 50 POAA Cone. ng/mL 0.00 0.00 0.00 0.00 0.150 0.00 264 261 37.4 62.6 156 80.9 Concentration of POAA ug/mL or % Ree. <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ 106% 104% 0.0943 0.125 10.4 4.85 Filenames BIks Grp 1 Grp 3 MS, MSD Mean POAA ug/mL <LOQ <LOQ <LOQ 105% 0.110 7.60 POAA S062299003-5 & 44-46 062299022-23 062399024-25 062299040-41 RSD Std. Dev. MS/MSD RPD NA NA NA 1% 19.8 0.0216 51.2 3.90 Dilutions 1/1 1/1 1/50 1/1 POAA = Perfluorooctanoate Date Entered/By: Date Verified/ By: 06/23/99, 06/25/99, 11/06/00 LAC 08/04/99 GML, 11/09/00 HOJ ETS-8-5.1 Sera Week32 FACT-TOX-026 Covance# 6329-231 Study: Product NumbertTest Substance): Matrix: Method/Revision: Analytical Equipment System Number: Instrument Software/Version: Filename: R-Squared Value: Slope: Y-Intercept: Date of Extraction/Analyst: Date of Analysis/Analyst: Date of Data Reduction/Analyst: Sam ple Data W eek 32 MONKEY SERA Group Dose Sample # Method Blk H20 Blk-1 H20 Blk-2 Matrix Blk Rabbit Sera Blk-1 Rabbit Sera Blk-2 Matrix Blk Monkey Sera Blk-1 Monkey Sera Blk-2 QC - 250 ppb MKS06119-MS-1 MKS06119-MSD-1 Group 1 I05718M Control, 0.0 mg/kg/day I05720M Group 3 10 mg/kg/day I05712M 105716M Limit of Quantitation (LOQ): 0.0137 ug/mL Correction factors not applicable for MS/MSD QC data 6 Month Capsule Toxicity Study with APFO in Cynomolgus Monkeys T-6889.2 (POAA) Monkey Sera ETS-8-4.1 &ETS-8-5.1 Soup 020199, Amelia 062498 MassLynx 3.2 See Attachments See Attachments See Attachments See Attachments 06/11/99 RWW 06/22/99, 06/23/99 DRB/MEE 06/23/99,06/24/99, 11/06/00 DRB/MEE/KJH Concentration of POAA ug/mL or % Ree. <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ 106% 104% 0.0943 0.125 10.4 4.85 Mean POAA ug/mL <LOQ <LOQ <LOQ 105% 0.110 7.60 RSD Std. Dev. MS/MSD RPD NA NA NA i% 19.8 0.0216 51.2 3.90 POAA = Perfluorooctanoate Date Entered/By: Date Verified/ By: 06/23/99,06/25/99,11/06/00 LAC 08/04/99 GML, 11/09/00 HOJ ETS-8-5.1 Sera Week32 FACT-TOX-026 Covance# 6329-231 Study: Product Number(Test Substance): Matrix: Method/Revision: Analytical Equipment System Number: Instrument Software/Version: Filename: R-Squared Value: Slope: Y-Intercept: Date of Extraction/Analyst: Date of Analysis/Analyst: Date of Data Reduction/Analyst: 6 Month Capsule Toxicity Study with APFO in Cynomolgus Monkeys T-6889.2 (POAA) Monkey Sera ETS-8-4.1 & ETS-8-5.1 Soup 020199, Amelia 062498 MassLynx 3.2 See Attachments See Attachments See Attachments See Attachments 06/11/99 RWW 06/22/99,06/23/99 DRB/MEE 06/23/99,06/24/99, 11/06/00 DRB/MEE/KJH Sam ple Data W eek 34 MONKEY SERA Group Dose Sample # Extraction Voi. Ratio POAA Std Correction Factor POAA Dilution Factor POAA Cone. ng/mL Concentration of POAA ug/mL or % Ree. Method Blk Matrix Blk Matrix Blk QC - 250 ppb H20 BIk-1 H20 Blk-2 Rabbit Sera Blk-1 Rabbit Sera Blk-2 Monkey Sera Blk-1 Monkey Sera Blk-2 MKS06119-MS-1 MKS06119-MSD-l 1.0 0.9582 1.0 0.9582 1.0 0.9582 1.0 0.9582 1.0 0.9582 1.0 0.9582 1.0 NA 1.0 NA 1 0.00 1 0.00 1 0.00 1 0.00 1 0.150 1 0.00 1 264 1 261 <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ 106% 104% Group 1 Control, 0.0 mg/kg/day I05718M I05720M 0.43 0.9582 1 30.5 0.45 0.9582 1 49.0 0.0680 0.104 Group 3 10 mg/kg/day I05712M I05716M 0.76 0.9582 50 86.4 0.64 0.9582 50 33.3 5.45 2.49 Limit of Quantitation (LOQ): 0.0137 ug/mL Correction factors not applicable for MS/MSD QC data Filenames Blks Grp 1 Grp 3 MS, MSD Mean POAA ug/mL <LOQ <LOQ <LOQ 105% 0.0861 3.97 POAA S062299003-5 & 44-46 062299024-25 062399028-29 062299040-41 Dilutions 1/1 1/1 1/50 1/1 RSD Std. Dev. MS/MSD RPD NA NA NA i% 29.7 0.0256 52.7 2.09 POAA = PerfVuorooctanoate Date Entered/By: Date Verified/ By: 06/23/99, 06/25/99, 11/06/00 LAC 08/04/99 GML, 11/09/00 HOJ F.TS-8-5.1 Sera Week34 FACT-TOX-026 Covance# 6329-231 Study: Product Number(Test Substance): Matrix: Method/Revision: Analytical Equipment System Number: Instrument Software/Version: Filename: R-Squared Value: Slope: Y-Intercept: Date of Extraction/Analyst: Date of Analysis/Analyst: Date of Data Reduction/Analyst: Sam ple Data W eek 34 MONKEY SERA Group Dose Sample # Method Blk H20 Blk-1 H20 Blk-2 Matrix Blk Rabbit Sera Blk-1 Rabbit Sera Blk-2 Matrix Blk Monkey Sera Blk-1 Monkey Sera Blk-2 QC - 250 ppb MKS06119-MS-1 MKS06119-MSD-l Group 1 105718M Control, 0.0 mg/kg/day I05720M Group 3 I05712M 10 mg/kg/day I05716M Limit of Quantitation (LOQ): 0.0137 ug/mL Correction factors not applicable for MS/MSD QC data 6 Month Capsule Toxicity Study with APFO in Cynomolgus Monkeys T-6889.2 (POAA) Monkey Sera ETS-8-4.1 &ETS-8-5.1 Soup 020199, Amelia 062498 MassLynx 3.2 See Attachments See Attachments See Attachments See Attachments 06/11/99 RWW 06/22/99, 06/23/99 DRB/MEE 06/23/99,06/24/99, 11/06/00 DRB/MEE/KJH Concentration of POAA ug/mL or % Ree. <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ 106% 104% 0.0680 0.104 5.45 2.49 Mean POAA ug/mL <LOQ <LOQ <LOQ 105% 0.0861 3.97 RSD Std. Dev. MS/MSD RPD NA NA NA 1% 29.7 0.0256 52.7 2.09 POAA = Perfluorooctanoate Date Entered/By: Date Verified/ By: 06/23/99,06/25/99,11/06/00 LAC 08/04/99 GML, 11/09/00 HOJ Sera Week34 FACT-TOX-026 Covance# 6329-231 Study: Product Number(Test Substance): Matrix: Method/Revision: Analytical Equipment System Number: Instrument Software/Version: Filename: R-Squared Value: Slope: Y-Intercept: Date of Extraction/Analyst: Date of Analysis/Analyst: Date of Data Reduction/Analyst: 6 Month Capsule Toxicity Study with APFO in Cynomolgus Monkeys T-6889.2 (POAA) Monkey Sera ETS-8-4.1 & ETS-8-5.1 Soup 020199, Amelia 062498 MassLynx 3.2 See Attachments See Attachments See Attachments See Attachments 07/13/99 MCH 07/14/99,07/21/99 GML/DRB 07/15/99,07/22/99, 11/06/00 GML/DRB/KJH Filenames Blks Grp 1 Grp 3 MS, MSD Sam ple Data W eek 36 M ONKEY SERA Group Dose Sample # Extraction Voi. Ratio Method Blk H20 Blk-1 H20 Blk-2 Matrix Blk Rabbit Sera Blk-1 Matrix Blk Rabbit Sera Blk-2 Monkey Sera Blk-1 QC - 250 ppb Monkey Sera Blk-2 MKS07139-MS-1 MKS07139-MSD-l Group 1 105718M Control, 0.0 mg/kg/day I05720M Group 3 I05712M 10 mg/kg/day I05716M Limit of Quantitation (LOQ): 0.0137 ug/mL Correction factors not applicable for MS/MSD QC data 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 0.45 0.48 0.64 0.65 POAA Std Correction Factor 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 NA NA 0.9582 0.9582 0.9582 0.9582 POAA Dilution Factor 1 1 1 1 1 1 1 1 1 1 10 10 POAA Cone. ng/mL 0.00 0.00 0.00 0.00 0.00 0.00 228 208 37.3 71.4 273 96.3 Concentration of POAA ug/mL or % Ree. <LOQ (0.0248 ug/mL) <LOQ (0.0248 ug/mL) <LOQ (0.0248 ug/mL) <LOQ (0.0248 ug/mL) <LOQ (0.0248 ug/mL) <LOQ (0.0248 ug/mL) 92% 84% 0.0795 0.142 4.08 1.42 Mean POAA ug/mL <LOQ <LOQ <LOQ 88% 0.111 2.75 POAA S071499003, 4, 16-17, & 39, 40 071499021-22 072199096-97 071499018-19 Box 99-130 Dilutions 1/1 1/1 1/10 1/1 RSD Std. Dev. MS/MSD RPD NA NA NA 9% 40.1 0.0445 68.4 1.88 POAA = Perfluorooctanoate Date Entered/By: Date Verified/ By: 07/20/99, 07/23/99, 11/06/00 LAC 08/04/99 GML, 11/09/00 HOJ ETS-8-5.1 Sera Week36 FACT-TOX-026 Covance# 6329-231 Study: Product Number(Test Substance): Matrix: Method/Revision: Analytical Equipment System Number: Instrument Software/Version: Filename: R-Squared Value: Slope: Y-Intercept: Date of Extraction/Analyst: Date of Analysis/Analyst: Date of Data Reduction/Analyst: Sam ple Data W eek 36 M ONKEY SERA Group Dose Sample # Method Blk H20 Blk-1 H20 Blk-2 Matrix Blk Rabbit Sera Blk-1 Rabbit Sera Blk-2 Matrix Blk Monkey Sera Blk-1 Monkey Sera Blk-2 QC - 250 ppb MKS07139-MS-1 MKS07139-MSD-1 Group 1 I05718M Control, 0.0 mg/kg/day I05720M Group 3 I05712M 10 mg/kg/day I05716M Limit of Quantitation (LOQ): 0.0137 ug/mL Correction factors not applicable for MS/MSD QC data 6 Month Capsule Toxicity Study with APFO in Cynomolgus Monkeys T-6889.2 (POAA) Monkey Sera ETS-8-4.1 & ETS-8-5.1 Soup 020199, Amelia 062498 MassLynx 3.2 See Attachments See Attachments See Attachments See Attachments 07/13/99 MCH 07/14/99,07/21/99 GML/DRB 07/15/99,07/22/99, 11/06/00 GML/DRB/KJH Concentration of POAA ug/mL or % Ree. <LOQ (0.0248 ug/mL) <LOQ (0.0248 ug/mL) <LOQ (0.0248 ug/mL) <LOQ (0.0248 ug/mL) <LOQ (0.0248 ug/mL) <LOQ (0.0248 ug/mL) 92% 84% 0.0795 0.142 4.08 1.42 Mean POAA ug/mL <LOQ <LOQ <LOQ 88% o.m 2.75 RSD Std. Dev. MS/MSD RPD NA NA NA 9% 40.1 0.0445 68.4 1.88 POAA = Perfluorooctanoate Date Entered/By: Date Verified/By: 07/20/99, 07/23/99, 11/06/00 LAC 08/04/99 GML, 11/09/00 HOJ ETS-8-5.1 Sera Week36 FACT-TOX-026 Covance# 6329-231 Study: Product Number(Test Substance): Matrix: Method/Revision: Analytical Equipment System Number: Instrument Software/Version: Filename: R-Squared Value: Slope: Y-Intercept: Date of Extraction/Analyst: Date of Analysis/Analyst: Date of Data Reduction/Analyst: 6 Month Capsule Toxicity Study with APFO in Cynomolgus Monkeys T-6889.2 (POAA) Monkey Sera ETS-8-4.1 & ETS-8-5.1 Soup 020199, Amelia 062498 MassLynx 3.2 See Attachments See Attachments See Attachments See Attachments 07/13/99 MCH 07/14/99, 07/21/99 GML/DRB 07/15/99, 07/22/99, 11/06/00 GML/DRB/KJH Filenames Blks Grp 1 Grp 3 MS, MSD Sam ple Data W eek 38 MONKEY SERA Group Dose Sample # Extraction Voi. Ratio Method Blk H20 Blk-1 H20 Blk-2 Matrix Blk Rabbit Sera Blk-1 Rabbit Sera Blk-2 Matrix Blk Monkey Sera Blk-1 Monkey Sera Blk-2 QC - 250 ppb MKS07139-MS-1 MKS07139-MSD-1 Group 1 105718M Control, 0.0 mg/kg/day I05720M Group 3 I05712M 10 mg/kg/day I05716M Limit of Quantitation (LOQ): 0.0137 ug/mL Correction factors not applicable for MS/MSD QC data 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 0.63 0.49 0.75 0.75 POAA Std Correction Factor 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 NA NA 0.9582 0.9582 0.9582 0.9582 POAA Dilution Factor 1 1 1 1 1 1 1 1 1 i 10 1 POAA Cone. ng/mL 0.00 0.00 0.00 0.00 0.00 0.00 228 208 46.8 59.8 221 659 Concentration of POAA ug/mL or % Ree. <LOQ (0.0248 ug/mL) <LOQ (0.0248 ug/mL) <LOQ (0.0248 ug/mL) <LOQ (0.0248 ug/mL) <LOQ (0.0248 ug/mL) <LOQ (0.0248 ug/mL) 92% 84% 0.0711 0.117 2.83 0.842 Mean POAA ug/mL <LOQ <LOQ <LOQ 88% 0.0941 1.84 POAA S071499003, 4, 16-17, & 39, 40 071499027-28 071499030, 072199098 071499018-19 Box 99-130 Dilutions 1/1 1/1 1/1&10 1/1 RSD Std. Dev. MS/MSD RPD NA NA NA 9% 34.5 0.0324 76.5 1.40 POAA = Perfluorooctanoate Date Entered/By: Date Verified/ By: 07/20/99, 07/23/99, 11/06/00 LAC 08/04/99 GML, 11/09/00 HOJ ETS-8-5.1 Sera Week38 FACT-TOX-026 Covance# 6329-231 Study: Product Number(Test Substance): Matrix: Method/Revision: Analytical Equipment System Number: Instrument Software/Version: Filename: R-Squared Value: Slope: Y-Intercept: Date of Extraction/Analyst: Date of Analysis/Analyst: Date of Data Reduction/Analyst: Sam ple Data W eek 38 M ONKEY SERA Group Dose Sample # Method Blk H20 Blk-1 H20 Blk-2 Matrix Blk Rabbit Sera Blk-1 Rabbit Sera Blk-2 Matrix Blk Monkey Sera Blk-1 Monkey Sera Blk-2 QC - 250 ppb MKS07139-MS-1 MKS07139-MSD-l Group 1 105718M Control, 0.0 mg/kg/day I05720M Group 3 I05712M 10 mg/kg/day 105716M Limit of Quantitation (LOQ): 0.0137 ug/mL Correction factors not applicable for MS/MSD QC data 6 Month Capsule Toxicity Study with APFO in Cynomolgus Monkeys T-6889.2 (POAA) Monkey Sera ETS-8-4.1 &ETS-8-5.1 Soup 020199, Amelia 062498 MassLynx 3.2 See Attachments See Attachments See Attachments See Attachments 07/13/99 MCH 07/14/99, 07/21/99 GML/DRB 07/15/99,07/22/99, 11/06/00 GML/DRB/KJH Concentration of POAA ug/mL or % Ree. <LOQ (0.0248 ug/mL) <LOQ (0.0248 ug/mL) <LOQ (0.0248 ug/mL) <LOQ (0.0248 ug/mL) <LOQ (0.0248 ug/mL) <LOQ (0.0248 ug/mL) 92% 84% 0.0711 0.117 2.83 0.842 Mean POAA ug/mL <LOQ <LOQ <LOQ 88% 0.0941 1.84 RSD Std. Dev. MS/MSD RPD NA NA NA 9% 34.5 0.0324 76.5 1.40 POAA = Perfluorooctanoate Date Entered/By: Date Verified/By: 07/20/99, 07/23/99,11/06/00 LAC 08/04/99 GML, 11/09/00 HOJ F.TS-8-5.1 Sera Week38 FACT-TOX-026 Covance# 6329-231 Study: Product Number(Test Substance): Matrix: Method/Revision: Analytical Equipment System Number: Instrument Software/Version: Filename: R-Squared Value: Slope: Y-Intercept: Date o f Extraction/Analyst: Date o f Analysis/Analyst: Date o f Data Reduction/Analyst: 6 Month Capsule Toxicity Study with APFO in Cynomolgus Monkeys T-6889.2 (POAA) Monkey Sera ETS-8-4.1 & ETS-8-5.1 Soup 020199, Amelia 062498 MassLynx 3.2 See Attachments See Attachments See Attachments See Attachments 07/13/99 MCH 07/14/99, 07/21/99 GML/DRB 07/15/99,07/22/99, 11/06/00 GML/DRB/KJH Filenames Blks Grp 1 Grp 3 MS, MSD Sam ple D ata W eek 40 M ONKEY SERA Group Dose Sample # Extraction Voi. Ratio Method Blk H20 Blk-1 H20 Blk-2 Matrix Blk Rabbit Sera Blk-1 Rabbit Sera Blk-2 Matrix Blk Monkey Sera Blk-1 Monkey Sera Blk-2 QC - 250 ppb MKS07139-MS-1 MKS07139-MSD-1 Group 1 105718M Control, 0.0 mg/kg/day I05720M Group 3 I05712M 10 mg/kg/day 105716M Limit of Quantitation (LOQ): 0.0137 ug/mL Correction factors not applicable for MS/MSD QC data 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 0.37 0.50 0.49 0.36 POAA Std Correction Factor 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 NA NA 0.9582 0.9582 0.9582 0.9582 POAA Dilution Factor 1 1 I 1 1 1 1 1 1 1 2 1 POAA Cone. ng/mL 0.00 0.00 0.00 0.00 0.00 0.00 228 208 27.8 39.4 452 225 Concentration of POAA ug/mL or % Ree. <LOQ (0.0248 ug/tnL) <LOQ (0.0248 ug/mL) <LOQ (0.0248 ug/mL) <LOQ (0.0248 ug/mL) <LOQ (0.0248 ug/mL) <LOQ (0.0248 ug/mL) 92% 84% 0.0719 0.0756 1.77 0.600 Mean POAA ug/mL <LOQ <LOQ <LOQ 88% 0.0738 1.18 POAA S071499003, 4, 16-17, & 39, 40 071499033-34 071499036, 072199099 071499018-19 Box 99-130 Dilutions 1/1 1/1 1/1&2 1/1 RSD Std. Dev. MS/MSD RPD NA NA NA 9% 3.47 0.00256 69.8 0.827 POAA = Perfluorooctanoate Date Entered/By: Date Verified/ By: 07/20/99, 07/23/99, 11/06/00 LAC 08/04/99 GML, 11/09/00 HOJ ETS-8-5.1 Sera Week40 FACT-TOX-026 Covance# 6329-231 Study: Product Number(Test Substance): Matrix: Method/Revision: Analytical Equipment System Number: Instrument Software/Version: Filename: R-Squared Value: Slope: Y-Intercept: Date of Extraction/Analyst: Date of Analysis/Analyst: Date of Data Reduction/Analyst: Sam ple Data W eek 40 M ONKEY SERA Group Dose Sample # Method Blk H20 Blk-1 H20 Blk-2 Matrix Blk Rabbit Sera Blk-1 Matrix Blk Rabbit Sera Blk-2 Monkey Sera Blk-1 Monkey Sera Blk-2 QC - 250 ppb MKS07139-MS-1 MKS07139-MSD-1 Group 1 I05718M Control, 0.0 mg/kg/day I05720M Group 3 I05712M 10 mg/kg/day I05716M Limit of Quantitation (LOQ): 0.0137 ug/mL Correction factors not applicable for MS/MSD QC data 6 Month Capsule Toxicity Study with APFO in Cynomolgus Monkeys T-6889.2 (POAA) Monkey Sera ETS-8-4.1 & ETS-8-5.1 Soup 020199, Amelia 062498 MassLynx 3.2 See Attachments See Attachments See Attachments See Attachments 07/13/99 MCH 07/14/99,07/21/99 GML/DRB 07/15/99,07/22/99, 11/06/00 GML/DRB/KJH Concentration of POAA ug/mL or % Ree. <LOQ (0.0248 ug/mL) <LOQ (0.0248 ug/mL) <LOQ (0.0248 ug/mL) <LOQ (0.0248 ug/mL) <LOQ (0.0248 ug/mL) <LOQ (0.0248 ug/mL) 92% 84% 0.0719 0.076 1.77 0.600 Mean POAA ug/mL <LOQ <LOQ <LOQ 88% 0.0738 1.18 RSD Std. Dev. MS/MSD RPD NA NA NA 9% 3.47 0.00256 69.8 0.827 POAA = Perfluorooctanoate Date Entered/By: Date Verified/ By: 07/20/99, 07/23/99, 11/06/00 LAC 08/04/99 GML, 11/09/00 HOJ FT<s-R-S t Sera Week40 ETS-8-5.1 FACT-TOX-026 Covance# 6329-231 Sera QC Summary - TO X026 11/13/1998 01/08/1999 04/29/1999 05/12/1999 05/14/1999 06/11/1999 07/13/1999 RBS11138-MS RBS11138-MSD RBS01089-MS RBS01089-MSD I05709M-MS I05714M-MS RBS05129-MS-3 RBS05129-MSD-3 RBS05129-MS-4 RBS05129-MSD-4 RBS05129-MS-7 RBS05129-MSD-7 RBS05129-MS-8 RBS05129-MSD-8 RBS05149-MS-11 RBS05149-MSD-11 RBS05149-MS-12 RBS05149-MSD-12 RBS05149-MS-9 RB S05149-MSD-9 MKS05149-MS-3 MKS05149-MSD-3 MKS06119-MS-l MKS06119-MSD-1 MKS07139-MS-1 MK.S07139-MSD-1 PFOA 109% 100% 100% 98% 79% 108% 80% 79% 92% 85% 103% 106% 103% 127% 116% 95% 99% 80% 104% 109% 83% 91% 106% 104% 92% 84% ** ** ** ** ** ** Bracketed by a CCV not witliin method criteria (31%). NA = Not Applicable NR = Not Reported Average of Monkey Spikes Standard Deviation Grubbs outliers? Number of monkey spikes 93% Used in Final Report 11% No 8 Used in Final Report Average of All Spikes Standard Deviation Grubbs outliers? Number of spikes 97% Data not used 12% No 26 Data not used QC Summary Study: Product Number(Test Substance): Matrix: Method/Revision: Analytical Equipment System Number: Instrument Software/Version: Date of Extraction/Analyst: Date of Analysis/Analyst: Date of Data Reduction/Analyst: Sam ple D ata F A C T -T O X -026 Covance# 6329-231 6 Month Capsule Toxicity Study with APFO in Cynomolgus Monkeys T-6889.2 (POAA) Monkey Liver Filename: See List to Right FACT-M-1.1 & FACT-M-2.1 R-Squared Value: See Attachments Soup 020199 Slope: See Attachments MassLynx 3.2 Y-Intercept: See Attachments 06/21/99 SAH 06/23/99, 07/28/99, 10/05/99 DRB/IAS 06/25/99, 07/29/99, 10/06/99, 11/02/00 DRB/IAS/KJH Filenames Blks Grp 1 Grp 2 Grp 3 Grp 4 MS, MSD POAA Dilutions S062399047-48 & 89-90 1/1 062399059-62 1/1 062399065-68 1/100 062399071-74 1/50,100 062399077-80,82 100599077 1/1,2,10,100,200,500 062399085-86 1/1 reanalyzed on 06/24/99 with confirmation of low recoveries M O N K EY LIV ER W eek 27 Group Sample # Dose Initial Wt. g Method Blk Matrix Blk QC - 250 ppb H20 Blk-3 H20 Blk-4 Rabbit Liver Blk-3 Rabbit Liver Blk-4 I05709M-MS-3 I05709M-MSD-3 Group 1 Control 0.0 mg/kg/day 105709M Wk27 105714M Wk27 I05715M Wk27 I05725M Wk27 Group 2 Mid Dose 3.0 mg/kg/day 5721M replacement I05702M Wk27 I05706M Wk27 I05717M Wk27 I05723M Group 3 Mid-High Dose 10 mg/kg/day I05707M Wk27 I05708M Wk27 I05710M Wk27 I05719M Wk27 Group 4 U l g l l jL /u ac 30 mg/kg/day I05703M Wk27 iTvD jC T/ Av y/T-rNivifi U7 1 , ' ) 7/ 10571 lMWk27 I05713M Wk27 I05722M Wk27 I05724M Moribund POAA = Perfluorooctanoate Limit of Quantitation (LOQ) = 75.5 ppb Correction Factors not applicable to MS/MSD QC data 1.0000 1.0000 1.0000 1.0000 1.0153 1.0153 1.0153 0.9931 1.0073 1.0006 1.0104 1.0002 0.9931 1.0163 1.0143 1.0061 1.0045 1.0048 1.0036 1 1.0040 1.0039 1.0143 1.0117 1.0164 Total Mass of Liver g NA NA NA NA NA NA NA NA . NA NA NA NA NA NA NA NA NA NA NA XT A i^ rv NA NA NA NA POAA Std POAA POAA POAA Concentration Correction Factor 0.9582 Cone. ng/g 13.0 Dilution Factor 1 Calc. Cone. " g/g 12.4 of POAA ug/g or % Ree. <LOQ 0.9582 2.33 1 2.23 <LOQ 0.9582 14.0 1 13.5 <LOQ . 0.9582 16.3 1 15.6 <LOQ NA 318 1 218 74% NA 282 1 183 62% 0.9582 96.7 1 91.3 0.0913 0.9582 56.0 1 54.0 <LOQ 0.9582 0.9582 238 43.9 1 . 226 0.226 1 42.0 <LOQ 0.9582 160 100 15206 15.2 0.9582 193 100 18531 18.5 0.9582 0.9582 117 173 100 11307 11.3 100 16301 16.3 0.9582 232 100 21917 21.9 0.9582 132 50 6294 6.29 0.9582 0.9582 92.9 197 100 8864 8.86 100 18753 18.8 0.9582 137 10 1309 1.31 rv n c o ' i U .7 J O 1679 10 16026 16.0 0.9582 226 1 216 0.216 0.9582 441 200 83299 83.3 0.9582 0.9582 746 327 2 1414 1.41 500 154369 154 Overall dilution = 1:100. 1 uL ofthe 1:10 dilution was injected on 6/23/99. LAC 11/03/00 - Mean POAA ng/g <LOQ <LOQ 68% 0.117 15.3 14.0 * 42.8 Date Entered/By: Date Verified/ By: 06/24/99,06/25/99,07/29/99, 10/07/99, 11/03/00 LAC 08/03/99 GML, 11/08/00 HOJ RSD Std. Dev. MS/MSD RPD NA NA 18% 62.4 0.0730 19.7 3.02 54.1 7.55 148 63.3 F A C T -M -2.0 Lvr062199 5/23/2001 F A C T -T O X -026 Covance# 6329-231 Study: Product Number(Test Substance): Matrix: Method/Revision: Analytical Equipment System Number: Instrument Software/Version: Date o f Extraction/Analyst: Date o f Analysis/Analyst: Date o f Data Reduction/Analyst: Sam ple D ata 6 Month Capsule Toxicity Study with APFO in Cynomolgus Monkeys T-6889.2 (POAA) Monkey Liver Filename: SeeAttachments FACT-M-1.1 & FACT-M-2.1 R-Squared ValuSee Attachments Soup 020199 MassLynx 3.2 Slope: Y-Intercept: SeeAttachments SeeAttachments 06/21/99 SAH 06/23/99, 07/28/99, 10/05/99 DRB/IAS 06/25/99,07/29/99, 10/06/99, 11/02/00 DRB/IAS/KJH M ONKEY LIV ER W eek 27 Group Dose Sample # POAA Calc. Cone, Method Blk H20 Blk-3 ng/g 12.4 H20 Blk-4 2.23 Matrix Blk Rabbit Liver Blk-3 13.5 Rabbit Liver Blk-4 15.6 QC - 250 ppb I05709M-MS-3 I05709M-MSD-3 218 183 Group 1 I05709M Wk27 91.3 Control I05714M Wk27 54.0 0.0 mg/kg/day I05715M Wk27 I05725M Wk27 226 42.0 Group 2 Mid Dose 3.0 mg/kg/day 5721M replacement I05702M Wk27 I05706M Wk27 I05717M Wk27 I05723M 15206 18531 11307 16301 Group 3 Mid-High Dose I05707M Wk27 I05708M Wk27 21917 6294 10 mg/kg/day I05710M Wk27 I05719M Wk27 8864 18753 Group 4 High Duse 30 mg/kg/day I05703M Wk27 T A f*)A 4X4- t m .A - t 1 V J /O H iV l VV1< U .I 10571 lMWk27 1309 16026 216 I05713M Wk27 83299 I05722M Wk27 I05724M Moribund 1414 154369 POAA = Perfluorooctanoate Limit of Quantitation (LOQ) = 75.5 ppb Correction Factors not applicable to MS/MSD QC data Concentration Mean RSD of POAA POAA Std. Dev. ug/g or % Ree. ug/g MS/MSD RPD <LOQ <LOQ <LOQ NA <LOQ <LOQ <LOQ NA 74% 62% 68% 18% 0.0913 <LOQ 0.226 62.4 <LOQ 0.117 0.0730 15.2 18.5 11.3 19.7 16.3 15.3 3.02 21.9 6.29 8.86 54.1 18.8 14.0 7.55 1.31 16.0 0.216 83.3 1.41 148 154 42.8 63.3 ` Overall dilution = 1:100. 1 uL of the 1:10 dilution was injected on 6/23/99. LAC 11/03/00 Date Entered/By: Date Verified/ By: 06/24/99, 06/25/99, 07/29/99,10/07/99, 11/03/00 LAC 08/03/99 GML, 11/08/00 HOJ FA C T -M -2.0 Lvr062199 5/23/2001 Study: Product Number(Test Substance): Matrix: Method/Revision: Analytical Equipment System Number: Instrument Software/Version: Date o f Extraction/Analyst: Date o f Analysis/Analyst: Date o f Data Reduction/Analyst: Sample Data FACT-T OX-026 Covance# 6329-231 6 Month Capsule Toxicity Study with APFO in Cynomolgus Monkeys T-6889.2 (POAA) Monkey Liver Filename: FACT-M-1.1 & FACT-M-2.1 R-Squared Value: Soup020199 Slope: MassLynx 3.2 Y-Intercept: 7/13/99 SEE 07/13/99 DRB 07/14/99, 11/02/00, 11/06/00 DRB/KJH See List to Right See Attachments See Attachments See Attachments Filenames Blks Grp 1 Grp 2 Grp 3 MS, MSD POAA A071399003-4, 30-31 071399019-20 072899016 071399023-24 071399015-16 Box 99-131 MONKEY LIVER Week 20 and 40 Group Sample # Dose Method Blk H20 Blk-11 H20 Blk-12 Matrix Blk Rabbit Liver Blk-11 Rabbit Liver Blk-12 QC - 250 ppb I05718M-MS-11 I05718M-MSD-12 Group 1, Wk40 0.0 mg/kg/day 105718M I05720M Group 2, Wk20 I05721M Group 3, Wk40 10 mg/kg/day 105712M 105716M POAA = Perfluorooctanoate Limit of Quantitation (LOQ) = 75.5 ppb Correction Factors not applicable to MS/MSD QC data Initial Wt. g 1.0000 1.0000 1.0000 1.0000 0.9933 0.9933 0.9933 0.9997 1.0083 1.0157 0.9965 Total Mass of Liver g NA NA NA NA NA NA NA NA NA NA NA POAA Std Correction Factor 0.9582 0.9582 0.9582 0.9582 NA NA 0.9582 0.9582 0.9582 0.9582 0.9582 POAA Cone. "g/g 9.55 0.00 3.08 2.95 353 345 14.0 22.5 193 154 86.6 POAA Dilution Factor 1 1 1 1 1 1 1 1 100 1 1 POAA Calc. Cone, "g/g 9.15 0.00 2.95 2.83 341 334 13.5 21.6 . 18321 146 83.3 Concentration of POAA ug/g or % Ree. <LOQ <LOQ <LOQ <LOQ 113% 111% <LOQ <LOQ 18.3 0.146 0.0833 Mean POAA "g/g <LOQ <LOQ 112% <LOQ NA 0.114 Date Entered/By: Date Verified/ By: 07/26/99, 11/02/00, 11/03/00, 11/06/00 LAC 08/03/99 GML, 11/08/00 HOJ Dilutions 1/1 1/1 1/100 1/1 1/1 RSD Std. Dev. MS/MSD RPD NA NA 2% NA NA 38.6 0.0441 . FA C T -M -2.0 LvrW k20-40 5/23/2001 FACT-TOX-026 Covance# 6329-231 Study: Product Number(Test Substance): Matrix: Method/Revision: Analytical Equipment System Number: Instrument Software/Version: Date of Extraction/Analyst: Date of Analysis/Analyst: Date of Data Reduction/Analyst: Sample Data 6 Month Capsule Toxicity Study with APFO in Cynomolgus Monkeys T-6889.2 (POAA) Monkey Liver FACT-M-1.1 & FACT-M-2.1 Soup020199 MassLynx 3.2 7/13/99 SEE 07/13/99 DRB 07/14/99, 11/02/00, 11/06/00 DRB/KJH MONKEY LIVER Week 20 and 40 Group Dose Sample # POAA Calc. Cone, Method Blk Matrix Blk QC - 250 ppb H20 Blk-11 H20 Blk-12 Rabbit Liver Blk-11 Rabbit Liver Blk-12 I05718M-MS-11 I05718M-MSD-12 ng/g 9.15 0.00 2.95 2.83 341 334 Group 1, Wk40 0.0 mg/kg/day 105718M I05720M 13.5 21.6 Group 2, Wk20 I05721M 18321 Group 3, Wk40 10 mg/kg/day I05712M I05716M 146 83.3 POAA = Perfluorooctanoate Limit o f Quantitation (LOQ) = 75.5 ppb Correction Factors not applicable to MS/MSD QC data Concentration of POAA ug/g or % Rec. <LOQ <LOQ <LOQ <LOQ 113% 111% <LOQ <LOQ . 18.3 0.146 0.0833 Mean POAA ug/g <LOQ <LOQ 112% <LOQ NA 0.114 RSD Std. Dev. MS/MSD RPD NA NA 2% 0.00 NA NA 38.6 0.0441 Date Entered/By: 07/26/99, 11/02/00, 11/03/00,11/06/00 LAC Date Verified/ By: 08/03/99 GML, 11/08/00 HOJ Filename: R-Squared Valu Slope: Y-Intercept: FA C T-M -2.0 LvrW k20-40 5/21/2001 FACT-TOX-026 Covance# 6329-231 Liver QC Summary - TOX026 06/21/1999 07/13/1999 I05709M-MS-3 I05709M-MSD-3 I05718M-MS-11 105718M-MSD-12 NA =Not Applicable NR =Not Reported Average Standard Deviation Grubbs outliers? Number of spikes PFOA 74% 62% 113% 111% 90% Used in Final Report 26% No 4 Used in Final Report FACT-M-2.0 QC Summary 5/23/2001 Study: Product Number(Test Substance): Matrix: Method/Revision: Analytical Equipment System Number: Instrument Software/Version: Filename: R-Squared Value: Slope: Y-Intercept: Dates o f Extraction/Analyst: Dates o f Analysis/Analyst: Date o f Data Reduction/Analyst: Sample Data MONKEY URINE QC SAMPLES Group Dose Sample # Method Blk MKU08049 H 2 0 Blk-1 MKU08049 H 20 Blk-2 MKU08059 H 2 0 Blk-1 MKU08059 H 20 Blk-2 MKU08179 H 2 0 Blk-1 MKU08179 H 20 Blk-2 MKU08209 H 2 0 Blk-1 MKU08209 H 20 Blk-2 MKU09219 H 2 0 Blk-1 MKU09219 H 20 Blk-2 Matrix Blk MKU08049 Urine Blk-1 MKU08049 Urine Blk-2 MKU08059 Urine Blk-1 MKU08059 Urine Blk-2 MKU08179 Urine Blk-1 MKU08179 Urine Blk-2 MKU08209 Urine Blk-1 MKU08209 Urine Blk-2 MKU09219 Urine Blk-1 MKU09219 Urine Blk-2 QC-MS 61.6 ppb MKU08049 MS-1 MKU08049 MSD-1 MKU08049 MS-2 MKU08049 MSD-2 MKU08059 MS MKU08059 MSD MKU08179 MS 1-1 MKU08179 MSD 1-1 MKU08179 MS 1-2 MKU08179 MSD 1-2 MKU08179 MS 1-3 MKU08179 MSD 1-3 MKU08179 MS 1-4 MKU08179 MSD 1-4 MKU08209 MS 1-1 MKU08209 MSD 1-1 MKU08209 MS 1-2 MKU08209 MSD 1-2 M KU09219 M S-1 MKU09219 MSD-1 Limit of Quantitation (LOQ): 0.0182 ug/mL FACT-TOX-026 Covance 6329-231 6 Month Capsule Toxicity Study with APFO in Cynomolgus Monkeys T-6889.2 (POAA) Monkey Urine Blks 08/04/99 ETS-8-96.0 & ETS-8-97.0 Blks 08/05/99 Soup 020199, Madeline 040198, Amelia 062498 Blks 08/17/99 MassLynx 3.2, 3.4 Blks 08/20/99 See listing to the right Blks 09/21/99 See Attachments Box Filenames POAA 081399004-5, A000927018,32 A 000927034, A001017017-18, 19 A 081999003,4, S082499003-4 082399003-4, 0908006,8 092299003-4, 092899059-60 99-160-1, 99-162-3, 99-166-7 Dilutions 1/1 1/1 1/1 1/1 1/1 See Attachments See Attachments 08/04/99, 08/05/99, 08/17/99, 08/20/99,09/21/99 SEE/RWW/MCH/SAL 08/13/99, 08/16/99, 08/19/99, 08/23/99,09/08/99, 09/22/99, 09/28/99, 09/27/00, 10/17/00 PTF/DRB/SAH/GML/IAS/MMH 08/18/99, 08/20/99, 08/24/99, 09/09/99,09/27/99, 09/29/99, 09/29/00, 10/23/00, 11/06/00 PTF/IAS/GML/DRB/MEE/MMH/KJH Ext. Vol. = 1 since curves are extracted at the same ratio as the specimens, 2 m L initial and 0.5 mL final volumes. Extraction Vol. 1 1 1 1 1 1 1 1 l 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 POAA Std Correction Factor 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA POAA Dilution Factor 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 POAA Cone. ng/mL 18.6 18.4 20.8 7.52 6.17 0.00 0.00 6.64 6.90 0.270 5.13 8.00 8.23 33.0 5.24 31.2 0.00 14.4 2.88 0.00 100 75.1 77.1 61.4 70.5 67.8 75.0 82.4 83.1 80.5 63.8 64.0 63.0 63.3 47.5 46.1 62.5 62.2 58.3 60.1 NA - Not Analyzed * Confirmed results. LAC 10/26/00 Concentration of POAA ug/mL or % Rec. 0.0178 0.0177 0.0199 <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ 0.0316 <LOQ 0.0299 <LOQ <LOQ <LOQ <LOQ 133% 92% 95% 70% 91% 87% 92% 104% 105% 101% 74% 74% 73% 73% 66% 63% 90% 89% 92% 95% POAA = Perfluorooctanoate C 8F 17CO O Mean POAA ug/mL 0.0177 NA NA NA NA NA NA NA NA <LOQ * * 112% 82% 89% 98% 103% 74% 73% 64% 90% 94% MS/MSDs 08/04/99 MS/MSDs 08/05/99 MS/MSDs 08/17/99 MS/MSDs 08/17/99 MS/MSDs 08/20/99 MS/MSDs 08/20/99 MS/MSDs 09/21/99 RSD Std. Dev. MS/MSD RPD 0.00703 NA NA NA NA NA NA NA NA NA 36% 31% 5% 12% 4% 1% 1% 4% 0% 3% Date Entered/By: Date Verified/ By: 08/20/99, 08/23/99, 08/24/99, 09/10/99, 09/13/99, 09/16/99, 09/17/99, 09/28/99, 10/10/00, 10/26/00, 11/07/00 GML/LAC 09/25/00 LAC, 10/26/00 KJH, 11/08/00 HOJ POAA A000927045-48, A001017020-21 A000927049,52 081999016-19 A000927059-62, A001017024 082399016-17 A001017025-26 092299016-17 Dilutions 1/1 1/1 1/1 1/1 1/1 1/1 1/1 FACT-TOX-026 Covance 6329-231 Study: Product Number(Test Substance): Matrix: Method/Revision: Analytical Equipment System Number: Instrument Software/Version: Filename: R-Squared Value: Slope: Y-Intercept: Dates o f Extraction/Analyst: Dates o f Analysis/Analyst: Date o f Data Reduction/Analyst: MONKEY URINE QC SAMPLES Group Sample # Dose Method Blk MKU08049 H 2 0 Blk-1 M KU08049 H 2 0 Blk-2 M KU08059 H 2 0 Blk-1 MKU08059 H 2 0 Blk-2 M KU08179 H 2 0 Blk-1 MKU08179 H 2 0 Blk-2 MKU08209 H 2 0 Blk-1 MKU08209 H 2 0 Blk-2 MKU09219 H 2 0 Blk-1 MKU09219 H 2 0 Blk-2 Matrix Blk MKU08049 Urine Blk-1 MKU08049 Urine Blk-2 MKU08059 U rine Blk-1 MKU08059 U rine Blk-2 M KU08179 Urine Blk-1 MKU08179 Urine Blk-2 MKU08209 U rine Blk-1 MKU08209 Urine Blk-2 M KU09219 Urine Blk-1 M KU09219 Urine Blk-2 QC-MS MKU08049 MS-1 61.6 ppb MKU08049 MSD-1 MKU08049 MS-2 MKU08049 MSD-2 MKU08059 MS MKU08059 MSD MKU08179 M S 1-1 MKU08179 MSD l-l MKU08179 M S 1-2 MKU08179 M SD 1-2 M K U 0879M S 1-3 MKU08179 M SD 1-3 MKU08179 MS 1-4 MKU08179 M SD 1-4 MKU08209 M S 1-1 MKU08209 M SD 1-1 MKU08209 M S 1-2 MKU08209 M SD 1-2 MKU09219 MS-1 M KU09219 MSD-1 Limit o f Quantitation (LOQ): 0.0182 ug/mL 6 Month Capsule Toxicity Study with APFO in Cynomolgus Monkeys T-6889.2 (POAA) Monkey Urine ETS-8-96.0 & ETS-8-97.0 Soup 020199, Madeline 040198, Amelia 062498 MassLynx 3.2, 3.4 See Attachments See Attachments See Attachments See Attachments 08/04/99, 08/05/99, 08/17/99, 08/20/99, 09/21/99 SEE/RWW/MCH/SAL 08/13/99, 08/16/99, 08/19/99, 08/23/99, 09/08/99, 09/22/99, 09/28/99, 09/27/00, 10/17/00 PTF/DRB/SAH/GML/IAS/MMH 08/18/99, 08/20/99, 08/24/99, 09/09/99, 09/27/99, 09/29/99, 09/29/00, 10/23/00, 11/06/00 PTF/IAS/GML/DRB/MEE/MMH/KJH Sample Data POAA C oncentration Cone. of POAA ng/mL ug/mL o r % Ree. 18.6 0.0178 18.4 0.0177 20.8 0.0199 7.52 <LOQ 6.17 <LOQ 0.00 <LOQ 0.00 <LOQ 6.64 <LOQ 6.90 <LOQ 0.270 <LOQ 5.13 <LOQ 8.00 <LOQ 8.23 <LOQ 33.0 0.0316 5.2 <LOQ 31.2 0.0299 0.00 <LOQ 14.4 <LOQ 2.88 <LOQ 0.00 <LOQ 100 133% 75.1 92% 77.1 95% 61.4 70% 70.5 91% 67.8 87% 75.0 92% 82.4 104% 83.1 105% 80.5 101% 63.8 74% 64.0 74% 63.0 73% 63.3 73% 47.5 66% 46.1 63% 62.5 90% 62.2 89% 58.3 92% 60.1 95% NA = Not Analyzed * Confirmed results. LAC 10/26/00 M ean POAA ug/m L RSD Std. Dev. MS/MSD RPD 0.0177 0.00703 NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA <LOQ * * 112% NA 36% 82% 31% 89% 5% 98% 12% 103% 4% 74% 1% 73% 1% 64% 4% 90% 0% 94% 3% POAA = Perfluorooctanoate C8F17COO- Date Entered/By: Date Verified/ By: 08/20/99, 08/23/99, 08/24/99, 09/10/99, 09/13/99, 09/16/99, 09/17/99, 09/28/99, 10/10/00, 10/26/00, 11/07/00 GML/LAC 09/25/00 LAC, 10/26/00 KJH, 11/08/00 HOJ nr FACT-TOX-026 Covance 6329-231 Study: Product Number(Test Substance): Matrix: Method/Revision: Analytical Equipment System Number: Instrument Software/Version: Filename: R-Squared Value: Slope: Y-Intercept: Dates of Extraction/Analyst: Dates of Analysis/Analyst: Date of Data Reduction/Analyst: Sam ple Data M ONKEY URINE W eek 2 Group Dose Sample # Group 1 0.0 mg/kg/day I05709M 105714M I05715M 105718M I05720M I05725M Group 2 3.0 mg/kg/day I05702M I05706M I05717M I05723M Group 3 10 mg/kg/day I05707M I05708M I05710M I05712M I05716M I05719M Group 4 30 mg/kg/day I05703M I05704M 105/11M I05713M I05722M I05724M Limit of Quantitation (LOQ): 0.0182 ug/mL 6 Month Capsule Toxicity Study with APFO in Cynomolgus Monkeys T-6889.2 (POAA) Monkey Urine Filenames ETS-8-96.0 & ETS-8-97.0 Soup 020199, Madeline 040198, Amelia 062498 Grp 1 MassLynx 3.2 Grp 2 See listing to the right Grp 3 See Attachments Grp 4 See Attachments Box See Attachments 08/04/99, 08/05/99, 08/17/99, 08/20/99, 09/21/99 SEE/RWW/MCH/SAL 08/19/99, 09/08/99, 09/09/99, 09/15/99 GML/PTF/IAS/MMH 08/20/99, 09/09/99, 09/13/99, 09/16/99, 11/06/00 GML/MMH/KJH POAA 081999022-23, 082399020-21,092299026, 092799050 090999018-21 090899049,78, 091099019-21, 091599150 091599035, 122-126 99-160-1,99-162-3, 99-166-7 Ext. Vol. = 1 since curves are extracted at the same ratio as the specimens, 2 mL initial and 0.5 mL final volumes. Extraction Vol. 1 1 1 1 1 0.9 1 1 1 i 1 1 1 1 1 1 1 1 1 1 1 1 POAA Std Correction Factor 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 09582 0.9582 0.9582 0.9582 POAA POAA Dilution Cone. Factor ng/mL 1 8.27 1 0.00 1 12.0 1 10.6 1 10.3 i 4.36 1000 57.1 1000 62.7 1000 51.1 1000 136 2000 215 1000 208 500 257 250 254 1000 252 1000 300 2000 274 8000 106 8000 123 8000 110 8000 128 8000 176 NA = Not Analyzed/Not Applicable Concentration of POAA ug/mL or % Rec. <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ 54.7 60.1 48.9 130 412 199 123 60.9 242 288 525 811 941 846 983 1350 POAA = Perfluorooctanoate C8F17COO Mean POAA ug/mL <LOQ 73.5 221 909 Date Entered/By: Date Verified/ By: 08/23/99, 08/24/99, 09/09/99, 09/13/99, 09/16/99, 09/17/99,09/28/99, 11/07/00 GML/LAC 09/25/00 LAC, 11/08/00 HOJ Dilutions 1/1 1/1000 1/500, 1/250, 1/1000, 1/2000 1/2000, 1/8000 RSD Std. Dev. MS/MSD RPD NA NA 51.9 38.1 56.2 124 29.6 269 ETC Q 07 O 1 JrineW k2 FACT-TOX-026 Covance 6329-231 Study: Product Number(Test Substance): Matrix: Method/Revision: Analytical Equipment System Number: Instrument Software/Version: Filename: R-Squared Value: Slope: Y-Intercept: Dates of Extraction/Analyst: Dates of Analysis/Analyst: Date of Data Reduction/Analyst: M ONKEY URINE W eek 2 Group Dose Sample # Group 1 I05709M 0.0 mg/kg/day I05714M I05715M I05718M I05720M I05725M Group 2 I05702M 3.0 mg/kg/day I05706M I05717M I05723M Group 3 I05707M 10 mg/kg/day I05708M I05710M I05712M I05716M I05719M Group 4 05703M 30 mg/kg/day I05704M 1U3/11M I05713M I05722M I05724M Limit of Quantitation (LOQ): 0.0182 ug/mL 6 Month Capsule Toxicity Study with APFO in Cynomolgus Monkeys T-6889.2 (POAA) Monkey Urine ETS-8-96.0 & ETS-8-97.0 Soup 020199, Madeline 040198, Amelia 062498 MassLynx 3.2 See Attachments See Attachments See Attachments See Attachments 08/04/99, 08/05/99, 08/17/99, 08/20/99, 09/21/99 SEE/RWW/MCH/SAL 08/19/99, 09/08/99, 09/09/99, 09/15/99 GML/PTF/IAS/MMH 08/20/99, 09/09/99, 09/13/99, 09/16/99, 11/06/00 GML/MMH/KJH Sam ple Data POAA Concentration Cone. of POAA ng/mL ug/mL or % Rec. 8.27 <LOQ 0.00 <LOQ 12.0 <LOQ 10.6 <LOQ 10.3 <LOQ 4.36 <LOQ 57.1 . 54.7 62.7 60.1 51.1 48.9 136 130 215 412 208 199 257 123 254 60.9 252 242 300 288 274 525 106 811 123 941 110 846 128 983 176 1350 NA = Not Analyzed/Not Applicable Mean POAA ug/mL RSD Std. Dev. MS/MSD RPD <LOQ 73.5 NA NA 51.9 38.1 56.2 221 124 29.6 909 269 POAA = Perfluorooctanoate C8F17COO- Date Entered/By: Date Verified/ By: 08/23/99,08/24/99, 09/09/99, 09/13/99, 09/16/99, 09/17/99, 09/28/99, 11/07/00 GML/LAC 09/25/00 LAC, 11/08/00 HOJ FT 9.8.Q 7 O UrineWk2 FACT-TOX-026 Covance 6329-231 Study: Product Number(Test Substance): Matrix: Method/Revision: Analytical Equipment System Number: Instrument Software/Version: Filename: R-Squared Value: Slope: Y-Intercept: Dates of Extraction/Analyst: Dates of Analysis/Analyst: Date of Data Reduction/Analyst: Sam ple D ata M ONKEY URINE W eek 4 Group Dose Sample # Group 1 I05709M 0.0 mg/kg/day 105714M I05715M I05718M I05720M I05725M Group 2 I05702M 3.0 mg/kg/day I05706M I05717M I05721M Group 3 10 mg/kg/day I05707M I05708M 105710M I05712M I05716M I05719M Group 4 30 mg/kg/day I05703M I05704M 1U5/11M I05713M I05722M I05724M Limit of Quantitation (LOQ): 0.0182 ug/mL 6 Month Capsule Toxicity Study with APFO in Cynomolgus Monkeys T-6889.2 (POAA) Monkey Urine ETS-8-96.0 & ETS-8-97.0 Soup 020199, Madeline 040198, Amelia 062498 MassLynx 3.2 See listing to the right See Attachments See Attachments Filenames Grp 1 Grp 2 Grp 3 Grp 4 Box POAA 081999024-25, 082399022-3,092799045,51 090999022-28 090899057,79, 091099022-27 091599043-45, 129-130, 092799047 99-160-1, 99-162-3, 99-166-7 See Attachments 08/04/99, 08/05/99, 08/17/99, 08/20/99, 09/21/99 SEE/RWW/MCH/SAL 08/19/99, 08/23/99, 09/08/99, 09/09/99, 09/10/99, 09/15/99, 09/27/99 GML/PTF/IAS/MMH/MEE/IAS 08/20/99, 08/24/99, 09/09/99, 09/13/99, 09/16/99, 09/28/99, 11/06/00 GML/MMH/IAS/KJH Ext. Vol. = 1 since curves are extracted at the same ratio as the specimens, 2 mL initial and 0.5 mL final volumes. Extraction Voi. POAA Std Correction Factor 1 0.9582 1 0.9582 1 0.9582 0.80 0.9582 1 0.9582 i 0.9582 1 0.9582 1 0.9582 1 0.9582 1 0.9582 1 0.9582 1 0.9582 1 0.9582 1 0.9582 1 0.9582 1 0.9582 1 0.9582 1 0.9582 1 0.9582 1 0.9582 1 0.9582 1 0.9582 NA = Not Analyzed/Not Applicable A = Below LOQ POAA Dilution Factor 1 1 1 1 1 5 1000 1000 1000 1000 1000 1000 1000 250 500 1000 4000 2000 000 2000 1000 1000 POAA Cone. ng/mL 10.6 4.17 0.00 22.5 11.8 175 57.7 57.9 62.6 50.9 204 283 276 149 277 253 99.9 24.8 194 232 308 86.6 Concentration of POAA ug/mL or % Ree. <LOQ <LOQ <LOQ 0.0270 <LOQ 0.839 55.3 55.5 60.0 48.7 195 271 264 35.8 133 242 383 47.6 186 444 295 83.0 POAA = Perfluorooctanoate CgF17COO Mean POAA ug/mL 0.152 54.9 190 240 RSD Std. Dev. MS/MSD RPD NA A 0.337 8.43 4.62 48.1 91.6 67.1 161 Date Entered/By: Date Verified/ By: 08/23/99, 08/24/99, 09/09/99, 09/13/99, 09/16/99, 09/17/99, 09/28/99, 11/07/00 GML/LAC 09/25/00 LAC, 11/08/00 HOJ Dilutions 1/1, 1/5 1/1000 1/500, 1/250, 1/1000 1/1000, 1/4000, 1/2000 FTfs-S-97 O UrineWk4 FACT-TOX-026 Covance 6329-231 Study: Product Number(Test Substance): Matrix: Method/Revision: Analytical Equipment System Number: Instrument Software/Version: Filename: R-Squared Value: Slope: Y-Intercept: Dates of Extraction/Analyst: Dates of Analysis/Analyst: Date of Data Reduction/Analyst: M ONKEY URINE W eek 4 Group Dose Sample # Group 1 I05709M 0.0 mg/kg/day I05714M 105715M 105718M I05720M I05725M Group 2 3.0 mg/kg/day I05702M I05706M I05717M I05721M Group 3 I05707M 10 mg/kg/day I05708M I05710M I05712M I05716M I05719M Group 4 I05703M 30 mg/kg/day I05704M 105711M I05713M I05722M I05724M Limit of Quantitation (LOQ): 0.0182 ug/mL 6 Month Capsule Toxicity Study with APFO in Cynomolgus Monkeys T-6889.2 (POAA) Monkey Urine ETS-8-96.0 & ETS-8-97.0 Soup 020199, Madeline 040198, Amelia 062498 MassLynx 3.2 See Attachments See Attachments See Attachments See Attachments 08/04/99, 08/05/99, 08/17/99, 08/20/99, 09/21/99 SEE/RWW/MCH/SAL 08/19/99, 08/23/99, 09/08/99, 09/09/99, 09/10/99, 09/15/99, 09/27/99 GML/PTF/IAS/MMH/MEE/IAS 08/20/99, 08/24/99, 09/09/99, 09/13/99, 09/16/99, 09/28/99, 11/06/00 GML/MMH/IAS/KJH Sam ple Data POAA Cone. ng/mL Concentration of POAA ug/mL or % Rec. Mean POAA ug/mL RSD Std. Dev. MS/MSD RPD 10.6 4.17 0.00 22.5 11.8 175 57.7 . 57.9 62.6 50.9 204 283 276 149 277 253 99.9 24.8 194 232 308 86.6 <LOQ <LOQ <LOQ 0.0270 <LOQ 0.839 55.3 55.5 60.0 48.7 195 271 264 36 133 242 383 47.6 186 444 295 83.0 0.152 A 54.9 NA 0.337 8.43 4.62 48.1 190 91.6 67.1 240 161 NA = Not Analyzed/Not Applicable POAA = Perfluorooctanoate A = Below LOQ C8F17COO- Date Entered/By: Date Verified/ By: 08/23/99, 08/24/99, 09/09/99, 09/13/99, 09/16/99, 09/17/99, 09/28/99, 11/07/00 GML/LAC 09/25/00 LAC, 11/08/00 HOJ ETC 0 7 n I JrineWk4 FACT-TOX-026 Covance 6329-231 Study: Product Number(Test Substance): Matrix: Method/Revision: Analytical Equipment System Number: Instrument Software/Version: Filename: R-Squared Value: Slope: Y-Intercept: Dates of Extraction/Analyst: Dates of Analysis/Analyst: Date of Data Reduction/Analyst: Sam ple Data M ONKEY URINE W eek 6 Group Dose Sample # Group 1 0.0 mg/kg/day I05709M I05714M I05715M I05718M I05720M I05725M Group 2 3.0 mg/kg/day I05702M I05706M I05717M I05721M Group 3 10 mg/kg/day I05707M I05708M I05710M I05712M I05716M I05719M Group 4 30 mg/kg/day I05703M I05704M 103 / 11M I05713M I05722M I05724M Limit of Quantitation (LOQ): 0.0182 ug/mL 6 Month Capsule Toxicity Study with APFO in Cynomolgus Monkeys T-6889.2 (POAA) Monkey Urine Filenames ETS-8-96.0 & ETS-8-97.0 POAA Soup 020199, Madeline 040198, Amelia 062498 Grp 1 081999028-29, 082399026-27, 092799052, 65 MassLynx 3.2 Grp 2 090999028-34 See listing to the right Grp 3 091599151-157, 160 See Attachments Grp 4 091599051-52, 131-133 See Attachments Box 99-160-1,99-162-3 See Attachments 08/04/99, 08/05/99, 08/17/99, 08/20/99, 09/21/99 SEE/RWW/MCH/SAL 08/19/99, 08/23/99, 09/09/99, 09/15/99, 09/27/99 GML/PTF/IAS/MMH/MEE/IAS 08/20/99, 08/24/99, 09/13/99, 09/16/99, 09/28/99, 11/06/00 GML/MMH/IAS/KJH Ext. Vol. = 1 since curves are extracted at the same ratio as the specimens, 2 mL initial and 0.5 mL final volumes. Extraction Voi. 1 1 1 1 1 1 ,1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 POAA Std Correction Factor 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 POAA POAA Dilution Cone. Factor ng/mL 1 179 1 0.00 1 4.66 1 26.5 1 14.9 1 131 1000 138 1000 58.0 1000 55.5 1000 22.8 1000 164 1000 203 1000 156 250 227 1000 122 1000 98.4 4000 92.3 4000 50.3 4000 111 1000 75.5 1000 328 NA NA NA = Not Analyzed/Not Applicable Concentration of POAA ug/mL or % Ree. 0.172 <LOQ <LOQ 0.0254 <LOQ 0.126 132 55.6 53.2 21.8 157 195 150 54.4 117 94.3 354 193 427 72.3 314 NA POAA = Perfluorooctanoate C8FI7COO* Mean POAA ug/mL 0.0587 65.7 128 272 Date Entered/By: Date Verified/ By: 08/23/99, 08/24/99, 09/13/99, 09/16/99, 09/17/99, 09/28/99,11/07/00 GML/KJH 09/25/00 LAC, 11/08/00 HOJ Dilutions 1/1 1/1000 1/1000, 1/250 1/1000, 1/4000 RSD Std. Dev. MS/MSD RPD 122 0.0716 71.4 46.9 39.1 50.0 51.6 140 PT-R-Q7 O UrineWk6 FACT-TOX-026 Covance 6329-231 Study: Product Number(Test Substance): Matrix: Method/Revision: Analytical Equipment System Number: Instrument Software/Version: Filename: R-Squared Value: Slope: Y-Intercept: Dates of Extraction/Analyst: Dates of Analysis/Analyst: Date of Data Reduction/Analyst: M ONKEY URINE W eek 6 Group Dose Sample # Group 1 I05709M 0.0 mg/kg/day 105714M I05715M I05718M I0572OM I05725M Group 2 I05702M 3.0 mg/kg/day I05706M 105717M I05721M Group 3 I05707M 10 mg/kg/day I05708M I05710M I05712M I05716M I05719M Group 4 I05703M 30 mg/kg/day I05704M 1U3/11M 105713M I05722M I05724M Limit of Quantitation (LOQ): 0.0182 ug/mL 6 Month Capsule Toxicity Study with APFO in Cynomolgus Monkeys T-6889.2 (POAA) Monkey Urine ETS-8-96.0 & ETS-8^97.0 Soup 020199, Madeline 040198, Amelia 062498 MassLynx 3.2 See Attachments See Attachments See Attachments See Attachments 08/04/99, 08/05/99, 08/17/99, 08/20/99, 09/21/99 SEE/RWW/MCH/SAL 08/19/99, 08/23/99, 09/09/99, 09/15/99, 09/27/99 GML/PTF/IAS/MMH/MEE/IAS 08/20/99, 08/24/99, 09/13/99, 09/16/99, 09/28/99, 11/06/00 GML/MMH/IAS/KJH Sam ple Data POAA Concentration Mean RSD Cone. of POAA POAA Std. Dev. ng/mL ug/mL or % Rec. ug/mL MS/MSD RPD 179 0.172 0.00 <LOQ 4.66 <LOQ 26.5 0.0254 14.9 <LOQ 122 131 0.126 0.0587 0.0716 . 138 132 58.0 55.6 55.5 53.2 71.4 22.8 21.8 65.7 46.9 164 157 203 195 156 150 227 54.4 122 117 39.1 98.4 94.3 128 50.0 92.3 354 50.3 193 111 427 75.5 72.3 328 314 51.6 NA NA 272 140 NA = Not Analyzed/Not Applicable POAA = Perfluorooctanoate C8F17COO- Date Entered/By: Date Verified/ By: 08/23/99, 08/24/99, 09/13/99, 09/16/99, 09/17/99, 09/28/99, 11/07/00 GML/KJH 09/25/00 LAC, 11/08/00 HOJ FTS-R-97 O UrineWk6 FACT-TOX-026 Covance 6329-231 Study: Product Number(Test Substance): Matrix: Method/Revision: Analytical Equipment System Number: Instrument Software/Version: Filename: R-Squared Value: Slope: Y-Intercept: Dates of Extraction/Analyst: Dates of Analysis/Analyst: Date of Data Reduction/Analyst: Sam ple Data M ONKEY URINE W eek 8 Group Dose Sample # Group 1 0.0 mg/kg/day I05709M I05714M I05715M I05718M I05720M I05725M Group 2 3.0 mg/kg/day I05702M I05706M I05717M I05721M Group 3 10 mg/kg/day I05707M I05708M I05710M I05712M I05716M I05719M Group 4 30 mg/kg/day I05703M I05704M I57TTM I05713M I05722M I05724M Limit of Quantitation (LOQ): 0.0182 ug/mL . 6 Month Capsule Toxicity Study with APFO in Cynomolgus Monkeys T-6889.2 (POAA) Monkey Urine Filenames ETS-8-96.0 & ETS-8-97.0 POAA Soup 020199, Madeline 040198, Amelia 062498 Grp 1 081999030-31, 082399028-29, 092299029, 092799053 MassLynx 3.2 Grp 2 090999035-40 See listing to the right Grp 3 090899074, 091099028-34 See Attachments Grp 4 082499061, 0915099056,136-138 See Attachments Box 99-160-1, 99-162-3, 99-166-7 See Attachments 08/04/99, 08/05/99, 08/17/99, 08/20/99, 09/21/99 SEE/RWW/MCH/SAL 08/19/99, 08/23/99, 09/08/99, 09/09/99, 09/10/99, 09/15/99, 09/22/99, 09/27/99 GML/PTF/IAS/MMH/MEE/IAS 08/20/99, 08/24/99, 09/09/99, 09/13/99, 09/16/99, 09/27/99, 09/28/99, 11/06/00 GML/MMH/MEE/IAS/KJH Ext. Vol. = 1 since curves are extracted at the same ratio as the specimens, 2 mL initial and 0.5 mL final volumes. Dilutions 1/1 1/1000 1/500, 1/250, 1/1000 1/50, 1/10, 1/1000, 1/2000 Extraction Vol. 1 1 1 1 1 1 1 1 1 i 1 1 1 1 1 1 1 0.95 0.80 1 1 NA POAA Std Correction Factor POAA Dilution Factor 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 1 1 1 1 1 1 1000 1000 1000 1000 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 1000 1000 1000 400 1000 1000 0,9582 0.9582 U.9582 0.9582 0.9582 0.9582 2000 1000 0 1000 2000 NA NA = Not Analyzed/Not Applicable A = Below LOQ POAA Cone. ng/mL 13.5 0.790 1.31 32.0 15.2 8.61 49.2 78.7 40.0 28.7 240 230 311 196 200 230 87.0 279 302 268 101 NA Concentration of POAA ug/mL or % Ree. <LOQ <LOQ <LOQ 0.0307 <LOQ <LOQ . 49.2 75.4 38.3 27.5 230 221 298 75.2 192 220 167 282 3.62 257 193 NA POAA = Perfluorooctanoate C8F17COO* Mean POAA ug/mL 0.0161 47.6 206 180 RSD Std. Dev. MS/MSD RPD 58.3 A 0.00940 43.2 20.6 35.5 73.1 60.5 109 Date Entered/By: Date Verified/ By: 08/23/99, 08/24/99, 08/26/99, 09/09/99, 09/13/99, 09/16/99,09/17/99, 09/28/99, 11/07/00 GML/LAC 09/25/00 LAC, 11/08/00 HOJ ETC Q 07 O I JrineWkS FACT-TOX-026 Covance 6329-231 Study: Product Number(Test Substance): Matrix: Method/Revision: Analytical Equipment System Number: Instrument Software/Version: Filename: R-Squared Value: Slope: Y-Intercept: Dates of Extraction/Analyst: Dates of Analysis/Analyst: Date of Data Reduction/Analyst: M ONKEY URINE W eek 8 Group Dose Sample # Group 1 0.0 mg/kg/day Group 2 3.0 mg/kg/day Group 3 10 mg/kg/day Group 4 30 mg/kg/day I05709M 105714M 105715M I05718M I05720M I05725M I05702M I05706M I05717M I05721M I05707M I05708M I05710M I05712M I05716M I05719M I05703M I05704M 1 U 3 / 1 1JVL 105713M 05722M I05724M Limit of Quantitation (LOQ): 0.0182 ug/mL 6 Month Capsule Toxicity Study with APFO in Cynomolgus Monkeys T-6889.2 (POAA) Monkey Urine ETS-8-96.0 & ETS-8-97.0 Soup 020199, Madeline 040198, Amelia 062498 MassLynx 3.2 See Attachments See Attachments See Attachments See Attachments 08/04/99, 08/05/99, 08/17/99, 08/20/99, 09/21/99 SEE/RWW/MCH/SAL 08/19/99, 08/23/99, 09/08/99, 09/09/99, 09/10/99, 09/15/99, 09/22/99, 09/27/99 GML/PTF/IAS/MMH/MEE/IAS 08/20/99, 08/24/99, 09/09/99, 09/13/99, 09/16/99, 09/27/99, 09/28/99, 11/06/00 GML/MMH/MEE/IAS/KJH Sam ple Data POAA Cone. ng/mL 13.5 0.790 1.31 32.0 15.2 8.61 . 49.2 78.7 40.0 28.7 240 230 311 196 200 230 Concentration of POAA ug/mL or % Ree. <LOQ <LOQ <LOQ 0.0307 <LOQ <LOQ 49.2 75.4 38.3 27.5 230 221 298 75.2 192 220 87.0 167 279 282 52 3.62 268 257 101 193 NA NA NA = Not Analyzed/Not Applicable A = Below LOQ Mean POAA ug/mL 0.0161 A 47.6 206 180 POAA = Perfluorooctanoate C8F17COO- RSD Std. Dev. MS/MSD RPD 58.3 0.00940 43.2 20.6 35.5 73.1 60.5 109 Date Entered/By: Date Verified/ By: 08/23/99, 08/24/99, 08/26/99, 09/09/99, 09/13/99, 09/16/99, 09/17/99, 09/28/99, 11/07/00 GML/LAC 09/25/00 LAC, 11/08/00 HOJ FTS-R-Q7 n UrineWk8 FACT-TOX-026 Covance 6329-231 Study: Product NumbertTest Substance): Matrix: Method/Revision: Analytical Equipment System Number: Instrument Software/Version: Filename: R-Squared Value: Slope: Y-Intercept: Dates of Extraction/Analyst: Dates of Analysis/Analyst: Date of Data Reduction/Analyst: Sam ple D ata M ONKEY URINE W eek 10 Group Dose Sample # Group 1 I05709M 0.0 mg/kg/day I05714M 105715M I05718M I05720M 05725M Group 2 I05702M 3.0 mg/kg/day I05706M 105717M I05721M Group 3 I05707M 10 mg/kg/day I05708M I05710M I05712M 105716M 105719M Group 4 30 mg/kg/day I05703M I05704M 1UD / 1 1 M I05713M I05722M I05724M Limit of Quantitation (LOQ): 0.0182 ug/mL 6 Month Capsule Toxicity Study with APFO in Cynomolgus Monkeys T-6889.2 (POAA) Monkey Urine Filenames ETS-8-96.0 & ETS-8-97.0 POAA Dilutions Soup 020199, Madeline 040198, Amelia 062498 Grp 1 081999034-35, 082399032-33, 092299030, 092799054 1/1 MassLynx 3.2 Grp 2 090999041-44 1/1000 See listing to the right Grp 3 090899072-73,80, 091099035-39 1/500, 1/250, 1/1000 See Attachments Grp 4 081399017, 091599059,63,140, 092399022 1/20, 1/1000, 1/2000, 1/4000 See Attachments Box 99-160-1,99-162-3,99-166-7 See Attachments 08/04/99, 08/05/99, 08/17/99, 08/20/99, 09/21/99 SEE/RWW/MCH/SAL 08/13/99, 08/19/99, 08/23/99, 09/08/99, 09/09/99, 09/10/99, 09/15/99, 09/22/99, 09/23/99, 09/27/99 GML/PTF/IAS/MMH/MEE/IAS 08/18/99, 08/20/99, 08/24/99, 09/09/99, 09/13/99, 09/16/99, 09/24/99, 09/27/99, 09/28/99, 11/06/00 PTF/DRB/GML/MMH/MEE/IAS/KJH Ext. Vol. = 1 since curves are extracted at the same ratio as the specimens, 2 mL initial and 0.5 mL final volumes. Extraction Vol. 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 POAA Std Correction Factor 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0 .9 5 2 0.9582 0.9582 0.9582 POAA Dilution Factor POAA Cone. ng/mL 1 10.9 1 0.00 1 1.08 1 23.5 1 11.8 1 37.5 1000 1000 1000 1000 1000 1000 1000 250 500 500 4000 1000 20 2000 1000 NA 34.7 . 66.5 45.5 20.0 284 263 243 188 197 322 295 342 7377 57.0 235 NA NA = Not Analyzed/Not Applicable A = Below LOQ Concentration of POAA ug/mL or % Ree. <LOQ <LOQ <LOQ 0.0225 <LOQ 0.0359 33.3 63.7 43.6 19.2 272 252 233 45.1 94.3 154 1131 328 1.41 109 225 NA Mean POAA ug/mL 0.0177 39.9 A 175 359 POAA = Perfluorooctanoate C8Fl7COO' RSD Std. Dev. MS/MSD RPD 64.1 0.0114 46.9 18.7 52.7 92.3 125 449 Date Entered/By: Date Verified/ By: 08/20/99, 08/23/99, 08/24/99, 09/09/99, 09/13/99, 09/16/99, 09/17/99, 09/27/99, 09/28/99, 11/06/00, 11/07/00 GML/LAC 09/25/00 LAC,, 11/08/00 HOJ FT-8-Q7 0 UrineWklO FACT-TOX-026 Covance 6329-231 Study: Product Number(Test Substance): Matrix: Method/Revision: Analytical Equipment System Number: Instrument Software/Version: Filename: R-Squared Value: Slope: Y-Intercept: Dates of Extraction/Analyst: Dates of Analysis/Analyst: Date of Data Reduction/Analyst: M ONKEY URINE W eek 10 Group Dose Sample # Group 1 0.0 mg/kg/day I05709M I05714M 105715M I05718M I05720M I05725M Group 2 3.0 mg/kg/day I05702M I05706M I05717M 105721M Group 3 I05707M 10 mg/kg/day I05708M I05710M I05712M 105716M I05719M Group 4 30 mg/kg/day I05703M I05704M I57TTM I05713M I05722M I05724M Limit of Quantitation (LOQ): 0.0182 ug/mL 6 Month Capsule Toxicity Study with APFO in Cynomolgus Monkeys T-6889.2 (POAA) Monkey Urine ETS-8-96.0 & ETS-8-97.0 Soup 020199, Madeline 040198, Amelia 062498 MassLynx 3.2 See Attachments See Attachments See Attachments See Attachments 08/04/99, 08/05/99, 08/17/99, 08/20/99, 09/21/99 SEE/RWW/MCH/SAL 08/13/99, 08/19/99, 08/23/99, 09/08/99, 09/09/99, 09/10/99, 09/15/99, 09/22/99, 09/23/99, 09/27/99 GML/PTF/IAS/MMH/MEE/IAS 08/18/99, 08/20/99, 08/24/99, 09/09/99, 09/13/99, 09/16/99, 09/24/99, 09/27/99, 09/28/99, 11/06/00 PTF/DRB/GML/MMH/MEE/IAS/KJH Sample D ata POAA Concentration Cone. of POAA ng/mL ug/mL or % Rec. 10.9 <LOQ 0.00 <LOQ 1.08 <LOQ 23.5 0.0225 11.8 <LOQ 37.5 0.0359 . 34.7 33.3 66.5 63.7 45.5 43.6 20.0 19.2 284 272 263 252 243 233 188 45.1 197 94.3 322 154 295 1131 342 328 7X7 ' 1.41 57.0 109 235 225 NA NA NA = Not Analyzed/Not Applicable Mean POAA ug/mL RSD Std. Dev. MS/MSD RPD 0.0177 A 39.9 64.1 0.0114 46.9 18.7 52.7 175 92.3 125 359 449 POAA = Perfluorooctanoate C8F17COO- Date Entered/By: Date Verified/ By: 08/20/99, 08/23/99, 08/24/99, 09/09/99, 09/13/99, 09/16/99, 09/17/99, 09/27/99, 09/28/99, 11/06/00, 11/07/00 GML/LAC 09/25/00 LAC,, 11/08/00 HOJ CTC.fi.Q7 n TIrineWklO FACT-TOX-026 Covance 6329-231 Study: Product Number(Test Substance): Matrix: Method/Revision: Analytical Equipment System Number: Instrument Software/Version: Filename: R-Squared Value: Slope: Y-Intercept: Dates of Extraction/Analyst: Dates of Analysis/Analyst: Date of Data Reduction/Analyst: Sam ple Data M ONKEY URINE W eek 12 Group Dose Sample # Group 1 I05709M 0.0 mg/kg/day I05714M 105715M 105718M I05720M I05725M Group 2 3.0 mg/kg/day . I05702M I05706M I05717M I05721M Group 3 I05707M 10 mg/kg/day I05708M 105710M I05712M 105716M I05719M Group 4 I05703M 30 mg/kg/day I05704M 1U5/11M I05713M I05722M I05724M Limit of Quantitation (LOQ): 0.0182 ug/mL 6 Month Capsule Toxicity Study with APFO in Cynomolgus Monkeys T-6889.2 (POAA) Monkey Urine Filenames ETS-8-96.0 & ETS-8-97.0 POAA Soup 020199, Madeline 040198, Amelia 062498 Grp 1 081999036-37, 082399034-35, 092299033, 092799057 MassLynx 3.2 Grp 2 090999047-50 See listing to the right Grp 3 082499016-21 See Attachments Grp 4 081399018-19, 091599143-145 See Attachments Box 99-160-1,99-162-3 See Attachments 08/04/99, 08/05/99, 08/17/99, 08/20/99, 09/21/99 SEE/RWW/MCH/SAL 08/13/99, 08/19/99, 08/23/99, 08/24/99, 09/09/99, 09/15/99, 09/22/99, 09/27/99 GML/SAH/MMH/IAS 08/18/99, 08/20/99, 08/24/99, 08/25/99, 09/13/99, 09/16/99, 09/27/99, 09/28/99, 11/06/00, 11/13/00 PTF/DRB/GML/MMH/MEE/IAS/KJH Ext. Vol. = 1 since curves are extracted at the same ratio as the specimens, 2 mL initial and 0.5 mL final volumes. Extraction Vol. 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 POAA Std Correction Factor POAA Dilution Factor 0.9582 1 0.9582 1 0.9582 1 0.9582 1 0.9582 1 0.9582 1 0.9582 1000 0.9582 1000 0.9582 1000 0.9582 1000 0.9582 1000 0.9582 1000 0.9582 1000 0:9582 1000 0.9582 1000 0.9582 1000 0.9582 2000 0.9582 2000 U.9582 20 0.9582 2000 0.9582 20 0.9582 NA NA = Not Analyzed/Not Applicable A - Below LOQ POAA Cone. ng/mL 14.7 1.49 1.96 18.9 19.2 15.6 ,68.2 65.6 40.5 27.8 177 150 381 270 148 135 91.3 88.2 1L 8 127 70.6 NA Concentration of POAA ug/mL or % Rec. <LOQ <LOQ <LOQ 0.0181 0.0184 <LOQ 65.3 62.8 38.8 26.6 170 144 365 259 142 130 175 169 0 ./25 243 1.35 NA POAA = Perfluorooctanoate C8F17COO Mean POAA ug/mL 0.0141 48.4 201 118 Date Entered/By: Date Verified/ By: 08/20/99,08/23/99,08/24/99,08/26/99,09/13/99,09/16/99,09/17/99,09/28/99, 11/06/00, 11/07/00, 11/14/00 GML/LAC 09/25/00 LAC, 11/08/00 HOJ, 11/15/00 HOJ Dilutions 1/1 1/1000 1/1000 1/ 20, 1/2000 RSD Std. Dev. MS/MSD RPD 46.1 A 0.00648 38.9 18.8 46.0 92.7 93.8 111 FT-8-Q7 n U rineW kl2 FACT-TOX-026 Covance 6329-231 Study: Product Number(Test Substance): Matrix: Method/Revision: Analytical Equipment System Number: Instrument Software/Version: Filename: R-Squared Value: Slope: Y-Intercept: Dates of Extraction/Analyst: Dates of Analysis/Analyst: Date of Data Reduction/Analyst: M ONKEY URINE W eek 12 Group Dose Sample # Group 1 I05709M 0.0 mg/kg/day I05714M I05715M I05718M I05720M I05725M Group 2 I05702M 3.0 mg/kg/day I05706M I05717M I05721M Group 3 10 mg/kg/day I05707M I05708M I05710M I05712M I05716M I05719M Group 4 I05703M 30 mg/kg/day 05704M 105711M I05713M I05722M I05724M Limit of Quantitation (LOQ): 0.0182 ug/mL 6 Month Capsule Toxicity Study with APFO in Cynomolgus Monkeys T-6889.2 (POAA) Monkey Urine ETS-8-96.0 & ETS-8-97.0 . Soup 020199, Madeline 040198, Amelia 062498 MassLynx 3.2 See Attachments See Attachments See Attachments See Attachments 08/04/99, 08/05/99, 08/17/99, 08/20/99, 09/21/99 SEE/RWW/MCH/SAL 08/13/99, 08/19/99, 08/23/99, 08/24/99, 09/09/99, 09/15/99, 09/22/99, 09/27/99 GML/SAH/MMH/IAS 08/18/99, 08/20/99, 08/24/99, 08/25/99, 09/13/99, 09/16/99, 09/27/99, 09/28/99, 11/06/00, 11/13/00 PTF/DRB/GML/MMH/MEE/IAS/KJH Sam ple Data POAA Concentration Cone. of POAA ng/mL ug/mL or % Ree. 14.7 <LOQ 1.49 <LOQ 1.96 <LOQ 18.9 0.0181 19.2 0.0184 15.6 <LOQ , 68.2 65.3 65.6 62.8 40.5 38.8 27.8 26.6 177 170 150 144 381 365 270 259 148 142 135 130 91.3 175 88.2 169 3T3 0.725 127 243 70.6 1.35 NA NA NA = Not Analyzed/Not Applicable Mean POAA ug/mL RSD Std. Dev. MS/MSD RPD 0.0141 48.4 46.1 A 0.00648 38.9 18.8 46.0 201 92.7 118 POAA = Perfluorooctanoate C8F17COO- 93.8 111 Date Entered/By: Date Verified/ By: 08/20/99,08/23/99,08/24/99,08/26/99,09/13/99,09/16/99,09/17/99,09/28/99, 11/06/00, 11/07/00, 11/14/00 GML/LAC 09/25/00 LAC, 11/08/00 HOJ, 11/15/00 HOJ F.TS-8-Q7 O UrineW kl2 FACT-TOX-026 Covance 6329-231 Study: Product Number(Test Substance): Matrix: Method/Revision: Analytical Equipment System Number: Instrument Software/Version: Filename: R-Squared Value: Slope: Y-Intercept: Dates of Extraction/Analyst: Dates of Analysis/Analyst: Date of Data Reduction/Analyst: Sam ple Data M ONKEY URINE W eek 14 Group Dose Sample # Group 1 0.0 mg/kg/day I05709M 105714M I05715M I05718M I05720M I05725M Group 2 . 3.0 mg/kg/day I05702M I05706M I05717M I05721M Group 3 10 mg/kg/day I05707M I05708M I05710M I05712M 105716M I05719M Group 4 30 mg/kg/day I05703M I05704M 105711M I05713M I05722M I05724M Limit of Quantitation (LOQ): 0.0182 ug/mL 6 Month Capsule Toxicity Study with APFO in Cynomolgus Monkeys T-6889.2 (POAA) Monkey Urine Filenames ETS-8-96.0 & ETS-8-97.0 . POAA Soup 020199, Madeline 040198, Amelia 062498 Grp 1 081999040, 082399052-3, 090899020, 092299034-35 MassLynx 3.2 Grp 2 090999051-56 See listing to the right Grp 3 090899021-028 See Attachments Grp 4 081399023-25,71, 092399019 See Attachments Box 99-160-1, 99-162-3, 99-166-7 See Attachments 08/04/99, 08/05/99, 08/17/99, 08/20/99, 09/21/99 SEE/RWW/MCH/SAL 08/13/99, 08/19/99, 08/23/99, 09/08/99, 09/09/99, 09/22/99, 09/23/99 GML/PTF/IAS/MMH/MEE/IAS 08/18/99, 08/20/99, 08/24/99, 09/09/99, 09/13/99, 09/24/99, 09/27/99, 11/06/00 PTF/DRB/GML/MMH/MEE/KJH Ext. Vol. = 1 since curves ate extracted at the same ratio as the specimens, 2 mL initial and 0.5 mL final volumes. Extraction Vol. 0.15 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 i 1 1 1 POAA Std Correction Factor 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 POAA Dilution Factor POAA Cone. ng/mL 1 1.19 1 2.88 1 4.36 1 23.4 250 199 1 6.52 1000 . 61.2 1000 136 1000 42.2 1000 23.9 1000 1000 1000 1000 1000 1000 133 178 193 52.5 120 191 10 18.9 1000 163 10 6.86 500 280 10 116 NA NA NA = Not Analyzed/Not Applicable Concentration of POAA ug/mL or % Ree. <LOQ <LOQ <LOQ 0.0224 47.7 <LOQ 58.7 131 40.4 22.9 127 171 185 50.3 115 183 0.181 156 <LOQ 134 1.11 NA POAA = Perfluorooctanoate C,,F17COO- Mean POAA ug/mL 7.96 63.1 139 72.9 Date Entered/By: Date Verified/ By: 08/20/99,08/23/99,08/24/99,09/09/99,09/13/99,09/16/99,09/17/99,09/27/99,09/28/99, 11/06/00, 11/07/00 GML/LAC 09/25/00 LAC, 11/08/00 HOJ Dilutions 1/1, 1/250 1/1000 1/1000 1/10,1/500, RSD Std. Dev. MS/MSD RPD 245 19.5 74.8 47.2 37.7 52.2 115 84.0 CTC fi 0 7 A U rineW kU FACT-TOX-026 Covance 6329-231 Study: Product Number(Test Substance): Matrix: Method/Revision: Analytical Equipment System Number: Instrument Software/Version: Filename: R-Squared Value: Slope: Y-Intercept: Dates of Extraction/Analyst: Dates of Analysis/Analyst: Date of Data Reduction/Analyst: M ONKEY URINE W eek 14 Group Dose Sample # Group 1 I05709M 0.0 mg/kg/day I05714M I05715M 105718M I05720M I05725M Group 2 . I05702M 3.0 mg/kg/day I05706M 105717M I05721M Group 3 I05707M 10 mg/kg/day I05708M I05710M I05712M 105716M 105719M Group 4 30 mg/kg/day I05703M I05704M 1UD/11M I05713M I05722M I05724M Limit of Quantitation (LOQ): 0.0182 ug/mL 6 Month Capsule Toxicity Study with APFO in Cynomolgus Monkeys T-6889.2 (POAA) Monkey Urine ETS-8-96.0 & ETS-8-97.0 Soup 020199, Madeline 040198, Amelia 062498 MassLynx 3.2 See Attachments See Attachments See Attachments See Attachments 08/04/99, 08/05/99, 08/17/99, 08/20/99, 09/21/99 SEE/RWW/MCH/SAL 08/13/99, 08/19/99, 08/23/99, 09/08/99, 09/09/99, 09/22/99, 09/23/99 GML/PTF/IAS/MMH/MEE/IAS 08/18/99, 08/20/99, 08/24/99, 09/09/99, 09/13/99, 09/24/99, 09/27/99, 11/06/00 PTF/DRB/GML/MMH/MEE/KJH Sam ple Data POAA Concentration Cone. of POAA ng/mL ug/mL or % Ree. 1.19 <LOQ 2.88 <LOQ 4.36 <LOQ 23.4 0.0224 199 47.7 6.52 <LOQ 61.2 58.7 136 131 42.2 40.4 23.9 22.9 133 127 178 171 193 185 52.5 50.3 120 115 191 183 18.9 0.181 163 156 6.86 <LOQ 280 134 116 1.11 NA NA NA = Not Analyzed/Not Applicable Mean POAA ug/mL RSD Std. Dev. MS/MSD RPD 245 7.96 19.5 74.8 63.1 47.2 37.7 139 52.2 115 72.9 84.0 POAA = Perfluorooctanoate C8F17COO- Date Entered/By: Date Verified/ By: 08/20/99, 08/23/99, 08/24/99, 09/09/99, 09/13/99, 09/16/99, 09/17/99, 09/27/99, 09/28/99, 11/06/00,11/07/00 GML/LAC 09/25/00 LAC, 11/08/00 HOJ FT<5-R-Q7 O UrineWkM FACT-TOX-026 Covance 6329-231 Study: Product Number(Test Substance): Matrix: Method/Revision: Analytical Equipment System Number: Instrument Software/Version: Filename: R-Squared Value: Slope: Y-Intercept: Dates of Extraction/Analyst: Dates of Analysis/Analyst: Date of Data Reduction/Analyst: Sam ple Data M ONKEY URINE W eek 16 Group Dose Sample # Group 1 I05709M 0.0 mg/kg/day I05714M I05715M I05718M I05720M I05725M Group 2 3.0 mg/kg/day I05702M I05706M I05717M I05721M Group 3 I05707M 10 mg/kg/day I05708M I05710M I05712M I05716M I05719M Group 4 I05703M 30 mg/kg/day I05704M 105 / M I05713M I05722M I05724M Limit of Quantitation (LOQ): 0.0182 ug/mL 6 Month Capsule Toxicity Study with APFO in Cynomolgus Monkeys T-6889.2 (POAA) Monkey Urine Filenames ETS-8-96.0 & ETS-8-97.0 POAA Soup 020199, Madeline 040198, Amelia 062498 Grp 1 081999042-43, 082399054-55, 092299036, 092799058 MassLynx 3.2 Grp 2 090999057-62 See listing to the right Grp 3 090899029-036 See Attachments Grp 4 081399029,31,72, 091599080, 092399018 See Attachments Box 99-160-1, 99-162-3, 99-166-7 See Attachments 08/04/99, 08/05/99, 08/17/99, 08/20/99, 09/21/99 SEE/RWW/MCH/SAL 08/13/99, 08/19/99, 08/23/99, 09/08/99, 09/09/99, 09/15/99, 09/22/99, 09/23/99, 09/27/99 GML/PTF/IAS/MMH/MEE/IAS 08/18/99, 08/20/99, 08/24/99, 09/09/99, 09/13/99, 09/16/99, 09/24/99, 09/27/99, 09/28/99, 11/06/00 PTF/DRB/GML/MMH/MEE/IAS/KJH Ext. Vol. = 1 since curves are extracted at the same ratio as the specimens, 2 mL initial and 0.5 mL final volumes. Extraction Vol. 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 POAA Std Correction Factor 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 OE952 0.9582 0.9582 0.9582 POAA POAA Dilution Factor Cone. ng/mL 1 17.8 1 1.62 1 1.24 1 99.4 1 38.2 1 8.01 . 1000 39.1 1000 65.1 1000 76.2 1000 29.3 1000 172 1000 196 1000 169 1000 44.6 1000 102 1000 188 10 18.7 500 15 500 10 NA 283 79.3 234 244 NA NA = Not Analyzed/Not Applicable Concentration of POAA ug/mL or % rec. <LOQ <LOQ <LOQ 0.0952 0.0366 <LOQ 37.4 62.4 73.1 28.1 165 187 162 42.7 98.1 181 0.179 136 0.380 112 2.34 NA POAA = Perfluorooctanoate C8FI7COO Mean POAA ug/mL 0.0299 50.2 139 50.2 Date Entered/By: Date Verified/By: 08/20/99, 08/23/99, 08/24/99, 09/09/99, 09/13/99, 09/16/99, 09/17/99, 09/27/99, 09/28/99, 11/06/00, 11/07/00 GML/LAC 09/25/00 LAC, 11/08/00 HOJ Dilutions 1/1 1/1000 1/1000 1/1,1/10, 1/500, 1/5 RSD Std. Dev. MS/MSD RPD 113 0.0339 41.8 21.0 40.9 57.0 135 67.9 FTS-R-Q7 n U rineW kl6 FACT-TOX-026 Covance 6329-231 Study: Product Number(Test Substance): Matrix: Method/Revision: Analytical Equipment System Number: Instrument Software/Version: Filename: R-Squared Value: Slope: Y-Intercept: Dates of Extraction/Analyst: Dates of Analysis/Analyst: Date of Data Reduction/Analyst: M ONKEY URINE W eek 16 Group Sample # Dose Group 1 0.0 mg/kg/day I05709M 105714M I05715M I05718M I05720M I05725M Group 2 3.0 mg/kg/day I05702M I05706M 105717M I05721M Group 3 I05707M 10 mg/kg/day I05708M I05710M 105712M I05716M I05719M Group 4 30 mg/kg/day I05703M I05704M I57TTM I05713M I05722M I05724M Limit of Quantitation (LOQ): 0.0182 ug/mL 6 Month Capsule Toxicity Study with APFO in Cynomolgus Monkeys T-6889.2 (POAA) Monkey Urine ETS-8-96.0 & ETS-8-97.0 Soup 020199, Madeline 040198, Amelia 062498 MassLynx 3.2 See Attachments See Attachments See Attachments See Attachments 08/04/99, 08/05/99, 08/17/99, 08/20/99, 09/21/99 SEE/RWW/MCH/SAL 08/13/99, 08/19/99, 08/23/99, 09/08/99, 09/09/99, 09/15/99, 09/22/99, 09/23/99, 09/27/99 GML/PTF/IAS/MMH/MEE/IAS 08/18/99, 08/20/99, 08/24/99, 09/09/99, 09/13/99, 09/16/99, 09/24/99, 09/27/99, 09/28/99, 11/06/00 PTF/DRB/GML/MMH/MEE/IAS/KJH Sam ple Data POAA Cone. ng/mL 17.8 1.62 1.24 99.4 38.2 8.01 39.1 65.1 76.2 29.3 172 196 169 44.6 102.4 188 18.7 283 ' 793 234 244 NA Concentration of POAA ug/mL or % ree. <LOQ <LOQ <LOQ 0.0952 0.0366 <LOQ 37.4 62.4 73.1 28.1 165 187 162 42.7 98.1 181 0.179 136 (080 112 2.34 NA Mean POAA ug/mL 0.0299 50.2 139 50.2 RSD Std. Dev. MS/MSD RPD 113 0.0339 41.8 21.0 40.9 57.0 135 67.9 NA = Not Analyzed/Not ApplicablePOAA = Perfluorooctanoate C8F17COO- Date Entered/By: Date Verified/ By: 08/20/99, 08/23/99, 08/24/99, 09/09/99,09/13/99, 09/16/99, 09/17/99, 09/27/99, 09/28/99, 11/06/00, 11/07/00 GML/LAC 09/25/00 LAC, 11/08/00 HOJ C T C Q 0*7 A TIrineW kl6 FACT-TOX-026 Covance 6329-231 Study: Product Number(Test Substance): Matrix: Method/Revision: Analytical Equipment System Number: Instrument Software/Version: Filename: R-Squared Value: Slope: Y-Intercept: Dates of Extraction/Analyst: Dates of Analysis/Analyst: Date of Data Reduction/Analyst: Sam ple Data M ONKEY URINE W eek 18 Group Dose Sample # Group 1 0.0 mg/kg/day I05709M 105714M 105715M 105718M I05720M I05725M . Group 2 3.0 mg/kg/day I05702M I05706M I05717M I05721M Group 3 I05707M 10 mg/kg/day I05708M I05710M I05712M 105716M I05719M Group 4 I05703M 30 mg/kg/day I05704M 1U5/11M I05713M I05722M I05724M Limit of Quantitation (LOQ): 0.0182 ug/mL 6 Month Capsule Toxicity Study with APFO in Cynomolgus Monkeys T-6889.2 (POAA) Monkey Urine Filenames ETS-8-96.0 & ETS-8-97.0 POAA Soup 020199, Madeline 040198, Amelia 062498 Grp 1 081999060-61, 082399058-59, 092299037, 092799059 MassLynx 3.2 Grp 2 090999063-65, 091599118 See listing to the right Grp 3 090899037-044 See Attachments Grp 4 081399035,38, 091599081,87, 092399017 See Attachments Box 99-160-1, 99-162-3, 99-166-7 See Attachments 08/04/99, 08/05/99, 08/17/99, 08/20/99, 09/21/99 SEE/RWW/MCH/SAL 08/13/99, 08/19/99, 08/23/99, 09/08/99, 09/09/99, 09/15/99, 09/22/99, 09/23/99, 09/27/99 GML/PTF/IAS/MMH/MEE/IAS 08/18/99, 08/20/99, 08/24/99, 09/09/99, 09/13/99, 09/16/99, 09/24/99, 09/27/99, 09/28/99, 11/06/00 PTF/DRB/GML/MMH/MEE/IAS/KJH Ext. Vol. = 1 since curves are extracted at the same ratio as the specimens, 2 mL initial and 0.5 mL final volumes. Dilutions 1/1 1/500, 1/1000 1/1000 1/1,1/5, 1/10, 1/500 Extraction Vol. 1 1 1 0.9 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 POAAStd Correction Factor 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 POAA POAA Dilution Cone. Factor ng/mL 1 11.0 1 14.3 1 7.25 1 5.32 1 78.5 1 4.78 . 1000 38.3 1000 60.8 1000 45.6 500 25.0 1000 164 1000 238 1000 203 1000 78.9 1000 213 1000 269 10 0.00 500 355 1 210 5 84.8 10 174 NA NA NA = Not Analyzed/Not Applicable Concentration of POAA ug/mL or % Rec. <LOQ <LOQ <LOQ <LOQ 0.0752 <LOQ 36.7 58.3 43.7 12.0 157 228 194 75.6 204 258 <LOQ 170 0.20i 0.406 1.67 NA POAA = Perfluorooctanoate C8F17COO Mean POAA ug/mL 0.0256 37.7 186 43.1 RSD Std. Dev. MS/MSD RPD 96.8 0.0248 51.3 19.3 34.3 63.9 196 84.6 Date Entered/By: Date Verified/ By: 08/20/99,08/23/99,08/24/99,09/13/99,09/16/99,09/17/99,09/28/99, 11/06/00, 11/07/00 GML/LAC 09/25/00 LAC, 11/08/00 HOJ FT.8.Q7 O U rineW kl8 FACT-TOX-026 Covance 6329-231 Study: Product Number(Test Substance): Matrix: Method/Revision: Analytical Equipment System Number: Instrument Software/Version: Filename: R-Squared Value: Slope: Y-Intercept: Dates of Extraction/Analyst: Dates of Analysis/Analyst: Date of Data Reduction/Analyst: M ONKEY URINE W eek 18 Group Dose Sample # Group 1 0.0 mg/kg/day I05709M I05714M I05715M 105718M I05720M I05725M Group 2 3.0 mg/kg/day I05702M I05706M I05717M I05721M Group 3 10 mg/kg/day I05707M I05708M I05710M I05712M 105716M I05719M Group 4 30 mg/kg/day I05703M I05704M 1UD / 1 1 M I05713M I05722M I05724M Limit of Quantitation (LOQ): 0.0182 ug/mL 6 Month Capsule Toxicity Study with APFO in Cynomolgus Monkeys T-6889.2 (POAA) Monkey Urine ETS-8-96.0 & ETS-8-97.0 . Soup 020199, Madeline 040198, Amelia 062498 MassLynx 3.2 See Attachments See Attachments See Attachments See Attachments 08/04/99, 08/05/99, 08/17/99, 08/20/99, 09/21/99 SEE/RWW/MCH/SAL 08/13/99, 08/19/99, 08/23/99, 09/08/99, 09/09/99, 09/15/99, 09/22/99, 09/23/99, 09/27/99 GML/PTF/IAS/MMH/MEE/IAS 08/18/99, 08/20/99, 08/24/99, 09/09/99, 09/13/99, 09/16/99, 09/24/99, 09/27/99, 09/28/99, 11/06/00 PTF/DRB/GML/MMH/MEE/IAS/KJH Sample Data POAA Concentration Cone. of POAA ng/mL ug/mL or % Rec. 11.0 <LOQ 14.3 <LOQ 7.25 <LOQ 5.32 <LOQ 78.5 0.0752 4.78 <LOQ 38.3 36.7 60.8 58.3 45.6 43.7 25.0 12.0 164 157 238 228 203 194 78.9 75.6 213 204 269 258 0.0 <LOQ 355 170 210 0.201 84.8 0.406 174 1.67 NA NA NA = Not Analyzed/Not Applicable Mean POAA ug/mL RSD Std. Dev. MS/MSD RPD 0.0256 37.7 96.8 0.0248 51.3 19.3 34.3 186 63.9 43.1 POAA = Perfluorooctanoate C8F17COO- 196 84.6 Date Entered/By: Date Verified/ By: 08/20/99, 08/23/99, 08/24/99, 09/13/99, 09/16/99, 09/17/99, 09/28/99, 11/06/00, 11/07/00 GML/LAC 09/25/00 LAC, 11/08/00 HOJ FTS-8-Q7 0 U rineW kl8 FACT-TOX-026 Covance 6329-231 Study: Product Number(Test Substance): Matrix: Method/Revision: Analytical Equipment System Number: Instrument Software/Version: Filename: R-Squared Value: Slope: Y-Intercept: Dates of Extraction/Analyst: Dates of Analysis/Analyst: Date of Data Reduction/Analyst: Sam ple D ata 6 Month Capsule Toxicity Study with APFO in Cynomolgus Monkeys T-6889.2 (POAA) Monkey Urine Filenames ETS-8-96.0 & ETS-8-97.0 POAA Soup 020199, Madeline 040198, Amelia 062498 Grp 1 081999062-63, 082399060-61, 092299040, 092799060 MassLynx 3.2 Grp 2 090999069-72 See listing to the right Grp 3 082499049-53, 092899073 See Attachments Grp 4 081399044,73, 09159982,89-90 See Attachments Box 99-160-1,99-162-3 See Attachments 08/04/99, 08/05/99, 08/17/99, 08/20/99, 09/21/99 SEE/RWW/MCH/SAL 08/13/99, 08/19/99, 08/23/99,09/09/99, 09/15/99, 09/22/99, 09/27/99, 09/28/99 GML/MMH/IAS/MEE 08/18/99, 08/20/99, 08/24/99,09/13/99, 09/16/99, 09/27/99, 09/28/99,09/29/99, 11/06/00, 11/13/00 PTF/DRB/GML/MMH/MEE/IAS/KJH Ext. Vol. = 1 since curves are extracted at the same ratio as the specimens, 2 mL initial and 0.5 mL final volumes. Dilutions 1/1 1/1000 1/1000 1/1,1/10,1/20,1/500 M ONKEY URINE W eek 20 Group Dose Sample # Extraction Vol. POAA Std Correction Factor POAA Dilution Factor POAA Cone. ng/mL Concentration of POAA ug/mL or % Rec. Mean POAA ug/mL RSD Std. Dev, MS/MSD RPD Group 1 I05709M 0.0 mg/kg/day 105714M I05715M 105718M I05720M I05725M Group 2 I05702M 3.0 mg/kg/day I05706M I05717M I05721M Group 3 10 mg/kg/day I05707M I05708M+ I05710M+ I05712M+ I05716M+ I05719M+ Group 4 30 mg/kg/day I05703M I05704M 105/11M I05713M I05722M I05724M Limit of Quantitation (LOQ): 0.0182 ug/mL 1 0.9582 1 17.7 <LOQ 1 0.9582 1 7.61 <LOQ 1 0.9582 1 13.4 <LOQ 1 0.9582 1 22.3 0.0213 1 0.9582 1 0.9582 1 1 47.1 5.20 0.0451 <LOQ 0.0211 1 0.9582 . 1000 1 0.9582 1000 51.0 69.0 48.9 66.1 1 0.9582 1000 46.2 44.3 i 0.9582 1000 51.1 48.9 52.1 1 0.9582 5000 344 1648 * 1 0.9582 1000 378 362 1 0.9582 1000 185 177 1 0.9582 1000 61.9 59.3 1 0.9582 1000 109 104 1 0.9582 1000 18.8 18.0 ** 144 1 0.9582 1 379 0.363 1 0.9582 500 258 124 1 0.9582 1 140 0.134 1 0.9582 500 195 93.3 1 0.9582 20 122 2.33 1 0.9582 NA NA NA 44.0 NA = Not Analyzed/Not Applicable POAA = Perfluorooctanoate 61.3 0.0130 18.5 9.63 93.9 135 136 59.9 ** Cone, was below the LOQ C8F17COO' + CCVs analyzed prior to these samples were not within +/- 30% criteria. LAC 11/16/00 Date Entered/By: 08/20/99,08/23/99,08/24/99, 08/25/00, 09/13/99, 09/16/99, 09/17/99, 09/28/99, 09/29/99, 11/06/00, 11/07/00, 11/14/00 GML/LAC Date Verified/ By: 09/25/00 LAC, 11/08/00 HOJ, 11/15/00 HOJ * This sample was not diluted 1/5000; a 2uL injection of D1000 was analyzed instead. All other injections were 10 uL. This provided a 1/5 dilution of D1000 for a total of D5000. A 1/4000 dilution of I05707M was also analyzed, but was just out of calibration range. GML/KJH 9/29/99 T3XC_C_07 n IIrineWk20 FACT-TOX-026 Covance 6329-231 Study: Product Number(Test Substance): Matrix: Method/Revision: Analytical Equipment System Number: Instrument Software/Version: Filename: R-Squared Value: Slope: Y-Intercept: Dates of Extraction/Analyst: Dates of Analysis/Analyst: Date of Data Reduction/Analyst: 6 Month Capsule Toxicity Study with APFO in Cynomolgus Monkeys T-6889.2 (POAA) Monkey Urine ETS-8-96.0 &ETS-8-97.0 Soup 020199, Madeline 040198, Amelia 062498 MassLynx 3.2 See Attachments See Attachments See Attachments See Attachments 08/04/99, 08/05/99, 08/17/99, 08/20/99, 09/21/99 SEE/RWW/MCH/SAL 08/13/99, 08/19/99, 08/23/99, 09/09/99, 09/15/99, 09/22/99, 09/27/99, 09/28/99 GML/MMH/IAS/MEE 08/18/99, 08/20/99, 08/24/99, 09/13/99, 09/16/99, 09/27/99, 09/28/99, 09/29/99, 11/06/00, 11/13/00 PTF/DRB/GML/MMH/MEE/IAS/KJH Sam ple Data M ONKEY URINE W eek 20 Group Dose Sample # Group 1 0.0 mg/kg/day I05709M I05714M I05715M 105718M I05720M I05725M Group 2 3.0 mg/kg/day I05702M I05706M I05717M I05721M Group 3 10 mg/kg/day I05707M I05708M+ I05710M+ I05712M+ 105716M+ I05719M+ Group 4 I05703M 30 mg/kg/day I05704M 1U5/ i iM I05713M I05722M I05724M Limit of Quantitation (LOQ): 0.0182 ug/mL POAA Concentration Cone. ng/mL of POAA ug/mL or % Rec. 17.7 <LOQ 7.61 <LOQ 13.4 <LOQ 22.3 0.0213 47.1 0.0451 5.20 <LOQ 51.0 48.9 69.0 66.1 46.2 44.3 51.1 48.9 344 1648 378 362 185 177 61.9 59.3 109 104 18.8 18.0 379 0.363 258 124 140 0.134 195 93.3 122 2.33 NA NA NA = Not Analyzed/Not Applicable Mean POAA ug/mL RSD Std. Dev. MS/MSD RPD 0.0211 52.1 * ** 144 61.3 0.0130 18.5 9.63 93.9 135 136 44.0 59.9 POAA = Perfluorooctanoate ** Cone, was below the LOQ C8F17COO- + CCVs analyzed prior to these samples were not within +/- 30% criteria. LAC 11/16/00 Date Entered/By: 08/20/99, 08/23/99, 08/24/99, 08/25/00, 09/13/99, 09/16/99, 09/17/99, 09/28/99, 09/29/99, 11/06/00, 11/07/00, 11/14/00 GML/LAC Date Verified/ By: 09/25/00 LAC, 11/08/00 HOJ, 11/15/00 HOJ * This sample was not diluted 1/5000; a 2uL injection of D1000 was analyzed instead. All other injections were 10 uL. This provided a 1/5 dilution of D1000 for a total of D5000. . A 1/4000 dilution of I05707M was also analyzed, but was just out of calibration range. GML/KJH 9/29/99 ETS-8-97.0 UrineWk20 FACT-TOX-026 Covance 6329-231 Study: Product Number(Test Substance): Matrix: Method/Revision: Analytical Equipment System Number: Instrument Software/Version: Filename: R-Squared Value: Slope: Y-Intercept: Dates of Extraction/Analyst: Dates of Analysis/Analyst: Date of Data Reduction/Analyst: 6 Month Capsule Toxicity Study with APFO in Cynomolgus Monkeys T-6889.2 (POAA) Monkey Urine Filenames ETS-8-96.0 & ETS-8-97.0 POAA Soup 020199, Madeline 040198, Amelia 062498 Grp 1 081999066-67, 082399064-5, 092299041, 092799061 MassLynx 3.2 Grp 2 090999075-77, 091599119 See listing to the right Grp 3 082499056-58,60-61, 091099040 See Attachments Grp 4 091599093-94, 092399016,24-25 See Attachments Box 99-160-1,99-162-3 See Attachments 08/04/99, 08/05/99, 08/17/99, 08/20/99, 09/21/99 SEE/RWW/MCH/SAL 08/19/99, 08/23/99, 08/24/99, 09/09/99, 09/15/99, 09/22/99, 09/23/99, 09/27/99 GML/SAH/PTF/MMH/IAS/MEE 08/20/99, 08/24/99, 08/25/99, 09/13/99, 09/16/99, 09/24/99, 09/27/99, 09/28/99, 11/06/00, 11/13/00 PTF/DRB/GML/MMH/MEE/IAS/KJH Sam ple D ata Ext. Vol. = 1 since curves are extracted at the same ratio as the specimens, 2 mL initial and 0.5 mL final volumes. M ONKEY URINE W eek 22 Group Dose Sample # Extraction Vol. POAA Std Correction Factor POAA Dilution Factor POAA Cone. ng/mL Concentration of POAA ug/mL or % Rec. Mean POAA ug/mL Group 1 0.0 mg/kg/day Group 2 3.0 mg/kg/day Group 3 10 mg/kg/day Group 4 30 mg/kg/day I05709M I05714M I05715M 105718M I05720M I05725M I05702M I05706M I05717M I05721M I05707M+ I05708M+ I05710M+ I05712M I05716M+ I05719M+ I05703M I05704M 105711M 105713M 105722M I05724M 1 0.9582 1 1 0.9582 1 1 0.9582 1 1 0.9582 1 1 0.9582 1 1 0.9582 1 1 0.9582 1000 1 0.9582 500 1 0.9582 1000 1 0.9582 1000 1 0.9582 1000 1 0.9582 1000 1 0.9582 1000 1 0.9582 1000 1 0.9582 1000 1 0.9582 1000 1 0.9582 1 1 0.9582 1000 1 0.95S2 1 1 0.9582 1000 1 0.9582 20 1 0.9582 NA 35.9 29.9 7.09 14.8 21.7 8.21 46.7 112 197 NA 60.6 175 246 90.7 150 266 367 266 348 247 45.3 NA 0.0344 0.0287 <LOQ <LOQ 0.0208 <LOQ 44.7 53.7 189 NA 58.0 168 235 86.9 144 255 0.352 255 0.334 237 0.868 NA 0.0231 95.8 158 98.5 Limit of Quantitation (LOQ): 0.0182 ug/mL NA = Not Analyzed/Not Applicable POAA = Perfluorooctanoate C8F17COO + CCVs analyzed prior to these samples were not within +/- 30% criteria. LAC 11/16/00 Date Entered/By: 08/23/99, 08/24/99, 08/25/99, 09/13/99, 09/16/99, 09/17/99, 09/27/99, 09/28/99, 11/07/00, 11/14/00 GML/LAC Date Verified/ By: 09/25/00 LAC, 11/08/00 HOJ, 11/15/00 HOJ Dilutions 1/1 1/500, 1/1000 1/1000 1/1,1/10,1/20, 1/1000 RSD Std. Dev. MS/MSD RPD 29.8 0.00688 84.4 80.8 49.7 78.4 136 134 FTQ-H.Q7 A UrineWk22 FACT-TOX-026 Covance 6329-231 Study: Product Number(Test Substance): Matrix: Method/Revision: Analytical Equipment System Number: Instrument Software/Version: Filename: R-Squared Value: Slope: Y-Intercept: Dates o f Extraction/Analyst: Dates of Analysis/Analyst: Date of Data Reduction/Analyst: 6 Month Capsule Toxicity Study with APFO in Cynomolgus Monkeys T-6889.2 (POAA) Monkey Urine ETS-8-96.0 & ETS-8-97.0 . Soup 020199, Madeline 040198, Amelia 062498 MassLynx 3.2 See Attachments See Attachments See Attachments See Attachments 08/04/99, 08/05/99, 08/17/99, 08/20/99, 09/21/99 SEE/RWW/MCH/SAL 08/19/99, 08/23/99, 08/24/99, 09/09/99, 09/15/99, 09/22/99, 09/23/99, 09/27/99 GML/SAH/PTF/MMH/IAS/MEE 08/20/99, 08/24/99, 08/25/99, 09/13/99, 09/16/99, 09/24/99, 09/27/99, 09/28/99, 11/06/00, 11/13/00 PTF/DRB/GML/MMH/MEE/IAS/KJH Sam ple Data M ONKEY URINE W eek 22 Group Dose Sample # POAA Cone. ng/mL Concentration of POAA ug/mL or % Rec. Mean POAA ug/mL RSD Std. Dev. MS/MSD RPD Group 1 0.0 mg/kg/day Group 2 3.0 mg/kg/day Group 3 10 mg/kg/day I05709M I05714M I05715M 105718M I05720M I05725M I05702M I05706M I05717M I05721M I05707M+ I05708M+ I05710M+ I05712M 105716M+ I05719M+ 35.9 0.0344 29.9 0.0287 7.09 <LOQ 14.8 <LOQ 21.7 0.0208 8.21 <LOQ 46.7 44.7 112 53.7 197 189 NA NA 60.6 58.0 175 168 246 235 90.7 86.9 150 144 266 255 0.0231 95.8 158 29.8 0.00688 84.4 80.8 49.7 78.4 Group 4 30 mg/kg/day I05703M I05704M 105711M I05713M I05722M I05724M Limit o f Quantitation (LOQ): 0.0182 ug/mL 367 0.352 266 255 348 0.334 247 237 45.3 0.868 NA NA 98.5 NA = Not Analyzed/Not Applicable POAA = Perfluorooctanoate 136 134 C8F17COO- + CCVs analyzed prior to these samples were not within +/- 30% criteria. LAC 11/16/00 Date Entered/By: 08/23/99, 08/24/99, 08/25/99,09/13/99, 09/16/99, 09/17/99, 09/27/99, 09/28/99, 11/07/00, 11/14/00 GML/LAC Date Verified/ By: 09/25/00 LAC, 11/08/00 HOJ, 11/15/00 HOJ CTC.5. -? n t JrineWk22 FACT-TOX-026 Covance 6329-231 Study: Product Number(Test Substance): Matrix: Method/Revision: Analytical Equipment System Number: Instrument Sofitware/Version: Filename: R-Squared Value: Slope: Y-Intercept: Dates of Extraction/Analyst: Dates of Analysis/Analyst: Date of Data Reduction/Analyst: 6 Month Capsule Toxicity Study with APFO in Cynomolgus Monkeys T-6889.2 (POAA) Monkey Urine Filenames ETS-8-96.0 & ETS-8-97.0 POAA Soup 020199, Madeline 040198, Amelia 062498 Grp 1 081999068-69, 082399066-67, 092299042-43 MassLynx 3.2 Grp 2 090999078-82 See listing to the right Grp 3 082499064-65,67-69, 091099041 See Attachments Grp 4 081399055,65, 091599083,95-96 See Attachments Box 99-160-1, 99-162-3 See Attachments 08/04/99, 08/05/99, 08/17/99, 08/20/99, 09/21/99 SEE/RWW/MCH/SAL 08/13/99, 08/19/99, 08/23/99, 08/24/99, 09/09/99, 09/10/99, 09/15/99, 09/22/99 GML/SAH/PTF/MMH/IAS 08/18/99, 08/20/99, 08/24/99, 09/13/99, 09/16/99, 09/27/99, 11/06/00, 11/13/00 PTF/DRB/GML/MMH/MEE/KJH Sam ple D ata Ext. Vol. = 1 since curves are extracted at the same ratio as the specimens, 2 mL initial and 0.5 mL final volumes. M ONKEY URINE W eek 24 Group Dose Sample # Extraction Vol. POAA Std Correction Factor POAA Dilution Factor POAA Cone. ng/mL Concentration of POAA ug/mL or % Ree. Mean POAA ug/mL Group 1 0.0 mg/kg/day Group 2 3.0 mg/kg/day Group 3 10 mg/kg/day Group 4 30 mg/kg/day I05709M I05714M 105715M I05718M I05720M I05725M I05702M I05706M I05717M I05721M I05707M+ I05708M+ I05710M I05712M+ I05716M+ I05719M+ I05703M I05704M 105711M I05713M I05722M I05724M 0.4 0.9582 1 0.9582 1 0.9582 1 0.9582 1 0.9582 1 0.9582 1 0.9582 1 0.9582 1 0.9582 i 0.9582 1 0.9582 1 0.9582 1 0.9582 1 0.9582 1 0.9582 1 0.9582 0.9582 0.9582 0.9582 0.9582 1 0.9582 1 0.9582 1 1 1 1 1 1 1000 1000 1000 1000 1000 1000 1000 1000 1000 1000 1 500 1 500 10 NA 7.42 6.32 4.53 3.10 22.2 5.39 38.4 55.6 50.9 NA 192 226 222 56.3 117 170 319 324 112 259 25.9 NA <LOQ <LOQ <LOQ <LOQ 0.0213 <LOQ 36.8 53.3 48.7 NA 184 216 212 54.0 112 163 0.306 155 0.107 124 0.248 NA 0.0125 46.3 157 56.0 Limit of Quantitation (LOQ): 0.0182 ug/mL NA = Not Analyzed/Not Applicable POAA = Perfluorooctanoate A = Below LOQ C8F17COO' + CCVs analyzed prior to these samples were not within +/- 30% criteria. LAC 11/16/00 Date Entered/By: 08/20/99, 08/23/99, 08/24/99, 08/25/99, 09/13/99, 09/16/99, 09/17/99, 09/28/99, 11/06/00, 11/07/00, 11/14/00 GML/LAC Date Verified/ By: 09/25/00 LAC, 11/08/00 HOJ, 11/15/00 HOJ Dilutions 1/1 1/1000 1/1000 1/1,1/10,1/500 RSD Std. Dev. MS/MSD RPD 60.1 A 0.00749 18.4 8.52 40.3 63.3 138 77.2 FTC-R.Q7 H UrineWk24 FACT-TOX-026 Covance 6329-231 Study: Product Number(Test Substance): Matrix: Method/Revision: Analytical Equipment System Number: Instrument Software/Version: Filename: R-Squared Value: Slope: Y-Intercept: Dates of Extraction/Analyst: Dates of Analysis/Analyst: Date of Data Reduction/Analyst: 6 Month Capsule Toxicity Study with APFO in Cynomolgus Monkeys T-6889.2 (POAA) Monkey Urine ETS-8-96.0 & ETS-8-97.0 . Soup 020199, Madeline 040198, Amelia 062498 MassLynx 3.2 See Attachments See Attachments See Attachments See Attachments 08/04/99, 08/05/99, 08/17/99, 08/20/99, 09/21/99 SEE/RWW/MCH/SAL 08/13/99, 08/19/99, 08/23/99, 08/24/99, 09/09/99, 09/10/99, 09/15/99, 09/22/99 GML/SAH/PTF/MMH/IAS 08/18/99, 08/20/99, 08/24/99, 09/13/99, 09/16/99, 09/27/99, 11/06/00, 11/13/00 PTF/DRB/GML/MMH/MEE/KJH Sam ple Data M ONKEY URINE W eek 24 Group Dose Sample # Group 1 I05709M 0.0 mg/kg/day 105714M I05715M 105718M I05720M I05725M . Group 2 I05702M 3.0 mg/kg/day I05706M I05717M I05721M Group 3 I05707M+ 10 mg/kg/day I05708M+ I05710M I05712M+ 105716M+ 05719M+ Group 4 I05703M 30 mg/kg/day I05704M 105711M I05713M I05722M I05724M Limit of Quantitation (LOQ): 0.0182 ug/mL POAA Concentration Mean Cone. of POAA POAA ng/mL ug/mL or % Rec. ug/mL 7.42 <LOQ 6.32 <LOQ 4.53 <LOQ 3.10 <LOQ 22.2 0.0213 5.39 <LOQ 0.0125 A 38.4 36.8 . 55.6 53.3 50.9 48.7 NA NA 46.3 192 184 226 216 222 212 56.3 54.0 117 112 170 163 157 319 0.306 324 155 112 0.107 259 124 25.9 0.248 NA NA 56.0 NA = Not Analyzed/Not Applicable POAA = Perfluorooctanoate RSD Std. Dev. MS/MSD RPD 60.1 0.00749 18.4 8.52 40.3 63.3 138 77.2 A = Below LOQ C8F17COO- + CCVs analyzed prior to these samples were not within +/- 30% criteria. LAC 11/16/00 Date Entered/By: 08/20/99, 08/23/99, 08/24/99, 08/25/99, 09/13/99, 09/16/99, 09/17/99, 09/28/99, 11/06/00, 11/07/00, 11/14/00 GML/LAC Date Verified/By: 09/25/00 LAC, 11/08/OOHOJ, 11/15/00 HOJ FTS-R-97 O UrineWk24 FACT-TOX-026 Covance 6329-231 Study: Product Number(Test Substance): Matrix: Method/Revision: Analytical Equipment System Number: Instrument SoftwareA/ersion: Filename: R-Squared Value: Slope: Y-Intercept: Dates of Extraction/Analyst: Dates of Analysis/Analyst: Date of Data Reduction/Analyst: 6 Month Capsule Toxicity Study with APFO in Cynomolgus Monkeys T-6889.2 (POAA) Monkey Urine Filenames ETS-8-96.0 & ETS-8-97.0 POAA Dilutions Soup 020199, Madeline 040198, Amelia 062498 Grp 1 081999072-73, 082399070-71, 092299044, 092799064 1/1 MassLynx 3.2 Grp 2 090999083-85 1/1000 See listing to the right Grp 3 082499072-75, 091099042, 092799046 1/1000 See Attachments Grp 4 081399066, 091599099-100, 092399015,23 1/1,1/10,1/250 See Attachments Box 99-160-1,99-162-3 See Attachments 08/17/99 SEE/RWW/MCH/SAL 08/13/99, 08/19/99, 08/23/99, 08/24/99, 09/09/99, 09/10/99, 09/15/99, 09/22/99, 09/23/99, 09/27/99 GML/SAH/PTF/MMH/IAS/MEE 08/18/99, 08/20/99, 08/24/99, 08/25/99, 09/13/99, 09/16/99, 09/24/99, 09/27/99, 09/28/99, 11/06/00, 11/13/00 PTF/DRB/GML/MMH/MEE/IAS/KJH Sam ple D ata Ext. Vol. = 1 since curves are extracted at the same ratio as the specimens, 2 mL initial and 0.5 mL final volumes. M ONKEY URINE W eek 26 Group Dose Sample # Extraction Vol. POAA Std Correction Factor POAA Dilution Factor POAA Cone. ng/mL Concentration of POAA ug/mL or % Rec. Mean POAA ug/mL RSD Std. Dev. MS/MSD RPD Group 1 0.0 mg/kg/day Group 2 3.0 mg/kg/day Group 3 10 mg/kg/day I05709M I05714M 105715M 105718M I05720M I05725M I05702M I05706M I05717M I05721M I05707M+ I05708M+ I05710M I05712M+ 105716M+ I05719M 1 1 1 1 1 1 1. 1 1 1 1 1 1 1 1 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 1 1 1 1 1 1 1000 1000 1000 1000 1000 1000 1000 1000 1000 1000 21.7 10.0 7.19 8.53 83.2 4.29 38.6 55.9 67.0 NA 268 69.6 53.0 75.5 107 112 0.0208 <LOQ <LOQ <LOQ 0.0797 <LOQ 37.0 53.6 64.2 NA 256 66.6 50.8 72.3 103 107 . 0.0268 51.6 109 98.7 0.0265 26.6 13.7 68.8 75.2 Group 4 30 mg/kg/day I05703M I05704M 105711M I05713M I05722M I05724M 1 0.9582 1 1 0.9582 250 1 0.9582 1 1 0.9582 250 1 0.9582 1 1 0.9582 NA 233 241 71.2 158 225 NA 0.223 57.7 0.0682 37.8 0.215 NA 141 19.2 27.0 Limit of Quantitation (LOQ): 0.0182 ug/mL NA = Not Analyzed/Not Applicable POAA = Perfluorooctanoate C8F17COO- + CCVs analyzed prior to these samples were not within +/- 30% criteria. LAC 11/16/00 Date Entered/By: 08/23/99, 08/24/99, 08/25/99, 09/13/99, 09/16/99, 09/17/99, 09/27/99, 09/28/99, 11/06/00, 11/07/00, 11/14/00 GML/LAC Date Verified/ By: 09/25/00 LAC, 11/08/00 HOJ, 11/15/00 HOJ etc q m n I IrinpWk76 FACT-TOX-026 Covance 6329-231 Study: Product Number(Test Substance): Matrix: Method/Revision: Analytical Equipment System Number: Instrument Software/Version: Filename: R-Squared Value: Slope: Y-Intercept: Dates o f Extraction/Analyst: Dates o f Analysis/Analyst: Date o f Data Reduction/Analyst: 6 Month Capsule Toxicity Study with APFO in Cynomolgus Monkeys T-6889.2 (POAA) Monkey Urine ETS-8-96.0 & ETSr8-97.0 Soup 020199, Madeline 040198, Amelia 062498 MassLynx 3.2 See Attachments See Attachments See Attachments See Attachments 08/17/99 SEE/RWW/MCH/SAL 08/13/99, 08/19/99, 08/23/99, 08/24/99, 09/09/99, 09/10/99, 09/15/99, 09/22/99, 09/23/99, 09/27/99 GML/SAH/PTF/MMH/IAS/MEE 08/18/99, 08/20/99, 08/24/99, 08/25/99, 09/13/99, 09/16/99, 09/24/99, 09/27/99, 09/28/99, 11/06/00, 11/13/00 PTF/DRB/GML/MMH/MEE/IAS/KJH Sam ple Data M ONKEY URINE W eek 26 Group Dose Sample # POAA Cone. ng/mL Concentration of POAA ug/mL or % Rec. Mean POAA ug/mL RSD Std. Dev. MS/MSD RPD Group 1 0.0 mg/kg/day Group 2 3.0 mg/kg/day Group 3 10 mg/kg/day Group 4 30 mg/kg/day I05709M 105714M I05715M I05718M I05720M I05725M I05702M I05706M 105717M I05721M I05707M+ I05708M+ I05710M I05712M+ I05716M+ I05719M I05703M I05704M 10571IM I05713M I05722M I05724M 21.7 0.0208 10.0 <LOQ 7.19 <LOQ 8.53 <LOQ 83.2 0.0797 98.7 4.29 <LOQ 0.0268 0.0265 38.6 37.0 55.9 53.6 67.0 64.2 26.6 NA NA 51.6 13.7 268 256 69.6 66.6 53.0 50.8 75.5 72.3 107 103 68.8 112 107 109 75.2 233 0.223 241 57.7 71.2 0.0682 158 37.8 225 0.215 141 NA NA 19.2 27.0 Limit o f Quantitation (LOQ): 0.0182 ug/mL NA = Not Analyzed/Not Applicable POAA = Perfluorooctanoate C8F17COO- + CCVs analyzed prior to these samples were not within +/- 30% criteria. LAC 11/16/00 Date Entered/By: 08/23/99, 08/24/99, 08/25/99, 09/13/99, 09/16/99, 09/17/99, 09/27/99, 09/28/99, 11/06/00, 11/07/00, 11/14/00 GML/LAC Date Verified/ By: 09/25/00 LAC, 11/08/00 HOJ, 11/15/00 HOJ FT-S-Q7 H I IrineWk26 FACT-TOX-026 Covance 6329-231 Study: Product Number(Test Substance): Matrix: Method/Revision: Analytical Equipment System Number: Instrument Software/Version: Filename: R-Squared Value: Slope: Y-Intercept: Dates of Extraction/Analyst: Dates of Analysis/Analyst: Date of Data Reduction/Analyst: Sample Data MONKEY URINE Week 28 Group Dose Sample # Group 1 105718M 0.0 mg/kg/day I05720M Group 3 I05712M 10 mg/kg/day I05716M Limit of Quantitation (LOQ): 0.0182 ug/mL 6 Month Capsule Toxicity Study with APFO in Cynomolgus Monkeys T-6889.2 (POAA) Monkey Urine ETS-8-96.0 & ETS-8-97.0 Soup 020199, Madeline 040198, Amelia 062498 MassLynx 3.2 Filenames Grp 1 POAA 081999074-75 Dilutions 1/1 See listing to the right See Attachments See Attachments See Attachments 08/17/99 SEE/RWW/MCH/SAL Grp 3 Box 082499052-53 99-160-1 1/10 08/19/99, 08/24/99 GML/SAH/PTF 08/20/99, 08/25/99,11/06/00 GML/PTF/KJH Ext. Vol. = 1 since curves are extracted at the same ratio as the specimens, 2 mL initial and 0.5 mL final volumes. Extraction POAA Std Vol. Correction Factor 1 0.9582 1 0.9582 1 0.9582 1 0.9582 NA = Not Analyzed/Not Applicable POAA Dilution Factor 1 1 10 10 POAA Cone. ng/mL 228 11.9 32.8 35.5 POAA = Perfluorooctanoate CsF ,7COO Concentration of POAA ug/mL or % Rec. 0.218 <LOQ 0.314 0.340 Mean POAA ug/mL 0.118 0.327 Date Entered/By: 08/23/99, 08/26/99, 11/07/00 GML/LAC Date Verified/ By: 09/25/00 LAC, 11/08/00 HOJ RSD Std. Dev. MS/MSD RPD 0.142 0.0182 F.TS-8-97.0 U rine W k28 FACT-TOX-026 Covance 6329-231 Study: Product Number(Test Substance): Matrix: Method/Revision: Analytical Equipment System Number: Instrument Software/Version: Filename: R-Squared Value: Slope: Y-Intercept: Dates of Extraction/Analyst: Dates of Analysis/Analyst: Date of Data Reduction/Analyst: MONKEY URINE Week 28 Group Dose Sample # Group 1 105718M 0.0 mg/kg/day I05720M Group 3 105712M 10 mg/kg/day 105716M Limit of Quantitation (LOQ): 0.0182 ug/mL 6 Month Capsule Toxicity Study with APFO in Cynomolgus Monkeys T-6889.2 (POAA) Monkey Urine ETS-8-96.0 & ETS-8-97.0 Soup 020199, Madeline 040198, Amelia 062498 MassLynx 3.2 See Attachments See Attachments See Attachments See Attachments 08/17/99 SEE/RWW/MCH/SAL 08/19/99, 08/24/99 GML/SAH/PTF 08/20/99,08/25/99, 11/06/00 GML/PTF/KJH Sample Data POAA Concentration Cone. of POAA ng/mL ug/mL or % Rec. 228 0.218 11.9 <LOQ 32.8 0.314 35.5 0.340 . NA = Not Analyzed/Not Applicable Mean POAA ug/mL RSD Std. Dev. MS/MSD RPD 0.118 0.142 0.327 0.0182 POAA = Perfluorooctanoate C8F17COO- Date Entered/By: Date Verified/' By: 08/23/99, 08/26/99, 11/07/00 GML/LAC 09/25/00 LAC, 11/08/00 HOJ FTS-R-Q7 0 U rineW k28 FACT-TOX-026 Covance 6329-231 Study: Product Number(Test Substance): Matrix: Method/Revision: Analytical Equipment System Number: Instrument Software/Version: Filename: R-Squared Value: Slope: Y-Intercept: Dates of Extraction/Analyst: Dates of Analysis/Analyst: Date o f Data Reduction/Analyst: Sample Data MONKEY URINE Week 30 Group Dose Sample # Group 1 105718M 0.0 mg/kg/day I05720M Group 3 I05712M 10 mg/kg/day 105716M Limit of Quantitation (LOQ): 0.0182 ug/mL 6 Month Capsule Toxicity Study with APFO in Cynomolgus Monkeys T-6889.2 (POAA) Monkey Urine ETS-8-96.0 & ETS-8-97.0 Soup 020199, Madeline 040198, Amelia 062498 MassLynx 3.2 See listing to the right See Attachments Filenames Grp 1 Grp 3 Box POAA 081999078-79 091599018,159 99-160-1 Dilutions 1/1 1/ 1&10 See Attachments See Attachments 08/17/99 SEE/RWW/MCH/SAL 08/19/99, 09/15/99 GML 08/20/99,09/16/99,11/06/00 GML/KJH Ext. Vol. = 1 since curves are extracted at the same ratio as the specimens, 2 mL initial and 0.5 mL final volumes. Extraction POAA Std Vol. Correction Factor 1 0.9582 1 0.9582 1 0.9582 1 0.9582 NA = Not Analyzed/Not Applicable POAA Dilution Factor POAA Cone. ng/mL 1 2.73 1 0.00 1 290 10 46.5 POAA = Perfluorooctanoate C8F17COO Concentration of POAA ug/mL or % Rec. <LOQ <LOQ 0.278 0.445 Mean POAA ug/mL <LOQ 0.361 Date Entered/By: 08/23/99, 09/16/99, 09/17/99, 11/07/00 GML/LAC Date Verified/ By: 09/25/00 LAC, 11/08/00 HOJ RSD Std. Dev. MS/MSD RPD NA 0.118 FTS-8-97.0 U rine W k30 FACT-TOX-026 Covance 6329-231 Study: 6 Month Capsule Toxicity Study with APFO in Cynomolgus Monkeys Product Number(Test Substance): T-6889.2 (POAA) Matrix: Monkey Urine Method/Revision: 8/23/99 GML, 8/24/99 GML, 9/ETS-8-96.0 & ETS-8-97.0 Analytical Equipment System Number: Soup 020199, Madeline 040198, Amelia 062498 Instrument Software/Version: MassLynx 3.2 Filename: See Attachments R-Squared Value: See Attachments Slope: See Attachments Y-Intercept: See Attachments Dates o f E x tr a c ti o n /A n a l y s t : 08/17/99 SEE/RWW/MCH/SAL Dates of Analysis/Analyst: 08/19/99, 09/15/99 GML Date of Data Reduction/Analyst: 08/20/99,09/16/99, 11/06/00 GML/KJH Sample Data MONKEY URINE Week30 Group Dose Sample # Group 1 0.0 mg/kg/day Group 3 10 mg/kg/day 105718M I05720M 105712M 105716M Limit of Quantitation (LOQ): 0.0182 ug/mL POAA Concentration Cone. ng/mL of POAA ug/mL or % Rec. 2.73 <LOQ 0.00 <LOQ 290 0.278 46.5 0.445 NA - Not Analyzed/Not Applicable Mean POAA ug/mL RSD Std. Dev. MS/MSD RPD <LOQ NA 0.361 0.118 POAA = Periluorooctanoate C8F17COO- Date Entered/By: 08/23/99,09/16/99,09/17/99,11/07/00 GML/LAC Date Verified/ By: 09/25/00 LAC, 11/08/00 HOJ ETS-8-97.0 Excel 97 Urine Wk30 TOX-026-urine23 l-4H.xls 5/23/2001 FACT-TOX-026 Covance 6329-231 Study: Product Number(Test Substance): Matrix: Method/Revision: Analytical Equipment System Number: Instrument Software/Version: Filename: R-Squared Value: Slope: Y-Intercept: Dates of Extraction/Analyst: Dates of Analysis/Analyst: Date of Data Reduction/Analyst: Sample Data MONKEY URINE Week 32 Group Dose Sample # Group 1 0.0 mg/kg/day 105718M I05720M Group 3 I05712M 10 mg/kg/day 105716M Limit of Quantitation (LOQ): 0.0182 ug/mL 6 Month Capsule Toxicity Study with APFO in Cynomolgus Monkeys T-6889.2 (POAA) Monkey Urine ETS-8-96.0 & ETS-8-97.0 Filenames Soup 020199, Madeline 040198, Amelia 062498 MassLynx 3.2 Grp 1 See listing to the right Grp 3 See Attachments Box See Attachments See Attachments 08/17/99 SEE/RWW/MCH/SAL 08/19/99, 09/15/99 GML 08/20/99,09/16/99,11/06/00 GML/KJH POAA 081999080-81 091599019-20 99-160-1 Dilutions 1/1 1/1 Ext. Vol. = 1 since curves are extracted at the same ratio the specimens, 2 mL initial and 0.5 mL final volumes. Extraction POAA Std Vol. Correction Factor 1 0.9582 1 0.9582 1 0.9582 1 0.9582 NA = Not Analyzed/Not Applicable POAA Dilution Factor POAA Cone. ng/mL 1 4.68 1 5.14 1 164 1 74.3 POAA = Perfluorooctanoate C8F17COO Concentration of POAA ug/mL or % Rec. <LOQ <LOQ 0.157 0.0712 Mean POAA ug/mL <LOQ 0.114 Date Entered/By: Date Verified/ By: 08/23/99, 09/16/99, 11/07/00 GML/LAC 09/25/00 LAC, 11/08/00 HOJ RSD Std. Dev. MS/MSD RPD NA 0.0608 CTC_S_Q7 O U rine W k32 FACT-TOX-026 Covance 6329-231 Study: Product Number(Test Substance): Matrix: Method/Revision: Analytical Equipment System Number: Instrument Software/Version: Filename: R-Squared Value: Slope: Y-Intercept: Dates of Extraction/Analyst: Dates of Analysis/Analyst: Date of Data Reduction/Analyst: MONKEY URINE Week 32 G roup D ose Sam ple # G roup 1 0.0 mg/kg/day 105718M I05720M G roup 3 105712M 10 mg/kg/day 105716M Limit of Quantitation (LOQ): 0.0182 ug/mL 6 Month Capsule Toxicity Study with APFO in Cynomolgus Monkeys T-6889.2 (POAA) Monkey Urine ETS-8-96.0 & ETS-8-97.0 Soup 020199, Madeline 040198, Amelia 062498 MassLynx 3.2 See Attachments See Attachments See Attachments See Attachments 08/17/99 SEE/RWW/MCH/SAL 08/19/99,09/15/99 GML 08/20/99,09/16/99,11/06/00 GML/KJH Sample Data POAA C oncentration C one. of POAA ng/m L ug/m L or % Rec. 4.68 <LOQ 5.14 <LOQ 164 0.157 74.3 0.0712 NA = Not Analyzed/Not Applicable M ean POAA ug/m L RSD S td. Dev. M S/M SD R PD <LOQ NA 0.114 0.0608 POAA = Perfluorooctanoate C8F17COO- Date Entered/By: Date Verified/' By: 08/23/99, 09/16/99, 11/07/00 GML/LAC 09/25/00 LAC, 11/08/00 HOJ FTS-8-97 0 U rineW k32 FACT-TOX-026 Covance 6329-231 Study: Product Number(Test Substance): Matrix: Method/Revision: Analytical Equipment System Number: Instrument Software/Version: Filename: R-Squared Value: Slope: Y-Intercept: Dates of Extraction/Analyst: Dates of Analysis/Analyst: Date o f Data Reduction/Analyst: Sample Data MONKEY URINE Week 34 Group Sample # Dose Group 1 105718M 0.0 mg/kg/day I05720M Group 3 I05712M 10 mg/kg/day 105716M Limit of Quantitation (LOQ): 0,0182 ug/mL 6 Month Capsule Toxicity Study with APFO in Cynomolgus Monkeys T-68B9.2 (POAA) Monkey Urine ETS-8-96.0 & ETS-8-97.0 Filenames Soup 020199, Madeline 040198, Amelia 062498 MassLynx 3.2 Grp 1 See listing to the right Grp 3 See Attachments Box See Attachments See Attachments 08/17/99 SEE/RWW/MCH/SAL 08/19/99, 09/15/99 GML 08/20/99, 09/16/99, 11/06/00 GML/KJH POAA 081999084-85 091599021-22 99-160-1 Dilutions 1/1 1/1 Ext. Vol. = 1 since curves are extracted at the same ratio as the specimens, 2 mL initial and 0.5 mL final volumes. Extraction POAA Std Vol. Correction Factor 1 0.9582 1 0.9582 1 0.9582 1 0.9582 NA = Not Analyzed/Not Applicable POAA Dilution Factor POAA Cone. ng/mL 1 0.00 1 0.00 1 104 1 149 POAA = Perfluorooctanoate C8F17COO Concentration of POAA ug/mL or % Rec. <LOQ <LOQ 0.0993 0.142 Mean POAA ug/mL <LOQ 0.121 Date Entered/By: 08/23/99, 09/16/99, 11/07/00 GML/LAC Date 'verified/ By: 09/25/00 LAC, 11/08/00 HOJ RSD Std. Dev. MS/MSD RPD NA 0.0305 F.TS-R-97.0 U rineW k34 FACT-TOX-026 Covance 6329-231 Study: Product Number(Test Substance): Matrix: Method/Revision: Analytical Equipment System Number: Instrument Software/Version: Filename: R-Squared Value: Slope: Y-Intercept: Dates of Extraction/Analyst: Dates of Analysis/Analyst: Date of Data Reduction/Analyst: MONKEY URINE Week34 Group Sample # Dose Group 1 I05718M 0.0 mg/kg/day I05720M Group 3 I05712M 10 mg/kg/day 105716M Limit of Quantitation (LOQ): 0.0182 ug/mL 6 Month Capsule Toxicity Study with APFO in Cynomolgus Monkeys T-6889.2 (POAA) Monkey Urine ETS-8-96.0 & ETS-8-97.0 Soup 020199, Madeline 040198, Amelia 062498 MassLynx 3.2 See Attachments See Attachments See Attachments See Attachments 08/17/99 SEE/RWW/MCH/SAL 08/19/99,09/15/99 GML 08/20/99,09/16/99, 11/06/00 GML/KJH Sample Data POAA Concentration Cone. of POAA ng/mL ug/mL or % Rec. 0.00 <LOQ 0.00 <LOQ 104 0.0993 149 0.142 NA = Not Analyzed/Not Applicable Mean POAA ug/mL RSD Std. Dev. MS/MSD RPD <LOQ NA 0.121 0.0305 POAA = Perfluorooctanoate C8F17COO- Date Entered/By: Date Verified/ By: 08/23/99,09/16/99, 11/07/00 GML/LAC 09/25/00 LAC, 11/08/00 KOJ FT-S-Q7 0 UrineW k34 FACT-TOX-026 Covance 6329-231 Study: Product Number(Test Substance): Matrix: Method/Revision: Analytical Equipment System Number: Instrument Software/Version: Filename: R-Squared Value: Slope: Y-Intercept: Dates of Extraction/Analyst: Dates of Analysis/Analyst: Date of Data Reduction/Analyst: Sample Data MONKEY URINE Week 36 Group Sample # Dose Group 1 I05718M 0.0 mg/kg/day I05720M Group 3 105712M 10 mg/kg/day 105716M Limit of Quantitation (LOQ): 0.0182 ug/mL 6 Month Capsule Toxicity Study with APFO in Cynomolgus Monkeys T-6889.2 (POAA) Monkey Urine ETS-8-96.0 & ETS-8-97.0 Soup 020199, Madeline 040198, Amelia 062498 MassLynx 3.2 See listing to the right See Attachments Filenames Grp 1 Grp 3 Box POAA 081999086-87 091599025-26 99-160-1 Dilutions 1/1 1/1 See Attachments See Attachments 08/17/99 SEE/RWW/MCH/SAL 08/19/99,09/15/99 GML 08/20/99, 09/16/99, 11/06/00 GML/KJH Ext. Vol. = 1 since curves are extracted at the same ratio as the specimens, 2 mL initial and 0.5 mL final volumes. Extraction Vol. POAA Std Correction Factor 1 0.9582 1 0.9582 1 0.9582 1 0.9582 NA = Not Analyzed/Not Applicable A = BelowLOQ POAA Dilution Factor POAA Cone. ng/mL 1 0.00 1 18.4 1 40.1 1 64.6 POAA = Perfluorooctanoate C8F17COO` Concentration of POAA ug/mL or % Rec. <LOQ 0.0176 0.0384 0.0619 Mean POAA ug/mL 0.0117 0.0502 Date Entered/By: 08/23/99, 09/16/99, 11/07/00 GML/LAC Date Verified/ By: 09/25/00 LAC, 11/08/00 HOJ RSD Std. Dev. MS/MSD RPD A 0.00841 0.0166 FT<\-8-Q7 0 Urine W k36 FACT-TOX-026 Covance 6329-231 Study: Product Number(Test Substance): Matrix: Method/Revision: Analytical Equipment System Number: Instrument Software/Version: Filename: R-Squared Value: Slope: Y-Intercept: Dates of Extraction/Analyst: Dates of Analysis/Analyst: Date of Data Reduction/Analyst: MONKEY URINE Week 36 Group Sample # Dose Group 1 105718M 0.0 mg/kg/day I05720M Group 3 105712M 10 mg/kg/day 105716M Limit of Quantitation (LOQ): 0.0182 ug/mL 6 Month Capsule Toxicity Study with APFO in Cynomolgus Monkeys T-6889.2 (POAA) Monkey Urine ETS-8-96.0 & ETS-8-97.0 Soup 020199, Madeline 040198, Amelia 062498 MassLynx 3.2 See Attachments See Attachments See Attachments See Attachments 08/17/99 SEE/RWW/MCH/SAL 08/19/99,09/15/99 GML 08/20/99,09/16/99, 11/06/00 GML/KJH Sample Data POAA Cone. ng/mL Concentration of POAA ug/mL or % Rec. 0.00 <LOQ 18.4 0.0176 40.1 0.0384 64.6 0.0619 NA = Not Analyzed/Not Applicable A = Below LOQ Mean POAA ug/mL RSD Std. Dev. MS/MSD RPD 0.0117 A 0.00841 0.0502 0.0166 POAA = Perfluorooctanoate C8F17COO- Date Entered/By: Date Verified/ By: 08/23/99,09/16/99, 11/07/00 GML/LAC 09/25/00 LAC, 11/08/00 HOJ FTS-8-97 0 U rine W k36 FACT-TOX-026 Covance 6329-231 Study: Product Number(Test Substance): Matrix: Method/Revision: Analytical Equipment System Number: Instrument Software/Version: Filename: R-Squared Value: Slope: Y-Intercept: Dates of Extraction/Analyst: Dates o f Analysis/Analyst: Date of Data Reduction/Analyst: Sample Data MONKEY URINE Week 38 Group Dose Sample # Group 1 105718M 0.0 mg/kg/day 105720M Group 3 I05712M 10 mg/kg/day 105716M Limit of Quantitation (LOQ): 0.0182 ug/mL 6 Month Capsule Toxicity Study with APFO in Cynomolgus Monkeys T-6889.2 (POAA) Monkey Urine ETS-8-96.0 & ETS-8-97.0 Soup 020199, Madeline 040198, Amelia 062498 MassLynx 3.2 See listing to the right See Attachments Filenames Grp 1 Grp 3 Box POAA 081999090-91 091599027-28 99-160-1 Dilutions 1/1 1/1 See Attachments See Attachments 08/17/99 SEE/RWW/MCH/SAL 08/19/99, 09/15/99 GML 08/20/99,09/16/99,11/06/00 GML/KJH Ext. Vol. = 1 since curves are extracted at the same ratio as the specimens, 2 mL initial and 0.5 mL final volumes. Extraction Vol. POAA Std Correction Factor 1 0.9582 1 0.9582 1 0.9582 1 0.9582 NA = Not Analyzed/Not Applicable POAA Dilution Factor POAA Cone. ng/mL 1 0.00 1 0.00 1 37.2 1 22.1 POAA = Perfluorooctanoate C8F17COO Concentration of POAA ug/mL or % Rec. <LOQ <LOQ 0.0357 0.0212 Mean POAA ug/mL <LOQ 0.0284 Date Entered/By: 08/23/99, 09/16/99, 11/07/00 GML/LAC Date Verified/' By: 09/25/00 LAC, i 1/08/00 HOJ RSD Std. Dev. MS/MSD RPD NA 0.0102 ETS-8-97.0 UrineWk38 FACT-TOX-026 Covance 6329-231 Study: Product Number(Test Substance): Matrix: Method/Revision: Analytical Equipment System Number: Instrument Software/Version: Filename: R-Squared Value: Slope: Y-Intercept: Dates o f Extraction/Analyst: Dates o f Analysis/Analyst: Date of Data Reduction/Analyst: MONKEY URINE Week 38 Group Sample # Dose Group 1 105718M 0.0 mg/kg/day I05720M Group 3 I05712M 10 mg/kg/day I05716M Limit of Quantitation (LOQ): 0.0182 ug/mL 6 Month Capsule Toxicity Study with APFO in Cynomolgus Monkeys T-6889.2 (POAA) Monkey Urine ETS-8-96.0 & ETS-8-97.0 Soup 020199, Madeline 040198, Amelia 062498 MassLynx 3.2 See Attachments See Attachments See Attachments See Attachments 08/17/99 SEE/RWW/MCH/SAL 08/19/99,09/15/99 GML 08/20/99,09/16/99, 11/06/00 GML/KJH Sample Data POAA Cone. ng/mL 0.00 0.00 37.2 22.1 Concentration of POAA ug/mL or % Rec. <LOQ <LOQ 0.0357 0.0212 Mean POAA ug/mL RSD Std. Dev. MS/MSD RPD <LOQ NA 0.0284 0.0102 POAA = Perfluorooctanoic Acid Anion C8F17COO Date Entered/By: rL\/_aic. -XvTc-.r.:uirit c- uj // ory, :.. 08/23/99, 09/16/99, 11/07/00 GML/LAC aa M r /p a \jy /z.j/w tl /*\ cr>, ni i /iuaoo//awa tntu<~jt F.TS-8-97.0 U rineW k38 FACT-TOX-026 Covance 6329-231 Study: Product Number(Test Substance): Matrix: Method/Revision: Analytical Equipment System Number: Instrument Software/Version: Filename: R-Squared Value: Slope: Y-Intercept: Dates of Extraction/Analyst: Dates of Analysis/Analyst: Date of Data Reduction/Analyst: Sample Data MONKEY URINE Week 40 Group Dose Sample # Group 1 I05718M 0.0 mg/kg/day I05720M Group 3 105712M 10 mg/kg/day 105716M Limit of Quantitation (LOQ): 0.0182 ug/mL 6 Month Capsule Toxicity Study with APFO in Cynomolgus Monkeys T-6889.2 (POAA) Monkey Urine ETS-8-96.0 & ETS-8-97.0 Soup 020199, Madeline 040198, Amelia 062498 MassLynx 3.2 See listing to the right See Attachments Filenames Grp 1 Grp 3 Box POAA 081999092-93 091599031-32 99-160-1 Dilutions 1/1 1/1 See Attachments See Attachments 08/17/99 SEE/RWW/MCH/SAL 08/19/99,09/15/99 GML 08/20/99,09/16/99, 11/06/00 GML/KJH Ext. Vol. = 1 since curves are extracted at the same ratio as the specimens, 2 mL initial and 0.5 mL final volumes. Extraction Vol. POAA Std Correction Factor i 0.9582 i 0.9582 i 0.9582 i 0.9582 NA = Not Analyzed/Not Applicable POAA Dilution Factor POAA Cone. ng/mL 1 0.00 1 0.00 1 34.0 1 17.7 POAA = Perfluorooctanoate C8F17COO Concentration of POAA ug/mL or % Rec. <LOQ <LOQ 0.0325 <LOQ Mean POAA ug/mL <LOQ 0.0254 Date Entered/By: Date 'verified/ By: 08/23/99,09/16/99, 11/07/00 GML/LAC 09/25/00 LAC, 11/08/00 HOJ RSD Std. Dev. NA 0.0101 F.TS-8-97.0 Urine W k40 FACT-TOX-026 Covance 6329-231 Study: Product Number(Test Substance): Matrix: Method/Revision: Analytical Equipment System Number: Instrument Software/Version: Filename: R-Squared Value: Slope: Y-Intercept: Dates of Extraction/Analyst: Dates of Analysis/Analyst: Date of Data Reduction/Analyst: MONKEY URINE Week40 Group Sample # Dose Group 1 105718M 0.0 mg/kg/day I05720M Group 3 I05712M 10 mg/kg/day 105716M Limit of Quantitation (LOQ): 0.0182 ug/mL 6 Month Capsule Toxicity Study with APFO in Cynomolgus Monkeys T-6889.2 (POAA) Monkey Urine ETS-8-96.0 & ETS-8-97.0 Soup 020199, Madeline 040198, Amelia 062498 MassLynx 3.2 See Attachments See Attachments See Attachments See Attachments 08/17/99 SEE/RWW/MCH/SAL 08/19/99,09/15/99 GML 08/20/99,09/16/99, 11/06/00 GML/KJH Sample Data POAA Cone. ng/mL Concentration of POAA ug/mL or % Ree. 0.00 <LOQ 0.00 <LOQ 34.0 0.0325 17.7 <LOQ NA = Not Analyzed/Not Applicable Mean POAA ug/mL RSD Std. Dev. MS/MSD RPD <LOQ NA 0.0254 0.010 POAA = Perfluorooctanoate C8F17COO- Date Entered/By: Date Verified/ By: 08/23/99,09/16/99, 11/07/00 GML/LAC 09/25/00 LAC, 11/08/00 HOJ CTC.O.7 n Urine W k40 ETS-8-97.0 FACT-TOX-026 Covance 6329-231 Urine QC Summary - TOX026 08/04/1999 08/05/1999 08/17/1999 08/20/1999 09/12/1999 MKU08049 MS-1 MKU08049 MSD-1 MKU08049 MS-2 MKU08049 MSD-2 MKU08059 MS MKU08059 MSD MKU08179 MS 1-1 MKU08179 MSD 1-1 MKU08179 MS 1-2 MKU08179 MSD 1-2 MKU08179 MS 1-3 MKU08179 MSD 1-3 MKU08179 MS 1-4 MKU08179 MSD 1-4 MKU08209 MS 1-1 MKU08209 MSD 1-1 MKU08209 MS 1-2 MKU08209 MSD 1-2 MKU09219 MS-1 MKU09219 MSD-1 PFOS 133% 92% 95% 70% 91% 87% 92% 104% 105% 101% 74% 74% 73% 73% 66% 63% 90% 89% 92% 95% NA =Not Applicable NR=Not Reported ^outlier by Grubbs test at 0.05 confidence . Average Standard Deviation Grubbs outlier included? Number of spikes 88% Used in Final Report 17% Yes 20 Average Standard Deviation Grubbs outlier included? Number of spikes 86% Data not used 13% No 19 QC Summary 3M Medical Department Study: T-6889.3 Analytical Report: FACT TOX-026 LRN-U2782 Appendix G: Example Calculations Formula Used for Sera Analyses in Study FACT TOX-026 AR (ng/mL) x DF x SC x FV (mL) x 1.0 pg = Reported Concentration pg/mL) EV (mL) 1000 ng Calculation Used for Group 2, Week 27, Animal ID 105702M 500 ng/mL x 100 x 0.9582 x 1 mL x 1.0 pg = 47.9 pg/mL 1 mL 1000 ng AR-- Analytical result from MassLynx summary DF-- Dilution factor SC--POAA salt correction constant (0.9582) FV--Final extract volume (1.0 mL unless otherwise noted) EV--Volume of sera extracted Formula Used for Liver Analyses in Study FACT TOX-026 AR (ng/g) x 9 curve (1) x SC x DF x 1.0 mg = [POAA] sample (pg/g) 9 sample 1000 ng (1) 9 curve is assumed to be: 1 g liver 5m L H 20 Calculation Used for Group 2, Week 27, Animal ID 105702M 160 ng/g x l g / 5 m L x 0.9582 x 100 x 1.0 pg = 15.2 pg/g 1.0104 g /5 mL Ong AR-- Analytical result from MassLynx summary 9 curve--Density of the liver standard curve, assumed to be lg liver/ 5 ml water 9 sample--Density of the liver sample (g sample/ 5 mL H20 ) SC--POAA salt correction constant (0.9582) DF-- Dilution factor 3M Environmental Laboratory Page 38 3M Medical Department Study: T-6889.3 Analytical Report: FACT TOX-026 LRN-U2782 Formula Used for Urine Analyses in Study FACT TOX-026 AR (ng/mL) x DF x SC x FV (mL) x 1.0 pg = Reported Concentration pg/mL) EV (mL) 1000 ng Calculation Used for Group 2, Week 26, Animal ID 105702M 38.6 ng/mL x 1000 x 0.9582 x 1 mL x 1.0 pg = 37.0 pg/mL 1 mL 1000 ng AR--Analytical result from MassLynx summary DF--Dilution factor SC--POAA salt correction constant (0.9582) ER--Extraction ratio (equal to 1.0 unless otherwise noted) FY--Final extract volume (1.0 mL unless otherwise noted) EV--Volume of urine extracted 3M Environmental Laboratory Page 39 3M Medical Department Study: T-6889.3 Analytical Report: FACT TOX-026 LRN-U2782 Appendix H: Contract Lab Report This appendix includes the following contract laboratory report: Centre Analytical Laboratories, 26-Week Capsule Toxicity Study with Ammonium Perfluorooctanoate (APFO/POAA) in Cynomolgus Monkeys, (110 pages) 3M Environmental Laboratory Page 40 ANALYTICAL REPORT STUDY TITLE 26-Week Capsule Toxicity Study with Ammonium Periluorooctanoate (APFO/POAA) in Cynomolgus Monkeys DATA REQUIREMENTS Analytical Method Requirements STUDY DIRECTOR Paul Lieder, 3M !6-^JU ir1mdL~-\ L~ PRINCIPAL INVESTIGATOR Emily R. Stauffer, Centre ANALYTICAL REPORT COMPLETION DATE April 2, 2001 PERFORMING LABORATORY / TESTING FACILITY Centre Analytical Laboratories, Inc. (Centre) 3048 Research Drive State College, PA 16801 Phone: 814-231-8032 STUDY SPONSOR 3M Toxicology Services - Medical Department 3M Center, Building 220-2E-02 P.O. Box 33220 St. Paul, MN 55133-3220 PROTECT IDENTIFICATION Sponsor Protocol Number: FACT-TOX-026 Centre Study Number: 023-011 Total Pages: 110 Centre Study No.: 023-011 Sponsor Protocol No: FACT-TOX-026 GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT Centre Study Number 023-011, entitled "26-Week Capsule Toxicity Study with Ammonium Perfluorooctanoate (APFO/POAA) in Cynomolgus Monkeys," conducted for 3M Toxicology Services - Medical Department, was performed in compliance with US EPA Good Laboratory Practice Standards (40 CFR 792) by Centre Analytical Laboratories, Inc. with the following exceptions: 1. The reference substances (analytical standards) used in this study (PFOA/POAA and THPFOS) have not been characterized under GLP's 160.105 (a). 2. In Set 102000AD (extracted 10/20/00 and analyzed 10/23/00), dilutions were performed on samples and not documented on the sample extraction and analysis tracking sheet as required by 792.130 (e). -Emrly R.'Stauffer PrincipaHnvestigator Centre Analytical Laboratories, Inc. Paul Lieder, Ph.D., DABT Study Director 3M Toxicology Services Centre Analytical Laboratories, Inc. Date Date Page 2 of 110 Centre Study No.: 023-011 Sponsor Protocol No: FACT-TOX-026 QUALITY ASSURANCE STATEMENT Centre Analytical Laboratories' Quality Assurance Unit reviewed Centre Study Number 023-011 entitled, "26-Week Capsule Toxicity Study with Ammonium Perfluorooctanoate (APFO/POAA) in Cynomolgus Monkeys". All phases were reviewed for conduct according to Centre Analytical Laboratories' Standard Operating Procedures, the Study Protocol, and all applicable Good Laboratory Practice Standards. All findings were reported to the Study Director and to management. Phase 1. Protocol Review Date Inspected 10/5/00 Date Reported to Centre Management 11/2/00 Date Reported to Study Director and Sponsor Management 11/17/00 2. Extraction 10/12/00 11/2/00 11/17/00 3. Raw Data Review 11/2-20/00 12/21/00 12/28/00 4. Draft Report Review 12/14,15,18/00 12/21/00 12/28/00 5. Final Report Review 3/15/01 3/19/01 3/19/01 _______ --j Naomi Lovallo Sr. Quality Assurance Auditor ______ Y/a, [ o ( D ate Centre Analytical Laboratories, Inc. Page 3 of 110 Centre Study No.: 023-011 Sponsor Protocol No: FACT-TOX-026 CERTIFICATION OF AUTHENTICITY This report, for Centre Study Number 023-011 entitled, "26-Week Capsule Toxicity Study with Ammonium Perfluorooctanoate (APFO/POAA) in Cynomolgus Monkeys", is a true and complete representation of the raw data for the study. Submitted by: Centre Analytical Laboratories, Inc. 3048 Research Drive State College, PA 16801 (814) 231-8032 Principal Investigator, Centre: feafily'R. Stauffer Scientist ^ Centre Analytical Laboratories, Inc. 01 Centre Analytical Laboratories, Inc. Facility Management: Laboratory Manager Centre Analytical Laboratories, Inc. D ate Study Director, 3M: fiLttf/PUv________ Paul Lieder, Ph.D., DABT 3M Toxicology Services M Date m Centre Analytical Laboratories, Inc. Page 4 of 110 Centre Study No.: 023-011 Sponsor Protocol No: FACT-TOX-026 STUDY IDENTIFICATION 26-Week Capsule Toxicity Study with Ammonium Perfluorooctanoate (APFO/POAA) in Cynomolgus Monkeys SPONSOR PROTOCOL NUMBER: FACT-TOX-026 TYPE OF STUDY: Toxicity TEST SYSTEM: Cynomolgus monkeys TEST MATERIAL: Ammonium perfluorooctanoate (APFO/POAA) SPONSOR: 3M Toxicology Services - Medical Department 3M Center, Building 220-2E-02 St. Paul, MN 55133-3320 STUDY DIRECTOR: From 11/12/98 to 2/10/00: Kris Hansen, Ph.D. 3M Environmental Technology and Safety Services Phone: (651)778-6018 From 2/10/00 to Present: Paul H. Lieder, Ph.D., DABT 3M Toxicology Services Phone: (651) 737-2678 TESTING FACILITY: Centre Analytical Laboratories, Inc. 3048 Research Drive State College, PA 16801 PRINCIPAL INVESTIGATOR From 9/19/00 to 10/23/00: Enaksha Wickremesinhe Ph.D., Centre Analytical Laboratories, Inc. Phone: (814) 231-8032 From 10/23/00 to Present: Emily R. Stauffer Centre Analytical Laboratories, Inc. Phone: (814) 231-8032 ANALYTICAL PHASE TIMETABLE: Study Initiation Date: Analytical Start Date: Analytical Termination Date: 11/12/98 09/27/00 10/26/00 Centre Analytical Laboratories, Inc. Page 5 of 110 Centre Study No.: 023-011 Sponsor Protocol No: FACT-TOX-026 PROJECT PERSONNEL The Study Director for this project was Paul Lieder at 3M Toxicology Services and the Principal Investigator for this project at Centre Analytical Laboratories, Inc. was Emily Stauffer. The following personnel from Centre Analytical Laboratories, Inc., were associated with various analytical phases of the study: Name Enaksha Wickremesinhe Emily Stauffer Karen Smith David Bell Tiffany Proctor Angela Morgan Ed Carnes Rickey Keller Lawrence Ord David Shirk Title Group/Team Leader Scientist Scientist Scientist Technician Technician Sample Custodian Sample Custodian Sample Custodian Sam ple Custodian Centre Analytical Laboratories, Inc. Page 6 of 110 Centre Study No.: 023-011 Sponsor Protocol No: FACT-TOX-026 TABLE OF CONTENTS Page TITLE PAGE..........................................................................................................................1 GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT..............................2 QUALITY ASSURANCE STATEMENT............................................................................ 3 CERTIFICATION OF AUTHENTICITY...... ...................................................................... 4 STUDY IDENTIFICATION..................................................................................................5 PROJECT PERSONNEL.......................................................................................................6 TABLE OF CONTENTS.......................................................................................................7 LIST OF TABLES................................................................................................................. 8 LIST OF FIGURES................................................................................................................9 LIST OF APPENDICES......................................................................................................10 1.0 SUMMARY.................................................................................................................. 11 2.0 OBJECTIVE.................................................................................................................. 11 3.0 INTRODUCTION.........................................................................................................11 4.0 TEST SYSTEM....................................... 11 5.0 REFERENCE MATERIAL..........................................................................................12 6.0 EXPERIMENTAL DESIGN.........................................................................................13 7.0 DESCRIPTION OF ANALYTICAL METHOD.........................................................13 7.1 Extraction Procedure...................................................................................................13 7.2 Preparation of Standards and Fortification Solutions................................................13 7.3 Chromatography.......................................................................................................... 14 7.4 Instrument Sensitivity.................................................................................................14 7.5 Description of Instrument and Operating Conditions................................................15 7.6 Quantitation and Example Calculation...................................................................... 16 8.0 RESULTS AND DISCUSSION....................................................................................18 9.0 CIRCUMSTANCES THAT MAY HAVE AFFECTED THE DATA.......................18 10.0 RETENTION OF DATA AND SAMPLES............................................................... 18 11.0 TABLES...................................................................................................................... 19 12.0 FIGURES....................................................................................................................38 13.0 APPENDICES............................................................................................................ 46 Centre Analytical Laboratories, Inc. Page 7 of 110 Centre Study No.: 023-011 Sponsor Protocol No: FACT-TOX-026 LIST OF TABLES Page Table I. Summary of POAA residues in Matrix Blanks and Matrix Zero Blanks.......... 20 Table II. Summary of POAA recoveries in Fortified Samples......................................... 22 Table HI. Summary of POAA residues in Week 2 .............................................................23 Table IV. Summary of POAA residues in Week 4 ............................................................24 Table V. Summary of POAA residues in Week 6..............................................................25 Table VI. Summary of POAA residues in Week 8 ............................................................26 Table VII. Summary of POAA residues in Week 1 0 ........................................................ 27 Table VIII. Summary of POAA residues in Week 1 2 .......................................................28 Table IX. Summary of POAA residues in Week 1 4 .........................................................29 Table X. Summary of POAA residues in Week 16...........................................................30 Table XT. Summary of POAA residues in Week 1 8 .........................................................31 Table XU. Summary of POAA residues in Week 2 0 ....................................................... 32 Table XHI. Summary of POAA residues in Week 2 2 ........................................................33 Table XTV. Summary of POAA residues in Week 24........................................................34 Table XV. Summary of POAA residues in Week 2 6 ........................................................35 Table XVI. Summary of POAA residues in Week 2 8 -3 4................................................ 36 Table XVH Summary of POAA residues in Weeks 3 6 -4 0 ............................................. 37 Centre Analytical Laboratories, Inc. Page 8 of 110 Centre Study No.: 023-011 Sponsor Protocol No: FACT-TOX-026 Figure 1. LIST OF FIGURES Page Typical Calibration Curve for POAA............................................................. 39 Figure 2. Typical Mean Response Factor for THPFOS.................................................40 Figure 3. Figure 4. Chromatogram Representing a 5 ng/mL extracted standard for POAA and 250 ng/mL extracted standard for THPFOS...................................................41 Chromatogram Representing a 125 ng/mL extracted standard for POAA and 250 ng/mL extracted standard for THPFOS..................................................42 Figure 5. Chromatogram Representing Control Monkey Feces for POAA and THPFOS (Centre ID: 0009684 Blank A, Set: 101200A).............................................. 43 Figure 6. Chromatogram Representing Control Monkey Feces Fortified with 50 ng/g of POAA and 500 ng/g of THPFOS (Centre ID: 0008545 Spk A, Set: 101200A)................................................. 44 Figure 7. Chromatogram of Monkey Feces Sample from Grp 2 in Week 12 (Centre ID: 0008548, Set: 101200A )............................................................................... 45 Centre Analytical Laboratories, Inc. Page 9 of 110 Centre Study No.: 023-011 Sponsor Protocol No: FACT-TOX-026 LIST OF APPENDICES Page Appendix A Study Protocol FACT-TOX-026 (Centre Study No. 023-011) entitled 26-Week Capsule Toxicity Study with Ammonium Perfluorooctanoate (APFO/POAA) in Cynomolgus Monkeys and Amendments and Deviations................................................................................................... 47 Appendix B Determination of Fluorochemical Residues in Monkey/Rat Feces by LC/MS/MS (Revision 2) Method #00M-023-003, revision 2 and Deviations and Modifications..............................................................84 Centre Analytical Laboratories, Inc Page 10 of 110 Centre Study No.: 023-011 Sponsor Protocol No: FACT-TOX-026 1.0 SUMMARY The purpose of this study was to analyze residues of perfluorooctanoate (APFO/POAA) in the feces of cynomolgus monkeys as specified in 3M Protocol FACT-TOX-026. The analytical method used for this study was entitled, "Determination of Fluorochemical Residues in Monkey/Rat Feces by LC/MS/MS, revision 2" (Centre method number: 00M-023-003, revision 2). Besides APFO/POAA (referred to as POAA hereafter), all of the samples were analyzed for residues of PFOS, PFOSA, PFOSAA, M570, M556, EtFOSE-OH, and PFOSEA. However, as requested by the Principal Analytical Investigator at 3M, only the results for POAA will be detailed in this report. The limit of quantification for POAA in monkey feces was 10 ppb. Residues ranging from non-detected levels to 242 pg/g were found in the monkey feces samples. Fortification recoveries ranged from 56-170% with an average of 117% and relative standard deviation of 19%. 2.0 OBJECTIVE The objective of this study was to determine levels of perfluorooctanoate (APFO/POAA) in specimens of feces of monkeys using the analytical method entitled "Determination of Fluorochemical Residues in Monkey/Rat Feces by LC/MS/MS, revision 2." 3.0 INTRODUCTION The study was initiated on November 12, 1998, when the study director signed the protocol FA C T-TO X -026. The complete protocol and am endm ents can be found in Appendix A. The analytical start date was September 27, 2000, and the analytical termination date was October 26, 2000. This report details the results of the residues of POAA detected in monkey feces, using the analytical method entitled, "Determination of Fluorochemical Residues in Monkey/Rat Feces by LC/MS/MS (revision 2)." Complete details of the analytical methodology can be found in Appendix B. 4.0 TEST SYSTEM The 300 monkey feces samples analyzed in this study were received frozen on dry ice from 3M Environmental Laboratory St. Paul, MN on August 29, 2000 and stored frozen Centre Analytical Laboratories, Inc. Page 11 of 110 Centre Study No.: 023-011 Sponsor Protocol No: FACT-TOX-026 upon receipt. The samples were then logged in on August 30, 2000 by Centre personnel and transferred to different frozen storage locale. The control rat feces used for standards and blanks was purchased from Lampire Biological Laboratories, Inc., Pipersville, PA and received at Centre chilled on blue ice on February 29, 2000 and October 10, 2000, logged in by Centre personnel and placed in frozen storage (<-10C). Sample login and chain of custody information can be found in the raw data package associated with this study. Storage records will be kept at Centre Analytical Laboratories, Inc. and a true copy of the storage records can be found in the raw data package associated with this study. 5.0 REFERENCE MATERIAL The analytical standard POAA (as the ammonium salt) was received at Centre on July 6, 2000 and the surrogate standard THPFOS was received on July 17, 2000 from 3M Environmental Technology and Services. Characterization of the reference material POAA will be the responsibility of the sponsor. The available information for the reference materials is listed below. The reference materials were stored at room temperature. Compound POAA THPFOS Centre Control No. 00-023-048 00-023-053 Batch No. 332 53406 Puritv (%) Expiration Date TBD 07/06/01 TBD 01/01/10 Molecular structures of POAA and THPFOS are given below. POAA Chemical Name Molecular weight C7F15COO' = = Perfluorooctanoate 413 Note: The neutral molecule and standard form which POAA (anion) is derived from is ammonium perfluoroctanoate [C7F15COONH4], molecular weight 431. THPFOS Chemical Name: 4-H, perfluorooctane sulfonic acid Molecular weight: 428 1-H, 1-H, 2-H, 2-H, C8F l3S 0 3H Centre Analytical Laboratories, Inc. Page 12 of 110 Centre Study No.: 023-011 Sponsor Protocol No: FACT-TOX-026 6.0 EXPERIMENTAL DESIGN Samples were extracted according to sampling day. Each set of samples contained two matrix blanks, two matrix blanks spiked with the surrogate standard only (zero blank), two matrix samples fortified with known amounts of POAA and with the surrogate standard, and 12-22 samples fortified with the surrogate standard. The extracts were analyzed by LC/MS/MS. Samples with residues outside the linear range of the calibration curve were diluted and re-analyzed. 7.0 DESCRIPTION OF ANALYTICAL METHOD Analytical method entitled "Determination of Fluorochemical Residues in Monkey/Rat Feces by LC/MS/MS (Revision 2)" was used for this study. 7.1 Extraction Procedure One gram 0.05 of sample was weighed into 20 mL polyethylene scintillation vials and fortified (if necessary) using disposable micropipettes. Ten mL of acetonitrile was added to the vial, capped tightly, and placed on a wrist-action shaker for ~ 30 min. The samples were filtered through a glass acrodisc filter. The filtered extract was passed through a conditioned carbon SPE column and collected. The columns were then eluted with ~10 mL acetonitrile followed by ~20 mL 90:10 acetonitrile:2% ascorbic acid in methanol. The combined extracts were evaporated down to almost dryness with a rotary evaporator and then re-constituted with methanol, making final volume 2 mL. The samples were analyzed using electrospray LC/MS/MS. 7.2 Preparation of Standards and Fortification Solutions Standard solutions were prepared on August 15, 2000, September 18, 2000 and September 26-27, 2000 as specified in Centre Analytical Laboratories' analytical method entitled, "Determination of Fluorochemical Residues in Monkey/Rat Feces by LC/MS/MS (Revision 2)." Individual stock standard solution of EtFOSE-OH, PFOSAA, M570, M556, PFOSEA, PFOS, PFOSA, and POAA was prepared at a concentration of 100 pg/mL by dissolving 10 mg of the standard (corrected for purity and salt content where appropriate) in methanol. From this solution, a mixed 10 pg/mL fortification standard solution was prepared by taking 10 mL of the each stock and bringing the volume up to 100 mL with methanol. Also, a stock standard of THPFOS was prepared at 100 pg/mL by dissolving 10 mg of the standard in methanol. An individual 10 pg/mL solution of THPFOS was prepared in the same fashion described above. Centre Analytical Laboratories, Inc. Page 13 of 110 Centre Study No.: 023-011 Sponsor Protocol No: FACT-TOX-026 A mixed 2.5 pg/mL fortification standard was prepared by taking 25 mL of the 10 pg/mL mixed fortification solution and bringing the volume up to 100 mL with methanol. An individual fortification solution of THPFOS was prepared in the same manner. A 0.5 pg/mL mixed standard was prepared by taking 20 mL of the 2.5 pg/mL mixed standard and bringing to 100 mL with methanol. To make the 0.1 pg/mL mixed fortification standard, 20 mL of the 0.5 pg/mL mixed standard was brought to 100 mL with methanol. Calibration standards were extracted according to the same procedure as the samples. Standards were fortified prior to extraction according to the following table: Cone, of Fortification Weight of Mixed Volume (pL) Control Fortification Sample (g) Solution (pg/ml) 0.05 0 . 1 100 1 . 0 0 . 1 200 1 . 0 0.5 100 1 .0 0.5 200 1 .0 2.5 100 1 .0 2.5 200 1 .0 * 2.5 pg/ml THPFOS fortification solution. Fort. Level of Voi. of Extracted Surrogate Calibration Standard* Standard added (pL) (ppb) 10 200 20 200 50 200 100 200 250 200 500 200 The stock standard solution and all fortification and calibration standard solutions were stored in a refrigerator (4 2C) when not in use. Documentation of standard preparation and extraction can be found in the raw data associated with this report. 7.3 Chromatography Quantification of POAA and THPFOS was accomplished by LC/MS/MS analysis using electrospray LC/MS/MS. The retention times of POAA and THPFOS were - 5 . 0 min. and - 5.0 min., respectively, with no significant interfering peaks in the control matrices corresponding to either of the analyte retention times. 7.4 Instrument Sensitivity The smallest standard amount injected during the chromatographic run was equivalent to 5 ng/mL of POAA and 250 ng/mL of THPFOS in the monkey feces matrix. Centre Analytical Laboratories, Inc. Page 14 of 110 Centre Study No.: 023-011 Sponsor Protocol No: FACT-TOX-026 7.5 Description of Instrument and Operating Conditions A Micromass Quattro Ultima LC/MS/MS coupled to a Hewlet Packard HPLC system was used. Data acquisition and processing were performed using Masslynx 3.4 software. Detailed operating conditions are listed below: Instrument: Micromass Quattro Ultima ELECTROSPRAY ION SOURCE: Capillary: 3.0 kV Hexapole 2: 0.3 V Hexapole 1: 0.1 V Source Block Temp.: 100C Aperture 1: 0.2 V Desolvation Temp.: 350C ANALYZER: LM Res 1: 10.5 V H M R es 1: 10.5 V E n erg y 1: 1.0 V Entrance: -2 V Exit: 2 V LM Res 2: 13.0 V HM Res 2: 13.0 V E n e rg y 2: 2 .0 V Multiplier: 6 50 V GAS FLOWS AND PRESSURE: Desolvation N2 R o w Rate: ~ 650 L/hr Nebuliser N2 R ow Rate: - 1 5 0 L/hr Gas Cell Pressure: -0 .0 0 3 mbar Computer: COMPAQ Professional Workstation AP200 Software: M icrosoft Windows NT: Version 4 Build 1381: Service Pack 5 Micromass Limited: Masslynx 3.4 Build 004 HPLC Equipment: Hewlett Packard (H P) Series 1100 HP Binary (or Quaternary) Pump HP Autosampler HP Vacuum Degasser HP Column Oven HPLC Column: Column Temperature: Mobile Phase (A ) : Mobile Phase ( B ) : Genesis C-8, 5 cm x 2.1 mm i.d. x 4 p 35C 2 mM Ammonium Acetate in Type I W ater Methanol Gradient: Injected Volume: Time (min) 0.0 0.4 1.0 7.0 7.5 10.5 1 1 .0 14.5 15.0 10 pL %A 60.0 60.0 1 0 .0 1 0 .0 0.0 0.0 60.0 60.0 60.0 % B Flow Rate (mL/min) 40.0 0.3 40.0 0.3 90.0 0.3 90.0 0.3 1 0 0 .0 0.3 1 0 0 .0 0.4 40.0 0.4 40.0 0.4 40.0 0.3 Centre Analytical Laboratories, Inc. Page 15 of 110 Ions m onitored: Analyte POAA THPFOS Transition Monitored 413 -->369 4 2 7 -* 80 Centre Study No.: 023-011 Sponsor Protocol No: FACT-TOX-026 Dwell (secs) Coll Energy (eV) Cone (V ) 0.1 10 30 0.1 35 34 7.6 Quantitation and Example Calculation Ten microliters of sample or extracted calibration standard was injected into the LC/MS/MS. The peak area was measured and the standard curve was generated (using 1/x weighted linear regression) by Masslynx software using at least six concentrations of standards. The surrogate standard, THPFOS, was only used to monitor the efficiency of the extraction procedure and was not used for quantitation of POAA. The residue concentration in monkey feces was determined using the following equations: Equations 1 and 2 were used to calculate the amount of analyte found (in ppb, based on peak area) using the standard curve generated by the Masslynx software program. Equation 1: Analyte found (ng/mL) = (Peak area - intercept) slope Equation 2: Analyte found (pg/g) = analyte found (ng/mL) x final vol. (mL) x DF x 1( pg) sample wt:. (g) x 1000 (ng) where DF = dilution factor. For samples fortified with known amounts of analytes prior to extraction, use Equation 3 to calculate the percent recovery. E qu atio n 3: Recovery (% ) = ((anal, found (ng/mL) - (anal, found in corresponding sample (ng/mL)/DF) x FV (mL) x DF) x 100 amount added (ng) An example of a calculation using an actual sample analyzed with extracted standards follows: Monkey feces sample Centre ID 0008434 Spk A (Set: 100200A), fortified with 50 ng of POAA. Where: peak area = 53080 intercept = 3141.11 slope = 1928.40 dilution factor =1 ng added (fort level) = 50 Centre Analytical Laboratories, Inc. Page 16 of 110 final vol. sample wt. Centre Study No.: 023-011 Sponsor Protocol No: FACT-TOX-026 = 2 mL = 0.97 g From equation 1: Analyte found (ng/mL) = f53080- 3141.111 1928.40 = 25.9 ng/mL From equation 2: Analyte found (pg/g) = 25.9 ng/mL x 2 mL x 1 x 1 pgl 0.97 g x 1000 ng = 0.0534 pg/g From equation 3: % Recovery = (25.9 ng/mL x 2 mL x 11 x 100 50 ng = 104% Note: This example calculation was done using rounded numbers, and therefore may be slightly different from the values shown in the RAW DATA. The amount of surrogate standard THPFOS found was calculated using the following equation: Analyte found (ng/mL): peak area mean response factor Other statistical methods used in analyzing this data were: (*,- - x ) 2 Standard Deviation = Mean = x = n Relative Standard Deviation (RSD or Coefficient o f Variation (C V )) = Standard Deviation x 100% Mean Centre Analytical Laboratories, Inc. Page 17 of 110 Centre Study No.: 023-011 Sponsor Protocol No: FACT-TOX-026 8.0 RESULTS AND DISCUSSION Although, the majority of the control samples did not contain significant interferences, a couple of samples did contain residues that were comparable to control group levels. A summary of residues found in all of the matrix blanks and matrix zero blanks is detailed in Table I. Fortification recoveries ranged from 56-170% with an average of 117% and relative standard deviation of 19% (n = 30). A summary of all of the fortification recoveries can be found in Table II. Residues of perfluorooctanoate ranging from non-detected levels to 242 pg/g were found in the monkey feces samples. The residues found in all of the samples plus the averages and standard deviations for each group at each interval are detailed in Tables III-XVII. It was established that ion suppression of the surrogate standard occurred in samples that contained high residues of perfluorooctanoate. Samples were diluted prior to the initial analysis to address the ion suppression of the surrogate standard. Assuming that matrix spike studies form a suitable indication of endogenous analyte recovery, the data detailed in this report can be considered accurate to within one standard deviation of the average fortified sample recovery. The average fortified sample recovery was 117% with a standard deviation of 22%. Typical calibration curves and chromatograms representing standards, controls, fortifications, and samples are depicted in Figures 1-7. 9.0 CIRCUMSTANCES THAT MAY HAVE AFFECTED THE DATA There are no circumstances that may have affected the quality or integrity of the data presented in this report. 10.0 RETENTION OF DATA AND SAMPLES When the final report is complete, all original study-specific paper data generated by Centre Analytical Laboratories, Inc. will be shipped to the sponsor. This does not include facility-specific raw data such as instrument logs, however exact copies of temperature logs will be submitted. Exact copies of all raw data, as well as a signed copy of the final analytical report and all original facility-specific raw data, will be retained in the Centre Analytical Laboratories, Inc. archives for the period of time specified in 40 CFR 792. Retained samples of reference substances are archived by the sponsor. Centre Analytical Laboratories, Inc. Page 18 of 110 Centre Study No.: 023-011 Sponsor Protocol No: FACT-TOX-026 11.0 TABLES Centre Analytical Laboratories, Inc. Page 19 of 110 Centre Study No.: 023-011 Sponsor Protocol No: FACT-TOX-026 Table I. Summary of POAA residues in Matrix Blanks and Matrix Zero Blanks ND = Not Detected and NQ = Not Quantifiable Sponsor ID na na na na na na na na na na na na na na na na na na na na na na na na na na na na na na na na na na na na Centre ID 0001345-48 Blank A 0001345-48 Blank B 0001345-48 Zero Blank C 0001345-48 Zero Blank D 0001345-48 Blank A 0001345-48 Blank B 0001345-48 Zero Blank C 0001345-48 Zero Blank D 0001344 Blank A 0001344 Blank B 0001344 Zero Blank C 0001344 Zero Blank D 0001344 Blank A 0001344 Blank B 0001344 Zero Blank C 0001344 Zero Blank D 0009684 Blank A 0009684 Blank B 0009684 Zero Blank C 0009684 Zero Blank D 0009684 Blank A 0009684 Blank B 0009684 Zero Blank C 0 0 0 9 6 8 4 Zero Blank D 0009684 Blank A 0009684 Blank B 0009684 Zero Blank C 0009684 Zero Blank D 0009684 Blank A 0009684 Blank B 0009684 Zero Blank C 0009684 Zero Blank D 0009684 Blank A 0009684 Blank B 0009684 Zero Blank C 0009684 Zero Blank D Set Number 100200A 100200A 100200A 100200A 100600A 100600A 100600A 100600A 100900A 100900A 100900A 100900A 101000A 101000A 101000A 101000A 101100A 101100A 101100A 101100A 101200A 101200A 101200A 101200A 101300A 101300A 101300A 101300A 101600AR 101600AR 101600AR 101600AR 101700A 101700A 101700A 101700A Extraction Date 10/2/00 10/2/00 10/2/00 10/2/00 10/6/00 10/6/00 10/6/00 10/6/00 10/9/00 10/9/00 10/9/00 10/9/00 10/10/00 10/10/00 10/10/00 10/10/00 10/11/00 10/11/00 10/11/00 10/11/00 10/12/00 10/12/00 10/12/00 10/12/00 10/13/00 10/13/00 10/13/00 10/13/00 10/16/00 10/16/00 10/16/00 10/16/00 10/17/00 10/17/00 10/17/00 10/17/00 A n alysis D ate 10/3/00 10/3/00 10/3/00 10/3/00 10/6-7/00 10/6-7/00 10/6-7/00 10/6-7/00 10/10/00 10/10/00 10/10/00 10/10/00 10/10-11/00 10/10-11/00 10/10-11/00 10/10-11/00 10/12-13/00 10/12-13/00 10/12-13/00 10/12-13/00 10/13-14/00 10/13-14/00 10/13-14/00 10/13-14/00 10/14-15/00 10/14-15/00 10/14-15/00 10/14-15/00 10/18-19/00 10/18-19/00 10/18-19/00 10/18-19/00 10/17-18/00 10/17-18/00 10/17-18/00 10/17-18/00 A n alyte Found (pg/g) NQ NQ NQ NQ NQ NQ NQ NQ 0.0453 0.0212 0.0913 0.0259 0.108 NQ NQ 0.0129 NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ Centre Analytical Laboratories, Inc. Page 20 of 110 Centre Study No.: 023-011 Sponsor Protocol No: FACT-TOX-026 Table I (cont.) Summary of POAA residues in Matrix Blanks and Matrix Zero Blanks ND = Not Detected and NQ = Not Quantifiable Sponsor ID na na na na Na Na Na Na Na na na na na na na na na na na na na na na na Centre ID 0009684 Blank A 0009684 Blank B 0009684 Zero Blank C 0009684 Zero Blank D 0009684 Blank A 0009684 Blank B 0009684 Zero Blank C 0009684 Zero Blank D 0009684 Blank A 0009684 Blank B 0009684 Zero Blank C 0009684 Zero Blank D 0009684 Blank A 0009684 Blank B 0009684 Zero Blank C 0009684 Zero Blank D 0009684 Blank A 0009684 Blank B 0009684 Zero Blank C 0009684 Zero Blank D 0009684 Blank A 0009684 Blank B 0009684 Zero Blank C 0009684 Zero Blank D Set Extraction Analysis Number Date Date 101800A 10/18/00 10/19-20/00 101800A 10/18/00 10/19-20/00 101800A 10/18/00 10/19-20/00 101800A 10/18/00 10/19-20/00 101900A 10/19/00 10/20-21/00 101900A 10/19/00 10/20-21/00 101900A 10/19/00 10/20-21/00 101900A 10/19/00 10/20-21/00 102000A 10/20/00 10/22-23/00 102000A 10/20/00 10/22-23/00 102000A 10/20/00 10/22-23/00 102000A 10/20/00 10/22-23/00 102300AR 10/23/00 10/25/00 102300AR 10/23/00 10/25/00 102300AR 10/23/00 10/25/00 102300AR 10/23/00 10/25/00 102400A 10/24/00 10/24-25/00 102400A 10/24/00 10/24-25/00 102400A 10/24/00 10/24-25/00 102400A 10/24/00 10/24-25/00 102500A 10/25/00 10/26/00 102500A 10/25/00 10/26/00 102500A 102500A 10/25/00 10/25/00 10/26/00 10/26/00 AVERAGE: STANDARD DEVIATION: Analyte Found (ug/g) NQ NQ NQ NQ NQ ND NQ NQ ND ND ND ND NQ NQ NQ NQ ND ND ND ND ND ND ND ND 0.0113 0.0175 For residues recorded as ND, zero was used to calculate the average and standard deviation and 0.0100 was used for those recorded as NQ. Centre Analytical Laboratories, Inc. Page 21 of 110 Centre Study No.: 023-011 Sponsor Protocol No: FACT-TOX-026 Table II. Summary of POAA recoveries in Fortified Samples Sponsor ID Centre ID Set Extraction Analysis Amount % Number Date Date Added (ng) Recovery I05709M Grp 1 Wk 2 105714M Grp 1 W k2 I05720M Grp 1 Wk 4 I05725M Grp 1 Wk 4 I05720M Grp 1 Wk 6 I05725M Grp 1 Wk 6 I05709M Grp 1 Wk 8 105714M Grp 1 Wk 8 I05715M Grp 1 Wk 10 105718M Grp 1 Wk 10 I05720M Grp 1 Wk 12 I05725M Grp 1 Wk 12 I05709M Grp 1 Wk 14 105714M Grp 1 Wk 14 105715M Grp 1 Wk 16 I05718M Grp 1 Wk 16 I05720M Grp 1 Wk 18 I05725M Grp 1 Wk 18 I05709M Grp 1 Wk 20 105714M Grp 1 Wk 20 I05720M Grp 1 W k22 I05725M Grp 1 Wk 22 I05709M Grp 1 Wk 24 105714M Grp 1 W k24 105718M Grp 1 W k26 I05 7 2 0 M Grp 1 W k 2 6 105718M Grp 1 Wk 28 I05720M Grp 1 Wk 30 105718M Grp 1 Wk 36 I05720M Grp 1 Wk 38 0008434 Spk A 0008435 Spk B 0008460 Spk A 0008461 Spk B 0008482 Spk A 0008483 Spk B 0008499 Spk A 0008500 Spk B 0008522 Spk A 0008523 Spk B 0008545 Spk A 0008546 Spk B 0008562 Spk A 0008563 Spk B 0008585 Spk A 0008586 SpkB 0008608 Spk A 0008609 SpkB 0008625 Spk A 0008626 Spk B 0008650 Spk A 0008651 SpkB 0008666 Spk A 0008667 Spk B 0008689 Spk A 0008690 Spk B 0008706 Spk A 0008711 SpkB 0008722 Spk A 0008726 Spk B 100200A 10/2/00 10/3/00 50 100200A 10/2/00 10/3/00 250 100600A 10/6/00 10/6-7/00 50 100600AD 10/6/00 10/7/00 1000 10Q900A 10/9/00 10/10/00 50 100900A 10/9/00 10/10/00 2000 101000A 10/10/00 10/10-11/00 50 101000A 10/10/00 10/10-11/00 10000 101100A 10/11/00 10/12-13/00 50 101100A 10/11/00 10/12-13/00 10000 101200A 10/12/00 10/13-14/00 50 101200A 10/12/00 10/13-14/00 20000 101300A 10/13/00 10/14-15/00 50 101300A 10/13/00 10/14-15/00 100,250 101600AR 10/16/00 10/18-19/00 50 101600AR 10/16/00 10/18-19/00 100,250 101700A 10/17/00 10/17-18/00 50 101700A 10/17/00 10/17-18/00 200,250 101800A 10/18/00 10/19-20/00 50 101800A 10/18/00 10/19-20/00 200,250 101900AD 10/19/00 10/22/00 50 101900A 10/19/00 10/20-21/00 200,250 102000A 10/20/00 10/22-23/00 50 102000AD 10/20/00 10/23/00 200,250 102300AR 10/23/00 10/25/00 50 102300AR 10/23/00 10/25/00 250 102400A 10/24/00 10/24-25/00 50 102300AD 10/24/00 10/26/00 100,250 102500A 10/25/00 10/26/00 50 102300AD 10/25/00 10/26/00 10,000 AVERAGE STANDARD DEVIATION RELATIVE STANDARD DEVIATION 104 88 119 137 116 139 105 133 98 128 87 115 72 128 114 128 127 125 120 141 130 96 108 170 56 129 123 119 128 125 117 22 19 Centre Analytical Laboratories, Inc. Page 22 of 110 Centre Study No.: 023-011 Sponsor Protocol No: FACT-TOX-026 Table III. Summary of POAA residues in Week 2 Sponsor ID I05709M Grp 1 Wk 2 105714M Grp 1 W k2 105715M Grp 1 W k2 105718M Grp 1 W k2 I05720M Grp 1 Wk 2 I05725M Grp 1 Wk 2 Centre ID 0008434 0008435 0008436 0008437 0008438 0008439 Set Number 100200A 100200A 100200A 100200A 100200A 100200A Extraction Analysis Date Date 10/2/00 10/3/00 10/2/00 10/3/00 10/2/00 10/3/00 10/2/00 10/3/00 10/2/00 10/3/00 10/2/00 10/3/00 Average: Standard Deviation: Analyte Found (ufi/g) NQ NQ NQ NQ NQ NQ NQ NQ I05702M Grp 2 Wk 2 I05706M Grp 2 Wk 2 I05717M Grp 2 Wk 2 I05723M Grp 2 Wk 2 0008440 0008441 0008442 0008443 100200AD 100200AD 100200AD 100200AD 10/2/00 10/4/00 10/2/00 10/4/00 10/2/00 10/4/00 10/2/00 10/4/00 Average: Standard Deviation: 14.0 12.1 1.67 1.94 7.43 6.54 I05707M Grp 3 Wk 2 I05708M Grp 3 Wk 2 I05710M Grp 3 Wk 2 I05712M Grp 3 Wk 2 I05716M Grp 3 Wk 2 I05719M Grp 3 Wk 2 0008444 0008445 0008446 0008447 0008448 0008449 100200ADD 100200AD 100200AD 100200AD 100200AD 100200AD 10/2/00 10/5/00 10/2/00 10/4/00 10/2/00 10/4/00 10/2/00 10/4/00 10/2/00 10/4/00 10/2/00 10/4/00 Average: Standard Deviation: 31.7 20.9 18.2 9.69 5.41 6.50 15.4 10.2 I05703M Grp 4 Wk 2 I05704M Grp 4 Wk 2 105711M Grp 4 Wk 2 105713M Grp 4 Wk 2 I05722M Grp 4 Wk 2 I05724M Grp 4 Wk 2 0008450 0008451 0008452 0008453 0008454 0008455 100200AD 100200ADD 100200ADD 100200AD 100200ADD 100200ADD 10/2/00 10/4/00 10/2/00 10/5/00 10/2/00 10/5/00 10/2/00 10/4/00 10/2/00 10/5/00 10/2/00 10/5/00 Average: Standard Deviation: 18.6 28.3 41.2 17.4 28.2 206 56.6 73.7 ND = Not Detected NQ = Not Quantifiable For residues recorded as ND, zero was used to calculate the average and standard deviation and 0.0100 was used for those recorded as NQ. Centre Analytical Laboratories, Inc. Page 23 of 110 Centre Study No.: 023-011 Sponsor Protocol No: FACT-TOX-026 Table IV. Summary of POAA residues in Week 4 Sponsor ID I05709M Grp 1 Wk 4 105714M Grp 1 W k4 105715M Grp 1 W k4 105718M Grp 1 W k4 I05720M Grp 1 Wk 4 I05725M Grp 1 Wk 4 Centre ID 0008456 0008457 0008458 0008459 0008460 0008461 Set Number 100600A 100600A 100600A 100600A 100600A 100600A Extraction Analysis Date Date 10/6/00 10/6/00 10/6/00 10/6/00 10/6/00 10/6/00 10/6-7/00 10/6-7/00 10/6-7/00 10/6-7/00 10/6-7/00 10/6-7/00 Average: Standard Deviation: Analyte Found (pg/g) NQ 0.0403 NQ NQ NQ 0.0481 0.0214 0.0178 I05702M Grp 2 Wk 4 I05706M Grp 2 Wk 4 I05717M Grp 2 Wk 4 I05721M Grp 2 Wk 4 0008462 0008463 0008464 0008465 100600A 100600AD 100600A 100600AD 10/6/00 10/6/00 10/6/00 10/6/00 10/6-7/00 10/7/00 10/6-7/00 10/7/00 Average: Standard Deviation: 1.48 28.0 4.07 8.22 10.4 12.0 I05707M Grp 3 Wk 4 I05708M Grp 3 Wk 4 10 5 7 10M Grp 3 Wk 4 I05712M Grp 3 Wk 4 10 5 7 16M Grp 3 Wk 4 10 5 7 19M Grp 3 Wk 4 0008466 0008467 0008468 0008469 0008470 0008471 100600AD 100600AD 100600AD 100600AD 100600AD 100600AD 10/6/00 10/6/00 10/6/00 10/6/00 10/6/00 10/6/00 10/7/00 10/7/00 10/7/00 10/7/00 10/7/00 10/7/00 Average: Standard Deviation: 30.8 24.0 12.2 9.72 26.3 37.2 23.4 10.6 I05703M Grp 4 Wk 4 I0 5 7 0 4 M Grp 4 W k 4 10 5711M Grp 4 Wk 4 105713M Grp 4 Wk 4 I05722M Grp 4 Wk 4 I05724M Grp 4 Wk 4 0008472 0008473 0008474 0008475 0008476 0008477 100600AD 100600AD 100600AD 100600AD 100600AD 100600AD 10/6/00 10/6/00 10/6/00 10/6/00 10/6/00 10/6/00 10/7/00 10/7/00 10/7/00 10/7/00 10/7/00 10/7/00 Average: Standard Deviation: 21.6 17.9 17.1 33.8 18.5 2 3 .2 22.0 6.23 ND = Not Detected NQ = Not Quantifiable For residues recorded as ND, zero was used to calculate the average and standard deviation and 0.0100 was used for those recorded as NQ. Centre Analytical Laboratories, Inc. Page 24 of 110 Centre Study No.: 023-011 Sponsor Protocol No: FACT-TOX-026 Table V. Summary of POAA residues in Week 6 Sponsor ID I05709M Grp 1 Wk 6 105714M Grp 1 W k6 105715M Grp 1 Wk 6 105718M Grp 1 W k6 I05720M Grp 1 Wk 6 I05725M Grp 1 Wk 6 Centre ID 0008478 0008479 0008480 0008481 0008482 0008483 Set Number 100900A 100900A 100900A 100900A 100900A 100900A Extraction Date 10/9/00 10/9/00 10/9/00 10/9/00 10/9/00 10/9/00 Analysis Date 10/10/00 10/10/00 10/10/00 10/10/00 10/10/00 10/10/00 Average: Standard Deviation: Analyte Found (pg/g) NQ NQ NQ 0.0147 NQ NQ 0.0108 0.00192 I05702M Grp 2 Wk 6 I05706M Grp 2 Wk 6 I05717M Grp 2 Wk 6 I05721M Grp 2 Wk 6 0008484 0008485 0008486 0008487 100900A 100900AD 100900A 100900AD 10/9/00 10/9/00 10/9/00 10/9/00 10/10/00 10/11/00 10/10/00 10/11/00 Average: Standard Deviation: 1.63 31.8 2.35 12.6 12.1 14.1 I05707M Grp 3 Wk 6 I05708M Grp 3 Wk 6 I05710M Grp 3 Wk 6 105712M Grp 3 Wk 6 I05716M Grp 3 Wk 6 I05719M Grp 3 Wk 6 0008488 0008489 0008490 0008491 0008492 0008493 100900AD 100900AD 100900AD 100900AD 100900AD 100900AD 10/9/00 10/9/00 10/9/00 10/9/00 10/9/00 10/9/00 10/11/00 10/11/00 10/11/00 10/11/00 10/11/00 10/11/00 Average: Standard Deviation: 18.3 16.8 30.0 17.7 37.4 19.4 23.3 8.46 I05703M Grp 4 Wk 6 I05 7 0 4 M Grp 4 W k 6 105711M Grp 4 Wk 6 I05713M Grp 4 Wk 6 I05722M Grp 4 Wk 6 0008494 0008495 0008496 0008497 0008498 100900ADD 100900AD 100900AD 100900AD 100900AD 10/9/00 10/9/00 10/9/00 10/9/00 10/9/00 10/12/00 10/11/00 10/11/00 10/11/00 10/11/00 Average: Standard Deviation: 242 68.6 75.7 9.17 110 101 86.7 ND = Not Detected NQ = Not Quantifiable For residues recorded as ND, zero was used to calculate the average and standard deviation and 0.0100 was used for those recorded as NQ. Centre Analytical Laboratories, Inc. Page 25 of 110 Centre Study No.: 023-011 Sponsor Protocol No: FACT-TOX-026 Table VI. Summary of POAA residues in Week 8 Sponsor ID I05709M Grp 1 Wk 8 105714M Grp 1 Wk 8 105715M Grp 1 Wk 8 105718M Grp 1 Wk 8 I05720M Grp 1 Wk 8 I05725M Grp 1 Wk 8 Centre ID 0008499 0008500 0008501 0008502 0008503 0008504 Set Number 101000A 101000A 101000A 101000A 101000A 101000A Extraction Analysis Date Date 10/10/00 10/10/00 10/10/00 10/10/00 10/10/00 10/10/00 10/10-11/00 10/10-11/00 10/10-11/00 10/10-11/00 10/10-11/00 10/10-11/00 Average: Standard Deviation: Analyte Found (jig/g) 0.0214 0.157 0.0102 0.0159 0.0104 0.254 0.0782 0.103 I05702M Grp 2 Wk 8 I05706M Grp 2 Wk 8 I05717M Grp 2 Wk 8 105721M Grp 2 Wk 8 0008505 0008506 0008507 0008508 101000A 101000AD 101000A 101000AD 10/10/00 10/10/00 10/10/00 10/10/00 10/10-11/00 10/11-12/00 10/10-11/00 10/11-12/00 Average: Standard Deviation: 3.24 22.8 3.41 8.37 9.46 9.21 I05707M Grp 3 Wk 8 I05708M Grp 3 Wk 8 105710M Grp 3 Wk 8 105712M Grp 3 Wk 8 105716M Grp 3 Wk 8 105719M Grp 3 Wk 8 0008509 0008510 0008511 0008512 0008513 0008514 101000AD 101000AD 101000AD 101000AD 101000AD 101000AD 10/10/00 10/10/00 10/10/00 10/10/00 10/10/00 10/10/00 10/11-12/00 10/11-12/00 10/11-12/00 10/11-12/00 10/11-12/00 10/11-12/00 Average: Standard Deviation: 51.7 17.8 76.3 7.64 40.8 51.8 41.0 25.0 I05703M Grp 4 Wk 8 I05 7 0 4 M Grp 4 W k 8 105711M Grp 4 Wk 8 105713M Grp 4 Wk 8 I05722M Grp 4 Wk 8 0008515 0008516 0008517 0008518 0008519 101000AD lOlOOOA D 101000A 101000AD 101000AD 10/10/00 lO/lO/OO 10/10/00 10/10/00 10/10/00 10/11-12/00 10/11-12/00 10/10-11/00 10/11-12/00 10/11-12/00 Average: Standard Deviation: 50.2 21.0 1.69 21.2 89.4 36.7 34.2 ND = Not Detected NQ = Not Quantifiable For residues recorded as ND and NQ, zero was used to calculate the average and standard deviation and 0.0100 was used for those recorded as < 0.0100. Centre Analytical Laboratories, Inc. Page 26 of 110 Centre Study No.: 023-011 Sponsor Protocol No: FACT-TOX-026 Table VII. Summary of POAA residues in Week 10 Sponsor ID I05709M Grp 1 Wk 10 105714M Grp 1 Wk 10 I05715M Grp 1 Wk 10 105718M Grp 1 Wk 10 I05720M Grp 1 Wk 10 I05725M Grp 1 Wk 10 Centre ID 0008520 0008521 0008522 0008523 0008524 0008525 Set Number 101100A 101100A 101100A 101100A 101100A 101100A Extraction Date 10/11/00 10/11/00 10/11/00 10/11/00 10/11/00 10/11/00 Analysis Date 10/12-13/00 10/12-13/00 10/12-13/00 10/12-13/00 10/12-13/00 10/12-13/00 Average: Standard Deviation: Analyte Found (pg/g) NQ NQ NQ NQ NQ NQ NQ NQ I05702M Grp 2 Wk 10 I05706M Grp 2 Wk 10 105717M Grp 2 Wk 10 105721M Grp 2 Wk 10 0008526 0008527 0008528 0008529 101100A 101100A 101100A 101100AD 10/11/00 10/11/00 10/11/00 10/11/00 10/12-13/00 10/12-13/00 10/12-13/00 10/13/00 Average: Standard Deviation: 1.67 2.64 2.09 9.45 3.96 3.68 I05707M Grp 3 Wk 10 I05708M Grp 3 Wk 10 I05710M Grp 3 Wk 10 105712M Grp 3 Wk 10 105716M Grp 3 Wk 10 105719M Grp 3 Wk 10 0008530 0008531 0008532 0008533 0008534 0008535 1 0 1 1 0 0 AD 101100AD 101100 AD 1 0 1 1 0 0 AD 101100AD 101100AD 10/11/00 10/11/00 10/11/00 10/11/00 10/11/00 10/11/00 10/13/00 10/13/00 10/13/00 10/13/00 10/13/00 10/13/00 Average: Standard Deviation: 31.0 18.7 44.1 10.1 5.39 46.9 26.0 17.4 I05703M Grp 4 Wk 10 I0 5 7 0 4 M Grp 4 W k 10 I05711M G rp4 Wk 10 105713M Grp 4 Wk 10 I05722M Grp 4 Wk 10 0008536 0008537 0008538 0008539 0008540 101100AD 101100AD 101100A 101100AD 1 0 1 1 0 0 AD 10/11/00 10/1 l/OO 10/11/00 10/11/00 10/11/00 10/13/00 10/13/00 10/12-13/00 10/13/00 10/13/00 Average: Standard Deviation: 32.6 45.5 0.635 80.3 81.1 48.0 34.0 ND = Not Detected NQ = Not Quantifiable For residues recorded as ND, zero was used to calculate the average and standard deviation and 0.0100 was used for those recorded as NQ. Centre Analytical Laboratories, Inc. Page 27 of 110 Centre Study No.: 023-011 Sponsor Protocol No: FACT-TOX-026 Table V ili. Summary of POAA residues in Week 12 Sponsor ID I05709M Grp 1 Wk 12 105714M Grp 1 Wk 12 105715M Grp 1 Wk 12 105718M Grp 1 Wk 12 I05720M Grp 1 Wk 12 I05725M Grp 1 Wk 12 Centre ID 0008541 0008542 0008543 0008544 0008545 0008546 Set Number 101200A 101200A 101200A 101200A 101200A 101200A Extraction Date 10/12/00 10/12/00 10/12/00 10/12/00 10/12/00 10/12/00 Analysis Date 10/13-14/00 10/13-14/00 10/13-14/00 10/13-14/00 10/13-14/00 10/13-14/00 Average: Standard Deviation: Analyte Found (pg/g) 0.0209 NQ NQ 0.232 NQ 0.0160 0.0498 0.0894 I05702M Grp 2 Wk 12 I05706M Grp 2 Wk 12 105717M Grp 2 Wk 12 105721M Grp 2 Wk 12 0008547 0008548 0008549 0008550 101200AD 101200A 101200A 101200AD 10/12/00 10/12/00 10/12/00 10/12/00 10/14/00 10/13-14/00 10/13-14/00 10/14/00 Average: Standard Deviation: 13.6 3.21 1.68 10.1 7.15 5.65 I05707M Grp 3 Wk 12 I05708M Grp 3 Wk 12 105710M Grp 3 Wk 12 105712M Grp 3 Wk 12 105716M Grp 3 Wk 12 105719M Grp 3 Wk 12 0008551 0008552 0008553 0008554 0008555 0008556 101200AD 101200AD 101200A 101200AD 101200AD 101200A 10/12/00 10/12/00 10/12/00 10/12/00 10/12/00 10/12/00 10/14/00 10/14/00 10/13-14/00 10/14/00 10/14/00 10/13-14/00 Average: Standard Deviation: 19.9 9.68 4.54 8.86 14.5 4.06 10.3 6.07 I05703M Grp 4 Wk 12 I0 5 7 0 4 M Grp 4 W k 12 105711M Grp 4 Wk 12 I05713M G rp4 Wk 12 I05722M Grp 4 Wk 12 0008557 0008558 0008559 0008560 0008561 101200AD 101200AD 101200A 101200AD 101200A 10/12/00 10/12/00 10/12/00 10/12/00 10/12/00 10/14/00 10/14/00 10/13-14/00 10/14/00 10/13-14/00 Average: Standard Deviation: 34.2 105 0.508 16.3 3.87 32.0 42.9 ND = Not Detected NQ = Not Quantifiable For residues recorded as ND, zero was used to calculate the average and standard deviation and 0.0100 was used for those recorded as NQ. Centre Analytical Laboratories, Inc. Page 28 of 110 Centre Study No.: 023-011 Sponsor Protocol No: FACT-TOX-026 Table IX. Summary of POAA residues in Week 14 Sponsor ID I05709M Grp 1 Wk 14 105714M Grp 1 Wk 14 105715M Grp 1 Wk 14 105718M Grp 1 Wk 14 I05720M Grp 1 Wk 14 I05725M Grp 1 Wk 14 Centre ID 0008562 0008563 0008564 0008565 0008566 0008567 Set Number 101300A 101300A 101300A 101300AD 101300A 101300A Extraction Date 10/13/00 10/13/00 10/13/00 10/13/00 10/13/00 10/13/00 Analysis Date 10/14-15/00 10/14-15/00 10/14-15/00 10 / 15/00 10/14-15/00 10/14-15/00 Average: Standard Deviation: Analyte Found (ug/g) 0.0276 NQ NQ 0.768 NQ NQ 0.139 0.308 I05702M Grp 2 Wk 14 I05706M Grp 2 Wk 14 105717M Grp 2 Wk 14 105721M Grp 2 Wk 14 0008568 0008569 0008570 0008571 101300A 101300AD 101300AD 101300AD 10/13/00 10/13/00 10/13/00 10/13/00 10/14-15/00 10/15/00 10 / 15/00 10/15/00 Average: Standard Deviation: 4.22 9.25 7.12 9.42 7.50 2.43 I05707M Grp 3 Wk 14 I05708M Grp 3 Wk 14 I05710M Grp 3 Wk 14 I05712M Grp 3 Wk 14 105716M Grp 3 Wk 14 105719M Grp 3 Wk 14 0008572 0008573 0008574 0008575 0008576 0008577 101300AD 101300AD 101300A 101300AD 101300AD 101300AD 10/13/00 10/13/00 10/13/00 10/13/00 10/13/00 10/13/00 10/15/00 10/15/00 10/14-15/00 10/15/00 10/15/00 10/15/00 Average: Standard Deviation: 82.2 10.3 4.88 8.30 20.2 37.6 27.2 29.4 I05703M Grp 4 Wk 14 I0 5 7 0 4 M Grp 4 W k 14 105711M Grp 4 Wk 14 105713M Grp 4 Wk 14 I05722M Grp 4 Wk 14 0008578 0008579 0008580 0008581 0008582 101300A 101300AD 101300A 101300AD 101300A 10/13/00 10/13/00 10/13/00 10/13/00 10/13/00 10/14-15/00 10/15/00 10/14-15/00 10/15/00 10/14-15/00 Average: Standard Deviation: 3.33 55.9 0.363 37.0 2.91 19.9 25.2 ND = Not Detected NQ = Not Quantifiable For residues recorded as ND, zero was used to calculate the average and standard deviation and 0.0100 was used for those recorded as NQ. Centre Analytical Laboratories, Inc. Page 29 of 110 Centre Study No.: 023-011 Sponsor Protocol No: FACT-TOX-026 Table X. Summary of POAA residues in Week 16 Sponsor ID I05709M Grp 1 Wk 16 105714M Grp 1 Wk 16 105715M Grp 1 Wk 16 105718M Grp 1 Wk 16 I05720M Grp 1 Wk 16 I05725M Grp 1 Wk 16 Centre ID 0008583 0008584 0008585 0008586 0008587 0008588 Set Number 101600AR 101600AR 101600AR 101600AR 101600AR 101600AR Extraction Date Analysis Date 10/16/00 10/16/00 10/16/00 10/16/00 10/16/00 10/16/00 10/18-19/00 10/18-19/00 10/18-19/00 10/18-19/00 10/18-19/00 10/18-19/00 Average: Standard Deviation: Analyte Found (|ig/g) 0.104 0.0159 0.193 NQ NQ NQ 0.0572 0.0762 I05702M Grp 2 Wk 16 I05706M Grp 2 Wk 16 105717M Grp 2 Wk 16 105721M Grp 2 Wk 16 0008589 0008590 0008591 0008592 101600AD 101600AD 101600AR 101600AD 10/16/00 10/16/00 10/16/00 10/16/00 10/19/00 10/19/00 10/18-19/00 10/19/00 Average: Standard Deviation: 6.83 7.25 3.52 9.92 6.88 2.62 I05707M Grp 3 Wk 16 I05708M Grp 3 Wk 16 105710M Grp 3 Wk 16 I05712M G rp3 Wk 16 105716M Grp 3 Wk 16 105719M Grp 3 Wk 16 0008593 0008594 0008595 0008596 0008597 0008598 101600AD 101600AR 101600AD 101600AD 101600AD 101600AD 10/16/00 10/16/00 10/16/00 10/16/00 10/16/00 10/16/00 10/19/00 10/18-19/00 10/19/00 10/19/00 10/19/00 10/19/00 Average: Standard Deviation: 20.0 5.04 68.0 13.2 36.3 45.7 31.4 23.3 I05703M Grp 4 Wk 16 I0 5 7 0 4 M Grp 4 W k 16 105711M Grp 4 Wk 16 I05713M Grp 4 Wk 16 I05722M Grp 4 Wk 16 0008599 0008600 0008601 0008602 0008603 101600AR 101600AD 101600AR 101600AD 101600AR 10/16/00 10/16/00 10/16/00 10/16/00 10/16/00 10/18-19/00 10/19/00 10/18-19/00 10/19/00 10/18-19/00 Average: Standard Deviation: 2.61 68.2 0.327 17.8 2.18 18.2 28.8 ND = Not Detected NQ = Not Quantifiable For residues recorded as ND, zero was used to calculate the average and standard deviation and 0.0100 was used for those recorded as NQ. Centre Analytical Laboratories, Inc. Page 30 of 110 Centre Study No.: 023-011 Sponsor Protocol No: FACT-TOX-026 Table XL Summary of POAA residues in Week 18 Sponsor ID I05709M Grp 1 Wk 18 105714M Grp 1 Wk 18 105715M Grp 1 Wk 18 105718M Grp 1 Wk 18 I05720M Grp 1 Wk 18 I05725M Grp 1 Wk 18 Centre ID 0008604 0008605 0008606 0008607 0008608 0008609 Set Number 101700A 101700A 101700A 101700AD 101700A 101700A Extraction Date 10/17/00 10/17/00 10/17/00 10/17/00 10/17/00 10/17/00 Analysis Date 10/17-18/00 10/17-18/00 10/17-18/00 10/19/00 10/17-18/00 10/17-18/00 Average: Standard Deviation: Analyte Found (pg/g) NQ NQ 0.0153 1.49 NQ NQ 0.258 0.604 I05702M Grp 2 Wk 18 I05706M Grp 2 Wk 18 I05717M Grp 2 Wk 18 105721M Grp 2 Wk 18 0008610 0008611 0008612 0008613 101700A 101700A 101700AD 101700A 10/17/00 10/17/00 10/17/00 10/17/00 10/17-18/00 10/17-18/00 10/19/00 10/17-18/00 Average: Standard Deviation: 1.62 1.76 16.4 3.08 5.72 7.15 I05707M Grp 3 Wk 18 I05708M Grp 3 Wk 18 105710M Grp 3 Wk 18 105712M Grp 3 Wk 18 I05716M Grp 3 Wk 18 105719M Grp 3 Wk 18 0008614 0008615 0008616 0008617 0008618 0008619 101700AD 101700A 101700AD 101700AD 101700AD 101700AD 10/17/00 10/17/00 10/17/00 10/17/00 10/17/00 10/17/00 10/19/00 10/17-18/00 10/19/00 10/19/00 10/19/00 10/19/00 Average: Standard Deviation: 41.1 4.48 22.9 9.50 16.3 9.26 17.3 13.3 I05703M Grp 4 Wk 18 I05704M Grp 4 Wk 18 105711M Grp 4 Wk 18 I05713M Grp 4 Wk 18 I05722M Grp 4 Wk 18 0008620 0008621 0008622 0008623 0008624 101700A 101700AD 101700A 101700AD 101700A 10/17/00 10/17/00 10/17/00 10/17/00 10/17/00 10/17-18/00 10/19/00 10/17-18/00 10/19/00 10/17-18/00 Average: Standard Deviation: 0.895 38.1 0.159 70.9 0.609 22.1 31.7 ND = Not Detected NQ = Not Quantifiable For residues recorded as ND, zero was used to calculate the average and standard deviation and 0.0100 was used for those recorded as NQ. Centre Analytical Laboratories, Inc. Page 31 of 110 Centre Study No.: 023-011 Sponsor Protocol No: FACT-TOX-026 Table XII. Summary of POAA residues in Week 20 Sponsor ID Centre ID Set Number Extraction Date Analysis Date Analyte Found (ug/g) I05709M Grp 1 Wk 20 105714M Grp 1 W k20 105715M Grp 1 Wk 20 105718M Grp 1 W k20 I05720M Grp 1 Wk 20 I05725M Grp 1 Wk 20 0008625 0008626 0008627 0008628 0008629 0008630 101800A 101800A 101800A 101800AD 101800A 101800A 10/18/00 10/18/00 10/18/00 10/18/00 10/18/00 10/18/00 10/19-20/00 10/19-20/00 10/19-20/00 10/20/00 10/19-20/00 10/19-20/00 Average: Standard Deviation: NQ NQ NQ 2.65 NQ NQ 0.450 1.08 I05702M Grp 2 Wk 20 I05706M Grp 2 Wk 20 105717M Grp 2 Wk 20 105721M Grp 2 Wk 20 0008631 0008632 0008633 0008634 101800AD 101800A 101800A 101800AD 10/18/00 10/18/00 10/18/00 10/18/00 10/20/00 10/19-20/00 10/19-20/00 10/20/00 Average: Standard Deviation: 6.91 4.33 2.41 13.6 6.81 4.89 I05707M Grp 3 Wk 20 I05708M Grp 3 Wk 20 105710M Grp 3 Wk 20 105712M Grp 3 Wk 20 105716M Grp 3 Wk 20 105719M Grp 3 Wk 20 0008635 0008636 0008637 0008638 0008639 0008640 101800AD 101800AD 101800AD 101800AD 101800AD 101800AD 10/18/00 10/18/00 10/18/00 10/18/00 10/18/00 10/18/00 10/20/00 10/20/00 10/20/00 10/20/00 10/20/00 10/20/00 Average: Standard Deviation: 35.0 24.9 81.3 25.5 27.4 120 52.4 39.5 I05703M Grp 4 Wk 20 I05704M Grp 4 Wk 20 105711M Grp 4 Wk 20 I05713M Grp 4 Wk 20 I05722M Grp 4 Wk 20 0008641 0008642 0008643 0008644 0008645 101800A 101800A 101800AD 101800A 101800AD 10/18/00 10/18/00 10/18/00 10/18/00 10/18/00 10/19-20/00 10/19-20/00 10/20/00 10/19-20/00 10/20/00 Average: Standard Deviation: 2.11 0.778 134 0.286 51.7 37.8 58.1 ND = Not Detected NQ = Not Quantifiable For residues recorded as ND, zero was used to calculate the average and standard deviation and 0.0100 was used for those recorded as NQ. Centre Analytical Laboratories, Inc. Page 32 of 110 Centre Study No.: 023-011 Sponsor Protocol No: FACT-TOX-026 Table XIII. Summary of POAA residues in Week 22 Sponsor ID I05709M Grp 1 Wk 22 105714M Grp 1 W k22 105715M Grp 1 W k22 105718M Grp 1 Wk 22 I05720M Grp 1 Wk 22 I05725M Grp 1 Wk 22 Centre ID 0008646 0008647 0008648 0008649 0008650 0008651 Set Number 101900A 101900A 101900A 102000AD 101900A 101900AD Extraction Date Analysis Date 10/19/00 10/19/00 10/19/00 10/19/00 10/19/00 10/19/00 10/20-21/00 10/20-21/00 10/20-21/00 10/23/00 10/20-21/00 10/22/00 Average: Standard Deviation: Analyte Found (pg/g) 0.0129 0.0422 NQ 90.8 0.0106 1.84 15.5 36.9 I05702M Grp 2 Wk 22 I05706M Grp 2 Wk 22 I05717M Grp 2 Wk 22 0008652 0008653 0008654 101900AD 101900AD 101900AD 10/19/00 10/19/00 10/19/00 10/22/00 10/22/00 10/22/00 Average: Standard Deviation: 7.82 17.1 16.6 13.8 5.22 I05707M Grp 3 Wk 22 I05708M Grp 3 Wk 22 I05710M Grp 3 Wk 22 I05712M Grp 3 Wk 22 I05716M Grp 3 Wk 22 105719M Grp 3 Wk 22 0008655 0008656 0008657 0008658 0008659 0008660 101900AD 101900AD 101900AD 101900AD 101900AD 101900AD 10/19/00 10/19/00 10/19/00 10/19/00 10/19/00 10/19/00 10/22/00 10/22/00 10/22/00 10/22/00 10/22/00 10/22/00 Average: Standard Deviation: 36.1 69.4 23.7 13.7 35.3 58.9 39.5 21.0 I05703M Grp 4 Wk 22 I05704M Grp 4 Wk 22 105711M Grp 4 Wk 22 105713M Grp 4 Wk 22 I05722M Grp 4 Wk 22 0008661 0008662 0008663 0008664 0008665 101900A 101900AD 101900A 101900AD 101900A 10/19/00 10/19/00 10/19/00 10/19/00 10/19/00 10/20-21/00 10/22/00 10/20-21/00 10/22/00 10/20-21/00 Average: Standard Deviation: 1.95 83.9 1.30 35.8 2.80 25.2 36.0 ND = Not Detected NQ = Not Quantifiable For residues recorded as ND, zero was used to calculate the average and standard deviation and 0.0100 was used for those recorded as NQ. Centre Analytical Laboratories, Inc. Page 33 of 110 Centre Study No.: 023-011 Sponsor Protocol No: FACT-TOX-026 Table XIV. Summary of POAA residues in Week 24 Sponsor ID I05709M Grp 1 Wk 24 105714M Grp 1 W k24 105715M Grp 1 W k24 105718M Grp 1 W k24 I05720M Grp 1 Wk 24 I05725M Grp 1 Wk 24 Centre ID 0008666 0008667 0008668 0008669 0008670 0008671 I05702M Grp 2 Wk 24 I05706M Grp 2 Wk 24 I05717M Grp 2 Wk 24 0008672 0008673 0008674 I05707M Grp 3 Wk 24 I05708M Grp 3 Wk 24 105710M Grp 3 Wk 24 105712M Grp 3 Wk 24 105716M Grp 3 Wk 24 105719M Grp 3 Wk 24 0008675 0008676 0008677 0008678 0008679 0008680 I05703M Grp 4 Wk 24 I05704M Grp 4 Wk 24 105711M Grp 4 Wk 24 105713M Grp 4 Wk 24 I05722M Grp 4 Wk 24 0008681 0008682 0008683 0008684 0008685 Set Number 102000A 102000AD 102000A 102000A 102000A 102000A Extraction Date Analysis Date 10/20/00 10/20/00 10/20/00 10/20/00 10/20/00 10/20/00 10/22-23/00 10/23/00 10/22-23/00 10/22-23/00 10/22-23/00 10/22-23/00 Average: Standard Deviation: Analyte Found (pg/g) 0.0261 2.82 NQ 0.0310 NQ 0.207 0.517 1.13 102000A 102000AD 102000A 10/20/00 10/20/00 10/20/00 10/22-23/00 10/23/00 10/22-23/00 Average: Standard Deviation: 4.14 12.40 2.12 6.22 5.45 102000AD 102000AD 102000AD 102000AD 102000AD 102000AD 10/20/00 10/20/00 10/20/00 10/20/00 10/20/00 10/20/00 10/23/00 10/23/00 10/23/00 10/23/00 10/23/00 10/23/00 Average: Standard Deviation: 30.4 53.7 58.4 10.0 65.3 25.3 40.5 21.8 102000A 102000AD 102000A 102000AD 102000A 10/20/00 10/20/00 10/20/00 10/20/00 10/20/00 10/22-23/00 10/23/00 10/22-23/00 10/23/00 10/22-23/00 Average: Standard Deviation: 0.534 99.6 0.310 71.9 0.685 34.6 47.7 ND = Not Detected NQ = Not Quantifiable For residues recorded as ND, zero was used to calculate the average and standard deviation and 0.0100 was used for those recorded as NQ. Centre Analytical Laboratories, Inc. Page 34 of 110 Centre Study No.: 023-011 Sponsor Protocol No: FACT-TOX-026 Table XV. Summary of POAA residues in Week 26 Sponsor ID I05709M Grp 1 Wk 26 105714M Grp 1 Wk 26 105715M Grp 1 W k26 105718M Grp 1 W k26 I05720M Grp 1 Wk 26 I05725M Grp 1 Wk 26 Centre ID 0008686 0008687 0008688 0008689 0008690 0008691 Set Number 102300AR 102300AR 102300AR 102300AR 102300AR 102300AR Extraction Date 10/23/00 10/23/00 10/23/00 10/23/00 10/23/00 10/23/00 Analysis Date 10/25/00 10/25/00 10/25/00 10/25/00 10/25/00 10/25/00 Average: Standard Deviation: Analyte Found (pg/g) NQ 0.0317 NQ 0.0227 NQ 0.0186 0.0172 0.00892 I05702M Grp 2 Wk 26 I05706M Grp 2 Wk 26 105717M Grp 2 Wk 26 0008692 0008693 0008694 102300AR 102300AR 102300AR 10/23/00 10/23/00 10/23/00 10/25/00 10/25/00 10/25/00 Average: Standard Deviation: 4.44 2.48 1.85 2.92 1.35 I05707M Grp 3 Wk 26 I05708M Grp 3 Wk 26 10 57 10M Grp 3 Wk 26 10 57 12M Grp 3 Wk 26 10 5 7 16M Grp 3 Wk 26 I05719M Grp 3 Wk 26 0008695 0008696 0008697 0008698 0008699 0008700 102300AD 102300AD 102300AD 102300AD 102300AD 102300AD 10/23/00 10/23/00 10/23/00 10/23/00 10/23/00 10/23/00 10/26/00 10/26/00 10/26/00 10/26/00 10/26/00 10/26/00 Average: Standard Deviation: 108 46.6 10.7 19.1 15.9 57.5 43.0 36.9 I05703M Grp 4 Wk 26 I05704M Grp 4 Wk 26 1 0 5 7 1 1M Grp 4 W k 2 6 105713M Grp 4 Wk 26 I05722M Grp 4 Wk 26 0008701 0008702 0008703 0008704 0008705 102300AR 102300AR 102300AD 102300AD 102300AR 10/23/00 10/23/00 10/23/00 10/23/00 10/23/00 10/25/00 10/25/00 10/26/00 10/26/00 10/25/00 Average: Standard Deviation: 0.238 3.77 NQ 47.4 0.107 10.3 20.8 ND = Not Detected NQ = Not Quantifiable For residues recorded as ND, zero was used to calculate the average and standard deviation and 0.0100 was used for those recorded as NQ. Centre Analytical Laboratories, Inc. Page 35 of 110 Centre Study No.: 023-011 Sponsor Protocol No: FACT-TOX-026 Table XVI. Summary of POAA residues in Week 28-34 Sponsor ID Centre ID Set Number Extraction Date Analysis Date Analyte Found (|ig/g) 105718M Grp 1 Wk 28 I05720M Grp 1 Wk 28 105718M Grp 1 Wk 30 I05720M Grp 1 Wk 30 105718M Grp 1 Wk 32 I05720M Grp 1 Wk 32 105718M Grp 1 Wk 34 I05720M Grp 1 Wk 34 0008706 0008707 0008710 0008711 0008714 0008715 0008718 0008719 105712M Grp 3 Wk 28 105716M Grp 3 Wk 28 I05712M Grp 3 Wk 30 I05716M Grp 3 Wk 30 105716M Grp 3 Wk 32 I05716M Grp 3 Wk 32 105716M Grp 3 Wk 34 105716M Grp 3 Wk 34 0008708 0008709 0008712 0008713 0008716 0008717 0008720 0008721 102400A 102300AD 102400A 102400A 102400A 102400A 102400A 102400A 102400A 102400A 102400A 102400A 102400A 102400A 102400A 102400A 10/24/00 10/24/00 10/24/00 10/24/00 10/24/00 10/24/00 10/24/00 10/24/00 10/24-25/00 10/26/00 10/24-25/00 10/24-25/00 10/24-25/00 10/24-25/00 10/24-25/00 10/24-25/00 Average: Standard Deviation: 10/24/00 10/24/00 10/24/00 10/24/00 10/24/00 10/24/00 10/24/00 10/24/00 10/24-25/00 10/24-25/00 10/24-25/00 10/24-25/00 10/24-25/00 10/24-25/00 10/24-25/00 10/24-25/00 Average: Standard Deviation: 0.0675 2.09 NQ 0.0123 NQ 0.0260 NQ NQ 0.279 0.732 1.23 0.381 0.380 0.498 0.285 0.0894 0.130 0.104 0.387 0.372 ND = Not Detected NQ = Not Quantifiable For residues recorded as ND, zero was used to calculate the average and standard deviation and 0.0100 was used for those recorded as NQ. Centre Analytical Laboratories, Inc. Page 36 of 110 Centre Study No.: 023-011 Sponsor Protocol No: FACT-TOX-026 Table XVII. Summary of POAA residues in Weeks 36-40 Sponsor ID 105718M Grp 1 Wk 36 I05720M Grp 1 Wk 36 105718M Grp 1 Wk 38 I05720M Grp 1 Wk 38 105718M Grp 1 W k40 I05720M Grp 1 Wk 40 Centre ID 0008722 0008723 0008726 0008727 0008730 0008731 Set Number 102500A 102500A 102500A 102500A 102500A 102500A Extraction Date 10/25/00 10/25/00 10/25/00 10/25/00 10/25/00 10/25/00 Analysis Date 10/26/00 10/26/00 10/26/00 10/26/00 10/26/00 10/26/00 Average: Standard Deviation: Analyte Found (pg/g) NQ 0.0216 ND NQ NQ NQ 0.0103 0.00684 I05712M Grp 3 Wk 36 I05716M Grp 3 Wk 36 105712M Grp 3 Wk 38 105716M Grp 3 Wk 38 I05716M Grp 3 Wk 40 105716M Grp 3 Wk 40 0008724 0008725 0008728 0008729 0008732 0008733 102500A 102500A 102500A 102500A 102500A 102500A 10/25/00 10/25/00 10/25/00 10/25/00 10/25/00 10/25/00 10/26/00 10/26/00 10/26/00 10/26/00 10/26/00 10/26/00 Average: Standard Deviation: 0.0944 0.0306 0.0327 NQ 0.0103 0.0235 0.0336 0.0313 ND = Not Detected NQ = Not Quantifiable For residues recorded as ND, zero was used to calculate the average and standard deviation and 0.0100 was used for those recorded as NQ. Centre Analytical Laboratories, Inc. Page 37 of 110 Centre Study No.: 023-011 Sponsor Protocol No: FACT-TOX-026 12.0 FIGURES Centre Analytical Laboratories, Inc Page 38 of 110 Figure 1. Centre Study No.: 023-011 Sponsor Protocol No: FACT-TOX-026 Typical Calibration Curve for POAA I Centre Analytical Laboratories, Inc. Page 39 of 110 Figure 2. Centre Study No.: 023-Oil Sponsor Protocol No: FACT-TOX-026 Typical Mean Response Factor for THPFOS Centre Analytical Laboratories, Ine. Page 40 of 110 Figure 3. Centre Study No.: 023-011 Sponsor Protocol No: FACT-TOX-026 Chromatogram Representing a 5 ng/mL extracted standard for POAA and 250 ng/mL extracted standard for THPFOS 8: THPFOS XCK31200-1, 5 ng/mL Std 1012 30A -1002 S m {S G , 2x2) 10(P %: Ch 1 .0 0 2 .0 0 9 : POAA XCK)1200-1, 5 ng/mL Std 1 0 1 2(3Q A -1002 S m (S G , 2 x2 ) 10CH 3 .0 0 %: nJ 1 , i , . . . i | . i i i j i . i i 1 .0 0 2 .0 0 I. :- Oo :o 5.17 22471^^ 13-0ct-2000 18:33:01 LC/MS/MS #6 M R M o f 9 C h a n ne ls E S 427 > 80 2 .2 8 e 5 A rea 4 .0 0 5 .0 0 6.00 ' 7.00 1 ` ! ' ' i ' ' T im e 8 .00 5.19 4S22S~^ 13-0ct-2000 18:33:01 LC/MS/MS #6 M R M o f 9 C h a n ne ls E S 413 > 369 3 .9 3 e 5 A rea 4.00 ' 5.00 "---- ----6 .0 0 7 .0 0 8 .0 0 rime Centre Analytical Laboratories, Inc. Page 41 of 110 Figure 4. Centre Study No.: 023-011 Sponsor Protocol No: FACT-TOX-026 Chromatogram Representing a 125 ng/mL extracted standard for POAA and 250 ng/mL extracted standard for THPFOS 8: THPFOS XC1C11200-5,125 ng/mL Std 1 0 1 2(D 0A-1006 S m (S G ,2 x 2 ) 10O] % L r 1 ' 111111 i 11 11 11111 111 1 1111'"n 1 .0 0 2 .0 0 3 .0 0 4 .0 0 1 5.00 6 .0 0 9 : POAA XCK)1200-5,125 ng/mL Std 1012 30A-1006 S m (SG , 2x2) 100 %- Al r- .............. 'I | 1 . i 1| . 1. I j . 1. | . | 1 .0 0 2 .0 0 3 .0 0 1 4 .0 0 5.00 6 .0 0 13-0ct-2000 19:39:49 LC/MS/MS #6 M R M o f 9 C h a n ne ls E S 427 > 80 1 .6 5 e 5 . A rea t i * * j ! 1 1 1j 7 .00 8 .0 0 I m e 13-0ct-2000 19:39:49 LC/MS/MS #6 M R M o f 9 C h a n ne ls ES413 >369 6 .2 0 e 6 A rea 7 .0 0 8 .0 0 Centre Analytical Laboratories, Inc. Page 42 of 110 Figure 5. Centre Study No.: 023-011 Sponsor Protocol No: FACT-TOX-026 Chromatogram Representing Control Monkey Feces for POAA and THPFOS (Centre ID: 0009684 Blank A, Set: 101200A) 8: THPFOS 9: POAA Centre Analytical Laboratories, Inc. Page 43 of 110 Centre Study No.: 023-011 Sponsor Protocol No: FACT-TOX-026 Figure 6. Chromatogram Representing Control Monkey Feces Fortified with 50 ng/g of POAA and 500 ng/g of THPFOS (Centre ID: 0008545 Spk A, Set: 101200A) 8: THPFOS 9: POAA Centre Analytical Laboratories, Inc. Page 44 of 110 Figure 7. Centre Study No.: 023-011 Sponsor Protocol No: FACT-TOX-026 Chromatogram of Monkey Feces Sample from Grp 2 in Week 12 (Centre ID: 0008548, Set: 101200A) 8: THPFOS 9: POAA 0008548 DF = 10 101200A-1026 Sm (SG, 2x2) 100- %- ~ i 'i ! 11 i 1 ri 1.00 2.00 15.21 1149713*1 14-0ct-2000 01:14:21 LC/MS/MS #6 MRM of 9 Channels ES413 >369 9.78e6 A r e a 3.00 , 1 .... t 4.00 . . ... 1 . 5.00 6.00 , -- i : i ' Time 7.00 8.00 Centre Analytical Laboratories, Inc. Page 45 of 110 Centre Study No.: 023-011 Sponsor Protocol No: FACT-TOX-026 13.0 APPENDICES Centre Analytical Laboratories, Inc Page 46 of 110 Centre Study No.: 023-011 Sponsor Protocol No: FACT-TOX-026 APPENDIX A Study Protocol FACT-TOX-026 (Centre Study No. 023-011) 26-Week Capsule Toxicity Study with Ammonium Perfluorooctanoate (APFO/POAA) in Cynomolgus Monkeys and Amendments and Deviations Centre Analytical Laboratories, Inc. Page 47 of 110 Centre Study No.: 023-011 Sponsor Protocol No: FACT-TOX-026 Protocol.XFACT- TO X-026 S tudy Title : 6-Month Capsule Toxicity Study with Ammonium Perfluorooctanoate (APFO/POAA) in Cynomolgus Monkeys - PROTOCOL A u th o r Lisa Clemen D ate: October 12,1998 Perform ing Laboratory 3M Environmental Technology & Safety Services 3M Environmental Laboratory 935 Bush Avenue ' St,Paul, MN 55106 . Laboratory Project Identification FACT-TOX-026 Exact Copy cf Cri~ -.1 3M Environmental Laboratory Centre Analytical Laboratories, Inc. Page 1of 9 Page 48 of 110 Centre Study No.: 023-011 Sponsor Protocol No: FACT-TOX-026 Protocol # FACT- TOX-026 S tudy Identification 6-Month Capsule Toxicity Study with Ammonium Perfluorooctanoate (APFO/POAA) in Cynomolgus Monkeys 1 Test M aterial i Ammoniumperfluorooctanoate (.APFO/POAA) Sponsor 3MToxicologyServices - Medical Department 3MCenter, Building 220-2E-02 P.O. Box 33220 ' St. Paul; MN 55133-3220 Sponsor Representative Paul Lieder, Ph.D., DABT 3MToxicology.Services Building 220-2E-02 651-737-2678 . ' Study Director \ . Study Location(s) In vivo Testing Facility Analytical Testing Laboratory Kristen Hansen, Ph.D. 3MEnvironmental Technology and Safety Services Building 2-3E-09 651-778-6018 Covance Laboratories, Inc. 3301 Kinsman Boulevard Madison, Wisconsin 53704 3MEnvironmental Laboratory Building 2-3E-09 935 Bush Avenue St. Paul, MN 55106 ' Proposed Study Timetable Analytical Start Date Analytical Termination Date November 12,1998 May 12,1999 3MEnvironmental Laboratory Centre Analytical Laboratories, Inc. Page 2 of9 . Page 49 of 110 Centre Study No.: 023-011 Sponsor Protocol No: FACT-TOX-026 Protocol ftFACT-TOX-026 1. S tudy : Six month capsule toxicity study with ammoniumperfluorooctanoate (APFO/POAA) in cynomolgus monkeys. 2. P urpose The analytical portion of this study is designed to determine levels of (APFO/POAA) in the liver and serum of cynomolgus monkeys. Based on these results additional tissues or fluids may be analyzed. The in-life portion of this study was conducted at Covance Laboratories, study #6329231. ' . ' 3. Regulatory Compliance ' This study will be conducted in accordance with the United States Environmental Protection Agency Good Laboratory Practices Standards, 40 CFR 792. However, analysis of the test material mixture for concentration, solubility, homogeneity, and stability will not be conducted, and is the responsibility of the Sponsor. 4. Quality Assurance .. The 3M Environmental Laboratory QualityAssurance Unit will audit the protocol, study conduct, and final report in accordance with the Good Laboratory Practice Standards and 3M Environmental Laboratory Standard OperatingProcedures. ' ' 5. Test Material 5.1 Identification Ammoniumperfluorooctanoate (APFO/POAA) 5 .2 Source 3M Specialty Chemical Division 5 .3 ' P h y sica l Description Gelatin capsules 5.4 P u rity and Stability Determined by the Sponsor 5 .5 s to ra g e Conditions Room temperature . 5.6 R eserve Sam ples Responsibilityof the Sponsor 5 .7 D isposition Specimens will be retained per GLP regulation 5.8 S afety Precautions Refer to MSDS for chemicals used. Wear appropriate laboratory attire, and follow adequate precautions for handling biological materials and preparing samples for analysis. ' 3M Environmental Laboratory Centre Analytical Laboratories, Inc. Page 3 of 9 Page 50 of 110 Centre Study No.: 023-011 Sponsor Protocol No: FACT-TOX-026 Protocol # FAC T- TO X-C2S 6. Control Material' 6.1 Id e n tific atio n Monkey liver and serum ' 6.2 S ou rce Covance Laboratories, Inc. 6.3 P h y s ic a l D escription Liver and serum 6 .4 P u rity a n d S ta b ility Not applicable ; 6 .5 S to ra g e C onditions Frozen at -20 C 10 C 6 .6 R es e rv e S am ples Not applicable 6.7 D ispo sitio n Biological tissues and fluids are retained per GLP regulation 6 .8 S a fe ty Precautions Refer to MSDS for chemicals used. Wear appropriate laboratory attire, an'd-followadequate precautions for handling biological materials and preparing samples for analysis. 7. Reference Material 7.1 Identification Ammoniumperfluorooctanoate (APFO/POAA), lot #377 or #245 7.2 S o u rce 3M Specialty Chemicals 7.3 P h y s ic a l D escription White powder - 7.4 P u rity a n d Stability Responsibility of the Sponsor 7.5 S to ra g e Conditions Room temperature 7.6 R e s e rv e Sam ples Not applicable 7.7 D ispo sitio n Retained as per GLP regulation and 3M Environmental Laboratory policy 7.8 \ S a fety Precautions Refer to MSDS for chemicals used. Wear appropriate laboratory attire, and followadequate precautions for handling biological materials and preparing samples for analysis. 8. Test S ystem Cynomolgus monkeys were used as the test system, and were maintained and dosed as described in Covance protocol #6329-231. Group 1control animals did not receive the test substance. Groups 2, 3, and 4 received the test substance daily for 26 weeks, in increasing concentration per group. Two animals each from Groups 1, 3, and 4 were designated as recovery animals and were allowed a 13 week recovery period after cessation of treatment. 3M Environmental Laboratory Centre Analytical Laboratories, Inc. Page 4 of 9 Page 51 of 110 Centre Study No.: 023-011 Sponsor Protocol No: FACT-TOX-026 Protocol HFACT- TOX-026 9. S pecimen Receipt The 3M Environmental Laboratory will receive samples of the following body tissues and fluids from the indicated points in the study: Body tissue/fluid Collected Shipped Expected # of samples Serum- all animals 7 days post treatment, every two weeks thereafter . Packed on dryice for shipping Urine, feces - recovery animals Liver-all animals After 6,30, and 90 days Packed on dry ice for recovery shipping Artermination of the Shipped with final study ' serum samples on dry ice 264 frommain study 78 additional from recovery 18.urine, 18 feces 22 Total number of test animals: 16 Total number of control animals: 6 Samples sent to 3M Environmental Laboratories will be received and tracked according to applicable Standard Operating Procedures. ' - 10. P repara tory.Methods 10.1 FACT-M-1.0, Extraction of Potassium Perfluorooctanesulfonate or Other Anionic Fluorochemical Surfactant from Liver for Analysis Using HPLC-Electrospray/Mass Spectrometry ' 10.2 FACT-M-3.1, Extraction of PotassiumPerfluorooctanesulfonate or Other . Fluorochemical Compounds from Serum or Other Fluid for Analysis Using HPLC- \ Electrospray/Mass Spectrometry . 10.3 If preparatory methods other than those listed above are used, an amendment to this protocol will be written. 11. Analytical Methods . 11.1 FACT-M-2.0, Analysis of Fluorochemicals in Liver Extracts Using HPLCElectrospray/Mass Spectrometry 11.2 FACT-M-4.1, Analysis of Potassium Perfluorooctanesulfonate or Other Fluorochemicals in Serumor Other Fluid Extracts Using HPLC-Electrospray/Mass Spectrometry 11.3 If analytical methods other than those listed above are used, an amendment to this protocol will be written. 3M Environmental Laboratory Centre Analytical Laboratories, Inc. Page 5 of 9 Page 52 of 110 Centre Study No.: 023-0,11 Sponsor Protocol No: FACT-TOX-026 Protocol HFACT-TOX-025 12. Data Quality Objectives The number of spikes/duplicates, use 6f surrogates, and information on other data quality indicators is included in the analytical methods. In addition, the following criteria will be met: 12.1 L in e a rity r2> 0.980 12.2 Lim its o f detection / quantitation 12.2.1 Method Detection Limit (MDL) for APFO/POAA . a. Serum: 5 ppb , b. Liven 24 ppb 12.2.2 Practical Quantitation Limit (PQL) - Equal to the lowest standard in the calibration curve 12.3 D u p lic a te accep tab le p recisio n < 30% for the method 12.4 S p ik e acceptable reco veries 70% -130% ' 12.5 U se o f confirm atory m ethods Indeterminate samples will be re-analyzed 12.6 D em onstration o f specificity Chromatographic retention time, mass spectral daughter ion characterization 13. S ub-Contracted Analysis .- . All analyses as detailed in this protocol will be performed at 3M Environmental Laboratories, Building 2-3E-09, 935 Bush Avenue, St. Paul, MN 55106. 14. S tatistical Anal ysis . Averages and standard deviations will be calculated. The statistical methods that will be used are described below: . 14.1 D ata transform ations an d analysis Data will be reported as the concentration . (weight/weight or weight/vol) of APFO/POAAor metabolite per tissue or fluid, or of APFO/POAA or metabolite per unit of tissue or fluia. ' 14.2 S ta tis tica l analysis Statistics used may include regression analysis of concentrations over time, and standard deviations calculated for the concentrations within each dose group. If necessary, simple statistical tests, such as Student's t test, may be applied to evaluate statistical difference. 15. Report A report of the results of the study will be prepared by 3MEnvironmental Laboratory. The report will include, but not be limited to, the following, when applicable: ` 15.1 Name and address of the facility performing the study 15-2 Dates upon which the study was initiated and completed 3/VfE nvironm ental Laboratory Page 6 of 9 Centre Analytical Laboratories, Inc. Page 53 of 110 Centre Study No.: 023-011 Sponsor Protocol No: FACT-TOX-026 Protocol FACT- TOX-026 15.3 A statement of compliance by the Study Director addressing any exceptions to Good Laboratory Practice Standards ' 15.4 Objectives andprocedures as stated in the approved protocol, including any changes in the original protocol i 15.5 The test substance identification by name, chemical abstracts number or code number, strength, purity, and composition or other appropriate characteristics, if provided by the Sponsor 15.6 Stability and the solubilityof the test-substances under the conditions of administration, if providedby the Sponsor 15.7 A description of the methods used to conduct the test(s) 15.8 A description of the test system 15.9 A description of anycircumstances that'mayhave affected the quality or the integrity ' of the data 15.10 The name of the StudyDirector and the names of other scientists, professionals, and supervisory personnel involved in the study 15.11 A description of the transformations, calculations, or operations performed on the data, a summary and analysis of the analytical chemistry data, and a statement of the conclusions drawn fromthe analyses 15.12 Statistical methods used toevaluate the data, if applicable 15.13 The signed and dated reports of each of the individual scientists or other professionals involved in the study, if applicable 15.14 The location where rawdata and the final report are to be stored 15.15 A statement prepared by the Quality Assurance Unit listing the dates that study \ inspections and audits were made, and the dates of any findings reported to the Study Director and Management If it is necessary to make corrections or additions to a final report after it has been accepted, the changes will be made in the formof-an amendment issued bythe Study Director. The amendment will clearly identify the part of the final report that is being amended, the reasons for the amendment, and will be signed by the Study Director. 16. Location o f Raw Data, Records, and Final Report Original data, or copies thereof, will be available at 3MEnvironmental Laboratory to facilitate audits of the study during its progress and before acceptance of the final report. When the final report is completed, all original paper data, including those items listed below, will be retained in the archives of 3M Environmental Laboratory for at least aperiod of time as specified by regulation, and as established by 3MEnvironmental Laboratory Standard Operating Procedures. 3M Environmental Laboratory Centre Analytical Laboratories, Inc. Page 7 of 9 Page 54 of 110 Centre Study No.: 023-011 Sponsor Protocol No: FACT-TOX-026 Protocol KFACT-TOX-Q26 16.1 The following raw data and records will be retained in the study folder in the snidy/project archives according to 3M Environmental Laboratory Standard Operating Procedures: 16.1.1 Approved protocol and amendments 16.1.2 Study correspondence 16.1.3 Shipping records 16.1.4 Raw data 16.1.5 Approved final report (original signed copy) . ' . 16.1.6 Electronic copies of data 16.2 The following supporting records will be retained separately from the study folder in ' the archives according to 3M Environmental Laboratory. Standard Operating ' Procedures: 16.2.1 Training records ' 16.2.2 Calibration records 16.2.3 Instrument maintenance logs 16.2.4 Standard Operating Procedures, Equipment Procedures, and Methods ' 17. S a m p l e R e te n tio n Specimens will be maintained in the laboratory specimen archives for at least a period of time as specified by regulation, and as established by 3M Environmental Laboratory Standard Operating Procedures. 18. P rottocol Amendments and dev iations _' Planned changes to the protocol will be in the form of written amendments signed by the Study D ire c to r a n d th e S p o n so r's R e p resen tativ e. A m e n d m e n ts w ill b e c o n s id e re d as p a rt o f th e protocol and will be attached to the final protocol. All changes-to the protocol will be indicated in the final report. Any other changes wilibe in the form of written deviations, signed by the Study Director and filed with the raw data. 19. Attachments 19.1 A tta c h m e n t A Preparatory, and analytical methods 3M Environmental Laboratory Centre Analytical Laboratories, Inc. Page 8 of 9 Page 55 of 110 Centre Study No.: 023-011 Sponsor Protocol No: FACT-TOX-026 20. Signatures Paul Lieder, Ph.D, DABT, Sponsor Representative Protocol HFACT-TOX-026 Date Knsten Hansen, Ph.D., 3M Environmental Laboratory Study Director Date AI 3M Environmental Laboratory Centre Analytical Laboratories, Inc. Page 9 of 9 Page 56 of 110 Centre Study No.: 023-011 Sponsor Protocol No: FACT-TOX-026 S tudy Title 26-W eek Capsule Toxicity Study with Ammonium Perfluorooctanoate (APFO) in Cynomolgus Monkeys PROTOCOL AMENDMENT NO. 1 Am endm ent Date: July 23, 999 Perform ing Laboratory 3M Environmental Technology & Safety Services 3M Environmental Laboratory 935 Bush Avenue - St. Paul, MN 55106 Laboratory Project Identification ET&SS FACT-TOX026 LERNU27S2 . 3MEnvironm ental Laboratory Centre Analytical Laboratories, Inc. Exact Copy of Original Initial---- aDlabtaico Page 57 of 110 Centre Study No.: 023-011 Sponsor Protocol No: FACT-TOX-026 Protocol FACT-TOX025 Amendment No. 1 This amendment modifies the following portion(s) of the protocol: f: 1. P R O TO C O L r e a d s : Section 10.0 and 11.0 list the following methods to use for extraction and analysis: > FACT-M-1.0 "Extraction o f Potassium Perfluorooctanesulfonate or Other Anionic Fluorochemical Surfactant from Liver for Analysis Using PPLC-Electrospray/Mass Spectrometry" . FACT-M-3.1 "Extraction of Potassium Perfluorooctanesulfonate or Other Fluorochemical ' Compounds from Serum or Other Fluid for Analysis Using HPLC-Electrospray/Mass ' Spectrometry" .. . FACT-M-2.0 "Analysis ofFluorochemicals in Liver Extracts Using HPLC-Electrospray/Mass Spectrometry" ,, FACT-M-4.1 "Analysis o f Potassium Perfluorooctanesulfonate or Other Fluorochemicals in Serum or Other Fluid Extracts Using HPLC-Electrospray/Mass Spectrometry" A m e n d TO r e a d : The extraction and analytical methods FACT-M-3.1 and FACT-M-4.1, respectively, were updated on 04/27/99 to: ' ETS-8-4.1 "Extraction o f Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds from Serum for Analysis Using HPLC-Electrospray/Mass Spectrometry" ETS-8-5.1 "Analysis o f Potassium Perfluorooctanesulfonate or Other Fluorochemicals in Serum Extracts Using HPLC-Electrospray/Mass Spectrometry" R E A S O N : The methods were updated to replace the extraction solvent ethyl acetate with a different extraction solvent MTBE (methyl tert butyl ether), POAA and Monoester were. removed from the standard mix, and M556 was added to the standard mix. The analytical method was updated to include linear regression with 1/x weighting and a few minor changes in the HPLC 1100 instrument parameters. . 2. P r o t o c o l r e a d s : Section 10.0 and -11.0 list the following methods to use for extraction and analysis: FACT-M-1.0 "Extraction of Potassium Perfluorooctanesulfonate or Other Anionic Fluorochemical Surfactant from Liver for Analysis Using HPLC-Electrospray/Mass Spectrometry" ETS-8-4.1 "Extraction o f Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds from Serum or Other Fluid for Analysis Using HPLC-Electrospray/Mass Spectrometry" FACT-M-2.0 "Analysis ofFluorochemicals in Liver Extracts Using HPLC-Electrospray/Mass Spectromeuy" ETS-8-5.1 "Analysis o f Potassium perfluorooctanesulfonate or Other Fluorochemicals in Serum or Other Fluid Extracts Using HPLC-Electrospray/Mass Spectrometry" 3M Environmental Laboratory Centre Analytical Laboratories, Inc. Page 58 of 110 Centre Study No.: 023-011 Sponsor Protocol No: FACT-TOX-026 Protoccl FACT-TOX026 i Amendment No. 1 Amend TO read: The extraction and analytical methods FACT-M-1.0 and FACT-M-2.0, respectively, were updated on 07/22/99 to: ' '1 ' ETS-8-6.0 "Extraction o f Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds from Liver for Analysis Using HPLC-Electrospray/Mass Spectrometry" ETS-8-7.0 "Analysis o f Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds in Liver Extracts Using HPLC-Electrospray/Mass Spectrometry" `\ Reason: The methods were updated to replace the extraction solvent ethyl acetate with a different extraction solvent M l BE, POAA and Monester were removed from the standard mix, and M556 was added to the-standard mix. The analytical method was .updated to include linear regression with 1/x weighting and a few minor changes in the HPLC 1100 instrument parameters. 3. PROTOCOL reads: Section 10.0 and 11.0 list the following methods to use for extraction and analysis: '' ETS-8-6.0 "Extraction of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds from Liver for Analysis Using HPLC-Electrospray/Mass Spectrometry"ETS-8-4.1 "Extraction o f Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds from Serum or Other Fluid for Analysis Using HPLC-Electrospray/Mass Spectrometry" ETS-8-7.0 "Analysis of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds in Liver Extracts Using HPLC-Electrospray/Mass Spectrometry" ETS-8-5.1 "Analysis of Potassium Perfluorooctanesulfonate or Other Fluorochemicals in Serum or Other Fluid Extracts Using HPLC-Electrospray/Mass Spectrometry" .\ ' . Amend to read: Additional extraction and analytical methods, listed below, were added to the protocol: ETS-8-96.0 "Extraction o f Potassium Perfluorooctanesulfonafe or other fluorochemical compounds from Urine for Analysis Using HPLC-Electrospray/Mass Spectrometry" ETS-8-97.0 "Analysis of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds in Urine Extracts Using HPLC-Electrospray/Mass Spectrometry" R E A S O N : These methods were developed and validated for urine extraction and analysis after the original protocol was written and approved. 2M Environmental Laboratory Centre Analytical Laboratories, Inc. Page 59 of 110 Centre Study No.: 023-011 Sponsor Protocol No: FACT-TOX-026 ' Protocol FACT-TOX026 Amendment No. 1 4. P r o t o c o l R E A D S : Section 2.0 lists serum and liver as the matrices of interest for determination o f POAA. ' ' A M E N'D t o r e a d : Uirine will be included for determination o f POAA. R e a s o n : The methods were developed and validated for extraction and analysis o f urine after the original protocol was written and approved. 5. P r o t o c o l r e a d s : Section 6.0 lists monkey serum and liver as the control matrices received from Covance Laboratories. A M E N D TO r e a d : Control matrix monkey urine was obtained from Covance Laboratories and is maintained at a temperature o f -20 C - 10 C. All traceability information for this matrix will be included in the final report. R E A S O N : Addition of control urine matrix to the analytical protocol. 6. P r o t o c o l R E A D S : Section 12.2 lists monkey serum and liver method detection limits. A m e n d t o r e a d : Method detection limit for urine is 6 ppb. ' R E A S O N : Addition of method detection limit for urine matrix. "\ ' Amendment Approval /P a d tr ib a d i' Paul Lieder, Ph.D., DABT, Sponsor Representative id f'jz------ ------------- Kris J. Hansen, Ph.D., Study Director 3M Environmental Laboratory Centre Analytical Laboratories, Inc. Date Page 60 of 110 Centre Study No.: 023-011 Sponsor Protocol No: FACT-TOX-026 Study Title 6-Month Capsule Toxicity Study with Ammonium Perfluorooctanoate (APFO/POAA) in' Cynomolgus Monkeys PROTOCOL AMENDMENT N O .2 Amendment Date: 20 January 2000 Performing Laboratory 3M Environmental Technology & Safety Services 3M Environmental Laboratory ' 9 3 5 Bush A v e n u e . St. Paul, MN 55106 - Laboratory Project Identification ET&SS LRN-U2782 . FACT TOX-026 Covance Study: 6329-231 3M Medical Department Study: T-6889.3 3M Environmental Laboratory Centre Analytical Laboratories, Inc. Page 61 of 110 Centre Study No.: 023-011 Sponsor Protocol No: FACT-TOX-026 Protocol LRN-U2782 Amendment Number 2 This am endm ent m odifies the fo llo w in g portion(s) o f the protocol: 1. P r o to c o l r e a d s : The study director for the present study was identified in the protocol as Kristen J. Hansen, Ph.D. A mend to read: The role o f study director for the present study was reassigned to Paul Lieder, Ph.D., as o f 20 January 2000. The previous study director, Kristen Hansen,- has been reassigned to the role o f Principle Analytical Investigator. Reason: ' ' The role o f study director was reassigned in an effort to ensure compliance w ith Good Laboratory Practice Standards that outline study personnel requirements (refer to 40 CFR Part 792). ' 2. P r o t o c o l r e a d s : ' The sponsor for the present study was identified as Paul Lieder. A mend to read: The role o f sponsor for the present study was reassigned to John L. Butenhoff, Ph.D., as of 20 January 2000. R easo n: ;, To ensure that the study director does not also carry the duties of study sponsor, the sponsor role was reassigned. In this manner, personnel responsibilities and ' workload are more evenly balanced. 3. Protocol reads: 17. Sam ple Retention: Specimens will be maintained in the laboratory specimen archives fo r at least a period of tim e as specified by regulation, and as established by 3M Environm ental Laboratory Standard Operating Procedures. A mend to read: . 17. Specim en Retention: Specim ens will be maintained in the 3M Environmental Laboratory specim en archives. Any specimens sent to sub-contract laboratories will be returned to the 3M Environmental Laboratory upon completion o f analysis and submission o f the sub contract laboratory(s) final report. Specimens analyzed at sub-contract laboratories will be returned with the following documentation: the signed original chain o f custody and records of storage conditions while at the sub-contract facility. Reason: To define in detail the appropriate disposition o f specimens analyzed at subcontract laboratories. 3M Environmental Laboratory Centre Analytical Laboratories, Inc. Page 62 of 110 Centre Study No.: 023-011 Sponsor Protocol No: FACT-TOX-026 Protocol LRN-U2782 Amendment Number 2 4. Protocol reads: Section 16 states that the following raw data and records will be retained in the study folder in the archives according to AMDT-S-8: Approved protocol and am endm ents; study correspondence; shipping records; raw data; approved final report (original signed copy); and electronic copies o f data. Additionally, Section 16 states th a t supporting records to be retained separately from the study folder in the archives according to AMDT-S-8 w ill include at least the following: Training records; calibration records; instrument m aintenance logs; Standard Operating Procedures, Equipm ent Procedures, and M ethods; and appropriate specimens. A mend to read: Section 16 states: "The original data, or copies thereof, will be available at the 3M Environm ental Laboratory to facilitate audits o f the study during its progress and before acceptance of the final report. W hen the final report is completed, all original paper data, including: approved protocol and amendments, study correspondence, . shipping records, raw data, approved final report, and electronic copies of data will be retained in the archives o f the 3M Environm ental Laboratory. All corresponding training records, calibration records, instrum ent maintenance logs, standard operating procedures, equipm ent procedures, and methods will be retained in the archives o f the facility performing each analysis. Reason: . To direct subcontract laboratories in the disposition o f the items listed above. \ 3M Environmental Laboratory Centre Analytical Laboratories, Inc. Page 63 of 110 1 Centre Study No.: 023-011 Sponsor Protocol No: FACT-TOX-026 Amendment Approval Protocol LRN-U2782 Amendment Number 2 John Butenhoff, Ph.D., Sponsor Representative & L L _ ^ Kristen J. Hansen, Ph.D., Outgoing Study Director Date I !- F < .b - 2/>on Date ' /O, co p Date \ 3M Environmental Laboratory Centre Analytical Laboratories, Inc. Page 64 of 110 Centre Study No,: 023-011 Sponsor Protocol No: FACT-TOX-026 Study Title 6-Month Capsule Toxicity Study with Ammonium Perfluorooctanoate (APFO/POAA) in Cynomolgus Monkeys PROTOCOL AMENDMENT NO. 3 Am endm ent Date: 20 April 2000 Perform ing Laboratories L iv e r , Serum , a n d Ur in e A n a ly s e s 3M Environmental Technology and Safety Services Fluorine Analytical Chemistry Team Building 2-3E-09,935 Bush Avenue SL Paul, MN 55106 . , Fec es A nalM es Centre Analytical Laboratories, Inc. 304S Research Drive State College, PA 16S51 L a b o ra to ry P ro je c t Id e n tific a tio n ET&SS LRN-U2782 FACT TOX-026 Covance Study: 6329-231 3M Medical Department Study: T-6889.3 3M Environmental Laboratory Centre Analytical Laboratories, Inc. Exact Copy of Original _ '-At- OuIzlIoq i..itial Data Page 65 of 110 Centre Study No.: 023-011 Sponsor Protocol No: FACT-TOX-026 Protocol LRN-U2732 Amendment Number 3 This amendment m odifies the fo llo w in g portion(s) o f the p ro toco l: 1. P r o t o c o l r e a d s : . : Tne amended section 2.0 text states that this study is designed to 'determine levels o f APFO/POAA in the liver, serum, and urine of cynomolgus monkeys. A mend to read: ' This study is designed to determine levels of APFO/POAA in the liver, serum, feces, and urine of cynomolgus monkeys. , i Reason: . '. The analysis of fecal tissue for the target chemical and/or its analytes was added to the scope of the study following the issuance o f the protocol. Feces extraction and analytical methods were not validated and approved prior to protocol approval. ' 2. Protocol reads: . The amended section 6.0 lists monkey liver, serum, and urine. A mend to read: . _ Add: monkey feces with a physical description of feces. Reason: ! Analysis of fecal tissue for the target chemical and/or its analytes was added to the scope of the study following the issuance of the original protocol. :' _ 3. Protocol Reads: The amended Section 12.2.1 items a., b., and c., list the Method Detection Limits for matrices analyzed in this study. A mend to read: The method detection limits for all compounds and matrices \vill be taken from the methods used for extraction and analysis. Reason: . The method detection limits are specific to the 3M Environmental Laboratory. This statement w a s a d d e d to a llo w for sub-contracted analyses, revised methods, and added matrices. 4. P rotocol Reads: Section 13. lists the laboratories that will be conducting analyses for this study. A mend to read: Add: Centre Analytical Laboratories, Inc., 3048 Research Drive, State College, PA 16801 Reason: Feces analyses were added to the scope of this study. The sub-contract laboratory performing analyses was not in the original protocol. 3M Environmental Laboratory Centre Analytical Laboratories, Inc. Page 66 of 110 Centre Study No.: 023-011 Sponsor Protocol No: FACT-TOX-026 Protocol LRN-U2782 Amendment Number 3 5. Protocol Reads: ' Sections 10.3 and 11.3 state that if methods other than those listed axe used in this study, an amendment will be written to include the new methods. A mend to read: ! The feces extraction and analytical method used by Centre Analytical Laboratories will be; 00M-023-O03 (Revision 2), "Determination of Fluorochemical Residues in Monkey/Rat Feces by LC/MS/MS." Reason: The sub-contract laboratory performing feces analyses was added to the scope of this study; this method was'not validated and approved prior to protocol approval. Amendment Approval John L. Butenhoff, Ph.D., Sponsor Representative Paul Lieder, Ph.D., Study Director Date /?,P.OOD Date 3M Environmental Laboratory Centre Analytical Laboratories, Inc. Page 67 of 110 Centre Study No.: 023-011 Sponsor Protocol No: FACT-TOX-026 3M : Study Title 26-W eek Capsule Toxicity Study with Ammonium Perfluorooctanoate (APFO) in Cynomolgus Monkeys PROTOCOL AMENDMENT NO. 4 Amendment Date: August 2,2000 Performing Laboratories Covance Laboratories Inc. 3301 Kinsman Boulevard Madison, W I53704 3M Environmental Technology & Safety Services 3M Environmental Laboratory 935 Bush Avenue St. Paul, MN 55106 L a b o r a to r y P r o je c t Id e n tific a tio n FACT-TOX-026 ' LIMSU2782 Covance 6329-231 3M Medical Department Study: T-6889.3 Centre Analytical Laboratories, Inc. c.'set Copy Uc Page 68 of 110 Centre Study No.: 023-011 Sponsor Protocol No: FACT-TOX-026 Protocol TOX-026 Amendment 4 This amendment modifies the following portion(s) of the protocol: 1. P rotocol reads: S t u d y Tit l e : "6-Month Capsule Toxicity protocol for the analytical phase o f the study). (on the title page and page 2 o f the A m end to r ea d : S t u d y Tit l e : "26-Week Capsule Toxicity [..]" (on the title page and page 2 o f the protocol for the analytical phase o f the study). R easo n: - To correct the title for the analytical phase o f the study. 2. P rotocol reads: Data Quality Objectives (Section 12., page 6) A m end to r ea d : Add to this section: LOQ in feces o f 10 ng/g Rea so n : ' To specify the analytical limits fc-r feces analyses. 3 . P r o t o c o l r e a d s : (page 2) S t u d y D ir e c t o r : Peter J. Thomford and Paul Lieder Te s t in g F a c il it y : Covance Laboratories S p o n so r : APME AdHoc APFO Toxicology Working Group and 3M Toxicology Services - Medical Department S p o n so r R e p r e se n t a t iv e : David Farrar and John Butenhoff A m en d to r ea d : ~' S t u d y D ir e c t o r : Paul Lieder Te s t in g F a c il it y : 3M Tdxicology Services - Medical Department S p o n s o r : APME AdHoc APFO Toxicology Working Group (including 3M Toxicology Services - Medical Department) S p o n s o r Re p r e s e n t a t iv e : David Farrar (in-life) and John Butenhoff (analytical) Rea so n : To reassign the testing facility, in order to abide by the GLP requirement for one study director. To clarify the responsibilities o f all parties included in this study. Centre Analytical Laboratories, Inc. Page 69 of 110 Centre Study No.: 023-011 Sponsor Protocol No: FACT-TOX-026 Protocol TO X-026 Amendment 4 4. P rotocol reads: , P r in c ip a l A n a l y t ic a l I n v e s t ig a t o r : Kristen Hansen, Ph.D. (Amendment No 2 to TOX 026, Section 1.) - Am end to read: Add; P r in c ip a l A n a l y t ic a l I n v e s t ig a t o r s ;. Kristen Hansen, Ph.D. a n d Enaksha Wickremesinhe, Ph.D. Reaso n: . To specify the PAI at Centre. ' 5. P rotocol r ea d s: A n a l y t ic a l T e r m in a t io n D a t e : May 12,1999 (under Proposed Study Timetable, page A m en d to read: A n a l y t ic a l T e r m in a t io n D a t e : September 30,2000 Rea so n : To allow for the analyses o f all samples. 6. P rotocol r ea d s: ' L o c a t io n o f Ra w D a t a , R e c o r d s , a n d F in a l R e p o r t (Section 16., page 7) A m end to r ea d : Add: After issuing their final report, Centre will forward all original study-specific raw data to 3M Environmental Laboratories, together with copies of appropriate facilityspecific raw data applicable to this study. Centre will maintain a copy of the applicable study-specific raw data, protocol and analytical report in the Centre archives. Re a so n : To specify the archival requirement for the portion of the data developed by Centre. 7. P r o t o c o l r e a d s : S a m p l e R e t e n t io n (Section 17., page 8) Am end to read: After the analytical report on feces is signed by the PAI, all feces specimens of this study will be returned to 3M Environmental Laboratory. These specimens may then be discarded by written direction of the study director. Specimens of blood, plasma, red blood cells, serum, bile, and urine may also be discarded by written direction of the study director after Centre Analytical Laboratories, Inc. Page 70 of 110 Centre Study No.: 023-011 Sponsor Protocol No: FACT-TOX-026 Protocol TO X-026 Amendment 4 the analytical report for the 3M Environmental Laboratory analyses is signed by the studv director. , ' R eason: T o specify the handling o f the above biological specimens, and to define when the quality assurance verification is considered complete. Centre Analytical Laboratories, Inc. Page 71 of 110 Centre Study No.: 023-011 Sponsor Protocol No: FACT-TOX-026 Amendment Approval Protocol TOX-026 Amendment 4 ____ $ 2 ^ -- John Butenhoff, Ph.D. Sponsor Representative, Analytical Phase ' Date Paul.L ied er, Ph.D., DABT 3M Study Director _ /c r Se r r Date f/z /t.o o linaksha Wickremesinhe, Ph.D. Date Centre Analytical Laboratories, Principal Analytical Investigator Centre Analytical Laboratories, Inc. Page 72 of 110 Centre Study No.: 023-011 Sponsor Protocol No: FACT-TOX-026 Study Title 26-W eek Capsule Toxicity Study with Ammonium Perfluorooctanoate (APFO) in Cynomolgus Monkeys ' . ' PROTOCOL AMENDMENT NO. 5 Amendment Date: October 12,2000 Performing Laboratory 3M Environmental Technology & Safety Services 3M Environmental Laboratory 935 Bush Avenue S t Paul, MN 55106 Laboratory Project Identification FACT-TOX-026 ' ET&SS LRN U2782_ Covance 6329-231 . 3M Medical Department Study: T-6889.3 3M Environmental Laboratory Centre Analytical Laboratories, Inc. Exact Copy of Original Initial Data Page 73 of 110 I Centre Study No.: 023-011 Sponsor Protocol No: FACT-TOX-026 ' , Protocol FA C T TO X-026 . Amendment No. 5 This amendment modifies the following portion(s) of the protocol: 1- P r o t o c o l r e a d s : Pnncipal Analytical Investigators: Kristen Hansen, PhD . and Enaksha Wickremesinhe ' 2. A mend to read: . Principal Analytical Investigators: Kristen Hansen, PhD . and Emily Stauffer. i Reason: To change role o f the Principal Analytical Investigator at Centre'Analytical Laboratories 3M Environmental Laboratory Centre Analytical Laboratories, Inc. Page 74 of 110 Centre Study No.: 023-011 Sponsor Protocol No: FACT-TOX-026 , , Protocol FA C T TO X-026 Amendment No. 5 Amendment Approval John L. Butenhoff, Ph.D. Sponsor Representative, Analytical Phase fo /ri/ O . Date i ' C io d lllA u h P aul Lieder, Ph.D,, DBT 3M Study Director l h ih o Date 1'M o o Date Centre Analytical Laboratories, Principal Analytical Investigator 3ME nvironm ental Laboratory Centre Analytical Laboratories, Inc. Page 75 of 110 3M Centre Study No.: 023-011 Sponsor Protocol No: FACT-TOX-026 ! . Study Title 26-Week Capsule Toxicity Study with Ammonium Perfluorooctanoate (APFO/POAA) in ' Cynomolgus Monkeys PROTOCOL AMENDMENT NO. 2 Amendment Date: January 11,2001 Performing Laboratories Covance Laboratories Inc. 3301 Kinsman Boulevard Madison, W I5 3 7 04 3M Environmental Technology & Safety Services 3M Environmental Laboratory 935 Bush Avenue St. Paul, MN 55106 ! . Laboratory Project Identification Covance Study: 6329-231 Proposal No. w6806a 3M Medical Department Study: T-6889.3 ET&SS LRN-U2782 FACT TOX-026 i 3M Environmental Laboratory Centre Analytical; Laboratories, Inc. i cY.jj,'..:.; Di:a Page 76 of 110 Centre Study No.: 023-011 Sponsor Protocol No: FACT-TOX-026 Covance $tudy 6329-231 Amendment 2 I. ,. T h is a m e n d m e n t m o d ifies th e fo llo w in g p o rtio n (s ) o f th e p ro to c o l: i' 1. Protocol reads: 3M Analytical Phase Protocol, Section 9. S pecimen Receipt It was expected that there would be 18 feces specimens and 18 urine specimens from the 1 recovery animals collected and shipped for analysis. Amend t o read: 1 There were 300 feces specimens collected and sent to Centre Analytical Laboratories for analysis. There were 300 urine specimens collected and sent to 3M Environmental Laboratory for analysis. R e a s o n : Feces and urine specimens were.collected every two weeks during the main part of the study, in addition to the samples pulled from the recovery animals. 2 . Protocol reads: Covance P rotocol 6329-231, page 2 The study location is designated as Covance Laboratories, Inc. Amend t o read: The testing facility is 3M Toxicology Services--Medical Department, 3M Center, Building 220-2E-02, P.O. Box 33220, St. Paul, MN 55133-3220. The test sites are Covance Laboratories, Inc., 3301 Kinsman Boulevard, Madison, W I53704 for the in-life phase and 3M Environmental Technology & Safety Services, 3M Environmental Laboratory, 935 Bush Avenue, St. Paul, MN 55106 for the analytical phase. R E A S O N : GLPs allow for only one study location per study. The study director and sponsor reside at 3M Toxicology Services. 3. Protocol reads: 3M Analytical Phase P rotocol TOX-026 and the Covance 6329-231: Originally there were two protocols for the same study. Amend to read: Amendment to the Covance Protocol 6329 -231: Covance Protocol 6329-231 is the one protocol for this study. Attached to this amendment are the protocol FACT TOX-026 and Amendments 1-5 for the analytical phase of the study. All of the following study, protocol and project identifications pertain to the same study. Covance Study 6329-231 (In-Life Phase) Proposal No. w6806a FACT-TOX-026 (Analytical Phase) LRN # U2782 3M Medical Department Study: T-6889.3 . R e a s o n : GLPs allow for only one protocol for a study. All study, protocol and project identifications are listed to provide a documented link between documents. i 3M Environmental Laboratory I. Centre Analytical Laboratori.es, Inc. Page 77 of 110 Centre Study No.: 023-011 Sponsor Protocol No: FACT-TOX-026 Covance Study 6329-231 Amendment 2 4. P r o t o c o l r e a d s : A m e n d e d 3 M A n a l y t ic a l P h a s e T O X -026 A m e n d m e n t # 4: Peter Thomford was removed from position as study director, but his status was not redefined. (i A mend to read: i Dr. Peter Thomford will take bn the position of Principal In-Life Investigator. l' R E A S O N : GLPs allow only pne study director for a study. Dr. Thomford's role needs to be redefined once he was removed as study director. 5. P r o t o c o l r e a d s : 3M A n a l y t ic a l P h a s e TOX-026, s e c t io n 4.0 a n d 3 M A m e n d m e n t . #3 to TOX-026: Amendment #3 adds Centre to list of performing laboratories. A m e n d t o r e a d : S e c t io n 4 o f TOX-026, A n a l y t ic a l P h a s e : Centre Analytical Labs Quality Assurance Unit will audit the data they provide for the study, at their facility. Reason: To clarify that 3M QAU will not be responsible for auditing Centre's data, as the protocol currently states. 6. P r o t o c o l r e a d s : 3M A n a l y t ic a l P h a s e P r o t o c o l F A C T T O X -0 26 , S e c t i o n 7. R e f e r e n c e M a t e r ia l: Protocol states that the reference material used will be lot #377 or #245. A mend to read: Ammonium perfluorooctanoate, lot #332, is the reference material used at Centre Analytical Labs. ' Reason: ' When protocol was written, it was anticipated that lot #377 or #245 would be used as reference material. Feces analyses performed at Centre Labs were not included in the original protocol but was added in Amendment 3. Centre actually used lot #332 for the reference. 3M Environmental Laboratory Centre Analytical Laboratories, Inc. Page 78 of 110 Centre Study No.: 023-011 Sponsor Protocol No: FACT-TOX-026 Covance Study 6329-231 Amendment 2 Amendment Approval ' Paul Lieder, Ph.D., Study Director Pis Feb o ) Date 3M E nvironm ental Laboratory Centre Analytical Laboratories, Inc. Page 79 of 110 Centre Study No.: 023-011 Sponsor Protocol No: FACT-TOX-026 3048 Research Drive, State College, PA 16801. Phone: (814) 231-8032, Facsimile: (814)231-1253 PROTOCOL DEVIATION Deviation Number: 1 . Date of Occurrence: 10/3/00 Centre Study Number 023-011 Sponsor Protocol Number FACT-TOX-026 DESCRIPTION OF DEVIATION I. 12.4 Page 6: Accepted recoveries of 66% for PFOSAA, 54% for M570, and 62% for M 556 for Centre sample 0008435 Spk B in Set 100200A. 1. Protocol deviation issued Recorded By/Date: ACTIONS TAKEN i r__ amendment issued SOP revision, e tc . ^ S v . f ~ ' y lol5(od IMPACT ON THE STUDY I. No negative impact on the study. Principal Ininstigator Signature Dan Sponsor Representative Signature /o Date STUDY PWttTCiL CAL QAU Review______tifi frier; Centre Analytical Laboratories, Inc. / Febmary 12, 1998/2 ( I Page 80 of 110 Centre Study No.: 023-011 Sponsor Protocol No: FACT-TOX-026 C e n tr e A n a ly tic a l L a b o r a t o r i e s , In c . 3048 Research Drive, State College, PA 16801. Phone: (814) 231-8032, Facsimile: (814) 231-1253 PROTOCOL DEVIATION Deviation Number: 2 Date o f Occurrence: (1 ) 10/07/00. (2) 10/10/00, (3) 10/10-11/00, (4) 10/10-11/00 Centre Study Number. 023-011 Sponsor Protocol N um ber FACT-TOX-026 ' DESCRIPTION OF DEVIATION 1. 12.4 Page 6: A ccepted recovery o f 1 3 7 % for PO A A for Centre sam ple 0 0 0 8 4 6 1 Spk B in Set 100600AD. . 2. 12.4 Page 6: A ccepted recovery o f 1 3 9 % for PO A A for Centre sam p le 0 0 0 8 4 8 2 Spk B in Set 100900A. . 3. 12.4 Page 6: Accepted recovery o f 133% for POAA for Centre sam ple 0 0 0 8 5 0 0 Spk B in Set 101000A. ; 4. 12.4 Page 6 : Accepted r value o f 0 .9 7 0 4 4 4 for EtFOSE-OH calibration curve in Set 10 1 0 0 0 . ACTIONS taken i e amendment issued. SOP revision, etc.. 1-4. Protocol deviation issued. Recorded By/Date: ii uj / IMPACT ON THE STUDY 1-?. No negative impact on the study. 4. N o negative impact on the study because still met method requirements for linearity. Principal Investigator Signature Sponsor-Representative Signature &LtaCL CAL QAU Review n Date /O J ^ r / Z o c Date Hji?FO f J C u llltflio t) 2oq/ February 12, 1998/2 Centre Analytical Laboratories, Inc. Page 81 of 110 Centre Study No.: 023-011 Sponsor Protocol No: FACT-TOX-026 C e n tr e A n a ly tic a l L a b o r a t o r i e s , In c . 3048 Research Drive, Slate College, PA 16801. Phone: (814) 231-8032, Facsimile: (814) 231-1253 PROTOCOL DEVIATION Deviation Number 3 Date of Occurrence: (1 ) 10/18-19/00, (2) 10/19-20/00, (3) 10/20-21/00, (4 ) 10/22-23/00, (5 ) 10/23/00 (6 ) 10/25/00 Centre Study Number 023-011 Sponsor Protocol Number: FACT-TOX-026 DESCRIPTION OF DEVIATION 1. 12.4 Page 6: Accepted recovery of 136% for PFOSEA for Centre sample 0008585 Spk A in Set 101600AR. ' 2. 12.4 Paae 6: Accepted recovery of 141% for POAA for Centre sample 0008626 Spk B in Set 10I800A. ~ 3. 12.4 Page 6: Accepted recovery of 138% for EtFOSE-OH for Centre sample 0008650 Spk A and 63% for PFOSAA and 57% for PFOSEA for Centre sample 0008651 Spk B in Set 101900A. 4. 12.4 Page 6: Accepted recovery of57% for PFOS for Centre sample 0008667 Spk B in Set 102000A. ' 5. 12.4 Page 6: Accepted recovery of 170% for POAA for Centre sample 0008667 Spk B in Set 102000AD.' .. 6. 12.4 Page 6: Accepted recovery of 56% for POAA for Centre sample 0008689 Spk A in Set 102300AR. 1-6. Protocol deviation issue ACTIONS TAKEN i.e.. amendment issued. SOP revision, etc.. Recorded By/Date: A n 'P fer' IMPACT ON THE STUDY 1-6. No negative impact on the study. Pnncfpat Inve^gatorSignature ?. Sponsor Representative Signature ^TU-Di iifr- DHt'DL CAL QAU Review. ______fCfr/A Allibi______________ ___ nAz\ OO Date /o Date h'V Io M *r February 12. 1998/2 Centre Analytical Laboratories, Inc. Page 82 of 110 Centre Study No.: 023-011 Sponsor Protocol No: FACT-TOX-026 C e n tr e A n a ly tic a l L a b o r a t o r i e s , In c . 3048 Research Drive, State College, PA 16801. Phone: (814) 231-8032, Facsimile: (814) 231-1253 PROTOCOL DEVIATION Deviation Number 4 Date of Occurrence: (1 & 2) Entire Study Centre Study Number. 023-011 Sponsor Protocol Number. FACT-TOX-026 DESCRIPTION OF DEVIATION 1. 1 Amendment No. 3: Throughout the study, samples were analyzed for EtFOSE-OH, PFOSAA M570, M556, PFOSEA, PFOS, PFOSA, and POAA, instead of only for POAA /. ' 2. 7 Reference Material Page 4: The lot of POAA used for this study was 332. ACTIONS TAKEN i.e.- amendment issued. SOP revision, etc.. 1-2. Protocol deviation issued. Recorded By/Date: F Wl K 'jfij) IMPACTON THE STUDY 1-2. No negative impact on the study. Pnncipal I n s tig a to r Signature Sponsjor Representative Signature rn.d.fcC'liJl J A ''/ 0Al(r CAL QAU Review. J2t[2A|o? Date /O Tese?/ Date ' Vtt, <~4-U}!crc February 12. 1998/2 Centre Analytical Laboratories, Inc. Page 83 of 110 Centre Study No.: 023-011 Sponsor Protocol No: FACT-TOX-026 APPENDIX B Determination of Fluorochemical Residues in Monkey/Rat Feces by LC/MS/MS (Revision 2) Method #00M-023-003, revision 2 and Deviations and Modifications Centre Analytical Laboratories, Inc. Page 84 of 110 Centre Study No.: 023-011 Sponsor Protocol No: FACT-TOX-026 TITLE Determination o f Fluorochemical Residues in Monkey/Rat Feces by LC/MS/MS (Revision 2) ! . AUTHORS i Enaksha Wickremesinhe, Shaozhi Zheng, and John Flaherty DATE ISSUED June 02,2000 SPONSOR 3M Environmental Technology and Safety Services' - Building 2-3E-09 PO Box 33331 St. Paul, MN 55133-3331 PERFORMING LABORATORY Centre Analytical Laboratories, Inc. (Centre) 3 04S Research Drive . State College, PA 16801 - CENTRE STUDY NUMBER 023-003 CENTRE METHOD NUMBER 00M-023-003, revision 2 TOTAL NUMBER OF PAGES 20 Centre Analytical Laboratories, Inc. Page 85 of 110 Centre Study No.: 023-011 Sponsor Protocol No: FACT-TOX-026 Ceacre Method No. : OQM-023-003, revision 2 MANAGEMENT APPROVAL The work cited here was performed'in conformance with applicable standard ODeratin procedures and general GLP's. : *3 Principal Investigator Centre Analytical Laboratories, Inc Shaozhr Zheng '' Principal Investigator Centre Analytical Laboratories, Inc. --C>2 -v~0 Date . glm F lJ a T /J o h n Flaherty / Laboratory Manager Centre Analytical Laboratories, Inc. Date 2 -JuaAT-oo ' Richard A ^rszzh, Ph.D. President Centre Analytical Laboratories, Inc. Date di'1i-MNC-CO - K iu. -- Kns Hansen, Ph.D. Group Leader 3M Environmental Laboratory / ('/O Date , Centre Analytical Laboratories, Inc. Study # 023-003 Centre Analytical Laboratories, Inc. Page 2 Page 86 of 110 Centre Study No.: 023-011 Sponsor Protocol No: FACT-TOX-026 o->IO . Centre Method N o.: 00M-O23-003, revision 2 ' . TABLE O F CONTENTS T IT L E P A G E ................................................................................................... ;...........- ..... ................ 1 M A N A G E M E N T A P P R O V A L ................................................................................................ 2 T A B L E O F C O N T E N T S ............................. ..................................................................................... 3 1. S U M M A R Y ...................................................................................................................................4 F L U O R O C H E M IC A L S T A N D A R D S ...................................................................................4 . C H E M IC A L S A N D SU P P L IE S ...............................................................................................7 3 . 1 . Chemicals..................................................................................................... ^ 3.2. FLUOROCHEMICAL STANDARDS........................................... ...........................`.7 3 .3 .;Equipment and Supplies....................................................... - ............................7 3 .4 . Solutions................................................................................................................. 3.5. P reparation of Stock, Fortification, and Calibration Solutions.........9 3.5.1. Stock solutions.....................................................................................9. 3 .5 .2 . F ortification S o lu tio n s......................... ............................... ............................ 9 3 .5 .3 . Calibration Standards...... .................................................................. 1 0 4 . M E T H O D ......................................................................................................................................... 4.1. Flow Diagram.................................................................................................... . 4 .2 . Sample Processing............................................................................................ .... 4 .3 . Sample Preparation.......................................................................................... 11 4.3.1. Matrix Blanks.............................................................. - ........................12 4 . 3 . 2 . M atrix Zero B la n k s ...... :.................................................................................1 2 4 .3 .3 . Calibration Stan dards................... 12 4 .3 .4 . Continuing Calibration Verification Standards................................... 12 4 . 3 .5 . Q C R ecovery S a m p les................................................................................... .. 4.4. Extraction.................................. 12 4 .4 .1 S P E Column Preparation.................................................................................1 3 4 . 4 . 2 S ilanization o f Pear-Shaped F la s k s.............................................................. 13 4 . 4 . 3 Standardization o f S P E C o lu m n s........................... .....................................13 .4.5. A n a l y s is b y L C /M S /M S ......................... 14 4 .5 .1 . LC /M S/M S S ystem and O perating C o n d ition sfE lectrosp ray)...........1 4 . 4 .5 .2 . Example Tune File Parameters........................................................... . 4 .5 .3 . Calibration Procedures.......................................................................1 6 4.5.4. Sample Analysis............................. - .................. ...............................17 : 4 . 6 . Performance Criterla................................................................................................1 8 4 . 7 . T ime Required for Analysis.................................................................................... 1 8 5. C A L C U L A T IO N S .................................................................................................................... 1 3 5 . 1 . Analyte F o u n d ............................................................................................................. I 3 5 . 2 . Q C Re c o v e r y ................................................................................................................ .. 5 . 3 . Mean Response Factor...............................................................................................1 9 6 . S A F E T Y ......................................................................................................................................1 9 A T T A C H M E N T 1 ............................................................................ ................................................2 0 Centre Analytical Laboratories, Inc. Study # 023-003 Centre Analytical Laboratories, Inc. Page 3 Page 87 of. 110 Centre Study No.: 023-011 Sponsor Protocol No: FACT-TOX-026 . 1. SU M M A R Y _ Centre Method N o.: 00M-023-003, revision 2 T h is docum ent details a m ethod o f analysis for residues o f perfluorooctane sulfonate TM F o s ). perfluorooctane su lfon ylam id e (PFO SA ), perfluorooctane su lfonylam idoethvl- acstate (PFO SA A ), perfluorooctanesulfonylethylam ide (PFO SEA ), perfluorooctanoate (P O A A ), 2(N-ethyIperfiuorooctanesulfonam ido)-ethyl alcohol (Et-FOSE-OH) M 5 7 0 and M 5 5 6 in m on k ey feces. The chem ical form ulas o f the analytes are given in S e -t'o ? o f this method. . "1n" fo r sa m p le extraction, to add the option o f hom ogenizing feces sam ples before w eig h !TM to cla rify the preparation o f QC recovery sam ples and to correct som e t v p o e r a ^ f i errors in m ethod 00M -023-003 revision 1. Rat feces is being added to the m ethod h o w ev er, it w a s validated for the analysis o f PFOS only. Therefore, this m ethod s h o u ld be u sed for the analysis o f PFO S only, in rat feces. R esid u es o f these fluorochem icals are extracted from feces w ith acetonitrile (A C N ) T he A C N extract is filtered and passed through a carbon solid-phase extraction (S P E ) colu m n A p p r o x im a te ly 3 - 4 drops o f 1-octanol are added to the extract and evap o ra ted to n ea r d ryn ess usin g rotary evaporation. Each extract is then reconstituted w ith m eth anol Q uantification o f PFOS, P F O S A P F O S A A P F O S E A Et-FOSE-OH, M 556, M 5 7 0 td P O A A is accom plished b y liquid chromatography/tandem m ass spectrom etry (L C /M S/M S) analysis using selected reaction m onitoring (SR M ). T h e p r o p o sed lim it o f quantitation (L O Q ) for this m eth od is 1 0 pp b ea ch f o r P F O 5? P F O S A , PFO SA A , PFO SEA , Et-FOSE-OH, M 556, M 570, and P O A A . T his is based on s tu d ie s con d u cted during the d ev elo p m en t o f this m eth od u sin g co n tro l m o n k e y f e *s 2. FLUOROCHEMICAL STANDARDS M olecu lar structures o f PFOS, P F O S A PFO SA A , P F O S E A Et-FO SE-O H M 5 7 0 , and P O A A are given below , ' ' PFOS M o le cu la r w eight: 4 9 9 (C sF 17S 0 3~ ) o ' C^fjyS-- O' q Chemical Name56Perrluorooctane sulfonate : N ote: T he neutral m olecule and standard form that the.P F O S (a n io n ) is d erived from is p o ta ssiu m perflu orooctane su lfon ate [C ,F ,- S 0 3K ], m o le c u la r w e ig h t 5 3 8 Centre Analytical Laboratories, Inc. Study H023-003 Page 4 Centre Analytical Laboratories, Inc. Page 88 of 110 ; Centre Study No.: 023-011 Sponsor Protocol No: FACT-TOX-026 pfosa M olecular weiaht: 499 Centre Method No. : 00M-023-003, revision 2 Cr17S -- NH2 0 Chemical Name = Perfluorooctane sulfonylamide PFOSAA M olecu lar weight: 585 / ch2cooh CsrS-- N \ ch2ch3 Chemical Name = perfluorooctane sulfonylamidoethylacetat PFOSEA M olecular weight: 527 /H Car]7S-- N \ CH2CH3` . . ' Chemical Name = Perfluorooctanesulfonylethylamide POAAM olecular weight: 413 C 7r15COO- Chemical Name = Perfluorooctanoate N ote: T h e neutral m olecule and standard form that the P O A A (a n io n ) is d e r iv e d fro m is am m o n iu m p erflu oroctanoate [C 7F ,5C O O N H 4], m o le c u la r w e ig h t 4 3 1 6 Centre Analytical Laboratories, Inc. Study # 02J-G03 Centre Analytical Laboratories, Inc. Page 5 Page 89 of 110 Centre Study No.: 023-011 Sponsor Protocol o: FACT-TOX-026 . Centre Method No.: OOM-023-003,revision 2 Et-FO SE-O H M olecu lar weight: 571 o(I CjrtjS-- N 0II NCH2Ob ; .! t Chemical Name = 2(N-ethy!peffluorooctanesulfonamido)-ethyl alcohol M 556 M olecular w eight.-557 o / ch2ccoh CarS-- N II . ' . Chemical Name = M556 M 570 M olecu lar w eisht: 571 y ch2ccoh Car)7S-- N \ ch3 Ch*zncal Name = W570 th pfo s M olecular weight: 42S 1-H, 1-H, 2-H, 2-H, C8F 13SO3H Chemical Name = 4-H, perilucrooctane sulfonic acid Centre Analytical Laboratories, Inc. Study * 013-003 Centre Analytical Laboratories, Inc. Page 6 Page 90 of 110 Centre Study No.: 023-011 Sponsor Protocol No: FACT-TOX-026 tase Method No. : OOM-023-003, revision 2 3. CHEMICALS AND SUPPLIES 3.1. C h e m ic a l s ' C hem ical G rade Source C atalog N o. A cetonitrile (ACN) Carbon 120/400 mesh M ethanol, (M eOH ) 1-O ctanol A m m onium Acetate L -A scorb ic acid T oluene . D im etyldichlorosilane W ater HPLC (V W R ) J. T. B aker - S u p e lco ' EPLC (V W R ) J. T . B a k er Reagent A ldrich Chem ical Reagent Sigm a-A ldrich Reagent Sigm a-A ldrich R eagen t (V W R ) J. T.-Baker R eagent. S u p e lco Type I in house JT 9017-3 57210-U JT 9093-2 111-87-5 A -7330 L -5960 JT 9460-3 3-3009 - (T y p e I w ater = electrical resistivity, m in im u m o f 1 6 .6 7 M Q /cm at 2 5 C , fro m a L abconco wateipro workstation) ' 3 .2 . F lu o r o c h em ic a l S t a n d a r d s St-andard PFOS PFOSA PFOSAA PFOSEA Et-FOSE-OH M 556 M 570 POAA ' THPFOS ' Source 3M 3M 3M 3M 3M 3M 3M 3M 3M . " '" 3 .3 . E q u ipm e n t a n d S u pplies _____________________ E q u ip m e n t__________ ___________ B a la n ce, analytical, (disp lay at lea st 0 .0 0 0 1 s:) B alance, top loading, (display at least 0.01 g ) Rotary evaporator ' R otary evaporator trap Pear-shaped flasks (1 2 5 m L) 20-m L SPE tubes (cat. no. N 0 5 7 1 7 7 ) w ith fri ts V acuum pump V isiprep vacuum manifold < Centre Analytical Laboratories, Inc. Study # 023-003 S o u rce M ettler M ettler Buchi Buchi Pyrex S u p e lc o B uchi Su oelco 'p Centre Analytical Laboratories, Inc. Page 91 of 110 Centre Study No.: 023-011 Sponsor Protocol No: FACT-TOX-026 Cintre Method No. : OQM-023-003, revision 2 W rist-action shaker 20-m L polyethylene scintillation vials w ith lids 20-m L syringe ' 25-m m diameter glass fibre acrodisc (cat # 4 5 2 3 ) D isp osab le pipets, test tubes etc. D isp osab le micropipets, 1 0 0 - 2 0 0 pL. 125-m L LDPE narrow-mouth bottles 2-m L clear HPLC vial kit (cat # 5 1 8 1 - 3 4 0 0 ) Standard lab equipment (class A pipets and volum etric flask, graduated cylinders, etc.) LC/M S/M S and HPLC system s Burrell W heaton (V W R ) VW R Gelm an VW R VW R N alsene Iff v a r io u s A s described in Section 4.5.1 N otes:' '' ' 1. In order to avoid contamination, the use o f disp osab le containers/tubes/pipeis etc. is high ly recom m ended. 2. Teflon or teflon-lined containers or equipment, in clu d in g ' teflon-lined H PLC vial caps should not b e used. 3 3. Pear-shaped flasks are silanized before use. 4 . It m ay b e necessary to check the solv en ts (m eth anol) fo r the presence o f contaminants (especially P O A A ) b y L C /M S/M S - before use. Certain lot num bers have been found to be unsuitable for use. . 5. Disposable m icropipets or pipets should be used to aliquot standard solutions, w hen preparing standards and sa m p le s for extraction. 6. Equivalent m aterials m ay be substituted for those sp ec ifie d in this method. H owever, the use o f carbon from S u p elco is . strongly recommended. 3.4. Solutions ', 1. 5 0 m M Ammonium Acetate: D issolve 3 .8 5 g o f am m onium acetate in 1 L o f type I water. 2 . 2 m M Ammonium Acetate: D ilute 4 0 m L o f the 1 0 0 m M am m on iu m acetate solution in a liter o f type I water, for m ob ile phase A. 3. Saturated Ascorbic A cid in Methanol: D isso lv e ~ 10 g ascorbic acid in 1 0 0 m L methanol. 4 . Tne 2 % Ascorbic A cid in Methanol: D isso lv e 2 g ascorbic acid in 1 0 0 m L methanol. N o te : The volumes show n are provided for guidance; alternative v o lu m es m a y b e prepared. C iatre Analytical Laboratories, Inc. Study i" 023-003 Centre Analytical Laboratories, Inc. Page 8 Page 92 of 110 Centre Study No.: 023-011 Sponsor Protocol No: FACT-TOX-026 v. a Meiod No. : OOM`023-003, revision 2 3.5. Preparation of Stock, Fortification, and Calibration ' Solu.tions i. A n a ly tica l standards are used for hvo purposes: (I) to fortify control m atrix sa m p le s designated to b e extracted and processed as calibration standards that w ill b e then used to calibrate the response o f the detector u sed in the analysis- and P ) to fortify other control matrix sam p les to determ ine analytical recovery. ' T h e absolute volum es o f the standards m ay be varied b y the analyst as lon- a s the correct proportions o f solute to solven t are m aintained. T he solutions cited b e lo w are g iv en a s an example; alternative concentrations m ay be prepared i f needed 3.5.1. S to ck solutions . T o prepare stock solutions o f 1 0 0 pg/m L each PFO S, PFO SA , P F 0 3 A A PFO SEA , Et-FOSE-OH, M 556, M 570. P O A A , and the surrogate standard THPFOS, w eigh out 10 m g o f analytical standard (corrected for purity and percent salt, i f necessary) rad dilute to 1 0 0 m L w ith m ethanol in a 1 0 0- m L volum etric flask. Prepare a separate solution for each analyte. T h ese stock solutions (in 125-m L LDPE bottles) are to be stored in a refrigerator at 2 C to 6 C and are stable for a m axim um period o f 6 m onths from the date o f preparation. ' - 3.5.2. F ortification S o lu tio n s a. 10 pg/mL M ixed Fortification Solution - P ip et 10.0 m L each o -'P F O S PFO SA , PFO SA A , P F O S E A , -Et-FOSE-OH, M 5 5 6 , M 5 7 0 , and" P O A A stock solution to a 100 m L volum etric flask. B rin g up to v o lu m e w ith methanol. ` b. '2.5 pg/mLMixedFortification Solution-Pip^t 25.0 mL of the lo pa/mL m ixed fortification solution to a 100-m L volum etric flask and bring up to volum e with methanol. c. 0 .5 pg/mL M ixed Fortification Solution - Pipet 2 0 .0 m L o f the 2 .5 p s/m L ' m ixed fortification solution to a 100-m L volum etric flask and bruw u o to volum e with methanol. ` d. 0.1 pg/mL M ixed Fortification Solution - P ip et 2 0 .0 m L o f the 0 .5 p s/m L m ixed fortification solution to a 100-m L volum etric flask and brin u o to volum e with methanol. * F o llo w Steps a and b above to m ake a 2 .5 pg/m L solu tion o f the su rro ^a t- standard (TH PFO S) from the sto c k s o M o n . =~ Analytical Laboratories, Inc. Study #023-003 p. Centre Analytical Laboratories, Inc. Page 93 of 110 Centre Study No.: 023-011 Sponsor Protocol No: FACT-TOX-026 ' 'Centre Method No. : 00M-023-003, revision 2 Store all fortification standard solution s (in 125-m L L D P E b ottles) in. a refrigerator at 2C to 6 C for a m axim um period o f 6 m onths from the date o f preparation. ' 3.5.3. C alibration Sta n d a rd s Prepare LC/M S/M S calibration standards b y fortifying w eigh ed out aliq uots o f th e sam e con trol f e c e s sam p le (o r a c o m b in ed "b u lk " con trol fe c e s s a m p le ) before they are processed through the extraction procedure. . T he follow in g is a typical exam ple; additional concentrations m ay be prepared as needed. It is recom m en ded to prepare the "fortification so lu tio n s so that th e volum e o f fortification is' betw een 5 0 and 2 0 0 p L and to u se disp osab le m icropipetsTor liquoting fortification volum es. Cone, o f M ixed Fortification F o rtific a tio n V olum e (pL) W eight o f Control Sam ple (g) Solution (pg/m l) NA NA 0.1 1 0 0 . 0.1 2 00 0.5 . 100 0.5 200 2.5 100 2.5 200 10 100 1.0 1.0 1.0 ' 1.0 1.0 1.0 . 1.0 1.0 * 2.5 pg/m l THPFOS fortification solution. Cone, o f Extracted C a lib r a tio n Standard (pph) NA 10 20 * 50 100 250 500 1000 V ol. o f Surrogate Standard* added (uL ) 200 200 ' 200 200 200 200 200 200 .Calibration standard preparations m a y b e stored in a refrig era to r at 2 C to 6C , i f needed, and used over a period o f tim e as lon g as its stab ility has b een v e r ifie d . ' Centre Analytical Laboratories, Inc. Study # 0234)03 Centre Analytical Laboratories, Inc. Page 10 Page 94 of 110 Centre Study No.: 023-011 Sponsor Protocol No: FACT-TOX-026 Centre Method No. : 00M-023-003, revision 2 4. METHOD 4.1. Flow D iagram T he flo w diagram o f the m ethod is given below , follow ed by a d "tail"d d escrip tion o f each step. " ' M ethod F low Diagram W eigh 1.0 g o f sam ple (fo rtify sam p les designated as calibration standards and QC recoveries) '' Extract w ith A C N and filter >1 i ' Carbon Solid-Phase Extraction 4- i Concentrate to alm ost dryness -I i ' Adjust final volu m e (2 .0 m L) l' J.C M S M S ' 4.2. Sample Processing ' A ll s a m p le s are receiv ed frozen and w ill b e kept fro zen (b e lo w -10C) u n til tim e o f extraction . T h e rat feces do n o t n eed any processin g, h ow ever, th e m o n k e y fe ce s m a y need to be honiogenized in order to be able to w eigh out th e sp e c ifie d am ounts. 4.3. Sample Preparation - Prepare the follow in g blank and calibration sam ples (S ection s 4.3.1 to 4 3 4 ) from sa m e con trol fe ce s sam ple (or a com b in ed "bu lk" control fe ce s sam p le) Centre Analytical Laboratories, Inc. Study # 023-003 Centre Analytical Laboratories, Inc. Page 11 Page 95 of 110 Centre Study No.: 023-011 Sponsor Protocol No: FACT-TOX-026 Centre Method No. : 00M-023-003, revision 2 4.3.1. M a trix B la n k s Prepare two blank sam ples w ithout any fortifications, per set o f sam ples analyzed. T hese w ill b e used as the matrix blank. : ~ 4.3.2. M a trix Z ero B la n k s Prepare two matrix blank sam ples fortified w ith the surrogate, per set o f sam ples analyzed. T hese w ill b e used as the m atrix zero blanks. 4.3.3. C alibration S ta n d a rd s Prepare calibration standards as describ ed in S e c tio n 3 .5 .3 . ' 4.3.4. C ontinuing C alibration V erification S ta n d a rd s T w o o f the .extracted calibration standards, a low -m id and a m id-high lev e l standard, w ill be.used as continuing calibration verification (C C V ) standards 4.3.5. O C R eco very S a m p le s Prepare QC recovery sam ples to represent every control m atrix included in the study. Prepare at least one QC recovery sam p le in the low -range, and o n e in the high-range, to approxim ately bracket the exp ected range o f an a ly te in the sam ples. For sets containing m ore than 2 0 sam ples, prepare an additional Q C recovery sam ple for every group o f 10 additional sam p les (for e x a ir o le i f o n e set has 2 4 sam ples., then. 3 lev e ls o f Q C recovery sam p les w ill b e prepared) Prepare these additional Q C recovery sam ples to bracket the range o f an alyte found in the sam ples, as appropriate. '. N ote: An exam ple o f a sam ple extraction w orksheet is attached as Attachm ent!. * 4 .4 . E x tr a c tio n N o te : A . Preparation/conditioning o f SPE colum ns is described in S e c tio n 4 4 1 B . Silanization o f pear-shaped flasks is described in Section 4 .4 .2 . C. Evaluation/standardization o f SPE colum ns (w h en a different su n o lier is used etc.) is described in Section 4 .4 .3 . `* a. W eigh approximately 1.0 g ( 0 .0 5 g) o f sam ple in to 2 0 m L p o ly e th y le n e scintillation vial (Note: designated sam ples need to be fortified w ith appropriate aliquots o f fortification standards). . b. A dd 10 mL o f AC N (note: break-up any clum ps u sin g a spatula or s la s s rod) shake on a wrist-action shaker for 3 0 m inutes. ~' C ease Analytical Laboratories, Inc. Study # 023-003 Page 12 Centre Analytical Laboratories, Inc. Page 96 of 110 Centre Study No.: 023-011 Sponsor Protocol No: FACT-TOX-026 '. Centre Method No. t OOM-023-CO3, revision 2 c. Let the vials sit for - 5 m inutes, carefully decant, directly on to a 2 0 m L disposable syringe barrel connected to a glass acrodisc (G elm an) filter. d . T ransfer the filtrate to a co n d itio n ed S P E colu m n (s e e S e c tio n 4 .4 . 1). C o lle - t the elu te in a 1 2 5- m L "sila n iz e d ''(s e e S e c tio n 4 . 4 . 2 ) pear-shaped flask. N ote: T he 2 0 m L SPE colum n fits w ell inside the m outh (sleeve) o f the pear-shaped flask. M ake sure that the pear-shaped flask is w e ll supported and w ill not topple. N o vacuum is needed for this step e. A dd 10 m L o f A C N and then 2 0 m l o f 90:10 A C N : 2 % ascorbic acid in M eO H to the SPE colum n and collect the eluate in to the sam e pear sh aped flask, com bining the eluates. ' f. A d d 3 - 4 drop o f 1-octan ol to the pear sh aped -flask and rotary e v a p o ra te to ' 'near dryn ess (a m in u te am ou n t o f 1-octanol w ill rem a in ) at a red u ced p r e ssu re o f - 40C . g . Reconstitute b y adding 2 .0 m L m ethanol (final volu m e = 2 m L ) and sw irl/m ix to dissolve. Transfer the extract to H PLC vials usin g disp osab le pioets. 4.4.1 S P E C olum n P reparation . . Pack 20-m L SPE tubes w ith 2 g carbon. Condition colum ns w ith 10 m L o f saturated ascorbic acid in M eO H follow ed by. 10 m L A C N . D iscard all w ashes. D o not allow the colum n to dry. N ote: The SPE colum ns m ay be packed and conditioned at any p oin t U se the vacuum m anifold to condition the colum ns. D o not let the SPE colum ns to run dry at any tim e. 4.4.2 Silanization o f P ear-Shaped F la sk s' . Silan ize the pear-shaped flasks before use, b y rinsing w ith a 3 0 % dim ethyl- dichlorosilane in toluene solution follow ed by a toluene rinse and fin a lly a m ethanol rinse. . 4.4.3 Standardization o f S P E C olum ns W hen using carbon' from a different supplier, SP E colu m n s sh ou ld be standardized in the follow ing manner, prior to analyzing sam ples: a. U sin g a m ixed standard w ith a concentration b etw een 1 0 0 and 5 0 0 rW m L (ea ch o f all eight analytes) fo llo w the elu tion s c h e m e s a s outlined in st<*ns 2 and 3. C ollect all eluting fractions. . Centre Analytical Laboratories, Inc. Study # 023-003 mp . , Centre Analytical Laboratories, Inc. Page 97 of 110 Centre Study No.: 023-011 Sponsor Protocol No: FACT-TOX-026 i Centre Method No.: OOM-023-003, revision 2 b. C ollect a post-elution fraction, after step 3, eluting with an additional 2 0 m L o f 9 0 : 1 0 ( A C N : 2 % ascorbic acid in M eOH). c. A djust the final volu m e o f all the fractions to 10 m L w ith m ethanol. 1 d. A n alyze all the fractions by LC/MS/MS. ' e. I f the target fraction contains a minimum o f 8 5 % o f the resp ective analytes, it m ay b e considered acceptable. f. I f the "post-elution" fraction contains significant am ount o f a n a ly tes, the target elu tion volu m e m ay be increased. 4.5. A n a l y s i s .b y LC/MS/MS 4.5.1. L C /M S /M S S y stem a n d O perating C onditions (E lectro sp ra y) M a ss Spe: Interface: C om puter Software: M icrom ass Quattro Ultim a (M icrom ass) Electrospray (Micromass) Harvard infusion pump . CO M PA Q Professional Workstation A P 20 0 Window's NT, M assLynx 3.3 HPLC: H ew lett Packard (HP) Series 1 1 00 H P Quat Pump HP Vacuum Degasser H P Autosampler HP Colum n Oven -N o te : A 4 x 10 m m hypercarb drop-in guard cartridge (K e y sto n e, part # 8 4 4 0 1 7 - 4 0 0 ) is attached on-line after-the purge valve and before th e sa m p le in jector p ort to trap any resid ue contam inants that m a y b e in the m o b ile p h a s e and/or H PLC system. . H PL C Colum n: G enesis C , (Jones Chromatograph}), 2.1 m m x 5 0 m m , 4 u Colum n Temp.: 3 5 C Injection V oi.: 10 pL _. - - M obile Phase (A): 2 m M Am m onium Acetate in Type I w ater M obile Phase (B): M ethanol Time 0 0.4 1.0 7.0 7.5 9.0 9.5 13.5 14.0 %A 60 60 10 10 0 0 60 . 60 ' 60 % B Flow Rate rmlVminl 40 0.300 40 0.300 90 0.300 90 0.300 100 0.300 100 0.400 40 40 ' 0.400 0.400 40 0.300 "Centre Analytical Laboratories, Inc. Study 023-003 Page 14 Centre Analytical Laboratories, Inc. Page 98 of 110 Centre Study No.: 023-011 Sponsor Protocol No: FACT-TOX-026 .. Centre Method N o .: 00M-O23-0O3', revision 2 It m ay b e n ecessary to ad ju st th e H P L C gradien t in order to o p t im iz e instrument performance. C olum n s w ith different d im en sion s (e.g. 2 .1 m m x 3 0 mm) and also columns from different manufacturers (K eyston e B e ta sil C ,s etc.) can be used provided equivalent chrom atography is obtained. Ions monitored: A n alvte M ode PFOS PFOSA PFOSAA PFOSEA * Et-FOSE-OH POAA ' M 556 M 570 THPFOS negative negative negative negative negative negative negative negative negative Parent Ion 499 498 584 526 630' 413 556 570 427 Product Ion 99 78 526 . 169 59 369 498 419 80 A pproxim ate R etention T im e fm m ) 5.3 * 6.0 5.7 6.7 '' 6.7 5.1 5.4 5.6 ' 5.1 N ote that the retention tim es m a y v ary sligh tly, on a d ay-to-d ay b a s is , depending on the batch o f m ob ile phase, etc. 4.5.2. E xa m p le T une F ile P a ra m e ters .. The follow ing values are provided as an exam ple. A ctu a l v a lu e s m a y v a ry from instrument to instrument. A ls o these'valu es m a y b e ch a n g ed from tim e to time in order to optimize for greatest sensitivity. Tne mass spectrometer is tuned usin g a solution o f each analyte at - 0.5 pg/mL, prepared via dilution o f the stock solution in m eth anol. T h e so lu tio n is infused (us.ing a `T ' e l e c t o r ) at 1 0 p L /m in in to a 0 . 2 m L /m in str e a m o f mobile phase consisting o f 4 0% m ethanol and 60% 2 mNf am m onium * a ceta te. The analytes are initially tuned for the parent io n and th en tu n ed fo r th e product ion. The optimized param eters are saved as a "tune file ". T h is tun e file is then used during routine analysis. A nalvte PFOS PFOSA PFOSAA PFOSEA Et-FOSE-OH POAA M 556 M 570 THPFOS Dwell (si 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 Centre Analytical Laboratories, Inc. Study # 023-003 C ollision E n ersv feV l 43 * 28 20 28 31 11 28 20 35 C o n e CV) 76 34 37 49 30 25 50 60 34 Centre Analytical Laboratories, Inc. Page 99 of 110 Centre Study No.: 023-011 Sponsor Protocol No: FACT-TOX-026 J Q- Cratre Method No. : 0OM-O23-OO3, revision 2 Source Capillary Hexapole 1 Aperture 1 Hexapole 2 Source Block Temp. Desolvation Temp. A nalvzer L M R esl H M R esl Energy 1 Entrance Exit LM R es2 H M R es2 Energy 2 M ultip lier Gas Flow s Cone Gas - Desolvation' Pressures Gas Cell .' : Set 2.56 kV 0.5.V 0.2 V 0.8 V 100C ' 400C Set 13.0 V 13.0 V 0.7 V -2 V IV . 11.0 V 11.0 V 1.0 V 650V ' Set _ ~150L /hr ~ 700L/hr Read back ~ 3.0e-3 mbar N ote: An alternative L C /M S/M S system m ay b e used once dem on strated . to be equivalent. 4 .5 .3 . Calibration P ro c ed u res a. In ject an aliquot (10 p L ) o f ea ch calibration standard into th e L C /M S /M S sy stem . Include standards corresponding to six or m ore co n cen tra tio n le v e ls (ranging from the L O Q to the h ig h est concentration le v e l to b e in c lu d e d in the a n alysis) in an a n a ly tica l set. b. U s e a linear y = m x + b fu n ction f o r quantification. L in ear stan dard c u r v es are generated for e a ch a n alyte b y lin ea r reg ressio n usin' l/ x w eighting o f peak area versus calibration standard concentration usim the W indow s NT, M assLynx 3.3 (or equivalent) software system A n y extracted standards that frJl o u tsid e the 7 0 to 1 3 0 % , based o n th e a v e ra g e response factor o f the surrogate standard, m u st be exclu d ed fro m ' the calibration curve. Centre Analytical Laboratories, Inc. Study # 023-003 Page 16 Centre Analytical Laboratories, Inc. Page 100 of 110 Centre Study No.: 023-011 Sponsor Protocol No: FACT-TOX-026 Centre Method No. : 00M-023-003, revision 2 c. The correlation coefficient (R ) for calibration curves generated m ust be > 0 .9 8 (R 2> 0 .9 6 ). I f calibration results fa ll ou tsid e these lim its, take appropriate steps to adjust instrument operation, and reanalyze the relevant sets o f samples. ' 4.5.4. S a m p le A nalysis a. Each set o f sam ples analyzed m ust include matrix blanks (w ithout sum ogate standard), martrix zero blanks (w ith the surrogate standard), a set o f extracted standards, and QC recovery sam ples. ' b. In ject art aliquot (1 0 p L ) o f each standard/sam pJe/Q C reco very/co n tro l into the LC/MS/MS system . B egin a set o f sam ples with an entire set o f calibrations.' Include tw o continuing calibration verification (C C V ) standards (one from the low-mid and on e from the m id-high range) after every 5 to 10 samples, at the m o st . c. D eterm ine the concentration o f each sam ple/Q C recovery/control from the standard curve,, based on the peak area o f each analyte. T he standard responses must bracket responses o f the residue found in each sam ple set. Sam ples that fall outside the range o f the standard curve m ust b e diluted appropriately and re-analyzed. d. Evaluate the ongoing acceptability o f instrum ental analysis based on the C C V results. S am p les an alyzed w ill b e a ccep ta b le as lo n g a s b oth C C V 's fall w ithin 3 0 % o f their true value. C alculate the C C V 'con centration found using the linear calibration curve. I f the C C V recovery is outsid e 7 0 % to 130% , re-analyze sam ples analyzed after the last acceptable check. e. M onitor the response o f the surrogate standard (S S ) for every sam ple, .extracted standard, blank, and QC r e c o v er y sample. The r e sp o n se is calculated using the "average response factor" function u sin g M assL yn x 3.3 (or equivalent). Evaluate the acceptab ility-of the sam ple extraction process based on the concentration o f the S S found in all extracted standards,, QC samples, blanks, and sam ples. T h e average resp on se factor for all samples, blanks, extracted standards, and Q C recovery sam p les m ust fall between 6 0 % to 14 0% ( 4 0 % ). T hose deviating b y greater than 3 0 % (outside 7 0 % to 1 3 0 % range) m ust be ch eck ed for p o ssib le errors and m ay need to be re-extracted. A ny responses deviatin g by greater than 4 0 % (falling outside the 6 0 % to 1 4 0 % range) w ill require re-extraction. f. I f analysis is delayed, sam ples m u st b e stored refrigerated at approxim ately 2C to 6C until analysis, and analyzed preferably w ith in a week. Ceatre Analytical Laboratories, Inc. Study ft 023-003 Pa3e 17 Centre Analytical Laboratories, Inc. Page 101 of 110 CentreiStudy No.: 023-011 Sponsor Protocol No: FACT-TOX-026 Csntr= Method No. : 00M-023-003, revision 2 4.6. Performance Criteria T he follow ing two criteria should be m et before the initial analysis o f sam p les e s p e c ia lly w h en u s in g d ifferen t in stru m en tation set-u p s than th o s e c ite d in this' m ethod. F irst Criterion - R un a standard solu tion on L C /M S/M S corresponding to the estim ated LOQ (the 1 0 ppb extracted standard) and obtain a signal to n o ise ratio o f at least 9:1 for all analytes. I f this criterion cannot b e m et, op tim ize and c h a n c e instrum ent operating parameters. S eco n d Criterion - R u n a set o f standards o f six or m o re con cen tration le v e ls ranging from the proposed LO Q up to the high est concentration lev e l to be included in the analysis. Generate a calibration curve for each analyte and obtain a linear regression w ith a correlation coefficien t o f at least 0 .9 8 for each analyte 4.7. Time Required for Analysis a set o f 1 4 sam ples can b: taken through the extraction procedure in approxim ately six hours b y one person. T he L C /M S/M S analysis ( P - 1 4 standards and 14 sam ples) w ill take approxim ately sev en hours. 5- CALCULATIONS 5.1. Analyte Found C alculate the amount o f analyte found (in pp b ) using the standard curve sen era ted b y the M ass Lynx softw are program using Equation T. Correction for sam p le w e ig h t (1 g ) and final v o lu m e (2 m L ) is n o t req uired sin c e th e sa m p le s and standards are extracted the sam e way. E q u a tio n 1: (peak area - intercept) x D F A n alyte found (ppb) = slope W here D F = dilution factor, if sam ples w ere diluted. Centre Analytical Laboratories, Inc. Study tt 023-003 Centre Analytical Laboratories, Inc. Page IS Page 102 of 110 Centre Study No.: 023-011 Sponsor Protocol No: FCT-TOX-026 Centre Method No. : OOM-023-003, revision12 5.2. QC Recovery < : F o r sam p les fortified w ith know n am ounts o f analytes prior to extraction, calculate the percent recovery from Equation 2. Equation 2: : 1 % Recovery = i an alyte foun d (ppb) - analyte found in m atrix blank (ppfa) ^ . -amount analyte added (ppb) ' 5.3. M e a n R e s p o n s e F a c t o r T h e m ean response factor (M R F) is calculated b y the M ass L y n x softw are program b y averaging the m ean response -for all calibration standards (response/am ount added) and dividing by the am ount added. T h e actual concen tration from each injection is then calculated b y dividing th in d ivid u al response b y the M RF. The recovery for each injection is the percent o f the actual concentration to the amount added. This calculation is perform ed' b y the M assL yn x 3 .3 (or equivalent) software. 6. SAFETY AJl sam p les m ust be treated as potential bioh azard s and appropriate u n iversal precautions m ust be taken (fellow standard operating procedures for the h an d lin g o f b ioflu id s). T hese include, but are not lim ited to, the use o f d ou b le layers o f latex or v in y l gloves, goggles, dust m ask, and lab coat. A ll disp osab le m aterials m u st b e treated as biohazards and handled accordingly. A ceton itrile, methanol, dim ethyldichlorosilane and toluene are h ig h ly fla m m a b le. Open and use under fum e hood only. ' A ll organic waste and solvent w aste must be segregated and d isp osed o f accordingly. : ' Centre Analytical Laboratories, Inc. Study # 023-003 Centre Analytical Laboratories, Inc. Page 19 Page 103 of 110 Centre Study No.: 023-011 Sponsor Protocol No: FACT-TOX-026 CcnteMethod No. : 00M-023-CO3, revision 2 ATTACHMENT!. i /J55*5SN. C entre Analytical L a b o ra to rie s. Inc. S04I Rcscutl) Drive, Sate CoJIeje,PA 16101. Pbone(3H3M032 Fi*; (Sl-)2j PREPARATION OF SAMPLES FOR EXTRACTION AND ANALYSIS C-atre Analytical Laboratories, Inc. Study it 023-003 Centre Analytical Laboratories, Inc. _ Page 20 Page 104 of 110 Centre Study No.: 023-011 Sponsor Protocol No: FACT-TOX-026 C e n tr e A n a ly tic a l L a b o r a t o r ie s , In c . 3048 Research Drive, State College, PA 16801. Phone: (814) 231-8032, Facsimile: (814)231-1253 METHOD DEVIATION Deviation Num ber 1 . Date o f Occurrence: 10/04-05/00 . Page 1 ' Centre Study N um ber 023-011 Sponsor Protocol Num ber FACT-TC>X-026 DESCRIPTION OF DEVIATION /! D eviations to m ethod titled "Determination o f Fluorochemical Residues in M onkey/Rat F eces by LC/M S/M S (R ev isio n 2 ) ", Centre method number 00 M -023-003, revision 2: 1. Section 4 . 5 . 4 . d : CC V recoveries o f 60 % , 5 4 % , 6 5 % , 66% , 5 7 % , and 6 0 % were accepted for EtFOSE-OH in Set 100200A R . ACTIONS TAKEN i.e.. amendment issued. SOP revision, etc.. 1. M ethod d eviation issued,. Recorded By/Date: 1. N o negative im pact ` J iid iA - ffc r lo fcfo o IMPACT ON THE STUDY Principal Investigator Signature VSK-.c A/-Q CAL QAU Review rz,,-LQ0 Date ivUth/i A- _________ February 12, 1998/2 Centre Analytical Laboratories, Inc. Page 105 of 110 Centre Study No.: 023-011 Sponsor Protocol No: FACT-TOX-026 C e n tre A n a ly tic a l L a b o r a t o r ie s , In c . 3045 Research Drive, State College, PA 16801. Phone: (814) 231-8032, Facsimile: (814)231-1253 METHOD DEVIATION Page 1 Deviation Number 2 Date of Occurrence: (1) 10/10/00, (2) 10/10/00, (3) 10/11-12/00 Centre Study Number 023-011 Sponsor Protocol Number: FACT-TOX-026 DESCRIPTION OF DEVIATION Deviations to method titled "Determination of Fluorochemical Residues in Monkey/Rat Feces by LC/MS/MS (Revision 2)", Centre method number 00M-023-003, revision 2: 1. Section 4.5.4.d: CCV recoveries of 50%, 51%, 59%, 60%, 61%, and 61% were accepted for EtFOSE-OH in Set 100900A. ' 2. Section 4.5.4.e: Surrogate recovery of 46% was accepted for Centre-sample 0008495 in Set 100900A. - 3. Section 4.5.4.e: Surrogate recoveries of 57% and 56% were accepted for Centre samples 0008514 and 0008515, respectively in Set 101000AD. ACTIONS TAKEN i.e.. amendment issued. SOP revision, etc. I 1. M/ ethod deviation iss Recorded By/Date: ^ IMPACT ON THE STUDY 1. No negative impact CAL QAU Review Date February 12. 1998/2 Centre Analytical Laboratories, Inc. Page 106 of 110 Centre Study No.: 023-011 Sponsor Protocol No: FACT-TOX-026 C e n tr e A n a ly tic a l L a b o r a t o r i e s , In c . 3048 Research Drive, Slate College, PA 16801. Phone: (814) 231-8032, Facsimile: (814)231-1253 METHOD DEVIATION Page 1 Deviation Number: 3 Date of Occurrence: (1) 10/18-19/00,(2) 10/17-18/00.(3) 10/22-23/00.(4) 10/25/00. (5) 10/24-25/00, (6) 10/26/00 Centre Study Number: 023-011 Sponsor Protocol Number FACT-TOX-026 DESCRIPTION OF DEVIATION Deviations to method titled "Determination of Fluorochemicai Residues in Monkey/Rat Feces by LC/MS/MS (Revision 2)", Centre method number 00M-023-003, revision 2: 1. Section 4.5.4.e : Surrogate recovery of 53% was accepted for Centre sample 0008595 in Set 101600AR. 2. Section 4_5.4.d: CCV recovery of 153% was accepted for EtFOSE-OH in Set 101700A. &3. Section 4_5.4.d e: In Set 102000A, a surrogate recovery of 149% was accepted for Centre sample 0008681 and CCV recoveries of 54%, 59%, 54%, 58%, and 55% for EtFOSE-OH were accepted. 4. Section 4 -5.4.d & e: In Set 102300AR. a surrogate recovery of 54% was accepted for Centre sample 0008690 Spk B and one of 51 % was accepted for 000S703. CCV recoveries of 54%, 51 %, 51 %, 53%, and 57% for EtFOSE-OH were accepted. 5. Section 4.5.4.d: CCV recoveries of 62%, 63%, and 55% were accepted for EtFOSE-OH in Set 102400A. 6. Section 4.5.4.d: CCV recoveries of 66%. 64%, 43%. and 43% were accepted for EtFOSE-OH in Set 102500A ACTIONS TAKEN i.e.. amendment issued. SOP revision, etc... 1-6. Method deviation issded. Recorded By/Date: iJ tzftlc IMPACT ON THE STUDY 1-6. No negative impact Principal Investigator Signature CAL QAU Review. IZZl DO. Date __ M i t t i l yelv February 12. 1998/2 Centre Analytical Laboratories, Inc. Page 107 of 110 Centre Study No.: 023-011 Sponsor Protocol No: FACT-TOX-026 C e n tr e A n a ly tic a l L a b o r a t o r i e s , In c . 3048 Research Drive, Slate College, PA 16801. Phone: (814) 231-8032, Facsimile: (814)231-1253 METHOD DEVIATION Page 1 Deviation Number: 4 Date of Occurrence: (1) 8/15/00 and 9/26/00, (2) Entire study Centre Study Number 023-011 Sponsor Protocol Number: F A C T -T O X -026 ' ` DESCRIPTION OF DEVIATION ; Deviations to method titled "Determination of Fluorochemical Residues in Monkey/Rat Feces by LC/MS/MS (Revision 2)", Centre method number 0OM-O23-0O3, revision 2: 1. Section 3.5.1: Did not correct stock standard solutions for % purity. 2. Section 4.3.5: Only prepared 2 QC samples for sets that contained more than 20 samples. ACTIONS TAKEN i.e.. amendment issued. SO P revision, etc.. 1-2. Method deviaition issuedit--- Recorded By/Date:: JZ/b0b / I 1-2. No negative impact IMPACT ON THE STUDY '5'i'AH A l(r February 12, 1998/2 Centre Analytical Laboratories, Inc. Page 108 of 110 Centre Study No.: 023-011 Sponsor Protocol No: FACT-TOX-026 C e n tre A n a ly tic a l L a b o r a t o r i e s , In e . _______ 3048 Research Drive. Statt College. PA 16801._______ Phon (814)231-8032. Facsimile: f814)231-1253_______ METHOD MODIFICATION FORM Page 1 of 2 Modification No.: 1 Study Director: Paul Lieder Centre Study Number 023-011 Sponsor Protocol Number FACT-TOX-026 Method Title: Determination of Fluorochemical Residues in Monkey/Rat Feces by LC/MS/MS (Revision 2) Centre Method Number 00M-O23-003, revision 2 MODIFICATION: '. 1. Section 3.4 Solutions The 100 mM ammonium acetate solution should be 50 mM ammounium acetate. 2. . Section 4.5.4 Sample Analysis (Item e.) Should'read as follows: Monitor the response of the surrogate standard (SS) for every sample, extracted standard, blank, and QC recovery sample. The concentration is calculated using the "average response factor" function from MassLynx 3.3 software (or equivalent). Evaluate the acceptability of the sample extraction process based on the concentration of the SS found in all extracted standards, QC samples, blanks, and samples. The percent recovery for all samples, blanks, extracted standards, and QC recovery samples must fall befween 60% and 140%. Those deviating by greater than 30% must be checked for possible errors and may need re extracted. Any recoveries deviating by greater than 40% (falling outside the 60% to 140% range) will require re-extraction. 3. Section 5.1 Analyte Found: Remove second sentence. . 4. Section5.1 Equations Modify Equation 1 to: Analyte found (ng/mL) = fPeak area - intercept) slope i Change Equation 2 to: / Analyte found (ppb) = f.inalvte found fng/mL) x final vqI. fmL) x DF) sample wt. (g) Add Equation 3 (where DF = dilution factor ofspk, FV = final volume): Recovery (% ) = _____ ((anal, found (ng/mL) - (anal, found in corresponding sample fng/mD/DF) x FV (mL) x DF) x 100_____ amount added (ng) 5. Section 5.3 Mean Response Factor: Modify first sentence to read: The mean response factor (MRF) is calculated by the MassLynx software program by averaging the response of the surrogate standard for all calibration standards and dividing by the amount added. Centre Analytical Laboratories, Inc. . Page 109 of 110 Centre Study No.: 023-011 Sponsor Protocol No: FACT-TOX-026 C e n tr e A n a ly tic a l L a b o r a t o r i e s , In c . 3048 Research Drive. State College. PA 16801. Phont: (814)231-8032. Facsimile: (814)231-1253 METHOD MODIFICATION FORM Page 2 of 2 Modification No.: 1 Study Director. Paul Lieder Centre Study Number: 023-011 Sponsor Protocol Number FACT-TOX-026 Method Title: Determination of Fluorochemical Residues in Monkey/Rat Feces by LC/MS/MS (Revision 2) Centre Method Number OOM-023-003, revision 2 MODIFICATION (continued'): 6. Section 4.1 Flow Diagram: The statement under Weigh 1.0 g of sample should read: (fortify designated samples with appropriate fortification solutions and surrogate standard) 7. Section 4.4.a Extraction: The Note should read as follows: (Note: designated samples need to be fortified with appropriate aliquots of fortification standards and all samples except matrix blanks need to be fortified with the surrogate standard). JUSTIFICATION: 1. To correct for a typographical error. 2. To clarify SS acceptable criteria. " 3. Remove incorrect statement because calculations did include sample weight and final volume. 4. Modify calculations to correspond with those used in study. ! 5. Clarify calculation of mean response factor i' 6. -7. Clarify that all samples except the matrix blanks need to be fortified with the surrogate standard. EFFECT ON STUDY: No negative impact Originator: W fH iU . J Principal Investigator 7--<~ Sponsor Management: (/ a /y / J , . 75f.iT- rL n oD Date -, Date Date wy / ^ . / February 12, 1998/3 Centre Analytical Laboratories, Inc. Page 110 of 110 3M Medical Department Study: T-6889.3 3M Medical Department Study: T-6889.3 Appendix I: Report Signature Page Analytical Report: FACT-TOX-026 LRN-U2782 Analytical Report: FACT TOX-026 LRN-U2782 Paul Lieder, Ph.D., DABT, Study Director & Date Johrn_. Butenhoff, Ph.D., Sponsor Representative -2 ^ 0 I Date William Reagen, Ph.D., Laboratory Manager Kris J. Hansen, Ph.D., Analytical Investigator Date Q IiilO I Date 3M Environmental Laboratory 3MEnvironmental Laboratory Page 41 Page 409 3M Medical Department Study: T-6889.3 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 Analytical Report: FACT TOX-026 LRN-U2782 Appendix J: Amendment 1 to FACT TOX-026 Final Report TOX-026 Final Study Report Amendment 1 Study number: TOX-026 Study title: 26-Week Capsule Study with Ammonium Perfluorooctanoate (APFO/POAA) in Cynomolgus Monkeys Study Director: Paul Lieder, Ph.D. Amendment date: June 15, 2001 Amendment number: 1 This amendment modifies the following portion of the final report: GLP Study--Quality Assurance Statement, page 4: The dates 5/3/01,5/8/01 and 5/9/01 need to be added to the draft report inspection dates. Statement of Data Quality, page 16: The sentence, `The average fortified sample recovery for sera was 93% with a standard deviation of 11%" should read, "The average fortified sample recovery for sera was 94% with a standard deviation of 11%." A draft version of page 16 with the 93% value was inadvertently inserted into the final report and still included the draft watermark. The only revision without the draft watermark is the final version, which lists the recovery value correctly as 94%. The correct page 16 was inserted into the report. Location of Archives, page 8: The statement, "Specimens analyzed at Centre Analytical Laboratories will be returned to 3M Environmental Laboratory upon completion of analysis and submission of the subcontract final report" should be deleted. In its place should be the following sentence, "Feces specimens will be returned from Centre Analytical Laboratories for archiving at the 3M Environmental Laboratory and will be maintained at 3M Environmental Laboratory according to GLP requirements." Other changes to the TOX-013 report include: The cover page was updated to reflect the total number of pages and the title was changed to say "Amended Analytical Report Title." The Table of Contents was updated to reflect the added amendment. 3M Environmental Laboratory 3MEnvironmental Laboratory Page 42 Page 410 3M Medical Department Study: T-6889.3 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 Analytical Report: FACT TOX-026 LRN-U2782 Approved by: --------------- Kristen H. Hansen,`Ph.D., Principal Analytical Investigator if/M /O f Date Paul Lieder, Ph.D., Study Director John Butenhoff, Ph.D., Sponsor's Representative Date *7f u ( Date Bill Reagan, Ph.D., Environmental Laboratory Manager Date 3M Environmental Laboratory 3 M E n v ir o n m e n ta l L a b o r a to r y Page 43 Page 411