Document aBgg8b6M0dMzgpq14MVkoZXne

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 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 Juant seig1n1in, g2001 Performing Laboratories Liver, Serum and Urine Analyses Feces Analyses 3M Environmental Laboratory Building2-3E-09, 935 BushAvenue St. Paul, MN 55106 Centre Analytical Laboratories, Inc. 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 #4#0# 8 3M Environmentaj Laboratory 3M Environmental Laboratory Page 1 Page 1 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 This page has been reserved for specific country requirements. 3M Environmental Laboratory 3M Environmental Laboratory Page 2 Page 2 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 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, FACT TOX-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. Exceptionsto GLP compliance: 0 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. 0 Labels of reagents and solutions did not contain all of GLP requirements. 0 It is unknown if an in-phase inspection was conducted. The audit report could not be located. @dIU I Paul Lieder, Ph.D., Study Director , ` Date Kris`J. Hansen, Ph.D., Principal Analytical lnvestigator William Reagen, Ph.D., Laboratory Manager s- q G " John L. Butenhoff, Ph.D., Sponsor Representative Date c16/a&-/,. Date * g,-/ Date 3M Environmental Laboratory 3M Environmental Laboratory Page 3 Page 3 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 GLP Study-Quality Assurance Statement Inspection Dates Phase Date Reported to Management Study Director 10/26/98 Protocol 10/27/98 I 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, 511 010 1 See Appendix H for individual contractor quality assurance statements. 10/27/98 10/9/00, 10/30/00 12/21/00, 511 010 1 QAU RepreGativc9 Date 3M Environmental Laboratory 3M Environmental Laboratory Page 4 Page 4 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 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 Characterizationand Control Matrices.......................................... 18 Appendix B: Protocol, Protocol Amendments and Deviation Summary ............................ 20 Appendix C: Extraction and Analytical Methods ............................................................... 2616 FACT-M-1.I,Extractionof Potassium Perfluorooctanesulfonateor Other Fluorochemical Compounds from Liver for Analysis Using HPLC-Electrospray/Mass Spectrometry, (15 pages)............................................................................................................................... 2617 FACT-M-2.1, Analysis of Fluorochemicals in Liver Extracts Using HPLC- Electrospray/Mass Spectrometry, (9 pages) ..................................................................... 8221 ETS-8-6.0, "Extraction of Potassium Perfluorooctanesulfonateor other Fluorochemical Compounds from Liver for Analysis using HPLC-Electrospray/Mass Spectrometry", (14 pages)................................................................................................................................ 921 ETS-8-7.0, "Analysis of Potassium Perfluorooctane-sulfonate or other Fluorochemicals in Liver Extracts Using HPLC-Electrospray/Mass Spectrometry", (10 pages) ...................12015 3M Environmental Laboratory 3M Environmental Laboratory Page 5 Page 5 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 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)............................................................................................................................... 21115 FACT-M-3.1, Extraction of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds from Serum or Other Fluid for Analysis Using HPLC-Electrospray/Mass Spectrometry, (17 pages) ................................................................................................. 21129 ETS-8-5.1, Analysis of Potassium Perfluorooctanesulfonateor Other Fluorochemicals in Serum Extracts Using HPLC-Electrospray/MassSpectrometry, (9 pages) ....................... 21146 FACT-M-4.1, Analysis of Potassium Perfluorooctanesulfonate or Other Fluorochemicals in Serum or Other Fluid Extracts Using HPLC-Electrospray/Mass Spectrometry, (9 pages)............................................................................................................................... 12515 ETS-8-96.0, Extraction of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds from Urine for Analysis Using HPLC-Electrospray/Mass Spectrometry, (14 pages)............................................................................................................................... 21164 ETS-8-97.0, Analysis of Potassium Perfluorooctanesulfonateor Other Fluorochemical Compounds in llrine Extracts using HPLC-Electrospray/Mass Spectrometry/Mass Spectrometry, (10 pages) ................................................................................................. 21178 Appendix D: MethodValidation Summary (TOX134)........................................................ 21288 Appendix E: Data Summary Tables .................................................................................. 21389 Appendix F: Data Spreadsheets....................................................................................... 32703 Appendix G: Example Calculations................................. ..................................................32895 Appendix H: Contract Lab Report..................................................................................... 42097 Centre Analytical Laboratories, 26-Week Capsule Toxicity Study with Ammonium Perfluorooctanoate (APFO/POAA) in Cynomolgus Monkeys, (110 pages)....................... 42098 Appendix I: Report Signature Page .................................................................................. 44108 Appendix J: Amendment 1 to FACT TOX-026 Final Report.............................................. 42 3M Environmental Laboratory 3M Environmental Laboratory Page 6 Page 6 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 List of TabIes 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.................................................... 12828 Table 6. LOQ Values Used in FACT TOX-026 Analyses by Method and Usage Dates...1.2839 Table 7. Recovery Summaries of Fortified Samples in FACT TOX-026 ........................... 12839 Table 8. Recovery Summaries of Fortified Samples in FACT TOX-026 ........................... 12940 Table 9. Recovery Summaries of Fortified Samples in FACT TOX-026 ........................... 12951 Table 10. TOX 026 Data Summary of POAA Concentration-Serum (pg/mL) .................12962 Table 11. TOX 026 Data Summary of POAA Concentration-Urine (pg/mL) ...................12894 Table 12. TOX 026 Data Summary of POAA Concentration-Liver (vg/g)....................... 13906 Table 13. TOX 026 Data Summary of POAA Concentration from Centre Analytical Laboratory-Feces (vg/g) .................................................................................... 13906 Table 14. FACT TOX-026 Individual POAA Results per Sample-Serum (pg/mL)...........13928 Table 14 (continued). FACT TOX-026 Individual POAA Results per Sample-Serum (pg/mL) ................................................................................................................ 13939 Table 15. FACT TOX-026 Individual POAA Results per Sample-Urine (vg/mL).............23040 Table 15 (continued). FACT TOX-026 Individual POAA Results per Sample-Urine (vg/mL) ................................................................................................................ 23051 Table 16. FACT TOX-026 Individual POAA Results per Sample-Liver (pg/g) ................23063 3M Environmental Laboratory 3M Environmental Laboratory Page 7 Page 7 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 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 Represenfafive Analytical Chemistry Laboratories Liver, Serum and Urine Analyses Feces Analyses 3M Environmental Laboratory Centre Analytical Laboratories, Inc Kristen J. Hansen, Ph.D.,Analytical lnvestigafor Emily Stauffer, Analytical lnvestigafor 3M Environmental Laboratory Contributing Personnel David R. Barnidge, Ph.D.' Lisa A. Clemen Rhonda S. Dick' Kelly J. Dorweilei Mark E. Ellefsori Sara E. Estes' Barb A. Gramenz' Sarah A. Heimdal' Cari S. Hewitt' Marlene M. Heying' 'Contract lab professirnaJserviceemployees 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 rep rt have been archived a the 3M Environmental L.aborat0r-y. 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 Laboratoriesfor archiving at the 3M Environmental Laboratory and will be maintained at 3M Environmental Laboratory according to GLP requirements. 3M Environmental Laboratory 3M Environmental Laboratory Page 8 Page 8 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 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 monkeyswere 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) Population I I Selected for Study Group I Selected for Recovery Group I Group 1 6 6 2 Control Group 2 Low-dose 4 4 0 (3 mg/kg/day) Group 3 Mid-dose 6 6 2 (10 mg/kg/day) Group 4 High-dose 6 6 0 (30/20 mg/kg/day)" 3M Environmental Laboratory 3M Environmental Laboratory Page 9 Page 9 3M Medical Department Study: T-6889.3 3M Medical Department Study: 1-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 Analytical Report: FACT TOX-026 LRN-U2782 Specimen 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 Laboratoryto 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 beginningon 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 EnvironmentalLaboratory received liver, serum, urine and feces specimens collected at predeterminedtime 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 &O"C and -20C *1O"C. 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 Laboratorywill be maintained at the laboratory for a period of time as specified by the regulations and will be kept at -50C *20"C or -20C *lO"C. 3M Environmental Laboratory 3M Environmental Laboratory Page 70 Page 10 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 Chemical Characterization of Test Substance Ammonium Petfluorooctanoate CAS Number: 3825-26-1 Chemical Formula: C7FI5C0iNH4+ 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. Procurement One lot of ammonium perfluorooctanoate (Lot number 332) was obtained from 3M Specialty Chemicals. Stability Studies No stability samples were sent for analysis. Dose ConfirmationAnalyses 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 3M Environmental Laboratory Page 11 Page 11 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 Method Summaries Centre Analytical 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 EnvironmentalLaboratory Following is a brief description of the methods used during this analytical phase by the 3M EnvironmentalLaboratory. 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. PREPARATORMYETHODS ETS-8-6.0, "Extraction of Potassium Perfluorooctanesulfonateor Other Fluorochemical Compounds from Liver for Analysis using HPLC-Electrospray/Mass Spectrometry" Liver samples were homogenized in water. An aliquot of each homogenatewas 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.O 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 Perfluorooctanesulfonateor Other Fluorochemical Compounds from Serum for Analysis using HPLC-Electrospray/Mass Spectrometry" Serum samples were extracted using an ion-pairing extraction procedure. An ionpairing 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.OmL 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 vm nylon filter using a 3 cc plastic syringe into glass autovials. 3M Environmental Laboratory 3M Environmental Laboratory Page 12 Page 12 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 3M Medical Department Study: T-6889.3 Analytical Report: FACT TOX-026 LRN-U2782 ANAL MTCAL METHODS 0 ETS-8-7.0, "Analysis of Potassium Perfluorooctanesulfonateor other Fluorochemicals in Liver Extracts Using HPLC-Electrospray/Mass Spectrometry" 0 ETS-8-5.1,"Analysis of Potassium Perfluorooctanesulfonate or other Fluorochemicals in Serum Extracts Using HPLC-Electrospray/Mass Spectrometry" 0 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 (C7FI5COi)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 l / x weighting. In urine analyses, each curve is plotted with an internal standard using a quadratic fit and l / 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 3M Environmental Laboratory Page 13 Page 13 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 ANALYTICAELQUIPMENT 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@BetasilTMCIS2x50 mm (5 pm) Column temperature: Ambient Mobile phase components: Component A: 2mM ammonium acetate Component B: methanol Flow rate: 300 pUmin Injection volume: 10 pL Solvent Gradient: Time (minutes) 0.0 1 .o 4.5 6.5 7.0 %B 40% 40% 95% 95% 40% Mass Spectrometer: Micromass@APVMass Quadrupole system Software: Mass LynxrM3.2-3.4 Cone Voltage: 15-60 V Collision Gas Energy: 20-40 eV Mode: Electrospray Negative Source Block Temperature: 150C *lO"C Electrode: Z-spray Analysis Type: Multiple Reaction Monitoring Spectrometer (MRM) Quattro IITMTriple Table 2. Target Ions Monitored in 3M Environmental Laboratory Analyses I Target Analyte I POAA I Primary Ion (AMU) I Product Ion (AMU) I I 41 3.0 I 119.0, 169.0, 219.0 I THPFOS 427.0 80.0 3M Environmental Laboratory 3M Environmental Laboratory Page 14 Page 14 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 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: 0 Linearity: The coefficient of determination (r2)equal to or greater than 0.980 0 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: 70-1 30% for sera, 60-1 40% 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 EnvironmentalLaboratoryprotocol for FACT TOX-026 (see Appendix B) were met with the exceptions noted in this report. Summary of Quality Control Analyses Results 0 Linearity: The coefficient of determination (r2)of the standard curve was 20.980. 0 Calibration Standards: For sera and liver analyses, quantitation of the target analyte u s e d linear regression analysis, weighted l / x , of t w o extracted matrix c u r v e s bracketing each group of samples, or of a single curve preceding the samples. Urine data was determined using a l / 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 responsemonitoring mode of the instrument (see Appendix C, Analytical Methods). 0 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 3M Environmental Laboratory Page 15 Page 15 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 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. 0 Precision: Not specifically determined within the course of this phase of the study. 0 Matrix Spikes: Sera-Matrix spike samples were preparedfrom 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 backgroundlevels 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. Statement 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 spik.e 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 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. 0 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 3M Environmental Laboratory Page 16 Page 16 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 0 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. 0 Precision: Not specifically determined within the course of this phase of the study. 0 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. 0 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. Statement 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 11Yo.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 Sample Results 0 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. 0 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 3M Environmental Laboratory Page 16 Page 17 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 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 3M Environmental Laboratory Page 17 Page 18 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 A: Chemical Characterization and Control Matrices Control Matrix Rabbit Serum TN-A-2052 Rabbit Serum TN-A-2307 Rabbit Liver TN-A-0808 Rabbit Serum TN-A-2391 Rabbit Serum TN-A-2573 Source Expiration Date Storage Conditions I Chemical Lot# I Physical Description Sigma Chemicals Sigma Chemicals 01/01/2010 01/01/2010 -20C f 10C -20C f 10C I 107H8409 I 118H8418 I I Rabbit Serum I Rabbit Serum I Coming Hazelton Sigma Chemicals Sigma Chemicals 01/01/2010 01/01/2010 01/01/2010 -20C f 10C -20C f 10C -20C f 10C FOOO11 I 118H8418 I 118H8418 I Rabbit Liver I Rabbit Serum I Rabbit Serum I I Control Matrix I Rabbit Liver TN-A-0806 I Rabbit Liver TN-A4802 I Rabbit Serum 99062-055 I Monkey Urine 99062-067 I Monkey Urine 99062-076 I Source Corning Hazelton Coming Hazelton Sigma Chemicals N/R Covance Expiration Date 01/01/2010 01/01/2010 01/01/2010 01/01/2010 01/01/2010 I I I I I I I Storage Conditions I I I I I I I Chemical Lot # -20C f 10C FOOOO9 -20C f 10C F00005 -20C f 10C 1181-18418 -20C f 10C I06015 -20C rt 10C 6329-262 I Phvsical Descriotion I Rabbit Liver I Rabbit Liver I Rabbit Serum I Monkev Urine I Monkev Urine I Control Matrix I Source Expiration Date Monkey Urine 99062-078 I Covance I 01/01/2010 Storage Conditions -20C f 10C Chemical Lot # 6329-262 Physical Description Monkey Urine 3M Environmental Laboratory 3M Environmental Laboratory Page 18 Page 19 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 Table 4. Characterizationof Test and Reference Substances in Study FACT TOX-026 I Testsubstance I Reference Substances I Laboratory LInacb.oratories' 3M EnvironmentalLab 3M EnvironmentalLab Centre Analytical Laboratories Chemical Name APFO (Ammonium Perfluorooctanoate) TC+~$Glqj .__ 'O (Ammonium luorooctanoate) TN-A-1479 SE-030 APFO (Ammonium Perfluorooctanoate) TN-A-0497 SD022 APFO (Ammonium Perfluorooctanoate) TCR-99030-30 Source Expiration Date Storage Conditions Chemical Lot # Physical Description I Purity 3M Specialty Chemical N/R Ambient temperature 332 White powder 95&95.2% ecialty Chemicals 2010 3M SpecialtyChemicals 1/1/2010 3M Specialty Chemicals N/R Ambient temperature Ambient temperature Ambient temperature 245 N/R 332 White powder TBD** Light-colored powder I TBD** White powder 95.0% I Analytical Reference Substances I I Laboratory Centre Analytical Laboratories 3M Environmental Lab Chemical Name THPFOS SE037 THPFOS SD028 1 1 I Expiration Date 1/01/2010 Ambient temperature 1/1/2010 Ambient temperature Chemical Lot # 53406 59909 Purity Brown waxy solid TBD** Brown powder I NA* 3M Environmental Laboratory 3M Environmental Laboratory Page 19 Page 20 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 B: Protocol, Protocol Amendments and Deviation Summary 3M Environmental Laboratory 3M Environmental Laboratory Page 20 Page 21 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 Protocol #FACT-TOX-O26 Study Title 6-Month Capsule Toxicity Study with Ammonium Perfluorooctanoate (APFOPOAA) in Cynomolgus Monkeys PROTOCOL Author Lisa Clemen Date: October 12, 1998 Performing Laboratory 3M Environmental Technology & Safety Services 3M Environmental Laboratory - 935 Bush Avenue St. Paul, MN 55106 Laboratory Project Identification FACT-TOX-026 3/14Environmental Laboratory 3M Environmental Laboratory Page 7 of 9 Page 22 - .- . ~ 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 Protocol #FACT-TOX-026 Study Identification 6-Month Capsule Toxicity Study with Ammonium Perfluorooctanoate (APFOPOAA) in Cynomolgus Monkeys Test Material Ammonium perfluorooctanoate ' (APFOPOAA) 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 65 1-737-2678 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. 330 1 Kinsman Boulevarg 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 3M Environmental Laboratory Page 2 of 9 Page 23 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 Protocol #FACT-TOX-026 1. STUDY Six month capsule toxicity study with ammonium perfluorooctanoate(APFOPOAA) in cynomolgus monkeys. 2. PURPOSE The analyticalportion 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. REGULATORCYOMPLIANCE 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. QUALITYASSURANCE 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. TESTMATERIAL 5.1 ldenfification Ammonium perfluorooctanoate(APFOPOAA) 5.2 Source 3M Specialty Chemical Division 5.3 Physical Description Gelatin capsules 5.4 Purity and Stabirify 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 3M Environmental Laboratory Page 3 of 9 Page 24 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 Protocol #FACT-TOX-O26 6. CONTROL MATERIAL 6.7 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 -20 "C k 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. REFERENCMEATERIAL 7.7 ldentification 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 biological materials and preparing samples for analysis. - 8. TESTSYSTEM 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 Environmenfal Laboratory 3M Environmental Laboratory Page 4 of 9 Page 25 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 Protocol #FACT-TOX-0L2R6N-U2782 9. SPECIMERNECEIPT 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 Packed on dry ice for shipping Shipped with final serum samples on dry 264 from main study 78 additional from recoverv 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.PREPARATORMYETHODS IO.1 FACT-M-1.O, Extraction of Potassium Peffluorooctanesulfonate or Other Anionic Fluorochemical Surfactant from Liver for Analysis Using HPLC-ElectrosprayNass Spectrometry 10.2 FACT-M-3.1,Extraction of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds from Serum or Other Fluid for Analysis Using HPLC- ElectrosprayMass Spectrometry . 10.3 If preparatory methods other than those listed above are used, an amendment to this protocol will be written. - 11.ANALYTICAMLETHODS 11.1 FACT-M-2.0, Analysis of Fluorochemicals in Liver Extracts Using HPLCElectrosprayNass Spectrometry 11.2 FACT-M-4.1,Analysis of Potassium Peffluorooctanesulfonate or Other Fluorochemicals in Serum or Other Fluid Extracts Using HPLC-ElectrosprayNass Spectrometry 11.3 If analytical methods other than those listed above are used, an amendment to this protocol will be written. 3M Environmental Laboratory 3M Environmental Laboratory Page 5 of 9 Page 26 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 Protocol #FACT-TOX-026 72. DATAQUALITOYBJECTIVES 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: 72.7 Linearity r2 2 0.980 72.2 Limits of detection/ quantitation 12.2.1Method Detection Limit (MDL) for APFOPOAA a. Serum: 5ppb b. Liver: 24ppb 72.2.2Practical Quantitation Limit (PQL) - Equal to the lowest standard in the calibration curve 72.3 Duplicate acceptable precision e 30% for the method 72.4 Spike acceptable recoveries 70% - 130% 72.5 Use of confirmatory methods Indeterminatesamples will be re-analyzed 72.6 Demonstration of specificity Chromatographicretention time, mass spectral daughter ion characterization 73.SUB-CONTRACTAENDALYSIS 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.STATISTICAALNALYSIS 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 (weighvweight or weighdvol) of APFOPOAA or metabolite APFOPOAA or metabolite per unit of tissue or fluid. per -tissue or fluid, or of 74.2 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. 75.REPORT A report of the results of the study will be prepared by 3M EnvironmentalLaboratory.The report will include, but not be limited to, the following, when applicable: 75.7 Name and address of the facility performing the study 75.2 Dates upon which the study was initiated and completed 3M EnvironmentaI Laboratory 3M Environmental Laboratory Page 6 of 9 Page 27 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 Protocol #FACT-TOX-026 75.3 A statement of compliance by the Study Director addressing any exceptions to Good Laboratory Practice Standards 75.4 Objectives and procedures as stated in the approved protocol, including any changes in the original protocol 75.5 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 75.6 Stability and the solubility of the test substances under the conditions of administration, if provided by the Sponsor 75.7 A description of the methods used to conduct the test(s) 75.8 A description of the test system 75.9 A description of any circumstances that may have affected the quality or the integrity of the data 75.70 The name of the Study Director and the names of other scientists, professionals, and supervisorypersonnel involved in the study 75.7 7 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 75.72 Statistical methods used to evaluate the data, if applicable 75.73 The signed and dated reports of each of the individual scientists or other professionals involved in the study, if applicable 75.74 The location where raw data and the final report are to be stored 75.75 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 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. 76.LOCATIONOF RAWDATA,RECORDSA,ND FINALREPORT Original data, or copies thereof, will be available at 3M EnvironmentalLaboratory to facilitate audits of the study during its progress and before acceptance of the final rep6rt. 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 EnvironmentalLaboratory Standard Operating Procedures. 3M Environmental Laboratory 3M Environmental Laboratory Page 7 of 9 Page 28 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 Protocol #FACT-TOX-026 16.1 The following raw data and records will be retained in the study folder in the study/projectarchives accordingto 3M EnvironmentalLaboratory 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 16.7.5 Approved final report (original signed copy) 16.1.6 Electronic copies of data 76.2 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 762.2 Calibration records 16.2.3 Instrument maintenance logs 16.2.4 Standard Operating Procedures, Equipment Procedures, and Methods 17.SAMPLERETENTION 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 EnvironmentalLaboratory Standard Operating Procedures. 18.PROTOCOL AMENDMENTASND DEVIATIONS Planned changes to the protocol will be in the form of written amendments 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 proto-col 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. ATTACHMENTS 19.1 Attachment A Preparatory and analytical methods 3M Environmental Laboratory 3M Environmental Laboratory Page 8 of 9 Page 29 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 Protocol #FACT-TOX-026 20. SIGNATURES PdldfA+ Paul Lieder, Ph.D, DABT, Sponsor Representative ///30/W Date l!w/A/b L Knsten Hansen, Ph.D., 3M Environmental Laboratory Study Director I// z/3Y Date 3M Environmental Laboratory 3M Environmental Laboratory Page 9 of 9 Page 30 3M Medical Department Study: T-6889.3 t c Analytical Report: FACT-TOX-026 LRN-U2782 . - 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 Environmental Technology & Safety Services 3M Environmental Laboratory 935 Bush Avenue St. Paul, MN 5 5 106 Laboratory Project Identification ET&SS FACT-TOX026 LIRN U2782 3M Environmental Laboratory 3M Environmental Laboratory Page 31 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 Protocol FACT-TOXO26 Amendment No. 1 This amendment modifies the following portion(s) of the protocol: 1. f ROTOCOL READS; Section 10.0 and 11.O list the followingmethods to use for extraction and analysis: FACT-M-1.O "Extraction of Potassium Perfluorooctanesulfonateor Other Anionic Fluorochemical Surfactant from Liver for Analysis Using HPLC_Electrospray/Mass Spectrometry" FACT-M-3.1 "Extraction of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds from Serum or Other Fluid for Analysis Using HPLC-ElectrosprayMass Spectrometry" FACT-M-2.0 "Analysis of Fluorochemicals in Liver Extracts Using HPLC-ElectrosprayMass Spectrometry" FACT-M-4.1 "Analysts of Potassium Perfluorooctanesulfonateor Other Fluorochemicalsin Serum or Other Fluid Extracts Using HPLC-ElectrosprayMass Spectrometry" AMENDTO 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 Perfluorooctanesulfonateor Other Fluorochemical Compounds from Serum for Analysis Using HPLC-ElectrosprayMass Spectrometry" ETS-8-5.1 "Analysis of Potassium Perfluorooctanesulfonate or Other Fluorochemicals in Serum Extracts Using HPLC-ElectrosprayMass 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 llx weighting and a few minor changes in the HPLC 1100 instrument parameters. 2. f ROTOCOL READS: Section 10.0 and 11.O list the followingmethods to use for extraction and analysis: FACT-M-1.O"Extraction of Potassium Perfluorooctanesulfonateor Other Anionic Fluorochemical Surfactant from Liver for Analysis Using HPLC-ElectrosprayMass Spectrometry" ETS-8-4.1 "Extraction of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds from Serum or Other Fluid for Analysis Using HPLC-ElectrosprayMass Spectrometry" FACT-M-2.0 "Analysis of Fluorochemicals in Liver Extracts Using HPLC-ElectrosprayMass Spectrometry" ETS-8-5.1 "Analysis of Potassium Perfluorooctanesulfonateor Other Fluorochemicals in Serum or Other Fluid Extracts Using HPLC-ElectrosprayMass Spectrometry" 3M Environmental Laboratory 3M Environmental Laboratory Page 32 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 Protocol FACT-TOX026 Amendment No. 1 AMENDTO READ: The extraction and analytical methods FACT-M-1.O and FACT-M-2.0, respectively, were updated on 07/22/99 to: ETS-8-6.0 "Extraction of Potassium Perfluorooctanesulfonateor Other Fluorochemical Compounds from Liver for Analysis Using HPLC-Electrosprayhlass Spectrometry" ETS-8-7.0 "Analysisaf Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds in Liver Extracts Using HPLC-ElectrosprayMass Spectrometry" REASONT: he 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 llx weighting and a few minor changes in the HPLC 1100 instrument parameters. 3. PROTOCOL READS: Section 10.0 and 11.O list tlle 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-ElectrosprayMass Spectrometry"ETS-8-4.1 "Extraction of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds from Serum or Other Fluid for Analysis Using HPLC-Electrosprayklass Spectrometry" ETS-8-7.0 "Analysis of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds in Liver Extracts Using HPLC-ElectrosprayMass Spectrometry" ETS-8-5.1 "Analysis of Potassium Perfluorooctanesulfonate or Other Fluorochemicals in Serum or Other Fluid Extracts Using HPLC-Electrosprayh4ass Spectrometry" AMENDTO 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-ElectrosprayMass Spectrometry" ETS-8-97.0 "Analysis of Potassium Perfluorooctanesulfonateor Other Fluorochemical Compounds in Urine Extracts Using HPLC-Electrosprayh4assSpectrometry" REASONT: hese methods were developed and validated for urine extraction and analysis after the original protocol was written and approved. 3M Environmental Laborafory 3M Environmental Laboratory L Page 33 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 Protocol FACT-TOXO26 Amendment No. 1 4. PROTOCOL READS: Section 2.0 lists serum and liver as the matrices of interest for determination of POAA. AMENDTO READ: Urine will be included for determinationof POAA. REASONT: he 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. AMI" TO READ; Control matrix monkey urine was obtained from Covance Laboratories and is maintained at a temperature of -20 OC 10 ' C . All traceability information for this matrix will be included in the final report. REASON:Addition of control urine matrix to the ahalytical protocol. 6. PROTOCOL READS; Section 12.2 lists monkey serum and liver method detection limits. AMENDTO READ: Method detection limit for urine is 6 ppb. REASONA: ddition of method detection limit for urine matrix. Amendment Approval Sponsor Representative 1997 u QL*dd,3, Date Kris J. Hansen, Ph.D., Study Director 3M Environmental Laboratory 3M Environmental Laboratory ?/4/sg Date Page 34 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 - Study Title 6-Month Capsule Toxicity Study with Ammonium Perfluorooctanoate(APFOFOAA) in Cynomolgus Monkeys PROTOCOL AMENDMENT NO. 2 Amendment Dafe: 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 3M Environmental Laboratory Page 35 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 Protocol LRN-U2 782 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. AMENDTO READ; 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 of study director was reassigned in an effort to ensure compliance 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. AMENDTO READ: The role of sponsor for 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 was reassigned. In this manner, personnel responsibilities and workload are more evenly balanced. 3. PROTOCOL READS: 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. AMENDTO READ: 17. Specimen Retention: Specimens will be maintained in the 3M Environmental Laboratory specimen archives. Any specimens sent to sub-contract laboratories will be returned to the 3M Environmental Laboratory upon completion of analysis and submission of the subcontract 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 of specimens analyzed at subcontract laboratories. 3M Environmental Laboratory 3M Environmental Laboratory Page 36 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 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 amendments; study correspondence; shipping records; raw data; approved final report (original signed copy); and electronic copies of data. Additionally, Section 16 states that supRorting 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; caIibration records; instrument maintenance logs; Standard Operating Procedures, Equipment Procedures, and Methods; and appropriate specimens. AMENDTO READ: Section 16 states: "The original data, or copies thereof, will be available at the 3M Environmental Laboratory to facilitate audits of the study during its progress and before acceptanee of the final report. When 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 of the 3M Environmental Laboratory. All corresponding training records, calibration records, instrument maintenance logs, standard operating procedures, equipment procedures, and methods will be retained in the archives of the facility performing each analysis. REASON: To direct subcontract laboratories in the disposition of the items listed above. 3M Environmenfal Laboratory 3M Environmental Laboratory Page 37 3M Medical Department Study: T-6889.3 t Amendment Approval Analytical Report: FACT-TOX-026 LRN-U2782 Protocol LRN-U2782 Amendment Number 2 John Butenho8 Ph.D., Sponsor Representative Date Kristen J Hunsen, Ph.D., Outgoing Study Director Date Paul Lieder, Ph.D., Incoming Study Director Date 3M Environmental Laboratory 3M Environmental Laboratory Page 38 3M Medical Department Study: T-6889.3 c Analytical Report: FACT-TOX-026 LRN-U2782 Study Title 6-Montch Capsule Toxicity Study with Ammonium Perfluorooctanoate (APFO/POAA) in Cynomolgus Modeys PROTOCOL AMENDMENT NO. 3 - Amendmevt Date: 20 April 2000 Performing Laboratories 3M Environmental Technology and Safety Services Fluorine Analytical Chemistry Team Building 2-3E-09,935 Bush Avenue St. Paul, MN 55 106 Centre Analytical Laboratories, Inc. 3048 Research Drive 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 3M Environmental Laboratory Page 39 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 Protocol LRN42782 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. AMENDTO 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, s e w , and urine. AMENDTO 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. PROTOCORLEADS: The amended Section 12.2.1 items a., b., and c., list the Method Detection Limits for matrices analyzed in this study. AMENDTO READ: The method detection limits for all compounds and matrices will be taken fiom 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. PROTOCORLEADS: Section 13. lists the laboratories that will be conducting analyses for this study. AMENDTO 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 3M Environmental Laboratory Page 40 3M Medical Department Study: T-6889.3 * Analytical Report: FACT-TOX-026 LRN-U2782 Protocol LRN-U2782 Amendment Number 3 5. PROTOCORLEADS: 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. AMENDTO READ: The feces extraction and analytical method used by Centre Analytical Laboratories will be; 00M-023-003 (Revision 2), "Determination of Fluorochemical Residues in MonkeyRat Feces by LCIMSlMS." 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. ButenhofJ; Ph.D., Sponsor Representative Date Paul Lieder, Ph.D., Study Director Date 3M Environmental Laboratory 3M Environmental Laboratory Page 41 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) in Cynomolgus Monkeys PROTOCOL AMENDMENT NO. 4 Amendment Date: August 2,2000 Performing Laboratories Covance LaboratoriesInc. 3301 Kinsman Boulevard Madison, WI 53704 3M EnvironmentalTechnology& Safety Services 3M EnvironmentalLaboratory 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 3M Environmental Laboratory Page 42 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 Protocol TOX-026 Amendment 4 This amendment modifies the following portion(s) of the protocol: 1. PROTOCOL READS: STUDYTITLE:"6-Month Capsule Toxicity [..I" (on the title page and page 2 of the protocol for the analytical phase of the study). AMENDTO READ: STUDYTITLE:"26-Week Capsule Toxicity [..I" (on the title page and page 2 of the protocol for the analytical phase of the study). REASON: To correct the title for the analyticalphase of the study. 2. PROTOCOL READS: DATAQUALITYOBJECTIVE(SSection 12., page 6) AMENDTO READ: Add to this section: LOQ in feces of 10 ng/g REASON: To specify the analytical limits for feces analyses. 3. PROTOCORLEADS: (page 2) STUDYDIRECTORPe: ter J. Thomford and Paul Lieder TESTINFGACILITYC: ovance Laboratories SPONSOR:APME AdHoc APFO Toxicology Working Group and 3M Toxicology Services - Medical Department SPONSORREPRESENTATIVDEav: id Farrar and John Butenhoff AMENDTO READ: STUDYDIRECTORP:aul Lieder TESTZNFGACILITY3:M Toxicology Services - Medical Department SPONSORA: PME AdHoc APFO Toxicology Working Group (including 3M Toxicology Services - Medical Department) SPONSOREPRESENTATIVDEa:vid 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 responsibilitiesof all parties included in this study. 3M Environmental Laboratory Page 43 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 Protocol TOX-026 Amendment 4 4. PROTOCOL READS: PRINCIPALANALYTICAINLVESTIGATOR:Kristen Hansen, Ph.D. (Amendment No 2 to TOX 026, Section 1.) AMENDTO READ: Add: PRINCIPALANALYTICAILNVESTIGATORS:Kristen Hansen, Ph.D. and Enaksha Wickremesinhe, Ph.D. REASON: To spec@ the PAI at Centre. 5. PROTOCOL READS: ANALYTICATLERMINATIODNATE:May 12, 1999 (under Proposed Study Timetable, page 2) AMENDTO READ: ANALYTICATLERMINATIODNATE:September 30,2000 REASON: To allow for the analyses of all samples. 6. PROTOCOL READS: LOCATIOONF RAWDATA,RECORDSA,ND FINALREPORT(Section 16., page 7) AMENDTO READ: Add: After issuing their final report, Centre will forward all original study-specific raw data to 3M EnvironmentalLaboratories,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. PROTOCOL READS: SAMPLERETENTIO(NSection 17., page 8) AMENDTO READ: After the analyticalreport on feces is signed by the PAI, all feces specimens of this study will be returned to 3M EnvironmentalLaboratory. 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 3M Environmental Laboratory Page 44 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 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 of the above biological specimens, and to define when the quality assurance verification is considered complete. 3M Environmental Laboratory Page 45 3M Medical Department Study: T-6889.3 Amendment Approval Analytical Report: FACT-TOX-026 LRN-U2782 Protocol TOX-026 Amendment 4 John Butenhos Ph.D. Date Sponsor Representative, Analytical Phase Paul Lieder, Ph.D., DABT Date 3M Study Director fiaksha Wickremesinhe, Ph.D. Date Centre Analytical Laboratories, Principal Analytical Investigator 3M Environmental Laboratory Page 46 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 i 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 3M Environmental Laboratory Page 47 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 PrOtOCOl FACT TOX-026 J 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. AMENDTO 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 3M Environmental Laboratory Page 48 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 Protocol FACT TOXL-0R2N6-U2782 J Amendment No. 5 Amendment Approval John L. Butenhofi Ph.D. Date Sponsor Representative, Analytical Phase Paul Lieder, Ph.D.,DABT 3M Study Director ldt5/D 0 Date J ' 2-100 Date Centre Analytical Laboratories, Principal Analytical Investigator 3M Environmental Laboratory 3M Environmental Laboratory Page 49 3M Medical Department Study: T-6889.3 Record of Deviation Analytical Report: FACT-TOX-026 LRN-U2782 DProtocol cw-w L3as- 23! Method CI Equipment Procedure CJ Other: I 111. Actions Taken: IV. Impact on Study/ Project __ - __ _^- - _ _ _ _ - - _ I _ _____-~---I------^-- -- - - - -. Date Form ETS-4-8.0 3M Environmental Laboratory Deviation No. (assigned by Study Director or Project Lead at the end of study or project) Page 50 3M Medical Department Study: T-6889.3 Record of Deviation Analytical Report: FACT-TOX-026 LRN-U2782 1. Identification Study-/Project N6.- - __ -- - __ __- -_ - _ _ - - _ __ - - __ Deviation Type FACT- n>aX-SOP C . ~ u o n a L329- 231 66 Method a Equipment Procedure (Check one) ClProtocol 0 Other: - Document Number FALT- yr\- '4.1 Date@)of occurrence II IIdq3, II/z.r/Qa, 12/03/qd i Odod94. Ol/d94 11. Description: Ol/Sf/4? Required ................. Procedure/process: ~ ...Ae.4h.d ....... .................................... ....... .- .......................... .......... .....L . H . c ~d,c..Jd he......pL.bLd - -___I .......................................... Ur.i.,..a ..Iine~.......~........ -~ ... ..................... ...... ............. ...................................................................................................... ............................................... .-....... ........ ...................................................................... ................................................................................................. -_____~__.__.I_ - ~ h.&d pL&.d wt;&Ld !.IN.. Actual Pr__o__c_.l_e-_-d__.l_u"re/process: .... CLXLML~.~ .... . .. . . u J . ; ~.....l.i.~~a r.... .r c ~ c ~ . ~.I. ~.... i ~ . n . ......... .- ................... ................................................... ___^______ .......... .................................. ___.______..I_ ... ....................... .... ................... .-........ -- 7h. ..... 1.J ..... Ill. Actions Taken: (such as amendment issued, SOP revision, etc.) -__II_ _-_l_l_________I____ll-lll____l_" . J IV. Impact on Study / Project Form ETS-4-8.0 3M Environmental Laboratory (assigned by Study Director or Project Lead at the end of study or project) Page 51 3M Medical Department Study: T-6889.3 Record of Deviation I. Identification I Analytical Report: FACT-TOX-026 LRN-U2782 Deviation Type (Check one) I Document Number a SOP JZJ Protocol Ffl~7.m -3, I + IJMethod IJEquipment Procedure CI Other: Date(s) of oqcuq-ence w I Ill. Actions Taken: (such as amendment issued, SOP revision, etc.) QA ~ f h h d b~ ~ht P F d ' b C d be iSJked. 3M Environmental Laboratory Form ETS-4-8.0 3M Environmental Laboratory Date Deviation No. 3 (assigned by Study Director or Project Lead at the end of study or project) Page 52 3M Medical Department Study: T-6889.3 Record of Deviation Analytical Report: FACT-TOX-026 LRN-U2782 1. Identification I Study / Project No. Deviation Type (Check one) T~~~~~ C D V L f l e e L3a5-231 a SOP RMethod Cl Equipment Procedure 0 Protocol Cl Other: I Document Number ET^ 8 ,Y. 1 (. k7.s- 2~ S,I Date(s) of occurrence Y Ia4l4q 11. Description: L Ill. Actions Taken: (such as amendment issued, SOP revision, etc.) Th- t d b d s hill h, rcu;d tO .I ~I*L +his Compofllnf. IIIRecorded By .-_ _ .. Date I IV. Impact on Study / Project I Date 3M Environmental Laboratory Form ETS-4-8.0 3M Environmental Laboratory Deviation No. CI (assigned by Study Director or Project Lead at the end of study or project) Page 53 3M Medical Department Study: T-6889.3 Record of Deviation 1. Identification Analytical Report: FACT-TOX-026 LRN-U2782 Deviation Type FACT-TU~ a02lp SOP (Check one) ClProtocol __ Document Number - - CDvonGL 6329- 231 ,lQ Method 0Equipment Procedure 0 Other: ' Date@)of occukence M~Ihod ACT - d a k s +L.Q.~~ . &Ud IGLd l bc .-wtd as 1L.. Requ..i..red Procedure/process: .. =EL!.. D.. .................................................................................................................................................................................................... ..,. ............ ..... ... ....... ....... .... . ........ .................................................. QX solue/ll. __ MecL$.-..k*l:.-h.x-a~-'dLQrL.h.Tw.E) 4% Actual Proc_ed_ure_/_p__r__o_c__e__sI_s_:....I" ..... .......... .- - ...... .........W.*J. .. ..u.re.k........R.5..... -- .- .. .... ......... ............-exLcccA~3n......- 4olveA I.............................................................. .... .............. ................................................................................................................................................................................................................................ Ill. Actions Taken: _..________ .. ..... d-&AkIV. Impact on Study/ Project -. j Date ................ Id lrloo 3M Environmental Laboratory Page 54 3M Medical Department Study: T-6889.3 Record of Deviation Analytical Report: FACT-TOX-026 LRN-U2782 I. Identification - " _ Study / Project No. Deviation Type (Check one) EACY:- Pak .SPOJPL ClProtocol LDULifdlc3Ra - a31 Method a Equipment Procedure a Other: .. ... ".................... ............... Document Number FACT- IT- 2.0 ................... . _ . . . ......-i... ................................................ ............ . . . . . . . . . . . . ' Date(s) of occurrence d d j 94, 5 1 r d 9 4 , 5 1 r 3 / 9 < .. biz'+/??, 151f?/91r, S/V.~?'I, L / r l - / ? 9 , b11ziS4, L / a j / q 9 , : 7111199. ? 1 2 i / 9 S 11. Description: Required P_ rocedure/process: ~ ~ M... dhad FA-CIz.P..:.U I;r\eor '&@a .......... dd ~ c.u.c% -...-............... ... In>_iU....hP, &kd. ~ ~ . i n % . ............................ ___I___.._______ I..... - .. _ ................... ................................. _ ... ~ _ ............ .- __........ .......... ........ ...... ............ ._ . ....- ....... -................ .................. -. ................. ....... ^ ........................................... ......... - .................................................................................. ............ ........ .......... _ ~-_l_l__ ._ ~ ................... _............... . ....." ............ ...... ....... ......-...... . . . . . . . . . . . . . . . . . . . . . . I___ ___._ -_-_.__-I_^--..^ - .......-. . . . . . . . . . . . . . - ........ .... ......... .......... ................................ - __ _-_^_ Ill. Actions Taken: (such as a_m_ _e_ ndment issued, SOP revi_si_on_, etc.) ~ _ I I Thrr d t v i a h - wOJ ~f~rlkkn.__ - _- -- - . ~ Recorded By . .. ! Date @hA LIV. Impact on Study / Project 3 M Environmental Laboratoiy a Form ETS-4-8.0 3M Environmental Laboratory Deviation No. b (assigned by Study Director or Project Lead at the end of study or project) Page 55 3M Medical Department Study: T-6889.3 Record of Deviation Analytical Report: FACT-TOX-026 LRN-U2782 1. /dentification I - - . ___ __ - Study / Project No. Deviation Type _F_Ac-r-_ P Xa- 0S2O(PP - @Protocol C~UQWL LXN - a31. IJMethod CI Equipment Procedure' # Other: Require.d.........P........r....o. cedure/pr_o....cess: T....... h..... ..Il&J ............. wi1.1 b.ewed zo d .+la "_~ ........~ _. .............. . .............. ... ........................................................................... Qn.0.l-.LL....... ................. ____^_..__-- I._ ........._ ............. -__ ^......~ ................................................................................. ...- . . .... Recorded By &&A _-__I I-.-l___^-I._l _ cQe.me*, IV. Impact on Study/ Project __ - . .-. "- ......... " " ........ ... ..................... j Date I lYl00 Form ETS-4-8.0 3M Environmental Laboratory Deviation No. 7 (assigned by Study Director or Project Lead at the end of study or project) Page 56 3M Medical Department Study: T-6889.3 Record of Deviation Analytical Report: FACT-TOX-026 LRN-U2782 MProtocol CuvanLe d324- 231 0 Method 0 Equipment Procedure Cl Other: I ..... ........... .... . ... _._I.... .. _-_ ....... - 111. Actions Taken: de -~___;-- - j - ~ - - _ _ --_(s_uch-a- s amendmeI nt_ i-ssued, SOP ~ revis_i_on, I_ etc.) - -- ~ VIQ 'CM War wriUen. _- __ I.............................................................. .............................................................................. ................................................................................................................. ................. -. ..................... .............................. ... .......... -. . . . . . . . . . . . ............. ........... ... .......... ...... I1 o l h A Recorded By .............. hIV. Impact on Study/ Project Date . . . . . . . . . . . . . . . . IYLS __ ___ Authorized By - ~ __ __ -- -t j h 11/17/d Date 3M Environmental Laboratory Form ETS-4-8.0 3M Environmental Laboratory Deviation No. 8 (assigned by Study Director or Project Lead at the end of study or project) Page 57 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 Record of Deviation IIStudy / Project No. I .F~...L.TZ..a-DA: ..L..CQ.~..&. Deviation Type ...... ............................. . 1. IdenMication ---- -- - ___ 0a.L SOP D Method ............._........................ flProtoco1 0 Other: ~.~.+?..:..J.J-J D Equipment Procedure ...._....... ......... ................................................. Document Number ............. ..AL.........I $ r L u.. Req.u.....i..red Procedure/process_ : _ .......... .... ..... .. ..&Jiw--..-cx. ...IIG..b.-.-WLh.ad ..... ....... . . ............ FA.LT:..-@..:..-...!................:.P.......... I ~ h . 7.d~e.v;iiL & ... - _.. Ill. Actions Taken: (such _.l_-__l____..-_I.-- as amendment issued, --.-__ -._...^_____I__ S_O_P__ _r_e_ v..i_ s.i._ .o....n...,. e-tc.)-.-...... - ....~ u a . J....~ i > . n.......................................................... .. ........ .............. ........ .. - ..... - .-........... .-........................................................................ . ... .. IRecorded By . .................................................................... ... __ .. ............................... ~ Date .-. . . . I IV. Impact on Study/ Project fd/J& * __ - .- - . -__ - - ___- _-- - Authorized By (Study Director / Projeci Lead) f7?&7. /,!O/W/ .. .- Date YELd/ SPWSW., J D ) \ ~ h L h o F C SL,dd DfiLcLr: Pc,l*\ L t d e p 3M Environmental Laboratory Deviation No. 9 Form ET'S-4-8.0 (assigned by Study Director or Project Lead at the end of study or project) 3M Environmental Laboratory Page 58 3M Medical Department Study: T-6889.3 Record of Deviation Analytical Report: FACT-TOX-026 LRN-U2782 1. Identification . ..-.............."....... .......... - - Study / Project No. Deviation Type (Check one) ..... ...... .......... ......_............. ..... .-................... ................................. ............................................... a FA_ C- T_ -TOx-Oab SOP a a Cpvcnte b3as--231 Method Equipment Procedure .- .............. ClProtocol Cl Other: __ - _- -Document Number Et5 - 8- 97 0 ___ - __ / Date(s) of occurrence ' 08/Ob/qY C 08/lO/44 11. Description: R.........e.....q......uired Procedure/process: . d ~ Q LhS s-Ldurd.. L & I LT ....i"2ekho ........... -kc- c i . a d s w inhn.~! POAA ........................................ ............ .. ...... .....UK~........ ............C A SI....? bc PlutJuc US;,,& c quc2d.mh.c ......................................... ll~0llk4SiQ ..... n~ Actual ........ Pro.cedure/process: . . i_ ......C.......mcf?c\trcki.fi......i..-. ....WaA .....pl&d iljiq .......... cJLJ~JR~~.x:Lmx!s.&A .and . ......-...~s...iyj ........... lhmr..... reglhssi U L ............................................................................................. ... -- P.O.QG ... cQ.~&................. ................................. Recorded By ; Date I . .............. ........ ......... .. ._ ........... Authorized By @udy Direct@/ Project Lead) ; Date ..... 3M Environmental Laboratory Form ETS-4-8.0 3M Environmental Laboratory Deviation No. lo (assigned by Study Director or Project Lead at the end of study or project) Page 59 3M Medical Department Study: T-6889.3 Record of Deviation Analytical Report: FACT-TOX-026 LRN-U2782 1. Identification ................. ..... __ ... . _ _ - Study / Project No. ....................... kP.CT.-.%X.,.. Deviation Type (Check one) . . . . . . . . . . . . ........................... .- - ..... ................................................................ a .b .O.$SOP.............. DProtocol d3 2s 2 El. C.0.wQn.....a....................................................3 1_......................................................... Method IJEquipment Procedure C! Other: ....................................... ................................ ..... ....... .- Document Number ET3 - 8- 93.D ........-........................... 11. ....................................................................................................................... 1 Date(s) of occurrence ! /Ti ! 8/24 Description: Required Procedure/process: ._ ................................................................................. IL SLQ$J d.~~-hVfi-.W& ..Q CI1GI~~ .......tndLo$ - z ~ Q M - I . pQ......mid.rw.+ ~ ~ f i L i u . ~ . ~ ~ C l ,JA d' e- ID J Q ~ P ~ . c . . o m ow bnid. _ ~ ~ ~ ~ . - ~ ~ ~ - ~ . . . ~ ~ ~ ~ . .P. ". _d ~ ~ . l - . . _ J ~ ~ ~ .-Al.-&J one iJkl&a ea1I l o ~ G ~ ~ n " Z?o:!:.&+.L. .-_IP.h.Ycq ...... J&d !aC!enhLLbo _ ....... - . ............. ......................................... ......... .. ! e . .~.........._. 0 ..... ..................................................... - I Actual Procedure/mocess: 1 ...................... ........ ................................................................................................................................................................................................................................................... I __ I- _- Authorized By -- -_ _(Study Director /Pr$ect Lead) - .- Date 3M Environmental Laboratoly F o ET~S-4-8.0 3M Environmental Laboratory Deviation NO. I1 (assigned by Study Director or Project Lead at the end of study or project) Page 60 3M Medical Department Study: T-6889.3 I Study/ Project No. - Record of Deviation 1. Identification NProtocol Cl Other: Analytical Report: FACT-TOX-026 LRN-U2782 Required Procedure-/process: .-% p.A.cal L&A..~u,~ __I -.......- LJJ .---.a:-Q~...Q n.Bph.8..........urina, ............... e 4x... 4a.c...1.................. ..._ ......... ........................... ................................................................................................................................ ..$ &LA ' 4 ----.-----I._. ?... .... .. ...---I___ 6 .... .LdGJ Ill. Actions Taken: (such as amendment is-s-ued, SOP revision, ---- etc.) -- ....... ......... -. . . . . . . . . . " . . W n. ............................................................ ............................................................... .. ............ Recorded By &&A h ......................................... ............................................................ ................................................. i Date IV. Impact on Study / Project I Authorized By (Study-Director /Project Lead) Date 4bB Jpunsor TO^, B,I&,FC j L d d D;,&&, 3M Environmental Laboratoiy Form ETS-4-8.0 la P c J Le& Deviation No. (assigned by Study Director or Project Lead at the end of study or project) 3M Environmental Laboratory Page 61 3M Medical Department Study: T-6889.3 Record of Deviation Analytical Report: FACT-TOX-026 LRN-U2782 Study / Project No. - _-__ ClProtocol Cl Other: occurrence Required Procedure/process: ........-..I I I._. .. plO.bd -% ...... .....-hf.....i....W-.A.L..... ~ Cxbfl..Ql..............J~.iln..d.ar.d., .^ ,~ ........................... .- ........................ ................. 8h .c+ @dr& .--___ ................... -. .. &:..L,.. .he;$led.. '4 ...- .................. ................................................... - ......... ... ,., .......Uf ................. .....-........... ................................................ ...-.. . . - .... -. .... .. -. .. .............................................................. 3M Environmental Laboratory Page 62 3M Medical Department Study: T-6889.3 Record of Deviation Analytical Report: FACT-TOX-026 LRN-U2782 I Study / Project No. 1 Deviation Type (Check one) Document Number fl&@G tl SOP a Protocol 0 M e t h o d m Equipment Procedure c7 Other: Date(s) of occurrence m4+& Ill. Actions Taken: (such as amendment issued, SOP revision, etc.) 92- I I I RecordedBy Date I IV. Impact on Study/ Project Form ETS-4-8.0 3M Environmental Laboratory Deviation No. (assigned by Study Director or Project Lead at the end of study or project) Page 63 3M Medical Department Study: T-6889.3 Record of Deviation Analytical Report: FACT-TOX-026 LRN-U2782 1. Identification study / Project No. -FACTLTOT-~OZ-B- Covance 6329-231 -- - - - - Deviation Type aSOP-- x Method a Equipment Procedure (Check one) DProtocol 0 Other: ! .... ..._............... ._ ~ . .......... ._ Document Number(s): ETS-8-5.1, ETS-8-7.0, and ETS-8-97.0 ....... ........... ._ .. ~ .................... Date(s) of occurrence: Entire study /I. Description: ............................ ... ...R........e.. qui- red Pro- cedure/process: _ The method describes the calculations Imatrix concentration. ~ ~ ~ ~~ .. fI-- _ - _ _ _ ~ ._ ".- that should .. _ ..- . be used to convert extract ................................................................................. ~ _. ... .............. concentration to .....~ .. ........ ................... ..A....c. tual Procedur.e...../._pr_o_ce~ss-:_ _ _ _ __ ~ _ ..- ._ ............ ............ ._........................... ............. The actual calculation used varied somewhat from that written in the method. One additional factor was added for salt correction. This accommodatesthe mass differencebetween the analytical standard (C7F15COONH4) and the target analyte (C7Fl5COO-) determined after the studv was completed. I ..... ........ __ __ ~ ~ Ills Actions Taken: ............ This deviation was writte(snu. ch amendment issued, SOP revision, etc.) .......... -__......... ...-_..._......~-_.--___..______II.. as .... ............................................................................................................... ....................... ......................................... I............... Recorded Bv ___ __ - .. ~ ............... ................-.... Date IV. Impact on Study / Project ............ .. ~ ~ ..... .... ~ ..... ~ ............................ ........ ............. .&3 The updated calculations accommodate new information and are an improvement. No adverse ...a...f.f..e.. ct on th..e. study. ~ ........ ............ . ..O Z.-./. ./.z. ./.Q. . . .1. . -. . 3M Environmental Laboratoly Form ETS-4-8.0 3M Environmental Laboratory Deviation No. td (assignedby StudyDirector or Project Lead at the endof studyor project) Page 64 3M Medical Department Study: T-6889.3 Record of Deviation Analytical Report: FACT-TOX-026 LRN-U2782 1. Identification Study / Project No. FACT-TOX-026 Covake 6329-231 I a X 0 Deviation Type (Check one) SOP Method ClProtocol Cl Other: Equipment Procedure Document Number(s): ETS-8-7.0, Date(s) of occurrence: ...................................................................... ......................................................... i .................................................................................................................... Required Proc..edure/pro.cess: ......... ............. ._ .................................... ................................................... -T-.hese method. s state.t._hat. matrix spike. percent recoveries must be within k 30% of the spiked -. ........ ............. _.-............................... _................................................ Actual Procedure/process: ... ..... .................................................................................................................. ....... ~ .................. .I. ........... ............................ . The matrix spike percent recoveries for liver and wine data were k 40% of the spiked ~ ................ ....................... ................................................................................. concentration. ____ ___^_____ ~ __.__I__-__I_ ~" _ll_.__l_l__.__l .. - . ... . _ _I _ _I This deviation was written. __ ...(..s..u. c.h.....a.. s ..a.....m.. e.n_.d....m... e.n..t...issu..e...d..., SOP re......v......i....s....ion, etc.....)............................................ ~ ..................................................................................................... _--- - -. .._. . . . . . -. ~ ........ .__ ..... -. ............. ..................................................... _ .......... .... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I Recorded By Date -- - - __ - - .-- - I-V__.- Impac.t-o-n- S--t-udy /__ P.- -roject - -- Data quality stated in the final report w_ _ ill reflect actual recovery of the matrix spikes. No adverse impacton the.st_u_d_y-.- - __-_.- 3M Environmental Laboratory Form ETS-4-8.0 3M Environmental Laboratory Deviation No. 15 (assigned by StudyDirectoror ProjectLead attheendof study or project) Page 65 3M Medical Department Study: T-6889.3 Record of Deviation Analytical Report: FACT-TOX-026 LRN-U2782 1. Identification Study / Project No. IDeviation Type I (Check one) FACT-TOX-026 Covance 6329-231 0 SOP 0 Method 0 Equipment Procedure XProtocol 0 Other: - - ____ __ - -- -_- - __ - - I Document Number(s): Date(s) of occurrence: Protocol FACT-TOX-026 Entire Study 11. Description: Required Procedu- re-/pr-o-cess: I . - ._ - _ " _ _ I . I_ Methods ETS-8-7.0 and ETS-8-97._0_ state that matrix spike percent recoveries mustbe within k 30% of the st>ikedconcentration. - - Actual Procedure/process: The matrix spike percent recoveries for liver and urine data were L 40% of the spiked " concentration. ........................................................... ... .... ~. ... ............................................... ..I ", _ _ , , . .. ....... ... .......... I Ill. Actions Taken: (such as amendment-is-s-u-ed,SO- P -r_e- visi_o-_n, etc_._) - --- - - - I _I This deviason was written. JIRecorded By Date IV. Impact on Study / Project Data quality stated in the final report will reflect actual recovery of the matrix spikes. No adverse-impacton the study. - 4-h OC//5/oI 3M Environmental Laboratory Form ETS-4-8.0 3M Environmental Laboratory Deviation No. I? (assignedby StudyDirector or Project Lead atthe endof study or project) Page 66 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 C: Extraction and Analytical Methods This appendix includes the following 3M Environmental Laboratory methods: Liver FACT-M-1.1, Extraction of Potassium Perfluorooctanesulfonateor 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-84.0, "Extraction of Potassium Perfluorooctanesulfonateor other Fluorochemical Compounds from Liver for Analysis using HPLC-Electrospray/Mass Spectrometry", (14 pages) ETS-8-7.0, "Analysis of Potassium Perfluorooctane-sulfonateor other Fluorochemicals in Liver Extracts Using HPLC-Electrospray/MassSpectrometry", (10 pages) Serum ETS-8-4.1, Extraction of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compoundsfrom Serumfor 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 PotassiumPerfluorooctanesulfonateor Other Fluorochemicals in Serum Extracts Using HPLC-Electrospray/MassSpectrometry, (9 pages) FACT-MQ.1, Analysis of Potassium Perfluorooctanesulfonateor Other Fluorochemicalsin Serum or Other Fluid Extracts Using HPLC-Electrospray/Mass spectrometry, (9 pages) Urine ETS-8-96.0, Extraction of Potassium Perfluorooctanesulfonateor Other Fluorochemical Compounds from Urine for Analysis Using HPLC-Electrospray/Mass Spectrometry, (14 pages) ETS-8-97.0, Analysis of PotassiumPerfluorooctanesulfonateor Other Fluorochemical Compounds in Urine Extracts using HPLC-Electrospray/MassSpectrometry/Mass Spectrometry, (10 pages) 3M Environmental Laboratory 3M Environmental Laboratory Page 21 Page 67 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 3M ENVIRONMENTLAALBORATORY METHOD EXTRACTIOONF POTASSIUM PERFLUOROOCTANESULFONATEOR OTHER FLUOROCHEMICCAOLMPOUNDSFROM LIVERFOR ANALYSIUSSING HPLC-ELECTROSPRAY/MSAPSESCTROMETRY Method Number: FACT-M-1.1 Author: Lisa Clemen, Glenn Langenburg Approved By: Adoption Date: 05/26/98 Revision Date: 06103 I49 Laboratory Manager Date Groub Leader duw & A&a Technical Reviewer Date ~lnii~~ Date \ 9 0 SCOPEAND APPLICATION 3 1 Scope: This method is for the extraction of potassium perfluorooctanesulfonate (PFOS) or 80 other fluorochemical compounds from liver. Ilk Applicable Compounds: Fluorochemical surfactants or other fluorinated compounds. @ Matrices: Rabbit, rat, bovine, and monkey liver or other liver as designated in the validation 0, Smreport. p> Microsoft 6.0195 3M Environmental Laboratory FACT-M- 1.1 Extraction of PFOS from Liver Page 1 of 15 Page 68 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 2.0 SUMMARY OF METHOD 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.O 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) C,F,,SO,3.2 PFOSA: perfluorooctane sulfonylamide C,F17S02NH, 3.3 PFOSAA: perfluorooctane sulfonylamido (ethy1)acetate C,Fl,S02N(CH2CH,)CH2CO; 3.4 EtFOSE-OH: 2(N-ethylperfluorooctanesu1fonamido)-ethylalcohol C8F, S0,N(CH2CH,)CH2CH20H 3.5 POAA: perfluorooctanoate(anion of ammonium salt) C7Fl,COO3.6 PFOSEA: perfluorooctane sulfonyl ethylamide C,F17S0,N(CH2CH3)H 3.7 FC-807 monoester C,F,,S02N(CH2CH3)CH,CH20-P03H) 3.8 Surrogate standard lH,1Hy2H,2Hperfluorooctane 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 Ultra-Turrax with T25 grinder attachment for grinding/dispersing/emulsifying 6.1.2 Vortex mixer, VWR, Vortex Genie 2 6.1.3 Centrihge, Mistral 1000 or IEC 6.1.4 Shaker, Eberbach or VWR 6.1.5 Nitrogen evaporator, Organomation 6.1.6 Balance, (+ 0.100 g) 3M Environmental Laboratory FACT-M-l .1 Extraction of PFOS from Liver Page 2 of 15 Page 69 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 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 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 mrn 7.16 Crimp cap autovials Note: Prior to using glassware and bottles, rinse 3 times with methanol and 3 times with Milli- Q" water. 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-QTMor equivalent; all water used in this method should be Milli-QTMwater and may be provided by a Milli-Q TOC PlusTMsystem. 8.2 Sodium hydroxide (NaOH), J.T Baker or equivalent 8.3 Tetrabutylammonium hydrogen sulfate (TBA), Kodak or equivalent 8.4 Sodium carbonate (NqCO,), J.T. Baker or equivalent 8.5 Sodium bicarbonate (NaHCO,), 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 3M Environmental Laboratory FACT-M- 1.1 Extraction of PFOS fiom Liver Page 3 of 15 Page 70 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 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 C,F,,SO,H) 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 1000ml beaker containing 500 ml Milli-QTMwater, mix until all solids are dissolved. Store in a 1 L Nalgene bottle. 8.11.2 1N sodium hydroxide (NaOH): Dilute 10NNaOH 1:lO. 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-Q" water. Adjust to pH 10 using approximately 44 to 54 ml of 1ONNaOH and dilute to volume with Milli-QTMwater. 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 1N NaOH solution. 8.11.4 0.25M Sodium carbonatehodium bicarbonate buffer (Na$O,/NaHCO,): Weigh approximately26.5 g of sodium carbonate (N%CO,) and 21.O g of sodium bicarbonate (NaHCO,) into a 1 L volumetric flask and bring to volume with Milli- QTMwater. 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.OO ppm PFOS, 1.02ppm PFOSA, 0.987 ppm PFOSAA, and 1.10ppm 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 tPg/ml)* 8.12.5 Dilute the stock solution with methanol for a working standard 1 solution of approximately 50 ppm. 3M Environmental Laboratory FACT-M- 1.1 Extraction of PFOS from Liver Page 4 of 15 Page 71 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 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,l-H, 2-H,2-HYC,F,,SO,H 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 10-20ppm. Record the actual volume transferred. 8.14 Liver homogenate preparation Note: Thefollowing procedure will be much easier to perform withfrozen liver tissue. Prevent tissuefrom thawing; keep stored on ice until excising a portion of it. 8.14.1 Weigh 40 g of blank or control liver into a 250 ml Nalgene bottle containing 100 mls Milli-Qm 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 MilliQTMwater, 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.O 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 of livedm1 of homogenate) can be calculated as follows: 8.14.3 Jgrarns (E) of liver1 x raw. weight of 1.O ml of homogenate (density) (g/ml)] {[grams (g) of liver] + [grams (g) ofwater]} 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 MilliQTMwater, and rinse with another 2.5 ml of MilliQm 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. (Donotperform 8.3.2for the sample liver homogenates). 3M Environmental Laboratory FACT-M- 1.1 Extraction of PFOS from Liver Page 5 of 15 Page 72 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 9.0 SAMPLHEANDLING 9.1 All livers are received frozen and must be kept frozen until the extraction is performed. * 10.0 QUALITCYONTROL 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.O ml aliquots of Milli-QTMwater 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 fiom 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 CALIBRATIOANND 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. 3M Environmental Laboratory FACT-M- 1.1 Extraction of PFOS from Liver Page 6 of 15 Page 73 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 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 concentrationsand 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-1.1-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 concentrationto fall within the calibration curve range 10ppb - 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. 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.O 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. 3M Environmental Laboratory FACT-M- 1.1 Extraction of PFOS from Liver Page 7 of 15 Page 74 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 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 11.1or 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 m10.5 M TBA and 2 ml of the 0.25 M sodium carbonatehodium bicarbonate buffer. 12.10Using 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. 12.15Add 1.Oml or appropriatevolume of methanol to each centrifuge tube using a graduated pipette. Methanol volume equals the initial volume of liver homogenate used for the extraction. 12.16Vortex mix for 30 seconds. 12.17Attach 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 DATAANALYSIASND CALCULATIONS 13.1 Calculations: 13.1.1 Calculate actual concentrations of PFOS, or other appropriate fluorochemical, in calibration standards using the following equation: 3M Environmental Laboratory FACT-M- 1.1 Extraction of PFOS from Liver Page 8 of 15 Page 75 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 ml of Standard x Concentration of Standard ( d m l ) - Concentration of Blank Liver Homogenate (g/ml) (see 8.14.2) Final Concentration (pg/g) of PFOS in Liver See Attachment D for a sample form to calculate the concentrations of standards. 14.0 METHODPERFORMANCE 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 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 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-1. 19.0 AFFECTEDDOCUMENTS 19.1 FACT-M-2.1, "Analysis of Liver Extracts for Fluorochemicals using HPLC-Electrospray Mass Spectrometry" 3M Environmental Laboratory FACT-M- 1.1 Extraction of PFOS from Liver Page 9 of 15 Page 76 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 20.0 REVISIONS ,: Revision Number. Reason For Revision 1 Validation of method to include 7Jluorochemicals, new APIlMS(MS) systems, monkey liver cross validation, improvements to ion pairing extraction, MDL study, updates in record keeping and storing policies, etc. Revision 08/01/98 3M Environmental Laboratory FACT-M-1.1 Extraction of PFOS from Liver Page 10 of 15 Page 77 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 Surrogate Std. FC Mix Std FC Mix Std FC Mix Std Comments I approx. ppm approx. 0.5 ppm approx. 5 ppm approx. 50 ppm actual ppm actual ppm actual ppm actual ppm #W #W #W #W Blank Std # Extraction Method/Revision: amount = Date & Initials Shake 20 min. Shaker Speed Centrifuge 20-25 min. Centrifuge speed: Remove a 4 ml aliquot of organic layer I Put on Nitrogen Evaporator to dryness Add methanol Volume Temperature: ml TN-A- Vortex 30 sec. Filter using a 3cc B-D syringe with a 0.2pm 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 3M Environmental Laboratory FACT-M- 1.1 Extraction of PFOS from Liver Page 11 of 15 Page 78 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 Compound PFOS PFOSA PFOSAA EtFOSE-OH POAA PFOSEA Monoester MDL (ppb) 11.8 6.06 55.7 58.7 23.7 16.2 dV LOQ (ppb) 37.4 19.3 177 187 75.5 51.7 n/v Linear Calibration Range (LCR) Approximate Concentrations to be used for preparing the Standard Calibration Curve 38 ppb - 1000ppb 20 ppb - 1200 ppb 180ppb-lOOOppb 190ppb-1800ppb 76 ppb - 1800 ppb 62 ppb - 1200ppb Monoester was not detectablelquantifiable at the spiked concentrations. Compound PFOS PFOSA PFOSAA EtFOSE-OH POAA PFOSEA Monoester MDL (ppb) 24.7 20.7 dV dv dV dV dV LOQ (ppb) 78.7 65.8 dv dv dv dv n/v Linear Calibration Range (LCR) Approximate Concentrations to be used for preparing the Standard Calibration Curve 62 ppb - 1200 ppb 20 ppb - 1200ppb 62 ppb - 1200 ppb 120 ppb - 1200 ppb 62 ppb - 1200ppb 120 ppb - 1200 ppb Monoester was not detectablelquantifiable at the spiked concentrations. Compound PFOS PFOSA PFOSAA EtFOSE-OH POAA PFOSEA Monoester MDL (ppb) dV 27.4 dV dv dV dV dV LOQ (ppb) dv 87.1 dv dv dv dv n/v Linear Calibration Range (LCR) Approximate Concentrations to be used for preparing the Standard Calibration Curve 59 ppb - 1200ppb 28 ppb - 1200 ppb 120ppb - 1200ppb 58 ppb - 1200 ppb 120ppb - 1200ppb 120ppb - 1200ppb Monoester was not detectablelquantifiable 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: MDLLOQ Values 3M Environmental Laboratory FACT-M- 1.1 Extraction of PFOS from Liver Page 12 of 15 Page 79 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 PFOS PFOSA I PFOSAA EtFOSE-OH Rabbit Bovine Rat Monkey Rabbit Bovine Rat Monkey Rabbit Bovine Rat Monkey Rabbit Bovine 11.8ppb 37.4 ppb I d d = not determined' 24.7ppb 78.7 ppb d v = not valid3 Standard 38 PPb 60 PPb 62 PPb 59 PPb 1000 ppb 1200 ppb 1200 ppb 1200 ppb 6.06 ppb dd 20.7 ppb I 27.4ppb 55.7 ppb dd dV dV 58.7 ppb dd 19.3 ppb dd 65.8 ppb I 87.1 ppb I 177 ppb dd dV dV 187 ppb dd 20 PPb 30 PPb 6 PPb 6 PPb 180 ppb 120 ppb 62 PPb 120 ppb 190 ppb 120 ppb 1200 ppb 1200 ppb j 1 - 1200 ppb 1900 ppb 1200 ppb 1200 ppb 1200 ppb 1800 ppb 1200 ppb POAA Rabbit Bovine Rat Monkey 1 23.7ppb dd dV dV I I I 75.5 ppb 76PPb I d d 1 120ppb 1 dV dV 120 ppb 1800 ppb I200 ppb , 1 - Upper Limit chosen where the value was within the Linear Calibration Range (LCR) but did not excessively weight the standard curve or affect Repeatability & Reproducibility values. I ~~ 2 - ~~ Not ~~ determined refers to no sample was analyzed for this data. 3 - Not valid refers to data from the analysis failed to meet specific criteria for a valid MDL/LOQ I determination. komDound I Liver Prepared Range of Matrix Range of Average Standards Curve I (ppb)(ngk) 5.95 - 1790 (ppb)(ng/g) 5.95 - 1790 Bovine 6.00 - 1200 1 7 6.22 - 1240 I Monkey 5.93 - 1190 6.00 - 1200 6.22 - 1240 5.93 - 1190 Range of Low Std. Curve nla nla Range of High Std. Curve 119 - 1790 nla nla nla Attachment C: LOQ summary 3M Environmental Laboratory FACT-M- 1.1 Extraction of PFOS from Liver Page 13 of 15 Page 80 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 ComDound 1 I I ICornpound I I I I I I EtFOSE-OH I I I I I I I I I I I I I t ComDound Liver Prepared Matrix Range of I Standards Rabbit Bovine Rat 6.14 - 1230 Monkey PFOSEA Range of Average 123 - 1230 Range of Low Std. Curve (ppb)(ng/g) ~ nla nla ~ nla nla Monoester was not detectable/quantifiable in liver matrix for the concentration range of 4.94 - 1450 ppb. Attachment C: LOQ s u m m a r y 3M Environmental Laboratory FACT-M- 1.1 Extraction of PFOS from Liver Page 14 of 15 Page 81 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 5.01 4.97 5.00 4.90 4.95 4.94 50.1 49.7 50.0 49.0 49.5 49.4 0.030 0.004 0.169 0.169 ~~~ Validated Ranges --~~ Approximate Concentrations Liver PFOS PFOSA PFOSAA EtFOSE-OH POAA Rabbit Bovine 40- l000ppb 20- 1200ppb 180- 1900ppb 190- 1800ppb 80- 1800ppb GO - 1200ppb 30 - 1200 ppb 120 - 1200ppb 120- 1200ppb 80 - 1200ppb Rat G0-1200ppb 70-1200ppb 60-1200ppb 120-1200ppb G O - 1200ppb Mnnkev h O - 1 2 0 O ~ ~ b90-12OO~Db 1 2 0 - 1 2 0 0 ~ ~GbO - 1 2 0 0 ~ ~1b20-12OODDb PFOSEA G O - 12OOppb 30 - 1200 ppb 120- 1200ppb 120-12OOD~b Attachment D: Calibration standard concentration worksheet 3M Environmental Laboratory FACT-M- 1.1 Extraction of PFOS from Liver Page 15 of 15 Page 82 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 3M ENVIRONMENTLAALBORATORY METHOD ANALYSIS OF FLUOROCHEMICIANLLSIVEREXTRACTUSSING HPLC-ELECTROSPRAYMASPSES CTROMETRY Method Number: FACT-M-2.1 Author: Lisa Clemen Approved By: Adoption Date: 05/26/98 Revision Date: O b [ 0 3 1 4 4 Laboratory Manager Date Group Leader C/r 193 Date Technical Reviewer k/Dh Date %rxn "% 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. a Applicable Compounds: Potassium perfluorooctanesulfonate, anionic fluorochemical surfactants, or other ionizable compounds. 0Matrices: Rabbit, rat, bovine, and monkey livers or other livers as designated in the e-validation report. 3 2 Word 7.0.1195 3M Environmental Laboratory FACT-M-2.1 Analysis of Liver Extract Using ESMS Page 1 of 9 Page 83 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 2.0 SUMMARYOF METHOD 2.1 This method describes the analysis of fluorochemical surfactants extracted from liver using HPLC-electrospray/massspectrometry. 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 atmosphericpressure (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 (MSMS): The API platforms are equipped with quadrupole mass selective detectors. Ions are selectively discriminatedby mass to charge ratio ( d z ) and subsequently detected. A single MS may be employed for ion detection or a series (MSNS) for more specific fragmentation information. 3.4 Conventional vs. %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 with similar systems (i.e. Z-spray components are compatible with other Z - spray systems, etc.) 3.5 Mass Lynx Software: System s o h a r e 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 I1 or Quattro I1 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 7.0.1195 3M Environmental Laboratory FACT-M-2.1 Analysis of Liver Extract Using ESMS .Page 2 of 9 Page 84 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 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 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 for perfluorooctanoate(POAA), teflon should not be used for sample storage or any part of instrumentationthat 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 HP1100 low pulse solvent pumping system and autosampler. 7.0 SUPPLIESAND 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 REAGENTASND STANDARDS 8.1 Reagents 8.1.1 Methanol, HPLC grade or equivalent. 8.1.2 ASTM, Type I water, Milli-QTMwater, all water used in this method should be Milli-Q'" 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-1.1. 9.0 SAMPLHE ANDLING 9.1 Fresh matrix standards are prepared with each analysis. Extracted standards and samples are stored in capped autovials or capped 15 mL centrihge 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. 3M Environmental Laboratory FACT-M-2.1 Analysis of Liver Extract Using ESMS Page 3 of 9 Page 85 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 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 concentrationsmay 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 calculatepercent difference. 11.o 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 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 100ppb 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 MFW. Set Ionization Mode as appropriate and mass to 499 or other appropriatemasses. A h l l scan is usually collected along with the SIRS. Save acquisition method. If MSMS instruments are employed, additional product ion fragmentation information may be collected. See Micromass 3M Environmental Laboratory FACT-M-2.1 Analysis of Liver Extract Using ES/MS Page 4 of 9 Page 86 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 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 HPllOO/autosamplerat 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 = 1 Time I MeOH I 2.0 mM I Ammonium acetate 0.00 min. 40% 60% 8.0 min. 90% 10% 11.Omin. 90% 10% 12.0 min. 40% 60% 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 prob`e and replace the stainless steel capillary. 12.3.4 Set HPLC pump to "On". Set the flow to 10 - 5OO'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: 3M Environmental Laboratory FACT-M-2.1 Analysis of Liver Extract Using ES/MS Page 5 of 9 Page 87 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 12.3.6.1 Drying gas 250-400 litershour 12.3.6.2 ESI nebulizing gas 10-15 litershour 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 Carefblly 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 ESMS 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 DATAANALYSIS AND CALCULATIONS 13.1 Calculations: 13.1.1 Calculate matrix spike percent recoveries using the following equation: % Recovery = Observed Result - Backmound Result x 100 Expected Result 13.1.2 Calculate percent difference using the following equation: % Difference = Expected Conc. - Calculated Conc. x 100 Expected Conc. 13.1.3 Calculate actual concentrationof PFOS anion in total liver (mg): (ug PFOS anion calc. fiom std curve) ( g of liva used for analysis I x Total mass of liver (g) lOOOug/l mg 14.0 METHODPERFORMANCE 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 3 value for the calibration curve must be 0.980 or better. 3M Environmental Laboratory FACT-M-2.1 Analysis of Liver Extract Using ESMS Page 6 of 9 Page 88 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 14.3 Matrix Spikes 14.3.1 Matrix spike percent recoveries are must be within L 30% of the spiked concentration. 14.4 Continuing Calibration Verifications 14.4.1 Continuing calibration verification percent recoveries must be k 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 l/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 TABLESD, IAGRAMFS,LOWCHARTASN,D VALIDATIONDATA 17.1 Attachment A: FACT-M-2.1 Data reporting spreadsheet 3M Environmental Laboratory FACT-M-2.1 Analysis of Liver Extract Using ESMS Page 7 of 9 Page 89 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 18.0 REFERENCES 18.1 FACT-M-1.1,"Extraction of Potassium Perfluorooctanesulfonate or Other Fluorochemical compounds fiom Serum for Analysis Using HPLC-ElectrosprayMass Spectrometry 18.2 ETS-9-24.0, "Operation and Maintenance of the Micromass Atmospheric Pressure Ionization/Mass Spectrometer Quattro I1 triple quadrupole Systems" 18.3 The validation report associated with this method is FACT-M-1.1-V & 2.1-V-1.. 19.0 AFFECTEDOCUMENTS 19.1 FACT-M-1.1, "Extraction of Potassium Perfluorooctanesulfonatefiom Liver for Analysis Using HPLC-Electrospray/MassSpectrometry" 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 Clarificationof solvent ramp. Section 17.1 Changed from attachment B to A. Revision 05/04/99 3M Environmental Laboratory FACT-M-2.1 Analysis of Liver Extract Using E S N S Page 8 of 9 Page 90 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 Laboratory Study # Study: Test Material: Matriflinal Solvent: MethodRevision: Analytical Equipment System Number: Instrument SoftwareNersion: Filename: R-Squared Value: Slope: Y Intercept: Date of ExtractiodAnalyst: Date of AnalysisIAnalyst: 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 Conc. (ugh&): Calculated by dividing the initial volume from the concentration Attachment A: Data Sheet 3M Environmental Laboratory FACT-M-2.0 Analysis of Liver Extract Using ES/MS Page 9 of 9 Page 91 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 3M ENVIRONMENTLAALBORATORY EXTRACTIOOFNPOTASSIUMPERFLUOROOCTANESULFOONRAOTTEHER FLUOROCHEMICAL COMPOUNDS J?ROM LIVER FOR ANALYSIS USING HPLC- ELECI'ROSPRAYMSAPSESCTROMETRY Method Number: ETS-8-6.0 Author: Lisa Clemen,Robert Wynne Approved By: Revision Date: 4 Group Leader -;L/l'tlS.9 Date XUX 9, 0 rc 0 0 1.o SCOPE AND APPLICATION 1.1 Scope: Thismethod is for the extraction of potassiumperfluorooctanesulfonate(PFOS) or 1 - 3 9, Word 6.0195 3M Environmental Laboratory ETS-8-6.0 Extraction of PFOS fiom Liver Page 1of 14 Page 92 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 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-tert-butylether (Mtl3E). In this method, seven fluorochemicals can be extracted: PFOS,PFOSA, PFOSAA, EtFOSE-OH,.P.FOSEA, 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.O mL methanol then filtered through a 3 cc plastic syringe attached to a 0.2 j.mnylon 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) C,F,,SO, 3.2 PFOSA:perfluorooctane sulfonylamide C$,,SO,NH, 3.3 PFOSAA. perfluorooctanesulfonylamido(ethy1)acetateC,F,,SO,N(CH,CH,)CH,CO, 3.4 EtFOSE-OH: 2(N-ethylperfluorooctane su1fonamido)-ethyl alcohol C$ ,,S0,N(CH2CH,)CH,CH20H 3.5 PFOSEA perfluorooctane sulfonyl ethylamide C,,F,,SO,N(CH,CH,)H 3.6 M556:C,F,,SO,N(H)(CH,COOH) 3.7 Surrogate standard: lH-lH-2H-2H perfluorooctane sulfonic acid 4.0 WARNINGS AND CAUTIONS . 4.1 Health and Safety Warnings: 4.1.1 Use universal precautions, especially laboratorycoats, goggles, and gloves when handling animal tissue, which may contain pathogens, 5.0 INTERFERENCES 5.1 There are no interferencesknown at this t h e . 6.0 EOUIPMENT 6.1 The following equipment is used while performing this method. Equivalent equipment is acceptable. 6.1.1 6.1.2 6.1.3 6.1.4 Ultra-Turrax " 2 5 Grinder for ginding liver samples Vortex mixer, VWR, Vortex Genie 2 Centrifuge, Mistral 1000 or IEC Shaker, Eberbach or V W R ETS-8-6.0 Extraction ofPFOS from Liver Page 2 of 14 3M Environmental Laboratory Page 93 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 6.1.5 Nitrogen Evaporator, Organomation 6.1.6 Balance (sensitivityto 0.100 g) 7.0 SUPPLIES AND MATERIALS 7.1 Gloves 7.2 Dissecting scalpels 7.3 Eppendorf or disposablepipettes 7.4 Nalgene bottles, capableof holding 250 mL and 1L 7.5 Volumetric flasks, glass, type A 7.6 I-CHEM vials, 40mL glass 7.7 Plastic sampule vials, Wheaton, 6mL (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, disposableplastic, 3 cc 7.14 Syringe filters, nylon, 0.2 p,25 mm 7.15 T h e r 7.16 Crimp cap autovids and caps 7.17 Crimpers Note: Prior to using glassware and bottles, rinse 3 times with methanol and 3 times with MilliQm water. Rinse syringes a minimumof 9 times with methanol, 3 rinses f!rom 3 separate vials. 8.0 REAGENTASND STANDARDS 8.1 Type I reagent grade water, Milli-QTMor equivalent; all water used in this method should be M W Q m water and be provided by a Milli-Q TOC Plusm system 8.2 Sodium hydroxide (NaOH), J.T Baker or equivalent 8;3 Tetrabutylammonium hydrogen sulfate(TEIA),Kodak or equivalent 8.4 Sodium carbonate (Na$O,), J.T. Baker or equivalent 8.5 Sodium bicarbonate (NaHCO,), J.T.Baker or equivalent 8.6 Methyl-tert-butyl ether, Omnkolv, glass distilled or HPLC grade 8.7 Methanol, Omnisolv, glass distilled or HPLC grade 8.8 Liver, frozen from supplier 8.9 Dry ice fkom supplier 8.10 Fluorochemicalstandards 8.10.1 PFOS (3M Specialty Chemical Division), molecular weight = 538 3M Environmental Laboratory ETS-8-6.0 Extraction ofPFOS from Liver Page 3 of 14 Page 94 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 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 SpecialtyChemical 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,l-H, 2-H, 2-H C8Fl3SO3Hm) olecular weight = 428 8.10.8 Other fluorochemicals, as appropriate 8.11 Reagent preparation NOTE: When preparing largervolumes thanlisted in reagent, standard, or surrogate preparation, adjust accordingly. 8.11.1 10N sodium hydroxide (NaOH): Weigh approximately200 g NaOH. Pour into a 1000mLbeaker containing 500 mL Milli-Qm water, mix until all solids are dissolved. Storein a 1L Nalgene bottle. 8.11.2 1 N sodium hydroxide (NaOH): Dilute 10N NaOH 1:lO. Measure 10mL of 10N NaOH solutioninto a 100mL volumetric flask and dilute to volume using Milli-Q" water. Store in a 125mLNalgene bottle. 8.11.3 0.5 M tetrabutylammoniumhydrogen sulfate (TBA): W.eigh approximately 169 g of TBA into a 1 L volumetric containing 500 rnL Milli-QTMwater. Adjust to pH 10using approximately44to 54mL of 10N NaOH W l e adding the last mL of NaOH, add slowly because the pH changes abruptly). Dilute to volume with Milli-Q" water. Storein a 1L Nalgene bottle. 8.11.3.1 TBA requires a check prior to each use to ensure pH = 10. Adjust as needed using 1N NaOH solution. 8.11.4 0.25 M sodium carbonate/sodium bicarbonate buffer (Na&O,/NaHCO,): Weigh approximately26.5 g of sodium carbonate (Na&OJ and 21.0 g of sodium bicarbonate (NaHCO,) into a 1 L volumetric flask and bring to volume with Milli- Qm water. Store in a 1L 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 fluorochemicd standards are acceptable (for example, one working standard solution containing 1.OO ppm PFOS,1.02ppm 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 1000ppm (Pg/mL). 8.12.5 Dilute the stock solutionwith methanol for a working standard 1solutionof approximately 50 ppm. 3M Environmental Laboratory ETS-8-6.0 Extraction of PFOS from Liver Page 4 of 14 Page 95 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 8.12.6 Dilute the stock solution with methanol for a working standard 2 solution of approx. 5 .O 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 50-60mg of surrogate standard 1-HJ-H, 2-H,2-H, CsFI3SO3Hinto a 50 mlvolumetric flaskand record the actual weight. 8.1.3.2 Bring to volume with methanol for a surrogate stock of approximately 1000-1200 PPm. 8.13.3 Prepare a surrogateworking standard. Transfer approximately 1.0 ml of surrogate stock to a 10mlvolumetric flask and bring to volume with methanol for a working standard of 10-20 ppm. Record the actual volume transferred. 9.0 SAMPLHEANDLING 9.1 All samples are received fiozen and must be kept fiozen until the extraction is performed. 10.0 OUALITYCONTROL 10.1 Matrix blanks and method blanks 10.1.1 An aliquot of 1.0 mL methanol is usedas a solvent blank. 10.1.2 Extract two 1.0 mL aliquots ofMilli-QTMwater following thisprocedure and use as method blanks. 10.1.3 Extract two 1.0mL 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 ofthe 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 Continuingcalibrationverifications 10.3.1 Prepare continuing calibrationverification 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 verificationsare prepared and extracted. 3M Environmental Laboratory ETS-8-6.0 Extraction of PFOS from Liver Page 5 of 14 Page 96 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 10.3.3 Prepare each continuing calibrationverification 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 g of liver into a 250 mL Nalgene bottle containing 200 m L s Milli-QTMwater. 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. 11.13 Refer to 13.0 to calculate the actual density of liver homogenateand the concentrationof solid liver tissue dispersed in 1.0 mL of homogenate solution. 11.1.5 Add 1 mL of homogenate to a 15 mL centrifbge tube. Re-suspend solution by shakingbetween aliquots while preparing a total of eighteen 1mL aliquots of homogeneous solution in 15 mL centrifugetubes. 11.1.6 Two 1mL, aliquots, or otherappropriate volume, serve as matrixblanks. ' 11.1.7 Typicdly use the standard concentrations and spiking amounts listed in Table 1, at the end of this section, to spike, in duplicate, two standard m e s , 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 Use Attachment C as an aid in calculating the concentrationsofthe working standards. .Referto 13.0 to calculate actual concentrationsof 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 - 1OOOppb. 3M Environmental Laboratory ETS-8-6.0 Extraction of PFOS from Liver Page 6 of 14 Page 97 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 11.3 Extract spiked liver homogenates following 12.14-12.25 of this method. Use these standards to establish each initial curve on the mass spectrometer. Table 1 Approximate SpikingAmounts for CalibrationStandards Working Standard (Approx. Conc.) - 0.50 pprn 0.50 pprn 0.50 ppm I 0.50 ppm 0.50 ppm 5.0 ppm 5.0 ppm 5.0 m m trl Approx. final conc. of PFOS in liver - Blank 2 0.005ppm 4 0.010 ppm 10 0.025 ppm 20 0.050 ppm 40 0.100 ppm 10 0.250 ppm 20 0.500 ppm 30 0.750 ppm 12.0 PROCEDURE 12.1 Obtain frozen liver samples. 123 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 m L s 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 samplewith 2.5 m L s water using a pipette. 12.9 Take the grinder apart and clean it with methanol after each sample. Refer to AMDT-EP- 22. 12.10 Cap the sample and vortex for 15 seconds. Label the sampulevial with the studynumber, weight, liver ID, date and analyst initials. . 3M Environmental Laboratory ETS-8-6.0 Extraction ofPFOS from Liver Page 7 of 14 Page 98 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 12.11 Pipette 1.0 mL, or other appropriate volume, of homogenate into a 15 mL polypropylene centrifuge tube. Label the centrifugetube 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 Mini-Q'" 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 m a h with the appropriateamount of standard as described in 11.1,or Table 1 of that section, for the calibrationcurve 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. If not, adjust accordingly. 12.17 To each sample, add 1mL 0.5 M TBA and 2 mL of the 0.25 M sodium carbonatdsodium bicarbonate buffer. 12.18 Using an Oxford Dispenser, add 5 mL methyl-tert-butyl ether. 12.19 Cap each sample and put on the shaker at a setting of 300 rpm,for 20minutes. 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 4.0mL of the organic layer to the fresh 15mL centrifugetube. 12.23 Put each sample on the analytical nitrogen evaporatoruntil dry, approximately 1to 2 hours. 12.24 Add 1.O mL, to each centrifuge tube using a graduated pipette. 12.25 Vortex mix 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) perf'orming the extraction. 12.28 Cap and store extracts at room temperature or at approximately4 "Cuntil analysis. 12.29 Complete the extractionworksheet, attached to this document, and tape in study notebook or include in study binder, as appropriate. 3M Environmental Laboratory ETS-8-6.0 Extraction of PFOS from Liver Page 8 of 14 Page 99 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 13.0 DATAANALYSIASND CALCULATIONS 13.1 Calculations: 13.1.1 Calculatethe average density of the liver homogenate by recording each mass of ten separate 1.O mL aliquots of homogenate. Average density (mg/mL) =Average mass ( m d of the aliauots 1.0 mL aliquot 13.1.2 Calculatethe amount of liver (mg) per 1.0 mLhomogenate (or concentration of dispersed solid tissue per mL of homogenate suspension)using the following equation: g of Liver x Average den&* of homogenate (mdmL) (g of Liver + g of Water) * refer to 13.1.1 for details. 13.1.3 Calculate actual concentrationsofPFOS and other fluorochemicalsin calibration standards using the following equation: & of Standardx Concentration (ua/mL) =Final Concentration (pg/g or m@g) mg Liver/ 1 mL homogenate* of PFOS in Liver *refer to 13.1.2 for details. 14.0 METFIOD PERFORMANCE 14.1 The method detection limit (MDL) is analyte and matrix specific, Referto MDL report for specificMDL and limit of quantitation (LOQ)values (refer to Attachments B and C). 14.2 The followkg 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 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 performancecriteria. 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 pipettewaste is disposed in broken glass containers located in the laboratory. 3M Environmental Laboratory ETS-8-6.0 Extraction of PFOS from Liver Page 9 of 14 Page 100 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 16.0 RECORDS 16.1 Completethe extraction worksheet attached to this method, and tape in the study notebook or include in the 3-ring studybinder, as appropriate. 17.0 TABLESD,IAGRAMFSL,OWCHARTSA,ND VALIDATION DATA 17.1 Attachment A, Extraction worksheet 17.2 Attachment B, MDLLOQ values and sumniary 17.3 Attachment C, Calibration standardcalculation and concentrationworksheet 18.0 REFERENCES 18.1 The validation report associatedwith this method is ETS-8-6.0& 7.0-V-1. 18.2 AMDT-EP-22,"Routine Maintenanceof Ultra-TurraxT-25" 18.3 FACT-M-1.1, ``Extraction ofPFOS or Other Anionic Fluorochdcal Surfactants from Liver for Analysis Using HPLC-Electrospray/Mas Spectrometry" 19.0 AFFECTEDOCUMENTS 19.1 ETS-8-7.0, "Analysis of Liver Extracts for Fluorochemicalsusing HPLC-Electrospray Mass Spectrometry" 20.0 REVISIONS Revision Number. .. Reason For Revision Revision 7 Date 3M Environmental Laboratory ETS-8-6.0 Extraction of PFOS from Liver Page 10 of 14 Page 101 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 Study # Matrix Box # may Date SpikedlAnalyst ccv MS MSD Surrogate Std approx. ppm actual ppm # FC Mix Std approx. 0.5 pprn actual ppm # FC Mix Std approx. 5 ppm actual ppm # FCMixStd approx. 50 ppm actual pprn # Comments I I r r - I I I I I 1 I I I ioaenate: Std # . Liver amount = R Attachment B: MDLJLOQ Values 3M Environmental Laboratory ETS-8-6.0 Extraction of PFOS from Liver Page 1 of 16 Page 102 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 Compound PFOS PFOSA PFOSPLA EtFOSE-OH M556 PFOSEA MDL (ppb) 8.45 3.50 24.6 108 82.3 33.9 LOQ @pb) 26.9 11.1 78.3 345 262 108 Linear CalibrationRange (LCR) Approximate concentrationsto be used for preparingthe Standard Calibration Curve 30 ppb - 1200 ppb 12ppb - 1200ppb 30 ppb - 1200ppb 60 ppb - 900ppb* 60 ppb - 1200 ppb 30 ppb- 1200ppb MDWLOQ values in rat, bovine, and monkey liver were not statisticallydetermined. Two curves in each of these matrices were extracted and analyzedwiththe 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 equivalentto those values as determined for the rabbit liver. Refer to LOQ Summary and MDL study in ETS-8-6.0 & 7.0-V-1for mer information * EtFOSE-OH estimates only for MDL and LOQ. Did not meet criteria for validation. Rabbit 6.19 - 1237 12 - 1200 12~1200.~-~1,2 - 300 -_ . 12-300. 60- 1200 - '-601200 . Liver matrix Rabbit Prepared range of standards (PPb) (ng/mL) 6.16- 1232 ' Rangeof average curve (PP~)(ndmL) 12 - 1200 LCR&, m.' r ave curve (wb)@g/mL) 30.-.1200 '' Range of low std curve . (ppb) (nglmL) 30-900 st&: . 'LcR;&m- ,.. Range of ' i high std . .curve.; curve (mbj..(.n. g/m.L). (ppb) (ndmL) -6. 0. .-9.00..-..''N/A .. 'LCRfrom. ' highstd. curve .(ppb) (nglmL) NIA Attachment B: h4DULOQ Values 3M Environmental Laboratory ETS-8-6.0 Extraction of PFOS from Liver Page 12 of 16 Page 103 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 Liver matrix Rabbit ' Prepared range of Rangeof average standards curve LCRfrom ave curve 31 - 900 Rangeof low std curve N/A LCRbm low std curve N/A Rangeof high std curve N/A LCRfrom high std curve NIA Liver matrix Rabbit Prepared range of standards (ppb) (ndmL) 6.17 - 1235 Rangeof average curve (ppb) (ng/rnL) 31 - 1200 LCRfrom ave curve (mbl.(ndmL) 31 - 1200-a Rangeof low std curve (PPW(nelmL) N/A LCRGom low std curve. (mb)(ng/mL) N/A . Compou ad: M556 Liver IMtKiX Rabbit Attachment C Standard Calculations 3M Environmental Laboratory ETS-8-6.0 Extraction of PFOS from Liver Page 13 of 14 Page 104 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 Ion Pair Standard Curves - Tissue Prep date@): Analyte(s): Sample matrix: Methodhevision: 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 Calculated concentrations of standards in the sample matrix 120 299 599 898 1198 120 299 599 898 1198 120 299 599 898 1198 120 299 599 898 1198 120 299 599 898 1198 120 299 599 898 1198 - Validated ranges amroximateconcentrations Liver PFOS 1 PFOSA I PFOSAA Rabbit 5-1000ppb I 5-1000ppb I 5-1000ppb Bovine Estimates only, use rabbit values. Rat Estimates only, use rabbit values. Monkey Estimatesonly,use rabbit values. I EtFOSEOH I I 5-1000ppb I nglmL 0.500 POAA I 5-1000ppb PFOSEA S-IOOOppb Attachment C: Standard Calculations 3M Environmental Laboratory ETS-8-6.0 Extraction of PFOS from Liver Page 14 of 14 Page 105 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 3M ENVIRONMENTLAALBORATORY METHOD ANALYSIS OF POTASSIUMPERFLUOROOCTANESULFONATEOR OTHER F L ~ ~ R ~ ~ ~ E MINI L~IAVELRESXTRACTSUSING HPLC-ELECTROSPRAY/MASPSESCTROMETRY Method Number: ETS-8-7.0 Author: LisaClemen, Glenn Langenburg Adoption Date: 0 712L Revision Date: Nfi Approved By: Group Leader L, Technical Reviewer 3 1 14/33 Date 07l,?J/?? Date I 7. 1.0 SCOPE AND APPLICATION 1.1 Scope: This method is for the analysisof liver extracts for fluorochemicalsurfactantsusing 4 HPLC-electrospray/mas spectrometry. . 1.2 Applicable Compounds:Fluorochemicalsurfactants or other fluorinated compounds, or other ionizable compounds. 1.3 Matrices: Rabbit, rat, bovine, monkey liver, or other tissues as designated in the validation report. Word 6/95 ETS-8-7.0 Analysis of Liver Extract Using ES/MS Page I of 10 3M Environmental Laboratory Page 106 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 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 characteristicof a particular fluorochemical, such as the periluorooctanesulfonate PFOS) anion,d z =499. Additionally,samples may be analyzedusing a tandem mass spectrometerto further verifythe identityof a compound by detecting daughter ions of the selected parent ion. 3.0 DEFINITIONS 3.1 Atmospheric Pressure Ionization (MI):The Micromass Q u a m IItriple quadrupole systems allow for various methods of ionization by utilizing various soufces,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 @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), TandemMass Spectrometer ( M S M S ) : The API Quattro II triple quadrupolemass spectrometeris equippedwith two quadrupole mass selectivedetectors and a collision cell. Ions are selectively discriminated by mass to charge ratio ( d z )and subsequentlydetected. 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 hgments may be analyzed in the second quadrupole. 3.4 Conventional vs. Zspray probe interface: The latest models of Micromass Quattro I1 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. Thoughthe configuration is different, the methods ofoperation, 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 I1 triple quadrupolesystems. CurrentlyMassLynx 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 II triple quadrupoleMassLynx or MassLynxNT 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. When engaged, theprobe employs a voltage of approximately 5000 Volts. 3M Environmental Laboratory ETS-8-7.0 Analysis of Liver Extract Using ESMS Page 2 of 10 Page 107 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 4.1.2 When handling samples or solventswear appropriateprotective 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 HP1 I00 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 instnunentation that comes in contact with the sample or extract. 6.0 EOUIPMENT 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 11triple quadrupole Mass Spectrometerequippedwith an electrospray ionization source. 6.1.2 HP1100 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 100psi (house air system) 7.1.2 HPLC analyticalcolumn, specifics to be determinedby the analyst and documented in the raw data 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-QTMwater (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-Qm water, mix until all solids are dissolved. Store at room temperature. ETS-8-7.0 Analysis of Liver Extract Using ESMS Page 3 of 10 3M Environmental Laboratory Page 108 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 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 SAMPLEHANDLING 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 approximately4" C, until analysis can be performed. 10.0 OUAL~TCYONTROL 10.1 Method BIanks and Matrix Blanks 10.1.1 Solvent blanks, method blanks, and ma& 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 samplep. With a minimum of 2 spikes per batch. 10.2.4 Matrix spike and matrix spike duplicate concentrationswill 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 veri@ the continued accuracy of the calibration curve. 10.3.2 Analyze a mid-range calibration standard every tenth sample,with a minimumof one per batch. 11.0 CALIBRATION AND STANDARDIZATION 11.1 Analyze the extracted matrix standardsprior to and following each set of sample extracts. The average of two standard curves will be plotted by linear regression (y =mx +b), weighted l/x, not forced throughthe 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. 3M Environmental Laboratory ETS-8-7.0 Analysis of Liver Extract Using ES/MS Page 4 of 10 Page 109 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 11.3 For purposes ofaccuracywhen quantitating low levels of analyte, it may be necessary to use the low end of the calibration curve rather than the fullrange of the standard curve. Example: when attempting to quantitate approximately 10 ppb of analyte, generate a calibration curve consisting of the standards *om 5 ppb to 100ppb rather than the full range of the curve (5 ppb to 1000ppb). 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 ofthe alphabet startingwith a 12.1.1.2 Assign a method (MS file) for acquiring 12.1.1.3 Assign an HPLC pro&arn (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 thenmass spectrometerheadings and select SIR (Single Ion Recording) or MRM (Multiple Reaction Monitoring). Set Ionization Mode as appropriate and mass to 499 or other appropriatemasses. A fill scan is usually collectedalong with the SIRs. Save acquisitionmethod. If MSMS instruments are employed, additionalproduct ion fragmentationinformationmay be collected. Refer to Micromass MassLynx GUIDE TO DATA ACQUISITION for additional information and MRM. 12.1.3 Typically the analytical batch runsequencebegins 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 HPl lOO/autosamplerat 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 hject/sample = 1 12.2.2.3 Cycle time = 9 minutes , 3M Environmental Laboratory ETS-8-7.0 Analysis of Liver Extract Using ESMS Page 5 of 10 Page 110 3M Medical Department Study: T-6889.3 12.2.2.4 Solvent ramp conditions Analytical Report: FACT-TOX-026 LRN-U2782 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 11 Triple Quadrupole Mass SpectrometerFitted with an AtmosphericPressure Ionization Source," for more details. 12.3.2 Check the solvent level inreservoirs and refill if necessary. 123.3 Check the stainless steel capillary at the end of the probe. Use an eyepieceto 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 stainlesssteel capillary. 123.4 Turn on the nitrogen. 12.3.5 Open the tune page. Clicks on operateto initiate sourceblock 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 Uymin 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 equilibratefor approximately 10minutes. 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 litenhour 12.3.7.2 ESI nebulizing gas 10-15 litershow 12.3.7.3 HPLC constant flow mode flow rate 10-500 pL,/min 12.3.7.4 Pressure ~ 4 0 b0ar (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 ESlMS Page 6 of 10 3M Environmental Laboratory Page 111 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 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. 123.9 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 DATAANALYSIASNDCALCULATIONS 13.1 Calculations: 13.1.4 Calculate matrix spike percent recoveries using the following equation: % Recovery = - ObservedResult Backmound Result x 100 Expected Result 13.1.5 Calculatepercent differenceusing the followingequation: % Difference = Exuected Conc. - Calculated Conc. x 100 Expected Conc. 13.1.6 Calculate actual concentrations in matrix (pg/g): (ring of PFOS calc. from std. Curve x Dilution Factor) (Initial Weirtht of Liver (E) Final Volume (mL) x 1up 1000 ng 14.0 METHODPERFORMANCE 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 $ value for the calibrationmust be 0.980 orbetter. 14.4 Matrix Spikes 14.4.1 Matrix spikepercent recoveriesmust be Within f 30% of the spiked concentration. 14.5 Continuing CalibrationVerification 14.5.1 Continuingcalibration verificationpercent recoveries must be within +,30% of the spiked concentration. 14.6 If criteria listed in the method performance section are not met, maintenancemay be performed on the system and samples reanalyzed or other actions as determined by the analyst. Document all actions in the appropriate logbook. 3M Environmental Laboratory ETS-8-7.0 Analysis ofLiver Extract Using ESMS Page 7 of 10 Page 112 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 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 q d tape into the instrument runlog. 16.3 Plot the calibration curve by linear regression, weighted L/x,then print these graphs and store in the study folder. 16.4 Print data integration surnmary, integration method, and chromatogramsfiomMassLynx and store in the study folder. 16.5 Summarize data using suitable software (Excel 5.W) and store in the study folder, refer to Attachment A for an example of a summary,spreadsheet. 16.6 Back up electronicdata to appropriatemedium. Record in study notebook the filename and location of backup electronicdata. 17.0 TABLESD,IAGRAMFSL, OWCHARTANSD, VALIDATION DATA 17.1 Attachment A ETS-8-7.0Data summary spreadsheet 18.0. REFERENCES 18.1 FACT-M-2.1,"Extraction of PotassiumPerfluorooctanesulfonateor Other Fluorochemical Compounds from Liver for Analysis Using HPLC-EIectrospray/Mas Spectrometry" 18.2 ETS-9-24.0",Operation and Maintenanceof the Micromass AtmosphericPressure Ionization/MassSpectrometerQuattro II triple quadrupole Systemsy' 183 The validation report associatedwith this method is ETS-8-6.0 8z 7.0-V-1 19.0 AFFECTEDOCUMENTS 19.1 ETS-8-6.0, "Extraction of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds from Liver or Fluid for Analysis Using HPLC-ElectrosprayMass Spectrometry" 3M Environmental Laboratory ETS-8-7.0 Analysis of Liver.Extract Using ES/MS Page 8 of 10 Page 113 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 20.0 REVISIONS Revision Number Reason For Revision Revision - Date 3M Environmental Laboratory ETS-E -7.0 Analysis of Liver Extract Using ESMS Page 9 of 10 Page 114 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 Laboratory Study # Study: Test Material: MatridFinal Solvent: MethodlRevision : Analytical Equipment System Number: Instrument SoftwareNersion: Filename: R-Squared Value: Slope: Y Intercept: Date of ExtractidAnalyst: . Date of Analysidhalyst Group Dose Sample# Concentration n&!k ' lnitinl Wt. Uilution Factor Final Conc. ug/g Slope: Taken tiom linearregresson equaaon. Attachment A: Summary Spreadsheet ETS-8-7.0 Analysis of Liver ExtractUsing ESMS 3M Environmental Laboratory Page 10 of 10 Page 115 3M Medical Department Study: T-6889.3 3M ENVIRONMENTALALBORATORY Analytical Report: FACT-TOX-026 LRN-U2782 METHOD EXTRACTIONOPOFTASSIUM PERFLUOROOCTANESULFONATE OR OTHER FLUOROCHEMICCAOLMPOUNDSFROM SERUM FOR ANALYSIS USING HPLC- ELECTROSPRAYMASS SPECTROMETRY Method Number: ETS-8-4.1 Adoption Date: 03/01/99 Author: Lisa Clemen, Glenn Langenburg Revision Date: q / a 7/49 Approved By: Laboratory Manage; Date Group Leader Technical Reviewer Date o&l4 9 Date 0 SCOPE AND APPLICATION I $1 Scope: This method is for the extraction of potassium perfluorooctanesulfonate (PFOS) T or other fluorochemical compounds from serum. e P, 3 2 Applicable compounds: Fluorochemical surfactants or other fluorinated compounds. 'F-3 Matrices: Rabbit, rat, bovine, monkey, and human serum or other fluids as designated in --. the validation report. Word 6/95 3M Environmental Laboratory ETS-8-4.1 Extraction of PFOS from Serum Page 1 of 14 Page 116 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 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-tert-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.O 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 DEFINITIONS 3.1 PFOS: perfluorooctanesulfonate (anion of potassium salt) C,F,,SO, 3.2 PFO'SA: perfluorooctane sulfonylamide C,F,,SO,NH, 3.3 PFOSAA: perfluorooctane sulfonylamido (ethy1)acetate C,F,,SO,N(CH,CH,)CH,CO,' 3.4 EtFOSE-OH: 2(N-ethylperfluorooctane su1fonamido)-ethyl alcohol CEF,,SO,N(CH,CH,)CH,CH,OH 3.5 PFOSEA: perfluorooctane sulfonyl ethylamide C,F,,SO,N(CH,CH,)H 3.6 M556: C,F,,SO,N(H)(CH,COOH) 3.7 Surrogate standard: 1H-lH-2H-2H perfluorooctanesulfonic 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 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 3M Environmental Laboratory ETS-8-4.1 Extraction of PFOS from Serum Page 2 of 14 Page 117 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 6.1.4 Nitrogen evaporator, Organomation 6.1.5 Balance (k 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 Centrifbge 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-Qm 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-QTMor equivalent; all water used in this method should be Milli-QTMwater and may be provided by a Milli-Q TOC Plusm system 8.2 Sodium hydroxide (NaOH), J.T Baker or equivalent 8.3 Tetrabutylammoniurn hydrogen sulfate(TBA), Kodak or equivalent 8.4 Sodium carbonate (NqCO,), J.T. Baker or equivalent 8.5 Sodium bicarbonate (NaHCO,), 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 3M Environmental Laboratory ETS-8-4.1 Extraction of PFOS from Serum Page 3 of 14 Page 118 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 8.9.3 PFOSAA (3M Specialty Chemical Division), molecular weight = 585 8.9.4 EtFOSE-OH (3M SpecialtyChemical Division), molecular weight = 570 8.9.5 PFOSEA (3M SpecialtyChemical Division), molecular weight = 527 8.9.6 M556 (3M Specialty Chemical Division), molecular weight = 557 8.9.7 Surrogate standard: 4-H, perfluorooctanesulfonic acid (1-H,1-H, 2-H, 2-H C,F,,SO,H) 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 10 N so_diumhydroxide (NaOH): Weigh approximately200 g NaOH. Pour into a 1000 mL beaker containing 500 mL Milli-QTMwater, 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:lO. Measure 10 mL of 10 , N NaOH solution into a 100 mL vohunetric flask and dilute to volume using Milli-QTMwater. Store in a 125 mL Nalgene bottle. 8.10.3 0.5 M tetrabutylammoniumhydrogen sulfate (TBA): Weigh approximately 169 g of TBA into a 1 L volumetric containing 500 mL Milli-QTMwater. Adjust to pH 10 using approximately44 to 54 rnL of 10 N NaOH (While adding the last mL of NaOH, add slowly because the pH changes abruptly). Dilute to volume with Milli-Qm 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/sodiumbicarbonate buffer (NqCOJNaHCO,): Weigh approximately 26.5 g of sodium carbonate (NqCO,) and 21.O g of sodium bicarbonate (NaHCO,) into a 1 L volumetric flask and bring to volume with MilliQm 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.OO pprn PFOS, 1.02ppm 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 1000ppm (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. 3M Environmental Laboratory ETS-8-4.1 Extraction of PFOS from Serum Page 4 of 14 Page 119 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 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,l-H, 2-H, 2-H, C,F,,SO,H 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 SAMPLHEANDLING 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 OUAL~TCYONTROL 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-QTMwater 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 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 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. 3M Environmental Laboratory ETS-8-4.1 Extraction of PFOS from Serum Page 5 of 14 Page 120 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 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.O 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 concentrationsof the working standards. See Section 13.0 to calculate actual concentrationsof 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 mass spectrometer. 3M Environmental Laboratory ETS-8-4. I Extraction of PFOS from Serum Page 6 of 14 Page 121 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 Working standard (approx. conc.) I k - I 0.500 ppm 0.500 ppm I 5.00 m m I 5.00 ppm 5.00 ppm 50.0 ppm . 50.0 ppm 50.0 ppm 50.0 ppm PL - 10 20 5 10 20 5 10 15 20 , Approx. final conc. of analyte in matrix I -Blank 0.005 ppm 0.010 ppm I 0.025 tmm 0.050 ppm 0.100 ppm 0.250 ppm 0.500 ppm 0.750 ppm 1.00 ppm 12.0 PROCEDURE 12.1 Obtain fiozen samples and allow to thaw at room temperature or in a lukewarm waterbath. 12.2 Vortex mix for 15 seconds, then transfer 1.O mL or other appropriatevolume to a 15 mL polypropylene centrifuge tube. 12.3 Return unused samples to fi-eezer 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-tert-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. 3M Environmental Laboratory ETS-8-4.1 Extraction of PFOS from Serum Page 7 of 14 Page 122 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 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.O mL of methanol to each centrihge tube using a graduated pipette. 12.18 Vortex mix fix 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 e.xtracts at room temperature or at approximately4 "Cuntil 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 DATAANALYSIS 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 (ua /mL) - mL of standard + mL of surrogate standard + initial matrix volume (mL) Final Concentration (pg/mL) of PFOS in matrix 14.0 METHODPERFORMANCE 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. 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. 3M Environmental Laboratory ETS-8-4.1 Extraction of PFOS from Serum Page 8 of 14 Page 123 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 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, MDLLOQ values and summary 17.3 Attachment C, Calibration standard concentrationworksheet 18.0 REFERENCES 18.1 The validation report associated with this method is ETS-8-4.0 & 5.0-V-1. 18.2 FACT-M-3.1, "Analysis of Serum or Other Fluid Extracts for Fluorochemicals using HPLC-Electrospray Mass Spectrometry" 19.0 AFFECTEDDOCUMENTS 19.1 ETS-8-5.1, "Analysis of Serum or Other Fluid Extracts for Fluorochemicals using HPLC-Electrospray Mass Spectrometry" 20.0 REVISIONS 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.OmL; not adjusted for initial volumes less than 1.0 mL. Revision - Date 04/02/99 3M Environmental Laboratory ETS-8-4.1 Extraction of PFOS from Serum Page 9 of 14 Page 124 3M Medical Department Study: T-6889.3 Extraction Worksheet ETS-8-4.1 Analytical Report: FACT-TOX-026 LRN-U2782 Study # Matrix Box # may 1 1 1 1 I I I I I I Surrogate Std FC-Mix approx. ppm approx. 0.5 pm actual ppm ~ t u a l ppm FC-Mix 1 FC-Mix I Comments I approx. 5 pprn approx. 50 ppm #actual ppm F t u a l ppm # DateSpikedAnaiyst ccv MS MSD Serum Extraction Method Dispense 5 mL of methyl-t-butyl ether Shake 20 min. TN-AShaker speed: Cont. Cal. Verifications used same matrix as for std curve. Attachment A 3M Environmental Laboratory ETS-8-4.1 Extraction of PFOS from Serum Date & Initials Page 10 of 14 Page 125 3M Medical Department Study: T-6889.3 MDLLOQ values for rabbit serum Analytical Report: FACT-TOX-026 LRN-U2782 MDLLOQ values in rat, bovine, monkey, and human serum, and monkey plasma were not statistically determined. Two curves in each of these matriceswere extractedand analyzedwith 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 studyin ETS-8-4.0 & 5.0-V-1for further information. ! Attachment B:MDLLOQ Summary 3M Environmental Laboratory ETS-8-4.1 Extraction of PFOS from Serum Page 11 of 14 Page 126 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 Compound: PFOS Full Range 0.995 - 978 1 I owc curve 1 I Highcurve I I l/x 4.9i-248 97.8-978 0.995-978 Compound: PFOSA Full Range Low Curve 0.993 - 976 4.93 - 97.6 1iX I 0.993-976 LCR from curve (PPb) (ng/mL) 24.8 - 978 ~ 4.94 - 248 97.8 - 978 4.94 - 978 4.93 - 976 4.93 - 97.6 24.8 - 978 1 4.93 -976 YORecovery Range RSD Range 83-108 85-104 85-106 94-1 11 4.67-11.0 5.34- 12.0 4.84-9.80 4.60- 10.5 % Recovery 7 I I 88-103 I I 87-105 5.10-14.7 9.85-14.7 5.08-13.9 5.10- 14.5 Compound: PFOSAA Prepared range Rabbit Serum of standards (PPb) W m L ) Full Range 1 LowCurve I High curve 0.991 -974 4.92-247 49.2 - 974 1/X 0.991 - 974 LCR from curve bPb) 24.7-974 9.74-247 YORecovery Range I 81-111 I I 97-107 1 RSD Range 4.18-10.6 -1I 6.38-21.8 ~ 97.4 - 974 9.74 - 974 85-108 95-1 15 4.33-12.5 4.1 1-23.2 Attachment B: MDLILOQ Summary 3M Environmental Laboratory ETS-8-4.1 Extraction of PFOS from Serum Page 12 of 14 Page 127 3M .Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 Compound: EtFOSE-OH I I Full Range I I h ow curve 0.993 - 976 4.93 -97.6 I Highcurve 1 49.3-976 1/X 0.993 - 493 LCR from curve (PPb) (ng/mL) 49.3 - 976 9.76 - 97.6 97.6 - 976 9.76 - 976 Range Range 77-110 97-107 90-109 86-1 11 I 11..2-25.5 I 14.1-21.3 1 11.5-19.6 11.1-21.2 Rabbit Serum Prepared range of standards @Pb) ( n d d ) Full Range Low Curve I Highcurve I 1/X 0.993 - 976 4.93 - 248 49.3-976 0.993 - 976 LCR from curve @Pb) (ng/mL) 24.8 - 976 9.76 - 248 49.3 - 976 9.76 - 976 Range 96- 106 91-1 10 86-106 95-117 10.1-16.2 11.8-19.5 I I I 10.2-18.2 I 1 10.1-19.1 Compound: M556 I Preparedrange I Rabbit Serum of standards @Pb) (ng/mL) LCRfiom I %Recovery I Range RSD Range Full Range 0.993 - 976 24.8 - 976 88-106 4.82-17.9 Low Curve 4.93 - 97.6 9.76 - 97.6 100- 105 5.95-18.2 High curve 97.6 - 976 I 0.993 -976 97.6 - 976 9.76 -976 1 81-1 11 97-110 5.1 1-9.74 1 4.77-19.5 Attachment B: MDL/LOQ Summary 3M Environmental Laboratory ETS-8-4.1 Extraction of PFOS from Serum Page 13 of 14 Page 128 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 Ion Pair Standard Curves - Fluids Prep date(s): Standard number: Analyte(s): Equipment number: Sample matrix: Final solvent and TN: Blank fluidhdentifier: Methodrevision: Target analyte(s): FC mix std approx. 0:500 ppm: FC mix std approx. 5 .OO ppm: FC mix std approx. 50.0 ppm: Surrogate std approx. 100 ppm: PFOS Std conc - ug/mL 0.500 0.500 5.00 5.00 5.00 I 50.0 j 50.0 1 50.0 I 50.0 I PFOSA Std conc ug/d 0.507 0.507 5.07 5.07 5.07 50.1 50.1 50.1 50.1 PFOSAA EtFOSE PFOSEA SCd conc . Std conc Std conc ug/mL u g l d ug/mL 0.532 0.501 0.521 0.532 0.501 0.521 5.32 5.01 5.21 ' 5.32 5.01 5.21 I 5.32 I 5.01 5.21 1 53.2 50.1 52.1 1 1 53.2 j 50.1 i 52.1 I 1 53.2 I 50.1 I 52.1 1 53.2 50.1 52.1 1 M556 Std conc ug/mL 0.501 0.501 5.01 5.01 5.01 50.1 50.1 50.1 50.1 All All Am't Final vol spikedmL mL 0.010 1.015 0.020 1.025 0.005 1.010 0.010 1.015 0.020 I .025 0.005 1.010 0.010 1.015 0.015 1.020 0.020 , 1.025 4.93 5.00 5.24 9.76 9.89 10.4 24.8 25.1 26.3 49.3 i 50.0 j 52.4 97.6 1 98.9 1 104 248 25 1 263 493 500 I 524 73 5 746 I 782 976 989 1 1038 I 4.94 I 5.0 1 ' 5.13 9.78 9.93 10.2 24.8 1 25.2 25.8 49.4 50.1 51.3 97.8 99.3 102 248 252 25 8 494 501 513 737 i 749 766 978 1 993 1017 100 Surrogate Final conc ng/mL 5 00 0.005 Serum Rabbit I I PFOS 5.00-1000 PFOSA 5.00-1000 PFOSAA I 5.00-1000 EtFOSE-OH PFOSEA M556 5.00-1000 I 5.00-1000 I 5.00-1 000 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. Attachment C: Ion Pair Standard Curves ETS-8-4.1 Extraction of PFOS from Serum 3M Environmental Laboratory Page 14 of 14 Page 129 3M Medical Department Study: T-6889.3 c. 3M ENVIRONMENTLAALBORATORY Analytical Report: FACT-TOX-026 LRN-U2782 METHOD mum EXTRACTIOONF POTASSIUMPERnUOROOCTANESULFONATE OR OTHER FLUOROCHEMIC~COMPOUNDS FROM SERUM OR OTHER FOR ANALYSIS USING HPLC-ELECTROSPRAYMASPSESCTROMETRY Method Number: FACT-M3.1 - Author: Lisa Clemen, Glenn Langenburg Adoption Date: 04/22/98 Revision Date: i d / 01 I 9 8 Approved By: LVJL Labodhory Manager ' Groip Leader Date IZCl4 B Date Technical Reviewer . Date 1 1.i : 0 1&5.i- 1.9 Scope: This method is for the extraction of potassium perfluorooctanesulfonate (PFOS) or other fluorochemical compounds fiom serum or other fluid. Applicable compounds: Fluorochemical surfactants or other fluorinated compounds. Matrices: Rabbit, rat, bovine, and monkey serum, rat whole blood, and rat milk curd. Word 6/95 3M Environmental Laboratory FACT-M-3.1 Extraction of PFOS from Serum and Other Fluids Page 1 of 17 Page 130 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 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 fluorochemicalswere 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.O 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) C,F,,SO, 3.2 PFOSA: perflunrooctane sulfonylamide C,F,,SO,NH, 3.3 PFOSAA: perfluorooctane sulfonylamido (ethy1)acetateC8F,,S0,N(CH2CH,)CH2CO; 3.4 EtFOSE-OH: 2(N-ethylperfluorooctane su1fonamido)-ethyl alcohol C,F,',SO,N( CH,CH,)CH,CH,OH 3.5 POAA: perfluorooctanoate (anion of ammonium salt) C,F,,COO- 3.6 PFOSEA: perfluorooctane sulfonyl ethylamide C,F,,SO,N(CH,CH,)H 3.7 FC-807 monoester C8F,,S02N(CH,CH3)CH2CH,0-P0,H) 3.8 Surrogate standard: 1H-lH-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) 3M Environmental Laboratory FACT-M-3.1 Extraction of PFOS from Serum or Other Fluid Page 2 of 17 Page 131 3M Medical Department Study: T-6889.3 I Analytical Report: FACT-TOX-026 LRN-U2782 7.0 SUPPLIESA N D MATERIALS 7.1 Gloves 7.2 Eppendorf or disposable pipettes 7.3 Electronic pipettor, Eppendorf or equivalent 7.4 Graduated pieettes 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, 15mL 7.i 1 Labels 7.12 Syringes, capable of measuring 5 pL to 50 pL 7.13 Syringes, disposableplastic, 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-QRyw' ater. Rinse syringes a minhum of 9 times with methanol, 3 rinses from 3 separate vials. 8.0 REAGENTS AND STANDARDS 8.1 Type I reagent grade water, Milli-QTMor equivalent; all water used in this method should be Milli-Q"" water and may be provided by a Milli-Q TOC PlusRys'ystem 8.2 Sodium hydroxide (NaOH), J.T Baker or equivalent 8.3 Tetrabutylammonium hydrogen sulfate(TBA), Kodak or equivalent 8.4 Sodium carbonate (N+CO,), J.T. Baker or equivalent 8.5 Sodium bicarbonate (NaHCO,), 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.io Fluorochemical standards 8.10.1 PFOS (3M Specialty Chemical Division), molecular weight = 538 8.10.2 PFOSA (3MSpecialty Chemical Division), molecular weight = 499 3M Environmental Laboratory FACT-M-3.1 Extraction of PFOS from Serum or Other Fluid Page 3 of 17 Page 132 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 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 = 43 1 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 fluorochemicalcomponents. The monoester moleciilar weight = 650 8.10.8 Surrogate standard: 4-H, perfluorooctane sulfonic acid (1-H,l-H, 2-H, 2-H C,F,,SO,H) 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. Pour into a 1000mL beaker containing 500 mL Milli-Qm 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 10mL of 10 N NaOH solution into a 100 mL volumetric flask and dilute to volume using Mi1li-Q"l 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-Qm water. Adjust to pH 10 using approximately44to 54 mL of 10 N NaOH and dilute to volume with Milli-Qm 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/sodiumbicarbonate buffer (NqCO,/NaHCO,): Weigh approximately 26.5 g of sodium carbonate (Na&O,) and 21.O g of sodium bicarbonate (NaHCO,) into a 1 L volumetric flask and bring to volume with MilliQm 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 fluorochemicalstandards, as appropriate. Multicomponent fluorochemical standards are acceptable (for example, one working standard solution containing 1.OO 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 (PLg/ml)* 8.12.5 Dilute the stock solution with methanol for a working standard 1 solution of approximately 50 pprn. 3M Environmental Laboratory FACT-M-3.1 Extraction of PFOS from Serum or Other Fluid Page 4 of 17 Page 133 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 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-H71-H72-H, 2-H, C,F,SO,H 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 mi of surrogate stock to a 50 ml volumetric flask and bring to volume with methanol for a -working standard of 10-20ppm. Record the actual volume transferred. 9.0 SAMPLEHANDLING 9.1 All samples are received fiozen and must be kept fiozen until the extraction is performed. 5 10.0 QUALITCYONTROL 10.1 Matrix blanks and method blanks 10.1.1 Extract two 1.0 mL aliquots of the appropriatematrix (serum or blood, with blood samples diluted 1:1with Milli-Qm water) following this procedure and use as matrix blanks. See 11.1.4. 10.1.2 Extract two 1.Oml aliquots of Milli-QTMwater 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 1ast 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. 3M Environmental Laboratory FACT-M-3.1 Extraction of PFOS from Serum or Other Fluid Page 5 of 17 Page 134 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 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 centrihge tube as in step 12.9, then add 1.O mL of the diluted matrix sample to each tube. 11.1.1 Transfef 1 mL-ofserum or 1 mL of blood (blood is diluted 1:l with Milli-QTM water) to a 15 mL centrihge 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. 11.112 If most sample volumes are less than 1.OmL, 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 centrifbge tubes, mix or shake between aliquots. 11.1.4 Two 1mL aliquots, or other appropriatevolume, 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 concentrationto 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. 3M Environmental Laboratory FACT-M-3.1 Extractionof PFOS from Serum or Other Fluid Page 6 of 17 Page 135 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 Table 1 Approximate spiking amounts for standards and spikes using 1.0 ml of matrix Working standard Approx. final conc. of (approx. conc.) analyte in matrix - - Blank 0.500 ppm 10 0.005 ppm 0.500 ppm 20 . 0.010ppm 5.00 ppm 5 0.025 ppm 5.00 m m I* 10 0.050 uum I& 5.00 ppm 20 0.100 ppm 5QOppm . 5 0.250 ppm 50.0 upm ** 50.0 ppm I I 10 0.500 uum I I 1- 15 I 0.750 uum 50.0 ppm 20 1.OO pprn , Working standard (approx. conc.) - 0.500 ppm 0.500 ppm 5.00 ppm 5.00 ppm 5.00 ppm 50.0 ppm PL Approx. final conc. of analyte in matrix Blank 5 0.005 ppm 10 0.010 ppm 2.5 0.025 ppm 5 0.050 ppm 10 0.100 ppm 2.5 0.250 uum I 50.0 ppm I 10 I 1.OO ppm I 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.OmL with Milli-Qm 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 fieezer after extraction amount has been removed. 3M Environmental Laboratory FACT-M-3.1 Extraction of PFOS from Serum or Other Fluid Page 7 of 17 Page 136 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 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 cdibration 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 1mL 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.OmL or other appropriatevolume of methanol to each centrifbge 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 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) performing the extraction. 12.19 Cap and store extracts at approximately4 "Cuntil analysis. 12.20 Complete the extraction worksheet, attached to this document, and tape in the study notebook or include in study binder, as appropriate. 3M Environmental Laboratory FACT-M-3.1 Extraction of PFOS from Serum or Other Fluid Page 8 of 17 Page 137 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 13.0 DATAANALYSIASND CALCULATIONS 13.1 Calculations 13.1.1 Calculate actual concentrationsof PFOS, or other applicable fluorochemical, in calibration standards using the following equation: mL of standard x concentration of standard (un /mL) - mL of standard + mL of surrogate standard + initial matrix volume (mL) Final Concentration (pg/mL) of PFOS in matrix 14.0 METHODPERFORMANCE 14.1 The method detection lirnjt (MDL) is analyte and matrix specific. Refer to MDL report for specific M L andlimit 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. * 14.2.1 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 calibrationcheck samples to determine the continued accuracy of 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, MDLLOQ 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-M3.1 & 4.1-V-1. 3M Environmental Laboratory FACT-M3.1 Extraction of PFOS from Serum or Other Fluid Page 9 of 17 Page 138 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 19.0 AFFECTEDOCUMENTS 19.1 FACT-M-4.1, "Analysis of Serum or Other Fluid Extracts for Fluorochemicals using HPLC-Electrospray Mass Spectrometry" 20.0 REVISIONS Revision Number 1 - Reason For Revision - Validation of method to include 7 fluorochemicals, an additional matrix, new A P I / M S ( M S ) systems, monkey serum cross validation, improvements to ion pairing extraction, MDL study, updates in record keeping and storing policies, etc. Revision - Date 07/01/98 3M Environmental Laboratory FACT-M3.1 Extraction of PFOS from Serum or Other Fluid Page 10 of 17 Page 139 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 Study # Sample Number set # H,O Blank Blank FC-Mix approx. 0.5 ppm actual ppm #W FC-Mix approx. 5 ppm actual ppm #W . FC-Mix #W Date and Comments I I I - I I Add methanol Volume ml TN-A- 1 Filter using a 3cc B-D syringe with a 0 . 2 p m SRI filter into a 1.5 ml autosample vial MS/MSD/- Cont. Checks: Spiked uL of a ppm std ( 1 ) for a final concentration of ppm. MS/MSD used sample . Cont. Checks used same matrix as for std curve. Surrogate Standard: Spdced uL of a ppm std ( ) to all samples, standards, and blanks Attachment A: Extraction worksheet FACT-M-3.1 Extraction of PFOS from Serum or Other Fluid 3M Environmental Laboratory Page 1 1 of 17 Page 140 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 Compound MDL (ppb) PFOS 1.38 PFOSA 2.23 PFOSAA 2.84 EtFOSE-OH 3.90 POAA 4.3 1 PFOSEA 1.09 Monoester 149 ~ - I Compound I -- PFOS I MDL 1.27 PFOSA 2.14 PFOSAA 2.32 EtFOSE-OH 3.25 POAA 1.20 PFOSEA 1.84 Monoester 149 LOQ (ppb) 4.39 7.09 9.04 12.4 13.7 3.48 248 Linear Calibration Range (LCR) Approximate concentrations to be used for preparing the Standard Calibration Curve 5 ppb - 1000 ppb 10 ppb - 1000 ppb 10 ppb - 1000ppb 15 ppb - 1000 ppb 15 ppb - 750 ppb 25 ppb - 1000 ppb 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-M3.1 & 4.1-V-1 for specifics. I LOQ 4.04 6.81 7.38 10.3 3.81 5.86 248 I Linear Calibration Range (LCR) Approximate concentrations to be used for preparing the Standard Calibration Curve 10 ppb - 1000 ppb 25 ppb - 1000 Ppb 10 ppb - 1000 ppb 50 ppb - 1000 ppb 5 ppb - 1000 ppb 10 ppb - 1000 ppb MDL and LOQ are estimates only. No valid htDL was determinable from MDL study. Any quantitation performed for monoester will be an estimate onlv. Please refer to FACT-M-3.1 & 4.1-V- 1 for sDecifics. Compound MDL (ppb) PFOS 2.1 1 PFOSA 5.04 PFOSAA 2.34 EtFOSE-OH 11.3 POAA 4.64 PFOSEA 3.71 Monoester 149 LOQ @pb) 6.70 16.0 7.45 35.8 14.8 11.8 248 Linear Calibration Range (LCR) Approximate concentrations to be used for preparing the Standard Calibration Curve 25 ppb - 1000 ppb 25 ppb - 1000ppb 260 ppb - 1000 ppb 50 ppb - 1000 ppb 15 ppb - 1000 ppb 15 ppb - 1000 ppb 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-1 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-1for specifics. Attachment A: Extraction worksheet FACT-M-3.1 Extraction of PFOS from Serum or Other Fluid 3M Environmental Laboratory Page 12 of 17 Page 141 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 MDLLOQ values for Monkey Serum: Compound MDL LOQ Linear Calibration Range (LCR) (ppb) (ppb) Approximate concentrations to be used for preparing the Standard Calibration 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 PFOSA - only. Please refer to FACT-M-3.1 & 4.1-V-1 for specifics. 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-M3.1 & 4.1-V-1 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-1 for specifics. - EtFOSE-OH 3.90 12.4 . MDL and LOQ are estimates only. No valid MDL was determinable from h4DL study, Any quantitation performed for EtFOSE-OH will be an estimate only. Please refer to FACT-M-3.1 & 4.1-V-1 for specifics. POAA 4.3 1 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-M3.1 & 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 estimate only. Please refer to FACT-M3.1 & 4.1-V-1 for specifics. Monoester 149 248 MDL and LOQ are estimates only. No valid MDL was determinable from MDL study. Any quantitationperformed for EtFOSE-OH will be an estimate only. Please refer to FACT-M-3.1 & 4.1-V- 1 for specifics. I Compound MDL @pb) PFOS 1.25 PFOSA 1.77 PFOSAA 17.3 EtFOSE-OH 7.89 POAA 4.73 PFOSEA 24.2 Monoester 5 8.0 LOQ (ppb) 3.96 5.65 55.0 25.1 15.1 77.1 185 Linear Calibration Range (LCR) Approximate Concentrations to be used for preparing the Standard Calibration Curve 5 ppb - 1000 ppb 10 ppb - 1000ppb 55 ppb - 1000ppb 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-1 for specifics. 15 ppb - 1000 ppb 80 ppb - 1000 ppb MDL and LOQ are estimates only. No valid MDL was determinable from MDL study. Any quantitation performed for monoester will be an estimate onlv. Please refer to FACT-M-3.1 & 4.1-V-1 for suecifics. Please see Attachment C (LOQ Summary) and MDL study in FACT-M-3.1 & 4.1-V-1 for specifics. Attachment A: Extraction worksheet FACT-M-3.1 Extraction of PFOS from Serum or Other Fluid 3M Environmental Laboratory Page 13 of 17 Page 142 3M Medical Department Study: T-6889.3 ~~~ Analytical Report: FACT-TOX-026 LRN-U2782 Ion Pairing Extraction of Fluorochemicals from Serum and Analysis by API/MS(MS) Summary Table: Limits of Quantitation Compound Matrix Rabbit 1.38 ppb Bovine 2.1 1 ppb 1.27 ppb Monkey dd PFOSA Rabbit Bovine 2.23 ppb 5.04 ppb Rat 2.14 ppb Monkey nld PFOSM Rabbit 2.84 ppb Bovine 2.34 ppb Rat - . 2.32 ppb Monkey n/d EtFOSE-OH Rabbit 3.90 ppb Bovine 11.3 ppb Rat 3.25 ppb Monkey dd POM Rabbit 4.31 ppb Bovine 4.64 ppb Rat 1.20 ppb Monkey nld PFOSEA Rabbit 1.03 ppb Bovine 3.71 ppb Rat 1.84 ppb Monkey nld Monoester' Rabbit 149 ppb Bovine 149 ppb Rat 149 ppb Monkey dd 1. Values for monoester are estimates only. 4.39 ppb 6.70 ppb nld I 7.09 ppb 16.0 ppb 6.81 ppb dd 9.04 ppb 7.45 ppb 7.38 ppb n/d 12.4 ppb 35.8 ppb * 10.3 ppb dd 113.7 ppb 14.8 ppb 3.81 ppb n/d 3.48 ppb 11.8 ppb 5.86 ppb n/d 474.0 ppb 474.0 ppb 474.0 ppb nld Approximate linear range' Low std High std 25 PPb 10 PPb 25 DDb 10ppb 25 ppb 25 ppb 10ppb 15 PPb 5 PPb 1000 ppb 1000 ppb 1000 DDb I I 1 1OOOppb I lOOOppb I 1OOOppb I I l000ppb 1000 ppb 1000 ppb 1000 ppb 1000 ppb 1000 ppb 1000 ppb I 750 ppb 1OOOppb I 25ppb 5 ppb 10ppb 5 PPb 250 ppb 250 ppb 250 ppb 100 ppb I I I 1OOOppb 1 lOOOib I lOOOppb I 1000 ppb 1000 ppb 1000 ppb 1000 ppb 1000 ppb Attachment C: LOQ Summary 3M Environmental Laboratory FACT-M-3.1 Extraction of PFOS from Serum or Other Fluid Page 14 of 17 Page 143 .3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 1 Compound:PFOSA Prepared matrix range of standards (ppb)(ng/W Rabbit 5.00 - 1470 Range of average curve (ppb)(ng/mL) 5.00 - 1470 1I Bovine It 5.00 - 1470 5.00 - 1470 5.00 - 1470 5.00 - 1470 5.00 - 1470 LCR from ave curve (ppb)(ng/mL) 9.89- 1000 Range of low std curve (ppb)(ng/mL) 5.00 - 251 25.1 - 1000 5.00 - 98.9 50.0 - 1000 9.89 - 500 98.9 - 1000 I 25.1 - 500 LCR from low std curve (ppb)(ng/mL) n/a Range of high std curve (ppb)(ng/mL) 98.9 - 1470 LCR from high std curve (ppb)(ng/mL) . 98.9 - 1000 da 98.9 - 1470 98.9 - 1000 25.1 - 500 98.9 - 1470 98.9 - 1000 25.1 - 500 I 98.9 - 1470 - 7 Compound: PFOSAA Compound: EtFOSE-OH Prepared range of Rabbit 4.94 - 1450 4.94 - 1450 4.94 - 1450 4.94 -1450 I 4.94-248 97.8 - 1000 4.94 - 248 1 97.8 - 1450 248 -1000 1 9.78 - 248 97.8 - 1450 da I Attachment C: LOQ Summary 3M Environmental Laboratory FACT-M-3.1 Extraction of PFOS from Serum or Other Fluid Page 15 of 17 Page 144 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 Compound: POAA matrix range of standards Range of average curve (ppb)(ng/W LCR from ave curve (PpbXndmL) Range of low std curve (ppb)(ndmL) 5.13- 1510 I Bovine I Rat 5.01 - 1480. 5.13 - 1510 I 5.01-1480 5.13 - 1510 1 Monkey 5.01-1480 5.13 - 1510 25.8-1000 102- 1000 51.3 - 1000 102 - 1000 I 5.13-258 5.13 -258 5.13 - 102 5.13 - 102 LCR from low std curve (ppb)(ndmL) da Range of high std curve (ppb)(ng/mL) 102 - 1510 5.13-258 102- 1510 5.13 - 102 102- 1510 5.13 - 102 I 102 - 1510 LCR from high std curve (ppb)(ndmL) da 258- 1000 102 - 1000 258 - 1000 Compound: PFOSEP range of I Bovine I Rat 1 Monkey 5\13- 1510 1 5.13 - 1510 I 5.13 - 1510 I 5.13 - 1510 Compound: Monoester 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 3M Environmental Laboratory FACT-M-3.1 Extraction of PFOS from Serum or Other Fluid Page 16 of 17 Page 145 3M Medical Department Study: T-6889.3 n L Analytical Report: FACT-TOX-026 LRN-U2782 Ion Pair Standard Curves - Fluids Prep date(s): Standard number: Analyte(s): Equipment number: Sample matrix: Final solvent and TN: Blank fluididentifier: Methodhevision: Target analyte(s): FC mix std approx. Or500 ppm: W398-641 FC mix std approx. 5.00 ppm: W3 98-640 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 Std conc Std conc ug/& ug/& Std conc ug/& Std conc ~g/& Std conc ug/d Std conc ug/d Std conc ug/mL spiked& Volume Calculated concentrations of standards in the sample matrix PFOS PFOSA PFOSAA EtFOSE POAA PFOSEA Monoester Final Final conc Final conc Final conc Final conc Final conc Std conc conc ng/mL ng/mL ng/mL nghL ng/mL ng/mL Surrogate Std conc ng/mL All Am't spiked w-1 ' Sera Rabbit Bovine Rat -- Monkey PFOS 5-1000 ppb 25-1000 ppb 10-1000 ppb Estimates only. PFOSA 10-1000 ppb 25-1000 ppb 25-1000 ppb Use values for PFOSAA 10-1000 ppb 263-1000 ppb 10-1000 ppb 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 3M Environmental Laboratory Page 17 of 17 Page 146 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 3M ENVIRONMENTLAALBORATORY METHOD ANALYSIS OF POTASSIUM PERFLUOROOCTANESULFONATEOR OTHER FLUOROCHEMICALSIN SERUM EXTRACTS USING HPLC-ELECTROSPRAYMSAPSESCTROMETRY Method Number: -ETS-8-5.1 Adoption Date: 03/01/99 Revision Date: lasp9 Group Leader . +/2% Date .d 1 b SCOPE AND APPLICATION 1 3Scope: This method describes the analysis of serum extracts for fluorochemicalsurfactants 0using HPLC-electrospray/massspectrometry. L3:Applicable Compounds: Fluorochemical surfactants or other fluorinated compounds, or 3 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 3M Environmental Laboratory ETS-8-5.1 Analysis of Serum Extract Using ESMS Page 1 o f 9 Page 147 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 2.0 SUMMARY OF METHOD 2.1 This method describes the analysis of fluorochemical surfactantsextracted 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 DEFINITIONS 3.1 Atmospheric Pressure Ionization (API): The Micromass Quattro 11triple 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 transferied 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 (MSMS): The API Quattro 11triple quadrupole systems are equipped with quadrupole mass selective detectors. Ions are selectively discriminated by mass to charge ratio ( d z ) and subsequently detected. A single MS may be employed for ion detection or a series (MUMS) for more specific fi-agmentationinformation. 3.4 Conventional vs. %spray probe interface: The latest models of Micromass Quattro I1 triple quadrupole systems (post 1998) utilize a "2-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 configurationis 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 @.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 I1 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 I1 triple quadrupole 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. 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. 3M Environmental Laboratory ETS-8-5.1 Analysis of Serum Extract Using ES/MS Page 2 of9 Page 148 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 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 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 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. Document any modifications inthe raw data as method deviations. 6.1.1 Micromass Quattro I1 triple quadrupole Mass Spectrometerequipped with an electrospray ionization source 6.1.2 , HP1100 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 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 REAGENTS AND STANDARDS 8.1 Reagents 8.1.1 Methanol, HPLC grade or equivalent 8.1.2 Milli-QTMwater, all water used in this method should be Milli-QTMwater 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 SAMPLHEANDLING 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. 3M Environmental Laboratory ETS-8-5.1 Analysis of Serum Extract Using ES/MS Page 3 of 9 Page 149 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 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 OUALITCYONTROL 10.1 Solvent Blanks, 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 samples, with a ' minimum of 2 spikes per batch. 10.2.4 Matrix spike and matrix spike duplicate concentrationswill fall in the mid-range of the initial calibration curve. Additional spike concentrationsmay 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.O CALIBRATIOANND 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), weighted l/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 100ppb rather than the fill range of the curve (5 ppb to 1000ppb). This will reduce inaccuracy attributed to linear regression weighting of high concentration standards. 3M Environmental Laboratory ETS-8-5.1 Analysis of Serum Extract Using ESMS Page 4 of 9 Page 150 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 Time 0.00 min. 8.50 min. 11.O min. 12.0 min. MeOH 40% 90% 90% 40% 2.0 mM Ammonium acetate 60% 10% 10% 60% 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. 3M Environmental Laboratory ETS-8-5.1 Analysis of Serum Extract Using ES/MS Page 5 o f 9 Page 151 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 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 onthe 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 litershour 12.3.6.2 ESI nebulizing gas 10-15 litershour 12.3.6.3 HPLC'constant flow mode, flow rate 10- 500 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 ESMS 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 DATAANALYSIS AND CALCULATIONS 13.1 Calculations: 13.1.4 Calculate matrix spike percent recoveries using the following equation: % Recovery = Observed Result - Backmound Result x 100 Expected Result 13.1.5 Calculate percent difference using the following equation: % Difference = Expected Conc. - Calculated Conc. x 100 Expected Conc. 13.1.6 Calculate actual concentration of PFOS, or other fluorochemical, in matrix (PdmL): (na of PFOS calc. from std. Curve x Dilution Factor) x 1 DQ (Initial Volume of matrix (mL) + mL of Surrogate Standard) 1000 ng Final Volume (mL) 3M Environmental Laboratory ETS-8-5.1 Analysis of Serum Extract Using ESMS Page 6 of 9 Page 152 . 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 14.0 METHODPERFORMANCE 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 Solventblanks, 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 I? 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 k 30% of the spiked concentration. 14.5 Continuing Calibration Verifications , I 14.5.1 Continuing calibration verification percent recoveries must be k 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 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 fiom 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 l/x, then print these graphs and store in the study folder. 16.4 Print data integration summary, integration method, and chromatograms, fiom MassLynx, and store in the study folder. 3M Environmental Laboratory ETS-8-5.1 Analysis of Serum Extract Using E S M S Page 7 of 9 Page 153 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 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 TABLESD, IAGRAMFS,LOWCHARTANSD, VALIDATION DATA 17.1 Attachment A: -ETS-8-5.1 Data summary spreadsheet. 18.0 REFERENCES 18.1 FACT-M-4.1, "Extraction of Potassium Perfluorooctanesulfonateor Other Fluorochemical compounds fiom Serum for Analysis Using HPLC-ElectrosprayMass Spectrometry 18.2 ETS-9-24.0, "Operation and Maintenance of the Micromass Atmospheric Pressure Ionization/Mass Spectrometer Quattro I1 triple quadrupole Systems" 18.3 The validation report associated with this method is ETS-8-4.0 & 5.0-V-1. 19.0 AFFECTEDDOCUMENTS 19.1 ETS-8-4.1, "Extraction of Potassium Perfluorooctanesulfonateor Other Fluorochemical Compounds f?om Serum for Analysis Using HPLC-ElectrosprayMass Spectrometry" 20.0 REVISIONS Revision Number. 1 Reason For Revision Section 6.1.2 Clarificationof HP1100 system components. Section 11.1 Average of two curves, not standard values, are used for plotting linear regression and added the l/x weighting of the curve. Section 12.2.2.4 Clarificationof solvent ramp. Section 17.1 Changed from attachment B to A. Revision - Date 04/02/99 3M Environmental Laboratory ETS-8-5.1 Analysis of Serum Extract Using ESIMS Page 8 of 9 Page 154 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 Laboratory Study # Study: Test Material: Matriflinal Solvent: MethodRevision: Analytical Equipment System Number: Instrument SoftwareNersion: Filename: R-Squared Value: Slope: Y Intercept: Date of Extractiodhalysh Date of AnalysidAnalyst: Sample#: Taken from the study folder. Concentration (ug//mL): Taken from the MassLynx integration summary. Initial Volume (d) Ta: ken from the study folder. Dilution Factor: Taken from the study folder. Final Conc. (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 3M Environmental Laboratory Page 9 of 9 Page 155 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 3M ENVIRONMENTLAALBORATORY METHOD ANALYSIS OF POTASSIUM PERFLUOROOCTANESULFONATEOR OTHER FLUOROCHEMICAINLSSERUM OR OTHER EXTRACTUSSING HPLC-ELECTROSPRAY~MSAPESSCTROMETRY Method Number: -FACT-M-4.1 - Author: Lisa Clemen, Glenn Langenburg Approved By: Adoption Date: 4/22/98 Revision Date: Io - 1 -q 8 Laboratory Manager - Group Leader Date 7Iz.j /49 Date . ) -6 tcn ", g -I > J 1.0 SCOPE AND APPLICATION Scope: This method is for the analysis of extracts from serum or blood for fluorochemical surfactants using HPLC-electrospray/mas spectrometry. &:2Applicable Compounds: Fluorochemical surfactants or other fluorinated compounds, or J' other ionizable compounds. f -T 3 Matrices: Rabbit, rat, bovine, or monkey serum and rat whole blood or milk curd. Word 6.0.1195 3M Environmental Laboratory FACT-M4.1 Analysis of Serum or Fluid Extract Using E S N S Page 1 of 9 Page 156 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 2.0 SUMMARY OF METHOD 2.1 This method describes the analysis of fluorochemicalsurfactants 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 A P I / M S N S 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 atmosphericpressure (i.e.'notunder 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 ( M S M S ) : The API platforms are equipped with quadrupole mass selective detectors. Ions are selectively discriminated by mass to charge ratio ( d z ) and subsequently detected. A single MS may be employed for ion detection or a series ( M S N S ) for more specific fragmentation information. 3.4 Conventional vs. %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 with similar systems (Le. 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 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 I1 or Quattro I1 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 6.0.1195 3M Environmental Laboratory FACT-M-4.1 Analysis of Serum or Fluid Extract Using ES/MS Page 2 of 9 Page 157 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 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 HP1100 will initiate automatic shutdown. 4.2.2 Do not run solvent pumps to dryness. c 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 HP1100 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 centrifbge tubes 8.0 REAGENTASND STANDARDS 8.1 Reagents 8.1.1 Methanol, HPLC grade or equivalent 8.1.2 Milli-Q" water, all water used in this method should be Milli-Qm 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 SAMPLEHANDLING 9.1 Fresh matrix standards are prepared with each analysis. Extracted standards and samples are stored in capped autovials or capped 15 ml centrifugetubes 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. 3M Environmental Laboratory FACT-M4.1 Analysis of Serum or Fluid Extract Using ESMS Page 3 of 9 Page 158 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 10.0 OUALITCYONTROL 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 calibratiop curve. . 10.2.3 See Section 13 to calculatepercent 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 acceptablecalibration standard will be used. The remaining samples must be reanalyzed. 10.3.2 See Section 13 to calculate percent difference. 11.0 CALIBRATIONAND 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 (?)for the calibration curve using MassLynx or other suitable software. 11.2 The ? value for the data should be 0.980 or greater. Lower values may be acceptable at the discretion of the analyst and documented approval of 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 calibrationcurve 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 1000ppb). 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-lastdigit of year-sample number, assign a method (MS) for acquiring, and type in sample descriptions. 3M Environmental Laboratory FACT-M-4.1 Analysis of Serum or Fluid Extract Using ES/MS Page 4 of 9 Page 159 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 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 MSMS 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 analyticalbatch run sequencebegins 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 HP1lOO/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 InjecVsample = 1 12.2.2.3 Cycle time = 15 minutes 12.2.2.4 Solvent ramp = Time 0.00 min. 7.5 min. 11.O min. 11.5 min. MeOH 45% 90% 90% 45% 2.0mM-- ~ Ammonium acetate 55% 10% 10% 55% Note: In this instrument configuration, the runmust 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. 3M Environmental Laboratory FACT-M4.1 Analysis of Serum or Fluid Extract Using ESMS Page 5 of 9 Page 160 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 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 litershour 12.3.6.2 ESI nebulizing gas 10-15 litershour 12.3.6.3 HPLC constant flow mode flow rate 10- 500 pL/min 12.3.6.4 -Pressure HPLC is <400 bar (This parameter operating correctly.) is not set, it is a guide to ensure the 12.3.7 Carefully guide`theprobe 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. I 12.3.9 Using the cross-flow counter electrode in the E S N S source is recommended for the analysis of biological matrices. 12.3.1OClick 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 DATAANALYSIS 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 Conc. - Calculated Conc. x 100 Expected Conc. 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 ug: (Initial Volume of matrix (ml) + ml of Surrogate Standard) 1000 ng Final Volume (mL) 14.0 METHODPERFORMANCE 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. 3M Environmental Laboratory FACT-M4.1 Analysis of Serum or Fluid Extract Using ES/MS Page 6 of 9 Page 161 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 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 calibrationcurve. 14.3 Matrix Spikes_ 14.3.1 Matrix spikes are analyzed with each sample set and the percent recoveries are expected to fall within f30% 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 k 30% of the spikea 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 PREVENTIONAND 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 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 fiom 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 TABLESD, IAGRAMFSL, OWCHARTANSD, VALIDATION DATA 17.1 Attachment A: FACT-M-4.1 Data reporting spreadsheet 17.2 The validation report associated with this method is FACT-M3.1 & 4.1-V-1. 3M Environmental Laboratory FACT-M4.1 Analysis of Serum or Fluid Extract Using E S M S Page 7 of 9 Page 162 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 18.0 REFERENCES 18.1 FACT-EP-3.O, "Operation and Maintenance of the Micromass Atmospheric Pressure IonizatiodMass SpectrometerPlatform Systems" 19.0 AFFECTEDDOCUMENTS 19.1 FACT-M-3.1, "Extraction of Potassium Perfluorooctanesulfonateor Other Fluorochemical Compounds &om Serum or Fluid for Analysis Using HPLC-ElectrospraylMass Spectrometry" 20.0 REVISIONS Revision Number. 1 - . Reason For Revision Validation of mei`hod to include 7fluorochemicals addition of whole blood matrix,surrogate standard, new API/MS(MS) systems, monkey sera cross validation, MDL study, updates in record keeping and storing policies, etc. Revision - Date 07101I98 3M Environmental Laboratory FACT-M-4.1 Analysis of Serum or Fluid Extract Using ESMS Page 8 of 9 Page 163 3M Medical Department Study: T-6889.3 r Attachment A Analytical Report: FACT-TOX-026 LRN-U2782 Laboratory Study # Study: Test Material: Matrix/Final Solvent: MethodiRevision: L Analytical Equipment System Number: Instrument SoftwareNersion: Filename: R-Squared Value: Slope: Y Intercept: Date of ExtractiodAnalysG Date of Analysis/Analyst: Ll-- Group Sample# 1 Concentration Ug/d Initial Vol. d. Dilution Factor Final Conc. ug/d Slope: Taken from linea Group/Dose: Taken fro1 Sample#: Taken from tl Concentration (ug/mL): Initial Volume (mL): T Dilution Factor: Taken Final Conc. (ug/mL): C regression equation. I 3M Environmental Laboratory FACT-M-4.0 Analysis of Serum or Fluid Extract Using E S M S Page 9 of9 Page 164 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 3M ENVIRONMENTLAALBORATORY METHOD EXTRACTION-OPOFTASSIUM PERFLUOROOCTANESULFONATEOR OTHER FLUOROCEIEMICALCOMPOUNDS FROM URnvE FOR ANALYSIS USING HPLC- ELECTROSPRAY/MSAPSECSTROMETRYLWASS SPECTROMETRY - Method Number: ETS-8-96.0 Adoption Date: 3/28-44 Revision Date: Author: Lisa Clemen, Glenn Langenburg Approved By: Group Leader T:chnical Reviewer WZ~l95 Date Date 1.6) SCOPE AND APPLICATION Scope: This method is for the extractionof potassium perfluorooctanesulfonate(PFOS) or other fluorochemical compounds fiom urine. Applicable compounds: Fluorochemicals or other fluorinatedcompounds. '-7 Matrices: Human,rat, and monkey urine or other fluids as designated in the validation c2. report. 91, Word 6/95 3M Environmental Laboratory ETS-8-96.0 Extraction of PFOS from Urine Page 1 of 14 Page 165 3M Medical Department Study: T-6889.3 . Analytical Report: FACT-TOX-026 LRN-U2782 2.0 SUMMARY OF METHOD 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-tert-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 Feagent 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 method. -extracts are analyzed following method ETS-8-97.0 or other appropriate 3.0 DEFINITIONS 3.1 PFOS: perfluorooctanesulfonate (anion of potassium salt) C,F,,SO; 3.2 PFOSA: perfluorooctane sulfonylamide C,F,,SO,NH, 3.3 PFOSAA: perfluorooctane sulfonylamido (ethy1)acetate C,F,,SO,N(CH,CH,)CH,CO; 3.4 EtFOSE-OH: 2(N-ethylperfluorooctane su1fonamido)-ethyl alcohol C,F ,S O,N(CH,CH,)CH,CH,OH 3.5 M556: C,F,,SO,N(H)(CH,COOH) 3.6 M570: C,F,,SO,N(CH,)CH,COOH 3.7 POAA: perfluorooctanoateC,F,,COO3.8 Surrogate standard THPFOS: lH-lH-2H-2H perfluorooctane sulfonic acid, used as an internal standard in this method. 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 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 halytes. 3M Environmental Laboratory ETS-8-96.0 Extraction of PFOS from Urine Page 2 of 14 Page 166 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 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-Qm 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-Qm or equivalent; all water used in this method should be Milli-QTMwater and may be provided by a Milli-Q TOC PlusTMsystem 8.2 Sodium hydroxide (NaOH), J.T Baker or equivalent 8.3 Tetrabutylammonium hydrogen sulfate(TBA),Kodak or equivalent 8.4 Sodium carbonate (NaJO,), J.T. Baker or equivalent 8.5 Sodium bicarbonate (NaHCO,), J.T. Baker or equivalent 3M Environmental Laboratory ETS-8-96.0 Extraction of PFOS fiom Urine Page 3 of 14 Page 167 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 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,l-H, 2-H, 2-H C,F,,SO,H) 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 10 N sodium hydroxide (NaOH): Weigh approximately 200 g NaOH. Pour into a 1000 ml beaker containing 500 ml Milli-Q" 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 10N NaOH solution into a 100 ml volumetric flask and dilute to volume using MilliQ" 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-Q" water. Adjust to pH 10 using approximately44 to 54 ml of 10N NaOH (While adding the last ml of NaOH, add slowly because the pH changes abruptly). Dilute to volume with Milli-Q" water. Store in a 1L Nalgene bottle. 8.10.3.1 TBA requires a check prior to each use to ensure pH = 10.0. Adjust as needed using 1N NaOH solution. 8.10.4 0.25 M sodium carbonate/sodium bicarbonate buffer (NqCOJNaHCO,): Weigh approximately26.5 g of sodium carbonate (NqCO,) and 21.O g of sodium bicarbonate (NaHCO,) into a 1 L volumetric flask and bring to volume with MilliQ" 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 fluorochemicalstandards, as appropria.te. Multicomponent fluorochemical standards are acceptable (for example, one working standard solution containing 1.OO ppm PFOS, 1.02 ppm PFOSA, 0.987 ppm PFOSAA, and 1.10 ppm EtFOSE-OH.) 3M Environmental Laboratory ETS-8-96.0 Extraction of PFOS from Urine Page 4 of 14 Page 168 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 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 1000ppm (PLg/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 Gpproximately 50-60 mg of surrogate standard 1-H,l-H, 2-H, 2-H, C,F,,SO,H 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 1ml 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 SAMPLHEANDLING 9.1 All samples are received frozen and must be kept fiozen until the extraction is performed. 9.2 Allow samples to thaw to room temperature prior to extraction. 10.0 QUALITCYONTROL 10.1 Solvent Blanks, Method blanks and matrix blanks 10.1.1 An aliquot of 2.0 ml methanol is used as a solvent blank. 10.1.2 Extract two 2.0 ml aliquots of Milli-Q" water followingthis 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. 3M Environmental Laboratory ETS-8-96.0 Extraction of PFOS from Urine Page 5 of 14 Page 169 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 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 calibrationverification 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 samplds. For example, if a sample set = 34, four checks are prepared and extracted. 10.3.3 Prepare each continuing calibrationverification fkom the same matrix used to prepare the initial curve. 10.3.4 The expected concentrationswill 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 - 1000ppb). 11.o 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 concentrationsand 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, W2067, which lists the working ranges and the Linear Calibration Range (LCR) 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. 3M Environmental Laboratory ETS-8-96.0 Extraction of PFOS from Urine Page 6 of 14 Page 170 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 Table 1 Approximate spiking amounts for standards and spikes Working standard I I I Using 2.0 ml of matrix PL Approx. final conc. Approx. final conc. (approx. conc.) -- of analyte in matrix Of analyte in solvent Blank - Blank 12.0 PROCEDURE 12.1 Obtain fiozen 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 fieezer after extraction amounts have been removed. 12.4 Record the initial volume on the sample weightlvolume worksheet. . See Attachment D. The originalweightholume worksheet is included in the study binder. 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 iIS 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 m10.5 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. 3M Environmental Laboratory ETS-8-96.0 Extractionof PFOS from Urine Page 7 of 14 Page 171 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 12.13 Centrihge 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 mi 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 centrifugetube 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 12.19 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. 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 refiigerated 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 DATAANALYSIASND 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 METHODPERFORMANCE 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. 3M Environmental Laboratory ETS-8-96.0 Extraction of PFOS from Urine Page 8 of 14 Page 172 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 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 TABLESD,IAGRAMSF,LOWCHARTASN,D VALIDATION DATE 17.1 Attachment A,-Extraction worksheet 17.2 Attachment B, MDLLOQ values and summary 17.3 Attachment C, Calibration standard concenpation worksheet 17.4 Attachment D, Sample weightholume worksheet 18.0 REFERENCES 18.1 The validation report associated with this method is FACT-'FOX-131, W2067. 19.0 AFFECTEDOCUMENTS 19.1 ETS-8-97.0, "Analysis of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds in Urine Extracts Using HPLC-Electrospray Mass Spectrometryhfass Spectrometry" 20.0 REVISIONS Revision Number Reason For Revision - Revision Date 3M Environmental Laboratory ETS-8-96.0 Extraction of PFOS from Urine Page 9 of 14 Page 173 3M Medical Department Study: T-6889.3 Extraction Worksheet ETS-8-96.0 Study # Surrogate Std r L DateSpikedlAnalyst Analytical Report: FACT-TOX-026 LRN-U2782 FC-Mix approx. 50 ppm actual ppm # Comments - - - - Pipette Matrix ,spike with appropriate surrogate or FC-Mix Volume ml 1 Shake 20 min. Centrifuge 20-25 min. Remove a 4 ml aliquot of organic layer Put on Nitrogen Evaporator to dryness Add methanol Volume Shaker speed: Centrifuge speed: Temperature: rnl TN-A- Filter using a 3cc B-D syringe with a 0.2um filter into an autosample vial Cont. Cal. Verifications used same matrix as for std curve. I Attachment A 3M Environmental Laboratory ETS-8-96.0 Extraction of PFOS from Urine Page 10 of 14 Page 174 3M Medical Department Study: T-6889.3 MDLLOQ values for human urine Analytical Report: FACT-TOX-026 LRN-U2782 n/d = not determined NOTE: to - calculate MDL, LOQ, and LCR values in ug/ml of urine divide the above values by 4. MDLLOQ 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 hrther information. Attachment B: MDLLOQ Summary 3M Environmental Laboratory ETS-8-96.0 Extraction of PFOS from Urine Page 11 of 14 Page 175 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 Human Urine Full Range Prepared range of standards (ppb) (ng/ml) dd LCR from curve (PPW (ng/ml) dd % Recovery Range dd Low Curve dd - High curve dd dd .dd dd dd 1/X 2.5ppb-15OOppb 15ppb-15OOp~b 75-1 16 RSD Range dd dd dd +I- 30% Human Urine Full Range Prepared range of standads (ppb) (ng/ml) dd LCR from curve (PPb) (ng/ml) dd Low Curve dd dd High curve 1K quadratic dd 2.5ppb - 1500 ppb dd 50 ppb - 1500 ppb % Recovery Range dd dd dd 86- 105 RSD Range dd dd dd +I- 30% Attachment B: MDLLOQ Summary 3M Environmental Laboratory ETS-8-96.0 Extraction of PFOS from Urine Page 12 of 14 Page 176 3M Medical Department Study: T-6889.3 c Analytical Report: FACT-TOX-026 LRN-U2782 Prep date(s): Analyte(s): Sample matrix: Ion Pair Standard Curves -Urine Standard number: Equipment number: Final solvent and TN: Blank fluid/identifier: Box Number: PFOS PFOSA PFOSAA EtFOSE POAA M556 M570 All All Std conc Std conc Std conc Std conc Std conc Std conc Std conc Am't Final vol ' PFOS PFOSA PFOSAA EtFOSE POAA M556 Final conc Final conc Final conc Final conc Final conc Final conc ng/ml ng/ml ng/d ng/ml 1.25 1 1.25 I 1.25 1 1.25 2.48 2.49 2.48 I 2.49 6.17 6.18 I 6.17 6.18 12.5 12.5 12.5 1 12.5 24.8 24.9 24.8 1 24.9 61.7 61.8 1 61.7 1 61.8 125 125 I 125 125 ng/ml 1.24 2.48 6.15 12.4 24.8 61.5 124 ng/ml I 1.25 I 2.48 1 6.17 12.5 24.8 61.7 125 I 187 187 1 187 187 , 186 187 I 1 1 248 372 249 372 1 248 372 I I 249 372 I 248 371 I 248 372 M570 Final conc ng/ml 1.25 2.49 6.18 12.5 24.9 61.8 125 187 249 3 72 Surrogate Std conc ng/ml 100 Surrogate Final conc ndml 125 All Am't spiked ml 0.0025 Calculated PFOS Final conc nglml 5.00 10.0 25.0 50.0 100 250 500 750 1000 1500 concentrations of standards in methanol extract PFOSA PFOSAA EtFOSE POAA Final conc Final conc Final conc Final conc ng/ml 5.01 ng/ml 5.00 ng/ml 5.01 ng/ml 4.99 10.0 10.0 10.0 10.0 25.1 25.0 25.1 25.0 50.1 50.0 50.1 49.9 100 100 100 100 25 1 250 25 1 250 501 500 501 499 752 750 752 749 1002 1000 1002 998 1503 1500 1503 1497 (0.5 ml final M556 Final conc nglml 5.00 10.0 25.0 50.0 100 250 500 750 1000 1500 volume) M570 Final conc ng/ml 5.01 10.0 25.1 50.1 100 25 1 50 1 ~752- 1002 1503 Surrogate Std conc ng/ml 100 Surrogate Final conc ng/ml 500 All Am't spikedml 0.0025 Attachment C: Standard Calculation Sheet ETS-8-96.0 Extraction of PFOS from Urine 3M Environmental Laboratory Page 13 of 14 Page 177 3M Medical Department Study: T-6889.3 4 Prep Date(s): Analyst(s): - Sample Matrix: Methodmevision: Target Analyte(s): Analytical Report: FACT-TOX-026 LRN-U2782 Study Number: Equipment Number: Final Solvent & TN Number: Matrix Blankndentifier: Box: Summary of method: Notes: Attachment D: Weightivolume Sheet 3M Environmental Laboratory ETS-8-96.0 Extraction of PFOS from Urine Page 14 of 14 Page 178 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 3M ENVIRONMENTLAALBORATORY METHOD ANALYSIS OF POTASSIUMPERnUOROOCTANES~l~ONATOER OTaER FLUOROCHEMICAL COMPOUNDSIN URINE EXTRACTUSSING HPLC-ELECTROSPRAY/MASPSESCTROMETRYMASS SPECTROMETRY Method Number: ETS. -8-97.0 Author: Lisa Clemen, Robert Wynne, Glenn Langenburg Adoption Date: 3-Z.6-W Revision Date: )\1p Approved By: - V I Laboratory Manager Group Leader Date WW39 Date $ethnical Reviewer Date X L?J (-3 I-+ 3 3 2 . 0 SCOPEAND APPLICATION "k"0 d.1Scope: This method describes the analysis of urine extracts for fluorochemicalsusing HPLC-electrospray/mass spectrometry. 2 2 Applicable Compounds: Fluorochemicals or other ionizable compounds. 3 . 3 Matrices: Human,rat, and monkey urine, or other fluids as designated in the validation 9, report. Word 6/95 3M Environmental Laboratory ETS-8-97.0 Analysis of Urine Extract Using ESMS Page 1of 10 Page 179 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 2.0 SUMMARY OF METHOD 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 characteristicof 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 dcompound by detecting daughter ions of the parent ion. 3.0 DEFINITIONS 3.1 Atmospheric Pressure Ionization (MI):The Micromass Quattro 11triple quadrupolesystems 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 ronization (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 (MSMS): The API Quattro 11triple quadrupole mass spectrometeris equipped with two quadrupole mass selective detectors and a collision cell. Ions are selectively discriminated by mass to charge ratio ( d 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 I1 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 (Le., 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 I1 triple quadrupole systems. Currently MassLynx has WindowsNT 4.0 versions. For more details see the manual specific to the instrument (Micromass Quattro I1 triple quadrupole MassLynx NT User's Guide). 4.0 WARNINGS AND CAUTIONS 4,l 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. 3M Environmental Laboratory ETS-8-97.0 Analysis of Urine Extract Using ESMS Page 2 of 10 Page 180 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 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 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 should not be used for sample storage or any part of instrumentation that comes in contact with the sample or extract. - 6.0 EOUIPMENT 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 quadrupole Mass Spectrometer equipped with an electrospray ionization source HP1100 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 nitrogen regulated to approximately 100 psi. (House air system) 7.1.2 High purity grade argon regulated to approximately6 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 centrihge tubes 8.0 REAGENTS AND STANDARDS 8.1 Reagents 8.1.1 Methanol, HPLC grade or equivalent 8.1.2 Milli-QTMwater (ASTM type I), all water used in this rnethod should be ASTM type I, or equivalent, and may be provided by a Milli-C! 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-QTMwater, mix until all solids are dissolved. Store at room temperature . 3M Environmental Laboratory ETS-8-97.0 Analysis of Urine Extract Using ESMS Page 3 of 10 Page 181 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 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 SAMPLHEANDLING 9.1 Fresh matrix stqdards are prepared with each analysis. Extracted standards and samples are stored in capped autovials or capped 15 mL centrifuge-tubesuntil analysis. 9.2 If analysis will be delayed, extracted standards and samples can be refiigerated at approximately 4" C, or at room temperature, until analysis can be performed. 10.0 QUALITCYONTROL 10.1 Solvent Blanks,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 solvent blank, method bid, 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 duplicateper 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 concentrationsmay 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 l/x, not forced through zero, with IS reference (surrogate is used as an internal standard) using MassLynx or other suitable software. 3M Environmental Laboratory ETS-8-97.0 Analysis of Urine Extract Using ESMS Page 4 of 10 Page 182 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 Compound PFOS POAA Weighting I Il/X 1/X Regression Fit II Linear Quadratic Response type II IS reference IS reference 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 two digits of yearincreasing letter of the alphabet starting with a 12.1.1.2 Assign a method ( M S 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 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 sequencebegins 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. 3M Environmental Laboratory ETS-8-97.0 Analysis of Urine Extract Using ESMS Page 5 of 10 Page 183 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 12.2.2 Set-up the HP 11OO/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 InjecVsample = 1 12.2.2.3 Cycle time = 9.0 minutes 12.2.2.4 Solvent ramp = Time Ammonium acetate 1.O rnin. 40% 60% I 4.5 Gin. I 95% I 5% 1 I I I I6.5min. 1 95% I 5% I I7.0min. I 40% I 60% I I 9.0min. I 40% I 60% I 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 I1 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. Ifthe 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 corning 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 litershour 3M Environmental Laboratory ETS-8-97.0 Analysis of Urine Extract Using ES/MS Page 6 of 10 Page 184 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 12.3.7.2 ESI nebulizing gas 10-15 litershour 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" 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 DATAANALYSIS AND CALCULATIONS 13.1 Calculations: 13.1.4 Calculate matrix spike percent recovehes 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 Conc. - Calculated Conc. x 100 Expected Conc. 13.1.6 Calculate actual concentrationof PFOS, or other fluorochemical, in matrix (pg/mL): InplmL of PFOS calc. from std. Curve x Dilution Factor) x Initial Volume of matrix h L ) Final Volume (d) 1 un 1000 ng 14.0 METHODPERFORMANCE 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 r' 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 k 30% of the spiked concentration. 3M Environmental Laboratory ETS-8-97.0 Analysis of Urine Extract Using E S M S Page 7 of 10 Page 185 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 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. I 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 a linear or quadratic fit, referenced to the internal standard (surrogate), weighted l/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 TABLESD, IAGRAMFSL, OWCHARTASN,D VALIDATIONDATA 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 IonizationMass Spectrometer Quattro I1 triple quadrupole Systems'' 18.2 The validation report associated with this method is FACT-TOX-131, W2067 3M Environmental Laboratory ETS-8-97.0 Analysis of Urine Extract Using ES/MS Page 8 of 10 Page 186 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 19.0 AFFECTEDDOCUMENTS 19.1 ETS-8-96.0, "Extraction of Potassium Perfluorooctanesulfonateor Other Fluorochemical Compounds from Urine for Analysis Using HPLC-Electrospray/'MassSpectrometry" 20.0 REVISIONS Revision Number. L Reason For Revision - Revision 3M Environmental Laboratory ETS-8-97.0 Analysis of Urine Extract Using ESMS Page 9 of 10 Page 187 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 Laboratory Study # Study: Test Material: Matrix/Final Solvent: MethodRevision: - Analytical Equipment System Number: Instrument SoftwareNersion: Filename: R-Squared Value: Slope: Y Intercept: Date of ExtractiodAnalyst Date of Analysis/Analyst: 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 Conc. (ug/mL): Calculated by dividing the initial volume from the concentration Attachment A: Summary Spreadsheet ETS-8-97.0 Analysis of Urine Extract Using ESMS 3M Environmental Laboratory Page 10 of 10 Page 188 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 D: Method Validation Summary (TOX134) Table 5. TOX134 Method Validation Results Summary I Parameters I Linear Range: I LOQ: ~~ ~~~ I Results ~ ~~~ ~ I - 5-1500 vg/ml(based on method, some run-to-run variability can be expected) I - 10 pg/mL (based on method, some run-to-run variability can be expected) ~ Precision: In ter-assay 64% Infra-assay <21% System Precision <3% Accuracy (Monkey sera): 88-94% Reproducibility: <8.5'/0 3M Environmental Laboratory 3M Environmental Laboratory Page 22 Page 189 3M Medical Department Study: T-6889.3 3M Medical Department Study: T-6889.3 Appendix E: Data Summary Tables Analytical Report: FACT-TOX-026 LRN-U2782 Analytical Report: FACT TOX-026 LRN-U2782 FACT-M-4.1 ETS-8-5.1 ETS-8-5.1 ETS-8-97.0 FACT-M-2.1 1011 1/98 4/26/99 4/26/99 7/28/99 6/03/99 0.0137 0.0137 0.01 37 11/16/98to 1/27/99(Day 9, Wk4, Wk 12, Wk 14) 4130199 to 6/23/99 (Wk6-Wk IO,Wk I 6 W k 3 4 ) 7/14/99 to 7/21/99 (Wk 36-40) 0.0182 8119/99 to 9/28/99 (Wk 2-W k 40) 0.0755 6/23/99 to 10/5/99 (Wk20, Wk27, Wk 40) Table 7. Recovery Summaries of Fortified Samples in FACT TOX-026 I Serum __ ~ _ _ ~ Sample Name ~~ I I MKS05149-MS-3 84 I I MKS05149-MSD-3 91 I I 105709M-MS 79 I I 105714M-MS 108 MKSO6119-MS-1 106 MKSO6119-MSD-1 __ __ ~~ MKS07139-MS-1 MKS07139-MSD-1 104 I 92 84 Average 94 Standard Deviation 11 3M Environmental Laboratory 3M Environmental Laboratory Page 23 Page 190 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 Table 8. Recovery Summaries of Fortified Samples in FACT TOX-026 Urine I Sample Name MKU08049 MS-1 133 MKU08049 MSD-1 92 I I MKU08049MS-2 95 I 1 MKU08049MSD-2 70 I I MKU08059 MS 91 I 1 MKU08059MSD 87 I MKU08179MS 1-1 I 92 MKU08179 MSD 1-1 MKU08179 MS 1-2 MKU08179 MSD 1-2 I MKU08179 MS 1-3 1 74 MKU08179 MSD 1-3 74 MKU08179 MS 1-4 73 I 1- MKU08179 MSDI-4 73 I I MKU08209MS 1-1 66 I I MKU08209 MSD 1-1 63 MKU08209 MS 1-2 MKU08209 MSD 1-2 MKU09219 MS-I I MKU09219MSD-1 1 95 ~ ~~~ ~~ Average 88 Standard Deviation 17 3M Environmental Laboratory 3M Environmental Laboratory Page 24 Page 191 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 Table 9. Recovery Summaries of Fortified Samples in FACT TOX-026 I Liver I I SampleName I Rezvery I I I I 105709M-MS-3 74 I I I 105709-MSD-3 62 I I I 105718M-MS-11 113 I 105718M-MSD-12 I 1 1 1 I I I I Average 90 Standard Deviation 26 3M Environmental Laboratory 3M Environmental Laboratory Page 25 Page 192 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 Day 9 Week 4 Week 6 Week 8 Week 10 Week 12 Week 14 0.0613 * 0.0472 (n=6) 0.206 f 0.105 (n=6) 0.103~0.0113 (n=6) <LOQ (n=6) 0.126 f 0.0348 (n=6) 0.123 rt 0.0507 (n=6) 0.162 f 0.0643 (n=6) 126 f 36.1 (n=5) 98.4 * 42.P (n=3) 102 f 33.6 (n=4) 94.7 f 27.3 (n=4) * 105 37.7 (n=4) 79.6 * 25.9 (n=4) 90.0 f 28.9 (n=4) 189 * 48.9 (n=Ci) 172f.71.9 (n=Ei) 95.8 f 20.6 (n=t3) 97.6 f 23.5 (n=6) 93.6 * 13.7 (n=6) 90.6 f 24.5 (n=6) 92.2 f 27.6 (n=6) 1597 f 2392 (n=6) 1084 f 1839 (n=6) 145 f 21.6 (n=5) 166 f 98.9 (n=5) 237 f 158 (n=5) 140 2 77.5 (n=5) 79.4 f 28.3 (n=5) 3M Environmental Laboratory 3M Environmental Laboratory Page 26 Page 193 3M Medical Department Study: T-6889.3 3M Medical Department Study: T-6889.3 Week 38 Week 40 Group 1 * 0.0 mglkglday Average SD 0.0941 f 0.0324 (n=2) 0.0738f 0.00256 (n=2) Group 2 * 3 mgn<glday Average SD NS NS Analytical Report: FACT-TOX-026 LRN-U2782 Analytical Report: FACT TOX-026 LRN-U2782 Group 3 10 mglkglday Average f SD 1.84f 1.40 (n=2) 1 .I 8f 0.827 (n=2) Group 4 30 mglkglday Average t SD NS NS 3M Environmental Laboratory 3M Environmental Laboratory Page 27 Page 194 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 Group 1 * 0.0 mglkglday Average SD Week 2 <LOQ (n=6) Week 4 Week 6 Week 8 0.152 f 0.337 (n=6) 0.0587 f 0.071 6 (n=6) I 0.0161 f 0.00940 (n=6) Week 10 0.0177 f 0.01 14 (n=6) 1 I Week 12 Week14 0.0141 * 0.00648 (n=6) 7.96 f 19.5 (n=6) Week 16 0.0299 f 0.0339 (n=6) Week 18 I Week20 1 II Week22 0.0256 f 0.0248 (n=6) 0.021 1 f 0.0130 (n=6) 0.0231 0.00688 (n=6) Week 24 I Week26 1 II Week28 0.0125 f 0.00749 (n=6) 0.0268 f 0.0265 (n=6) 0.118*0.142 (n=2) Week 30 <LOQ (n=2) Week 32 <LOQ (n=2) I I Week 34 Week36 <LOQ (n=2) 0.01 17 f 0.00841 (n=2) Week 38 <LOQ (n=2) Group 2 3 mglkglday Average t SD 73.5 f 38.1 54.9 f 4.62 (n=4) 65.7 f 46.9 (n=4) 47.6 f 20.6 (n=4) 39.9 f 18.7 (n=4 48.4 f 18.8 (n=4 63.1 f 47.2 (n=4) 50.2 f 21.O (n=4) 37.7 f 19.3 (n=4) 52.1 rt9.63 (n=4) 95.8 f 80.8 (n=3) 46.3 f 8.52 (n=3) 51.6 f 13.7 (n=3) NS NS NS NS NS NS Group 3 * 10 mglkglday Average SD 221 f 124 (n=Ei) 190 f 91.6 (n=E;) I 128 f 50.0 (n=6) 206 f 73.1 175 f 92.3 201 f 92.7 (n=6) I 139 f 52.2 (n=6) 139 f 57.0 (n=6) 186 f 63.9 (n=6) 144 f 135 I (n=6) 158 f 78.4 I (n=6) 157 f 63.3 (n=6) 109 f 75.2 I (n=6) 1 (n=2:) 0.361 f 0.118 (n=2) 0.1 14 f 0.0608 (n=2:) 0.121 f 0.0305 (n=2:) I 0.0502 f 0.0166 (n=2) 0.0284 f 0.0102 (n=2) Group 4 30 mglkglday Average SD 909 f 269 (n=6) 240 f 161 (n=6) 272 rt 140 (n=5) 180 f 109 118f111 (n=5) 72.9 f 84.0 (n=5) 50.2 f 67.9 (n=5) 43.1 f84.6 (n=5) 44.0 f 59.9 (n=5) 98.5 f 134 (n=5) 56.0 f 77.2 (n=s) 19.2 f 27.0 (n=5) NS NS NS NS NS NS 3M Environmental Laboratory 3M Environmental Laboratory Page 28 Page 195 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 Group 1 0.0 mgkglday Average t SD Group 2 3 mglkglday Average t SD Group 3 10 mglkglday Average t SD Group 4 30 mglkglday Average t SD cLOQ (n=2) NS 0.025(4nf=20).0101 NS I I I I I <LOQ--Bdour limit of quantitation NS-No sample NOTE Results are eqxessed as grouwgenderaverages *the standarddeviationassociatedwith that grouwgender. NOTE: It is not possible to verify true recoveryof endogenous analvne from tissues without radio-labeled reference material. The only measurement of accuracyawilable 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 3M Environmental Laboratory Page 29 Page 196 3M Medical Department Study: T-6889.3 3M Medical Department Study: T-6889.3 Week 20 Week 27 Week 40 Group 1 * 0.0 mgkglday Average SD NS 0.1 17 rt: 0.0730 (n=4) cLOQ (n=2) Group 2 * 3 mgkglday Average SD 18.3 (n=1) 15.3 f 3.02 (n=4) NS Analytical Report: FACT-TOX-026 LRN-U2782 Analytical Report: FACT TOX-026 LRN-U2782 Group 3 * 10 mgkglday Average SD NS 14.0 f '7.55 (n=4 0.1 14 f 0.0441 (n=2) Group 4 * 30 mgkglday Average SD NS 42.8 f 63.3 (n=6) NS Week 2 cLOQ (n=6) 7.43 f 6.54 (n=4) 15.4 f '10.2 (n=6) 56.6 f 73.7 (n=6) Week 4 I * Week I II I 1 Week8 0.0214 f 0.0178 (n=6) 0.0108 f 0.001 92 (n=6) 0.0782 f 0.103 (n=6) 10.4 f 12.0 (n=4) 12.1 14.1 (n=4) 9.46 f 9.21 (n=4) 23.4 f 'I 0.6 (n=6) 23.3 + 8.46 (n=6) 41.Of 25.0 (n=6) 22.0 f 6.23 (n=6) 36.7 f 34.2 (n=5) 1 I Week 10 Week12 cLOQ (n=6) 0.0498 f 0.0894 (n=6) 3.96 f 3.68 (n=4 7.1 5 f 5.65 (n=4 26.0 f 17.4 48.0 f 34.0 (n=6) (n=5) 10.3 f 6.07 (n=6) 1 32.0 f 42.9 (n=5) Week 14 0.139 f 0.308 (n=6) 7.50 f 2.43 (n=4) 27.2 f ;!9.4 19.9 f 25.2 * Week 16 I I I Week18 0.0572 f 0.0762 (n=6) 0.258 f 0.604 (n=6) 6.88 2.62 (n=4) 5.72 f 7.15 (n=4 31.4 f 23.3 17.3 f 13.3 (n=6') 18.2 f 28.8 22.1 f 31.7 (n=5) ~ Week 20 0.450 f 1.08 (n=6) 6.81 f 4.89 (n=4) 52.4 f 39.5 (n=6) 37.8 f 58.1 (n=5) I 3M Environmental Laboratory 3M Environmental Laboratory Page 30 Page 197 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 Group 1 * 0.0 mgkglday Average SD Week 22 15.5 f 36.9 (n=6) Week 24 I Week26 1 I Week2&34 I II Week3640 0.517i 1.13 (n=6) 0.0172 i 0.00892 (n=6) 0.279 f 0.732 (n=8) 0.0103 i 0.00684 (n=6) Group 2 3 msncglday Average t SD 13.8 f 5.22 (n=3) 6.22 i 5.45 (n=3) 2.92 f 1.35 (n=3) NS NS Group 3 * 10 m@g/day Average SD 39.5 i 21.o (n=6) 40.5 i 21.8 (n=6) 43.0 i 36.9 I (n=6) I I 0.0336 i 0.0313 (n=6) Group 4 * 30 mglkglday Average SD 25.2 i 36.0 (n=5) 34.6 f 47.7 (n=5) 10.3 f 20.8 (n=5) NS NS N W osample <LOGtBdow limit of quantitation NOTE: Results are expressed as grouwgender averages f the standard deviation associated with that grouwgender. NOTE: It is not possibleto verify true recoveryof endogenousanalyte from tissues without radio-labeled reference material. The only measurementof 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 recovetywas 117% with a standard deviation of 22%. 3M Environmental Laboratory 3M Environmental Laboratory Page 31 Page 198 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 Table 14. FACT TOX-026 IndividualP O M Results per Sample-Serum (pg/mL) AnimallDI Day9 I Week4 I Week6 I Week8 Week10 I Week12 ~ 0.191 0.207 75.9 64.0 114 85.3 74.8 55.5 154 114 97.4 94.0 72.8 56.9 87.9 79.0 103 131 102 84.4 108 98.4 313 235 128 103 67.4 32.2 208 185 467 143 Week 14 ~ 0.275 0.156 0.0981 0.153 0.1 82 0.1 05 72.7 112 58.4 117 68.0 103 61.3 79.2 134 108 84.4 109 68.2 36.9 98.8 Week16 0.275 <LOQ <LOQ <LOQ <LOQ Week18 0.263 0.244 0.0950 0.165 0.193 Week201 Week22 0.342 0.393 0.212 0.157 0.115 0.106 0.224 0.269 0.242 0.372 Week24 E E <LOQ <LOQ <LOQ 72.6 16.0 11.4 43.4 66.8 33.3 57.5 60.2 103 23.8 69.6 I 99.5 13.4 107 122 89.1 60.2 9.67 61.9 63.7 61.4 73.2 46.4 75.2 87.7 81.8 1 180 13.7 137 168 116 91.3 10.1 91.4 106 82.3 86.5 11.3 107 85.2 115 68.2 68.8 38.9 16.4 20.4 113 20.0 92.5 92.7 90.4 13.3 19.0 2.39 4.29 I 1 167 27.3 322 96.3 181 I 57.7 I I 45.9 I 1 32.9 I I 86.0 1 1 1 17.0 -No extract remaining *Sample evaporated to &ness, tentative value Shaded areas-No sample aAnimal105706M, Group 2, Week 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%. 3M Environmental Laboratory 3M Environmental Laboratory Page 32 Page 199 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 Table 14 (continued). FACTTOX-026 Individual POAA Results per Sample-Serum (VglmL) Group 1 0.0 mg/kg/day Control -- Animal ID Week26 105709M 0.471 105714M 105715M 105718M 0.108 1 0.101 I 0.206 105720M 0.308 2W6&ee2k7 0.377 0.219 0.0945 0.206 0.308 Week 28 Week 30 Week 32 Week 34 I I I I II 0.153 0.209 I 0.127 I 1 0.161 0.0943 0.0680 0.125 0.104 Week361 0.0795 0.142 Week38 0.071 1 0.117 1 Week40 0.071 9 0.0756 Group 2 3.0 mg/kg/day Low Dose 105721M replacement Group 3 10 mg/kg/day Mid-Dose 4.08 2.83 1.77 1.42 0.842 0.600 Group 4 30 mg/kg/day High Dose 105713M 299 184 1 I 105722M 15.8 6.65 105724M 489 U laded areas-No samDle NOTE: L is not possible tdverify 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 recoverywas 94% with a standard deviation of 11%. 3M Environmental Laboratory 3M Environmental Laboratory Page 33 Page 200 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 Table 15. FACT TOX-026 Individual POAA Results per Sample-Urine Animal ID1 Week2 I Week4 I Week6 I Week8 Group 1 3.0mgkg/day Control (pg/mL) Group 2 3.0 m@g/day Mid Dose Group 3 10 mgkglday Aid-high Dose 105719M 288 242 94.3 220 Group 4 105703M 525 383 354 167 30 mg/kg/day 105704M 811 47.6 193 282 High Dose 105711M 941 186 427 3.62 105713M 846 444 72.3 257 I I 105722M 983 295 105724M 1350 83.0 I 314 193 -0Q-Below lin of quantitation Shaded area-No sample *Thissamplewas not diluted 1/5000;a 2pL injectionof DlOOO was analyzed instead.All other injectionswere 1OpL. This provides a 1/51dilutionof DlOOO for a totalof 05000.A 1/4000 dilution of 105707M was also analyzed, but was just out of calibration range. NOTE: It is not possible to verily true recoveryof 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%. Week 26 0.0208 <LOO cLOQ <LOQ 0.0797 <iOQ 37.0 53.6 64.2 256 66.6 50.8 72.3 103 107 0.223 57.7 0.0682 37.8 0.21 5 3M Environmental Laboratory 3M Environmental Laboratory Page 34 Page 201 3M Medical Department Study: T-6889.3 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) Analytical Report: FACT-TOX-026 LRN-U2782 <LOQ-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 dewation of 17% 3M Environmental Laboratory 3M Environmental Laboratory Page 35 Page 202 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 animal. 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 !he liver data are accurate to within one standard deviation of the average fortified sample recovery. The average fortifiedsample recoverywas 90% with a standard deviation of 26%. 3M Environmental Laboratory 3M Environmental Laboratory Page 36 Page 203 3M Medical Department Study: T-6889.3 3M Medical Department Study: T-6889.3 Appendix F: Data Spreadsheets Analytical Report: FACT-TOX-026 LRN-U2782 Analytical Report: FACT TOX-026 LRN-U2782 3M Environmental Laboratory 3M Environmental Laboratory Page 37 Page 204 3M Medical Department Study: T-6889.3 FACT-TOX-026 Covance# 6329-231 Study: Product NumbeflTest Substance): Matrix: MethodRevision: Analytical Equipment System Number: 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 Filenames Blks Grp 1 Grp 2 Grp 3 Grp 4 MS, MSD Analytical Report: FACT-TOX-026 LRN-U2782 POAA A111698003,4&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 111 Day 9 MONKEY SERA Group Dose Sample ## Method Blk Matrix Blk QC - 500 ppb H 2 0 Blk- 1 H 2 0 Blk-2 Rabbit Sera Blk-l Rabbit Sera Blk-2 RBSll138-MS RBSll138-MSD Group 1 Conhol 0.0 mgkgiday Group 2 Low Dose 3.0 mgkglday Group 3 Mid-High Dose 10 mgkgiday I05709M I05714M I05715M 105718M I05720M I05725M I05702M I05706M I05717M I05721M I05723M 105707M I05708M I05710M I05712M I05716M I05719M Group 4 High Dose . 30 mgkgiday 105703M I05704M 105711M I05713M I05722M I05724M Limit of Quantitation (LOQ): 0.0137 ug/mL Extraction Vol. Ratio NA NA 1 .oo 1.oo 1.oo 1.OO 0.50 0.50 0.60 0.50 0.30 0.50 0.70 0.50 0.60 0.30 0.50 0.60 0.50 0.60 0.50 0.50 0.40 0.50 0.40 0.60 0.30 0.40 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 POAA Conc. ng/mL <LOQ <LOQ <LOQ 14.4 530 489 Concentration of POAA ug/rnL or % Rec. <LOQ <LOQ <LOQ 0.0138 109% ** 100% ** 1 33.7 1 71.4 1 4.68 1 46.4 1 8.23 1 26.0 750 89.2 750 123 750 96.8 NR NR 75 497 1000 120 1000 64.5 1000 90.1 1000 112 1000 119 1000 115 0.0645 0.137 cLOQ 0.0889 <LOQ 0.0499 91.6 177 116 NR 119 230 103 173 178 228 220 5000 2000 2000 2000 1 529 81.8 . 350 71.8 47257 6338 313 1676 229 151 * 2000 183 876 *Tentative value, sample evaporated to dryness. POAA = Pefluorooctanoate Date EnterediBy: Date Verified By: 11/21/98, 12/07/98, 10/10/00 LAC 06/21/99 EAD, lO/ll/OO KJH, 11/09/00 HOJ Mean POAA ug/d <LOQ <LOQ 104% 0.0613 126 189 1591 FACT-M-4.1 3M EEnxvceilr9o7 nmental Laboratory Sera Day9 TOX-026-sera23 1-8C.xls RSD Std. Dev. MS/MSD RPD NA NA 8% 77.1 0.0472 28.7 36.1 25 9 48.9 150 2392 5/23/200P1age 205 3M Medical Department Study: T-6889.3 FACT-TOX-026 Covance# 6329-231 6 Month Capsule Toxicity Study with APFO in Cynomolgus Monkeys T-6889.2 (POAA) Monkey Sera FACT-MJ. 1 & FACT-M4.1 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, 11130198, 12/07/98,09/29/00 HOJKJH Group Dose Sample # Method Blk H20 Blk-l H20 Blk-2 Matrix Blk Rabbit Sera Blk-1 QC - 500 p.p.b Rabbit Sera Blk-2 RBS 1 1 138-MS RBSl1138-MSD Group 1 Control I I05709M I05714M 0.0 mg/kg/day I05715M I05718M I05720M I05725M Group 2 I05702M Low Dose I05706M I05717M I05721M I05723M Group 3 I05707M Mid-High Dose I05708M 10 mgikglday I05710M I05712M I05716M I05719M Group 4 I05703M High Dose I05704M 30 mgikglday . I05711M I05713M I05722M I05724M imit of Quantitation (LOQ): 0. 137 ug/mL Correction factors not applicable for MSiMSD QC data Concentration of POAA u d m L or % Rec. <LOQ 0.0138 109% 100% 0.0645 0.137 <LOQ 0.0889 <LOQ 0.0499 91.6 !230 173 178 228 220 6338 313 1676 229 151 876 Mean POAA udmL <LOQ <LOQ ** ** 104% RSD Std. Dev. MSlMSD RPD NA NA 8% I 0.0613 126 77.1 0.0472 28.7 36.1 25.9 189 48.9 * 1597 **Bracketed by a CCV not 150 2392 xated to dryness. hin method criteria (31%) POAA = Perfluorooctanoate Date EnterediBy: 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 FACT-M-4.1 3M EExncveli9r7onmental Laboratory Sera Day3 TOX-026-sera231-8C.xls Analytical Report: FACT-TOX-026 LRN-U2782 5/23/20P01age 206 3M Medical Department Study: T-6889.3 FACT-TOX-026 Covance# 6329-231 Study: Product Nwnber(Test Substance): Matrix: MethodRevision: Analytical Equipment System Number: Instrument SoftwareNersion: Filename: R-Squared Value: Slope: Y-Intercept: Date of Extraction/Analyst: Date of AnalysidAnalyst: 6 Month Capsule Toxicity Study with APFO in CynomolgusMonkeys 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 Date of Data ReductiodAnalyst: Sample Data 11/17/98, 11/30/98, 12/07/98, 01/07/99, 09/29/00 HOJ/KJH Week 4 MONKEY SERA Sample # Factor Dilution Conc. of POAA ug/mL or % Ref. H 2 0 Blk-l 0.9582 H20 Blk-2 0.9582 Rabbit Sera Blk-1 0.9582 FGroup 1 Control 0.0 mgkg/day Group 2 Low Dose Rabbit Sera Blk-2 0.9582 RBSll138-MS RBSl1138-MSD 1 .oo NA I05709M I 0.50 I 0.9582 I05702M I05706M 0.60 0.9582 0.40 0.9582 3.0 mglkglday 105717M 0.50 0.9582 tMid-High Dose IO mglkgiday Group 4 High Dose I05721M I05723M I05710M I05712M I05716M I05719M I05703M IOS704M 0.40 0.9582 0.9582 0.30 0.9582 0.40 0.9582 0.60 0.9582 0.50 0.9582 0.40 0.9582 0.40 0.9582 30 mglkglday 10571IM 0.30 0.9582 I05713M 0.30 0.9582 I05722M 0.50 0.9582 I Limit of Quantitation (LOQ): 0 I05724M 0.50 0.9582 1 1 489 156 I 120 49.1 109 52.2 1 59.5 750 I 80.8 I 1875 804 I 1 750 98.7 250 23576 NR - 150 150 150 E - 150 150 200 200 5000 301 60 20893 200 2000 214 0.0138 109% 100% 0.299 0.230 0.0785 0.347 0.167 0.114 96.8 361 1 142 56.5 .4802 66.7 822 Correction factors not applicable for MSMSD QC data **Bracketedby a CCV not within method criteria (31%) POAA = Perfluorooctanoate Date EnteredBy: 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, l O / l l / O O KJH, 11/09/00 OJ Analytical Report: FACT-TOX-026 LRN-U2782 Filenames Blks Grp 1 Grp 2 Grp 3 Grp 4 Grp 4 MS, MSD POAA AI 11698003,4 & 79, 80 1 11698045-50 12039866-68,01049902 1 112498041-46 112498049-50,52 12039881,83010499020 1 11698075-76 Dilutions Ill l/l 1/250, 750, 1875 1/150 11200 1/60, 2000, SO00 111 <LOQ <LOQ I I *I 104% Std. Dev. MSIMSD RPD NA NA 0.206 0.105 * 98.4 43.4* 43.4 98.4; 42.7* 42.7 180 977 1757 41.7 172 71.9 1084 I 170 1839 FACT-M-4.1 3M EEnxcveil r97onmental Laboratory Sera Week4 TOX-026-sera23 1-8C.xls 5/23/20P01age 207 3M Medical Department Study: T-6889.3 FACT-TOX-026 Covance# 6329-231 Analytical Report: FACT-TOX-026 LRN-U2782 Week 4 MONKEY SERA Group Dose Sample # Method Blk H20 Blk-l Matrix Blk H 2 0 Blk-2 Rabbit Sera Blk-1 QC - 500 ppb Rabbit Sera Blk-2 RBS11138-MS Group 1 RBSll138-MSD 105709M Control I057 14M 0.0 mgikglday I05715M 105718M 105720M I05725M Group 2 105702M Low Dose I05706M I05717M I05721M Group 3 Mid-High Dose I05723M 105707M I05708M IO mgikgiday I05710M I05712M I05716M I05719M Group 4 105703M High Dose 105704M 30 mglkgiday I05711M I05713M I05722M 105724M Limit of Quantitation (LOQ): 0.0137 ug/mL Concentration Mean of POAA POAA u g h L or % Rec. ug/mL <LOQ <LOQ <LOQ <LOQ 0.0138 109% 100% 0.299 <LOQ ** ** 104% 0.230 0.0785 0.347 0.167 0.114 0.206 96.8 361 1 * 142 98.4* 56.5 NR 977 197 84.6 268 E 1 I9 194 172 286 251 4802 66.7 273 822 1084 *Anomaly, was not included in these values. RSD Std. Dev. MSlMSD RPD NA NA 8% 51.1 0.105 43.4* 42.7* 180 1757 41.7 11.9 170 1839 FACT-M-4.1 3M EEnxvceilr9o1 nmental Laboratory Sera Week4 TOX-026-sera231-8C.xIs 5/23/20P01age 208 3M Medical Department Study: T-6889.3 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 Sera MethodRevision: ETS-8-4.1 and ETS-8-5.1 Analytical Equipment System Number: Amelia 062498, Soup 020199 Instrument Sofhvare/Version: MassLynx 3.2 Filename: See Attachments R-Squared Value: See Attachments Slope: See Attachments Y-Intercept: See Attachments Date of ExtractiodAnalyst: 05/12/99 SAH Date of Analysis/Analyst: 06/03/99, 06/07/99, 06110199, 06116/99 HOJISAWMEWLAC Date of Data ReductiodAnalyst: 06/04/99, 06/08/99, 06111/99, 06117199, 11/06/00 HOJILACKJH Sample Data Week 6 MONKEY SERA I Group I Dose Sample # Extraction Vol. Ratio POAA Std Correction POAA Dilution POAA Cone. Concentration of POAA Method Blk Matrix Blk QC - 250 ppb H20 Blk-3 H20 Blk-4 Factor Factor ng/mL u g / d or O h Rec. - 1.oo 0.9582 1 0.00 <LOQ (0.0983 ug/mL) 1 .oo 0.9582 0.00 <LOQ (0.0983 ug/mL) I .oo 0.9582 0.00 <LOQ (0.0983 ug1mL) 1.oo 0.9582 1.00 - 0.00 <LOQ (0.0983 ug1mL) 199 80% RBS05129-MSD-3 1.00 1.00 RBSOS 129-MSD-4 1.00 199 79% 229 92% 214 85% Group 1 Control I05714M 0.54 0.9582 0.74 0.9582 56.3 <LOQ (0.0983 ug/mL) 100 0.126 0.0 mgkgiday I05715M 0.70 0.9582 22.5 <LOQ (0.0983 ug/mL) I05718M 0.55 0.9582 33.0 <LOQ (0.0983 ug/mL) Group 2 Low Dose 105720M I05725M I I05702M I05706M - 0.56 0.9582 3.15 <LOQ (0.0983 ug/mL) 0.67 0.9582 21.0 <LOQ (0.0983 ug/mL) 0.70 0.9582 100 493 67.5 0.55 0.9582 100 3.0 nigkglday 105717M 0.56 0.9582 100 I05721M 0.65 0.9582 1000 I05723M NR 0.9582 1 0.72 0.9582 1000 Mid-High Dose I05708M 0.55 0.9582 100 IO mgkgiday I05710M 0.53 0.9582 100 I05712M 0.82 0.9582 1000 I057 16M 0.67 0.9582 100 I05719M 0.40 0.9582 100 Group 4 I I05703M 0.49 0.9582 200 High Dose I05704M 0.39 0.9582 100 450 111 30 mglkgiday 10571 1M 0.61 0.9582 1000 102 161 I05713M I05722M Limit of Quantitation (LOQ): 0.0137 ug/mL - - 0.59 0.9582 1000 95.1 154 0.49 0.9582 1000 70.0 137 Correction factors not applicable for MSMSD QC data NR = Sample not received nor reported. POAA = Perfluorooctanoate Date Enteremy: 06/08/99, 06114199, 06117199, 11/06/00 LAC Date Verified/ By: 06/21/99 EAD, 11/09/00 HOJ Blks Grp 1 Grp 2 Grp 3 Grp 4 MS, MSD Mean POAA ug/d <LoQ <LOQ 80% I 89% 0.103 145 ETS-8-5.1 3M EEnxcveli9r7onmental Laboratory Sera Week6 TOX-026-sera23 1-8C.xls Analytical Report: FACT-TOX-026 LRN-U2782 Filenames POAA A061099031,32 & 99, 100 061099043-50 060799015-1 8,64 060799022-23,27-28,67-68 060799031-32,71-73,06l6990115 061099051-54 Dilutions 1/1 l/l 11100&1000 1/1OO& 1000 1/100,200,&1000 1/1 RSD Std. Dev. MSlMSD RPD NA NA 0% I 7% 11.0 0.0113 14.9 21.6 5/23/20P01age 209 3M Medical Department Study: T-6889.3 FACT-TOX-026 Covance# 6329-231 Study: Product Number(Test Substance): Matrix: MethodRevision: Analytical Equipment System Number: Instrument SofhvareNersion: Filename: R-Squared Value: Slope: Y-Intercept: Date of ExhactiodAnalyst: Date of AnalysidAnalyst: Date of Data ReductiodAnalyst: Sample 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, Soup 020199 MassLynx 3.2 See Attachments See Attachments See Attachments See Attachments 05/12/99 SAH 06/03/99, 06/07/99, 06110199, 06116/99 HOJ/SAHIMEE/LAC 06/04/99, 06/08/99, 06/11/99, 06/17/99, 11/06/00 HOJLACKJH Group Dose Method Blk Matrix Blk QC - 250 ppb Group 1 Control 0.0 mgkglday Group 2 Low Dose Mid-High Dose I O mgkglday Group 4 High Dose 30 mg/kg/day Sample # H20 Blk-3 H 2 0 Blk-4 Rabbit Sera Blk-3 Rabbit Sera Blk-4 RBSOS 129-MS-3 RBS05129-MSD-3 RBS05129-MS-4 RBSOS 129-MSD-4 105709M I057 14M I05715M 105718M 105720M 105725M 1 1 I05702M I05706M I05717M I05721M I05723M Concentration of POAA u%mL or % Ree. <LOQ (0.0983 ug/mL) <LOQ (0.0983 ug/mL) <LOQ (0.0983 ug/mL) CLOQ (0.0983 ug/mL) 80% 79% 92% 85% <LOQ (0.0983 ug/mL) 0.126 <LOQ (0.0983 ug/mL) CLOQ (0.0983 ug/mL) <LOQ (0.0983 uglmL) <LOQ (0.0983 ug/mL) 67.5 115 83.7 143 NR I05708M 78.8 105710M 103 I057 12M 87.9 I05716M 86.2 I05719M 135 I05703M 162 105704M 111 105711M 161 105713M 154 I05722M 137 Mean POAA u%d <LOQ <LOQ 80% 89% RSD Std. Dev. MWMSD RPD NA NA 0% 7% 0.103 11.0 0.01 13 1 32.8 102 33.6 21.5 95.8 20.6 14.9 145 21.6 ETS-8-5.1 3M EEnxvceilr9o7 nmental Laboratory Sera Week6 TOX-026-sera23 1-8C.xls Analytical Report: FACT-TOX-026 LRN-U2782 5/23/200P1age 210 3M Medical Department Study: T-6889.3 FACT-TOX-026 Covance# 6329-231 Analytical Report: FACT-TOX-026 LRN-U2782 Study: Product Number(Test Substance): Matrix: MethodlRevision: Analytical Equipment System Number: Instrument SofhvareNersion: Filename: R-Squared Value: Slope: Y-Intercept: Date of ExhactiodAnalyst: Date of AnalysidAnalyst: Date of Data ReductiodAnalyst: Sample Data Week 8 MONKEY SERA Group Sample # Method Blk H20 Blk-3 H20 BIk-4 Matrix Blk II QC - 250 ppb Rabbit Sera Blk-3 I RabbitSeraBlk-4 I RBSO5129-MS-3 RBS05129-MSD-3 RBS05129-MS-4 RBS05129-MSD-4 Group 1 I05709M Control 0.0 mg/kg/day 1057 14M I05715M 105718M I05720M 105725M Group 2 Low Dose I05702M I05706M 3.0 mgkgiday I057 17M I05721M I05723M tiroup 3 105707M Mid-High Dose I05708M 10 mgkglday 105710M I05712M 105716M I05719M Group 4 I05703M High Dose I05704M 30 mg/kg/day 10571IM I05713M I05722M Limit of Quantitahon (LOQ): 0.0137 ug/mL 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, 06111/99, 11/06/00 HOJ/KJH Blks Grp 1 Grp 2 Grp 3 Grp 4 MS, MSD Filenames POAA A061099031,32 & 99, 100 061099057-62 060799038-41 060799044-46,SO-51,80 060799054-56,8445 061099051-54 Dilutions 111 1/1 1/100 1/100&1000 1/100&1000 111 Extraction Vol. Ratio I 1.oo I 1.oo 1 1.oo 1 1.oo 1 .oo 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 POAA Dilution Factor I 1 11 I 11 1 POAA Cone. ng/mL 0.00 0.00 0.00 0.00 Concentration of POAA ug/mL or O h Ree. I I <LOQ (0.0983 ug/mL) <LOQ(O.O983ug/mL) I <LOO (0.0983 udmL) I - , <LOQ (0.0983 ug/mL) I NA 1 1 1 199 79% NA I l l 229 I 92% NA 1 214 85% 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 1 1 1 1 100 100 100 100 1 100 100 100 1000 100 100 100 100 100 1000 1000 86.2 <LOQ (0.0983 ug/mL) 0.00 <LOQ (0.0983 ug/mL) 0.00 <LOQ (0.0983 ug/mL) 39.3 CLOQ (0.0983 ug/mL) 4.97 <LOQ (0.0983 ug/mL) 431 76.4 529 101 419 70.5 707 130 NR NR 588 97.2 446 67.8 379 88.5 62.7 82.3 597 102 567 133 659 175 513 117 684 102 61.8 100 213 334 Mean POAA ug/mL RSD Std. Dev. MSIMSD RPD I <LOQ NA 1 <LOQ NA 80% I 0% 1 1 89% 1 NA 97.6 I 24.1 23.5 59.6 166 98.9 NR = Sample not received nor reported. POAA = Perfluorooctanoate Date EnteredBy: Date Verified/ By: 06/08/99,06/14/99, 11/06/00 LAC 06/21/99 EAD, 11/09/00 HOJ ETS-8-5.1 3M EEnxvceilr9o7nmental Laboratory Sera Week8 TOX-026-sera231-8C.xls 5/23/20P01age 211 3M Medical Department Study: T-6889.3 FACT-TOX-026 Covance# 6329-231 Study: Product Number(Test Substance): Matrix: MethodiRevision: Analytical Equipment System Number: Instrument Sofhvare/Version: Filename: R-Squared Value: Slope: Y-Intercept: Date of ExtractioniAnalyst: Date of Analysis/Analyst: Date of Data ReductiodAnalyst: Sample Data W e e k 8 MONKEY SERA Matrix Blk QC - 250 ppb H20 BIk-4 I Rabbit Sera Blk-3 I Rabbit Sera Blk-4 I RBSO5129-MS-3 RBS05129-MSD-4 Group 1 I05709M Control I05714M 0.0 mgikgiday I05715M I05718M I05720M I05725M Group 2 I05702M Low Dose I05706M I05717h4 I05721M I05723M Group 3 I05707M Mid-High Dose I05708M 10 mgikg/day IO57 1OM 105712M IO571 6M IO57 19M Group 4 I05703M High Dose I05704M 30 mg/kg/day I05711M I05713M 105722M .imit of Quantitation (LOQ): 0.0137 ug/mL Correction factors not applicable for MS/MSD QC data 6 Month Capsule Toxicity Study with AF'FO 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, 06111/99, 11/06/00 HOJKJH Concentration of POAA ug/mL or % Rec. :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 ugh&) :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 Mean POAA ug/mL <LOQ cLOQ 80% 89% RSD Std. Dev. MS/MSD RPD NA NA 0% 7% NA <LOQ NA I 28.8 NR = Sample not received nor reported. POAA = Perfluorooctanoate Date EnteredBy: Date Venfiedi By: 06/08/99,06/14/99, 11/06/00 LAC 06/21/99 EAD, 11/09/00 HOJ ETS-8-5.1 3M EEnxcvelir97onmental Laboratory Sera Week8 TOX-026-sera23 I -8C.xls Analytical Report: FACT-TOX-026 LRN-U2782 5/23/20P01age 212 3M Medical Department Study: T-6889.3 FACT-TOX-026 Covance# 6329-231 Analytical Report: FACT-TOX-026 LRN-U2782 3M Study: Product Number(Test Substance): Matrix: MethodRevision: Analytical Equipment System Number: 6 Month Capsule Toxicity Study with APFO in Cynomolgus Monkeys T-6889.2 (POAA) Monkey Sera ETS-84.1 and ETS-8-5.1 Madeline 041098 Instrument Software/Version: MassLynx 3.2 Filename: R-Squared Value: See Attachments See Attachments Slope: Y-Intercept: Date of ExtractiodAnalyst: Date of Analysis/Analyst: Date of Data ReductiodAnalyst: Sample Data See Attachments See Attachments 05113199 SFWJCP 05/17/99, 05/19/99,06/10/99 MEEiKJWHOJ 05118/99, 05/20/99,06/1 1/99, 11/06/00 KJWMEWHOJ Week 10 MONKEY SERA Group Dose Method Blk Matrix Blk Matrix Blk Group 1 Control 0.0 mgikgiday Group 2 Low Dose 3.0 mglkgiday Group 3 Mid-High Dose 10 mglkglday Group 4 High Dose 30 mglkglday Sample # Extraction Vol. Ratio POAA Std Correction POAA Dilution H 2 0 Blk-7 H20 Blk-8 Rabbit Sera Blk-7 Rabbit Sera Blk-8 Monkev Sera Blk-7 Monkey Sera Blk-8 RBS05129-MSD-7 I 1.00 I 0.9582 I 1 1.00 0.9582 1 1.00 0.9582 1 1.00 0.9582 1 1 .oo 0.9582 1 1 .oo 0.9582 1 1.00 I NA 1 RBS05129-MSD-8 I05709M I057 14M I05715M I05718M 105720M I05725M I05702M I05706M 0.54 I05717M 0.42 105721M I05723M I05707M I05708M I05710M I05712M I05716M 0.66 I05719M I05703M 0.50 I05704M 0.73 I05711M 0.68 I05713M 0.55 I05722M 0.53 1 0.9582 1 0.9582 1 0.9582 1 0.9582 1 0.9582 1 0.9582 1 0.9582 100 0.9582 100 0.9582 100 0.9582 100 0.9582 NR 0.9582 100 0.9582 100 0.9582 100 0.9582 100 0.9582 100 0.9582 100 0.9582 250 0.9582 250 0.9582 250 0.9582 250 0.9582 500 POAA Cone. ng/mL 7.56 0.00 30.0 32.8 85.4 47.4 257 265 258 317 85.5 90.7 75.7 76.6 70.9 51.5 412 64 1 328 532 NR 559 585 560 740 703 708 654 392 191 477 517 Concentration of POAA ug/rnL or % Rec. <LOQ <LOQ * 0.0288 0.0315 * 0.0819 * 0.0454 * 103% * 106% * 103% * 127% * 0.191 0.105 0.0919 0.129 0.128 0.110 75.9 114 74.8 154 NR 97.4 72.8 87.9 103 102 108 313 128 67.4 208 467 Correction factors not applicable for MSiMSD QC data eting these data was outside criteria. NR = Sample not received nor reported. POAA = Perfluorooctanoate E = Sample evaporated, not analyzed. Date EnteredBy: Date Verified By: ETS-8-5.1 05/20/99, 05/25/99, 06/14/99, 11106100, 11109100 LAC 06/21/99 EAD, 06/22/99 LAC, 11/09100 HOJ Sera Week10 EEnxcveli9r7onmental Laboratory TOX-026-sera231-8C.xls Blks Grp 1 Grp 2 Grp 3 Grp 4 MS, MSD 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 OS 1999016-19 051999022-27 051999030-34,052499058 051799102-105,061099073 Dilutions 1/1 111 1/100 1/100 1 /250&500 1/1 RSD Std. Dev. MSlMSD RPD NA 0.00192 0.0258 3% 20% 27.7 0.0348 36.0 37.7 14.6 13.7 66.8 158 5123/20P01age 213 3M Medical Department Study: T-6889.3 Study: Product NumberfTest Substance): Matrix: Method/Revision: Analytical Equipment System Number: Instrument SohardVenion: Filename: R-Squared Value: Slope: Y-Intercept: Date of ExtractiodAnalyst: Date of AnalysidAnalyst: Date of Data ReductioniAnalyst: Sample Data Week 10 MONKEY SERA Group Sample # Dose Method Blk Matrix Blk Matrix Blk QC - 250 ppb Group 1 Control 0.0 mg/kg/day Group 2 Low Dose Group 3 did-High Dose 10 mgkglday Group 4 High Dose 30 mgkglday H 2 0 Blk-7 H20 Blk-8 Rabbit Sera Blk-7 Rabbit Sera Blk-8 Monkey Sera Blk-7 Monkey Sera Blk-8 RBS05129-MS-7 RBSOS 129-MSD-7 RBSOS 129-MS-8 RBSOS 129-MSD-8 I05709M I05714M I057 15M I05718M I05720M I05725M I05702M I05706M I05717M 105721M I05723M I05707M I05708M I05710M I05712M 105716M I05719M I05703M I05704M I0571 1M I05713M I05722M FACT-TOX-026 Covance# 6329-231 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 MEUKJWHOJ 05/18/99,05/20/99, 06111/99, 11/06/00 KJWMEWHOI Concentration of POAA ug/mL or % Rec. <LOQ <LOQ I 0.0288 0.0315 I 0.0819 I 0.0454 * 103% * 106% * 103% * 127% * 0.191 0.105 0.0919 0.129 0.128 0.110 75.9 114 74.8 154 NR 97.4 Mean POAA ug/mL 0.0301 0.0636 104% 115% 0.126 1 05 RSD Std. Dev. MSlMSD RPD 0.00192 0.0258 3% 20% 27.7 0.0348 36.0 37.7 I 313 128 67.4 208 467 14.6 1 66.8 237 158 ETS-8-5.1 3M EEnxcvelir97onmental Laboratory Analytical Report: FACT-TOX-026 LRN-U2782 5/23/20P01age 214 3M Medical Department Study: T-6889.3 FACT-TOX-026 Covance# 6329-231 Study: Product Number(Test Substance): Matrix: MethodRevision: Analytical Equipment System Number: Instrument SoftwareNerjion: Filename: R-Squared Value: Slope: Y-Intercept: Date of ExtractiodAnalyst: Date of Analysis/Analyst: Date of Data ReductiodAnalyst: 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 SAWJCP 01/21/99, 01/27/99 HOJiMEE 01/25/99, 01/28/99, 10/02/00, lO/ll/OO KJWMEE Analytical Report: FACT-TOX-026 LRN-U2782 Filenames Blks Grp 1 Grp 2 Grp 3 Grp 4 MS, MSD POAA M012199003,4 & 79, 80 012199016-2 1 012 199024-27 012199030-35,012799026 012199039-40, 012799027-29 012199074-75 Dilutions l/l 1/1 11150 11150, U400 1/50, 1/150, 1/250, 1/400, 1/500 111 Group Dose Method Blk Matrix Blk QC - 250 ppb Group 1 Control 0.0 mg/kg/day Low Dose 3.0 mgkglday 10 mg/kgiday Group 4 High Dose 30 mg/kg/day Sample # H 2 0 Blk-l H 2 0 Blk-2 Rabbit Sera Blk-l Rabbit Sera Blk-2 I I RBS01089-MS RBSOlO89-MSD I I05709M I 105714M I05715M I05718M I05720M I05725M Extraction Vol. Ratio NA NA 1.oo 1.00 1.00 1.00 0.50 0.70 0.60 0.40 0.60 0.50 POAA Std Correction Factor 0.9582 0.9582 0.9582 0.9582 I NA NA I 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 I 1 I05706M I05717M I05721M I05723M 0.60 0.9582 0.9582 0.9582 1.oo 0.9582 I05710M I05712M I05716M I057 19M I05703M I05704M I05711M I05713M I05722M IOSi24M 0.50 0.9582 0.70 0.9582 0.60 0.9582 0.50 0.9582 0.70 0.9582 0.60 0.9582 0.60 0.9582 0.50 0.9582 0.70 0.9582 1 .oo 0.9582 POAA Dilution Factor 1 1 1 1 1 1 1 1 1 1 1 150 150 150 150 NR 150 150 150 400 150 150 500 150 50 400 400 NR Correction factors not applicable for MSiMSD QC data POAA = Perfluorooctanoate Date EnteredBy: Date Verified/ By: 01/27/99, 01/28/99, 10/10/00, 10/13/00 LAC 06/21/99 EAD, 10111\00, 10/19/00KJH, 11/09/00 HOJ Cone. <LOQ 247 243 108 73.0 40.2 58.2 87.9 45.6 268 358 272 397 NR 329 239 276 240 354 344 345 433 405 243 263 NR of POAA ug/mL or % Rec. <LOQ <LOQ <LOO <LOQ 100% 98% 0.207 0.0994 0.064 0.139 0.140 0.0869 64.0 85.3 55.5 1 I4 NR 94.0 56.9 79.0 131 84.4 98.4 235 103 32.2 185 143 NR <LOQ <LOQ 99% 0.123 79.6 90.6 140 Std. Dev. MSlMSD RPD NA 2% ~~ 41.4 0.0507 32.6 25.9 27.1 24.5 55.5 77.5 FACT-M-4.1 3M EEnxcvelir97onmental Laboratory Sera Week12 TOX-026-sera23 1-8C.xls 5/23/20P01age 215 3M Medical Department Study: T-6889.3 Study: FACT-TOX-026 Covance# 6329-231 6 Month Capsule Toxicity Study with APFO in Cynomolgus Monkeys Analytical Report: FACT-TOX-026 LRN-U2782 Group Dose Matrix Bik QC - 250 ppb Group 1 Control 0.0 mglkgiday Group 2 Low Dose Group 3 Mid-High Dose 10 mg/kg/day Sample # Concentration of POAA ug/mL or % Rec. II I 1 H20Blk-2 Rabbit SeraBlk-1 <LOQ <LOQ I I I I Rabbit SeraBlk-2 1 RBS01089-MS <LOQ 100% I RBS01089-MSD 98% I05709M 0.207 I05714M 0.0994 I05715M 0.0639 I05718M 0.139 I05720M 0.140 I05725M 0.0869 I05702M 64.0 I05706M 85.3 I05717M 55.5 I05721M 114 I05723M NR I05707M 94.0 I05708M 56.9 I05710M 79.0 Mean POAA u%mL <LOQ <LOQ 99% 0.123 79.6 I05719M 98 90.6 Group 4 I05703M 235 I High Dose I05704M 103 30 mgkg/day 105711M 32.2 I05713M 185 I05722M 143 I05724M h4 140 Correction factors not applicable for MS/MSD QC data POAA = Perfluorooctanoate Date EnteredBy: Date Venfiedl By: 01/27/99, 01/28/99, 10/10/00, 10/13/00 LAC 06/21/99 EAD, 10/11/00, 10/19/00KJH, 11/09/00 HOJ RSD Std. Dev. MSMSDRPD NA NA 2% 41.4 0.0507 32.6 25.9 27.1 24.5 55.5 FACT-M-4.1 3M EEnxcveli9r7onmental Laboratory Sera Week12 TOX-026-sera231-8C.xls 5/23/200P1age 216 3M Medical Department Study: T-6889.3 FACT-TOX-026 Covance# 6329-231 Analytical Report: FACT-TOX-026 LRN-U2782 Study: 6 Month Capsule Toxicity Study with APFO in Cynomolgus Monkeys Product Number(Test Substance): T-6889.2 (POAA) Matrix: Monkey Sera MethodRevision: FACT-M-3.1 & FACT-M-4.1, ETS-8-5.1 Analytical Equipment System Number: Madeline 041098 Inshument SofhvareNersion: MassLynx 3.1 Filename: See Attachments R-Squared Value: See Attachments Slope: See Attachments Y-Intercept: See Attachments Date of ExtractiodAnalyst: 01/08/99 SAWJCP Date of Analysis/Analyst: 01/21/99,01/27/99 HOJiMEE Date of Data ReductiodAnalyst: 01/25/99,01/28/99, 10/02/00, 10111/00 KJWMEE Sample Data Week 14 MONKEY SERA Group Dose Sample # Extraction Vol. Ratio POAA Std Correction - POAA POAA Dilution Cone. Concentration of POAA Factor Factor ng/mL ug/mL or O h Rec. Method Blk Matrix Blk QC - 250 ppb H20 Blk-1 I H20Blk-2 Rabbit Sera Blk-1 I I Rabbit Sera Blk-2 RBSOIO89-MSD I I I NA 0.9582 1 I NA 0.9582 1 1.00 0.9582 I 1 1.oo 0.9582 1 1.00 NA 11 <LOQ <LOQ <LOQ <LOQ 247 243 <LOQ <LOQ <LOQ 98% Group 1 144 0.275 Control I05714M 0.50 0.9582 1 81.6 0.156 0.0 mgkglday 105715M 0.50 0.9582 1 51.4 0.0981 105718M 0.50 0.9582 1 80.3 0.153 Group 2 Low Dose 3.0 mgikgiday 105720M 105725M - 105706M I05717M I :::: 1 1 :i 105721M 0.30 0.9582 1 57.3 0.50 0.9582 1 55.0 381 0.50 0.9582 200 294 0.9582 306 0.9582 428 0.9582 NR 0.182 0.105 72.7 112 58.4 117 NR Group 3 0.9582 178 68.0 Mid-High Dose 105708M 270 10 mg/kg/day 105710M 0.9582 322 105712M 0.9582 415 Group 4 105716M 105719M 105703M 0.9582 0.9582 0.9582 - 316 45 1 295 High Dose 105704M 0.50 0.9582 150 380 30 mgkglday 105711M 0.9582 150 215 . 105713M 0.50 0.9582 129 105722M I05724M 0.50 0.9582 150 346 0.9582 rux NR imit of Quantitation (LOQ): 0 1137 ug/mL Comection factors not applicable for MSiMSD QC data Filenames Blks Grp 1 Grp 2 Grp 3 Grp 4 MS, MSD Mean POAA ug/mL I <LOQ 99% 0.162 90.0 POAA M012199003,4 & 79, 80 012 199045-50 012 199053-56,012799033 012199061-62,64 012799034-36 012199067-71 012199074-75 RSD Std. Dev. MSIMSD RPD I NA 2% 39.8 0.0643 32.1 28.9 Dilutions 111 l/l 1/100,1/200 1/100,1/200 11150 l/l POAA = Pertluorooctanoate Date Enteremy: Date Verified/ By: 01/27/99, 01/28/99, 10/10/00, 10/13/00 LAC 06/21/99 EAD, 10/11/00, 10/19/00KJH, 11/09/00 HOJ FACT-M-4.1 3M EEnxcveli9r7onmental Laboratory Sera Week14 TOX-026-sera231-8C.xls 5/23/20P01age 217 3M Medical Department Study: T-6889.3 Study: Product Number(Test Substance): Matrix: MethodRevision: Analytical Equipment System Number: Instrument SofhvareNersion: Filename: R-Squared Value: Slope: Y-Intercept: Date of ExtractioniAnalyst: Date of Analysis/Analyst: Date of Data ReductiodAnalyst: FACT-TOX-026 Covance# 6329-231 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 SAWJCP 01/21/99, 01/27/99 HOJiMEE 01/25/99,01/28/99, 10/02/00, 10/11/00 KJWMEE Group Dose Method Blk Matrix Blk QC - 250 ppb Group 1 Control 0.0 mgkgiday Group 2 Low Dose Group 3 Mid-High Dose IO mgkgiday Sample # H20 Blk-l H 2 0 Blk-2 Rabbit Sera Blk-l Rabbit Sera Blk-2 RBSOl089-MS RBS01089-MSD I05709M I05714M I05715M I05718M I05720M 105725M I05702M I05706M I05717M I05721M I05723M I05707M i j I05708M I05710M 105712M I05716M I05719M Concentration of POAA u g h L or YORee. <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 Mean <LOQ 99% 0.162 High Dose I05704M 109 30 mgkg/day I0571 1M 68.2 I05713M 36.9 I I I05722M 98.8 I05724M iux 79.4 Limit of Quantitation (LOO): 0.0137 udmL Correction factors not applicable for MSiMSD QC data POAA = Perfluorooctanoate Date EnteredBy: Date Verifiedi By: 01/27/99, 01/28/99, 10/10/00, 10/13/00 LAC 06/21/99 EAD, lO/ll/OO, 10/19/00 KJH, 11/09/00 HOJ I RSD Std. Dev. MSIMSD RPD 1 NA I I NA I 2% 39.8 0.0643 35.6 28.3 FACT-M-4.1 3M EEnxvceilr9o7 nmental Laboratory Sera Week14 TOX-026-sera23 1-8C.xls Analytical Report: FACT-TOX-026 LRN-U2782 5/23/20P01age 218 3M Medical Department Study: T-6889.3 FACT-TOX-026 Covance# 6329-231 Study: Product Number(Test Substance): Matrix: MethodiRevision: Analytical Equipment System Number: Instrument SoftwareNersion: Filename: R-Squared Value: Slope: Y-Intercept: Date of ExtractiodAnalyst: Date of AnalysidAnalyst: Date of Data ReductiodAnalyst: Sample Data Week 16 MONKEY SERA 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 SRPIJCP 05/17/99, 05/19/99,06/10/99 MEE 05/18/99, 05/20/99,06/11/99, 11/06/00 KJWMEUHOJ Group Method Blk Matrix Blk Mahix Blk QC - 250 ppb Control 0.0 mg/kg/day Sample # H 2 0 Blk-7 H20 Blk-8 Rabbit Sera Blk-7 Rabbit Sera Blk-8 Monkey Sera Blk-7 Monkey Sera Blk-8 RBS05129-MS-7 RBSOS 129-MSD-8 IO5 7 14M I05715M 105718M I05720M I05725M Extraction Vol. Ratio 1 .oo 1 .oo 1 .oo 1.00 1.00 1 .oo 1 .oo 0.54 0.66 0.64 0.51 0.50 POAAStd Correction Factor 0.9582 0.9582 0.9582 0.9582 0.9.582 0.9582 NA NA NA NA 0.9582 0.9582 0.9582 0.9582 0.9582 POAA Dilution Factor 1 1 1 1 1 1 1 1 1 I 1 POAA Conc. ng/mL 7.56 0.00 30.0 32.8 85.4 47.4 257 265 258 317 155 Concentration of POAA ug/mL or Ye Rec. <LOQ <LOQ 0.0288 0.0315 0.0819 0.0454 106% 127% <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) I05702M 55.3 Low Dose I05706M 0.9582 72.6 3.0 mgikglday I05717M 0.9582 43.4 I iO5RiM 0.52 0.9582 103 I05723M NR 0.9582 NR Group 3 I I05707M I 0.57 0.9582 99.5 Mid-High Dose I05708M 0.68 0.9582 100 427 60.2 IO mgkgiday IO57 1 OM 0.56 0.9582 100 428 73.2 I05712M 0.59 0.9582 500 222 180 I I05716M 0.57 I05719M 0.64 0.9582 0.9582 100 543 100 578 91.3 86.5 Group 4 I I05703M I 0.57 0.9582 250 162 68.2 High Dose I05704M 0.75 0.9582 250 355 1 I3 30 mgkglday I05711M 0.60 0.9582 IO 835 13.3 105713M 0.74 0.9582 250 516 167 I05722M 0.54 0.9582 250 130 57.7 105724M NR 0.9582 NR NR NR Blks Grp 1 Grp 2 Grp 3 Grp 4 MS, MSD Mean POAA ug/mL * <LOQ * 0.0301 * 0.0636 104% 115% 0.128 68.6 98.5 83.9 Analytical Report: FACT-TOX-026 LRN-U2782 Filenames POAA M0.51799028, 29, 98-99, & 109, 110 061099065-69, 72 OS199903740 05199904345,47-48,052499059 OS 1999051-52,54-55,06l099074 051799102-105,06l099073 Dilutions l/l 111 1/100 1/100 1/10&250 1/1 RSD Std. Dev. MSIMSD RPD NA 0.00192 20% 56.4 0.0721 37.9 26.0 43.0 42.3 69.7 58.5 ETS-8-5.1 Date Verifiedi By: 06/21/99 EAD, 06/22/99 LAC, 11/09/00 HOJ 3M EEnxcvelir97onmental Laboratory Sera Week16 TOX-026-sera231-8C.xls 5/23/20P01age 219 3M Medical Department Study: T-6889.3 FACT-TOX-026 Covance# 6329-231 Study: Product Number(Test Substance): Matrix: Method/Revision: Analytical Equipment System Number: Instrument SofhvareNersion: Filename: R-Squared Value: Slope: Y-Intercept: Date of ExtractiodAnalyst: Date of Analysis/Analyst: Date of Data ReductiodAnalyst: Sample Data Week 16 MONKEY SERA Group Dose Sample # 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 RES05 129-MSD-7 I RBS05129-MS-8 RBS05129-MSD-8 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/l8/99,05/20/99,06/1l/99, 11/06/00 KJWMEEIHOJ Concentration of POAA ug/mL o r O h Rec. <LOQ <LOQ * 0.0288 0.0315 0.0819 * I 0.0454 t 103% * 106% * 103% * 127% * Mean POAA ug/mL cLOQ 0.0301 0.0636 104% 115% RSD Std. Dev. MS/MSD RPD NA 0.00192 0.02578 3% I 20% Control 0.0 mgkg/day Group 2 Low Dose Group 3 Mid-High Dose IO mgkglday Group 4 High Dose 30 mgkglday I057 14M <LOQ (0.0983 ug/mL) I05715M <LOQ (0.0983 ug/mL) I05718M <LOQ (0.0983 ug/mL) I05720M <LOQ (0.0983 ug/mL) I05725M <LOQ (0.0983 ug/mL) 0.128 I05702M 55.3 I05706M 72.6 I05717M 43.4 I05721M 103 105723M NR 68.6 I05707M 100 I05708M 60.2 I05710M 73.2 I05712M 180 I05716M 91.3 I05719M 86.5 98.5 I05703M 68.2 IOSiO4M I I3 10571IM 13.3 I05713M 161 I I05722M I 57.7 I05724M NR I I 83.9 56.4 0.0721 37.9 26.0 43.0 42.3 69.1 58.5 3M ETS-8-5.1 EEnxvceilr9o7 nmental Correction factors not applicable POAA = Peffluorooctanoate Date EnteredBy: Date Verified/ By: Laboratory for MS/MSD QC data 05/20/99, 06/14/99, 11/06/00 LAC 06/21/99 EAD, 06/22/99 LAC, 11/09/00 r - E = Sample evaporated, not analyzed. * CCV bracketing these data was not within HOJ Sera Week16 TOX-026-sera23 1-8C.xls criteria. Analytical Report: FACT-TOX-026 LRN-U2782 5/23/20P01age 220 3M Medical Department Study: T-6889.3 FACT-TOX-026 Covance# 6329-231 Analytical Report: FACT-TOX-026 LRN-U2782 Study: 6 Month CapsuleToxicity Study with APFO in Cynomolgus Monkeys Product Number(Test Substance): T-6889.2 (POAA) Matrix: Monkey Sera MethodRevision: ETS-84.1 and ETS-8-5.1 Analytical Equipment System Number: Madeline 041098, Amelia 062498 Instrument SoftwareNersion: MassLynx 3.2 Filename: See Attachments R-Squared Value: See Attachments Slope: See Attachments Y-Intercept: See Attachments Date of ExtractiodAnalyst: 05/14/99 SEE Date of AnalysidAnalyst: 05/18/99, 05/24/99,05/25/99, 06/07/99 IEEiHOJISAH Date of Data ReductiodAnalyst: 05/19/99, 05/25199,05127/99, 06/08/99 MEEMOJ Sample Data Week 18 MONKEY SERA I Group I Dose Sample # Extraction Vol. Ratio POAA Std Correction POAA Dilution POAA Cone. Concentration of POAA Factor Factor ng/mL ug/mL or % Rec. I NA 0.9582 1 0.00 NA 0.9582 1 1 0.00 <LOQ <LOQ 1.00 1 .oo 1.oo 1.00 1.00 RBS05149-MSD-12 1.00 0.9582 1 1.76 II NA 1 1 1 236 NA 1 1 1 247 NA 1 201 <LOQ 95% 99% 80% 0.63 0.9582 1 173 0.263 Control 105714M 0.57 0.9582 1 145 0.244 0.0 mgkglday I057 1SM 0.57 0.9582 1 56.5 0.0950 I05718M 0.48 0.9582 1 82.7 0.165 I05720M 0.43 0.9582 1 86.7 0.193 I05725M 0.52 0.9582 1 75.1 0.138 Group 2 I I05702M 0.69 0.9582 50 160 11.1 Low Dose I05706M 0.63 0.9582 50 210 16.0 i 3.Omgkgiday I Group 3 Mid-High Dose 10 mgkglday Group 4 High Dose 105717111 0.45 0.9582 250 125 66.8 105721M 0.47 0.9582 so 234 23.8 I05707M 0.52 0.9582 so 145 13.4 I05708M 0.68 0.9582 50 137 9.67 I05710M 0.48 0.9582 so 465 46.4 105712M 0.60 0.9582 so 171 13.7 105716M 0.70 0.9582 50 147 10.1 105719M 0.75 0.9582 50 178 11.3 I05703M 0.46 0.9582 50 660 68.8 I05704M 0.66 0.9582 50 . 276 20.0 30 mgkglday I05711M 0.32 0.9582 50 127 19.0 I05713M 0.70 0.9582 50 399 27.3 I 105722M 0.60 0.9582 50 575 45.9 Limit of Quantitation (LOQ): 0 )I37ug/mL Correction factors not applicable for MSIMSD QC data Filenames Blks Grp 1 Grp 2 Grp 3 Grp 4 MS, MSD ug/mL <LOQ 105% 90% 0.183 36.2 POAA POAA M051899013, I4 & 89,90 05 1899026-33 052599086-87,89 60799061 052599092-97 0 5 2 4 9 9 0 3 0 , 3 2 , 3 4 , 0 5 2 5 9 9 1 0 1 , 103 051899084-87 Std. Dev. MS/MSD RPD I NA 1 20% 21% 34.8 0.0637 I1 58.5 21.2 Dilutions 111 Ill 1150&250 1/50 1/50 111 POAA = Perfluorooctanoate Date Enteremy: Date Verified/ By: 05/19/99, 05/25/99, 05/27/99, 06/08/99 LAC 06/21/99 EAD ETS-8-5.1 3M EEnxcveli9r7onmental Laboratory Sera Week18 TOX-026-sera23 1-8C.xls 5/23/20P01age 221 3M Medical Department Study: T-6889.3 FACT-TOX-026 Covance# 6329-231 Study: Product Numbe<Test Substance): Matrix: MethodiRevision: Analytical Equipment System Number: Instrument SoftwareIVersion: Filename: R-Squared Value: Slope: Y-Intercept: Date of ExtractiodAnalyst: Date of AnalysisiAnalyst: Date of Data ReductiodAnalyst: Sample Data Week IS MONKEY SERA Group Dose Sample # Method Blk Matrix Blk QC - 250 ppb H20 Blk-l H20 BIk-2 Rabbit Sera Blk-l Rabbit Sera Blk-2 RBS05149-MS-11 RBS05149-MSD-11 RBSO5149-MS-12 RBSOS 149-MSD-12 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 05118199,05/24/99, 05/25/99, 06/07/99 MEE/HOJ/SAH 05/19/99,05/25/99, 05/27/99, 06/08/99 MEEMOJ Concentration Mean RSD of POAA POAA Std. Dev. ug/mL or % Ree. ug/mL MSlMSD RPD <LOQ <LOQ <LOQ NA <LOQ cLOQ <LOQ NA I I I 116% 95% 105% 20% I 99% I I 80% 90% 21% Control 0.0 mgkglday I057 14M I05715M I05718M I05720M I05725M 0.0950 0.165 0.193 0.138 0.183 34.8 0.0637 Low Dose Group 3 Mid-High Dose 10 mgkgiday Group 4 High Dose 30 mglkglday I05706M I05717M I0572 1M I05707M 105708M 105710M I05712M I05716M IO5719M 105703M I05704M I0571 IM 105713M I05722M 16.0 66.8 86.5 23.8 29.4 25.4 13.4 9.67 46.4 13.7 10.1 82.1 11.3 17.4 14.3 68.8 20.0 19.0 27.3 58.5 45.9 36.2 21.2 Correction factors not applicable for M S N S D QC data POAA = Perfluorooctanoate Date EnteredIBy: Date Verifiedi By: 05/19/99, 05/25/99, 05/27/99,06/08/99 LAC 06121199 EAD ETS-8-5.1 3M EExncevli9r7onmental Laboratory Sera Week18 TOX-026-sera23 1-8C.xls Analytical Report: FACT-TOX-026 LRN-U2782 5/23/20P01age 222 3M Medical Department Study: T-6889.3 Study: Product Nwnber(Test Substance): Matrix: MethodRevision: Analytical Equipment System Number: Instrument SofhvareNersion: Filename: R-Squared Value: Slope: Y-Intercept: Date of ExtractiodAnalyst: Date of AnalysisiAnalyst: Date of Data ReductiodAnalyst: FACT-TOX-026 Covance# 6329-231 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 MEWHOJISAHlLAC 05/19/99,05/25199,05/27/99, 06111/99,06/17199, 11/06/00 MEE/HOJ/LAC/KJH Filenames Blks Grp 1 Grp 2 Grp 3 Grp 4 MS, MSD Analytical Report: FACT-TOX-026 0.250746269 LRN-U2782 POAA M051899013,14 & 89,90 051899055-62 052599107-1 IO 052499045,061099075-76,061699018-20 052499051,53,55,061699021-22 051899084-87 Dilutions 111 111 1/50 1/50&500 1/50&500 111 Group Dose Method Blk I Matrix Blk I QC - 250 ppb Group 1 Control 0.0 mgkglday Group 2 Low Dose 3.O mgkglday Group 3 Mid-High Dose 10 mgkgiday Group 4 High Dose 30 mgkglday Sample # Extraction Vol. Ratio H20 BIk-1 NA I H 2 0 Blk-2 NA I Rabbit SeraBlk-l 1.oo I I Rabbit SeraBlk-2 1.oo I I RBSO5149-MS-11 1 .oo RBS05149-MSD-11 RBS05 149-MS-12 RBS05149-MSD-12 I05709M 0.59 I05714M 0.66 I05715M 0.62 I05718M 0.33 I05720M I05725M I05702M I 0.58 0.73 105706M 0.65 I05717M 0.63 I05721M 0.24 I05707M 0.43 I05708M 0.70 I05710M 0.42 I05712M 0.65 I05716M 0.55 105719M 0.62 I05703M 0.49 I05704M 0.53 I05711M 0.38 I05713M 0.68 I05722M 0.36 POAA Std POAA POAA Concentration Mean Correction Dilution Cone. of POAA POAA Factor Factor ng/mL ug/mL or O h Rec. ug/mL 0.9582 1 0.00 <LOQ 0.9582 1 0.00 <LOQ <LOQ 0.9582 1 8.69 <LOQ 0.9582 1 1.76 <LOQ <LOQ I I NA 1 290 NA 11 236 NA I l l 247 I 116% 95% 99% I I 105% I 1 NA 1 201 80% 90% 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 1 146 1 74.3 1 77.2 1 134 1 126 50 271 50 155 50 43 8 50 348 50 964 50 905 50 660 500 186 500 105 500 138 50 398 500 102 50 18.9 500 456 50 248 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 0.224 33.0 96.6 91.1 RSD Std. Dev. MSlMSD RPD NA NA 20% 21% 32.6 0.0730 78.8 26.0 27.5 26.6 132 129 Date EnterediBy: Date Verified/ By: 05120/99, 05/25/99, 05\27/99, 06114/99,06117/99, 11/06/00 LAC 06/21/99 EAD, 06/22/99 LAC, 11/09/00 HOJ ETS-8-5.1 3M EEnxcveli9r7onmental Laboratory Sera Week20 TOX-026-sera23 1-8C.xls 5/23/20P01age 223 3M Medical Department Study: T-6889.3 Study: Product Number(Test Substance): Matrix: MethodIRevision: Analytical Equipment System Number: Instrument SoftwareiVersion: Filename: R-Squared Value: Slope: Y-Intercept: Date of ExtractiodAnalyst: Date of AnalysidAnalyst: Date of Data ReductioniAnalyst: FACT-TOX-026 Covance# 6329-231 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 05118/99, 05/24/99, 05/25/99, 06/10/99, 06/16/99 MEWHOJISAWLAC 05/19/99, 05125199, 05/27/99, 0611 1/99, 06/17/99, 11/06/00 MEEMOJILACIKJH Group Dose Method Blk QC - 250 ppb Group 1 Control 0.0 mg/kgiday Group 2 Low Dose Group 3 Mid-High Dose 10 mgikglday Group 4 High Dose 30 mgikgiday Sample # H20 Blk-1 H 2 0 Blk-2 I I RabbitSeraBlk-2 I I RBS05149-MS-11 I RBS05149-MSD-11 I RBS05149-MS-12 RBS05149-MSD-12 I05709M I057 14M I05715M I05718M I05720M I05725M I05702M I05706M I05717M I05721M I05707M I05708M I05710M I05712M I05716M I05719M I05703M I05704M 105711M I05713M I05722M Concentration of POAA u g h L or % Rec. <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 <LOQ CLOQ 105% 90% Std. Dev. MSlMSD RPD I I NA NA I 20% I 21% 0.224 33.0 32.6 0.0730 78.8 26.0 I 132 97.7 129 POAA = Perfluorooctanoate Date EnterediBy: Date Venfiedi By: 05/20199, 05/25/99,05/27/99, 06/14/99, 06117/99, 11/06/00 LAC 06/21/99 EAD, 06/22/99 LAC, 11/09/00 HOJ ETS-8-5.1 3M EEnxcveli9r7onmental Laboratory Sera Week20 TOX-026-sera231-8C.xls Analytical Report: FACT-TOX-026 LRN-U2782 5/23/20P01age 224 3M Medical Department Study: T-6889.3 FACT-TOX-026 Covance# 6329-231 Study: Product Numbe<Test Substance): Matrix: MethodRevision: Analytical Equipment System Number: Instrument SoAwareiVersion: Filename: R-Squared Value: Slope: Y-intercept: Date of ExtractiodAnalyst: Date of AnalysidAnalyst: Date of Data ReductiodAnalyst: 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, 05/26/99,05/28199,06/10/99 HOJMEE 05/24/99. 05/27/99,06/01/99,06/11l99,11106100HOJ/MEE/KJH Analytical Report: FACT-TOX-026 LRN-U2782 Filenames Blks Grp 1 Grp 2 Grp 3 Grp 4 MS, MSD POAA A052099035,36 & 98,99 052099047-51,061099086 052699025-27 052699028-32,34-35,052899104 052699036-42 061099095-96 Dilutions 111 111 l/lOO 11100&200 1/10&100 111 Group Dose Method Blk Matrix Blk QC - 250 p..pb Control 0.0 mgkglday Group 2 Low Dose 3.0 mgkglday Mid-High Dose 10 mg/kg/day I High Dose 30 mgkglday Sample # Extraction Vol. Ratio H20 Blk-9 1.0 H 2 0 Blk-10 1.o Rabbit Sera Blk-9 1.o Rabbit Sera Blk-10 1.o RBS05 149-MS-9 1.o RBSOS 149-MSD-9 1.o I05714M 105715M I05718M I05720M I05725M 0.28 I05702M 0.75 I05706M 0.32 I05717M 0.68 I 1 105708M 0.55 105710M 0.65 105712M 0.54 I 105716M I 0.46 I05 7 19M 0.60 I05704M 0.28 I0571 1M IO57 13M I05722M 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 POAA POAA Dilution Cone. Factor ng/mL 1 0.00 1 42.5 1 0.00 1 32.9 I 1 261 1 273 Concentration of POAA ug/rnLor%Rec. <LOQ 0.0407 <LOQ 0.03 15 104% 109% I I ; 1 :";I," 1 111 1 120 0.157 0.106 0.269 0.372 1 0.00 <LOQ (0.0983 ug/mL) 100 547 69.8 100 352 105 100 408 57.5 100 533 122 100 366 63.7 100 595 87.7 200 472 Mean POAA ug/mL NA NA 107% 0.232 77.6 Correction factors not applicable for MSMSD QC data POAA = Perfluorooctanoate Date EnterediBy: Date Verifiedi By: 05/25/99, 05/27/99, 06/02/99, 06/14/99, lI/O6/00 LAC 06/21/99 EAD, 06/22/99 LAC, 11/09/00 HOJ RSD Std. Dev. MSIMSD RPD NA NA 4% 56.5 0.131 32.1 24.9 ETS-8-5.1 3M EExncveli9r7onmental Laboratory Sera Week22 TOX-026-sera23 1 -8C.xls 5/23/20P01age 225 3M Medical Department Study: T-6889.3 FACT-TOX-026 Covance# 6329-231 Study: Product Number(Test Substance): Matrix: MethodRevision: Analytical Equipment System Number: Instrument SoftwareiVersion: Filename: R-Squared Value: Slope: Y-intercept: Date of ExtractiodAnalyst: Date of Analysis/Analyst: Date of Data ReductiodAnalyst: Sample Data Week 22 MONKEY SERA I Group I Dose Sample # I Method Blk Matrix Blk QC - 250 ppb I H20 Blk-9 H20 Blk-IO Rabbit Sera Blk-9 Rabbit Sera Blk-10 RBS05149-MS-9 Group 1 Control 0.0 mg/kg/day Group 2 Low Dose I05709M I05714M I05715M 105718M I05720M I05725M I I05702M I05706M I0571 7M 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 HOJIMEE 05/24/99, 05/27/99, 06/01/99, 0611 1/99, 11/06/00 HOJIMEEIKJH Concentration of POAA ug/mL or % Rec. <LOQ 0.0407 iL0Q 0.0315 104% 1- 109% 0.393 0.157 0.106 0.269 0.372 <LOQ (0.0983 ug/mL) Mean POAA ug/mL NA NA 107% RSD Std. Dev. MSIMSD RPD NA NA 4% 177.6 :::; Mid-High Dose I05708M 63.7 IO mgkglday I0571OM 87.7 I05712M I05716M Group 4 High Dose I05704M 92.7 30 mgkgiday 10571 1M I I05713M 96.3 I05722M 86.0 Limit of Quantitahon (LOQ): 0.0137 ug/mL Correction factors not applicable for MS/MSD QC data 77.8 58.6 45.6 POAA = Perfluorooctanoate Date EnterediBy: Date Verifiedi 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 ETS-8-5.1 3M EEnxcveil r97onmental Laboratory Sera Week22 TOX-026-sera23 1-8C.xls Analytical Report: FACT-TOX-026 LRN-U2782 5/23/20P01age 226 3M Medical Department Study: T-6889.3 Study: Product Number(Test Substance): Matrix: MethodRevision: Analytical Equipment System Number: FACT-TOX-026 Covance# 6329-231 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 POAA Analytical Report: FACT-TOX-026 LRN-U2782 .. .. Dirunons 111 111 1/100&200 moo I l l , 10,20,&200 1/1 0.0 mgkghiay Group 2 Low Dose 3.0 mgkgiday Group 3 Mid-High Dose 10 mgkglday Group 4 High Dose 30 mgkgiday I05715M 0.31 0.9582 1 5.55 <LOQ (0.0983 ug/mL) 105718M 0.47 0.9582 1 55.5 <LOQ (0.0983 ug/mL) I05720M 0.29 0.9582 1 77.5 <LOQ (0.0983 ug/mL) NA I05725M 0.38 0.9582 1 5.08 <LOQ (0.0983 ug/mL) <LOQ NA I05702M 0.61 0.9582 100 64.9 10.2 I05706M 0.69 0.9582 200 527 146 I05717M 0.43 0.9582 100 270 60.2 95.3 72.2 68.8 I05707M 0.71 0.9582 100 660 89.1 I05708M 0.64 0.9582 100 410 61.4 I05710M 0.61 0.9582 100 521 81.8 I05712M 0.54 0.9582 100 656 1I6 105716M 0.59 0.9582 100 507 82.3 23.3 105719M 0.54 0.9582 100 646 115 I05703M 0.57 0.9582 20 606 20.4 90.9 21.2 I05704M 0.72 0.9582 100 679 90.4 I05711M 0.52 0.9582 IO 233 4.29 I05713M 0.63 0.9582 200 596 181 119 I05722M 0.43 . 0.9582 IO 765 17.0 62.7 74.3 POAA = Perfluorooctanoate Date EnteredBy: Date Verified/ By: 05/25/99, 05/27/99, 06/02/99, 1 1/06/00LAC 06/21/99 EAD, 11/09/00 HOJ ETS-8-5. I 3M EEnxcveli9r7onmental Laboratory Sera Week24 TOX-026-sera23 1-8C.xls 5/23/20P01age 227 3M Medical Department Study: T-6889.3 FACT-TOX-026 Covance# 6329-231 Study: Product NumbeflTest Substance): Matrix: MetbodiRevision: Analytical Equipment System Number: Instrument SoftwareNersion: Filename: R-Squared Value: Slope: Y-Intercept: Date of ExtractiodAnalyst: Date of AnalysisIAnalyst: Date of Data ReductiodAnalyst: Sample Data Week 24 MONKEY SERA Group Dose Sample # Method Blk Matrix Blk QC - 250 ppb Group 1 Control 0.0 mgkglday Group 2 Low Dose Group 3 Mid-High Dose 10 mgkglday Group 4 High Dose 30 mgkglday H 2 0 Blk-9 H 2 0 Blk-IO Rabbit Sera Blk-9 Rabbit Sera Blk-10 RBSO5149-MS-9 RBS05 149-MSD-9 I05709M I05714M I05715M I0571 8M 105720M I05725M 105702M I05706M I05717M 105707M I05708M I05710M 105712M 105716M 105719M 105703M 105704M I0571 1M I05713M I05722M 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 05l20199, 05126199, 05128199 HOJISAWMEE 05/24/99, 05/27/99, 06/01/99, 11/06/00 HOJIMEEiKJH Concentration of POAA ug/mL or % Rec. <LOQ 0.0407 <LOQ 0.0315 104% 109% Ext Ext <LOQ (0.0983 ugImL) <LOQ (0.0983 uglmL) <LOQ (0.0983 ug/mL) <LOQ (0.0983 uglmL) 10.2 146 60.2 89.1 61.4 81.8 116 82.3 115 20.4 90.4 4.29 181 17.0 Mean POAA Ug/d 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 Correction factors not applicable for MS/MSD QC data POAA = Perfluorooctanoate Date EnteredBy: Date Verifiedi 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 3M EExncveli9r7onmental Laboratory Sera Week24 TOX-026-sera23 1-8C.xls Analytical Report: FACT-TOX-026 LRN-U2782 5/23/20P01age 228 3M Medical Department Study: T-6889.3 FACT-TOX-026 Covance# 6329-231 Analytical Report: FACT-TOX-026 LRN-U2782 Filenames Blks Grp 1 Grp 2 Grp 3 Grp 4 MS, MSD POAA A051799055, 56 & 95,96 051799067-70 052699015-17 052099120-121, 123 & 052699020 051799086,052099111,126 & 052699021-22 061099093-94 Dilutions 111 111 11200 11100&200 111,10,20,100,&500 111 0.0 mgkglday Group 2 Mid Dose 3.0 mgkglday Group 3 Mid-High Dose 10 mgkglday Group 4 High Dose 30 mgkglday 105715M 105718M 105720M 105725M IO5702M 105706M I057 1 7M 105707M 105708M 105710M 105719M 105703M 105704M 105711M 105713M 105722M 0.57 0.9582 1 60.3 0.50 0.9582 1 108 0.50 0.9582 1 161 0.52 0.9582 1 33.8 0.62 0.9582 200 403 0.61 0.9582 200 450 0.61 0.9582 200 279 0.49 0.9582 100 399 0.58 0.9582 100 335 0.65 0.9582 200 327 0.38 0.9582 100 316 0.37 0.9582 10 429 0.70 0.9582 100 451 0.36 0.9582 1 565 0.63 0.9582 500 393 0.51 0.9582 20 422 POAA = Peffluorooctanoate Date Entere&'Eiy: Date Verified/ By: 05i19i98, 051'27199, 06il4199, 1li06iOO LAC 06/21/99 EAD, 06/22/99 LAC 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 0.209 118 77.4 77.8 74.5 0.156 23.3 27.5 21.8 16.9 162 126 ETS-8-5.1 3M EEnxcveli9r7onmental Laboratory Sera Week26 TOX-026-sera231-8C.xls 5123120P01age 229 3M Medical Department Study: T-6889.3 FACT-TOX-026 Covance# 6329-231 Study: 6 Month Capsule Toxicity Study with APFO in Cynomolgus Monkeys Product Numbe<Test Substance): T-6889.2 (POAA) Matrix: Monkey Sera MethodIRevision: ETS-8-4.1 & ETS-8-5.1 Analytical Equipment System Number: Amelia 062498 Instrument SoftwareNersion: MassLynx 3.2 Filename: See Attachments R-Squared Value: See Attachments Slope: See Attachments Y -Intercept: See Attachments Date of ExtractiodAnalyst: 04/29/99, 05114199 SAH Date of AnalysidAnalyst: 05117/99, 05/20/99, 05/26/99, 06/10/99 HOJISAWMEE Date of Data ReductiodAnalyst: 05119199, 05124199, 05/27/99, 06111199, 11/06/00 HOJIMEWKJH Sample Data Week 26 MONKEY SERA Sample # Concentration of POAA ug/mL or % Rec. Mean POAA u%d RSD Std. Dev. MSlMSD RPD H 2 0 Blk-3 <LOQ (0.0250 ug/mL) Method Blk H20 Blk-4 <LOQ (0.0250 ug/mL) <LOQ NA Rabbit Sera Blk-3 <LOQ (0.0250 ug/mL) Matrix Blk Rabbit Sera Blk4 <LOQ (0.0250 ug/mL) <LOQ NA MKSOS 149-MS-3 83% MKSO5 149-MSD-3 Group 1 I05709M Control I057 14M 0.0 mglkgiday I057 ISM I05718M I05720M I05725M Group 2 I05702M Mid Dose I05706M I05717M Group 3 I05707M Mid-High Dose I05708M IO mgkglday I05710M I05719M Group 4 I05703M High Dose I05704M 30 mglkgiday I05711M I05713M 105722M 15.8 77.8 126 37 ug/mL Correction factors not applicable for MS/MSD QC data POAA = Perfluorooctanoate Date EnlerediBy: Date Verified/ By: 05/19i98,05/2ii99, 06/14/99, 1l/06/00 LAC 06/21/99 EAD, 06/22/99 LAC ETS-8-5.1 3M EEnxcveil r9o7 nmental Laboratory Sera Week26 TOX-026-sera23 I-8C.xls Analytical Report: FACT-TOX-026 LRN-U2782 5/23/20P01age 230 3M Medical Department Study: T-6889.3 FACT-TOX-026 Covance# 6329-231 Analytical Report: FACT-TOX-026 LRN-U2782 Study: 6 Month Capsule Toxicity Study with APFO in Cynomolgus Monkeys Product NurnberlTest Substance): T-6889.2 (POAA) Matrix: Monkey Sera MethodRevision: ETS-8-4.1 & ETS-8-5.1 Filenames Analytical Equipment System Number: Amelia 062498 Instrument SoftwareNersion: Filename: R-Squared Value: Slope: MassLynx 3.2 See Attachments See Attachments See Attachments Blks Grp 1 Grp 2 Grp 3 Y-Intercept: Date of ExtractiodAnalyst: Date of Analvsis/Analvst: See Attachments Grp 4 04/29/99 SAH MS, MSD 04/30/99,05/03/99. 05/04/99. 05/C '99 KJWMEUHOJ Date of Data ReductiodAnalyst: Sample Data 05/03/99,05/04/99, 05/05/99, 05/25/99, 1 1/02/00, 11/06/00 HOJiKJH Week 26.27 MONKEY SERA Sample # Extraction Vol. Ratio POAAStd Correction Factor POAA Dilution Factor POAA Cone. nglmL Concentration of POAA ug/mL or % Rec. H20 Blk-l NA H20 Blk-2 NA Rabbit Sera Blk-1 1 .o Rabbit Sera Blk-2 1 .o I05709M-MS 1 .o I05714M-MS 1 .o I05709M Wk27 1 .o I05714M Wk27 1 .o I05715M Wk27 1 .o I05718M Wk26 0.50 105720111Wk26 0.50 I05725M Wk27 1 .o 0.9582 0.9582 0.9582 0.9582 NA NA 0.9582 0.9582 0.9582 0.9582 0.9582 0.9582 1 0.00 <LOQ (0.00983 ug/mL) 1 0.00 <LOQ (0.00983 ug/mL) 1 0.00 <LOQ (0.00983 ug/mL) 1 0.00 cLOQ (0.00983 ug/mL) 1 592 79% 1 500 108% 1 393 0.377 1 229 0.219 1 98.7 0.0945 1 108 0.206 1 161 0.308 1 142 0.136 I I ::: I ::: I05702MWk27 47.9 I05706M Wk27 1 .o 0.9582 100 673 64.5 105717M Wk27 1 .o 0.9582 44.1 I05721M Moribund 1 .o 0.9582 46.0 I05723M 1 .o 0.9582 100 626 60.0 Group 3 I05707M Wk27 121 I I I Mid-High Dose I05708M Wk27 1.0 0.9582 100 335 32.1 IO mgikgiday I05710M Wk27 1 .o 0.9582 100 497 47.6 I05712M Wk26 0.50 0.9582 101 1 1 ::: I iiz 105716111Wk26 0.60 0.9582 78.1 I05719M Wk27 1 .o 0.9582 100 680 65.2 Group 4 I05703M Wk27 High Dose 105704M Wk27 0.9582 30 mgkgiday 10571IM Wk27 0.9582 I05713M Wk27 0.9582 I05722M Wk27 0.9582 I I05724M Moribund Limit of Quantitahon (LOQ): 0 37 ug/mL 0.9582 1000 7.48 58.6 0.877 184 6.65 489 Correction factors not applicable for MSMSD QC data POAA A050399003,4 & 42,43 043099065-70 050399016-18,36-37 050399021-28,050599l16 050399029-32, OS04990l6,05OS99l I7 043099073-74 Mean POAA ug/d <LOQ <LOQ 94% RSD Std. Dev. MSIMSD RPD NA NA 17% 0.223 52.5 47.1 0.105 17.4 9.14 44 7 74.1 33.1 151 51.5 17.6 Dilutions l/l 1/1 1/100 1/100&1000 1/100&1000 1/1 POAA = Perfluorooctanoate Date EnteredBy: Date Verifiedi 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 ETS-8-5.1 3M EEnxcvelir91onmental Laboratory Sera Week26,27 TOX-026-sera23 1 -8C.xls 5/23/20P01age 231 3M Medical Department Study: T-6889.3 Study: Product Number(Test Substance): Matrix: MethodRevision: Analytical Equipment System Number: Instrument SoftwareiVersion: Filename: R-Squared Value: Slope: Y-Intercept: Date of ExtractiodAnalyst: Date of AnalysisiAnalyst: Date of Data ReductiodAnalyst: Group Dose Method Blk Matrix Blk QC - 250 ppb Group 1 Control 0.0 mgkgiday Group 2 I Mid Dose Group 3 I 10 mg/kg/day Group 4 High Dose 30 mg/ks/day Sample # H 2 0 Blk-1 H20 Blk-2 Rabbit Sera Blk-l Rabbit Sera Blk-2 I05709M-MS I05714M-MS I05709M Wk27 I05714M Wk27 I05715M Wk27 I05718M Wk26 I05720M Wk26 105725111Wk27 I05702M Wk27 105706M Wk27 I05717M Wk27 I05721M Moribund I05723M I05707M Wk27 I05710M Wk27 I05712M Wk26 I05716M Wk26 I05719M Wk27 I05703M Wk27 I05704M Wk27 I0571IM Wk27 I05713M Wk27 I05722M W 7 IOS724M Moribund FACT-TOX-026 Covance# 6329-231 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 04130199, 05/03/99, 05/04/99, 05/05/99 KJH/MEE/HOJ 05/03/99, 05/04/99,05/05/99, 05/25/99, 1 1/02/00, 11/06/00 HOJiKJH Concentration of POAA ug/mL or % Rec. <LOQ (0.00983 ug/mL) <LOQ (0.00983 ugimL) <LOQ (0.00983 ug1mL) <LOQ (0.00983 ug/mL) 79% 108% 0.377 0.219 0.0945 0.206 I 0.308 0.136 47.9 64.5 44.1 I 46.0 60.0 121 47.6 101 78.1 65.2 7.48 58.6 0.877 184 6.65 489 <LOQ Std. Dev. MS/MSD RPD I NA 94% I 17% +I 0.223 0.105 17.4 POAA = Perfluorooctanoate Date EnteredBy: 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 ETS-8-5.1 3M EEnxcvelir97onmental Laboratory Sera Week26,27 TOX-026-sera23 1-8C.xls Analytical Report: FACT-TOX-026 LRN-U2782 5/23/20P01age 232 3M Medical Department Study: T-6889.3 FACT-TOX-026 Covance# 6329-231 Study: Product Number(Test Substance): Matrix: MethodIRevision: Analytical Equipment System Number: Instrument SohareNersion: Filename: R-Squared Value: Slope: Y-Intercept: Date of ExtractiodAnalyst: Date of Analysis/Analyst: Date of Data ReductiodAnalyst: Sample Data Week 28 MONKEY SERA 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 DRBiMEE 06/23/99, 06/24/99, 11/06/00 DRBiMEEiKJH POAA SO62299003-5 & 44-46 062299016-17 06239901 6-1 7 062299040-41 Analytical Report: FACT-TOX-026 LRN-U2782 Dilutions 1/1 1/1 1/50 111 Limit of Quantitation (LOQ): 0.0137 ug/mL Correction factors not applicable for MSiMSD QC data POAA = Periluorooctanoate Date EnteredBy: Date Verifiedi By: 06/23/99, 06/25/99, 11/06/00 LAC 08/04/99 GML, 11/09/00 HOJ ETS-8-5.1 3M EEnxcveli9r7onmental Laboratory Sera Week28 TOX-026-sera231-8C.xls 5/23/20P01age 233 3M Medical Department Study: T-6889.3 FACT-TOX-026 Covance# 6329-231 Study: Product Number(Test Substance): Matrix: MethodiRevision: Analytical Equipment System Number: Instrument SoftwareNersion: Filename: R-Squared Value: Slope: Y-Intercept: Date of ExtractiodAnalyst: Date of AnalysisiAnalyst: Date of Data ReductioniAnalyst: Sample Data Week 28 MONKEY SERA Group Dose Sample # Method Blk Matrix Blk Matrix Blk I QC - 250 ppb Group 1 Control, 0.0 mglkgiday Group 3 I 10 mglkgiday H 20 Blk-l H 20 Blk-2 Rabbit Sera Blk-1 Rabbit Sera Blk-2 Monkey Sera Blk-1 I Monkey SeraBlk-2 I MKS06119-MS-1 MKS06119-MSD-1 105718M I05720M I057 12M I05716M Correction factors not applicable for MSMSD Q C 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 DRBIMEE 06/23/99, 06/24/99. 11/06/00 DRBIMEEKJH Concentration of POAA ug/mL or % Ree. <LOQ <LOQ <LOQ <LOQ I I <LOQ <LOQ I 106% I 104% 0.153 0.209 30.9 21.0 Mean POAA ug/mL <LOQ <LOQ <LOQ 105% 0.181 25.9 RSD Std. Dev. MSIMSD RPD NA NA I I NA I 1% 21.6 0.0391 27.2 7.07 POAA = Pertluorooctanoate Date Enteremy: Date Verified/ By: 06/23/99, 06/25/99, 11/06/00 LAC 08/04/99 GML, 11/09/00 HOJ Analytical Report: FACT-TOX-026 LRN-U2782 ETS-8-5.1 3M EEnxcveli9r7onmental Laboratory Sera Week28 TOX-026-sera23 1-8C.xls 5/23/20P01age 234 3M Medical Department Study: T-6889.3 FACT-TOX-026 Covance# 6329-231 Analytical Report: FACT-TOX-026 LRN-U2782 Study: 6 Month Capsule Toxicity Study with APFO in Cynomolgus Monkeys Product Number(Test Substance): T-6889.2 (POAA) Matrix: Monkey Sera MethodRevision: ETS-8-4.1 & ETS-8-5.1 Analytical Equipment System Number: Soup 020199, Amelia 062498 Instrument SoftwareNersion: MassLynx 3.2 Filename: See Attachments R-Squared Value: See Attachments Slope: See Attachments Y-Intercept: See Attachments Date of ExtractiodAnalyst: 06/11/99 RWW Date of AnalysidAnalyst: 06/22/99,06/23/99 DRBiMEE Date of Data ReductiodAnalyst: 06123/99,06124199, 11/06/00 DRBiMEEiKJH Sample Data Week 30 MONKEY SERA Sample # Extraction Vol. Ratio POAA Std Correetion POAA Dilution POAA Conc. Concentration of POAA Factor Faetor ng/mL ug/mL or % Rec. I II I I I I Matrix Blk H20 Blk-1 1.0 0.9582 1 0.00 H20 Blk-2 1.0 0.9582 1 0.00 Rabbit Sera Blk-l I 1.0 I 0.9582 I 1 1 0.00 I Rabbit Sera BIk-2 1.0 0.9582 1 0.00 <LOQ <LOQ <LOO <LOQ Filenames Blks Grp 1 Grp 3 MS, MSD Mean POAA ug/mL I <LOQ I I <LOQ POAA SO62299003-5 & 44-46 062299018-19 062399020-21 062299040-41 RSD Std. Dev. MS/MSD RPD II NA I NA Dilutions 111 111 1/50 111 MKS06119-MSD-I 1 .o 105718M 0.65 105720M 0.62 I I Grour, 3 I05712M 0.75 10 mgikglday 105716M 0.55 Limit of Quantitation (LOO): 0 Correction factors not applicable for MSiMSD QC data POAA = Perfluorooctanoate Date EnteredBy: Date Verifiedi By: 06/23/99, 06/25/99. 11/06/00 LAC 08/04/99 GML, 11/09/00 HOJ NA 0.9582 0.9582 0.9582 0.9582 1 261 1 86.1 1 104 50 234 50 88.4 104% 0.127 0.161 14.9 7.70 105% 0.144 11.3 1 % 16.6 0.0238 45.2 5.11 ETS-8-5.1 3M EEnxcveilr9o7 nmental Laboratory Sera Week30 TOX-026-sera231-8C.xls 5/23/20P0a1 ge 235 3M Medical Department Study: T-6889.3 Study: Product Number(Test Substance): Matrix: MethodIRevision: Analytical Equipment System Number: Instrument SoftwareNersion: FACT-TOX-026 Covance# 6329-231 6 Month Capsule Toxicity Study with U F O in Cynomolgus Monkeys T-6889.2 (POAA) Monkey Sera ETS-8-4.1 & ETS-8-5.1 Soup 020199, Amelia 062498 MassLynx 3.2 Analytical Report: FACT-TOX-026 LRN-U2782 Group Dose Sample # Method Blk Mafxix Blk Matrix Blk I QC - 250 ppb Group 1 Control, 0.0 mg/kg/day Group 3 I IO mg/kg/day H20 Blk-l H20 BIk-2 Rabbit SeraBlk-l Rabbit Sera Blk-2 I Monkey Sera Blk-l I Monkey SeraBlk-2 MKS06119-MS-I MKS061I9-MSD-1 105718M 105720M I05712M I05716M Correction factors not applicable for MSMSD QC data Concentration of POAA uglmL or % Rec. <LOQ <LOQ <LOQ <LOQ I I <LOQ <LOQ I 106% I 104% 0.127 0.161 14.9 7.70 POAA = Perfluorooctanoate Date EnteredBy: Date Verified/ By: 06/23/99, 06/25/99, 11/06/00 LAC 08/04/99 GML, 11/09/00 HOJ Mean POAA ug/mL <LOQ <LOQ <LOQ 105% 0.144 11.3 RSD Std. Dev. MSIMSD RPD NA NA I NA I 1% 16.6 0.0238 45.2 5.11 ETS-8-5.1 3M EEnxcveli9r7onmental Laboratory Sera Week30 TOX-026-sera231-8C.xls 5/23/20P01age 236 3M Medical Department Study: T-6889.3 FACT-TOX-026 Covance# 6329-231 Analytical Report: FACT-TOX-026 LRN-U2782 Study: Product Number(Test Substance): Matrix: MethodlRevision: Analytical Equipment System Number: Instrument SoftwareNersion: Filename: R-Squared Value: Slope: Y-Intercept: Date of ExtractiodAnalyst: Date of AnalysidAnalyst: Date of Data ReductiodAnalyst: Sample Data Week 32 MONKEY SERA Group Sample # Dose Method Blk Matrix Blk Matrix Blk QC - 250 ppb Group 1 Control, 0.0 mgkgiday Group 3 IO mgkgiday H20 Blk-l H20 Blk-2 Rabbit Sera Blk-1 Rabbit Sera Blk-2 Monkey Sera Blk-I I Monkey SeraBlk-2 I MKS06119-MS-I MKS06119-MSD-1 105718M I05720M 105712M IO571 6M 6 Month Capsule Toxicity Study with APFO in Cynomolgus Monkeys T-6889.2 (POAA) Monkey Sera ETS-8-41 & 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 DRBIMEE 06/23/99,06/24199, 11/06/00 DRBIMEEKJH Extraction POAA Std Vol. Ratio Correction Factor 1 .o 0.9582 1.0 0.9582 1.0 0.9582 1.0 0.9582 I I 1.0 0.9582 1 .o 0.9582 I 1.0 I NA 1 .o NA 0.38 0.9582 0.48 0.9582 0.72 0.9582 0.80 0.9582 POAA I POAA I Dilution Cone. Factor ng/mL 1 0.00 1 0.00 1 0.00 1 0.00 1 0.150 1 0.00 1 264 1 261 Concentration of POAA ug/mL or % Rec. <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ 106% 104% 1 62.6 50 156 50 80.9 0.125 10.4 4.85 Filenames Blks Grp 1 Grp 3 MS, MSD I Mean POAA ug/mL <LOQ <LOQ <LOQ 105% 0.110 7.60 POAA SO62299003-5 & 44-46 062299022-23 062399024-25 062299040-41 I RSD Std. Dev. MSlMSD RPD NA NA NA 1% 0.0216 51.2 3.90 Dilutions 111 111 1/50 111 ETS-8-5.1 3M EEnxcvelir97onmental Laboratory Sera Week32 TOX-026-sera23 1-8C.xls 5/23/20P01age 237 3M Medical Department Study: T-6889.3 FACT-TOX-026 Covance# 6329-231 Study: Product Number(Test Substance): Matrix: MethodRevision: Analytical Equipment System Number: Instrument SofhvareNersion: Filename: R-Squared Value: Slope: Y-Intercept: Date of ExtractiodAnalyst: Date of AnalysisIAnalyst: Date of Data ReductiodAnalyst: Sample Data Week 32 MONKEY SERA I Grouo I Dose Samole # Method Blk Matrix Blk Matrix Blk OC - 250 u.u.b I H20 Blk-l H20 Blk-2 I Rabbit SemBlk-l Rabbit Sera Blk-2 II Monkey SeraBlk-1 Monkey SeraBlk-2 I MKS06119-MS-1 MKS061I9-MSD-1 Group I Control, 0.0 rngikgiday I05718M I05720M I Group 3 10 mgkglday I05712M I05716M 6 Month Capsule Toxicity Study with APFO in CynomolgusMonkeys 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 DREVMEEIKJH I Concentration I Mean I RSD of POAA POAA Std. Dev. ug/mL or % Ree. ug/mL MSlMSD RPD <LOQ I 1 I <LOQ <LOQ NA I <LOO I I I I I <LOQ <LOQ NA I I I I <LOQ <LOQ <LOQ NA I 106% I I 104% 105% 1% 0.0943 19.8 0.125 0.110 0.0216 10.4 51.2 4.85 7.60 3.90 Analytical Report: FACT-TOX-026 LRN-U2782 ETS-8-5.1 3M EnExvcierl 9o7nmental Laboratory Sera Week32 TOX-026-sera231-8C.xls 5/23/20P01age 238 3M Medical Department Study: T-6889.3 Study: Product NumbeNTest Substance): Matrix: MethodlRevision: Analytical Equipment System Number: Instrument SoftwareIVersion: Filename: R-Squared Value: Slope: Y -Intercept: Date of ExtractiodAnalyst: Date of AnalysisIAnalyst: Date of Data ReductiodAnalyst: Sample Data Week 34 MONKEY SERA FACT-TOX-026 Covance# 6329-231 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 DRBiMEE 06/23/99,06/24/99, 11/06/00 DRBIMEEIKJH Analytical Report: FACT-TOX-026 LRN-U2782 Filenames Blks Grp 1 Grp 3 MS, MSD POAA SO62299003-5 & 44-46 062299024-25 062399028-29 062299040-41 Dilutions 111 111 1/50 Ill Limit of Ouantitation (.LOO.),: 0.0137 udmL Correction factors not applicable for MSMSD QC data POAA = Periluorooctanoate Date EnteredBy: Date Verified/ By: 06/23199,06/25/99, 11/06/00 LAC 08/04/99 GML, 11/09/00 HOJ ETS-8-5.1 3M EEnxcveilr97onmental Laboratory Sera Week34 TOX-026-sera231-8C.xls 5/23/20P0a1 ge 239 3M Medical Department Study: T-6889.3 FACT-TOX-026 Covance# 6329-231 Study: Product Number(Test Substance): Mahix: MethodiRevision: Analytical Equipment System Number: Instrument SofhvareIVenion: Filename: R-Squared Value: Slope: Y-Intercept: Date of ExtractioniAnalyst: Date of AnalysdAnalyst: Date of Data ReductioniAnalyst: Sample Data Week 34 MONKEY SERA Group Dose Sample # Method Blk Matrix Blk Matrix Blk I QC - 250 ppb Group 1 Control, 0.0 mgkglday Group 3 IO mgkglday H 2 0 Blk-l H 2 0 Blk-2 Rabbit Sera Blk-l Rabbit Sera Blk-2 Monkey Sera Blk-l I Monkey SeraBIk-2 I MKS06119-MS-1 MKS06119-MSD-I I05718M I05720M I05712M I05716M 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 DRBiMEE 06/23/99, 06/24/99, 11/06/00 DRBiMEE/KJH Concentration Mean RSD of POAA POAA Std. Dev. ug/mL or O h Rec. ug/mL MSIMSD RPD <LOQ <LOQ <LOQ NA <LOQ <LOQ <LOQ NA I 1 I <LOQ <LOQ <LOQ NA I 106% I I 104% 105% 1% 0.0680 29.7 0.104 0.0861 0.0256 I 5.45 52.1 2.49 3.97 2.09 Correction factors not applicable for MSIMSD QC data POAA = Perfluorooctanoate Date Enteremy: Date Verified/ By: 06/23/99, 06/25/99, I1/06/00 LAC 08/04/99 GML, 11/09/00 HOJ Analytical Report: FACT-TOX-026 LRN-U2782 ETS-8-5.1 3M EEnxcveli9r7onmental Laboratory Sera Week34 TOX-026-sera23 1-8C.xls 5/23/20P01age 240 3M Medical Department Study: T-6889.3 FACT-TOX-026 Covance# 6329-231 Analytical Report: FACT-TOX-026 LRN-U2782 Study: Product Number(Test Substance): Matrix: MetbodlRevision: Analytical Equipment System Number: Instrument Software/Version: Filename: R-Squared Value: Slope: Y-Intercept: Date of ExtractiodAnalyst: Date of AnalysidAnalyst: Date of Data ReductiodAnalyst: Sample Data Week 36 MONKEY SERA Group Sample # Dose Method Blk Matrix Blk Matrix Blk QC - 250 ppb Group 1 Coneol, 0.0mgfltg/day H20 Blk-l H 2 0 Blk-2 Rabbit Sera Blk-1 Rabbit Sera Blk-2 Monkey Sera Blk-l Monkey Sera Blk-2 MKS07139-MS-1 MKS07 139-MSD-1 I I057 18M I05720M IO mgkglday IO571 6M 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 GMWDRB 07/15/99, 07/22/99, 11/06/00 GMWDRBKJH Filenames Blks Grp 1 Grp 3 MS, MSD Extraction Vol. Ratio 1.o 1.0 1.0 1 .o 1 .o 1.0 1.0 1.0 0.45 0.48 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 IO IO 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/mLor%Rec. <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 071499021-22 072 199096-97 071499018-19 Box 99-130 Dilutions 1/1 l/l 1/10 111 RSD Std. Dev. MSlMSD RPD NA NA NA 9% 40.1 0.0445 68.4 1.88 Correction factors not applicable for MS/MSD QC data POAA = Perfhorooctanoate Date Enteremy: Date Verified By: 07120199, 07/23/99, 11/06/00 LAC 08/04/99 GML, 11/09/00 HOJ ETS-8-5.1 3M EEnxcvelir97onmental Laboratory Sera Week36 TOX-026-sera23 1-8C.xls 5/23/20P01age 241 3M Medical Department Study: T-6889.3 FACT-TOX-026 Covance# 6329-231 Study: Product Number(Test Substance): Matrix: MethodiRevision: Analytical Equipment System Number: Instnnnent SoftwareNersion: Filename: R-Squared Value: Slope: Y-Intercept: Date of ExbactiodAnalyst: Date of AnalysislAnalyst: Date of Data ReductiodAnalyst: Sample Data Week 36 MONKEY SERA H 2 0 Blk-2 Matrix Blk Rabbit Sera Blk-l Rabbit Sera Blk-2 Matrix Blk Monkey Sera Blk-l QC - 250 ppb Monkey Sera Blk-2 MKS07139-MS- 1 MKSO7I 39-MSD-1 Group 1 I05718M Control, 0.0 mg/kg/day I05720M Group 3 I057 12M 10 mglkgiday I05716M imit of Quantitation (LOO): 0.0137 u.&C 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 GMUDRB 07/15/99, 07/22/99, 11/06100 GMUDRBIKJH Concentration of POAA ug/mL or % Rec. <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 RSD Std. Dev. MSlMSD RPD NA NA NA 9% 40.1 0.0445 68.4 1.88 POAA = Perfluorooctanoate Date EnterediBy: Date Verified By: 07l20199, 01/23/99, lI/O6/00 LAC 08/04/99 GML, 11/09/00 HOJ Analytical Report: FACT-TOX-026 LRN-U2782 ETS-8-5.1 3M EEnxvceilr9o7 nmental Laboratory Sera Week36 TOX-026-sera23 I-8C.xls 5/23/20P01age 242 3M Medical Department Study: T-6889.3 FACT-TOX-026 Covance# 6329-231 Analytical Report: FACT-TOX-026 LRN-U2782 Filenames Blks Grp 1 Grp 3 MS, MSD POAA S071499003,4, 16-17, & 39, 40 071499027-28 07 1499030,072I99098 071499018-19 Box 99-130 Dilutions l/l 1/1 1/1&10 1/1 Group Dose Sample # Extraction Vol. Ratio Method Blk H 2 0 Blk-l 1.0 H20 Blk-2 1.0 Matrix Blk Rabbit Sera Blk-l 1.0 Rabbit Sera Blk-2 1 .o Matrix Blk Monkey Sera BIk-I 1.0 QC - 250 ppb Monkey Sera Blk-2 1.0 MKSO7139-MS-1 1.0 MKS07139-MSD-1 1.0 Group 1 I05718M 0.63 Confxol,0.0 mglkgiday I05720M 0.49 Group 3 I05712M 0.75 10 mgkg/day I05716M 0.75 Limit of Quantitation (LOQ): 0.0137 ug/mL Correction factors not applicable for MSMSD QC data POAA = Perfluorooctanoate Date EnterediBy: Date Verified/ By: 07/20/99, 07/23/99, 11/06/00 LAC 08/04/99 GML, 11/09/00 HOJ 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 IO 1 POAA Cone. ng/mL 0.00 0.00 0.00 0.00 0.00 0.00 228 208 46.8 59.8 22 1 659 Concentration of POAA ug/mL o r % 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% 0.071 1 0.117 2.83 0.842 Mean POAA ug/mL <LOQ <LOQ <LOQ 88% 0.0941 1.84 RSD Std. Dev. MSIMSD RPD NA NA NA 9% 34.5 0.0324 76.5 1.40 ETS-8-5.1 3M EEnxcveilr97onmental Laboratory Sera Week38 TOX-026-sera231-8C.xls 5/23/20P01age 243 3M Medical Department Study: T-6889.3 Study: FACT-TOX-026 Covance# 6329-231 6 Month Capsule Toxicity Study with APFO in Cynomolgus Monkeys Analytical Report: FACT-TOX-026 LRN-U2782 Group Dose Sample # Concentration of POAA Mean POAA RSD Std. Dev. MSlMSD RPD Limit of Ouantitation (.LOO.),: 0.0137 ug/mL Correction factors not applicable for MSMSD QC data POAA = Periluorooctanoate Date EnterediBy: 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 3M EEnxvceilr9o7nmental Laboratory Sera Week38 TOX-026-sera231-8C.xls 5/23/20P01age 244 3M Medical Department Study: T-6889.3 FACT-TOX-026 Covance# 6329-231 Analytical Report: FACT-TOX-026 LRN-U2782 Study: Product Number(Test Substance): Matrix: MethodlRevision: Analytical Equipment System Number: Instrument SoftwareNersion: Filename: R-Squared Value: Slope: Y-Intercept: Date of ExtractiodAnalyst: Date of Analysis/Analyst: Date of Data ReductiodAnalyst: Sample Data Week 40 MONKEY SERA Group Sample # Dose Method Blk H20 Blk-1 H20 Blk-2 Matrix Blk Rabbit Sera Blk-1 Rabbit Sera Blk-2 Matrix Blk Monkey Sera Blk-1 QC - 250 ppb Monkey Sera Blk-2 MKS07139-MS-1 MKS07139-MSD-1 Group 1 I05718M Control, 0.0 mgikglday I05720M Group 3 I05712M 10 mg/kg/day I05716M imit of Quantitation (LOQ): 0 1137 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 GMUDRB 07/15/99, 07/22/99, 11/06/00 GMWDRBKJH Filenames Blks Grp 1 Grp 3 MS, MSD Concentration Mean of POAA POAA Factor ug/mL or O h Ree. ug/mL <LOQ (0.0248 ug/mL) 0.9582 <LOQ (0.0248 ug/mL) <LOQ 0.9582 <LOQ (0.0248 ug/mL) 0.9582 0.9582 0.9582 <LOQ (0.0248 ug/mL) <LOQ 0.00 <LOQ (0.0248 ug/mL) <LOQ (0.0248 ug/mL) I <LOQ 1 228 92% I 208 84% 88% 0.37 0.9582 1 27.8 0.50 0.9582 1 39.4 0.0756 0.0738 0.49 0.9582 2 452 1.77 0.36 0.9582 1 225 0.600 1.18 POAA S071499003,4, 16-17, & 39,40 071499033-34 071499036,072199099 071499018-19 Box 99-130 Dilutions 1/1 111 1/1&2 1/1 RSD Std. Dev. MSlMSD RPD NA NA I NA I 9% 0.00256 69.8 0.827 POAA = Periluorooctanoate Date EnterediBy: Date Verified/ By: 07120199, 07/23/99, 11/06/00 LAC 08/04/99 GML, 11/09/00 HOJ ETS-8-5.1 3M EEnxcveil r9o7 nmental Laboratory Sera Week40 TOX-026-sera231-8C.xls 5/23/20P01age 245 3M Medical Department Study: T-6889.3 FACT-TOX-026 Covance# 6329-231 Study: Product Number(Test Substance): Matrix: Method'Revision: Analytical Equipment System Number: Instrument SoftwareNersion: Filename: R-Squared Value: Slope: Y-Intercept: Date of ExtractiodAnalyst: Date of AnalysisiAnalyst: Date of Data ReductiodAnalyst: Sample 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 GMLiDRB 07/15/99, 07/22/99, 11/06/00 GMLiDRBKJH Group Dose Method Blk I Matrix Blk I Matrix Blk I QC - 250 p..pb Group 1 Control, 0.0 mgkglday Group 3 10 mgkgiday Sample # Concentration of POAA ug/mL or % Ree. H20 Blk-1 <LOQ (0.0248 ug/mL) H 2 0 Blk-2 I<LOQ (0.0248 ug/mL)I I Rabbit Sera Blk-1 k L O Q (0.0248 u-dmL.)I I Rabbit SeraBlk-2 I<LOQ (0.0248 ug/mL)I I Monkey SeraBlk-1 I<LOQ (0.0248 ug/mL)I I I Monkey SeraBlk-2 <LOQ (0.0248 ug/mL)I I MKS07139-MS-1 I 92% I MKS07139-MSD-1 84% I05718M 0.0719 105720M 0.076 I05712M 1.77 105716M 0.600 Mean POAA ug/mL <LOQ <LOQ <LOQ 88% 0.0738 1.18 RSD Std. Dev. MSlMSD RPD I NA I I NA I I NA I 9% 3.47 0.00256 69.8 0.827 Analytical Report: FACT-TOX-026 LRN-U2782 ETS-8-5.1 3M EEnxcveli9r7onmental Laboratory Sera Week40 TOX-026-sera231-8C.xls 5/23/20P01age 246 3M Medical Department Study: T-6889.3 FACT-TOX-026 Covance# 6329-231 Sera QC Summary - TOX026 11/13/1998 01/08/1999 04/29/1999 05/12/1999 05/14/1999 0611 111999 07/1311999 RBS11138-MS RBSl1138-MSD RBS01089-MS RBS01089-MSD I05709M-MS IOS714M-MS RBS05129-MS-3 RBS05129-MSD-3 RBSOS 129-MS-4 RBS05 129-MSD-4 RBS05129-MS-7 RBS05129-MSD-7 RBS05129-MS-8 RBS05 129-MSD-8 RBS05149-MS-11 RBS05149-MSD-11 RBS05149-MS-12 RBS05 149-MSD-12 RBS05149-MS-9 RBS05 149-MSD-9 MKS05149-MS-3 MKS05 149-MSD-3 MKS06119-MS-I MKS06119-MSD-I MKS07139-MS-1 MKS07139-MSD-1 PFOA 109% ** 100% ** 100% 98% 79% 108% 80% 79% 92% ** 85% ** 103% ** 106% ** 103% 121% 116% 95% 99% 80% 104% 109% 83% 91% 106% 104% 92% 84% Average of Monkey Spikes I Standard Deviation Grubbs outliers? Number of monkey spikes 93% Used in Final Report 11% No 8 Used in Final Report Standard Deviation Grubbs outliers? Number of spikes 12% 26 Data not used Analytical Report: FACT-TOX-026 LRN-U2782 ETS-8-5.1 3M EEnxvceilr9o7nmental Laboratory QC Summary TOX-026-sera231-8C.xls 5/23/20P01age 247 3M Medical Department Study: T-6889.3 FACT-TOX-026 Covance# 6329-231 Study: Product Number(Test Substance): Matrix: MethodRevision: Analytical Equipment System Number: Instrument SoftwareNersion: Date of Extraction/Analyst: Date of AnalysisIAnalyst: Date of Data ReductiodAnalyst: Sample Data 6 Month Capsule Toxicity Study with APFO in Cynomolgus Monkeys T-6889.2 (POAA) Monkey Liver Filename: See List to Right FACT-M- I . 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 DRBIIAS 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 Analytical Report: FACT-TOX-026 LRN-U2782 POAA SO62399047-48 & 89-90 Dilutions 1/1 062399059-62 l/l 062399065-68 1/100 06239907 1-74 1/50,100 062399077-80,82 100599077 1/1,2,10,100,200,500 062399085-86 111 reanalyzed on 06/24/99 with confirmation of low recoveries MONKEY LIVER Week 27 Group Sample # Dose Method Blk Matrix Blk QC - 250 ppb Group 1 Control 0.0 mg/kg/day Group 2 Mid Dose 3.0 mg/kg/day 572 1M replacement Group 3 Mid-High Dose 10 mg/kg/day Group 4 LJ:-l. 111 p "n"a.=...,. 30 rng/kg/day POAA = Perfluorooctanoate H 2 0 Blk-3 H 2 0 Blk-4 Rabbit Liver Blk-3 Rabbit Liver Blk-4 I05709M-MS-3 I05709M-MSD-3 I05709M Wk27 105714M Wk27 I05715M Wk27 I05725M Wk27 I05702M Wk27 I05706M Wk27 I05717M Wk27 I05723M I05707M Wk27 105708M Wk27 105710M Wk27 105719M Wk27 105703M Wk27 !05704M W i 2 7 I0571 1M Wk27 105713M Wk27 105722M Wk27 I05724M Moribund Initial Wt. g 1.oooo 1 .oooo 1 .oooo 1.oooo 1.0153 1.0153 1.0153 0.993 1 1.0073 1.0006 1.0104 1.0002 0.993 1 1.0163 1.0143 1.006 1 1.0045 1.0048 1.0036 !.e036 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 :A NA NA NA NA POAA Std Correction POAA Conc. POAA Dilution POAA Calc. Conc. Concentration of POAA Factor ngk Factor n& up/g or % Rec. 0.9582 13.0 1 12.4 <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 238 1 226 0.226 0.9582 43.9 0.9582 160 0.9582 193 0.9582 117 0.9582 173 1 42.0 <LOQ 100 15206 15.2 100 1853 1 18.5 100 11307 11.3 100 16301 16.3 0.9582 232 0.9582 132 0.9582 92.9 100 21917 21.9 50 6294 6.29 100 8864 8.86 0.9582 197 100 I O 0.9582 137 ,,, n "co? 1 <-I,, ".7JOL IU17 IU 0.9582 226 1 18753 1309 i 6026 216 18.8 .,,.1.31 I0.U 0.216 0.9582 44 1 200 83299 83.3 0.9582 746 2 1414 1.41 0.9582 327 500 154369 154 *Overall dilution = 1: 100. 1 uL of the 1: 10 dilution was injected on 6/23/99. LAC 1I/O3/00 Mean POAA ug/g <LOQ <LOQ 68% 0.117 15.3 14.0 * 42.8 RSD Std. Dev. MSMSD RF'D NA NA 18% 62.4 0.0730 19.7 3.02 54.1 7.55 148 63.3 Date EnteredBy: 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 FACT-M-2.0 3M EEnxcvelir9o7 nmental Laboratory LvrO62199 TOX-026-liver23 1-6C.xls 5/23/2001 3:27 PPMage 248 3M Medical Department Study: T-6889.3 FACT-TOX-026 Covance# 6329-231 Analytical Report: FACT-TOX-026 LRN-U2782 Study: Product Number(Test Substance): Matrix: MethodlRevision: Analytical Equipment System Number: Instrument SoftwareNersion: Date of ExtractiodAnalyst: Date of AnalysisiAnalyst: Date of Data ReductiodAnalyst: Sample Data 6 Month Capsule Toxicity Study with APFO in Cynomolgus Monkeys T-6889.2 (POAA) Monkey Liver Filename: See Attachments FACT-M-I. 1 & FACT-M-2.1 R-Squared ValuSee Attachments soup020199 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 MONKEY LIVER Week 27 Group Sample # POAA Dose Calc. Conc. ng/g Method Blk H 2 0 Blk-3 12.4 H 2 0 Blk-4 2.23 Matrix Blk Rabbit Liver Blk-3 13.5 Rabbit Liver Blk-4 15.6 QC - 250 ppb I05709M-MS-3 218 I05709M-MSD-3 I83 Group 1 I05709M Wk27 91.3 Control I05714M Wk27 54.0 0.0 mgkg/day I05715M Wk27 226 I05725M Wk27 42.0 Group 2 I05702M Wk27 15206 Mid Dose 105706M Wk27 18531 3.O mgkgiday 572 1M replacement 105717M Wk27 I05723M 11307 16301 Group 3 I05707M Wk27 21917 Mid-High Dose I05708M Wk27 6294 10 mg/kg/day I05710M Wk27 8864 I05719M Wk27 18753 Group 4 ,n*ig`_uLn uu.x. . I05703M Wk27 ,AC-IA"., .,n~*_l IUJ IU'ilVl V V K L I 1309 iG26 30 mgkglday I05711MWk27 216 I05713M Wk27 83299 I05722M wk27 1414 105724M Moribund 154369 ` O M= Perfluorooctai ate Limit of Quantitation (LOQ) = 75.5 ppb Correction Factors not applicable to MSiMSD QC data Concentration of POAA uglg or % Ree. <LOQ <LOQ <LOQ <LOQ 74% 62% 0.0913 <LOQ 0.226 <LOO 15.2 18.5 11.3 16.3 21.9 6.29 8.86 18.8 1.31 iG.0 0.216 83.3 1.41 154 'Overall dilution = 1: 10 Mean <LOQ I RSD Std. Dev. M S M S D RF'D I NA 68% 18% 0.117 1 0.0730 19.7 1 148 42.8 63.3 1 uL of the 1: I O dilution was injecl 1 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 FACT-M-2.0 3M EEnxvceilr9o7nmental Laboratory LvrO62199 TOX-026-liver23 1-6C.xls 5/23/2001 3:27 PPMage 249 3M Medical Department Study: T-6889.3 Study: Product Number(Test Substance): Matrix: Methodkvision: Analytical Equipment System Number: Instrument SoftwareNersion: Date of Extraction/Analyst: Date of Analysis/Analyst: Date of Data Reduction/Analyst: Sample Data FACT-TOX-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: Soup020 199 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 Analytical Report: FACT-TOX-026 LRN-U2782 POAA AO71399003-4,30-31 071399019-20 0728990 16 07 1399023-24 07 1399015-16 Box 99-13 1 Dilutions 1/1 1/1 1/100 1/1 111 Group Dose Method Blk Matrix Blk QC - 250 ppb Group 1, Wk40 0.0 mgikdday Group 2, Wk20 Group 3, Wk40 10 mg/kg/day Sample # H 2 0 Blk-11 H 2 0 Blk-12 Rabbit Liver Blk-11 Rabbit Liver Blk-12 105718M-MS-11 IO571 8M-MSD-12 I057 ISM I05720M I05721M 105712M I057 16M Initial Wt. g 1 .oooo 1.oooo 1 .oooo 1.oooo 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 Conc. np/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. Conc. np/g 9.15 0.00 2.95 2.83 341 334 13.5 21.6 18321 146 83.3 Concentration of POAA uglg or % Rec. <LOQ <LOQ <LOQ <LOQ 113% 111% <LOQ <LOQ 18.3 0.146 0.0833 Mean POAA ugk <LOQ <LoQ 112% <LOQ NA 0.114 RSD Std. Dev. MSMSD RPD NA NA 2% NA NA 38.6 0.0441 Date EnteredBy: Date Verified/ By: 07/26/99, 11/02/00, 11/03/00, 11/06/00 LAC 08/03/99 GML, 11/08/00 HOJ FACT-M-2.0 3M EEnxvceilr9o7nmental Laboratory LvrWk20-40 TOX-026-liver23 1-6C.xls 5/23/2001 3:27 PPMage 250 3M Medical Department Study: T-6889.3 FACT-TOX-026 Covance# 6329-231 Study: Product Number(Test Substance): Matrix: MethodIRevision: Analytical Equipment System Number: Instrument SoftwareNersion: Date of ExtractiodAnalyst: Date of AnalydAnalyst: 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 Sample # P0.4.4 Dose Calc. Conc. np/g Method Blk H 2 0 Blk-11 9.15 H 2 0 Blk-12 0.00 Matrix Blk Rabbit Liver Blk-11 2.95 - Rabbit Liver Blk-12 2.83 QC 250 p.p.b I05718M-MS-11 341 I057 18M-MSD-12 334 Group 1, Wk40 I I05718M I 13.5 0.0 mg/kg/day 105720M Group 2, Wk2O I05721M 1832 1 Group 3, Wk40 I057 12M 10 mg/kg/day I057 16M 83.3 POAA = Perfluorooctanoate Limit of Quantitation (LOQ) = 75.5 ppb Correction Factors not applicable to MS/MSD Q C data Concentration 1 of POAA ug/g or % Rec. <LOQ <LOQ <LOQ 111% I <LoQ 0.146 0.0833 Mean POAA ugk <LOQ <LOQ 112% <LOQ 0.114 RSD Std. Dev. MSMSD FWD I NA NA 2% NA 38.6 0.0441 Date E n t e r e w y : Date Verified/ By: 07/26/99, 11/02/00, 1 1/03/00, 11/06/00 LAC 08/03/99 GML, 11/08/00 HOJ Analytical Report: FACT-TOX-026 LRN-U2782 Filename: R-Squared Valu Slope: Y-Intercept: FACT-M-2.0 3M EEnxcveil r9o7 nmental Laboratory LvrWk20-40 TOX-026-liver23 1-6C.xls 5/23/2001 3:27 PPMage 251 3M Medical Department Study: T-6889.3 FACT-TOX-026 Covance# 6329-231 Liver QC Summary - TOX026 0612 111999 0711311999 I05709M-MS-3 I05709M-MSD-3 I05718M-MS-11 I057 18M-MSD-12 NA = Not Applicable NR =Not Reported Standard Deviation Grubbs outliers? Number of spikes PFOA 74% 62% 113% 111% Used in Final Report 26% 4 Used in Final Report Analytical Report: FACT-TOX-026 LRN-U2782 FACT-M-2.O 3M EEnxvcierlo9n7mental Laboratory QC Summary TOX-026-liver231-6C.xls 512312001 3:27 PPMage 252 3M Medical Department Study: T-6889.3 FACT-TOX-026 Covance 6329-231 Study Product Number(Test Substance) Matrix. MethodiRevision Analytical Equipment System Number Instrument SoftwareNersion Filename R-Squared Value: Slope Y-Intercept: Dates of ExtractioniAnalystDates of Analysis/Analyst Date of Data Reduction/Analyst: Sample Data MONKEY URINE QC SAMPLES 6 Month Capsule Toxicity Study with APFO in Cynomolgus Monkeys T-6889 2 (POAA) Monkey Urine Blks 08/04/99 Filenames POAA 081399004-5, A000927018,32 Dilutions I/l ETS-8-96 0 & ETS-8-97 0 Soup 020199, Madeline 040198, Amelia 062498 MassLynx 3 2, 3.4 See listing to the right See Attachments Blks 08/05/99 Blks 08/17/99 Blks 08/20/99 Blks 09/21/99 Box A000927034, A001017017-18, 19 111 A081999003,4, SO82499003-4 111 082399003-4,0908006.8 1/1 0922990034,092899059-60 l/l 99-160-1,99-162-3, 99-166-7 See Attachments See Attachments 08/04/99, 08/05/99,08/17/99, 08/20/99,09/21199SEWRWWiMCWSAL 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/DREUSAWGML/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/GMLIDRBiMEEMH/KJH Ext Vol = 1 sincecurvesare extracted at the same ratio as the specimens,2 mL initial and 0.5 mL final volumes MS/MSDs 08/04/99 MSMSDs 08/05/99 MSMSDs 08/17/99 MSMSDs 08/17/99 MSMSDs 08/20/99 MSMSDs 08/20/99 MSMSDs 09/21/99 Analytical Report: FACT-TOX-026 LRN-U2782 POAA A000927045-48, A00IO17020-21 A000927049,52 08 1999016-I9 A000927059-62,AOOlOI7024 082399016-I7 A001017025-26 092299016-17 Dilutions l/l 1/1 l/l l/l 111 l/l l/l Limit of Quantitation(LOQ) 0 0182 udmL NA =Not Analyzed * Confirmedresults LAC 10/26/00 POAA = Perfluormctanoate C8F,,C00- Date Enteremy 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. 10126100, 11/07/00 GMULAC 09/25/00 LAC, 10/26/00KJH, 11/08/00HOJ ETS-8-97 0 3M EEnxvceli9r7onmental Laboratory QC TOX-026-urine2314H XIS 5/23/2P00a1ge 253 3M Medical Department Study: T-6889.3 Study Product Number(Test Substance) Matrix Methdevision Analytical Equipment System Number Instrument SoftwareNersion: Filename. R-Squared Value Slope Y-Intercept: Dates of ExtractiodAnalyst Dates of AnalysisiAnalyst Date of Data ReductiodAnalyst FACT-TOX-026 Covance 6329-231 Analytical Report: 6 Month Capsule Toxicity Studywith 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/l7/99,08/20/99,09/21/99SEWRWWMCWSAL 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 08/18/99, 08120199,08/24/99,09/09/99,09/27/99,09/29/99, 09/29/00, 10/23/00, 11/06/00 PTF/DRB/SAWGMLAAS/MMH PTFAAS/GMUDRB/MEEMMHKJH FACT-TOX-026 LRN-U2782 Date EnteredIBy Date Verifiedi By MKU08179 MSD 1-1 82.4 MKU08179MS 1-2 83 1 MKU08179 MSD 1-2 80 5 MK308179MS 1-3 63.8 MKU08179 MSD 1-3 64 0 MKU08179 MS 1-4 63.0 MKU08179 MSD 1-4 63.3 MKU08209 MS 1-1 47 5 MKU08209 MSD 1-1 46 1 MKU08209 MS 1-2 62 5 MKU08209 MSD 1-2 62 2 MKU09219 MS-I 58 3 MKU09219 MSD-I 60 1 104% 105% 101% 74% 74% 73% 73% 66% 63% 90% 89% 92% 95% 98% 12% 103% 4% 74% 1% 73% 1% 64% 4% 90% 0% 94% 3% 08/20/99,08/23/99,08/24/99,09/10/99. 09/13/99, 09/16/99, 09/17/99. 09/28/99, lO/lO/OO, 10126100, 11/07/00 GMLLAC 09/25/00 LAC, 10/26/WKJH, 11/08/00HOJ ETS-8-97 0 3M EEnxcveli9r7onmental Laboratory QC TOX-026-urine23 1 4 H XIS 5/23/2P001age 254 3M Medical Department Study: T-6889.3 FACT-TOX-026 Covance 6329-231 Analytical Report: FACT-TOX-026 LRN-U2782 Study: Product Number(Test Substance): Matrix: MethodRevision: Analytical Equipment System Number: Instrument SofhvareNersion: Filename: R-Squared Value: Slope: Y-Intercept: Dates of ExtractiodAnalyst: Dates of AnalysisIAnalyst: Date of Data ReductiodAnalyst: Sample Data I Group Dose I IGroup 1 I 0.0 mgkgiday Group 2 3.0 mgkglday I Group 3 Sample # I05709M I057 14M I05715M I05718M 105720M 105725M I05702M I05706M I05717M I05723M I05707M I05708M IO571 OM I05712M I05716M I05719M I05704M iujniM I05713M I05722M I05724M 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 POAA Soup 020199, Madeline 040198, Amelia 062498 Grp 1 081999022-23,082399020-21,092299026,092799050 MassLynx 3.2 Grp 2 090999018-21 See listing to the right Grp 3 090899049,78,09 1099019-21,091599150 See Attachments See Attachments Grp 4 Box 091599035, 122-126 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 SEEIRWWIMCWSAL 08/19/99, 09/08/99,09/09199, 09/15/99 GMWPTFIIASIMMH 08/20/99, 09/09/99, 09/13/99, 09/16/99, 11/06/00 GMWMMWKJH Ext. Vol. = 1 since c w e s are extracted at the same ratio as the specimens, 2 mL initial and 0.5 mL final volumes. Extraction Vol. 1 1 POAA Std Correction 0.9582 I 0.9582 0.9582 POAA Dilution 1 0.9 0.9582 1 1 0.9582 1000 1 0.9582 1000 1 0.9582 1000 1 0.9582 1000 I I I 1 0.9582 2000 1 0.9582 1000 1 0.9582 500 1 0.9582 1000 1 0.9582 8000 I U.Y>L(L nuuu 0.9582 8000 0.9582 8000 1 0.9582 8000 POAA Cone. ng/mL 8.27 0.00 12.0 10.6 10.3 4.36 57.1 62.7 51.1 136 215 208 257 254 252 300 274 106 i23 110 128 176 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 94i 846 983 1350 POAA = Perfluorooctanoate Mean POAA ug/mL <LOQ 73.5 22 1 909 Dilutions 1/1 1/1000 11500, 11250, 1/1000, 1/2000 112000, l/8000 RSD Std. Dev. MSIMSD RPD NA NA 51.9 38.1 56.2 124 29.6 269 Date EnteredBy: Date Verified/ By: 08/23/99, 08/24/99, 09/09/99, 09/13/99, 09/16/99, 09117/99,09/28/99, 11/07/00 GMLiLAC 09/25/00 LAC, 11/08/00 HOJ ETS-8-97.0 3M EEnxcveilr9o7 nmental Laboratory UrineWk2 TOX-026-urine23I4H.xls 5/23/20P01age 255 3M Medical Department Study: T-6889.3 Study: Product Number(Test Substance): Matrix: MethodIRevision: Analytical Equipment System Number: Instrument SofhvareiVersion: Filename: R-Squared Value: Slope: Y-Intercept: Dates of ExtractioniAnalyst: Dates of AnalysisIAnalyst: Date of Data ReductiodAnalyst: MONKEY URINE Week 2 Group Dose Group 1 0.0 mgkgiday Group 2 3.0 mg/kg/day Group 3 10 mg/kg/hy Group 4 Sample # 105709111 I05714M 105715M 105718M I05720M I05725M I05702M I05706M I057 17M 105723M 105707M I05708M I0571OM I05712M IO5716M I05 7 19M 105703M 105704M iO57iiM I05713M I05722M 105724M FACT-TOX-026 Covance 6329-231 Analytical Report: FACT-TOX-026 LRN-U2782 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 MassLynn 3.2 See Attachments See Attachments See Attachments See Attachments 08/04/99, 08/05/99, 08117/99, 08/20/99, 09/21/99 SEEIRWWMCHISAL 08/19/99, 09/08/99, 09/09/99, 09/15/99 GMLIPTFIIASMMH 08/20/99,09/09/99, 09/13/99, 09/16/99, 11/06/00 GMUMMWKJH Sample Data POAA Cone. ng/mL 8.27 0.00 12.0 10.6 10.3 4.36 57.1 62.7 51.1 136 215 208 257 254 252 300 274 106 IL3 110 128 176 Concentration of POAA ug/mL or % Ree. <LOQ <LOQ <LOQ iL0Q cLOQ <LOQ 54.7 60.1 48.9 130 412 199 123 60.9 242 288 525 81 I 94i 846 983 1350 Mean POAA ug/mL <LOQ 73.5 22 1 909 RSD Std. Dev. MSlMSD RPD NA NA 51.9 38.1 56.2 124 29.6 269 ETS-8-97.0 3M EEnxvceilr9o7 nmental Laboratory UrineWk2 TOX-026-urine23 1-4H.xls 5/23/2P00a1 ge 256 3M Medical Department Study: T-6889.3 FACT-TOX-026 Covance 6329-231 Analytical Report: FACT-TOX-026 LRN-U2782 MONKEY URINE Week 4 Group Sample # Dose Group 1 105709M 0.0 mgikglday I057 14M I05715M 105718M I05720M I05725M Group 2 I05702M 3.0 mgikgiday I05706M I05717M I0572 1M Group 3 I05707M 10 mgikgiday I05708M I057 1OM 105712M I05716M I05719M Group 4 I05703M 30 mgkgiday I05704M io5iiiki I05713M 105722M 105724M Limit ofQuantitation (LOQ): 0.0182 ug/mL Extraction POAA Std Vol. Correction Factor 1 0.9582 1 0.9582 I 0.9582 0.80 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 I 0.9582 1 0.9582 1 0.9582 1 0.9582 i 0.9582 1 0.9582 1 0.9582 1 0.9582 NA = Not Analyzemot Applicable POAA Dilution Factor 1 1 1 I 1 5 1000 1000 1000 1000 1000 1000 1000 250 500 1000 4000 2000 i Uou 2000 1000 1000 POAA Cone. nglmL 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'Y Rec. <LOQ <LOQ <LOQ 0.0270 iL0Q 0.839 55.3 55.5 60.0 48.7 195 271 264 35.8 133 242 383 47.6 I86 444 295 83.0 POAA = Periluorooctanoate Mean POAA ug/mL 0.152 54.9 190 240 RSD Std. Dev. MSMSD RPD NA A 0.337 8.43 4.62 48.1 91.6 67.1 161 ETS-8-97.0 3M EEnxcveil r9o1 nmental Laboratory UrineWk4 TOX-026-urine23 1-4H.xls 5/23/20P01age 257 3M Medical Department Study: T-6889.3 FACT-TOX-026 Covance 6329-231 Analytical Report: FACT-TOX-026 LRN-U2782 Study: Product Number(Test Substance): Matrix: MethodiRevision: Analytical Equipment System Number: Instrument SoftwareNersion: Filename: R-Squared Value: Slope: Y-Intercept: Dates of ExtractiodAnalyst: Dates of AnalysisIAnalyst: Date of Data ReductiodAnalyst: MONKEY URINE Week 4 I Group I Sample # Group 1 0.0 mgkglday I05709M I05714M I057 15M I057 18M I05720M I05725M Group 2 3.0 mgkg/day 105702M 105706M I05717M I05721M Group 3 10 mgkglday IOS707M 105708M I05710M 105712M 105716M I05719M Group 4 I05703M 30 mg,kg/day 105704M 105711M 1057 13M 105722M 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, OW17/99, 08120/99,09/21/99 SEEIRWWIMCWSAL 08/19/99,08/23/99, 09/08/99, 09/09/99,09/10/99,09/15/99, 09/21/99 GMWPTF/IASiMMWMEEiIAS 08/20/99,08/24/99,09/09/99,09/13/99, 09/16/99, 09/28/99, 11/06/00 GMMMWIASIKJH Sample Data POAA Cone. ng/mL 10.6 Coneentration of POAA ug/mL or % Ree. <LOQ 0.0270 <LOQ 0.839 57.7 55.3 57.9 55.5 62.6 60.0 50.9 48.7 204 195 Mean POAA Ug/d 0.152 54.9 RSD Std. Dev. MS/MSD RPD 1 NA A 0.337 I 8.43 4.62 277 253 242 99.9 383 I 24.8 47.6 194 186 I232 444 308 295 86.6 83.0 A = Below LOQ 190 I 240 48.1 I 91.6 II 67.1 161 C8F17C00- Date EnteredBy: Date Verifiedi By: 08/23/99, 08124199,09/09/99,09/13/99, 09/16199,09/17/99,09/28/99,11/07/00 GMLILAC 09/25/00 LAC, 11/08/00 HOJ ETS-8-97.0 3M EEnxvceilr9o1nmental Laboratory UrineWk4 TOX-026-urine231-4H.xls 512312P00a1ge 258 3M Medical Department Study: T-6889.3 FACT-TOX-026 Covance 6329-231 Analytical Report: FACT-TOX-026 LRN-U2782 Study: Product Number(Test Substance): Matrix: Methomevision: Analytical Equipment System Number: Instrument SoftwareNersion: Filename: R-Squared Value: Slope: Y-Intercept: Dates of ExtractionIAnalyst: Dates of AnalysislAnalyst: Date of Data ReductiodAnalyst: Sample Data MONKEY URINE Week 6 Group Sample # Dose Group 1 0.0 mgikglday Group 2 3.0 mgikgiday Group 3 IO mgkglday 105709M I057 14M 105715M 105718M I05720M 105725M 105702M 105706M 105717M 105721M 105707M 105708M 105710M 105712M 105716M 105719M 105704M iO57iiM 105713M 105722M 105724M 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 POAA 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, 08117199,08/20199, 09/21/99 SEEYRWWMCWSAL 08/19/99, 08/23/99, 09109199,09115199,09/27/99 GMLPTFIIASIMMWMEUIAS 08/20199, 08/24/99, 09/13/99, 09/16/99, 09/28/99, 11/06/00 GMIJMMHIIASIKJH Ext. Vol. = I 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 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 Conc. 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 4W0 50 3 ._. 111 1000 75.5 1000 328 NA NA NA = Not AnalyzedNot Applicable Concentration of POAA ug/mL or O h Rec. 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 Mean POAA ug/mL 0.0587 65.7 272 Dilutions 111 l/lOOO 1/1000, 11250 1/1OW, 114000 RSD Std. Dev. MSlMSD RPD 122 0.0716 71.4 46.9 51.6 140 Date EnteredIBy: Date Verified/ By: 08/23/99, 08/24/99, 09/13/99,09/16/99, 09/17/99, 09/28/99, 11/07/00 GMUKJH 09/25/00 LAC, 11/08/00 HOJ ETS-8-97.0 3M EEnxcveilr97onmental Laboratory UrineWk6 TOX-026-urine231-4H.xls 5/23/20P01age 259 3M Medical Department Study: T-6889.3 Study: Product Number(Test Substance): Matrix: MethodIRevision: Analytical Equipment System Number: Instrument SofiwareNersion: Filename: R-Squared Value: Slope: Y-Intercept: Dates of ExtractiodAnalyst: Dates of AnalysisiAnalyst: Date of Data ReduchodAnalyst: MONKEY URINE Week 6 Group Dose Grow 1 I Sample # I05709M IO5714M I05715M I05718M 105720M 105725M 3.0 mgkglday Group 3 IO mgkgiday Group 4 30 mgkglday I05706M I057 17M I05721M I05707M 105708M 105710M 305712M 105716M 105719M 105703M 105704M i057i i ivi 105713M 105722M 105724M FACT-TOX-026 Covance 6329-231 Analytical Report: FACT-TOX-026 LRN-U2782 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 08/04199,08/05/99, 08117/99, 08/20/99, 09/21/99 SEEIRWWiMCWSAL 08/19/99, 08/23/99, 09/09/99, 09115/99, 09/27/99 GMWPTFflASIMMHIMEEnAS 08/20/99, 08/24/99, 09/13/99, 09116/99, 09128199, 11/06/00 GMLiMMWIASIKJH Sample Data POAA I I Cone. ng/mL I 179 I 0.00 4.66 26.5 14.9 131 Concentration of POAA ug/mL or % Ree. 0.172 <LOQ <LOQ 0.0254 , <LOO 0.126 58.0 55.6 55.5 53.2 22.8 21.8 164 157 203 195 156 150 227 54.4 122 117 98.4 94.3 92.3 354 50.3 193 ... 111 427 75.5 72.3 328 314 NA NA 0.0587 65.7 128 Std. Dev. MSlMSD RPD 122 0.0716 71.4 46.9 39.1 50.0 Date EnteredBy: Date Verified/ By: 08/23199,08124/99, 09/13/99, 09/16/99, 09/17199,09/28/99, 11/07/00 GMLIKJH 09/25/00 LAC, 11/08/00 HOJ ETS-8-97.0 3M EEnxcveli9r7onmental Laboratory UrineWk6 TOX-026-urine23 1-4H.xls 5/23/20P01age 260 3M Medical Department Study: T-6889.3 FACT-TOX-026 Covance 6329-231 Analytical Report: FACT-TOX-026 LRN-U2782 Study: Product Number(Test Substance): Mahix: MethodiRevision: Analytical Equipment System Number: Instrument SoftwareNersion: Filename: R-Squared Value: Slope: Y-Intercept: Dates of ExtractionlAnalyst: Dates of AnalysidAnalyst: Date of Data ReductionlAnalyst: vlONKJ3Y URINE Week 8 Group Sample # Dose Group 1 I05709M 0.0 mgikglday 105714M 105715M 105718M 105720M I05725M Group 2 I05702M 3.0 mgikgiday 105706M I05717M 105721M Group 3 I05707M 10 mgkgiday I05708M I05710M I05712M I05716M I05719M Group 4 I05703M 30 mgikgiday I05704M iosn iM 105713M 105722M I05724M .imit ofQuantitation (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/MCWSAL 08/19/99, 08/23/99, 09/08/99,09/09/99,09/10/990,9115/99, 09/22/99, 09/27/99 GMLPTFIIASIMMWMEWIAS 08/20/99,08/24/99, 09/09/99,09/13/99, 09116/99,09/27/99,09/28/99, 11/06/00 GMUMMHIMEWIASIKJH Dilutions Ill l/lOOO 11500, 11250, l/lOOO 1/50, 1/10, 1/1000, 1/2000 Extraction Vol. 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0.95 0.80 1 1 NA POAA Std POAA Correction Factor 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 0.9582 1000 1000 0.9582 400 0.9582 1000 0.9582 1000 0.9582 2000 0 9582 1000 0.9582 io 0.9582 IO00 0.9582 2000 0.9582 NA NA =Not Analyzemot Applicable POAA Conc. 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 % Rec. <LOQ <LOQ <LOQ 0.0307 <LOQ <LOQ 49.2 75.4 38.3 27.5 230 22 1 298 75.2 192 220 167 282 3.62 257 193 NA POAA = Pertluorooctanoate Mean POAA ug/mL 0.0161 47.6 206 180 RSD Std. Dev. MSIMSD RPD 58.3 A 0.00940 43.2 20.6 35.5 73.1 60.5 109 Date EnterediBy: Date Verified/ By: 08/23/99, 08/24/99, 08/26/99, 09/09/99, 09/13/99, 09/16/99,09/17/99,09/28/99,1 1/07/00 GMJJLAC 09/25/00 LAC, 11/08/00HOJ ETS-8-97.0 3M EEnxvceilr9o7 nmental Laboratory UrineWk8 TOX-026-urine23 1-4H.xls 5/23/2P00a1 ge 261 3M Medical Department Study: T-6889.3 FACT-TOX-026 Covance 6329-231 Analytical Report: FACT-TOX-026 LRN-U2782 Study: Product NumbeflTest Substance): Matrix: MethodIRevision: Analytical Equipment System Number: Instrument SoftwareNersion: Filename: R-Squared Value: Slope: Y-Intercept: Dates of ExtractiodAnalyst: Dates of AnalysisIAnalyst: Date of Data ReductiodAnalyst: MONKEY URINE Week 8 I I I Group I Dose 0.0 mgikgiday I Group 2 I 3.0 mgikglday Group 3 10 mgkglday I Group 4 I I 30 mgikgiday Sample # I05714M I05715M I05718M I05720M I05725M I05702M I05706M I05717M I05721M I05707M I05708M I05710M I05712M I05716M I05719M I05703M --I05704..M. IU3/111U I05713M 105722M Limit of Quantitation (LOQ): 0.01 82 ugImL 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 SEEiRWWiMCWSAL 08/19/99, 08/23/99, 09/08/99,09/09/99, 09/10/99, 09/15/99, 09/22/99, 09/27/99 GMLTTFIIASiMMWMEEIIAS 08120199, 08/24/99, 09/09/99, 09/13/99,09/16/99, 09/27/99, 09/28/99, 11106100 GMUMMWMEWIASIKJH Sample 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 87.0 279 302 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 I92 220 167 282 3.62 Mean POAA ug/mL 0.0161 A 47.6 206 A = Below LOQ 180 'OAA = Perfluorooctanoatl C8F17C00- RSD Std. Dev. MSlMSD RPD 58.3 0.00940 43.2 20.6 35.5 73.1 60.5 1 a9 Date EnterediBy: 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 GMULAC 09/25/00 LAC, 11/08/00 HOJ ETS-8-97.0 3M EEnxcveilr97onmental Laboratory UrineWk8 TOX-026-urine231-4H.xls 5/23/20P01age 262 3M Medical Department Study: T-6889.3 FACT-TOX-026 Covance 6329-231 Analytical Report: FACT-TOX-026 LRN-U2782 Study: Product Numbe<Test Substance): Matrix: MethodRevision: Analytical Equipment System Number: Instrument SofhvareNersion: Filename: R-Squared Value: Slope: Y-Intercept: Dates of ExtractiodAnalyst: Dates of AnalysisIAnalyst: Date of Data ReductionlAnaIyst: Sample Data Group Dose Group 1 0.0 nigkglday I Group 2 3.0 mgkglday Group 3 IO mgkglday Sample # I05709M I057 14M I057 15M 105718M I05720M I05725M 105702M 105706M I05717M 105721M 105707M 105708M I0571OM I057 12M I05716M 105719M 105713M 105722M I05724M 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 POAA 081999034-35,082399032-33,092299030,092799054 Dilutions 111 MassLynx 3.2 Grp 2 09099904 1-44 l/lOOO See listing to the right Grp 3 090899072-73,80,09 1099035-39 1/500, 11250, 1/1000 See Attachments See Attachments Grp 4 Box 081399017,09 1599059,63,140,092399022 99-160-1,99-162-3, 99-166-7 1/20, 111000, 112000, 114000 See Attachments 08/04/99, 08/05/99, 08/17/99, 08/20/99,09/2 1/99 SEEIRWWIMCWSAL 08/13/99, 08/19/99, 08/23/99, 09/08/99, 09/09/99, 09110199,09115199, 09/22/99, 09/23/99,09/27/99 GMLIPTFIIASIMMWMEEIIAS 08118/99, 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 PTFIDFWGMLIMMWMEE/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. I 1 1 1 I 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 POAA Dilution Factor 1 1 1 1 1 1 1000 1000 1000 1000 1000 1000 1000 250 500 500 4000 1000 LU 2000 1000 NA POAA Cone. ng/mL 10.9 0.00 1.08 23.5 11.8 37.5 34.7 66.5 45.5 20.0 284 263 243 188 197 322 295 342 /3./ 57.0 235 NA Concentration of POAA ug/mL or % Rec. <LOQ <LOQ <LOQ 0.0225 <LOQ 0.0359 33.3 63.7 43.6 19.2 272 252 233 45.1 94.3 154 328 i.4i 109 225 NA Mean POAA ug/mL RSD Std. Dev. MSlMSD RPD 64.1 0.0177 A 0.01 14 46.9 39.9 18.7 52.7 175 92.3 125 359 449 Date EnteredlBy: Date Verified By: 08120199, 08/23/99, 08/24/99,09/09/99, 09/13/99. 09116/99,09/17/99, 09/27/99, 09/28/99, 11/06/00, 11/07/00 GMLLAC 09/25/00 LAC,, 11/08/00 HOJ ETS-8-97.0 3M EEnxcveilr9o7 nmental Laboratory UrineWklO TOX-026-urine231-4H.xls 5/23/20P0a1 ge 263 3M Medical Department Study: T-6889.3 FACT-TOX-026 Covance 6329-231 Analytical Report: FACT-TOX-026 LRN-U2782 Study: Product Numher(Test Substance): Matrix: MethodRevision: Analytical Equipment System Number: Instrument SoftwareNersion: Filename: R-Squared Value: Slope: Y-Intercept: Dates of ExtractionlAnalyst: Dates of AnalysisIAnalyst: Date of Data ReductionlAnalyst: MONKEY URINE Week 10 Group Dose Sample # Group 1 0.0 mglkgiday I05709M I05714M I05715M 105718M 105720M I05725M 3.0 mglkglday Group 3 10 mglkglday I I05706M I05717M I05721M I05707M 105708M I05710M I05712M I05716M I05719M I05704M i057i iili I05713M 105722M 105724M 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, 08117/99, 08/20/99,09/21/99 SEWRWWMCWSAL 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 GMWPTFIIASMMWMEEnAS 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 PTFIDRFVGMUMMWMEWIASiKJH Sample Data POAA Cone. nglmL 10.9 0.00 1.08 23.5 11.8 37.5 66.5 45.5 20.0 284 263 243 188 197 322 NA Concentration of POAA uglmL or % Rec. <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 i.4i 109 225 NA 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 92.3 359 CSF17C00- 125 449 xooctanoate Date EnterediBy: Date Verified By: 08/20/99, 08/23/99, 08124199,09109199,09/13/99, 09/16/99, 09/17/99, 09/27/99, 09/28/99, 1I/O6/00, 11/07/00 GMLILAC 09/25/00 LAC,, 11/08/00 HOJ ETS-8-97.0 3M EEnxcveilr9o7 nmental Laboratory UrineWklO TOX-026-urine23 1-4H.xls 5/23/2P00a1 ge 264 3M Medical Department Study: T-6889.3 FACT-TOX-026 Covance 6329-231 Analytical Report: FACT-TOX-026 LRN-U2782 Study: Product Number(Test Substance): Matrix: Methomevision: Analytical Equipment System Number: Instrument Software/Version: Filename: R-Squared Value: Slope: Y-Intercept: Dates of ExtractioniAnalyst: Dates of AnalysisiAnalyst: Date of Data ReductioniAnalyst: Sample Data MONKEY URINE Week 12 Group Sample # Dose Group 1 0.0 mgkglday 105709M 105714M I05715M I05718M I05720M I05725M 3.0 mg/kg/day Group 3 IO mgkgiday I I05706M 105717M I0572 1M I05707M I05708M I057 1OM I05712M I05716M 105719M 105713M I05722M 105724M 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 MassLyux 3.2 See listing to the right See Attachments See Attachments Filenames Grp 1 Grp 2 Grp 3 Grp 4 Box POAA 081999036-37,082399034-35,092299033,092799057 090999047-50 082499016-21 081399018-19,091599143-1457 99-1 60-1.99-1 62-3 See Attachments 08/04/99, 08/05/99,08/17/99, 08120199, 09/21/99 SEEIRWWIMCWSAL 08/13/99, 08/19/99,08/23/99,08/24/99, 09/09/99, 09/15/99,09/22/99, 09/27/99 GMUSAWMMWIAS 08/18/99, 08/20/99,08/24/99,08/25/99, 09/13/99,09/16/99, 09/27/99, 09/28/99, lI/O6/00, 11/13/00 PTF/DRBIGMUMMWMEE/IASiWH Ext. Vol. = 1 since curves are extracted at the same ratio as the specimens, 2 mL initial and 0.5mL final volumes. Extraction VOl. 1 POAA Std Correction Factor 0.9582 POAA Dilution Factor 1 1 0.9582 1 I : I I 0.9582 1000 0.9582 1000 1 0.9582 1000 1 0.9582 1000 0.9582 1000 0.9582 1000 1 0.9582 1000 U.Y>XL LU 1 0.9582 2000 1 0.9582 20 1 0.9582 NA POAA Concentration Conc. of POAA ng/mL u g / d or % Rec. 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 I 88.2 169 37.8 0.725 I 127 243 70.6 1.35 NA NA Mean POAA Ug/d 0.0141 48.4 201 118 C8F,,CO0 Date EnteredBy: Date Verifiedi By: 08/20/99, 08/23/99,08/24/99, 08/26/99, 09113/99, 09/l6/99,09/17/99,09/28/99, 11/06/00, 11/07/00, 11/14/00 GML/LAC 09/25/00 LAC, 11/08/00 HOJ, 11/15/00 HOJ Dilutions 111 1/1000 l/lOOO 1/20, 1/2000 RSD Std. Dev. MSlMSD RPD 46.1 A 0.00648 38.9 18.8 46.0 92.7 II 93.8 111 ETS-8-97.0 3M EEnxvceilr9o7 nmental Laboratory UrineWkl2 TOX-026-urine23 1-4H.xls 5/23/20P0a1 ge 265 3M Medical Department Study: T-6889.3 FACT-TOX-026 Covance 6329-231 Analytical Report: FACT-TOX-026 LRN-U2782 Study: Product Number(Test Substance): Matrix: Method/Revision: Analytical Equipment System Number: Instrument SoftwareiVenion: Filename: R-Squared Value: Slope: Y-Intercept: Dates of ExtractiodAnalyst: Dates of AnalysisiAnalyst: Date of Data ReductiodAnalyst: MONKEY URINE I tGirrooup Dose II GGrroouupp 11 0.0 mgkg/day Group 2 3.0 mgkgiday Group 3 10 mgkglday Group 4 30 mgikglday Week 12 Sample # I05709M I05714M I05715M I05718M I05720M I05725M I05706M IO5717M I05721M I05708M I05710M I05 7 12M I0571 6M I05719M 105704M Limit of Quantitation (L( I057 13M I05722M I05724M 8): 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 SEJYRWWMCWSAL 08/13/99,08/19/99,08/23/99,08/24/99, 09/09/99, 09/15/99, 09/22/99, 09/27/99 GMUSAWMMWIAS 08118/99,08/20/99,08/24/99,08/25/99, 09/13/99, 09\16/99, 09/27/99,09/28/99, 11/06/00, 11/13/00 PTFII)RB/GMWMMWMEE/IAS/KJH Sample Data Cone. 15.6 I 65.6 40.5 27.8 150 381 270 148 135 88.2 NA = Not Anal Cnneentration of POAA ug/mL or % Ree. <LOQ <LOQ <LOQ 0.0181 0.0184 <LOQ 65.3 62.8 38.8 26.6 170 144 365 259 142 130 175 169 0.725 243 1.35 NA :&Not Applicable Mean POAA udmL 0.0141 A 48.4 201 1 1 8 ~~~ OAA = Perfluorooctano; C8F17C00- RSD Std. Dev. MS/MSD RPD 46.1 0.00648 38.9 18.8 46.0 92.7 93.8 111 Date EnterediBy: 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 GMULAC 09/25/00 LAC, 11/08/00 HOJ, 11/15/00HOJ ETS-8-97.0 3M EEnxcveli9r7onmental Laboratory UrineWkl2 TOX-026-urine23 1-4H.xls 5/23/20P01age 266 3M Medical Department Study: T-6889.3 FACT-TOX-026 Covance 6329-231 Analytical Report: FACT-TOX-026 LRN-U2782 Study: Product Number(Test Substance): Matrix: MethodlRevision: Analytical Equipment System Number: Instrument SoftwareNersion: Filename: R-Squared Value: Slope: Y-Intercept: Dates of ExtractiodAnalyst: Dates of Analysis/Analyst: Date of Data ReductiodAnalyst: Sample Data 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 08l999040,082399052-3,090899020,092299034-35 MassLynx 3.2 See listing to the right See Attachments See Attachments Grp 2 Grp 3 Grp 4 Box 090999051-56 090899021-028 081399023-25,71,092399019 99-160-1, 99-162-3, 99-166-7 See Attachments 08/04/99, 08/05/99, 08117/99,08/20/99, 09/21/99 SEWRWWMCWSAL 08/13/99, 08/19/99, 08/23/99,09/08/99,09/09/990, 9/22/99, 09/23/99 GMLiPTF/IASMMH/MEE/IAS 08/18/99, 08120199, 08/24/99,09/09/99,09/13/99, 09/24/99,09/27/99, 11/06/00 PTF/DRB/GMLMMH/MEEKJH Ext. Vol. = 1 since curves are extracted at the same ratio as the specimens, 2 mL initial and 0.5 mL final volumes Group Dose Group 1 0.0 mgikgiday Group 2 3.0 mgkglday Group 3 IO mgikglday Group 4 30mglkg/day I I Sample # I05709M I05714M I057 15M I05718M I05720M I05725M I05702M I05706M I05717M I05721M I05707M I05708M I057 1OM I05712M I05716M I05719M I05703M I05704M IOSIllM 105713M I05722M I05724M Extraction VOl. 0.15 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 POAA Dilution Factor 1 1 1 1 250 1 1000 1000 POAA Concentration Conc. of POAA ng/d ug/mL or % Rec. 1.19 <LOQ 2.88 <LOQ 4.36 <LOQ 23.4 0.0224 199 47.7 6.52 <LOQ 61.2 58.7 131 1 40.4 I 23.9 22.9 1000 178 1000 193 1000 52.5 1000 120 1000 191 IO 18.9 1000 163 IU NA I NA 171 185 50.3 115 183 0.181 156 <iOQ 134 1.11 NA C8F,,CO0 Mean POAA ug/mL 7.96 63.1 139 72.9 Date EnterediBy: Date Verified/ By: 08/20/99, 08/23/99, 08/24/99, 09/09/99, 09/13199,09/16/99,09/17/99,09/27/99, 09/28/99, 11/06/00, 11/07/00 GMLnAC 09/25/00 LAC, 11/08/00 HOJ Dilutions 111, 1/250 1/1000 l/looO 1/10,11500, l/lOOO RSD Std. Dev. MSlMSD RPD 245 19.5 I 74.8 47.2 37.7 52.2 1I 115 84.0 ETS-8-97.0 3M EEnxcveil r9o7 nmental Laboratory UrineWkl4 TOX-026-urine23 1-4H.xls 5/23/2P00a1ge 267 3M Medical Department Study: T-6889.3 Study: Product NumbeflTest Substance): Matrix: MethodiRevision: Analytical Equipment System Number: Instrument SoRwareNenion: Filename: R-Squared Value: Slope: Y-Intercept: Dates of ExtractioniAnalyst: Dates of AnalysisiAnalyst: Date of Data ReductioniAnalyst: MONKEY URINE Week 14 Group Sample # Dose Group 1 I05709M 0.0 rng/kg/day I05714M I05715M I05718M I05720M I05725M Group 2 I05702M 3.0 mg/kg/day 105706M 105717M I05721M Group 3 I05707M 10 mg/kg/day I05708M I05710M I05712M I05716M I05719M Group 4 I05703M 30 mgkgiday 105704M iO57i iivi I057 13M I05722M I05724M Limit of Quantitation (LOQ): 0.0182 ugImL FACT-TOX-026 Covance 6329-231 Analytical Report: FACT-TOX-026 LRN-U2782 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 SEEIRWIMCWSAL 08/13/99, 08/19/99, 08/23/99, 09/08/99, 09/09/99,09/22/99, 09/23/99 GMLIPTFIIASIMMWMEEIIAS 08/18/99, 08120199, 08/24/99,09/09/99, 09/13/99, 09/24/99, 09/27/99, 11/06/00 P T F I D R B / G M L M M W M E E H POAA Cone. ng/mL Concentration of POAA 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 <iOQ 280 134 116 1.11 NA NA NA = Not Analyzemot Applicable Mean POAA ug/mL RSD Std. Dev. MSIMSD RPD 245 7.96 19.5 74.8 63.1 47.2 37.7 139 52.2 115 72.9 84.0 POAA = Pertluorooctanoate Date Enteremy: Date Verified By: 08120199, 08/23/99.08/24/99, 09/09/99, 09/13/99, 09/16/99, 09117199, 09/27/99,09/28/99, 11/06/00, 11/07/00 GMULAC 09/25/00 LAC, 11/08/00HOJ ETS-8-97.0 3M EEnxcveilr9o7 nmental Laboratory UrineWkl4 TOX-026-urine231-4H.xls 5/23/20P0a1 ge 268 3M Medical Department Study: T-6889.3 FACT-TOX-026 Covance 6329-231 Analytical Report: FACT-TOX-026 LRN-U2782 Study: Product Numbe<Test Substance): Matrix: MethodIRevision: Analytical Equipment System Number: Instrument SoftwareIVersion: Filename: R-Squared Value: Slope: Y-Intercept: Dates of ExtractiodAnalyst: Dates of AnalysisiAnalyst: Date of Data ReductioniAnalyst: Sample Data MONKEY URINE Week 16 Group Sample # Dose Group 1 I05709M 0.0 mgkglday 105714M I05715M I05718M I05720M I05725M Group 2 105702M 3.0 mgkglday I05706M I05717M I05721M Group 3 I05707M 10 mglkglday I05708M I05710M 105712M I05716M I05719M Group 4 I05703M 30 mgkglday I05704M iO57iiM I05713M I05722M 105724M imit of Quantitation (LOQ): 0.0182 ug/mL 6 Month Capsule Toxicity Skdy 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 POAA 08199904243,082399054-55,092299036,092799058 MassLynx 3.2 Grp 2 090999057-62 See listing to the right Grp 3 090899029-036 See Attachments See Attachments Grp 4 Box 081399029,31,72,091599080,092399018 99-160-1,99-162-3, 99-166-7 See Attachments 08/04/99, 08/05/99, 08117199,08/20/99,09/21/99 SEEIRWWIMCWSAL 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 GMWTFIIASIMMHIMEWIAS 08118/99, 08/20199, 08/24199,09/09/99, 09/13199,09/16/99, 09/24/99,09/27/99, 09/28/99, 11/06/00 PTFIDREVGMUMMWMEWIASAUH 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 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 POAA Dilution Cone. Factor ng/d 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 -2-83 5 ir.3 500 234 10 244 NA NA NA =Not AnalyzedNot Applicable Concentration of POAA ug/mL or Oh 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 Mean POAA ug/mL 0.0299 50.2 139 50.2 Dilutions 111 1/1000 1/1000 l/l,l/lO, 11500, 115 RSD Std. Dev. MSlMSD RPD 113 0.0339 41.8 21.0 40.9 57.0 135 67.9 ETS-8-97.0 3M EEnxcveli9r7onmental Laboratory UrineWkl6 TOX-026-urine231-4H.xls 5123/20P01age 269 3M Medical Department Study: T-6889.3 FACT-TOX-026 Covance 6329-231 Analytical Report: FACT-TOX-026 LRN-U2782 Study: Product Number(Test Substance): Matrix: MethodiRevision: Analytical Equipment System Number: Instrument SofhvareiVersion: Filename: R-Squared Value: Slope: Y-Intercept: Dates of ExtractiodAnalyst: Dates of AnalysisIAnalyst: Date of Data ReductiodAnalvst: 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, 08117199,08120199, 09/21/99 SEEIRWWIMCWSAL 08113/99,08/19/99, 08/23/99,09/08/99, 09/09/99, 09/15/99, 09/22/99,09/23/99, 09/27/99 GMWPTFIIASIMMHIMEEIIAS 08118/99,08/20/99, 08/24/99, 09/09/99, 09/13/99, 09/16/99, 09/24/99, 09/27/99, 09/28/99, 11/06/00 PTFIDREVGMLIMMWMEUIASiKJH Sample Data Group Dose Group 1 0.0 mgkgiday GrouD 2 I 3.0 mgikdday Group 3 IO mgikgiday -Grow 4 30 mgikdday 1 Sample # I05709M I05 7 14M I05715M I05718M I05720M I05725M I05702M I05706M I05717M I05721M I05707M I05708M I057 1OM I057 12M I05716M I05719M I05703M I05704M i05ii IM I05713M I05722M I05724M POAA Cone. n%d 17.8 1.62 1.24 99.4 I 38.2 8.01 I 39.1 65.1 76.2 29.3 172 196 169 44.6 102.4 188 18.7 NA Concentration of POAA ug/mL or % rec. <LOQ cLOQ cLOQ 0.0952 0.0366 <LOQ 37.4 62.4 73.1 28.1 !: Mean POAA ug/mL 0.0299 I 150.2 139 0.179 I 136 I 0.380 112 I I 2.34 NA 50.2 RSD Std. Dev. MS/MSD RPD 113 0.0339 41.8 21.0 40.9 57.0 135 61.9 Date EnterediBy: Date Verified/ By: C8F17C00- 08/20/99,08/23/99, 08/24/99, 09/09/99,09/13/99,09/16/99,09117/99, 09/27/99, 09/28/99, 11106100, 11/07/00 GMLLAC 09/25/00 LAC, 11/08/00 HOJ ETS-8-97.0 3M EEnxvceilr9o7 nmental Laboratory UrineWkl6 TOX-026-urine23 1-4H.xls 5/23/20P01age 270 3M Medical Department Study: T-6889.3 FACT-TOX-026 Covance 6329-231 Analytical Report: FACT-TOX-026 LRN-U2782 Study: Product Number(Test Substance): Mabix: Method/Revision: Analytical Equipment System Number: Instrument SoftwareNersion: Filename: R-Squared Value: Slope: Y-Intercept: Dates of ExtractiodAnalyst: Dates of AnalysislAnalyst: Date of Data ReductiodAnalyst: Sample Data MONKEY URINE Week 18 Group Sample # Dose 0.0 mgkglday 3.0 mgkglday I Group 3 10 mgkglday 30 mgkg- lday 1 I05714M I05715M 105718M I05720M I05725M I05706M I0571 7M I05721M I05707M I05708M I05710M I057 12M I05716M I05719M I05704M i057i i M I05713M I05722M I05724M 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 Grp 2 POAA 081999060-61,082399058-59,092299037,092799059 090999063-65,091599118 See listing to the right See Attachments See Attachments Grp 3 Grp 4 Box 090899037-044 081399035,38,091599081,87,092399017 99-160-1, 99-162-3, 99-166-7 See Attachments 08104199, 08/05/99, 08/17/99, 08120/99,09121199 SEWRWWIMCWSAL 08/13/99, 08/19/99, 08/23/99,09108/99,09/09/99, 09/15/99, 09/22/99, 09/23/99,09/27199 GMWPTFIIASIMMWMEEIIAS 08118/99,08/20/99, 08/24199,09109/99, 09/13199,09116/99, 09/24/99, 09/27/99, 09/28/99, 11/06/00 PTFIDRB/GMLIMMWMEEIIAS/KJH Ext. Vol. = 1 since curves are extracted at the same ratio as the specimens, 2 mL initial and 0.5mL final volumes. Dilutions 111 M O O , 1/1000 1/1000 1/1,1/5, 1/10, 11500 Extraction VOl. 1 1 1 0.9 1 1 1 1 1 I: 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. ndmL 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 I I 500 355 LIU 5 84.8 10 174 NA NA Concentration of POAA udmL or % Rec. <LOQ <LOQ <LOQ 0.0752 <LOQ 36.7 58.3 43.7 12.0 157 228 194 75.6 204 258 <LOQ 170 0.iOi 0.406 1.67 NA Mean POAA udmL 0.0256 37.7 186 43.1 RSD Std. Dev. MSlMSD RPD 96.8 0.0248 51.3 19.3 34.3 63.9 196 84.6 Date EnterediBy: Date Verified By: 08/20/99, 08/23/99,08124/99, 09/13/99. 09116/99, 09117/99,09/28/99, 11/06/00, 11/07/00 GMLILAC 09/25/00 LAC, 11/08/00 HOJ ETS-8-97.0 3M EEnxvceilr9o7 nmental Laboratory UrineWkl8 TOX-026-wine23 1-4H.xls 5/23/2P00a1ge 271 3M Medical Department Study: T-6889.3 FACT-TOX-026 Covance 6329-231 Analytical Report: FACT-TOX-026 LRN-U2782 Study: Product Number(Test Substance): Matrix: MethodRevision: Analytical Equipment System Number: Instrument SoftwareNersion: Filename: R-Squared Value: Slope: Y-Intercept: Dates of ExVactiodAnalyst: Dates of AnalysidAnalyst: Date of Data ReductiodAnalyst: MONKEY URINE Week 18 Group Sample #I Dose 0.0 nigkglday Group 2 I 3.0 mgkglday Group 3 IO mgkglday Group 4 30 mgkglday 105714M 105715M 105718M I05720M I05725M I05702M I05706M I05717M I05721M 105707M 105708M I05710M I05712M I05716M 105719M 105703M 105704M i057i iivi 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 MassLynn 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 SEEiRWWiMCWSAL 08/13/99, 08/19199,08/23199, 09/08/99, 09/09/99, 09/15/99, 09/22/99, 09/23/99, 09/27/99 GMLIPTFIIASIMMHIMEEAS 08/18/99, 08/20199,08124/99, 09/09/99, 09/13/99, 09/16/99, 09/24/99,09/27/99, 09/28/99, 11/06/00 PTFIDRBIGMLIMMHIMEEIIASKJH Sample Data POAA Cone. ng/mL 11.0 14.3 7.25 5.32 78.5 4.78 38.3 60.8 45.6 25.0 164 238 203 78.9 213 269 0.0 355 ii0 Concentration of POAA ug/mL or % Ree. <LOQ <LOQ <LOQ <LOQ 0.0752 <LOQ 36.7 58.3 43.7 12.0 157 228 194 75.6 204 258 <LOQ 170 0.2Oi Mean POAA Ug/d 0.0256 43.1 RSD Std. Dev. MS/MSD RPD 96.8 0.0248 196 84.6 Date EnteredBy: 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 GMWLAC 09/25/00 LAC, 11/08/00 HOJ ETS-8-97.0 3M EEnxcvelir97onmental Laboratory UrineWkl8 TOX-026-urine23 1-4H.xls 5/23/20P01age 272 3M Medical Department Study: T-6889.3 FACT-TOX-026 Covance 6329-231 Analytical Report: FACT-TOX-026 LRN-U2782 Study: Product Number(Test Substance): Matrix: MethodiRevision: Analytical Equipment System Number: Instrument SoftwareNersion: Filename: R-Squared Value: Slope: Y-Intercept: Dates of ExtractiodAnalyst: Dates of Analysis/Analyst: Date of Data ReductiodAnalyst: Sample Data 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 081999062-63,082399060-61,092299040,092799060 090999069-72 082499049-53,092899073 08 1399044,73,09159982,89-90 99-160-1,99-162-3 See Attachments 08/04/99, 08/05/99, 08/17/99,08/20/99,09/2l/99 SEERWWMCWSAL 08/13/99, 08/19/99, 08/23/99,09/09/99, 09/15/99, 09/22/99, 09/27/99,09/28/99 GMLMMWIASMEE 08118/99, 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/MEEIIAS/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 111 l/lOOO l/lOOO 1/1,1/10,1/20,1/500 Group Dose Group 1 0.0 mgkgiday Group 2 3.0 mgkglday Group 3 IO mgikglday Group 4 30 mgikgiday I Sample # 105709M 105714M I05715M 105718M 105720M 105725M 105702M 105706M 105717M 105721M 105707M 105708M+ 105710M+ I057 12M+ 105716M+ 105719M+ 105703M I05704M IO5illM 105713M 105722M 105724M Extraction Vol. 1 1 1 1 1 1 I 1 1 1 1 1 1 1 1 1 1 1 Ij 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 1 1 1 1 1 1 1000 1000 1000 1000 5000 1000 1000 1000 1000 1000 1 500 20 NA POAA Conc. ng/d 17.7 7.61 13.4 22.3 47.1 5.20 51.0 69.0 46.2 51.1 344 378 185 61.9 109 18.8 379 195 122 NA Concentration of POAA u g / d or % Rec. <LOQ <LOQ <LOQ 0.0213 0.0451 <LOQ 48.9 66.1 44.3 48.9 1648 * 362 177 59.3 104 18.0 I* 0.363 124 93.3 2.33 NA Mean POAA ug/d 0.021 1 52.1 144 44.0 , RSD Std. Dev. MSlMSD RPD 61.3 0.0130 18.5 9.63 93.9 135 136 59.9 ** Conc. was below the LOQ C8F,,CO0 + CCVs analyzed prior to these samples were not within +/- 30% criteria. LAC 11/16/00 Date EnterediBy: 08/20/99,08/23/99,08/24/99, 08/25/00, 09113/99, 09/16/99, 09117/99, 09/28/99, 09/29/99, 11/06/00, 11/07/00, 11/14/00 GMULAC Date Verified/ By: 09/25/00 LAC, 11/08/00 HOJ, 11/15/00 HOJ * This sample was not diluted 115000; a 2uL injection of DlOOO was analyzed instead. All other injections were IO uL. This provided a 1/5 dilution of DlOOO for a total of D5000. A 1/4000.dilutionof 105707M was also analyzed, but was just out of calibration range. GMLKJH 9/29/99 ETS-8-97.0 3M EEnxvceilr9o7 nmental Laboratory UrineWk20 TOX-026-urine231-4H.xls 5/23/2P00a1 ge 273 3M Medical Department Study: T-6889.3 FACT-TOX-026 Covance 6329-231 Analytical Report: FACT-TOX-026 LRN-U2782 Study: Product NumbeflTest Substance): Matrix: MethodlRevision: Analytical Equipment System Number: Instrument SoftwareNersion: Filename: R-Squared Value: Slope: Y-Intercept: Dates of ExtractiodAnalyst: Dates of AnalysisiAnalyst: Date of Data ReductiodAnalyst: MONKEY URINE Week 20 I Group Dose Group 1 0.0 mg/kg/day Group 2 3.0 mgikglday Sample # I05709M I057 14M I05715M I05718M I05720M I0572SM I05702M I05706M I05717M I05721M 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 SEWRWWIMCWSAL 08/13/99, 08/19/99,08/23/99,09/09/99, 09/15/99, 09/22/99. 09/27/99,09/28/99 GMLIMMWIASIMEE 08/18/99, 08120199, 08/24/99,09/13/99, 09/16/99,09/27/99, 09/28/99, 09/29/99, 11/06/00, 11/13/00 PTFDRB/GMUMMH/MEEIIAS/KJH Sample Data POAA Conc. ng/d 17.7 7.61 13.4 22.3 47.1 5.20 51.0 69.0 I 46.2 51.1 Concentration of POAA ug/mL or YORec. cLOQ <LOQ CLOQ 0.0213 0.045 1 CLOQ 48.9 66.1 44.3 48.9 Mean POAA ug/mL RSD Std. Dev. MSlMSD RPD 0.021 1 52.1 61.3 0.0130 18.5 9.63 10 mgikglday Group 4 30 mgikglday I05708M+ I0571OM+ I05712M+ I05716M+ I057 19M+ I05703M ----... I05704M , IUJ I I l l V l I05713M I05722M I05724M 378 185 61.9 109 18.8 379 ..^ 258 14u 195 122 NA 362 177 59.3 104 18.0 *I 0.363 124 O.i% 93.3 2.33 NA 136 44.0 59.9 ** Conc. was below the LOQ + CCVs analyzed prior to these samples were not within +/- 30% criteria. LAC 11/16/00 C8F17COO- Date EnteredBy: 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 GMLLAC Date Verified/ By: 09/25/00 LAC, 11/08/00 HOJ, 11/15/00 HOJ * This sample was not diluted li5000; a 2uL injection of DlOOO was analyzed instead. All other injections were 10 uL. This provided a 1/5 dilution of DlOOO for a total of D5000. A 1/4000 dilubon of I05707M was also analyzed, but was just out of calibration range. GMLKJH 9/29/99 ETS-8-97.0 3M EEnxcvelir97onmental Laboratory UrineWk2O TOX-026-urine231-4H.xls 5/23/20P01age 274 3M Medical Department Study: T-6889.3 FACT-TOX-026 Covance 6329-231 Analytical Report: FACT-TOX-026 LRN-U2782 Study: Product Number(Test Substance): Matrix: MethodRevision: Analytical Equipment System Number: Instrument SoftwareNersion: Filename: R-Squared Value: Slope: Y-Intercept: Dates of ExtractiodAnalyst: Dates of AnalysisIAnalyst: Date of Data ReductiodAnalyst: Sample Data MONKEY URINE Week 22 Group Sample # Dose Group 1 0.0 mglkglday I05709M I05714M I05715M I05718M I05720M 105725M 3.0 mgkgiday Group 3 10 mg/kg/day 105706M I05717M 105721M I05707M+ 105708M+ I0571OM+ I05712M 105716M+ I05719M+ 30 mglkglday I05704M iO5711M IO57 13M 105722M I05724M imit of Quantitation (LOQ): 0.0182 uglmL 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 081999066-67,082399064-5,092299041,09279906l 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 SEEIRWWIMCWSAL 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/SAHlPTFIMMWIASIMEE 08/20/99,08/24/99, 08/25/99, 09/13/99, 09/16/99, 09/24/99,09/27199, 09/28/99, 11/06/00, 11/13/00 PTFIDRBIGMUMMHlMEEIIASiKTH 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 I 1 1 1 I I 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 Dilution Factor 1 1 1 1000 POAA Cone. ng/mL 35.9 29.9 14.8 271.0.79 8.21 197 NA 175 Concentration of POAA ug/mL or % Ree. 0.0344 0.0287 <LOQ <LOQ 0.0208 <LOQ 53.7 189 NA 168 Mean POAA u%d 0.0231 95.8 1 0.9582 246 235 1 0.9582 90.7 86.9 1 0.9582 150 144 1 0.9582 1000 266 255 158 0.9582 367 0.352 0.9582 1000 266 255 0.9582 348 0.334 0.9582 247 237 0.9582 45.3 0.868 NA NA 98.5 NA = Not Analyze1 Dilutions 111 1/500, 1/1000 1/1000 111,1110,1120, l/lOOO RSD Std. Dev. MS/MSD RPD 1I 29.8 0.00688 I 84.4 I 80.8 49.7 78.4 136 134 + CCVs analyzed prior to these samples were not within +/- 30% criteria. LAC 11/16/00 Date EnterediBy: 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 GMLILAC Date Verified By: 09/25/00 LAC, 1II08lOO HOJ, 11/15/00 HOJ ETS-8-97.0 3M EEnxcveli9r1onmental Laboratory UrineWkZ2 TOX-026-urine231-4H.xls 5/23/20P01age 275 3M Medical Department Study: T-6889.3 FACT-TOX-026 Covance 6329-231 Analytical Report: FACT-TOX-026 LRN-U2782 Study: Product NumbeflTest Substance): Matrix: MethodIRevision: Analytical Equipment System Number: Instrument Software/Version: Filename: R-Squared Value: Slope: Y-Intercept: Dates of ExtractiodAnalyst: Dates of Analysis/Analyst: Date of Data ReductiodAnalvst: MONKEY URINE Week 22 Group Dose Sample # Group 1 0.0 mgkgiday Grouo 2 3.0 mgkgiday Group 3 10 mgkgiday 105709M I05714M I05715M I05718M I05720M I05725M I05702M I05706M 105717M I05721M I05707M+ I05708M+ 10571OM+ I05712M I05716M+ I057 19M+ I05704M I05711M I05713M I05722M I05724M 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, 08/20/99,09/2l/99 SEWRWW/MCWSAL 08/19/99,08/23/99, 08/24/99, 09/09/99,09/l5/99, 09/22/99, 09/23/99, 09/27/99 GML/SAWPTFIMMWIAS/MEE 08/20/99,08/24199,08/25/99, 09/13/99, 09/16/99, 09/24/99, 09/27/99, 09/28/99, lI/O6/00, 11/13/00 PTF/DFWGMUMMHIMEWIAS/KJH Sample Data POAA Cone. nun& 35.9 29.9 7.09 14.8 21.7 8.21 46.7 112 197 NA 60.6 175 246 90.7 I I 150 266 Concentration of POAA ug/mL or % Rec. 0.0344 0.0287 <LOQ <LOQ 0.0208 <LOQ 44.7 53.7 189 NA 58.0 168 235 86.9 144 255 266 255 348 0.334 241 237 45.3 0.868 NA NA Mean POAA Ug/d 0.023 1 95.8 158 98.5 RSD Std. Dev. MSlMSD RPD 29.8 0.00688 84.4 80.8 49.7 78.4 136 134 C8Fl7COO- + CCVs analyzed prior to these samples were notwithin +/- 30% criteria. LAC 11/16/00 Date EnterediBy: 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 GMULAC Date Verified By: 09/25/00 LAC, 11/08/00 HOJ, 11/15/00 HOJ ETS-8-97.0 3M EEnxcveilr9o7 nmental Laboratory UrineWk22 TOX-026-urine231-4H.xls 5/23/20P01age 276 3M Medical Department Study: T-6889.3 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: MethodRewsion: Analytical Equipment System Number: Instrument SoftwareiVersion: Filename: R-Squared Value: Slope: 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 081999068-69,082399066-67,09229904243 090999078-82 082499064-65,67-69,09109904l 081399055,65, 09 1599083,9596 99-160-1,99-162-3 Y-Intercept: Dates of ExtractiodAnalyst: Dates of Analysis/Analyst: Date of Data ReductiodAnalyst: Sample Data MONKEY URINE Week 24 See Attachments 08/04/99, 08/05/99, 08/17/99, 08/20/99, 09/21/99 SEWRWWIMCHISAL 08/13/99, 08/19/99,08/23/99, 08/24/99, 09/09/99, 09/10/99,09/15/99, 09/22/99 GMWSAWPTFIMMWIAS 08/18/99, 08/20/99, 08/24/99,09/13/99,09/16/99,09/27/99, 11/06/00, 11/13/00 PTFIDRBIGMLIMMHJMEEIH Ext. Vol. = 1 since curves are extracted at the same ratio as the specimens, 2 mL initial and 0.5 mL final volumes. Group Dose Sample # Extraction VOl. POAA Std Correction Factor POAA Dilution Factor POAA Conc. n%d Concentration of POAA u g / d or % Ref. Mean POAA ug/d Group 1 0.0 mgkglday I05709M 105714M 105715M 0.4 0.9582 1 7.42 1 6.32 1 4.53 <LOQ <LOQ <LOQ 105718M 1 3.10 <LOQ I05720M 1 22.2 0.0213 I05725M 0.9582 1 5.39 <LOQ 0.0125 Group 2 3.0 mgkgiday I05702M I05706M 105717M 0.9582 1000 38.4 36.8 0.9582 1000 55.6 53.3 0.9582 1000 50.9 48.7 I05721M 0.9582 1000 NA NA 46.3 Group 3 I05707M+ 1 0.9582 1000 192 184 IO mgkgiday I05708M+ 0.9582 1000 226 216 I05710M 0.9582 1000 222 212 105712M+ 1000 56.3 54.0 105716M+ 1000 117 112 105719M+ 1 0.9582 1000 170 163 157 Group 4 30 mgkglday I05704M 10571 1M 1 319 1 0.9582 500 324 1 0.9582 1 112 0.306 155 0.107 I05713M 1 0.9582 500 259 124 I05722M 1 0.9582 IO 25.9 0.248 I05724M 1 0.9582 NA NA NA 56.0 .imit of Quantitation A = Below LOQ C,F,,COO- + CCVs analyzed prior to these samples were not within +/- 30% criteria. LAC 11/16/00 Date EnterediBy: 08120199, 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 GMIJLAC Date Verified/ By: 09/25/00 LAC, 11/08/00HOJ, 11/15/00 HOJ Analytical Report: FACT-TOX-026 LRN-U2782 Dilutions 111 1/1000 l/lOOO l/l,l/l0,l/500 RSD Std. Dev. MSlMSD RPD 60.1 A 0.00749 18.4 8.52 40.3 63.3 138 77.2 ETS-8-97.0 3M EEnxcveilr9o7 nmental Laboratory UrineWk24 TOX-026-urine231-4H.xls 5/23/20P0a1 ge 277 3M Medical Department Study: T-6889.3 Study: Product Number(Test Substance): Matrix: Method/Revision: Analytical Equipment System Number: Instrument SoftwareNersion: Filename: R-Squared Value: Slope: Y-Intercept: Dates of ExtractionlAnalyst: Dates of Analysis/Analyst: Date of Data ReductiodAnalyst: MONKEY URINE Week 24 Group Dose Sample # Group 1 I05709M 0.0 mgkg/day I05714M 105715M I05718M I05720M I05725M Group 2 I05702M 3.0 mgkg/day I05706M I05717M I05721M Group 3 I05707M+ 10 mgkg/day I05708M+ I0571OM 105712M+ 105716M+ 105719M+ Group 4 I05703M 30 mgkgiday I05704M I05711M I05713M I05722M I05724M Limit of Quantitation (LOQ): 0.0182 ug/mL FACT-TOX-026 Covance 6329-231 Analytical Report: FACT-TOX-026 LRN-U2782 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 SEERWWIMCWSAL 08/13/99,08/19/99, 08/23/99,08/24/99, 09/09/99, 09/10/99, 09/15/99, 09/22/99 GMUSAWPTFIMMWIAS 08118/99,08/20/99, 08/24/99,09/13/99, 09/16/99,09/27/99, 11/06/00, 11/13/00 PTFIDREUGMUMMWMEEIKJH POAA Concentration Mean Conc. of POAA POAA ng/mL uglmL 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 I63 157 319 0.306 324 155 112 0.107 259 124 25.9 0.248 NA NA 56.0 NA =Not Analyzemot Applicable POAA = Pertluorooctanoate RSD Std. Dev. MSlMSD RPD 60.1 0.00749 18.4 8.52 40.3 63.3 138 77.2 ETS-8-97.0 3M EEnxvceilr9o7 nmental Laboratory UrineWk24 TOX-026-urine231-4H.xls 5/23/2P00a1ge 278 3M Medical Department Study: T-6889.3 FACT-TOX-026 Covance 6329-231 Analytical Report: FACT-TOX-026 LRN-U2782 Study: Product Number(Test Substance): Matrix: MethodiRevision: Analytical Equipment System Number: Instrument SofhvareNersion: Filename: R-Squared Value: Slope: Y-Intercept: Dates of ExtractiodAnalyst: Dates of AnalysidAnalyst: Date of Data ReductiodAnalyst: Sample Data IONKEY URINE Week 26 Group Sample # Dose Group 1 0.0 mgkglday Group 2 3.0 mgikglday Group 3 10 mgkglday Group 4 30 mgkgiday I05709M I05714M I05715M 105718M I05720M I05725M I05702M I05706M I05717M I05721M I05707M+ I05708M+ I057 1OM I057 12M+ 105716Mc I05719M I05703M I05704M I05711M I05713M I05722M I05724M 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 See listing to the right See Attachments See Attachments Grp 2 Grp 3 Grp 4 Box 090999083-85 082499072-75,091099042,092799046 081399066,091599099-100,092399015,23 99-160-1, 99-162-3 l/lOOO 1/1000 l/l,1/l0,l/250 See Attachments 08/17/99 SEWRWWIMCWSAL 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 GMUSAWPTFhiMHlIASIMEE 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/GMLMMWMEEIIAS/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 0.9582 0.9582 0.9582 0.9582 POAA Dilution Factor 1 1 1 1 1 1 1000 1000 1000 1000 1000 1000 1000 1000 1000 1000 1 250 1 250 1 NA POAA Concentration Cone. of POAA ndmL u d m L or % Rec. 21.7 0.0208 10.0 <LOQ 7.19 <LOQ 8.53 iLOQ 83.2 0.0797 4.29 <LOQ 38.6 37.0 55.9 53.6 67.0 64.2 NA NA 268 256 69.6 66.6 53.0 50.8 75.5 72.3 107 103 112 107 233 0.223 241 57.7 71.2 0.0682 158 37.8 I 225 NA 0.215 NA Mean POAA UdmL 0.0268 51.6 109 19.2 RSD Std. Dev. MSlMSD RPD 98.7 0.0265 26.6 13.7 68.8 75.2 141 27.0 + CCVs analyzed prior to these samples were not within +/- 30% criteria. LAC 11/16/00 . Date EnterediBy: 08/23/99,08/24/99, 08/25/99,09/13/99,09/16/99,09/l7/99, 09/27/99, 09/28/99, 11/06/00, 11/07/00, 11/14/00 GMLLAC Date Verified/ By: 09/25/00 LAC, 11/08/00 HOJ, 11/15/00 HOJ ETS-8-97.0 3M EEnxcveil r97onmental Laboratory UrineWkZ6 TOX-026-urine231-4H.xls 5/23/20P0a1 ge 279 3M Medical Department Study: T-6889.3 FACT-TOX-026 Covance 6329-231 Analytical Report: FACT-TOX-026 LRN-U2782 Study: Product Numbe<Test Substance): Matrix: MethodJRevision: Analytical Equipment System Number: Instrument SoftwareNersion: Filename: R-Squared Value: Slope: Y-Intercept: Dates of ExIxactiodAnalyst: Dates of AnalydAnalyst: Date of Data ReductiodAnalyst: MONKEY URINE Week 26 Group Sample # Dose Group 1 I05709M 0.0 mglkgiday 105714M I05715M I05718M 105720M I05725M Group 2 105702M 3.0 mglkgiday I05706M I05717M 105721M Group 3 I05707M+ 10 mglkglday I05708M+ 105710M 105712M+ 105716M+ 105719M Group 4 105703M 30 mgkgiday I05704M I0571 IM I05713M I05722M I05724M .imit 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 SEEIRWWIMCWSAL 08/13/99, 08/19/99, 08/23/99, 08/24/99, 09/09/99,09110/99, 09/15/99, 09/22/99, 09/23/99,09/27/99 GML/SAWPTF/MMWIASIMEE 08/18/99, 08120199, 08/24/99, 08/25199,09/13/99, 09/16/99, 09/24/99,09/27/99, 09/28/99, 11/06/00, 11/13/00 PTF/DRB/GMUMMHlMEEIIAS/KJH Sample Data POAA Concentration Mean RSD Cone. og/mL of POAA ug/mL o r % Rec. POAA ug/mL Std. Dev. MS/MSD RPD 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 NA =Not AnalyzediNot Applicable POAA = Perfluorooctanoate ETS-8-97.0 3M EEnxvceilr9o7 nmental Laboratory UrineWkZ6 TOX-026-urine231-4H.xls 512312P00a1 ge 280 3M Medical Department Study: T-6889.3 FACT-TOX-026 Covance 6329-231 Analytical Report: FACT-TOX-026 LRN-U2782 Study: Product Number(Test Substance): Matrix: Methodkvision: Analytical Equipment System Number: Instrument SoftwareNersion: Filename: R-Squared Value: Slope: Y-Intercept: Dates of ExtractiodAnalyst: Dates of Analysis/Analyst: Date of Data Reduction/Analyst: Sample Data MONKEY URINE Week 28 Group Sample # Dose Group 1 0.0 mg/kg/day Group 3 10 mg/kg/day I057 18M 105720M 105712M I05716M 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 See listing to the right Grp 1 Grp 3 POAA 081999074-75 082499052-53 Dilutions 1/1 1/10 See Attachments Box 99- 160-1 See Attachments See Attachments 08/11/99 SEEIRWWIMCWSAL 08/19/99,08/24/99 GMUSAWPTF 08/20/99,08/25~99,11/06/00GML/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 VOl. 1 1 1 1 POAA Std Correction Factor 0.9582 0.9582 0.9582 0.9582 POAA Dilution Factor 1 1 10 10 POAA Conc. ndmL 228 11.9 32.8 35.5 Concentration of POAA udmL or % Rec. 0.218 <LOQ 0.314 0.340 Mean POAA UdmL ~~ 0.118 0.327 RSD Std. Dev. MS/MSD RF'D 0.142 0.0182 Date EnteredlSy: ."rerif,e& By; 08/23/99,08/26/99, 11/07/00 GMLILAC nn , I ) c !An UYILJIUU r A r. LAL, 3 1 3 l l Um 8 l (UnUn rnlG 1 ETS-8-97.0 3M EEnxvceilr9o7nmental Laboratory UrineWk28 TOX-026-urine23 1-4H.xls 5/23/2P00a1ge 281 3M Medical Department Study: T-6889.3 Study: Product Number(Test Substance): Matrix: MethodRevision: Analytical Equipment System Number: Instrument SoftwareNersion: Filename: R-Squared Value: Slope: Y-Intercept: Dates of Extraction/Analyst: Dates of AnalysidAnalyst: Date of Data Reduction/Analyst: FACT-TOX-026 Covance 6329-231 Analytical Report: FACT-TOX-026 LRN-U2782 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 OS/ 17/99 SEE/RWWIMCWSAL 08/19/99,08/24/99 GMUSAWPTF 08/20/99,08/25/99, 11/06/00 GML/PTF/KJH Sample Data Dose Group 1 0.0 mg/kg/day Group 3 10 mg/kg/day I05718M I05720M 105712M I057 16M Cone. ng/mL 228 11.9 32.8 35.5 of POAA ug/mL or % Ree. 0.2 18 <LOQ 0.314 0.340 POAA ug/mL 0.118 0.327 Std. Dev. MS/MSD RPD 0.142 0.0182 Date EnteredBy: Date Verifiedi By: 08/23/99,08/26/99, 11/07/00 GMLLAC 09/25/80 LAC, i i/08/60 IiOi ETS-8-97.0 3M EEnxvceilr9o7nmental Laboratory UrineWk28 TOX-026-urine23 1-4H.xls 5/23/20P0a1 ge 282 3M Medical Department Study: T-6889.3 FACT-TOX-026 Covance 6329-231 Analytical Report: FACT-TOX-026 LRN-U2782 Study: Product Number(Test Substance): Matrix: Method/Revision: Analytical Equipment System Number: Instrument SoftwareNersion: 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 30 Group Sample # Dose ~ Group 1 0 0 mg/kg/day Group 3 10 mg/kg/day I05718M I05720M 105712111 I05716M 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 Filenames POAA Dilutions MassLynx 3.2 Grp 1 08 1999078-79 1/1 See listing to the right See Attachments Grp 3 Box 091599018, 159 99- 160-1 1/1&10 See Attachments See Attachments 08/17/99 SEERWWIMCWSAL 08/19/99,09/15/99 GML 08/20/99,09/16/99,11/06/00 GMLKJH 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 POAA Std Correction Factor 0 9582 0 9582 0 9582 0 9582 POAA Dilution Factor 1 1 1 10 POAA Cone. ne/mL 2 73 0 00 290 46 5 Concentration of POAA ue/mL or % Rec. <LOQ <LOQ 0 278 0 445 Mean POAA ue/mL <LOQ 0 361 RSD Std. Dev. MS/MSD RPD NA 0 118 Date Enteremy: Date Verified/ By: 08/23/99,09/16/99,09/17/99, 11/07/00 GMLLAC 09/25/00 LAC, 11/08/00 HOJ ETS-8-97.0 3M EEnxvceilr9o7nmental Laboratory UrineWk30 TOX-026-urine23 1-4H.xls 5/23/2P00a1ge 283 3M Medical Department Study: T-6889.3 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: 8123199 GML, 8/24/99 GML, 9ETS-8-96.0 & ETS-8-97.0 Analytical Equipment System Number: Soup 020199, Madeline 040198, Amelia 062498 Instrument SoRwareNersion: MassLynx 3.2 Filename: See Attachments R-Squared Value: See Attachments Slope: See Attachments Y-Intercept: See Attachments Dates of Extractiodhalyst: 08/17/99 SEEIRWWlMCWSAL Dates of AnalysidAnalyst: Date of Data ReductiodAnalyst: 08/19199,09/15/99 GML 08/20/99, 09/16/99, 11/06/00 GMJJKJH Sample Data MONKEY URINE Week 30 Group Dose Sample # POAA Conc. ng/mL Concentration of POAA ug/mL or % Rec. Mean POAA ug/mL RSD Std. Dev. MSlMSD RPD Group 1 0.0 mg/kg/day Crow 3 I05718M I05720M 1~~ 057~12M I05716M 2.73 0.00 I 290 46.5 <LOQ <LOQ 0.278 0.445 <LoQ 0.361 NA 0.118 Date EnteredBy: Date Verified By: 08/23/99,09/16199,09/17/99, 11/07/00 GMLLAC 09/25/00 LAC, 11/08/00HOJ C8F 17COO- Analytical Report: FACT-TOX-026 LRN-U2782 ETS-8-97.0 Excel 97 3M Environmental Laboratory UrineWk30 TOX-026-urine23l 4 H . x l s 512312001 Page 284 3M Medical Department Study: T-6889.3 FACT-TOX-026 Covance 6329-231 Study: Product Number(Test Substance): Matrix: Method/Revision: Analytical Equipment System Number: Instrument SoftwareNersion: Filename: R-Squared Value: Slope: Y-Intercept: Dates of Extraction/Analyst: Dates of AnalydAnalyst: Date of Data Reductiodhalyst: 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 SEEIRWWIMCWSAL 08/19/99,09/15/99 GML 08/20/99,09/16/99, 11/06/00 GML/KJH POAA 081999080-81 09 1599019-20 99-1 60-1 Analytical Report: FACT-TOX-026 LRN-U2782 Dilutions 1/1 1/1 Group Dose GrouD 1 0.0 mgkgiday Group 3 Sample # 105718M I05720M 105712M Extraction VOl. 1 1 1 I POAA Std Correction Factor 0.9582 0.9582 0.9582 POAA Dilution Factor 1 1 1 POAA Cone. ng/d 4.68 5.14 164 Concentration of POAA u g / d or % Rec. <LOQ <LOQ 0.157 Mean POAA ug/d <LOQ RSD Std. Dev. MSMSD RPD NA Date Enteremy: Date Verified/ By: 08/23/99,09/16/99, 11/07/00 GMLLAC 09/25/00 LAC, 11/08/00HOJ ETS-8-97.0 3M EEnxvceilr9o7nmental Laboratory UrineWk32 TOX-026-urine23 1-4H.xls 5/23/2P00a1ge 285 3M Medical Department Study: T-6889.3 Study: Product Number(Test Substance): Matrix: MethodRevision: Analytical Equipment System Number: Instrument SoftwareNersion: Filename: R-Squared Value: Slope: Y-Intercept: Dates of ExtractiodAnalyst: Dates of AnalydAnalyst: Date of Data ReductiodAnalyst: FACT-TOX-026 Covance 6329-231 Analytical Report: FACT-TOX-026 LRN-U2782 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 Dose Group 1 0.0 mg/kg/day Group 3 10 mg/kg/day I05718M I05720M I057 12M I057 16M Conc. ng/mL 4.68 5.14 164 74.3 of POAA ug/mLor%Rec. <LOQ <LOQ 0.157 0.0712 POAA ug/mL <LOQ 0.114 Std. Dev. MSMSDRPD NA 0.0608 Date EnteredBy: Date Vcrifit& By. 08/23/99,09/16/99, 11/07/00 GMLLAC 09/25/00 LAC, 11/88/00 HOJ ETS-8-97.0 3M EEnxvceilr9o7nmental Laboratory UrineWk32 TOX-026-urine23 1-4H.xls 5/23/2P00a1ge 286 3M Medical Department Study: T-6889.3 FACT-TOX-026 Covance 6329-231 Analytical Report: FACT-TOX-026 LRN-U2782 Study: Product Number(Test Substance): Matrix: Method/Revision: Analytical Equipment System Number: Instrument SoftwareNersion: 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 34 Group Sample # Dose Group 1 0.0 mg/kg/day Group 3 10 mg/kg/day I057 18M I05720M I05712M I057 16M 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 POAA Dilutions MassLynx 3.2 See listing to the nght Grp 1 Grp 3 081999084-85 1/1 09 1599021-22 111 See Attachments Box 99-160-1 See Attachments See Attachments 08/17/99 SEEIRWWIMCWSAL 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. 1 1 1 1 POAA Std Correction Factor 0.9582 0.9582 0.9582 0.9582 POAA Dilution Factor 1 1 1 1 POAA Conc. ng/a 0.00 0.00 104 149 Concentration of POAA ug/mL. or % Rec. <LOQ <LOQ 0.0993 0.142 Mean POAA ug/mL <LOQ 0.121 RSD Std. Dev. MSMSD RPD NA 0.0305 Date Enteremy: Date Verified/ a y : 08/23/99, 09/16/99, 11/07/00 GMLLAC 89/25/88 LAC, i i/08/00 KG1 ETS-8-97.0 3M EEnxvceilr9o7nmental Laboratory UrineWk34 TOX-026-urine23 1-4H.xls 5/23/2P00a1ge 287 3M Medical Department Study: T-6889.3 Study: Product Number(Test Substance): Matrix: MethodlRevision: Analytical Equipment System Number: Instrument SoftwareNersion: Filename: R-Squared Value: Slope: Y-Intercept: Dates of ExtractiodAnalyst: Dates of Analysis/Analyst: Date of Data ReductiodAnalyst: FACT-TOX-026 Covance 6329-231 Analytical Report: FACT-TOX-026 LRN-U2782 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 SEEIRWW/MCWSAL 08/19/99,09/15/99 GML 08/20/99,09/16/99, 11/06/00 GMLKJH Sample Data Dose Group 1 0.0 mg/kg/day Group 3 10 rngikdday I05718M I05720M I05712M I057 16M Conc. ng/mL 0.00 0.00 104 149 of POAA ug/mL or % Rec. <LOQ <LOQ 0.0993 0.142 POAA ug/mL <LOQ 0.121 Std. Dev. MS/MSD RPD NA 0.0305 Date Enteremy: Date Verifiedi By: 08/23/99,09/16/99, 11/07/00 GMLLAC 09/25/00 LAC, i i/OS/OO KO; ETS-8-97.0 3M EnExvcierl 9o7nmental Laboratory UrineWk34 TOX-026-urine23 1-4H.xls 5/23/2P00a1ge 288 3M Medical Department Study: T-6889.3 FACT-TOX-026 Covance 6329-231 Analytical Report: FACT-TOX-026 LRN-U2782 Dilutions 1/1 111 Group Dose Sample # Extraction VOl. Group 1 I05718M 1 0.0 mg/kg/day I05720M 1 Group 3 I05712M 1 10 mg/kg/day 105716M 1 A = Below LOQ Date Enteremy: Date Verified/ By: 08/23/99,09/16/99, 11/07/00 GMLLAC 09/25/00 LAC, 11/08/00HOJ POAA Std Correction Factor 0.9582 0.9582 0.9582 0.9582 POAA Dilution Factor 1 1 1 1 C8F,,CO0 POAA Conc. ng/mL 0.00 18.4 40.1 64.6 Concentration of POAA ug/mL or % Rec. <LoQ 0.0176 0.0384 0.0619 Mean POAA ug/d 0.01 17 0.0502 RSD Std. Dev. MSMSD RPD A 0.00841 0.0 166 ETS-8-97.0 3M EEnxvceilr9o7nmental Laboratory UrineWk36 TOX-026-urine23 1-4H.xls S/23/2P00a1ge 289 3M Medical Department Study: T-6889.3 Study: Product Number(Test Substance): Matrix: MethodiRevision: Analytical Equipment System Number: Instrument SoftwareNersion: Filename: R-Squared Value: Slope: Y-Intercept: Dates of Extraction/Analyst: Dates of Analysis/Analyst: Date of Data Reductiodhalyst: FACT-TOX-026 Covance 6329-231 Analytical Report: FACT-TOX-026 LRN-U2782 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 SEERWWIMCWSAL 08/19/99, 09/15/99 GML 08/20/99, 09/16/99, 11/06/00 GMLKJH Sample Data Dose Group 1 0.0 mg/kg/day Group 3 10 mg/kg/day Date Enteremy: Date Verified/ By: I057 18M I05720M 105712M I057 16M Cone. ng/mL 0.00 18.4 40.1 64.6 of POAA ug/mL or % Ree. <LOQ 0.0176 0.0384 0.0619 POAA ug/mL 0.0117 A 0.0502 Std. Dev. MSMSD RPD 0.00841 0.0166 A = Below LOQ 08/23/99,09/16/99, 11/07/00 GMLLAC 09/25/00 LAC, 11/08/00 HOJ C8F17C00- ETS-8-97.0 3M EEnxvceilr9o7nmental Laboratory UrineWk36 TOX-026-urine23 1-4H.xls 5/23/2P00a1ge 290 3M Medical Department Study: T-6889.3 FACT-TOX-026 Covance 6329-231 Analytical Report: FACT-TOX-026 LRN-U2782 Study: Product Number(Test Substance): Matrix: MethodlRevision: Analytical Equipment System Number: Instrument SoftwareNersion: Filename: R-Squared Value: Slope: Y-Intercept: Dates of Extraction/Analyst: Dates of Analysisihalyst: Date of Data ReductiodAnalyst: Sample Data MONKEY URINE Week 38 I Group Dose Sample # Group 1 0.0 rngkdday Group 3 10 mg/kg/day I057 18M I05720M I05712M I057 16M 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 POAA Dilutions MassLynx 3.2 Grp 1 08 1999090-91 1/1 See listing to the right See Attachments Grp 3 09 1599027-28 1/1 Box 99-160-1 See Attachments See Attachments OS/ 17/99 SEEIRWWMCWSAL 08/19/99, 09/15/99 GML 08/20/99, 09/l6/99,11/06/00 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. I 1 1 1 1 POAA Std Correction Factor 0.9582 0.9582 0.9582 0.9582 POAA Dilution Factor 1 1 1 1 POAA Cone. ng/mL 0.00 0.00 37.2 22.1 Concentration of POAA u g / d or YORec. <LOQ <LOQ 0.0357 0.02 12 Mean POAA ug/d <LOQ 0.0284 RSD Std. Dev. MS/MSD RPD NA 0.0102 Date EnteredBy: Date V-erifiediBy: 08/23/99,09/16/99, 11/07/00 GMLLAC 09/25/00 LAC, i ii08iOO KOJ ETS-8-97.0 3M EEnxvceilr9o7nmental Laboratory UrineWk38 TOX-026-urine23 1-4H.xIs 5/23/20P01age 291 3M Medical Department Study: T-6889.3 Study: Product Number(Test Substance): Matrix: MethodRevision: Analytical Equipment System Number: Instrument SoftwareNersion: Filename: R-Squared Value: Slope: Y-Intercept: Dates of Extraction/Analyst: Dates of AnalysdAnalyst: Date of Data ReductiodAnalyst: Group Dose Group 1 0.0 mg/kg/day Group 3 10 mg/kg/day Sample # I057 18M 105720M I05712M 105716M FACT-TOX-026 Covance 6329-231 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/MCWSAL 08/19/99,09/15/99 GML 08/20/99,09/16/99, 11/06/00 G M W H 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 <LOQ 0.0284 RSD Std. Dev. MSMSD RPD NA 0.0102 Date Enteremy: Ddic Vciifieil; By. 08/23/99,09/16/99, 11/07/00 GML/LAC 09/25/00LAC, i 1/08/00 EO; Analytical Report: FACT-TOX-026 LRN-U2782 ETS-8-97.0 3M EEnxvceilr9o7nmental Laboratory UrineWk38 TOX-026-urine23 1-4H.xls 5/23/20P0a1 ge 292 3M Medical Department Study: T-6889.3 Study: Product Number(Test Substance): Matrix: Method/Revision: Analytical Equipment System Number: Instrument SofiwareNersion: Filename: R-Squared Value: Slope: Y-Intercept: Dates of Extractiodhalyst: Dates of AnalysidAnalyst: Date of Data ReductiodAnalyst: Sample Data MONKEY URINE Week 40 Group Sample # Dose Group 1 0.0 mg/kg/day Group 3 10 mgkg/day 105718M 105720M 105712M I057 16M FACT-TOX-026 Covance 6329-231 Analytical Report: FACT-TOX-026 LRN-U2782 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 POAA Dilutions MassLynx 3.2 Grp 1 081999092-93 1/1 See listing to the right Grp 3 091599031-32 1/1 See Attachments Box 99-1 60-1 See Attachments See Attachments 08/17/99 SEEfRWWfMCH/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 rnL initial and 0.5 mL final volumes. Extraction Vol. 1 1 1 1 POAA Std Correction Factor 0.9582 0.9582 0.9582 0.9582 POAA Dilution Factor 1 1 1 1 POAA Conc. ndmL 0.00 0.00 34.0 17.7 Concentration of POAA u d m L or % Rec. <LOQ <LOQ 0.0325 <LOQ Mean POAA udmL <LOQ 0.0254 RSD Std. Dev. NA 0.0101 Date Enteremy: yT. a k Verified/ By. 08/23/99,09/16/99, 11/07/00 GMLLAC 09/25/00 LAC, 11/08/00EO; ETS-8-97.0 3M EEnxvceilr9o7nmental Laboratory UrineWk40 TOX-026-urine231-4H.xls 5/23/2P00a1ge 293 3M Medical Department Study: T-6889.3 FACT-TOX-026 Covance 6329-231 Analytical Report: FACT-TOX-026 LRN-U2782 Study: Product Number(Test Substance): Matrix: MethodiRevision: Analytical Equipment System Number: Instrument SoftwareNersion: Filename: R-Squared Value: Slope: Y-Intercept: Dates of Extraction/Analyst: Dates of AnalysisiAnalyst: Date of Data ReductionIAnalyst: 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 Group Dose Group 1 Sample # I05718M POAA Conc. ng/mL. 0.00 Concentration of POAA ug/mL or % Rec. <LOQ Mean POAA ug/mL RSD Std. Dev. MS/MSD RPD I I 10mgikgIday I057 16M Limit of Quantitation (LOO): 0.0182 ug/mL I I I I I 17.7 <LOQ NA = Not Analyzernot Applicable 0.0254 0.010 POAA = Perfluorooctanoate CSF17C00- Date EnteredBy: Dare Veriiiedi By: 08/23/99,09/16/99, 11/07/00 GMLLAC iE/iMO LAC, i iiO8iOO ECX ETS-8-97.0 3M EEnxvceirl 9o7nmental Laboratory UrineWk40 TOX-026-urine23 1-4H.xls 5/23/2P00a1ge 294 3M Medical Department Study: T-6889.3 FACT-TOX-026 Covance 6329-231 Urine QC Summary - TOX026 08/04/1999 08/05/1999 0811711999 08/20/1999 0911 211999 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 MKU08I79 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 level Average Standard Deviation Grubbs outlier included? Number of spikes Used in Final Report 17% 20 ETS-8-97.0 3M EEnxvcierlo9n7mental Laboratory Standard Deviation 13% Grubbs outlier included? Number of spikes 19 QC Summary TOX-026-urine231-4H.xls Analytical Report: FACT-TOX-026 LRN-U2782 5/23/20P01age 295 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 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) 1OOOng 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 1000ng AR- Analytical result from MassLynx summary DF- Dilution factor SC-POAA salt correction constant (0.9582) FV-Final extract volume (1.O mL unless otherwisenoted) EV-Volume of sera extracted Formula Used for Liver Analyses in Study FACT TOX-026 AR (ng/g) x a curve ( I ) x SC x DF x 3 sample (I) a curve is assumed to be: 1 g liver 5 mL H20 1.0 pg = [POAA] sample (pg/g) 1000 ng Calculation Used for Group 2, Week 27, Animal ID 105702M 160ng/g x 1 g / 5 m L ~ 0 . 9 5 8 21 ~0- 0 ~- 1.Opg = 15.2pg/g 1.0104g/ 5 mL I 000 ng AR- Analytical result from MassLynx summary a curve-Density of the liver standard curve, assumed to be l g liver/ 5 ml water a sample-Density of the liver sample (g sample/ 5 rriL HzO) SC-POAA salt correction constant (0.9582) DF- Dilution factor 3M Environmental Laboratory 3M Environmental Laboratory Page 38 Page 296 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 Formula Used for Urine Analyses in Study FACT TOX-026 AR (ng/mL) x DF x SC x FV (mL) x 1.0 UE == Reported Concentration pg/mL) EV (mL) 1OOOng 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 ~g = 37.0yg/mL 1 mL 1000ng AR-Analytical result from MassLynx summary DF-Dilution factor SC-POAA salt correction constant (0.9582) ER-Extraction ratio (equal to 1.O unless otherwise noted) FV-Final extract volume (1.O mL unless otherwise noted) EV-Volume of urine extracted 3M Environmental Laboratory 3M Environmental Laboratory Page 39 Page 297 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 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 3M Environmental Laboratory Page 40 Page 298 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 ANALYTICAL REPORT STUDY TITLE 26-Week Capsule Toxicity Study with Ammonium Perfluorooctanoate(APFOPOAA) in Cynomolgus Monkeys DATA REOUIREMENTS Analytical Method Requirements STUDY DIRECTOR Paul Lieder, 3M 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 S5133-3220 PROJECT IDENTIFICATION Sponsor Protocol Number: FACT-TOX-026 Centre Study Number: 023-011 Total Pages: 110 3M Environmental Laboratory Page 299 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 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 (APFOPOAA) 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 (PFOAROAA and THPFOS) have not been characterized under GLP's 5160.105 (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 5792.130 (e). Principaldnvestigator Centre Analytical Laboratories, Inc. Paul Lieder, Ph.D:, DABT Study Director 3M Toxicology Services Centre Analytical Laboratories, Inc. 3M Environmental Laboratory Ylz/o, Date .5YYbJ Date Page 2 of 110 Page 300 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 Centre Study No.: 023-01I Sponsor Protocol No: FACT-TOX-026 OUALITYASSURANCE STATEMENT Centre Analytical Laboratories' Quality Assurance Unit reviewed Centre Study Number 023-011entitled, "26-Week Capsule Toxicity Study with Ammonium Peffluorooctanoate (APFOPOAA) 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 11/2/100 Date Reported to Study Director and SDonsor 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 3/15/0 1 3/19/0 1 3/19/0 1 Naomi Lovallo Sr. Quality Assurance Auditor Centre Analytical Laboratories,Inc. 3M Environmental Laboratory Page 3 of 110 Page 301 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 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 (APFOPOAA) 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: E&ly\R. Scientist 6S uffei Centre Analytical Laboratories, Inc. Centre Analytical Laboratories, Inc. Facility Management: F o h n Flaherty Date Laboratory Manager Centre Analytical Laboratories, Inc. Study Director, 3M: Paul Lieder, Ph.D., DABT 3M Toxicology Services </?/a/ Date Centre Analytical Laboratories, Inc. 3M Environmental Laboratory Page 4 of 110 Page 302 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 Centre Study No.: 023-01 1 Sponsor Protocol No: FACT-TOX-026 STUDY IDENTIFICATION 26-Week Capsule Toxicity Study with Ammonium Perfluorooctanoate(APFOROAA) in Cynomolgus Monkeys SPONSOR PROTOCOL NUMBER: FACT-TOX-026 TYPE OF STUDY: Toxicity TEST SYSTEM: Cynomolgus monkeys TEST MATERIAL: Ammonium perfluorooctanoate (APFOROAA) SPONSOR: 3M Toxicology Services - Medical Department 3M Center, Building 220-2E-02 St. Paul, MN 55133-3320 STUDY DIRECTOR: From 11/12/98to 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 10/23/00to 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. 3M Environmental Laboratory Page 5 of 110 Page 303 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 Centre Study No.: 023-01I 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 Groupmeam Leader Scientist Scientist Scientist Technician Technician Sample Custodian Sample Custodian Sample Custodian Sample Custodian Centre Analytical Laboratories, Inc. 3M Environmental Laboratory Page 6 of 110 Page 304 3M Medical Department Study: T-6889.3 . Analytical Report: FACT-TOX-026 LRN-U2782 Centre Study No. 023-011 Sponsor Protocol No: FACT-TOX-026 TABLE OF CONTENTS TITLE PAGE ...................................................................................................................... 1 GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT ............................. 2 QUALITY ASSURANCE STATEMENT.......................................................................... 3 CERTIFICATIONOF 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 EXPERIMENTALDESIGN...................................................................................... 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 CIRCUMSTANCESTHAT 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. 3M Environmental Laboratory Page 7 of 110 Page 305 3M Medical Department Study: T-6889.3 . Analytical Report: FACT-TOX-026 LRN-U2782 Centre Study No. 023-011 Sponsor Protocol No: FACT-TOX-026 LIST OF TABLES Pag;e Table 1. Summary of POAA residues in Matrix Blanks and Matrix Zero Blanks ...........20 Table II. Summary of POAA recoveries in Fortified Samples......................................... 22 Table IIl. Summary of POAA residues in Week 2 ........................................................... 23 Table W. 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 10....................................................... 27 Table VIII. Summary of POAA residues in Week 12...................................................... 28 Table IX. Summary of POAA residues in Week 14 ........................................................ 29 Table X. Summary of POAA residues in Week 16.......................................................... 30 Table XI. Summary of POAA residues in Week 18 ........................................................ 31 Table XII. Summary of POAA residues in Week 20 ....................................................... 32 Table XIII. Summary of POAA residues in Week 22...................................................... 33 Table XIV . Summary of POAA residues in Week 24...................................................... 34 Table XV. Summary of POAA residues in Week 26 ....................................................... 35 Table XVI. Summary of POAA residues in Week 28-34 ................................................ 36 Table XVII. Summary of POAA residues in Weeks 36-40 ............................................. 37 Centre Analytical Laboratories. Inc. 3M Environmental Laboratory Page 8 of 1 10 Page 306 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 Centre Study No.: 023-01 1 Sponsor Protocol No: FACT-TOX-026 Figure 1. Figure 2. Figure 3. Figure 4. Figure 5. Figure 6 . Figure 7. LIST OF FIGURES PaRe Typical Calibration Curve for POAA ............................................................ 39 Typical Mean Response Factor for THPFOS ................................................ 40 Chromatogram Representing a 5 ng/mL extracted standard for POAA and 250 ng/mL extracted standard for THPFOS .................................................. 41 Chromatogram Representing a 125 n g / d extracted standard for POAA and 250 ng/mL extracted standard for THPFOS .................................................. 42 Chromatogram Representing Control Monkey Feces for POAA and THPFOS (Centre ID: 0009684 Blank A, Set: 101200A).............................................. 43 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 Chromatogram of Monkey Feces Sample from Grp 2 in Week 12 (Centre ID: 0008548, Set: 101200A) ............................................................................... 45 Centre Analytical Laboratories, Inc. 3M Environmental Laboratory Page 9 of 1 10 Page 307 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 Centre Study No.: 023-011 Sponsor Protocol No: FACT-TOX-026 LIST OF APPENDICES Appendix A Study Protocol FACT-TOX-026 (Centre Study No. 023-011) entitled 26-Week Capsule Toxicity Study with Ammonium Peffluorooctanoate D(AePviF.aOti.oPnOsA....A...)..i.n....C...y..n..o..m....o..l.g..u..s...M....o..n..k..e..y..s...a..n..d...A...m...e..n..d...m...e..n..t.s...a..n..d........ .47 Appendix B Determination of Fluorochemical Residues in Monkeymat Feces by LC/MS/MS (Revision 2) Method #00M-023-003, revision 2 and Deviations and Modifications............................................................. 84 Centre Analytical Laboratories, Inc. 3M Environmental Laboratory Page 10of 110 Page 308 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 Centre Study No.: 023-01I Sponsor Protocol No: FACT-TOX-026 1.0 SUMMARY The purpose of this study was to analyze residues of peffluorooctanoate (APFOPOAA) 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 Monkeymat Feces by L C N S N S , revision 2" (Centre method number: 00M-023-003, revision 2). Besides APFOPOAA (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 peffluorooctanoate (APFOPOAA) in specimens of feces of monkeys using the analytical method entitled "Determination of Fluorochemical Residues in Monkeymat Feces by LCNSIMS, revision 2." 3.0 INTRODUCTION The study was initiated on November 12, 1998, when the study director signed the protocol FACT-TOX-026. The complete protocol and amendments 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 Monkeymat 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. 3M Environmental Laboratory Page 11 of 110 Page 309 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 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 (<-1O"C). 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. ComDound POAA THPFOS Centre Control No. 00-023-048 00-023-053 - Batch No. 332 53406 Purity (%I TBD TBD Expiration Date 07/06/0 1 01/01/ 10 Molecular structures of POAA and THPFOS are given below. POAA Chemical Name - Molecular weight = Perfluorooctanoate 413 Note: The neutral molecule and standard form which POAA (anion) is derived from is ammonium perfluoroctanoate[C7F15COOW], molecular weight 43 1. THPFOS Chemical Name: 4-H, perfluorooctanesulfonic acid Molecular weight: 428 I-H, 1-H, 2-H, 2-H, CgF13S03H Centre Analytical Laboratories, Inc. 3M Environmental Laboratory Page 12 of 1 10 Page 310 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 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 Monkeymat Feces by LC/MSNS (Revision 2)" was used for this study. 7.1 Extraction Procedure One gram 4 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 9O:lO 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 Monkeymat 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 punty 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. 3M Environmental Laboratory Page 13 of 110 Page 311 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 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: Conc. of Mixed Fortification Solution (a) Fortification Volume Weight of Control Sample (8) f 0.05 Fort. Level of Vol. of Extracted Surrogate (a) Calibration Standard* Standard added 0.1 100 1.o 10 200 0.1 200 1.o 20 200 0.5 100 1.o 50 200 0.5 200 1.o 100 200 2.5 100 1.o 250 200 2.5 200 1.o 500 200 * 2.5 pg/d THPFOS fortification solution. The stock standard solution and all fortification and calibration standard solutions were stored in a refrigerator (4" f 2OC) 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. 3M Environmental Laboratory Page 14 of 110 Page 312 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 Centre Study No.: 023-01 I Sponsor Protocol No: FACT-TOX-026 7.5 Description of Instrument and Operating Conditions A Micromass Quattro Ultima LCIMSIMS 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 ANALYER: LM Res 1: 10.5 V HM Res 1: 10.5 V Energy 1: 1.0 V Entrance: -2 V Exit: 2 V LM Res 2: 13.0 V HM Res 2: 13.0 V Energy 2:2.0 V Multiplier: 650 V GAS FLOWS AND PRESSURE: Desolvation N2Flow Rate: -650 uhr Nebuliser N2Flow Rate: -150 L/hr Gas Cell Pressure: -0.003 mbar Computer: COMPAQ Professional Workstation AP2!00 Software: Microsoft Windows NT: Version 4 Build 1381: Service Pack 5 Micromass Limited: Masslynx 3.4 Build 004 HPLC Equipment: Hewlett Packard (HP)Series 1100 HP Binary (or Quaternary) Pump HP Autosampler 1 8 Vacuum Degasser IBColumn 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 Water Methanol Gradient: Injected Volume: Time (min) %A 0.0 60.0 0.4 60.0 1.o 10.0 7.0 10.0 7.5 0.0 10.5 0.0 11.0 60.0 14.5 60.0 15.0 60.0 10 pL %B 40.0 40.0 90.0 90.0 100.0 100.0 40.0 40.0 40.0 Flow Rate (mL/min) 0.3 0.3 0.3 0.3 0.3 0.4 0.4 0.4 0.3 Centre Analytical Laboratories, Inc. 3M Environmental Laboratory Page 15 of 110 Page 313 3M Medical Department Study: T-6889.3 Ions monitored : Analvte POAA THPFOS Transition Monitored 413 3 369 427 80 Analytical Report: FACT-TOX-026 LRN-U2782 Centre Study No.: 023-011 Sponsor Protocol No: FACT-TOX-026 Dwell Csecs) Coll Energy CeV) Cone CV) 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 l/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. Eauation 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( un) sample wt. (8) 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. Eauation 3: Recovery (%) = ((anal.found (ng/mL)- (anal. found in corresponding sample (ng/mL)lDF)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: 1001;!00A),fortified with 50 ng of POAA. Where: peak area 53080 intercept slope dilution factor - 3141.11 1928.40 - 1 ng added (fort level) = 50 Centre Analytical Laboratories, Inc. Page 16 of 110 3M Environmental Laboratory Page 314 3M Medical Department Study: T-6889.3 final vol. sample wt. Analytical Report: FACT-TOX-026 LRN-U2782 Centre Study No.: 023-011 Sponsor Protocol No: FACT-TOX-026 --- 02.m97Lg From equation 1: Analyte found (ng/mL) = 153080- 3141.111 1928.40 = 25.9 ng/mL From equation 2: Analyte found (pg/g) = ~25.9ng/mLx2mLxlxlU~ 0.97 g x 1000 ng = O.O534pg/g From equation 3: % Recovery = (25.9 ndmL x 2 mL x 1) 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: Standard Deviation = 1i=l n-1 Mean = -x = - c : = , x i n Relative Standard Deviation (RSD or Coeficient of Variation (CV))= Standard Deviation x 100% Mean Centre Analytical Laboratories, Inc. 3M Environmental Laboratory Page 17 of 110 Page 315 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 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 11. 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 111-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-specificraw 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. 3M Environmental Laboratory Page 18 of 110 Page 316 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 Centre Study No.: 023-011 Sponsor Protocol No: FACT-TOX-026 11.0 TABLES Centre Analytical Laboratories, Inc. 3M Environmental Laboratory Page 19 of 110 Page 317 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 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 0001345-48Zero Blank D 0001345-48 Blank A 0001345-48 Blank B 0001345-48 Zero Blank C 0001345-48Zero 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 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 Number 100200A 100200A 100200A 100200A 100600A 100600A 100600A 100600A 100900A 100900A 100900A 100900A 101000A 101000A lOl000A lOl000A 101l00A 101l00A 101 IOOA 101lOOA 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/OO 10/6/OO 10/6/OO 10/6/OO 10/9/OO 10/9/OO 10/9/00 10/9/00 1o/ 10/00 1o/ 10/00 1o/ 10/00 1o/ 10/00 10111/00 10/11/00 10111/00 10/11/00 10/12/00 1o/ 12/00 1o/ 12/00 1o/ 12/00 1O/ 13/00 10/13/00 10/13/00 10/13/00 1O/ 16/00 1O/ 16/00 1O/16/00 1O/ 16/00 10/17/00 10/17/00 1O/ 17/00 1O/ 17/00 Analysis Date 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 1o/ 10/00 1o/ 10/00 1o/ 1o/oo 1o/ 10/00 10/10-11/00 10/10-11/00 10/10-11/00 10/10-11/00 1O/ 12-13/00 1O/ 12-13/00 10/12-13/00 10/12-13/00 1O/ 13-14/00 1O/ 13-14/00 1O/ 13-14/00 10/13-14/00 10/14-15/00 1 0/14- 15/00 10/14-15/00 10/14-15/00 1O/ 18-19/00 1O/ 18-19/00 1O/ 18-19/00 10/18-19/00 1O/ 17-18/00 10/17-18/00 10/17-18/00 1O/ 17-18/00 Analyte Found (pg/g) .I NQ NQ NQ NQ NQ NQ NQ NQ 0.0453 0.0212 0.09 13 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. 3M Environmental Laboratory Page 20 of 110 Page 318 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 Centre Study No.: 023-01 1 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 Centre Set Extraction Analysis Analyte na 0009684 Blank A 101800A 1o/18/00 10/19-20/00 NQ na 0009684 Blank B 101800A 10/18/00 10/19-20/00 NQ na 0009684 Zero Blank C 101800A 10/18/00 10/19-20/00 NQ na 0009684 Zero Blank D 101800A 1o/ 18/00 10/19-20/00 NQ Na 0009684 Blank A 101900A 1o/19/00 10120-21/00 NQ Na 0009684 Blank B 101900A IO/19/00 10/20-21/00 ND Na 0009684 Zero Blank C 101900A 1o/ 19/00 10/20-21/00 NQ Na 0009684 Zero Blank D 101900A 1o/19/00 10/20-21/00 NQ Na 0009684 Blank A 102000A 10/20/00 10/22-23/00 ND na 0009684 Blank B 102000A 10/20/00 10/22-23/00 ND na 0009684 Zero Blank C 102000A 10/20/00 10122-23/00 ND na 0009684 Zero Blank D 102000A 10/20/00 10122-23/00 ND na 0009684 Blank A 102300AR 10/23/OO 10/25/00 NQ na 0009684 Blank B 102300AR 10/23/00 10/25/OO NQ na 0009684 Zero Blank C 102300AR 10/23/00 10/25/00 NQ na 0009684 Zero Blank D 102300AR 10/23/00 10/25/OO NQ na 0009684 Blank A 102400A 10/24/OO 10/24-25/00 ND na 0009684 Blank B 102400A 10/24/00 10/24-25/OO ND na 0009684 Zero Blank C 102400A 10/24/00 10/24-25/00 ND na 0009684 Zero Blank D 102400A 10/24/00 10/24-25/00 ND na 0009684 Blank A 102500A 10/25/00 10/26/OO ND na 0009684 Blank B 102500A 10/25/00 10/26/00 ND na 0009684 Zero Blank C 102500A 10/25/00 10/26/00 ND na 0009684Zero Blank D 102500A 10/25100 10/26/OO ND AVERAGE: 0.0113 STANDARD DEVIATION: 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. 3M Environmental Laboratory Page 21 of 110 Page 319 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 Centre Study No.: 023-011 Sponsor Protocol No: FACT-TOX-026 Table 11. Summary of POAA recoveries in Fortified Samples Sponsor ID Centre ID Set Number Extraction Date Analysis Date Amount 96 Added (ng) Recovery I05709M Grp 1 Wk 2 0008434 Spk A 100200A 10/2/00 10/3/00 50 104 I05714M Grp 1 Wk 2 0008435 Spk B 100200A 10/2/00 10/3/00 250 88 I05720M Grp 1 Wk 4 0008460 Spk A 100600A 10/6/OO 10/6-7/00 50 119 105725M Grp 1 Wk 4 0008461 Spk B 100600AD 10/6/00 10/7/00 1000 137 I05720M Grp 1Wk 6 0008482 Spk A 100900A 10/9/OO 1o/10/00 50 116 105725MGrp 1 Wk 6 0008483 Spk B 100900A 10/9/00 1o/1o/oo 2000 139 I05709M Grp 1 Wk 8 0008499 Spk A lOl000A 1o/ 10/00 10/10-11/00 50 105 I05714M Grp 1Wk 8 0008500 Spk B lOl000A 1o/ 1o/oo 10/10-11/00 loo00 133 I05715M Grp 1Wk 10 0008522 Spk A 101l00A 10/11/00 10/12-13/00 50 98 I05718M Grp 1 Wk 10 0008523 Spk B 101l00A 10/11/00 1O/ 12-13/00 loo00 128 I05720M Grp 1 Wk 12 0008545 Spk A 101200A 1o/12/00 10/13-14/00 50 87 I05725M Grp 1 Wk 12 0008546 Spk B 101200A 10/12/00 10/13-14/00 2 m 115 I05709M Grp 1Wk 14 0008562 Spk A 101300A 1O/13/00 10/14-15/00 50 72 I05714M Grp 1Wk 14 0008563 Spk B 101300A 10/13/00 10/14-15/00 100,250 128 105715MGrp 1Wk 16 0008585 Spk A 101600AR 10/16/00 1O/ 18-19/00 50 114 I05718M Grp 1 Wk 16 0008586 Spk B 101600AR 1O/ 16/00 1O/ 18-19/00 100,250 128 I05720M Grp 1 Wk 18 0008608 Spk A 101700A 10117/00 10/17-18/00 50 127 105725MGrp 1 Wk 18 0008609 Spk B 101700A 1O/ 17/W 10/17-18/00 200.250 125 I05709M Grp 1 Wk 20 0008625 Spk A 101800A 10/18/00 1O/ 19-20/00 50 120 I05714M Grp 1 Wk 20 0008626SpkB 101800A 1o/18/00 10/19-20/00 200,250 141 I05720M Grp 1Wk 22 0008650 Spk A 101900AD 1O/ 19/00 10/22/00 50 130 I05725M Grp 1 Wk 22 0008651 Spk B 101900A 1O/ 19/00 10/20-21/00 200,250 96 I05709M Grp 1 Wk 24 0008666 Spk A 102000A 10/20/00 10/22-23/00 50 108 I05714M Grp 1 Wk 24 0008667 Spk B 102000AD 10/20/00 10/23/00 200,250 170 I05718M Grp 1 Wk 26 0008689 Spk A 102300AR 10/23/OO 10/25/OO 50 56 I05720M Grp 1 W k 26 OOO8690 Spk B 102300AR 10/23/00 10/25/00 250 129 105718MGrp 1Wk 28 0008706 Spk A 102400A 10/24/OO 10/24-25/00 50 123 I05720M Grp 1Wk 30 0008711 Spk B 102300AD 10/24/00 10/26/00 100,250 119 I05718M Grp 1 Wk 36 0008722 Spk A 102500A 10/25/00 10/26/OO 50 128 105720MGrp 1 Wk 38 0008726 Spk B 102300AD 10/25/OO 10/26/00 10.000 _12_ 5 ~ AVERAGE: 117 STANDARD DEVIATION: 22 RELATIVE STANDARD DEVIATION: 19 Centre Analytical Laboratories, Inc. 3M Environmental Laboratory Page 22 of 110 Page 320 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 Centre Study No.: 023-01 I Sponsor Protocol No: FACT-TOX-026 Table 111. Summary of POAA residues in Week 2 Sponsor Centre Set Extraction Analysis Analyte 105709MGrp 1 Wk2 0008434 100200A 10/2/00 10/3/00 NQ I05714M Grp 1 Wk 2 0008435 100200A 10/2/00 10/3/00 NQ 105715M Grp 1 Wk 2 0008436 100200A 10/2/00 10/3/OO NQ I05718M Grp 1Wk 2 0008437 100200A 10/2/00 10/3/00 NQ 105720M Grp 1 Wk 2 0008438 100200A 10/2/00 10/3/00 NQ 105725M Grp 1 Wk 2 0008439 100200A 10/2/00 10/3/00 NQ Average: NQ Standard Deviation: NQ 105702M Grp 2 Wk 2 0008440 100200AD 10/2/00 10/4/00 14.0 I05706M Grp 2 Wk 2 0008441 100200AD 10/2/00 10/4/OO 12.1 I05717M Grp 2 Wk 2 0008442 100200AD 10/2/00 10/4/OO 1.67 I05723M Grp 2 Wk 2 0008443 100200AD 10/2/00 10/4/OO 1.94 Average: 7.43 Standard Deviation: 6.54 I05707M Grp 3 Wk 2 0008444 100200ADD 10/2/00 10/5/00 31.7 105708M Grp 3 Wk 2 0008445 100200AD 10/2/00 10/4/00 20.9 I0571OM Grp 3 Wk 2 0008446 100200AD 10/2/00 10/4/00 18.2 I05712M Grp 3 Wk 2 0008447 100200AD lOIU00 10/4/OO 9.69 I05716M Grp 3 Wk 2 0008448 100200AD 10/2/00 10/4/OO 5.41 I05719M Grp 3 Wk 2 0008449 100200AD 10/2/00 10/4/00 6.50 Average: 15.4 Standard Deviation: 10.2 I05703M Grp 4 Wk 2 0008450 100200AD 10/2/00 10/4/OO 18.6 105704M Grp 4 W k 2 0008451 100200ADD 10/2/00 10/5/00 28.3 105711M Grp 4 Wk 2 0008452 100200ADD 10/2/00 10/5/OO 41.2 I05713M Grp 4 Wk 2 0008453 100200AD 10/2/00 10/4/OO 17.4 I05722M Grp 4 Wk 2 0008454 100200ADD 10/2/00 10/5/OO 28.2 I05724M Grp 4 Wk 2 0008455 100200ADD 10/2/00 10/5/00 206 Average: 56.6 Standard Deviation: 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. 3M Environmental Laboratory Page 23 of 110 Page 321 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 Centre Study No.: 023-01I Sponsor Protocol No: FACT-TOX-026 Table IV. Summary of POAA residues in Week 4 Sponsor Centre Set Extraction Analysis Analyte I05709M Grp 1 Wk 4 I05714M Grp 1 Wk 4 I05715M Grp 1 Wk 4 I05718M Grp 1 Wk 4 I05720M Grp 1 Wk 4 I05725M Grp 1 Wk 4 0008456 0008457 0008458 0008459 0008460 0008461 100600A 100600A 100600A 100600A 100600A 100600A 10/6/00 10/6/OO 10/6/00 10/6/00 10/6/00 10/6/OO 10/6-7/00 1016-7/00 10/6-7/00 10/6-7/00 10/6-7/00 10/6-7/00 Average: Standard Deviation: NQ 0.0403 NQ NQ NQ 0.048 1 0.0214 0.0178 I05702M Grp 2 Wk 4 0008462 100600A 10/6/OO 10/6-7/00 1.48 I05706M Grp 2 Wk 4 0008463 100600AD 10/6/00 10/7/00 28.0 I05717M Grp 2 Wk 4 0008464 100600A 10/6/OO 10/6-7/00 4.07 105721MGrp 2 Wk 4 0008465 100600AD 10/6/OO 10/7/00 8.22 Average: 10.4 Standard Deviation: 12.0 I05707M Grp 3 Wk 4 0008466 100600AD 10/6/OO 10/7/OO 30.8 I05708M Grp 3 Wk 4 0008467 100600AD 10/6/OO iomo 24.0 I057 10M Grp 3 Wk 4 0008468 100600AD 10/6/OO 10/7/00 12.2 I05712M Grp 3 Wk 4 0008469 100600AD 10/6/OO 10/7/00 9.72 I05716M Grp 3 Wk 4 0008470 100600AD 10/6/OO 10/7/OO 26.3 I05719M Grp 3 Wk 4 0008471 100600AD 10/6/OO 10/7/OO 37.2 Average: 23.4 Standard Deviation: 10.6 I05703M Grp 4 Wk 4 0008472 100600AD 10/6/00 10/7/OO 21.6 105704M Grp 4 W k 4 0008473 1oO600AT3 10/6/oO 10/7/00 17.9 I05711M Grp 4 Wk 4 0008474 100600AD 10/6/00 10/7/00 17.1 I057 13M Grp 4 Wk 4 0008475 100600AD 10/6/OO 10/7/OO 33.8 I05722M Grp 4 Wk 4 0008476 100600AD 10/6/OO 10/7/00 18.5 I05724M Grp 4 Wk 4 0008477 100600AD 10/6/OO 10/7/00 23.2 Average: 22.0 Standard Deviation: 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. 3M Environmental Laboratory Page 24 of 110 Page 322 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 Centre Study No.: 023-011 Sponsor Protocol No: FACT-TOX-026 Table V. Summary of POAA residues in Week 6 Sponsor Centre Set Extraction Analysis 105709M Grp 1 Wk 6 I05714M Grp 1 Wk 6 I05715M Grp 1 Wk 6 I05718M Grp 1 Wk 6 I05720M Grp 1 Wk 6 105725M Grp 1 Wk 6 0008478 0008479 0008480 0008481 0008482 0008483 I05702M Grp 2 Wk 6 105706M Grp 2 Wk 6 I05717M Grp 2 Wk 6 I05721M Grp 2 Wk 6 0008484 0008485 0008486 0008487 100900A 100900A 100900A 100900A 100900A 100900A 100900A 100900AD 100900A 100900AD 10/9/OO 10/9/00 10/9/OO 10/9/00 10/9/OO 10/9/OO 1o/ 10/00 1o/ 10/00 1o/10/00 1o/ 10/00 1o/ 10/00 1o/ 10/00 Average: Standard Deviation: 10/9/OO 10/9/OO 10/9/OO 10/9/OO 1o/ 10/00 10111/00 10110/00 10/11/00 Average: Standard Deviation: I05707M Grp 3 Wk 6 105708M Grp 3 Wk 6 I05710M Grp 3 Wk 6 I05712M Grp 3 Wk 6 I05716M Grp 3 Wk 6 I057 19M Grp 3 Wk 6 I05703M Grp 4Wk 6 I05704M Grp 4 W k 6 I057 11M Grp 4 Wk 6 I05713M Grp 4 Wk 6 105722MGrp 4 Wk 6 0008488 0008489 0008490 0008491 0008492 0008493 0008494 0008495 0008496 0008497 0008498 100900AD 100900AD 100900AD 100900AD 100900AD 100900AD 100900ADD 1009OOAD 1009OOAD 100900AD 100900AD 10/9/00 10/9/00 10/9/OO 10/9/OO 10/9/OO 10/9/OO 10/11/00 10111/00 10111/00 10111/00 10111/00 10111/00 Average: Standard Deviation: 10/9/00 10/9/00 10/9/OO 10/9/OO 10/9/00 1o/ 12/00 1011 1/00 10/11/00 10/11/00 10111/00 Average: Standard Deviation: 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. Analyte NQ NQ NQ 0.0147 NQ NQ 0.0108 0.00192 1.63 31.8 2.35 12.6 12.1 14.1 18.3 16.8 30.0 17.7 37.4 19.4 23.3 8.46 242 68.6 75.7 9.17 110 101 86.7 Centre Analytical Laboratories, Inc. 3M Environmental Laboratory Page 25 of 110 Page 323 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 Centre Study No.: 023-01I Sponsor Protocol No: FACT-TOX-026 Table VI. Summary of POAA residues in Week 8 SpQIlSQr ID I05709M Grp 1 Wk 8 I05714M Grp 1 Wk 8 105715M Grp 1 Wk 8 I05718M Grp 1 Wk 8 I05720M Grp 1 Wk 8 I05725MGrp 1 Wk 8 Centre ID 0008499 0008500 0008501 0008502 0008503 0008504 I05702M Grp 2 Wk 8 I05706M Grp 2 Wk 8 I05717M Grp 2 Wk 8 I05721MGrp 2Wk 8 0008505 0008506 0008507 0008508 Set Number 101000A lOl000A lOl000A lOl000A 101000A lOl000A lOl000A lOl000AD lOl000A lOl000AD Extraction Date 1o/ 10/00 1o/ 10/00 1o/ 1o/oo 1o/ 10100 1o/ 10/00 1o/ 10/00 Analysis Date 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 (pg/g) 0.0214 0.157 0.0102 0.0159 0.0104 0.254 0.0782 0.103 1 01 1 0100 10/10/oo 1o/ 1o/oo 10/10/00 10/10-11/00 10/11-12/00 10/10-11/00 10111-12/00 Average: Standard Deviation: ~ 3.24 22.8 3.41 8.37 9.46 9.21 I05707M Grp 3 Wk 8 0008509 lOl000AD 10/10/00 10/11-12/00 51.7 I05708M Grp 3 Wk 8 0008510 lOl000AD 10/10/00 10/11-12/00 17.8 I05710MGrp 3 Wk 8 0008511 lOl000AD 10/10/00 10/11-12/00 76.3 105712M Grp 3 Wk 8 0008512 lOl000AD 10/10/00 10/11-12/00 7.64 105716M Grp 3 Wk 8 0008513 lOl000AD 10/10/00 10/11-12/00 40.8 I05719MGrp 3 Wk 8 0008514 lOl000AD 10/10/00 10/11-12/00 51.8 Average: 41.0 Standard Deviation: 25.0 105703M Grp 4 Wk 8 105704M Grp 4 W k 8 105711MGrp 4Wk 8 105713MGrp 4Wk 8 105722M Grp 4 Wk 8 00085 15 0008516 OOO85 17 0008518 00085 19 lOl000AD lOl000AD lOl000A lOl000AD lOl000AD 10/10/00 10/11-12/00 10110/00 1o/1o/oo 10/11-12/00 10/10-11/oo 10/10/00 10/11-12/00 10/10/00 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. 3M Environmental Laboratory Page 26 of 110 Page 324 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 Centre Study No.: 023-01 1 Sponsor Protocol No: FACT-TOX-026 Table VII. Summary of POAA residues in Week 10 Sponsor Centre Set Extraction Analysis Analyte 105709MGrp 1 Wk 10 0008520 lOllOOA 10/11/00 10/12-13/00 NQ 105714M Grp 1 Wk 10 0008521 lOllOOA 10/11/00 10/12-13/00 NQ 105715MGrp 1 Wk 10 0008522 lOllOOA 10/11/00 1O/ 12-13/00 NQ 105718M Grp 1 Wk 10 0008523 lOllOOA 10/11/00 10/12-13/00 NQ I05720MGrp 1 Wk 10 0008524 101lOOA 10/11/00 1O/ 12-13/00 NQ 105725M Grp 1 Wk 10 0008525 101l00A 10/11/00 1O/ 12-13/00 NQ Average: NQ Standard Deviation: NQ 105702MGrp 2 Wk 10 0008526 101l00A 10111/00 1O/ 12-13/00 1.67 105706M Grp 2Wk 10 0008527 lOll00A 10/11/00 1O/ 12-13/00 2.64 105717MGrp 2Wk 10 0008528 101l00A 10/11/00 10/12-13/00 2.09 105721M Grp 2 Wk 10 0008529 lOll00AD 10/11/00 10/13/00 9.45 Average: 3.96 Standard Deviation: 3.68 105707MGrp 3Wk 10 0008530 lOll00AD 10/11/00 10/13/00 31.0 105708MGrp 3Wk 10 0008531 101l00AD 10/11/00 10/13/00 18.7 I05710MGrp 3Wk 10 0008532 101l00AD 10111/00 1O / 13/00 44.1 105712MGrp 3Wk 10 0008533 101l00AD 10111/00 10/13/00 10.1 105716MGrp 3Wk 10 0008534 101l00AD 10111/00 10/13/00 5.39 105719MGrp 3Wk 10 0008535 101100AD 10/11/00 10/13/00 46.9 Average: 26.0 Standard Deviation: 17.4 105703MGrp 4Wk 10 105704M Grp 4 W k 10 10571IMGrp 4 W k 10 105713MGrp 4Wk 10 105722MGrp 4Wk 10 0008536 0008537 0008538 0008539 0008540 101 l00AD 1011 0 0 0 101 lOOA 101 l00AD 101 l00AD 1011 1/00 10/11/00 1011 1/00 10111/00 1011 1/00 10113/00 10/13/00 1O/12-13/00 10/13/00 1O/ 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. 3M Environmental Laboratory Page 27 of 110 Page 325 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 Centre Study No.: 023-011 Sponsor Protocol No: FACT-TOX-026 Table VIII. Summary of POAA residues in Week 12 Sponsor Centre Set Extraction Analysis Analy te I05709M Grp 1 Wk 12 I05714M Grp 1 Wk 12 105715M Grp 1 Wk 12 I05718M Grp 1 Wk 12 I05720M Grp I Wk 12 105725M Grp 1 Wk 12 0008541 0008542 0008543 0008544 0008545 0008546 101200A 101200A 101200A 101200A 101200A 101200A 1o/ 12/00 10/12/00 1o/ 12/00 1o/ 12/00 1o/ 12/00 1o/ 12/00 1O/ 13-14/00 1O/ 13-14/00 1O/ 13- 14/00 1O/ 13-14/00 1O/ 13-14/00 1O/ 13-14/00 Average: Standard Deviation: 0.0209 NQ NQ 0.232 NQ 0.0 160 0.0498 0.0894 I05702M Grp 2 Wk 12 0008547 101200AD 10/12/00 1O/ 14/00 13.6 I05706MGrp 2Wk 12 0008548 101200A 1o/ 12/00 1O/ 13-14/00 3.21 105717MGrp 2Wk 12 0008549 101200A 10112/00 10113-14/00 1.68 I05721M Grp 2 Wk 12 0008550 101200AD 10/12/00 101 14/00 10.1 Average: 7.15 Standard Deviation: 5.65 105707M Grp 3 Wk 12 0008551 101200AD 10/12/00 1O/ 14/00 19.9 I05708MGrp 3Wk 12 0008552 101200AD 10/12/00 1O/ 14/00 9.68 I05710MGrp 3 Wk 12 0008553 101200A 1o/12/00 1O/ 13-14/00 4.54 I05712M Grp 3 Wk 12 0008554 101200AD 10/12/00 1O/ 14/00 8.86 I05716M Grp 3 Wk 12 0008555 101200AD 10/12/00 10/14/00 14.5 105719MGrp 3 Wk 12 0008556 101200A 10/12/00 1O/ 13-14/00 4.06 Average: 10.3 Standard Deviation: 6.07 I05703M Grp 4 Wk 12 105704M Grp 4 W k 12 I05711M Grp 4 Wk 12 105713M Grp 4Wk 12 I05722M Grp 4Wk 12 0008557 0008558 0008559 0008560 0008561 101200AD 101200AD 101200A 101200AD 101200A 10/12/00 10/12/00 1o/12/00 10/12/00 1o/ 12/00 10/14/00 1O/14/00 1O/13-14/00 1O/ 14/00 1O/ 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. 3M Environmental Laboratory Page 28 of 110 Page 326 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 Centre Study No.: 023-011 Sponsor Protocol No: FACT-TOX-026 Table IX. Summary of POAA residues in Week 14 -Sponsor ID I 105709MGrp 1 Wk 14 I05714M Grp 1 Wk 14 105715MGrp 1 Wk 14 I05718M Grp 1 Wk 14 105720M 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 1011 3/00 10/13/00 10/13/00 10/13/00 - Analysis Date 10/14-15/00 1O/ 14-15/00 10/14-15/00 10/15/00 10/14-15/00 10/14-15/00 Average: Standard Deviation: Analyte Found(pa 0.0276 NQ NQ 0.768 NQ NQ 0.139 0.308 105702M Grp 2 Wk 14 0008568 101300A 10/13/00 1O/ 14-15/00 4.22 I05706M Grp 2 Wk 14 0008569 101300AD 10/13/00 10/15/00 9.25 I05717M Grp 2 Wk 14 0008570 101300AD 10/13/00 1O/ 15/00 7.12 I05721M Grp 2 Wk 14 0008571 101300AD 10/13/00 10/15/00 9.42 Average: 7.50 Standard Deviation: 2.43 I05707M Grp 3 Wk 14 0008572 101300AD 10/13/00 10/15/00 82.2 I05708M Grp 3 Wk 14 0008573 101300AD 10/13/00 10/15/00 10.3 105710M Grp 3 W k 14 0008574 101300A 10/13/00 10/14-15/00 4.88 I05712M Grp 3 Wk 14 0008575 101300AD 10/13/00 10/15/00 8.30 I05716M Grp 3 Wk 14 0008576 101300AD 10/13/00 10/15/00 20.2 105719M Grp 3 Wk 14 0008577 101300AD 10/13/00 10/15/00 37.6 Average: 27.2 Standard Deviation: 29.4 I05703M Grp 4 Wk 14 I05704M Grp 4 W k 14 I0571 1M 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 1O/13/00 10/14-15/00 1O/15/00 10/14-15/00 1O/ 15/00 10/14-15/00 Average: Standard Deviation: 3.33 55.9 0.363 37.0 2.9 1 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. 3M Environmental Laboratory Page 29 of 110 Page 327 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 Centre Study No.: 023-01 I Sponsor Protocol No: FACT-TOX-026 Table X. Summary of POAA residues in Week 16 Sponsor Centre Set Extraction Analysis Analyte I05709M Grp 1 Wk 16 105714M Grp 1 Wk 16 105715MGrp 1 Wk 16 105718M Grp 1 Wk 16 I05720M Grp 1 Wk 16 105725M Grp 1 Wk 16 0008583 0008584 0008585 0008586 0008587 0008588 101600AR 101600AR 101600AR 101600AR 101600AR 101600AR 10/16/00 10/16/00 10/16/00 10/16/00 10/16/00 10/16/00 10118-19/00 10/18-19/00 1O/ 18-19/00 1O/ 18-19/00 10118-19/00 10118-19/00 Average: Standard Deviation: 0.104 0.0159 0.193 NQ NQ NQ 0.0572 0.0762 I05702M Grp 2Wk 16 I05706M Grp 2 Wk 16 I05717M Grp 2 Wk 16 I05721M Grp 2Wk 16 0008589 0008590 0008591 0008592 101600AD 101600AD 101600AR 101600AD 10/16/00 10/16/00 10/16/00 10/16/00 1o/ 19/00 1O/ 19/00 1o/ 18-19/00 1O/ 19/00 Average: Standard Deviation: 6.83 7.25 3.52 9.92 6.88 2.62 105707MGrp 3 Wk 16 I05708M Grp 3 Wk 16 105710M Grp 3 Wk 16 I05712M Grp 3 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 10118-19/00 1O/ 19/00 10/19/00 1O/ 19/00 1O/ 19/00 Average: Standard Deviation: 20.0 5.04 68.0 13.2 36.3 45.7 31.4 23.3 105703M Grp 4Wk 16 105704M Grp 4 W k 16 I05711M Grp 4W k 16 105713M Grp 4Wk 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 1O/ 18-19/00 1o/ 19/00 1O/ 18-19/00 1O/ 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. 3M Environmental Laboratory Page 30 of 110 Page 328 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 Centre Study No.: 023-01I Sponsor Protocol No: FACT-TOX-026 Table XI. Summary of POAA residues in Week 18 Sponsor Centre Set Extraction Analysis Analyte I05709M Grp 1 Wk 18 I05714M Grp 1 Wk 18 105715M Grp 1 Wk 18 105718M Grp 1Wk 18 I05720M Grp 1 Wk 18 105725M Grp 1 Wk 18 0008604 0008605 0008606 0008607 0008608 0008609 101700A 101700A 101700A 101700AD 101700A 101700A 1O/ 17/00 10/17/00 1O/ 17/00 10/17/00 10/17/00 101 1 7/00 10117-18/00 1O/ 17-18/00 1O/ 17-18/00 1o/ 19/00 10/17-18/00 10/17-18/00 Average: Standard Deviation: NQ NQ 0.0153 1.49 NQ NQ 0.258 0.604 I05702M Grp 2 Wk 18 0008610 101700A 10/17/00 1O/ 17-18/00 1.62 I05706M Grp 2 W k 18 0008611 101700A 10/17/00 1O/ 17-18/00 1.76 I05717M Grp 2 Wk 18 0008612 101700AD 10/17/00 1o/ 19/00 16.4 105721M Grp 2 Wk 18 0008613 101700A 10/17/00 10/17-18/00 3.08 Average: 5.72 Standard Deviation: 7.15 I05707M Grp 3 Wk 18 0008614 101700AD 10/17/00 1o/ 19/00 41.1 I05708M Grp 3 Wk 18 0008615 101700A 10/17/00 1O/ 17-18/00 4.48 I05710M Grp 3 Wk 18 0008616 101700AD 10/17/00 1o/ 19/00 22.9 I05712M Grp 3 Wk 18 0008617 101700AD 10/17/00 1o/ 19/00 9.50 105716M Grp 3 W k 18 0008618 101700AD 10/17/00 1o/ 19/00 16.3 105719M Grp 3 Wk 18 0008619 101700AD 10/17/00 1o/ 19/00 9.26 Average: 17.3 Standard Deviation: 13.3 I05703M Grp 4 Wk 18 I05704M Grp 4 W k 18 I05711M Grp 4Wk 18 I05713M Grp 4 Wk 18 I05722M Grp 4 Wk 18 0008620 0008621 0008622 0008623 0008624 101700A 101700AD 101700A 101700AD 101700A 1O/ 17/00 10/17/00 10/17/00 10/17/00 1O/ 17/00 1O/ 17-18/00 1o/ 1 9/00 10/17-18/00 1o/ 19/00 1O/ 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. 3M Environmental Laboratory Page 3 1 of 110 Page 329 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 Centre Study No.: 023-01 I Sponsor Protocol No: FACT-TOX-026 Table XII. Summary of POAA residues in Week 20 Sponsor - ID Centre ID I05709M Grp 1 Wk 20 I05714M Grp 1 Wk 20 105715M Grp 1 Wk 20 I05718M Grp 1 Wk 20 I05720M Grp 1 Wk 20 105725M Grp 1 Wk 20 0008625 0008626 0008627 0008628 0008629 0008630 Set Numbe=r Extraction Date Analysis Date Analyte Foun=d (@g) 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 1O/ 19-20/00 Average: Standard Deviation: NQ NQ NQ 2.65 NQ NQ 0.450 1.08 I05702M Grp 2 Wk 20 0008631 101800AD 10/18/00 10/20/00 6.91 I05706M Grp 2 Wk 20 0008632 101800A 10/18/00 10119-20/00 4.33 I05717M Grp 2 Wk 20 0008633 101800A 10/18/00 10/19-20/00 2.41 105721M Grp 2 Wk 20 0008634 101800AD 10/18/00 10/20/00 13.6 Average: 6.81 Standard Deviation: 4.89 I05707M Grp 3 Wk 20 0008635 101800AD 10/18/00 10/20/00 35.0 I05708M Grp 3 Wk 20 0008636 1018OOAD 10/18/00 10/20/00 24.9 I05710M Grp 3 Wk 20 0008637 101800AD 10/18/00 10/20/00 81.3 I05712M Grp 3 Wk 20 0008638 101800AD 10/18/00 10/20/00 25.5 I05716M Grp 3 Wk 20 0008639 101800AD 10/18/00 10/20/00 27.4 I05719M Grp 3 Wk 20 0008640 1018OOAD 10/18/00 10/20/00 120 Average: 52.4 Standard Deviation: 39.5 I05703M Grp 4 Wk 20 I05704M Grp 4 Wk 20 I05711M Grp 4 Wk 20 I05713M Grp 4 Wk 20 I05722M Grp 4 Wk 20 OOO8641 0008642 0008643 0008644 0008645 101800A 101800A 101800AD 101800A 101800AD 10/18/OO 10/18/00 10/18/00 10/18/00 10/18/00 1O/ 19-20/OO 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. 3M Environmental Laboratory Page 32 of 110 Page 330 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 Centre Study No.: 023-01 I Sponsor Protocol No: FACT-TOX-026 Table XIII. Summary of POAA residues in Week 22 Sponsor Centre Set Extraction Analysis Analyte I05709M Grp 1 Wk 22 I05714M Grp 1 Wk 22 105715MGrp 1Wk 22 I05718M Grp 1 Wk 22 I05720M Grp 1 Wk 22 105725M Grp 1 Wk 22 0008646 0008647 0008648 0008649 0008650 0008651 101900A 101900A 101900A 102000AD 101900A 101900AD I05702M Grp 2 Wk 22 I05706M Grp 2 Wk 22 I05717M Grp 2 Wk 22 0008652 0008653 0008654 101900AD 101900AD 101900AD I05707M Grp 3 Wk 22 I05708M Grp 3 Wk 22 I05710M Grp 3 Wk 22 105712MGrp 3 Wk 22 I05716M Grp 3 Wk 22 I05719M Grp 3 Wk 22 0008655 0008656 0008657 0008658 0008659 0008660 101900AD 101900AD 101900AD 101900AD 101900AD 101900AD I05703M Grp 4 Wk 22 105704MGrp 4 Wk 22 I05711M Grp 4 Wk 22 I05713M Grp 4 Wk 22 I05722M Grp 4Wk 22 0008661 0008662 0008663 0008664 0008665 101900A 101900AD 101900A 101900AD 101900A 1O/ 19/00 1O/ 19/00 1O/ 19/00 10/19/00 1O/ 19/00 10/19/00 10/20-21/00 10/20-21/00 10120-21/00 10/23/00 10/20-21/00 10/22/00 Average: Standard Deviation: 10/19/00 10/19/00 10/19/00 10/22/00 10/22/00 10/22/00 Average: Standard Deviation: 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: 1O/ 19/00 10/19/00 10/19/00 10/19/00 1o/ 19/00 10/20-21/00 10/22/00 10/20-21/00 10/22/00 10/20-2 1/00 Average: Standard Deviation: 0.0129 0.0422 NQ 90.8 0.0106 1.84 15.5 36.9 7.82 17.1 16.6 13.8 5.22 36.1 69.4 23.7 13.7 35.3 58.9 39.5 21.0 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. 3M Environmental Laboratory Page 33 of 110 Page 331 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 Centre Study No.: 023-01I Sponsor Protocol No: FACT-TOX-026 Table XIV. Summary of POAA residues in Week 24 Sponsor Centre Set Extraction Analysis Analyte I05709M Grp 1 Wk 24 I05714M Grp 1 Wk 24 105715M Grp 1 Wk 24 I05718M Grp 1 Wk 24 I05720M Grp 1Wk 24 105725M Grp 1 Wk 24 0008666 0008667 0008668 0008669 0008670 0008671 105702M 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 I05710M Grp 3 Wk 24 I05712M Grp 3 Wk 24 I05716M 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 I05711M Grp 4 Wk 24 I05713M Grp 4 Wk 24 I05722M Grp 4 Wk 24 0008681 0008682 0008683 0008684 0008685 102000A 102000AD 102000A 102000A 102000A 102000A 102000A 102000AD 102000A 102000AD 102000AD 102000AD 102000AD 102000AD 102000AD 102000A 102000AD 102000A 102000AD 102000A 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: 10/20/00 10/20/00 10/20/00 10/22-23/00 10/23/00 10/22-23/00 Average: Standard Deviation: 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/OO 10/23/00 Average: Standard Deviation: 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: 0.0261 2.82 NQ 0.03 10 NQ 0.207 0.517 1.13 4.14 12.40 2.12 6.22 5.45 30.4 53.7 58.4 10.0 65.3 25.3 40.5 21.8 0.534 99.6 0.3 10 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. 3M Environmental Laboratory Page 34 of 110 Page 332 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 Centre Study No.: 023-01 I Sponsor Protocol No: FACT-TOX-026 Table XV. Summary of POAA residues in Week 26 Sponsor Centre Set Extraction Analysis Analyte I05709M Grp 1 Wk 26 I05714M Grp 1 Wk 26 105715M Grp 1Wk 26 I05718M Grp 1 Wk 26 I05720M Grp 1 Wk 26 105725M Grp 1 Wk 26 0008686 0008687 0008688 0008689 0008690 0008691 102300AR 102300AR 102300A.R 102300AR 102300AR 102300AR 10/23/00 10/23/00 10/23/00 10/23/00 10/23/00 10/23/00 10/25/00 10/25/00 10/25/00 10/25/00 10/25/00 10/25/00 Average: Standard Deviation: NQ 0.03 17 NQ 0.0227 NQ 0.0186 0.0172 0.00892 105702M Grp 2 Wk 26 0008692 102300AR 10/23/00 10/25/OO 4.44 I05706M Grp 2 Wk 26 0008693 102300AR 10/23/00 10/25/00 2.48 I05717M Grp 2 Wk 26 0008694 102300AR 10/23/00 10/25/00 1.85 Average: 2.92 Standard Deviation: 1.35 I05707M Grp 3 Wk 26 0008695 102300AD 10/23/00 10/26/00 108 I05708M Grp 3 Wk 26 0008696 102300AD 10/23/00 10/26/00 46.6 I05710M Grp 3 Wk 26 0008697 102300AD 10/23/00 10/26/00 10.7 105712MGrp 3 Wk 26 0008698 102300AD 10/23/00 10/26/00 19.1 I05716M Grp 3 Wk 26 0008699 102300AD 10/23/00 10/26/OO 15.9 I05719M Grp 3 Wk 26 0008700 102300AD 10/23/00 10/26/OO 57.5 Average: 43.0 Standard Deviation: 36.9 I05703M Grp 4 Wk 26 I05704M Grp 4 Wk 26 I05711M Grp 4 W k 26 I057 13M 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/OO 10/26/OO 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. 3M Environmental Laboratory Page 35 of 110 Page 333 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 Centre Study No.: 023-011 Sponsor Protocol No: FACT-TOX-026 Table XVI. Summary of POAA residues in Week 28-34 Sponsor Centre Set Extraction Analysis Analyte I05718M Grp 1 Wk 28 I05720M Grp 1 Wk 28 I05718M Grp 1 Wk 30 I05720M Grp 1 Wk 30 105718M Grp 1 Wk 32 I05720M Grp 1Wk 32 I05718M Grp 1 Wk 34 I05720M Grp 1 Wk 34 0008706 0008707 00087 10 0008711 0008714 0008715 0008718 0008719 102400A 102300AD 102400A 102400A 102400A 102400A 102400A 102400A 10/24/00 10/24/OO 10/24/00 10/24/OO 10/24/00 10/24/00 10/24/00 10/24/OO 10/24-25/00 10/26/OO 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 I05712M Grp 3 Wk 28 I05716M Grp 3 Wk 28 I05712M Grp 3 Wk 30 I05716M Grp 3 Wk 30 I05716M Grp 3 Wk 32 I05716M Grp 3 Wk 32 I05716M Grp 3 Wk 34 I05716M Grp 3 Wk 34 0008708 0008709 0008712 0008713 0008716 0008717 0008720 0008721 102400A 102400A 102400A 102400A 102400A 102400A 102400A 102400A 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: 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. 3M Environmental Laboratory Page 36 of 110 Page 334 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 Centre Study No.: 023-011 Sponsor Protocol No: FACT-TOX-026 Table XVII. Summary of POAA residues in Weeks 36-40 Sponsor Centre Set Extraction Analysis Analyte I05718M Grp 1 Wk 36 I05720M Grp 1 Wk 36 I05718M Grp 1 Wk 38 I05720M Grp 1 Wk 38 I05718M Grp 1 Wk 40 I05720M Grp 1Wk 40 0008722 0008723 0008726 0008727 0008730 0008731 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/OO 10/26/00 10/26/OO 10/26/OO 10/26/W 10/26/00 Average: Standard Deviation: NQ 0.02 16 ND NQ NQ NQ 0.0103 0.00684 I05712M Grp 3 Wk 36 I05716M Grp 3 Wk 36 I05712M Grp 3 Wk 38 I05716M Grp 3 Wk 38 I05716M Grp 3 Wk 40 I05716M 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/OO 10/26/OO 10/26/OO 10/26/OO 10/26/OO 10/26/OO Average: Standard Deviation: 0.0944 0.0306 0.0327 NQ 0.0103 0.0235 0.0336 0.03 13 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. 3M Environmental Laboratory Page 37 of 110 Page 335 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 Centre Study No.: 023-01 1 Sponsor Protocol No: FACT-TOX-026 12.0 FIGURES Centre Analytical Laboratories, Inc. 3M Environmental Laboratory Page 38 of 110 Page 336 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 Centre Study No.: 023-011 Sponsor Protocol No: FACT-TOX-026 Figure 1. Typical Calibration Curve for POAA Compound 9 name: POAA Coefficientof Determination: 0.995377 , Calibration curve: 6743.59 x + 12362.0 Response type: External Std, Area Curve type: Linear, Origin: Exclude, Weighting: l/x, Axis trans: None X / ' ' ' / I )',I 50.0 " 100.0 a 150.0 ' I' 200.0 I I I I ng/mL 250.0 Centre Analytical Laboratories, Inc. 3M Environmental Laboratory Page 39 of 110 Page 337 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 Centre Study No.: 023-011 Sponsor Protocol No: FACT-TOX-026 Figure 2. Typical Mean Response Factor for THPFOS :ompound 8 name: THPFOS qesponse Factor: 84.6032 qesponse type: ExternalStd, Area : w e type: RF 2.42ed .. . -c qesponse 0c I * n a ' l 4 I c I ng/mL 50.0 100.0' z I a i50.0 ' ' 200.0 250.0 Centre Analytical Laboratories, Inc. 3M Environmental Laboratory Page 40 of 110 Page 338 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 Centre Study No.: 023-0111 Sponsor Protocol No: FACT-TOX-026 Figure 3. Chromatogram Representing a 5 n g h L extracted standard for POAA and 250 ng/mL extracted standard for THPFOS 8 : THPFOS XC101200-1,5 ng/mL Std - 101200A-1002 Sm(SG, 2x2) 100- 5.1 7 %- 13-Oct-2000 18:33:011 LC/MS/MS #6 MRM of 9 Channels ES- 427 > 80 2.28e5 Area O ' U J. I I . , , ll..I.I.I,l..III.I.l.lll ....l....l..I.l/,,, . I I I l . I . I I I . . I Time L 1 . b O " " "2".0"0' " " ' 3.0I 0 4.00 5.00 6.00 7.00 8.00 101200A-1002Sm (SG, 2x2) I ':I, ,, , , ,, ,.) , ,~ 1 .oo 2.00 3.00 I-Oct-2000 18:33:01 LC/MS/MS #6 MRM of 9 Channels ES- 413 > 369 3.93e5 4.00 A( _ I , , ' " ' I . - * . , .- . ,. I . , . , , , I " " 7 . . .LOO 6.00 7.00 8.00 .,.. .. Area Time Centre Analytical Laboratories, Inc. 3M Environmental Laboratory Page 41 of 110 Page 339 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 Centre Study No.: 023-011 Sponsor Protocol No: FACT-TOX-026 Figure 4. Chromatogram Representing a 125 ng/mL extracted standard for POAA and 250 ng/mL extracted standard for THPFOS ' XC101200-5,125 ng/mL Std 101200A-1006 Sm (SG, 2x2) 100- o/- 159.51975 13-Oct-2000 19:39:49 LC/MS/MS #6 M R M of 9 Channels ES- 427 > 80 1.65e5 Area 0 ""I"' ' ' ' l " ~ r I ' , , _1 . . I . I . . ' ' I ' . ' ' 1 . ..I 0 I ' ' ' ' ( . . . . l . . l . I . . , , l , . , , I , / , , l . . 1 Time i . o o ' ' I " '2' .'0'0 3.00 4.00 5.00 6.00 7.00 8.00 6.20e6 Area %- Centre Analytical Laboratories, Inc. 3M Environmental Laboratory Page 42 of 110 Page 340 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 Centre Study No.: 023-01 1: Sponsor Protocol No: FACT-TOX-026 Figure 5. Chromatogram Representing Control Monkey Feces for POAA and THPF0,S (Centre ID:'0009684Blank A, Set: 101200A) 1009684 Blank A 101200A-1009 Sm (SG, 2x2) .oo . . . . I ' ' ~ ' I ' ' . ~ / ' ' ~ 1 2.50 0110- c' ' 3484 13-Oct-2000 20:29:53 LC/MS/MS # 6 MRM of 9 Channels ES427 > 80 6.79e3 . , I I: . ,,-, , , , , , , , ~ , 8.00 Time 4.87e4 Area Centre Analytical Laboratories, Inc. 3M Environmental Laboratory Page 43 of 110 Page 341 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 Centre Study No.: 023-01L1RN-U2782 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 0008545 Spk A, 50 ppb 1 Ot %- %- 13-Oct-2000 21:36:49 LC/MS/MS #6 1.30e5 Area 158555 1.34e6 Area Centre Analytical Laboratories, Inc. 3M Environmental Laboratory Page 44 of 110 Page 342 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 Centre Study No.: 023-011 3 Sponsor Protocol No: FACT-TOX-026 Figure 7. Chromatogram of Monkey Feces Sample from Grp 2 in Week 12 (Centre ID: 0008548, Set: 101200A) 0008548 DF = 10 101200A-1026Sm (SG,2x2) - 14-Oct-2000 01 :14:21 LC/MS/MS #6 MRM of 9 Channels ES- 5.17 427 > 80 2.03e4 Area .oo I , I * Z ' I " , 1 2.60"' 9 : Po- 10008548 DF = 10 '3.I0'0 ' I " ' 4.'00' " I '*,I-'/ 5.00 6.bO ' P'- 7.00 I" I a I 8.00 Time 14-Oct-2000 01:14:21 1 149713- 9.78eE .Are2 Centre Analytical Laboratories, Inc. 3M Environmental Laboratory Page 45 of 110 Page 343 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 Centre Study No.: 023-011 Sponsor Protocol No: FACT-TOX-026 13.0 APPENDICES Centre Analytical Laboratories, Inc. 3M Environmental Laboratory Page 46 of 110 Page 344 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 Centre Study No.: 023-01I 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 (APFOPOAA) in Cynomolgus Monkeys and Amendments and Deviations Centre Analytical Laboratories, Inc. 3M Environmental Laboratory Page 47 of 110 Page 345 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 Centre Study No.: 023-011 Sponsor Protocol No: 'FACT-TOX-026 Study Title 6-Month Capsule Toxicity Study with Ammonium Perfluorooctanoate (APFOLPOU) in Cynomolgus Monkeys -.- c PROTOCOL Author Lisa Clemen Date: October 12, 199s Performing Laboratory 3 M Environmental Technolo,oy & Safety Services 31.41 Environmental Laboratory - 935 Bush Avenue St,Paul,MB 55106 Laboratory Project Identification FACT-TOX-026 3M EnvironmentalLabora!ory Centre Analytical Laboratories, Inc. 3M Environmental Laboratory I Page 1 o f 9 Page 48 of 110 Page 346 3M Medical Department Study: T-6889.3 . . . -..... Analytical Report: FACT-TOX-026 LRN-U2782 Centre Study No.: 023-011 Sponsor Protocol No: FACT-TOX-026 .. . . . I ............... . . . ..... ....... .. 6-Month Capsule Toxicity Study with Ammonium Perfluorooctanoate,( A P F O P O U ) in . Cynomolgus Monkeys I Test Material I .Ammonium periluorooctmoate (APFOIPOA.4) Sponsor -.- L 3 1ToxicologyServices - Medical Department 3M Center, Bailding 220-2E-02 P.O. Box 33220 . St. Paul; hlN 55133-3220 sponsor Representative Paul Lieder, Ph.D., DAQT 3M Toxicology.Services Building 220-2E-02 651-737-267s . Study Director ', . study Location(s) In vivo Testing Facility Analytical Testing Laboratory Kristen Hansen..Ph.D~-. 3 M Environmental Technology and Safety Services Building 2-3E-09 65I-77s-6018 Covancc Laboratories,Inc. 3 0 1 Khsnpn Boulevard Madison, Wisconsin 53704 3 M Environmental Laboratory Building 2-3E-09 935 Bush Avenue St. Paul, M N 55106 Proposed Study Timetable Analytical Start Date Analytical TerminationDate November 11,199s M3y 12,1999 3M EnvircnmentalLaboratoiy Centre Analytical Laboratories, Inc. 3M Environmental Laboratory Page 2 of 9 Page 49 of 110 Page 347 3M Medical Department Study: T-6889.3 . . . . .. . . Analytical Report: FACT-TOX-026 LRN-U2782 Centre Study No.: 023-01 1 Sponsor Protocol No: FACT-TOX-026 ... .. . . . ..... t PrOfOCd %FACT-TOX-026 I 7. STUDY Six month capsule toxicity study with m o n i u m perfluorooctmoate (APFOPOAA)in cynomolgus monkeys. I 2. PURPOSE The analytical ponion of this study is designed to determine levels of(APFOPOtL4) in the liver and serum of cynomolgus monkeys. Based on these results additional tissues or fluids may be analyzed. The in-life ponion of this study was conducted at Covance Laboratories, study t632923 1. 3. REGULATORY COMPUANCE This study will be conductea;h accordance with the United States Environmental Protection Agency Good Lboratory 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. QUAL~TYASSURANCE Tine 3hl Environmentd Laboratory Quality Assurance Unit will audit the protocol, study conduct, and final report in accordmce with the Good Laboratory Practice Standards and 3 M Environmental Laboratory Stmdard Operating Procedures. 5. TESTMATERIAL 5.7 identification ,4mrnonium perfluorooctanoate (APFOPOAA) 5.21 Source 3b1Specialty Chemical Division 5.3 !Physical Description Gelatin capsules 5.4 Purity and stability Determinedby the Sponsor 5.5 Stofage Conditjons Room temperature 5.6 Reserve Samples Responsibility of the Sponsor 5.7 Disposition Specimens will be retained per GLP replation 5.8 SafetyPrecautions Refer to MSDS for chernicds used. Wear appropriate laboratory attire, and follow adequats precautions for handling biolo$cd materials and preparing samples for analysis. I 3M Environmental Laborabry Centre Analytical Laboratories, Inc. 3M Environmental Laboratory Page 3 of 9 Page 50 of 110 Page 348 3M Medical Department Study: T-6889.3 ._.__---.....-~-....-.--A Analytical Report: FACT-TOX-026 LRN-U2782 Centre Study No.: 023-0 1 I Sponsor Protocol No: FACT-TOX-026 _._ - . .--. . . - . . -- PrOtOCOl XFACT-TOX-026 6. COiVrROL MATERIAL 6.1 Identification Monkey liver and serum 6.2 Source Covance Laboratories. hc. 6.3 Physical Description Liver and serum 6.4 Purity and Stability Not applicable 6.5 Storage Conditions Frozen at -20 "C k 10 "C 6.6 Reserve Samples Not applicable 6.7 Disposition Biological tissues and fluids are retained per GLP regulation 6.8 .SafetyPrecautions Refer to MSDS for chemicals used. Wear appropriate laboratory attire, all-follow adequate precautions for handling biological materials and preparing samples for analysis. 7. REFERENCMEATERIAL 7.7 IdentificafjonAmmonium perfluorooctanoate (MFOPOAA),lot $377 or $24j 7.2 Source 3M Specialty Chemicals 7.3 Physical Description White powder - 7.4 Purity and stability Responsibility of the STonsor 7.5 Storage Conditions Room temperature 7.6 Reserve Samples Not applicable 7.7 Disposition Retained as per GLP replation and 3M Environmental Laboratory 1 7.8 SafetyPrecautions Refer to MSDS for chemicals used. Wear appropiate laboratory attire, and follow adequate precautions for handling biological materials and preparing samples for analysis. - 8. TESTSYSTEM Cynomolgus monkeys were used 3s the test system, and were maintained and dosed as described in Covance protocol #6329-23 1. 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 desi,onated as recovery animals and were allowed a 13 week recovery period after cessation of treatment. Centre Analytical Laboratories, Lnc. 3M Environmental Laboratory Page 4 of 9 Page 51 of 110 Page 349 3M Medical Department Study: T-6889.3 . .... ' Analytical Report: FACT-TOX-026 LRN-U2782 Centre Study No.: 023-011 Sponsor Protocol No: FACT-TOX-026 . . . ... . . . . . . .. .. -. .-._. . . . - . . . . .I_. ._-. . Protocol#FACT-TO%-026 9. SPECIMEN RECEIPT The 3hl Environmental Laboratory will receive Samples of the following body tissues and fluids from the indicated points in the study: Serum - all animals 7 days post treatment, Packed on dry ice for 264 from main every two weeks shipping study thereafter 78 additional Urine, feces - recovery After 6,30,and 90 days Packed on dry ice for I animals recovery 1 shipping - I Liver ail animals I Artemination of the Shipped with final from recovery 1 I8urine, 18 I feces 1 3-2 serum samples on dry 1 ice 1 - 10.PREPARAF~MREYTHODS 70.7 FACT-M-1.O,Exuaction of Potassium PefflUorOoctaneSulfOnateor Other Anionic Fluorochemical Surfactant from Liver for Analysis Using HPLC-Electrospray/&fas Spectrometry 70.2 FACT-M-3.1,Extraction of Potassium Perfluorooctanesu~fonateor Other .. Ruorochemical Compounds from Serum or Other Fluid for Analysis Using HPLC-\; Electrosprayfilass Spectrometry 10.3 Ifpreparatory methods other than those listed above are used, an amendment to this protocol will be written. -. 11.ANALMlCAi METHODS . 11.1 FACT-M-2.0,Analysis of Fluorochemicals in Liver Extracts Using HPLcElectrosprayhfass Spectrometry 11.2 FACT-h1-4.1, Analysis of Potassium Perfluorooctanesulfonateor Other Fluorochemicals in Serum or Other Fluid Extracts Using HPLC-Electrosprav~~ass Spectrometry 11.3 If analytical methods other than those listed above are used, an amendment to this protocol will be written. 3hf EnvironmentalLaboratory Centre Analytical Laboratories, Inc. 3M Environmental Laboratory Page 5 of 9 Page 52 of 110 Page 350 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 Centre Study No.: 023-0,ll Sponsor Protocol No: FACTiTOX-026 ..- ._ _._ -- .. -- . .- ... ... PrOtOCOl #FACT-TOX-025 I' 72.DATA QUALIOTBYJECTIVES 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 m&: 72.1 Linearity rz 2 0.980 I 12.2Lh i t s o fdetection / quantitation 12.2.1Method Detection Limit ODL)for MFOPOAA !. a. Serum: 5ppb b. Liver: 24ppb 12.22Practical Quantitation Limit (PQL) -Equal to the lowest standard in the . calibration curve i _c 12.3Duplicate acceptable precision < 30% for the method 12.4 Spike acceptable recoveries 70% - 130% 12.5 Use o f confirmatorymethods Indeterminate samples will be re-analyzed 12.6 Demonstration o fspecificity Chromatographic retention time, m a s spectral daughter ion characterization 13.SUB-CONTRACATNEADLYSIS AI1 analyses as detailed in this protocol will be performed at 3M Environmental Laboratories, Building 2-3E-09,935 Bush A-venue,St. Paul, blX 55106. 14.STATISTICAL ANALYSIS Averag? 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 '. (weighvweight or weighVvo1) ofAPFO/l'OAA Or metabolite per tissue or fluid, or of APFOPOAA or cletabolite per unit of tissue or fluia. 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 statisticaldifference. 15.REPORT A repon of the results of the study will be prepared by 3M Environmental Laboratory. The reDOa 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 31vEnvironmental Laboratory Page 6 of 9 Centre Analytical Laboratories, Inc. 3M Environmental Laboratory Page 53 of 110 Page 351 3M Medical Department Study: T-6889.3 . . .. . . . . .- .- . -.. .- . Analytical Report: FACT-TOX-026 LRN-U2782 Centre Study No.: 023-01I Sponsor Protocol No: FACT-TOX-026 .. . -- .. .. -_ . .. -. .. . _.-.-.--.-- I PrOtOCOl $FACT-TOX-026 15.3 A stitement of compliance by the Study Director addressing any excepiom to Good Laboratory Practice Standards 15.4 Objectives ahd procedures as stated in the approved protocol, including my changes in the original protocol I 75.5 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 75.6 Stability and &e solubility of the test.substances under the conditions of administration, if provided by the Sponsor 75.7 A description of the methods used to conduct the test(s) 75.8 A description of.[& test system 75.9 A description of any circumstances that h a y have affected the quality or the inteFity .of the data 75.70 The name of the Study Director and the n w e s Of other scientists, professionals, and syervisory personnel involved in the s m d ~ 15.7 7 A description of the transformations, CdCUhtiOnS, or operations performed on the data, a summary and analysis of the andyticd chemistry data, and a statement of the conclusions drawn from the analyses 15.72 Statistical methods used to evaluate the data, if applicable 75.73 The signed and dated reports of each of the individual scientists or other professionals involved in the study, if applicable 75.74 The location where raw data and the find report are to be stored 75.75 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 Manto,ement If it is necessary to make corrections or additions to a finalreport after it has been accepted, the chanses will be made in &e form of an amendment issued b f i e Study Director. The amendment will clearly identify the part of the find report that is being amended, the reasons for the amendment, and will be siFed by the Study Director. 312.1Environmental Laboratory Centre Analytical Laboratories, Inc. 3M Environmental Laboratory Page 7 of 9 Page 54 of 110 Page 352 3M Medical Department Study: T-6889.3 ...-........,. ... Analytical Report: FACT-TOX-026 LRN-U2782 Centre Study No.: 023-01I Sponsor Protocol No: FACT-TOX-026 . . . . . . . . - . ................ ........... .............. I Protocol #FACT-TOX-026 16.1The following raw data and records will be retained in the study folder in the snidy/project archives according to 3 M Environmental Laboratoj Standard Operating Procedures: 7 6.7.1 Approved protocol and amendments I 16.1.2 Study correspondence 76.1.3 Shipping records 16.1.4 Raw data 16.1.5 'Approved final report (original s i y e d copy) 76.7.6 Electronic copies of data 16.2The following supporting records will be retained separately from the study folder in. the archives accordin,. to 3 M Environmental Laboratory.Standard Operating Procedures: 16.2.7 Training records ' 16.2.2 Calibration records 16.2.3 Instrument maintenjmce loss 162.4 Stmdxd Operating Procedures, Equipment Procedures, and Methods - SAMPLE REENTION Specimens will be maintained in the laboratory specimen archives for at l e u t a period of time as specified by regulation, and as established by 3 M Environmental Laboratory Standard Operating Proctdures. 18.PRdTOCOL AMENDMENTASND DEVIATIONS Planned changes to the protocol will be in the fonn of written amendments 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 chan,oesto the protocol will be indicated in the final report. ~ n otyher changes will'be in the form of written deviations, signed by the Study Director and filed with the raw data. 3Lf EnvironmentalLaboratory Centre Analytical Laboratories,Inc. 3M Environmental Laboratory Page 8 of 9 Page 55 of 110 Page 353 3M Medical Department Study: T-6889.3 - .. . . , . Analytical Report: FACT-TOX-026 LRN-U2782 Centre Study No.: 023-011 Sponsor Protocol No: FACT-TOX-026 .- --_ ... . - - ... - - . . , _ -. . . .. . .... .... ~ P a d Lieder, Ph.D, DABT,Sponsor Represen'rat''we ///3dPf Date- %sten Hansen, PbD., 3M Environmentd Laboratory Study Director 11/ I ~ 1Y 5 Date 3M EnvironmentalLabomtory Centre Analytical Laboratories, Inc. 3M Environmental Laboratory Page 9 of 9 Page 56 of 110 Page 354 3M Medical Department Study: T-6889.3 . . . .. .- .. . - Analytical Report: FACT-TOX-026 LRN-U2782 Centre Study No.: 023-01I Sponsor Protocol No: FACT-TOX-026 --.. - -.-...-..- - . . .. __._.__..__ ._,__ _ _-.-- . . ---._ . -.. .. ! Study Title - . 26-Week Capsule Toxicity ShidywithArnxnoniUmPerfluorooctanoate (MFO) in Cynomolgus Monkeys PROTOCOL AMENDMENT NO. 1 Amendment Date: July23, 1999 \ t Performing Laboratory 3M Environmental Technology & Safety Services - 3M Environmental Labontory 935 Bush Avenue St. Paul, IvfN 55106 Laboratory Project Identification ETBSS FACT-TOX026 L R V U27S2 3M Environmental Laboratory Centre Analytical Laboratories, Inc. .~ 3M Environmental Laboratory Page 57 of 110 Page 355 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 Centre Study No.: 023-011 Sponsor Protocol No: FACT-TOX-026 PrOtCCOl FACT-Tox026 Amendment NO. I This amendment modifies the following portion(s) of the protocol: ' I 1. PROTOCORL ~ O S S: ection 10.0 and 11.O list the following methods to.use for extractibn and analysis: 1 FACT-M-1.0 `Txbction of Potassium Perfluorooctanesulfonateor Other Anionic Fluorochemical Surfactant ftom Liver for Analysis Using kPLC-ElectrosprayMass Spectrometry" FACT-M3.1 ``Extractionof Potassium Perfluorooctanesu~fonatoer Other Fluorochemicd Compounds from Serum or Other Fluid for Analysis Uskg HPLC-Electrospray/Mas Spectrometry" - FACT-M-2.0 "Analysis of Fluorochemicals in Liver Exkcts Using HPLC-Electr~~pray/M~s Spectrometry" i c . FACT-M4.1 "&alysis of Potassium Peduorooctanesulfonateor Other FluorocheGcals in Serum or Other Fluid Extracts Using HPLC-EIectrosprayMass Spectrometry" AMENDTO READ: n e extraction and analytical methods FACT-M-3.1 and FACT-M-4.1, respectively, were updated on 04/27/99 to: ETS-84.1 ``Extraction ofpotassium Perfluorooctanesulfonateor Other Fluorochemical Compounds fiom S e m for h d y s i s Using EPLC-ElectrospnyMass Spectrometry" ETS-8-5.1 ``Analysis ofPot;issim Perfluorooctanesulfonateor Other Fluorochemicalsin Serum - Extracts Using HPLC-ElectrosprayMSS Sp=tromeb"' REdSON: The methods were updated to replace the extraction solvent ethyl acetate with a different extraction solvent MTBE (methyl fertbutyl ether), P O M 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 k weighting and a few minor ch'angesin the HPLC 1100instrument parameters. 2. PROTOCOL READS: Section 10.0 and-11.0 list the f o l l 0 6 g methods to use for extraction and analysis: FACT-M-1 .O "Extraction ofPot&um Perfluorooctanesulfonateor Other Anionic Fluorochemical Surfactant from Liver for Analysis Using HPLC-Electrospray/Mass Spectrometry'' ETS-S-3.1 ``Extraction of Potassium Perfluorooctanesulfonateor Other Fluorochemical Compounds from Serum or Other Fluid for Analysis Using HPLC-Electrospray/iCizss Spectrometry" FACT-hVI-2.0 ``halysis of FluorochemidS in Liver Extracts Using HPLC-Electrospray/&fas Spectrometry'' ETS-8-5.1 "Analysis ofPot&m Pefluorooctanesulfonate or Other Fluorochemicals in Serum or Other Fluid Extrac& usingHpLC-Elcctrosprayfifas Spectrometry" . ... - - - . . ..-- . 3M Environmental Laboratory Page 58 of 110 Page 356 3M Medical Department Study: T-6889.3 . - .._..-.._. .- . . Analytical Report: FACT-TOX-026 LRN-U2782 Centre Study No.: 023-01I Sponsor Protocol No: FACT-TOX-026 . . .. . .- . - ....-.... ... - --..____.. . ..- . .- .-.-. -- I PrOtOCCl FACT-TOXO26 Amendment No. 7 AMENDTO READ: n e extraction and analytical methods FACT-M-1.0 and FACT-hi-2.0, respectively, were updated on 07/22/99 to: ETS-8-6.0 "Extraction of Potassium PerfIuorooctanesulfonateor Other Fluorochem&l Compounds from Liver for Analysis Using HPLC-Electrospmyhlass Spectrometry" ETS-8-7.0"Analysis of Potassium Perfluorooctanesulfonateor Other Fluorochernical Compounds in Liver Extracts Using HPLC-Electrospmyhhs Spectrometry" REASONT:he methods were updated td replace the extraction solvent ethyl acetatewith a different extraction solvent MTBE, POAA and Monster were removed fiom the standard miy, and M556 was added to thestandard mix. The analytical mehod wa.updated to include linear r e p s i o n with I/x weighting and a few minor changes in the HPLC 1100instrument parameters. 3. PROTOCOLREADS: Section 10.0and 11.0list the followingmethods to use for extraction and analysis: ETS-8-6.0 "Extraction of Potassium Perfluorooctanesdfonate or Other Fluorochemical Compounds from Liver for Analysis Using HPLC-ElectrospmyMass Spectrometry"ETS-8-4.1 "Extraction of Potassium Peduorooctanesulfonate or OtherFluorochemical Compounds from Serum or Other Fluid for Analysis Using HPLC-ElectrOspmYMas Spectrometry" ETS-8-7.0 "Analysis ofPot&um Perfluorooctanesulfonateor Other Fhorochemicd Compounds in Liver Extracts Using HPLC-ElectrospraylMass Spectrometry" ETS-8-5.1"Analysis of Potassium Pe~uorooctanesulfonateor Other Fluorochemicals in S e r u m or Other Fluid Extracts usins WLC-Electrosprayhlass Spectrometry" \ AMEND t o READ: Additional extraction and adytical methods, listed below, were added to the protocol: ETS-$96.0 "Extraction of Potassium Perfluorooctanesulfonaleor other fluorochemical compounds fromUrine for Analysis Using HPLC-ElectrospraylMas Spectrometry" ETS-8-97.0 ``hdysis of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds in Urine Extracts Using HPLC-Electrosprayfilas Spectrometry" REASON: These methods were developed and validated for urine extraction and analysis after the original protocol was written and approved. X i Environmental Laboratory Centre Analytical Laboratories, Inc. 3M Environmental Laboratory Page 59 of 110 Page 357 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 Centre Study No.: 023-01 I Sponsor Protocol No: FACT-TOX-026 .. . .... -. . . _. PrOtOCOl FACT-TOXO26 Amendment No. 1 . 4. PROTOCORLEADS: Section 2.0 lists serum and liver as the matrices of interest for determination of POAA. AMENDTO READ: U&e Will be included for determination of POk4. I REASONn: e metho& were developed and validated for extraction and analysis of urine after the on,ginal protocol was written and approved. 5. PROTOCOL READS: Section 6.0 lists monkey serum k d liver as the control matrices received fiom Covance Laboratones. AMENDTO READ: ControI mfiy monkey urine was obiained fiom Covance Laboratories and is maintained at a temperame of -20O C 10 'C. All traceabilityinformation for this matrix \vi11 be included in the final report. REASONA: ddition of control urine matrix to the andyti~dprotocol. 6. PROTOCORLEADS: Section 12.2 lists monkey serum and liver method detection limits. REASONA: ddition ofmethod detection limit for urine matrix. \ I Amendment Approval Sponsor Representative QLd,-7,/997 Date ILLJS. Hansen, Ph.D., Study Director 3124 Environmental Laboratory Centre Analytical Laboratories,Inc. 3M Environmental Laboratory 7k/93 Date Page 60 of 110 Page 358 3M Medical Department Study: T-6889.3 I .. .. ...-.-- .. . Analytical Report: FACT-TOX-026 LRN-U2782 Centre Study No.: 023-011 Sponsor Protocol No: FACT-TOX-026 - .. . . ._ - - - - ........._ ~ ----- I .-- c Study Title 6-hfonth Capsule Toxicity Study with h O n i U m Perfluorooctanoate (APFO/POfi) in Cynomolgus Monkeys PROTOCOLAMENDMENTNO.2 Amendment Date: 20 January 2000 , Performing Laboratory ! 3hl Environmental Technology & Safety Services 3 M Environmental Laboratory . . 935 Bush Avenue St. Paul, MX 55106 - Laboratory Project Identification E T S S LRN-U27S2 FACT TOX-026 Covmce Study: 6329-23 1 3 M Medic4 Department Study: T-6SS9.3 3M Environmental Laboratory I Centre Analytical Laboratories, Lnc. 3M Environmental Laboratory Page 61 of 110 Page 359 3M Medical Department Study: T-6889.3 .. .- . . . Analytical Report: FACT-TOX-026 LRN-U2782 Centre Study No.: 023-011 Sponsor Protocol No: FACT-TOX-026 . . ..._._ _. - .. -. -- - -...I.^ . _ _ _ _ _ _ _ _ _ _ . _-_. _ _ _ _ . . - .-.._ _~ . _ _ . .. _-.. PrOtOCOl LRN-U2782 Amendment Number 2 This amendment modifies the following portion(s) of the protocol: 1. PROTOCORLEADS: The study director for the present study was identifiedin the protocol as Kristen J. Hansen, Ph.D. AMENDTO READ: The role of study director forthe 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 of study directorwas reassigned in an effortto ensure compliance with Good Laboratory Practice Standards that outline study personnel requirements (referto 40 CFR Part 792). 2. PROTOCORLEADS: The sponsor forthe present study was identified as Paul Lieder. AMENDTO READ: The role of sponsor for the present study was reassigned to John L. Butenhoff, Ph.D., a s of 20 January 2000. REASON: To ensure that the study director does not also CSnY the duties of study sponsor, the sponsor role was reassigned. In this manner, personnel responsib3ities and workload are more evenly balanced. 3. PROTOCORLEADS: 17. Sample Retention: Specimens will be maintained in the laboratory specimen archives for at least a period of time as specifiedby regulation, and as established by 3M Environmental Labdratory Standard Operating Procedures. AMENDTO READ: 17. Specimen Retention: Specimens will be maintained in the 3M Environmenfal Laboratory specimen archives. Any specimens sent to sub-contract laboratories will be returned to the 3M Environmental Laboratory upon completion of analysis and submission of t h e subcontract laboratory(s)finalreport. Specimens analyzed at sub-contract laboratories will be returned with the followingdocumentation: 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 of specimens analyzed at subcontract laboratories. 3M Environmental Laboratory Centre Analytical Laboratories, Inc. .-. 3M Environmental Laboratory Page 62 of 110 Page 360 3M Medical Department Study: T-6889.3 ~ ._ Analytical Report: FACT-TOX-026 Centre Study No.: LR. N-U2782 023-01 I Sponsor Protocol No: FACT-TOX-026 Protocof LRN42782 Amendment Number 2 , 4. PROTOCORLEADS: I Section 16 states that 1.,e following raw data and records will b - retained in the study folder in the archives according to AMDT-S-8: Approved protocol and amendments; study correspondence: shipping records; raw data; approved final report (original signed copy); and electronic copies of data. Additionally,Section 16 states that supporting records to be retained Separatelyfrom 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, Equipment Procedures, and Methods: and appropriate specimens. AMEND TO READ: Section 16 states: "The original data, or copies thereof, Will be available at the 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: approved protocol and amendments, study correspondence, shipping records, raw data, approved final report, and electronic copies of data will be retained in the archives of the 3M Environmental Laboratory. All corresponding training records, calibration records, instrument maintenance logs, standard operating procedures, equipment procedures, and methods will be retained in the archives of the facilityperformingeach analysis. REASON: To direc-t s-ubcontract laboratories in the disposition of the items listed above. 3M Environmental Laboratory Centre Analytical Laboratories,Inc. 3M Environmental Laboratory Page 63 of 110 Page 361 3M Medical Department Study: T-6889.3 . ... - ... Analytical Report: FACT-TOX-026 LRN-U2782 Centre Study No.: 023-01I Sponsor Protocol No: FACT-TOX-026 . ..- . -.....- .- . .... - r .. . .. .-. . . . ...- ..-.-.. Amendment Approval - i Kristen J Hansen, Ph.D., Outgoing Study Director i 11- F<b - a00 Date ' iaymector - Date ! 3:v Environmental Laboratory Centre Analytical Laboratories,Inc. 3M Environmental Laboratory Page 64 of 110 Page 362 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 Centre Study No,: 023-01 1 Sponsor Protocol No: FACT-TOX-026 . . ........ . - ... . . . . . . . . . . . ... ....... _I. .- - . . . . . . I . Study Title 6-Month Capsule Toxicity Study Rith Ammonium Perfluorooctmoate (APFOPOAA) in Cynomolgus Monkeys .i PROTOCOL AMENDMENT NO. 3 Amendment Date: 20 April 2000 Performing Laboratories ~- 3M Environmental Technology 3nd Safety Services FluoMe Analytical Chcmimy Team Building 2-3E-09,935 Bush Avenue SL Paul, MN 55106 3045 Research Drive State College, PA 16S31 -. Laboratory ProjectIdentification ET&SS LRN-U2782 FACT TOX-026 C O V U CS~tudy: 6329-231 3h.1Medical Department Study: T-6SS9.3 3M Environmental Laboratory Centre Analytical Laboratories, Inc. 3M Environmental Laboratory Ewct Copy of Original Page 65 of 110 Page 363 3M Medical Department Study: T-6889.3 - ._ Analytical Report: FACT-TOX-026 LRN-U2782 Centre Study No.: 023-01 1 Sponsor Protocol No: FACT-TOX-026 . . I ..- . . .. . . ,. I AmP reOntdCmCednLtRNNu-mUb2e7r832 This amendment modifies the following portion(s) of the protocol: 1. PROTOCORLEADS: Tine amended section 2.0 text states that this study is designed to betermine levels of APFOPOAA in the liver, serum, and urine of cynomolgus monkeys. AMENDTO READ: ! 'Ihis study is designed to determine levels ofA m / P O - 4 4 in the live:, serum, feces, and . urine of cynomolgus monkeys. REASON:. The analysis offecal tissue for the target chemical andor its anal).tes was added to the scope of the study following the issuance of the protocol. Feces extraction and analytical methods we:e not validated md'approved prior to protocol approval. ' 2. PROTOCORLEADS: The amended section 6.0 lists modey liver, serum and urine. AMENDTO READ: Add: monkey feces with a physical description of feces. REASON: Analysis of fecal tissue for the target chemical andor its malytes was added to the scope of the study following the issuance of the original protocol. 3. PROTOCORLEADS: The amended Section 12.3.1 items a., b., and c., list the Method Detection Limits for matrices analyzed in this study. AXEND 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 deiection limits are specific to the 3 M Environmental Laboratory. This statement was added to allow for sub-contractedanalyses, revised -methods, and added matrices. 4. PROTOCORLEADS: Section 13. lists the labontones that will be conducting analyses for this study. AMENDTO READ: Add: Centre Analq.tical Labontories, Inc., 3035 Research Drive, State College, PA 16801 REASON: Feces analyses were added to the scope of this study. ?he sub-contract laboratory performing analyses was not in the original protocol. 3 4 Environmentd Laboratory I Centre Analytical Laboratories, Inc. 3M Environmental Laboratory Page 66 of 110 Page 364 3M Medical Department Study: T-6889.3 .... - . Analytical Report: FACT-TOX-026 LRN-U2782 Centre Study No.: 023-01 I Sponsor Protocol No: FACT-TOX-026 - . . . . - ........ I ..... . . .- .. .. ............ ....... -.. ..-- --. P:otocol LRN-U27&?2 I Amendment Number 3 5. PROTOCOL REdDS: Sections 10.3 and i 1.3 State that if methods other than those listed are used in this study, an amendment will be written to include the new methods. . AMENDTO READ: The feces extraction and analytical method used by Centre AnaIyticaI Laboratories will be; OOM-023-003 (Revision 2), ``Determination of Ruorochemical Residues in iMonkey/Rat Feces by LCMSNS." REASON: The sub-contract laboratory performing feces analyses was added to the scope of tGs study; this method was'not validated and approved prior to protocol ipproval. . .- .* c Amendment Approval John L. Butenhog Ph.D., Sponsor Representative Date 3ibf Environmental Laboratory I Centre Analytical Laboratories, Inc. 3M Environmental Laboratory Page 67 of 110 Page 365 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 Centre Study No.: 023-01 I Sponsor Protocol No: FACT-TOX-026 . . ... __ - .-..-..-. - ... Study Title I 26-Week Capsule Toxicity Study with Ammonium Perfluorooctanoate (UFO) I in Cynomolgus Monkeys i PROTOCOL AIEiWMEiyT NO. 4 -.-. Amendment Date: , Aupst 2,2000 Performing Laboratories Covance Laboratories Inc. 3301 Kinsman Boulevard Madison, WI 53704 3M Environmental Technology & Safety Services 3M EnvironmentalLaboratory 935 Bush Avenue St. Paul, MN 55106 - Laboratory Project Identification FACT-TOX-026 LMS U2782 Covance 6329-231 3 M Medical Department Study: T-6589.3 - . Centre Analytical Labo_r_atories, Inc. 3M Environmental Laboratory Page 68 of 110 Page 366 3M Medical Department Study: T-6889.3 .. I . . . ........................ Analytical Report: FACT-TOX-026 LRN-U2782 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. PROTOCOL READS: STUDYTITLE;"6-Month Capsule Toxicity [..I" (on the title page and page 2 of the I protocol for the analytical phase of the study). AMEVDTO m: STUDY TITLE"26-'Week Capsule Toxicity [..I" (On the title page and page 2 of the protocol for the analytical phase of $e study). REASON: To correct the title for the analytical phase of the study. 1. PROTOCOwL s : DATAQUALITOYBJECTIVE(SSection 1 2 , page 6) AMENDTO REm: Add to this section: LOQ in feces of 10 ng/g REASON: TOspecify &e analyticallimits f;-r feces analyses. 3. PROTOCORLE ~ S (:page 2) STUDYDIRECTOPeRte:r J. Thornford and Paul Lieder TESTINFGACILITYC:ovance Laboratories SPOXSOR:A P E AdHoc APFO Toxicology Working Group and 3M Toxicology Services - Medical Department SPONSOR R E P R E S E ~ V AD~a:vid Farrar and John Butenhoff AMEND TO =AD: - STUDYDIRECTOPRau:l T E S ~FGACILITY3:M Lieder T6xicology Services - Medical Department SPONSOR:~ p A mbo c A~PFO Toxicology Working Group (including 3 M Toxicolog Services - Medical Department) SPO~VSO&RPR.ESE,VTATTVE: David Farrar (in-life) and John Butenhoff (analytical) REASOX: To r e s s i g 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. Centre Analytical Laboratories, Inc. 3M Environmental Laboratory Page 69 of 1 10 Page 367 3M Medical Department Study: T-6889.3 . . . . . . ... - . . .. Analytical Report: FACT-TOX-026 LRN-U2782 Centre Study No.: 023-01 1 Sponsor Protocol No: FACT-TOX-026 . . . . . ~ . . . - .. - . . . .____L... I Protocol TOX-026 Amendment 4 4. PROTOCOLRWS: PRINCIPALANALYTICALZIWSTIGATOR: fisten Hmsen, PhD. (Amendment No 2 to TOX 026, Section 1.) REASON: To specify the PAI at Centre. 5. PROTOCOLRE,WS: .--ANALYTIW TERMINATION DUE: May 12, 1999 (under Proposed study Timetable, page 2) AMENDTO READ: AV.4LYTIC.u TER.W,VUIONDATE:September 30,2000 REASON: TOallow for the analyses of all samples. - .. 6. PROTOCOLREADS: LOCATIONOFRAWDATAR, ECORDS,AND.FIN~LcRE~oRT(Section 16., page 7) A,MEND TO READ: Add: After issuing their fmal report, Centre Will forward all original study-specific data to 3 M EnvironmentalLaboratories, 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. PROTOCOLREADS: S A M P L E R E T E N ~ O N 1(7S.,~p~ag~e ~8)~ ~ Centre Analytical Laboratories, Inc. 3M Environmental Laboratory ' Page 70 of 110 Page 368 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 Centre Study No.: 023-011 Sponsor Protocol No: FACT-TOX-026 .. .- ...... ...... . ... . , _ . _. . PrOtOCO/ TOX-026 I Amendment 4 the analytical repoi for the 3M Environmental Laboratory analyses is signed by the study director. REASO,~:To specify the handling of the above biological specimens, and to define when &e quality assurance verification is considered complete. .- . Centre Analytical Laboratories, Inc. .. ... 3M Environmental Laboratory Page 71 of 110 Page 369 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 Centre Study No.: 023-011 Sponsor Protocol No: FACT-TOX-026 _ _ _ -.. .. - ---- -. - ..-.. .. .. . Amendment Approval PrOtOCOl TOX-026 Amendment 4 . Sponsor Representative, Analytical Phase' Lieder, Ph.D., DABT Date 3M Study Director ~ .. Centre Analytical Laboratories, Principal Analytical Investigator , Centre Analytical Laboratories, Inc. 3M Environmental Laboratory Page 72 of 110 Page 370 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 Centre Study No.: 023-01 1 Sponsor Protocol No: FACT-TOX-026 . .. ... _ _ .. -. ~ . ... _ _ ,._. _.. . . Sfudy Title I 26-Week'Capsule Toxicity Study withA r a m O n i U m Perfluorooctanoate(QFo) in Cynomolgus Monkeys . -' PROTOCOL AMENDMENT NO. 5 i L Amendment Date: OCtObG 12,2000 Performing Laboratory 3M Environmental TechnoIogy & Safety Services 3M EnvironmentalLaboratory 935 Bush Avenue St.P a l , MN 55106 Laboratory Project Identification' FACT-TOX-026 ET&SS LRNU2782 Covauce 6329-23 1 -. 3M Medical Department Study:'T-6889.3 Centre Analytical Laboratories, Inc. 3M Environmental Laboratory Page 73 of 110 Page 371 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 Centre Study No.: 023-011 Sponsor Protocol No: FACT-TOX-026 - . .. .. . . . - . . - ..-. . . . . J J.ProtocAolmFeAnCdTmTeOntXN-o0.256 I This amendment modifies the following portion(s) of the protocol: 1. `PROTOCORLEADS: Principal hdytical Investigators:Kristen H m ~ nP,bD. and Enabha Wickremesinhe, PbD. 3M Environmental Laboratory Centre Anaiytical Laboratories, Inc. 3M Environmental Laboratory Page 74 of 1 10 Page 372 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 Centre Study No.: 023-01 1 Sponsor Protocol No: FACT-TOX-026 --. . . - ..., ' J J PfOtOCAOlmFeAnCdmT eTnOt XN-o0.256 Amendment Approval * ?&?* John L. Butenhog Ph.D. Date . Sponsor Representative, Analytical Phase I Qd?L$A.-- - Paul Lieder, Ph.D., DABT 3M Study Director / P / t 5/> 0 Date Date Centre Analytical Laboratories, Principal Analytical investigator 3M Envimnrnenfal Laboratory Centre Analytical Laboratories, Inc. 3M Environmental Laboratory Page 75 of 110 Page 373 3M Medical Department Study: T-6889.3 . - . . . ~. . Analytical Report: FACT-TOX-026 LRN-U2782 Centre Study No.: 023-011 Sponsor Protocol No: 'FACT-TOX-026 . . - . . -.-- . - .. ---- ' Study Title .. 26-Week CapsuleToxicity Study with Ammonium Perfluorooctanoate(ApFO@OAA)in Cynomolgus Monkeys PROTOCOL AMENDMENT NO. 2 Amendment Date: January 11,2001 Performing Laboratories Cov'ance Laboratories Inc. 3301 Kinsman Boulevard Madison, WI 53704 3M EnvironmentalTechnology & Safety Services 3M Environmental Laboratory 935 Bush Avenue St. Paul, h4N 55106 1 Laboratory Project Identification I Covance Study: 6329-231 ProposalNO.w6806a 3M Medical Department Study: T-6889.3 ET&SS LRN-U2782 FACT TOX-026 , I 3M Environmental Laboratory -LA-: .-.4 *? .I:?;' c ; i.:..,.. -, -A- 0 J / I 4 2 ( !;:?!a L I E25 -- . - - _ Centre Analytical,Laboratories, Inc. -. 3M Environmental Laboratory -, Page 76 of 110 Page 374 3M Medical Department Study: T-6889.3 . .~ ........... Analytical Report: FACT-TOX-026 LRN-U2782 Centre Study No.: 023-011 Sponsor Protbcol No: FACT-TOX-026 .... ~ _.. - - ~ Covance $tudy 6329-231 Amendment 2 I This amendment modifies the following portion(s) of the protocol: 7. PROTOCOL READS: 3M ANALY77CAL PHASE PROTOCOL, SECTION 9. SPECIMEN RECEIPT It was expected that there would be 18 feces specimens and 18 unne specimens from the recovery animals collected and shipped for analysis. I AMENDTO READ: I 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. REASON: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 PROTOCOL 6329-231, PAGE 2 The study location is desigated as Covance Laboratones, Inc. AMENDTO 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, WI 53704 for the in-life phase and 3M Environmental Technology & SafetyServices,3M Environmental Laboratory, 935 Bush Avenue, St. Paul, MN 55106 for the analytical phase. REASON:G U Sallow for only one study location per study. The study director and sponsor reside at 3M Toxicology Services. 3. PROTOCOL READS: 3M ANALYTICAL PHASE PROTOCOL TOX-026 AND THE COVANCE 6329-237: Originally there were two protocols for the same study. AMENDTO READ: AMENDMENTTO 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. CovanFe Study 6329-231 &-Life Phase) ' Proposal No. w6806a FACT-TOX-026 (Analytical Phase) LRN t U2782 3M Medical Department Study: T-6SS9.3 REASON: GLPs allow for only one protocol for a study. All study, protocol and project identifications are listed to provide a documented link between documents. 3M Environmental Laboratory Centre Analytical Laboratories, Inc. -.- - -I --..____ _._ ___. 3M Environmental Laboratory Page 77 of 110 Page 375 3M Medical Department Study: T-6889.3 . . ... . .. . ~ I Analytical Report: FACT-TOX-026 LRN-U2782 Centre Study No.: 023-01 I Sponsor Protocol No: FACT-TOX-026 .... .- - - - ... .._. _ _ _ _ _ . _ _ _ _ _ _ 8 Covance Study 6329-231 I Amendment 2 I 4. PROTOCOL READS: AMENDED 3M ANALYTICAL PHASE TOX-026 AMENDMEN#T4: Peter Thornford was removed from position as study director, but his status was not redefined. 4 I AMENDTO READ: 1 Dr. Peter Thornford will take bn the position of Principal In-Life Investigator. I REASON:GLPs allow only qne study director for a study. Dr.Thomford's role needs to be re,defined once he was removed as study director. 5. PROTOCOL READS: 3M ANALYTICAPLHASE TOX-026,SECTION 4.0 AND 3M AMENDMENT #3 TO TOX-026: 1 Amendment #3 adds Centre to list of performing laboratories. AMENDTO R&D: SEcnoN 4 OF TOX-026,ANALMICAL'PHASE: 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. PROTOCOL READS: 3M ANALYTICAL PHASE PROTOCOL FACT TOX-026, SECTION7. REFERENCMEATERIAL: Protocol states that the reference material used will be lot #377 or #245. AMENDTO 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 materid. Feces analyses performed at Centre Labs were not included in the original protocol but was added in Amendment 3. Centre actuallyused lot #332 for the reference. i 3M EnvironmentalLaboratory Centre Analytical Laboratories, Inc. 3M Environmental Laboratory Page 78 of 110 Page 376 3M Medical Department Study: T-6889.3 . . .. . . - . , .~. Analytical Report: FACT-TOX-026 LRN-U2782 Centre Study No.: 023-011 Sponsor Protocol No: FACT-TOX-026 . .. .. . . I - . - - ~ -... ~ I Amendment Approval .. i Cbvance Study 6329-231 Amendment 2 I ' I John L. Butenhoff, Ph.D., Sponsor~epreSenfutive Date I i - . Paul Lieder, Ph.D., Study Director 0 6 f i h a( Date 3M Environmental Laboratory Centre Analytical Laboratories, Inc. 3M Environmental Laboratory Page 79 of 110 Page 377 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 Centre Study No.: 023-011 Sponsor Protocol No: FACT-TOX-026 - \ Cc=ntre Analytical Laboratories, 1nc. 3048 RsrJrch Drive,State College, PA 16801. Phone: (814) 231-8032, Facsimile: (814)231-1253 PROTOCOL DEVIATION Deviation Number: 1 . Date of Occurrence: lM/W Centre Study Number:023-011 Sponsor Protocol Number:FAm-TOx-026 DESCRIPTION OF DEVIATION . 6 12.4 Page 6: Accepted recoveries of 66% for PFOSM. 54% for M570,and 62%for M556 for :entre sample 0008435 Spk B in Set 1007OA. j.e.. amendment issued. SOP revision.etc., . Protocol deviation i s s u e k J IMPACT ON THE STUDY . No negative impact on the study. ~ ~ ~~ ponsoARepresentative Signature Dare Centre Analytical Laboratories, Inc. 3M Environmental Laboratory Page 80 of 110 Page 378 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 Centre Study No.: 023-011 Sponsor Protocol No: FACT-TOX-026 .. - \ CEntrez Analytical Laborator k s . Inc. 3048 Raearch Drive, State College, PA 16801. Phone: (814) 231-8032, Facsimile: (814) 231-1253 PROTOCOL DEVIATION Deviation Number: 2 Date of Occurrence: (1) 10/07/00.(2) 10/1w00.(3) 10/10-11/00, (4) 10110-11/00 Centre Study Number.023-011 Sponsor Protocol Number: FACT-TOX-026 DESCRIPTION OF DEVIATION . 5 12.4 Page 6: Accepted recovery of 137% forPOAA for Centre sample 0 0 0 ~ 6 S1pk B in Set mAD. !. 8 12.4 Page 6: Accepted recovery of 139% for POAA for Centre sample 0008482 Spk B in Set 00900A. . 5 12.4 Page 6: Accepted recovery of 133%forPOAA for Centre sample 0008500Spk B in Set OloOoA. . 8 12.4 Page 6: Accepted r' value of 0.970444for EtFOSE-OH calibrationcurve in Set lol()O()A. ACTIONS TAKEN j.c.. amendment issued. SOP revision. ert... c;3,$tu& IZJ, S, lCt) I, I M P K T ON THE STUDY -j.No negative impact on the study. . Nb negative impact on the study because still met method requirements for linearity. nicipal Inkstigator Signature & Centre Analytical Laboratories, Xnc. 3M Environmental Laboratory Page 81 of 110 Page 379 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 Centre Study No.: 023-01 I Sponsor Protocol No: FACT-TOX-026 CEntrE Analytical L a b o r a t o r i e s . Inc. 3 0 4 Research Drive, Sutc College. PA 16801. Phone: (81.1) 231-8032, F~aimile:(814) 231-1253 PROTOCOL DEVIATION Deviation Number: 3 Centre Study Number: 023-011 SponsorProtocol Number: FACT-TOX-026 DESCRIPTION OF DEVIATION 1. 8 12.4 Page 6: Accepted recovery of 136% for PFoSEA for Centre sample 0?08585 Spk A in Set 1016ooAR. 2. 8 12.4 Page 6: Accepted recovery of 141%for PoAA for Centre Sample 0008626 Spk B in Set 101SOOA. 3. 8 12.4 Page 6: Accepted recovery of 138%forEGOSE-OH for Centre sample 0008650Spk A Ind 63% for PFOSM and 57% for PFOSEA for Centre Sample 0008651 Spk B in Set 1019OOA. 1. 0 12.4 Page 61 Accepted recovery of574b for PFOS for Centre sample 0008667 Spk B in Set lO2000A. 5. 8 12.4 Page 6: Accepted recovery of 170%for P o M for Centre Sample0008667Spk B in Set 102OOOA.D.- 5. 5 11.4 Page 6: Accepted recovery of 56% for POAA for Centre Sample0008689 Spk A in Set 102300AR. I i.c.. amendment issued. sop vision. CIC, I 1-d. Protocol deviation issugd, - IMPACT ON THE STUDY .-6. NO negative impact on the study. Centre Analytical Laboratories,Inc. 3M Environmental Laboratory Page 82 of 110 Page 380 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 Centre Study No.: 023-011 Sponsor Protocol No:FACT-TOX-026 3048 Rexarch Drive, State Collqc. PA 16801. Phone: (814) 231-8032, Facsimile: (811) 231.1233 PROTOCOL DEVIATION Deviation Number: 4 Date of Occurrence: (1 & 2) Entire Study Centre Study Number: 023-011 Sponsor PrOtWOl Number: F.4m-ToX-026 DESCRIPTION OF DEVIATION 1. 9 1 Amendment No. 3: Throughout the study. samples were analyzed for ELFOSE-OH, PFOSAA, M570.M556. PFOSEA, PFOS. PFOSA. and POAA, instead of only for PO& ! I 2. 9 7 Reference Material Page 4: Ttie lot of POAA used for this study was 332. ACTIONS TAKEN j.e.. amendment issued. SOP revision. etc. 1-2. Protocol deviation i y y d . lecorded Bymate: v (I & 121,4/d IMT'ACTDN THE STUDY Centre Analytical Laboratories, Inc. 3M Environmental Laboratory Page 83 of 1 10 Page 381 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 Centre Study No.: 023-011 Sponsor Protocol No: FACT-TOX-026 APPENDIX B Determination of Fluorochemical Residues in Monkeymat Feces by LC/MS/MS (Revision 2) Method #OOM-023-003, revision 2 and Deviations and Modifications Centre Analytical Laboratories, Inc. 3M Environmental Laboratory Page 84 of 110 Page 382 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 Centre Study No.: 023-011 Sponsor Protocol No: FACT-TOX-026 . .. . ...-.--. . .. ... -- . .__._ . I . ..- . .. ._. --l- . TITLE Determination ofFluorochemica1 Residues in Monkeykt Fecis by LC&[Sb1s (Revision 2 ) AUTHORS i Enaksha Wichemesinf;e, Shaozhi Zheng, and John Flaherty - . DATE ISSUED June 02,2000 SPONSOR . 3M Environmental Technology and Safety ServicesBuilding 2-3E-09 POBox33331 . St. Paul, MN55133-3331 PERFORXUJG LtlBOR4TORY Centre AnaIyticaI Labolatoria, Inc. (Centre) 3045 Research Drive . State College, PA 16801 -'. -. . CENTRE STUDY NUMBER 023-003 . OOM-023-003, revision 2 TOT-= h-L.%lBER OF PAGES 20 -- , Centre Analytical Laboratories, Inc. 3M Environmental Laboratory Page 85 of 110 Page 383 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 Centre Study No.: 023-01 1 Sponsor Protocol No: FACT-TOX-026 Principal Investigator Centre Analytical Laboratories, Inc P;incipd hveitigator Centre Analytical Laboratories, Inc. Laboratory Manager. Centre h d y t i c a l Laboratories, Inc. President w . Centre Analytical Labontones, hc. Group Leader 3hf Environmental Labontory .... Centre Analytical Laboratories, Inc. 3M Environmental Laboratory Page 86 of 110 Page 384 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 Centre Study No.: 023-011 Sponsor Protocol No: FACT-TOX-026 _- Caue MethodNO.: OOM-023-003,revision 2 I I . TABLE OF COiVTENTS TITLE PAGE ............................... .................................................... ....:.......... ^..........1 $,f*AGE,QZm )_PpROVa ........................................................................................... 7 T ~ L OEF CONTENTS..................................................................................................... 3 1. sulMMARy................................................................................................................. 4 -7. F L U O R O C ~ S~TCA~~IJXRD..S...................................................................... 4 3. C ~ ~ C SUP~PLESS.................................................................................. 7 3.1. C H E ~ C P..J...S..... ..................................................................... :................7 3 3.I. F L ~ ~ RSTAONDARwDS..~.........~........~.......................................................... 7 3.3..'EQUIP- ANI)SWPLm........................................................... ............ 7 3.4. SOLU~O.N..S........... ................................................................................ 8 3.j. p ~ p k q ~OnFSoT~OC~KF,oRIT~ICATON, C.4LE3L4nON SoLmONs.........9 3.5.1.'Stock~ 0 1....~.......~......0.......~.............................................................. 9 3.j.7-. FortificationSolutipns.................................................... I....................... 9 3.j.3. Calibration Stm&& .......................................................................... 10 4. METHOD .................................................................................................................. 11 4.1. FLOWDL4GW.M........... ............................................................................ 11 4.7-. SAMPLEPROCESSING......................................................................................... 11 4.3. S r t M P L E p ~ p ~ n..O.....N................................................................................ 11 4.3.1. Ma~Bl&s......................... ..........................*............_................. 12 4.3.3-. M ~ &Zero Blm! ............................................................................. 1. 4.3.3. Calibration Stmd& .......................................................................... 12 . 4.3.4. conh,,ksCabbration Verification Standards................................... 12 3.j. QC Recovery SmpIes......................................................................... 12. 4.4. E x r ; ~ 4 a.o....~........................... ..................................................................... 12 4.4.1 SPE col~preparatio.n..................................................................... 13 4.4.2 Silaniation Ofpew-Shaped F l a b ...................................................... 13 4.4.3 Stmdx&ation of SPE CO]? ......................................................... 13 4.5.~ , a Y s r sBY LC/MS~I.S................................................................................ 14 4.5.1. L C ~ ~ SSyst~emSand Openhg -COnditi~(E1ecbovay)...........14 4.5.7-. ~~~~l~ Tme File parameters.....:...................................................... 15 4.5.3. Calib&n procedures ................................................ ....................... 16 4.5.4. sample Analysis ........................... ................................. ........... 17 : 4.6. PE,JO~WNCEm R L 4................................................................................... 18 4.7. T mQqmaD FOR .q?J,\LysIs......................................................................... 1s 5. C.4.LaiATIONS .......................... ........................................................................... 1s 5.1. A N A LFo~mQ........................ ....................................................................... 1s 5.2. j.3. QC f Rv;czovS~~-r?P.y...~F..O .A....~...~oN ...R............S...............~........................................................................................................................................ 19 19 6. S.UETY ............................ ........................................................................................ 19 A T T X C ` M N T 1...................... ..;.................................................................................... 20 Centre Analytical Laboratories,h c . 3M Environmental Laboratory Page 87 of.110 Page 385 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 Centre Study No.: 023-01 I * Sponsor Protocol No: FACT-TOX-026 .- . Centre Analytical Laboratories, Inc. 3M Environmental Laboratory Page 88 of 110 Page 386 3M Medical Department Study: T-6889.3 _ _ .I Analytical Report: FACT-TOX-026 LRN-U2782 ' Centre Study No.: 023-01I Sponsor Protocol No: FACT-TOX-026 - - - ... .._ .. . . - . . PFOSA Moltcuiar weight: 499 0 ChemicalXu'ync =Pcrfluoroocmc sulfonylamide .. PFOSAA Molecular weight: 585 - PFOSEA Molecular wei$t: 527 -- ' PO.44- Molecular weight: 413 c7F15CoCP Chemic.11 Nan: = Ptrfluorooctanottc - . _. I Centre Analytical Laboratories, Inc. 3M Environmental Laboratory Page 5 Page 89 of 110 Page 387 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 Centre Study No.: 023-01I Sponsor Protocol fio: FACT-TOX-026 \ Et-FOSE-OH . Molecular wei$t: 571 I . M 556 . Molecular weizh:. 557 . Chcmicil Name = M556 M 570 hfolecular weight: 571 THPFOS Molecular wei$t: 47s 1-H, 1-H,2-H, 2-H, CgF1:SO;H Centre Analytical Laboratories, Inc. 3M Environmental Laboratory Page 6 Page 90 of 110 Page 388 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 Centre Study No.: 023-011 ' Sponsor Protocol No: FACT-TOX-026 . .. .. - .. .. .. ... , ._. _... ... .-. . .-A ,.J 3. CBEMlCALS AM> SUPPLIES 3.1. CHEMICALS I Chemical AcetonifAle (Am Grade HPLC Source (t7"iR) J. T. Baker Catalog No. JT9017-3 Carbon 120/400 mesh Methanol (MeOH) b32LC SupeIco (VWR) J. T. Baker 57210-U JTgog3-2 1-Octanol Reagent Aldrich Chemical 111-87-5 Ammonium Acetate Reagent Sipa-Aldrich A-7330 .. L-Ascorbic acid R e a p t Si-ma-Aldrich L-5960 . Toluene - ' Reagent , QWR) J.T.-Baker JT9460-3 DimetyldichIorosiIae Reagent . Supelco 3-3009 Water . Type1 in house (Type I water = electricalresistivity, minimumof16-67MWcm at %.C, from a Labconco waterpro workstation) 3.2. 'FLUOROCHEMICAL STAXDARDS Stndard PFOS PFOSA PFOSAA PFOSEA E t-FOSE-OH Id556 M570 P-OM * 'THPFOS Source 3M 3M 3M 3b.I 3M 3M 3M 3M . 3M- - . * . 3.3. EQUIPMENTANDSUPPLIES Equip men t Balance, analytical,(display at least 0.0001p) Balance, top loading, (display at k S t 0.01 9 ) Rotary evaporator . Rotary evapontor f n p Pear-shaped flasks (125 mL) 20-mt SPE tubes (cat. no. NO57157) with h t s Vacuum pump Visiprep vacuum manifold C e n ~ c&lytic31 Labontorits,Inc Study % 023-003 Source Mettle.t_ MettIer Ecchi Buchi Pyrex Scipelco Buchi Supelco Page 7 Centre Analytical Laboratories, Inc. 3M Environmental Laboratory Page 91 of 110 Page 389 3M Medical Department Study: T-6889.3 I .. . . ..._ - /. Analytical Report: FACT-TOX-026 LRN-U2782 Centre Study No.: 023-011 Sponsor Protocol No: FACT-TOX-026 . .. - . ... . ... ----- I C c z f ~Me ethod NO.: O O M - O ~ - O O ~ , 2 I ', ... ' . P JVristlaction shaker 20-p.~,polyethyleae scintillation vids with lids Burrell Wheaton (VtVR) .....I' 20-mL syringe ' 25-mm diameterglass fibre acrodisc ( a t $3523) Disposable pipets, test tubes etc. Disposable micropipea, 100- 200 &. \;wR . GeIman VUiR vt\rR 125-mLLDPE narrow-mouthbottles 2 - d clear HPLC vial kit (cat S 5181->400) Standard lab equipment (class A pipets and volumetric flask, graduated cylinders, etc.) Nalgene HP vu;ous LC/MSM and HPLC systems As described in Section 4.5.1 * - Notes: 1. 2. 3. 4. 5. 6. . ... .- .. . . Centre Analytical Laboratories,Inc. 3M Environmental Laboratory Page 92 of 110 Page 390 3M Medical Department Study: T-6889.3 I Analytical Report: FACT-TOX-026 LRN-U2782 Centre Study No.: 023-011 Sponsor Protocol No: FACT-TOX-026 _. .. ~ . _ . _ . . _. - - ... .... ... . ., Centre Analytical Laboratories, h c . 3M Environmental Laboratory Page 93 of 110 Page 391 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 Centre Study No.: 023-011 Sponsor Protocol No: FACT-TOX-026 3.3.3. Calibration Standards Prepare Lm1SM.S calibration standards by forti&: weighed out aiiquots of the same control feces sample (or a combined "bulk'?control feces before they are processed thr~ughthe extraction procedure. The following is a typical example; additional concentrations may be as needed It is recommended to prepare the 'fortificationsolutions SO that the volume of fortification is'between 50 and 200 pL and to use &sposable micropipets-for aiiquoting fortiiicationV O ~ I I ~ S . 0.1 100 - 1.0 0.1 200 . 1.0 - 0.5 100 1.o 0.5' 200 1.o 2.5 100 . . 1.0 . 2.5 200 1.0 10 100 1.o * 2.5 pJm1 THTFOE fortificationsolution, &VU 1.0_ ?nn 2UV 20 - ' 100 50 200 100 200 250 200 500 -m.""n 1000 200 -_ . _ - Centre Analytical Laboratories, Inc. .- -_ 3M Environmental Laboratory Page 10 Page 94 of 1 10 Page 392 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 Centre Study No.: 023-01 1 Sponsor Protocol No: FACT-TOX-026 .. - -. . . _ _ . . .-. -. ..-.-.. . ._,.. . . .._ . _I _.'-. -;. 4- METBOD 4.1. FLOWD W G U M The flow dia-gam of the method is given below, . description of each step. Method Flow D i a m Weigh 1.Og of simple - (fortiesamples designated as calibration standards an1 I J. 'J Extract with ACN and filter J . ! Carbon Solid-Phase Extraction .J J Concentrate to ahnost dryness J J Adjust hal volume (2.0 mL) J J. 7-;2kf S M S QC recoveries) - 4.2. S.A~MPLEPROCESSING . .. All samples are received f?ozen,and will be k q t rozene@elow-loot) uti1 the of extraction. The rat feces do not need any processing, however, &e monkey feces may need to be honiogenized in order to be able to weigh out &e specified mounts. 4.3. SAMPLPER E P ; Z R ~ O N Prepare the following blan.. and calibrationsamples (Sections 4.3.1 to 4.3.3) eom s m e control feces sample (or a combined "bulk" control feces smpie). . .- .. Centre Analytical Laboratories, Inc. 3M Environmental Laboratory P3pe 1.1 Page 95 of 110 Page 393 3M Medical Department Study: T-6889.3 . .. . .. . Analytical Report: FACT-TOX-026 LRN-U2782 Centre Study No.: 023-011 Sponsor Protocol No: FACT-TOX-026 ' . .. .. - - . .--- .-.. . . - .... .. .... -. I . .-~ , . . . . . . -.... . .... -. . .. _. .. - . . Centre Analytical Laboratories, Inc. 3M Environmental Laboratory P q c I2 Page 96 of 110 Page 394 3M Medical Department Study: T-6889.3 . .- .. Analytical Report: FACT-TOX-026 LRN-U2782 Centre Study No.: 023-011 Sponsor Protocol No: FACT-TOX-026 > -... . . .... __.-_. I _ _.. . . Centre Analytical Laboratories, Inc. ... - . . .. 3M Environmental Laboratory Page 97 of 1 10 Page 395 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 Centre Study No.: 023-01 1 Sponsor Protocol No: FACT-TOX-026 ,I . ' Y. .. I .. i I b. Collect a post-elution h t i o n , after Step 3,' eluting with an additional 20 m~ of 9o:io (.4m:2% ascorbicacid in MeOH). c. Adjust the final vO1ume G f d1 the hCtionS to 10dtvi& methanol. , d. Analyze all the fractions by LCMShfS. . e. If. the target hction contains a m k h ~ mof 85% of &e respective arialytes, it may be considered acctptable. f If the "post-elution" hction contains significant 2irnount of aalytes, the tarset elution volume may be increxed 4.5. ANALYSISL.CBhYIS/MS 4.5.I. LC M S k SS'sfem and Operating Conditions(Elecpospray) M a s Spec:. . Inttrfacc: Compute:: Sofiware: Micromass Quattro Ultima (Micromass) Electrospray (Micromas) Hanard infusion pump COMP.4Q ProfessionalWorkstationAp700 Windows hi,MasLynu 3.3 HPLC: Hewlett Packard (HP) Series 1100 HP Quat Pump KP Vacuum Degasser HP Autosampler . HP Column Oven - Note: A 4 x 10 mm hypercarb drop-in F a d c a h d s e (Keystone, p u t 844017-400) is attached on-line after.the purse valve ad before the injector port to trap any residue contdmnts thar may be in the mobile phase andor HPLC system. HPLC Column: Cofumn Temp.: hjection Vol.: Genesis C, (Jones Chromato-mphy), 2.1 mm x 50 m,dU 35" C 10 pL - ..._. MobilePhase (.A):? &,Lmoni& AcetateinTyde I water Mobile Phase (B):Methanol - T- ine - % A %B 0 60 40 0.4 60 1.0 10 i.0 10 7.5 0 9.0 0 0.5 60 11.5 60 14.0 60 40 90 50 100 100 40 40 ' 40 0.300 0.300 o._:oo 0.300 o.:oo 0.100 0.400 0.: 00 Centre Analytical Laboratories, Inc. 3M Environmental Laboratory Page 98 of 1 10 Page 396 3M Medical Department Study: T-6889.3 . . .. . . . - -.- . . . Analytical Report: FACT-TOX-026 LRN-U2782 Centre Study No.: 023-011 Sponsor Protocol No: FACT-TOX-026 . .. - ... . ! - -.. . . .. ... . _ .. , .. - .. I ' It may be nectssary to adjust the HPLC gneent in order to optimhz instrument performance. Columns with different dimensions (e.3. 2.1 mm 30 mm) aid also columns from diffe:ent manufacturers (Keystone Betasil c,, etc.) can be used provided equivalent chromatographyis obtained. I Ions monitored: - Analvte - Mode Parent Ion Product Approximate Ion Retention Time (mini PFOS . PFOSA .PFOSAA PFOSEA . - Et-FOSE-OH POAA * M556 negative negative negative negative negative negative negative 499 498 584 526 630 . 413 556 99 78 526 169 59 . . 369 . 498 5.3 \ 6.0 5.7 6.7 6.7 5.1 5.4 M570 negative 570 419 5.6 . THPFOS negative 427 80 5.1 Xote that the retention times may vary sti$tly, on a day-to-by basis, dependingon the batch of mobile phase, etc. 4.5.2. fiainple Tune FiIe Parameters The f o l i o ~ ~vaglues are provided as an example. Actual values may vary from b e n t to instrument. Also these values may be changed fion t h e to time in order to optimize for greatest sensitivity. - Analvte PFOS PFOSA PFOSAA PFOSEA Et-FOSE-OH POAA M556 M570 THPFOS 'Dwell (SI 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 Collision Ener43 25 20 28 31 11 28 20 35 (em . ' Cone 76 34 74, 7 49 30 25 50 60 34 Page 15 3M Environmental Laboratory Page 99 of 110 Page 397 3M Medical Department Study: T-6889.3 2 I ' '. Source Capillary Hexapole 1 Aperture 1 Hexapole 2 Source Block Temp. Desolvation Temp. Analner LMRes 1 HMRa 1 -Energy 1 Entrance Exit LMRes 2 HMRS 2 Energy 2 Multiplier Gas Flows Cone Gas - Desolvation Pressures Gascell . ' Analytical Report: FACT-TOX-026 LRN-U2782 Centre Study No.: 023-01I Sponsor Protocol No: FACT-TOX-026 . .. -. . - - - . .. . . ..._ CCIIK Method NO.: 00hf-023-003,revision 2 I Set 2.56kV 0.s.v 0.2 v 0.8 v 100C 400C -Set 13.0 v 13.0 V 0.7 V -2 v 1v 11.ov 11.ov 1.0 v 650 v -7OOh . -Read back 3.0e-3 mbar -- - ._ . - . .. Centre Analytical Laboratories, Inc. 3M Environmental Laboratory Page 16 Page ,100of 110 Page 398 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 Centre Study No.: 023-01I Sponsor Protocol No: FACT-TOX-026 1 . Centre Mebod NO.: 00M-023-003r,evision 2 /-. :?I h.2` .- .. . . . . - Centre Analytical Laboratories, Inc. .. . .. 3M Environmental Laboratory Page 101 of 110 Page 399 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 Centre:Study No.: 023-011 Sponsor Protocol No: FACT-TOX-026 . . . - . . .. .. .- . Centre M e h d No.: 00M-023-003, revision 2 L. . 3. CALCULATIONS 5.1. AVALYTFEorm Equation 1: Aialyte found (ppb) = (peak arc3 -intercept) x DF slope Centre Analytical Laboratories, Inc. . _. 3M Environmental Laboratory Psgz 1s Page 102 of 110 Page 400 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 Centre Study No.: 023-011 Sponsor Protocol No: FACT-TOX-026 . . . _ . _ _ .. _ . . . i I o/o Recovery = i - anaifle found (ppb) i dyte found in matrix blan!! @pb)x 100 amount analytt added (ppb) . 5.3. ih&mRESPONSE FACTOR . 6. SAFETY. Centre Analytical Laboratories, Inc. 3M Environmental Laboratory Page 103 of 110 Page 401 3M Medical Department Study: T-6889.3 .. . ..... . Analytical Report: FACT-TOX-026 LRN-U2782 Centre Study No.: 023-01 1 Sponsor Protocol No: FACT-TOX-026 .. ~..- -.- - - -. _._ . . ..._ ..- , . _!' ATTACBGMENT 1. a\ I C m t r e Analytical Labaratories. Inc %r-or- - -.. . ~ . . . . - . . . . Centre Analytical Laboratories, Inc. 3M Environmental Laboratory Page 20 Page 104 of 110 Page 402 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 Centre Study No.: 023-011 Sponsor Protocol No: FACT-TOX-026 CEntrE Analyticai Laboratories, Inc- 3048 Research Drive,State College, PA 16801. Phone: (814) 231-8032, Facsimile: (814)U1-133 METHOD DEVIATION DeviationNurnber: 1 . Date of Occurrence: 10/04-05/00 . Page 1 Centre Study Number: 023-011 Sponsor Protocol Number: FAcT-TbXO26 DESCRIPTION OF DEVIATION t Deviations to method titled "Determination of Fluorochemicd Residues in Monkeymat Feces by LC/MS&fS (Revision 2)". Centre method number OOM-023-003, revision 2 1. Section 4.5.4.d : cm moveries of 60% 54%.65%.66%.57%. and 60% w e n accepted for WOSE-OH in Set 100200AR. ACTIONS TAKEN i.e.. amendment issued. SOP revision. etc., 1. Method deviation issued- , I L,No negative impact a IMPACTON THE STUDY Centre Analytical Laboratories, Inc. 3M Environmental Laboratory Page 105 of 110 Page 403 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 Centre Study No.: 023-01 1 Sponsor Protocol No: FACT-TOX-026 mti!!!hCentrE Analytical L a b o r a t o r i E s . inc- METHOD DEVIATION Deviation Number: 2 Page 1 Date of Occurrence: (1) 10/10/oo. (2) 10/1Q/OO,(3) 10/11-12/00 Centre StudyNumber: 023-011 Sponsor Protocol NurnbPr: FAcT-Tox-026 DESCRIPTION OF DEVIATION I Deviations to method titled "Determination of FluorcchemicalResidues in Monkeymat Feces by ~c/MsIMS(Revision2)", Centre method number 00M-023-003, revision 2: 1. Section 4.5.4.d : CCV recoveries of 50%. 51%,59%.60`% 614, and 61% were accepted for EGOSE-OH in Set 100900A. 2. Section 4.5.4.e: Surro,oaterecovery of 46% ws 100900k accepted for Cen-tresample 0008495 in Set 3. Section4.5.4.e: Surrogaterecoveries of 57% and 56% were accepted for Centre samples lo08514 and 0008515. respectively in Set 1010CCJAD. I I 1. No negative impact ACTIONS TAKEN &e.. amendment issued. SOP revision. efc... w w ma,h!@ IMPACTONTHESTUDY A February I?. 1995/2 Centre Analytical Laboratories, Inc. 3M Environmental Laboratory Page 106 of 110 Page 404 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 Centre Study No.: 023-011 Sponsor Protocol No: FACT-TOX-026 Ca-rtre Analytical Laboratories, Inc. 3048 Reseucb Drive, StJte College, PA 16801. Phone: (814) 231-8032,Facsimile: (814)231-lls3 DESCRIF'TION OF DEVIATION I Ieviations to method titled "Determination of Ruorochemical Residues in Monkey/Rat Feces by .C/MS/MS(Revision 2)". Centre method number OOM-023-003,revision 2: . Section 4.5.4.e : S ~ g p t ree ~ v e r yof 53% was accepted for Centre sample 0008595 in Set 1 0 ~ j C x M ~ . . Section45.4.d CCV recovery of 153% w s accepted for EGOSE-OH in Set 101700k . Section 45.4.d & e: In Set IUXK!OA. a surrogate WOvery Of 149% was accepted for Centre sample 008681 and CCV recoveries of54%.59%.%%, 58% and 55% for EtFOSE-OH were accepted. ,. ccv . Section 4.5.4.d & e: In Set IU2j00AR. a surrogate f e c o v e o~f 54% W ~ aSccepted for Centre sample 008690Spk B and one of51% was accepted for 0005703. reCOveflCS-Of54%. 5 1%. 51%. 532,and 7% for EtHISE-OH were accepted. . Section 4.5.4.d: CCV recoveries of 62%, 632. and 55% were accepted for ELFOSE-OH in Set 0?400k . Section 4.5.4.d: CCV recoveries of 66%. 64%. 43%.and 43% were accepted for EtFOSE-OH in Set D2500A I ACTIONS TAKEN i.e.. amendment issued. SOP revision. CfC... -6. NO negative impact iZ/\Q!& IMPACT ON THE STUDY Centre Analytical Laboratories, Inc. 3M Environmental Laboratory Page 107 of 110 Page 405 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 Centre Study No.: 023-011 Sponsor Protocol No: FACT-TOX-026 CEntrF Analytical L a b o r a t o r k s . inc, 3048 Research Drive, State College, PA 16801. Phone: (514) 231-5032 Facsimile: (814)=1.153 Page I METHOD DEVIATION 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: FAm-TOX-026 DESCRIPTION OF DEVIATION , Ieviations to method titied "Determination of Ruorochemical Residues in Monkeymat Feces by .c/MS/MS (Revision 2)", Centre method number OOM-023-003. revision 2: . Section 3.5.1: Did not correct stock standard solutions for % punty. .I Section4.3.5: Only prepared 2 QC samples for sets that contained more than 20 samples. - ACTIONS TAKEN i.e.. amendment issued. SOP revision. etc... -2. Method deviation issued, !ecorded Bymate: - I t -2. No negative impact I n I M P A n ON THE STUDY Centre Analytical Laboratories, Inc. 3M Environmental Laboratory Page 108 of 110 Page 406 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 Centre Study No.:023-011 Sponsor Protocol No: FACT-TOX-026 CEntre Analytical Laboratorks, inc. 3028 Raearcb Drive. State Collnc. PA 16801. Phonc: f81W31-8035. F ~ ~ i m i lfe8:14)231-1253 METHOD MODIFICATION FORM Page I of : Modification No.: 1 Studv Director: Paul Lieder Centre Studv Number 023-011 SDonsor Protocol Number: FACT-TOX426 Method Title: Determination of FluomchemicalResidues in Monkeymat Feces by LC/MS/Ms (Revision 2) Cenue Mehod Number:00M-023-003,revision 2 I I 1 MODIFICATION: 1. Section 3.4 ! j o \ u t i o ~ 100 m~ Ynmonium acetate solution should be 50 mM ammounium acetate. 2. . Section 45.4 Sample Analysis (Item e.) Should'read as follows: Monitor the response of the sunogatc standud (SS)for e v W s;lmple. extracted S t d u d , blank, and QC recovery sample. The concentration is calculated using the "average response factor" function from MassLynx 3.3 software(or equivalent). Evaluate the acceptabilityOf the Sample extraction process b a e d on h e concentration of the SS found in a11 extracted standards. Samples. blanks, and samples. The percent recovery for ~ J saI mptes. blanks, extnctcd stathrds. and QC recovery samples must fall bcrween 60% and 140%. Thase deviating by greater than 30% must checked for possible errors and may need re- extractid. Any rxoveries deviating by peater lhan 40% (fallingoutside the 60%to 140% range) will require rc;cxmction. 3. Section 5.1 Analytc Found Removesecond sentence. 4. Section 5.1 Equations Modify Equation 1 to: &alpe found ( n d d )=(Peak area - interceotl I I Change Equation 2 to: slope Add Equation 3 (where DF = dilution factor of spk. W = final volume): I R C C O V C(7~6~)= - ((anal. found (n=/rnL) (anal, found in correswndinn sample IndrnL)IDFl X FV (ax)DF) x I 0 0 amount added (ng) 5. Section 5.3 Mean Response Factor: Modify first sentence to read: The mean response factor ( b m is calculared by the M s s L y s~ofrwarcprogram by averaging the response of the surrogate srandxd for d l cslibrarion standards and dividing by the mount added. Centre Analytical Laboratories, Inc. . 3M Environmental Laboratory Page 109 of 110 Page 407 3M Medical Department Study: T-6889.3 Analytical Report: FACT-TOX-026 LRN-U2782 Centre Study No.: 023-011 Sponsor Protocol No: FACT-TOX-026 Centre Analytical Laboratorks, Inc, 3W Rarareh Drive. State Collae. PA 16801. Phone: ~814231.8032Facsimile: (814)131.12S3 METHOD MODIFICATIONFORM Page 2 of I Centre StudyNumber.02341 1 Modification No.: 1 Studv Director: Paul Lieder Swnsor Protocol Number: FAa-ToX-026 Method Title: Determination of FluorochemicalResidues in MonkeylRatFeces by L c / M s / ~ s (Revision2) CentreMethod Number:OOM-023-003. revision 2 MODIFICATION (continuedk I 6. Section 4.1 Flow Diagram: The statement under Weigh 1.0 g of sample should read: (fortify designat& samples with appropriate fortification solutions and surrogate standard) 7. Section 4.4.a Extraction: The- should read as ~OIIOWS: &&: designated m p l e s need to be fortified with lppropnate aliquou of fortification standuds and dl samplesucept m u i x blanks need to be fortified With the surrogatestandard). NSTIFICATION: 1. To correct for P typographical error. 2. TOclarify SS acceptablecriteria. 3. Remove incorrect smtement because calculations did include sample weight and final volume. 4. Modify cakulations to comespond with those used in study. I 5. Clarify cdchation o f m a n response factor 4 6-7. Clarify that all wmo!es except the matrix t!anks need to be fortified with the surroeatesundud. EFFECT ON STUDY: NO negative impact Originator: a*5 t d k Principal Investigator: d&3axIviw Sponsor Management: ~f*/,5L.d ,+-iDY -oLLtc-ioc 121191og Date nlzl100 Date I C m &Hi Date -I/. 4 0 d ?*)February DAG I?, 199813 Centre Analytical Laboratories, Inc. 3M Environmental Laboratory Page 110 of 110 Page 408 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 LRN42782 PaGI Lieder, Ph.1D., DABT, Study Director Date Johd.Butenhoff, Ph.D., Spdzsor Representathe Date . "s ~~ William Reagen, Ph.D., Laboratory Manager Kris J. H h s e n , Ph.D.,' Ahalyfjcal lnvesfigafor Date 05-fzy/ 0 f Date 3M Environmental Laboratory 3M Environmental 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: 0 GLP Studyy-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. 0 Statement of Data Quality, page 16: The sentence, "The average fortified sample recovery for sera was 93% with a standard deviation of 11Yo"should read, "The average fortified sample recovery for sera was 94% with a standard deviation of 11Yo.'' 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. 0 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 Laboratoriesfor archiving at the 3M Environmental Laboratory and will be maintained at 3M Environmental Laboratory according to GLP requirement:;." 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 3M Environmental Laboratory Page 42 Page 410 3M Medical Department Study: T-6889.3 3M Medical Department Study: T-6889.3 Approved by: Analytical Report: FACT-TOX-026 LRN-U2782 Analytical Report: FACT TOX-026 LRN-U2782 / Kristen H. Hansen,`Ph.D., Principal Analytical lnvestigator Date Paul Lieder, Ph.D., Study Directo; L AJohn Butenhoff, TPh.D., Sponsor's Reflesenta tive y&sfl---y Bill Reagan, Ph.D., Environmental Laboratory Manager Date Date Date 3M Environmental Laboratory 3M Environmental Laboratory Page 43 Page 411