Document BRooOrD4r7D963p7RY32mNX6j

AR226-1751 STUDY TITLE Extraction of Potassium Perfluorooctanesulfonate from Quail Serum and Quail Liver for Analysis Using HPLC-ElectrosprayMass Spectrometry DATA REQUIREMENTS Analytical Method Requirements STUDY DIRECTOR Sean Gallagher STUDY COMPLETED ON September 06,2001 -F'ERFORMING LABORATORY / TESTING FACILITY Centre Analytical Laboratories, Inc. (Centre) 3048 Research Drive State College, PA 16801 Phone: 814-231-8032 STUDY SPONSOR 3M Environmental Technology and Safety Services Building 2-3E-09 PO Box 33331 St. Paul, MN 55133-3331 PROJECT Centre Protocol Number: OOP-023-041 Centre Study Number: 023-041 Total Pages: 102 GoOD LaBQBBTpBy PRACTICE COMPLIANCE STATEMENT Cenw Study Number 023-041, entitled "Extraction of Potassium Perfluoro0c~n~:suUonaftme m Quail Senun and Quat1 Liver far Andysi8 UslnB Hac- Electrospray/M,ass Spectrometry," conducted for 3M Environmental Techno1ogy and Safety Servico:~,was performed in compliance with US EPA TSCA Good Xaboratary Practice Standadds (40CFR Part 792)by Centre AnalyticalLahatones, Inc. n principd hveutfiator Centre Analytical Laboratanas, Inc. Sean Gallaghtv Study Director Wildlife International.Ltd. Dsatle oldl Date 3MknvlronmentalTLhnology and Safety S m i c c s Centre Anatyticd Laboratories, ne. Page 2 of 102 Centre Study No.: 023-041 OUALITY ASSURANCE STATEMENT Centre Analytical Laboratories' Quality Assurance Unit reviewed Centre Study Number 023-041, entitled, "Extraction of Potassium Perfluorooctanesulfonatefrom Quail Serum and Quail Liver for Analysis Using HPLC-ElectrosprayMass Spectrometry". 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 2. Extraction 3. Raw Data & Summary Table Review 4. Raw Data and Draft Report Review 5. Final Report Review Date Inspected Date Reported to Date Reported to Centre Study Director and Management Sponsor ManaPement 12/19/00 12121/00 12/28/00 12/19/00 12121/00 12/28/00 2/28,3/1/01 3/19/01 312310 1 3/23-2610 1 41910 1 51710 1 91610 1 91610 1 91610 1 `.LA-1 Naomi Lovallo Sr. Quality Assurance Auditor Centre Analytical Laboratories, Inc. Page 3 of 102 Centre Study No.:a 3 4 4 1 CERTIFICATION OF AUTHENTlCITY ' I l i a eport, far Centre Study Numba 023-041, i s a true and complete representation of the raw data for the study. Submitted by: Cenm Analytical Laboratories,Inc, 3048 Research D r i v e State College,PA 16801 (814) 231-8032 Principal Invczitigator,Centre: Scientist Ccntre Analytical Laboratories, Inc. Centre Analfllcal Laboratories,Inc.Facility Management: / Richard A. y & z z i e v . President Centre AnaIylical Laboratories,Jnc, Study Directoa, Wildlife Intarnational: Sean Gallagha Wildlife Inminational,Ltd, Sponsor Repixsentatlve,3M: Centre halyrical Laboaatories, Inc. E'afic4 of 102 Centre Study No.: 023-041 STUDY IDENTIFICATION Extraction of Potassium Perfluorooctanesulfonate from Quail Serum and Quail Liver for Analysis Using HPLC-ElectrosprayMass Spectrometry CENTRE PROTOCOL NUMBER: OOP-023-041 CENTRE STUDY NUMBER: 023-041 TYPE OF STUDY: Analytical SAMPLE MATRIX: Quail Serum and Quail Liver TEST SUBSTANCE: Perfluorooctanesulfonate (PFOS) SPONSOR: 3M Environmental Technology and Safety Services Building 2-3E-09 PO Box 33331 St. Paul, MN 55 133-3331 STUDY DIRECTOR: Sean Gallagher Wildlife International, Ltd. 8598 Commerce Drive Easton, MD 21601 SPONSOR REPRESENTATIVE: Rochelle Robideau 3M Environmental Technology and Safety Services Building 2-3E-09 PO Box 33331 St. Paul, MN 55133-3331 TESTING FACILITY: Centre Analytical Laboratories, Inc. 3048 Research Drive State College, PA 16801 ANALYTICAL PHASE TIMETABLE: Study Initiation Date: Experimental Start Date: Experimental Termination Date: Study Completion Date: 12./08/00 12./18/00 03/08/01 09/06/01 Centre Analytical Laboratories,Inc. Page 5 of 102 Centre Study No.: 023-041 PROJECT PERSONNEL The Study Director for this project was Sean Gallagher at Wildlife International, Ltd. The following personnel from Centre Analytical Laboratories, Inc., were associated with various phases of the study: Name Emily Stauffer Dave 13ell Melissa Kennedy Whitsel John Filaherty Tiffany Proctor Angela Morgan Michelle Arjmand Lawrence Ord Rickey Keller Shawn Robb Karen Risha Scientist Scientist Supervisor Operations Manager Technician Technician Technician Sample Custodian Sample Custodian Sample Custodian Scientist Centre Analytical Laboratories, Inc. Page 6 of 102 . Centre StudyNo. 023-041 TABLE OF CONTENTS Paae TITLE PAGE ....................................................................................................................... 1 GOOD LAB0:RATORYPRACTICE COMPLIANCE STATEMENT.............................. 2 QUALITY ASSURANCE STATEMENT .......................................................................... 3 CERTIFICAT1:ONOF AUTHENTICITY........................................................................... 4 STUDY IDENTIFICATION................................................................................................. 5 PROJECT PERSONNEL...................................................................................................... 6 TABLE OF CONTENTS...................................................................................................... 7 LIST OF TAB:LES............................................................................................................... 8 LIST OF FIGURES............................................................................................................. 10 LIST OF APPENDICES.................................................................................................... 11 1.0 SUMMAFLY................................................................................................................ 12 2.0 OBJECTWE ............................................................................................................... 12 3.0 INTRODUCTION....................................................................................................... 12 4.0 TEST SYSTEM .......................................................................................................... 13 5.0 REFERENCE MATERIAL, ......................................................................................... 13 6.0 DESCRIPTION OF ANALYTICAL METHOD ......................................................... 14 6.1 Extraction Procedure ................................................................................................ 14 6.2 Preparation of Standardsand Fortification Solutions................................................ 14 6.3 Chromatography ........................................................................................................ 15 6.4 Instrumeint Sensitivity............................................................................................... 15 6.5 Description of Instrument and Operating Conditions............................................... 15 6.6 Quantitation and Example Calculation..................................................................... 16 7.0 EXPERIMIENTAL DESIGN ...................................................................................... 18 8.0 RESULTS ................................................................................................................... 18 9.0 CONCLlJSIONS...................................................................................................... 18 10.0 RETENTION OF DATA AND SAMPLES ............................................................. 19 Centre AnalyticalLaboratories. Inc. Page 7 of 102 Centre Study No.: 023-041 LIST OF TABLES Table I. Summaryof PFOS in Reagent Blanks............................................................ 21 Table 11. Summary of PFOS in Quail Liver Blanks ...................................................... 22 Table 111. Summary of PFOS in Quail Serum Blanks .................................................... 22 Table IV. Summary of PFOS Recoveries in Quail Liver ................................................ 23 Table V. Summary of PFOS Recoveries in Quail Serum.............................................. 24 Table VI. Summary of PFOS in Adult Liver Samples at 0 ppm a.i. (Test Termination 19 Weeks) ..................................................................................25 Table VII. Summary of PFOS in Adult Liver Samples at 1.8 ppm a.i. (Test Termination 6 Weeks) ....................................................................................26 Table VI11 Summary of PFOS in Adult Liver Samples at 6.2 ppm a.i. (Test Termination 6 Weeks) ................................................................* ...................27 Table E. Summary of PFOS in Adult Liver Samples at 17.6 ppm a.i. (Test Teimination 19 Weeks) ..................................................................................28 Table X. Summary of PFOS in Juvenile Liver Samples at 0 ppm a.i. (Test Teirmination 12 Weeks) ..................................................................................29 Table XI. Summary of PFOS in Juvenile Liver Samples at 1.8 ppm a.i. (Test Termination 12 Weeks) ..............................................................,,...................30 Table XII. Smmmary of PFOS in Juvenile Liver Samples at 6.2 ppm a.i. (Test Teimination 12 Weeks) ..............................................................#. ..................31 Table XIII. Summary of PFOS in Juvenile Liver Samples at 17.6 ppm a.i. (Test Teimination 12 Weeks) ..............................................................*. .................32 Table XIV. Smmary of PFOS in Adult Serum Samples at 0 ppm a.i. (Test Teimination 19 Weeks) ..................................................................................33 Table XV. S u l m a r y of PFOS in Adult Serum Samples at 1.8 ppm a.i. (Test Termination 6 Weeks) ....................................................................................34 Table XVI. S u m a r y of PFOS in Adult Serum Samples at 6.2 ppm a.i. (Test Termination 6 Weeks) .................................................................................... 35 Table XVII. Summary of PFOS in Adult Serum Samples at 17.6 ppm a.i. (Test Termination 19 Weeks) ..................................................................................36 Table XVIII. Sh.unmary of PFOS in Juvenile Serum Samples at 0 ppm a.i. (Test Teimination 12 Weeks) ..................................................................................37 Table XIX. Summary of PFOS in Juvenile Serum Samples at 1.8 ppm a.i. (Test Teimination 12 Weeks) ..................................................................................38 Table XX. Smmary of PFOS in Juvenile Serum Samples at 6.2 ppm a.i. (Test Termination 12 Weeks) ..................................................................................39 Centre Analytical Laboratories, Inc. Page 8 of 102 Centre Study No.: 023-041 LIST OF TABLES (continued) PaRe Table X X I . Summary of PFOS in Juvenile Serum Samples at 17.6 ppm a.i. (Test Termination 12 Weeks) ................................................................................ ..40 Table XXII. Summary of Percent Moistures for Quail Liver Samples............................. 41 Centre Analytical Laboratories, Inc. Page 9 of 102 Centre Study No.: 023-041 Figure 1. LIST OF FIGURES PaRe Typical Calibration Curve for PFOS .............................................................. 43 Figure 2. Chaomatogram Representing a 0.1 ng/mL standard for PFOS ....................... 44 Figure 3. Chromatogram Representing a 1.0 ng/mL standard for PFOS ....................... 45 Figure 4. Chromatogram Representing a Reagent Blank for PFOS (Centre ID Reagent Blank A, Set: 122000A)............................................... "..................4.6 Figure 5. Chromatogram Representing Control Quail Serum for PFOS (Centre ID: 0011424Blank A, Set: 122000A).............................................. "..................4.7 Figure 6. Chromatogram Representing Control Quail Liver for PFOS, (Centre ID: 0007870 Blank A, Set: 121900A) .................................................................. 48 Figure 7. Chromatogram Representing Control Quail Serum Fortified with 500 ng/'mL of PFOS (Centre ID: 0011424 Spk A, Set: 122000A)........................ 49 Figure 8. Chromatogram Representing Control Quail Liver Fortified at 10 ppb with PFOS (Centre ID: 0011423 Spk A, Set: 121800A)................................ 50 Figure 9. Chromatogram Representing Juvenile Quail Serum Sample (Centre ID: 0007965, Sponsor ID: 9544, Set: 122000A)................................................... 51 Figure 10. Chromatogram Representing Juvenile Quail Liver Sample (Centre ID: 0007926, Sponsor ID: 9544, Set: 121800B)................................................... 52 Centre Analytical Laboratories, Inc. Page 10 of 102 Centre Study No.: 023-041 LIST OF APPENDICES PaRe Appendix A Study Protocol OOP-023-041 (Centre Study No. 023-041) and Amendmentsand Deviations.....................................................................53 Centre Analytical Laboratories,Inc. Page 11 of 102 Centre Study No.: 023-04I 1.0 SUMMARY Centre Analytical Laboratories, Inc. (Centre) extracted quail liver samples and quail serum samples for the determination of perfluorooctanesulfonate (PFOS) according to protocol OOP-023-041 (Appendix A). The limit of quantation for quail liver was 10 ppb and for quail serum 10 ng/mL. These were determined in the method verification study (Centre study 023-054). PFOS in the quail liver samples ranged from non-quantifiable levels (a peak was detected at the corresponding analyte retention time but was less than the lowest concentration of the calibration standards (0.0001 pg/mL)) to 8.37 pg/g. These results are reported on a wet weight basis. Residues of PFOS in the quail serum samples ranged from 0.00212 pg/mL levels to 263 pg/mL. The average percent recovery k standard deviation for PFOS in serum samples was 108% f 10%. The average percent recovery k standard deviation for PFOS in liver samples was 95% f 11%. 2.0 OBJECTIVE The objective of this study was to determine levels of perfluorooctanesulfonate (PFOS) in specimens of quail liver and quail serum using the analytical methods described in protocol OOP-023-041. 3.0 INTRODUCTION This report details the results of the analysis for the determination of PFOS in quail serum and quail liver, using the analytical methods entitled, "Extraction of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds From Serum for Analysis Using HPLC-ElectrosprayMass Spectrometry" and "Extraction of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds From Liver for Analysis Using HPLC-EClectrosprayMass Spectrometry" with the modifications listed in Section 6. The study was initiated on December 8, 2000, when the study director signed Centre protocol number OOP-023-041. The experimental start date was December 18,2000, and the experimental termination date was March 8,2001. Centre AnalyticidLaboratories,Inc. Page 12 of 102 Centre Study No.: 023-041 4.0 TEST SYSTEM The control quail serum and liver used for the matrix blanks and matrix fortifications was received on November 28,2000 fiom Wildlife International Ltd., Easton, MD. The control quail liver was assigned the Centre ID of 001 1423 and the control quail serum was assigned the Centre ID of 0011424. Fifty-six quail serum samples and sixty quail liver samples were received at Centre on August 10, 2000. The samples were stored frozen until they were logged in by Centre personnel on .August 16, 2000 and stored frozen. An additional twenty quail serum samples and twenty quail liver samples were received on February 23, 2001 and stored frozen until they were logged in by Centre personnel on February 26,2001. 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 truie copy of the storage records can be found in the raw data package associated with this study. 5.0 REFERENCE MATERIAL The analytical standard PFOS was received at Centre on June 3, 2000 from 3M Environmental Technology and Services. The available information for the reference material is listed below. The reference material was stored frozen. Stability was not assessed in this study but the reference material was stored according to the certificate of analysis and was used prior to expiration. Thle solubility of the reference material was not relevant to this study. Compound PFOS Centre Control No. TCR No. Purity (%) Expiration Date 00-023-042 TCR 00017-46 97.9 08/3 1/O1 The molecular structure of PFOS is given below. PFOS Chemical Name Molecular weight = Perfluorooctanesulfonate 499 (CsF17S03-) Note: `fie neutral molecule and standard form fiom which PFOS (anion) is derived, is potassium perfluorooctanesulfonate [C8F17SO&], molecular weight 538. Centre Analytical Laboratories, Inc. Page 13 of 102 Centre Study No.: 023-041 6.0 DESCRIPTION OF ANALYTICAL METHOD Analytical meithods entitled "Extraction of Potassium Perfluorooctanesulfonate or Other Fluorochemiceil Compounds From Serum for Analysis Using HPLC-Electrosprayhlass Spectrometry" and "Extraction of Potassium Perfluorooctanesulfonate or Other Fluorochemiczil Compounds From Liver for Analysis Using HPLC-Electrosprayhlass Spectrometry" with the modifications listed below were used for this study. 6.1 Extractioni Procedure - A 100 pL aliquot of the serum and 0.50 g sample of liver was used for the extraction - procedure. After fortification of appropriate samples, the samples were vortexed for 15 seconds. One mL of 0.5 M TBA was added to the liver samples and 0.5 mL was added to the serum samples. Two mL of 0.25 M sodium carbonate/sodium bicarbonate was added to the liver samples and 1 mL was added to the serum samples. Five mL of MTBE was added shaker - to both for 20 - the serum and liver samples. min. and then centrifuged for Each sample 15 min. Four was mL placed on wrist-action of the organic layer was taken and dried on an N-Evap and then reconstituted with 1 mL of methanol. Each sample was analyzed by LC/MS/MS electrospray. 6.2 Preparation of Standards and Fortification Solutions Standard solutions were prepared on September 18, 2000 as specified in Centre Analytical Laboratories' protocol OOP-023-041. An individual stock standard solution of PFOS was prelmred at a concentration of 100 pg/mL by dissolving 10 mg of the standard (corrected for purity and salt content) in methanol. From this solution, a 1.0 pg/mL fortification standard solution was prepared by taking 1 mL of the stock and bringing the volume up to 100 mL with methanol. The 0.1 pg/mL fortification standard was prepared by taking 10 mL of the 1 .O &mL fortification standard and bringing the volume up to 100 mL with methanol. A 0.01 pg/mL standard was prepared by taking 10 mL of the 0.1 pg/mL standard and bringing to 100 mL with methanol. A set of standards containing PFOS was prepared by dilution of the 0.1 pg/mL and 0.01 pg/mL solutions in the following manner. Initial Conc. (pg/mL)' 0.1 0.1 0.1 0.01 0.01 ' 0.01 ofPFOS Centre Analytic(a1Laboratories, Inc. Volume (mL) 5 2 1 5 2 1 Diluted to (mL) 100 100 100 100 100 100 Final Conc. (pg/mL) 0.005 0.002 0.001 0.0005 0.0002 0.0001 Page 14 of 102 Centre Study No.: 023-041 The stock startdard solution and all fortification and calibration standard solutions were stored in a refrigerator (4" k 2OC) when not in use. Documentation of standard preparation can be found in the raw data associated with this report. 6.3 Chromatography Quantification of PFOS was accomplished by LC/MS/MS. 'The retention time of PFOS was -LC/MS/MS 4.4 min. analysis using electrospray Peaks were detected in the control matricles corresponding to the analyte retention time and some of the amounts detected were :significantenough to alter the results reported in this study. 6.4 Instrument Sensitivity The smallest standard amount injected during the chromatographic run was equivalent to 0.0001 pg/mL of PFOS. 6.5 Description of Instrument and Operating Conditions Mass Spec: PE SCIEX API 3000 Biomolecular Mass Analyzer Interface: SCIEX TurboIon Spray Liquid Introduction Interface Harvard infusion pump Computer: Dell Ultrascan P1110 Software: HPLC: PE SciexAnalyst 1.1 Windows NT Hewlett Packard (HP) Series 1100 HP Quat Pump HP Vacuum Degasser HP Autosampler HP Column Oven HPLC Co1umn:Genesis CS(Jones Chromatography), 2.1 mni x 50 mm, 4p Column Temp.: 35" C Injection Vol.: 10 pL Mobile Phase (A): 2 mM Ammonium Acetate in ASTM type I water Mobile Phase (B): Methanol Flow Rate: 0.3 mL/min. Centre Analytical Laboratories, Inc. Page 15 of 102 Centre Study No.: 023-041 Time %A %B 0 60 40 1.o 0 100 7.0 0 100 7.5 60 40 11.0 60 40 Ions monitored: Analyte PFOS Mode negative + Transition Monitored 499 99 Approximate Retention Time (min) 4.40 Tune File Parameters Controls IS -1ospray DP-Declustering Potential FP-Focusing Potential EP-Entrance Potential CE-Collision Energy CXP-Collision Cell Exit Potential DF-Deflector CEM-Channel Electron Multiplier Set -4200.0 -51.O -230.0 10.0 -70 -5 300.0 2800.0 Gas Flows Nebulizer Gas Curtain Gas Collision Gas TIS Temperature - Set 12 13 4 350C 6.6 Quantitation and Example Calculation Ten microliters of sample or calibration standard was injected into the LC/MS/MS. The peak area was measured and the standard curve was generated (using l h fit weighted linear regression) by Analyst software using six concentrations of standards. The concentration for quail serum and quail liver was determined from the following equations: Use Equation 1 to calculate the amount of analyte found (in ng/mL, based on peak area) using the standard curve (linear regression parameters) generated by the Analyst software program. Then use Equation 2 to calculate the amount of analyte found in pg/g (pg/mL for serum samples). Centre Analytical Laboratories, Inc. Page 16 of 102 Centre Study No.: 023-041 Eauation 1: Analyte found (ng/mL) = (Peak area - intercept) slope Eauation 2: Analyte found (pg/g) = (anal. found (ng/mL) x FV (mL) x DF x EV (mL)) x (AV (mL) x sample wt. (g)) 1000 ng Where: FV = Final Volume DF = Dilution Factor EV = Extraction Volume AV = Aliquot Volume For samples fortified with known amounts of PFOS prior to extraction, use Equation 3 to calculate the percent recovery. Equation 3: Recovery (%) := (anal. found (ug/g;)- avg;.analvte in ctrl (ugdg;)) x 1000 ng;x 100% amount added (ng/g) x 1 pg An example of a calculation using an actual sample follows: Quail liver sample Centre ID 0007870 Spk A (Set: 121900A), fortified at 1000 ng/g with PFOS. Where: peik area intercept slope dilution factor - 11483 - 463 -- 2790 - - 100 ng/g added (fort level) = 1000 ng/g avg. amt in controls = 0 (Not quantifiable) final volume - 1 mL extraction volume -- 5 mL aliiquot volume - 4 mL sample weight -- 0.5 g From equatioin 1: Analyte found (ng/mL) = 111483- 4631 2790 From equatioin 2: Analyte found (&g) = 3.9 n g / d = J3.9 ndmL x 1 mL x 100 x 5 mL) x lllg (4 mL x 0.5 g) 1000 ng = 0.975 pg/mL Centre Analytical Laboratories, Inc. Page 17 of 102 Centre Study No.: 023-041 From equation 3: %Recovery = (0.975 udmL - 0 DdrnL) x (1000 nn/lp,a) x 100% 1000 ng/g = 98% 7.0 EXPERIMENTAL DESIGN - Each set of samples (liver or serum) consisted of one reagent blank, one matrix blank, two matrix blanks fortified at known concentrations, and 20 samples. Each sample was extracted using the appropriate method and then analyzed in duplicate. 8.0 RESULTS The PFOS found in the reagent blanks are listed in Table I. The PFOS in the quail liver blanks and quail serum blanks are given in Tables I1 & 111. Individual recoveries for PFOS in the liver samples are detailed in Table IV. The average percent recovery f standard deviation for PFOS in liver samples was 95% f 11%. Individual recoveries for PFOS in the serum samples are detailed in Table V. The average percent recovery f standard deviation for PFOS in serum samples was 108% f 10%. PFOS in the quail liver samples ranged fiom non-quantifiable levels (a peak was detected at the corresponding analyte retention time but was less than the lowest concentration of the calibration standards (0.0001 pg/mL)) to 8.37 pg/g. Individual results are listed in Tables VI-XIII. The results are reported on a wet weight basis. PFOS in the quail serum samples ranged fiom 0.00212 pg/mL levels to 263 pg/mL. Individual results are listed in Tables XIV-XXI. The average percent moistures for the quail liver samples are given in Table XXII. 9.0 CONCLUSIONS The quail liver and serum samples were successfully extracted and analyzed according to protocol OOP-023-041. Centre Analyticial Laboratories, Inc. Page 18 of 102 Centre Study No.: 023-041 10.0 RETENTION OF DATA AND SAMPLES When the final report is complete, all original paper data generated by Centre Analytical Laboratories, Inc. will be shipped to the sponsor. This does not include facility-specific raw data such as instrument logs, however exact copies of temperature logs will be submitted. Exact copies of all raw data, as well as a signed copy of the final analytical report and all original facility-specific raw data, will be retained in the Centre Analytical Laboratories, Inc. archives for the period of time specified in 40 CFR Part 792. Retained samples of reference substances are archived by the sponsor. Centre Analytical Laboratories, Inc. Page 19 of 102 Centre Study No.: 023-041 TABLES Centre Analytical Laboratories,Inc. Page 20 of 102 Centre Study No.: 023-041 Table I. Summary of PFOS in Reagent Blanks Sponsor ID na na na na na na na na na na na na na na na na Centre ID Reagent Blank A Reagent Blank A* Reagent Blank A Reagent Blank A* Reagent Blank A :Reagent Blank A* Reagent Blank A ]ReagentBlank A* Reagent Blank A ]ReagentBlank A* Reagent Blank A ]ReagentBlank A* Reagent Blank A ]Reagent Blank A* Reagent Blank A Reagent Blank A* * Duplicate injection Set Number 121800A 121800A 12 1800B 121800B 121900A 121900A 121900B 121900B 122000A 122000A 122000B 122000B 030501A 030501A 030601A 030601A Extraction Analysis Date Date 12/18/00 12/18/00 12118/00 12/18/00 12/19/00 12/19/00 12119/00 12119/00 12/20/00 12/20/00 12119/00 12/19/00 12/20/00 12/20/00 12/21/00 12/21/00 12121-22100 12121-22/00 12/22/00 12/22/00 12/20/00 12/20/00 31510 1 3/5/0 1 12122-23/00 12122-23/00 316-710 1 3/6-7/0 1 31610 1 31610 1 3/7/0 1 31710 1 AVERAGE: STANDARD DEVIATION: Analyte Found (pg/mL) NQ NQ NQ NQ NQ NQ NQ NQ 0.000200 0.000200 NQ NQ ND ND NQ NQ NQ NQ NQ = Not Quantifiable (A peak was detected at the corresponding analyte retention time but was less th,anthe lowest concentration of the calibration standards (0.0001 pg/mL)) ND = Not Detected For values recorded as ND,zero was used to calculate the average and standard deviation. For values recorded as NQ, 0.00005 pg/mL (half the value of the lowest calibration standard, 0.0001 pg/mL) was used to calculate the average and standard deviation. LOQ = 0.01 pg/g for liver and 0.01 pg/mL for serum Centre Analytical Laboratories, Inc. Page 21 of 102 ~~ Centre Study No.: 023-041 Table 11. Summary of PFOS in Quail Liver Blanks Sponsor ID na na na Na 454 454 456 456 Centre ID 001 1423 Blank A 0011423Blank A* 0011423 Blank A 0011423 Blank A* 0007870 Blank A 0007870 Blank A* 0007872 Blank A 0007872 Blank A* Set Number 121800A 121800A 121800B 1218OOB 121900A 121900A 030601A 030601A Extraction Date Analysis Analyte Date - Found (pg/g) 12118/00 12118/00 12/18/00 12118/00 12119/00 12119/00 12/20/00 12/20/00 NQ NQ 0.001 13 0.00109 12119/00 12119/00 31610 1 31610 1 12121/00 12121/00 31710 1 31710 1 AVERAGE: NQ NQ 0.00120 0.00 110 0.000590 STANDARD DEVIATION: 0.000578 Table 111. Summary of PFOS in Quail Serum Blanks Sponsor ID na na na na na na na na Centre ID ,0011424Blank A 0011424 Blank A* 0011424 Blank A 001 1424 Blank A* 0011424 Blank A 0011424Blank A* 10011424Blank A 0011424Blank A* * Duplicate injection Set Number 121900B 121900B 122000A 122000A 122000B 122000B 030501A 030501A Extraction Analysis Analyte Date Date Found (pg/mL) 12/19/00 12/19/00 12/20/00 12/20/00 12121-22/00 12121-22/00 12/22/00 12/22/00 0.0136 0.0144 0.0138 0.0151 12/20/00 12122-23/00 12/20/00 12122-23/00 31510 1 316-7101 31510 1 316-7/01 AVERAGE: STANDARD DEVIATION: 0.0127 0.0109 0.00555 0.00559 0.0115 0.00384 NQ = Not Quantifiable (A peak was detected at the corresponding analyte retention time but was less than the lowest concentration of the calibration standards (0.0001 pg/mL)) For values recorded as NQ, 0.00005 pg/g (half the value of the lowest calibration standard, 0.0001 pg/mL) was used to calculate the average and standard deviation. LOQ = 0.01 prJg for liver and 0.01 pg/mL for serum Centre Analytic,alLaboratories, Inc. Page 22 of 102 Centre Study No.: 023-041 Table IV. Summary of PFOS Recoveries in Quail Liver Sponsor ID na na na na na na na na 454 454 454 454 456 456 456 456 Centre ID 0011423 Spk A 0011423 SpkA* 0011423 Spk B 0011423 SpkB* 0011423 Spk A 0011423 Spk A* 001 1423 Spk B 001 1423 Spk B* 0007870 Spk A 0007870 Spk A* 0007870 Spk B 0007870 Spk B* 0007872 Spk A 0007872 Spk A* 0007872 Spk B 0007872 Spk B* Set Number 121800A 121800A 121800A 121800A 121800B 121800B 121900A 121900A 121900A 121900A 121900A 121900A 030601A 030601A 030601A 030601A Extraction Analysis Amt Date Date Added (ng/g) 12/18/00 12/18/00 12/18/00 12/18/00 12/18/00 12/19/00 12/19/00 12/19/00 12/19/00 12/20/00 10 10 2000 2000 1000 12/18/00 12/20/00 1000 12/18/00 12120-21/00 20000 12/18/00 12120-21/00 20000 12/19/00 12/21/00 1000 12/19/00 12/21/00 1000 12/19/00 12/19/00 3/6/01 12/21/00 12/21/00 31710 1 20000 20000 20 3/6/01 31710 1 20 3/6/01 31710 1 200 3/6/01 31710 1 200 AVERAGE: STANDARD DEVIATION: RELATIVE STANDARD DEVIATION: YO Recovery 102 104 101 103 113 110 80 80 99 103 85 84 93 86 82 84 94 11 12 * Duplicate injection LOQ = 0.01 pg/g for liver and 0.01 pg/mL for serum Centre Analytic,alLaboratories, Inc. Page 23 of 102 Centre Study No.: 023-041 Table V. Summary of PFOS Recoveries in Quail Serum Sponsor Centre Set Extraction Analysis Amt YO ID na 001 1424 Spk A 121900BD 12/19/00 12123-24/00 500 103 na 0011424 Spk A* 121900BD 12/19/00 12123-24/00 500 106 na 001 1424 Spk B 121900BD 12/19/00 12123-24/00 100000 90 na 0011424 Spk B* 121900BD 12/19/00 12123-24/00 100000 93 na 0011424 Spk A 122000A 12/20/00 12/22/00 500 106 na 001 1424 Spk A* 122000A 12/20/00 12/22/00 500 103 na 001 1424 Spk B 122000A 12/20/00 12/22/00 100000 97 na 001 1424 Spk B* 122000A 12/20/00 12/22/00 100000 103 na 0011424 Spk A 122000B 12/20/00 12122-23/00 500 108 na 0011424 Spk A* 122000B 12/20/00 12122-23/00 500 110 na 0011424 Spk B 122000B 12/20/00 12122-23/00 100000 113 na 001 1424 Spk B* 122000B 12/20/00 12122-23/00 100000 98 na 0011424 Spk A 030501A 3/5/01 316-7101 100 122 na 001 1424 Spk A* 030501A 3/5/01 316-7101 100 122 na 0011424 Spk B 030501A 3/5/01 316-710 1 1000 107 na 0011424 Spk B* 030501A 3/5/01 316-710 1 1000 115 AVERAGE: 106 STANDARD DEVIATION: 9 RELATIVE STANDARD DEVIATION: 9 * Duplicate injection LOQ = 0.01 pg/g for liver and 0.01 p g / d for serum Centre Analytical Laboratories, Inc. Page 24 of 102 Centre Study No.: 023-041 Table VI. Summary of PFOS in Adult Liver Samples at 0 ppm a i . (Test Termination 19 Weeks) Sponsor ID 45 1 451* 452 452* 453 453* 454 454* 455 455* 456 456* 457 457* 458 458* 459 459* 460 460* Centre ID 0007867 0 0 0 7 8 6 7 0007868 0007868 0007869 0007869 0007870 0007870 000787 1 000787 1 0007872 0007872 0007873 0007873 0007874 0007874 0007875 0007875 0007876 0007876 Set Number 121800AD 121800AD 121800A 121800A 121800AD 12 1800AD 121800A 121800A 121800AD 121800AD 121800A 121800A 121800AD 1218OOAD 121800A 121800A 121800AD 121800AD 121800A 121800A Extraction Analysis Date Date 12118/00 12/20/00 12118/00 12/20/00 12118/00 12118/00 12/18/00 12119/00 12/19/00 12/20/00 12/18/00 12/2.0/00 12118/00 12118/00 12118/00 12119/00 12119/00 12/20/00 12118/00 12/20/00 12118/00 12118/00 12118/00 12/19/00 12/20/00 12/20/00 12118/00 12120100 12118/00 12119/00 12118/00 12119/00 12118/00 1212 0100 12118/00 1212 0100 12/18/00 12119/00 12118/00 12/19/00 AVERAGE: STANDARD DEVIATION: Analyte Found (pg/g) 0.2 15 0.2 15 NQ NQ 0.303 0.304 NQ NQ 0.1 14 0.1 11 NQ NQ 0.0762 0.0739 0.0 103 0.0 104 0.236 0.235 0.001 17 0.001 17 0.0953 0.113 * Duplicate injection NQ = Not Quantifiable (A peak was detected at the corresponding analyte retention time but was less than the lowest concentration of the calibration standards (0.0001 pg/mL)) For values recorded as NQ, 0.00005 pg/g (half the value of the lowest calibration standard, 0.0001 pg/mL) was used to calculate the average and standard deviation. LOQ = 0.01 pg/g for liver and 0.01 pg/mL for serum -- ~~ Centre Analytical Laboratories, Inc. Page 25 of 102 Centre Study No.: 023-041 Table VII. Summary of PFOS in Adult Liver Samples at 1.8 ppm a.i. (Test Termination 6 Weeks) Sponsor ID EO1-0259-20780 E0 1-0259-20780* E01-0259-2078 1 EO 1-0259-20781* EO1-0259-20782 EO 1-0259-20782* EO1-0259-20783 E01-0259-20783* EO 1-0259-20784 EO 1-0259-20784* EO1-0259-20790 EO 1-0259-20790* E01-0259-20791 EO 1-0259-20791* EO 1-0259-207512 E01-0259-20792* EO 1-0259-207513 E01-0259-20793* EO 1-0259-20704 EO 1-0259-20794* Centre ID 0102997 0102997 0102998 0 102998 0 102999 0 102999 0103000 0103000 0 103001 0103001 0103007 0103007 0 103008 0 103008 0103009 0103009 0 103010 0 1030 10 010301 1 010301 1 * Duplicate injection Set Number 03060 1AD 03060 1AD 030601AD 030601AD 030601AD 030601AD 03060 1AD 030601AD 030601AD 030601AD 030601AD 030601AD 03060 1AD 03060 1AD 030601A 030601A 030601AD 03060 1AD 03060 1AD 03060 1AD Extraction Analysis Date Date 31610 1 3/8/0 1 31610 1 3/810 1 31610 1 3/8/0 1 31610 1 3/8/0 1 31610 1 3/8/0 1 31610 1 3/8/01 31610 1 3/8/0 1 31610 1 3/8/0 1 31610 1 3/8/0 1 31610 1 3/8/0 1 31610 1 3/810 1 31610 1 3/8/0 1 31610 1 3/8/0 1 31610 1 31aio 1 31610 1 31710 1 31610 1 31710 1 31610 1 3/8/01 31610 1 3/8/0 1 31610 1 3/8/0 1 31610 1 3/8/0 1 AVERAGE: STANDARD DEVIATION: Analyte Found (pglg) 0.800 0.722 0.149 0.134 0.341 0.311 0.455 0.422 0.0988 0.0843 0.210 0.210 0.177 0.191 0.0124 0.0121 0.0381 0.0394 0.0629 0.0720 0.227 0.224 LOQ = 0.01 pFJg for liver and 0.01 pglmL for serum Centre Analytkal Laboratories, Inc. Page 26 of 102 Centre StudyNo.: 023-041 Table VIII. Summary of PFOS in Adult Liver Samples at 6.2 ppm a.i. (Test Termination 6 Weeks) Sponsor ID EO 1-0259-20785 E01-0259-207885* EO 1-0259-20786 EO 1-0259-2078,6* EO1-0259-207137 EO 1-0259-20787* EO 1-0259-20788 EO 1-0259-20788* EO 1-0259-20789 E01-0259-20789* EO1-0259-20795 E01-0259-20795* EO1-0259-207!)6 EO 1-0259-20796* EO 1-0259-207!)7 EO 1-0259-20797* E01-0259-207!28 E01-0259-20798* E01 -0259-207!29 EO 1-0259-20799* * Duplicate injection Centre ID 0 103002 0103002 0103003 0103003 0103004 0 103004 0103005 0103005 0 103006 0 103006 0 1030 12 0103012 01030 13 01030 13 0 103014 0103014 0 103015 0103015 0 103016 0103016 Set Number 03060 1AD 030601AD 030601AD 030601AD 030601AD 030601AD 030601AD 030601AD 030601AD 030601AD 030601AD 03060 1AD 03060 1AD 03060 1AD 030601AD 03060 1AD 030601AD 030601AD 030601AD 030601AD Extraction Date Analysis Date 31610 1 3/8/01 31610 1 31810 1 31610 1 3/8/01 3/6/01 3/8/01 3/6/01 3/8/01 31610 1 3/8/01 3/6/01 3/8/01 3/6/01 3/8/01 3/6/01 3/8/01 31610 1 3/8/01 31610 1 3/8/01 3/6/01 3/8/01 3/6/01 31810 1 31610 1 31810 1 3/6/01 3/8/01 31610 1 31810 1 3/6/01 3/8/01 3/6/01 3/8/01 3/6/01 3/8/01 31610 1 3/8/01 AVERAGE: STANDARD DEVIATION: Analyte Found (pg/g) 1.15 1.20 1.18 1.19 1.43 1.42 1.60 1.51 1.06 1.17 0.216 0.208 0.115 0.0934 0.0801 0.0797 0.187 0.218 0.0901 0.0954 0.715 0.606 LOQ = 0.01 plJg for liver and 0.01 pg/mL for serum Centre Analytical Laboratories, Inc. Page 27 of 102 Centre Study No.: 023-041 Table IX. Summary of PFOS in Adult Liver Samples at 17.6 ppm a i . (Test Termination 19 Weeks) Sponsor ID 48 1 481* 482 482* 483 483* 484 484* 485 485* 486 486* 487 487* 488 488* 489 489* 490 490* Centre ID 0007877 0007877 0007878 0007878 0007879 0007879 0007880 0007880 0007881 000788 1 0007882 0007882 0007883 0007883 0007884 0007884 0007885 0007885 0007886 0007886 Set Number 121800AD 121800AD 121800AD 121800AD 1218OOAD 1218OOAD 121800AD 121800AD 121800AD 121800AD 121800AD 121800AD 121800AD 121800AD 121800AD 121800AD 121800AD 121800AD 121800AD 121800AD Extraction Analysis Date Date 12118/00 12/20/00 12/18/00 12/20/00 12/18/00 12/20/00 12118/00 12/20/00 12118/00 12118/00 12/20100 12/20/00 12118/00 12/20/00 12118/00 12118/00 12/18/00 12/18/00 12/20/00 12/20/00 12/20/00 12/20/00 12118/00 12118/00 12/20/00 12120/00 12118/00 12118/00 12/20/00 12/20/00 12/18/00 12118/00 12118/00 12/20/00 12/20/00 12/20/00 12118/00 12/20/00 12118/00 12/20/00 AVERAGE: STANDARD DEVIATION: Analyte Found (pg/g) 1.05 1.05 4.10 4.17 8.37 8.30 1.41 1.43 1.54 1.51 0.4 15 0.392 1.17 1.15 0.51 1 0.509 2.42 2.48 0.351 0.360 2.13 2.39 * Duplicate injection LOQ = 0.01 ppJg for liver and 0.01 pg/mL for serum Centre Analytical Laboratories, Inc. Page 28 of 102 Centre Study No.: 023-041 Table X. Summary of PFOS in Juvenile Liver Samples at 0 ppm a.i. (Test Termination 12 Weeks) Sponsor ID 9503 9503* 9506 9506* 9508 9508* 9512 9512* 9515 9515* 9518 9518* 9520 9520* 9522 9522* 9525 9525* 9526 9526* Centre ID 0007907 0007907 0007908 0007908 0007909 0007909 00079 10 0007910 00079 1 1 0007911 0007912 00079 12 00079 13 00079 13 0007914 0007914 00079 15 00079 15 0007916 0007916 * Duplicate injection Set Number 121800B 12 1800B 12 1800B 12 1800B 12 1800B 121800B 121800B 121800B 121800B 12 1800B 12 1800B 121800B 121800B 121800B 121800B 121800B 121800B 12 1800B 12 1800B 12 1800B Extraction Analysis Date Date 12118/00 12/20/00 12/18/00 12/20/00 12118/00 12/20/00 12/18/00 12/20/00 12118/00 12/20/00 12118/00 12/20/00 12118/00 12/20/00 12118/00 12/20/00 12/18/00 12/20/00 12118/00 12/20/00 12118/00 12/20/00 12118/00 12/20/00 12118/00 12/20/00 12118/00 12/20/00 12118/00 12/20/00 12118/00 12/20/00 12118/00 12/20/00 12/18/00 12/20/00 12118/00 12/20/00 12118/00 12/20/00 AVERAGE: STANDARD DEVIATION: Analyte Found (pg/g) NQ NQ NQ NQ NQ NQ NQ NQ 0.000785 0.000755 NQ NQ 0.000734 0.000672 0.0007 18 0.000656 NQ NQ NQ NQ 0.000251 0.000316 NQ = Not Quantifiable (A peak was detected at the corresponding analyte retention time but was less than the lowest concentration of the calibration standards (0.0001 pg/mL)) For values recorded as NQ, 0.00005 pg/g (half the value of the lowest calibration standard, 0.0001 pg/mL) was used to calculate the average and standard deviation. LOQ = 0.01 pg/g for liver and 0.01 p g / d for serum Centre Analytical Laboratories, Inc. Page 29 of 102 Centre Study No.: 023-041 Table XI. Summary of PFOS in Juvenile Liver Samples at 1.8 ppm ah. (Test Termination 12 Weeks) Sponsor ID 9528 9528* 9529 9529* 9530 9530* 953 1 953 1* 9533 9533* 9535 9535* 9540 9540* 954 1 9541* 9543 9543* 9544 9544* Centre ID 0007917 0007917 00079 18 00079 18 00079 19 00079 19 0007920 0007920 000792 1 0007921 0007922 0007922 0007923 0007923 0007924 0007924 0007925 0007925 0007926 0007926 Set Number 121800B 12 1800B 121800B 121800B 12 1800B 12 1800B 121800B 121800B 121800B 121800B 12 1800B 121800B 121800B 12 1800B 121800B 121800B 121800B 121800B 121800B 121800B Extraction Analysis Date Date 12/18/00 12/20/00 12118/00 12/20/00 12118/00 12/20/00 12118/00 12/20/00 12/18/00 12/20/00 12/18/00 12/20/00 12/18/00 12/20/00 12118/00 12/20/00 12118/00 12/20/00 12118/00 12/20/00 12118/00 12/20/00 12118/00 12/20/00 12118/00 12/20/00 12118/00 12/20,/00 12118/00 12/2OlOO 12118/00 12/20/00 12/18/00 12/20/00 12118/00 12/20/00 12118/00 12/20/00 12118/00 12/20/00 AVERAGE: STANDARD DEVIATION: Analyte Found (pg/g)- 0.0130 0.0128 0.00295 0.00295 0.00904 0.00876 0.00417 0.00435 0.01 19 0.01 17 0.01 13 0.01 14 0.00635 0.00623 0.00305 0.00297 0.0126 0.0126 0.005 14 0.00521 0.00792 0.00393 * Duplicate injection LOQ = 0.01 ppJg for liver and 0.01 &mL for serum Centre Analytical Laboratories, Inc. Page 30 of 102 Centre Study No.: 023-041 Table XII. Summary of PFOS in Juvenile Liver Samples at 6.2 ppm a i . (Test Termination 12 Weeks) Sponsor ID 9548 9548* 9549 9549* 955 1 9551* 9552 9552* 9555 9555* 9559 9559* 956 1 9561* 9562 9562* 9563 9563* 9564 9564* Centre ID 0007927 0007927 0007928 0007928 0007929 0007929 0007930 0007930 000793 1 000793 1 0007932 0007932 0007933 0007933 0007934 0007934 000793 5 000793 5 0007936 0007936 * Duplicate injection Set Number 12 1900A 12 1900A 121900A 121900A 12 1900A 121900A 121900A 121900A 12 1900AD 121900AD 121900AD 121900AD 121900AD 121900AD 121900A 121900A 121900AD 12 1900AD 121900A 121900A Extraction Analysis Date Da1:e 12119/00 12121IO0 12119/00 1212 1/00 12119/00 1212 1IO0 12119/00 12121IO0 12/19/00 12/19/00 12119/00 12119/00 12119/00 12119/00 12119/00 12119/00 12/19/00 12121/00 12121IO0 1212 1IO0 12121IO0 12/23/00 12/23/00 12/23/00 12/23/00 12/23/00 12/19/00 12/23/00 12119/00 12121IO0 12119/00 12121IO0 12/19/00 12/23/00 12119/00 12/23/00 12119/00 12121IO0 12/19/00 12121IO0 AVERAGE: STANDARD DEVIATION: Analyte Found (pg/g) 0.00303 0.00305 0.0100 0.0105 0.0123 0.0125 0.003 16 0.00302 0.0410 0.0408 0.0340 0.0341 0.0446 0.0445 0.01 16 0.0121 0.0193 0.0197 0.00184 0.00201 0.0182 0.0156 LOQ = 0.01 pglg for liver and 0.01 pglmL for serum Centre Analytical Laboratories, Inc. Page 31 of 102 Centre Study No.: 023-041 Table XIII. Summary of PFOS in Juvenile Liver Samples at 17.6 ppm a.i. (Test Termination 12 Weeks) Sponsor ID 9567 9567* 9570 9570* 9575 9575* 9579 9579* 9580 9580* 9581 9581* 9582 9582* 9583 9583* 9584 9584* 9588 9588* Centre ID 0007937 0007937 0007938 0007938 0007939 0007939 0007940 0007940 0007941 0007941 0007942 0007942 0007943 0007943 0007944 0007944 0007945 0007945 0007946 0007946 * Duplicate injection Set Number 121900A 121900A 121900AD 121900AD 12 1900A 121900A 12 1900AD 12 1900AD 12 1900AD 12 1900AD 12 1900AD 121900AD 121900AD 12 1900AD 121900AD 121900AD 12 1900AD 121900AD 121900AD 12 1900AD Extraction Date Analysis Date 12119/00 12121IO0 12119/00 12121100 12/19/00 12/23/00 12119/00 12/23/00 12119/00 12/21/00 12119/00 12121/00 12119/00 12/23/00 12119/00 12/23/00 12119/00 12/23/00 12/19/00 12/23/00 12/19/00 12/23/00 12/19/00 12/23/00 12119/00 12/23/00 12/19/00 12/23100 12119/00 12/23/00 12119/00 12/23/00 12119/00 12/23/00 12119/00 12/23/00 12/19/00 12/23/00 12119/00 12/23/00 AVERAGE: STANDARD DEVIATION: Analyte Found (pg/g) 0.00904 0.009 12 0.0424 0.0429 0.00629 0.00623 0.0880 0.0898 0.0461 0.0441 0.0492 0.045 1 0.0747 0.0733 0.156 0.132 0.0983 0.101 0.0350 0.0370 0.0593 0.0417 LOQ = 0.01 pg/g for liver and 0.01 pg/mL for serum Centre Analytkal Laboratories, Inc. Page 32 of 102 Centre Study No.: 023-041 Table XIV. Summary of PFOS in Adult Serum Samples at 0 ppm a.i. (Test Termination 19 Weeks) Sponsor ID 45 1 451* 452 452* 453 453* 454 454* 455 455* 456 456* 457 457* 458 458* 459 459* Centre ID 0007887 0007887 0007888 0007888 0007889 0007889 0007890 0007890 0007891 0007891 0007892 0007892 0007893 0007893 0007894 0007894 0007895 0007895 Set Number 121900BD 121900BD 121900B 121900B 121900BD 12 1900BD 12 1900B 121900B 121900B 121900B 12 1900BD 12 1900BD 12 1900BD 12 1900BD 12 1900BD 12 1900BD 12 1900BD 12 1900BD Extraction Analysis Date Date 12/19/00 12119/00 12/19/00 12/23-24/00 12123-24/00 12121-22/00 12/19/00 12/19/00 12/19/00 12/21-22/00 12123-24/00 12/23-24/00 12/19/00 12119/00 12/19/00 12/19/00 12119/00 12/19/00 12119/00 12121-22/00 12121-22100 12121-22/00 12121-22/00 12/23-24fOO 12/23-24/00 12123-24/00 12/19/00 12119/00 12/19/00 12/19/00 12/19/00 12123-24/00 12/23-24100 12123-24/00 12/23-24/00 12/23-24/00 AVERAGE: STANDARD DEVIATION: Analyte Found (pg/mL) 11.6 11.8 0.0102 0.00909 9.04 10.4 0.00274 0.00212 0.0125 0.0123 6.32 6.46 6.58 6.35 0.105 0.102 8.75 8.50 4.78 4.64 * Duplicate injection LOQ = 0.01 pg/g for liver and 0.01 pg/mL for serum Centre Analytkal Laboratories, Inc. Page 33 of 102 Centre Study No.: 023-041 Table XV. Summary of PFOS in Adult Serum Samples at 1.8 ppm a.i. (Test Termination 6 Weeks) Sponsor ID EO 1-0259-20752 E01-0259-2075;!* EO 1-0259-20753 E01-0259-20753* EO1-0259-2075.4 EO 1-0259-207541* EO 1-0259-20755 EO 1-0259-20755* EO 1-0259-207516 EO 1-0259-20756* EO1-0259-2076:2 EO1-0259-20762!* EO 1-0259-2076.3 EO 1-0259-207638 * E01-0259-20764 EO 1-0259-20764.* E01-0259-2076.5 E01-0259-20765* EO 1-0259-20766 E01-0259-207661* Centre ID 0 102969 0102969 0 102970 0 102970 010297 1 010297 1 0 102972 0 102972 0 102973 0102973 0 102979 0 102979 0102980 0 102980 0102981 0102981 0 102982 0102982 0 102983 0 102983 Set Number 03050 1AD 030501AD 030501AD 030501AD 03050 1AD 030501AD 030501AD 030501AD 03050 1AD 030501AD 030501AD 030501AD 030501AD 03050 1AD 030501AD 030501AD 030501AD 03050 1AD 030501AD 030501AD Extraction Analysis Date Date 3/5/01 317-810 1 3/5/01 317-810 1 3/5/01 317-810 1 3/5/01 317-8/01 3/5/01 317-8/01 31510 1 317-810 1 3/510 1 317-810 1 3/5/01 317-8/01 3/5/01 317-810 1 31510 1 317-810 1 3/901 317-8/01 3/5/0 1 317-810 1 3/5/01 317-8101 3/5/01 317-8/01 31510 1 317-810 1 3/5/01 317-810 1 3/5/01 317-8101 3/5/01 317-8/01 31510 1 317-810 1 3/5/01 317-8101 AVERAGE: STANDARD DEVIATION: Analyte Found (pg/mL) 10.9 10.9 7.92 8.01 16.4 16.0 35.8 33.1 24.3 23.7 1.50 1.89 5.01 4.98 1.57 1.84 0.134 0.117 10.5 9.79 11.2 10.7 * Duplicate injection LOQ = 0.01 pgJg for liver and 0.01 pg/mL for serum Centre Analyticid Laboratories, Inc. Page 34 of 102 Centre Study No.: 023-041 Table XVI. Summary of PFOS in Adult Serum Samples at 6.2 ppm a i . (Test Termination 6 Weeks) Sponsor ID E01 -0259-20757 EO 1-0259-20757* EO 1-0259-20758 EO 1-0259-20758* EO1-0259-2075'9 EO1-0259-20759* EO 1-0259-207610 EO 1-0259-20760* E01-0259-2076 1 E01-0259-20761* E01-0259-2076'7 EO1-0259-20767* EO 1-0259-20768 EO1-0259-207613* EO 1-0259-2076'3 EO 1-0259-2076S'* EO 1-0259-20770 EO 1-0259-20770* E01-0259-2077 1 E01-0259-20771* Centre ID 0 102974 0 102974 0102975 0102975 0 102976 0 102976 0 102977 0 102977 0 102978 0 102978 0102984 0 102984 0102985 0 102985 0102986 0102986 0102987 0 102987 0102988 0102988 * Duplicate injection Set Number 030501AD 030501AD 030501AD 030501AD 030501AD 030501AD 030501AD 03050 1AD 030501AD 030501AD 030501AD 030501AD 030501AD 030501AD 030501AD 030501AD 030501AD 030501AD 03050 1AD 030501AD Extraction Analysis Date Date 31510 1 31510 1 31510 1 31510 1 317-810 1 317-810 1 317-810 1 317-810 1 3/5/01 31510 1 31510 1 317-8101 317-8101 317-8101 31510 1 31510 1 31510 1 31510 1 31510 1 36/01 31510 1 317-8101 317-810 1 317-810 1 317-8101 317-8101 317-810 1 317-810 1 31510 1 317-8101 31510 1 317-8101 31510 1 317-8/01 31510 1 317-8101 31510 1 317-8101 31510 1 317-8101 AVERAGE: STANDARD DEVIATfON: Analyte Found (pg/mL) 52.2 56.1 102 94.7 80.4 84.6 69.2 62.9 84.6 86.7 4.57 4.07 2.72 2.61 4.02 3.87 3.24 3.3 1 2.10 1.70 40.3 39.7 LOQ = 0.01 pgJg for liver and 0.01 pg/mL for serum Centre Analytic(a1Laboratories, Inc. Page 35 of 102 Centre Study No.: 023-041 Table XVII,, Summary of PFOS in Adult Serum Samples at 17.6 ppm a i . (Test Termination 19 Weeks) Sponsor ID 48 1 481* 482 482* 484 484* 485 485* 487 487* 488 488* 489 489* 490 490* Centre ID 0007896 0007896 0007897 0007897 0007898 0007898 0007899 0007899 0007900 0007900 000790 1 000790 1 0007902 0007902 0007903 0007903 Set Number 12 1900BD 121900BD 12 1900BD 121900BD 12 1900BD 12 1900BD 121900BD 121900BD 121900BD 121900BD 121900BD 12 1900BD 030501AD 030501AD 121900BD 121900BD Extraction Analysis Date Date 12/19/00 12123-24100 12/19/00 12119/00 12123-24/00 12/23-24,100 12/19/00 12123-24/00 12/19/00 12/19/00 12123-24/00 12123-24,100 12/19/00 12/23-24,100 12/19/00 12/19/00 12123-24/00 12123-24/00 12119/00 12/19/00 12119/00 12123-24/00 12123-24/00 12/23-24100 31510 1 3/7-8101 31510 1 317-810 1 12119/00 12123-24/00 12/19/00 12123-24/00 AVERAGE: STANDARD DEVIATION: Analyte Found (pglmL) 260 263 43.1 42.4 118 106 179 172 133 127 15.8 16.8 232 228 6.87 6.80 122 93.0 * Duplicate injecti.on LOQ = 0.01 pg/g for liver and 0.01 pg/mL for serum Centre Analytical Laboratories, Inc. Page 36 of 102 Centre Study No.: 023-041 Table XVIII. Summary of PFOS in Juvenile Serum Samples at 0 ppm a i . (Test Termination 12 Weeks) Sponsor ID 9503 9503* 9506 9506* 9508 9508* 9512 9512* 9515 9515* 9518 9518* 9520 9520* 9525 9525* 9526 9526* Centre ID 0007947 0007947 0007948 0007948 0007949 0007949 0007950 0007950 000795 1 000795 1 0007952 0007952 0007953 0007953 0007954 0007954 0007955 0007955 Set Number 122000A 122000A 122000A 122000A 122000A 122000A 22000A 22000A 22000A 22000A 22000A 122000A 122000A 122000A 122000A 122000A 122000A 122000A Extraction Analysis Date Date 12/20/00 12/22/00 12/20/00 12/20/00 12/22/00 12/22/00 12/20/00 12/22/00 12/20/00 12/20/00 12/22/00 12/22/00 12/20/00 12/20/00 12/22/00 12/22/00 12/20/00 12/20/00 12/20/00 12/20/00 12/20/00 12/20/00 12/22/00 12/22/00 12/22/00 12/22/00 12/22/00 12/22/00 12/20/00 12/22/00 12/20/00 12/20/00 12/22/00 12/22/00 12/20/00 12/22/00 AVERAGE: STANDARD DEVIATION: Analyte Found (pgJmL) 0.00486 0.00385 0.00718 0.00754 0.00365 0.003 19 0.0043 1 0.00457 0.00789 0.00528 0.00378 0.00481 0.00367 0.00395 0.00552 0.00438 0.00354 0.00388 0.00477 0.00142 * Duplicate injection LOQ = 0.01 pg/g for liver and 0.01 pg/mL for serum Centre Analytical Laboratories, Inc. Page 37 of 102 Centre Study No.: 023-041 Table XIX. Summary of PFOS in Juvenile Serum Samples at 1.8 ppm a i . (Test Termination 12 Weeks) Sponsor ID 9528 9528* 9529 9529* 9530 9530* 953 1 953 1* 9533 9533* 9535 9535* 9540 9540* 954 1 954 1* 9543 9543* 9544 9544* Centre ID 0007956 0007956 0007957 0007957 0007958 0007958 0007959 0007959 0007960 0007960 0007961 0007961 0007962 0007962 0007963 0007963 0007964 0007964 0007965 0007965 Set Number 122000D 122000D 122000A 122000A 122000A 122000A 122000A 122000A 122000A 122000A 122000A 122000A 122000A 122000A 122000A 122000A 122000A 122000A 122000A 122000A Extraction Analysis Date Date 12/20/00 12/20/00 12/20/00 12/20/00 12/20/00 12/20/00 12/20/00 12/20/00 12/20/00 12/20/00 12/20/00 12/20/00 12/20/00 12/20/00 12/24/00 12/24/00 12/22/00 12/22/00 12/22/00 12/22/00 12/22/00 12/22/00 12/22/00 12/22/00 12/22/00 12/22/00 12/22/00 12/22/00 2/20/00 12/22/00 2/20/00 12/22/00 2/20/00 12/22/00 2/20/00 12/22/00 12/20/00 12/22/00 12/20/00 12/22/00 AVERAGE: STANDARD DEVIATION: Analyte Found (pg/mL) 0.0762 0.0670 0.0273 0.0284 0.00785 0.00824 0.0480 0.0476 0.0488 0.0494 0.0416 0.0413 0.0192 0.0189 0.0321 0.0334 0.00969 0.00983 0.0158 0.0166 0.0324 0.0198 * Duplicate injection LOQ = 0.01 pg/g for liver and 0.01 pg/mL for serum Centre Analytical Laboratories, Inc. Page 38 of 102 Centre Study No.: 023-041 Table XX. Summary of PFOS in Juvenile Serum Samples at 6.2 ppm a i . (Test Termination 12 Weeks) Sponsor ID 9548 9548* 9549 9549* 955 1 9551* 9552 9552* 9555 9555* 9559 9559* 9561 9561* 9562 9562* 9563 9563* 9564 9564* Centre ID 0007966 0007966 0007967 0007967 0007968 0007968 0007969 0007969 0007970 0007970 000797 1 000797 1 0007972 0 0 0 7 9 7 2 0007973 0007973 0007974 0007974 0007975 0007975 Set Number 122000D 122000D 122000D 122000D 122000D 122000D 122000B 122000B 122000D 122000D 122000D 122000D 122000D 122000D 122000D 122000D 122000D 122000D 122000D 122000D Extraction Analysis Date Date 12/20/00 12/24/00 12/20/00 12/24/00 12/20/00 12/24/00 12/20/00 12/24/00 12/20/00 12/24/00 12/20/00 12/24/00 12/20/00 12/22-23/00 12/20/00 12122-23/00 12/20/00 12/24/00 12/20/00 12/24/00 12/20/00 12/24/00 12/20/00 12/24/00 12/20/00 12/24/00 12/20/00 12/24/00 12/20/00 12/24/00 12/20/00 12/24/00 12/20/00 12/24/00 12/20/00 12/24/00 12/20/00 12/24/00 12/20/00 12/24/00 AVERAGE: STANDARD DEVIATION: Analyte Found (pgjmL) 0.137 0.160 0.382 0.390 0.222 0.208 0.0542 0.0535 0.243 0.218 0.161 0.168 0.185 0.171 0.23 1 0.229 0.259 0.254 0.171 0.173 0.203 0.0838 * Duplicate injection LOQ = 0.01 pg/g for liver and 0.01 pg/mL for serum Centre Analytical Laboratories, Inc. Page 39 of 102 Centre Study No.: 023-041 Table XXI. Summary of PFOS in Juvenile Serum Samples at 17.6 ppm a.i. (Test Termination 12 Weeks) Sponsor ID 9567 9567* 9570 9570* 9575 9575* 9579 9579* 9580 9580* 9581 9581* 9582 9582* 9583 9583* 9584 9584* 9588 9588* Centre ID 0007976 0007976 0007977 0007977 0007978 0007978 0007979 0007979 0007980 0007980 0007981 0007981 0007982 0007982 0007983 0007983 0007984 0007984 0007985 0007985 Set Number 122000D 122000D 122000D 122000D 122000D 122000D 122000D 122000D 122000D 122000D 122000D 122000D 122000D 122000D 122000D 122000D 122000D 122000D 122000D 122000D Extraction Analysis Date Date 12/20/00 12/24/00 12/20/00 12/24/00 12/20/00 12/24/00 12/20/00 12/24/00 12/20/00 12/24/00 12/20/00 12/24/00 12/20/00 12/24/00 12/20/00 12/24/00 12/20/00 12/24/00 12/20/00 12/24/00 12/20/00 12/24/00 12/20/00 12/24/00 12/20/00 12/24/00 12/20/00 12/24/00 12/20/00 12/24/00 12/20/00 12/24/00 12/20/00 12/24/00 12/20/00 12/24/00 12/20/00 12/24/00 12/2OlOO 12/24/00 AVERAGE: STANDARD DEVIATION: Analyte Found (pdmL) 0.329 0.355 0.483 0.512 0.241 0.252 0.406 0.424 0.287 0.254 0.295 0.285 0.459 0.473 0.521 0.524 0.553 0.564 0.532 0.509 0.413 0.114 * Duplicate injectlion LOQ = 0.01 pgjg for liver and 0.01 pg/mL for serum Centre Analytical Laboratories, Inc. Page 40 of 102 Centre Study No.: 023-041 Table XXII. Summary of Percent Moistures for Quail Liver Samples Sponsor ID 45 1 452 453 454 455 456 457 458 460 482 484 486 488 490 9506 9508 95 12 9518 9520 9522 9526 9530 9540 9543 9544 9548 955 1 9552 9555 Centre ID 0007867 0007868 0007869 0007870 0007871 0007872 0007873 0007874 0007876 0007878 0007880 0007882 0007884 0007886 0007908 0007909 00079 10 0007912 0007913 00079 14 00079 16 0007919 0007923 0007925 0007926 0007927 0007929 0007930 000793 1 YOMoisture 66.7 64.4 68.4 65.6 64.1 71.8 59.5 71.2 69.0 55.0 60.6 72.5 61.5 65.1 70.7 72.1 67.4 73.2 73.5 68.3 67.9 70.0 66.7 57.1 55.8 59.4 81.3 53.5 65.0 Sponsor ID 9559 956 1 9562 9563 9564 9570 9579 9584 9588 IO 1-0259-20780 :01-0259-2078 1 :01-0259-20782 101-0259-20783 ;01-0259-20784 101-0259-20785 101-0259-20786 :01-0259-20787 :O 1-0259-20788 :O 1-0259-20789 .01-0259-20790 ,O1-0259-20791 f01-0259-20792 ,01-0259-20793 ,01-0259-20794 01-0259-20795 0 1-0259-20796 01-0259-20797 01-0259-20798 01-0259-20799 Centre ID 0007932 0007933 0007934 0007935 0007936 0007938 0007940 0007945 0007946 0 102997 0102998 0102999 0103000 010300 1 0 103002 0103003 0103004 0 103005 0103006 0103007 0 103008 0103009 0103010 010301 1 0 103012 01030 13 0103014 0103015 0103016 YOMoisture 78.3 80.6 57.8 63.4 34.6 85.2 88.7 59.6 65.2 70.3 57.8 70.6 67.0 67.7 64.5 72.3 67.6 68.0 68.0 69.3 70.9 67.0 65.3 74.5 65.7 71.7 72.8 71.3 72.1 Centre Analytical Laboratories, Inc. Page 41 of 102 Centre Study No.: 023-041 FIGURES Centre Analytical Laboratories, Inc. Page 42 of 102 1.564 1.4e4 I 1.3e4 1.2e4 l.le4 1 .Oe4 9000.0 n -!l5 8ooo*o 7000.0 i 6000.C ., . . e ` 5000.C 4000.C 3000.C e e 8 2000.c I , 1ooo.c 0.c w P % c 0 h) : I ! ! ' ,/ " e e cn E Y Z 0.. 0 w h) b f: Centre Study No.:023-041 Figure 2. Chromatogram Representing a 0.1 ng/mL standard for PFOS pc 9 ' Conc.nLrazion: 0.100 233, I Acq. D a t m : Acq. T h : 12/22/00 10:37:10 AH 220 : I ' Modified: Y, . ' Bunching Factor: 1 Uoise Thr.shold: 1.91 AI-. Threshold: 9.55 Num. S-ths: 1 RT M i n d a : 30.0 sc. Exp.cteg RT: 4.51 .in Sap. WIdth: 0.20 Sep. Heiqhr: 0.01 m. Exp. Peak Ratio: 5.00 Ad). Ratio: 4.00 Exp. v.1. Ratio: 3.00 Uao R.latrv. RT: No 200, 1 180. I too! 1 i14D.I l1Z0O0. Inc. Type: Uanurl I Retention T i m : 4.49 -in Ax.: Heiqhr: 21.0183e9r10002 COUcpa.tI W I Start T h : 4.40 min End Tim: 4.59 .in 40 1 I 20 1 Centre Analytical Laboratories, Inc. ....... . . . -. . . . - ... . , . ........... Page 44 of 102 Centre Study No.:023-041 Figure 3. Chromatogram Representing a 1.0 ng/mL standard for PFOS . . - . .. . Concentrac&m: A m . Dare: ACq. r1m: 1.000 12f22/00 11:15:09 M I4cdifi.d: Y, . Bunching Factor: 1 NOISa ThraShold: 1.91 ASea Thr*ahold: 9.55 N U . S-Chs: 1 ?W. ln'dIow: 30.0 m c &pQct.d 111: 4.51 .in Sap. Width: Sop. WeIqht: B h p . Peak b c i o : f EXP. Adj. b t ~ o : Exp. Val. b r i o : f US. R*laLIve RT: 0.20 0.01 5.00 4.00 3.00 No I n t . type: R.tention T h : A:.I Hoiqhz: st.rt T h : End Tim: H.nu.1 4.50 4881.6 i.i3.+om 4.40 4.61 .in esunt. cp. rin rin 1181 1100. 1000: 900. 800 700 600 do0 400 100 200 100 0 1- 0.5 1.0 1.5 2.0 . 2.5 10 1.5 40 Tmo. mn Centre Analytical Laboratories, Inc. Page 45 of 102 . . -. . , ..-. .. - .. . I ..,?.? . . " , . .__ - , ., . . - . Centre Study No.: 023-041 Figure 4. Chromatogram Representing a Reagent Blank for PFOS (Centre ID Reagent Blank A, Set: 122000A) Sa.plc Index: ;1 S l l p l . Type: Concentration: t Acq. Date: Acq. time: 8 Bl."k 0.000 12122100 12:05:48 R( 4wi 450 nodifiad: Y.S Bunching Factor: 1 Noise 1hz.rhold: 1.91 AT.a Threshold: 9.S5 - Nur. SmothS: RT Window: Expected RT: 1 3 0 . 0 s.e 4.Sl .in 5.p. Width: 0.20 Sep. Height: 0.01 wlp. Peak Flatdo: 5.00 wlp. Mj. Ratio: 4.00 Exp. V a l . Ratio: 3.00 US. R.l.ti". n1: No 400 550 so0 B -250 - zoo Int. Type: Retention T i m : :..IA Height: stazt Tip.: End Time: wmu.1 4.50 2299.8 4.50.*002 4.39 4.61 .in counts Epa mi" min 150 100 50 - .. 0' - 0.5 1.0 9.5 2.0 2.l 3.0 3.5 4.0 I-- "A. Centre Analytical Laboratories, Inc. .. . . .. --. ,. . . Page 46 of 102 .. .. . . .. Centre Study No.:023-041 Figure 5. Chromatogram Representing Control Quail Serum for PFOS (Centre ID: 0011424Blank A,Set: 122000A) Sample Index: Sample Type: conc.mtration: Acq. Oat.: Acq. I&: 10 Bhnk 0.000 12/??/00 12:Jl:OO M 1083 1000 Wifi*d: Bunchinq h c t o r : Nois. Thr.rhold: Area Ihz*ahold: Nun. S-tha: RT Window: Expected RT: Sap. Width: S e i . Height: Exp. Peak Ratlo: Em. Mt. Ratio: Yea 1 1.91 9.55 1 30.0 4.51 0.20 0.01 5.00 4.00 SC. Din . 8(. i 900 800 100 600. so0 . + Xnt. Typ.: 1 R.t.ntion Tim: 41:..IAHeight: I s t * r t 1Ln: i End T h : I hnual 4.50 4110.1 1.03.*003 4.39 4.63 mitn counts cpr Din min 400; too: 1 I 1 P 0.5 t.0 1,s 2.0 2.5 3.0 3.5 4.0 Tim. m n Centre Analytical Laboratories, Inc. Page 47 of 102 Centre Study No.: 023-041 Figure 6. Chromatogram Representing Control Quail Liver for PFOS, (Centre ID: 0007870 Blank A, Set: 121900A) 1 Acq. O w * : ACq. T I M : 12/21/00 11:29:44 W Uodified: Bunchinq Faccoc: Nois. 1hr.shold: Area Thr.sho1d: Yea 1 1.91 9.55 Nun. 5-tha: RT Window: Exp.ct.d RT: S.p. Width: Sap. Height: 5 Exp. Peak Ratio: Exp. Mi. Ratio: Exp. Val. Ratio: f Use Ralative RT: 1 30.0 a= 4.51 .in 0.20 0.01 s.00 4.00 3.00 No I n t . Typ.: htmtion Tim: A:.I Height: SCJCC Tim: End TIM: . Manual 4.48 402.8 1.01.+002 4.37 4.61 rin COUIILS cpr rin .in i 1 4.37 . . ,.4.64 ' .I 6 ~ .,'* ' 3 . 1 7 5.57 . 6.47 Centre Analytical Laboratories, Inc. .. Page 48 of 102 .. - . . . . ~ .. - - ^ . *- Centre Study No.: 023-041 Figure 7. Chromatogram Representing Control Quail Serum Fortified with 500 ng/mL of PFOS (Centre ID: 0011424 Spk A, Set: 122000A) : Sampl. Typ.: ! concencration: Acq. Dace: I Acq. Tim: a 2.000 12/22/00 12:56:17 R1 1 1600. Uodified: Yes Bunching Factor: 1 Noise Threshold: 1.91 .IA Threshold: 9.55 1600 1400 ! NU.. Smooths: 1 RT window: Expected 112: 3 0 . 0 s.c 4.51 min 1200 4 Sep. Width: 0.20 i Sap. HeLghC: GYP. Pk. R.110: EXP. Mi. Ratto: exp. V a l . ruc10: Use R.1atlve RT: 0.01 5.00 4.00 3.00 NO 1000 = I 4 a00 ; Int. Type: R.cencion Tim: k..: I(.nUJl 4.49 7570.8 dn C0""tI I 600. I Height: 5t a r t Tim: End Tim: 1.79e*003 4- .- 4-04.62 cpr -4. . mm 400. 200 3 i 0 I i 1 -.._---_-_ -_-c --. --_._.__- 0.5 1.0 1.5 2.0 2.5 3.0 1.5 4.0 d 5.0 5.5 6.0 6.5 7.0. 1.5 Centre Analytical Laboratories, Inc. Page 49 of 102 Centre Study No.: 023-041 Figure 8. Chromatogram Representing Control Quail Liver Fortified at 10 ppb with PFOS (Centre ID: 0011423 Spk A, Set: 121800A) I-I. m.. a Centre Analytical Laboratories, Inc. Page 50 of 102 Centre Study No.:023-041 Figure 9. Chromatogram Representing Juvenile Quail Serum Sample (Centre ID: 0007965, Sponsor ID: 9544, Set: 122000A) 1 i Acq. Acq. Dace: Tim: 12/22/00 10:29:16 RI ! Modified: YO8 . Bunching factor: 1 1 Nois. Threshold: 1.91 Area Threshold: $.55 I Num. Smoochr: 1 I RT Window: 1 fxwcc.d RI: 30.0 aoc 4.51 l i n ' Sob. Widch: 0.20 I S.D. Hoiaht: 0.01 1200 1100 1000 900 800 700 600 500 400 JW 200, 1W; - 0' 0 5 1.0 1.5 2.0 2.5 J.0 3.1 4.0 - _4.67 .- 5.0 __-___ .-,-.---.--L--* .__C__r ._-/.. ~ 5.5 6.0 6.5 7.0 7.5 Centre Analytical Laboratories, Inc. Page 51 of 102 Centre Study No.: 023-041 Figure 10. Chromatogram Representing Juvenile Quail Liver Sample (Centre ID: 0007926, Sponsor ID:9544, Set: 121800B) I S-1. Index: 59 I con;*ntr;iion: Acq. mc.: 1 Acq. Tim: I U/A 12/20/00 .01:33:11 i I4diti.d: ..I . Dunchins Factor: 1 I WOia. Thr.shold: 1.91 As'.. Thrashold: 1 Nun. S l w t h a : . j RT Nindw: 9.55 1 30.0 Expactad RT: I Sep. Nidth: , Sep. Height: 4.51 0.20 0.01 I rXp. Peak Ratio: Exp. Ad). Patio:' U p . V 8 l . Ratio: 5.00 4.00 3.00 1 Us. Relativ. RT: lo pw au mi" 1 2101 1I 1dOO. j,MOO 1400. I troo! . I 1000 ! 800 600 400 - zoo. 0 05 10 15 20 25 30 35 40 5 5.0 5.5 6.0 6.5 7.0 7.5 Tnna.mu, ~ Centre Analytical Laboratories, Inc. Page 52 of 102 __ Centre StudyNo.:023-041 APPENDIX A Study Protocol OOP-023-041 (Centre Study No. 023-041) and Amendments and Deviations Centre Analytical Laboratories, Inc. Page 53 of 102 - ... - . . . . . ..: >.: .. __._.l .c.- .. . . .-..L:.--L*L--.--- c 1 I . .. . . -...I. - -. . . .. . . . ... . . . .... CentreStudyNo.:'623-041 -.._-.--_____-___ __,______ Centre protocol No.OOPM3-041 . (-- $.v- STUDY PROTOCOL I STUDYTITLE EXTRACTIONOFPOTASSNM PERFLUOROOCT&ULFONATE FROM QUAILSERUM AND QUAELIVER FOR ANALYSIS USING HPLC- . El,ECTROSPRAY/MASS SPECTROMETRY - SPONSOR 3M En&onmental Technology and Safety Services Building 2-3E-09 PO Box 33331 St. Paul, MN 25133-3331 - - DATAREQUIREMENTS - h-dytical Method Requirements TESTING LABORATORY Centre Analytical Laboratories. Inc. (Centrc) 3048 Rescarch Drive - Stat+Coll~g,PA16801 Phone 814-231-8032 PROTOCOL IDENTIFICATION M E R I , centre Analytical Laboratories, bc. -Centre Analytical Laboratpries, Inc. ........ ........ ~-.-~.~. . . . . . . .-.-,--.., - .. -. Page 1 of 43 ........ ..... . . . . . . .. . .... ^_-.-- ... . Centre Study No.: 023-041 .-- _- .____ I_ I ? Centre Protocol No. OOPM3-Wl Title:EXTRACTION OF POTASSIUMP E R F L U O R O O a ~ W O N A T EFROM QUAIL SERUM AND QUAE LIVERFOR ANALYSIS USING WLC- . ELECTROSPRAY/MASS SPECTROMETRY TABLE OF CONTENTS Page TABLEOFC 0 " T S ..... .............................................................................................. 2 ~ 1. PURPOSE ............................................................................ I . x. ................. 3 . 2. TESTMATERIALS;.............................................................................................. 3 3. SPONSOR............................................................................................................... 4 4. T'ESTINGFACILlTY AND PERFORMINGLABORATQRY .......... :.......4 5. PROPOSED~ E U M & I T A LTIME-FRAhiE................1.................................. 4 6. JUSTIFICATION FOR "J-E SELECTIONOF THE TEST SYSTJ34.................4 7. . SAMPLEPROCESSING, STORAGEAND IDENTIFICATION ................:.......5 8. ANALYTICAL-OD U.................................................................................. 5 9. EXPERIMENTAI. DESIGN........................................... :...................... 10 ... ~ ._ 10. PROTOCOLAMENDMENTSAND DEWATIONS........... ...................io ; 11. RECORDS ............. :................................................................................. 11 12. QUALXIY ASSURANCE.......................................................... 1............. 12 13. DATA AND REPORT................... ..................................... 12 14. ARCHIVE STATEMENT........................................ ........................................... 13 i5. REFERENcEs.....1...............,............................... 13 . 16. PROTOCOLAPPROVAL....................................................... i.............. 14 . APPENDIX:A N A L m C L METHODS...................................... ................................ 15 I .... -.......... I Centre Analytical Laboratoria, Ihc. ' Centre Analytical Laboratories, Inc. Page 2 Of 43 page's5 of 102 ... .. . . .. f .. . . s .. . . . ,:.. .. .. . , .. , . , ... . .. . . . . _. _. .:. .... .:"_:: :... : '.:. .::.: ..._. ,, . . . :. . Centre Study No.: 023-041 i , CentreProtocol No.OOP-023-041 $tu& Title: I EXTRACI'ION OF POTASSIUMPERFLUOROOC'I'ANESUNATE FROM QUAIL SERUM AND QUALLIVER FOR ANALYSIS USING HPLC- ELECl'ROSPRAYMSS SPECTROMEI'RY ' 1. PURPOSE The purpose of this study is to analyze quail serum samples and quail liver samples forresidues of peffluomctanesulfonate(PFOS) using methods entitled, "ix&tion Of Pbtassium PertIuorooctanesulfonate Or Other Fluomchemical Compounds From. Serum For Analysis Using HPLC-Electrosprayhfas Spectrometry" (3Mmethod number ETS-84.1) and "Extraction Of Potassium . Peffluorooctanesulfonate Or Other Fluomchemical Compounds From Liver For Analysis Using HPLC-Electro~pray/%4aS~pectrometry" (3M method number ETS-8-6.0) with the modifcationslisted in Section 8. The QualityAssurance Unit of Centre Analytical Laboratories,Inc.. will audit the study for compliance with EPA TSCA Good Laboratory Practice standards 40 CFR Part 792 (1). 2 TESTMATERIALS The following analytical standards will be used: . . Test Material PFOS - TCfj Number TCR0001748 .Purity (%) 97.9 Expiration Date 08/31/01 I . .. ... ., i PFOS ChemicalNme: W A C Name: CAS Number: Molecular Weight: 0 0 &gg The neutral molecule and staildard form that PFOS (anion) is derived from is perfluorooctanesulfonatepotassiuin salt [C8nSO3Kl, molecularweight 538. Centre Analytical Laboratories. Jnc. * Page 3 of 43 Centre Analytical Laboratories, Inc.' Page 56 of 102 ' .. . . 2.. . . . . . ..... . ,. ~ .. . . . . . . . . . . _.-. . . . . . . .. . . . ... . . . . . Centre Study No.: 023-041 .. . . . . . .-. .- .... .- .... .. -'- ~ ..____-__.----..-- I 1 Centre ProtocolNo.oOP-o2u)41 .' A record of test and refemce substance receipt, storage conditions, and a record of use will be maintained at Cencre Analytical Laboratories, Inc. Forms - documenting chain-of-custody and shipping records 'for tmcking of the test substanceswill be included as part of the raw data package. All standadtest substances and any pnpand solutions must be identifiedwith a unique label or number on the containeror cross-referencedto the container. A current MSDS for the chemical(s) used in this study will be maintained at the testingfacility. . 3. SPONSOR 3M EnvironmentalTechnologyand SafetyServices Building 2-3E-09 PO Box 33331 .- St.Paul, MN 55133-3331 RSTocUhDelYleDRIoRbEidCeaTuOR - i- TEIEPHOm 651-778-7065 f '*..- FAX NUMBER: 651-778-6176 . 4. TESTING FACILITY AND PERFORMINGLABOFTORY Centre Analytical Laboratories, Inc. (Centre) 3048 Research Drive I .. State College, PA 16801 PRINCIqAL INVESTIGATOR: Emily R. Stauffer, Centre TELEPHONE: 814-23 1-8032 \- FAX NUMBER. 814-231-1580 5. PROPOSEDEXPENMENTALTIME-FRAME Experimental Start Date Experimental Termination Date Report Issued Dec. 11,2000 Dec. 15,2000 De. 30,2000 6. JUSTIFICATIONFOR THESELECTION OFTHE TEST SYSTEM ! Quail serum samples arid liver samples collected by Wildlife International,Ltd. will be used as the test systems for this study. ' Quails represent wild bird i populations and they arc an EPA recommended species because they do well in a ._. laboratory environment. Quail habitats include bushy pastures, p i x y roadsides, I' Page 4 of 43 I ckntre Analytical Laboratories, ~ n c . --- 1 I .. I -- - I .... .- .. Centre Study No.: 023-041 . __-.--.--..---I-.. . . . - - .I .......*..___._II._..__. _______ Ceria Protocol No.00PM3-041 farmlands;and open woodlands. and they are considered land grazers. The use of these matrices for the study was to determine if PFOS was deposited in the liver and serum of mating adults that were fed PFOS. A complete description of the test system along with all of the in-life parameters is documaited in the sponsor's protocol no. 454/0106oO/QP/SuB454, Project No.454-1,Mthat is associatedwith this analyticalprotocol. 7. SAMPLE PROCESSING,STORAGEAND IDENTIFICATION No pmcmsing will be q u i d for the mum samples. Due to the small mount of sample available,the liversampleswill not be homogenizedprior to extraction; . however, they will be mashingthan by hand . homogenized by laying the as best as possible. samples on a flat surface and Each sample will be assigned a unique Sample identification number at Centre. with will be used for tracking and identification of the samples. The samples will be stored in a temperature-monitored freezer. maintained at < -10" C. except when .removed for extraction and analysis as described in the method. The sampleswill be kept isolated from the tcst.substanceduringstorage. . . Sample receipt and storage location and conditions during the _study will be doymented.' All samples and any nSulting Sample extracts will be identified with a unique label or sample number. Such identification will be either on the container or cioss-referenced to the container. 8. . &AL~CALME"HOD The saum samples will be analyzed according to the analytical method titled "Exfraction Of Potassium Peffluorooctanesdfonate Or Other l?Iuorochemical Compounds -From Serum For Analysis Using HXC-Elemspray/Mass Spectrometry"(3Mmethod'number ETS-8-4.1) and the control liver sampleswill be according to the method titled "Extraction ot Potassium ' . Peffluorooctanesulfonate Or.0ther.Flu~hemicaCl ompounds Frpm Liver For Analysis Using HPLC-ElcctmsprayIMass Spectrometry" (3M method number ETS-8-6.0). The followingmodifications will be applied to each method: 1. Then will be no surrogate standard used in the extraction prkcdun. The stock,fortification,and calibrationstandardswill be prepared as follows: I Stock Solution Prepan a stock solution of PFOS at 100 @nL by weighing out 10.0 mg of analytick standard (corrected for percent salt and percent purity). Adjust final volume to 100 d,with methanol in a 100-d, volumetric flask. Store this stock solution (in 125-mL LDPE bottles) in a refrigerator at 2C to 6C for a . maximum period of 6 months from the date of preparation. Centn Analytical Laborataim. Inc. Page 5 of43 Centre Analytical Laboratories, Inc. Page 58 of 102 - . ... . . .- - - . . . ... . .. Centre Study No::'023-041 - . .. - .... .. ._ . _.__--.-.-...--- ---.--. -. - -...---..--i----- .-____-_I-.____.__ ____ *, I .- -4 G t r e ProtocolNo.oopM3-041 Fortif3cation Solutions a 1.0 udmLForo'fication - Solution Pipette 1.0 mL of the 100 p@mL stock I solution into a 100 mL volumetric flask. Bring up to volume with methanol. . . - b. QJ&@ Fomfimon Solution Pipette 10.0 mL of the 1.0 pg/d . fortification solution into a 100-mL volum.etric flask arld bring up to __- _J- Vamm-K- e-_ -.- - c. 0.01 && .. Forhficahon Sohh'on I Pipette 10.0 mL of the 0.1 pg/mL fortification solution into a 100-mL volumetric flask and bring up to volume with methanol Ston all fortification standard solutions (in 125-mL mPE bottles) in a kfrigerator at 24C to 6C for a maximum period of 6 months from the date of pnparation. Calibration Standards Prepare six Lc/MsIMscalibration standards in methailol via dilution of . the 0.1 pg/mL and the 0.01 pglmL fodfication solutions. ~ This is a typical example; additional concentrations may be p n p m d as needed. . Initial Conc. (&mL) Volume (mE) Diluted to (mL) Final Conc. ( p d m ~ ) - 0.1 5 100 0,005 0.1 -2 100 0.002 . 0.1 1 100 0.001 0.01 5 100 0.0005 0.01 2 100 , 0.0002 0.01 1 100 0.0001 Store all calibration standard S O ~ U ~ ~ (Oin~ S125-mL LDPE bottles) in a . refrigerator at 2C to 6C for a maximum period of 6 months from the date of preparation. - 2. The liver samples will not be pro~essedto make the homogenate described in the method Section 12.2 to 12.12. Due to limited sample available, samples will be mashed on a flat surface by hand prior to extraction. 3. The sample weight for-liverwill be 0.50 g (*0.05) and the sample volume for serum will be 100 pL. The amount of 0.5 M TBA added to the m u m k p l e will be 0.5 mL and the amount of 0.25 M sodium carbonatdsodium - bicarbonate buffer added will be 1.0 mL Samples will be placed on a wrist- action shaker for -'20 min. at level 10 and then centrifuged for 15 min @ .-3000 rpm. The sampleswill only be filtmd with 0.2 pm nylon mesh filterif Page 6 Of 43 Centre Analytical Laboratories, Inc. -..I - .- Page 59 of 102 .. .. .i. . . .... 1 I ' ,' . .. . . .. . .. . . . , . _ _ _.. - . Centre Study No.: 023-041 Centre protocol No.OOP-023-041 necessary. Samples will be reconstituted in a final volume of 1 I& of methanol for both matrices. 4. The samples will be analyzed under the following conditions: LC/MS/MS System and Op&ating Conditions(TurboIonspray) Mass Spec: PE SCIEX API 3000 BiomolecularMass Analyzer ~ Interface: SCIEX TurboIon SprayLiquid IntroductionInterface - . _ Harvard infusion pump . Computer: Power Macintosh 0 3 Software: PE SciexAnalyst 1.1 Windows NT HPLC: .- HewlettP a c w (HP) Series 1100 W Quat Pump - s.Ip Vacuum Degasser HPAutosampler HPColumnOven . HPLC Co1umn:Gmesis CS(JonesChromatography),2.1 -mx 50 mm.4p . ColumnTemp.: 35OC . Injection Vol.: 10pL MobilePhase (A):. 2 mMAmmonium Acetate in ASTM type I water Mobile Phase (B): Methanol Flow Rate: 0.3 mL.!.min. nnGu!&B .. 0 60. .40 1.0 0 . - 100 7.0 0 ' 100 7.5 40. 60 11.0 40 - 60 It may be necessary to adjust the HPLC gddient in order to optimize instrument performance. Columns with different dimensions (e.g., 2.1 mm x 30 d)and columns from diffmnt manufacturers (Keystone Betasil Cls etc.) can be used, provided equivalent chromatography is obtained. ~ o n smonitored Centre Analytical Laboratories, ~ n c . .. . Page 7 of 43 I Page 60 of lOi _ _ . _. . . . ... .. . . . .. 1 'I , . !I - . - i.-:... ._ I ' . ... . . .. Centre Study No.: 023-041 - -.--' --.-------. ~ --__...-.- . . - 1 a n h e protocolNo.OOP423-041 Analvte PFOS ' w negative + .. I Tm10onMonitond 499 99 ' Cm Approximate Retention Time 4.20 On a day-today basis, the ntention times may vary slighhtlydependingon the batch of mobile phase, etc. Example Tune Fiie Parameters The following vslue~are p p v i a~s an example. AC~&values may vary from instrumentto instlument. Also. these values may be chmiged from time to time in order to optimize for greatest sensitivity. The mass specmmctcr is tuned using a 0.5pg/mL PFOS solution, prepared via dilution of the stock solution in methanol. .The solution is infused (using a 'T' connector) at 10 wmni into a 0.2 d i n i n stream of mobile phase consisting of 4096 methanol and 60% 2 mM ammonium acetate. The analytes arc initially tuned for the parent ion and then tuned for the product ion. Once the instrument is tuned, the optimized parameters are saved as a "tune file". This tune file is then used during routineanalysis. The tuning proceduremay b.repeated as necessary . to ensure optimal seositivity. controls - Is-Iospray OR-Orifice - RNG-FocusRing Q O - Q d 0 R d -Offset IQL inter quad 1 lens ST-Stubbies ROl-QllidlRod Offset 1 Q 2 - hq~uad 2 lens ROZ-Quad2 d offset ST3-Stubbia' . R03-Qd 3 rodOffset DF-CEMDeflection Plate CEM-Channel Electron Multiplier . QasFlowt Nebulizer Gas qntainGas CdlisionGas- - TIS Temperature &' 4000.0 41.0 . . -270.0- - 10.0 9.3 -15.0 . .93 20.0 -84.0 100.0 86.0 . 300.0 2800.0 sa' 12 13 4 350OC , i I ., Calibration Prkdures - a Inject the same VO~W (bet;veen 10 to 20 &)of each calibration standard (prepared in MeOH).into the u=IMsIMs. Centre Analytical Laborataier,Inc. Page 8 of 43 , I Centre Analytical Laboratories, Inc. I 9 .. .. .._.__.__..__ _._. - -.--- Centre Study No.: 023-041 ............... CentreProtocol No.OOPM3W1 b. Use line& l/x weighted standard Curves for quantification. Enear standard curves are generated for each set by linear r e m i o n using the appropriate softwaresystem. Any calibration standards falling outside f 30% based on its calculated concentration,must be excludedfrom the calibration c u m . However, the total number of calibration standards that may be excluded must not exceed 30% of the total number of standards injected. c. The corklation coefficient (r) for calibration curves generated must be 20.9925 ((20.985). If calibrationresults fall outside t h a limits, then appropriate steps should be taken to adjust instrument ' operation,and the relevant set of samplesmust be reanalyzed Sample Analysis Inject the same volume used for the calibration standards (between 10 to 20 p ~ ) of each sample, fortification,-control,etc. into the Lc/MS/Ms.Each sample must be analyzed in duplicate. a. standards comsponding to included in an analytical set. a- t least six concentrationi must be - b. Inject an entire set of stan- (six) at the beginning of the a n and inject standards interspersed about every 5-10sample-s. All sample injectionsmustbe bracketed byptandard injections. c. Each set of-samples analyzed (not to exceed 25) must include at least one reagent control (extraction solvents orily), at least om matrix . control, and two matrix control Samples fortified at known concentrationsand carriedthrough the P K X ~ W Cto v d f y recovery. d. The concentration of- each SarnpleD fortification, control, etc. is determined from the standard curve based on the peak area of the . n d ~ ein all standards injected during a set. The standard nsponsts must bracket responses of the residue found in the sample set. .If - necessary, dilute the samples and r e - a n a l p to give a rcspon& within the standard curve range. e. Fortification recovcxies within 80 to 12096 arc acceptable for fortificationsat the LOQ level. Recoveries between 80 to 120% are acceptable for fortifications at levels greater than the LOQ? Failure to mreaentaiiyhscissedcthrietearfifaecrteeqdusiaremsplaens. inv-estigation of cause and a full f. Samples in which no peaks arc detected at the comsponding andyte retention times will be nportod as ND (not detected). Samples in which peaks arc detected at the corresponding analyte retention times Page 9 of 43 Centre Analytical Laboratories, Inc. ... - .................. ... Page 62 of 102 .... Centre Study No.: 023-041 ; but arc less than 3:l signal:noise will be reponed as NQ (not quantifiable). ... g. Background levels of analyte found in controhlank samples that comspond to values below the LOQ, but are still quantifiable, will be used toc o w fortificationrecoveries. h. ' If samplesare not loaded on the insrnunentto be analyzzdthe day they I arc extracted, samples must be stored refrigerated at approximately . 2OC to 6C until analysisand analyzedpreferably within'a week. . A 9. E-RIMEWAL DESIGN Samples obtained by Wildlife International L.td. have k n shipped to Centre AnalyticalLaboratories;Inc. for analysis. AU samples will'be analyzed according to methods, 'Zxtraction.of Potassium Peffluorooctanesulfonatcor Other Fluomchemical Compounds From Serum for Analysis Using HPLC-Eiectrospray/Mass Spectrometry" and "Extraction of Potassium Pduorooctanesidfonate or Other Fluomchemical Compounds From Liver for Analysis Using H P L C - ~ e c t r o s p yS~pectrometry" with the modificationsdetailed in Section 8. -. Methods to control bias' will include assay- of untread control saniples, foaifcation of untreated control samplw to obtain recovery data, and replicate . .analysisof fortifiedsamplesto providean indication of reproducibility. . . The aveige recovery and relative standarddeviation of the fortified samples will be calculated. Also, the average residue found and standard deviation for each - matrix will be calculated. Moisture dctmninationwill be p&onnd bn all liver samples accordingto Centre SOP CAL-lOBll1with the followingmodifications: - 1. The sampleweight will be 0.5 g. - 2. The drying time will be 12 hours.- _-._._..- - 10. PROTOCOLMNDMlW'I"'I' AND DEVIATIONS I Any deviations from the protocol or from the analytical method as provided will be documentedand reported promptly to the Study Director. Planned changes to this protocol or to the analyticalmethod will be made in writing as an amendment and approved by the Principal Investigator and the Study Director. Any . amendments or deviations will be appended to.this protocol and included in the final report. I Centre Analytical Laboratuiies. Inc. . Page 10 of43 Centre Analytical Laboratories, Inc. - . . . .. . .. -.. . . .. . ..~ . Page 63 of 102 .- Centre Study No.:023-041 -i 1 I i i (......'.....,........ . I CantreProtocol No.OOP-023-041 : - Planned changes to the approved protocol shall be documentedby amendments that clearly describe the change,justification for the change, and impact on the study. Amendments will be signed and dated by the Principal Investigator and Study Director. Copies of mendments will be sent to the quality assuranceunit. mol deviations: Protocol deviations, which an one time and unplanned ' deviationsfrom the protocol,shall be documentedin the study records,noting the nature of the deviation, potential effect or impact on the study, and corrective action if required Protocol deviations an signed and dated by the Principal Investigatorand Study Directorand reviewed by QAU. 11. RECORDS -. Records to be maintained inclu&..but are not limited the following (as appropxiate): . 1. Sampletracking shctt(s). 2. S&nple receipt reco.rds,storagehistory,and chains of custody 3. History and preparationof standards (stock,fortification,&libration) 4. 5. Descriptionof any modifications to Instrumentrun sheets, bench-sheets tohrelomgesthod- 6.Analyticaldata tables .- 7. All chromatographic and instrumentalconditions 8. Sample extraction &d analysis dates . 9. A completelisting of studypersonnel, signaturesand initials 10.Chronologicalpresentationof all study correspondence 11. Any other data necessary for the reconstruction of the study . All chromatograms will contain the following: a. Sample identification. date, arrow or Other indication of the area of ' intenst, and injection number comsponding to the run. b. Additionally, fortifications will include the fortification level of the tlllalyte. c. Analytical standard Chromatograms will additionally: include the concentration(e-g., p#mL n#mJ-, ppb, ppf etc.). Each data set will contain information on temperatures,, flow rates, column pmameters, gases, instmmait parameters, and instrument type, etc. The Centre study number will also be recorded on the first chromatogram of each daily analytical run. I -. - cenlnAnalyticalLaboratcic&he. Centre Analytical Caboratories, Inc. .. . . -. .. . __.... ... -.... . . . _ _ . .. Page 11 of 43 i i i Page 64 of 102: .. . -- Centre Study No.: 023-041 ~ - - "_ --CZmtm pmtocol No. OOP-023-041. ( Ceria Q A Unit wili review the protocol, audit the study conduct and the study documentation (including raw data and final report) to ascertain that all QNGLP procedures a~ adhered to. Centre QA Unit will inspect the study at intervals adequate to assure compliance to GLP's, and will report the findings of the audits to the-PrincipalInvestigator, CmtreManagement,and the StudyDirector. I 13. DATAANDREPORT ' 1. All raw data and the original sigind @Dtocol-Willbe maintained in the study file. This data includes the protocol' amendments, protocol deviations, laboratory notebooks, analytical standard Solution preparation, sample chain of custody sheets. sample work sheets, chromatograms, calibration curves, and any other appropriate data generated. Raw. data not wed will be stored in the study file. The reason for exclusion will be documented. -2. A final report will be issued by Centre. approved and signG by the Study Director and sufficient for submission to WA. The report contents should ~ - indude, but not limited to: - . .. (i 1. Objectives and pmedtgcs stated in the p:&ol 2. Analytical and statistiealmethods used 3. Test materials identified by name, lot, purity. and other charirctcristics 4. Name of testing facility and study initiation and completion dates . 5. Statementprepared and signed by the qualify assuranceunit 6. Tablescontainingall applicabledata 7. All chromatographicand instrumental conditions 8. A complete listing of Centre study personnel - Centre will send a copy of the draft report to the Study Erector. who will rehm , the 'cpoa with comments. Centxe Analytical will makc revisions and finalize the . *port with the approval of the StudyDirector. The Centre Q A unit will conduct an audit of the final draft report and data files to assure accuracy and GLPcompliance. The final report must be approved by the Study Director prior to signature. A statement of QAU inspections and a statement of GLP compliance will be included in the final report. Any corrections or additions to the final report shall be in the form of an amendment by the Principal Investigator. The amendment shall clea$y identify the p k of the final report that is being comcted and the'justification for correction. The amendment shall be signed and dated by the principal Investigatorand the StudyDimtor. .. Centre Analytical Laboratories, Inc. I Page 12 of 43 Page 65 of 102 *I 'I . -.'.-. . ' . .__ . .- . Centre Study No.: 023-041 . CentreRotocol No.OoP-023-041 I REPORT DISTRIBUTION: Original: Study -tor Copy: Centre Archives 14. AR-STATEMJ" Study records to be maintainee Rcc.ords to be maintained for the study include all raw data, observations worded. during the conduct of the study, documentation, chromatograms, specimen tracking information, and study related correspondence. This includes a description of equipment used during the conduct of the study. All characterizationdata and any shippingrecords shaH be retained. Document archives: Upon completion of the study, the study records, pmtmoi and amendments, and the final report and amendments shall be retained in the 3M document archives. If it is necessary to substitute a COPY for an original record, it will be certified as an exact copy. Centre will retain facility-relatedoriginals and a copy ' of the find report and raw data package. 1 U.S.Environmental Prot&tion Agency. Toxic Substances Control Act (TSCA). - Good LaboratoryPractice-Standards.Final Rule, 40 CFRPart 792. , I I Centre AnalyticalLaboratories,hc. Centre Analytical Laboratories, Inc. __ . __ _. - Page 13 of 43 Page 66 of 102 . I . . . . .. . , ... . . . . . . . . . .... . - .. . . - . .. ... . . . Centre Study No.: 023-041 -..---.- .- --_.-_ _-_' _..._. _ --L Centre Protocol No. OOP-oU-041 16. PROTOCOL APPROVAL This protocol was audited by the Quality Assurance Unit of Centre Analytical Laboratories, Inc. Quality Assurance Reviewer, Centre Principai Investigator, Centre -Facility Management, Centre ohn Flaherty / Fgboratory Manager Study Director, 3M .. .. . ._. ...... Centre Analytical Laboratories, Inc. . . .._ . Page 14 of 43 Page 67 of 102 . .~ .. .... ... ... -.. . . . . . ... .. . . . . . . . . . . . . . . . . . . . Centre Study No.: 023-041 ................................ --------.I APPENDIX: ANALYTICAL METHODS A. "Extractionof Potassium Pduorooctanesulfonateor OthqFluorochemical Compounds From Senun for Analysis Using HPLC-Electrospray/Mass Spectrometrf` B. ``Extractionof Potassium PerfluorooctanesuIfonattor Other Fluomchemical Compoun+ From Liver for Analysis Using l"LC-Elcctmspray/hh,s `Spectrometry" ..i.e'.. .' ` I *.. . Cena ~nalyticr~l d m i a~n,c. .... .... . . . ... ..... ~ . .. Centre Analytical Laboratories, Inc. ....... .. Page 15 of 43 .. .. ... 1 . . . P. a.ge. 68 of 102i _.. . Centre-StudyNo.: 023-041 I i _--- ~ -_ .. Ccntrc Aiialyticd Laboratories, hc. . . .. _.__I--.-... .- . ...-P-ag-e_1.6._of43 .. .I Centre Analytical Laboratories, Inc. .--I.. . .- - -- _-- Page 69 of 102' ~- .. . .-.. . .. . . ,~ .. . , Centre Study No.: 023-041 I a n a ~ n a l y t i db. -. ~ qhc. .,.. . .- : . _..- .- ....-.... Centre Analytical Laboratories, Inc. - . . ,: _. . I ' . Page 17 of 43. . .. . Page 70 of 102 - .. . . . . ..., .. .. . ,. . .. . . .. . ..... . . . . . . . .. _ _. . . . . . . Centre Study No.: 023-041 ,.e' 1 I. I.... ' \. . I .. ctpenAnalytid,Labomtoriab.or..atories,Inc. , , . ,_. . .: . , -. . Centre Analytical Laboratories, Inc. . _. Page i8 Of 43 Page 71 of 102 ' , ......... .... -. 1 .; . . * A*- (7- - .......... .... *---. ......... .- ....... .~ . . . . . . . . . . . . . ~ ,Centre Study No.:023-041 -.--- . - i - c-. .I k... Centre Analytical Labomttxics, Inc. .. . . . . . . . . ... .. . . . Pagel9of43 Centre Analytical I,aboratories, Inc. .... ....... - _ - _ _ _ _ _ _ - _ . . C I Page 72 of 102 Centre Study No.: 023-041 .. .. .. ... i .?.....-. .. . . . . . ........ . . . . . . . . .... .r.-.:.r.--. . I .. I . -Analytical -.--.l&m.t.c&s!fnr .. Centre Analytical Laboratories, Inc. ......... .. . . . . . . . . Page 20. of 4.3 . .. - .. ....... _. Page 73 of 102 Centre.StudyNo.: 023-041 ._ .. Centre Analytical Laboratories, Inc. . .. .. . .. .. . Page 74 of 102 *, ``:mmS ..... . . .. . . . . . . .~ .. , .-2. 1 ...... -:_.I. . .. .. -..~... Centre StudyNo.:023-041 .. Centre~nalyticaLl ab~ratoriuh. . ... Centre Analytical Laboratories, Inc. I ,' Page 22 of 43 Page 75 of ioi i...-: , . . . ...... .... . _. . . . . . . . . . . . . Centre Study No.:023-041 I Centre Analytical Laboratories, Inc. ._.. . . . . . __ ... ._- . . . . . -. . ........ -...._-.-- .. Page 23 of 43 . . . . . .- . Page 76 of 02 - ......... .. .. Centre Study No.: 023-041 'C_... _. Centce ApalyticalLaboratories,Inc. Centre Analytical Laboratories, Inc. . , *_ Page 24 of 43 ' Page 77 of 102 ' . - .. I '. . ... . . . . . .- . . . . . . . . . . . . . . ...... Centre Study No.: 023-041 .. ...... I Centre Anal++ Laboratories, Inc. Centre Analytical Laboratories, Inc. .- ... ............... ' Page 25 of 43 Page 78 of 102 ... . . - . .. . . . . . . . . . . . . . . . . . .................. . Centre Study No.: 023-041 J-( , .: * : . . . . . . . . . . ...... . .. . .. . . ,. .* . . . . . . . . . .- centre Analytical Laboratories, Inc. .. ' t .. CentreAnalytical Laboratories, ~ n c . ............... ......... .._' Page 26 of 43 .. . f' I .* Centre Study No.: 023-041 -.... ( . I" w.; . .. . . . . .. . * . . .. . .. . .-. ... .. - . . - - ... .-. . .. . ...i. . . . .. .. .. . . . . . .*:-:: ., .. .. ....... .... ." . 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I' __----.-_. - .... -... ......-- - .-.-. .-.-. i---.-..- - . .. . e . Centre :StudyNo.: 023-041 . . . . . . . - ........ . ...,. - --..e-- , . - h a #l H, s r e t e e e l . N O . - O - - . - - ~ - .. .. .. Centre Analytical LaboratMies, Inc. Centre Analytical Laboratories, Inc. . . . . . . -._. . . . . . . . . . . . _ . . -- .... .: Page 31 of 43 Page 84 of 102 .. *. . - .- I.' . . .. .. .. Centre Study NO.: 023-041 i ... .L .I centre Analyticrl Laboratories, Inc. .. Centre Analytical Laboratories, Inc. ... _.... - . ...... *I ' .. Page 32 of 43 Page 85 of 102. Centre Study No.: 023-041 /'. t.: -' :. . . ..,.:........ ., . .. ...j..;.. . c-....... ,...,...?. - .: ytidLaboratories.In~. . . Centre Analytical Laboratories, Inc. - _ ._ : ....... . Page 33 of43 Page 86 of 102 . . . . . . . -. ........... , .-....__..-_, .. ..... -.--.--.- 'Centre Study No.: 023-041 -_-.__ -----.-----_I_ ........ .... . . . . - .... . . Centre Analytical.Laboratories,Inc. - ..- . . . . . .. ,.. .. .- . - _ _ ... ....... .. - .. .... ! >. : I Page 34 of 43 ' ........ Page 87 of 102 . . .~ .. ... ... . . . . . -. . . . . . . . . . . . . . . . . . . . . ~ ........ .. - .. . . ......... . ...._- . . . . I.. . . ,! - - . . . .. - ......... . . ..... .........-.- _--_Centre StudyNo.:023-041 ! ..--.-&Ratocol No.AlOP-O2&04L--.- ' - ,I --.- .. Centre Analytical Laboratories, Inc. ... .... .- .... Page 88 of 102 - . i-` I ... Centre Study No.: 023-041 , . . . . . - . . . . . .. .* I . .. .. . , -. . . Y ..&. . . . .. ....... .. ....... Centre Analytical Laboratories, Inc. ...... I - ., Page 36 of 43 Page 89 of 102 ..... .... ' . - --.-- I .- . . *. . . . . . . . . . . . . Centre Stydy No.: 023-041 ..... - . ._--. ! _.-, .. i,,' .( - centreAnalytical La&atories. Inc. . ' I . t. . . .-... ............. . - .. * .- ........... Clentre Analytical Laboratories, ~ n c . -. . .-. ., . . . . . . . . . . . . . . . . Page 37of 43 . .-- ... ... ! Page 90 of loa . . -.. . Centre,StudyNo.: 023-041 ................ ....... ....... :entre Analytical Laboratories, Inc. . . ........_...._......-. ... ............ Page 38 of 43 ... a Page 91 of 102 ..... . 4 .~ __._ i .... ..... __----- .. .... Centre Study No.:023-041 .-.- .---^-- -c.-.-r .. . . .. . . I 1 . ? Centre Analytical Laboratcniu. Inc. . Centre Analytical Laboratories, Inc. .- . . . . . ....___.._._._. . . ..... - . . . . . . . . . . . . . . . . .. ... ... .-. . . . I. Page 39 of 43 Page 92 of 102 . a .. i . .............. ...... .. - Centre Study No.: 023-041 -- . __. ......... - - . . _ _ _ . . . _ . . . . ...... .. . "wmocolNo.a p w - - . .... L- . . I . .- .. .. I ' ctnaAnalytical i#&qato&s, bc. .. -. Page.4..0of43 . ... . . .............. .................. Centre Analytical Laboratories, Inc. -. . _ . .. Page 93 of 102 ._.- Centre Study No.: 023-041 ; . I 1 .. . Centre Analytical Labomoriq, Inc. Centre Analytical Laboratories,.Inc. Page 41 of 43 ' -. .. -I Page 94 of 102 . ! .- , .. ' I CeneAnalyticalLaboratories, hc... . . . .... Centre Analytical Laboratories, Inc. ....... --_........... Page 42 of 43 .. Page 95 of 102 __ , .*- I .............. * ' - - .- RW'.,', v- Centre Study No:: 023-041 .......... -----. _. .. . Ceria Rotocol No.qOP-023-041' . ..... .. ... .. I ------ *...C.....-S....-.h...l I & d. W. . . . . . . ~ o f P FRaSs4h-6o.0l D L i * p . , h~cI40f14 . !. . . . ,.' . -. .---....-..-.-..-.-.-_)'-_. ..-...... -.. . . . . . ..._"... . .. . . . . . Centre Analytical Laboratories, Inc. ..... ... Page 96.of102 SEP. 6. 2001 11:39AM ENVIBONENTAL LAB 2 31 09 .. Centre Study No.: 023-041 NO. 5647 P. 2 PROTOCOL AMENDMENT Page 1 of Amendment Number: 1 EfYcctivcDate: 12/18100 . IICentt= Study Number 923-041 CentreProtocol N u m k pOP-023-041 AMlmEQm 1. Change the but paragraph to read as follows: Moimun detumination will be performedon all liver samples according to AOAC - - officialMethod 950,44SectionB. 1. The #ample weight will be 0.5 2 The drying &e will be 12-14 (a). g. ho with un. the following modifications: RATIONALE 1. At the requestof the rponsor,maishue deierminatiomwill be done for every liver sample. Dw to Iimited sample she, the sample weight used will be -0.5 g insread of ., -2 g, The drying time nquired for such a s d sample size docs not have to be 16-18 how, EO the sample0 will be dried for 12-14 hours ACT ON l"ES W Y 1. No negative impact on the study. &ds;nt&a;(Study Direct&) Signature Dak' 1 ' E C E I M D T I M SEP. 6. . . ._ 1:Pffl Centre Analytical Laboratories, Inc. Page 97 of 102 SIP. 6. 2001 11:39M ENVIBONUNTAL LAB 2 31 09 .. Centre Study No.: 023-041 NO. 5647 P. 5 I i ~ ~ I V E D T I MSEP. 6. 1:- .. , . .. .. I.. .........- .. .,_.. ., .., ._ , . . . . . . . , ,, . , .. . .,. .. , . Centre Analytical Laboratories, Inc. .- Page 98 of 102 Centre Study No.: 023-041 CEntrts 'Analytical 3048RuarrL Drive, State Cdlege, PA 16801. Pbone: (814)231-8032. Faaimtie: (814)231-1253 Centre StudyNumber:Q23-041 PROTOCOL AMENDMENT AmendmentNumber: 2 Date of Occurrence: QW24/0J, Centre Protocol Number: QOP-073-041 DESCRIPTION OF AMENDEDSECTION 1. 53: SPONSOR @ENDEDTO 1. Change the study director fiom Rochelle Robideau at 3M Environmental Technology and Safety Servicesto Scan Gallagherat Wildlife International, LTD, 8598 Commerce Drive, Easton, MD 21601, phone: 410-822-8600, fax: 410-822-0632. - 2. ChangeRochelleRobideautothe SponsorRepresentative. RATIONALE 1-2. By request of Rochelle Robideau (fax dated OSnlllol included in correspondence section of the raw data associated with this study) 1-2. No negative impact on the study. L e*' * ( r IncomingStudy DDiirectoor Signature &??/ Date Date Centre Analytical Laboratories, Inc. Page 99 of 102 Centre Study No.: 023-041 SIP. 6. 2001 ll:4OW 9 ENVIBOMNTAL LAB 2 3E 09 NO. 5647 P. 8 - \ ;I Centre AnaIyticai ! Laboratories, Inc, I I 4 I I PROTOCOL DEVIATION i DeVidmNumba: 1 f I I DIjscRlpnONOFDEVIATION . 4 Amendment #1: Did not pafonnmoisturedetumination on the followingsamplesh u e ! me waa no umpla available &a ex[rocfioll: i I -sJialm -iahlcn2 oo07875 459 o(107920 953 1 0007879 483 ooo7921 9533 0007881 485 m7922 9535 - ! ow7883 487 0041924 9541 OOO7885 489 mgza 9549 * 9503 0041937 9s6l ooo7911 9515 0007941 9580 1 0007915 9525 m 2+ 9581 0007917 9sta ooo7943 9582 ! oOol918 9529 m944 9583 i AcnoNs TAKEN ! ! i i i *. .._.,___--_.. . . . . . . . . . . . . . . . . . ..-. Centre Analytical Laboratories, Inc. . . . . ............... ... Page 100 of 102 SEP. 6. 2001 11:40AM ENVIBONMENTAL LAB 2 3E 09 _ - ... Centre Study No.: 023-041 NO. 5647 P. 7 PROTOCOL DEVIATION DESQUPTION OF DEVIATION - 1. 0 Amenrimat #I: Weighsdh r than 0.5 gfor the miamredetumination011 the following ramplea because it WLI rtl of rhc rampiervrilabkafter d o n : sumawm 6007913 00071167 maao om927 0041936 OW7938 aaLLQ!l!&i&u 9sm 0.34 451 0.30 484 0.33 9548 0.32 - 9564 0.26 -95m 0.27 i C Centre Analytical Laboratories, Inc. .. _.__....- ...--...,. . Page 101 of 102 - r SEP. 6. 2001 11:40AM ENVIBONMENTAL LAB 2 3E 09 Centre Study No.: 023-041 NO. 5647 P. 6 - \ Centre Analytical Laboratoriss, Inc. PROTOCOL DEVIATION DavirrionNUmbsr: 3 , ECEIMD TIM SEP. 6 . 1:m Centre Analytical Laboratories, Inc. ... ... ._. . Page 102 of 102