Document 2RvQXRDLNdYLQGN85LMxRgB9N

STUDY TITLE Extraction of Potassium Perfluorooctanesulfonate from Red Blood Cells and Serum for Analysis Using HPLC-Electrospray/Mass Spectrometry DATA REQUIREMENTS Analytical Method Requirements STUDY DIRECTOR Sean Gallagher ANALYTICAL REPORT DATE June 2, 2003 PERFORMING LABORATORY / TESTING FACILITY Exygen Research 3058 Research Drive State College, PA 16801 Phone: 814-272-1039 STUDY SPONSOR 3M Environmental Technology and Safety Services Building 2-3E-09 POBox 33331 St. Paul, MN 55133-3331 PROTECT Exygen Protocol Number: 01P-023-066 Exygen Study Number: 023-066 Total Pages: 98 0 00832 Exygen Study No.: 023-066 GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT Exygen Study Number 023-066, entitled "Extraction of Potassium Perfluorooctanesulfonate from Red Blood Cells and Serum for Analysis Using HPLCElectrospray/Mass Spectrometry," conducted for 3M Environmental Technology and Safety Services, was performed in compliance with US EPA TSCA Good Laboratory Practice Standards (40 CFR Part 792) by Exygen Research. l Sean Gallagher Study Director Wildlife International, Ltd. A M Date Date Date Exygen Research Page 2 of 98 C 'iQ s a a Exygen Study No.: 023-066 QUALITY ASSURANCE STATEMENT Exygen's Quality Assurance Unit reviewed Exygen Study Number 023-066, entitled, "Extraction of Potassium Perfluorooctanesulfonate from Red Blood Cells for Analysis Using HPLC-Electrospray/Mass Spectrometry". All reviewed phases were inspected for conduct according to Exygen's 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 Date Inspected Date Reported to Date Reported to Exygen Study Director and Management Sponsor Management 1. Protocol Review 5/22/01 6/4/01 6/15/01 2. Extraction, Fortification 5/22/01 6/4/01 6/15/01 3. Raw Data Review 6/4,6,8/01 7/13/01 9/7/01 4. Raw Data Review 8/7-9/01 8/21/01 9/7/01 5. Draft Report Review 11/15,16,19/01 12/05/01 02/21/02 6. Final Report Review 04/11,21/03 06/02/03 06/02/03 Lydia. affer Quali Assurance Auditor 0 / (.jl 3 Date Exygen Research Page 3 of 98 C0-0S34 Exygen Study No.: 023-066 CERTIFICATION OF AUTHENTICITY This report, for Exygen Study Number 023-066, is a true and complete representation of the raw data for the study. Submitted by: Exygen Research 3058 Research Drive State College, PA 16801 (814) 272-1039 Principal Investigator, Exygen: Date Exygen Facility Management: Date Study Director, Wildlife International: ___ ____________________________________ Sean Gallagher ' Wildlife International, Ltd. Sponsor Representative, Entrix: \\^ Date Exygen Research Page 4 of 98 G0QS35 Exygen Study No.: 023-066 STUDY IDENTIFICATION Extraction of Potassium Perfluorooctanesulfonate from Red Blood Cells and Serum for Analysis Using HPLC-Electrospray/Mass Spectrometry EXYGEN PROTOCOL NUMBER: 01P-023-066 EXYGEN STUDY NUMBER: 023-066 TYPE OF STUDY: Analytical SAMPLE MATRIX: Mallard and Quail Red Blood Cells and Serum TEST SUBSTANCE: Perfluorooctanesulfonate (PFOS) SPONSOR: 3M Environmental Technology and Safety Services Building 2-3E-09 PO Box 33331 St. Paul, MN 55133-3331 STUDY DIRECTOR: Sean Gallagher Wildlife International, Ltd. 8598 Commerce Drive Easton, MD 21601 SPONSOR REPRESENTATIVE: John Newsted Entrix, Inc. 4295 Okemos Road, Suite 101 Okemos, MI 48864 TESTING FACILITY/PRINCIPAL: Emily R. Decker INVESTIGATOR Exygen Research 3058 Research Drive State College, PA 16801 ANALYTICAL PHASE TIMETABLE: Study Initiation Date: Experimental Start Date: Experimental Termination Date: Analytical Report Date: 04/23/01 05/18/01 07/28/01 06/02/03 Exygen Research Page 5 of 98 G00836 Exygen Study No.: 023-066 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 Decker Dave Bell Karen Risha Ling Ling Liu Marcia Swartzwelder Xiaoming Zhu Mitra Arjmand Lawrence Ord Ed Carnes Shawn Robb Title Scientist Scientist Scientist Technician Technician Technician Technician Sample Custodian Sample Custodian Sample Custodian Exygen Research Page 6 o f 98 O'?0 8 3 7 Exygen Study No.: 023-066 TABLE OF CONTENTS Page TITLE PAGE........................................................................................................................... 1 GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT..............................2 QUALITY ASSURANCE STATEMENT.............................................................................3 CERTIFICATION OF AUTHENTICITY............................................................................. 4 STUDY IDENTIFICATION.................................................................................................. 5 PROJECT PERSONNEL....................................................................................................... 6 TABLE OF CONTENTS....................................................................................................... 7 LIST OF TABLES...................................................................................................................S LIST OF FIGURES............................................................................................................... 10 LIST OF APPENDICES.......................................................................................................11 1.0 SUMMARY................................................................................................................... 12 2.0 OBJECTIVE................................................................................................................... 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 Standards and Fortification Solutions................................................ 14 6.3 Chromatography...........................................................................................................15 6.4 Instrument Sensitivity..................................................................................................15 6.5 Description of Instrument and Operating Conditions................................................ 15 6.6Quantitation and Example Calculation.......................................................................16 7.0 EXPERIMENTAL DESIGN......................................................................................... 18 8.0 RESULTS....................................................................................................................... 18 9.0 CONCLUSIONS............................................................................................................19 10.0 RETENTION OF DATA AND SAMPLES............................................................... 19 Exygen Research Page 7 of 98 c o o sn s Exygen Study No.: 023-066 Table I. LIST OF TABLES Page Summary of PFOS in Reagent Blanks............................................................ 21 Table II. Summary of PFOS in Mallard Red Blood Cell Blanks.................................. 22 Table D3. Summary of PFOS in Mallard Serum Blanks................................................. 22 Table IV. Summary of PFOS in Quail Red Blood Cell Blanks......................................23 Table V. Summary of PFOS in Quail Serum Blanks..................................................... 23 Table VI. Summary of PFOS Recoveries in Mallard Red Blood C ells......................... 24 Table VH Summary of PFOS Recoveries in Mallard Serum ..........................................25 Table VIII. Summary of PFOS Recoveries in Quail Red Blood Cells.............................. 26 Table IX. Summary of PFOS Recoveries in Quail Serum.............................................. 27 Table X. Summary of PFOS in Mallard Red Blood Cell Samples at 0 ppm a.i. for Week 5 ...............................................................................................................28 Table XI. Summary of PFOS in Mallard Red Blood Cell Samples at 10 ppm a.i. for Week 5 ...............................................................................................................29 Table XII. Summary of PFOS in Mallard Red Blood Cell Samples at 50 ppm a.i. for Week 5 ...............................................................................................................30 Table XIH. Summary of PFOS in Mallard Red Blood Cell Samples at 0 ppm a.i. for Week 1 0 .............................................................................................................30 Table XIV. Summary of PFOS in Mallard Red Blood Cell Samples at 10 ppm a.i. for Week 1 0 .............................................................................................................31 Table XV. Summary of PFOS in Mallard Red Blood Cell Samples at 0 ppm a.i. for Week 1 5 .............................................................................................................32 Table XVI. Summary of PFOS in Mallard Red Blood Cell Samples at 10 ppm a.i. for Week 1 5 .............................................................................................................33 Table XVII. Summary of PFOS in Mallard Red Blood Cell Samples at 50 ppm a.i. for T E R M ................................................................................................................. 33 Table XVIDL Summary of PFOS in Mallard Serum Samples at 0 ppm a.i. for Week 5 ... 34 Table XIX. Summary of PFOS in Mallard Serum Samples at 10 ppm a.i. for Week 5.... 35 Table XX. Summary of PFOS in Mallard Serum Samples at 50 ppm a.i. for Week 5.... 35 Table XXI. Summary of PFOS in Mallard Serum Samples at 0 ppm a.i. for Week 10.... 36 Exygen Research Page 8 of 98 COOS39 Exygen Study No.: 023-066 Table XXII. Summary of PFOS in Mallard Serum Samples at 10 ppm a.i. for Week 10....................................................................................................................... 37 Table XXIH. Summary of PFOS in Mallard Serum Samples at 0 ppm a.i. for Week 15..........................................................................................................38 Table XXIV. Summary of PFOS in Mallard Serum Samples at 10 ppm a.i. for Week 15.........................................................................................................39 Table XXV. Summary of PFOS in Mallard Serum Samples at 50 ppm a.i. for TERM .............................................................................................................39 Table XXVI. Summary of PFOS in Quail Red Blood Cell Samples at 0 ppm a.i. for Week 5 ..................................................................................................... 40 Table XXVII. Summary of PFOS in Quail Red Blood Cell Samples at 10 ppm a.i. for Week 5 ............................................................................................................41 Table XXVIEL Summary of PFOS in Quail Red Blood Cell Samples at 50 ppm a.i. for W eek5.......................................................................................................... 42 Table XXIX. Summary of PFOS in Quail Red Blood Cell Samples at 0 ppm a.i. for Week 10......................................................................................................... 42 Table XXX. Summary of PFOS in Quail Red Blood Cell Samples at 10 ppm a.i. for Week 10......................................................................................................... 43 Table XXXI. Summary of PFOS in Quail Red Blood Cell Samples at 0 ppm a.i. for Week 15......................................................................................................... 44 Table XXXH. Summary of PFOS in Quail Red Blood Cell Samples at 10 ppm a.i. for Week 15.................................................................................................. 45 Table XXXHI. Summary of PFOS in Quail Red Blood Cell Samples at 50 ppm a.i. for TERM..................................................................................................... 45 Table XXXIV. Summary of PFOS in Quail Serum Samples at 0 ppm a.i. for Week 5.... 46 Table XXXV. Summary of PFOS in Quail Serum Samples at 10 ppm a.i. for Week 5 ...47 Table XXXVI. Summary of PFOS in Quail Serum Samples at 50 ppm a.i. for Week 5 ........................................................................................................ 47 Table XXXVEL Summary of PFOS in Quail Serum Samples at 0 ppm a.i. for Week 10...................................................................................................... 48 Table XXXVHI. Summary of PFOS in Quail Serum Samples at 10 ppm a.i. for Week 10..................................................................................................... 49 Table XXXIX. Summary of PFOS in Quail Serum Samples at 0 ppm a.i. for Week 15.. 50 Table XL. Summary of PFOS in Quail Serum Samples at 10 ppm a.i. for Week 1 5 ..... 51 Table X II. Summary of PFOS in Quail Serum Samples at 50 ppm a.i. for TERM ........51 Exygen Research Page 9 of 98 G4034G Exygen Study No.: 023-066 LIST OF FIGURES Page Figure 1. Typical Calibration Curve for PFOS.................................................................53 Figure 2. Chromatogram Representing a 0.1 ng/mL standard for PFO S....................... 54 Figure 3. Chromatogram Representing a 1.0 ng/mL standard for PFO S........................55 Figure 4. Chromatogram Representing a Reagent Blank for PFOS (Exygen ID Reagent Blank A, Set: 051801A )................................................56 Figure 5. Chromatogram Representing Control Quail Red Blood Cells for PFOS (Exygen ID: 0107376 Blank A, Set: 051801A)............................................. 57 Figure 6. Chromatogram Representing Control Quail Serum for PFOS, (Exygen ID: 0107375 Blank A, Set: 072301C)............................................. 58 Figure 7. Chromatogram Representing Control Quail Red Blood Cells fortified with 10 ng/mL of PFOS (Exygen ID: 0107376 Spk A, Set: 051801DR)..... 59 Figure 8. Chromatogram Representing Control Quail Serum fortified with 10 ng/mL of PFOS (Centre ID: 0107375 Spk A, Set: 072301D)......... 60 Figure 9. Chromatogram Representing Quail Red Blood Cell Sample (Exygen ID: 0107084, Sponsor ID: 454-108-401 Wk5, Set: 051801DR)..........................61 Figure 10. Chromatogram Representing Quail Serum Sample (Exygen ID: 0107207, Sponsor ID: 454-108-407-Wkl5, Set: 072301DD)....................................... 62 Exygen Research Page 10 of 98 Exygen Study No.: 023-066 LIST OF APPENDICES Page Appendix A Study Protocol 01P-023-066 (Exygen Study No. 023-066) and Amendments and Deviations.......................................................................63 Exygen Research Page 11 of 98 Exygen Study No.: 023-066 1.0 SUMMARY Exygen Research (Exygen) extracted mallard and quail red blood cell samples and serum samples for the determination of perfluorooctanesulfonate (PFOS) according to protocol 01P-023-066 (Appendix A). The limit of quantitation for mallard red blood cells and serum was 10 ng/mL. The limit of quantitation for quail red blood cells and serum was 10 ng/mL. The LOQ for each matrix was determined in a method verification study performed at Centre (Centre Study: 023-065). PFOS in the mallard red blood cell samples ranged from non-quanitifiable levels to 418 pg/mL. PFOS in the mallard serum samples ranged from non-detected levels to 685 pg/mL. PFOS in the quail red blood cell samples ranged from non-quantifiable levels to 361 pg/mL. PFOS in the quail serum samples ranged from non-quantifiable levels to 514 pg/mL. The average percent recoveries standard deviation for PFOS in mallard red blood cell samples and serum samples were 90% 9% and 99% 13%, respectively. The average percent recovery standard deviation for PFOS in quail red blood cell samples and serum samples were 100% 10% and 102% 20%, respectively. 2.0 OBJECTIVE The objective of this study was to determine levels of perfluorooctanesulfonate (PFOS) in specimens of mallard and quail red blood cell samples and serum samples using the analytical method described in protocol 01P-023-066. 3.0 INTRODUCTION This report details the results of the analysis for the determination of PFOS in mallard and quail red blood cell samples and serum samples, using the analytical method entitled, "Extraction of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds From Serum for Analysis Using HPLC-Electrospray/Mass Spectrometry". The study was initiated on April 23, 2001, when the study director signed Exygen protocol number 01P-023-066. The experimental start date was May 18, 2001, and the experimental termination date was July 28, 2001. Exygen Research Page 12 of 98 Exygen Study No.: 023-066 4.0 TEST SYSTEM The control mallard and quail red blood cells and serum used for the matrix blanks and matrix fortifications were received chilled on blue ice on May 17, 2001 from Wildlife International Ltd., Easton, MD. They were assigned the following Exygen EDnumbers: Matrix Quail Serum Quail Blood Mallard Serum Mallard Blood Exvgen ID 0107375 0107376 0107377 0107378 Seventy-seven quail red blood cell samples and seventy-six quail serum samples were received chilled on blue ice at Exygen on May 17, 2001 and logged in by Exygen personnel and placed in frozen storage. Seventy-three mallard red blood cell samples and seventy-two mallard serum samples were received chilled on blue ice at Exygen on May 17, 2001 and logged in by Exygen personnel and placed in frozen storage. Sample log-in and chain of custody information can be found in the raw data package associated with this study. Storage records will be kept at Exygen and a true copy of the storage records can be found in the raw data package associated with this study. 5.0 REFERENCE MATERIAL The analytical standard PFOS was received at Exygen 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. Compound PFOS Exygen Control No. TCR No. Purity (%) Expiration Date 00-023-042 TCR 00017-46 97.9 08/31/01 Exygen Research Page 13 o f 98 Exygen Study No.: 023-066 The molecular structure of PFOS is given below. PFOS Chemical Name Molecular weight O Perfluorooctanesulfonate 499 (C8F17S 0 3~) c8f17s -- O- I o Note: The neutral molecule and standard form from which PFOS (anion) is derived, is potassium perfluorooctanesulfonate [C8F 17SO3K], molecular weight 538. 6.0 DESCRIPTION OF ANALYTICAL METHOD Analytical method entitled "Extraction of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds From Serum for Analysis Using HPLC-Electrospray/Mass Spectrometry" was used for this study. 6.1 Extraction Procedure A 100 pL aliquot of the red blood cells and serum was used for the extraction procedure. After fortification of appropriate samples, the samples were vortexed for ~ 15 seconds. An aliquot of 0.5 milliliter of 0.5 M tetrabutylammonium hydrogen sulfate was added to the samples. One milliliter of 0.25 M sodium carbonate/sodium bicarbonate was added to the samples. Five milliliters of MTBE were added to the samples. Each sample was placed on wrist-action shaker for ~ 20 min. and then centrifuged for ~ 15 min. Four milliliters of the organic layer were taken and dried on a nitrogen evaporator and then reconstituted with 1 milliliter of methanol. Each sample was analyzed by LC/MS/MS electrospray. 6.2 Preparation of Standards and Fortification Solutions Standard solutions were prepared on March 14, 2001 as specified in Exygen protocol 01P-023-066. An individual stock standard solution of PFOS was prepared 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. A 0.1 pg/mL fortification standard was prepared by taking 10 mL of the 1.0 pg/mL fortification standard and bringing the volume up to 100 mL with methanol. A 0.01 Exygen Research Page 14 of 98 0^0545 Exygen Study No.: 023-066 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 serial dilution of the 0.1 pg/mL and 0.01 pg/mL solutions in the following manner: Initial Cone. (pg/mL)1 Volume (mL) Diluted to (mL) Final Cone. (pg/mL) 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 1of PFOS The stock standard solution and all fortification and calibration standard solutions were stored in a refrigerator (4 2C) when not in use. Documentation of standard preparation can be found in the raw data associated with this report. 6.3 Chromatography Quantification of PFOS was accomplished by LC/MS/MS electrospray. The retention time of PFOS was - 4.4 min. Peaks were detected in the control matrices corresponding to the analyte retention time, but the amounts detected were only significant enough to alter several fortification recoveries and the rest were less than the lowest calibration standard (0.0001 pg/mL). 6.4 Instrument Sensitivity The smallest standard amount injected during the chromatographic run had a concentration of 0.0001 pg/mL of PFOS. 6.5 Description of Instrument and Operating Conditions Instrument: PE SCBEX API 3000 Biomolecular Mass Analyzer Interface: SCIEX Turboion Spray Liquid Introduction Interface Computer: Dell UltraScan PI 110 Software: HPLC: PE SciexAnalyst version 1.1 Windows NT, version 4 Hewlett Packard (HP) Series 1100 HP Quat Pump HP Vacuum Degasser Exygen Research Page 15 of 98 C 'tO SA G Exygen Study No.: 023-066 HP Autosampler HP Column Oven HPLC Column:Genesis Cg (Jones Chromatography), 2.1 mm x 50 mm, 4p Column Temp.: 35 C Injection Voi.: 10 pL Mobile Phase (A): 2 mM Ammonium Acetate in ASTM type I water Mobile Phase (B): Methanol Flow Rate: 0.3 mL/min. Time % A % B 0 60 40 1.0 0 100 7.0 0 100 7.5 60 40 11.0 60 40 Ions monitored: Analvte PFOS Mode Transition Monitored negative 499 99 Approximate Retention Time (mini 4.20 Tune File Parameters Controls IS-Iospray 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 -51.0 -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 were injected into the LC/MS/MS. The peak area was measured and the standard curve was generated (using 1/x fit weighted Exygen Research Page 16 of 98 c rT> f-s\ i-2JA.-7* Exygen Study No.: 023-066 linear regression) by Analyst software using six concentrations of standards. The concentration was determined from the equations below. Equation 1 calculated 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 Equation 2 calculated the amount of analyte found in pg/mL (the equations for serum are shown). Equation 1: Analyte found (ng/mL) = (Peak area - intercept! slope Equation 2: Analyte found (pg/mL) = ((anal, found (ng/mL) x FV (mL) x DF x EV fmL)) x 1 pg (AV (mL) x sample vol. (mL)) 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, Equation 3 calculated the percent recovery. Equation 3: Recovery (%) = ((anal, found (pg/mL) - avg. anal, in Ctrl (pg/mL)) x (1000 ng/1 pg)) x 100 amount added (ng/mL) An example of a calculation using an actual sample follows: Mallard serum sample Centre ID 0003803 Spk A (Set: 112100B), fortified at 10 ng/mL with PFOS, where: peak area = 6558 intercept = 515 slope = 7740 dilution factor =1 ng added (fort level) 1ng avg. amt in controls 0 (Not quantifiable) final volume 1 mL extraction volume 5 mL aliquot volume 4 mL sample weight (volume) 0.1 mL Exygen Research Page 17 of 98 C005*.8 Exygen Study No.: 023-066 From equation 1: Analyte found (ng/mL) = f6558- 5151 7740 From equation 2: Analyte found (pg/mL) 0.8 ng/mL = (0.8 ng/mL x 1 mL x 1 x 5 mLl x 1 pg (4 mL x 0.1 mL) 1000 ng = 0.00976 pg/mL From equation 3: % Recovery = ('(0.00976 ug/mL x - 0 ug/mLl x 1000) x 100 10 ng/mL = 98% 7.0 EXPERIMENTAL DESIGN Each set of samples (red blood cells 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 amount of PFOS found in the reagent blanks are listed in Table I. The amount of PFOS in the mallard red blood cells and serum blanks are given in Tables II & III. The amount of PFOS in the quail red blood cells and serum are given in Tables IV & V. Peaks were detected in the control matrices corresponding to the analyte retention time. The amounts detected were only significant enough to alter several fortification recoveries and as a result are reported. The rest of the samples were less than the lowest calibration standard (0.0001 pg/mL) and not quantifiable. Individual recoveries for PFOS in the mallard red blood cell and serum samples are detailed in Tables VI & VII. The average percent recoveries standard deviations for PFOS in mallard red blood cell and serum samples were 90% 9% and 99% 13%, respectively. Individual recoveries for PFOS in the quail red blood cell and serum samples are detailed in Tables VIII & IX. The average percent recoveries standard deviations for PFOS in mallard red blood cell and serum samples were 100% 10% and 102% 20%, respectively. PFOS in the mallard red blood cell samples ranged from non-detected levels to 418 pg/mL. Individual results are listed in Tables X-XVII. PFOS in the mallard serum Exygen Research Page 18 of 98 Exygen Study No.: 023-066 samples ranged from non-detected levels to 685 pg/mL. Individual results are listed in Tables XVIII-XXV. PFOS in the quail red blood cell samples ranged from non-detected levels to 361 pg/mL. Individual results are listed in Tables XXVI-XXXIII. PFOS in the quail serum samples ranged from non-detected levels to 514 pg/mL. Individual results are listed in Tables XXXIV-XLI. 9.0 CONCLUSIONS The mallard and quail red blood cell and serum samples were successfully extracted and analyzed according to protocol 01P-023-066. 10.0 RETENTION OF DATA AND SAMPLES When the final report is complete, all original paper data generated by Exygen 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 Exygen archives for the period of time specified in 40 CFR Part 792. Retained samples of reference substances are archived by the sponsor. Exygen Research Page 19 of 98 cro sso Exygen Study No.: 023-066 TABLES Exygen Research Page 20 of 98 n ossi Exygen Study No.: 023-066 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 na na na na na na na na na na Centre ID Reagent Blank A Reagent Blank A* Reagent Blank A2 Reagent Blank A2* Reagent Blank A Reagent Blank A* Reagent Blank A Reagent Blank A* Reagent Blank A Reagent Blank A* Reagent Blank A Reagent Blank A* Reagent Blank A Reagent Blank A* Reagent Blank A3 Reagent Blank A3* Reagent Blank A Reagent Blank A* Reagent Blank A2 Reagent Blank A2* Reagent Blank A Reagent Blank A* Reagent Blank A2 Reagent Blank A2* Reagent Blank A3 Reagent Blank A3* Set Number 051801A 051801A 051801B 051801B 052I01A 052101A 052101B 052101B 052201A 052201A 052201B 052201B 052201C 052201C 071901A 071901A 072001A 072001A 072001B 072001B 072301A 072301A 07230IB 072301B 072301C 072301C Extraction Date 5/18/01 5/18/01 5/18/01 5/18/01 5/21/01 5/21/01 5/21/01 5/21/01 5/22/01 5/22/01 5/22/01 5/22/01 5/22/01 5/22/01 7/19/01 7/19/01 7/20/01 7/20/01 7/20/01 7/20/01 7/23/01 7/23/01 7/23/01 7/23/01 7/23/01 7/23/01 Analysis Analyte Date Found (ue/mL) 5/18-19/01 5/18-19/01 NQ NQ 5/19/01 5/19/01 NQ NQ 5/22/01 5/22/01 5/22/01 5/22/01 5/23/01 5/23/01 5/23/01 NQ NQ NQ NQ NQ NQ NQ 5/23/01 5/23/01 NQ NQ 5/23/01 7/19-20/01 NQ ND 7/19-20/01 7/21/01 7/21/01 7/21-22/01 7/21-22/01 7/25/01 7/25/01 ND ND ND NQ ND NQ NQ 7/24/01 7/24/01 7/24-25/01 7/24-25/01 AVERAGE: NQ NQ NQ NQ NQ STANDARD DEVIATION: NQ * 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)) ND = Not Detected (A peak was not detected at the corresponding analyte retention time) 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/mL for red blood cells and serum Exygen Research Page 21 o f 98 Exygen Study No.: 023-066 Table II. Summary of PFOS in Mallard Red Blood Cell Blanks Sponsor ID control control control control control control control control Centre ID 0107378 Blank A 0107378 Blank A* 0107378 Blank A 0107378 Blank A* 0107378 Blank A2 0107378 Blank A2* 0107378 Blank B 0107378 Blank B* Set Number 052101B 052101B 052201A 052201A 052201B 05220IB 052201C 052201C Extraction Analysis Analyte Date Date Found (ue/mL) 5/21/01 5/21/01 5/22/01 5/22/01 5/22/01 5/22/01 5/22/01 5/22/01 5/22/01 5/23/01 5/23/01 5/23/01 5/23/01 5/23/01 NQ NQ NQ NQ 0.00224 0.00184 NQ 5/22/01 5/23/01 AVERAGE: STANDARD DEVIATION: NQ 0.000548 0.000927 Table III. Summary of PFOS in Mallard Serum Blanks Sponsor ID control control control control control control Centre ID 0107377 Blank A3 0107377 Blank A3* 0107377 Blank A 0107377 Blank A* 0107377 Blank A2 0107377 Blank A2* Set Number 071901A 071901A 072001A 072001A 072001B 072001B Extraction Analysis Analyte Date Date Found (ug/mL) 7/19/01 7/19-20/01 7/19/01 7/19-20/01 7/20/01 7/21/01 7/20/01 7/21/01 7/20/01 7/21-22/01 7/20/01 7/21-22/01 AVERAGE: STANDARD DEVIATION: 0.00325 0.00331 0.00277 0.00312 0.00188 0.00214 0.00275 0.000605 * 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)) LOQ = 0.01 pg/mL for red blood cells and serum 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. Exygen Research Page 22 of 98 cro ssa Exygen Study No.: 023-066 Table IV. Summary of PFOS in Quail Red Blood Cell Blanks Sponsor ID control control control control control control control control Centre ID 0107376 Blank A 0107376 Blank A* 0107376 Blank A2 0107376 Blank A2* 0107376 Blank A 0107376 Blank A* 0107376 Blank A 0107376 Blank A* Set Number 051801A 051801A 051801B 051801B 052101A 052101A 052201C 052201C Extraction Analysis Analyte Date Date Found (pg/mL) 5/18/01 5/18-19/01 5/18/01 5/18-19/01 5/18/01 5/19/01 5/18/01 5/19/01 5/21/01 5/22/01 5/21/01 5/22/01 5/22/01 5/23/01 5/22/01 5/23/01 AVERAGE: STANDARD DEVIATION: NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ Table V. Summary of PFOS in Quail Serum Blanks Sponsor ID control control control control control control Centre ID 0107375 Blank A 0107375 Blank A* 0107375 Blank A2 0107375 Blank A2* 0107375 Blank A3 0107375 Blank A3* Set Extraction Analysis Analyte Number Date Date Found (pg/mL) 072301AD 072301 AD 07230IB 07230IB 072301C 072301C 7/23/01 7/23/01 7/23/01 7/23/01 7/23/01 7/23/01 7/26/01 7/26/01 7/24/01 7/24/01 7/24-25/01 7/24-25/01 0.00309 0.00272 NQ NQ NQ NQ AVERAGE: STANDARD DEVIATION: 0.00100 0.00148 * 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)) LOQ = 0.01 pg/mL for red blood cells and serum 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. Exygen Research Page 23 of 98 COO554 Exygen Study No.: 023-066 Table VI. Summary of PFOS Recoveries in Mallard Red Blood Cells Sponsor ID control control control control control control control control control control control control control control Centre ID 0107378 Spk A 0107378 Spk A* 0107378 Spk B 0107378 SpkB* 0107378 Spk A 0107378 Spk A* 0107378 SpkB 0107378 SpkB* 0107378 Spk A2 0107378 Spk A2* 0107378 Spk B2 0107378 Spk B2* 0107378 SpkB 0107378 SpkB* Set Number 052101B 052101B 052101B 052101B 052201A 052201A 052201A 052201A 05220IB 05220IB 05220IB 052201B 052201C 052201C Extraction Analysis Amt Date Date Added (ng/mL) 5/21/01 5/21/01 5/21/01 5/21/01 5/22/01 5/22/01 5/22/01 5/22/01 5/22/01 5/23/01 10 10 50000 50000 10 5/22/01 5/23/01 10 5/22/01 5/23/01 50000 5/22/01 5/23/01 50000 5/22/01 5/22/01 5/23/01 5/23/01 10 10 5/22/01 5/22/01 5/22/01 5/23/01 5/23/01 5/23/01 50000 50000 50000 5/22/01 5/23/01 50000 AVERAGE: STANDARD DEVIATION: RELATIVE STANDARD DEVIATION: % Recovery 85 88 91 95 84 82 84 82 80 76 101 105 102 101 90 9 11 * Duplicate injection LOQ = 0.01 ng/mL for red blood cells and serum Exygen Research Page 24 of 98 C00555 Exygen Study No.: 023-066 Table VII. Summary of PFOS Recoveries in Mallard Serum Sponsor ID control control control control control control control control control control control control Centre ID 0107377 Spk A3 0107377 Spk A3* 0107377 Spk B3 0107377 Spk B3* 0107377 Spk A 0107377 Spk A* 0107377 Spk B 0107377 Spk B* 0107377 Spk A2 0107377 Spk A2* 0107377 Spk B2 0107377 Spk B2* Set Number 071901A 071901A 071901A 071901A 072001A 072001A 072001A 072001A 072001B 07200IB 072001B 07200IB Extraction Analysis Amt Date Date Added (ng/mL) 7/19/01 7/19/01 7/19/01 7/19/01 7/20/01 7/20/01 7/20/01 7/20/01 7/20/01 7/20/01 7/20/01 7/20/01 7/19-20/01 7/19-20/01 7/19-20/01 7/19-20/01 7/21/01 7/21/01 7/21/01 7/21/01 7/21-22/01 7/21-22/01 7/21-22/01 7/21-22/01 10 10 500000 500000 10 10 500000 500000 10 10 500000 500000 AVERAGE: STANDARD DEVIATION: RELATIVE STANDARD DEVIATION: % Recovery 90 89 111 112 76 82 117 97 107 110 92 99 99 13 13 * Duplicate injection LOQ = 0.01 jig/mL for red blood cells and serum Exygen Research Page 25 of 98 000556 Exygen Study No.: 023-066 Table VIII. Summary of PFOS Recoveries in Quail Red Blood Cells Sponsor ID control control control control control control control control control control control control control control Centre Set ID Number 0107376 Spk A 051801DR 0107376 Spk A* 051801DR 0107376 SpkB 051801DR 0107376 Spk B* 051801DR 0107376 Spk A2 051801DR 0107376 Spk A2* 051801DR 0107376 Spk B2 051801DR 0107376 Spk B2* 051801DR 0107376 Spk A 052101A 0107376 Spk A* 052101A 0107376 Spk B 052101A 0107376 SpkB* 052101A 0107376 Spk A 052201C 0107376 Spk A* 052201C Extraction Analysis Amt Date Date Added (ng/mL) 5/18/01 5/18/01 5/21-22/01 5/21-22/01 10 10 5/18/01 5/21-22/01 50 5/18/01 5/21-22/01 50 5/18/01 5/21-22/01 10 5/18/01 5/21-22/01 10 5/18/01 5/21-22/01 50 5/18/01 5/21-22/01 50 5/21/01 5/22/01 10 5/21/01 5/22/01 10 5/21/01 5/22/01 50000 5/21/01 5/22/01 50000 5/22/01 5/23/01 10 5/22/01 5/23/01 10 AVERAGE STANDARD DEVIATION RELATIVE STANDARD DEVIATION % Recovery 102 99 111 111 113 110 109 104 87 89 91 91 90 94 100 10 10 * Duplicate injection LOQ = 0.01 lig/mL for red blood cells and serum Exygen Research Page 26 of 98 Exygen Study No.: 023-066 Table IX. Summary of PFOS Recoveries in Quail Serum Sponsor ID control control control control control control control control control control control control Centre Set ID Number 0107375 Spk A 072301AR 0107375 Spk A* 072301AR 0107375 Spk B 072301AR 0107375 SpkB* 072301AR 0107375 Spk A2 072301D 0107375 Spk A2* 072301D 0107375 Spk B2 072301B 0107375 Spk B2* 07230IB 0107375 Spk A3 072301D 0107375 Spk A3* 072301D 0107375 Spk B3 072301C 0107375 Spk B3* 072301C Extraction Analysis Amt Date Date Added (ng/mL) 7/23/01 7/25/01 10 7/23/01 7/25/01 10 7/23/01 7/25/01 500000 7/23/01 7/25/01 500000 7/23/01 7/26-27/01 10 7/23/01 7/26-27/01 10 7/23/01 7/24/01 500000 7/23/01 7/24/01 500000 7/23/01 7/26-27/01 10 7/23/01 7/26-27/01 10 7/23/01 7/24-25/01 500000 7/23/01 7/24-25/01 500000 AVERAGE: STANDARD DEVIATION: RELATIVE STANDARD DEVIATION: % Recovery 64 62 104 107 112 113 124 126 106 105 100 101 102 20 20 * Duplicate injection LOQ = 0.01 fig/mL for red blood cells and serum Exygen Research Page 27 of 98 0 } 5 8 Exygen Study No.: 023-066 Table X. Summary of PFOS in Mallard Red Blood Cell Samples at 0 ppm a.i. for Week 5 Sponsor ID 454-109-3249-Wk5 454-109-3249-Wk5 * 454-109-3251-Wk5 454-109-3251-Wk5* 454- 109-3252-Wk5 454-109-3252-Wk5* 454-109-3253-Wk5 454-109-3253-Wk5* 454-109-3254-Wk5 454-109-3254-Wk5* 454-109-3255-Wk5 454-109-3255-Wk5* 454-109-3256-Wk5 454-109-3256-Wk5* Centre ID 0107230 0107230 0107231 0107231 0107232 0107232 0107233 0107233 0107234 0107234 0107235 0107235 0107236 0107236 Set Number 052101B 052101B 052101B 052101B 052101B 052101B 052101B 052101B 052101B 052101B 052101B 052101B 052101B 052101B Extraction Analysis Analyte Date Date Found (ue/mL) 5/21/01 5/22/01 NQ 5/21/01 5/22/01 NQ 5/21/01 5/21/01 5/21/01 5/22/01 5/22/01 5/22/01 0.00197 0.00167 NQ 5/21/01 5/21/01 5/22/01 5/22/01 NQ NQ 5/21/01 5/22/01 NQ 5/21/01 5/21/01 5/21/01 5/21/01 5/22/01 5/22/01 5/22/01 5/22/01 NQ NQ 0.00143 NQ 5/21/01 5/22/01 NQ 5/21/01 5/22/01 ______ NQ____ AVERAGE: 0.000401 STANDARD DEVIATION: 0.000706 * 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)) LOQ = 0.01 pg/mL for red blood cells and serum 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. Exygen Research Page 28 of 98 C00359 Exygen Study No.: 023-066 Table XI. Summary of PFOS in Mallard Red Blood Cell Samples at 10 ppm a.i. for Week 5 Sponsor ID 454-109-3289-Wk5 454-109-3289-Wk5* 454-109-3290-Wk5 454-109-3290-Wk5 * 454-109-3291 -Wk5 454-109-3291-Wk5* 454-109-3292-Wk5 454-109-3292-Wk5* 454-109-3293-Wk5 454- 109-3293-Wk5* 454- 109-3294-Wk5 454-109-3294-Wk5* 454-109-3295-Wk5 454-109-3295-Wk5* 454-109-3296-Wk5 454-109-3296-Wk5* Centre ID 0107237 0107237 0107238 0107238 0107239 0107239 0107240 0107240 0107241 0107241 0107242 0107242 0107243 0107243 0107244 0107244 Set Number 052101BD 052101BD 052101BD 052101BD 052101BD 052101BD 052101BD 052101BD 052101BD 052101BD 052101BD 052101BD 052101BD 052101BD 052101BD 052101BD Extraction Analysis Analyte Date Date Found (jig/mL) 5/21/01 5/21/01 5/21/01 5/21/01 5/21/01 5/21/01 5/21/01 5/21/01 5/23/01 5/23/01 5/23/01 5/23/01 5/23/01 5/23/01 5/23/01 5/23/01 24.4 24.7 11.2 10.0 11.8 12.3 15.1 13.1 5/21/01 5/21/01 5/21/01 5/21/01 5/21/01 5/21/01 5/21/01 5/23/01 5/23/01 5/23/01 5/23/01 5/23/01 5/23/01 5/23/01 30.1 30.8 4.88 3.95 18.9 18.8 26.4 5/21/01 5/23/01 28.5 AVERAGE: 17.8 STANDARD DEVIATION: 8.80 * Duplicate injection LOQ = 0.01 (ig/mL for red blood cells and serum Exygen Research Page 29 of 98 Exygen Study No.: 023-066 Table XII. Summary of PFOS in Mallard Red Blood Cell Samples at 50 ppm a.i. for Week 5 Sponsor ID 454-109-3329-Wk5 454-109-3329-Wk5* 454- 109-3330-Wk5 454-109-3330-Wk5* Centre ID 0107245 0107245 0107246 0107246 Set Number 052201CD 052201CD 052201CD 052201CD Extraction Analysis Date Date 5/22/01 5/24/01 5/22/01 5/24/01 5/22/01 5/24/01 5/22/01 5/24/01 AVERAGE: STANDARD DEVIATION: Analyte Found (pg/mL) 58.9 62.2 112 123 89 33 Table XIII. Summary of PFOS in Mallard Red Blood Cell Samples at 0 ppm a.i. for Week 10 Sponsor ID 454-109-3249-Wkl 0 454-109-3249-Wk10* 454-109-3250-Wk10 454-109-3250-WklO* 454-109-3251-Wk 10 454-109-3251-Wkl0* 454-109-3252-WklO 454-109-3252-Wk 10* 454- 109-3253-Wk10 454-109-3253-Wkl0* 454-109-3254-Wk 10 454-109-3254-WklO* 454-109-3255-Wkl0 454-109-3255-Wkl0* 454-109-3256-Wk 10 454-109-3256-Wkl0* Centre ID 0107250 0107250 0107251 0107251 0107252 0107252 0107253 0107253 0107254 0107254 0107255 0107255 0107256 0107256 0107257 0107257 * Duplicate injection Set Number 052201A 052201A 052201A 052201A 052201A 052201A 052201A 052201A 052201A 052201A 052201A 052201A 052201A 052201A 052201A 052201A Extraction Analysis Analyte Date Date Found (pg/mL) 5/22/01 5/22/01 5/22/01 5/22/01 5/22/01 5/22/01 5/22/01 5/22/01 5/22/01 5/23/01 5/23/01 5/23/01 5/23/01 5/23/01 5/23/01 5/23/01 5/23/01 5/23/01 NQ NQ NQ NQ NQ NQ NQ NQ NQ 5/22/01 5/23/01 NQ 5/22/01 5/23/01 NQ 5/22/01 5/22/01 5/22/01 5/22/01 5/22/01 5/23/01 5/23/01 5/23/01 5/23/01 5/23/01 NQ NQ NQ NQ 0.00245 AVERAGE: STANDARD DEVIATION: 0.000200 0.000600 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 (ig/mL)) LOQ = 0.01 pg/mL for red blood cells and serum 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. Exygen Research Page 30 of 98 cCO SGI Exygen Study No.: 023-066 Table XIV. Summary of PFOS in Mallard Red Blood Cell Samples at 10 ppm a.i. for Week 10 Sponsor ID 454- 109-3289-Wkl 0 454-109-3289-Wk 10* 454-109-3290-Wk10 454-109-3290-WklO* 454-109-3291-Wk10 454-109-3291-Wkl0* 454-109-3292-Wk10 454-109-3292-Wk10* 454-109-3293-Wk 10 454- 109-3293-Wkl 0* 454-109-3294-WklO 454-109-3294-WklO* 454-109-3295-Wkl0 454-109-3295-Wkl0* 454-109-3296-Wk 10 454-109-3296-Wk 10* Centre ID 0107258 0107258 0107259 0107259 0107260 0107260 0107261 0107261 0107262 0107262 0107263 0107263 0107264 0107264 0107265 0107265 * Duplicate injection Set Number 052201AD 052201AD 052201AD 052201AD 052201AD 052201AD 052201AD 052201AD 052201AD 052201 AD 052201AD 052201AD 052201 AD 052201AD 052201AD 052201 AD Extraction Analysis Date Date 5/22/01 5/22/01 5/22/01 5/22/01 5/22/01 5/22/01 5/22/01 5/22/01 5/22/01 5/22/01 5/22/01 5/22/01 5/22/01 5/22/01 5/22/01 5/22/01 5/23-24/01 5/23-24/01 5/23-24/01 5/23-24/01 5/23-24/01 5/23-24/01 5/23-24/01 5/23-24/01 5/23-24/01 5/23-24/01 5/23-24/01 5/23-24/01 5/23-24/01 5/23-24/01 5/23-24/01 5/23-24/01 AVERAGE: STANDARD DEVIATION: Analyte Found (ug/mL) 19.3 19.6 20.4 20.2 14.1 14.9 67.3 66.0 33.5 34.2 60.8 58.9 16.1 16.0 11.6 11.7 30.3 20.7 LOQ = 0.01 ng/mL for red blood cells and serum Exygen Research Page 31 of 98 coo C/J Exygen Study No.: 023-066 Table XV. Summary of PFOS in Mallard Red Blood Cell Samples at 0 ppm a.i. for Week 15 Sponsor ID 454-109-3249-Wkl5 454-109-3249-Wkl5* 454-109-3250-Wkl5 454-109-3250-Wkl5* 454-109-3251 -Wk15 454-109-3251-Wkl5* 454-109-3252-Wk 15 454-109-3252-Wkl5* 454-109-3253-Wkl5 454-109-3253-Wkl5* 454-109-3254-Wkl5 454-109-3254-Wk 15* 454-109-3255-Wk 15 454-109-3255-Wkl5* 454-109-3256-Wkl5 454-109-3256-Wkl5* 454- 109-3236-Wkl 5 454-109-3236-Wkl5* Centre ID 0107266 0107266 0107267 0107267 0107268 0107268 0107269 0107269 0107270 0107270 0107271 0107271 0107272 0107272 0107273 0107273 0107274 0107274 * Duplicate injection Set Number 052201B 052201B 05220IB 05220IB 05220IB 05220IB 05220IB 05220IB 05220IB 05220IB 052201B 05220IB 052201B 05220IB 052201B 05220IB 052201B 052201B Extraction Date Analysis Date 5/22/01 5/23/01 5/22/01 5/22/01 5/23/01 5/23/01 5/22/01 5/23/01 5/22/01 5/22/01 5/22/01 5/22/01 5/22/01 5/22/01 5/23/01 5/23/01 5/23/01 5/23/01 5/23/01 5/23/01 5/22/01 5/22/01 5/22/01 5/22/01 5/23/01 5/23/01 5/23/01 5/23/01 5/22/01 5/23/01 5/22/01 5/22/01 5/22/01 5/23/01 5/23/01 5/23/01 AVERAGE: STANDARD DEVIATION: Analyte Found (ug/mL) NQ NQ NQ NQ 0.00179 0.00188 NQ NQ NQ NQ ND ND NQ NQ NQ NQ NQ NQ 0.000243 0.000613 ND = No peak detected 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)) LOQ = 0.01 pg/mL for red blood cells and serum For values recorded as NQ, 0.00005 pg/mL (half the value of the lowest calibration standard, 0.0001 pg/mL) and for ND, zero was used to calculate the average and standard deviation. Exygen Research Page 32 of 98 Exygen Study No.: 023-066 Table XVI. Summary of PFOS in Mallard Red Blood Cell Samples at 10 ppm a.i. for Week 15 Sponsor ID 454-109-3289-Wkl5 454-109-3289-Wk15* 454- 109-3290-Wk 15 454-109-3290-Wkl 5* 454-109-3291-Wk15 454-109-3291-Wk15* 454-109-3292-Wk 15 454-109-3292-Wk15* 454- 109-3293-Wk 15 454-109-3293-Wkl5* 454-109-3294-Wk15 454-109-3294-Wkl5* 454- 109-3295-Wkl 5 454-109-3295-Wkl 5* 454-109-3296-Wkl5 454-109-3296-Wkl5* Centre ID 0107275 0107275 0107276 0107276 0107277 0107277 0107278 0107278 0107279 0107279 0107280 0107280 0107281 0107281 0107282 0107282 Set Number 052201BD 052201BD 052201BD 052201BD 052201BD 052201BD 052201BD 052201BD 052201BDR 052201BDR 052201BDR 052201BDR 052201BDR 052201BDR 052201BD 052201BD Extraction Analysis Analyte Date Date Found (ug/mL) 5/22/01 5/23-24/01 5/22/01 5/23-24/01 5/22/01 5/23-24/01 5/22/01 5/23-24/01 5/22/01 5/23-24/01 5/22/01 5/23-24/01 5/22/01 5/23-24/01 5/22/01 5/23-24/01 5/22/01 5/24/01 5/22/01 5/24/01 5/22/01 5/24/01 5/22/01 5/24/01 5/22/01 5/24/01 5/22/01 5/24/01 5/22/01 5/23-24/01 5/22/01 5/23-24/01 AVERAGE: STANDARD DEVIATION: 53.5 48.2 3.98 3.44 60.9 61.4 9.65 8.79 113 116 1.54 1.61 8.03 6.56 0.623 0.505 31.1 39.8 Table XVII. Summary of PFOS in Mallard Red Blood Cell Samples at 50 ppm a.i. for TERM Sponsor ID 454-109-3297-TERM 454-109-3297-TERM* 454-109-3298-TERM 454-109-3298-TERM* Centre ID 0107283 0107283 0107284 0107284 * Duplicate injection Set Number 052201CD 052201CD 052201BDR 052201BDR Extraction Date Analysis Date 5/22/01 5/24/01 5/22/01 5/24/01 5/22/01 5/24/01 5/22/01 5/24/01 AVERAGE: STANDARD DEVIATION: Analyte Found (ug/mL) 275 272 406 418 343 80 LOQ = 0.01 (ig/mL for red blood cells and serum Exygen Research Page 33 of 98 C993G4 Exygen Study No.: 023-066 Table XVIII. Summary of PFOS in Mallard Serum Samples at 0 ppm a.i. for Week 5 Sponsor ID 454-109-3249-Wk5 454-109-3249-Wk5* 454-109-3251 -Wk5 454-109-3251 -Wk5* 454-109-3252-Wk5 454-109-3252-Wk5* 454-109-3253-Wk5 454-109-3253-Wk5* 454-109-3254-Wk5 454-109-3254-Wk5* 454-109-3255-Wk5 454-109-3255-Wk5* 454-109-3256-Wk5 454-109-3256-Wk5* Centre ID 0107303 0107303 0107304 0107304 0107305 0107305 0107306 0107306 0107307 0107307 0107308 0107308 0107309 0107309 * Duplicate injection Set Number 072001A 072001A 072001A 072001A 072001A 072001A 072001A 072001A 072001A 072001A 072001A 072001A 072001A 072001A Extraction Date Analysis Date 7/20/01 7/20/01 7/20/01 7/20/01 7/20/01 7/20/01 7/20/01 7/20/01 7/20/01 7/20/01 7/20/01 7/20/01 7/20/01 7/20/01 7/21/01 7/21/01 7/21/01 7/21/01 7/21/01 7/21/01 7/21/01 7/21/01 7/21/01 7/21/01 7/21/01 7/21/01 7/21/01 7/21/01 AVERAGE: STANDARD DEVIATION: Analyte Found (pg/mL) 0.00414 0.00417 0.00566 0.00599 0.00878 0.00943 0.00516 0.00499 0.00432 0.00444 0.00462 0.00392 0.00544 0.00516 0.00544 0.00167 LOQ = 0.01 |ag/mL for red blood cells and serum Exygen Research Page 34 of 98 Exygen Study No.: 023-066 Table XIX. Summary of PFOS in Mallard Serum Samples at 10 ppm a.i. for Week 5 Sponsor ID 454-109-3289-Wk5 454- 109-3289-Wk5* 454-109-3290-Wk5 454-109-3290-Wk5* 454-109-329l-Wk5 454-109-3291-Wk5* 454- 109-3292-Wk5 454-109-3292-Wk5* 454-109-3293-Wk5 454-109-3293-Wk5* 454-109-3294-Wk5 454-109-3294-Wk5* 454-109-3295-Wk5 454-109-3295-Wk5* Centre ID 0107310 0107310 0107311 0107311 0107312 0107312 0107313 0107313 0107314 0107314 0107315 0107315 0107316 0107316 Set Number 072001AD 072001 AD 072001 AD 072001AD 072001 AD 072001 AD 072001AD 072001 AD 072001AD 072001 AD 072001AD 072001 AD 072001 AD 072001AD Extraction Analysis Date Date 7/20/01 7/20/01 7/20/01 7/20/01 7/20/01 7/20/01 7/20/01 7/22/01 7/22/01 7/22/01 7/22/01 7/22/01 7/22/01 7/22/01 7/20/01 7/20/01 7/20/01 7/20/01 7/20/01 7/20/01 7/20/01 7/22/01 7/22/01 7/22/01 7/22/01 7/22/01 7/22/01 7/22/01 AVERAGE: STANDARD DEVIATION: Analyte Found (us/mL) 128 120 79.6 94.2 104 110 83.4 85.8 59.4 76.6 60.6 57.5 73.3 76.9 86.4 22.2 Table XX. Summary of PFOS in Mallard Serum Samples at 50 ppm a.i. for Week 5 Sponsor ID 454-109-3329-Wk5 454-109-3329-Wk5* 454-109-3330-Wk5 454-109-3330-Wk5* Centre ID 0107317 0107317 0107318 0107318 Set Number 072001 AD 072001AD 072001AD 072001 AD Extraction Date Analysis Date 7/20/01 7/20/01 7/20/01 7/20/01 7/22/01 7/22/01 7/22/01 7/22/01 AVERAGE: STANDARD DEVIATION: Analyte Found (pg/mL) 376 400 320 290 347 50 * Duplicate injection LOQ = 0.01 |ag/mL for red blood cells and serum Exygen Research Page 35 of 98 ccosse Exygen Study No.: 023-066 Table XXI. Summary of PFOS in Mallard Serum Samples at 0 ppm a.i. for Week 10 Sponsor ID 454-109-3249-Wk 10 454-109-3249-Wk 10* 454-109-3250-Wkl 0 454-109-3250-WklO* 454-109-3251-WklO 454-109-3251-Wkl0* 454- 109-3252-Wkl0 454-109-3252-Wk 10* 454-109-3253-Wk 10 454-109-3253-WklO* 454-109-3254-WklO 454-109-3254-Wkl0* 454-109-3255-Wkl0 454-109-3255-WklO* 454-109-3256-Wk 10 454-109-3256-WklO* Centre ID 0107322 0107322 0107323 0107323 0107324 0107324 0107325 0107325 0107326 0107326 0107327 0107327 0107328 0107328 0107329 0107329 Set Number 072001B 072001B 072001B 07200IB 07200IB 07200IB 072001B 07200IB 07200IB 072001B 072001B 072001B 07200IB 072001B 072001B 07200IB Extraction Analysis Date Date 7/20/01 7/21-22/01 7/20/01 7/20/01 7/21-22/01 7/21-22/01 7/20/01 7/21-22/01 7/20/01 7/20/01 7/20/01 7/20/01 7/20/01 7/20/01 7/20/01 7/21-22/01 7/21-22/01 7/21-22/01 7/21-22/01 7/21-22/01 7/21-22/01 7/21-22/01 7/20/01 7/21-22/01 7/20/01 7/21-22/01 7/20/01 7/20/01 7/20/01 7/21-22/01 7/21-22/01 7/21-22/01 AVERAGE: STANDARD DEVIATION: Analyte Found (ue/mL) 0.00271 0.00372 0.00726 0.00666 0.00659 0.00530 0.00899 0.00940 0.00517 0.00482 ND ND 0.00337 0.00403 0.00419 0.00384 0.00476 0.00266 * Duplicate injection ND = No peak detected 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)) LOQ = 0.01 pg/mL for red blood cells and serum For values recorded as NQ, 0.00005 pg/mL (half the value of the lowest calibration standard, 0.0001 pg/mL) and for ND, zero was used to calculate the average and standard deviation. Exygen Research Page 36 of 98 C00S67 Exygen Study No.: 023-066 Table XXII. Summary of PFOS in Mallard Serum Samples at 10 ppm a.i. for Week 10 Sponsor ID 454-109-3289-WklO 454-109-3289-WklO* 454-109-3290-WklO 454-109-3290-WklO* 454-109-3291-Wkl0 454-109-3291-WklO* 454- 109-3292-Wk 10 454-109-3292-WklO* 454-109-3293-Wkl0 454- 109-3293-Wkl 0* 454- 109-3294-Wkl0 454-109-3294-Wk10* 454-109-3295-Wk 10 454-109-3295-Wk10* 454-109-3296-Wkl 0 454-109-3296-Wk 10* Centre ID 0107330 0107330 0107331 0107331 0107332 0107332 0107333 0107333 0107334 0107334 0107335 0107335 0107336 0107336 0107337 0107337 Set Number 072001BD 072001BD 072001BD 072001BD 072001BD 072001BD 072001BD 072001BD 072001BD 072001BD 072001BD 072001BD 072001BD 072001BD 072001BD 072001BD Extraction Analysis Date Date 7/20/01 7/22/01 7/20/01 7/22/01 7/20/01 7/22/01 7/20/01 7/22/01 7/20/01 7/22/01 7/20/01 7/22/01 7/20/01 7/22/01 7/20/01 7/22/01 7/20/01 7/22/01 7/20/01 7/22/01 7/20/01 7/22/01 7/20/01 7/22/01 7/20/01 7/22/01 7/20/01 7/22/01 7/20/01 7/22/01 7/20/01 7/22/01 AVERAGE: STANDARD DEVIATION: Analyte Found (ug/mL) 145 147 32.7 32.5 93.5 102 107 111 132 136 85.3 86.4 123 120 114 112 105 33.8 * Duplicate injection LOQ = 0.01 |ig/mL for red blood cells and serum Exygen Research Page 37 of 98 CC0S68 Exygen Study No.: 023-066 Table XXIII. Summary of PFOS in Mallard Serum Samples at 0 ppm a.i. for Week 15 Sponsor ID 454- I09-3236-Wk 15 454-109-3236-Wkl5* 454-109-3249-Wk 15 454-109-3249-Wk15* 454-109-3250-Wkl 5 454-109-3250-Wkl5* 454-109-3251-Wkl5 454-109-3251-Wkl5* 454-109-3252-Wkl5 454-109-3252-Wkl5* 454-109-3253-Wkl5 454-109-3253-Wkl5* 454-109-3254-Wkl 5 454-109-3254-Wkl5* 454-109-3255-Wkl 5 454-109-3255-Wkl5* 454-109-3256-Wkl5 454-109-3256-Wkl5* Centre ID 0107338 0107338 0107339 0107339 0107340 0107340 0107341 0107341 0107342 0107342 0107343 0107343 0107344 0107344 0107345 0107345 0107346 0107346 Set Number 071901A 071901A 071901A 071901A 071901A 071901A 071901A 071901A 071901A 071901A 071901A 071901A 071901A 071901A 071901A 071901A 071901A 071901A Extraction Analysis Date Date 7/19/01 7/19-20/01 7/19/01 7/19/01 7/19/01 7/19/01 7/19/01 7/19/01 7/19/01 7/19/01 7/19/01 7/19/01 7/19-20/01 7/19-20/01 7/19-20/01 7/19-20/01 7/19-20/01 7/19-20/01 7/19-20/01 7/19-20/01 7/19-20/01 7/19-20/01 7/19/01 7/19/01 7/19/01 7/19/01 7/19/01 7/19/01 7/19/01 7/19-20/01 7/19-20/01 7/19-20/01 7/19-20/01 7/19-20/01 7/19-20/01 7/19-20/01 AVERAGE: STANDARD DEVIATION: Analyte Found (ua/mL) ND ND 0.00729 0.0102 ND ND 0.00466 0.00441 ND ND 0.00649 0.00723 ND ND 0.0233 0.00699 0.0195 ND 0.000503 0.00616 * Duplicate injection ND = No peak detected 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)) LOQ = 0.01 pg/mL for red blood cells and serum For values recorded as NQ, 0.00005 pg/mL (half the value of the lowest calibration standard, 0.0001 pg/mL) and for ND, zero was used to calculate the average and standard deviation. Exygen Research Page 38 o f 98 C00S69 Exygen Study No.: 023-066 Table XXIV. Summary of PFOS in Mallard Serum Samples at 10 ppm a.i. for Week 15 Sponsor ID 454-109-3289-Wkl5 454-109-3289-Wkl5* 454-109-3290-Wkl5 454-109-3290-Wkl5* 454-109-3291-Wkl5 454-109-3291-Wkl5* 454-109-3292-Wk 15 454-109-3292-Wkl5* 454-109-3293-Wk15 454-109-3293-Wkl5* 454-109-3294-Wkl5 454-109-3294-Wkl5* 454- 109-3295-Wk 15 454-109-3295-Wkl 5 * 454- 109-3296-Wkl5 454-109-3296-Wkl5* Centre ID 0107347 0107347 0107348 0107348 0107349 0107349 0107350 0107350 0107351 0107351 0107352 0107352 0107353 0107353 0107354 0107354 Set Number 071901AD 071901AD 071901AD 071901 AD 071901AD 071901AD 071901AD 071901AD 071901AD 071901 AD 071901 AD 071901AD 071901AD 071901AD 071901AD 071901 AD Extraction Analysis Date Date 7/19/01 7/20-21/01 7/19/01 7/20-21/01 7/19/01 7/20-21/01 7/19/01 7/20-21/01 7/19/01 7/20-21/01 7/19/01 7/20-21/01 7/19/01 7/20-21/01 7/19/01 7/20-21/01 7/19/01 7/20-21/01 7/19/01 7/20-21/01 7/19/01 7/20-21/01 7/19/01 7/20-21/01 7/19/01 7/20-21/01 7/19/01 7/20-21/01 7/19/01 7/20-21/01 7/19/01 7/20-21/01 AVERAGE: STANDARD DEVIATION: Analyte Found (ug/mL) 152 162 10.1 8.75 152 142 12.1 11.6 128 132 6.73 4.52 117 116 9.52 11.3 73.5 67.3 Table XXV. Summary of PFOS in Mallard Serum Samples at 50 ppm a.i. for TERM Sponsor ID 454-109-3297-TERM 454-109-3297-TERM* 454-109-3298-TERM 454-109-3298-TERM* Centre ID 0107355 0107355 0107356 0107356 * Duplicate injection Set Number 072001 AD 072001AD 072001AD 072001AD Extraction Analysis Date Date 7/20/01 7/20/01 7/20/01 7/20/01 7/22/01 7/22/01 7/22/01 7/22/01 AVERAGE: STANDARD DEVIATION: Analyte Found (ug/mL) 685 601 664 601 638 43 LOQ = 0.01 [ig/mL for red blood cells and serum Exygen Research Page 39 of 98 000370 Exygen Study No.: 023-066 Table XXVI. Summary of PFOS in Quail Red Blood Cell Samples at 0 ppm a.i. for Week 5 Sponsor ID 454-108-361-Wk5 454-108-361-Wk5 * 454-108-362-Wk5 454- 108-362-Wk5* 454-108-363-Wk5 454-108-363-Wk5* 454-108-364-Wk5 454-108-364-Wk5* 454-108-365-Wk5 454-108-365-Wk5* 454-108-366-Wk5 454-108-366-Wk5* 454-108-368-Wk5 454- 108-368-Wk5* Centre ID 0107077 0107077 0107078 0107078 0107079 0107079 0107080 0107080 0107081 0107081 0107082 0107082 0107083 0107083 Set Number 051801A 051801A 051801A 051801A 051801A 051801A 051801A 051801A 051801A 051801A 051801A 051801A 051801A 051801A Extraction Analysis Date Date 5/18/01 5/18-19/01 5/18/01 5/18/01 5/18-19/01 5/18-19/01 5/18/01 5/18-19/01 5/18/01 5/18-19/01 5/18/01 5/18-19/01 5/18/01 5/18-19/01 5/18/01 5/18-19/01 5/18/01 5/18-19/01 5/18/01 5/18-19/01 5/18/01 5/18-19/01 5/18/01 5/18-19/01 5/18/01 5/18-19/01 5/18/01 5/18-19/01 AVERAGE: STANDARD DEVIATION: Analyte Found (ug/mL) NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ 0.00292 0.00264 NQ NQ 0.000440 0.000993 * 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)) LOQ = 0.01 pg/mL for red blood cells and serum 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. Exygen Research Page 40 of 98 Exygen Study No.: 023-066 Table XXVII. Summary of PFOS in Quail Red Blood Cell Samples at 10 ppm a.i. for Week 5 Sponsor ID 454-108-401-Wk5 454-108-401-Wk5* 454-108-402-Wk5 454-108-402-Wk5* 454-108-403-Wk5 454-108-403-Wk5* 454-108-404-Wk5 454-108-404-Wk5* 454- 108-405-Wk5 454-108-405-Wk5* 454-108-406-Wk5 454-108-406-Wk5* 454-108-407-Wk5 454-108-407-Wk5* 454-108-408-Wk5 454-108-408-Wk5* Centre ID 0107084 0107084 0107085 0107085 0107086 0107086 0107087 0107087 0107088 0107088 0107089 0107089 0107090 0107090 0107091 0107091 Set Number 051801DR 051801DR 051801DRD 051801DRD 051801DR 051801DR 051801DR 051801DR 051801DRD 051801DRD 051801DRD 051801DRD 051801DRD 051801DRD 051801DRD 051801DRD Extraction Date Analysis Date 5/18/01 5/18/01 5/18/01 5/18/01 5/18/01 5/18/01 5/18/01 5/18/01 5/18/01 5/18/01 5/18/01 5/18/01 5/18/01 5/18/01 5/18/01 5/18/01 5/21-22/01 5/21-22/01 5/22/01 5/22/01 5/21-22/01 5/21-22/01 5/21-22/01 5/21-22/01 5/22/01 5/22/01 5/22/01 5/22/01 5/22/01 5/22/01 5/22/01 5/22/01 AVERAGE: STANDARD DEVIATION: Analyte Found (ug/mL) 113 107 353 351 30.5 30.4 26.9 25.8 415 447 1050 1040 495 501 451 471 369 324 * Duplicate injection LOQ = 0.01 (ig/mL for red blood cells and serum Exygen Research Page 41 of 98 000872 Exygen Study No.: 023-066 Table XXVIII. Summary of PFOS in Quail Red Blood Cell Samples at 50 ppm a.i. for Week 5 Sponsor ID 454-108-441-Wk5 454-108-441-Wk5* 454-108-442-Wk5 454-108-442-Wk5* Centre ID 0107092 0107092 0107093 0107093 Set Number 052201CD 052201CD 052201CD 052201CD Extraction Date Analysis Date 5/22/01 5/24/01 5/22/01 5/24/01 5/22/01 5/24/01 5/22/01 5/24/01 AVERAGE: STANDARD DEVIATION: Analyte Found (us/mL) 166 163 108 103 135 34 Table XXIX. Summary of PFOS in Quail Red Blood Cell Samples at 0 ppm a.i. for Week 10 Sponsor ID 454-108-361 -WklO 454-108-361 -WklO* 454-108-362-WklO 454-108-362-Wk 10* 454-108-363-WklO 454- 108-363-Wkl0* 454-108-364-WklO 454-108-364-Wkl0* 454-108-365-Wkl0 454-108-365-WklO* 454- 108-366-Wk 10 454-108-366-WklO* 454-108-367-WklO 454-108-367-Wkl0* 454-108-368-Wk 10 454-108-368-Wkl0* Centre ID 0107100 0107100 0107101 0107101 0107102 0107102 0107103 0107103 0107104 0107104 0107105 0107105 0107106 0107106 0107107 0107107 * Duplicate injection Set Number 051801B 051801B 05180IB 051801B 051801B 051801B 051801B 051801B 051801B 051801B 05180IB 051801B 051801B 051801B 051801B 051801B Extraction Analysis Analyte Date Date Found (ue/mL) 5/18/01 5/18/01 5/18/01 5/19/01 5/19/01 5/19/01 NQ NQ NQ 5/18/01 5/18/01 5/18/01 5/19/01 5/19/01 5/19/01 NQ NQ NQ 5/18/01 5/18/01 5/19/01 5/19/01 NQ NQ 5/18/01 5/18/01 5/18/01 5/18/01 5/18/01 5/18/01 5/18/01 5/18/01 5/19/01 NQ 5/19/01 NQ 5/19/01 NQ 5/19/01 NQ 5/19/01 NQ 5/19/01 NQ 5/19/01 NQ 5/19/01 ________NQ______ AVERAGE: NQ STANDARD DEVIATION: NQ 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)) LOQ = 0.01 pg/mL for red blood cells and serum 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. Exygen Research Page 42 of 98 COOS73 Exygen Study No.: 023-066 Table XXX.Summary of PFOS in Quail Red Blood Cell Samples at 10 ppm a.i. for Week 10 Sponsor ID 454-108-401-WklO 454-108-401 -Wk10* 454-108-402-WklO 454-108-402-Wk10* 454-I08-403-Wkl0 454-108-403-WU0* 454-108-404-WklO 454-108-404-Wkl0* 454-108-405-Wkl0 454-108-405-WkI0* 454-108-406-Wk 10 454-108-406-Wkl0* 454-108-407-WklO 454-108-407-Wk10* 454-108-408-Wkl0 454-108-408-Wk 10* Centre ID 0107108 0107108 0107109 0107109 0107110 0107110 0107111 0107111 0107112 0107112 0107113 0107113 0107114 0107114 0107115 0107115 * Duplicate injection Set Number 051801DRD 051801DRD 051801DRD 051801DRD 051801DR 051801DR 051801DR 051801DR 051801DR 051801DR 051801DR 051801DR 051801DR 051801DR 051801DR 051801DR Extraction Analysis Analyte Date Date Found (ne/mL) 5/18/01 5/18/01 5/18/01 5/22/01 5/22/01 5/22/01 672 671 203 5/18/01 5/18/01 5/18/01 5/18/01 5/18/01 5/18/01 5/18/01 5/18/01 5/18/01 5/18/01 5/18/01 5/18/01 5/18/01 5/22/01 5/21-22/01 5/21-22/01 5/21-22/01 5/21-22/01 5/21-22/01 5/21-22/01 5/21-22/01 5/21-22/01 5/21-22/01 5/21-22/01 5/21-22/01 5/21-22/01 190 23.0 20.7 19.0 21.2 18.0 20.0 48.9 50.5 22.8 22.3 30.4 30.2 AVERAGE: STANDARD DEVIATION: 129 220 LOQ = 0.01 iag/mL for red blood cells and serum Exygen Research Page 43 o f 98 Exygen Study No.: 023-066 Table XXXI. Summary of PFOS in Quail Red Blood Cell Samples at 0 ppm a.i. for Week 15 Sponsor ID 454-108-361-Wkl5 454-108-361-Wkl5* 454-108-362-Wkl5 454-108-362-Wkl 5* 454- 108-363-Wkl 5 454-108-363-Wkl5* 454-108-364-Wkl5 454-108-364-Wk15* 454-108-365-Wkl5 454-108-365-Wkl5* 454-108-366-Wkl5 454-108-366-Wk 15* 454-108-367-Wkl5 454-108-367-Wkl5* 454-108-368-Wkl5 454-108-368-Wkl5* 454-108-333-Wkl5 454-108-333-Wkl5* Centre ID 0107116 0107116 0107117 0107117 0107118 0107118 0107119 0107119 0107120 0107120 0107121 0107121 0107122 0107122 0107123 0107123 0107124 0107124 * Duplicate injection Set Number 052101A 052101A 052101A 052101A 052101A 052101A 052101A 052101A 052101A 052101A 052101A 052101A 052101A 052101A 052101A 052101A 052101A 052101A Extraction Date Analysis Date 5/21/01 5/22/01 5/21/01 5/22/01 5/21/01 5/22/01 5/21/01 5/22/01 5/21/01 5/22/01 5/21/01 5/22/01 5/21/01 5/22/01 5/21/01 5/22/01 5/21/01 5/22/01 5/21/01 5/22/01 5/21/01 5/22/01 5/21/01 5/22/01 5/21/01 5/22/01 5/21/01 5/22/01 5/21/01 5/22/01 5/21/01 5/22/01 5/21/01 5/22/01 5/21/01 5/22/01 AVERAGE: STANDARD DEVIATION: Analyte Found (ue/r NQ NQ NQ NQ 0.00226 0.00328 0.00127 NQ 0.00705 0.00391 NQ NQ 0.00795 0.00816 NQ NQ 0.00130 NQ 0.00198 0.00304 ND = No peak detected 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)) LOQ = 0.01 pg/mL for red blood cells and serum For values recorded as NQ, 0.00005 pg/mL (half the value of the lowest calibration standard, 0.0001 pg/mL) and for ND, zero was used to calculate the average and standard deviation. Exygen Research Page 44 o f 98 Exygen Study No.: 023-066 Table XXXII. Summary of PFOS in Quail Red Blood Cell Samples at 10 ppm a.i. for Week 15 Sponsor ID 454- 108-401-Wkl 5 454-108-401-Wkl5* 454-108-402-Wk 15 454-108-402-Wkl5* 454-108-403-Wkl5 454-108-403-Wkl 5* 454-108-404-Wkl 5 454-108-404-Wkl5* 454-108-405-Wkl5 454-108-405-Wk 15* 454-108-406-Wk15 454-108-406-Wkl5* 454-108-407-Wkl5 454-108-407-Wkl 5* 454-108-408-Wkl5 454-108-408-Wkl5* 454-108-383-Wkl5 454-108-383-Wkl5* Centre ID 0107125 0107125 0107126 0107126 0107127 0107127 0107128 0107128 0107129 0107129 0107130 0107130 0107131 0107131 0107132 0107132 0107133 0107133 Set Number 052101AD 052101AD 052101AD 052101AD 052101AD 052101AD 052101AD 052101AD 052101AD 052101AD 052101 AD 052101AD 052101AD 052101AD 052201CD 052201CD 052101AD 052101AD Extraction Analysis Date Date 5/21/01 5/22-23/01 5/21/01 5/21/01 5/21/01 5/21/01 5/22-23/01 5/22-23/01 5/22-23/01 5/22-23/01 5/21/01 5/21/01 5/22-23/01 5/22-23/01 5/21/01 5/22-23/01 5/21/01 5/22-23/01 5/21/01 5/21/01 5/21/01 5/22-23/01 5/22-23/01 5/22-23/01 5/21/01 5/21/01 5/22-23/01 5/22-23/01 5/21/01 5/24/01 5/21/01 5/24/01 5/21/01 5/22-23/01 5/21/01 5/22-23/01 AVERAGE: STANDARD DEVIATION: Analyte Found (ug/mL) 72.2 69.1 2.36 1.65 21.2 18.8 1.30 1.22 15.8 17.9 3.61 3.23 13.3 9.60 1.24 1.55 56.7 54.1 20.3 24.8 Table XXXIII. Summary of PFOS in Quail Red Blood Cell Samples at 50 ppm a.i. for TERM Sponsor ID 454-108-409-TERM 454-108-409-TERM* 454-108-410-TERM 454-108-410-TERM* Centre ID 0107134 0107134 0107135 0107135 Set Number 052201CD 052201CD 052201CD 052201CD Extraction Date Analysis Date Analyte Found (ug/mL) 5/22/01 5/24/01 5/22/01 5/24/01 5/22/01 5/24/01 5/22/01 5/24/01 AVERAGE: STANDARD DEVIATION: 361 356 106 114 234 144 * Duplicate injection LOQ = 0.01 jig/mL for red blood cells and serum Exygen Research Page 45 of 98 CO0376 Exygen Study No.: 023-066 Table XXXIV. Summary of PFOS in Quail Serum Samples at 0 ppm a.i. for Week 5 Sponsor ID 454-108-361-Wk5 454-108-361-Wk5* 454-108-362-Wk5 454-108-362-Wk5* 454-108-363-Wk5 454-108-363-Wk5* 454-108-364-Wk5 454- 108-364-Wk5* 454-108-366-Wk5 454-108-366-Wk5* 454-108-368-Wk5 454- 108-368-Wk5* Centre ID 0107154 0107154 0107155 0107155 0107156 0107156 0107157 0107157 0107159 0107159 0107160 0107160 * Duplicate injection Set Number 072301AR 072301AR 072301AR 072301AR 072301AR 072301AR 072301AR 072301AR 072301AR 072301AR 072301AR 072301AR Extraction Date Analysis Date 7/23/01 7/23/01 7/23/01 7/23/01 7/23/01 7/23/01 7/23/01 7/23/01 7/23/01 7/23/01 7/23/01 7/23/01 7/25/01 7/25/01 7/25/01 7/25/01 7/25/01 7/25/01 7/25/01 7/25/01 7/25/01 7/25/01 7/25/01 7/25/01 AVERAGE: STANDARD DEVIATION: Analyte Found (pg/mL) 0.0282 0.0295 0.0149 0.0134 0.0198 0.0166 0.00510 0.00535 0.0135 0.0120 0.0138 0.0156 0.0156 0.00746 LOQ = 0.01 ng/mL for red blood cells and serum Exygen Research Page 46 o f 98 000577 Exygen Study No.: 023-066 Table XXXV. Summary of PFOS in Quail Serum Samples at 10 ppm a.i. for Week 5 Sponsor ID 454-108-402-Wk5 454-108-402-Wk5* 454- 108-403-Wk5 454-108-403-Wk5* 454-108-404-Wk5 454-108-404-Wk5* 454-108-405-Wk5 454-108-405-Wk5* 454-108-406-Wk5 454-108-406-Wk5* 454-108-407-Wk5 454-108-407-Wk5* 454-108-408-Wk5 454- 108-408-Wk5 * Centre ID 0107162 0107162 0107163 0107163 0107164 0107164 0107165 0107165 0107166 0107166 0107167 0107167 0107168 0107168 Set Number 072301AD 072301 AD 072301AD 072301AD 072301AD 072301AD 072301 AD 072301 AD 072301 AD 072301AD 072301AD 072301AD 072301AD 072301 AD Extraction Analysis Date Date 7/23/01 7/26/01 7/23/01 7/26/01 7/23/01 7/23/01 7/23/01 7/23/01 7/23/01 7/26/01 7/26/01 7/26/01 7/26/01 7/26/01 7/23/01 7/23/01 7/23/01 7/23/01 7/23/01 7/23/01 7/23/01 7/26/01 7/26/01 7/26/01 7/26/01 7/26/01 7/26/01 7/26/01 AVERAGE: STANDARD DEVIATION: Analyte Found (pg/mL) 45.8 45.6 115 128 104 98.3 138 129 106 107 42.4 36.3 77.6 84.4 89.8 35.0 Table XXXVI. Summary of PFOS in Quail Serum Samples at 50 ppm a.i. for Week 5 Sponsor ID 454-108-441-Wk5 454-108-441-Wk5* 454-108-442-Wk5 454-108-442-Wk5* Centre ID 0107092 0107092 0107093 0107093 Set Number 072301 AD 072301AD 072301 AD 072301 AD Extraction Analysis Date Date 7/23/01 7/26/01 7/23/01 7/26/01 7/23/01 7/26/01 7/23/01 7/26/01 AVERAGE: STANDARD DEVIATION: Analyte Found (ug/mL) 551 562 529 520 541 19.4 * Duplicate injection LOQ = 0.01 |ig/mL for red blood cells and serum Exygen Research Page 47 of 98 CO0S78 Exygen Study No.: 023-066 Table XXXVII. Summary of PFOS in Quail Serum Samples at 0 ppm a.i. for Week 10 Sponsor ID 454-108-361-Wkl0 454-108-361-Wkl0* 454-108-362-WklO 454-108-362-WklO* 454-108-363-WklO 454-108-363-Wkl0* 454-108-364-WklO 454-108-364-Wk10* 454-108-365-Wkl0 454- 108-365-WklO* 454-108-366-WklO 454-108-366-WklO* 454-108-367-WklO 454-108-367-Wkl0* 454-108-368-WklO 454-108-368-WklO* Centre ID 0107177 0107177 0107178 0107178 0107179 0107179 0107180 0107180 0107181 0107181 0107182 0107182 0107183 0107183 0107184 0107184 * Duplicate injection Set Number 072301B 072301B 072301B 072301B 072301B 072301B 07230IB 072301B 072301B 07230IB 072301B 072301B 072301B 072301B 07230IB 072301B Extraction Date Analysis Date 7/23/01 7/23/01 7/23/01 7/23/01 7/23/01 7/23/01 7/23/01 7/23/01 7/23/01 7/23/01 7/23/01 7/23/01 7/23/01 7/23/01 7/23/01 7/23/01 7/24/01 7/24/01 7/24/01 7/24/01 7/24/01 7/24/01 7/24/01 7/24/01 7/24/01 7/24/01 7/24/01 7/24/01 7/24/01 7/24/01 7/24/01 7/24/01 AVERAGE: STANDARD DEVIATION: Analyte Found (pg/mL) 0.00483 0.00489 0.00295 0.00249 0.00467 0.00521 0.00390 0.00375 0.00271 0.00292 0.00439 0.00437 0.00563 0.00542 0.00537 0.00526 0.00430 0.00105 LOQ = 0.01 |ig/mL for red blood cells and serum Exygen Research Page 48 of 98 0005579 Exygen Study No.: 023-066 Table XXXVIII. Summary of PFOS in Quail Serum Samples at 10 ppm a.i. for Week 10 Sponsor ID 454-108-401-Wk10 454-108-401-Wkl0* 454-108-402-Wkl0 454-108-402-Wk 10* 454-108-403-Wkl0 454-108-403-Wk10* 454-108-404-Wkl0 454-108-404-Wkl 0* 454- 108-405-Wkl 0 454-108-405-WklO* 454-108-407-Wkl0 454-108-407-Wkl0* 454-108-408-WklO 454-108-408-WklO* Centre ID 0107185 0107185 0107186 0107186 0107187 0107187 0107188 0107188 0107189 0107189 0107190 0107190 0107191 0107191 Set Number 072301DD 072301DD 072301DD 072301DD 072301 DD 072301DD 072301D 072301D 072301DD 072301DD 072301DD 072301DD 072301DD 072301DD Extraction Analysis Date Date 7/23/01 7/23/01 7/23/01 7/23/01 7/27-28/01 7/27-28/01 7/27-28/01 7/27-28/01 7/23/01 7/27-28/01 7/23/01 7/27-28/01 7/23/01 7/27-28/01 7/23/01 7/23/01 7/23/01 7/23/01 7/23/01 7/23/01 7/27-28/01 7/27-28/01 7/27-28/01 7/27-28/01 7/27-28/01 7/27-28/01 7/23/01 7/27-28/01 AVERAGE: STANDARD DEVIATION: Analyte Found (ug/mL) 213 207 53.6 53.7 148 177 19.4 20.3 157 147 105 110 97.0 93.7 114 63.4 * Duplicate injection LOQ = 0.01 |ig/mL for red blood cells and serum Exygen Research Page 49 of 98 Exygen Study No.: 023-066 Table XXXIX. Summary of PFOS in Quail Serum Samples at 0 ppm a.i. for Week 15 Sponsor Centre Set Extraction Analysis Analyte _ J 2 OE1______J2========^i^SL=========il========2^===!====^HSiM^Sil 454-108-361-Wkl5 0107192 072301C 7/23/01 7/24-25/01 NQ 454-108-361-Wkl 5* 0107192 072301C 7/23/01 7/24-25/01 NQ 454-108-362-Wk 15 0107193 072301C 7/23/01 7/24-25/01 NQ 454-108-362-Wkl5 * 0107193 072301C 7/23/01 7/24-25/01 NQ 454-108-363-Wkl5 0107194 072301C 7/23/01 7/24-25/01 0.00154 454- 108-363-Wk 15* 0107194 072301C 7/23/01 7/24-25/01 0.00160 454-108-364-Wkl5 0107195 072301C 7/23/01 7/24-25/01 NQ 454-108-364-Wkl5* 0107195 072301C 7/23/01 7/24-25/01 NQ 454-108-365-Wkl5 0107196 072301C 7/23/01 7/24-25/01 NQ 454- 108-365-Wkl5* 0107196 072301C 7/23/01 7/24-25/01 NQ 454-108-366-Wkl5 0107197 072301C 7/23/01 7/24-25/01 NQ 454-108-366-Wkl 5* 0107197 072301C 7/23/01 7/24-25/01 NQ 454-108-367-Wkl5 0107198 072301C 7/23/01 7/24-25/01 0.00227 454-108-367-Wkl 5* 0107198 072301C 7/23/01 7/24-25/01 0.00249 454-108-368-Wkl5 0107199 072301C 7/23/01 7/24-25/01 0.00253 454-108-368-Wkl5* 0107199 072301C 7/23/01 7/24-25/01 0.00255 454-108-333-Wkl5 0107200 072301C 7/23/01 7/24-25/01 0.00162 454-108-333-Wkl5* 0107200 072301C 7/23/01 7/24-25/01 0.00160 AVERAGE: 0.000928 STANDARD DEVIATION: 0.00105 * 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)) LOQ = 0.01 pg/mL for red blood cells and serum For values recorded as NQ, 0.00005 pg/mL (half the value of the lowest calibration standard, 0.0001 pg/mL) and for ND, zero was used to calculate the average and standard deviation. Exygen Research Page 50 o f 98 Exygen Study No.: 023-066 Table XL. Summary of PFOS in Quail Serum Samples at 10 ppm a.i. for Week 15 Sponsor ID 454-108-401-Wkl5 454-108-401-Wkl 5* 454-108-402-Wkl 5 454-108-402-Wkl5* 454-108-403-Wk15 454-108-403-Wkl5* 454-108-404-Wk15 454-108-404-Wkl5* 454-108-405-Wkl 5 454-108-405-Wk15* 454-108-406-Wk15 454-108-406-Wkl5* 454-108-407-Wkl5 454-108-407-Wkl5* 454-108-408-Wkl 5 454-108-408-Wkl5* 454-108-383-Wkl5 454-108-383-Wkl5* Centre ID 0107201 0107201 0107202 0107202 0107203 0107203 0107204 0107204 0107205 0107205 0107206 0107206 0107207 0107207 0107208 0107208 0107209 0107209 Set Number 072301DD 072301DD 072301DD 072301DD 072301DD 072301DD 072301DD 072301DD 072301DD 072301DD 072301DD 072301DD 072301DD 072301DD 072301DD 072301DD 072301DD 072301DD Extraction Analysis Date Date 7/23/01 7/23/01 7/23/01 7/23/01 7/23/01 7/23/01 7/23/01 7/23/01 7/23/01 7/23/01 7/23/01 7/23/01 7/27-28/01 7/27-28/01 7/27-28/01 7/27-28/01 7/27-28/01 7/27-28/01 7/27-28/01 7/27-28/01 7/27-28/01 7/27-28/01 7/27-28/01 7/27-28/01 7/23/01 7/23/01 7/23/01 7/27-28/01 7/27-28/01 7/27-28/01 7/23/01 7/23/01 7/23/01 7/27-28/01 7/27-28/01 7/27-28/01 AVERAGE: STANDARD DEVIATION: Analyte Found (ug/mL) 181 180 8.21 8.45 246 239 11.1 10.7 141 156 7.04 6.98 107 114 10.2 8.91 131 143 95.0 86.6 Table XLI. Summary of PFOS in Quail Serum Samples at 50 ppm a.i. for TERM Sponsor ID 454-108-410-TERM 454-108-410-TERM* Centre ID 0107211 0107211 * Duplicate injection Set Number 072301DD 072301DD Extraction Date Analysis Date 7/23/01 7/23/01 7/27-28/01 7/27-28/01 AVERAGE: STANDARD DEVIATION: Analyte Found (ug/mL) 476 514 495 26.9 LOQ = 0.01 ng/mL for red blood cells and serum Exygen Research Page 51 o f 98 ceoss2 Exygen Study No.: 023-066 FIGURES Exygen Research Page 52 of 98 C00SS3 Exygen Study No.: 023-066 Figure 1. Typical Calibration Curve for PFOS 051801A.rdb (PFOS): "Linear" Regression ("1 / x" weighting): y = 7822.67 x + 1176.24 (r = 0.9989155) Exygen Research Page 53 of 98 Exygen Study No.: 023-066 Figure 2. Chromatogram Representing a 0.1 ng/mL standard for PFOS I 3rf*Nir;*oaiaoiA-U)r &wnpIO: 'SUndarfl' FI*: *qu*| WtodMl* *MK Nam: "PFOS" UM*(N): m o w o armi' C e ra m e t *01 t y 'mC PFOS (C 03t40t-r Atvuisfon: " sa a p la tndaxi 1 S H * t( Type Standard C oB cantratlen: 3.100 C a lc u la te d Cone: 9.101 ng/aX ng/L 315 Acq. D atai Acq. Tina i 0 3 /1 1 /0 1 0 4 . 1 5 . 2S ftl 310 M odifiedi Bunching f a c to r . Mo 1 300- M aiao T h raa h o ld i A ta rh ia a h o ld i 1 .7 4 0 .0 0 296 Run. Saeoeha: XT Windowi 2 1 0 .0 290 Expactad m 4 .4 ] Sap. W idth Sap. H aight. 0 .2 0 0.01 Exp. Peek R a tio : Exp. Ad}. R a tio : Exp. V al. R atio : Ue R e l a t i v e RT> 3 .0 0 4.00 1 .0 0 Nn Int.Typai R atantien T ina' 1049.9 count: H aight i S ta rt T inat 2.90OO2 epa 4 . in End T in a : 4.10 In as 260275 270 205: 2002E5- 245 240- 4.4169 215210206 200 195 190 155 IK ITS 170 IBS I 160 155 145140: 135 130 125 120 IIS no 100 Exygen Research Page 54 of 98 C-0OSS Exygen Study No.: 023-066 Figure 3. Chromatogram Representing a 1.0 ng/mL standard for PFOS Exygen Research Page 55 of 98 COO356 Exygen Study No.: 023-066 Figure 4. Chromatogram Representing a Reagent Blank for PFOS (Exygen ID Reagent Blank A, Set: 051801A) IS unfM N urW O SiaolA-tia* Sarryta 10;-Raagtnl Stank- A takktam *PFOS' `4M.W99.Danw* Commsnl; `ftaagani Btanfc A- AmoUton: " Sf uame pl *la Ttnydpam>a Un1k0nown C ancantration N/A 206 C a lc u lated Conci kcq. D a t a > 05/16/01 206 Xcq. Timai 04i 09iIS m M odifiedi Bunching r a c to n V oiaa TH reeheldi A rea IT ireahcldt Mum. S m ee th ai R r window > t a p a c i a d RTs Sap. w idth Sap. H eighti Exp. peak R a tie i Exp. A di. R atioi Exp. Va2. R a tia i Uae R a la c lv a RTi No 1.74 B.00 2 1 0 .0 0 .1 0 0.01 5 .0 0 4 .0 0 1 .0 0 Ho cpa cpa aac in 200 too 16$ 160 Fita `qua)btoodwiT In c. Typoi R etention T iaai Height S t a r t Tim a tout Tim a t a n To Baaa 4.42 1099. count a 1 . t l a *002 cpa 4.29 1.57 m in 175 170 166- 160- 103' ISO- 146 140135- 130' 125' 120 115- HO I MS- 100 0 6306 4.41a s so ts' to 75 70 56 45 40 36 30 25 20 13 \ 13601 OS 5 2 0 9 6 3.0 3 5 7.seofr^ 1 5 0 35 30 ( S 70 75 Exygen Research Page 56 of 98 000337 Exygen Study No.: 023-066 Figure 5. Chromatogram Representing Control Quail Red Blood Cells for PFOS (Exygen ID: 0107376 Blank A, Set: 051801A) Exygen Research Page 57 of 98 000838 Exygen Study No.: 023-066 Figure 6. Chromatogram Representing Control Quail Serum for PFOS, (Exygen ID: 0107375 Blank A, Set: 072301C) Exygen Research Page 58 of 98 C 003S9 Exygen Study No.: 023-066 Figure 7. Chromatogram Representing Control Quail Red Blood Cells fortified with 10 ng/mL of PFOS (Exygen ID: 0107376 Spk A, Set: 051801DR) Exygen Research Page 59 of 98 to0S9O Exygen Study No.: 023-066 Figure 8. Chromatogram Representing Control Quail Serum fortified with 10 ng/mL of PFOS (Exygen ID: 0107375 Spk A, Set: 072301D) Exygen Research Page 60 of 98 CC0S9I Exygen Study No.: 023-066 Figure 9. Chromatogram Representing Quail Red Blood Cell Sample (Exygen ID: 0107084, Sponsor ID: 454-108-401 Wk5, Set: 051801DR) Exygen Research Page 61 of 98 Exygen Study No.: 023-066 Figure 10. Chromatogram Representing Quail Serum Sample (Exygen ID: 0107207, Sponsor ID: 454-108-407-W kl5, Set: 072301DD) 0107207 (454-108-407-Wk15) DF = 5000 072301DD-635 Sm (Mn. 3x2) 28-Jul-2001 00:11:27 LC/MS/MS #7 MHM of 1 Channel ES- Exygen Research Page 62 of 98 COOS93 Exygen Study No.: 023-066 APPENDIX A Study Protocol 01P-023-066 (Centre Study No. 023-066) and Amendments and Deviations Exygen Research Page 63 of 98 C ^0S94 Exygen Study No.: 023-066 Centre Protocol No. 01P-023-066 ANALYTICAL PHASE PROTOCOL ANALYTICAL PHASE TITLE EXTRACTION OF POTASSIUM PERFLUOROOCTANESULFONATE FROM RED BLOOD CELLS FOR ANALYSIS USING HFLC-ELECTROSPRAY/MASS SPECTROMETRY SPONSOR 3M Environmental Technology and Safety Services Building 2-3E-09 PO Box 33331 St. Paul, MN 55133-3331 DATA REQUIREMENTS Analytical Method Requirements PERFORMING LABORATORY Centre Analytical Laboratories, Inc. (Centre) 3048 Research Drive State College, PA 16801 Phone 814-231-8032 PROTOCOL IDENTIFICATION NUMBER 01P-023-066 i Centre Analytical Laboratories, Inc. Exygen Research Page 1 of 29 Page 64 of 98 C 00595 Exygen Study No.: 023-066 Centre Protocol No. 01P-023-066 Title: EXTRACTION OF POTASSIUM PERFLUOROOCTANESULFONATE FROM RED BLOOD CELLS FOR ANALYSIS USING HPLC-ELECTROSPRAY/MASS SPECTROMETRY TABLE OF CONTENTS _________________________________________________ Paee TABLE OF CONTENTS...................................................................................................2 1. PURPOSE........................................................................................................................... 3 2. REFERENCE MATERIAL...................................................................................3 3. SPONSOR............................................................................................................. 4 4. TESTING FACILITY................................................................................... ........4 5. PERFORMING LABORATORY____________________________________ 4 6. PROPOSED EXPERIMENTAL TIME-FRAME..................................................4 7. JUSTIFICATION FOR THE SELECTION OF THE TEST SYSTEM...................5 8. SAMPLE PROCESSING, STORAGE AND IDENTIFICATION......................._..5 9. ANALYTICAL METHOD............................................................................................. 5 10. EXPERIMENTAL DESIGN....................................................................................... . 10 11. PROTOCOL AMENDMENTS AND DEVIATIONS............................................. 11 12. RECORDS...... ................................................................................................................. 11 13. QUALITY ASSURANCE...................................................................... .......................12 14. DATA AND REPORT....................................................................................................12 15. ARCHIVE STATEMENT............................................................................................. 13 16. REFERENCES.................................................................................................................13 17. PROTOCOL APPROVAL............................................................................................ 14 APPENDIX: ANALYTICAL METHOD..................................................................... 15 j t i ! jI{ [ Iw f f !t | i Centre Analytical Laboratories, Inc. Exygen Research Page 2 of 29 Page 65 of 98 GOO8 ^ 6 Exygen Study No.: 023-066 Cent Protocol No. 01P-023-066 T itle: EXTRACTION OF POTASSIUM PERFLUOROOCTANESULFONATE FROM RED BLOOD CELLS FOR ANALYSIS USING HPLC-ELECTROSFRAY/MASS SPECTROMETRY 1. PURPOSE The purpose of this study is to analyze mallard and quail red blood cells samples for residues of perfluorooctanesulfonate (PFOS) using method entitled, "Extraction Of Potassium Perfluorooctanesulfonate Or Other Fluorochemical Compounds From Serum For Analysis -Using HPLC-Electrospray/Mass Spectrometry*' (3M method number ETS-8-4.1) with the modifications listed in Section 9. The analytical phase will be conducted under EPA TSCA Good Laboratory Practice standards 40 CFR Part 792 (1). 2. REFERENCE MATERIAL The following analytical standard will be used: Test Material PFOS TCR Number TCR00017-46 Purity (%) 97.9 Expiration Date 08/31/01 Chemical name and structure of the compound is presented below. PFOS Chemical Name: Perfluorooctanesulfonate IUPAC Name: 1-Octanesulfonic acid,l,1,2,2,3,3,4,4,5,5,6,6,7,7,8,8,8- heptadecafluoro-, potassium salt CAS Number 2795-39-3 MolecularWeight: 499 (CsFitSOI O C sF iyS O" O Note: The neutral molecule and standard from which the PFOS (anion) is obtained is perfluorooctanesulfonate potassium salt [CgFnS03K], molecular weight 538. A record of test and reference substance receipt, storage conditions, and a record of use will be maintained at Centre Analytical Laboratories, Inc. Forms Centre Analytical Laboratories, Inc. Page 3 of 29 Exygen Research Page 66 of 98 Exygen Study No.: 023-066 CentreProtocolNo. 01P-023-066 documenting chain-of-custody and shipping records for tracking of the test substances will be included as part of the raw data package. ' All standards/test substances and any prepared solutions must be identified with a unique label or number on the container or cross-referenced to the container. HAZARD INFORMATION A current MSDS for the chemical($) used in this study will be maintained at the testing facility. 3. SPONSOR 3M Environmental Technology and Safety Services Building 2-3E-09 PO Box 33331 S t Paul, MN 55133-3331 Sponsor Representative: Rochelle Robideau TELEPHONE: 651-778-7065 FAX NUMBER: 651-778-6176 4. TESTING FACILITY Wildlife International, Ltd. 8598 Commerce Drive Easton, MD 21601 STUDY DIRECTOR: Sean Gallagher TELEPHONE: 410-822-8600 FAX NUMBER: 410-822-0632 5. PERFORMING LABORATORY Centre Analytical Laboratories, Inc. (Centre) 3048 Research Drive State College, PA 16801 PRINCIPAL INVESTIGATOR: Emily R. Stauffer, Centre TELEPHONE: 814-231-8032 FAX NUMBER: 814-231-1580 6. PROPOSED EXPERIMENTAL TIME-FRAME Analytical Start Date Analytical Termination Date Report Issued April 23,2001 April 30,2001 May 31,2001 i.. I; f' i Centre Analytical Laboratories, Inc. Page 4 of 29 Exygen Research Page 67 of 98 C C 0S38 Exygen Study No.: 023-066 CentreProtocol No. 01P-023-066 7. JUSTIFICATION FOR THE SELECTION OF THE TEST SYSTEM Mallard and quail red blood cells samples collected by Wildlife International, Ltd. ( will be used as the analytical samples for this phase. Mallards and quails represent wild bird populations and they are EPA recommended species because they do well in a laboratory environment Quail habitats include bushy pastures, grassy roadsides, farmlands and open woodlands, and they are considered land grazers. Mallard habitats include ponds, lakes and marshes, and they are considered aquatic grazers. The use of these matrices for die study was to determine if PFOS was deposited in the whole blood of mating adults that were fed PFOS. A complete description of the test system along with all of the in-life parameters is documented in Mallard Protocol No. 454/010600/MP/SUB454, Project No. 454-105 and Quail Protocol No. 454/01060Q/QP/SUB454, Project No. 454-104. This project is the analytical portion of those studies. 8. SAMPLE PROCESSING, STORAGE AND IDENTIFICATION No processing will be required for the any of the samples. Each sample will be assigned a unique sample identification number at Centre, which will be used for tracking and identification of the samples. The samples will be stored in a temperature-monitored freezer, maintained at < -10s C, except when removed for extraction and analysis as described in the method. The samples will be kept isolated from the test substance during storage. Sample receipt and storage location and conditions during the study will be documented. All samples and any resulting sample extracts will be identified with a unique label or sample number. Such identification will be either on the container or cross-referenced to the container. 9. ANALYTICAL METHOD All samples will be analyzed according to the analytical method titled "Extraction Of Potassium Perfluorooctanesulfonate Or Other Fluorochemical Compounds From Serum For Analysis Using HPLC-Electrospray/Mass Spectrometry" (3M method numberETS-8-4.1) with the following modifications: 1. There will be no surrogate standard used in the extraction procedure. The stock, fortification, and calibration standards will be prepared as follows: Stock Solution Prepare a stock solution of PFOS at 100 pg/mL by weighing out 10.0 mg of analytical standard (corrected for percent salt and percent purity). Adjust final volume to 100 mL with methanol in a 100-mL volumetric flask. Store this Centre Analytical Laboratories, Inc. Exygen Research Page 5 of 29 Page 68 o f 98 Exygen Study No.: 023-066 Centre Protocol No. 01P-Q23-066 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. Fortification Solutions a. 1.0 ug/mL Fortification Solution - Pipette 1.0 mL of the 100 pg/mL stock solution into a 100 mL volumetric flask. Bring up to volume with methanol. b. Q.Lug/mL Fortification Solution - Pipette 10.0 mL of the 1.0 pg/ml fortification solution into a 100-mL volumetric flask and bring up to volume with methanol. c. 0.01 ue/mL Fortification Solution - 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 Store all fortification standard solutions fin 125-mL LDPE bottles) in a refrigerator at 2C to 6C for a maximum period of 6 months from the date of preparation. Calibration Standards Prepare six LC/MS/MS calibration standards in methanol via dilution of the 0.1 pg/mL and0.01 pg/mL fortification solutions. This is a typical example; additional concentrations may be prepared as needed. Initial Cone. (pg/mL) 0.1 0.1 0.1 0.01 0.01 0.01 Volume (mL) 5 2 1 5 2 1 Diluted to (mL) 100 100 100 100 100 100 Final Cone. (us/mL) 0.005 0.002 0.001 0.0005 0.0002 0.0001 Store all calibration standard solutions (in 125-mL LDPE bottles) in a refrigerator at 2C to 6C for a maximum period of 6 months from the date of preparation. 3. The sample volume for the red blood cells samples will be 100 pL. The amount of 0.5 M TBA added to the sample will be 0.5 mL and the amount of 0.25 M sodium carbonate/sodium bicarbonate buffer added will be 1.0 mL. The samples will only be filtered with 0.2 pm nylon mesh filter if necessary. 4. The samples will be analyzed under the following conditions: j, [( | V | ; t i | j 1 k Centre Analytical Laboratories, Inc. Exygen Research Page 6 of 29 Page 69 of 98 CO0900 00901 (50090 C00903 Exygen Study No.: 023-066 CentreProtocol No. 01P-Q23-066 10. EXPERIMENTAL DESIGN Samples obtained by Wildlife International Ltd. will be shipped to Centre Analytical Laboratories, Inc. for analysis. There are no known contaminants expected in the test system that would affect the results of this study. Only those samples designated by the Sponsor will be extracted and analyzed at Centre according to method, "Extraction Of Potassium Perfluorooctaneaulfonate Or Other Fluorochemical Compounds From Serum For Analysis Using HPLCElectrospray/Mass Spectrometry" (3M method number ETS-8-4.1) with the modifications listed in Section 9. Methods to control bias will include assay of untreated control samples, fortification of untreated control samples to obtain recovery data, and replicate analysis of fortified samples to provide an indication of reproducibility. Fortification will be made to the matrix prior to extraction at levels ranging from the LOQ to above the highest residue found. The fortification solution containing the test/reference substance will be applied to the designated samples via a Hamilton syringe, micropipette, pipet, or equivalent, to maintain consistency and accuracy. The average recovery and relative standard deviation of the fortified samples will be calculated. If necessary, apply a standard test for outliers. If an outlier exists, then it may be excluded from the statistical analysis. Also, the average residue found and standard deviation for each matrix will be calculated. The following equations will be used for the calculations. Equation 1: Analyte found (ng/mL) slope Equation 2: Analyte found (pg/mL) = .((apaL fq.uud_fog/mLj x FYImL) x fiE * EV. (mLll x Lug (AV (mL) x sample vol. (mL)) 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. C entre A nalytical L aboratories, Inc. Page 10 of 29 I s ! I . $ lK'- v. Exygen Research Page 73 of 98 (j.1 ^ .0 9 0 4 : Exygen Study No.: 023-066 Centre Protocol No. 0IP-023-066 Equation 3: Recovery (%) = ' ((anal, found (ug/mL) - avg. anal, in ctri (ue/mL)) x (1000 ng/1 ug)> x 100 amount added (ng/mL) 11. PROTOCOL AMENDMENTS AND DEVIATIONS Any deviations from the protocol or from the analytical method as provided will be documented and reported promptly to (he Study Director and Sponsor Representative. Planned changes to this protocol or to the analytical method will be made in writing as an amendment and approved by the Principal Investigator, the Study Director, and the Sponsor Representative. Any amendments or deviations will be appended to this protocol and included in the final report. ! Protocol amendments: Planned changes to the approved protocol shall be documented by amendments that cleariy describe die change, justification for the change, and impact on the study. Amendments will be signed and dated by the Principal Investigator, Study Director, and SponsorRepresentative. Copies of amendments will be sent to the quality assurance unit. 2. Protocol deviations: Protocol deviations, which are one time and unplanned deviations from the protocol, shall be documented in the study records, noting the nature of the deviation, potential effect or impact on the study, and corrective action if required. Protocol deviations are signed and dated by the Principal Investigator, Study Director, and Sponsor Representative and reviewed by QAU. 12. RECORDS Records to be maintained include, but are not limited the following (as appropriate): 1. Sample tracking sheet(s) 2. Sample receipt records, storage history, and chains of custody 3. History and preparation of standards (stock, fortification, calibration) 4. Description of any modifications to the method 5. Instrument run sheets, bench-sheets or logs 6. Analytical data tables 7. All chromatographic and instrumental conditions 8. Sample extraction and analysis dates 9. A complete listing of study personnel, signatures and initials 10. Chronological presentation of all study correspondence 11. Any other data necessary for the reconstruction of the study All chromatograms will contain the following: Centre Analytical Laboratories, Inc. Page I I o f 29 Exygen Research Page 74 of 98 Exygen Study No.: 023-066 Centre Protocol No. 01P-023-066 a. Sample identification, date, arrow or other indication of the area of interest, and injection number corresponding to the run. h. Additionally, fortifications will include the fortification level of the analyte. c. Analytical standard chromatograms will additionally include the concentration (e.g., pg/ml, ng/mL, ppb, ppt, etc.). Each data set will contain information on temperatures, flow rates, column parameters, gases, instrument parameters, and instrument type, etc. 13. QUALITY ASSURANCE Centre QA Unit will review the protocol, audit the study conduct and the study documentation (including raw data and final report) to ascertain that all GLP standards are met. 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 Principal Investigator, Centre Management, and the Study Director. 14. DATA AND REPORT 1. All raw data and the original signed protocol will be 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 used will be stored in the study file. The reason for exclusion will be documented. 2. A report will be issued by Centre, approved and signed by the Study Director and the Sponsor Representative and sufficient for submission to EPA. The report contents should include, but not limited to: 1. Objectives and procedures stated in the protocol 2. Analytical and statistical methods used 3. Reference materials identified by name, lot, purity, and other characteristics 4. Name of performing laboratory and analytical start and termination dates 5. Statement prepared and signed by the quality assurance unit 6. Tables containing all applicable data 7. All chromatographic and instrumental conditions 8. A complete listing of Centre study personnel Centre will send a copy of the draft report to the Study Director and Sponsor Representative, who will return the report with comments. Centre Analytical will make revisions and finalize the report with the approval of the Study Director and Sponsor Representative. The Centre QA unit will conduct an audit of the final draft report and raw data package to assure accuracy and GLP compliance. The Centre Analytical Laboratories, Inc. Page 12 of 29 Exygen Research Page 75 of 98 Exygen Study No.: 023-066 Centre Protocol No. 01P-023-066 report must be approved by the Study Director and Sponsor Representative 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 clearly identify the part of the final report that is being corrected and the justification for correction. The amendment shall be signed and dated by the Principal Investigator, Study Director and SponsorRepresentative. REPORT DISTRIBUTION: Original: Study Director Copy Sponsor Representative Copy: Centre Archives 15. ARCHIVE STATEMENT Study records to be maintained: Records to be maintained for the study include all raw data, observations recorded 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 characterization data and any shipping records shall be retained. Document archives: Upon completion of the study, the study records, protocol 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-related originals and a copy of the final report and raw data package. 16. REFERENCES 1 U.S. Environmental Protection Agency. Toxic Substances Control Act (TSCA). Good Laboratory Practice Standards. Final Rule, 40 CFR Part 792. i' V I' i \ Centre Analytical Laboratories. Inc. Exygen Research Page 13 of 29 [: Page 76 of 98 000907 Exygen Study No.: 023-066 CentreProtocolNo. 01P-023-066 17. PROTOCOL APPROVAL This protocol was audited by the Quality Assurance Unit of Centre Analytical Laboratories, Inc. Quality Assurance Reviewer, Centre J l`(" Date Principal Investigator, Centre lyDj^ker Sdentisi u Facility Management, Centre q jid o i Date1 r ' Operations Manager Study Director, Wildlife International, Ltd. ! ? Centre Analytical Laboratories, Inc. Exygen Research J Page 14 of 29 Page 77 of 98 000908 Exygen Study No.: 023-066 CentreProtocol No. 01P-023-066 APPENDIX: ANALYTICAL METHOD A. "Extraction of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds From Scrum for Analysis Using HPLC-Ekctrospray/Mass ' Spectrometry" 5 Centre Analytical Laboratories, Inc. Exygen Research Page 15 of 29 Page 78 of 98 a 909 Exygen Study No.: 023-066 . .C e n tn P rotocol N o. 01Pz023-066.. O 3M E n v ir o n m e n t a l l a b o r a t o r y _____ !------------- r f }r . . ! Method - Extraction of PotassiumPerfluorooctanesulfomate or Other \aU OT.OCBEM ICA L COMPOUNDS FROM SERUM FORANALYSES USING H P L C - - 7 1 $ v Elecirospray/Mass Spectrometry M ethod Number: ETS^8-4.1 - -, I. ! . Adoption D ate: 03/01/99 Author: Lisa Clemen, Glenn Langenburg Revision D ate: Y /3W ? Approved By. o kH A Laboratory Manager Group Leader _ J .. / A , V , - Date V ' .'W tf/z i/3 3 ikM > Technical Reviewer I` ^ '0 I--0- Sc o t aw>b m vm w ______L _ * ^*Sfbr *f. 4* 5#"i A 1.1 Scope: This method is for the extraction ofpotassium perfhiorooctanesulfonate (PFOS) or other duorochemical compounds from serum. 1-2 Applicable compounds: Fluorochemical surfactants or other Quoriruted compounds.'1 1-3 M atrices: Rabbit, rat, bovine, monkey, and human serum or other Quids as designated in the validation report ' j Word 6/95 j ETS4-4.1 ExtractionofPFOS fratti Serata CentreAnalytical Laboratories. Inc. Page 16 of 29 Exygen Research Page 79 of 98 00091 Exygen Study No.: 023-066 CentreProtocol No. 01P-023-Q66 ,* ; >> * 0 .Sum mary o f M ethod r vvsJE fcl .tThhiiss method it-T fl * die procedure for extracting potassium perfiuorooctanesulfonate " ) or ^ i ftuorochemicai surfoctantsfrom serum, or otherfluidi, using an ion _ ' ju d methyl-terf-butyl ether (MtBE). In this method, seven ^hiprocliriticaffwere extracted: PFOS,PFOSA,PFOSAA, EtFOSEOH, PFOSEA, MS56, and surrogate standard (see 3.0Definitions). An icmpairing reagent is added to ,the sample and the analyte ion pair is partitioned into MtBE. The MtBE extract is | n p re| and putonto a nitrogen evaporatoruntil dry. Each extract is reconstituted in 1.0 ! ' g lio fju tS ap o l, then filtered through a 3 cc plastic syringe attached to a 0.2 pm nylon filterfiS o-ilasajutovials.v; Z 2 These^bmptejKtracts'are analyzed following method ETS-8-J.I or other appropriate 3.0 Pranm oN S 3.1 PFOS: perihujrooc^ie^ ne (anion ofpotassium salt) C^FSO,' 3.2 PFOSAiperfluorooclane aulfonylamide C^jSC^NHi 23 PFOSAA: pcrfluaroocta^"iiU foc^nuda (ethyI)aceUic CjFnSO*N(CH|CHj)CH,CO," 3.4 EtFOS-OH: 2(N^thylperflu<JrpMtene sidforanrido>c%i alcohol 3.5 PFOSEA: prffuoroctane sulfonyl thylrmde C J jtSOjKCCHjCHJH 3.6 M556: ^,,S O ^H X O IjC O O H ) .Y % 3.7 Surrogate standard: 1H-1H-2H-2H perilnorooc^sulfonic acid ft-Pjy AKHIWWAW>CAVTiPgS. 4.1 H ealth ondsafety warnings *0W 4.1.1 Use universal precautions, especially laboratory coats, goggles, and gloves when handling animal tissue, which may contain pathogens. *' & fl.Iim T E I>Z H C Z 3 S.1 There are.no interferences known at this time. 6.0 Equipment__________ ;___________ __ 6.1 The following equipment is used while performing this method. Equivalent equipment is > acceptable. __ 6.1.1 Vortex mixer, VWR, Vortex Genie 2 . .* 6 .U Centrifuge, Mistral 1P00 or IEC o 6.1 J Shaker, Eberbach or VWR -- ETS4-4.1 ExtraeriooofPFOSfami Senna Fige 2 of 14 h s Centre Analytical Laboratories, Inc. Exygen Research Page 17 of 29 Page 80 of 98 (00911 Exygen Study No.: 023-066 . CentreProtocol No. 01P-023-066 l'^ ^ V ^ fi-I.4 Nitrogen evaporator, Organomation 4.1.5 Balance (0.100 g) ' 7.0 Suy'fLiEs and materials_________ !------- J . <* '7 Eppendarfor disposable pipettes 7-i Nalgcac bottles, capable o fbolding 250 m Land 1 L 7.4 ^-V jiilm etrio flasks, glass, type A '7.5* POHEM-vQs; glass, 40 mL glass 7.6 Centrifuge tulres^olypropylene, 15 mL 7.7 Labels ''A . 7.8 Oxford D ispcm crr^O jb 10.0 mL 7S Syringes, capable ojmeasming 5 |iL to 50 |iL * ; 7.10 Graduated pipettes ' ^ 7.11 Syringes, disposable plastic, 3 cc 7.12 Syringe filters, nylon, 0.2 pm ,25m in 7.13 Timer aL . :. 7.14 Crimp cap autovials and caps \' 7.15 c ^ p e is Note: Prior to using glassware and bottles, rinse 3 tunes with methanol and 3 times with MtUi-QTMwater. Rinse syringes a minimum o fprimes methanol, 3 rinses from 3 separate vials. M - R sacents amp Stanparps s.l Type I reagent grade water, Mflli^QTM or equivalent; all water tried in this method should be M illiOTM w aterand may be provided by a MiRK}TOC PlusTM system- &2 Sodium hydroxide (NaOH),J.T Baker or equivalent 0" 8J Tetrabutylansmonium hydrogen sulfateTBA), Kodak or equivalent * 8.4 Sodium carbonate (NajCOj), J.T. Baker or equivalent 8 J Sodium bicarbonate (NaHCOJ, J.T. Baker or quivalent 8.6 Methyl-T-Butyl Ether, Omnisolv, glass distilled orHPLC giade 8.7 Methanol, Omnisolv, glass distilled or HPLC grade 8.8 Seram or blood, frozen from supplier 8.9 Fluorocbtm ical standards 8.9.1 PFOS(3M Specialty Chemical Division), molecularweight-5 3 8 J 8J.2 PFOSA (3M Specialty Chemical Division), molecular weight - 499 ETS-H .r Extraction ofPFOS ftoraSmza i' ! Page 3 of 14 - .y j- Centre Analytical Laboratories, Inc. Page 18 of 29 iLt:i i j iII- Exygen Research Page 81 of 98 y '. Exygen Study No.: 023-066 CentreProtocol No. 01P-023-066 * . _. . .**; *' '. -re .: .1 . `-- * i . . ` . '' s j V '': . * V V . - . . * - ` Jr ' ` i . .. -- ] * * . . V . ! 'V 0 U J PFOSAA (3M Specialty Chemical Division), molecular w eight J8J 8-9.4 EtFQSB-OH (3M Specialty ChemicalDivision), molecular weight 570 t'.v &9.S FFOSEA.(3M Specialty Chemical Division^ molecularweight -3 2 7 f .8.9.6 M56 (3M Specialty Chemical Division), moleculsrweight " 537 ,35?8.9.7 Surrogate standard: 4-H,perfluorooettoe sulfonic add (1-H.l-H, 2-H, 2-H i y ^ C^F,jSOjH) molecularweight " 428 X .^ W J ^ H h e r Suorochemicala, as appropriate 8.10 -iRea'geat preparation ' 'S to d g y ; I y preparation, adjust accordingly. , or surrogate 8.10.1 1 0 $ sodium'hydroxide (NaOH): Weigh approximately 200 g NaOH. Pour into a . 1000 mL beaker1coiitaiiBng 500 mL hfiffi-Q1" water, mix until allsolids are dissolvd^Storo in a 1 L Nalgene bottle. 8.10.2 1N sodium hydroxide (NaOH): Dilute lONNaOH 1:10. .Measure 10 mL o f 10 .N NaOH sdhmou'mto a 100 mL volumtrie Sasicand dilute to volume using MilE-QTM w ateriS to re y s 123 mLNalgenebottle. 8.10.3 0.5 M tetiabutybm m ^rium '^rogen sulfite fIBA): Weigh approximately 169 g i ofTBA into a 1 L volumetric containing 500 mL Mli-QTM water. Adjust to pH \ 10 using approxim ately^ to 54 m |5>f lONNaOH (While adding the last mL o f NaOH, add slowlybecause th pHchanges abruptly). Dilute to volume with Mli-Q7" water. Store in sl-L N algeneW tle. ' 8.10.3.1TB A requires a check prior to e&fnise to ensure pH " 10. A djusts needed using IN NaOH s o h |i 8 ^ " v 8.10.4 0.25 M sodium carbonate/sodium bicarbonate buffer(Na,CO^NaHCO^: Weigh approximately 2SJ g o fsodium carbonate (N ijCO Jand 21.0 g o f sodium bicarbonate (NaHCOJ into a 1 L vohmtetrwflmk m d brmg to volume with M tlliQ" water. Store in a 1L Nalgene bottle. 8.11 Standards preparation 8.11.1 PreparePFO Sstandiidsforthe standard curve. 8.11.2 Prepare other fluorochemical standards, as appropriate. Mfficnponem fluorochemical standards are acceptable (fix: example, one wolfing standard solution containing 1.00 ppm PFOS, 1.02 ppm PFOSA, 0.987 ppm PFSA^and i 1.10 ppm EtFOSE-OH.) ' " . __ 8.113 Weigh approximately 100 mg ofPFOS into a 100 mL volumetric Sask'nd rehord ^ . I the actual w eight .i; - 8.11.4 Bring to volume with methanol for a stock standard ofapproximately 1000 ppm^""*** ' (pg/mL). . r\ 8.11.5 Dilute the stock solution with methanol for a working standard 1 soludonof approximately 50 ppm. o 8.11.6 Dilute working standard 1 with methanol for a working standard 2 solution o f approx: 5.0 ppm. : ETS-M.1 . PageS of 14 ' Exnsthn ofPFOS fromSanaa CentreAnalytical Laboratories, Inc. Page 19 of 29 111 \ Exygen Research Page 82 o f 98 00913 Exygen Study No.: 023-066 CentreProtocol No. 01P-023-066 . > 'r`: ) 8.11.7 M u te w rkirigstandard 1 with methanol for a working standard 3 solution o f approx.0.30ppm .: ' .. - 8-17 Surrogate stock standard preparation 12.1 Weigh ipproxim aiely J0-S0 mg o fsurrogate standard 1-H.l-H, 2-H, 2-H, ' ". C ^ ,i ^ H into a iO mL volumetric flask and record the actual weight yr M 2 3 Bring to volume with methanol for a surrogate stock o f approximately 1000-1200 ^PPm- ire a surrogate working standard. Transfer approximately 1 m Lof surrogate ;stock to a lOm L volumetric flask and bring to volume with methanol for a w offing standard o f 100 ppm. Record the actual volume transferred. 9-Q Sa m p l e H A trnrj ^ j g r t. 9.1 All samples are 92 . Allow samples cavedfrozen and must be kept frozen until the extraction is performed. Savor? room temprature prior to extraction. 10.0 Qualityrcifn ^ , <u vep > 10.1 Solvent Blanks, Method.km]cs and m atrix blanks 10.1.1 An aliquot o f 1.0 mLmemanol is used as a solvent blank. 10.1.2 Extract two 1.0 mLaUqrata o f . as method blanks. water following this procedure and y 10.13 Extract two l.O m L aliquots o f the serum follqwmg this procedure and use as m atrix blanks. See 11J.4 . . ^ 10.2 illcate samples to determine 1022 Prepare each spike using a sample chosen by the analyst,puiually the control matrix received with each sample se t ^ 10.23 Expected concentrations will foil in the mid-range ofthejnitial calibration curve. Additional spikes may be included and may foil in the low-rangefdid initial calibration curve. ' n r' 10.2.4 Prepare one matrix spike and matrix spike duplicate per 40 sam ple^jritb a K minimum of2 matrix spikes per batch. _ *' 10.3 Continning calibration checks "N , 103.1 Prepare continuing calibration check samples to ensure the accuracy of the initial calbiration curve. 1 0 3 3 Prepare, at a minimum, one continuing check per group oflO samples. Fpr v example, if a sample set 34, four checks are prepared and extracted. 1 0 3 3 Prepare each continuing calibration check &om the same matrix used to prepare O the initial curve. - ETS-W.1 . ExtractionofPFOSfromSenna Pate 5 of 14 Centre Analytical Laboratories, Inc. Page 20 of 29 Exygen Research Page 83 of 98 CO09 3 A Exygen Study No.: 023-066 CentreProtocal No. 01P-Q23-066 _ *. `.\V*-*' - `,. !.. ,\\f '? } X H -v - : ' 103.4 The expected concentrations 'will foil w ithinthe mid-range of the initial. calibration curve. Additional spikes may be.included that fill in the low-range o f the initial calibration curve. This is necessary,if the analyst must quantitate using <55 , only the low end of the calibration curve (for example, 5 ppb -1 0 0 ppb, rather than * sppf-ioooppb). ; 1L-Q_Caubration andStandardization------------------------------------------- :________ sp lll^ jP re jjs u -e m atrix calibration standards '^ U .l.l^ ra n sfe r 1 mL ofserum to a 15 mL centrifiige tube. 1 1 3 3 jlfm drtsam ple volumes am less than 1.0 m li, extractstandntis with matrix ? volumesequal to the sample volumes. Do not extract less than 030 mL of m atnirR reord each sample volume an the extraction sheet 11.13 'While prepajm^a total 'oftwenty aliquots hr 15 mL centrifiige tubes, mix or shake betw eeraiiquots:) ; 11.1.4 Two 1 mL ahquota/M'other appropriate volume, serve as matrix blanks. Typically use thesfendafd concentrations and spiking amounts listed in Table 1, at tire end o fthiasectitnC to spilce, in duplicate, two standard curves, for a total o f eighteen stimdardviyo matrix blanks, and two method blanks. - 1 1 3 3 Refer to validation reportETS-8-4.0 4s KTS-8-5.0-Y-1, which fists the working o ranges rad the Tinrag Catihration Range (L O t) for calibration curves. . 113.6 Use Attachment D as an aiJm calculating the concentrations ofthe working standards. See Section 13.0 tocalculate actual concentrations ofPROS in calibration standards. 11.2 To each standard, blank, or continuing checSOedd approm de amount of surrogate working standard for the concentration to foil within thojcatibiation curve range 5 ppb - 1000 ppb. 'W M ' 113 Extract spiked matrix standards foUawing 12.M 2.16vof.this method. Use these standarda ' to establish each initial curve on the mass spectrometer. o ETS-W.I CentreAnalytical Laboratories, Inc. Exygen Research PeiofM : - \ .--y w r Page 21 of 29 Page 84 of 98 6 -0 0 9 3 -5 Exygen Study No.: 023-066 Centre.ErotocolNo. 01P.-023t066 ? t i O u- , _ Table 1 . . .>. ; Approxim ate spiking amounts fo r standards and spikes .UsinR 1.0 mL of m atrix ' i " fo rk in g standard. (approx, cone.) ! * - : K . 0300 ppm . & v 0.500 ppm Siy-'A .O O ppm .5,Oftppm pL . 10 20 5. 10 Approx, fiial cone, of . analyte in matrix Blank ` 0.005 ppm 0.010 ppm 0.025 ppm 0.050 ppm j 5.00 bpm '^5^50.0 pp&v - ' 50-O pfSn A W.p&S& 50.0 ppm jt&r" \ 20 .5 10 15 . 20 0.10Qppm . 0250 ppm 0300 ppm 0.750 ppm - 1.00ppm O o UH PiwCTprnm 12.1 walerbath. lOWtL or in a lukewarm 123 Voiteat mix for 15 seconds, then transfer 1.0 mL Brother appropriate volume to a IS mL polypropylene ceahi& getube. 123 Return unused samples to freezer after extrsc^njunountshave been removed. 12.4 Record tho initial volume on the extraction worksheet 123 Label (he tube w ith the study number, sample ID. d4e m d& ilyinitials. See studied worksheet for documenting the remaining steps. 126 Spike ail samples, including blanks and standards, ready Sir extraction'with surrogate standard as described in 113. . W' )r 12.7 Spike each matrix with die appropriate amount o fstandard as deambed n i l l .l , or Table 1 in that section, for tbe calibration curve standards. Also prepare m a te spikearad continuing calibration standards. , . if p? \ 12.8 Vortex mix die standard curve samples, matrix spike samples,and continuing calibration samples for IS seconds. ] 129 Check to ensure the 03 M TBA reagent is at pH 10. If not, a rtist accordingly^ 12.10 To each sample, add 1 m2 0.5 M TBA and 2 mL of025M sodium carbonate/sodiuru^,-'^~ bicarbonate buffer. 12.11 Using an Oxford Dispenser, add 5 mL methyl-iert-butyl ether. .' 12.12 Cap each sample and put on the shaker at a setting o f300 ipm, for20 minutes. 1203 Centri&ge for 20 to 25 minutes at a setting o f3500 rpm, or until layers are well separated. , ETS4-4.1 . . : , Bxuacdon ofPFOS ftomSemra Pag 7 of 14 ! .. ____ Centre Analytical Laboratories, Inc. Page 22 of 29 Exygen Research Page 85 of 98 000916 Exygen Study No.: 023-066 -Centn Protocol No_01Ph023-Q66. 7g.; .- v. .' r T^ 0 12.14 Label a fiesh IS mL centrifuge tube with the same information as in 124. 1X15 Remove 4.0 mL o fthe organic layer to this clean 15 mL centri&ge tube. 1X1$ Put each sample on the analytical nitrogen evaporator until dry, approximately 1 to 2 V ,b0Un- .y ' . lX ITjjA dd 1.0 mL ofmethanol to each centrifoge tube using a graduated pipette. " 1X18 -Vortex mix for 30 seconds. 1X29 A ttach a OXjun nylon mesh filter to a 3 cc syringe and transfix the sample to this J |S yrQ tge| Filter into a I J mL glass autoviai or low-volume autovUl when necessary. 1X20 l& d A e.aato y ial with the study number, animalnumber and gender, sample timepoint, m atri^ IlM solvent, extraction date, and analyst(s) performing Ore extraction. 1221 Cap and storefxtrabts at room temperature or at approximately 4 C until analysis. 1222 Completethe extraction-worksheet, attached to this document, and tape in the study notebook or in ^ e ^ n y tu d y hinder, ss appropriate. $ -s 13...0 P ata Analysis and Calculat?ows 1X1 Calculations 13,1.1 Calculate actual coneenttations i f PPOS, oro tta appttcablefluorochanicaL In calibration standards sm^the following equation; .mL of standard x concentration1ofstahdard fua/mLl mL ofstandard *m L of surrogate standard * initial matrix volume (mL) Final Concentration (jig/mL) ofPFOS in matrix 14J1 Method Peupqumancx _________!---------- -------------------------- -- 14.1 The method detection limit (MDL) is analyte and naatii^spcctfic. Refer to MDL report for specific MDL and limit o fquantitation (LOQ) values'(see Attachments B and C). 142 The following quality control samples are extracted with each bitch o f samples to evaluate the quality o f the extraction and analysis.' y~ 142.1 Metho&blanks and matrix blanks. 1 4 2 2 Matrix spike and matrix spike duplicate samples to determine accuracyand ^ precision of the extraction. 1 4 2 2 Continuing calibration check samples to determine the continued accuracy'ofthe v , initial calibration curve. 142 Refer to section 14 o f ETS-8-S.1 for method performance criteria. ' "K ~ ,.}> . 1 ..a . : r * 15.Q Pollution Prevention andWastbManagement------------------------- .*" o 15.1 Sample waste is disposed in biohazard containers, flammable solvent waste is disposed in high BTU containers, and used glass pipette waste is disposed in broken glass containers located in the laboratory. ETS-J-U . ExmcdoaofPFOSfromSerna PageS of 14 Centre Analytical Laboratories, Inc. Page 23 of 29 Exygen Research Page 86 of 98 000917 Exygen Study No.: 023-066 CentreProtocol No. 01P-023-066 Y , j *. O I " 16.0 RtCOnns_______ =. , ________________ 1S.1 Complete the extraction worksheet attached to this method, a d tape in the study notebook or include in the 3-ring study binder, is appropriate. y; 17.0A _iS? tta.c h m \ nts * :________ :_______________________:-------- 17.1 Attachment A, Extraction worksheet n ^ 4^ M m e n t B, MDL/LOQ values and summary .173 \^ttachmenl C, Calibration standard concentration worksheet 18.0 RiwaigwfTB .d 18.1 The validation reportVassociated with this method is EXS-8-4.0 A5.0-V -l, 182 FACT-M-3.l,.`iABa|ysis of Seram or OtherFluid Extracts for Fluorochemicals using H P I-B ectrospray M assSpectrom etry" I M Am C T r a Documents $ 19.1 ETS-8-5.1, "Analysis o f Seram^orOtiier Fluid Extracts forFluorochemicals using HPLC-Electrospray Mass Spectrometry" v 20.0 Revisions :_________'K . Revision M im te 1 \ ' Reason For Revision^ Section 12.21 Changed to include sample stqrage'at room temperature. Section 12.13 Added the shakerspeed."?'' jr ' \ Section 12.17 Final volume is 1.0 mL; not adjustrf for initial volumes 'r less than 1.0 mL. .4 Revision Date 04/02/99 3* c 55' a? .. o ; ETS-S-4.1 . Extraction ofPFOS from Seram Page 9 of 14 Centre Analytical Laboratories, Inc. ; Exygen Research Page 24 of 29 Page 87 of 98 000918 Exygen Study No.: 023-066 Centre Protocol No. 01P-023-066 O Q O S t u d y # M atrix B o x # W VDav . . Dam Spiked/Aaahnt Sonatala sm Bppcox.' ppm actual : ppm # ' ; . P W C x - .. approx. OJ pm sant "ppm # FC-M ix i approx. ppm actual . ppm # FC -M ix approx. 50 ppm actual ppm # rw m n w rd ti \. > xiTSSa. mr * : . . CCV* MS 55 ' ' 1 "V. S i - w tit / S :i m/ '-x H' t#lf # V '*S 'r k " xS r & 'irnm i \ -t 4 m s 1 3 SS7 V iw . -a - . t J .V Ik * Cff ' - . a Vf s v ? -A, 4& * V d t^ * .^ . - ' / fei X- r- s * j V ; ' v- -X ` - - : jQ&- H - n*y 1 * .. - 1 * l i g n u m " '' -- -s a B U i* . .IM S ________ I i* uaivi r ? r i M ; h n T J IT iiL | K pem lm L o^0O 5N w C C hA U 3M N aH C O lbtfnr a s s r t iL E i _____ mL * = 2 S ------------------ P a t . C la W a la '*.** * Vokasc ,,,, a * * SkL v . Sid.* feV at 1 i ' 1 m il-- THAp ~ vjtt & S lam an O ii. Shalt aaead: A Cm iH flw ipe* >- -- Uw" t % m B' W B M & . t e s g Temoeramnu I A sd methanol Volume A , _____ :.... . , ? * - -------------------- 1 V 8 T O M .e e ._________________ r i : n v ? i . r r - r r r r o T n i a M mL Mtosamale ial ont Cat VeriOcftboiuusedn s x matrixas tor Hdcarve. "-- y zza ... *yr. r :------ ---- ------------ 1 Attachment A , . '. 1 . J ________ ' \,, ' t . EIS4-U ' f.*. ^ ExtrsctionofPFOSfttan Serein .. ________X - >>*. Centre Analytical Laboratories, Inc. Page 10 of 14 Page25 of29 Exygen Research Page 88 of 98 0^0919 Exygen Study No.: 023-066 Centre Protocol Na 01P-023-066 I.. r j -.s..** v-fci; . -IT. o - i ' # ' - Compound ISN , MDL LOQ Linear C alibration Range (LCR) . (ppb) ", (PPb) Approximate concentrations to be u e d for preparing the Standard C alibration C arre POS- " VIA 5.55 5ppb-lOOOppb - POSAX PfOSAA^ 1.51 4.79 5 ppb-lOOOppb 3.46 -20.5 5 ppb-lOOOppb -' EtFOSEOH \ 11.4 362 5 ppb-1000ppb M556** 6.03 m '5 npb-lOOOppb PFOSE tV).TJ1..nJLJU 182 5ppb-lOOOppb i t s IH ly lU V Il* * w j IIW MM^ r 11 ' ............. .... .............. -- ---- ^ detenained. tw o cmy^memch ofthese m ilrkei aw e extim ted^analyzrtndthtbe rabbit nian cwYntodetennineajuivalence. Responses in the rat, bovine, monfcy, sndbumnnwoe equivalentto the rabbittesponj^thendorvtheir MDL and LOQ will he the same values u determined in rabbit " -fW _ j Plewe see tOQ Summary indM D LrtS^ InETS-M.0 & 5jO-V-l for furtherinformation. ZXr-* .'>TJ N s# \ 1,S O Attachment B: MDL/LOQ Sommary ETS4M.1 .j.' Extraction ofPFOS from Serum/.*,- Centre Analytical Laboratories, Inc. Exygen Research FignU ofU Page 26 of 29 Page 89 of 98 C00920 Exygen Study No.: 023-066 entreProtocolNo. 01P-023-066 ----- S' ` jl-;:'" Rabbit Sem \: Futt,Range Prepared range o f standard] (ppb) (ntnL) ' .a ' >0.99J''-97S r Curvi/ s H igSrSm yT I VX .V.j 4.94-248 . |7.a-S7* LCRfrom % Recovery curve . Range R sn Range. .(ppb) ' (ng/mL) r a -97* 3-10 ' - 4.67-115) 494-24* . 5-104 334-125) 97.-97 3-10 . 434-930 4.94-97 94-Ul 440-103 Rabbit Serum Full Range Prepared radge" v LCRfrom - ofstsm^rds v J .carve to*) g ifa )a -s*fob) $ (ng/mL) .0993 - 976 \ i 493-*.976 r .r& t i> \ Lew Curve 493-97.6 % Recovery -Range 8-103 7-103 RSD Range * 3.10-14.7 933-14.7 High curve 1 .. 243-97 0.9-97 2 4 .i? ^ n , y # 5-1.02 493-976^ 7 -`" 5.0S-139 . 3.10-143 . Prepared range Rabbit Serum ofstandards . (ppb) (ng/mL) Full Range Low Curve High curve i 0991-974 . 492-247 49.2- 974 0991-974 LCRfrom curve - (ppb) (ng/mL) 24.7- 974 9.74-247 97.4-974* 9.74- 974 % Recovery R a n g e ^ f Zj--- 1-111 .97-107 *3-10 Jr 4.1-1CoL3d/'^ 638-2L8 .% 433-129 r 93-113 4.11-233 rtzP. >%p& o AtactacntS: MDL/LQQSummary ' ET5-8*4.I 1 . . ... Extnctiou ofPFOS fromSoma .. vy'i . ' ' . V'-*' Centre Analytical Laboratories, Inc. Exygen Research Page J2 of 14 Page 27 of 29 Page 90 of 98 C s-* > 0 9 3 - Exygen Study No.: 023-066 CentreProtocolNo. 01P-023-066 o 1 "'-Vf.--' i: 5; - Preparedn o te .Ribbit Serum ofstandards (ppb) {ag/mL) .V '-i ' / F^H aSg^ ' 0J93-976 ` LCRfrom * curve <ppb) (ng/mL) 493- 976 % Recovery Range 77-110 LowCuive 4J3-97.fi 9.76-97.fi 97-107 High cur\ve' : 1. > i - m l/x } r -' Si*?' 97.fi-97 . 9.7-976 Preparedrange . 1 LCRfrom Rabbit Scrum ` ofstSairds/'--' curve (pph) (ng/mL) t- ; Full Range 0593-976 . 90-109 16-111 %Recovery Range 96-106 Low Curve High curve 453 - 248 ' 493-976 9.7fid24 91-UO 493-976'' 3ci?w86-106 1/X . 0593-976 9.76-976 USD Range 113-253 14.1-20 lU-l9.fi 11.1-30 RSD Range 103-163 1U-19J 103-113 10.1-19.1 Rabbit Serum Full Range Prepared range ofstandards . (ppb) (ng/mL) 0J93-976 LCRfromcurve (ppb) (ng/mL) 24.1-976 Low Curve 4J3-97. 9.76-97. High curve 97.6- 976 97.6- 97 1/X 0J93 - 976 9.76- 976 % Recovery 7 Range`^ r R-RsaSnDge 11-106 100-105 . 11-111 #ri&. 4.g2-17J ? 195-113^, i f 1y 5.11-9.74 97-110 4.77-193 .; a; rS 1V ; fi}c: i rth$v, O Attachment B: MDULOQ Summary ETS-W.l Extraction of PPOS finer Scrum Centre Analytical Laboratories, Inc. Exygen Research Pagel3ofl4 I Page 28 of 29 Page 91 of 98 C00922 Exygen Study No.: 023-066 Centre Protocol No. 01P-023-066 : : ' o Prep ate(i); '; Ion Pair Standard Carres-Plaids ' . Standard nnmbtn . EqulgmaxtoaiabcR . Sknipl matrix; : ^'T Final solvent and IN: V |i\ ' - .a ' i 'M^^frerWonr'' * ! " Tjeanaljrtt(i)i . Blank Onld/ideaUOcn > **$** 0200 ppnu . FC mixstd n p pret1o0.oHp"pm5: ' Sorrotale rtdronrox^lQQ ppm: WOS POSrMiFOSAA. ZtFOSZ PFOSEA M556 Ail Stdcooe Stdconr^ StdoncSNstdcooo Stdcon . Sldeooa Ami n|ML a g /a L ' a if tS ? S a g h i. os/mL notai. ipadmL 0200 0.507 0J3Z.iT 0201 0221 0201 . 0.010 OSCO 0207- 0J32 A * l\ 0221 0201 0.020 3.00 s x n s a z i m s m h 321 521 0.005 3.00 i s n SJ2 5.01 i >521 SM 0.010 33 5.07 332 >3.01F ,521 521 0.020 503) 50.1 332 50.1 rF 3 50.1 0.005 300 30.1 332 50.1-3} 52.1 > 50.1. 0.010 30.0 30.1 332 30.0 30.1 332 30.1 ` _S2.1 J ti ^ 50.1 * 0.015 30.1 J Z I Y 5ai 0.(00 AU Fmalvol mL ' 1.015 1223 1.010 1.013 . 1.023 1.010 1.015 1.020 1.025 nos FFoS FOSAA StFOSB PrOSXA^M554 "- imrrvptt 5 nnleape ' Finalcone Findcom Finitene Finalcotte F^eon Slcone AralxpOced "S^L atfmL nirimL ncAnL * agtaL nsffiLJsSnituL mL 4SS 9.78 24.1 495 97.8 . 2a 3.00 9.19 23.1 50.0 98.9 25! 5.24 4.94 5.0! 5.13.1ft 100 0405 1<U 9.78 9.93 KUV 2SJ 24.1 25.2 254 52.4 49.4 504 514 Final6QM` 104 97 994 102 283 4 25 25 *> 4ss . 735 976 500 . 324 494 748 . -- s i-- 737 989 1038 971 5I 7jj4j9 5)3 766 1017 ST ' Scram rros- PFOSA PFOSAA ZlfOSEOH PFOSXA *w.M3M. j rS " 3M-IM0 1 5.00-I05T| 5-00-10 1 3A0-10M | SJOMOOO | 3.00-1000 Wtm Estantesonly. Usevahic*Ibrrabbit tal Enmaitaonly. Usevaluesferrabbit. . ik- donkey* Piuma Estimatesonly. Userabiesforrabbit . Humaa Estimatesonly. Usovaluesforrabbit r O AtttctmcalC; Ion Pair Centre Analytical Laboratories, Inc. s 14 of 14 >>V`V Page 29 of29 Exygen Research Page 92 of 98 0e0923 Exygen Study No.: 023-066 3048 Research Drive, State Collect, PA 16801. Phone: (814)331-8032, Facsimile: (814)231-1253 PROTOCOL AMENDMENT Page 1 of 1 Centre Study Number 023-066 Amendment Number: 1 Effective Date: 06/27/01 Centre Protocol Number 01P-023-Q66 DESCRIPTION OF AMENDED SECTION 1. 7: Justification ft the Selection of the Test System Change the first sentence to read: )EDTO Mallard and quail red blood cells and serum samples collected by Wildlife International, Ltd. will be used as the analytical samples for this phase. RATIONALE 1. At the request of the sponsor, all serum samples are to be analyzed with this study. IMPACT ON THESTUDY 1. No negative impact on the study. t rI. Jkf- -h J L L . Studivy DDiirrvecrttnorr SQilgrnmaathuinrea * Date Vf t/ o f Centre QAU Review ( February 12,1998/1 i Exygen Research Page 93 of 98 G0 0 9 2 4 Exygen Study No.: 023-066 Exygen Research Page 94 o f 98 0.00925 Exygen Study No.: 023-066 Centre Analytical Laboratories, Inc:. 3048 Research Drive, State Cofleje, PA 16801. Phones (814) 231-8032, Facsimiles (814) 231-1253 PROTOCOL DEVIATION Deviation Number 1 ` Date of Occurrence: (1,2) 05/23/01, (3) 05/24/01 Centre Study Number 023-066 Centre Protocol Number 01P-023-066 DESCRIPTION OF DEVIATION 1. 9 Analytical Method, Sample Analysis d.: Quantified Centre sample 0107261 and its duplicate injection when the concentrations were greater than that of the highest standard in Set 052201AD. {r @> 2. $ 9 Analytical Method Sample Analysis e.: Accepted recoveiry of 76%jthe duplicate injection of Centre sample 0107378 Spit A2 in Set 052201B. 3. 1. 9 Analytical Method, Sample Analysis d.: Quantified Centre samples 0107092 and 0107134 and their duplicate injections when the concentrations were greater than that of the highest standard in Set 052201CD. ACTIONS TAKEN i.e- amendment issued. SOP revision, etc... 1-3. Protocol deviation issued. Recorded By/Date:_________ IMPACT ON THE STUDY 1,3. No negative impact because the areas were < 15% greater than the highest standard. 2. No negative impact because the overall average of all of the fortifications were within the acceptable range. :ipal Investigator Signature Study Director Signature / Date1 sV4i Date Spctsor Management Signature D ate HL .e d ito r i CAL QAU Review. CL 9/y/d? W rfQ r e - itv*-ec) W - Vo SuspeoK d Febtuaty 12,1998/2 of''W v6. Exygen Research Page 95 of 98 00926 Exygen Study No.: 023-066 Centre Analytical Laboratories, Inc. 3048 Research Drive, State College, FA 16801. Phone: (814) 231-8032, Facsimile: (814) 231-1253 PROTOCOL DEVIATION Deviation Number: 2 ' Date of Occurrence: (1)5/18/01 Centre Study Number: 023-066 Centre Protocol Number: 01P-023-066 DESCRIPTION OF DEVIATION 1. 8 9 Analytical Method, 3. Sample volumes used for Centre samples 0107080,0107081,0107084, and 0107085 were 0.05 jtl~ 0.03 jtL, 0.025 jsL. and 0.025 jiL, respectively. IfJiJ m M jri 1. Protocol deviation issued. ACTIONS TAKEN i,&.mariam issued,SOPrevision,w - Recorded By/Date:________ IMPACT ON THE STUDY 1. No negative impact because the actual volume was used in the calculations. incipal Irttestigator Signature Dat1e $ - Study Director Signature Date z v fc fio fe 1 Date CAL QAU Review. UC.(^ February 12,1998/2 (W a b c r i H \oe-\Ncc u pecteP 0>*s o fH n t q |n k Exygen Research Page 96 of 98 099977 Exygen Study No.: 023-066 Centre Analytical Laboratories, Inc:. 3048 Research Drive, State College, PA 18801. Phone: (814) 231-8032, Facsimile: (814) 231-1253 PROTOCOL DEVIATION Deviation Number. 3 ' Date of Occurrence: (1) 7/23/01 Centre Study Number. 023-066 Centre Protocol Number. 01P-023-066 DESCRIPTION OF DEVIATION 1. 9 Analytical Method, 3. Sample volumes used for Centre samples 0107159,0107167,0107168, and 0107209 were 0.05 mL. ACTIONS TAKEN Le., amendmentissued. SOP revision,etc,. 1. Protocol deviation issued. Recorded By/Date:________ IMPACT ON THE STUDY 1. No negative impact because die actual volume was used in the calculations. kici\>al Investigator Signature Study Director Signature ' ^ 4/ S p o sar Management Signature Date sink Date Date C A L Q A U R eview ________ February 12,1998/2 1i:- Exygen Research Page 97 of 98 0-C09?8 en RESEARCH Precise Research. Exygen Study No.: 023-066 Exygen Research Page 98 of 98