Document n9J4g9Jde2kV0NJ6o6Zz1zJLX

3M General OMices Iw f f 3M & H Q f 3M Center St. Paul, MN 55144-1000 612 733 MIO A. VIB _ \0lOe, 0 1 7 H 0- G8 September 5, 2001 Document Processing Center (7407) Office o f Toxic Substances U.S. Environmental Protection Agency 401 M Street, SW Washington, DC 20460 Attn: TSCA Section 8(e) Coordinator 3?3 ( ( q -% Dear Section 8(e) Docket Coordinator: Re: TSCA 8(e) Supplemental Notice on Sulfonate-based Fluorochemicals With this letter, 3M is providing final reports and other supplemental information related to previous TSCA Section 8(e) notifications. Many o f the enclosed items are analytical reports providing blood serum and liver levels o f test materials for which the in-life report referring to administered doses has already been submitted to the 8(e) docket. In other cases where the 8(e) notification consisted o f preliminary data, we are submitting a final study report. All o f the enclosed items are already in EPA's possession and available in TSCA Docket AR-226. We believe, however, that placing these items in the 8(e) docket may allow for more convenient access to information directly related to previous 8(e) notifications by 3M. The table below lists the enclosed items and references the study or data which already has been the subject o f an 8(e) notification by 3M: Attached Submission Related Study/Data Already Filed Under 8(e) 1. Amended Analytical Study, 2(N-Ethylperfluorooctane Combined Oral (Gavage) Fertility, sulfonamido)-ethanol in Two Generation Rat Reproduction, Developmental and Perinatal/Postnatal Determination of the Presence and Concentration of PFOS, Reproduction Toxicity Study of N- M556, PFOSAA, and PFOSA in the Liver and PFOS, EtFOSE in Rats, 3M Reference No. Tj-o M556, PFOSAA, PFOSA and EtFOSE-OH in the Sera of 6316.5, June 30, 1999, full report Crl.CDBR VAF/Plus Rats Exposed to EtFOSE-OH, 3M submitted February 15, 2000 to Reference No. T-6316.5, Analytical Report TOX-013, LRN-U2095, June 11,2001. supplement earlier filing ~o no cn 32* Contain NO CBl CO CO o RECEIVED TSCA Section 8(e) Docket Coordinator Page 2 . Attached Submission Related Study/Data Already Filed Under 8(e) 2. Analytical Laboratory Report, Determination of the Presence and Concentration of Potassium Perfluorooctanesulfonate (CAS Number: 2759-39-3) in the Serum and Liver of Sprague-Dawley Rats Exposed to PFOS via Gavage, Laboratory Report No. U2006, Requestor Project No. 3M TOX 6295.9, October 27, 1999. 3. Report Amendment 1, Combined Oral (Gavage) Fertility, Developmental and Perinatal/Postnatal Reproduction Toxicity Study of PFOS in Rats, Argus Research Laboratories, Inc., Protocol 418-008, Sponsor's Study No. 6295.9, April 13,2000. Combined Oral (Gavage) Fertility, Developmental and Perinatal/Postnatal Reproduction Toxicity Study of PFOS in Rats, Argus Research Laboratories, Inc., Sponsor's Study No. 6295.9, June 10, 1999, full report submitted February 15, 2000 supplementing earlier filing 4. Analytical Report, Determination of the Presence and Concentration of Perfluorooctanesulfonate, Perfluorooctanesulfonylamide, M556, and M570 in the Liver and Sera Samples, 3M Environmental Laboratory Ref. No. U2636, TOX-028, February 23, 2001 13-Week Dietary Study of N-Methyl Perfluorooctanesulfonamido Ethanol (N-MeFOSE) in Rats, 3M Ref. No. T6314.1, Covance Study No. 6329-225, dated June 30, 2000, Section 8(e) filing July 24, 2000 5. Analytical Laboratory Report, Determination of the Concentration of PFOS, PFOSA, PFOSAA, and EtFOSEOH in the Sera and Liver of Crl.CDBR VAF/Plus Rats Exposed to N-EtFOSE, 3M Environmental Laboratory Report No. TOX-098, Laboratory Request No. U2402, 3M Ref. No. T-6316.7, February 6, 2001. Final Report, Oral (Gavage) Developmental Toxicity Study of 2(NEthylperfluorooctanesulfonamido)ethanol in Rats, 3M Reference No. T6316.7, December 17, 1998, submitted to Section 8(e) docket per letter of August 21, 2000 6. Analytical Laboratory Report on the Determination of the Presence and Concentration of Potassium Perfluorooctanesulfonate (PFOS) or another metabolite of 2(N-ethylperfluorooctanesulfonamido)-ethanol(N- EtFOSE) in Liver and Serum Specimens, 3M Environmental Laboratory Report No. TOX-097, Laboratory Request No. U2452, 3M Ref. No. T-6316.8, February 8,2001 Final Report, Oral (Stomach Tube) Developmental Toxicity Study of NEtFOSE in Rabbits, 3M Reference No. T-6316.8, January 11, 1999, submitted to Section 8(e) docket per letter of August 21,2000 7. Final Report, Alexander, B., Mortality Studies of Workers Employed at the 3M Decatur Facility, University of Minnesota, April 26, 2001. Preliminary data submitted to Section 8(e) docket in letter of December 15, 2000 TSCA Section 8(e) Docket Coordinator Page 3 . Attached Submission Related Study/Data Already Filed Under 8(e) 8. Final Report, Acute Oral Toxicity Screen with T-3290CoC Acute Oral Toxicity Screen with T- in Albino Rats, Safety Evaluation Laboratory, Riker 3290CoC in Albino Rats, Safety Laboratories, Inc., Project No. 0882AR0362, 3M Reference Evaluation Laboratory, Riker No. T-3290 (40 %K+PFOSAA in 3 % EtOH, 17 %IPA Laboratories, Inc., Project No. and 40 % H20, L-6778, F-6873, Lot 501), November 5, 0882AR0362, 3M Reference No. T- 1982 [B ibliography entry in Docket AR-226, fin a l report 3290 (40 %K+PFOSAA in 3 % EtOH, was to be m oved to TSCA 8(e) docket] 17 % IPA and 40 %H20, L-6778, F- 6873, Lot 501), November 5, 1982, submitted to Section 8(e) docket in August 21, 2000 self-audit letter (which erroneously refers to rabbits rather than rats) 9. Giesy, J.P., and K. Kannan, Accumulation of Perfluorooctanesulfonate and Related Fluorochemicals in Fish Tissue, Michigan State University, June 20, 2001. Preliminary data submitted to Section 8(e) docket May 26, 1999 10. Giesy, J.P., and K. Kannan, Accumulation of Perfluorooctanesulfonate and Related Fluorochemicals in Mink and River Otters, Michigan State University, June 20, 2001. 11. Giesy, J.P., and K. Kannan, Perfluorooctanesulfonate and Related Fluorochemicals in Oyster, Crassostrea Virginica, From the Gulf of Mexico and Chesapeake Bay, Michigan State University, June 20, 2001. 12. Giesy, J.P. and K. Kannan, Perfluorooctanesulfonate and Related Fluorochemicals in Fish-Eating Water Birds, Michigan State University, June 20, 2001. 13. Giesy, J.P. and K. Kannan, Accumulation of Perfluorooctanesulfonate and Related Fluorochemicals in Marine Mammals, Michigan State University, June 20, 2001. If you have any questions about this submission, please contact me at (651)737-4795. Sincerely, Enclosures Geoqe^n Adams Manager, 3M Corporate Product Responsibility 3M Medical Department Study: T-6314.1 Analytical Report: FACT TOX-028 LRN-U2636 Study Title Determination of the Presence and Concentration of PFOS, PFOSA, M556, and M570 in the 13-Week Dietary Study of CrliCD (SD) IGS BR Rats Analytical Laboratory Report Title Determination of the Presence and Concentration of Perfluorooctanesulfonate, Perfluorooctanesulfonylamide, M556 and M570 in Liver and Sera Samples Data Requirement Not Applicable Author 3M Environmental Laboratory Study Completion Data February 23, 2001 Performing Laboratories Liver and Sera Analyses Liver and Sera Extractions 3M Environmental Laboratory Building 2-3E-09, 935 BushAvenue St Paul, MN 55106 Pace Analytical Tier II Laboratory, Inc. 1700 Elm Street Suite 100 Minneapolis, MN 55414 Project Identification 3M Study: T-6314.1 Covance In-Life Study: 6329-225 Analytical Report: FACT-TOX-028 3M Laboratory Request No. U2636 Total Number of Pages 130 Co O A EPA-OTS 000811828S oDoanaaas 3M Environmental Laboratory -,M Co age 1 3M Medical Department Study: T-6314.1 BACK TO MAIN Analytical Report: FACT TOX-028 LRN-U2636 This page has been reserved for specific country requirements. 3M Environmental Laboratory Page 2 3M Medical Department Study: T-6314.1 BACK TO MAIN Analytical Report: FACTTOX-028 . LRN-U2636 GLP Compliance Statement Analytical Laboratory Report Title: Determination of the Presence and Concentration of PFOS, PFOSA, M556, and M570 in the 13-Week Dietary Study of Crl:CD (SD) IGS BR Rats Study Identification Numbers: T-6314.1, FACT-TOX-028, LRN-U2636 The determination of M570 in liver and sera specimens will be conducted according to documented, but not validated methods. This study was conducted in compliance with United States Food and Drug Administration (FDA) Good Laboratory Practice (GLP) Regulations for Nondinical Laboratory Studies, Final Rule 21 CFR Part 58, with the exceptions in. the bulleted list below. Exceptions to GLP compliance: Analytical standard stability was not yet determined. , Purity of the following analytical reference materials have not been completed at this time; PFOSA, M556 and M570. Occasionally, corrections were not made according to 21 CFR 58.130 (e). The electronic data systems have not been validated and there is not an electronic audit trail of corrections currently available. Authenticated hard copies o f chromatography and associated documents will be considered as the original raw data. Results of purity and stability analysis of the test material have not been completed by the sponsor. This information will be included in the final toxicology report. 7' ry ? ' Andrew Seacat Ph.D., Study Director 3/ a / Date Date Date 3M Environmental Laboratory Page 3 BACK TO MAIN 3M Medical Department Study: T-6314.1 , Analytical Report: FACT TOX-028 LRN-U2636 GLP Study--Quality Assurance Statement Analytical Laboratory Report Title: Determination of the Presence and Concentration of PFOS, PFOSA, M556, and M570 in the 13-Week Dietary Study of Crl:CD (SD) IGS BR Rats Study Identification Numbers: T-6314.1, FACT-TOX-028, LRN-U2636 This study has been inspected by the 3M Environmental Laboratory Quality Assurance Unit (QAU) as indicated in the following table. The findings were reported to the study director and laboratory management. . Inspection Dates Phase Date Reported to Management Study Director 10/30/00 Analysis 10/31/00 10/31/00 10/10/00 11/13-17/00,11/20/00, 11/27-28/00 12/19/00,12/21/00 12/27-12/28/01 02/05-02/06/01 Extraction Data Draft report #1 Draft report #2 10/11/00 11/28/00 01/02/01 02/07/01 10/11/00 11/28/00 01/02/01 02/07/01 7 /Z? to ( Date 3M Environmental Laboratory Page 4 BACK TO MAIN 3M Medical Department Study: T-6314.1 Analytical Report: FACT TOX-028 LRN-U2636 Table of Contents GLP Compliance Statement.................................................................................................3 GLP Study--Quality Assurance Statement......................................................................... 4 Study Personnel and Contributors....................................................................................... 8 Introduction and Purpose.................................................................................................... 9 Test System.................................................................................................................... 9 Specimen Collection and Analyses................................................................................. 10 Specimen Receipt and Maintenance................................................................................... 10 Chemical Characterization of Reference Materials..............................................................11 Method Summaries.............................................................................................................. 12 Preparatory Methods................................................................................................. 12 Analytical Methods......................................................................................................12 Analytical Equipment..................................................................................................13 Data Quality Objectives and Data Integrity..........................................................................14 Data Summary, Analyses, and Results...............................................................................14 Summary of Quality Control Analyses Results................................................................14 Statement of Data Quality...............................................................................................16 Summary of Analytical Sample Results.......................................................................... 16 Statistical Methods and Calculations................................................................................... 17 Statement of Conclusion......................................................................................................17 Appendix A: Control Matrices and Test Substance Characterization................................. 18 Test Substance Characterization.................................................................................... 18 Appendix B: Protocol, Amendments and Deviations........................................................... 19 Appendix C: Extraction and Analytical Methods................................................................. 45 ETS-8-4.1, "Extraction of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds from Serum for Analysis Using HPLC-Electrospray/Mass Spectrometry," (14 pages).............................................................................................................................46 ETS-8-6.0, "Extraction of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds from Liver for Analysis Using HPLC-Electrospray/Mass Spectrometry," (14 pages)................................................................................................................................... 60 ETS-8-5.1, "Analysis of Potassium Perfluorooctanesulfonate or Other Fluorochemicals in Serum Using HPLC-Electrospray/Mass Spectrometry," (9 pages)................................. 74 ETS-8-7.0, "Analysis of Potassium Perfluorooctanesulfonate or Other Fluorochemicals in LiverUsing HPLC-Electrospray/Mass Spectrometry," (10 pages)................................... 85 Appendix D: Data Tables..................................................................................................... 97 Appendix E: Data Spreadsheets.......................................................................................... 105 Appendix F: Example Calculations.......................................................................................124 Appendix G: Interim Certificate(s) of Analysis..................................................................... 126 3M Environmental Laboratory Page 5 BACK TO MAIN 3M M edical D epartm ent Study: T-6314.1 Analytical Report: FAC T TO X -028 LR N -U 2 6 36 Appendix H: Report Signature Page............................ ...................................................... 130 3M Environmental Laboratory Page 6 BACK TO MAIN 3M Medical Department Study: T-6314.1 Analytical Report: FACT TOX -028 LRN-U2636 List of Tables Table 1. Dosage Levels, Dose Groups and Group Composition for Study 6329-225...... 9 Table 2. Characterization of the Analytical Reference Materials in Study FACT-TOX-028 11 Table 3. Negative Ions Monitored in 3M Laboratory Analyses.......................................... 13 Table 4. Matrix Spike/Matrix Spike Duplicate results for M556 in Sera..............................15 Table 5. Matrix Spike/Matrix Spike Duplicate results for PFOSA and M556 inLiver........ 16 Table 6. Characterization of the Control Matrices Used for Liver and Sera Analyses in Study FACT-TOX-028.........................1...................................................................18 Table 7. Characterization of Test Substance in Study FACT-TOX-028............................. 18 Table 8. Rat Serum Week 5 Data for FACT-TOX-028....................................................... 97 Table 9. Rat Serum Week 14 Data for FACT-TOX-028..................................................... 98 Table 10. Rat Liver Week 5 Data for FACT-TOX-028........................................................ 99 Table 11. Rat Liver Week 14 Data for FACT-TOX-028......................................................100 Table 12. Data Summary of Average Week 5 Rat Serum Concentration (pg/mL) and Standard Deviation (SD) for FACT-TOX-028...................................................... 101 Table 13. Data Summary of Average Week 14 Rat Serum Concentration (pg/mL) and Standard Deviation (SD) for FACT-TOX-028...................................................... 101 Table 14. Data Summary of Average Week 5 Rat Liver Concentration (pg/g) and Standard Deviation (SD) for FACT-TOX-028......................................................102 Table 15. Data Summary of Average Week 14 Rat Liver Concentration (pg/g) and Standard Deviation (SD) for FACT-TOX-028......................................................102 Table 16. Approximate LOQ Values Used in FACT-TOX-028 Analyses by Matrix, Compound and Usage Dates.................................................................................103 Table 17. FACT-TOX-028 Rat Liver Week 5 Precision Data for PFOS and PFOSA.........103 Table 18. FACT-TOX-028 Rat Liver Week 5 Precision Data for M556 and M570............ 104 3M Environmental Laboratory Page 7 BACK TO MAIN 3M Medical Department Study: T-6314.1 Analytical Report: FACT TOX-028 LRN-U2636 Study Personnel and Contributors Study Director Andrew M. Seacat, Ph.D. 3M Toxicology Services Building 220-2E-02, 3M Center St. Paul, MN 55144-1000 Sponsor 3M Toxicology Services Building 220-2E-02 St. Paul, MN 55144-1000 John L. Butenhoff, Ph.D., Sponsor Representative Analytical Chemistry Laboratories Liver and Sera A nalyses 3M Environmental Laboratory Kristen J. Hansen, Ph.D., P rincipal A nalytical In v e s tig a to r Liver and Sera Extractions Pace Analytical Tier II Laboratory Inc. 3M Laboratory Contributing Personnel Lisa A. Clemen Kelly J. Dorweiler* Barb A. Gramenz* Sarah A. Heimdal* Marlene M. Heying* Harold O. Johnson ' Contract lab professional service employees Kelly J. Kuehlwein* Sally A. Linda* Joe Pilon* Thomas P. Wagner* Bob W. Wynne* Richard D. Youngblom* Location of Archives All original raw data, protocol, and analytical report have been archived at the 3M Environmental Laboratory. The test substance and analytical reference standard reserve samples, as well as the specimens pertaining to the analytical phase of this study are archived at the 3M Environmental Laboratory for a minimum of 10 years. 3M Environmental Laboratory Page 8 BACK TO MAIN 3M Medical Department Study: T-6314.1 Analytical Report: FACT TOX-028 LRN-U2636 Introduction and Purpose The purpose of the analytical phase of this study is to evaluate levels of PFOS, PFOSA, M556, and M570 In liver and sera specimens of the rats exposed to N-MeFOSE-OH (T-6314). The in-life phase of this study was conducted at Covance Laboratories, Inc., (Covance Study Number 6329 225). The in-life data, dose preparation and analysis are reported in Covance Study Number Report 6329-225. The analytical portion of the study was initiated on 23 August 2000. Test System The test system species and strain selected was the Crl:CD(SD) IGS BR rats received from Charles River Laboratories, Inc. Four dose groups comprised of 20 male and 20 female rats each (80 males and 80 females total) were fed daily nominal doses of O-(control), 3-(low dose), 30-(mid dose), or 100-(high dose) ppm of N-MeFOSE-OH (T-6314) in their feed for at least 13 weeks. After 4 weeks, liver and sera specimens were collected from five-animals/sex/dose group. At the end of the study (after week 13), liver and sera specimens were collected from all remaining animals in the test system. All liver and sera specimens were sent to the 3M Environmental Laboratory for analysis. Table 1. Dosage Levels, Dose Groups and Group Composition for Study 6329-225 Dose Group Number of Animals Dietary Levels3 1 (Control h) 20 male / 20 female 2 (Low Dose) 20 male / 20 female 3 (Mid Dose) 20 male / 20 female 4 (High Dose) 20 male / 20 female aDose Levels are expressed as ppm of N-MeFOSEI-OH. bControl Group animals received a basal diet only. 0 ppm 3 ppm 30 ppm 100 ppm 3M Environmental Laboratory Page 9 BACK TO MAIN 3M Medical Department Study: T-6314.1 Analytical Report: FAC T TO X -028 L R N -U 2 6 36 Specimen Collection and Analyses In the analytical study reported here, 160 liver (80 male and 80 female) and 80 (40 male and 40 female) sera specimens were collected during the course of the in-life phase of Covance Study 6329-225 and sent to the 3M Environmental Laboratory to be extracted and analyzed for PFOS, PFOSA, M556 and M570. The study director determined that it was appropriate only to analyze the liver samples that have corresponding sera samples. There was a total of 80 liver analyses and a total of 80 sera analyses for each of the four compounds. Blood specimens were allowed to clot at room temperature and centrifuged. Serum was then harvested and stored in a freezer set to maintain specimens at -60C to -80C until shipped to the 3M Environmental Laboratory. Liver specimens collected from each animal were flash frozen in liquid nitrogen and then stored in a freezer set to maintain specimens at -60C to -80C until shipped to the 3M Environmental Laboratory. Liver and sera specimens were shipped to the 3M Environmental Laboratory frozen and on dry ice 22 December 1998 and 13 October 1998, respectively. Liver and Sera samples were sent to Pace Analytical Tier II Laboratory, Inc. on 21 September 2000. The sera and livers were extracted using 3M Environmental Laboratory analytical methods beginning on 25 September 2000 and 10 October 2000, respectively. Analytical samples were extracted using an ion-pairing procedure. An ion-pairing reagent was added to the laboratory sample and the analyte ion pair was partitioned into methyl-ferf-butyl ether (MtBE). The MtBE extract was removed and put onto a nitrogen evaporator until dry. Each extracted laboratory sample was then reconstituted in 1.0 mL of methanol and filtered through a 3 cc plastic syringe attached to a 0.2 pm nylon filter into a glass autovial. Liver samples were homogenized prior to the extraction procedure. The extracts were returned 11 October 2000 to the 3M Environmental Laboratory after the extractions were completed. Sample analyses were performed by monitoring one product ion selected from a single primary ion characteristic of a particular fluorochemical using HPLC-ES/MS/MS in the multiple reaction monitoring. Analytical details are included in this report. The PFOS data reported has been adjusted to reflect the purity determined for the analytical reference material. (See table 2). Specimen Receipt and Maintenance The 3M Environmental Laboratory received liver and sera specimens on 13 October 1998 and 22 December 1998 that were collected during and at the end of the in-life phase of Covance Study 6329-225. All specimens were received frozen on dry ice and were immediately transferred to storage at -20C 10C. 3M Environmental Laboratory Page 10 3M Medical Department Study: T-6314.1 Chemical Characterization of Reference Materials BACK TO MAIN Analytical Report: FACTTOX-028 LRN-U2636 Table 2. Characterization of the Analytical Reference Materials in Study FACT-TOX-028 Material Identification Chemical Formula Source Expiration Date Storage Conditions Chemical Lot Number Physical Description Purity KPFOS* Potassium Perfluorooctane suifonate C 8F 17S 0 3 'K * 3 M Specialty Chem icals 01/01/2010 Room Tem perature Lot 171 Light colored powder 86.4% PFOSA Peril uorooctanesulfonylamide CgF17S O 2N H 2 3 M Specialty Chem icals 01/01/2010 Room Tem perature L-15709 Light yellow w a x y solid TBDb M556 M556 C aFffS C W H ) (C H 2C O O H ) 3M Specialty Chem icals 01/01/2010 Room Tem perature 3M # 113047-80 W hite powder TBDb M570 M 570 C 8F17S 02N (C H 3) (C H 2C O O H ) 3 M Specialty Chem icals 01/ 01/2010 Room Tem perature 3M # 118506-26 W hite powder TBDb TBD - To be determined a The target analyte is PFOS (Perfluorooctanesuifonate), C8 F17SO3' b The purity of the substances listed above were nominally determined by NMR analyses. Subsequent chemical characterization are occurring and this analytical report will be amended to indicate the purity of these substances when a certificate of analysis are issued. 3M Environmental Laboratory Page 11 BACK TO MAIN 3M Medical Department Study. T-6314.1 Analytical Report: FACT TOX-028 LRN-U2636 Method Summaries Following is a brief description of the methods used for extraction and analyses during this analytical study by the 3M Environmental Laboratory. Detailed descriptions of the methods used in this study are located in Appendix C. The preparatory methods used by the technicians at the Pace Analytical Tier II Laboratory, Inc. are methods from and developed by the 3M Environmental Laboratory. Preparatory Methods % ETS-8-4.1, "Extraction of Potassium Perfluorooctanesulfonate or other Fluorochemical Compounds from Serum for Analysis Using HPLC-Electrospray/Mass Spectrometry" ETS-8-6.0, "Extraction of Potassium Perfluorooctane-sulfonate or other Fluorochemical Compounds from Liver for Analysis using HPLC-Electrospray/Mass Spectrometry" Both extraction methods use an ion-pairing procedure. An ion-pairing reagent was added to the laboratory sample and the analyte ion pair was partitioned into methyl-te/f-butyl ether (MtBE). The MtBE extract was removed and put onto a nitrogen evaporator until dry. Each extracted laboratory sample was then reconstituted in 1.0 mL of methanol and filtered through a 3 cc plastic syringe attached to a 0.2 pm nylon filter into a glass autovial. Liver samples were homogenized prior to the extraction procedure. Analytical Methods % ETS-8-5.1, "Analysis of Potassium Perfluorooctanesulfonate or other Fluorochemicals in Serum Extracts Using HPLC-Electrospray/Mass Spectrometry" ETS-8-7.0, "Analysis of Potassium Perfluorooctanesulfonate or other Fluorochemicals in Liver Extracts Using HPLC-Electrospray/Mass Spectrometry" These methods describe the analysis of fluorochemical surfactants from liver and serum extracts using HPLC-ES/MS/MS. The analysis was performed by monitoring a product ion selected from a single primary ion characteristic of a particular fluorochemical. For example, molecular ion 499, selected as the primary ion for PFOS (CsF^SCV) analysis, was fragmented to produce ion 99 (FS03"). The characteristic ion 99 was monitored for quantitative analysis. 3M Environmental Laboratory Page 12 BACK TO MAIN 3M Medical Department Study: T-6314.1 Analytical Report: FAC T TO X -028 LR N -U 2 6 36 Analytical Equipment The analytical equipment settings used in the present analytical phase of this study may have varied slightly during data collection. The following is representative of the settings used during the analytical phase of this study. Liquid Chromatograph: Hewlett-Packard Series 1100 Liquid Chromatograph system Analytical column: Keystone BetasilTM C182x50 mm (5 pm) Column temperature: Ambient Mobile phase components: Component A: 2mM ammonium acetate Component B: methanol Flow rate: 300 pL/min Injection volume: 10 pL Solvent Gradient: 10.0 minutes Time (minutes) %B 0.0 10% 1.0 10% 5.5 95% 7.5 95% 8.0 10% Mass Spectrometer: Micromass API/Mass Spectrometer Quattro IITMTriple Quadrupole system Software: Mass LynxTM 3.4 Cone Voltage: 30-60 V Collision Gas Energy: 25-45 eV Mode: Electrospray Negative Source Block Temperature: 150C 10C Electrode: Z-spray Analysis Type: Multiple Reaction Monitoring (MRM) Table 3. Negative Ions Monitored in 3M Laboratory Analyses Target Analyte Primary Ion (a m u ) Product Ion (a m u ) PFOS 499 99 PFOSA 498 78 M556 556 169 M570 570 169 THPFOS* ` Internal Standard 427 80 3M Environmental Laboratory Page 13 BACK TO MAIN 3M Medical Department Study: T-6314.1 Analytical Report: FACTTOX-028 LRN-U2636 Data Quality Objectives and Data Integrity The following data quality objectives (DQOs) were indicated in the protocol for this study: Linearity: The coefficient of determination (r2) must be equal to or greater than 0.985 using either no weighting or 1/x weighting as appropriate. % Lim its o f Quantitation (LOQ): The LOQ was equal to the lowest acceptable standard in the calibration curve. % Acceptable Precision: Precision should be better than 30% for the method. % Acceptable Spike Recoveries: 70-130% unless otherwise documented. % Demonstration of Specificity: Compound identification will be substantiated by chromatographic retention time (within 5%) and by selection of a characteristic product ion from a characteristic primary ion. See table 3. Data Summary, Analyses, and Results Data quality objectives for the analytical phase of this study outlined in the 3M Environmental Laboratory protocol for FACT-TOX-028 were met with the exceptions noted in this report (see Appendix B). Summary of Quality Control Analyses Results % Linearity: The coefficient of determination (r2) of the standard curves was >0.985. % Calibration Standards: Calibration curves were prepared at concentrations within the linear range of the instrument and were not diluted. Quantitation of the target analyte was based on linear regression analysis (1/x weighting) of one or two extracted matrix curves bracketing each group of samples. High or low points on the curve may have been deactivated to provide a better linear fit over the curve range most appropriate to the data. Low curve points with peak areas less than two times that of the extraction blanks were deactivated to disqualify a data range that may have been significantly affected by background levels of the analyte. Occasionally, a single mid-range curve point that was an obvious outlier may have been deactivated. Quantitation of each analyte was based on the response of one specific product ion using the multiple response-monitoring mode of the instrument (see Appendix C, Analytical Methods). % Lim its o f Quantitation (LOQ): The LOQ is equal to the lowest acceptable standard in the calibration curve (defined as a standard within 30% of the theoretical value), and is at least two times the analyte peak area detected in the extraction blanks. (Appendix D, Table 16). v Blanks: All blanks were below the lower limit of quantitation for the compounds of interest. To simplify analyses that were complicated by endogenous levels of fluorochemicals in unexposed rat sera and liver, rabbit sera and liver were selected as a suitable surrogate matrices for the preparation of calibration curves and continuing calibration verification standards. Non-exposed rat sera was used as the control matrix for matrix spike studies. 3M Environmental Laboratory Page 14 BACK TO MAIN 3M Medical Department Study: T-6314.1 Analytical Report: F A C T T O X -0 2 8 LR N -U 2 6 36 Precision: Precision was determined by a triplicate analysis of one extract and was reproducible to within 5% for all analytes. Reproducibility: At the request of the study sponsor, data was collected to assess the reproducibility of extraction/homogeneity of distribution of the target analytes in liver of dosed test animals. This was conducted by extracting duplicate liver samples for each of six, group 2 animals; a separate homogenate was prepared for each of the 12 extractions. The samples were evaluated against an extracted curve and the percent difference in the levels of each analyte was calculated. Results of this analysis are presented in Appendix D, tables 17 and 18. Note that the samples were not spiked. Reproducibility was assessed based on the non spiked levels of analytes. Matrix Spikes: Matrix spike studies were prepared to approximate concentrations in Group I samples. Matrix spikes and matrix spike duplicates (MS/MSD) were extracted with each set of samples and analyzed during analytical runs at the 3M Environmental Laboratory. Sera Spike Recoveries: Two sets of MS/MSD were prepared with the sera samples. All matrix spikes were within acceptance criteria, with the exception of RTS09250-250 ppbMS-5 and -MSD-5 for M556. These spikes were evaluated at 135 and 140% recovery. Because all the other analyte spikes were within acceptance criteria, it is unlikely that these high recoveries are a result of spiking error. The high recovery results were confirmed, but cannot be explained. An additional four matrix spikes were prepared and analyzed. These four matrix spikes were within acceptance criteria. (See table 4). Table 4. Matrix Spike/Matrix Spike Duplicate results fo r M556 in Sera Matrix/Compound Original MS/MSD Recoveries (%) Re-extract MS/MSD Recoveries (%) Sera/M556 135 140 128 116 118 118 124 128 Liver Spike Recoveries: Two sets of MS/MSD were prepared with the liver samples. Of the four spikes, a single spike, C95991F/G1/wk5-MSD-250 ppb was low for PFOSA and M556. An additional four matrix spikes were prepared and analyzed. These four matrix spikes were within acceptance criteria for PFOSA. Some responses for M556 were still lower than expected. The average recovery for the 8 prepared matrix spikes was 66%. (See table 5). 3M Environmental Laboratory Page 15 3M Medical Department Study: T-6314.1 BACK TO MAIN A nalytical Report: FA C T T O X -028 L R N -U 2 6 36 Table 5. Matrix Spike/Matrix Spike Duplicate results for PFOSA and M556 in Liver Original Re-extract Matrix/Compound MS/MSD MS/MSD Recoveries (%) Recoveries (%) Liver/PFOSA Liver/M556 92 89 78 58 79 84 74 42 86 99 90 107 41 63 71 70 Internal Standards: The internal standard (IS), THPFOS was added to all samples and standards. THPFOS was not used for quantitation, but was used to monitor for gross instrument failure. The IS response of each analytical run was verified to determine that it did not vary more than 50% from the mean within each analytical run. IS responses deviated by > 50% in several sera and liver analyses. These responses were confirmed but cannot be attributed to a specific problem. This data is flagged in the raw data table but no further action is being taken, as more suitable internal standards (e.g. radio labeled material) are not available. Statem ent of Data Quality It is not possible to verify true recovery of endogenous analyte from tissues without radio labeled reference material. The only measurement of accuracy available at this time, matrix spike studies, indicates the data are quantitative to 30%. Determination of M556 levels in liver may be biased low and should be considered quantitative to 50%. Summary of Analytical Sample Results Samples from Control Animals: Low levels of some analytes were often detected in the sera and liver of some the control animals. These levels were significantly lower than those found in the low dose test animals. Samples from Dosed Animals: In general, analyte levels found in the sera and liver of the test animals increased with dose group. Analyte levels increased as dosage increased. Detailed sample data tables are presented in Appendices D and E. 3M Environmental Laboratory Page 16 BACK TO MAIN 3M Medical Department Study: T-6314.1 Analytical Report: FACTTOX-028 LRN-U2636 Statistical Methods and Calculations Statistical methods were limited to the calculation of means and standard deviations. See Appendix F for example calculations used to generate the liver and serum sample data in FACTTOX-028. When one or more sample results within a group was below the LOQ, calculations for average were done using the LOQ value in the calculation Statement of Conclusion Under the conditions of the present studies, the fluorochemicals PFOS, PFOSA, M556 and M570 were observed in the liver and sera of all rats dosed with the N-MeFOSE-OH during the in-life phase of the study. 3M Environmental Laboratory Page 17 3M Medical Department Study: T-6314.1 BACK TO MAIN Analytical Report: FACT TOX-028 LRN-U2636 Appendix A: Control Matrices and Test Substance Characterization Table 6. Characterization of the Control Matrices Used for Liver and Sera Analyses in Study FACT-TOX-028 Control Matrix Rabbit Liver Rat Semm Rabbit Serum Source Expiration Date Storage Conditions Chem ical Lot # Physical Description Covance Laboratory /0 1 0 1 / 2 0 1 0 -20C 10C F 0 4 0 5 1 -F e m a le Rabbit Liver Sigm a Chemicals Sigm a Chemicals /0 1 0 1 / 2 0 1 0 -20C 10C 19 H 8 9 2 9 2 Rat Serum /0 1 0 1 / 2 0 1 0 -20C 10C 118H8418 Rabbit Serum Test Substance Characterization N-Methyl Perfluorooctanesulfonamido Ethanol (N-MeFOSE-OH) CAS Number: 24448-09-7 Chemical Formula: C8F17S 02N ((CH3)(CH2CH2OH)) Molecular Weight: 557.0 Table 7. Characterization of Test Substance in Study FA C T-TO X-028 Test Substance Chemical Name Source Expiration Date N -M e th yl Perfluorooctanesulfonam ido -ethanol (N -M eFO S E -O H ) 3M Specialty Chemicals unknown Storage Conditions Am bient temperature Chemical Lot # T-6314 Based on information obtained from the in-life protocol. 3M Environmental Laboratory Page 18 3M Medical Department Study: T-6314.1 BACK TO MAIN Analytical Report: FAC T TO X -028 LR N -U 2 6 36 Appendix B: Protocol, Amendments and Deviations 3M Environmental Laboratory Page 19 3M Medical Department Study: T-6314.1 BACK TO MAIN Analytical Report: FACT TQX-028 S tu d y T itle Determination of the Presence and Concentration of PFOS, PFOSA, M556, and M570 in the 13-Week Dietary Study of Crl:CD(SID) IGS BR Rats ANALYTICAL PHASE PROTOCOL A n F D A G o o d L a b o ra to ry P ra c tic e (G LP ) C o m p lia n t S tu d y D ate: 15 August 2000 P e rfo rm in g L a b o rato ries 3M Environmental Technology & Safety Services 3M Environmental Laboratoiy 935 Bush Avenue St. Paul, MN 55106 Pace Analytical Services, Inc. 1700 Elm Street SE, Suite 100 Minneapolis, MN 55414 L a b o ra to ry P ro je c t Id e n tificatio n FACT Tox Number: Tox-028 LRN Number: U2636 3M Environmental Laboratory 3M Environmental Laboratory Page 1 o f 11 Page 20 3M Medical Department Study: T-6314^ BACK TO MAIN Analytical Report: FAC T TO X -028 Protocol Number: FACT-Tox-028 Lab Request Number (LRN): U2636 Study Identification Determination of the Presence and Concentration of PFOS, PFOSA, M556, andM570 in the 13-Week Dietary Study of Crl:CD(SD) IGS BR Rats T e s t A rtic le Sponsor S p o n s o r R e p re s e n ta tiv e S tu d y D ire c to r S tu d y L o c atio n (s) In vivo Testing Facility Analytical Testing Laboratories P ro p o s e d S tu d y T im e ta b le In-Life Experimental Start Date In-Life Termination Date Analytical Start Date Analytical Termination Date Study Completion Date N-Methyl Perfluorooctanesulfonamido Ethanol (N-MeFOSE, T-6314) 3M Toxicology Services-Medical Department 3M Center, Building 220-2E-02 St. Paul, MN 55144-1000 John L. Butenhoff, Ph.D. 3M Toxicology Services Telephone: (612) 733-1962 Andrew M. Seacat, Ph.D. 3M Toxicology Services Telephone: (612) 575-3161 Facsimile: (612) 733-1773 Covance Laboratories Inc. 3301 Kinsman Boulevard Madison, WI53704 3M Environmental Laboratory Building 2-3E-09 935 Bush Avenue St. P a u l,M N 55106 Pace Analytical Services, Inc. 1700 Elm Street SE, Suite 100 Minneapolis;, MN 55414 01 September 1998 03 December 1998 1 September 2000 1 December 2000 . 1 March 2001 3M Environmental Laboratory 3M Environmental Laboratory Page 2 o f 11 Page 21 BACK TO MAIN 3M Medical Department Study: T-6314/1 Analytical Report: FAC T TO X -028 111 LMN-WIWF Protocol Number: FACT-Tox-028 Lab Request Number (LRN): U2636 1. Study Groups of male and female Crl:CD (SD) IGS BR rats were exposed daily to basal diet, or N-MeFOSE (T-6314) in feed for 13 weeks. At the end of the in-life study phase, liver and sera specimens were sent to the 3M Environmental Laboratory for analyses to evaluate the presence and concentration of T-6314 metabolites; specifically, PFOS, PFOSA, M556, and M570 (see Table 1). The determination ofM570 in sera and liver specimens will be conducted according to documented, but not validated, melhods. Compound Identification Chem ical Form u la C A S Number M ole cu lar W eight (amu) PFOS Perfluorooctane- sulfonate C8F17SO3- 2795-39-3 PFOSA Perfluorooctane- sulfonylamide CaFi7S 0 2N(H2) 754-91-6 M556 M556 C8Fi7S0 2N(H) (CH2COOH) N/A M570 M570 C8F,7S0 2N(CH3) (CH2COOH) N/A 498.9 498.9 556.96 570.97 Table 1. Metabolites of N-MeFOSE (Test Article) to beAnalyzed in the Present Study 2. Purpose The analytical phase of this study is designed to evaluate levels of PFOS, PFOSA, M556, and M570 in liver and sera specimens of the rats exposed to T-6314. The in-life phase of the study was conducted at Covance Laboratories, Inc., (Covance Study number 6329-225). All liver and sera specimens will be extracted by Pace Analytical Services, Inc., and analyzed at the 3M Environmental Laboratory. Specimen matrices for this study will be serum and liver; data quality objectives for this study are indicated in section 12. 3. Reg u la to r y Compliance With the initial exception of M570 determination in sera and liver samples, this study will be conducted in accordance with the United States Food andDrag Administration Good Laboratory Practice Regulations forNonclinical Laboratory Studies, Final Rule 21 CFR Part 58. 4. Quality A ssurance The 3M Environmental Laboratory Quality Assurance Unit (QAU) will audit the study conduct, raw data, and final report to determine compliance with Good Laboratory Practice Regulations, this protocol, and 3M Environmental Laboratory Standard Operating Procedures. 3M Environmental Laboratory 3M Environmental Laboratory Page 3 o f 11 Page 22 BACK TO MAIN 3M Medical Department Study: T-6314.1 Analytical Report: FAC T TO X -028 . WN-imr Protocol Number: FACT-Tox-028 Lab Request Number (LRN): U2636 5 . Test A rticle 5.1 Id e n tific a tio n N-Methyl Perfluorooctanesulfonamido Ethanol (N-MeFOSE T-6314, CAS 24448-09-7) Note: Acetone was used as the vehicle to dissolve the test article before mixing with the diet. 5.2 P h y s i c a l D e s c r i p t i o n Amber waxy solid 5 .3 P u rity a n d S ta b ility Results of purity and stability analyses completed by the sponsor will be presented in the final report. 5 .4 S to ra g e C o n d itio n s T-6314 was stored at room temperature at Covance Laboratories during the in-life phase of the study. 5.5 R e s e r v e S a m p le s A reserve sample (approximately 5g) from each lot was set aside and stored at room temperature. Reserve samples were shipped to the Sponsor at the end of the in-life phase of the study. 5 .6 D is p o s a l Unused test article was returned to the sponsor at the end of the in-life phase of the study. The test article will be maintained in the 3M Environmental Laboratory specimen archives for a period of time specified by GLP regulation, or as long as the quality of the preparation affords evaluation. 6. Control Article 6.1 Id e n tific a tio n Certified Rodent Diet #5002 meal 6 .2 S o u rc e PMI Nutritional International 6 .3 P h y s ic a l D e sc rip tio n Meal (ad libitum, unless otherwise specified) 6 .4 P u rity and Stability There are no known contaminants in the diet that would interfere with this study. 6 .5 S to ra g e C o n d itio n s Room temperature 6 .6 R e s e rv e S am p le s Approximately lOOgwas taken from each dose preparation and stored at room temperature. 6.7 D i s p o s a l Unless used for dose analyses, samples were discarded one month after the completion of the in-life phase ofthe study by Covance Laboratories. 3M Environmental Laboratory 3M Environmental Laboratory Page 4 o f 11 Page 23 3M Medical Department Study: T-6314.1 BACK TO MAIN Analytical Report: FACT TO X-028 Protocol Number: FACT-Tox-028 Lab Request Number (LRN): U2636 7. Reference Materials PFOS PFOSA M556 M570 Id e n tific a tio n TCR Substance # Source P h y s ic a l D escription P u rity S tability Storage C onditions C hem ical L o t # Reserve Sam ples D isposal Safety Precautions (Attachm ent A) Perfluorooctanesulfonate Perfluorooctanesulfonylamide M556 M570 SD009, SD018, SD046 (equivalent lots) SE027 or SD029 (equivalent lots) SD014 SD040 3M Specialty Chemicals 3M Specialty Chemicals 3M Specialty 3M Specialty (R. Buckanin) Chemicals (T. Spawn) Chemicals (G. Moore) Light-colored powder White powder/chunks/ Light-yellow waxy solid W hite Powder W hite powder Purity of standards is currently under determination. Purity values will be included in the final report or in an amendment to the final report. unknown unknown unknown unknown Room temperature Room temperature Room temperature Room temperature Batch 171 o r 217 L-15709 3M# 113047-80 3M #N B 118506-26 A reserve sample from each batch/iot o f reference mate rial w ill be retained for 5 years after submission o f the final report or two years after completion if not submitted, but not longer than 10 years following the effective date of the final test rule (if applicable). Unused reference material will be retained for use by 3M Environmental Laboratory and will be discarded when the quality of preparation no longer affords evaluation. Refer to the M SDSfor PFOS Refer to the M SDSfor PFOSA Refer to the MSDS for M556 Refer to the MSDS for M570 Table 2. Reference Materials for FACTTox-028 8. Test System Four dose groups comprised of 20 male and 20 female Crl:CD (SD) IGS BR rats each, were exposed daily to 0- (control), 3- (low exposure), 30- (mid exposure), or 100- (high exposure) pg/g N-MeFOSE (T-6314) in their feed for at least 13 weeks (Table 3). Individuals in the control group were identified with an implantable microchip identification device. After 4 weeks, liver and sera specimens were collected from five animals/sex/dose group. At the end of the study (after week 13), liver and sera specimens were collected from all remaining animals in the test system. All liver and sera specimens were sent to the 3M Environmental Laboratory for analysis. 3M Environmental Laboratory 3M Environmental Laboratory Page 5 o f 11 Page 24 BACK TO MAIN 3M Medical Department Study: T-6314.1 1 mui&rA n a lytica l R eport: F A C T T O X -028 ......... Protocol N um ber FAC T-Tox-028 Lab Request Number (LRN): U2636 Dose Group N u m b e r o f A n im a l s D ie t a r y L e ve ls * 1 C o n tr o l1' 2 Low Exposure 3 M id Exposure 2 0 m a le / 2 0 fe m a le an im als 2 0 m a le / 2 0 fe m a le an im als 2 0 m a le / 2 0 fe m a le anim als 0 ppm 3 ppm 30 ppm 4 H igh Exposure 2 0 m a le / 2 0 fe m a le anim als 100 ppm a D o s e Lev els a re exp ressed a s ppm o f N -M e F O S E b C o n tro l g ro u p a n im a ls w ill re c e iv e a b a s a l d ie t only Table 3. Dosage Levels, Dose Groups and Group Composition in the 13-Week Dietary Study of T-6314 Iri Rats 9. Specimen Receipt Tissue specimens were received from Covance Laboratories during and at the end of the in life phase of study, on October 13, and December 22,1998. Liver and sera specimens were shipped by overnight courier andwere received frozen on dry ice. Specimens were identified with specimen numbers (lab request numbers or numbers assigned by Covance Laboratories) upon receipt, registered with the 3M Environmental Laboratory and transferred to a freezer for storage. All specimens sent to the 3M Environmental Laboratory were received and tracked according to the Sample Tracking System Standard Operating Procedures. Details of specimen inspection for damage, receipt, storage, identification, chain of custody protocols, and data will be presented in the final report. 10. Preparatory Methods Method numbers: 10.1 ETS-8-4, "Extraction of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds from Serum for Analysis Using HPLC-Electrospray/Mass Spectrometry" 10.2 ETS-8-6, "Extraction ofPotassium Perfluorooctanei-sulfonate or other Fluorochemical Compounds from Liver for Analysis using HPLC-Electrospray/Mass Spectrometry" Analytical samples are extracted using an ion-pairing extraction procedure. An ion pairing reagent is added to a laboratory sample and the analyte ion pairpartitioned into MtBE. The MtBE extract is then removed and put onto a nitrogen evaporator until dry. Each extracted laboratory sample is reconstituted in. 1.0 mL of methanol and filtered through a 3 cc plastic syringe attached to a 0.2 pm nylon filter into glass autovials. A modification will be made to the extraction method prior to the commencement of this study; an internal standard (THPFOS) will be added after the extracted laboratory sample is reconstituted in 1.0 mL of methanol. 11. Analytical Methods 11.1 ETS-8-5, "Analysis of Potassium Perfluorooctanesulfonate or Other Fluorochemical in Serum Extracts Using HPLC-Electrospray/Mass Spectrometry" 11.2 ETS-8-7, "Analysis of Potassium Perfluorooctane-sulfonate or other Fluorochemicals in Liver Extracts Using HPLC-Electrospray/Mass Spectrometry" 3M Environmental Laboratory Page 6 o f 11 3M Environmental Laboratory Page 25 3M Medical Department Study: T-6314.1 BACK TO MAIN Analytical Report: FAC T TO X -028 Protocol N um ber FACT-Tox-028 Lab Request Number (LRN): U2636 Analyses are performed by monitoring one or more product ions selected from a single primary ion characteristic of a particular fluorocheinical using HPLC-ESMSMS. For example, molecular ion 499, selected as the primary ion for PFOS (C8F17SO3-) analysis, is fragmented further to produce ion 99 (FSO3-). The characteristic ion 99 is monitored for quantitative analysis. The analysis of M570 in sera and liver matrix samples will be conducted according to the methods used by 3M Environmental Laboratory for the other target analytes. However, these methods have not been specifically validated for M570. 12. Data Quality Objectives The number of spikes/duplicates, use of internal standards, and information on other quality indicators are included in the analytical methods (Attachment B). As there were no sponsorrecommended data quality objectives, the following will be used: 12.1 L in e a rity The coefficient of determination (r2) of the standard curve must be equal to or greater than 0.985 using either no weighting or 1/x weighting, as appropriate. 12 .2 L im its o f Q u a n titatio n (L O Q ) The LOQ may be determined according to 40 CFR Part 136, Appendix B, or alternatively, the LOQ may be equal to the lowest acceptable standard in the calibration curve and die low curve point, which is within 30% of the calculated calibration curves, as appropriate. 12 .3 A c c e p ta b le P re c is io n <30% for the method 12 .4 A c c e p ta b le R e c o v e rie s 70-130% . Removal o f individual outliers from calculations may be done i f it is documented with a reasonable explanation. 1 2 .5 U s e o f C o n firm a to ry M e th o d s None. If a confirmatory method is used, an amendment to this protocol will be written. 1 2 .6 D e m o n s tra tio n o f S p e c ific ity , etc. For HPLC/MS/MS, compound identification will be substantiated by chromatographic retention time and by selection of a characteristic product ion from a characteristic primary ion (see Table 4). 3M Environmental Laboratory 3M Environmental Laboratory Page 7 o f 11 Page 26 3M Medical Department Study: T-6314.1 BACK TO MAIN Analytical Report: FAC T T Q X -028 Protocol Number: FACT-Tox-028 Lab Request Number (LRN): U2636 Compound PRIMARY ION PRODUCT Io n A p p r o x im a t e R e t e n t io n T im e (m in ) THPFOS (SS) PFOS M556 M570 PFOSA 427 499 556 570 498 80 99 169 169 78 7.2 7.3 7.5 7.7 8.0 Table 4. Compound Identification with HPLC/MS/MS 13. Statistical Methods and Calculations Statistical methods will be limited to the calculation of means and standard deviations. Examples of the calculations used in this study will be included in the final report. 14. Subcontract Laboratory Pace Analytical Services, Inc. (Pace--Tier II Facility, Minneapolis, MN) will be performing the extraction of samples for analyses of liver and serum specimens. All extracted samples will be shipped to the 3M Environmental Laboratory for analysis. Pace will follow all applicable 3M Environmental Laboratory Standard Operaling Procedures and the following Pace Standard Operating Procedures: PSS-Admin-01 PSS-DC-05 PSS-OPS-Ol PSS-OPS-02 PSS-OPS-03 PSS-OPS-04 PSS-OPS-05 PSS-OPS-06 PSS-OPS-07 PSS-OPS-08 PSS-OPS-13 PSS-OPS-15 PSS-OPS-16 PSS-OPS-31 PSS-OPS-32 ETS-9-40 (3M Lab) Quality System Lab Notebooks, Logbooks, and Phone Logs Use and Maintenance of Ultra-Turrax T25 Homogenizer Raw Data Hazardous Waste Disposal Use and Maintenance o f N-Evap Analytical Evaporator Use of Compressed Gases in the Laboratory Use and Maintenance ofNANOpure II Water Purification System Cleaning Non-disposable Volumetric Pipettes Laboratory Calculations Use and Maintenance of Lab Refrigerators, Freezers, and Ovens Glassware Cleaning Use and Maintenance of the Fisher Scientific Isotemp Freezer Use and Maintenance of Shakers (Use for Shaker Only) Operation, Maintenance and Calibration of Laboratory Centrifuges Operation and Maintenance of Shakers and Vortex Mixers (Use for Vortex Mixer Only) 3M Environmental Laboratory 3M Environmental Laboratory Page 8 o f 11 Page 27 BACK TO MAIN 3M Medical Department Study: T-6314.1 --------- nmrnrAnalytical Report: F A C T TO X -028 PSS-OPS-37 PSS-MTR-01 PSS-MTR-02 PSS-MTR-06 PSS-MTR-08 PSS-MC-01 PSS-ARC-02 PSS-DM-03 Protocol N um ber FACT-Tox-028 Lab Request Number (LRN): U2636 Operation, Maintenance, and Calibration of pH Meters and pH Electrodes Calibration of Certified Weight Set Calibration of Certified Thermometers/Thermocouples Verification of Calibration of the EppendorfPipettor Verification of Calibration and Use of .Analytical Balances Purchasing of Laboratory Supplies Disposition of Archive Materials Statistical Evaluation of Data 15. Report A report of the results of the study will be prepared by 3M Environmental Laboratory. When analyses are subcontracted to other laboratories, each laboratory will prepare an analytical report and submit a copy of it to the 3M Environmental Laboratory for inclusion as an attachment in the 3M Environmental Laboratory final report. Each report will include, but not be limited to, the following, when applicable: 15.1 Name and address ofthe facility performing the study. 15.2 Dates upon which the study was initiated and completed. 15.3 A statement of compliance by the Study Director addressing any exceptions to Good Laboratory Practice Standards. 15.4 Objectives and procedures as stated in the approved protocol, including any amendments to the original protocol. 15.5 The test article identification by name, chemical abstracts number or code number, strength, purity, and composition or other appropriate characteristics, if provided by the Sponsor. 15.6 Stability and the solubility of the test articles under the conditions of administration, if provided by the Sponsor. 15.7 A description ofthe methods used to conduct the test(s). 15.8 A description ofthe test system (not required in the contract lab analytical report). 15.9 A description of any circumstances that may have affected the quality or the integrity of the data. 15.10 The name of the Study Director andthe names of other scientists, professionals, and supervisory personnel involved in the study. 15.11 A description of the transformations, calculations, or operations performed on the data, a summary and analysis of the analytical chemistry data, and a statement of the conclusions drawn from the analyses. 15.12 Statistical methods used to evaluate the data, if applicable. 3M Environmental Laboratory 3M Environmental Laboratory Page 9 o f 11 Page 28 BACK TO MAIN 3M Medical Department Study: T-6314.1 --------------------------------------------------------------- ---------------------------- Analytical Report: FACT TOX-028 - 1LTIN-U&r Protocol N um ber FACT-Tox-028 Lab Request Number (LRN): U2636 15.13 The signed and dated reports of each of the individual scientists or other professionals involved in the study, if applicable. 15.14 The location where raw data and the final report art: to be stored. 15.15 A statement prepared by the quality assurance unit listing the dates that study inspections and audits were made and the dates of iny findings reported to the Study Director and Management. 15.16 If it is necessary to make corrections or additions to a final report after it has been accepted, the changes will be made in the form of an amendment issued by the Study Director. The amendment will clearly identify the part of the final report that is being amended, the reasons for the amendment, and will be signed by the Study Director. 16. Location of Ra w Data, Records, and Final Report The original data, or copies thereof, will be available at the 3M Environmental Laboratory to facilitate audits of the study during its progress andbefore acceptance of the final report. When the final report is completed, all original paper data, including: approved protocol and amendments, study correspondence, shipping records, raw data, approved final report, and electronic copies of datawill be retained in the archives of the 3M Environmental Laboratory. All corresponding training records, calibration records, instrument maintenance logs, standard operating procedures, equipment procedures, and methods will be retained in the archives of the facility performing each analysis. 16.1 The following raw data and records will be retained in the study folder in the archives according to 3M Environmental Laboratory Standard Operating Procedures. 16.1.1 Approved protocol and amendments 16.1.2 Study correspondence 16.1.3 Shipping records 16.1.4 Raw data 16.1.5 Approved final report (original signed copy) 16.1.6 Electronic copies of data 1 6 .2 The following supporting records will be retained separately from the study folder in the archives according to 3MEnvironmental Laboratory Standard Operating Procedures: 16.2.1 Training records 16.2.2 Calibration records 16.2.3 Instrument maintenance logs 16.2.4 Standard Operating Procedures, Equipment Procedures, and Methods 16.2.5 Appropriate specimens 17. Data / S pecimen Retention Specimens will be maintained in the 3M Environmental Laboratory specimen archives for a period of time specified by regulation, or as long as the quality of the preparation affords evaluation, but not longer than ten years following the effective date of the final test rule (if 3M Environmental Laboratory 3M Environmental Laboratory Page 10 o f 11 Page 29 BACK TO MAIN 3M M edical D epartm ent Study: T-6314.1 Analytical Report: FACT TO X -028 985? Protocol N um ber FACT-Tox-028 Lab Request Number (LRN): U2636 applicable), and as established by 3M Environmental Laboratory Standard Operating Procedures. 18. Protocol Amendments and Deviations Planned changes to the protocol will be in the form of written amendments signed by the Study Director and the Sponsor Representative. Amendments will be considered as part of the protocol and will be attached to the final protocol. All changes to the protocol will be indicated in the final report. Any other changes will be in the form of written deviations, signed by the Study Director and filed with the raw data. 19. A ttachments 1 9 .1 A t t a c h m e n t A : Material Safety Data Sheets (MSDS) for Reference Materials 1 9 .2 A t t a c h m e n t B : Extraction and Analytical Methods 20. SIGNATURES John L. Butenhoff, Ph.D., Sponsor Representative Date Andrew M. Seacat, Ph.D., Study Director Date 3M Environmental Laboratory 3M Environmental Laboratory Page 11 o f 11 Page 30 BACK TO MAIN 3M Medical Department Study: T-6314.1 ______________________ Anajytjcaj_ReEor^FACTTO^;^g8 " LRN-U2636 Protocol Number. FACT-Tox-028 Lab Request Number (LRN): U2636 Attachment A Ma te r ia l Sa fe ty Da ta S heets (MSDS) for Reference Materials 3M Environmental Laboratory 3M Environmental Laboratory Attachment A Page 31 BACK TO MAIN 3M Medical Department Study: T-6314.1 ^ . ............................................... Anajyticaj_Re2Ort^ATTO):028 " " LRN-U2636 Protocol N um ber FACT-Tox-028 Lab Request Number (LRN): U2636 Attachment B Extraction and A nalytical Methods 3M Environmental Laboratory 3M Environmental Laboratory Attachment B Page 32 3M Medical Department Study: T-6314.1 ---------------------------------------------------- BACK TO MAIN _ _ >^_ _ _ ^^^ _ A n a l^tic a U R e o rtJ jA C T | TO)028 LRN-U2636 S tu d y T itle Covance 6329-225 13-Week Dietary Toxicity Study with N-Methyl Perfluorooctanesulfonamido Ethanol (N- MeFOSE, T-6314) in Rats PROTOCOL AMENDMENT NO. 4 A m en d m en t D ate: August 17, 2000 P erfo rm in g L ab o rato ry 3M Environmental Technology & Safety Services 3M Environmental Laboratory 935 Bush Avenue St. Paul, MN 55106 L a b o ra to ry P ro je c t Id e n tificatio n Covance 6329-225 ET&SS FACT TOX-028 LRN Number: U2636 3M Environmental Laboratory 3M Environmental Laboratory Page 33 3M Medical Department Study: T-6314.1 BACK TO MAIN A nalytical Report: FACT TO X -028 Covance Protocol 6329-225 Amendment 4 This amendment modifies the following portion(s) of the protocol: 1. Protocol reads: Andrew M. Seacat is assigned to the role of sponsor representative (study monitor). Amend to read: The role of sponsor representative has been reassigned to John Butenhoff. Reason: The role of sponsor representative must be assigned to one person for both the analytical and in-life protocols. Since 3M is the sponsor for the study, the sponsor representative must be from 3M. 2. Protocol reads: The study director role is assigned to Peter J. Thomford from Covance Laboratories. Amend to read: The role of study director has been reassigned to Andrew Seacat. The role of principal in-life investigator has been reassigned to Peter Thomford. Reason: Per GLP regulations, only one study director may be assigned to a study. Since 3M is the sponsor of the study, the study director should be from 3M. 3. Protocol reads: The performing laboratory is listed as Covance Laboratories Inc., 3301 Kinsman Boulevard, Madison WI 53704-2595. Amend to read: Change the performing laboratory to 3M Environmental Laboratory, Building 2-3E-09,935 Bush Avenue, St. Paul MN 55106. Selected extractions will be performed at Pace Analytical Service, Inc. as per the PAL Reason: All analytical testing will be done at 3M Environmental Laboratory or PACE. 4. Protocol reads: The study location is specified as Covance Laboratories, Inc., 3301 Kinsman Boulevard, Madison WI 53704-2595. Amend to read: Change the study location to 3M Toxicology Services--Medical Department, 3M Center, Building 220-2E-02, St. Paul MN 55144-1000. 3M Environmental Laboratory 3M Environmental Laboratory Page 34 BACK TO MAIN 3M Medical Department Study: T-6314.1 ----------------------------------------------------------------- -- ------------ Analytical Report: FACT TOX-028 LHN-US g r Reason: 3M Toxicology Services is the sponsor of the study. Covance Protocol 6329-225 Amendment 4 5. PROTOCOL READS: There is no principal analytical investigator (PAI) listed for 3M Environmental Laboratory for FACT Tox-028. AMEND TOREAD: Kristen J. Hansen has been assigned to the role ofprincipal analytical investigator for the 3M Environmental Laboratory. Reason: No PAI had previously been assigned. 6. Protocol reads: The analytical phase protocol has not been written or approved. Amend to read: Add that the analytical phase protocol is attached to this list ofprotocol amendments. Reason: There are two phases to the study; the in-life phase and the analytical phase. The analytical phase protocol FACT TOX-028 is an amendment to the Covance protocol 6329-225. 3M Environmental Laboratory 3M Environmental Laboratory Page 35 3M Medical Department Study: T-6314.1______________ BACK TO MAIN ______ ^ ^ ^ ^ ^ ^ _ ^ _ A n a l^ t ic a l||Rej3ort FATT2&IJ28 L R N -U 2 6 3 6 Covance Protocol 6329-225 Amendment 4 Amendment Approval John Butenhoff, Ph.D., Sponsor Representative /t> 6 Date Andrew Seacat, Ph.D., Study Director / n/ ' ---- Kristen J Hansen, Ph.D., Principal Analytical Investigator g y is /a o Date g 2 ,3 /0 0 3M Environmental Laboratory 3M Environmental Laboratory Page 36 3M Medical Department Study: T-6314.1 3M Environmental Technology and Services PO Box 33331 Si. Paul, MN 55133-3331 612 778 6442 BACK TO MAIN Anal^rticalRejDom_FACTTOX^8 LR N -U 263'" S tu d y T itle Determination of the Pie<sence and Concentration of PFOS, M556, and M570 in the 13-Week Dietary Study of Crl:CD (SD) IGS BR Rats PROTOCOL AMENDMENT NO. 5 A m en d m en t D ate: January 05,2001 P e rfo rm in g L a b o ra to ry 3M Environr lental Technology & Safety Services 3M| Environmental Laboratory 935 Bush Avenue St. Paul, MN 55106 L abor, ito ry P ro je c t Id e n tific a tio n FACT-TOX-028 LRN Number U2636 3M Environmental Laboratory 3M Environmental Laboratory Page 37 BACK TO MAIN 3M Medical Department Study: T-6314.1 Analytical Report: F A C T TQ X -028 L R N -U 2 6 36 : FACT-TOX-028 \ Amendment 5 This amendment Clarifies identification of the test article. 1. Protocol reads: \ This study uses different abbreviations to identify the test article, N-Methyl Perfluoroctanesulfonamido Ethanol^ The abbreviations include N-MeFOSE-OH, N-MeFOSE, MeFOSE, MeFOS, and MeFOSE alcohol. Amend to: j The abbreviations N-MeFOSE-OH,iN-MeFOSE, MeFOSE, MeFOS, and MeFOSE alcohol are deemed to be equivalent for this study. Reason: \ To clarify the use of these different abbreviations. Amendment Approval (O'-*- f j z - Kristen J. Hansen PhD ., Principal Analytical Investigator 01/ o s /O ) Date Andrew Seacat, PhD ., Study Director Date 3M Environmental Laboratory 3M Environmental Laboratory Page 38 3M M edical D epartm ent Study: T-6314.1 BACK TO MAIN A nalytical Report: FACT T O X -028 LR N -U 2 6 36 Record of Deviation I. Id e n tific a tio n a! ProjectNo. Per T~-- tion Type (Check one) SOP Method O Equipment Procedure 3 Protocol Other: D.Dooccuumm eenn tt JNNuummbbeerr S k t l t t r t C t ) P i tf Psvofm ) j: Date(s) of occurrence jS A -u f Z&.o> i II. D e s c rip tio n : Required Proced.ure/process: r rroo Ft yprroo jJ M f t* r j r.. . / j ? J ' 7 y r . - J s - e A / t / r , . r r ri o Z-. r 0M & ce. j c ______ 7, Actual Procedure/process:......... .......................... ........ ........ .____________________ -- X f'J '-- 7 j TA~ <r>j2/'2-.^.L III. A c tio n s T a k e n : _ (such as amendment issued, SOP revision, etc.) . ' F / z j - sF-iTt-t-iF*L'*__aLJCtlj > .t\___ .L e i . i t a l i . A t - c i .i0 U i( < C A % e a f- ..t .fo fa iti..a te te n ...& > . Recorded By 14L C / X O A j ua'Uj IV . Im p a c t o n S tu d y /P r o je c t ; Date 9 //? / fyk 7f/// X J Authorized By (Study Director i Project Lead) t\ / ) Jp ^ ate T v&'jF' &/rz/F>> 4'P^A)AFar&Ff o/i Spenser Rej) - JcJmv BuMtefP ^tud^ D.iec-Wr - AAtirg.i^ 3M Environmental Laboratory Deviation No. Form ETS-4-8.0 (assigned by Study Director or Project Lead at the end of study or project) 3M Environmental Laboratory Page 39 3M Medical Department Study: T-6314.1 BACK TO MAIN Analytical Report: FACT TOX-028 LRN-U2636 Record of Deviation I. Id e n tific a tio n Study / Project No. -T O X -038 Deviation Type (Check one) SOP ^Method Equipment Procedure Protocol Other: Document Number 6ts- v h . I Date(s) of occurrence II. D e s c rip tio n : Required Procedure/process: T h gX tm cbOQ, s h ia fr.. fo r___ indicates Vne... CCV15_______________ Odt.__________ ______ __qJ L jh 5-pp.oa_S>iTZC0I___________________________ .' r _ Actual Procedure/process: ____Us j d a __ S A -S p rL ^ d a j^ De\Aad-mo III. A c tio n s T a k e n : (such as amendment issued, SOP revision, etc.) U3Q5, d ( M j j r r a r \ i e c [ on 't t i r s ..i o r c r i Recorded By KjJU U fr 3 . i C u u S ^ x j i n Date lo /v / o IV . Im p a c t o n S tu d y / P ro je c t jkxyte- Cd-t/z t L^ h- <Xo jLvW'sCciseJl (DCs. A t -A -f x x W ^ . _______________ ;_______________ A ft ICjOHIOQ___________________ Authorized By (Study Director / Project Lead) Date f fi { / i b ( C D Sportier 6&pT5Ikn ButtnKcW 3M Environmental Laboratory F o rm ETS-4- 8.0 Iu.lIa C)ntcl'or- l\n<lrfru> -Uc-u!" Deviation No. (assigned by Stu dy Director or Project Lead at the end o f study or project) 3M Environmental Laboratory Page 40 3M Medical Department Study: T-6314.1 BACK TO MAIN Analytical Report: FACT TOX-028 LRN-U2636 Record of Deviation I. Id e n tific a tio n Study / Project No. rox-oae.. Deviation Type (Check one) C c iu o jr \c ^ lS gS-L SOP ^Method Equipment Procedure Protocol Other: Document Number Date(s) of occurrence f : T S - % - q . t , e -ts- ^ - ip .o ?/as/ oo.j. IQfa loo II. D e s c rip tio n : Required Procedure/process: T u ^ e . JC u n + e j T r s __ C e c ^ L L c S ^ l... i h c . ^ . L - S t U OE S ^ Actual Procedure/process: T V >e Tu-p*. n a n o p L u r g ,...w ;a :te / " j s d ___ Z M /c J J S J i M o a o C L __________________________ :---------------------------- III. A c tio n s T a k e n : (such as amendment issued, SOP re vision, etc.) T h L T u r na jrx p L u re _.vol t a r __u ja s x S o a .. irr JfCrOy--- __ LCSxU1-L_j----------------------- ------- --------------------------------- The. reSAi t s _ .v o hC rQ__ ty.'ic^ f__C x ro e* -- Recorded By K iX U Date to 3 l O IV . Im p a c t o n S tu d y / P ro je c t Tffl* -- un4Cnc-t id.uAsLtj_o ^ x a ._~u'zjzhi^.. h h U//7-/0H Authorized By (Study Director / P r c j r r t f , -DI ate 1 V fo -'t* * I //A s / SpWiiier'. Tciui BuWi't'fW 3M E nvironm ental Laboratory Form E TS-4-8.0 S./iL.c\cr'' fl/tiiretC SfcOCrij- Deviation No. (assigned by Study Director or Project Leadat theendof study or project) 3M Environmental Laboratory Page 41 BACK TO MAIN 3M Medical Department Study: T-6314.1 Analytical Report: FACT TOX-028 LRN-U2636 Record of Deviation .id e n tific a tio n S tudy/P roject No. TO X 0028 (LIM S #U 2636) D eviation type (Check one) SOP X Method Protocol Other: Equipment Procedure D ocum ent num ber ETS-8-5.1 and E TS-8-7.0 D ate(s) o f occurrence 10/09/00,10/18/00 10/23/00 10/24/00 11/02/00 Required procedure/process:___________________________ ________________________ Section 14.2.1: Solvent blanks, method blanks, and matrix blanks must be below the lowest standard on the calibration curve. Actual procedure/process: Occasionally, the first solvent blank injected for a run was above the LOQ. Deviation written. Recorded by tyk 1T-loS/m) D ate In most places a high solvent blank was analyzed, additional solvent blanks or method blanks were analyzed immediately following the high blank. This second injection was typically w ell below the LOQ. Occasionally, the first injection of a run is high because it may immediately follow injection of a high standard from a previous run. For this reason, more than one blank is usually analyzed prior to the start of a calibration curve. These second and third, etc. blanks are below the LOQ and are more representative of the analytical conditions of the samples; the Authorized by . /yA ^ n <3 J . D ate j / (assignedby Study Director or Project Lead at the end of study or project) A ttachm ent A : R ecord o f D eviation 3M Environmental Laboratory E T S -4 -8 .0 Page 1 of 1 Page 42 BACK TO MAIN 3M M edical D epartm ent S p d y : T-6314.1 A nalytical Report: FAC T TO X -028 L R N -U 2 6 3 6 Record of Deviation I. Id e n tific a tio n Study / Project No. TOX028 Deviation Type (Check one) SOP Method Equipment Procedure Protocol Other: Document Number: ETS-8-7.0 Date(s) of occurrence: 10/23/00 II. D e s c rip tio n : Required Procedure/process: Analyze a mid-range calibration standard.... Actual Procedure/process: A 306 ppb calibration standard was used for sample run D001023a. Although 306 ppb is in the mid-range of the entire curve, many of the high curve points were deactivated in order to optimize a linear range appropriate to the data. As a result, the 306 ppb CCV was no longer at the mid-range, but at the same level as the highest point in the calibration curve. III. A c tio n s T a k e n : (such as amendment issued, SOP revision, etc.) A deviation was written. Recorded By: jjh litfo/oo Date: November 30,2000 IV . Im p a c t o n S tu d y / P ro je c t The CCVs were acceptable. Although this use of the high CCVs is notj^ ea ^ g w ^ fce,^ ^ \o3> followed for construction of the calibration curve, this deviation will4iave an averse impact on ' the data._______________ ;____________________ Lfh iU S o (fft) j-___ Authorized By (Study Director / Project Lead) Date SjP'-iitscr i 7 o1ia f t u W i k / i l 'i 'l u i a 3M Environmental Laboratory Form ETS-4-8.0 r~ OirtC-W . A ndi'icd ai Deviation No. (assigned by Study Director or Project Lead at the end of study or project) 3M Environmental Laboratory Page 43 3M M edical D epartm ent Study: T-6314,1 BACK TO MAIN Analytical Report: FAC T TO X-028 L R N -U 2 6 36 Record of Deviation Study / Project No. TOX028 1. Id e n tific a tio n Deviation Type (Check one) SOP Method Equipment Procedure Protocol Other: Document Number: ETS-8-5.1 i Date(s) of occurrence: 11/6/00 II. D e s c r ip tio n : Required Procedure/rocess: Cqntinuing calibration verification percent recoveries must be within 30% of the spiked concentration. Actual Procedure/process: A calibration curve was followed by 5 samples, a CCV and a closing calibration curve. TheCCV was at 25ppb and the samples were at 400+ppb. The linear, range of the of the curve optimized to the data was 49.6 to 740 ppb; as a resuit, the CCV was out ofthe calibration^^ samples were in the mid range of the curve and were immediately surrounded by two calibration curves, the data is sufficenfly'faacfe teid........ III. A c tio n s T aken : (such o s amendm en t issued, SOP revision, etc.) A deviation was written. Recorded By: Harold Johnson IV. Im p a c t o n S t u d y / P r o je c t No adverse impact on t t e ^ .................... // ! Date: j November 30, 2000 Authorized By (Study Director / Project Lead) .i Date Sponsor*: T*!''' to 3M E nvironm ental Laboratory Form E TS-4-8.0 Deviation No. L (assigned by Study Director or Project Lead at the end of study or project) 3M Environmental Laboratory Page 44 BACK TO MAIN 3M Medical Department Study: T-6314.1 Analytical Report: FACT TOX-028 LRN-U2636 Appendix C: Extraction and Analytical Methods This appendix includes the following methods: ETS-8-4.1, "Extraction of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds from Serum for Analysis Using HPLC-Electrospray/Mass Spectrometry," (14 pages) ETS-8-6.0, "Extraction of Potassium Perfluorooctanesul fonate or Other Fluorochemical Compounds from Liver for Analysis Using HPLC-Electrospray/Mass Spectrometry," (14 pages) ETS-8-5.1, "Analysis of Potassium Perfluorooctanesulfonate or Other Fluorochemicals in Serum Using HPLC-Electrospray/Mass Spectrometry," (9 pages) ETS-8-7.0, "Analysis of Potassium Perfluorooctanesulfonate or Other Fluorochemicals in LiverUsing HPLC-Electrospray/Mass Spectrometry," (10 pages) 3M Environmental Laboratory Page 45 3M Medical Department Study: T-6314.1 BACK TO MAIN Analytical Report: FACT TO X-028 L R N -U 2 6 36 3M Environmental Laboratory M ethod Extraction of P otassium P e r f l u o r o o c t a n e s u l f o n a t e o r Other HPLC-Fluorochem ical compounds from Serum f o r Analysis U sing Electrospray/M ass Spectrometry Method Number: lETS-8-4.1 Adoption Date: 03/01/99 Author: Lisa Clemen, Glenn Langenburg . Revision Date: ^ ^ Approved By: Laboratory Manager f j z ---- Group Leader Technical Reviewer Date y/zL /gj Date fw /ak/i-? Date 1.0 Scope and Application 1.1 Scope: This method is for the extraction ofpotassium perfluorooctanesulfonate (PFOS) or other fluorochemical compounds from serum. 1.2 Applicable compounds: Fluorochemical surfactsints or other fluorinated compounds. 1.3 Matrices: Rabbit, rat, bovine, monkey, andhumsin serum or other fluids as designated in the validation report. W ord 6/95 3M Environmental Laboratory ETS-8-4.1 Extraction of PFOS from Serum P a g e l o f 14 Page 46 3M Medical Department Study: T-6314.1 BACK TO MAIN Analytical Report: FACT TOX-028 LRN-U2636 2.0 Summary of Method______________________ ;____________ ______________________ 2.1 This method describes the procedure for extracting potassium perfluorooctanesulfonate (PFOS) or other fluorochemical surfactants from seram, or other fluids, using an ion pairing reagent and methyl-tert-butyl ether (MtBE). Inthis method, seven fluorochemicals were extracted: PFOS, PFOSA, PFOSAA, EtFOSE-OH, PFOSEA, M556, and surrogate standard (see 3.0 Definitions). An ion pairing reagent is added to the sample and the analyte ion pair is partitioned into MtBE. The MtBE extract is removed andput onto a nitrogen evaporator until dry. Each extract is reconstituted in 1.0 mL ofmethanol, then filtered through a 3 cc plastic syringe attached to a 0.2 pm nylon filter into glass autovials. 2.2 These sample extracts are analyzed following method ETS-8-5.1 or other appropriate methods. 3.0 Definitions ______ __________________________ ___________ _________ ;______ 3.1 PFOS: perfluorooctanesulfonate (anion ofpotassium salt) C8F17S03' 3.2 PFOSA: perfluorooctane sulfonylamide CgF^SOjNHj 3.3 PFOSAA: perfluorooctane sulfonylamido (ethyl)acetate C8F17S02N(CH2CH3)CH2C02` 3.4 EtFOSE-OH: 2(N-ethylperfluorooctane sulfonamido)-ethyl alcohol C8FlvS 0 2N(CH2CH3)CH2CH20H . 3.5 PFOSEA: perfluorooctane sulfonyl ethylamide C,,F,7S02N(CH2CH3)H 3.6 M556: C8F17S02N(H)(CH2C00H) . 3.7 Surrogate standard: 1H-1H-2H-2H perfluorooctane sulfonic acid 4.0 W arnings and Ca u t i o n s ________________________________________ 4.1 Health and safety warnings 4.1.1 Use universalprecautions, especially laboratory coats, goggles, and gloves when handling animal tissue, which may containpathogens. 5.0 Interferences_____________ |____________________________________________ 5.1 There are no interferences known at this time. 6.0 Equipment____________________________________________________________ 6.1 The following equipment is used while performing tliis method. Equivalent equipment is acceptable. 6.1.1 Vortex mixer, VWR, Vortex Genie 2 6.1.2 Centrifuge, Mistral 1000 orIEC 6.1.3 Shaker, Eberbach or VWR ETS-8-4.1 Extraction of PFOS from Scrum Page 2 o f 14 3M Environmental Laboratory Page 47 3M Medical Department Study: T-6314.1 BACK TO MAIN Analytical Report: FACT TOX-028 LRN-U2636 6.1.4 Nitrogen evaporator, Organomation 6.1.5 Balance ( 0.100 g) 7.0 Supplies and Materials________________ ;___________________ ^__________________ 7.1 Gloves 7.2 Eppendorfor disposable pipettes 7.3 Nalgene bottles, capable of holding 250 mL and 1 L 7.4 Volumetric flasks, glass, type A 7.5 I-CHEM vials, glass, 40 mL glass 7.6 Centrifuge tubes, polypropylene, 15 mL 7.7 Labels 7.8 Oxford Dispenser- 3.0 to 10.0 mL 7.9 Syringes, capable ofmeasuring 5 (xLto 50 pL . 7.10 Graduatedpipettes 7.11 Syringes, disposable plastic, 3 cc 7.12 Syringe filters, nylon, 0.2 pm, 25 mm 7.13 Timer 7.14 Crimp cap autovials and caps 7.15 Crimpers Note: Prior to using glassware andbottles, rinse 3 times with methanol and 3 times with Milli-QTMwater. Rinse syringes aminimum of 9 times with methanol, 3 rinses from 3 separate vials. 8.0 Reagents and Standards______________________ ._________________________ 8.1 Type I reagent grade water, Milli-QTM or equivalent; all water used in this method should be Milli-QTM water andmay be provided by a Milli-Q TOC PlusTM system 8.2 Sodium hydroxide (NaOH), J.T Baker or equivalent 8.3 Tetrabutylammonium hydrogen sulfate(TBA), Kodak or equivalent 8.4 Sodium carbonate (Na2C03), J.T. Baker or equivalent 8.5 Sodium bicarbonate (NaHC03), J.T. Baker or equivalent 8.6 Methyl-T-Butyl Ether, Omnisolv, glass distilled or HPLC grade 8.7 Methanol, Omnisolv, glass distilled orHPLC grade 8.8 Serum orblood, frozen from supplier 8.9 Fluorochemical standards 8.9.1 PFOS (3M Specialty Chemical Division), molecular weight = 538 8.9.2 PFOSA (3M Specialty Chemical Division), molecular weight = 499 ETS-8-4.1 Extraction of PFOS from Serum Page 3 of 14 3M Environmental Laboratory Page 48 3M Medical Department Study: T-6314.1 BACK TO MAIN Analytical Report: FACT TO X-028 L R N -U 2 6 3 6 8.9.3 PFOSAA (3M Specialty Chemical Division),, molecularweight = 585 8.9.4 EtFOSE-OH (3M Specialty Chemical Division), molecular weight = 570 8.9.5 PFOSEA (3M Specialty Chemical Division), molecular weight = 527 8.9.6 M556 (3M Specialty Chemical Division), molecular weight = 557 8.9.7 Surrogate standard: 4-H, perfluorooctane sulfonic acid (1-H.l-H, 2-H, 2-H C8FnS03H) molecular weight = 428 8.9.8 Other fluorochemicals, as appropriate 8.10 Reagent preparation NOTE: When preparing larger volumes than listed in reagent, standard, or surrogate preparation, adjust accordingly. 8.10.1 ION sodium hydroxide (NaOH): Weigh approximately 200 g NaOH. Pour into a 1000 mL beaker containing 500 mL Milli-QTM water, mix until all solids are dissolved. Store in a 1 L Nalgene bottle. 8.10.2 1 N sodium hydroxide (NaOH): Dilute 10N NaOH 1:10. Measure 10 mL of 10 N NaOH solution into a 100 mL volumetric flask and dilute to volume using .Milli-QTM water. Store in a 125 mL Nalgene bottle. 8.10.3 0.5 M tetrabutylammoniumhydrogen sulfate (TBA): Weigh approximately 169 g of TBA into a 1 L volumetric containing 500 mLMilli-QTM water. Adjust to pH 10 using approximately 44 to 54 mL of 10 N NaOH (While adding the last mL of ' NaOH, add slowly because thepH changes abruptly). Dilute to volume with Milli-QTM water. Store in a 1L Nalgene boltle. 8.10.3.1 TBA requires a check prior to each use to ensure pH = 10. Adjust as ' needed using I N NaOH solution. 8.10.4 0.25 M sodium carbonate/sodiumbicarbonate buffer (NajCOj/NaHCO,): Weigh approximately 26.5 g of sodium carbonate (NajCOj) and 21.0 g of sodium bicarbonate (NaHCO,) into a 1 L volumetric flask andbring to volume with MilliQTMwater. Store in a 1L Nalgene bottle. 8.11 Standards preparation ' 8.11.1 Prepare PFOS standards for the standard curve. 8.11.2 Prepare other fluorochemical standards, as appropriate. Multicomponent fluorochemical standards are acceptable (for example, one working standard solution containing 1.00 ppmPFOS, 1.02 ppm PFOSA, 0.987 ppmPFOSAA, and 1.10 ppm EtFOSE-OH.) 8.11.3 Weigh approximately 100 mg ofPFOS into a 100 mL volumetric flask and record the actual weight. 8.11.4 Bring to volume with methanol for a stock standard of approximately 1000 ppm . (pg/mL). 8.11.5 Dilute the stock solution with methanol fox a working standard 1 solution of approximately 50 ppm. 8.11.6 Dilute working standard 1with methanol for a working standard 2 solution of approx. 5.0 ppm. ETS-8-4.1 Extraction of PFOS from Slerum Page 4 of 14 3M Environmental Laboratory Page 49 3M Medical Department Study: T-6314.1 BACK TO MAIN Analytical Report: FACT TO X-028 L R N -U 2 6 36 8.11.7 Dilute working standard 1with methanol for a working standard 3 solution of approx. 0.50 ppm. 8.12 Surrogate stock standard preparation 8.12.1 Weigh approximately 50-60 mg of surrogate standard 1-H,1-H, 2-H, 2-H, C,F,3S03H into a 50 mL volumetric flask andrecord the actual weight. 8.12.2 Bring to volume with methanol for a surrogate stock of approximately 1000-1200 ppm. 8.12.3 Prepare a surrogate working standard. Transfer approximately 1 mL of surrogate stock to a 10 mL volumetric flask andbring to volume with methanol for a working standard of 100 ppm. Record the actual volume transferred. 9.0 Sample Handling ________ ;_____________________________ [________ ;___________ 9.1 All samples are received frozen and must be kept frozen until the extraction is performed. 9.2 Allow samples to thaw to room temperature prior to extraction. 10.0 Quality Control_____________________ ;_____________ _________________ 10.1 Solvent Blanks, Method blanks and matrix blanks . 10.1.1 An aliquot of 1.0 mL methanol is used as a solvent blank. 10.1.2 Extract two 1.0 mL aliquots ofMilli-QTM water following this procedure and use as method blanks. 10.1.3 Extract two 1.0 mL aliquots of the serum following this procedure and use as matrixblanks. See 11.1.4. 10.2 Matrix spikes 10.2.1 Prepare and analyze matrix spike andmatrix spike duplicate samples to determine the accuracy of the extraction. 10.2.2 Prepare each spike using a sample chosen by th analyst, usually the control matrix received with each sample set. 10.2.3 Expected concentrations will fall in the mid-range ofthe initial calibration curve. Additional spikes may be included and may fall in the low-range ofthe initial calibration curve. 10.2.4 Prepare one matrix spike andmatrix spike duplicate per 40 samples, with a minimum of 2 matrix spikes perbatch. 10.3 Continuing calibration checks 10.3.1 Prepare continuing calibration check samples to ensure the accuracy of the initial calibration curve. . 10.3.2 Prepare, at a minimum, one continuing check per group of 10 samples. For example, if a sample set = 34, four checks ire prepared and extracted. 10.3.3 Prepare each continuing calibration check from the same matrix used to prepare the initial curve. 3M Environmental Laboratory ETS-8-4.1 Extraction of PFOS from Serum Page 5 of 14 Page 50 3M Medical Department Study: T-6314.1 BACK TO MAIN Analytical Report: FACT TOX-028 LRN-U2636 10.3.4 The expected concentrations will fall within the raid-range of the initial calibration curve. Additional spikes may be included that fall in the low-range of the initial calibration curve. This is necessary if the analyst must quantitate using only the low end of the calibration curve (for example, 5 ppb- 100 ppb, rather than 5 ppb -1000 ppb). 11.0 Calibration and Standardization__________________ ;_________ ;_______________ 11.1 Prepare matrix calibration standards 11.1.1 Transfer 1 mL of serumto a 15 mL centrifuge tube. 11.1.2 Ifmost sample volumes are less than 1.0 mL, extract standards with matrix volumes equal to the sample volumes. Do not extract less than 0.50 mL of . matrix. Record each sample volume on the extraction sheet. 11.1.3 While preparing a total oftwenty aliquots in 15 mL centrifuge tubes, mix or shake between aliquots. 11.1.4 Two 1 mL aliquots, or other appropriate volume, serve as matrix blanks. Typically use the standard concentrations and spiking amounts listed in Table 1, at tire end of this section, to spike, in duplicate, two standard curves, for a total of eighteen standards, two matrix blanks, and two methodblanks. 11.1.5 Refer to validation report ETS-8-4.0 & ETS-8-5.0-V-1, which lists the working ranges and the Linear Calibration Range (LCR) for calibration curves. 11.1.6 Use Attachment D as an aid in calculating the concentrations ofthe working standards. See Section 13.0 to calculate actual concentrations of PFOS in calibration standards. 11.2 To each standard, blank, or continuing check, add appropriate amount of surrogate working standard for the concentrationto fall within the calibration curve range 5 ppb 1000 ppb. 113 Extract spiked matrix standards following 12.6-12.16 ofthis method. Use these standards to establish each initial curve on the mass spectrometer. 3M Environmental Laboratory ETS-8-4.1 Extraction of PFOS from Serum Page 6 of 14 Page 51 3M Medical Department Study: T-6314.1 BACK TO MAIN Analytical Report: FACT TOX-028 LRN-U2636 Table 1 Approximate spiking amounts for standards and spikes Using 1.0 mL of matrix Working standard pL Approx, final cone, of (approx, cone.) analyte in matrix - - Blank 0.500 ppm 10 0.005 ppm 0.500 ppm 20 0.010 ppm 5.00 ppm 5 0.025 ppm 5.00 ppm 10 . 0.050 ppm 5.00 ppm . 20 0.100 ppm 50.0 ppm 5 0.250 ppm 50.0 ppm 10 0.500 ppm 50.0 ppm 15 0.750 ppm 50.0 ppm 20 1.00 ppm 12.0 Procedure_____________________________ _______ ;________________ _____________ 12.1 Obtain frozen samples and allow to thaw at room temperature or in a lukewarm waterbath. 12.2 Vortex mix for 15 seconds, then transfer 1.0 mL or other appropriate volume to a 15 mL polypropylene centrifuge tube. 12.3 Returnunused samples to freezer after extraction,amounts have been removed. 12.4 Record the initial volume on the extraction worksheet. 12.5 Label the tube with the study number, sample ID, date and analyst initials. See attached worksheet for documenting the remaining steps. 12.6 . Spike all samples, including blanks and standards, ready for extraction with surrogate standard as described in 11.2. 12.7 Spike each matrix with the appropriate amount of standard as described in 11.1, or Table 1 in that section, for the calibration curve standards, Also prepare matrix spikes and continuing calibration standards. 1 12.8 Vortex mix the standardcurve samples, matrix spike samples, and continuing calibration samples for 15 seconds. 12.9 Check to ensure the 0.5 M TBA reagent is at pH 10. Ifnot, adjust accordingly. 12.10 To each sample, add 1 mL 0.5 M TBA and 2 mL of 0.25M sodium carbonate/sodium bicarbonate buffer. 12.11 Using an Oxford Dispenser, add 5 mL methyl-tert-butyl ether. 12.12 Cap each sample and put on the shaker at a setting of 300 rpm, for 20 minutes. 12.13 Centrifuge for 20 to 25 minutes at a setting of 3500 rpm, oruntil layers are well separated. ETS-8-4.1 Extraction of PFOS from Scram . Page 7 of 14 3M Environmental Laboratory Page 52 3M Medical Department Study: T-6314.1 BACK TO MAIN Analytical Report: FACT TO X -028 L R N -U 2 6 3 6 12.14 Label a fresh 15 mL centrifuge tube with the same information as in 12.5. 12.15 Remove 4.0 mL of the organic layer to this clean 15 mL centrifuge tube. 12.16 Put each sample on the analytical nitrogen evaporator until dry, approximately 1 to 2 hours. . 12.17 Add 1.0 mL of methanol to each centrifuge tube using a graduatedpipette. 12.18 Vortex mix for 30 seconds. 12.19 Attach a 0.2 pm nylon mesh filter to a 3 cc syringe and transferthe sample to this . syringe. Filter into a 1.5 mL glass autovial or low-volume autovial when necessary. 12.20 Label the autovial with the study number, animal number and gender, sample timepoint, matrix, final solvent, extraction date, and analyses) performingthe extraction. 12.21 Cap and store extracts atroom temperature or at approximately 4 C until analysis. 12.22 Complete the extractionworksheet, attached to this document, and tape in the study notebook or include in study binder, as appropriate. 13.0 Data Analysis and Ca l c u l a t i o n s _________________________ :______ ;_______ 13.1 Calculations 13.1.1 Calculate actual concentrations of PFOS, or other applicable fluorochemical, in calibration standards using the following equation: mL of standard x concentration of standard! (u.g/mT.t_________ ;______ _=> . . mL of standard + mL of surrogate standard H-initial matrix volume (mL) Final Concentration (pg/mL) ofPFOS in matrix 14.0 Method Performance___________________ ;___________________________________ 14.1 The method detection limit (MDL) is analyte and matrix specific. Refer to MDL report for specific MDL andlimit of quantitation (LOQ) values (see Attachments B and C). 14.2 The follow ing quality control samples are extracted with each batch o f samples to evaluate the quality of the extraction and analysis. 14.2.1 Method blanks andmatrix blanks. . 14.2.2. Matrix spike andmatrix spike duplicate samples to determine accuracy and precision ofthe extraction. 14.2.3 Continuing calibration check samples to determine the continued accuracy of the initial calibration curve. . 14.3 Refer to section 14 of ETS-8-5.1 for method performance criteria. 15.0 Pollution Prevention and Waste Management________________________ 15.1 Sample waste is disposed in biohazard containers, ilammable solvent waste is disposed in high BTU containers, and used glass pipette waste is disposed in broken glass containers located in the laboratory. ETS-8-4.1 Extraction of PFOS from Serum Page 8 of 14 3M Environmental Laboratory Page 53 3M Medical Department Study: T-6314.1 BACK TO MAIN Analytical Report: FACT TOX-028 LRN-U2636 16.0 Records_____________ ;_____________ ;__________________________________ ;______ 16.1 Complete the extraction worksheet attached to this method, and tape in the study notebook or include in the 3-ring study binder, as appropriate. 17.0 Attachments____________________________________ ;______________.____________ 17.1 Attachment A, Extraction worksheet 17.2 Attachment B, MDL/LOQ values and summary 17.3 Attachment C, Calibration standard concentration worksheet 18.0 References_______________________ ' ________________________________ ' 18.1 The validation report associated with this method is ETS-8-4.0 & 5.0-V-l. 18.2 FACT-M-3.1, "Analysis of Serum or Other Fluid Extracts for Fluorochemicals using HPLC-Electrospray Mass Spectrometry" 19.0 Affected Documents________ :______________ ;_________________________________ 19.1 ETS-8-5.1, "Analysis of Serum or OtherFluid Extracts for Fluorochemicals using HPLC-Electrospray Mass Spectrometry*' 20.0 RVISIONS _______ ,_______:___________________ ;___________________________ Revision Number 1 Reason ForRevision Section 12.21 Changedto include sample storage at room temperature. Section 12.13 Added the shaker speed. Section 12.17 Final volume is 1.0 mL; not adjusted for initial volumes less than 1.0 mL. Revision Date 04/02/99 3M Environmental Laboratory . ETS-8-4.1 Extraction o f PFOS from Serum Page 9 of 14 Page 54 BACK TO MAIN 3M Medical Department Study: T-6314.1 Analytical Report: FACT TO X-028 L R N -U 2 6 3 6 Extraction Worksheet ETS-8-4.1 S tudv# M atrix Box# W k/D av D ateSpiked/A nalyst CCV MS M SD Surrogate Std approx, ppm actual ppm # FC-M ix approx. 0.5 p m actual ppm # FC-M ix approx. 5 ppm actual ppm # FC-M ix approx. 50 ppm actual ppm # Comments ---- -' --------- - - '" Pinnlr________;_______________ Std #_______________________ amount- ' -. 'r " " .......tnL ___ Serum E x tractio n M ethod____________________________j_________________________________________________ D ate & In itia ls Vortex 15 see. Pipette Matrix__________________________ ' Volume______________ E k Pipette 1mL o f 0.5 M TBA, pH 10. pH - - Std. M_______ Pipette 2 mL o f 0.25 NaaCOVOJSM NaHCOl buffer Std. # Dispense 5 mL o f methyl-t-butyl ether___________________ TN-A- Shakc 20 min. Shakerspeed: O ntrifiige 20-25 min. Centrifuge speed: _____ Remove a 4 mL aliquot o f organic layer Put on Nitrogen Evaporator to drvness __________________Temperature:_______ Add methanol___________ Volume mL___________ TN-A- Vortex 30 sec,______ '- Filter using a 3cc B-D syringe with a0,2um filter into a 1.5 mL autosample vial______ Cont Cal. Verifications used same matrix as for std curve. Attachment A 3M"Environmental Laboratory ETS-8-4.1 Extraction of PFOS from Serum Page 10 of 14 Page 55 3M Medical Department Study: T-6314.1 BACK TO MAIN Analytical Report: FACT TO X-028 L R N -U 2 6 3 6 MDL/LOQ values for rabbit serum Compound MDL LOQ Linear Calibration Range (LCR) (ppb) (ppb) Approximate concentrations to be used for preparing the Standard Calibration Curve PFOS 1.74 5.55 5ppb-1000ppb PFOSA 1.51 4.79 5ppb-1000ppb PFOSAA 3.46 20.5 5 ppb - 1000 ppb EtFOSE-OH 11.4 36.2 5ppb-1000ppb M556 6.03 19.2 5 ppb -1000 ppb PFOSEA 5.71 18.2 5 ppb-1000 ppb MDL/LOQvalues inrat, bovine, monkey, andhumanserum, andmonkeyplasmawere not statistically determined. Two curves in eachofthese matriceswere extracted andanalyzedwiththe rabbit serum curves to determine equivalence. Responses intherat, bovine, monkey, andhumanwere equivalent to therabbitresponses, therefore, theirMDLandLOQwill be the samevalues as determinedinrabbit serum. Please see LOQ Summary andMDL study inETS-8-4.0 &5.0-V-l forfurtherinformation. Attachment B: MDL/LOQ Summary ETS-8-4.1 Extraction of PFOS from Ssrum Page 11 of 14 3M Medical Department Study: T-6314.1 BACK TO MAIN Analytical Report: FACT TOX 028 LRN-U2636 Compound: P FOS Preparedrange Rabbit Serum of standards (ppb) (ng/mL) Full Range Low Curve High curve 0.995 - 978 4.94 - 248 9 7 .8 -9 7 8 LCRfrom curve (ppb) (ng/mL) 24.8 - 978 4.94 - 248 9 7 .8 -9 7 8 %Recovery Range 83-108 85-104 85-106 1 /X 0.995 - 978 4.94 - 978 94-111 Compound: PFOSA Preparedrange Rabbit Serum ofstandards (ppb) (ng/mL) Full Range Low Curve High curve 1 /X . 0.993 - 976 4.93 - 97.6 24.8 - 976 0.993 - 976 LCRfrom curve (PPb) (ng/mL) 4.93 - 976 %Recovery Range 88-103 4.93 - 97.6 . . 87-105 2 4 .8 -9 7 8 4.93 - 976 93-102 94-103 Compound: PFOSAA .. Preparedrange Rabbit Serum of standards (ppb) (ng/mL) Full Range Low Curve High curve 0.991 - 974 4.92 - 247 4 9 .2 -9 7 4 LCRfrom curve (PPb) (ng/mL) 24.7 - 974 9 .7 4 -2 4 7 97.4 - 974 %Recovery Range 81-111 97-107 85-108 1 /X 0.99 1 -9 7 4 9.74 - 974 95-115 RSD Range 4 .6 7 -1 1 .0 5.34-12.0 4 .8 4 -9 .8 0 4.60-10.5 RSD Range 5.10-14.7 9.85-14.7 5.08-13.9 5.10-14.5 RSD Range 4 .1 8 -1 0 .6 6.38-21.8 4.33-12.5 4 .1 1 -2 3 .2 Attachment B: MDL/LOQ Summary ETS-8-4.1 Extraction of PFOS from Serum 3M Environmental Laboratory Page 12 o f 14 Page 57 3M Medical Department Study: T-6314.1 BACK TO MAIN Analytical Report: FACT TOX-028 LRN-U2636 Compound; EtFOSE-OH Prepared range Rabbit Serum of standards (ppb) (ng/mL) Full Range 0.993 - 976 LCR from curve (ppb) (ng/mL) . 49.3 -976 Low Curve 4.93 - 97.6 9.76-97.6 High curve 49.3 - 976 97.6 - 976 1/X 0.993 - 493 9.76 - 976 % Recovery Range 77-110 97-107 90-109 86-111 Compound: PFOSEA Prepared range Rabbit Serum o f standards (ppb) (ng/mL) Full Range Low Curve High curve 1/X 0.993-976 4.93 - 248 49.3 - 976 0.993 - 976 .LCR from curve (PPb) (ng/mL) 24,8 - 976 9.76 - 248 49.3 - 976 9.76-976 % Recovery Range 96-106 91-110 86-106 95-117 Compound: M556 Prepared range Rabbit Serum o f standards (ppb) (ng/mL) Full Range Low Curve High curve 1/X 0.993 - 976 4.93-97.6 97.6-976 0.993 - 976 LCR from curve (PPb) (ng/mL) 24.8 - 976 9.76-97.6 97.6 - 976 9.76 - 976 %Recovay Range 88-106 100-105 81-111 97-110 RSD Range 11.2-25.5 14.1-21.3 11.5-19.6 11.1-21.2 RSD Range 10.1-16.2 11.8-19.5 10.2-18.2 10.1-19.1 RSD Range 4 .8 2 -1 7 .9 5.95-18.2 5.11-9.74 4.77-19.5 Attachment B: MDL/LOQ Summary ETS-8-4.1 Extraction of PFOS from Serum 3M Environmental Laboratory 1 Page 13 of 14 Page 58 3M Medical Department Study: T-6314.1 BACK TO MAIN Analytical Report: FACT TO X -028 L R N -U 2 6 3 6 Ion Pair Standard Curves - Fluids Prep date(s): Standard number: Analyte(s): Equipment number: Sample matrix: Final solvent and TN: Blank fluld/identifier: Method/revision: Target analyte(s): FC mix std approx. 0.500 ppm: FC mix std approx. 5.00 ppm: . FC mix std approx. 50.0 ppm: Surrogate std approx. 100 ppm: Actual concentrations of standards in the FC mix PFO S P F O S A P F O S A A E tF O S E P F O S E A S td co n e S td co n e S td co n e S td cone S td co n e u g /m L u g /m L ug/m L u g /m L u g/m L 0 .5 0 0 0 .5 0 7 0 .5 3 2 0 .5 0 1 0.5 2 1 0 .5 0 0 0 .5 0 7 0 .5 3 2 0 .5 0 1 0 .5 2 1 5 .0 0 5 .0 7 5 .3 2 5.01 5 .2 1 5 .0 0 5 .0 7 5 .3 2 5.01 5 .2 1 5 .0 0 5 .0 7 5 .3 2 5.01 5 .2 1 5 0 .0 5 0 .1 5 3 .2 50.1 52.1 5 0 .0 5 0 .1 5 3 .2 50.1 5 2 .1 5 0 .0 5 0 .1 5 3 .2 50.1 5 2 .1 5 0 .0 5 0 .1 5 3 .2 5 0 .1 5 2 .1 M 55(i S td cone u g/m L 0 .5 0 1 0.501. 5 .0 1 5 .0 1 5 .0 1 5 0 .1 5 0 .1 5 0.1 5 0 .1 A ll A m 't spiked m L 0 .0 1 0 0 .0 2 0 0 .0 0 5 0.0 1 0 0.0 2 0 0 .0 0 5 0 .0 1 0 0 .0 1 5 0 .0 2 0 A ll F in al vo l mL 1 .0 1 5 1 .0 2 5 1 .0 1 0 1 .0 1 5 1 .0 2 5 1 .0 1 0 1 .0 1 5 1 .0 2 0 1 .0 2 5 Calculated concentrations of standards in the sample matrix PFOS PFOSA PFO SA A E tF O S E PFO SEA M 556 S u rro g ate F inal cone ng/m L 4 .9 3 F inal cone ng/m L 5 .0 0 Final cone ng/m L 5 .2 4 Final cone ng/m L 4 .9 4 F inal cone ng/m L 5.01 F inal cone ng/m L 5.13 Std cone ng/m L 100 9 .7 6 2 4 .8 4 9 .3 97.6 248 9 .8 9 25.1 5 0 .0 98.9 251 10.4 2 6 .3 5 2 .4 104 263 . 9.78 24.8 4 9 .4 97.8 248 9.93 10.2 2 5 .2 . 25.8 S urrogate 50.1 51.3 F inal cone 99.3 to :: ng/mL 252 25! 500 493 500 524 494 501 513 735 746 782 737 749 . 766 976 989 1038 978 993 1017 AU A m t spiked mL 0.005 Validated ranges --approximate concentrations Serum PFOS PFOSA PFOSAA EtFOSE-OH PFOSEA M556 Rabbit 5.00-1000 | 5.00-1000 | 5.00-1000 I- 5.00-1000 | 5.00-1000 | 5.00-1000 Bovine Estimates only. Use values for rabbit . Rat Estimates only. Use values for rabbit Monkey & Plasma Estimates only. Use values for rabbit Human Estimates only. Use values for rabbit. Attachment C: Ion Pair Standard Curves ETS-8-4.1 . Extraction of PFOS from Serum 3M Environmental Laboratory Page 14 of 14 Page 59 3M Medical Department Study: T-6314.1 BACK TO MAIN Analytical Report: FACT TO X -028 L R N -U 2 6 36 TM Environmental Laboratory Method E xtraction of P otassium Perfluorooctanesulfonate or other HPLC-Fluorochem ical Compounds from Liver for Analysis using ELECTROSPRAY/MASS SPECTROMETRY Method Number: ETS-8-6.0 Author: Lisa Clemen, Robert Wynne Adoption Date: o7|u.kt Revision Date: pJPc Approved By: i/l Laboratory Manager w .....*jf----a.l..t..A............ -----------*-------------------------- G roup Leader Technical Reviewer " V 2-v h i Date tW /to D ate o / h Ivi Date _ i. o Sc o p e a n d A p p l ic a t io n 1.1 Scope: This method is for the extraction ofpotassium perfluorooctanesulfonate (PFOS) or other fluorochemical compounds from liver. 1.2 Applicable Compounds: Fluorochemical surfactants or other fluorinated compounds. 1.3 Matrices: Rabbit, rat, bovine, andmonkey livers or ether tissues as designated in the validation report. W o rd 6 .0 /9 5 ETS-8-6.0 Extraction of PFOS from liv e r P age 1 o f 14 3M Environmental Laboratory Page 60 3M Medical Department Study: T-6314.1 BACK TO MAIN Analytical Report: FACT TO X -028 L R N -U 2 6 3 6 2.0 Summary of Method______________ ;______________ ________________________ ' 2.1 This method describes the procedure for extracting potassium perfluorooctanesulfonate (PFOS) or other fluorochemical surfactants from liver, or other tissues, using an ion pairing reagent and methyl-/ert-butyl ether (MtBE). In this method, seven fluorochemicals can be extracted: PFOS, PFOSA, PFOSAA, EtFOSE-OH,.PFOSEA, M556, and surrogate standard. An ion pairing reagent is added to the sample andthe analyte ion pair is partitioned into MtBE. The MtBE extract is transferred to a centrifuge tube andput onto a nitrogen evaporator until dry. Each extract is reconstituted in 1.0 mL methanol then filtered through a 3 cc plastic syringe attached to a 0.2 pmnylon filter into glass autovials. 2.2 These sample extracts are analyzed following method ETS-8-7.0 or other appropriate methods. 3.0 Definitions________________ ,__________________ _______________________________ 3.1. PFOS: perfluorooctanesulfonate (anion ofpotassium salt) C8F17S03 3.2 PFOSA: perfluorooctane sulfonylamide C8F17S02NH;; 3.3 PFOSAA: perfluorooctane sulfonylamido (ethyl)acetate C,Fi7S02N(CH2CH3)CH2C02 3.4 EtFOSE-OH: 2(N-ethylperfluorooctane sulfonamido)-ethyl alcohol C,F17S 0 2N(CH2CH3)CH2CH20H 3.5 PFOSEA: perfluorooctane sulfonyl ethylamide C8F17S02N(CH2CH3)H 3.6 M556: C8F17S02N(H)(CH2C00H) 3.7 Surrogate standard: 1H-1H-2H-2H perfluorooctane sulfonic acid 4.0 W a r n in g s a n d C a u t io n s ______________________________________ ;________________ ' 4.1 Health and Safety Warnings: 4.1.1 U se u n iv e rs a l p re ca u tio n s, e s p e c ia lly la b o ra to ry coats, g o g g le s, and g lo ve s w h e n handling animal tissue, which may contain pathogens. . 5.0 Interferences______ ;________________________________________________________ 5.1 There are no interferences known at this time. ' 6.0 Equipment______________________ ;________________ '__________________ _________ 6.1 The following equipment is used while performing this method. Equivalent equipment is acceptable. 6.1.1 Ultra-Turrax T25 Grinder for grinding liver samples 6.1.2 Vortex mixer, VWR, Vortex Genie 2 6.1.3 Centrifuge, Mistral 1000 or IEC 6.1.4 Shaker, Eberbach or VWR ' ETS-8-6.0 Extraction of PFOS from Liver Page 2 of 14 3M Environmental Laboratory Page 61 3M Medical Department Studyy: T-6314.1 BACK TO MAIN A. na.ly. .tica,l ,,Report: FACT TOX-028 L R N -U 2 6 3 6 6.1.5 Nitrogen Evaporator, Organomation 6.1.6 Balance (sensitivity to 0.100 g) 7.0 Supplies and Materials________________ ;_____________________:_________________ 7.1 Gloves 7.2 Dissecting scalpels 7.3 Eppendorf or disposable pipettes 7.4 Nalgene bottles, capable of holding 250 mL and 1 L 7.5 Volumetric flasks, glass, type A .. 7.6 I-CHEM vials, 40 mL glass . 7.7 Plastic sampule vials, Wheaton, 6 mL (or appropriate size) 7.8 Centrifuge tubes, polypropylene, 15 mL 7.9 Labels 7.10 Oxford Dispensor- 3.0 to 10.0 ml 7.11 Syringes, capable of measuring 5 pL to 50 pL 7.12 Graduated pipettes 7.13 Syringes, disposable plastic, 3 cc 7.14 Syringe filters, nylon, 0.2 pm, 25 mm . 7.15 Timer 7.16 Crimp cap autovials and caps 7.17 Crimpers Note: Prior to using glassware andbottles, rinse 3 times with methanol and 3 times with Milli- QTMwater. Rinse syringes aminimum of 9 times 'withmethanol, 3 rinses from 3 separate vials. . 8.0 R e a g e n t s a n d St a n d a r d s ____________________________'________________ ' 8.1 Type I reagent grade water, Milli-QTM or equivalent; all waterused in this method should be Milli-QTM water andbe provided by a Milli-Q TOC PlusTM system 8.2 Sodium hydroxide (NaOH), J.T Baker or equivalent 8.3 Tetrabutylammonium hydrogen sulfate(TBA), Kodak or equivalent 8.4 Sodium carbonate (NajCO,), J.T. Baker or equivalent 8.5 Sodium bicarbonate (NaHC03), J.T. Baker or equivalent 8.6 Methyl-iert-butyl ether, Omnisolv, glass distilled or HPLC grade 8.7 Methanol, Omnisolv, glass distilled orHPLC grade 8.8 Liver, frozen from supplier 8.9 Dry ice from supplier 8.10 Fluorochemical standards 8.10.1 PFOS (3M Specialty Chemical Division), molecular weight = 538 . ' ETS-8-6.0 Extraction of PFOS from Liver Page 3 of 14 3M Environmental Laboratory Page 62 3M Medical Department Study: T-6314.1 BACK TO MAIN Analytical Report: FAC T TO X -028 L R N -U 2 6 3 6 8.10.2 PFOSA (3M Specialty Chemical Division), molecular weight = 499 . 8.10.3 PFOSAA (3M Specialty Chemical Division), molecular weight = 585 8.10.4 EtFOSE-OH (3M Specialty Chemical Division), molecular weight = 570 8.10.5 PFOSEA (3M Specialty Chemical Division), molecular weight = 527 8.10.6 M556 (3M Specialty Chemical Division), molecular weight = 557 8.10.7 Surrogate standard: 4-H, perfluorooctane sulfonic acid (1-H.l-H, 2-H, 2-H C8F,3S03H) molecular weight = 428 8.10.8 Other fluorochemicals, as appropriate 8.11 Reagent preparation NOTE: When preparing largervolumes than listed in reagent, standard, or surrogate preparation, adjust accordingly. 8.11.1 ION sodium hydroxide (NaOH): Weigh approximately 200 g NaOH. Pour into a 1000 mL beaker containing 500 mL Milli-QTM water, mix until all solids are . dissolved. Store in a 1 L Nalgene bottle. 8.11.2 1 N sodium hydroxide (NaOH): Dilute 10 N NaOH 1:10. Measure 10 mL of 10 N NaOH solution into a 100 mL volumetric flask and dilute to volume using Milli-QTM water. Store in a 125 mL Nalgene: bottle. 8.11.3 0.5 M tetrabutylammonium hydrogen sulfate (TBA): Weigh approximately 169 g of TBA into a 1 L volumetric containing 500 mL Milli-QTM water. Adjust to pH 10 using approximately 44 to 54 mL of 10 N NaOH (While adding the last mL of NaOH, add slowly because the pH changes abruptly). Dilute to volume with Milli-QTM water. Store in a 1LNalgene bottle. 8.11.3.1 TBA requires a check prior to each: use to ensure pH = 10. Adjust as needed using 1N NaOH solution. 8.11.4 0.25 M sodium carbonate/sodiumbicarbonatebuffer (NajC03/NaHC03): Weigh approximately 26.5 g of sodium carbonate (NajCOj) and 21.0 g of sodium bicarbonate (NaHC03) into a 1 L volumetric flask andbring to volume with MilliQ TM -w ater. S tore in a 1 L N algene b o ttle . 8.12 Standards preparation 8.12.1 Prepare PFOS standards for the standard curve. 8.12.2 Prepare other fluorochemical standards, as appropriate. Multicomponent fluorochemical standards are acceptable (for example, one working standard solution containing 1.00 ppm PFOS, 1.02 ppm PFOSA, 0.987 ppm PFOSAA, and 1.10 ppm EtFOSE-OH.) . 8.12.3 Weigh approximately 100 mg of PFOS into a 100 mL volumetric flask and record the actual weight. 8.12.4 Bring to volume with methanol for a stock standard of approximately 1000 ppm (pg/mL). 8.12.5 Dilute the stock solution with methanol for a working standard 1 solution of approximately 50 ppm. 3M Environmental Laboratory ETS-8-6.0 Extraction of PFOS from Liver Page 4 o f 14 Page 63 3M Medical Department Study: T-6314.1 BACK TO MAIN Analytical Report: FACT TOX-028 LRN-U2636 8.12.6 Dilute the stock solution with methanol for aworking standard 2 solution of approx. 5.0 ppm. 8.12.7 Dilute the stock solution with methanol for aworking standard 3 solution of approx. 0.50 ppm. 8.13 Surrogate stock standard preparation 8.13.1 Weigh approximately 50-60 mg of surrogate standard 1-H.l-H, 2-H, 2-H, C,F|3S03H into a 50 ml volumetric flask aid record the actual weight. 8.13.2 Bring to volume with methanol for a surrogate stock of approximately 1000-1200 Ppm8.13.3 Prepare a surrogate working standard. Transfer approximately 1.0 ml of surrogate stock to a 10 ml volumetric flask andbring to volume with methanol for a working standard of 10-20 ppm. Record tie actual volume transferred. 9.0 Sample Handling_____________________________ ____ ___________________________ 9.1 All samples are received frozen and mustbe kept frozen until the extraction is performed. 10.0 Quality Control________________:___________________________________ 10.1 Matrix blanks and method blanks . 10.1.1 An aliquot of 1.0 mL methanol is used as a solvent blank. 10.1.2 Extract two 1.0 mL aliquots ofMilli-QTM water following this procedure and use as method blanks. 10.1.3 Extract two 1.0 mL aliquots of liver homogenate following this procedure and use as matrix blanks. Refer to 11.1.6. -- 10.2 Matrix spikes 10.2.1 Prepare and analyze matrix spike andmatrix spike duplicate samples to determine the accuracy ofthe extraction. 1 0 .2 .2 P repare each sp ike u s in g a sam ple chosen b y th e a n a lyst, u s u a lly a c o n tro l liv e r received with each sample set 10.2.3 Expected concentrations will fall in the mid-range of the initial calibration curve. Additional spikes may be included andmay fall in the low-range of the initial calibration curve. 10.2.4 Prepare one matrix spike andmatrix spike duplicate per 40 samples, with a minimum of 2 matrix spikes per batch. 10.3 Continuing calibration verifications 10.3.1 Prepare continuing calibration verification samples to ensure the accuracy of the initial calibration curve. 10.3.2 Prepare, at a minimum, one continuing calibration verification sample per group of 10 samples. For example, if a sample set = 34, four verifications are prepared and extracted. ' 3M Environmental Laboratory ETS-8-6.0 Extraction of PFOS from Liver Page 5 of 14 Page-84- 3M Medical Department Study: T-6314.1 BACK TO MAIN Analytical Report: FACT TO X-028 L R N -U 2 6 36 10.33 Prepare each continuing calibration verification from the same matrix used to prepare the initial curve. 103.4 The expected concentrations will fall within the mid-range of the initial calibration curve. Additional spikes may be included that fall in the low-range of the initial calibration curve. This is necessary if the analyst must quantitate using only the low end of the calibration curve (for example, 5 ppb - 100 ppb, rather than 5 ppb -1000 ppb). 11.0 Calibration and Standardization________ '_____________________.________ 11.1 Prepare matrix calibration standards 11.1.1 Weigh approximately 40 g of liver into a 250 mLNalgene bottle containing 200 mLs Milli-QTM water. Grind to ahomogeneous solution. 11.1.2 If 40 g is not available, use appropriate amounts of liver andwater to ensure a 1:5 ratio. . 11.13 Refer to 13.0 to calculate the actual density of liver homogenate and the concentration of solid liver tissue dispersed in 1.0 mL ofhomogenate solution. 11.1.5 Add 1 mL ofhomogenate to a 15 mL centrifuge tube. Re-suspend solution by shaking between aliquots while preparing a total of eighteen 1mL aliquots of homogeneous solution in 15 mL centrifuge tubes. 11.1.6 Two 1 mL aliquots, or otherappropriate volume, serve as matrixblanks. 11.1.7 Typically use the standard concentrations and spiking amounts listed in Table 1, at the end of this section, to spike, in duplicate, two standard curves, for a total of eighteen samples, two matrix blanks, and two method blanks. 11.1.8 Refer to validation reports ETS-8-6.0 and ETS-8-7.0-V-1 or Attachment B, which lists the working ranges and the Linear Calibration Range (LCR) for calibration curves. 11.1.9 Use Attachment C as an aid in calculating the concentrations ofthe working standards. .R e fe r to 13.0 to ca lcu la te a ctu a l co n ce n tra tio n s o f P FO S in c a lib ra tio n standards. 11.2 To each working standard, blank, or continuing verification, add appropriate amount of surrogate working standard for the concentration to fall within the calibration curve range 5 ppb --lOOOppb. 3M Environmental Laboratory ETS-8-6.0 Extraction of PFOS from Liver Page 6 of 14 ige 65 3M Medical Department Study: T-6314.1 BACK TO MAIN Analytical Report: FACT TO X-028 LR N -U 2 6 36 11.3 Extract spiked liver homogenates following 12.14-12.25 of this method. Use these standards to establish each initial curve on the mass spectrometer. Table 1 Approximate Spiking Amounts for Calibration Standards Working Standard (Approx. Cone.) - 0.50 ppm 0.50 ppm 0.50 ppm 0.50 ppm 0.50 ppm 5.0 ppm 5.0 ppm 5.0 ppm 50 ppm Ml '2 4 10 20 40 10 20 30 4 Approx, final cone, of PFOS in liver Blank 0.005 ppm 0.010 ppm 0.025 ppm 0.050 ppm . 0.100 ppm 0.250 ppm 0.500 ppm 0.750 ppm 1.00 ppm 12.0 Procedure_________________________________________________________________ 12.1 Obtain frozen liver samples. 12.2 Cut approximately 1 g of liver using a dissecting scalpel. This part of the procedure is best performed quickly, not allowing the liver to thaw. 123 Weigh the sample directly into a taredplastic sampule vial. 1 2 .4 R e c o rd th e liv e r w e ig h t in 'th e stu d y n o te b o o k. 12.5 Return unused liver portions to freezer. 12.6 Add 2.5 mLs of water to sampule vial. 12.7 Grind the sample. Put the grinderprobe in the sample and grind for about 2 minutes, or until the sample is homogeneous. 12.8 Rinse the probe into the samplewith 2.5 mLs water using a pipette. 12.9 Take the grinder apart and clean it with methanol after each sample. Refer to AMDT-EP22. 12.10 Cap the sample andvortex for 15 seconds. Label the sampule vial with the study number, weight, liver ID, date and analyst initials. 3M Environmental Laboratory ETS-8-6.0 Extraction of PFOS from Liver Page 7 o f 14 Page 66 Medical Department Study: T-6314.1 BACK TO MAIN Analytical Report: FACT TOX-028 LRN-U2636 12.11 Pipette 1.0 mL, or other appropriate volume, ofhomogenate into a 15 mL polypropylene centrifuge tube. Label the centrifuge tube with the identical information as the sampule vial. Refer to attached worksheet for documenting the remaining steps. 12.12 Pipette two 1 mL aliquots of Milli-QTM water to centrifuge tubes. These will serve as method blanks. 12.13 Spike all samples, including blanks and standards ready for extraction with surrogate standard as described in section 11.2. 12.14 Spike each matrix with the appropriate amount of standard as described in 11.1, or Table 1 ofthat section, for the calibration curve standards. Also prepare matrix spikes and continuing calibration standards. . 12.15 Vortex mix the standard curve samples, matrix spike samples, and continuing calibration samples for 15 seconds. 12.16 Check to ensure 0.5 M TBA reagent is at pH 10. Ifnot, adjust accordingly. 12.17 To each sample, add 1 mL 0.5 M TBA and 2 mL ofthe 0.25 M sodium carbonate/sodium bicarbonate buffer. 12.18 Using an Oxford Dispenser, add 5 mLmethyl-terf-butyl ether. 12.19 Cap each sample andput on the shaker at a setting of 300 ipm, for 20 minutes. 12.20 Centrifuge for 20 to 25 minutes at a setting of 3500 rpm, or until layers arewell separated. 12.21 Label a fresh 15 mL centrifuge tube with the same information as in 12.10. 12.22 Remove 4.0 mL of the organic layer to the fresh 15 mL centrifuge tube. 12.23 Put each sample on the analytical nitrogen evaporator until dry, approximately 1 to 2 hours. 12.24 Add 1.0 mL to each centrifuge tube using a graduatedpipette. 12.25 Vortex mix for 30 seconds. 1 2 .2 6 A tta c h a 0 .2 p m n y lo n m esh filte r to a 3 cc syrin g e and tra n s fe r th e sam ple to th is sy rin g e . Filter into a 1.5 mL glass autovial or low-volume autovial when necessary. 12.27 Label the autovial with the study number, animal number and gender, sample timepoint, matrix, final solvent, extraction date, andanalyst(s) performing the extraction. 12.28 Cap and store extracts at room temperature or at approximately 4 C until analysis. 12.29 Complete the extraction worksheet, attached to this document, and tape in study notebook or include in studybinder, as appropriate. Environmental Laboratory ETS-8-6.0 Extraction of PFOS from Liver Page 8 of 14 Page 67 3M Medical Department Study: T-6314.1 BACK TO MAIN Analytical Report: FACT TO X -028 L R N -U 2 6 36 13.0 D a t a A n a l y s is a n d C a l c u l a t io n s __________________________________________________ 13.1 Calculations: 13.1.1 Calculate the average density of the liver homogenate by recording each mass of ten separate 1.0 mL aliquots of homogenate. Average density (mg/mL) = Average mass ('mg') ofthe aliquots 1.0 mL aliquot 13.1.2 Calculate the amount of liver (mg) per 1.0 ml, homogenate (or concentration of dispersed solid tissue per mL of homogenate suspension) using the following equation: g of Liver x Average density* ofhomogenate fag/niD (gofLiver + g ofWater) * referto 13.1.1 for details. 13.1.3 Calculate actual concentrations ofPFOS and other fluorochemicals in calibration standards using the following equation: II.T. o f Standard x Concentration (ug /mL) = Final Concentration (pg/g or mg/kg) mg Liver/ 1 mL homogenate* ofPFOS in Liver *refer to 13.1.2 for details. 14.0 M e t h o d P e r f o r m a n c e ________________________________________________________ _-- _ 14.1 The method detection limit (MDL) is analyte and matrix specific. Refer to MDL report for specific MDL and limit of quantitation (LOQ) values (refer to Attachments B and C). 14.2 The following quality control samples are extracted with eachbatch of samples to evaluate th e q u a lity o f th e e x tra c tio n and an a lysis. 14.2.1 Method blanks andmatrixblanks. 14.2.2 Matrix spike andmatrix spike duplicate samples to determine accuracy and . precision of the extraction. 14.2.3 Continuing calibration verification samples: to determine the continued accuracy of the initial calibration curve. 14.3 Refer to section 14 of ETS-8-7.0 for method performance criteria. 1 5.Q P o l l u t io n P r e v e n t io n a n d W a s t e M a n a g e m e n t ___________________ _-- ------------------- 15.1 Sample waste is disposed in biohazard containers, ilammable solvent waste is disposed in * high BTU containers, and used glass pipette waste is disposed in broken glass containers located in the laboratory. 3M Environmental Laboratory ETS-8-6.0 Extraction of PFOS from Liver Page 9 of 14 Page 68 3M Medical Department Study: T-6314.1 BACK TO MAIN Analytica| Report; FACTTOX-028 LRN-U2636 16.0 R ecords_____________________________________________________;________ 16.1 Complete the extraction worksheet attached to this method, and tape in the study notebook or include in the 3-ring studybinder, as appropriate. 17.0 T ables. D iagrams. Flowcharts, and Validation Data________________________ 17.1 Attachment A, Extraction worksheet 17.2 Attachment B, MDL/LOQ values and summary 17.3 Attachment C, Calibration standardcalculation and concentrationworksheet 18-0 R eferences____________________________________________ '___________________ 18.1 The validation report associated with this method is ETS-8-6.0 & 7.0-V-l. . 18.2 AMDT-EP-22, "Routine Maintenance of Ultra-Turrax T-25" 18.3 FACT-M-1.1, "Extraction ofPFOS or Other Anionic Fluorochemical Surfactants from Liver for Analysis Using HPLC-Electrospray/Mass Spectrometry" 19. Affected Documents__________ ^_______________________________________ 19.1 ETS-8-7.0, "Analysis ofLiver Extracts for Fluorochemicals using HPLC-Electrospray Mass Spectrometry" 2 0 .0 R e v is io n s ____________ ___ _______________________ Revision NiimV.pr Reason For Revision Revision Date 3M Environmental Laboratory ETS-8-6.0 Extraction of PFOS from Liver Page 10 of 14 Page 69~ 3M Medical Department Study: T-6314.1 BACK TO MAIN Analytical Report: FACT TO X-028 L R N -U 2 6 3 6 S tu d v # M atrix Box # W k /D av D ate S p ik ed /A n aly st CCV MS M SD S u rro g ate S td approx, p p m actu al ppm # F C M ix S td ap p ro x . 0 .5 p p m actu al ppm # F C M ix S td approx. 5 ppm actu al p p m # F C M ix S td approx. 50 p p m actu al ppm # C o m m en ts - -- -- - - - -- '- - -` . - -. - - - - - - - - -- - - - - .- - - .- - - - - - .- - .- -- Blank Liver Homogenate: S td # Liver amount = . k ... L iver E x tra c tio n M e t h o d ... ....... ... ......................................... _ ____________________ _______________ D ate & In itials-- ----- Spike surrogate and Standard m ix. Vortex 15 sec. Pioette 1 m L o f Liver Solution Pipette 1 m L o f f0 .5 M T BA , pH 10. pH - . _________________________________________ ______ ______________ ________________________________________________ _-- .--.--------------------- Std. # . Pipette 2 m L o f 0.25 N a2C O i/0.25M N aH C O t Buffer D iS D en se 5ml o f M ethyl-t-B utyl Ether Std. # T N -A -__ ________________________________ ___ __________________ _____ : -- Shake 20 m in. Centrifuge 20-25 m in. Remove a 4 m L aliauot o f organic laver Put on N itrogen EvaDorator to drvness Add 1.0 m L o f M ethanol . . _______ S h a k e r S p eed ---------------------------------------- _----------------------------------------------------------------------- ------- CentrifURe Speed . ... -- Evaporator Temperature ----------------------------------- -- --------------------------------------------- -- T N -A - ---------------------- --------------------- --- ------------ Vortex 30 sec. .-- F ilte r using a 3cc B-D svringe w ith a 0.2um SRI filter into autosam ple vial C o n t Cal. V erifications u sed th e sam e m atrix as fo r the standard curve. -------------------------------------------------------------------------------------------------------- -------- --------- __________________________________________ _-- ---- ------------------------ Attachment B: MDL/LOQ Values 3M Environmental Laboratory . ETS-8-6.0 Extraction of PFOS from Liver Page 11 of 16 Page 70 3M Medical Department Study: T-6314.1 BACK TO MAIN Analytical Report: FACT TO X-028 L R N -U 2 6 36 MDL/LOQ values for rabbit liver Compound MDL LOQ Linear Calibration Range (LCR) (PPb) (PPb) Approximate concentrations to be used for preparing the Standard Calibration Curve PFOS 8.45 26.9 30 ppb - 1200 ppb PFOSA 3.50 11.1 12 ppb -1200 ppb PFOSAA 24.6 78.3 30 p p b -1200 ppb EtFOSE-OH 108 345 60 ppb - 900 ppb* M556 82.3 262 60 ppb - 1200 ppb PFOSEA 33.9 108 30 ppb- 1200 ppb MDL/LOQ values in rat, bovine, andmonkey liver were not statistically determined. Two curves in each of these matrices were extracted and analyzedwith the rabbit liver curves to determine equivalence. Responses in the rat, bovine, andmonkey liver curves were equivalent to the rabbit responses, therefore, their MDL and LOQ will be assumed to be equivalent to those values as determined for the rabbit liver. Refer to LOQ Summary andMDL studyin ETS-8-6.0 &7.0-V-l for furtherinformation * EtFOSE-OH estimates only for MDLandLOQ. Didnotmeet criteria forvalidation. Compound; PFOS__________________ __ ___________ ______________ Prepared R ange o f :/-L Q R 'fro ih ^ R ange o f :_E G ^S id^ R ange o f . L C R -frifr" L iver m atrix range o f standards (ppb) (ng/mL) average ' curve (ppb) (ng/m L) : (ppb)'(ng/niL)A lo w s td .1. curve ''VV'iOTryeTfj^ (ppb) (ng/mL> .f( p b )n B S n l)ii high std h ig h std curve r-' -'C u rv e ' :'. (ppb) (ng/m L) ' '(ppb)' ;(ng/mL) ; R abbit 6 .1 9 -1 2 3 7 12 -1 2 0 0 ; v s f ' 6 - 300 ; r :t 6 0 - 1200 Compound; PFOSA Prepared L iver m atrix range o f standards (ppb) (ng/mL) Range o f average curve (ppb) (ng/mL) ' .a v c rv e ''f 'if.-, . (bpbO.tn^mL)" Range of lo w Std curve (ppb) (ng/mL) ! ^ripwt'!.,:(pbb)Tg/rriL) ' R a n g e o f . :L C R :frrtt^ ; ' h ig h s td v ; ,,H igh std"- ; c u rv e . v - ' i c u i v i r ; (ppb) (ng/mL) v(jpb) .(ng/rnti) : R abbit 6.19 - 1237 12 - 1200 12V l20Q ;C . 1 2 - 3 0 0 : i2.'L3obv ; 6 0 - 1 2 0 0 '6 0 - 1 2 0 0 ; Compound; PFOSAA Prepared ` Range o f L iver m atrix range of standards (ppb) (ng/mL) average curve (ppb) (ng/mL) R abbit 6.16 -1 2 3 2 1 2 - 1200 L Q ^ftom . aye curve (ppb) (ng/mL) Range o f low std curve (ppb) (ng/ml.) LG RfrornloW stdV : curve (ppb) (ng/mL) Range of high std curve (ppb) (ng/mL) L C R from ' 'high std curve (ppb) (ng/mL) 30-1200 ; 3 0 -900 6 0 -9 0 0 N /A ' N /A Attachment B; MDL/LOQ Values 3M Environmental Laboratory ETS-8-6.0 . Extraction of PFOS from Liver Page 12 o f 16 Page 7 1 3M Medical Department Study: T-6314.1 BACK TO MAIN A nalytical Report: F A C T T O X -028 LRN-U2636 Compound: EtFOSE-OH L iv e r m atrix Prepared range o f standards (ppb) (ng/mL) Range of average curve (ppb) (ng/mL) R abbit 6.17 -1 2 3 5 3 1 -9 0 0 LCR from ve curve (ppb) (ng/mL) 3 1 -9 0 0 Range of low std curve (ppb) (ng/mL) N /A LCR from low std curve (ppb) (ng/mL) N /A Range of high std curve (ppb) (ng/mL) N /A L C R from high std curve (ppb) (ng/mL) N /A Com Dound: PFOSEA Prepared Range of L iver m atrix . range o f standards (ppb) (ng/mL) average curve (ppb) (ng/mL) R abbit 6.17 -1 2 3 5 31 - 1200 L C R from ave.curve- - (ppb). (ng/mL)- Range of low std curve (ppb) (ng/mL) 31 r 1?Q0-- N /A LUR from low std curve. (ppb). (ng/mL) Range of high std curve (ppb) (ng/mL) ' N /A - N /A L C R from high std curve." (ppb) (ng/mL) . N /A . ' Com Dound: M556 L iv e r m atrix Prepared range o f standards (ppb) (ng/mL) R abbit 6 .17-1235 Range of average curve (ppb) (ng/mL) 31 -1 2 0 0 L Q L froni'k (ppb)-(ngWdi)- Range of low std LGR''from :-: :lo w stdi. ' curve V r 'c u r v e ! ^ - (ppb) (ng/mL) -..(ppb) ;(ng/ttlr; Range of high std curve (ppb) (ng/mL) LC R from .h ig h s td V curvei ._ .(ppb) (ng/inLV .60.i n o l i t i N /A N /A A ttachm ent C: Standard C alculations ETS-8-6.0 Extraction of PFOS from Liver 3M Environmental Laboratory Page 13 o f 14 Paye 72 3M Medical Department Study: T-6314.1 BACK TO MAIN Analytical Report: FACT TO X -028 L R N -U 2 6 3 6 Ion Pair Standard Curves - Tissue Prep date(s): Analyte(s): Sample matrix: Method/revision: Target analyte(s): FC mix std approx. 0.500 ppm: FC mix std approx. 5.00 ppm: FC mix std approx. 50.0 ppm: Surrogate std approx. 100ppm: Standard number: Equipment number: Final solvent and TN: Blank liver/identifier: Actual concentrations of standards in the FC mix PFO S Std cone PFOSA Std cone PFOSAA Std cone E tFO SE PFO SE A Std cone Std cone ug/m L ug/m L ug/m L ug/m L ug/m L 0 .5 0 0 0.500 0 .5 0 0 0.500 0.500 0 .5 0 0 0 .5 0 0 0.500 0.500 0.500 0 .5 0 0 0 .5 0 0 0 .5 0 0 0.500 0.500 0 .5 0 0 0 .5 0 0 0 .5 0 0 0.500 0.500 0.500 0.500 0.500 0.500 0.500 5.00 5.00 5.00 5.00 5.00 5.00 5.00 5.00 5.00 5.00 5.00 5.00 5.00 5.00 5.00 50.0 50.0 50.0 50.0 50.0 M SSti Std cone ug/m L 0.500 0.500 0.500 0.500 0.5 0C' 5.00 5.00 5.00 50.0 Std cone ug/m L A ll A m t spiked mL 0.002 0.004 0.010 0.020 0.040 0.010 . 0.020 0.030 0.004 AU D ensity g 0.167 0.167 0.167 0.167 0.167 0.167 0.167 0.167 0.167 Calculated concentrations of standards in the sample matrix PFO S F in a l cone ng/g 5.99 12.0 29.9 PFOSA F in a l cone n g /g 5.99 12.0 29.9 PFOSAA Final cone n g /g 5.99 12.0 29.9 E tFO SE Final cone ng/g 5.99 12.0 29.9 PFOSEA F in a l cone ng/g 5.99 12.0 29.9 M 556 F in a l cone ng/g 5.99 12.0 29.9 Std cone n g /g 59.9 120 59.9 120 59.9 120 . 59.9 120 59.9 120 59.9 120 299 299 299 299 299 299 599 599 599 599 599 599 898 1198 898 1198 898 1198 898 1198 898 1198 898 1198 S urrogate Std cone ng/m L too S u rro g a te Final cone ng/mL 0.500 A ll A m 't spiked mL 0:005 Validated ranges - approximate concentrations L iver PFOS PFOSA PFOSAA R a b b it . 5-1000 ppb 5-1000 ppb 5-1000 ppb Bovine ` Estim ates only, use rabbit values. Rat Estim ates only, use rabbit values. M onkey Estim ates only, use rabbit values. E tFO SE -O H 5-1000 ppb POAA 5-1000 ppb PFOSEA 5-1000 ppb Attachment C: Standard Calculations ETS-8-6.0 Extraction of PFOS from Liver 3M Environmental Laboratory Page 14 of 14 Page 73 3M Medical Department Study: T-6314.1 BACK TO MAIN Analytical Report: FACT TO X-028 L R N -U 2 6 36 3M Environmentat. Laboratory Method Analysis of Potassium Perfluorooctanesulfonate or Other Fluorochemicals in Serum Extracts Using HPLC-Electrospray/M ass Spectrometry Method Number: ETS-8-5.1 Author: Lisa Clemen, Robert Wynne Approved By: ](H Laboratory Manager -- Adoption Date: 03/01/99 Revision Date: Date Group Leader __Cfrts*. A CJbmu-______________________________ Technical Reviewer Date oh/ u / i i Date 1.0 Scope and Application______________________________________________________ 1.1 Scope: This method describes the analysis of serum extracts for fluorochemical surfactants using HPLC-electrospray/mass spectrometry. ' 1.2 Applicable Compounds: Fluorochemical surfactants or other fluorinated compounds, or other ionizable compounds. 1.3 Matrices: Rabbit, rat, bovine, monkey, and human serum, or other fluids as designated in the validation report. Word 6/95 ETS-8-5.1 Analysis of Serum Extract Using ES/MS Page 1 o f 9 3M Medical Department Study: T-6314.1 BACK TO MAIN Analytical Report: FACT TO X -028 L R N -U 2 6 36 2.0 Summary of Me t h o d __________________________________________________ __ 2.1 This method describes the analysis of fluorochemical surfactants extracted from serum or other fluids, using HPLC-electrospray/mass spectrometry, or similar system as appropriate. The analysis is performed by monitoring a single ion characteristic of a particular fluorochemical, such as the perfluorooctanesulfonate (PFOS) anion, m/z= 499. Additionally, samples may be analyzed using a tandem mass spectrometer to further verify the identity of a compound by detecting daughter ions of the parent ion. 3.0 Definitions___________________________ ;__________________________;_____________ 3.1 Atmospheric Pressure Ionization (API): The Micromass Quattro II triple quadrupole systems allow for various methods of ionization by utilizing various sources, probes, and interfaces. These include but are not limited to: Electrospray Ionization (ESI), Atmospheric Pressure chemical Ionization (APcI), Thermospray, etc. The ionization process in these techniques occurs at atmospheric pressure (i.e., not under a vacuum). 3.2 Electrospray Ionization (ES, ESI): a method of ionization performed at atmospheric pressure, whereby ions in solution are transferred to the gas phase via tiny charged droplets. These charged droplets are produced by the application of a strong electrical field. 3.3 Mass Spectrometry, Mass Spectrometer (MS), Tandem Mass Spectrometer (MS/MS): The API Quattro II triple quadrupole systems are equipped with quadrupole mass selective detectors. Ions are selectively discriminated by mass to charge ratio (m/z) and subsequently detected. A single MS may be employed for ion detection or a series (MS/MS) for more specific fragmentation information. 3.4 Conventional vs. Z-spray probe interface; The latest models of Micromass Quattro II triple quadrupole systems (post 1998) utilize a "Z-spray" conformation. The spray emitted from a probe is orthogonal to the cone aperture. In the conventional conformation it is aimed directly at the cone aperture, after passing through a tortuous pathway in the counter electrode. Though the configuration is different, the methods of operation, cleaning, and maintenance are the same. However, Z-spray components and conventional components are not compatible with one another, but only with similar systems (i.e., Z-spray components are compatible with some other Z-spray systems, etc.) 3.5 Mass Lynx Software: System software designed for the specific operation of these Quattro II triple quadrupole systems. Currently MassLynx has Windows 95 and WindowsNT 4.0 versions. All versions are similar. For more details see the manual specific to the instrument (Micromass Quattro II triple quadrupole MassLynx or MsissLynx NT User's Guide). 4.0 Warnings and Cautions____________________________________________________ 4.1 Health and Safety 'Warnings: 4.1.1 Use caution with the voltage cables for the probe. When engaged, the probe employs a voltage of approximately 5000 Volts, 4.1.2 When handling samples or solvents wear appropriate protective gloves, eyewear, and clothing. ETS-8-5.1 A nalysis o f Serum E xtract U sing ES/M S Page 2 o f9 3M Page 75 3M Medical Department Study: T-6314.1 BACK TO MAIN Analytical Report: FACT TO X-028 L R N -U 2 6 36 4.2 Cautions: 4.2.1 Do not operate solvent pumps above capacity of 400 bar (5800 psi) back pressure. If the back pressure exceeds 400 bar, the HP1100 will initiate automatic shutdown. 4.2.2 Do not run solvent pumps to dryness. 5.0 I N T E R F E R E N C E S ___________________________________________________________________________________ 5.1 To minimize interferences when analyzing samples, teflon should not be used for sample storage or any part of instrumentation that comes in contact with the sample or extract. 6.0 Eq u i p m e n t _______________________________________________________________ 6.1 Equipment listed below may be modified in order to optimize the system. Document any modifications in the raw data as methoddeviations. 6.1.1 Micromass Quattro II triple quadrapole Mass Spectrometer equipped with an electrospray ionization source 6.1.2 HP1100 low pulse solvent pumping system, solvent degasser, column compartment, and autosampler 7.0 Supplies and Ma t e r i a l s ___________________ ;_______________________ 7.1 Supplies 7.1.1 High purity grade nitrogen gas regulated to approximately 100 psi (House air system) 7.1.2 HPLC analytical column, specifics to be determiined by the analyst and documented in the raw data. 7.1.3 Capped autovials or capped 15 mL centrifuge tubes 8.0 Reagents and Standards_______________________ 8.1 Reagents ____________________ 8.1.1 M ethanol, HPLC grade or equivalent 8.1.2 Milli-QTM water, all water used in this method should be Milli-QTM water or equivalent, and may be provided by a Milli-Q TOC Plus system or other vendor 8.1.3 Ammonium acetate, reagent grade or equivalent 8.2 Standards 8.2.1 Typically two method blanks, two matrix blanks, and eighteen matrix standards are prepared during the extraction procedure. See ETS-8-4.1. 9.0 Sample Handling____________________ '__________________________ ______________ 9.1 Fresh matrix standards are prepared with each analysis. Extracted standards and samples are stored in capped autovials or capped 15 mL centrifuge tubes until analysis. 3M Environmental Laboratory ETS-8-5.1 Analysis of Serum Extract Using ES/MS Page 3 o f 9 Page 76 3M Medical Department Study: T-6314.1 BACK TO MAIN Analytical Report: F A C T T O X -028 L R N -U 2 6 3 6 9.2 If analysis will be delayed, extracted standards and samples can be refrigerated at approximately 4 C, or at room temperature, until analysis can be performed. 10.0 Quality Control_____________________________________ ___________________ _ 10.1 Solvent Blanks, Method Blanks and Matrix Blanks 10.1.1 Solvent blanks, method blanks and matrix blanks are prepared and analyzed with each batch to determine contamination or carryover. 10.1.2 Analyze a method blank and a matrix blank prior to each calibration curve. 10.2 Matrix Spikes 10.2.1 Matrix spikes are prepared and analyzed to determine the matrix effect on the ' recovery efficiency. 10.2.2 Matrix spike duplicates are prepared and analyzedto measure the precision and the recovery for each analyte. 10.2.3 Analyze a matrix spike andmatrix spike duplicate per forty samples, with a minimum of 2 spikes per batch. 10.2.4 Matrix spike and matrix spike duplicate concentrations will fall in the mid-range of the initial calibration curve. Additional spike concentrations may fall in the lowrange of the initial calibration curve. 10.3 Continuing Calibration Verifications 10.3.1 Continuing calibration verifications are analyzed to verify the continued accuracy of the calibration curve. ' 10.3.2 Analyze a mid-range calibration standard after every tenth sample, with a minimum of one per batch. 11.0 Calibration and Standardization______________ ___________________ ;_________ 11.1 Analyze the extracted matrix standards prior to and following each set of extracts. The average of two standard curves will be plotted by linear regression (y = my + b), weighted 1/x, not forced through zero, using MassLynx or other suitable software. 11.2 If the curve does not meet requirements, perform routine maintenance or reextract the standard curve (ifnecessary) and reanalyze. 11.3 For purposes of accuracy when quantitating low levels of analyte, it may be necessary to use the low end of the calibration curve rather than the lull range of the standard curve. Example: when attempting to quantitate approximately 10 ppb of analyte, generate a calibration curve consisting offile standards from 5 ppb to 100 ppb rather than the full range of the curve (5 ppb to 1000 ppb). This will reduce inaccuracy attributed to linear regression weighting of high concentration standards. 3M Environmental Labmatuty ETS-8-5.1 Analysis of Serum Extract Using ES/MS Page 4 of 9 Page 77 3M Medical Department Study: T-6314.1 BACK TO MAIN Analytical Report: FACT T O X -028 L R N -U 2 6 36 12.0 Procedures _______________________;_____________________________________ 12.1 Acquisition Set up 12.1.1 Click on start button in the Acquisition Control Panel. Set up a sample list. Assign a filename using MO-DAY-last digit of year-sample number, assign a method (MS) for acquiring, and type in sample descriptions. 12.1.2 To create a method click on scan button in the Acquisition control panel and select SIR (Single Ion Recording) or MRM. Set Ionization Mode as appropriate and mass to 499 or other appropriate masses. A full scan is usually collected along with the SIRs. Save acquisition method. IfMS/MS insliuments are employed, additional product ion fragmentation information may be collected. See Micromass MassLynx GUIDE TO DATA ACQUISITION for additional information and MRM (Multiple Reaction Monitoring). . 12.1.3 Typically the analytical batch run sequence begins with a set of extracted matrix standards and ends with a set of extractedmatrix standards. 12.1.4 Samples are analyzed with a continuing calibration check injected after every tenth sample. Solvent blanks should be analyzed periodically to monitor possible analyte carryover and are not considered samples butmay be included as such. 12.2 Using the Autosampler 12.2.1 Set up sample tray according to the sample list prepared in Section 12.1.1. 12.2.2 Set-up the HP1100/autosampler at the following conditions or at conditions the analyst considers appropriate for optimal response. Record actual conditions in the instrument logbook: 12.2.2.1 Sample size = 10 pL injection 12.2.2.2 Inject/sample = 1 12.2.2.3 Cycle time = 13.5 minutes 12.2.2.4 Solvent ramp = ' Time 0.00 min. 8.50 min. 11.0 min. 12.0 min. MeOH 40% 90% 90% 40% 2.0 mM Ammonium acetate 60% . 10% 10% 60% 12.2.2.5 Press the "Start" button. 12.3 Instrument Set-up 12.3.1 Refer to ETS-9-24.0 for more details. 12.3.2 Check the solvent level in reservoirs and refill if necessary. 3M bnvirortinental Lauurmury ETS-8-5.1 Analysis of Serum Extract Using E5/MS Page 5 of 9 Page 78 3M Medical Department Study: T-6314.1 BACK TO MAIN Analytical Report: FACT TO X-028 L R N -U 2 6 36 12.3.3 Check the stainless steel capillary at the end of the probe. Use an eyepiece to check the tip. The tip should be flat with nojagged edges. If the tip is found to be unsatisfactory, disassemble the probe and replace the stainless steel capillary. 12.3.4 Set HPLC pump to "On". Set the flow to 10-500 uL/min or as appropriate. Observe droplets coming out of the tip of the probe. Allow to equilibrate for approximately 10 minutes. 12.3.5 Turn on the nitrogen. A fne mist should be expelled with no nitrogen leaking around the tip of the probe. Readjust the tip of the probe if no mist is observed. 12.3.6 The instrument uses these parameters at the following settings. These settings may change in order to optimize the response: 12.3.6.1 Drying gas 250-400 liters/hour 12.3.6.2 ESI nebulizing.gas 10-15 liters/hour 12.3.6.3 HPLC constant flow mode, flow rate 10 - 500 pL/min 12.3.6.4 Pressure <400 bar (This parameter is not set, it is a guide to ensure the HPLC is operating correctly.) 12.3.7 Carefully guide the probe into the opening. Insert probe until it will not go any further. Connect the voltage cables to the probe. 12.3.8 Print the tune page, with its parameters, and store it in the study binder with a copy taped into the instrument log. 12.3.9 Using the cross-flow counter electrode in the ES/MS source is recommended for the analysis ofbiological matrices. 12.3.10Click on start button in the Acquisition ControlPanel (this may vary among MassLynx versions, see appropriate MassLynx USER'S GUIDE). Press the start button. Ensure start and end sample number includes all samples to be analyzed. 13.0 Data Analysis and Calculations______________', ________________________ 13.1 Calculations: 13.1.4 Calculate m atrix spike percent recoveries using the following equation: %Recovery = Observed Result - Background Result x 100 Expected Result 13.1.5 Calculate percent difference using the following equation: %Difference = Expected Cone. - Calculated Cone, x 100 Expected Cone. 13.1.6 Calculate actual concentration of PFOS, or other fluorochemical, in matrix (pg/mL): fng of PFOS calc, from std. Curve x Dilution Factor) x 1 pg (Initial Volume of matrix imLl + mL of Surrogate Standard) 1000 ng Final Volume (mL) ETS-8-5.1 Analysis of Serum Extract Using ES/MS Page 6 of 9 3M Environmental Lauuratuiy Page 79 3M Medical Department Study: T-6314.1 BACK TO MAIN Analytical Report: F A C T T O X -028 L R N -U 2 6 3 6 14.0 M e t h o d P e r f o r m a n c e ______________________________________________________ _ 14.1 Method Detection Limit (MDL) and Limit of Quantitation (LOQ) are method, analyte, and matrix specific. Please see ETS-8-4.1, Attachment B, for a listing of current validated MDL and LOQ values. 14.2 Solvent Blanks, Method Blanks, and Matrix Blanks 14.2.1 Solvent blanks, method blanks, and matrix blanks values are must be below the lowest standard in the calibration curve 14.3 Calibration Curves 14.3.1 The r2value for the calibration curve must be 0.980 or better. 14.4 Matrix Spikes 14.4.1 Matrix spike percent recoveries are must be wilhin 30% of the spiked concentration. 14.5 Continuing Calibration Verifications 14.5.1 Continuing calibration verification percent recoveries must be 30% of the spiked concentration. 14.6 If criteria listed in this method performance section isn 't met, maintenance may be performed on the system and samples reanalyzed or other actions as determined by the analyst. Document all actions in the appropriate logbook. 14.7 If data are to be reported when performance criteriahave not been met, the data must be footnoted on tables and discussed in the text of the report. 15.0 Pollution Prevention and Waste Management _________ ______________ 15.1 Sample extract waste and flammable solvent is disposed in high BTU containers, and glass pipette waste is disposed in broken glass containers located in the laboratory. 16.0 R e c o r d s __________________________________'___________________ !_________________ 16.1 Each page generated for a study must have the following information included either in the header or hand written on the page: study or project number, acquisition method, integration method, sample name, extraction date, dilution factor (if applicable), and analyst. 16.2 Print the tune page, sample list, and acquisition method from MassLynx to include in the appropriate study folder. Copy these pages and tape into the instrument runlog. 16.3 Plot the calibration curve by linear regression, weighted 1/x, then print these graphs and store in the study folder. 16.4 Print data integration summary, integration method, and chromatograms, from MassLynx, and store in the study folder. 3M Environmental LaLiui alary ETS-8-5.1 Analysis o f Serum Extract Using ES/MS Page 7 of 9 Page 80 3M Medical Department Study: T-6314.1 BACK TO MAIN Analytical Report: FACT T O X -028 L R N -U 2 6 3 6 16.5 Summarize data using suitable software (Excel 5.0) and store in the study folder, see Attachment A for an example of a summary spreadsheet. 16.6 Back up electronic data to appropriate medium. Record in study notebook the file name and location of backup electronic data. 17.0 Tables. Diagrams. Flowcharts, and Validation Da t a _________________ 17.1 Attachment A: ETS-8-5.1 Data summary spreadsheet. 18.0 References___________________________ ;_____________________________________ _ 18.1 FACT-M-4.1, "Extraction of Potassium Perfluorooctanesulfonate or Other Fluorochemical compounds from Serum for Analysis Using HPLC-Electrospray/Mass Spectrometry 18.2 ETS-9-24.0, "Operation and Maintenance of the Micromass Atmospheric Pressure Ionization/Mass Spectrometer Quattro n triple quadrupole Systems" 18.3 The validation report associated with this method is ETS-8-4.0 & 5.0-V-l. 19.0 Affected Documents _______,_____________________________________ 19.1 ETS-8-4.1, "Extraction of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds from Serum for Analysis Using HPLC-Electrospray/Mass Spectrometry" 20.0 R e v is io n s _______________________________________________________________________ Revision Number. 1 Reason For Revision Section 6.1.2 Clarification of HP1100 system components. Section 11.1 Average of two curves, not standard values, are used for plotting linear regression and added the 1/x weighting of the curve. Section 12.2.2.4 Clarification of solvent ramp. Section 17.1 Changed from attachment B to A. Revision Date 04/02/99 3M Environmental Laboratory ETS-8-5.1 Analysis of Serum Extract Using ES/MS Page 8 of 9 Page 81 3M Medical Department Study: T-6314.1 BACK TO MAIN Analytical Report: FACT TO X-028 L R N -U 2 6 3 6 Laboratory Study # S tu d y : T est M aterial: M atrix/Final Solvent: M ethod/R evision: A nalytical E quipm ent System Num ber: Instrum ent Softw are/V ersion: . Filenam e: R -Squared V alue: Slope: Y Intercept: D ate o f E xtraction/A nalyst: D ate o f A nalysis/A nalyst G roup D ose Sam ple# C oncentration ug/m L In itia l V oi. mL D ilution F actor F inal C one. ug/m L S lo p e: T ak en from linear regression equation. G ro u p /D o se: T aken from the study folder. S am ple#: T aken from the study folder. C o n c e n tra tio n (ug/m L ): T aken from the M assLynx integration sum m ary. In itia l V o lu m e (m L ): T aken from the study folder. D ilu tio n F a c to r: T aken fro m the study folder. F in a l C o n e. (u g /m L ): C alculated by dividing the initial volum e from the concentration Attachment A: Summary Spreadsheet ETS-8-5.1 Analysis o f Serum Extract Using ES/MS . SM'bvirofrtniai Lauurawiy Page 9 of 9 Page 82 BACK TO MAIN 3M Medical Department Study: T-6314.1 Analytical Report: FACT TO X -028 Study #: FA CT-TO X-028 3M Environmental Lab -- Method Modification Method: ETS-8-5.1 "Analysis of Potassium Perfluorooetanesulfonate or Other Fluorochemicals in Sera Extracts Using HPLC-Electrospray/Mass Spectrometry" Section modified: Effective date ofmodifications: 10.3.2,14.5.1, add sections 14.3.2-14.3.6 April 26,1999 Section 10.3.2___________________________________'____________________ Method reads: 10.3.2 A nalyze a m id-range calibration standard after every tenth sam ple, w ith a m inim um o f one per batch. Modify method to read: . 10.3.2 A nalyze a m id-range calibration standard a t least after every ten sam ples, w ith a m inim um o f one per batch. Section 14.5.1________________________________________________________ Method reads: 14.5.1 C ontinuing calibration verification percent recoveries m ust be w ithin 30% o f the spiked concentration. Modify method to read: 14.5.1 A t least one continuing calibration verification per ten sam ples m ust show a percent recovery w ithin +/-30% o f the spiked concentration. Section 14.3.2_______________________________________________________ Method reads: NA Modify method to read: 14.3.2 T h e second (bracketing) calibration curve m ay be deactivated, i f instrum ental drift affects the data. T he first curve and acceptable calibration checks shall bracket usable data. 3M Environmental Laboratory Page 83 BACK TO MAIN 3M Medical Department Study: T-6314.1 iwrAnalytical Report: FACT TOX-028 Study#: FA CT-TO X-028 Section 14.3.3______ _____________________________________________ ;_____ Method reads: NA Modify method to read: 14.3.3 C alibration standards w ith peak areas less than 2 tim es the cuive m atrix blank should be deactivated to disqualify a data range that m ay be affected by background levels o f the analyte. Section 14,3.4________________________________________________________ Method reads: NA Modify method to read: 14.3.4 L ow o r high curve points m ay be deactivated to optim ize a linear range appropriate to th e data. Section 14.3.5________________________________________________________ Method reads: NA Modify method to read: 14.3.5 A curve point m ay be deactivated if it deviates m ore than 30% from the theoretical value w hen the curve is evaluated over a linear range appropriate to the data. Section 14.3.6________________________________________________________ Method reads: NA Modify method to read: 14.3.6 A valid calibration curve must contain at least 5 active points. . ----- ---------- Ulx /J? Signature of PAI and date Signature of Sponsor and date Signature of Study Director and date 3M Environmental Laboratory Page 84 3M Medical Department Study: T-6314.1 BACK TO MAIN Analytical Report: FACT T O X -028 L R N -U 2 6 3 6 3M Environmental Laboratory Method Analysis o f P otassium Perfluorooctanesulfonate or Other Fluorochem icals in Liv er Extracts Using H PLC-Electrospray/M ass Spectrometry Method Number: ETS-8-7.0 Author: Lisa Clemen, Glenn Langenburg Approved By: ' \ y /[ Laboratory Manager . u f^ t k * - -- - Group Leader A_______ _______________ Technical Reviewer Adoption Date: Revision Date: fJPc - Date Date o j /h /m Date 1.0 Scope and Application________:______;______________________________________ 1.1 Scope: This method is for the analysis ofliver extracts for fluorochemical surfactants using HPLC-electrospray/mass spectrometry. ' 1.2 Applicable Compounds: Fluorochemical surfactants or other fluorinated compounds, or other ionizable compounds. 1.3 Matrices: Rabbit, rat, bovine, monkey liver, or other tissues as designated in the validation report. Word 6/95 ETS-8-7.0 Analysis of Liver Extract Using ES/MS Page 1 of 10 3M Environmental LaLiuiaiuiy Page 85 3M Medical Department Study: T-6314.1 BACK TO MAIN Analytical Report: FACT TO X-028 L R N -U 2 6 3 6 2.0 Summary of Method__________________________ ____________ ;___________ 2.1 This method describes the analysis of fluorochemical surfactants extracted from liver using HPLC-electrospray/mass spectrometry, or similar system as appropriate. The analysis is performed by monitoring a single ion characteristic of aparticular fluorochemical, such as the perfluorooctanesulfonate (PFOS) anion, m/z = 499. Additionally, samples may be analyzed using a tandem mass spectrometer to further verify the identity of a compound by detecting daughter ions ofthe selected parent ion. 3.0 D e f in it io n s ______________________________ '__________________;__________________________ 3.1 Atmospheric Pressure Ionization (API): The Micromass Quattro II triple quadrupole systems allow for various methods of ionization by utilizing various sources, probes, and interfaces. These include but are not limited to: Electrospray Ionization (ESI), Atmospheric Pressure chemical Ionization (APcI), Thermospray, etc. The ionization process in these techniques occurs at atmospheric pressure (i.e. not under a vacuum). 3.2 Electrospray Ionization (ES, ESI): amethod of ionization performed at atmospheric pressure, whereby ions in solution aretransferred to the gas phase via tiny charged droplets. These charged droplets are producedby the application of a strong electrical field. 3.3 Mass Spectrometry, Mass Spectrometer (MS), Tandem Mass Spectrometer (MS/MS): The API Quattro II triple quadrupole mass spectrometer is equippedwith two quadrupole mass selective detectors and a collision cell. Ions are selectively discriminatedby mass to charge ratio (m/z) and subsequently detected. A single MS may be employed for ion detection or an ion may be selected in the first quadrupole, fragmented in the collision cell, and these fragments may be analyzed in the second quiidrupole. 3.4 Conventional vs. Z-spray probe interface: The latest models of Micromass Quattro II triple quadrupole (post 1998) utilize a"Z-spray" conformation. The spray emitted from a probe is orthogonal to the cone aperture. In the conventional conformation it is aimed directly at the cone aperture, afterpassing through a tortuous pathway in the counter electrode. Though the configuration is different, the methods of operation, cleaning, and maintenance are the same. However, Z-spray components and conventional components are not compatible with one another, but only with similar systems (i.e. Z-spray components are compatible with otherZ-spray systems, etc.) 3.5 Mass Lynx Software: System software designed for the specific operation of these Quattro II triple quadrupole systems. Currently MassLynx has Windows 95 and WindowsNT 4.0 versions. All versions are similar. Formore details refer to the manual specific to the instrument (Micromass Quattro II triple quadrupole MassLynx or MassLynx NT User's Guide). 4.0 W arnings and Cautions_____________________ . ________ 4.1 Health and Safety Warnings: 4.1.1 Use caution with the voltage cables for the probe. When engaged, the probe employs a voltage of approximately 5000 Volts. ETS-8-7.0 Analysis of Liver Extract Usin g ES/MS Page 2 of 10 3M Medical Department Study: T-6314.1 BACK TO MAIN Analytical Report: FACT TO X-028 L R N -U 2 6 3 6 4.1.2 Whenhandling samples or solvents wear appropriate protective gloves, eyewear, and clothing. ' 4.2 Cautions: . 4.2.1 Operate the solvent pumps below a back pressure o f400 bar (5800 psi). If the back pressure exceeds 400 bar, the HP1100 will initiate automatic shutdown. 4.2.2 Do not run solvent pumps to dryness. 5.0 Interferences________________ ;_______________ ___________________________ 5.1 To minimize interferences when analyzing samples, Teflon shall not be used for sample storage or any part ofinstrumentation that comes in contact with the sample or extract. 6.0 E quipment__________ :___________________________________;_________ ' 6.1 Equipment listed below may be modified in orderto optimize the system. Document any modifications in the raw data as method deviations. 6.1.1 Micromass Quattro II triple quadrupole Mass Spectrometer equipped with an electrospray ionization source. 6.1.2 HP1100 low pulse solvent pumping system, solvent degasser, column compartment, and autosampler 7.0 Supplies and Materials_____________ ;________________ :____________________ 7.1 Supplies . 7.1.1 High purity grade air regulated to approximately 100 psi (house air system) ' 7.1.2 HPLC analytical column, specifics to be deterniined by the analyst and documented in the raw data 7.1.3 Capped autovials or capped 15 ml centrifuge tubes 8.0 R eagents and St a n d a r d s ______________________________________________ _ 8.1 Reagents 8.1.1 Methanol, HPLC grade or equivalent . 8.1.2 Milli-QTM water (ASTM type I), all water used in this method should be ATSM type I, or equivalent, and be providedby a Milli-Q TOC Plus system or other vendor 8.1.3 Ammonium acetate, reagent grade or equivalent 8.1.3.1 When preparing different amounts than those listed, adjust accordingly. 8.1.3.2 2.0 mM ammonium acetate solution: Weigh approximately 0.300 g ammonium acetate. Pour into a 2000 mL volumetric container containing . 2000 mL Milli-QTM water, mix until all solids are dissolved. Store at room temperature. ETS-8-7.0 Analysis of Liver Extract Using ES/MS Page 3 of 10 3M EhvliuniMUiiial Labuialuiy Page 87 3M Medical Department Study: T-6314.1 BACK TO MAIN Analytical Report: F A C T TO X -028 L R N -U 2 6 36 8.2 Standards 8.2.1 Typically two methodblanks, two matrix blanks, and eighteen matrix standards are prepared during the extraction procedure. Refer to ETS-8-6.0. 9.0 Sample Handling______ ;__________ ________________________________________ 9.1 Fresh matrix standards are prepared with each analysis. Extracted standards and samples are stored in capped autovials or capped 15 ml centrifuge tubes until analysis. 9.2 If analysis will be delayed, extracted standards and stunples may be stored at room temperature, or refrigerated at approximately 4 C, until analysis canbe performed. 10.0 Quality Control_____________________ 10.1 Method Blanks and Matrix Blanks ____________________ ;________ . 10.1.1 Solvent blanks, methodblanks, and matrix blanks arepreparedand analyzed with each batch to determine contamination or canyover. 10.1.2 Analyze a method blank and a matrix blank prior to each calibration curve. 10.2 Matrix Spikes 10.2.1 Matrix spikes are prepared and analyzed to determine the matrix effect on the recovery efficiency. 10.2.2 Matrix spike duplicates are prepared and analyzed to measure the precision and the recovery for each analyte. 10.23 Analyze amatrix spike and matrix spike duplicate per forty sample^. With a minimum of 2 spikes per batch. 10.2.4 Matrix spike and matrix spike duplicate concentrations will fall in the mid-range of the initial calibration curve. Additional spike concentrations may fall in the lowrange of the initial calibration curve. 10.3 C o ntin u in g C alib ratio n Checks 10.3.1 Continuing calibration verifications are analyzed to verify the continued accuracy of the calibration curve. 10.3.2 Analyze amid-range calibration standard every tenth sample, with a minimum of one per batch. 11.0 Calibration and Standardization___________________________________ 11.1 Analyze the extracted matrix standards prior to and following each set of sample extracts. . The average of two standard curves will be plotted by linear regression (y = mx + b), weighted 1/x, not forced through the origin, using MassLynx or other suitable software. 11.2 If the curve does not meet requirements perform routine maintenance or reextract the standard curve (if necessary) and reanalyze. 3M'Ertvlroriiiieiiial Laburaiuiy ETS-8-7.0 Analysis of Liver Extract Using; ES/MS Page 4 of 10 Page 88 3M Medical Department Study: T-6314.1 BACK TO MAIN A nalytical R eport: FAC T T O X -028 L R N -U 2 6 3 6 11.3 For purposes of accuracywhen quantitating low levels of analyte, it may be necessary to use the low end ofthe calibration curve rather thanthe full range ofthe standard curve. Example: when attempting to quantitate approximately 10 ppb of analyte, generate a calibration curve consisting of the standards from 5 ppb to 100 ppb rather than the full range of the curve (5 ppb to 1000 ppb). This will reduce inaccuracy attributed to linear regression weighting ofhigh concentration standards,, 12.0 Procedures___________ '_________ ;______ '________ _________________________ 12.1 Acquisition Set up 12.1.1 Set up the sample list 12.1.1.1 Assign a sample list filename using MO-DAY-last digit of year-increasing letter of the alphabet starting with a 12.1.1.2 Assign a method (MS file) for acquiring 12.1.13 Assign an HPLC program (Inlet file) 12.1.1.4 Type in sample descriptions and vial position numbers 12.1.2 To create a method click on method in the Acquisition control panel then macs spectrometer headings and select SIR (Single Ion Recording) orMRM (Multiple Reaction Monitoring). Set Ionization Mode as appropriate and mass to 499 or other appropriate masses. A full scan is usually collected dong with the SIRs. Save acquisition method. IfMS/MS instruments are employed, additional product ion fragmentation informationmaybe collected. Refer to Micromass MassLynx GUIDE TO DATA ACQUISITION for additional information andMRM. 12.13 Typically the analytical batchrun sequence begins and ends with a set of extracted matrix standards. 12.1.4 Samples are analyzed with a continuing calibration verification injected standard after every tenth sample. Solvent blanks should be analyzed periodically to monitor possible analyte carryover and arenot considered samples but may be included as such. 12.2 Using the Autosampler 12.2.1 Set up sample tray according to the sample list prepared in Section 12.1.1. 12.2.2 Set-up the HP1100/autosampler at the following conditions or at conditions the analyst considers appropriate for optimal response. Record actual conditions in the instrument logbook: 12.2.2.1 Sample size = 10 pL injection 12.2.2.2 Inject/sample = 1 12.2.2.3 Cycle time = 9 minutes . E T S -8 -7 ,0 A nalysis o f L iver E xtract U sing ES/M S Page 5 o f 10 Page 89 3M Medical Department Study: T-6314.1 BACK TO MAIN Analytical Report: FACT TO X-028 ' LRN-U2636 12.2.2.4 Solvent ramp conditions Time MeOH 0.00 min. 1.0 min. 4.5 min. 6.5 min. 7.0 min. 9.0 mi. 40% 40% 95% 95% 40% 40% 2.0 mM /Nmmonium acetate 60% 60% 5% 5% 60% 60% 12.2.2.5 Press the "Start" button. 12.3 Instrument Set-up . 12.3.1 Refer to ETS-9-24.0, "Operation and Maintenance of the Micromass Quattro II Triple Quadrupole Mass SpectrometerFitted with an Atmospheric Pressure Ionization Source," for more details. 12.3.2 Check the solvent level in reservoirs and refill if necessary. 12.3.3 Check the stainless steel capillary at the end of the probe. Use an eyepiece to check the tip. The tip should be flat with nojagged edges. Ifthe tip is found to be unsatisfactory, disassemble the probe and replace the stainless steel capillary. 123.4 Turn on the nitrogen. 123.5 Open the tune page. Clicks on operate to initiate source block and desolvatioh heaters. . 123.6 Open the Inlet Editor. 123.6.1 Set HPLC pump to "On" 123.6.2 Set the flow to 10 - 500 uL/min or as appropriate 123.6.3 Observe droplets coming out of the tip of the probe. A fine mist should be expelled with no nitrogen leaking around the tip of the probe. Readjust the tip of the probe if no mist is observed 12.3.6.4 Allow to equilibrate for approximately 10 minutes. 123.7 The instrument uses these parameters at the following settings. These settings may change in order to optimize the response: 12.3.7.1 Drying gas 250-400 liters/hour 12.3.7.2 ESI nebulizing gas 10-15 liters/hour 123.7.3 HPLC constant flow mode flow rate 10 - 500 pL/min 12.3.7.4 Pressure <400 bar (This parameter is not set, it is a guide to ensure the HPLC is operating correctly.) 123.7.5 Source block temperature 150 123.7.6 Desolvation temperature 250 ETS-8-7.0 Analysis o f Liver Extract Using ES/MS Page 6 of 10 Laboratory Page 90 3M Medical Department Study: T-6314.1 BACK TO MAIN Analytical Report: FACT T O X -028 L R N -U 2 6 36 12.3.8 Print the tune page, with its parameters, and store it in the study binder with a copy taped into the instrument log. 12.3.9 Click on start button in the Acquisition Control Panel (this may vary among MassLynx versions, refer to appropriate MassLynx User's Guide). Ensure start and end sample number includes all samples to be analyzed. 13.0 Data Analysis and Calculations________________________ __________________ 13.1 Calculations: 13.1.4 Calculate matrix spike percent recoveries using the following equation: %Recovery- ObservedResult-Background Result x 100 Expected Result ' 13.1.5 Calculatepercent difference using the following equation: %Difference = Expected Cone. - Calculated Cone, x 100 Expected Cone. 13.1.6 Calculate actual concentrations in matrix (jrg/g): fag of PFOS calc, from std. Curve x Dilution Factor) x 1 ug (Initial Weight of Liver (gl 1000 ng Final Volume (mL) 14.0 Method Performance____________________________________________________ 14.1 Method Detection Limit (MDL) and Limit of Quantitation (LOQ) are method, analyte, and . matrix specific. Refer to ETS-8-6.0, Attachment B for a listing of current validated MDL and LOQ values. 14.2 Solvent Blanks, Method Blanks and Matrix Blanks 14.2.1 Solvent blanks, method blanks, andmatrix blanks must be below the lowest standard in the calibration curve. . 14.3 C a lib ra tio n Curves 14.3.1 The i2value for the calibrationmust be 0.980 orbetter. 14.4 Matrix Spikes 14.4.1 Matrix spike percent recoveries must be withiin 30% of the spiked concentration. 14.5 Continuing Calibration Verification 14.5.1 Continuing calibration verification percent recoveries must be within 30% of the . spiked concentration. 14.6 If criteria listed in the method performance section are not met, maintenance may be performed on the system and samples reanalyzed or other actions as determined by the analyst. Document all actions in the appropriate logbook. ETS-8-7.0 Analysis of Liver Extract Using ES/MS Page 7 o f 10 3M Medical Department Study: T-6314.1 BACK TO MAIN Analytical Report: FACT T O X -028 L R N -U 2 6 3 6 14.7 If data are to be reported when performance criteria have not been met, the data must be footnoted on tables and discussed in the text of the report. 15.0 Pollution Prevention and Waste Management_______ _______________________ 15.1 Sample extract waste and flammable solvent is disposed in high BTU containers, and glass pipette waste is disposed in broken glass containers located in the laboratory. 16.0 Records______ ________________ ;________ ;______________ ' , __________ 16.1 Each page generated for a study must have the following information included either in the header orhand written on the page: study or projectnumber, acquisition method, integration method, sample name, extraction date, dilution factor (if applicable), and analyst. 16.2 Print the tune page, sample list, and acquisition method from MassLynx to include in the appropriate study folder. Copy these pages and tape into the instrument runlog. 16.3 Plot the calibration curve by linear regression, weighted 1/x, then print these graphs and store in the study folder. 16.4 Print data integration summary, integration method, Eindchromatograms from MassLynx and store in the study folder. 16.5 Summarize data using suitable software (Excel 5.0+) and store in the study folder, refer to Attachment A for an example of a summary-spreadsheet. 16.6 Brick up electronic data to appropriate medium. Record in study notebook the file name and location ofbackup electronic data. 17.0 Tables. Diagrams. Flowcharts, and Validation Data________________________ 17.1 Attachment A: ETS-8-7.0 Data summary spreadsheet 18.0 R e f e r e n c e s ________________________________________________________ _ 18.1 FACT-M-2.1, "Extraction ofPotassium Perfluorooctanesulfonate or Other Fluorochemical Compounds fromLiver for Analysis Using HPLC-Electrospray/Mass Spectrometry" 18.2 ETS-9-24.0, "Operation andMaintenance of the Micromass Atmospheric Pressure Ionization/Mass Spectrometer Quattro II triple quadripole Systems" 18.3 The validation report associated with this method is ETS-8-6.0 & 7.0-V-l 19.0 Affected Do c u m e n t s _____________________________________ ________________ 19.1 ETS-8-6.0, "Extraction ofPotassium Perfluorooctanesulfonate or Other Fluorochemical Compounds from Liver or Fluid for Analysis Using HPLC-Electrospray/Mass Spectrometry" ETS-8-7.0 Analysis of Liver.Extract Using ES/MS Page 8 of 10 Page 92 3M Medical Department Study: T-6314.1 20.0 REVISIONS Revision Number BACK TO MAIN Analytical Report: FACT TO X-028 L R N -U 2 6 36 Reason ForRevision Revision Date 3M Environmental Laboratory E T S -8 -7 .0 A nalysis o f Liver Extract U sing ES/M S Page 9 o f 10 Page 93 3M Medical Department Study: T-6314.1 BACK TO MAIN Analytical Report: FACT TO X-028 L R N -U 2 6 36 Laboratory Study # Study: T est M aterial: M atrix/Final Solvent: M ethod/R evision: A nalytical Equipm ent System Num ber: Instrum ent Software/V ersion: Filenam e: . R -Squared V alue: Slope: Y Intercept: D ate o f E xtraction/A nalyst D ate o f A nalysis/A nalyst (roup D ose Sam ple# C oncentration n g /g in itia l W t. S D ilution F actor F inal C one, u g /g G ro u p /D o se: T aken from the study folder. Sam ple#: Taken from the study folder. C o n c e n tra tio n (ng/g): Taken from the M assLynx integration sum mary. In itia l W t. (g): T aken from the study folder. D ilution F a c to r: Taken from the study folder. F in a l C one, (ug/g): C alculated by dividing the initial volum e from the concentration Attachment A: Summary Spreadsheet ETS-8-7.0 Analysis of Liver Extract Using ES/MS 3M Environmental Laboratory Page 10 of 10 Page 94 3M Medical Department Study: T-6314.1 Study #: FA CT-TO X -028 BACK TO MAIN A nalytical R eport: F A C T TOX-OPfl L R N -U 2 6 36 3M Environmental Lab - Method Modification Method: ETS-8-7.0 "Analysis o f Potassium Perfluorooctanesulfonate or Other Fluorochemicals in Liver Extracts Using HPLC-Electrospray/Mass Spectrometry" Section modified: Effective date ofmodifications: 10.3.2,14.5.1, add sections 14.3.2-14.3.6 July 22,1999 Section 10.3.2______________________________ Method reads: '_____________________ 10.3.2 A nalyze a m id-range calibration standard after every ten th sam ple, w ith a m in im u m o f one p er batch. . Modify method to read: 10.3.2 A nalyze a m id-range calibration standard a t least after every ten sam ples, w ith a m inim um o f one p er batch. Section 14.5.1_________________________________________________________ Method reads: 14.5.1 C ontinuing calibration verification percen t recoveries m u st b e w ithin 30% o f th e spiked concentration. . Modify method to read: 14.5.1 A t least one continuing calibration verification p er ten sam ples m u st show a percen t recovery w ithin +/-30% o f the spiked concentration. Section 14.3.2__________ _______________________________________________ Method reads: NA Modify method to read: 14.3.2 T he second (bracketing) calibration curve m ay b e deactivated if instrum ental d rift affects th e data. The first curve and acceptable calibration checks shall bracket usable data. 3M Environmental Laboratory Page 95 3M Medical Department Study: T-6314.1 BACK TO MAIN Analytical Report: FACT TOX-028 Study #: FA CT-TO X-028 Section 14.3.3___________________________________________________ _ _ _ _ Method reads: NA Modify method to read: 14.3.3 C alibration standards w ith p eak areas less than 2 tim es the curve m atrix blank should be deactivated to disqualify a data range that m ay be affected by background levels o f the analyte. Section 14.3.4______________ ;__________________________________________ Method reads: NA Modify method to read: 14.3.4 L ow or high curve points m ay be deactivated to optim ize a linear range appropriate to the data. Section 14.3.5________________________________________________________ Method reads: NA Modify method to read: 14.3.5 A curve point m ay be deactivated i f it deviates m ore than 30% from the theoretical value w hen the curve is evaluated over a linear range appropriate to the data. Section 14.3.6___________________________________________________________ Method reads: NA Modify method to read: 14.3.6 A valid calibration curve must contain at least 5 active points. . - - Signature of PAI and date tijzyjo Signature of Sponsor and date Signature of Study Director and date 3M Environmental Laboratory Page 96 BACK TO MAIN 3M Medical Department Study: T-6314.1 Appendix D: Data Tables Analytical Report: FACT TOX-028 LRN-U2636 Table 8. Rat Serum Week 5 Data for FACT-TOX-028 Group Dose Sample # PFOS pg/mL PFOSA pg/mL M556 pg/mL C95911M 0.00937 <LOQ (0.00980) <LOQ (0.00980) C95912M <LOQ (0.00848) <LOQ (0.00980) <LOQ (0.00980) C95913M <LOQ (0.00848) <LOQ (0.00980) <LOQ (0.00980) Group 1 0 ppm C95914M C95915M C95991F C95992F C95993F C95994F C95995F <LOQ (0.00848) 0.0111 0.0298 0.0486 0.0431 0.0422 0.0387 <LOQ (0.00980) <LOQ (0.00980) <LOQ (0.00980) <LOQ (0.00980) <LOQ (0.00980) <LOQ (0.00980) <LOQ (0.00980) <LOQ (0.00980) <LOQ (0.00980) <LOQ (0.00980) <LCX) (0.00980) <LOQ (0.00980) <LOQ (0.00980) <LOQ (0.00980) Group 2 3 ppm C95931M C95932M C95933M C95934M C95935M C96011F C96012F C96013F C96014F C96015F 1.90 2.30 1.94 1.88 2.36 3.12 3.65 4.25 4.66 4.62 0.0196 0.0179 0.0211 0.0210 0.0279 0.0448 0.0394 0.0676 0.0618 0.0682 0.674 0.440 0.524 0.743 0.943 0.442 0.323 0.628 0.448 0.590 Group 3 C95951M C95952M C95953M C95954M C95955M 24.3 27.9 30.1 28.8 34.0 0.420 0.471 0.547 0.451 0.503 5.98 6.42 10.1 8.07 6.78 30 ppm C96031F C96032F C96033F C 96034F C96035F 41.0 52.8 41.6 38.7 50.2 0.649 0.775 0.869 0.498 0.857 4.34 3.40 7.57 7.98 5.87 Group 4 C95971M C95972M C95973M C95974M C95975M 48.2 47.1 56.2 53.2 55.8 0.599 0.660 0.745 0.777 0.658 10.2 11.3 15.9 14.5 12.0 100 ppm C96051F 63.8 1.27 10.6 C96052F 69.5 1.20 20.1 C96053F 75.2 1.23 17.0 C96054F 50.3 1.20 14.2 C96055F 58.1 1.41 12.3 It is not possible to verify true recovery of endogenous analyte from tissues without radio-labeled reference material. The only measurement of accuracy available at this time, matrix spike studies, indicate that the data quantitative to 30%. M570 p g/m L 0.00864 <LOQ (0.00495) <LOQ (0.00495) <LOQ (0.00495) 0.00963 0.00802 0.00862 0.00971 0.00949 0.0185 4.29 3.18 3.63 3.98 4.19 2.29 2.29 4.39 3.22 5.11 39.5 40.4 45.1 43.3 44.2 33.4 31.8 33.1 35.0 36.0 51.4 57.4 54.9 62.5 61.3 64.6 68.4 51.2 51.3 62.5 3M Environmental Laboratory Page 97 3M Medical Department Study: T-6314.1 BACK TO MAIN Analytical Report: FAC T TO X -028 LR N -U 2 6 36 Table 9. Rat Serum Week 14 Data for FACT-TOX-028 Group Dose Sam ple# PFOS pg/mL PFOSA pg/mL M556 pg/mL C95907M <LOQ (0.0215) <LOQ (0.00980) <LOQ (0.0249) C95908M 0.0226 <LOQ (0.00980) <LOQ (0.0249) C95910M <LOQ (0.0215) <LOQ (0.00980) <LOQ (0.0249) Group 1 0 ppm C95916M C95917M C95981F <LOQ (0.0215) <LOQ (0.0215) 0.0370 <LOQ (0.00980) <LOQ (0.00980) <LOQ (0.00980) <LOQ (0.0249) <LOQ (0.0249) <LOQ (0.0249) C95982F 0.0908 <LOQ (0.00980) <LOQ (0.0249) C95989F 0.0809 <LOQ (0.00980) <LOQ (0.0249) C95998F 0.0701 <LOQ (0.00980) <LOQ (0.0249) C95999F 0.0701 <LOQ (0.00980) <LOQ (0.0249) C95923M 3.45 0.0598 1.07 C95929M 4.38 0.0348 1.25 C95930M 4.12 0.0367 0.764 Group 2 3 ppm C95938M C95940M C96004F 4.10 3.65 6.89 0.0308 0.0364 0.0415 0.820 0.960 0.709 C96005F 9.41 0.0618 0.547 C96009F 10.2 0.0688 0.637 C96017F 8.57 0.0523 0.672 C96018F 11.3 0.0668 1.01 C95945M 44.7 0.663 9.00 C95946M 38.9 0.497 6.94 C95956M 36.9 0.453 8.22 Group 3 30 ppm C95957M C95959M C96028F 33.8 46.6 56.0 0.486 0.571 0.742 8.22 6.82 5.95 C96029F 79.9 0.780 7.08 C96036F 74.7 0.726 7.14 C96037F 65.6 0.649 7.30 C96039F C95966M 66.8 104 1.05 1.27 7.29 26.7 C95970M 134 0.784 22.7 C95976M 109 0.946 26.8 Group 4 100 ppm C95978M C95979M C96041F 110 101 178 0.890 0.905 1.25 26.9 22.6 23.9 C96043F 135 1.55 24.7 C96057F 238 1.33 21.3 C96058F 185 1.19 29.4 C96059F 262 0.969 30.0 It is not possible to verify true recovery of endogenous analyte from tissues without radio-labeled reference material. The only measurement of accuracy available at this time, matrix spike studies, indicate that the data quantitative to 30%. M570 p g /m L <LOQ (0.00980) <LOQ (0.00980) <LOQ (0.00980) <LOQ (0.00980) <LOQ (0.00980) <LOQ (0.00980) <LOQ (0.00980) <LOQ (0.00980) <LOQ (0.00980) <LOQ (0.00980) 3.07 2.99 4.35 3.54 3.90 3.75 3.28 3.05 4.06 3.53 23.4 20.1 22.7 20.5 16.4 24.1 21.3 21.4 18.2 22.2 32.6 16.7 20.6 22.2 26.3 25.0 27.0 23.2 26.7 21.0 3M Environmental Laboratory Page 98 3M Medical Department Study: T-6314.1 BACK TO MAIN Analytical Report: FACT TOX-028 LRN-U2636 Table 10. Rat Liver Week 5 Data for FACT-TOX-028 Group Dose Sample # PFOS pg/g PFOSA pg/g M556 pg/g M570 pg/g Group 1 0 ppm C95911M C95912M C95913M C95914M C95915M C95991F C95992F C95993F 0.134 0.134 0.124 0.104 0.158 0.0589 0.0399 0.0705 <LOQ (0.0122) <LOQ (0.0122) <LOQ (0.0122) <LOQ (0.0122) <LOQ (0.0122) <LOQ (0.0122) <LOQ (0.0122) <LOQ (0.0122) 0.00989 0.00683 0.00654 <LOQ (0.00611) <LOQ (0.00611) <LOQ (0.00611) <LOQ (0.00611) <LOQ (0.00611) C95994F C95995F 0.0741 0.0839 <LOQ (0.0122) <LOQ (0.0122) <LOQ (0.00611) 0.0102 Group 2 3 ppm C95931M C95932M C95933M C95934M C95935M C96011F C96012F C96013F C96014F C96015F 17.9 19.9 18.6 15.7 15.0 18.7 11.2 8.50 8.45 7.95 1.15 0.733 1.05 1.15 1.61 2.10 0.877 1.54 0.555 0.777 2.00 1.83 2.41 1.94 1.86 1.99 1.03 0.917 0.846 0.667 Group 3 C95951M C95952M C95953M C95954M C95955M 82.0 98.9 72.1 88.6 84.1 8.67 6.70 8.01 8.66 11.3 23.4 21.1 21.9 21.7 21.5 30 ppm C96031F C96032F C96033F C96034F C96035F 70.3 146 77.4 80.4 96.0 5.92 7.47 6.97 6.16 4 .2 5 10.8 17.6 11.5 15.9 11.6 C95971M 224 15.3 54.4 C95972M 141 10.6 45.7 C95973M 159 12.0 50.8 C95974M 221 15.4 62.7 Group 4 C95975M 216 13.3 55.8 100 ppm C96051F 197 12.3 34.7 C96052F 218 14.2 48.5 C96053F 275 14.4 46.0 C96054F 208 15.4 46.1 C96055F 218 14.5 29.4 It is not possible to verify true recovery of endogenous analyte from tissues without radio-labeled reference material. The only measurement of accuracy available at this time, matrix spike studies, indicate that the data quantitative to 30%. Determination o f M556 levels in liver may be biased low and should be considered quantitative to 50%. 0.0318 0.0213 0.0324 <LOQ (0.0122) 0.0159 0.0198 0.0129 0.0157 0.0238 0.0442 6.88 6.97 8.39 6.89 6.90 5.97 3.09 4.35 2.22 2.98 85.5 78.4 72.4 99.8 88.2 30.4 47.8 27.3 42.6 3 3 .3 163 138 132 176 157 114 141 124 138 123 3M Environmental Laboratory Page 99 3M Medical Department Study: T-6314.1 BACK TO MAIN Analytical Report: FACT TOX-028 LRN-U2636 Table 11. Rat Liver Week 14 Data for FACT-TOX-028 Group Dose Sample # PFOS pg/g PFOSA pg/g M556 pg/g Group 1 0 ppm C95907M C95908M C95910M C95916M C95917M C95981F 0.216 0.249 0.320 0.107 0.0945 0.0555 <LOQ (0.0122) <LOQ (0.0122) <LOQ (0.0122) 0.0125 <LOQ (0.0122) 0.0122 0.0164 0.0196 0.0151 <LOQ (0.0122) <LOQ (0.0122) <LOQ (0.0122) C95982F 0.160 0.0164 <LOQ (0.0122) C95989F 0.159 0.0153 <LOQ (0.0122) C95998F 0.146 0.0237 0.0246 C95999F 0.0768 <LOQ (0.0122) <LOQ (0.0122) C95923M 23.8 1.81 1.94 C95929M 25.7 10.8 1.45 C95930M 22.3 5.88 1.66 C95938M 29.5 5.01 1.39 Group 2 C95940M 29.3 7.88 1.88 3 ppm C96004F 17.8 1.47 1.03 C96005F 42.7 2.67 1.30 C96009F 45.6 2.25 1.42 C96017F 31.7 2.70 1.00 C96018F 47.0 2.07 1.94 C95945M 233 27.6 45.4 C95946M 234 18.9 31.5 C95956M 213 20.8 34.7 C95957M 195 16.7 37.8 Group 3 C95959M 311 24.2 50.4 30 ppm C96028F 203 14.2 11.4 C96029F 403 13.4 19.2 C96036F 336 11.7 15.3 C96037F 309 26.8 13.1 C 96039F 215 14 .7 1 3.6 C95966M C95970M 651 822 30.4 25.8 118 90.4 C95976M 729 32.3 103 C95978M 665 29.6 95.3 Group 4 C95979M 783 30.8 115 100 ppm C96041F 717 26.0 35.4 C96043F 610 24.7 39.5 C96057F 820 27.0 39.0 C96058F 759 24.6 30.8 C96059F 800 23.9 34.4 It is not possible to verify true recovery of endogenous analyte from tissues without radio-labeled reference material. The only measurement o f accuracy available at this tirr e, matrix spike studies, indicate that the data quantitative to 30%. Determination o f M556 levels in liver may be biased low and should be considered quantitative to 50%. M570 pg/g 0.0466 0.0388 0.0566 0.0827 0.0209 0.0183 0.0242 0.0606 0.125 0.0248 7.27 4.08 5.48 5.09 5.56 3.83 6.08 4.86 4.67 4.51 171 162 152 166 196 93.7 86.3 88.1 75.6 7 1 .4 347 229 278 259 346 136 148 139 106 108 3M Environmental Laboratory Page 100 BACK TO MAIN 3M Medical Department Study: T-6314.1 A nalytical Report: FA C T TO X -028 LR N -U 2 6 36 Table 12. Data Summary of Average Week 5 Rat Serum Concentration (|jg/mL) and Standard Deviation (SD) for FACT-TOX-028 Group Dose Group 1 0 ppm Group 2 3 ppm Group 3 30 ppm Sex (n=5) Male Female Male Female Male Female PFOS Average SD 0.00918 0.00113 0.0405 0.00691 2.08 0.232 4.06 0.664 29.0 3.52 44.9 6.24 PFOSA Average SD <LOQ NA <LOQ NA 0.0215 0.00380 0.0563 0.0134 0.478 0.0488 0.730 0.157 M556 Average SD <LOQ NA <LOQ NA 0.665 0.196 0.486 0.123 7.47 1.66 5.83 1.99 M570 Average SD 0.00662 0.00232 0.0109 0.00430 3.85 0.453 3.46 1.26 42.5 2.44 33.9 1.68 Group 4 100 ppm Male Female 52 4.23 63.4 9.71 0.688 0.0722 1.26 0.0889 12.8 2.34 14.9 3.77 It Is not possible to verify true recovery of endogenous analyte from tissues without radio-labeled reference material. The only measurement of accuracy available at this time, matrix spike studies, indicate that the data quantitative to 30%. NA = Not Applicable 57.5 4.59 59.6 7.92 Table 13. Data Summary of Average Week 14 Rat Serum Concentration (|jg/mL) and Standard Deviation (SD) for FACT-TOX-028 Group Dose Group 1 0 ppm Group 2 3 ppm Group 3 30 ppm Sex (n=5) Male Female Male Female Male Female PFOS Average SD 0.0217 0.000512 0.0698 0.0202 3.94 0.377 9.30 1.67 40.2 5.38 68.6 9.14 PFOSA Average SD <LOQ NA <LOQ NA 0.0397 0.0115 0.0582 0.0113 0.534 0.0840 0.790 0.154 M556 Average SD <LOQ NA <LOQ NA 0.973 0.197 0.714 0.174 7.84 0.936 6.95 0.568 M570 Average SD <LOQ NA <LOQ NA 3.57 0.571 3.53 0.393 20.6 2.72 21.4 2.11 Group 4 Male 111 13.0 0.958 0.182 25.2 2.26 100 ppm Female 200 50.5 1.26 0.212 25.9 3.73 It is not possible to verify true recovery of endogenous analyte from tissues, without radio-labeled reference material. The only measurement of accuracy available at this time, matrix spike studies, indicate that the data quantitative to 30%. NA = Not Applicable 23.7 6.06 24.6 2.51 3M Environmental Laboratory Page 101 BACK TO MAIN 3M Medical Department Study. T-6314.1 Analytical Report: FA C T TO X -028 L R N -U 2 6 36 Table 14. Data Summary of Average Week 5 Rat Liver Concentration (|jg/g) and Standard Deviation (SD) for FACT-TOX-028 Group Dose Group 1 0 ppm Group 2 3 ppm Sex (n=5) Male Female Male Female PFOS Average SD 0.131 0.0197 0.0655 0.0169 17.4 2.03 11.0 4.50 PFOSA Average SD <LOQ NA <LOQ NA 1.14 0.313 1.17 0.637 M556 Average SD 0.00710 0.00159 0.00693 0.00184 2.01 0.235 1.09 0.521 M570 Average SD 0.0227 0.00915 0.0233 0.0124 7.20 0.664 3.72 1.47 Group 3 30 ppm Male Female 85.2 9.77 94.0 30.6 8.67 1.67 6.15 1.23 21.9 0.878 13.5 3.08 84.9 10.4 36.3 8.62 Group 4 100 ppm Male Female 192 39.2 223 30.4 13.3 2.09 14.1 1.14 53.9 6.29 40.9 8.39 It is not possible to verify true recovery of endogenous analyte from tissues without radio-labeled reference material. The only measurement of accuracy available at this time, matrix spike studies, indicate that the data quantitative to 30%. Determination o f M556 levels in liver may be biased low and should be considered quantitative to 50%. NA = Not Applicable 153 18.2 128 11.5 Table 15. Data Summary of Average Week 14 Rat Liver Concentration (pg/g) and Standard Deviation (SD) for FACT-TOX-028 Group Dose Group 1 0 ppm Sex Male Female PFOS Average SD 0.197 0.0960 0.120 0 .0 4 9 8 PFOSA Average SD 0.0123 0.000114 0.0160 0 .0 0 4 7 0 M556 Average SD 0.0151 0.00310 0.0147 0 .0 0 5 5 6 M570 Average SD 0.0491 0.0229 0.0506 0 .0 4 4 8 Group 2 3 ppm Male Female 26.1 3.23 36.9 12.3 6.28 3.35 2.23 0.505 1.67 0.249 1.34 0.381 5.50 1.15 4.79 0.820 Group 3 30 ppm Male Female 237 44.1 293 84.0 21.6 4.32 16.2 6.1 39.9 7.79 14.5 2.98 169 16.4 83.0 9.23 Group 4 100 ppm Male Female 730 73.9 741 83.3 29.8 2.43 25.3 1.24 104 12.0 35.8 3.55 It is not possible to verify true recovery o f endogenous analyte from tissues without radio-labeled reference material. The only measurement of accuracy available at this time, matrix spike studies, indicate that the data quantitative to 30%. Determination of M556 levels in liver may be biased low and should be considered quantitative to 50%. NA = Not Applicable 292 53.0 128 19.3 3M Environmental Laboratory Page 102 BACK TO MAIN 3M Medical Department Study: T-6314.1 Analytical Report: FA C T TO X -028 LR N -U 2 6 36 Table 16. Approximate LOQ Values Used in FACT-TOX-028 Analyses by Matrix, Compound and Usage Dates Matrix Liver Sera Compound PFOS PFOSA M556 M570 PFOS PFOSA M556 M570 LOQ 0.0122 / 0.0306 pg/g 0.00611 / 0.0306 / 0.0122 pg/g 0.00611 / 0 .0 3 0 6 10.0122 pg/g 0 .0 1 2 2 / 0.0306 pg/g 0.00848 / 0.0215 pg/mL 0.00445/0.00980 pg/mL 0 .00980/0.0249 pg/mL 0 .0 0495/0.00980 pg/mL Usage Dates 10/23 to 11/02/00 10/23 to 11/02/00 10/23 to 11/02/00 10/23 to 11/02/00 10/02to 11/02/00 10/02to 11/02/00 10/02to 11/02/00 10/02to 11/02/00 Table 17. FACT-TOX-028 Rat Liver Week 5 Precision Data for PFOS and PFOSA Group Dose Group 2 3 ppm Sample # C95931M-1 C95931M-2 C95932M-1 C95932M-2 C95934M-1 C95934M-2 C96011F-1 C96011F-2 C96014F-1 C96014F-2 C96015F-1 C96015F-2 Average Percent Difference PFOS pg/g 15.5 15.8 17.9 20.4 15.5 14.5 21.6 21.0 20.1 17.3 17.5 17.7 PFOS Mean 15.7 19.2 15.0 21.3 18.7 17.6 PFOS Relative % Difference 1.92 13.1 6.67 2.82 15.0 1.14 6.76 PFOSA pg/g 0.754 1.12 0.732 0.605 1.01 1.00 0.764 0.956 0.511 0.392 0.940 0.821 PFOSA Mean PFOSA Relative % Difference 0.935 38.6 0.668 19.0 1.00 0.400 0.860 22.3 0.452 26.2 0.880 13.5 20.0 3M Environmental Laboratory Page 103 3M Medical Department Study: T-6314.1 BACK TO MAIN A nalytical Report: F A C T TO X -028 L R N -U 2 6 36 Table 18. FACT-TOX-028 Rat Liver Week 5 Precision Data for M556 and M570 Group Dose Group 2 3 ppm Sample # C95931M-1 C95931M-2 C95932M-1 C95932M-2 C95934M-1 C95934M-2 C96011F-1 C96011F-2 C96014F-1 C96014F-2 C96015F-1 C96015F-2 Average Percent Difference M556 pg/g 1.82 2.16 1.73 1.77 1.91 1.56 1.60 1.91 1.85 1.27 0.955 1.15 M556 Mean 1.99 1.75 1.56 1.05 M556 Relative % Difference 16.7 2.83 20.1 17.9 37.4 18.4 18.9 M570 pg/g 7.44 8.95 7.90 7.81 6.99 6.75 6.00 6.36 5.94 4.92 5.75 6.03 M570 Mean 8.20 M570 Relative % Difference 18.4 7.86 1.12 6.87 3.45 6.18 5.77 5.43 18.9 5.89 4.75 8.72 3M Environmental Laboratory Page 104 3M Medical Department Study: T-6314.1 Appendix E: Data Spreadsheets BACK TO MAIN Analytical Report: FACT TOX-028 LRN-U2636 3M Environmental Laboratory Page 105 3M Medical Department Study: T-6314.1 F A C T -T O X -021 Covjmcc# 6329*225 Analytical Report: FACTTOX-028 LRN-U2636 M y. Pfoduci W m C T cat SabaUacct M alr: M cthod/Setttoc Anat)1ical E f i i p u r t 3 )rio n Nub* : b a n n a i l So/uare/Vanjo: Flamec R-Squrod Value Slope: Y-lnUrdpt Dek* of ExUatiioo/Aeah* Ot* o Aa*1>ro/AaalyO: O t nf O t llrdurtnnlAaab* SimpleDili RATSERUMWEEK5 Group Dm S -a p k f U3S09250-H20 Blk-S-J J&S0925Q-5<fa SkJ-1 JtTSMlSfriara Blk-W *730*250-250 p^M SO -S CS5I11U C9S912U C919IJM C* 5W IF C9J99SF C tH U M C95931M C9591SM C960IIF * C9601JF CWOISF CW013F C95974M C96052F C96053F CMQS4F CM05SF C tn u o c - n i . FAC7-TOX-02* Ddtffiiaariaa r f l l > w i ^ r i C c o c r t a l i M r f f f C 8 , > B S * . M 5 , i i M 5 l f l l i a t i . W ^ : [ W B y t o j r r f C a C I i ( t t ) 1 6 i a i h t l T-6JI4 RMScnut E T d .lfcE T S 4 .il Ameba 06249* Ua*t)BX 14 SttBalaw OtoWm2SiXoXoI.(iwaLy/VwJK./KiqW/mW/oq,toni/,nomomhhauh tCVDMU. 10/W rf,10/12/30, 10.0K . 1074/30,1LQ6A30 UMHCJH id rtd la a Va*. L LOO 100 M 1.00 100 1.00 1.00 140 I jOO 1430 1430 1.00 100 1.00 1.00 1.00 1430 1.00 1.00 1430 14X3 1.00 1430 1.00 1430 14X3 100 1.00 1430 I jOO 1.00 1430 1430 1430 1410 1430 14X3 1.00 14X3 1.00 1.00 lu m catt Varillad NA NA NA NA NA NA NA NA NA NA NA NA Na NA NA HA NA Jadam lrO K 2ad aaalyaia OC 2nd aaal>iM OK 2a aaal)*i OK 2adaaah>OK 2nd aad)*i* OK 2ad aaalyii* C3K 2nd aaaljnia QIC 2nd a u h w OIL NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA 1*3 K O SFuriv Ck t m m T adar 0.(640 1.(640 0.1640 0.1640 0J64O 0.1640 NA NA 0(640 04640 04640 04640 0.1640 0.1640 OA440 0.1640 <3.(640 0.1640 04640 0.(640 0J640 04640 01640 04640 04640 0.(640 0.(640 0.1640 0(640 04640 0(640, 01640 04640 0.1640 04640 01640 0.1640 0.1640 04640 04640 04640 0.0640 01640 04640 04640 04640 01640 01640 r r o > PFOS im n ip i Case. Factar 1 1 _ r 0410 0.00 1 0.00 1 0.00 l 0170 1 0430 1 256 1 265 1 104 1 224 1 4.1 1 A66 L_ 1 1 no 541 54.2 1 4*4 1 4(4 1 441 10 220 10 367 >0 225 3 211 10 271 10 561 10 422 10 4*2 10 540 10 S il loo 212 100 325 100 341 100 315 100 354 loo 475 IDO 611 too 4(2 100 441 100 511 loo 551 too 545 100 650 100 (16 100 64* IOO 73 t 100 104 100 171 100 2 100 672 F ilw aM (ap4N*al) A0010050M AOOlOflHt* A001005006 A00 IOO5Q20 A001005007 A010O02I AQOIK5022 AOOI0H23 A00I005026 AOOIQQS027 A00100502I AO0I0O5O2 AOOIOOMJO A001P05031 AJ30I005034 ADOI005035 A00100S036 A00100J037 A00I025017 AOOIB2XU* AOOI02MI9 A0OI023O2O A001023021 AOOIQ25025 AOOIQ23Q26 ACO103027 AOOIMM24 ACO19232* AOOIO10019 A001010Q20 AD01010021 A00I010Q22 AOOIOK3021 A0010I0036 AD01010027 ACO1010024 A001010029 AOOI01003C A001010031 A23O1010034 A001010031 A001010Q56 AOOIOlorJ AMlOlOtMO A001010041 AOO1010042 A oautum ) A0Q1010044 Cauda Ira(Ua a pros a ^ u L a r V. Bw <U 3Q (942V -L) <LOO (942 ne/mLl < L O Q (* 4 2 a* /m L ) <LOO fl.12 na/atLl LOQ (9.12 af/nd.) < 0 0 15.12 wjW.1 10356 106 0530937 < tO Q ($.t2aS aL ) <LOQ(*42 af/mL) < O Q ( ,I2 *afcO.) 04111 0,029* 03)4(6 04431 0.0422 04317 IJ0 250 I.M 4* 256 1.12 345 421 446 4.62 244 Z7J 10.1 2(4 544 414 524 414 34.7 501 4*4 47.1 564 514 554 61.1 69J 754 504 5*3 U rna M as z AbL LOO 1940 M f o n <LOO1940 m AbL) < Q O (9.10 vafmL) 104 <LOQ *2 Anomala 0.0405 2-0* 4 06 294 44.9 511 63.4 BSD SU .D k U 1 A IS D K P D fam uM Varillad NA , NA ---------- f _______ 4 na , na NA NA NA NA NA NA NA NA 17.1 0.0069] 114 0.252 144 0664 12.1 _____ } - n 13.9 644 111 441 154 9.71 NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA C a aO tm U law reafirmad la w C a ota m dL aw Cmfinaed Law C iafim rilaa NA fnaHmiad la w CaaoaadLaw NA Omfinaad Lew C aafin o td law C a lm a d law Cm fi n a ad L a w C a afim a dla w Confirmad Law Cm fim adlaw Cm&imULew C o n fir m a d la * Confirmad law CwfiimadLaw rro sA DM h Portar 1 ____ J------1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 I 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 i 5 S 1 5 rro sA C aat n/w L 040 O tlO 0500 0450 040 0740 501 310 0700 0 .1 ) 0 0.410 ooo DITO 040 0)00 0.300 0.700 0.00 194 174 114 214 27.9 444 39.4 67-4 6(4 6(4 430 471 547 451 50) 619 775 (69 4M *57 5*9 660 745 777 651 254 240 247 240 2(2 IlklH M ( f a u l) A 0 0 100240) AOO300201* AOO1002006 AOI002D20 A01002007 AOO1002021 AD0IQ02022 AOO1002021 AOO1002026 AOO1002Q27 A001002432* A00I002O29 A001002030 A00I002O33 A00I002Q54 A0010Q3035 A001002036 AD01002017 AO0I0050M AODIDMJ A001005056 AQOIOOSQ37 A00100505* AD01005061 A001005062 A0OI0O5O63 AOO1003064 A 0 0 1005065 ADOIOIOOO A ooioioau AOOIOIOOH A0O1DIOOS3 AOO)0100*4 AOOIOIOOH ACO1010090 AOO1010091 AD0IQI0O92 AO0I010O9) A 00 0100*6 A001010097 AOO10)0091 A001010099 ADOlOtOlOO AOO1010075 AOO1010076 AOO1010077 AOO101007* AO0I0I0O79 Ciaran r a lb a afP fO S A aeA aL ar *A R n <LOQ (9 (0 o^/aL) <LOQ (9.H nfAaL) < 0 0 <9.(0 nc/aL) < O Q (9.(0 ag/mL) 121 124 <O Q {.IO */L > < Q (9.10 ag/nd.) LOQ (940 afAaL) < tO Q (940i(tel} < L O O (9(O attL ) < L O Q (*.ttaV fcL ) <U2Q (9.(0 0|AaL) < jO Q (l.(0aM .) LOQ(l.(Oa*/aiL) < 0 0 (9 4 0 atM .) 0 4 |* 6 04179 042II 00210 0 0279 aim * a0)94 04676 006(1 006(3 0420 0471 0547 0451 OSO) 0649 077) Ot* 049t 0.(57 0599 0440 0745 0777 04U 1.27 140 123 120 141 U na rro sA <oo <Q 125 <LOO LOO 00(5 00561 0471 07 0 6*1 1.16 * H ub ( c o o ) ceHraud ilk 10/35/30 uul>u. L /C 10111/00 O atc& trat'B r Date Vcofwd/ By. Pw iy EnhauW aifiefc HV1I/C0.1Q/IMM. 10/19X30,10OMM, H A M O LAC 11/36*0,11/37/30 KJH 12-70*0 LAC. 1277/00 tank M O SA * FuUiannrtanenilBiaamila I1U*CIFITSOJH(UJCH2COOH MS7I - CSFI7$02N(CH3)C112C00H BSD S4d.D e *. H SA U O V O NA NA NA NA 3 NA NA NA NA 17.7 0.003(0 2)4 00134 102 0.044! 21.5 0.157 10.5 0 0722 744 O O II9 3N/|Envronmeg!tt'Laboratory TnvJn-****.im I/4/2U01 Page 106 BAC K TO MAIN ill 3M Medical Department Study: T-6314.1 F A C T -T 0X -028 Covi ace* 329-235 Study" frodaci Nam tu(Tcd Subclase); WUrii: Mci*odfltcTHua: A auhiiud lyuipoeaf Syikat tna/uawnl SuOware/Veraiun: SKl-uSnqguucd Value Yluuupt 1*4=* e t F-XInstieu/Ajulv*: lMc* a t Ajulyai/AnakaC Dat* U llaU Rcduction/Aaalyit: Sample Data RAT SERUM WEEKS C raap Di m SaopU f ktclhudllft MuUix OH QC CfM f 1 4 PI (U1S0110-IDD filL-J-l RJJSMS0412O Blk-S-2 U lS M S O S d a Blt-5-l KKSMMO-Saa lUk-S-l RTStmSO-Scra DJk-5-1 RTSO230-SerBlk-S-2 RTSOT250 250 pp^U S-J RTSOWSO-ISO ppb-MSD-1 c m iiM C f lf 12M CiaapX 3ppm Gnau# 3 Mppm Croup 4 lOOppm C95991F C IJW c u n iM C9S9UM C9393414 CM !5M C960I2F CMOI3F C960I4F C960ISP C99IM C9592M C M fS IM C9S*S4M C9S935M C9603 IF C96C32F C96033F C96034F C 9 6 0 JJ F C9397IM CM 72*4 CU97U4 C9574(4 C9S97JM C960S1F C960J2F C960S3F CUW F m W M I * ETW-5.1 Amalia 063491 Manlyax 3.4 SaeBdow S D etcn aiu taa of the ftn c a c c la i CaxadnlM * ofPfO S, FFOSA, M556, ted 570 a tbe I J - W a i Dietary Study a f C itC D * (S>) 1G5 U Rat 0K M U SAlAUK/RWW iw iiA . io o s n o , i(Vinco, lo /i ic o . lo o ic o MMKACJH IO/D/OQ, 10094. 10/12/00,10/IU X ),1004X30, H O M O MMH/KW SuTm M Variiiad NA NA HA NA NA NA NA NA HA NA NA NA NA NA NA NA NA NA 2nd a a l i OK 2nd 0*1)11 OK 2nd arulyiif OK 2nd mjJyua OK b dm lvniO K 2nd analy OK 2nd analya OK 2nd aoajjruc OK 2 n d --a lyiaO K 2ad a n a li OK NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA MS DlhMiMI F id ar 1 1 l 1 1 ! 1 1 1 1 t 1 ______!.. ... 1 i i i i 10 10 10 10 to 10 10 10 10 10 100 too too too too too too 100 100 too too 100 100 100 too too too too 100 too M S C nc hM L 000 000 000 0.00 0.00 0.00 no M0 2-*4 000 000 0.00 04 000 0.00 QQ0 000 04 7.4 4441 S2.4 TO *41 M2 SO 1 44J 590 SJ 643 101* 10.7 47. 4J.4 340 73.7 79* M.7 102 113 13 143 120 106 201 170 142 111 flkaam * fopUaaat) A001002003 A00100201 AOO103200* A01002020 ADO1002007 ADO1002021 AOO1002022 A0010Q2021 A00100202 AOO1002027 AOO1002021 ADO100202 AOO10020XI AOO10020 33 AO01OO2O34 A0Q10Q2Q11 A0010Q20J* AOO1002077 AOO102 17 ADOI d i 11 ADO102 If A00102 2 0 ADO!023021 AOO1023023 ADO1023021 AOO1023027 AO1023021 AOO102302 A001010019 AWI010030 AOO[010021 ADQlOtOOa A001010023 ADO101002 ADO!010027 ADOIDI0021 A00101002 AOO]010030 A001010033 AOO1010034 A00101001S ADO101003 A001010037 A001010040 ADO1010041 A001010042 AOOI010043 A001010044 CoactaUatiaa aO IS K w / L o rY .Iac < O Q (9.U BftaL) <tO O f.M aa/M .I <LOQ (1JO *ifw L) < O Q fVIO a/mL) < O Q (9J0p*M .) <LDO i9.*0i**/raLl 133M 140* < O Q <9JO mr/mL) < O Q (9.10 apteL) < O Q ( I K ac/mL) <O Q (JO a#W .) < O Q (9.10 m faL ) <LOQ(10pYL) < O Q (I JO ap/mL) <LOQ ( t.U np/mL) < D Q ((M i|^ L ) <LQQ fS.10 apAnL) 0.674 0 440 0.524 0.741 8. *41 0.442 0J2* 0.421 0.441 0590 5.91 .42 10.1 .1 71 4.34 3.40 7.57 7.91 2J.I7 10 11.3 13J 14J 1X0 10. 20.1 17* 142 tx i U aat *4354 C/nL LOO <LOO <00 131H LOO to o 0.663 0.4*6 7.47 in 1X1 u . UD Sad. D ae M S/M SDUD NA NA NA .......... NA NA NA 4*6 NA NA NA NA 3 019 23.4 0-123 2X3 1.6 *14M.1 1U X34 23.4 1.77 a n c a te VaritW* NA NA NA MA NA NA NA NA NA NA NA NA NA NA NA KA NA NA la d an atn a O K 2ad acalynQK 2nd aaalysi* OK 2ad o i l n i i OK 2nd analymi OK 2 ad --al;h i OK SndaaafyOK 2nd aoalyiii OK 2nd aaahsis OK NA KA NA NA NA NA NA NA KNA* NA NA NA NA NA NA NA NA NA NA * llifhiecovcr) sa n frau d uii IX31C0 aaatyiii LAC 10/11*0 IfeicEjtfcnxbtty: a a c Verified/ D>: Purdy lriicicd/V aificd. 10/1 MM, lO /ll/M . 10/194. 10/26/X, ] IAKA La C I WD6A, IM IAO KJH 12/20/00 LAC. 1207CO m b W5S6* CIFl7502N(H)CH2COOH M3T0 - CtF17SQ2N(CH3)CH2COOH MS7 M ad-- F a c to r 1 1 1 1 1 | 1 | ------------ J------------ 1 1 I 1 1 1 1 | ID 10 10 10 >0 10 10 M 10 100 too 100 100 100 100 100 100 too 02 100 100 100 100 too 100 100 too too too M570 CMC efiaL ODO 03 03 0.00 0.00 03 211 24) IH 122 1.14 3.32 .* 0.02 1*2 9.71 .4 IIS 43 363 IM 419 229 229 4 122 SII 193 404 4SI 433 442 334 311 331 US330 314 37 5# 623 613 66 6*4 312 311 23 Analytical Report: FACT TOX-028 LRN-U2636 FUaaama (apttoaaJ) CaaoM niiaa a t USTI AOO1002003 AD0100201 ADO100200 <LOQ(4 5 nt/oA ) < O Q (4.93 a|AnL> AOO1002007 A0OIOQ2O21 <L O Q (4l H/mL) < 0 0 4-95 Rt/mL) A0OIOO3O2I ll% AOOID02027 ADO1002021 A0OIOO202I AMIDO JO ADO100203) < 0 Q (4.95 afAnL) 000963 000002 A M I00202S A001002036 AMIOCCOn ADO1023017 000971 0.00*49 0.0115 4X9 3*3 A00102M12I ADO1023023 A001023026 A M 1021027 ADO102)021 ADO102)029 A00I0I20I9 AMISI 2020 AOO1012021 A001012022 AOO1012023 AMI0I2021 AOO101202 AS010I20 A ooto;)*. ADDIO!2033 A M I012036 AM IO12017 AOO1OI203S AMI012039 4.10 229 22 4.3* >.22 311 39.3 40.4 4SI 43.) 442 31.4 *1.1 *30 3fi.fi 31.4 54.0 621 ------ in ------- A MI 012045 AMI 012046 AMI DI2047 3IJ 6X3 114*6 146 53 50* USD 14 Aar. 365 1.26 7.*2 3M Environmental Laboratory TOX-ozS-un223-lHjJi Page 107 3M Medical Department Study: T-6314.1 S iu J y. Product N u n to fT cA Suhrtancc): Mans McUtod/Rcvnioa: Analtica! ljuipouqu 5>rian N astier b u M u c at Sotittufc/Vdiias: Filettine: R-Squaicd Value Slope Y -lalauft Dutci of L0,1*1mWAajH it ule of AjuIi m /,Aa*I) . Dete o f il*U XodisctiWAestiT Sam ple D ata RAT SERUM W EEKS (329-325. FACT-TOX-tM I ET5-M -1AETS4-J.I Amelio 06249 Mo!}-34 09/23*0 SAUIUK/JtWW 1040430.10/0JAB. 10/104, 10/12*0,1003*0 MMH4CJH 1 0 * 5 * 0 ,1 0 * 9 * 0 ,1 0 /1 2 * 0 .1 0 /1 3 * 0 .1 0 0 4 * 0 .1l * M UUMUH FACT-TOX-02 Contee*6329-225 38. PFOSA.M J6. ami UTM b th o lJ -W o et Dietary Study ufC ltC D 9(S D ) K3S B K I a u Analytical Report: FACT TOX-028 LRN-U2636 Date Vaificii/ l)y. 1 1 * 6 * 0 .1 1 * 7 * 0 JUH runt>- Entercd/Vcnfwd: 120000 LAC, 1207*0 mmk 3l\fl Envi ronmental Laboratory TOX-02t#o*22J-lH.sii 1/40001 UMn> Page 108 BACK TO MAIN 3M Medical Department Study: T-6314.1 F A C T -T O X -O J C o v an ct# 63Z9-11S Analytical Report: FACT TOX-028 LRN-U2636 Huo>ha(TcstStixiy: FroJuci SuxlawJ: Matrix: Mcthod/Rcvwxw. C o n o c e 6329-223. FACT-TQX-021 P rmnnMioo b Ite Pio t o and C-ancen P itit c r ff f O S , PFOSA, MS36. a n i M570 in die IJ-W eA D idw y Sue* tfC ri.C D * (SD) ICS B R I * T-C3I4 JUi Soda ETS4-4.1 k ETS-I-S.I Analytical Kijutjxncnl Sri k m N um b* lmvumtnrSnOwan/Vcnien: Filename: R -Squared Value: Stupe: Y -Iii ia c crt: EUleufLiInclita/AiuJyM Soup 020199, Darei' 070799, A n clu (562496 M u t i , 3.4 SeaBatew So Attachment Sm Attacrirantt SmAttaccaceli ooso saUUK/rww Dales o f Andjiu/AojJyiL- 1 0 0 * 0 0 , ]/| IX. 10/I M . 10/114, 10774. ll/JM O MMH4UH Dole o f lN u Kaiueaa/Aa*lyX; Simple D iti 10164. 1011430,1072430.107000,114364 W M St/H RAT SERUM W EEK 14_________________________________________________________ te 171 C iw f ' Saapkl L U n d jic S vrrtftk FFOJPuriijr Dm MdhudUL MeUBlk RBS920-H2O Dtk-l-l RBSOT1S04 DO WL-t -1 RflSW230-Ser* Btk-1-1 Rii50925O-Sen B4-1-2 V et L 1.00 to o 100 100 Verified NA HA NA NA Carnada* Furie 0.1640 0*640 0.1640 04640 RT50925O-Sa Btfc-l-l K T S 0 n S 0 * cn B lk-t-2 140 100 NA NA 0.1640 01640 CC RT5W2S0-S0 pp*>-MS-l 140 RTSOVJ5O-IS0 ,,AA 4SD-I 1J NA NA C nupt C95907M 14 2nd AaatyaU OK 01640 Oppm C95906M CV59IOM 100 2nd AaaJyri* OK 1.00 2*4 Am 1)*u CMC 0.1640 0.1640 C9S9I6M CIJ9ITM CVS9I1F 1.00 2nd A ea lja i OK 100 IikIA u Kv OK 1.00 NA 01640 C9S9I2F C9S99f 1.00 NA to o HA 0.1640 01640 CM M IF C93999F LOO NA 100 NA 01640 O.I64 ' Cruuf J C95923M C95929M C9S930M C9S9UM 1.00 NA 1.00 NA 1.00 NA 1.00 NA 01640 OI&40 01610 0.1640 C95940M C960O4F C9600F 1.00 NA loo NA 1.00 NA 0.1640 Q PHA 04640 C9609F C96017F C9601IF 1.00 NA 14 Na 100 NA 01640 04640 01640 G nupi C9S94SM 1.00 CrmfinaadLow 04640 C$594614 C95956M C95957I4 C9599M C 960 2I F C96029F C960J6F 1.00 Confirmad Lew 14 Confirmed Lew 13 Confirmed Lou1.00 Confirmed Low 1.00 Confirmed Lew 1.00 C onfittati L oir 1.00 C o n fin ad Low 01640 0.1640 04640 0.1640 0.1640 01640. 04640 C96037F C96Q39F 1 0 0 Confirmed Low 1 0 0 C onfanad Low 04640 01640 G n u fi IOOppm C9S96M C95970M 1.00 C oofcocdL cw 100 ConfirmedLew- 046 04640 C95976M 1.00 C w d u c d L e w 01640 C9597IM 100 Confinen! Loir 01640 C9979M 1.00 Confirmed Lew 0.1640 C9604IF 1.00 C o n fesad Low 016 C9G04JF LOO C o n fesad Low 016 C9607F 1 0 0 Confirmed Low 046 C960JIF 0 9 7 Confirmed Low 01640 C960S9F LOO Confirmed Low 0 .1 6 l umi o / Qii-uiiuoo a-n Q > IVOS - 5 35 nML {p>\ PFOSA - 4.79 ppb, M3 5 - 13.2 ppb, 5 4 5 7 0 -9 * 0 ppb F FOI Dla tr ie Feder 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 10 10 IO IO 20 30 20 n 20 100 100 >00 100 100 100 li 100 100 100 200 200 200 200 200 200 200 loco 200 toc PFOS Ca ut/m L 0.00 000 000 040 040 0.00 224 222 740 264 741 0400 149 42.9 MS 934 ILI 11.2 399 307 477 473 423 399 343 309 496 634 311 430 427 391 339 tu Mi 164 760 77 5 773 629 634 517 1029 7*4 276 1039 301 TM -------T D 001009003 DOGI009019 D001Q09006 D00100S020 DOGI009007 D0M0W2! D001009022 IMO1009023 A 0 010271I7 A01271I A0OI027U A 001027120 AOO1027121 DOOIOM021 DOO1011034 EX 10HOJJ DOOIOH036 DOOIOHOJ7 A0IJ7119 AOO 1027(36 AOO1027137 AOO1027131 A M ICH I 9 A 001102016 AOQUQ017 Aoo n a ie i l A01102019 AOO1102020 SCOIO] 1030 S 001011031 S00 Hit 1012 *001011013 SOO1OII0M S0010U037 o m in n n ii *0010)1039 SOO1OII04O SOOlOliwi sooiontnj 0 0 Wl 1073 BOOM 11074 SOOIOI107J S 0 0 1 0 I (076 S0010II079 soaioiiuo *10130*6 SO1011C2 5001013041 Ceee--t n itrii e rrraa u riL irK lh c <LO Q (942 Df/edO <LDO 9 .II oxtaL l <LO Q C942n|M .) < X 3 0(9l2neA nL > <O Q (942.*A el <LOO V U oxhrU 90% I9K < O Q (24.9n*/raL ) 04226 < J 3 Q C24-9 egfteL) <LOQ (24.9 e^riiL} < 0 0 /2 4 9 AnL) 04370 0.0906 0.0609 04701 04701 3.45 431 4.12 4 10 165 619 941 10.2 157 111 44.7 m 36,9 331 460 560 ??.? 74.7 634 666 10 124 109 HO 101 171 113 23 US 262 Mmm PF04 Ui / h L <00 <00 - <OQ 90% 0.0226 < 0 0 - 1 Am cm Jt 0.0691 3.94 9J 402 666 111 200 RIO Sed. Dee, KS/MSD RTD NA NA NA NA _ NA NA 11 NA - NA 290 0.0202 lit 0.377 ILO U7 I3A m 13J 9.14 11.7 130 25J 303 tu rte p ti PFOSA 7FSA U n i a t VetMed M etri* (9 6 e.l Poetar acriiL NA 1 123 001 Di 1005 NA - 1 NA 1 0 0 0 D0010IIOI9 0 4 0 D00101I006 NA 1 0.00 D00101010 NA 1 0,00 DOO101*037 NA 1 040 000101U H NA 1 217 DOO1009022 NA 1 211 DOO1009023 NA 1 0.710 AOO1027117 NA 1 0.160 A00IO27U HA 0.620 A401027119 NA 1 0520 A001027120 NA f 0170 A 001027121 NA 1 0.0300 A01027I24 NA 1 0 4 0 AOO1027125 N A 1 0.00 AOO1027126 NA 1 0.00 A00J027I27 NA 1 0.0200 AOO102712* NA 1 594 AOD1027151 NA 1 144 A 001027(52 NA 1 367 A001Q27ISI NA 1 3 4 4 AOO1027150 NA 1 364 A001027149 NA 1 41.5 AOO1027161 NA 1 614 AOO1O27160 NA 64 A001027159 NA ! 52-3 AO0I027I5I NA 1 66.1 A001027157 Confirmed Low 20 112 *00100031 Coulea ed Low C onfinad Low 20 20 24.9 $001013052 22.7 $001013053 C ud ned Low Confirmad Low 20 20 24 J S00I013OS4 21.6 50010150)5 Confirmed Lew 20 >31 $00101305* 2 37.0 W VIUIJtUI Confirm ed L o w 20 J6.I $00101)0(0 C onfinad Low Confirmed Low 20 20 32.1 $00101)061 524 SOO1015062 Confinad Low Piallirea d 1rrr 3 5 aoototma251 SO0I013079 157 C onfinad Low 5 11 $001013011 C onfinad Low C o n fin a d Low S 3 171 SOOI013OI2 l i t $00)01)013 C onfinad Low 3 249 $001013066 Confirmad Low 3 310 S0010IK67 Confirmad Low 5 263 3OOI0I3II Confirmad Low 3 230 S0I013U9 Confirmed Low 3 rFQ S-i 194 $0010)3090 FFO U ^h iflu an o cttaad b iiaili Dalo liolcml/Oy: HVIfcOO, 10/19/00, 10/264, 10/314. 114174 LAC H - CIFJ 7S02N<HX:H2C00H M 3 7 *-C IF 17S 02N < C H l) C H 2C C X 3H D jU Verified! Oy. Peti? l^ ia aW aific * IMffOKJH I2/2TO LAC. 1 2774 mmk Ca-- uiriatfen ad PFOSA uT aL arH Rm < O Q (4.95 eAuL) < 0 0 4.9J pifad.) < O Q (4.95 affioL) <14)0/4 9) aefiuD < O Q (4 95eAaL) < 0 0 ( 4 W nifioU 95% 15% < O Q (9-10 ryAnL) < Q Q (9 * 0 b*AoL) < 0 Q (940 i^laL) <O Q (9M *fid3 <LOQ(9.W n*AL) < O Q (940 nffialA < O Q (910 o^fieL) < O Q (940 JoffinL) < 0 0 (910 ne/mL) 0 0596 0.0546 04367 O.OO* 0.0364 00415 40611 00661 0052) 046*1 0.663 0.497 0451 0416 0.571 47*2 v ./tu 0 726 0649 145 U7 4764 0.946 0490 0904 1-25 IJ5 1.33 1.19 0.969 In n PFOSA ut/m L ri-DQ <470 <oo 90% <00 <LOQ 0 0)97 00512 0334 0790 0954 1.26 USD M D ct. M M U D IT D NA NA NA NA NA NA 12% NA NA NA NA 26.9 00115 19 4 0011 157 00140 194 0 15 190 0.112 169 02)2 BACK TO MAIN V. 3M Environm ental Laboratory TOX -0U -eu22-lH .* Page 109 3M Medical Department Study: T-6314.1 PACT-TOX-OH Covanet* 6329-225 Analytical Report: FACTTOX-028 LRN-U2636 Study-. Produci Nenbe(TM* Sbetenos): Miroc Mahudttmiao: A /uM L al Equtpmaii S ) a Nun liu m acr* SeOnaic/Vsnuo: Fiksuma: K -S^jwcd Value: Sop: YDa-ItoeLt9oEj a: naienl/Mtfyit I W si A ul)iW A m |)it C W at Date JU JudW A ruJ)*: Sample Data O v .n o . 6329-225.FACT-raX-02* Q a B M i i f l i r f l f t a m i a l C ( m * i i i i u r y f O I , n W . M 5 5 l . w i M n O i i t e l V W i J : D i a ^ l i ^ r f C i t t p g O ) I O l W M i T-UI4 Rat S<no EIV4-4.1 S T U -5 1 Soup 020199, D \y 073799, Affidi. 062*94 Sm B d m teA nactam i So* ABactacats a t f u n o SALKJioitww UkM/oo,iviuno.wvoo.ioni*. io/ctao. iujk mmh/kih M U D O , M i n o . 1MXCC. i a v e v o , JU J&oo u u m c j h Creup mm Suyhf te m p a V attiU d NA NA NA NA NA NA MS56 1 1 t 1 1 1 MSS* COM. arfm L 0X0 4.41 OOQ 000 04 000 sia 2*9 5-3 D 031009005 D0010090U D001009006 DO01009020 DOOIOOM07 D 00I0 0902I DCO1009022 DOOI00902J C9J5I0M NA 1 0X0 D00009026 NA 1 0 0 0 DOOI0O9Q27 NA 1 000 D 001009024 NA 1 8-00 DOGI00902 OOQ DOOIOD90IO NA 0.00 000100905] 1 000 D 001009054 NA 1 o o o D001009015 OOQ D001009016 C9S999F 1 04 D001014037 2nd AmlyiiiOK IO 107 AO0I02T15 fP* 2nd A n d a ti CK NA IO 125 76.4 AO0I027D4 D0010ISCH2 2a d A a a l) O K 10 uo A 0 0 10271)6 D00I0IIO44 JT ' . NA IO 70* D001011O47 NA 54.7 D001014044 NA IO <1.7 DQ0I0IKM9 472 D0010IW 50 NA IO 101 001011051 20 450 SODI 011051 20 J47 5001011052 20 411 4001011055 20 4 S00101MS4 5 0 0 ) 0 J 1055 Confirmed Le 20 2*7 500101)054 fiaiff IITIM*1~ M *54 se sie :;:;; M 557 20 565 C e afirae d la * E 164 40010)1060 S00I0I506I *001013061 C9J966M CeofrmedLow Confirmed D m 200 200 200 200 154 m 154 154 so o io ix m 500101M32 S0010IM5) S0OI0IM34 5001011035 Coc rnm lD n*' C e cf ir sie d L ew 200 200 120 125 scom noii $00101)039 200 107 5001015040 C960S9F 141 Confirmed le w 200 ISO S00101KMI 5001015042 Ltui o f Quentoon (LOQ): FFOS - 5.55 ngM L (ppb). O S A - 4.7* ppfe.M 3 - J9.2 ppb, MITO - 9.IO ppb Cam--O rarti 4M556 ML r% Rm <LOQ (24* optai.) L O Q P*9rtad.) <tO Q (24.tapM g <LOQ f24* n a ta ti LOQ 0 * 9 eptad.) tO 0(24 9 n p to l) 12156 11656 <X X 5(24a|A nL) <LOQ p*.*opM L ) LOQ (24.9 aptaL ) LOQ (247 eptaL) <LOO (24f m MLI L O Q (J4*e*M L > LOQ (24 apM L) LOQ(24 9ptaiU LO Q(24l*M L> LOQ(24*n#AnL) 1.07 125 0.764 0.120 0.960 0.709 0 547 0.657 0672 101 9X0 694 422 122 612 591 7X6 7.14 750 729 267 22.7 264 26* 226 23.9 24-7 21.5 2*4 50.0 Mm MS56 <QQ <LOQ LOO 122% < rx} LOQ 0 971 0714 7.34 695 252 259 U> SO. Dm. MS/MSD RTO NA NA NA NA NA NA 9% NA NA NA NA 202 0.197 24J 0 174 11. 0 9J4 1.17 0564 9X0 2 ........ 14.4 3.7J M51t VerilDd DUe rectef NA 1 NA 1 NA 1 NA 1 NA 1 NA ____ !____ NA J NA 1 NA 1 NA 1 NA 1 NA 1 NA 1 NA 1 NA NA NA 2J Amh-W OK 1 1 1 2nd Aaalyi OC 2nd Arni** GK 10 10 NA 2ad Aanljai OK NA 10 NA M N A 10 NA 10 NA 10 N A 10 Cm fanedLo Confirmed Lew 100 100 Confirmed Lew C onfim i Uw Confirma* Lew C m finnadL nw 100 100 100 ioe Confirmed Lew Contened Lew 1O0 100 C en ten ed L o w C ontened Low 100 100 Coafissmi Low C a fim d U * 200 . 20) Confirmed Lew C ontened Lew 200 200 Contened La Confirmed Lew Confina! Low Confirmed Lew Coahzmmd Lew Confirmed Lew 200 200 200 200 200 200 MS7* Cm . 0 00 000 000 0X0 000 251 I2X 15.7 17J 10J 12.6 11.4 IS.I 235 110 15 2 554 175 305 406 1 234 201 241 214 161 103 111 125 1)5 105 Ite m (ep liem 4 D 0 0 1009 005 C e ee ran te 45197* M N L er*U U e_ <LOQ (9 40 apfad.) 1X01009006 DCCI0090ZO DOC1009007 P00100902I DCO1009022 <LOQC*40e*AnL) U3QX* <LOQ (9 40 o p ta i) 10296 DOO1014026 D 0 0 101 40 27 D 0 0 1 0 11021 DOO1 0 14G29 DOOIOIHZM 00)01403) D ooioiaoM DC01014015 D00I0I9O36 ooioiton AOOIQ27I39 LOQ (9.90 o p ta i) < L O Q (9 .9 0 cp fin li <LOO<*.0 n^mL) LOQ (9 ao np5aL) *d-OQ(9 40 apSeL) <LOQ (9JO a p U .) <LOQ(9.*0 opW O < L O Q (90n#A D 507 A0O1IU7IM DOOIO1*0*4 D00101*0*7 DOO10140*4 00010110*9 D00I0I4O5O DOMOltOSl 30010)3017 354 3.75 4X6 21.4 5001013024 *00101)027 *00101)0)1 S00I013O5) 500101103* S0010D03S 500I013OII S00I013X19 500101)041 500101)042 24.1 116 206 212 141 25.0 27.0 23.2 26.7 2IX 96m MSN uaML u sd StA Dm. M S/M SD R TD ----------- ---------------NA UX NA NA L O O LOQ 206 Jt-4 211 Dale E aM y: Dola VaiCtai/OyPuruv Enterod/VcriCed: l& flM . IM 9A 0.1CV 2M . I(VI DOO, l l 7 JOO LAG 1MTMWIH I2/20AX LAC, \IT V K li BACK TO MAIN l-TS-l-5.1 3M Environrrlft'fal L aboratory T 0X -0ll-m r12S -lH ja >!> P age 110 3MjMedical Department Study: T-6314.1 CFoAvCtaTc-tT*O63X1-90-2225 Study: Predust Nurbcr(Tcjl Subauacc): Matrix; Mctltod/RciiMOK Analytical Kifiipmctt Syrian Number Io rinano* Softwue/Venioo: Faentine C o w ic t 6329-225, FACT-TOX-tOI Dctew utioo of It* p iz ta tt and Conaeatrttian t/p f O S . PFOSA. 11556, and US7D m tba 13-Week Didmy Study r f C t t d (SD) J C S M JUl T -O I4 Hat Serum ETS-4-4. I A ETS-f-5.1 Soup 020199, Dovcy 070799, Antdio 062491 M in l jw 3.4 See AtUrisncnix R-Squwcd.Vatue: Slope Y-lrtcrxcpL Dale* of lixU aH ioo/A mliit ITalea of Analyaix/Analyu: SooA tu rin e n ta See AUacdnocntt SeeAtiedm orir 09/25/00 SAL/KJK/RWW KV09A, 10/1IX . 10/13/00. XVI*00, KV2TA0,11 0200 MMHKJH Dato of Data R uiw im /A nalyiL Sample Data 10/1400. IOtlMW, 1 0 0 3 0 0 , JO/JO0O. 110600 MMHKJH RAT SERUM WEEK 14 Crvup Dm M riiw d U iL Matrix Wk QC Cruuf | 0ppm Cfmsf 3ppm C ia f) 30 ppm G roup4 IQOppn Sanph* R JlS 0 9 2 M -l[ 2 O D lk -l- l RRStW lSWOOBBt-M EOS092JJ-Sa ffik-l-l RnS092M-So* Btt-f-2 RTS092SO-SamBlL-l-l RTSODIJO-Sera BIk-l-2 KTSO?230-250ppb-M3-1 RTSO2SO-2SOt>pb-MSD.l C95907M rro s Cm a tte l 0-00 05 0-00 000 0.00 0.00 224 222 7.10 26.2 C enelatkw r pro v t e L arS4 Rac < L O q (9 C 2 narinL) < L O O {9 * 2 a* A ri- 3 < O Q (9.42n*A aL ) <L O O (?-2nM oL LOQ (9.42 njAnL) < L O O 9 .lln aA n L ) 90% 9% < jOQ (24.9 np/mL) MImam p ro s e/mL 400 <LOO to o 1OT StA Dar, MStMSD RPB NA NA NA IH 5 9 4 IF 14.9 < tO O (243 nx/mL] LO O -1 Anonwh' 423 04070 NA u m C95929M 507 C959JOM 477 5 9JIM 475 C9S9MM 42) C90004F 399 C90OO5F 545 C960W 519 C9607K 496 C96014F 654 C95 94JM 514 C95946M C9956M 4 427 C95957M 391 5959M 539 C9602BF 64 C96019F *24 C96016F 464 C960J7F 760 C960J9F 771 C9966M 599 C95970M 775 C95976M 629 397IM 634 C9979M 517 C 9601 IF 1029 C96MJF 6057F 744 276 C960S4F 10)9 C960S9F 303 PFOS 5.55 n*/ruL (ppb). PFO SA- 4 .7 ? f 345 4M 4.12 4.10 165 4*9 *.41 103 4.57 113 447 319 369 33J 464 MM 79. 74.7 656 66| 104 134 109 110 101 394 9.27 402 616 111 1)5 2)4 115 200 M556 - 19.2 ppb, M 1 7 0 -9 .1 0 * 9.51 0377 110 1.67 13.4 J 34 IU 9 14 11.7 130 35.3 $ PFOSA C . c/mL US 02 0.00 0 .0 0 05 0.00 237 211 0.7 a* 0.6 032 0.2 0X000 0.00 aoo 000 0.0200 59.1 341 36.7 10J 364 415 <11 641 SU 661 331 24.9 217 24J 216 17.1 .? 363 315 52.6 2 157 119 171 III 249 310 265 230 194 XPFOftA u A a L *r 96 Ran < Q Q (4.95 ng/mL) 1 lO O (4.95 naAri.) <LOQ (4.95 ao/biL) tO O 44.95 a*teL ) <L O Q (4 .9 5 n f/m L ) LOO (4.95 na/tnL) 95H I5H < L0Q (9.W a*A nL) <LOQ (*10M ttay LOQ (9.10 ajAoLJ LOQ (9.40 x|AnL) < 0 0 f* IO uAnL> <LOQ(7JOe*AuL) LOQ (930 opta*) LOQ (9.40 nf/mL) tO Q ^lO iftat) < L O O (9 O n aA n L ) O.C590 0.0341 0 0367 0.0301 0-0364 10415 00611 0.0614 0.0523 00661 0.663 0497 0.45J 0.446 0571 0.742 0.7:0 0.724 049 1053 117 0.714 0146 0.490 0 905 115 115 1.13 1.19 0% ) Mean PFOSA LOO <00 LOO 9054 U30 <100 00397 0.0542 0514 0.790 0954 136 RO ltd . Da. MS/MSD RPD NA NA NA 12H NA NA NA NA 21 O.OIIS 194 0 011) 15.7 00140 19J 0.154 19 0 112 16.9 0112 MSN Cae*. 0 .0 0 441 000 0.00 0 .0 0 0.00 311 219 000 0.00 0 .0 0 0 .0 0 0 .0 0 ooo 0 .0 0 o0 000 ooo 107 125 76.4 12 960 70.9 $47 63.7 67.2 101 450 347 411 411 341 297 S 157 365 364 1)4 114 134 114 113 120 107 150 4M S56 w feL r H Rae < O Q (24.9 agfoL) < 0 0 (24.9 uA nL l 103(249 < 0 0 (24 9 aAnLl <O Q (24.9 |AaL) tOO (2 4 * tuAnU 12*54 1 1654 ICQ (24.9 ***.) LOQ (24.9 af^aL ) < C Q (2*3a*/w L ) < O Q (24.9 a p ta * ) LOO (24.9 aMnLJ LOQ (24 9 a#/roL) LOQ (24 J ateL ) LOQ(24.9 a^AaL) LOQ (24.9 afaL ) U 30a4 9nHL J7 135 0.764 0.120 0.960 0.709 0.547 0.637 0172 101 9.00 6.94 132 122 6.12 S.9S 7 . i 7.14 7.30 7.29 26.7 237 26.1 26.9 32 6 J 24.7 213 29.4 30 PFOSA Partiimmoctanfndfrnunida Date Gniaed/Dy: Date Verified/ By: 10/1MX). 10/1 MW. IO/260, IV3IA. 11/07W0 LAC DANDO U H M i 56 - CtF17SQ2N(H)CH2COQH M J70 - CIF17S02N(CH3)CH2COaH Purity lintercd/Veriiind: 12/20/00 LAC. 12/2700 ato Mana MS66 d te l LOO LOO LOO 12254 LOO 097J 07)4 714 695 153 23.9 BSD M .IW . NA NA NA 954 NA NA NA 203 0197 243 0 17* 11.9 0 936 1.17 0.561 *01 2.24 1.71 Analytical Report: FACT TOX-028 LRN-U2636 MS79 Cm C n m U atlm 4M57a 0.00 <LD Q(9.l0nAoL) 000 < 0 0 ( 9 .1 0 naAnl.) 0.00 <LOQl9.M&iAnL) 000 D0 000 260 10494 120 LOQ (9.40 n^AnL) l.7 LOQ (9.10 nf/mL) 171 <LOQ(9J0n*AnL) 10.9 126 11.4 <LOQ (9JO OfAnL) 11 1 LOQ 49.0 | M ) 233 (l.O 152 LOQ (9.H) n*/ml.) Mcm 1 M571 RIO SM. Dcv. <00 LOO NA NA 10356 )% UX3 NA 415 154 3.54 7*0 3 90 175 121 305 406 4.06 353 353 201 2J7 205 203 164 164 2D 213 214 112 11.2 222 222 I t i 32.6 44 101 2 0 4 111 111 2 6 ) IU J.53 0 3 9 1 214 2.11 105 2 1 0 24 6 2 .il BACK TO MAIN liT S IS l 3M E nvironm ental Laboratory TOX4J21 2 2 3 - n id i P age 111 BACK TO MAIN 53333 i 3M Medical Department Study: T-6314.1 CFtmACmTc-tT*O<3X2-90-2225 Analytical Report: FACT TOX-028 LRN-U2636 Hub<Tu Due al EuactiewAnalyst Due gfA a tty ^ A u h *: DuofDu*6a4ean'AulYiC SaapkE htta Ceua** 6329-225. FACT-T0X-2* I T4M4 H alli* E IH 4A A E 1U .14 Asalia 062496 * u d CeuuWio, ofPTOS, PFMA, U$54, wd MITO i . 4 . 1 3-Wk B u r y S U * ef C rtC M f$D) KH B * t u lO/tftOO SALUK B u i : 00-9 IO lito , IM40, 027*0. 103000.11ZOO. 124)1.00,12044 KJHMMHHOJ 102400, 1021/00, 103000, 103100. U-0600, 120400. 12054 MMH R A T L I V t R W t l K S _____________________________ ___________________ ___________ C ru p Dm *-- ** tam aau VariaC MWWl < M H M tt M Bk QC Cm pl trvm iffm G uy] *- O --f 4 100 P i* 10100400 B*-J 10IOO-H30-Mt-6 EBU0100-Levee | | . ] RBL10100-Uvea Bk4 C9WI lU O l'W k-U SZ ppb C939IIUO1/WU-MSD-250 get C9S991KH/WU-US-20 ppb CW9IFA31/WU-MSD-2JO pk CH9NF C l 59*2/ C15993F CW994F C9J991P C959UM CV5933M C95934M C9593SU CM oiir C96012/ C940IJF C960I4F C9601JF C9595IM C95932M C939SJM C95954M C9393SM C96031F C96032F C96013F C96034F C96035F CM972M C95971U C95974U C9597JM CMC5IF C96052F C96053F MA MA NA NA MA NA NA CM eeuJLu NA NA NA NA NA NA NA NA NA NA C--fceudHiik CmnMdKvfc Coa4edH*b CoUnWHMfe NA NA NA KA NA NA NA NA NA NA NA NA NA NA NA NA HA HA NA NA NA NA 1.0000 1.0000 1*000 10000 10031 1.0031 1-0392 i* m 1*031 1*107 1*464 *.*967 1.061T 1*392 1*313 1*371 M H 1*472 1*0)1 1*103 0.9*32 1*47* 1.0316 0*676 1.0071 1*027 05*9* 1*102 1*164 1*037 1*1*3 05129 1.0246 1*236 1*441 0*661 05671 1*416 1*10 0*7*4 0*650 1*120 1.0592 1*305 1.0497 09631 Total Mae* rfliaar NA NA NA NA NA NA NA NA NA NA NA NA HA NA NA NA NA NA NA NA NA NA HA HA HA NA NA HA NA HA NA NA NA NA NA NA NA NA NA NA NA NA NA NA C96033F NA 0.9613 NA ** L w i w y ii i f c u i l f c 1027-00 11 0 2 0 0 e u ly u . LAC 11,07*0 M ITI rrc u ru rt* C o a r.rtU r * 0-6640 0.6640 0.1640 V i------NA NA 0*640 0.6640 05640 05640 05640 05640 0.1640 05640 OJM6 0.6640 05640 05640 05640 05640 05640 05640 05640 5640 0 6640 05640 05640 0.0640 05640 0.6640 05640 0.0640 05640 05640 0.0640 05640 05640 05640 05640 0.0640 05640 05640 05640 05640 n.n rn i irY iiijiT taVnWA^ Purity r i w W i i i r i : 103400. 1031*0. 1107*0.120400 LAjGIH 11*400. 11,0400, 12/1 WOUH/LAC 123000 LAC. 12/2740 u f e P5O0 CMC 0*0 0.09 000 0.00 306 410 111 3JJ 136 156 151 1M 194 72.2 476 065 m 102 1040 1164 106* 952 914 427 261 1*7 1*4 1*0 1*1 230 170 201 200 167 131 177 114 212 323 11* 162 31* 330 400 329 627 , 496 m* F u rie 1 1 1 1 1 1 1 1 1 1 l 1 1 1 l l 20 20 20 20 20 SO 50 50 n 50 500 500 500 300 500 500 300 500 500 500 300 500 500 500 300 900 500 500 500 PfO CU C ut # 0*0 0.00 0.00 0.00 346 272 223 24* 134 4 124 104 156 S45 95 705 34.1 636 17913 1*697 115*4 15700 13017 16675 11210 6502 451 7*47 62031 90*4) 72137 66356 64140 7025* 146010 77366 60356 960 224219 141335 151735 221345 216024 1*7433 217397 273376 217713 F ln w (F*-- D001023006 AMI023052 0001023007 A001023033 A M IM I O l 4*012*1016 AQ0IM10I7 4*01201011 0001027020 0001027021 D001027022 DOC1077021 DODI027024 D001027027 D01027026 D001027029 D001077030 DOO1027031 A001030030 A0010300)1 ADO1030033 A001030033 A0010)0034 DOOI02347 00102)046 000102)049 010230 D001023051 00102301 000102300 001023021 DOO1023022 D001023023 0001023026 DO1023027 DOO023026 DOO1023029 DOO10230)0 000102)03) P001023034 00010230)3 DOO023036 DOO10230J7 DOO1023040 DOO1023041 DOO1023042 UUUIBdUU DOO1023044 _______________ ______________ ____________________________in tV iS W rrro - *% <LOQ (0.0103 u*i) LOO (0.0264 (*) LOQ [QM IOS a^t) LOO (0*264 s'il 116% 91% 79% 16% 0.134 0.134 0.124 0.104 0.156 0530* 023399 0*705 OJI741 0.063* I7J 1*5 1*5 15.7 IS* 11.7 115 Ut 6.45 753 650 965 72.1 165 64.1 703 146 77.4 60.4 MO 224 141 1 221 216 197 ZI 273 206 216 Mau PF srt LOO LOO IM% *2% USD M. D u WMSDRTO NA NA 25% 9% 0.131 ISO 0197 0.0633 23.1 0*169 11.7 17.4 203 41.1 11.0 450 11.3 135 9.77 125 ** 30. 20.4 192 395 135 271 30 4 uTrigMt Variarf NA NA NA NA NA NA NA NA NA NA HA NA NA NA NA NA HA NA NA NA NA NA NA NA KA HA MA NA NA HA KA NA HA NA NA NA MA NA NA NA MA NA -MA-- NA KA NA NA NA FFOCA PT06A Cue D W a 0.00 363 0510 000 177 234 17* 0.00 0.00 0.00 0*0 0*0 156 0.00 0*0 02 3.24 57.7 37* 521 605 *45 >04 442 77* 275 40* 17.6 135 165 17* 23.1 12.1 155 135 122 155 31* 20.7 23.7 115 JM -- 235 295 2*5 SU 2*5 l 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 20 20 20 20 20 20 20 20 20 300 500 300 300 300 500 300 300 MO 300 300 500 500 300 -5W 500 500 300 300 500 ffO M - ferihiienrU nettiuuli MS96- C6Fl TSCQNODCHCQH MS7-C*F17S02N(CH3)CH2C00H PFOOA C U Cue. zz C t II 6 FFOCA au FFOCA 0*0 15 510 0*0 276 266 222 143 0*0 0*0 000 0*0 o.oo 1.77 000 0*0 0*0 3.09 1149 733 1030 1149 D001023006 A001024004 DOO1023007 AMI 024003 D001027034 0001027033 DOOJ027036 DODI027034 DOO1027030 DOOI027021 001027B22 DOO1027023 DOO1027X2* DOO1027027 DOO1027021 DOOI027029 DOO1027030 DOOI027*31 DOO1027041 DOOI027042 DOOIOZ7043 D001027044 LOQ (0*0611 u*') <L0O(0 0)06 */) LOQ (0.00611uf/f) <4-00(0 0306 a*') 92% 19% 76% 36% " 000(0*122^5) LOQ (0.0122 a*'*) LOQ (0.0122 a*,'*) LOQ (0*122 a.'*) LOQ(0*122 nf/g) < 0 0 (0 0 1 2 2 * .' } LOQ(0.0)22 */*) LOQ (0*122 <*/} ^ 1 .1 5 0 733 1.03 1.15 " ------ LOO 66% 2101 *77 1336 333 777 1673 6702 014 *663 11267 3911 7469 6970 61 4241 13310 10611 12013 13443 13317 12270 14110 14363 13)76 14414 DOO1027041 D00IQ27O49 DOO10270 DOOI0270SI DM1827032 DMI8210I9 DOO1033020 DOO1823021 DOO1025022 DOO1023023 0001823006 000102)027 DM1023036 000102302* 000102)0)0 DOO1021033 DOO1025034 D001023033 DOO1023034 DOO1023037 D001023040 DOOI02304! DOO1023042 DOO102)04) D001023044 2.10 0.677 154 0533 0 777 657 6.70 0.01 *66 HJ 3.92 7.47 6,91 6.16 45$ 153 10.6 12.0 13.4 135 125 142 14.4 13.4 <4 3 1.17 6.67 6.IJ 1)5 14.1 un 646. Dee. NA NA 275 54.4 195 30.0 12) 15.7 6*5 3.14 ETM-7.0 E197 3M Environmental Laboratory TOX-C2S-bw221-7jdi Page 112 BACK TO MAIN 111 3M Medical Department Study: T-6314.1 CFmAtMCcTt-MTO<3X2-90-222*5 Analytical Report: FACT TOX-028 LRN-U2636 SMdy: I W N"biT*- 3 d M n > AsWytesJ E jfofmm t Sy+m Vmnbmr Dm ExttvMico'AattyiC DMxfAMlyiM'AMty Dm* i f D u s " --Vrlnn'*"*:" Sm #le D M C m 6329-225. FACT-TOX4m r'-m in .m m - p MJM b lM ETVL66 h ETS-V7.0 A - n 06249 >o f m n . TRISA, MJ36, u 4M570h l b 1 3 .W o * D m y * W r< C rtC D * (S D ) KB BC kite IO'lCTO SAL/UK JOLiOO. IWMO, 1027*0. IO34W0. 11*2*0, 12*1*0. I1*0 UUMMUftOJ 1024.00. IO2S00. 1020*0. 1021*0, 11*6*0. 12*4*0, 12*5*0 MMH RAT U V E W EEK 5 tot UBI 1104740 tm m fti fam fM MSM Dm V dW CMC rt Ua0>o4k Ik M I010Q-K2O Bk-5 19I0O-H2O-U-6 KBU0100-U - 5 HA HA MA o.co O 4.16 RBLlOKMMJiwBfc NA QC CT3411M 1/WE5-MS-250ppk NA 0.00 246 C95411MX71/WU-M3D-2M mM c n m r a i r w k i - u s - u o pph C9599IF<;i/Wt5-MS>.230 ppb MA NA NA 261 226 131 C95911M NA 494 tv m C959I2M CW IJU NA 690 NA 6 6 C939I4J4 C9J913M C55991F CMW2T CW99SF C95994T C9599SF NA 369 NA t 55 NA J J 0 NA 13* NA 0.00 NA 3.17 NA 107 3pfM CW9J2M C9J933M CV5SJ4M C954J5M C96011F CM0I2F NA 1W NA 42J NA 120 NA ICO NA M I NA 393 NA 20J C9MI3F NA 16.4 C9M14F C960ISF NA 16.7 NA 13.7 Q --,3 30ppm C9S951M CWMM C9S9JJM 2a4AMlybOK 2*4 AsHybOK 2*4 A*Nym 0K 476 425 446 C95954M CV59JSM CM031F C96032F C960S1F 2*4 Asslyii* DK 2*4 Aaaiyb 0K lU M yM O K 2*4 AaHyOK 2*dAw4yiuOK 42 440 221 346 227 C960J4F C9603JF 2a4 Aaolyai OK 2*4 AsNyst OK 314 241 G n~4 CVW71M NA 110 100 ppm C9S972M NA 69J cvstom NA 100 C9J97434 C9S97JM NA 127 NA MI C9603IF NA 73* C9MJ2F NA 102 C9605JF NA 90.4 C960J5F NA "2. HA 511 L sH rf QMasbarics (LOQJ nprfn.6 fe>piggy o s 0101*1 PFOS " 122 ,'(. 30* af/j. LAC 0121*1 ~ Law raanscytasfiraM dntil0 2 7 . mM ( I*2*0 sMlys. LAC 11*7*0 MH T i i i t t i i i i i i i t i i i i i 20 20 20 20 20 50 JO JO 50 JO JO JO so JO JO so JO JO 50 500 500 500 500 J00 500 J00 500 500 MS96 Ci Ic Cm c .. * * 0.00 4.16 9 2J7 250 210 120 911 643 634 344 1.46 312 IJI 1.00 3.90 102 1*99 1(26 2412 1*45 IMS l**l 1032 *17 646 667 23410 21120 21192 21747 21463 107SJ 17619 11466 15*21 11642 S4363 45719 50777 62727 55102 34722 46504 459(3 C it 29374 MS l '|r t - b D00IQ23006 A001023052 DOOI02J007 AOOIQ2305J D0010270J4 OOOIOJ703J A001103076 DOO1027031 D001027020 DOO1027021 0001027022 001027023 neo1027024 DOO1027027 D001027021 DOO1027024 0001027030 DOO1027031 0001027041 0001027042 01027043 01027*44 D001027045 0001023047 01023041 0001023049 0 1 0 2 3 0 JO 01023051 D00J027056 D001027057 0001027036 D001027059 D001Q77Q62 DOGI077063 01027064 DOO1027065 01027066 01003033 01023034 01023031 DOO1023036 DOO1023037 DOO1023040 DOO1023041 01023042 01023044 Cm M . I m TMSS6 iT i* * L C Q tO O lH *) LOO (0.0304 0*^1 <LOQ(02J122rt) <L00 rO-8306 V i ) 79% UH 74% 42% ** 0.0090* 0.006*3 0.M6S4 LOQ (060611 V i ) LOO (000611 *<) <L0Q (00061 ) * r t ) LOQ (090611 v fcj LOQ (0.00611 V i ) LOQ <0.00611 V i ) 00102 200 163 2.41 104 116 1.99 101 0517 0646 0,467 23.4 21.1 21.9 21.7 2I.J 10. 176 IIO IJ.9 116 546 41.7 596 42.7 356 34.7 46J 460 46.; 29.4 Meas UD * LOO LOO lt% M% 0.00719 0.00693 2.01 16 21J II.S SJJ 409 M .D *. JB M D V D NA NA 9% JJ% 33.4 9.0015* 366 0 00114 11.7 0235 476 0.521 4 0.171 226 3.09 11.7 629 iu-i (.39 DM EMiM'Am I ) 1026*0.1021*0. 11*7*0, 1 2 * 4 LACUM Dm V mM M oriyt 11*1*0.11*9*0. 1111*0 KJtilAC PMWy Em m V i M : 1220*0 LAC. 1227.00 wmk b iM * VriM HA NA NA NA NA NA NA HA NA NA NA HA NA NA HA NA HA NA NA NA NA NA NA NA 4A NA HA NA HA HA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA toTgW O iM * M97* MITI Cm . rt OJO 0 00 i 060 i 0.00 i 325 i 331 i 327 i 261 326 i 316 i 360 i IU 16* i 21. i 133 i 166 i 237 i 463 t 341 20 352 30 417 30 361 30 361 20 i l l 30 23 SO I7 J so 639 JO 61J J 174 SCO m 300 14 300 196 5 l it 500 623 500 941 500 536 500 4.1 500 6*4 500 JJO 500 36* 500 25* 500 356 500 M2 500 340 500 297 500 243 500 U l sao 242 500 MJ79 CHc Cm c * 3 ... 99 009 090 999 OH" 0 DM1923M6 01023052 D001023007 AOO1023052 r tB T O W frK b c LOQ (9.9106 o'j) < 00(00122 v i ) LOQ(0.0306V i ) LOO (ODI22 us'd 2*2 297 29 221.4 JM 21J 124 IM 15.9 19J AMI (00076 A 1103077 AMI 10307* A001103079 DM1027020 0001027021 DM1027023 DM1027023 DM1927024 D00IQ2T027 9f% 9*H 101% MH 0631 0.0213 06324 LOQ(06122 safe) 06159 129 IJ.7 336 443 6M2 69*7 1390 6MJ 6996 J97J 3094 4354 2219 2976 15517 TUO) 72359 99117 1*219 301*2 <7*22 272*4 42600 3JJI4 163074 137123 11135 17J791 1566 11410* 141493 133614 I1UM I22J74 DOO1027021 DM1027029 DM 1027030 0010270)1 DM1027941 DM1027942 DM1027943 DM1027944 DM1027945 DM1923947 001023041 DM1023949 DM1023050 DOO1023051 0001023019 DM1023020 DM1023021 DM1023022 DM1023023 DM1023026 DM1023027 DM102302S DM1023029 DOO10230JO DM1023033 DM1023034 DOO1023035 DOO10230)6 DOO1025037 001023040 DM1023941 DOO1023042 DOO1023043 DOO1023044 PPOC-PHftu FKBA-IVfl 06129 061 $7 0623 0.0*42 697 39 <69 567 369 299 996 M2 30.4 476 JSJ 163 131 132 176 157 141 124 13 _______ 2 ________ 1 uMji7o-- cc&* nm7ssoct2tnM<hciacutejcaoccooMoH LOO 99% 90% 3.72 49 363 153 12 M . Ds*. MVMfDRPO NA 23% 402 0.0124 147 10.4 162 11.9 11.5 ETV4-70 Estol 97 3M Environmental Laboratory TOX-02t-b>22i-7Ejtti 2202001 I0JPU Page 113 BACK TO MAIN HI 3NI Medicai Department Study: T-6314.1 FACT-TOX-021 Covaicc#6329-225 IPMredFaciNmIhi<TmSabauaaa^ Marte AMiytM) t q tirmm* Symtm N*Mr. Comm* 25-225. FACT-TOX-021 T-MU Ratinar ETS-4-.O * ET3-0-70 Am U*062490 o**a Pm m **4 rfPFOS. PFOSA. M336. aa MS ia * a 13-Wa*fcDiaawy ftr ty oCrtCD*(SD) IOS BX Km Fh I-S^uaMVaM: SmAuAmb a - , of AaalyartAadyat Dm of D*m RadaaiortAaalyat SMfkDtU lO'ltWO SALKJK 10/liCO, i m o , IIV274. IftOODO, 1142/00, ]2 4140,124* UHMUHW U 102*00, 102540, IO3OO0, 103100,114640.124*00. 124 MMH HAT LIVES W H X 3 G r^ Daaa -1 * * pro Cart. Case afPFOO MMM Marta Hk lOtQMUO Bfc4 10l06-H30-Mc-4 IBL10100-Ow Bfc-5 0.00 < O Q (000101 a*) 0.00 < O Q (0 0 2 6 4 -rt) . 0.00 <LOQ (0.00105 **'(> UU. 10lOO-Lmr B4-6 0.00 < 0 0 ( 0 0264 arta) QC C9J91LMCI /WfcJ-MS-230 ppb 3*0 C9M11M C1/WU-MSD-2M *rt> 272 C9>IF/Cl/Wfc5-US-230 ffk 223 C95991FA31AVU-M3D-250 ppt 24* 116% *1% 79% 46% Craa*l 134 0.134 C9S912M C9S9I3M 134 0.114 124 0.124 C959MU 104 0.104 C9S9ISM 13* O.I5I C9S99IF 54-* 00349 C95992F .* 0039 C9J993F C9S994F 70 5 0.0705 74.1 0.0741 C9S99F CcaapX 3ppm C95931M C95932M CMMJM 17*15 1*097 14594 17J 190 Hi C9J934M 13700 15.7 C93935M 15017 150 C9601IF 14675 12.7 C960I2F 11210 112 CM01IF CN0MF C960ISF 4502 IJO 4451 143 7947 7 91 Cm * ! rt- C9395IM C95932M CMMJM 2031 4M] 72137 42.0 9U 72.1 C95954M C95933M C960J1F 14554 14140 7025* 44.6 44 1 70J C96032F 146010 146 C96033F 7736 77.4 C960J4F C9W3SF 053* 96030 0 4 *6.0 C9397IM 21421* 224 100 C9WT2M 141325 Ml C95973*4 Q5F74U 154735 221345 159 221 C95975M C9605IF C960S2F C960JJF 21M24 197*31 2175*7 275376 216 197 211 275 CMCF C9605SF 2S7S2 217713 " 211 L -rta fQ iiiB M b (L0Q)n>4***d farparty aa014141 PF06 2-2 a'*. 306 a#1*. LAC 01/3141 -- t w M T M h d M IflgWOOart 114140 a a a fri. LAC 114740 MaaPFOi <00 <OQ 104% 2% o.ist 174 11.0 452 940 192 no ft* Dr*. MSMSDXm HA HA 23% 1)0 00197 2 11.7 2*1 41.1 430 MJ in SIS 4 20.4 102 1 304 DM E M r fM * tM iV d U M y Party E M V a i W : 10/2640,103140. 114740. 124040 tACTUH IJ4040,114040,12/1140 KJH/LAC 12/2040 LAC 12/7740 im h p ra u Cart. CaK. arta 163 0210 OOO 276 2a 223 0.00 OOO 0.00 OOO 000 I.T7 000 0.00 0.00 30* 114* 713 1050 1149 1607 210] 477 1536 333 777 4673 *702 0014 4663 112*7 3911 7469 6970 6159 4244 15310 10616 12013 15443 11317 12270 MHO 14363 3i< 14434 a* PFOSA .... ._ . < O Q (000611 a r t ) < 0 0 (00306 art*) < O Q (000611 a r t ) < 0 0 (00306 arti) 2% 9% 7t% 50% - < o q (00122) < O Q {00122 a r t) <OQ (00122^4) < O Q <00(22 a*) < O 0 (00122 arti) < O Q (00122 a r t ) < O Q (00122 a r t ) < O Q (00122 a r t) < O Q {OOI22 a r t ) < 00(00122 arti) 1-13 0.7JJ 103 1.13 1.61 2.10 OJT77 \M O iii 0.777 447 6.70 01 446 IIJ 932 747 477 *16 425 154 104 110 15.4 134 12-3 Mi 144 54 14.5 PRUA a rt <00 <00 *1% 60% <00 <0Q 1.14 1.17 1*7 6.15 I3J 14.1 UB M UR -jg ag p R T g - NA HA 3% 30% HA HA NA HA 27-3 O-JIJ 34.4 0437 1*3 147 200 t-23 13.7 2,09 3 1.14 M6S6 Cak-Caat. ----- J r f l 000 0.00 4.16 0.00 237 230 210 120 9.19 D 634 149 1.46 112 IJ3 to o 1.90 102 1999 1(2* 2412 1943 1(65 1991 1032 *17 46 667 23410 21120 21(92 21747 214(3 10733 17619 114(4 13921 11642 54361 4371* W777 62727 35102 34722 504 439(3 4*0*9 2*17* C aaataH a af MU6 9 r ta r % R> < O Q (i.om 9i) < 0 0 (0.03M art) < O Q (OOI22 *rt) <00(0.0306 art) 79% (4% 74% 42% * 0009(9 0006(3 000634 < 0 Q <000611 art) <OQ(000*II rtl) < O Q (0.00611 art) < 0 Q <000611 art) < 0 Q (0.00611 art) < 0 Q (0.00611 r t ) 001023 200 143 2.41 14* I.M 1.9* 103 0.917 0.M6 0.667 23.4 21.1 IIJ 21.7 21.) 104 174 11-4 13.9 114 34.4 45.7 30. 627 334 347 415 460 46.1 29.4 PPOIA.-P i da--life -- Ha MM6 - Cffl7>O2><(H)CH2C00H MS70 - C*F17JO2>*,CH3(CHIC00H <00 <LOO 1% 50% 0.00710 000*93 201 IO* 21J 135 33.9 40.9 Analytical Report: FACT TOX-028 LRN-U2636 MO a* Dot. Mk'MSDRFD HA NA J% 53% 224 000159 264 0.001(4 11.7 0435 474 0.S2I 400 0.171 224 3.00 11.7 629 204 49 M57* C r t Case. C aaiaaiM 4M370 OOO 000 OOO 0.00 292 297 2 22 314 214 324 99 13.9 194 I2J 15.7 234 442 <OQ(00306 art) < 0 0 (0 0 1 2 2 arta) < O Q (0.0306 a rt) < O O (00inartil M% 99% 101% 0% 00311 00211 00324 <LOQ(0.0(22 * rt) 00159 0019 00129 00131 00231 00442 641 1390 6M5 6096 3*13 3094 (29 649 690 JJ7 109 2*76 7(403 72339 99017 39 303(2 27294 a^xiin 313(4 143074 137423 1737*1 13660* 114100 141493 123614 13(4 122374 Ut 134 714 724 9*4 nj 30.4 474 274 424 334 163 131 176 137 114 Ml 124 I3t 123 M ta a MSI* 90% 0.0221 3.12 >64 1 MD a* Da*. 23% 000*15 0.0124 1.47 42 1902 I na ETS4-7 0 E n t9 7 3M Environmental Laboratory TOX-C2MTM,225-7jd. Page 114 3M Medical Department Study: T-6314.1 FACT-TOX-028 Covince#6329-225 Analytical Report: FACT TOX-028 LRN-U2636 Study: P r o d NumberfTeat Subriame): Matrix Analytical EquifUKatSyscB Nun kuVwcat S oftim /V ow c Date o Extractim/Analy*: OrteafAnilyaiWAmljut; DKc f Dala RodroW Anriyat: Sumpie Data Covante 6329-225, Fa CT-TOX-021 D d o n i n n o f be Proem* and Canomtratiaa of PFOS, PFCSA, M5S6. n j M S * m the 13-Wea* D ictsy S Jy of CflCD (SD) 1GS B T-6314 Sat L ira Filename SoeBdow ETS4-6.0 it ETM-7.0 R-Squarod Value See A dattam i* A neli. 062491 Slope Muotywc 3.4 Y-W oeq* 10/10*0 SALAUK * . 00-91 10/23*0,1 0/24*0,10/27*0,1000*0,10/31*0,11*2*0. 12*1*0.12*4*0 KJH/MMH/HQJ 10/24*0, 10/25*0, KV)O*0. 10/31*0. 11*1*0. 11*6*0, 12*4*0.12*5*0 MMH RAT LIVER WEEK 14 Cmup Dm Sanata Method Bik Matrix Bft QC Group 1 Oppn Croup 2 3 ffm C roup] 30 fpm Croup 4 I0I00-K2O EUk-5 IOIOO-JDO-Blk-6 R3L101DO-Lira B1k-3 RSL 10100-Lira Blk-6 C9591IM/GI/V/U MS-250 ppb C9911M/GI/WR3-MSD-250 n*> C9599IF/GI/WU-MS-250 ffb C9991F/O1/WkJ-MSD-250 ppb C9J907M C9590SM C95910M C9S916M C95917M C93911F C959K2F C9S9IJF C9S99IF C9SW9F C95923M C9J929M C95930M C9593CM C95940M C96004F C9600SF C96009F C96017F C960IIF C95945M C95946M C9S956M C9S97M C95959M C9602IF C96029F C96036F C96037F C9039F C93966M C95970M C9976M C3W5SM C T ^ C9604IF C9643F C96057F C9605IF C96059F SaraiU a VtrtSad NA NA NA NA NA NA NA Coofinnol Ltnv NA NA NA C a d n u tlro NA NA NA NA NA NA 2nd Aastysa OK Confirmed Lo 2nd Analyst OK C u fiw d lro C w fm o d Low 2nd Analysa QK CoufinaadLow NA C o sf ia w e d la w C onfined Low NA NA NA NA NA NA NA NA C a afin a ad L o w NA Ctnfinned Hi*h CiMlfim--1Ui|fc Confino High C o n fin a Vbh NA CooUniMd H i|l NA NA NA NA InUW W l 1.0000 1 0000 1.0000 1.0000 1.0051 1.0051 1-0392 1.0592 1.015 091)4 0.9960 1.0037 1 0530 10131 1.0103 10)16 09*45 1.0350 0.H79 10090 1.0401 1.0531 10654 1.0512 0.9951 1 0224 1.0117 1 0024 0.9*43 1 0351 1 0049 1.0200 1 0196 1.03*6 1011 1.0327 1.0304 1 0255 1.0019 1.0409 131242 V tt S.y04 0 9991 1.0059 10345 1.0466 1.0211 Tem Mu aTU rar NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA HA i\ NA NA NA NA NA F N IM Q C iro rtu ru a r O.S6 06* 0.16 01640 NA NA NA NA 01640 016 01644 0*640 01640 0.16 0.16 0.1640 016 0.1MB 0.16 0.1640 0.16 0.16 0.16 016 0.1640 OMO 0.16 016 0.16 0.16 0.16 0.1640 016 0 .1 6 0.1640 0.16 0.16 0.16 0.16 0.16 0.16 O .U tt u.aovu 01640 0 .1 6 0 .1 6 0.16 0 .1 6 PIO * Cape. rt 000 0.00 ooo 0.00 506 430 311 3 254 213 369 124 US 65.1 IU US 167 920 544 600 537 719 724 432 914 1071 7 1090 536 561 496 461 733 419 44 K3 736 511 754 991 164 795 ro 130 710 912 919 947 ro s M atta 1 t l l 1 1 1 1 l 1 1 1 1 1 1 1 1 ) 50 50 50 SO so 50 50 50 50 50 500 500 500 500 500 500 500 500 500 5 1000 1000 1000 1W IUUO lo c o 1000 1000 1000 1000 re s Cale CHc. 02 0.00 0.00 0.00 341 272 225 246 216 249 320 107 94.5 SSJ 160 15* 146 76. 23770 25672 22319 29470 29337 17761 42727 4S57 31734 46955 232106 233979 213047 195332 310671 203231 402143 335712 301777 215211 650557 122241 721917 665261 713402 717359 60900 COI 19 751530 799651 (uptfauaO EMO1023006 A0OIO23O32 D0010230O7 A001023053 A0OI2OIO15 AOQI20IOI6 A0OI2OI017 AOOI20101I A001102041 A01102049 A 0 0 1 102050 A00IIQ2O51 A001102052 A001102055 A001102056 AO0I1G2OS7 A001102051 A001102059 A001Q23I ACO1023094 A00I02433 AO0IO23096 A001023097 A0102403i AOO1023101 AOO1024040 AOO1023101 ADOIO24042 AOO1023065 A001023066 AD01D23067 A00102306I AOO1023069 A001023072 A001Q23073 A001023074 A001023075 AOO1023076 AOO1030016 AOO1010017 AOO1030011 AOO1030019 AOO1030Q20 A001030023 AO0IO30Q24 AOO1030025 AOO1030026 AOO1030027 rfPfO S m ft ar% Ra <LOQ(OJI05 (/() <LOO0O264a*/) <LOQ fOX105 up/j) <LOO (0.0264 aa/l) 11696 9194 7994 1694 0.216 0249 0320 0.107 0.0945 O.OS55 0.160 0.1S9 0.146 0076 23* 25.7 22.1 29.5 29.1 17. 42.7 45.6 33 7 47.0 233 234 211 195 311 203 403 336 309 215 4SI 22 729 663 713 717 610 20 739 00 Mam rro s _SQ <UX3 10494 >294 0 197 0 1 26.1 36.9 237 293 730 741 h 10/27*0 m d 11*2*0 aaalyris. LAC 11*7*0 4 CCVabfsActnqlfaeae dala w arn* wRNa-*/-3044. C o n rida analyzed Cray 5 a a q ita w c within a ile ia . LAC 11*1*0 DM. Enund/A aalyat Dala V erified/A nly* Pwity Entaed/Vcrified: 1 0 2 7 * 0 ,11 * 3 * 0 ,11 * 7 * 0 ,1 2 * 1 * 0 LAC/KJH I l * t * 0 , 11 * 9 * 0,12/11*0 KA4LAC 1 2*0*0 LAC, 12*7*0 m l 1054 - CIF17S02N(H)CH2C00H K 57t- C I F 1 7 S 0 2 N ( C H 3 ) C H 2 C O O H ssa> ltd. Dar. M U U D in NA NA 2594 9% 416 0.096 41.6 0.049 a 3 23 na 12 3 116 44! 2S.7 4.0 w 73. lia 13.3 UOSA Cauc. NA NA NA NA NA NA NA NA NA NA NA Confirmad Low NA NA NA NA NA NA NA NA NA NA NA Na NA NA NA NA 2nd A aahsa CtC NA CeufinadLew CouTuroadLow C ronm d Loar NA C a td n w d Low C rm fnm Low CaafinaadLow C ndirorilaw 2nd Analysa OK 2nd Analysa OK 2ad Analysa OK 2nd Analysa OK 2nd Analysa OK 2nd Analysa OK C onfina Low NA Contornad Low NA 000 363 o a io 0X0 277 269 23 176 11.0 7.97 10. as J2 12.4 16.6 15.6 23.3 595 19.3 545 306 264 420 77.3 131 115 13 104 549 391 411 341 3 295 271 242 5SJ 302 609 53 662 612 611 530 497 55* 514 490 UOSA M odas 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 20 20 20 20 20 20 50 50 50 50 50 50 50 sao 50 SO 50 50 50 50 50 SO 50 50 50 ITOSA Cale. C iac a i* 0.00 361 o a io 0 .0 0 27 26 223 165 il .1 2 10. as 524 12.2 164 15 3 23,7 575 1107 10007 5*71 5014 7177 1470 2672 2246 2702 2073 27604 in a a a o i 16702 24114 14171 13412 11709 26 IO 14723 30402 25136 12341 29630 30712 2599 24727 27040 24571 23949 (aptfcu.1) CUweeattad* t if o s a D001023006 AMI024004 DODI023007 AOO1024005 D00IO27D34 [011727035 EX) 027036 DOT102703 *110204 AOO!102049 AOO1102050 AMI 102051 AMI 102052 AOO1102055 AMI102056 AMI 102057 AMI 10205 AMI 102059 AD1024043 AD1024046 A1024047 AD102404 AD1024049 AO1024053 A1024054 AO1024055 A01O24O36 AO102057 S00103103S 5001031036 9001031037 9001031031 SO0IOJ1O39 5001031044 *1024062 AD0I024063 A M 1024065 500103104 5001031017 500103I0tS 5001031019 SODI031020 SM103102I 9001031026 SM I031027 SCOI031021 SMI 03102 5001031030 <LOQ (0.0061 lu f /f ) <LOQ (0.0306 a r t ) <LOQ(0.00611 V ) <LOQ (0.0306 ap/|) 9294 944 7*56 5194 1 0 Q (0.0122 up'll) <L0Q(0.0122 up/j) < O Q (0.0122 up/j) oom <LOO (0.0122 /> o .om 0.0164 0O15J 00237 *tO Q (0.0122 m b ) 1.11 10J 5.M 9.01 7.1* 147 167 205 ITO 2.07 27.6 1*9 . 16.7 242 HO 13.4 11.7 26. 147 ' 30.4 25 1 32.3 296 30 26.0 24.7 27.0 24.6 23 9 * 4 + + 4 4 4 4 4 4 4 4 4 4 4 H aas rro sA <LOQ 9194 6096 0 0123 0.0160 6.2 223 216 162 29 25 3 BACK TO MAIN tTS-O-7.0 E n d 97 3M Environmental Laboratory T O X -02l4 ira 225-7 E .xlj 2/20/2001 I7 4 PM Page 115 3I\| Medical Department Study: T-6314.1 FACT-TOX-02 Covane>-225 Analytical Report: FACT TOX-028 LRN-U2636 f r o J m Habu ( T q | SuIx M ct): iM iu H Softam/Vcn Dateaf ExtncbaUAjalyk: Dke tiAatiytu/Huly*: Due of Dm Rofectk>a/Aaal>*t SunpfeD ata Conacc 6329-225. FaCT.TQX-02 DcurraMisa o f* P i a o s and C a a M M n of PK38, PFOSA, M 3 , m i MJ70 k the I}-W Dsttery SCody of C rtC D * (SD) IG5 BRIL T-6314 Rf Li*tf F ib n c SocBdow ETS4-6.0 * E T S^7.0 W q u iro iV ^ Set A lutknou A n d 062491 Sk^c I M y n 1.4 Y-intowpC ityiom o s a l /k j k 10/23450. lOOMO. 1(1/27/00, ItWCK. MWWO, I IAQ/00. I2A01AW. 12W K ) KJH/UMHiHOJ 10/24/00, I(VI5/00,10/304)0,10/31490,1W W 0,11/064. I24M4, 124)34 UMH RSD SM. Dar. M S/M SD B TO NA NA 3% 30% 0.927 0.0001)4 29J 0.00470 51.1 1.15 22 0.505 20.0 412 37.3 6.1 1.14 lit 4.91 1-24 RAT LIVER WEEK 14 Cm* Dm * S o iilai tw n p ti Verified lai NBI13047-00 M5S6 Caac M556 M556 Calc. Caac Method BR Matrix B& l0100-H20BJk-3 | 0100-H2CVaik-6 RBUOIOO-Um Blk-5 RBLIO 100-Liiw BIk-6 NA 0.00 1 0X0 NA 0.00 000 NA 4.16 l 4.16 NA 000 1 0.00 QC CV591 IM/GI/Wk5-MS-250 n * NA 241 1 in C9S9llMA>lAVk5-MSD-250 ppb NA 161 1 250 C95991F/G I/Wk3-MS-250 ff* C9577IF/1/WV-MSD-250 ppb NA NA 226 1 144 1 210 120 G napl C9J907M NA Iti 1 16.4 C95906M NA 19.2 1 19.6 C939I0M NA ISO 1 15.1 C939I6M CoofaaodLaw 9.47 1 9.44 C9S917M NA 6.04 5.74 C95MIF NA 0.00 1 0.00 C939S2F NA 4.95 l 4.90 .C9S9I9F NA IIJ 1 il.l C95VW NA 24J 1 24.4 C9999F NA 3.1 1 J.07 Cm *2 C95923M NA 96.1 20 1945 C95929M NA 732 20 1450 C95930U C9593U4 NA M 2 20 1661 HA 7J.I 20 1317 C9S940M NA 100 20 mi C 96004? NA 53 9 20 1026 C96005F NA 64.7 20 1301 C96009F NA 72.7 20 >422 C96017F NA 50.9 20 1000 C 9601 IF NA 97.1 20 1931 C9S94SM NA 902 500 45379 Mppm C95946M NA 652 500 31490 C93956M C93957M NA 49.7 500 34445 NA 77.1 500 37714 C9599M NA 103 500 50397 C9602SF NA 23.6 500 11311 C96C29F NA 3*9 500 19211 C96036F NA 116 500 15300 C96037F C m S m td lm f 270 500 13092 C96039F NA 27.9 500 13593 C l*4 C95966M 2adA M tyiO K 236 300 117766 100 ppm C95970M C95976M 2nd Analyal OK 2nd Aaaiyau OK 1M 211 500 500 90379 103227 C9597U4 C" *"7**i 2nd AnaJyeuOK 2sdA=sh=sCK WT 22 S0Q CC 95251 1:4772 C 9604 IF 2 a d A a b k a O K 70.7 500 35357 C96043F NA 79.4 500 39467 C9607F 2nd AaalyaUOK 0.7 500 3*9*5 C9605IF Confirmed Low 64.5 500 30*14 C960S9F Confirmad Low 70.5 500 34439 L k t af Qwaditatka (LOQ) ifdalcd 1er parity an 01/31/01 PF06 12J a j/j. 30.6 n</* 1AC 01/31/01 Lowtccovcfy eoo&modwilk 10/27/00m i 114)24 analytic LAC 11/074 CCVi breetdiaj kee <Uu w o o l widin /- 30%. Carve da analyzed e ro y n n * k a a oe within criteri* LAC 11/014 Fiaaama (-pti-a-J) DOO1023006 A001Q23052 13001023007 A0IQ23OS3 DOO1027034 DO0J02703S A001102071 DOO102703 AOO1102041 AMI 102049 ADO1102050 A001102051 A001102052 AMI 102055 A 0 0 I 102056 AMI 102057 A M I 10005 AMI 102059 AMMB4043 301024046 AM1Q24047 AM 102404 AGO1024049 AM1Q24053 AM 1024054 A M 1024035 AMI 024056 AMI 024057 A001023065 AM 102X166 AMI 023067 AOOIQ23061 AOO1023069 A M 1023072 AOO1023073 AM 1023074 AM 1023071 AM 1023076 AM10Q4017 AM 1024011 A00102401* AOO1024020 AM10I24S2: AOO1024024 A0010230S7 AOO1024026 AM1023M9 AM 1023090 d caatrattaa - f 44554 <LOQ (0.0122 * / | ) <LOO (0.0306 na/al <LO Q (O O m rt) <LOO (0.0306 /) 79% 14% 74% 42% 0 0164 00196 0.0151 <LOQ(0.0122t* /|) 100(0.0122 uafr) <LOQ(0 0122 tif/g) <LOQ (0 9122 ug^> <LOQ (0.0122 /*) 0.0246 <LOQ(0 0 1 2 2 iirt) 1.94 1.45 166 139 in 1.01 1J0 1.42 1.00 1.94 45.4 31J 34.7 37-1 50.4 11.4 19J ISJ 13.1 13.6 111 90.4 103 955 ili 35.4 39J 39.0 30J 34.4 M to o to o 11% 5% 0.0351 00147 167 1.34 39.9 14 5 4 35. RSD SU. Dar. M S /K SD RH 3 - na Na 5% 55% 20.6 0.00310 37.9 000556 15.0 0.249 2S.S 0.3*1 I9J 7.79 20.5 2 9 UJ 12.0 9.9 3.55 NA NA NA NA NA NA NA NA NA NA NA C o n lim a d le w NA NA NA NA NA NA 2nd Aaaly OK Confinad Low 2adAaayw0K Confinad Loar C o n fin a ri Low 2nd A n a l i OK Confinad Lo NA Confirmadla C arinriLow NA NA NA NA NA NA NA NA O w fi n n riL n w NA 2nd A nab 0K 2nd A nab QK 2nd A nab 0K 2nd A nab 0K 2nd AnaivaiOK 2nd Analy OK NA 2nd A nab OK Confinad Law Confirmad low MSP Caac M )I OM 0.00 ODO OM 325 131 7 116 *7.3 ai 56.4 13 22.0 lt-6 24 5 61.7 123 23.6 144 2.4 114 107 11 04 121 99J 951 90.4 341 336 305 340 399 195 175 tu 156 146 696 477 56 534 690 273 299 2 222 221 M il P iatta l I 1 1 1 ._ r i i i i i i i i i i i i 50 50 50 50 SO 50 50 so so 500 500 500 500 500 500 500 500 500 500 500 500 500 500 500 500 500 SCO 500 500 D ka Eatand/Aimlyit: Date Vnifind/Aanlyit Pwily Eakred/V oifieit 10074, I W 34. 114)74)0.124)14 LAC/KJH 11/014. 114)94, 12/114 KJH/tAC 12004 LAC, 1207/00 m d i I! 1---------- l II! MSX Cale Caac M /f 0.00 000 0.00 0.00 292 297 2S9 2214 464 3*1 564 2.7 20.9 1*3 24J 04 125 24 7272 40*3 5477 5092 5559 31! 60*4 415 4674 4510 171106 16231 151547 166461 195724 9369 6277 U14! 73611 71394 347101 229004 2775*3 23US1 346110 136297 141390 139116 105962 IMI 17 D00IQ23O06 AM 1013052 DM1023007 A001023052 301102076 AMI 102077 A M 110207 AMI 102079 AMI 10204 AMI 102049 A M 1102050 A M 1102031 AMI 102052 AMI 102055 A M I102056 AM I 102057 AMI 10205 AMI 102059 AOO1024031 AOO1023094 AM 1024033 AM1Q23096 A M 10230*7 A M 102403* A M 1023101 A M H U 3 102 A M 102J1 0J AOO1023104 A M 1023065 AM1023066 A M 102306? A M 1023061 AMI02306* A M 1023072 A M 1023073 A M 1023074 A001023C75 A M 1023076 A M 1024017 AM 102401 A M I024019 A M I024020 A001024021 A M 1024024 AMI0230C7 AMI024026 A M 1023019 AMI 023090 Caacaatrafiea a f M570 <tOQ (0.0306 *%) <LO O (O O I2 2m /n ) tOQ (00306*9%) 90% 99% 101% 0% 0.0466 OHMS 0.0566 0 3*27 0 0209 0.0113 80242 0.0606 0.125 0004 7J7 4M 5.41 5.09 3.56 3.13 6.N 4.16 4.67 451 17| 162 152 166 196 93.7 63 *1 75.6 71.4 347 229 27 239 346 136 141 139 106 10 MS56 - C*F17SQ2N(H)CH2COQH MS70 CIF17S02N(CH3)CH2C00H Ilf to o <LOQ 9t% 90% RSD S U .D ct. MSABDVD NA NA 2% 23% 00491 46.6 0.0229 0.0506 16 0.0441 21. 3.50 MS 17.) 4.79 0.120 967 169 164 It.l 1.0 913 1*2 292 530 15 1 121 19J BACK TO MAIN E re-t.7 .0 Excel 97 3M Environmental Laboratory TOX-021-li*22$-7Exl* 2000001 12:42 PM Page 116 3I\I Medical Department Study: T-6314.1 FACT-TOX-02* Covuce#329-225 Analytical Report: FACT TOX-028 LRN-U2636 Study: Produrt NwwbqfTcB frihauncc) KUriic WetWRcvirioe Analytkai Fquipaaal SytoW Nanher h d w o l Softwe/Vna: M eGfExtracden/Auly*: M cofAaalyiii/'AuJyte M e of M a BwWtxWAariyte S cu p k D a ta Cerante 6329-225, FACT-TOX-02 D a ta u o a iio o tf Ik P tta a td O a M a J rte o c of PK3S. T-6314 Rat Liver F ila rn e ETS-S-d 0 A ETS-t-7.0 R -& p m d Value Arndt 062491 Sla: Maadyvx 3.4 Y -tak to tp c io/ioa sau n n i I0O3AX), 10/24/00. KV27AX). I0/30AX). 1031/00.ll/OWO.12AHAX), 124MW KJTOMMH/HOJ 10/24AU, HVJ5AB, IQOOAM, 10/31/00. IIAJIAX), 11/3600. UA>4A, I1AMA MMH 1S6. aid M570 m * e I J-Woek Dkucy Stady o(C j1 C D *(SO )IG 5 BR RU RAT LIVER WEEK 14 CfMV Sample f PFOS C am atretl-- Maaa BSD Daw C alc Caae. rrro s pros St*. Dar. aeta . MS/MSDXPD Method B k 10100-K2O Bk-3 IOIOO-HKVBlk-6 0.00 <LOQ (0.00105 / | ) 0.00 <LOQ (0 0164 a/i) <L0Q HA Matrix BB RBL10|Q0-Li*m BB-5 RBLlOlQIUiwBIt- 0.00 < O Q (0.00105 u f /|) 000 < O Q (0.0264/*) < 0 0 HA QC C9S911M/Gl/Wk5-M3-2S0 ppb 34 C9391 lM/3l/WkS-MSD-2S0 m 272 116H 91% 104% 23% CV5991F/G l/Wk5-MS-2M ppb 223 79% C95991F/Q1/W35-MSO-2O ppb 24 16% 12% 9% C95907M 21 0J1 C95906M 249 0249 C95910M C959I6M C939I7M 320 0320 107 0.107 3 0.093 4M 0.20 010 C939SIF 53 3 0.0333 C9W2F 160 0.160 C9J9I9F 159 0159 C9399IF 14 0.146 416 C93999P 761 0 0 7 0.120 0.0491 C rm 2 3PP* C95G1M C93929M C93930M 23770 23672 22319 Ui 25.7 223 C9393IM 29470 293 124 C95940M 29337 29.3 26.1 333 C96004F 17763 IT.* C96O0SF CM009F C96017F C9601IF 4272T 43557 31734 46935 42.7 43* 317 47.0 333 369 123 G nay] C93943M 232S06 233 C95946M 233979 234 C93956M 213047 213 CTJW7M 195332 193 11.6 C9S959M C96C2IF 310671 203231 III 203 237 44.1 C96029F 402S4J 403 C96036F 3337*2 336 C960J7F C96039P 30*777 21321* 309 215 21.7 293 *40 G taay 4 C9S966M *50537 6S1 100ppm C9S970M 1222a *22 C9S976M 72*917 729 C99MM *63261 665 10.1 C jjr'rlfl 734C2 73 730 o C96041F 717339 717 C96043F 609S* 610 C96057F 120119 120 C9605tf 75I30 759 113 C96059F 799631 *00 741 3.3 L u i t of Quantitattoo (TOO) updated far purity oa 01/31A)I PFOS 122 n*/|, 30 6 / * LAC 01/31/01 Lewrco*y coafinncdaitlt KV27AXI aod 1I/B2A)Q aatlyait. LAC 11/07/00 CCV bracked Iheae dU w r a t wxhin / 3044. C a m e ia aaalyaad way 5 w a p iti m m wHUd criteie LAC 11AW0 PPOSA Cate. Cape. ........... i t 0.00 3.63 0310 0.00 276 261 223 163 11.1 1.12 to * 12.5 334 123 164 13.1 23.7 5.73 1*07 10107 3*71 3014 7*77 1470 2672 2246 2700 2073 27604 IMS* 20*01 16702 24114 1417* 13412 11709 26*10 14723 30402 25*36 32341 29630 9UIM 25991 24727 27040 24371 23949 M e Edand/AutyM: Dele Vcriod/Aaalyd: Punty EaUted/Vcrified: 10/27/30. 11*3*0. 11/07/00. UAJ1AJ0 LaC/KJH llfOVOO. 11*9*0. 12/11*0 ICJH/1AC 12/20*0 LAC. 12/37*0 unfa C a atan tirria a Meas afPPOSA PPOSA MA Dar. /fear %Bac < 0 Q (0.00611 u r t ) r t MS/MSDKPO < C Q (0.0306 ta ) < O Q (0.00611 /* ) < to o HA ,<CX 2 p.0306 a,,} 92% LOO . . B * ____ *9% 91% m . 7*% S*H " 61% 30% < 0 0 (0 .0 1 2 2 /* ) < O Q (0.0122 */*) <LOQ (02)122 a*/*) 0.0125 0.927 < O Q (0.0122 aa/i) 00122 0.0123 0.000114 0.01*4 OD153 0-0237 < 0 0 ( 0 0122 /* ) 0.0160 29.5 0304X1 131 10.1 3 SOI 53.3 731 6.2* 3 J5 1.47 237 235 2.70 223 307 221 0.305 27.6 119 203 + 167 + 20.0 243 + 21.6 143 4J2 114 11.7 263 37.5 147 163 6.06 30.4 253 * 32J + 2*3 114 ju.a 29. 243 26.0 4- 24.7 + 270 + 243 + 4.91 239 + 25.3 134 PFOS - P g flug nat l a f dfeu te PFQ8A * Patflamnrtani nilftmoni.li M 55 6 -C lF |7 * aZ N (H )C H 2 C O O H M570 dFI7SCCN(CH3)CtOCOOH MSS6 C a le Caac. 0.00 0.00 4.16 0.00 2J7 250 2)0 120 16.4 193 13.1 9.44 3.74 0.00 490 tl.l 243 3.07 1943 1430 1663 13*7 11*1 1026 1301 1422 lot 193* 43379 31490 346*5 377*4 30397 113*1 m it 13)00 13092 11591 117766 90J79 103227 95231 114972 33337 39467 319*3 30114 34439 af R556 artar% K ac <LOQ (02)122 a*/*) <UX>(0 0306 >/() < O Q (0.0112 a*/*) < O Q (0.0306 a*/l 79% 14% 74% 42% 001*4 00196 O01SI < O Q (0.0122 u*/|) <430(0.0122 a*/*) < O Q (00122 */*) < 0 Q (0.0122 a*^) < O Q (0.0122 a*/*) 0.0246 < O Q (0.0122 a*/*) 1.94 1.4$ 166 1-Jf l.tt 1.03 1J0 1.42 IM 1.94 43.4 315 34.7 37.1 504 11.4 192 131 13.1 136 11* 904 10) 93J tts 35.4 39.3 39,0 30.1 34.4 " M au MS56 ta <oo <00 11% % 00131 0.0147 U7 134 399 145 104 33.1 BSD SU. Dar. M S /M SD R PD NA HA 3% 53% 20.6 0.003)0 37.9 0.00556 15.0 0249 2*5 OMI 19J 7.79 20.5 291 Il S 12.0 9.1 IJS MST* Calc C u e OOO 000 OOO OOO 292 297 2*9 22S.4 466 3*1 56.6 *17 209 1*3 242 606 125 24* 7272 40*1 5477 5092 3559 3*21 6064 4151 4674 4510 171306 16231* 151547 166461 195724 9)69 6277 *1143 75611 71394 347101 229004 2773*3 25MS3 346110 136297 141390 139116 105962 101117 C u cu m tfw f M570 a/* ae % Bac < Q (02)306 */*) < 0 0 (0.0122 ta ) < O Q (02)306 ta ) 9C% 99% 101% *0% 02)466 9-03M 00566 0.0*27 0 0209 o .o m 0.0242 0.0606 0.024* 4.0 3.09 5.56 4.16 451 132 166 196 93.7 163 SOI 73.6 714 347 229 27* 239 346 136 14* 3# 10* M au M S* <00 <00 9*% 90% BSD S U Dar. MSMSDBPD NA NA 2% 23% 00491 466 0.0229 0.0306 0.0441 S 479 0120 169 164 110 9J3 117 292 510 i a 19.1 BACK TO MAIN ET&4-7.0 Excel 97 3M Environmental Laboratory TOX-QZ-Ii*r22 S-7jd* 2/20/2001 12:42 PM Page 117 3M Medical Department Study: T-6314.1 FACT-TOX-OM C tw c r f fi3-225 Analytical Report: FACTTOX-028 LRN-U2636 rtodma MN D u ow M /A alyK Dm rf Dm rf D*e Red*litiA*ly* Sm pkDM i Cm m 129-223. FACT-TQX-02* P U n M n r f A . H-- m i Gmem**** rffFOS. ITOTA. 14136, >cd M370 ia * * tJ-Wk D m -y SWy rfCH.CO (SD)K3 BK Km T-63M b liw FI5 *A X A .E T V *-7 .0 f j -------- te am teaAm >n iirti Aamd634M, DrnrnyCWm U ud yw tiA 11/21*0 MC/SAL 1102* 0. 11/27*0 u m h ** V - Ia ta r a * I m # 00-1 iaA flaA m U - ----- - ] 107*0, II/2XAM MMH G r^ Dm* -- *** VrtM M mm Um M QC 1010 0- K2 0M -5 10100-K20-Hk-6 ULlOIOCMimHft-J ULIOIOO-LWIIM C9J9I2W1/WM-I4SOM n * CH l2U l/W k546nU 50 j** C9$92fX))/Wki4-MS-2H rp*> c w o r e i / M U-MSD-150 Pf* MA NA NA NA W Aw^mOK 2a6A m *iO C 2 a4 A aaf ew O K 24 A j4ym OK ' --- - " l t ' / wI ) T , J * " l---J -- PF0S-6.il / U C 0101*1 M dW l S 1.0000 1-0000 1JHOO 10000 #9971 0.1*71 I.OOOO 1.0000 PFO*Fw*7 ftH W f NA DOMO NA 0*600 NA OJ646 NA 0*640 NA NA NA NA NA NA NA NA KO* Orno. 000 0.00 J00 000 J7* 43* m 4*1 pro Facwr l l l 1 1 t i l PFM O fcC m Wi 000 01 000 0 00 221 2*2 111 293 Flwm * 01122047 A001122060 AMI 12204* A001122061 AMI 127097 AMU2709* AMI 127101 ami irridi -------- - - m o i <UJQ (0.00521^(1 <LOO (0.00321 u'i) 4 0 Q (0.0032* '() <LO0 (0 00321 M'tl 74% 94% 104% 90% III na . m it <U <LOO 4% 101% U lk MONTORTD NA NA 21% 1% W T f 1 NA NA NA NA la lA aa fe m O C 2a6 Aiudvm K 2*6A ulym OC M AsNymdK PFOOA C at $32 2.94 421 1.7 260 2*7 2*1 in rro tA r * r 1 1 1 1 l 1 1 1 rro u Cak-Ceac. $33 2.94 632 1.79 260 29* ITI 121 C iw iH ln rrrocA 01122047 AMI 122060 01122041 AMI 122061 AMI 127097 AOOI12709* AMI 127101 AMI 127102 <UX} (0.0306^1) ax iio w it 'ii <L OQt#1U06 ,i ) <lQO (0.0306 *%) 16% 9*% 90% 107% Mesa FFOU <yOQ <LOO *1% 99% MO M . Dcr. K m nm HA NA ii% 17% D w bnA M lyc O n VwtC'AmfelC PMyEataM/V artf** 12*1*0 LA 1211*0 UH 1200*0 LAC. BACK TO MAIN u- ETS-6-7.0 E u l 97 3M Environmental Laboratory TQX-62*4im22S-7ILid 2000001 12:56 PW Page 118 31^ Medical Department Study: T-6314.1 Mr ... MNk E fM M M a N M i --"-pD ik ifb ln M A iil) Ou. c iA-lyW A M lyK D w TOm RadM M A M y S M p k D iU Cm m 432*223. FACT-T0X-C2* T43I4 bu EI3-M.0 * ETSX70 Anta06249t. DvyO707V9 11/31/00 CK/SAL 11/32.00,11/27.00 MUH ti.3 7 , HOMO MMH HAT tJV T QC Dm M r* V iriU Irf NBMXM7-M M&M Cm . U M B ft 10100400 a t -j NA I0100-H204&-6 NA tteo B k RLlOlOO-UwBft-5 MA XBLIOIOO-LrarM- NA QC C939I2M331/W13-MS-230 pt 2*4 AaalyMOK CWI2M43l/Wki-USD-2S0 fob M M v -ac C*iTOPO 1/WkU-MS-230 rfk CTS2F/GI/Wkl*Msr>-2M pb 2nd AiuJnit OK L i r f q . - . w . tM(L O Q )y tfc l far pwny ,, 0I/3ID1 r K K - i . l l *'*. L C M 0.00 0.00 00 0.00 130 m 211 MSS Factor I 1 1 _______ !______ i i i i MSK C M Cm 0D0 000 ODO 0.00 123 IU 213 210 Dm . f . l i r - * '* --* r r Di*. Vb M A ^ j c r iy E jW ^ Y U : 1X01/00 U C IX I LOO UH 12/2000 UC. FACT-TOX-02* C*rocc4 6329-225 PfOSA. MS5.a d MJ70 ft* J3-W* Dmmjt JrWy of CitCDft (SO) KS Bit ItM raw i wr R -V >dV rfN Im B M -- --------------- Analytical Report: FACT TOX-028 LRN-U2636 n>M (f* M U0M22MT ADDI 1720*0 AMI122MI AO0I1Z2061 A00I1270*7 A0011770M ADOI 177101 AD01I27IQ2 ------- *rr" 'r <LOQ (O304 n'|) < j0 0 M 0 3 0 i .*'*> <L0Q (0.030 %) < D 0 (0.O306 a r t ) 41% 43H TIN 70% Um USM < to o <LOQ 32% 71% MD M D i.. M S M O in NA NA 4274 2% -, i VMM MA NA NA NA 2M .W p.0K 2*4 AadpnOK 2*4W r*.OK 2nd Atoka* OK tolCMOCIT-M M570 M37 C M . T *M 1 1-2 1 000 000 1 1*7 1 24* 1 243 t 241 1 MS70 O O .C M 4J3 3-2 ODO 000 174 22* 21 23 r ------- (P M AM1I2304T A0OU220M ADOII22044 ADOII 22061 AMI 1270*7 ADOl1270*4 AMI 127101 ADOI 127102 pfQ S 'P * . -----MUST* <LOQ (00122 <XOQ(0.0122 )> <l o o (om a m i > S9% 7*% 73% 00% M334 - O f l 7KHM(ti)CK2COOfi MJ70 - C4n7SQ2N(CH3)CH2COOH H n% sir MO M D n. 2*% ON BACK TO MAIN CTM-7.0 EW *7 3M Environmental Laboratory TQX-a244im22S-7Ejfc 2307001 12:30 PM Page 119 3N/ Medical Department Study: T-6314.1 FACT-Tox-a CnunH B M U Mr- Nod lW w { T t U ( t MMk Co*m * U29-223. FACT-TQX-oe D M m i T-4114 iM LM i ETW-4 0A ETX-4-7* A*i0t749*. Pw)070TW U M mM 11/2POO UK/5AL 11/22/00.M7TO MMH 11.7700. MU T O HUH *> rfPfOC, FfOSA. MJM. 4 M570 - fca 13-Waafc Dattry IM v c fQ tC D (ID) ICS M Rxa R-S^m W Value - ---<---- - MTUvnoc hw QC *-- ** IOIOWOOWk-5 IOIOO-HXVBa-4 RBUOlOOLnvHki KBL10|0 *4>Bt-6 C959l2Mtl/Wk5-MS-2SO py* C95!2M/GI/Wk5-MSD-2J0 *A C W F C l/WU4 ffk CT59f2F/Gl/Wkl4-UJI>-2 A IMITO LAC IMITO V H 117000 LAC P ft* Ml 0.00 00 eoo OH 223 2U 111 295 m irto* <LOQ (ODOUt M l) < U JO (0 .W J2 M ) <LOQ (0.00321 M f ) 4X)Q (01)0521 m /li _ 74% 94% |04% 99% M cm r * M . <LOO <LO0 101% no M Dn MS/MSDRTO MA 24% rroA Calc. Caac. 3J2 _ 2*4 I2N 2*0 .. ........** m o u Mm TM ** <2jOQ<0.0)06 M l) < ?2P.o.-*iL <LQ (O O M ^i) (0 0304 . y d 14% <100 MO 4*4. De*. M M U D Irtl MA MA 271 90% 107% 99% 17% IH M A -9 h w iiIm iiM nM. MJ70 - O f 175004(00 JOOCOQH MS CA C. Mc 0.00 oog 000 0.00 121 m 21J 210 ru s M cr% M _ <tOQ (0 030* i |) < 100(0 030* v i) <LQ(C0M*Tr l > <100(0030* U 41% u% 71% 70% <100 <100 71% Analytical Report: FACT TOX-028 L R N -U 2 6 3 6 no M .IM . MMOORTO MA NA 42% 2% USTI Q kC *4MST* 4 JJ------- 32* 0.0 0.0C 17* 229 21 2 <tOQ (04)122 t'i) <100(0.0122 s'il lOQ (0 011 2 ^ |) <100 (0 0122 u * i) 39% 74% 73% 10% <oo <ioo 44% 7*% MD M D ct. IOM D KTO MA MA 9% BAC K TO MAIN Ili ill rfV *-7J 3 Exal 97 3M Environmental Laboratory 70X42 t4mr22S-7Cjtfi 2002001 12J4 7W Page 120 BACK TO MAIN ii lii 3I\1 Medical Department Study: T-6314.1 FCT-TOX-02* C ovmm* C329-Z25 C tatrfAaipa'A tope Da t a *?>> D*U C m c a *329-225, FaCT-TOX D T-4JI4 bL w M dkM UN \im m R w w 11'IMO MHH m tm uh WO*. KHA. M3S*. m i U S * mt o l3-Wa* DoyS U * ofOLCD* (SO) IQS BJt f a t n in : Sm M m* c-- f UM K KBL13070WUO Bfc-l-l K BLI20W HX7I-1-2 UartcHk IU .IW 0 M Bfc-I RBLI2D7aM^wBk-2 QC C939I3MU1-US-M0 ( C959UU*J t-MSO-303 i( /i - 1 ! C W JiU -1 CW 1IU1 CW J2 U -1 CW32M2 C W M U -I C9J934U4 CUOI If-I C9W1IF-2 CM014F- C94414F-2 C M illF -i C9MI5F-2 U uH rQ *atiM to (UJQ) toM f a p a r s o l a 191 f f o e - 1 TM I t a VrtM filar ---------1 ____ MA 1.0M0 NA NA HA NA Na n e Na NA 100*0 1.0000 10444 IM H NA na na Na 12W12 na NA n m NA NA 14291 NA NA 14073 NA NA I.M31 Na - _NA L* NA NA 144*1 NA NA 14421 i---- --- NA 140*4 NA NA 14410 NA NA 141*4 NA NA 144 NA M rt LA Ctiai: m M trn ttf 1 cw m * 0.1*40 01*40 0.1*40 0.1*40 NA NA 0-1*40 1.1*40 04*40 04*40 04*40 04*40 04*40 04*40 4*40 4*40 4*40 0.1*40 tr a <w. - * IU 400 40 NANE NS NS Mi J7I 427 473 Ht 135 522 sor m 4JT 414 42? l i m 12777 LAC m i dfc-CMM. C-- W N rm o Mm mu ssss% * --* i 1*4 001211*10 41*0 i 4 COCI213423 tOO 190(05 *ofcl 04111 o* h o i 0 DOOUtMll <LOQ((.0I(5 Kfk) i NANE NANE ------MANI to o HA i M NA NA i NS NA NA NA NA 30 1511 D0012H01* 154 10 I5H2 boom ion IJJ ______l 7 ______ 1*7% 10 i m t DDO1211011 174 30590 DM12H019 20.4 192 l i n t 13471 D0OI2IK30 1J4 JO 14490 POOlJINm 145 110 *47% 21172 001213032 214 M 20*99 0001215031 210 214 2-17% 30 20075 D0P13I3Q54 20.1 10 1710* 00I2IM9T 174 10 17312 DO*213C3I 17J ia 1 5 - I7*U D00121M19 17.7 fVOS hrftKK 174 1130% t.7*% M * CW|79CUN(CW)CH2COaH Analytical Report: FACTTOX-028 LRN-U2636 rr NA NA NA NANE NA NA NA NA NA NA NA NA NA -.NA NA NA NA NA TTOAA 4 4M 940 NAVE NI M m 5*7 27.7 10.5 544 4J9 1*4 199 1*4 1.17 1 17.1 no*A Flrtar 1 1 1 1 1 t 29 2* 21 20 29 n M 50 10 H m *a CN tO w . Mk 40 040 040 NANE NI NS 734 lili 712 *01 109* 1092 7*4 4M 111 m 940 121 I g T j I WHOM 0001212011 < j0Q (9.00*11 'i ) NA NA DHI11M24 NA NA 9.79 U t 121192* 9.722 DMI2I29S0 ----------f u --------------- 0121)02 4M 00912(1034 411 901213031 D90I2I2029 4449 421 Na mu NA 4M A(-- M lh w ii . kiU n% NA NA NA nm 19.0% 4.400% 224% 2*.2% 114% 20.0% ETS-A-7.0 El*7 3N Environmental Laboratory TOX-0-W225-77L*1 m i ti2 t ru Page 121 BACK TO MAIN II il 3M Medical Department Study: T-6314.1 FACT-T0X-G2* Cavane* 329-225 mt KOS. HOEA. MSH. md U70 h a II-W M Di-- * JEMVrfCACD* (SD) IOS M bfe ET SM O E IU -T.t taiT E n K M M ly c ---- rr..--Saaapfc Data K A T U V I W - f c 5 Prariric Gtm &M4 tM d lk IM n tt U L 12P ttJU O flft.|.2 KBLlXmO-Lar M kl QC C9S9UU43I-UE-M0 I t 'l c m tiM o i-u stv io Q **tt C9S91IW-2 CWH2J4-I cw aiM -i CHN41U CW934M-1 C8601IF-1 cwaiiF-1 C9MI4F-J OM014Fj CMOiSF-l CM415M ireino w linV W UUH XVIVH UH i1 V iriM MSH MSM <W M dn S L . --ty*T . NA 9.00 1 ______ N . . NA -9< im 1 1 ____ t a a s ____ NANE NA N HA N* - 1 .... 1 \ NA *1.7 20 NA U* NA M i 21 NA M.4 2 NA 1*4 2* NA 17 NA M.4 NA 1 NA .4 NA M.4 M NA 20.4 S* NA ZJ* OACk l------ <**-- * 4 MAM k ff% li JO 1X0110*23 1.0 DOOI2UOII <0QfOJOCII a r t ) -- n a s ____ NA/NK M NA NA M MA MA 1121 DMI2U0M li n Doomjo 1 1 1721 D002IM 1.71 ira DM1IWZ7 ITT m i DM1212070 IJ1 i OBIII 11 U9I DWUU0J2 l-SO 14* 12 DOOI2IMU 1*1 II x n iJiw M MS 1244 DM12I10T7 1*7 M DMI2I30M 1149 DMI2IJ0M OJSS MS Ilf <230 <LOO NA 1.9* I.7S 174 ITS IS* 1.05 ______ * 4 NA ha _____ tf-TM 2.0% _____ J V - IT** J74M .** NA NA NA MANE NA NA NA NA NA NA NA NA NA NA NA _ NA NA NA MS7i Cm . wk . 4M - 9*f 0J0 NA/NE NS NS 174 < ss. 407 994 171 JJ7 IN US 127 121 1 KST0 1 1 1 1 1 1 20 20 20 . 20 fi s* so so J* so MIN Cafe. Cm c OM 4M 400 NANE NE 7441 N tt 7902 Till MO *752 MOI -- s*-- 4*20 1741 M27 NA Do o u in s* h u m , j m m lac WMS7* NA *.7S 40 Analytical Report: FACT TOX-028 LRN-U2636 M an MST* B akattn* 00*-- m NA NA s .n 4.7S% ETS--7jO E n d TI 3M Environmental Laboratory TOX-02A1jvh2S-7Ej 2/30/2ooi i * rw Page 122 BACK TO MAIN il Ht 3M Medical Department Study: T-6314.1 FACT-TOX-tM CvvmccA <329-225 **r- tr o tta H a >ii(T i SHriuct t Matix: Cb w 2M 23. FACT-TCK-S21 T-OI4 S a tiv a ETV- A ETX-4-7J riO fWMt n c --* iM riTFPI.m)BA. M3X . H MSTOaSa11-W* OMySriyrfO tC D (ra} KMBO Vika: l a A M t M t 11*7*0 I H M MUH IM W fM U T U V U W a f c 5 Pwftrii f MtM Bfc U U 297WLH2 0 Bfc-l-l oc -si lit" C9WIJU/GI-U1OQ0 y c n s it w o i- u s k -j o t /( C9S9MM-I C919J1M-2 C99UM-I .............- V W & i . . C9UIIF-I CWOMFJ CM0 I5F-I CfO lJf-J mx kCM L s im n k -----IM OAD SJ NANE NS NS 001*0 4 jOO(SAIS) u fel "LOQ(B01W *'*) NANE NA NA USI* . . 11? -- 179J* MJ 1)1 I7J . HWIM71 a i.. I * -11)73 KM* 20*75 in o f ITSU nui ...........* 210 lit 17.1 I7J 17.7 _____ 0122_____ . ________________ _ NA 11.7 ______ iII. . . lit ______ P- _______i i l no U * lK NA 192% I1.M* *07* 111% 159* USO* DM t m m i/Am t y * M VaiW AaNyit yaytaiIIV aH U : Urt WO. 1177*0 LAC 0 UOMO LAC. rroA OfcCM c 01 01 0.90 HWi NS NS 754 11IS 7)2 tot UH 1002 TM 0* SU JM *41 12] ^rm uT Mm mu <LOQ(*J0* ll rtJ <LOO0004I! *4'l ^LOQ(0D41) NANS NA NA 07M M2 0.7 B.tt 101 MO .1*4 M J II un 0*40 0J 2I <LOO <LOO NA in i OMI ______ L _ ______iss.-.. .....M S * ss: NA NA NA JM * 19.0* 0.400* - ___n * * ___ M J* U J* M9M CMc. C W oso 00 LO NANE NS NS IS2S 2157 1723 1773 1911 ISAS ISM 1912 USO IMO S >149 rii * 4 AQJMIICfe) -LOOPJMII u /0 <LOQ<OM*llrt) KVW NA NA 112 Ut 171 177 191 MO-----------1.91 LIS US tfO A mNirilinr ili iiii iiim Ii MS* * O f n * 02N(H)crac0 0 H U57* - CSFI7W 2K(CHJ>CH2COOIf Mm MW <LOO <oo NA 1.99 I.7S 1.74 I.7S IM Analytical Report: FACTTOX-028 LRN-U2636 N 1M il* NA NA NA 14.7* 2.UW sia Q C H OM 000 NS 17.9* 11.4* u n riMSTO NA SI Mm 0079 MA NA SJ9 4.73% ETS4-7.0 E ar7 3M Environmental Laboratory TOX-02*-kva225- 7Eji 24MOOI lU S FM Page 123 BACK TO MAIN 3M Medical Department Study: T-6314.1 Analytical Report: FACTTOX-028 LRN-U2636 Appendix F: Example Calculations Formula Used for Sera Analyses in Study FACT-TOX-028 AR (ng/mL) x DF x PCx FV (mL) x 1.0 pg = Reported Concentration (pg/mL) EV (mL) 1000 ng Calculation Used for Group 3, Week 5, Animal ID C96031F (PFOS) 475 ng/mL x 100 x 0.864x 1.0 mL x 1.0 ug = 41.0 pg/mL 1.0 mL 1000 ng AR-- Analytical result from MassLynx summary DF-- Dilution factor FV--Final extract volume (1.0 mL unless otherwise noted) EV-- Volume of sera extracted PC--Purity Correction (for PFOS, only) Formula Used for Liver Analyses in Study FACT-TOX-028 AR (ng/g) x 3 curve(1) x DFxPC x 1.0 ug = Reported Concentration (pg/g) 3 sample 1000 ng (1) 3 curve is assumed to be: 1 g liver 5 mL H20 Calculation Used for Group 3, Week 5, Animal ID C96032 (PFOS) 353 ng/g x l g/ 5 mL x 500 x 0.864 x 1.0 pg = 146 pg/g 1.0443 g/5mL 1000 ng AR-- Analytical result from MassLynx summary 3 curve-- Density o f the liver standard curve, assumed to be Ig liver/ 5 ml water 3 sample-- Density o f the liver sample (g sample/ 5 mL H20 ) DF-- Dilution factor PC-- Purity Correction (for PFOS, only) 3M Environmental Laboratory Page 124 BACK TO MAIN 3M Medical Department Study: T-6314.1 Analytical Report: FACT TOX-028 LRN-U2636 Formula Used for Liver Precision Analyses in Study FACT-TOX-028 |(Replicate l(ng/g) - Replicate 2(ng/g))| x 100% = Relative Percent Difference Average o f Replicates 1 and 2(ng/g) Calculation Used for Group 2, Week 5, Animal ID C95931M (PFOS) 1(15.5 n g /g - 15.8 ng/g)| x 100% = 1.92% (15.5 ng/g + 15.8 ng/g) / 2 3M Environmental Laboratory Page 125 3M Medical Department Study: T-6314.1 BACK TO MAIN Analytical Report: FACTTOX-028 LRN-U2636 Appendix G: Interim Certificate(s) of Analysis 3M Environmental Laboratory Page 126 BACK TO MAIN 3M M edical D epartm ent Study: T-6314.1 A nalytical R eport: FA C T TO X -028 LR N -U 2 6 36 Centre Analytical Laboratories, Inc. 3048 R esearch D rive Phone: (814) 231-8032 S tate C ollege, PA 16801 Fax:: (81 4 ) 2 3 1 -1 2 5 3 o r (81 4 ) 2 3 1 -1 5 8 0 INTERIM CERTIFICATE OFANALYSIS R e v is io n 1 (9 /7 /0 0 ) Centre Analytical Laboratories COA Reference #: 023-018B 3M Product: PFOS, Lot 171 Reference #: SD-009 __________________ Purity: 86.4% Test Name Specifications Result Purity1 vv:-: 86.4% Appearance White CrystallinePowder Conforms Identification ' :' '< ;': ' NMR Metals (ICP/MS) ' " .. > . > Positive 1. Calcium 2. Magnesium 3. Sodium 4. Potassium2 . : 1. 0.017 wt7wt.% >'V'-*1 `" 2. 0.007 wt./wt.% 3. 1.355 wt/wt.% 4. 6.552 wt./wt.% 5. Nickel 6. Iron 7. Manganese Total %Impurity (NMR) 5. 0.003 wt.Avt.% 6. 0.004 wt./wt.% 7. <0.001 wt./wt.% 1.00 wt./wt.% Total %Impurity (LC/MS) 10.60 wt./wt.% ' ' . ., , ' . ':':i ": ;'; r ' - Total %Impurity. ; - None Detected (GC/MS) Related Compounds - . POAA 0.30 wt./wt.% Residual Solvents (TGA) Purityby DSC None Detected Not Applicable1 Inorganic Anions (IQ 1. Chloride 2. Fluoride 1. <0.015 wt./wt.% 2. 0.27 wt./wt.% 3. Bromide 4. Nitrate 5. Nitrite 6. Phosphate 7. Sulfate4 Organic Acids5(IC) 1. TFA 2. PFPA 3. HFBA 4. NFPA Elemental Analysis6: , : ' . : 1. . -\ -!:> ; ;. ' :. . -- 3. <0.040 wt./wt.% 4. <0.009 wt./wt.% 5. <0.006 wt./wt.% 6. <0.007 wt./wt.% 7. 8.82 wt./wt.% 1. <0.1wt./wt.% 2. <0.1 wt./wt.% 3. <0.1 wt./wt.% 4. <0.25 wt./wt.% 1. Carbon 2. Hydrogen 3. Nitrogen 4. Sulfur 5. Fluorine 1. Theoretical Value = 17.8% 2. Theoretical Value = 0% 3. Theoretical Value = 0% 4. Theoretical Value = 5.95% 5. Theoretical Value = 60% 1. 12.08 wt/wt.% 2. 0.794 wt/wt.% 3. 1.61 wt/wt.% 4. 10.1 wt./wt.% 5. 50.4 wt/wt.% CO A 023-018B Page 1 o f3 3M EiiviiuiifMuiilal Laboratory Page 127 BACK TO MAIN 3M M edical D epartm ent Study: T-6314.1 Analytical Report: FAC T TO X -028 L R N -U 2 6 36 Centre Analytical Laboratories, Inc. _ 3048 Research Drive State College, PA 16801 Phone: (814) 231-8032 Fax: (814) 231-1253 or (814) 231-1580 INTERIM CERTIFICATE OF ANALYSIS Centre Analytical Laboratories COA Reference #: 023-018B Date of Last Analysis: 08/31/00 Expiration Date: 08/31/01 Storage Conditions: Frozen <-10C Re-assessment Date: 08/31/01 'Purity = 100%- (sum of metal impurities, 1.39%+LC/MS impurities, 10.60%+Inorganic Fluoride, 0.27%+NMR impurities, 1.00%+POAA, 0.30%) Total impurity from all tests = 13. 56% Purity = 100%-13.56% = 86.4% 2 ,, p. Potassium is expected in this salt form and is therefore not considered an impurity. 3Purity by DSC is generally not applicable to materials of low purity. No endothermwas observed for this sample. 4Sulfur in the sample appears to be converted to SO4and hence detected using the inorganic anion method conditions. The anion result agrees well with the sulfur determination in the elemental analysis, lending confidence to this interpretation. Based on the results, the SO4is not considered an impurity.5 5TFA HFBA NFPA PFPA Trifluoroacetic acid Heptafluorobutyric acid Nonofluoropentanoic acid Pentafluoropropanoic acid ''Theoretical value calculations based on the empirical formula, CgFnSOs'K* (MW=538) This work was conducted under EPA Good Laboratory Practice Standards (40 CFR 160). c COA023-018B 3M Environmental Laboratory Page 2 o f 3 Page 128 BACK TO MAIN 3M M edical D epartm ent S tudy: T-6314.1 Analytical R eport: FAC T TO X -028 L R N -U 2 6 36 Centre Analytical Laboratories, Inc. 3048 R esearch D rive Phone: (814) 231-8032 S tate C ollege, PA 16801 Fax: (814) 231-1253 or (814) 231-1580 INTERIM CERTIFICATE OF ANALYSIS Centre Analytical Laboratories COA Reference #: 023-018B LC/MS Purity Profile: Impurity C4 C5 C6 C7 Total wt./wt. % 1.03 1.56 6.38 1.63 10.60 Note: The C4 and C6 values were calculated using the C4 and C6 standard calibration curves, respectively. The C5 value was calculated using the average response factors from the C4 and C6 standard curves. Likewise, the C7 value was calculated using the average response factors fromthe C6 and C8 standard curves. r. Prepared By: David S. Bell Date Sdenrtist, Centri ytical Laboratories Reviewed By: C/*L. fit /A John Flaherty Date Laboratory Manager, Centre Analytical Laboratories COA023-018B 3M Environmental Laboratory Page 3 o f 3 Page 129 3M Medical Department Study: T-6314.1 Appendix H: Report Signature Page BACK TO MAIN Analytical Report: FACT TO X-028 L R N -U 2 6 36 Andrew M. Seacat, Ph.D., Study Director -------2-Z. T/--z---s--r/----- 1/------------- Date Cf ^ ^ John L. Butenhoff, Ph.D., Sponsor Representative Date 23 f lz . ---------Kristen J. Hansen, Ph.D., Principal Analytical Investigator 2 -/2 3 /o f Date -- William K. Reagen, Ph.D., Laboratory Manager 6 ^ 3 Date 3M Environmental Laboratory Page 130