Document 06v24djNEnj5N7DOj2B627x9J

3M Medical Department Study: T-6316.1 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 Analytical Report: FACT TOX-003 LRN-U2104 Study Title 104-Week Dietary Carcinogenicity Study with Narrow Range N-Ethyl PerfluorooctanesulfonamidoEthanol in Rats Analytical Laboratory Report Title Determination of the Presence and Concentration of PFOS, PFOSA, PFOSAA, EtFOSE-OH, M556, and PFOSEA in Serum and Liver Samples of Crl:CD@(SD)IGS BR Rats Exposed to N-Ethyl PerfluorooctanesulfonamidoEthanol Data Requirement Not Applicable Author 3M Environmental Laboratory Study Completion Date JuAntes0ig6n,in2g001 Performing Laboratories Liver and Serum Analyses JI 3M Environmental Laboratory Building 2-3E-09,935 Bush Avenue St. Paul, MN 55106 Project Identification 3M Medical Department Study: T-6316.1 Covance In-Life Study: 6329-228 Analytical Report: FACT TOX-003 3M Laboratory Request No. U2104 Total Number of Pages #2#1#1 3M Environmental Laboratory 3M Environmental Laboratory Page 1 Page 1 3M Medical Department Study: T-6316.1 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 Analytical Report: FACT TOX-003 LRN-U2104 This page has been reserved for specific country requirements. 3M Environmental Laboratory 3M Environmental Laboratory Page 2 Page 2 3M Medical Department Study: T-6316.1 3M Medical Department Study: T-6316.1 GLP Compliance Statement Analytical Report: FACT-TOX-003 LRN-U2104 Analytical Report: FACT TOX-003 LRN-U2104 Analytical Laboratory Report Title: Determination of the Presence and Concentration of PFOS, PFOSA, PFOSAA, EtFOSE-OH, M556, arid PFOSEA in Serum and Liver Samples of Crl:CD@(SD)IGS BR Rats Exposed to N-Ethyl PerfluorooctanesulfonamidoEthanol Study Identification Numbers: T-6316.1, FACT TOX-003, LRN-U2104 This study was conducted in compliance with United States Food and Drug Administration (FDA) Good Laboratory Practice (GLP) Regulations 21 CFR Part 58, with the exceptions in the bulleted list below. The analytical phase completed at the 3M Environmental Laboratory was performed in accordance with 3M Environmental Laboratory Standard Operating Procedures. Exceptions to GLP compliance: There were two study directors in this study. This study was designed as two separate studies. The in-life phase study was considered to end at the generation and shipment of specimens. The analytical study was considered to start at the receipt of these specimens for analysis. This resulted in having two separate study directors, one for each phase of the same study. However, since I the technical performance of each phase was entirely separate, no effect is expected from this exception. Sample storage stability will not be determined. Characterization of the analytical standards is underway, but has not yet been completed (21 CFR 58.105 (a)). Lot No. 59905 of the surrogate, THPFOS, will not be characterized for purity since quantities of this standa.rd are exhausted. Lot 936 of EtFOSE-OH, and Lot L2353 and TN-A-1886 of PFOSA will not be characterized since quantities of these standards are exhausted. The electronic data systems in use have not been validated and there is not an electronic audit trail of corrections currently available (21 CFR 58.130 (e)). Authenticated hard copies of chromatograms and associated documents will be considered as the original raw data. There were instances in the raw data where corrections were not made according to 21CFR58.130(e) GLP requirements. Data was crossed out with no reason given, or original entries were obscured or contained "write-overs" with no explanation, initial or date provided. Some reagents (00-022-2, 0.5M TBA solution and 00-003-28, 0.5M TBA solution) were not labeled with storage requirements. Persons responsible for equipment were not identified (021188-55 IEC Cetra GP*R and N-Evap.) (See next page for signatures) 3M Environmental Laboratoty 3M Environmental Laboratory Page 3 Page 3 3M Medical Department Study: T-6316.1 3M Medical Department Study: T-6316.1 GLP Compliance Statement continued Analytical Report: FACT-TOX-003 LRN-U2104 Analytical Report: FACT TOX-003 LRN-U2104 John L. Butenhoff, Ph. D., Studybirector A 0 &w; 7-CL Marvin T. Case, D.V.M., Ph.D., Sponsor Representative - " Krikten J. Hansen, Ph.D., PrincipalAnalytical lnvestigafor Date s,*z,1 , %#/ [late H-/3//D/ Date boratory Manager Date 3M Environmental Laboratory 3M Environmental Laboratory Page 4 Page 4 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 Inspection Dates - Ir 4/20/00 6/30/00, 7/5/00 4/23/014 / 3 0 / 0 1 4/30/01-vi c i d I iiun 4I 5/22/0 1 Phase I Date Reportedto Management Study Director Sample prep 8/8/00 8/8/00 Sample prep Data I I Report 7/21100 7/21/00 -7 I 5/2/01 5/2/01 1 5/23/01 5/23/01 3M Environmental Laboratory 3M Environmental Laboratory Page 5 Page 5 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 3M Medical Department Study: T-6316.1 Table of Contents Analytical Report: FACT TOX-003 LRN-U2104 GLP Compliance Statement ............................................................................................. 3 GLP Study-Quality Assurance Statement ...................................................................... 5 Study Personnel and Contributors.................................................................................... 8 Introduction and Purpose.................................................................................................. 9 Test System ................................................................................................................. 9 Specimen Collection and Analysis ............................................................................... 9 Specimen Receipt and Maintenance................................................................................. 10 Chemical Characterization of the Reference Standards ................................................... 11 Dose Confirmation Analyses........................................................................................ 11 Method Summaries........................................................................................................... 12 3M Environmental Laboratory ...................................................................................... 12 Preparatory Methods ............................................................................................... 12 Analytical Methods .................................................................................................. 13 Analytical Equipment............................................................................................... 13 Data Quality Objectives and Data Integrity ....................................................................... 14 Data Summary, Analyses, and Results.............................................................................. 15 Summary of Quality Control Analyses Results............................................................. 15 Statement of Data Quality ............................................................................................ 17 Summary of Sample Results........................................................................................ 17 Statistical Methods and Calculations ................................................................................. 17 Statement of Conclusion................................................................................................... 17 References ...................................................................................................... ;................17 Appendix A: Chemical Characterization and Control Matrices.......................................... 18 Appendix B: Protocol. Amendments and Deviations.......................................................... 19 Appendix C: Extraction and Analytical Methods ............................................................... 2707 Appendix D: Data Summary Tables.................................................................................. 12517 Appendix E: Data Spreadsheets....................................................................................... 12651 Appendix F: Example Calculations ................................................................................... 12864 Appendix G: Interim Certificates of Analysis..................................................................... 12876 Appendix H: Report Signature Page................................................................................. 22181 3M Environmental Laboratory 3M Environmental Laboratory Page 6 Page 6 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 3M Medical Department Study: T-6316.1 List of Tables Analytical Report: FACT TOX-003 LRN-U2104 Table 1. Test System Population Demographics for Study 6329-228 .............................. 9 Table 2. Characterization of the Analytical Reference Standards in Study FACT TOX-003 .............................................................................................................. 11 Table 3. Target Ions Monitored in 3M LaboratoryAnalyses.............................................. 14 Table 4. Matrix Spikes-Sera .......................................................................................... 16 Table 5. Matrix Spikes-Liver ........................................................................................... 16 Table 6. Characterization of the Control Matrices Used for Sera Analyses in Study FACT TOX-003.................................................................................................... 18 Table 7. Characterization of the Control Matrices Used for Liver Arialyses in Study FACT TOX-003 .................................................................................................... 18 Table 8.Characterization of Test Article in Study FACT TOX-003 ................................... 18 Table 9. FACT TOX-003 Data Summary for PFOS. PFOSA. PFOSAA. EtFOSE, M556 and PFOSEA-Rat Serum (pg/mL) ..................................................................... 21157 Table 9. (Continued) FACT TOX-003 Data Summary for PFOS, PFOSA. PFOSAA, EtFOSE, M556 and PFOSEA-Rat Serum (pg/mL) ............................................ 21258 Tjlble 10. FACT TOX-003 Data Summary for PFOS, PFOSA, PFOSAA, EtFOSE, M556 and PFOSEA-Rat Liver (pg/g)........................................................................... 21359 Table 10. (Continued) FACT TOX-003 Data Summary for PFOS, PFOSA. PFOSAA, EtFOSE. M556 and PFOSEA-Rat Liver (pg/g)................................. :................21460 3M Environmental Laboratory 3M Environmental Laboratory Page 7 Page 7 3M Medical Department Study: T-6316.1 3M Medical Department Study: T-6316.1 Study Personnel and Contributors Analytical Report: FACT-TOX-003 LRN-U2104 Analytical Report: FACT TOX-003 LRN-U2104 Study Director - John L. Butenhoff, Ph.D. 3M Corporate Toxicology Medical Department 3M Center Building 220-2E-02 St. Paul, MN 55144-1000 651-733-1962 Sponsor Marvin T. Case, D.V.M., Ph.D. 3M Corporate Toxicology - Medical Department 3M Center Building 220-2E-02 St. Paul, MN 5!~44-1000 Analytical Chemistry Laboratories Liver and Serum Analyses 3M Environmental Laboratory Kristen J. Hansen, Ph.D. Principal Analytical hvestigator 3M Lab Contributing Personnel [List all personnel alphabetically] David R. Barnidge, Ph.D.* Lisa A. Clemen Rhonda Dick* Kelly J. Doweiler* Mark E. Ellefson Barb A. Gramenz* Sarah A. Heimdal* Marlene M. Heying* Megan C. Holloway* *Contract lab professionalservice employees Harold 0. Johnson Kelly J. Kuehlwein* Ognjenka Krupljanin* Sally A. Linda* Ian A. Smith* Kathleen M. Stock* Anh-Dao Vo Bob W. Wynne* Richard 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. 3M Environmental Laboratory 3M Environmental Laboratory Page 8 Page 8 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 3M Medical Department Study: T-6316.1 Introduction and Purpose Analytical Report: FACT TOX-003 LRN-U2104 The purpose of the study is to determine the presence and concentration of PFOS, PFOSA, PFOSAA, EtFOSE-OH, M556, and PFOSEA in serum and liver specimens collected from Covance Study No.: 6329-228 titled: 104-Week Dietary Carcinogenicity Study with Narrow Range (98.1Yo)N-Ethyl Perfluorooctanesulfonamido Ethanol in Rats. The Covance in-life study was initiated on April 17, 1998. The analytical phase of the study was initiated on May 22, 1998. Test System A total of 140 males and 140 female rats were used as the test system. Table 1 outlines the rat population demographics and dosage levels for study 6329-228. The test system species and strain selected was the Crl:CD@(SD)IGS BR rat received from Charles River Laboratories, Inc., identified using an implanted microchip device. At the initiation of treatment the rats were approximately 6-8 weeks old and weighed Ibetween approximately 100-300 g. Table 1. Test System Population Demographicsfor Study 6329-228 ~ ~~ Number of Animals d Study Group Approximatte Dietary Levels of Male EtFOSE-OH (ppm) Group 8 Control Group 9 Treated 70 70 0 70 70 1 Specimen Collection and Analysis Specimens were collected by Covance (study 6329-228) and sent to the 3 M Environmental Laboratory for analysis. On week 4, 20 serum specimens and 20 liver specimens were collected from Groups 8 and 9 males and females. On week 14,20 serum specimens and 20 liver specimens were collected from Groups 8 and 9 males and females. On week 53, 20 serum specimens and 20 liver specimens were collected from Groups 8 and 9 males and females. On week 105, 40 liver specimens were collected from Groups 8 and 9 males and females. On week 105, 40 serum specimens were collected from Groups 8 and 9 males and females. The number and type of specimens collected for analyses in the analytical phase of this study are presented. Specimens Collected from Study Groups 8 and 9 Serum Specimens-100 specimens Liver Specimens-100 specimens Liver and sera specimens were shipped to the 3M Environmental Laboratory frozen and on dry ice. 3M Environmental Laboratoy 3M Environmental Laboratory Page 9 Page 9 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 3M Medical Department Study: T-6316.1 Analytical Report: FACT TOX-003 LRN-U2104 Sera and liver samples were extracted beginning on May 27, 1998 using an ion pairing reagent and either ethyl acetate or methyl-tert-butyl ether (MtBE). Liver samples were homogenized prior to the extraction procedure. Sample extracts were analyzed using high-pressure liquid chromatography-electrospray/tandem mass spectrometry (HPLC-EShSSMS) in the multiple reaction monitoring mode. PFOS, PFOSA, PFOSAA, EtFOSE-OH, M!j56, and PFOSEA levels were evaluated by external calibration using extracted curves. Analytiisal details are included in this report. Specimen Receipt and Maintenance The 3M Environmental Laboratory received liver and serum specimen.scollected at predetermined time points during and at the end of the in-life phase 01 Covance Study 6329-228 from Covance May 1998 through June 2000. All specimens were received frozen on dry ice and were immediately transferred to storage at -20C *lOC. Control matrices used in liver and sera analyses performed during TOX-003 were obtained from commercial sources and are presented in Appendix A. Samples analyzed at the 3M Environmental Laboratory will be maintained for a period of 10 years and will be stored at the laboratory at -20C *lOC. 3M Environmental Laboratory 3M Environmental Laboratory Page 10 Page 10 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 3M Medical Department Study: T-6316.1 Analytical Report: FACT TOX-003 LRN-U2104 Chemical Characterization of the Reference Standards Chemical characterization information on the analytical reference materials used in this study is presented in tabular form below. Table 2. Characterizationof the Analytical Reference Standards in Study FACTTOX-003 Potassium Perfluorooctanesulfonate `This lot is exhausted and cannot be characterized. N R 4 d recorded NA-Not applicable TBD-To be determined Target analyte is PFOS, C8F1,.93; %get analyteis C,F,~~{(CH:!CH,)(CH*C~~} `Unless othennriseindicated, at the time of quantitation. the puntyfor di anaiyteswas assumed to be 100%. dForreferencelot 617, a purityof 53.62% was usedfor PFOSAA result quantitation. Dose Confimtion Analyses Dose preparation methods and analysis were performed by Covance, ulsinga validated analytical method provided by Covance (MP-M312-MA), and are reported separately (Reference Covance 6329-228). 3M Environmental Laboratory 3M Environmental Laboratory Page I I Page 11 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 3M Medical Department Study: T-6316.1 Analytical Report: FACT TOX-003 LRN-U2104 Method Summaries Following is a brief description of the methods used during this analytical study by the 3M Environmental Laboratory. Detailed descriptions of the methods used in this study are located in Appendix C. Data collected prior to November 1999 was reworked in 2000 to accommodate improvements in data reduction methods. Both the original and "reworked" data are archived; reworked data is presented in the final results. The improved methods are documented in the form of method modifications. As the present study progressed, more advanced methods evolved and earlier methods were used with deviations until amendments to the protocol were written. Protocol and method deviations are located in Appendix 6 of this report. 3M Environmental Laboratory PREPARATORY MEnrODS FACT-M-1.O. "Extraction of Potassium Perfluorooctanesulfonate or Other Anionic Fluorochemical Surfactants from Liver for Analysis Using HPLC-Electrospray/Mass Spectrometry". This method was used for week 4 samples extracted on 6/9/98 and week 14 samples extracted on 7/17/98. FACT-MS.0, "Extraction of Potassium Perfluorooctanesulfonateor Other Anionic Fluorochemical Surfactants from Serum for Analysis using HPLC-Electrospray/Mass Spectrometry". This method was used for week 4 samples extracted on 5/27/98 and week 14 samples extracted on 7/15/98. ETS-8-4.1 , "Extraction of Potassium Perfluorooctanesulfonate or other Fluorochemical Compounds from Serum for Analysis using HPLC-Electrospray/MassSpectrometry". This method was used for week 53 samples extracted on 4/20/00, and week 105 samples extracted on 6/29/00. ETS-8-6.0, "Extraction of Potassium Perfluorooctanesulfonate or other Fluorochemical Compounds from Liver for Analysis using HPLC-Electrospray/MassSpectrometry". This method was used for week 53 and week 105 samples extracted on 4/14/00. An ion-pairing reagent was added to the sample and the analyte ion pair was partitioned into ethyl acetate (FACT-M-1.O and FACT-M-3.0) or MtBE (ETS-8-4.1 and ETS-8-6.0). The extract was transferred to a centrifuge tube and put onto a nitrogen evaporator until dry. Each extract was reconstituted in 1.OmL of methanol, and then filtered through a 3 cc plastic syringe attached to a 0.2 pm nylon filter into glass autovials. 3M Environmental Laboratory 3M Environmental Laboratory Page 12 Page 12 3M Medical Department Study: T-6316.1 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 Analytical Report: FACT TOX-003 LRN-LJ2104 ANAL~ C A MLETHODS 0 FACT-M-2.0, "Analysis of Liver Extracts for Fluorochemicals Usinig HPLC-Electrospray/Mass Spectrometry". 0 FACT-M4.0, "Analysis of Fluorochemicals in Serum Extracts Using HPLC-Electrospray/Mass Spectrometry". 0 ETS-8-5.1, "Analysis of Potassium Perfluorooctanesulfonateor other Fluorochemicals in Serum Extracts Using HPLC-Electrospray/MassSpectrometry". 0 ETS-8-7.0, "Analysis of Potassium Perfluorooctanesulfonate or other Fluorochemicals in Liver Extracts Using HPLC-Electrospray/Massspectrometry". The analyses were performed by monitoring one or more product lions selected from a single primary ion characteristic of a particular fluorochemical using HPLGES/MS/MS. For example, molecular ion 499, selected as the primary ion for PFOS (CeFI7SOi) analysis, was fragmented further to produce ion 99 (FSOS). The characteristic piPoduction 99 was monitored for quantitative analysis. AML Y77CAL EQUIPMENT The actual analytical equipment settings used in the present analytical'phase of this study varied slightly during actual 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@BetasilTMCIS2x50 mm (5 pm) Column temperature: Ambient Mobile phase components: Component A: 2mM ammonium acetate Component 6:methanol Flow rate: 300 pUmin Injection volume: 10 pL Solvent Gradient: 9.0 minutes Time (minutes) %B 0.o 40% 1 .o 40% 4.5 95% 6.5 95% 7.0 40% 9.0 40% 3M Environmental Laboratory 3M Environmental Laboratory Page 13 Page 13 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 3M Medical Department Study: T-6316.1 Analytical Report: FACT TOX-003 LRN-lJ2104 Mass Spectrometer: Micromass@API/Mass Spectrometer Quattro llTT"riple Quadrupole system Software: Mass Lynx'" 3.1 , 3.2, 3.3, 3.4 Cone Voltage: 30-60 V Collision Gas Energy: 25-45 eV Mode: Electrospray Negative Source Block Temperature: 150C *1O"C Electrode: Z-spray Analysis Type: Multiple Reaction Monitoring (MRM) Table 3. Target Ions Monitored in 3M LaboratoryAnalyses I Target tinalyte I Primary Ion (AM") T ~~ Produc- t I I PFOS I 499 I 80,991,130 I ~ PFOSA 498 PFOSAA 584 I EtFOSE-OH I 630 I 59 I I PFOSEA I 526 I 65 I M556 556 65,78, 83, 169 THPFOS 427 Data Quality Objectives and Data Integrity The following data quality objectives were indicated in the method performance section of ETS8-5.1 , Analysis of Potassium Perfluorooctanesulfonate or Other Fluorochemicals in Serum Extracts Using HPLC-Electrospray/Mass spectrometry and ETS-8-7.0, Analysis of Potassium Perfluorooctanesulfonateor Other Fluorochemicals in Liver Extracts Using HPLC-Electrospray/Mass Spectrometry: Linearity: The coefficient of determination (r2)equal to or greater than 0.980. Limits of Quantitation (LOQ): The LOQ is equal to the lowest acceptable standard in the calibration curve. Acceptable Precision: Precision is better than 30% for the method. Acceptable Spike Recoveries: 70-1 30%. 3M Environmental Laboratory 3M Environmental Laboratory Page 74 Page 14 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 3M Medical Department Study: T-6316.1 Data Summary, Analyses, and Results Analytical Report: FACT TOX-003 LRN-U2104 Data quality objectives for the analytical phase of this study outlined in the 3M Environmental Laboratory protocol for FACT TOX-003 (see Appendix B) were met with the exceptions noted in this report. Summary of Quality Control Analyses Results Linearity: The coefficient of determination (r2)of the standard curve was 20.980. Calibration Standards: Quantitation of the target analytes was based on linear regression analysis weighted l / x of a single, opening or two bracketing extracted matrix curves for each group of samples. Rat and rabbit sera were used for matrix curves in sera analyses, while rabbit liver was used for matrix curves in liver analyses. High or low points on the curve may have been deactivated to provide a better linear fit over the curve range most appropriate to the data. Low curve points with peak areas less than two times that of the extraction blanks were deactivated to disqualify a data range that may have been significantly affected by background levels of the analyte. Occasionally, a single mid-range curve point that was an obvious outlier may have been deactivated. Quantitation of each a.nalyte was based on the response of one or more specific product ion(s) using the multiple reaction monitoring mode of the instrument (see Appendix C, Analytical Methods). Calibration standards were prepared to run, undiluted, approximately within the linear range of the instrument (approximately 51000 ng/g). Limits of Quantitation (LOQ): The LOQ is equal to the lowest acceptable standard in the calibration curve (defined as a standard within *30% of the theoretical value), and is at least two times the analyte peak area detected in the surrogate matrix bllanks. (See Appendix D). 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 suitable surrogate matrices for method blanks. Precision: Instrumental precision was determined by replicate injections of a single serum extract from a related study (TOX-001). Instrumental precision was determined for PFOS, PFOSAA, and M556; variation was less than 8.5% for all targeted analytes. Matrix Spikes: Sera-Matrix spike samples were prepared from control rat sera along with each batch of sera samples. Samples were spiked between 75-250 ng/mL, levels that approximate the levels detected in Groups 8 and 9 samples, depending upon the analyte. All spikes were prepared to run, undiluted, within a ten-fold limit of the linear range of the calibration curve. For some analytes, samples for some high dose animals may be much higher than the range of these prepared spikes. Sera matrix spike recoveries are presented in tabular form below. 3M Environmental Laboratory 3M Environmental Laboratory Page 15 Page 15 3M Medical Department Study: T-6316.1 3M Medical Department Study: T-6316.1 Table 4. Matrix Spikes-Sera Analytical Report: FACT-TOX-003 LRN-U2104 Analytical Report: FACT TOX-003 LRN-U2104 unavailable and had to be synthesized. No M556 data is providedfor the Week 4 through Week 14. Liver-Matrix spike samples were prepared from a control group rat liver specimen along with each batch of liver samples. Samples were spiked between 100-500 ng/g, levels that approximate the levels detected in Groups 8 and 9 samples, depending on the analyte. All spikes were prepared to run, undiluted, within a ten-fold limit of the linear range of the calibration curve. For some analytes, samples for some high dose animals may b13 much higher than the range of these prepared spikes. Liver matrix spike recoveries are presented in tabular form below. Table 5. Matrix Spikes-Liver # of Spikes Prepared Average Spike Recovery 94% 65-1 37% 23% PFOSAA 12 96% 53-200% I 39% M556* 6 EtFOSE-OH 4 PFOSEA 6 ~~ 103% 118% 108% 77-1 54% 69-144% 3 39%) 27%) 'M556 was identified as a metabolite of EtFOSE-OHafter initiationof the analytical work. Standard material was unavailable and had to be synthesized. No M556 data or QC is provided for the Week 4 through Week 14 samples. 0 Surrogates: The surrogate (THPFOS) was added to all samples and standards, with the exception of Week 4 liver and sera samples. THPFOS was not used for quantitation, but was used to monitor for gross (*50%) instrument failure. 3M Environmental Laboratory 3M Environmental Laboratory Page 16 Page 16 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 3M Medical Department Study: T-6316.1 Analytical Report: FACT TOX-003 LRN-U2104 Statement of Data Quality It is not possible to verify true recovery of endogenous analyte from tissues without radio-labeled reference material. The only measurement of accuracy available at this time, matrix spike studies, indicate that the sera and liver data can be considered to be 'accurate to within one standard deviation of the average fortified samples recovery. Refer to Tables 4 and 5 for matrix spikes from each analyte and matrix. The results of quality control analyses (curve fit, CCVs, and MS/MSDs) for EtFOSE-OH and PFOSEA were inconsistent and indicate that data presented for this analyte should be considered to be qualitative only. Values for these analytes are presented in data tables in the spirit of full disclosure, but should not be used in any quantitative assessment of the data. Summary of Sample Results PFOS results (those obtained using lot # 171 and #193) and EtFOSE-OH results (those obtained with lot # SDO13) have been corrected for purity of the analytical reference material. Uncorrected results are noted in the data tables. 0 Samples from Control Animals: The target analytes were often detected in the sera and liver samples from the control animals. These levels were usually lower than those found in the low dose test animals. 0 Samples from Dosed Animals: In general, target analytes were present in the sera and liver samples in the dose group. Detailed sample data tables are presented in Appendices D and E. 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 FACT TOX-003. Statement of Conclusion Under the conditions of the present studies, the concentrations of PFOS, PFOSA, PFOSAA, EtFOSE-OH, M556, and PFOSEA were determined in the sera and liver of rats dosed with NEthyl Perfluoroctanesulfonamido Ethanol during the in-life phase of the study. References Covance Study No.: 6329-228 titled: 104-Week Dietary Carcinogenicity Study with Narrow Range (98.1Yo)N-Ethyl PerfluorooctanesulfonamidoEthanol in Rats 3M Environmental Laboratory 3M Environmental Laboratory Page 17 Page 17 3M Medical Department Study: T-6316.1 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 Analytical Report: FACT TOX-003 LRN-U2104 Appendix A: Chemical Characterization and Control Matrices Table 6. Characterizationof the Control Matrices Used for Sera Analyses in Study FACT TOX-003 Table 7. Characterizationof the Control Matrices Used for Liver Analyws in Stud!IFACT TOX-003 I I Rabbit Liver I Rabbit Liver I RabbitLiver Table 8. Characterization of Test Article in Study FACT TOX-003 ~ Test Article 1 Chemical Name II Source EtFOSE-OH N-Ethyl Perfluorooctanesulfonamidoethanol T-6316 I Expiration Date 11/26/01 Storage Conditions Ambient temperature Chemical Lot # (30035,30037,30039) Physical Description Purity Light yellow, waxy soliMlakes 97.4% - 3M Environmental Laboratory 3M Environmental Laboratory Page 18 Page 18 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 3M Medical Department Study: T-6316.1 Analytical Report: FACT TOX-003 LRN-U2104 Appendix B: Protocol, Amendments and Deviations 3M Environmental Laboratory 3M Environmental Laboratory Page 19 Page 19 3M Medical Department Study: T-6316.1 .. .' .. THE DEVELOPMENT SERVICES COMPANY Analytical Report: FACT-TOX-003 LRN-U2104 Sponsor: 3M St. Paul, Minnesota PROTOCOL - - ::. --I~ . * j~ = - * Study Title: 104-Week Dietary Carcinogenicity Study with Narrow Range (98.1%) N-Ethyl Perfluorooctanesulfonamido Ethanol in Rats If Date: April 1,1998 Performing Laboratory: Covance Laboratories Inc. 3301 Kinsman Boulevard Madison,Wisconsin 53704-2595 Laboratory Study Identification: Proposal No. 23 124A Covance 6329-228 $31947 - 6 3 i b . I 3M Environmental Laboratory Page 20 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 Covance 6329-228 Study 104-Week Dietary Carcinogenicity Study with Narrow Range (98.1%) N-Ethyl PerfluorooctanesulfonamidoEthanol in Rats Purpose To assess the carcinogenicity of the test material when administered in the diet to rats for at least 104 weeks Sponsor 3M Toxicology Services Building 220-2E-O2,3M Center St. Paul, Minnesota 55144-1000 Study Monitor Andrew M. Seacat, PhD 3M Telephone No.: 612.575.3161 Facsimile No.: 612.733.1773 Study Location Covance Laboratories Inc. 3301 Kinsman Boulevard Madison, Wisconsin 53704-2595 Mailing Address: PO Box 7545 Madison, Wisconsin 53707-7545 Study Director Peter J. Thomford, PhD Covance Laboratories Inc. Telephone No.: 608.241.7207 Facsimile No.: 608.242.2736 3M Environmental Laboratory . Page 21 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 Covance 6329-228 Toxicologist Thomas E. Ryan, BS Covance Laboratories Inc. Proposed Study Timetable In-Life Start Date: A p d 6 , 1998 In-Life End Date: April 5,2000 Regulatory Compliance This study will be conducted in compliance with the Food and Drug Administration Good Laboratory Practice Regulations as set forth in Title 21 of the US Code of Federal Regulations, Part 58, issued December 22, 1978 (effective June 20, 1979), and with any applicable amendments. Animal Care and Use Statement All procedures in this protocol are in compliance with the Animal Welfare Act I Regulations, 9 CFR 1-4. In the opinion of the Sponsor and study director, the study does not unnecessarily duplicate any previous work. Quality Assurance The protocol, study conduct, and final report will be audited by the Covance Quality Assurance Unit (QAU). The proliferation cell nuclear antigen evaluation, data, and report will be audited by the QAU of Pathology Associates International Test Material Identification T-6316 (Narrow Range N-Ethyl Perfluorooctanesulfonamido Ethanol, NEtFOSE) Lot Numbers The lot numbers will be maintained in the raw data. Purity 98.1% NEtFOSE (w/w) 3M Environmental Laboratory Page 22 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 Stability Responsibility of the Sponsor Covance 6329-228 Page 4 Storage Condilions At room temperature Charactenstics Informationon synthesis methods, composition, or other characteristics that define the test material is on file with the Sponsor. Reserve (Archive) Samples A reserve sample (approximately5 g) will be taken and stored at room temperature. This sample will be transferred to the Sponsor after completion of the in-life phase to be retained in accordance with 21 CFR 58.195. Disposition of Test Material I' After authorization from the Sponsor, any remaining test matt:rial will be returned to: Andrew M. Seacat, PhD 3M Toxicology Services Building 220-28-02,3M Center St. Paul, Minnesota 55144-1000 Telephone No.: 6 12.575.3161 Facsimile No.: 612.733.1733 Animals Species Rat Strain Crl:CD@'(SD)IGS BR Source Charles River Laboratories, Inc.. Raleigh, North Carolina 3M Environmental Laboratory Page 23 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 Age at Initiation of Treatment Preferably 6 weeks of age, but not more than 8 weeks of age Covance 6329-228 Page 5 Weight at Initiation of Treatment 100 to 300 g Number and Sex 140 males and 140 females Identification Implantable microchip identification device Husbandry Housing Individual (may be group-housed during acclimation) a Diet Certified Rodent Diet #5002,meal, (PMI Nutrition Internaiional) ad libitum, unless otherwise specified. The diet is routinely analyzed by the manufacturer for nutritional components and environmental contaminants. Results of specified nutrient and contaminant analyses are on file at Covance-Madison. Water Ad libitum. Samples of the water are routinely analyzed for specified microorganisms and environmental contaminants. The results are on file at Covance-Madison. Contaminants There are no known contaminants in the diet or water at levlAs that might interfere with this study. Environment Environmental controls for the animal room will be set to maintain 18 to 26"C, a reIative humidity of 30 to 70%, a 12-hour lightIl2-hour dark cycle, and a minimum of 10 room air changeshour. The lighddark cycle may be interrupted to accommodate study-related activities. 3M Environmental Laboratory Page 24 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 Acclimation At least 1 week Covance 6329-228 Page 6 Randomization Selection of animalsfor the study will be based on body weights, clinical observations, and other data as appropriate. Animals will be assigned to treatment groups using a computerized blocking procedure designed to achieve body weight balance with respect to treatment groups. At the time of randomizatioix, the weight variation of the animals of each sex used will not exceed & standard deviations of the mean weight, and the mean body weight for each group of each sex will not be statistically different at the 5.0% probability level. Justification Rats historically have been used in safety evaluation studies and are recommended by appropriate regulatory agencies. I' Group Designations and Dietary Levels Group' 8 (Contr01)C.~~ 9 (Treated)@ Number of Animals Male Female 70 70 70 70 Dietary Levels (ppm NEtFOSE)b .o 1 a Groups wdl be designated as Groups 8 and 9. This study will be located in the same animal room as Covance 6329-212. b T-6316 is 98.1% n-ethyl perfluorooctanesulfonamidoethanol (NEtFOSE); dose levels are expressed as ppm of NEtFOSE. c The control animals will receive the basal diet only. d Five animals/sex/group will be sacrificed during Weeks 4 arid 14 for hepatocellular proliferation rate measurementsand biochemical analyses (palmitoyl-CoA oxidation). e Ten animalslsedgroup will be designated as interim sacrificl=animals and will be sacrificed after at least 78 weeks of treatment. Dosing Procedures Method of Administration Dietary. Animals will receive test diet for at least 104 weeks. 3M Environmental Laboratory Page 25 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 Reason for Dosing Route The potential human exposure is by the oral route. Covance 6329-228 Page 7 Dose Preparation Dose preparations will be mixed at least once every 4 weeks according to the study-specific mixing procedure developed by Covance. Dose concentrations will be based on the NEtFOSE content as supplied; the Sponsor has stated the T-6316 is 98.1% NEtFOSE (w/w) as supplied. All dose preparations will be stored at room temperature. Retention Samples Samples (approximately 100 g) will be taken from each dose preparation and stored at room temperature. Unless used for analyses, these samples will be discarded at least 1 month after completion of the in-life phase. Dose Analyses J By Covance using a method supplied by the Sponsor and validated by Covance Homogeneity Homogeneity will be determined for the l-ppm diet preparation mixed for Week 1. One sample (approximately 100 g) each from the top, middle, and bottom of the diet preparation will be collected, divided into three subsamples for extraction and analysis, and analyzed for test material content. AD samples will be stored at room temperature until analyzed within 7 days of mixing. Homogeneity analy:iis will be repeated if the batch size changes by more than 30%. Stability The middle homogeneity sample will be analyzed on the day of mixing and serve as stabdity time 0. Two additional samples (approximately 100 g each) will be taken from the 1-ppm diet preparation mixed for Week 1 to establish stability. One sample will be stored at room temperature for at least 19 days, then analyzed. The other sample will be stored at room temperature after at least 32 days, then analyzed. 3M Environmental Laboratory Page 26 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 Covance 6329-228 Page 8 Dose Confirmation Samples (approximately 100 g each) will be collected from all dose preparations and analyzed in duplicate. Homogeneity samples collected from the middle of the dose preparation for Week 1 will be used for dose confirmatioiiresults. All samples will be stored at room temperature until analyzed. Observation of Animals Clinical Observations Each animal will be observed twice daily (a.m. and p.m.) for mortality and moribundity, frndings will be recorded as they are observed. Once prior to treatment and weekly thereafter, each animal will be removed from its cage and examined; abnormal findings or an indication of normal will be recorded. The following information on each grossly visible or palpalblemass will be recorded. 41 time of onset location size (small or large) appearance progression Body Weights Prior to treatment (at randomization), weekly for Weeks 1 through 17, once every 4 weeks thereafter, and at Week 105 Food Consumption Weekly for Weeks 1 through 16 and once every 4 weeks thereafter Clinical Pathology Frequency and Number of Animals Unscheduled Collection When possible, a blood f h will be made and held for possible fkture examination from animals sacnficed at unscheduled intervals. 3M Environmental Laboratory Page 27 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 Covance 6329-228 Page 9 Scheduled Collections Hematology, clinical chemistry, urinalysis, and urine chemistry will be done on 10 animakdsexlgroup during Weeks 14,27, and 53. A blood film will be made and held for possible future examination from animals at scheduled sacrifices after at least 78 and 104 weeks of treatment. Method of Collection Hematology, Clinical Chemistry, Urinalyses, and Urine Chemistry Animals will be fasted overnight; blood will be collected from a jugular vein. The anticoagulant will be potassium EDTA for hematology tests. Samples for clinical chemistry will be collected without anticoagulant. Urine will be collected chilled overnight (approximately 16 hours). Blood Films I' Blood films will be taken as part of the necropsy proced'ure. Tests Hematology red blood cell (erythrocyte) count hemoglobin hematocrit mean corpuscular volume mean corpuscular hemoglobin mean corpuscular hemoglobin concentration platelet count white blood cell (leukocyte) count differential blood cell count blood cell imorphology reticulocytic smear (made,but not examined) glucose urea nitrogen creatinine total protein albumin glob u h cholesterol total bilirubin Clinical Chemistry alanine aminotransferase gamma gluitamyltransferase aspartate aminotransferase calcium inorganic phosphorus sodium potassium chloride 3M Environmental Laboratory Page 28 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 Covance 6329-228 appearance volume specific gravity PH protein urobilinogen sodium potassium Urinalysis glucose ketones bilirubin blood microsclapic examination of sediment Urine Chemistry 16 hour excretion of: sodium potassium Serum Analyses Frequency and Number of Animals C f Five animals/sex/group during Weeks 4 and 14 (animals selected for hepatocellular proliferation and biochemical analyses), five animWse.dgroup (from animals selected for interim sacrifice) after at least 78 weeks of treatment,.and five animals/sex/group at the terminal sacrifice Method of Collection Animals will be fasted overnight; blood (approximately2 mL) will be collected from a jugular vein. Samples will be collected without itnticoagulant. Sample Handling Blood samples will be allowed to clot at room temperature and centfiged. Serum samples will be harvested and stored in a freezer set to maintain -60 to -80C. Samples will be packed on dry ice and shipped to: Kris J. Hansen, PhD 3M Environmental Technology and Safety Services 935 Bush Avenue Building 2-3E-09 St. Paul, Minnesota 55133-3331 Telephone No.: 612.778.6018 Facsimile No.: 612.778.6176 3M Environmental Laboratory Page 29 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 Covance 6329-228 Samples will be retained by the Sponsor for possible .Future analysis. Termination Unscheduled Sacrifices and Deaths Necropsies will be done. Animals to be sacrificed wiU be anesthetized with sodium pentobarbital, weighed, and exsanguinated. A blood film will be taken as part of the necropsy procedure for sacrificed animals. Scheduled Sacrifices Interim Sacrifices During Week 4, five animals/sexlgroup will be fasted overnight, bled for serum samples, anesthetized with sodium pentobarbital, weighed, and exsanguinated. The abdominal cavity of each animal will be opened, the liver will be removed and weighed, and liver samples will be collected. Animals will be discarded after liver collection. During Week 14, five animalslsedgroup will be fasted overnight, bled for serum samples, anesthetized with sodium pentobarbital, weighed, and exsanguinated. The abdominalcavity of each animal will be opened, the liver will'be removed, weighed, and liver samples collected. Animals will be discarded after liver collection. After at least 78 weeks of treatment, 10 animals/sex/group will be fasted overnight, bled for serum samples (five animals/sex/group), anesthetized with sodium pentobarbital, weighed, exsanguinated,and necropsied. A blood filmwill be taken as part of the necropsy procedure. Terminal Sacrifice After at least 104 weeks of treatment, the remaining anirnals w d be fasted overnight, bled for serum samples (five animals/sex/group), anesthetized with sodium pentobarbital, weighed, exsanguinated,and necropsied. A blood film will be taken as part of the necropsy procedure. 3M Environmental Laboratory Page 30 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 Postmortem Procedures Covance 6329-228 Page 12 Necropsy The necropsy will include an examination of the external features of the carcass; all external body orifices; the abdominal, thoracic, and cranial cavities; organs; and tissues. Cell Proliferation Tissue Collection and Immunohistochemical Evaluation At the Week 4 and 14 interim sacrifices, representative samples of the left lateral lobe of the liver and any macroscopic lesions of the liver will be collected and preserved in zinc formalin. After fixation, each sample of liver will be embedded in paraffin, and the paraffin blocks will be shipped to: Sandra R. Eldridge, PhD II Pathology Associates International 15 Worman's Mill Court, Suite I Frederick, Maryland 21701 Telephone No.: 301.663.1644, ext. 2201 Facsimile No: 301.663.8994 Proliferation cell nuclear antigen (PCNA) evaluation will be done on the samples. In addition, liver sections will be stained with hematoxylin and eosin and examined microscopically. Results will be provided for inclusion in the final report. Palmitoyl-CoA Oxidase Tissue Collection and Analyses At the Week 4 and 14 interim sacrifices,a sample (approximately500 mg) of the right lateral lobe of the liver will also be collected from each animizl and flash-frozen in liquid nitrogen. The liver tissue will be stored in a freezer set to maintain -60 to -80C until analyzed by Covance for palmitoyl-CoA oxidase activity. Organ Weights At the Week 79 and terminal sacrifices, the following organs (when present) will be weighed; paired organs will be weighed separately: 3M Environmental Laboratory Page 31 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 Covance 6329-228 Page 13 adrenal (2) brain kidney (2) liver lung O V U Y (2) spleen testis (2) thyroid (2:) with parathyroid uterus with cervix Organ-to-body weight percentages and organ-to-brain weight ratios will be calculated. Bone Marrow Smear From the femur of each animal at the Week 79 and terminal sacrifices only; made but not examined Additional Liver Sample Collection A portion of the liver will be collected from five animalslse:xlgroupat the interim and terminal sacrifices and stored in a freezer set to maintain -60 to -80C. Samples will be packed on dry ice and shipped to Kris J. Hansen, PhD, 3M Environmental 8 Technology and Safety Services. Samples will be retained by the Sponsor for possible future analysis. Tissue Preservation The following tissues (when present) from each animalwill be presemed in 10%neutral-buffered formalin: adrenal (2) brain cecum cervix colon duodenum epididymis (2) esophagus eye (2) femur with bone marrow (articular surface of the distal end) Harderian gland heart ileum jejunum kidney (2) lesions liver lung with mainstem bronchi lymph node (mesenteric) mammary gland (females only) ovary (2) pancreas pituitary prostate rectum sahvary gland [mandibular (2)] sciatic nerve seminal vesick: (2) skeletal muscle (thigh) 3M Environmental Laboratory Page 32 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 Covance 6329-228 Page 14 Skin spinal cord (cervical, thoracic, and lumbar) spleen sternum with bone marrow stomach testis (2) thymus thyroid (2) with parathyroid trachea urinary bladder uterus vagina Histopathology Tissues will be held for possible future examination. Reports One copy of each draft report will be sent to the Sponsor. The report for this study will be included as an appendix to the report for Covance 6329-212. The report will include the following information: Experimental Design and Methods r(1 Results dose analyses mortality clinical observations body weights body weight changes food consumption test material consumption clinical pathology results paltnitoyl CoA oxidase activities macroscopic observations cell proliferation assessments (provided by the Sponsor's designee) Statistical Evaluation body weights body weight changes food consumption survival rates clinical pathology values palmitoyl CoA oxidase activities Statistical methods will be those presented in Attachments Nos. 1 and 2. For each sex, Group 9 will be compared to Group 8 (Control). 3M Environmental Laboratory Page 33 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 Covance 6329-228 At the end of 1 year after issuance of the audited draft report, if no requested revisions or instructions to fmahze have been communicatedby the Sponsor, then the audited draft report will be considered 'fmal' and issued as the final report, .signedby the study director, and submitted to the Sponsor. Any modifications or changes to the audited draft report requested 1 year after issuance will be performed at additional cost to the Sponsor. Record Retention All raw data, documentation, records, protocol, specimens, and final report generated as a result of this study, including those items listed below, will be ,archivedin the storage facilities of Covance-Madison for a period of 1 year following submission of the final report to the Sponsor. All raw data stored on magnetic media, the protocol and protocol amendments, study correspondence, and the original report will be retained by Covance. One year after submissionof the final report, allof the aforementionedmaterials will be sent to the Sponsor, and a return fee will be charged. The Sponsor may elect to have the d materials retained in the Covance archives for an additional period of time, and Covance will charge a storage fee. If the Sponsor chooses to have Covance dispose of the materials, a disposal fee will be charged. protocol and protocol amendments dose preparation records in-life records animal receipt acclimation animal room maintenance randomizations dose administration clinical observations body weights food consumption sample collection clinical pathology records anatomical pathology records statistical analyses study correspondence tissue specimens (wet) blood and bone marrow slides final report (original signed copy) 3M Environmental Laboratory Page 34 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 Covance 6329-228 Page 16 The following supporting records will be retained at Covancc-Madison but will not be archived with the study data. feed analysis records water analysis records animal room environment records refrigerator and freezer temperature records room temperature records for test material storage instrument calibration and maintenance records PCNA evaluation data and paraffin blocks and tissue slides will be retained by Pathology Associates International. Serum and liver samples sent to the Sponsor will be retained by the Sponsor 3M Environmental Laboratory Page 35 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 PROTOCOL APPROVAL Covance 6329-228 Page 17 f j - Andrew M.Seacat, PhD Date Study Monitor 3M Covance Laboratories Inc. 3M Environmental Laboratory Page 36 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 Covance 6329-228 Page 18 Attachment No. 1 Statistical Analyses Levene's test will be done to test for variance homogeneity. In the case of heterogeneity of variance at p s 0.05, transformationswdl be used to stabilize the variance. Comparison tests will take variance heterogeneity into consideration. One-way analysis of variance (ANOVA) will be used (if applicable) to analyze body weights, body weight changes, food consumption, continuous clinical pathology values, and organ weight data. If the ANOVA is significant, Student':; t-test will be used for control versus treated group comparisons. If the ANOVA shows significancefor body weights at Week 1, one-way analysis of covariance (ANCOVA) will be used to analyze body weights, with initial body weights as the covariate. If the ANCOVA is significant, covariate-adjustedmeans will be used for control versus treated group comparisons. # Group comparisons (Group 9 versus Group 8) will be evaluated at the 5.0% two-tailed probability level. Only data collected on or after the first day of treatment'will be analyzed statisticaIly. 3M Environmental Laboratory Page 37 3M Medical Department Study: T-6316.1 .. . . , . Analytical Report: FACT-TOX-003 LRN-U2104 Attachment No. 2 Covance 6329-228 Page 19 Adjusted survival data are analyzed by the National Cancer Institute (NCI) lifetable package. The tests include: Graphical (Kaplan-Meier product-limit estimation curves), Cox-Tarone binary regression methods for trend and heterogeneity, and Gehan-Breslow nonparametric methods for trend and heterogeneity. t 3M Environmental Laboratory Page 38 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 W E D M L O P M E H T SERWCCI COMPAWV PROTOCOL AMENDMENT NO,.1 Covance 6329-228 104-Week Dietary Carcinogenicity Study with Narrow R,ange(98.1%) N-Ethyl Perfluorooctanesulfonamido Ethanol in Rats Sponsor: Study Monitor: Testing Facility: Study Director: 3M, St. Paul, Minnesota Andrew M. Seacat, PhD Covance Laboratories Inc., Madison, Wisc:onsin Peter J. Thomford, PhD This amendment modifies the following portions of the protocol: rcf Effective April 23,1998 1. Page 11, Termination. To reflect the decision to anesth.etizeanimalsto be sacrificed with carbon dioxide, delete the text in this section and replace with the following: Unscheduled Sacrifices and Deaths Necropsies will be done. Animals to be sacrificed will be anesthetized with carbon dioxide, weighed, and exsanguinated. A blood film will be taken as part of the necropsy procedure for sacSiced animals. 3M Environmental Laboratory Page 39 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 Scheduled Sacrifices Covance 6329-228 Protocol Amendment No.1 Page 2 Interim Sacrifices During Week 4, five animals/sex/group will be fasted Overnight, bled for senun samples, anesthetized with carbon dioxide, weighed, and exsanguinated. The abdominal cavity of each animal will be opened, the: liver vvlll be removed and weighed, and liver samples will be collected. Animals will be discarded after liver collection During Week 14, five animals/sex/group will be fasled overnight, bled for serum samples, anesthetized with carbon dioxide, weighed, and exsanguinated. The abdominal cavity of each animal will be opened, the liver will be removed and weighed, and liver samples will be collected. A~zimalswill be discarded after liver collection. i After at least 78 weeks of treatment, 10 animals/sex/'groupwill be fasted overnight, bled for serum samples (five animals/sex/group), anesthetized with carbon dioxide, weighed, exsanguinated,and necropsied. A blood film win be taken as part of the necropsy procedure. Terminal Sacridice After at least 104 weeks of treatment, the remaining i b a l s will-befasted overnight, bled for serum samples (five animaldsexlgroup), anesthetized with carbon dioxide, weighed, exsanguinated, and necropsied. A blood f j h win be taken as part of the necropsy procedure. 2 Page 12, Postmortem Procedures, Cell Proliferation Tissue Collection and Immunohistochemical Evaluation, Paragraph 1. To reflect the decision to collect liver samples for proliferation cell nuclear antigen evaluation at Week 14 only, delete the text in this paragraph and replace with the following: At the Week 14 interim sacrfice, representativesamples of the left lateral lobe of the liver and any macroscopic lesions of the liver will be collected and preserved in zinc formalin. 3M Environmental Laboratory Page 40 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 Effective April 28,1998 Covance 6329-228 Protocol Amendment No. 1 Page 3 3. Page 2, Alternate Study Monitor. To include the alternate study monitor in the protocol, add the following after "Study Monitor:" Alternate Study Monitor Marvin T. Case, DVM, PhD 3M Toxicology Services Telephone No.: 612.733.5180 Facsimile No.: 612.733.1773 4. Page 6, Group Designations and Dietary Levels, Folotnote e. To reflect the decision to change the Week 79 interim sacrifice to Week 53,delete the text in this footnote and replace with the following: Ten animalslsedgroup will be designated as interim sacrifice animals and will be dl sacrificed after at least 52 weeks of treatment. 5. Page 9, Clinical Pathology, Frequency and Number of Animals, Scheduled Collections, Paragraph 2. The Week 79 interim sacrifice will be moved to Week 53, therefore blood films will be prepared from arlimals sacrificed after 52 weeks of treatment. To reflect this decision, delete t.hetext in this paragraph and replace with the following: A blood film will be made and held for possible future e;r:aminationfrom a,nimahat scheduled sacrifices after at least 52 and 104weeks of treatment. 6. Page 10, Serum Analyses, Frequency and Number of Animals. The Week 79 interim sacrifice will be moved to Week 53,therefore serum samples will be collected from animals sacrificed after 52 weeks of treatment. To reflect this decision, delete the text in this section and replace with the following: 3M Environmental Laboratory Page 41 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 Covance 6329-228 Protocol Amendment No. 1 7. 8. 1f 9. Five animalslsexlgroupduring Weeks 4 and 14 (animals selected for hepatocellular proliferation and biochemical analyses), five Wals/se:x/group (from animals selected for interim sacrifice) after at least 52 weeks of treatment, and five animalslsexlgroup at the terminal sacrifice Page 11, Termination, Scheduled Sacrifices, Interinn Sacrifices, Paragraph 3. To reflect the decision to change the Week 79 interim sacrifice to Week 53,delete the text in this paragraph and replace with the following: After at least 52 weeks of treatment, 10 animals/sex/group will be fasted overnight, bled for serum samples (five an@aIdsex/group), anesthetized with carbon dioxide, weighed, exsanguinated, and necropsied. A blood Nm will be taken as part of the necropsy procedure. Page 12, Postmortem Procedures, Organ Weights. To reflect the decision to record organ weights at the Week 53 interim sacrifice only, delete the text m this section and replace with the following: At the Week 53 interim sacrifice, the following organs (when present) will be weighed; paired organs will be weighed separately: adrenal (2) brain kidney (2) liver lung ovary (2) spleen testis (2) thyroid (2) with parathyroid uterus with cervix Organ-to-body weight percentages and organ-to-brain weight ratios will be calculated. Page 13, Postmortem Procedures, Bone Marrow Smear. The Week 79 interim sacrifice will be moved to Week 53,therefore bone marro'w smears will be collected from animalssacrifked after 52 weeks of treatment. To reflect this decision, delete the text in this section and replace with the following: 3M Environmental Laboratory Page 42 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 Covance 6329-228 Protocol Amendment No. 1 Page 5 From the femur of each animal at the Week 53 and terminal sacrifices only; made but not examined 10. Page 14, Reports, Paragraph 1. To reflect the decision to provide unaudited summary reports after the Week 14 and 53 interim sac:nfices, delete the text in this paragraph and replace with the following: After the Week 14 and 53 interim sacrifices, unaudited summary reports will be sent to the Sponsor in conjunction with the summary reports for Covance 6329-212. The summary reports win include a brief description of methods and results and summary tables of in-life data, clinical pathology data, and anatomical pathology data. After completion of the study, one copy of each draft report will be sent to the Sponsor. The report for this study will be included as an appendix to the report for Covance 6329-212. The report will include the following information: d Effective May 15,1998 11. Page lo, Serum Analyses, Method of Collection. To reflect the decision to increase the volume collected for serum analyses at the Week 53 and 105 sacrifices, delete the text in this section and replace with following: Animals will be fasted overnight; blood (approximately 2 mL at the Week 4 and 14 sacrifces, approximately3 mL at the Week 53 and 105 sacrifices) will be c,ollected from a jugular vein. Samples Win be coUected without anticoagulant. 3M Environmental Laboratory Page 43 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 AMENDMENT APPROVAL Covance 6329-228 Protocol Amendment No.I p&@&J drew M. Seacat, PhD Study Monitor 3M - ?&?/k7 Date Covance Laboratories Inc. 3M Environmental Laboratory Page 44 3M Medical Department Study: T-6316.1 ... Analytical Report: FACT-TOX-003 LRN-U2104 covm PROTOCOL AMENDMENT NO. 2 COV~IIC63C29-228 1WWeek Dietary CarcinogenicityStudy with Narrow Range (98.1%) N-Ethyl Perfhorooctanesullbnamido Ethanol m Rats Sponsor: Study Monitor: Testing Facility: Study Director: 3M,St. Paul, Minnesota Andrew M. Seacat, PhD Covance Laboratories Inc., Madison, Wiscorsin Peter J: Thomford, PhD This amendment modifies the following portions of the protocol: Effective April 1,1998 1. Page 4. To mclude the vehicle used to dissolve the test mamial befork mixing . with the diet, add the following section. Vehicle IdentilScahion Acetone Lot Numbers The lot numbers will be maintained in the raw data. mts On file with the manufacturer Stability On file with the manufacturer 3M Environmental Laboratory Page 45 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 Storage Conditions At room temperature characteristics Informationon synthesismethods, composition, or other characteristics that define the vehicle is on file with the manufac:turer. Effective June 30,1998 2. Page 7, Dosing Procedure, Retention Samples. To r e k c t the clzcision to send retention samples of control diet to the Sponsor, delete tlle text m thissection and replace with the following: Samples (approximately 100 g) Win be taken from each dose preparation during the in-Me phase and stored at room temperature. Unless used for analyses, retention samples from the treated groups will be discarded at least 1month after. completion of the in-We phase. Samplesfrom control diels prepared for Weeks 14 and before will be shipped to: / Kris J. Hansen, PbD 3M EnvironmentalTechnology and Safety Services 935 Bush Avenue Building 2-313-09 St. Paul, Minnesota 55133-3331 Telephone No.: 651.778.6018 Facsimile No.: 651.778.6176 EBFecb've July 20,1998 3. Page2,Study Monitor and Alternate Study Monitor. To indicate the change m the area code, delete the text m these sections and replace with the following: Study Monitor Andrew M. Seacat, PhD 3M Telephone No.: 651.575.3161 Facsimile No.: 651.733.1773 3M Environmental Laboratory Page 46 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 Covance 6329228 Protocol Amendment No.2 Page 3 Alternate Study Monitor Marvin T. Case,DVM, PhD 3M Toxicology Services Telephone No.: 651.733.5180 Facsimile No.: 651.733.1773 4. Page 4, Disposition of Test Material. To indicate the change in the area code, delete thc text m these section and replace with the followiag: Aftcr authorization from the Sponsor, any remaining test material will be returned to: Andrew M. Seacat, PhD 3M Toxicology Services Building 220-2E-O2,3MCenter i . St. Paul, Minnesota 55144-1000 Telephone No.: 651575.3161 Facsimile No.: 651.733.1733 5. Page 10, SerumAnalyses, Sample Handiing. To indicate the change in the area code for the Sponsor, delete tbe text in these section and replace with the mowing: BIood sampbs Win be allowed to clot at room temperature itnd centrifuged. Serum samples willbe harvested and stored in a freezer set l:omaintain -60to -80 C. Sampleswill be packed on dry ice and shipped to: Kris J. Hansen, PhD 3M EnvironmentalTechnology and Safety Services 935 Bush Avenue Building 2-3E-09 St. Paul, Minnesota 55133-3331 Telephone No.: 651.778.6018 Facsimile No.: 651.778.6176 Samples Win be retained by the Sponsor for possible future analysis. 3M Environmental Laboratory Page 47 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 Covance 6329228 Protocol Ammdment No.2 Effective April 9,1999 6. Page 9, Clinical Pathology, Frequency and Number of Animals, Schedded Collections,Paragraph 2. Hematology tests will be done for animalssacrificed at Week 53 and blood filmswill not be necessary. Therefcre, delete this paragraph and replace with the following. A blood film will be made and held for possiile futureexaminationfrom animals at scheduled sacdice after at least 104weeks of treatment. 7. Page 11, Termination, Scheduled Sacrifices, Interim Sacrifices, Paragraph 3. To reflect the decision to collect clinical pathology samples fiom animals at the Week 53 interim sacrifice, delete the text in this paragraph and replace with the fonowing: I After at least 52 weeks of treatment, 10 animals/sex/group wiIl be fasted overnight, bled for c h i d pathology tests (4animals) and s e m samples (five animaWsex/group),anesthetized with carbon dioxide, weighed, exsbguinated, - and necropsied. 3M Environmental Laboratory Page 48 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 AMENDMENT APPROVAL C o m a 6329228 Protocol Amendment No. 2 Page 5 Audrew M. Seacat, PhD Study Monitor 3M ! ' ' ..;. .i Covkce Laboratories Inc. Date ' 3M Environmental Laboratory Page 49 3M Medical Department Study: T-6316.1 covm Analytical Report: FACT-TOX-003 LRN-U2104 PROTOCOL AMENDMENT NO.3 Covance 6329-228 104-Week Dietary Carcinogenicity Study with Narrow Range (98.1%) N-Ethyl Perfluorooctanesdfonamido Ethanol in Rats Sponsor: Study Monitor: Testing Facility: Study Director: 3M, St. Paul,Minnesota Andrew M. Seacat, PhD Covance Laboratories Inc., Madison,Wisconsin Peter .J. Thomford, PhD ~ ~- This amendment modifies the following portion of the protocol. .t Effective January 26,2000 1. Page 14, Postmortem Procedures,his to pa tho log^^. The Spqnsor has requested that all tissues fkom animalx in Group 8 and selected tissues from animalsin Group 9 be examined microscopically. To reflectthis decision, delete the text in this section and replace with the following. Tissues (as appropriate) fiom each animal in Group 8 willl be embedded m paraffin, sectioned, stained with hematoxylin and eosin, and examined microscopicaUy. Lesions, liver, lungs, kidneys, pancreas, thyroid, testes, and mammary glands (females) from each animal in Group 9 will be embedded in paraffin, sectioned, stained with hematoxylin and eosin, and examined microscopically. Parathyroids will be processed with the thyroids, but will not be examined. 3M Environmental Laboratory Page 50 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 Covance 6329-228 Protocol Amendment No.3 Page 2 Effective February 18,2000 2. Page 10, Serum Analyses, Frequency and Number of Animals. To reflect the decision to collect serum samples from 10 animalslsexlgroup at termination, delete the text in this section and replace with the following: Five animals/sex/groupduring Weeks 4 and 14 (animails selected for hepatocellular proliferation and biochemical analyses), five animaldsexlgroup (from animals selected for interim sacrifice) after at least 52 weeks of treatment, and 10 animals/sex/group at the terminal sacrifice 3. Page 11, Termination, Scheduled Sacrifices, Terminlal Sacrifice. To reflect the decision to collect senun samples from 10animalslsedgroupat termination, delete the text in this section and replace with the following: After at least 104 weeks of treatment, the remaining animals will be fasted overnight, bled for serum samples (10 animalslsexlgroup), anesthetized with sodium pentobarbital, weighed, exsanguinated, and necropsied. A,blood film will be taken as part of the necropsy procedure. 4. Page 13, Postmortem Procedures,Additional Liver Sample Collection. decision to collect liver samples from 10 animaldsex/group at termination, delete the text in this section and replace with the following: A portion of the liver will be collected from five animals/:sex/groupat the interim sacrifice and 10 animaIs/sex/group at the terminal sacrifice and stored in a freezer set to maintain -60 to -80C.Samples wdl be packed on d r y ice and shpped to Kris J. Hansen, PhD,3M Environmental Technology and Safety Services. Samples will be retained by the Sponsor for possible future analysis. 3M Environmental Laboratory Page 51 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 AMENDMENT APPROVAL, Covance 6329-228 Protocol Amendment No.3 Page 3 r - An3rew M. Seacat, PhD Date Study Monitor 3M Covance Laboratories Inc. 3M Environmental Laboratory Page 52 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 3M Environmental Laboratorv Protocol - Analytical Study Phase 104-Week Dietary Carcinogenicity Study with Narrow Range N-Ethyl PerfluorooctanesulfonamidoEthanol in Rats . In-Vivo Study Reference Number: Covance # 6329-228 @ IAC 3 / 2 7 / 9 9 Study Number: FACT-05-bWH osOSqa.2 Test Material: T-63 16 Name and Address of Sponsor: 3M Toxicology Services Building 220-2B-O2,3M Center St. Paul, MN 55144-1000 Name and Address of Testing Facility: 3M EnvironmentalTechnologyand Services 935 Bush Avenue St. Paul, MN 55106 Proposed Experimental Start Date: May 25,1998 Prdposed Experimental Termination Date: December 30,2001 Method Numbers and Revisions FACT-M-1.0, Extraction of Perfluorooctanesulfonate from Liver for Analysis using HPLC-ElectrosprayMass Spectrometry FACT-M-2.0, Analysis of Liver Extracts for Fluorochemicalsusing HPLC- ElectrosprayLMass Spectrometry FACT-M-3.0, Extraction of Perfluorooctanesulfonate fkom Sera for Analysis using HPLC-ElectrosprayMass Spectrometry FACT-M-4.0, Analysis of Sera Extracts for Fluorochemicalsusing HE'LC- ElectrosprayMass Spectrometry - Author: Lisa Clemen 5)22/ 4 R Kris J. kansen, PhD Date Dale Bacon Date Study Director Study Director Management drew M. Seacat, PhD Date Sponsor Representative 3M Environmental Laboratory Page 1 of 5 Page 53 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 1.0 PURPOSE 1.1 According to this analytical protocol, the 3M Environmental Laboratory will analyze the tissue and fluid samples from the Covance study number 6329-2`28,"104-Week Dietary Carcinogenicity with Narrow Range N-Ethyl Perfluorooctanesul fonamido Ethanol in Rats." The collected data will be provided to the sponsor for use in the assessment of toxicological effects of the test material when administered in the diets of rats for at least 104 weeks. 1.2 Data collected in the Environmental Laboratory will be considered non-quantitative screening data until future studies have been conducted to determine absolute recoveries of specific or general fluorochemical compounds. 2.0 REGULATORY COMPLIANCE 2.1 This analytical phase of the study will be conducted in accordance with the FDA Good Laboratory Practices Regulations 2 1 CFR 58, with the following exceptions: 2.1.1 The analytical phase is being conducted as a separate study and therefore has a separate Study Director, protocol, and fmal report, from those listed in the Covance protocol 6329-228. 2.1.2 The characterizationof the reference material, including purity, identity, and stability, are the responsibility of the sponsor. 2.1.3 Sample storage stability will not be determined. 3.0 , TEST MATERIALS 3.1 Control, and reference Materials and Matrices 3.1.1 Analytical Reference Material: T-63 16, from 3M ICP/I'CP Division 3.1.2 Analytical Reference Matrix: Rat liver, from Covance slnd rat serum, from Sigma Chemical Company. 3.1.3 Analytical Control Material: None. 3.1.4 Analytical Control Matrix: Rat liver, from Covance and rat serum, from Sigma Chemical Company. 3.2 Number of Test and Control Samples: Throughout the course of the study, liver and serum from 140 test animals and 140 control animals will be made available. Samples will be analyzed as requested by the sponsor or the study director. Other biological tissues (kidney, bile, dermal application site, and cellular fraction) will be available for analysis if deemed appropriate. 3.3 Identification of Test and Control Samples: The samples will be identified using the Covance animal identification number that consists of a letter and five-digit number, plus the tissue identity and day identity (serum). 3.4 Purity and Identity of Reference Material: To be determined by Sponsor. 3.5 Stability of Reference Material: To be determined by Sponsor. 3M Environmental Laboratory Page 2 of 5 Page 54 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 3.6 Storage Conditions for Reference Materials: Reference materials will be stored at room temperature (3.1.I), and samples will be stored at -20 f 1OmC (3.1.2, 3.1.4). Test and Control samples will be received according to AMDT-S- 10-0. 3.7 Disposition of Specimens: Biological tissues and fluids will be: retained per GLP Regulation for the time period required for studies longer than 28 days. 3.8 Safety Precautions: Refer to appropriate MSDS. Wear appropriate laboratory attire. Use caution when handling knives for cutting tissue samples. 4.0 EXPERIMENTAL - Overview 4.1 The tissues Erom animals dosed as described (Covance# 6329-22 S), will be available for analysis for fluorine-containingcompounds. At the discretion of the Study Director, a series of analytical tests can be performed. All high dose and control stera and livers will be analyzed initially using HPLC-electrospray mass spectrometry to identify fluorine-containing compounds of interest present in the sera and liver (if any). The screening for organic fluoride in liver via combustion may be performed to present definitive data for fluorine in the liver. Based on the findings from these analyses, additional samples, tissues, or fluids may be analyzed at the discretion of the Study Director to determine the presence of fluorochemicals in these matrices. 5.0 - EXPERIMENTAL Methods 5.1 Methods (attached): 5.1.1 FACT-M-1.0, "Extraction of Perfluorooctanesulfonate fiom Liver for Analysis using HPLC-ElectrosprayMass Spectrometry" 5.1.2 FACT-M-2.0, "Analysis of Liver Extracts for Fluorochemicals using HPLCElectrosprayMass Spectrometry'' 5.1.3 FACT-M3.0, "Extraction of Perfluorooctanesulfonatefrom Sen& for Analysis using HPLC-ElectrosprayMass Spectrometry'' 5.1.4 FACT-M-4.0, "Analysis of Serum Extracts for Fluorochemicals using HPLCElectrospraylMass Spectrometry'' 6.0 DATA ANALYSIS 6.1 Quality Control: Matrix spikes will be extracted and analyzed to determine accuracy of the method. Also, continuing calibration checks will be analyzed to determine response bias. 6.2 Transformations: Any transformations performed on data collected during the analytical phase of the study will be documented in the final report. 6.3 Statistics: At the discretion of the Study Director, statistics used may include regression analysis of serum concentrations with time, averages, and standard deviations of concentrations for the different dose groups. If necessary, simple tests such as the Student's t-test may be applied to determine statistical difference. Any statistical analysis performed will be documented in the final report. 6.4 Data Reporting: A final data package will be submitted to 3M Toxicology Services. The data package will include the following with additional data included as deemed appropriate. 3M Environmental Laboratory Page 3 of 5 Page 55 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 6.4.1 A summary of individual sample results, reported as a concentration (weighvweight, weightholume) of fluoride per tissue or fluid, or as the niass of a specific fluorochemical (HPLC-electrospray mass spectrometry) per unit of tissue or fluid. 6.4.2 A summary of quality control results (continuing calibration checks, method blanks, instrument blanks, matrix spikes, and matrix spike duplicates). 6.4.3 Certified copies or originals of the written validated methods. 6.4.4 Certified copies or originals of sample identification sheets sent fiom Covance. 6.4.5 Certified copies or originals of study specific raw data. 6.4.6 A summary of key personnel involved with the analytical phase of the study. 6.4.7 A signed QAU statement listing the dates of inspections 2nd reports of findings to management and Study Director. 7.0 MAINTENANCE OF RAW DATA AM)RECORDS 7.1 The following raw data and records (or certified copies thereof) vlrill be maintained in the study folder in the archives according to appropriate SOPs. 7.1.1 7.1.2 7.1.3 7.1.4 ' 7.1.5 7.1.6 7.1.7 Approved protocol Approved methods Data summaries Study correspondence Shipping records Raw data Electronic copies of data 7.2 Supporting records to be retained separately fiom the study folder in the archives according to 3M ET & SS SOPs, will include, but not necessarily be limited to the following: 7.2.1 7.2.2 7.2.3 7.2.4 7.2.5 7.2.6 Approved validation reports Training records Calibration records Instrument maintenance logs Standard operating procedures, equipment procedures, and methods Appropriate specimens 8.0 REFERENCES 8.1 Approved AMDT standard operating procedures. 8.2 Approved ETS standard operating procedures. 9.0 ATTACHMENTS 9.1 FACT-M-1.O, Extraction of Perfluorooctanesulfonatefrom Liver for Analysis using HPLCElectrosprayMass Spectrometry 3M Environmental Laboratory Page 4 of 5 Page 56 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 9.2 FACT-M-2.0, Analysis of Liver Extracts for Fluorochemicals using HPLCElectrosprayMass Spectrometry 9.3 FACT-M-3.0, Extraction of Perfluorooctanesulfonate from Senm for Analysis using HPLCElectrosprayNass Spectrometry 9.4 FACT-M-4.0, Analysis of Serum Extracts for Fluorochemicals using HPLCElectrosprayAdass Spectrometry 3M Environmental Laboratory Page 5 of 5 Page 57 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 Study Title 104-Week Dietary Carcinogenicity Study with Narrow Range N-Ethyl Perfluorooctanesulfonamido Ethanol in Rats PROTOCOL AMENDMENT NO. 1 Amendment Date: 20 January 2000 Performing Laboratory 3M Environmental Technology & Safety Services 3M Environmental Laboratory 935 Bush Avenue St. Paul, MN 55106 Laboratory Project Identification ET&SS LRN-U2104 FACT TOX-003 Covance Study: 6329-228 3M Medical Department Study: T-63 16.1 3M Environmental Laboratory 3M Environmental Laboratory Page 58 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 Protocol LRN-U21L0R4N-U2104 Amendment Number 1 This amendment modifies the following portion(s) of the protocol: 1. PROTOCORLEADS: The following methods will be used: FACT-M-1.O,Extraction of Perfluorooctanesulfonate from Liver for Analysis using HPLC-ElectrospraylMass Spectrometry FACT-M-2.0, Analysis of Liver Extracts for Fluorochemicals using HPLCElectrospray/Mass Spectrometry FACT-M-3.0, Extraction of Perfluorooctanesulfonate from Sera for Analysis using HPLC-Electrospray/Mass Spectrometry FACT-M-4.0, Analysis of Sera Extracts for Fluorochemicals using HPLCElectrospray/Mass Spectrometry AMENDTO READ: The following methods will be used: ETS-8-4.1, Extraction of Potassium Perfluorooctanesulfonate or Other Fluorochemical compounds from Serum for Analysis Using HPLCElectrospraylMass Spectrometry ETS-8-5.1, Analysis of Potassium Perfluorooctanesulfonateor Other Fluorochemicals in Serum Extracts Using HPLC-ElectrospraylMass Spectrometry ETS-8-6.0, Extraction of Potassium Perfluorooctanesulfonateor other Fluorochemical Compounds from Liver for Analysis using HPI-C-Electrospraylhlass Spectrometry ETS-8-7.0, Analysis of Potassium Perfluorooctanesulfonateor Other Fluorochemicals in Liver Extracts Using HPLC-Electrospray/F/lassSpectrometry REASON: The methods originally listed were superseded during the course of the study. 2. PROTOCORLEADS: There is no independent section of the protocol that addresses sample retention. AMENDTO READ: Specimens will be maintained in the 3M Environmental Laboratory specimen archives. Any specimens sent to sub-contract laboratories will be returned to the 3M - Environmental Laboratory upon completion of analysis and submission of the subcontract laboratory(s) final report. Specimens analyzed at sub-contract laboratories will be returned with the following documentation: the signed original chain of custody and records of storage conditions while at the sub-contract facility. REASON: To define in detail the appropriate disposition of specimens analyzed at subcontract laboratories. 3M Environmental Laboratory 3M Environmental Laboratory Page 59 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 Protocol LRN42L10R4N-U2104 Amendment Number 1 3. PROTOCORLEADS: Section 7 states that the following raw data and records will be retained in the study folder in the archives according to AMDT-S-8: Approved protocol and amendments; approved methods; data summaries; study correspondence; shipping records; raw data; and electronic copies of data. Additionally, Section 7 states that supporting records to be retained separately from the study folder in the archives according to AMDT-S-8 will include at least the following: Approved validation reports; training records; calibration records; instrument maintenance logs; Standard Operating Procedures, Equipment Procedures, and Methods; and appropriate specimens. AMENDTO READ: Section 7 states: "The original data, or copies thereof, will be available at the 3M Environmental Laboratory to facilitate audits of the study during its progress and before acceptance of the final report. M e n the final report is completed, all original paper data, including: approved protocol and amendments, study correspondence, shipping records, raw data, approved final report, and electronic copies of data will be retained in the archives of the 3M Environmental Laboratory. AI1 corresponding training records, calibration records, instrument maintenance logs, standard operating procedures, equipment procedures, and methods will be retained in the archives of the facility performing each analysis.'' REASON: To direct subcontract laboratories in the disposition of the items listed above. d 4. PROTOCOL READS: The study director for the present study was identified in the protocol as Kristen J. Hansen, Ph.D. AMENDTO READ: The role of study director for the present study was reassigned to John L. Butenhoff, Ph.D., as of 20 January 2000. The previous study director, Kristen Hansen, has been reassigned to the role of Principle Analytical Investigator. REASON: The role of study director was reassigned in an effort to ensure compliance with Good Laboratory Practice Standards that outline study personnel requirements (refer to 21 CFR Part 58). 5. PROTOCOL READS: The sponsor for the present study was identified as Andrew hl. Seacat, Ph.D. AMENDTO READ: The role of sponsor for the present study was reassigned to Marvin T. Case, D.V.M., Ph.D., as of 20 January 2000. REASON: The change was made at the request of the sponsor. 3M Environmental Laboratory 3M Environmental Laboratory Page 60 3M Medical Department Study: T-6316.1 Amendment Approval Analytical Report: FACT-TOX-003 Protocol LRN-U2L10R4N-U2104 Amendment Number 1 msor Representative /Af.& - Xristen J. Hansen, Ph.D., Outgoing Study Director , bate - 11- F-kb zuuu Date / b b L A)+'+,4J Mawin T. Case, D. K M , Ph.D., Incoming Sponsor Representative 5ate i +z- John L. Butenhog Ph.D., Incoming Study Director F&6mw%$ fd, iuwo Date 3M Environmental Laboratory 3M Environmental Laboratory Page 61 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 Study Title 104-Week Dietary Carcinogenicity Study with Narrow Range N-Ethyl PerfluorooctanesulfonamidoEthanol in Rats PROTOCOL AMENDMENT NO. 3 Amendment Date: May 4,2001 Performing Laboratory 3M Environmental Technology & Safety Services 3M Environmental Laboratory 935 Bush Avenue St. Paul, MN 55106 Project Identification 3M Medical Department Study: T-6316.1 Covance In-Life Study: 6329-228 Analytical Report: FACT TOX-003 3M Laboratory Request No. U2104 3M Environmental Laboratory 3M Environmental Laboratory Page 62 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 F'rotocol FACT TOX-003 Amendment No. 3 This amendment modifies the following portion(s) of the protocol: 1. PROTOCOL READS: PURPOSET:he FACT TOX-003 protocol does not clearly identlfythe intended analytes for the study. AMENDTO READ: PURPOSE: Rat sera and liver samples will be analyzed for PFOS, PFOSA, PFOSAA, PFOSEA, EtFOSE-OH and M556. REASON: The target analytes will be clearly specified. 3M Environmental Laboratory 3M Environmental Laboratory Page 63 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 rDrotocol FACT TOX-003 Amendment No. 3 Amendment Approval Lr L Marvin Case, D.M.V ,Ph. D.,Sponsor Representative John Butenhofi Ph.D., StudiDirector Date 3M Environmental Laboratory 3M Environmental Laboratory Page 64 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 Study Title 1WWeek Dietary Carcinogenicity Study with Narrow Range N-Ethyl PerfluorooctanesulfonamidoEthanol in Rat!; PROTOCOL AMENDMENT NO. 2 Amendment Date: January 8,2001 Performing Laboratory 3M Environmental Technology & Safety Services 3M Environmental Laboratory 935 Bush Avenue St. Paul, MN 55106 Laboratory Project Identification ET&SS LRN-U2104 FACT TOX-003 Covance 6329-228 3M Medical Department Study: T-6316.1 3M Environmental Laboratory 3M Environmental Laboratory Page 65 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 Protocol FACT TOX-0L0R3N-U2104 Amendment No. 2 This amendment modifies the following portion@)of the protocol: 1. PROTOCOL READS: 1.2 Data collected in the Environmental Laboratory will be considered non-quantitative screening data until future studies have been conducted to determine absolute recoveries of specific or general fluorochemical compounds. AMENDTO READS 1.2 If matrix spike studies provide accurate representation of recovery of endogenous levels of PFOS, PFOSA, PFOSAA, PFOSEA, M556,and EtFOSE-OH, the 3M .EnvironmentalLaboratory will provide semi-quantitative data for sera and liver samples collected from test animals. REASON: Due to improved analytical methods, the decision was made to change the purpose of the study results from non-quantitative to semi-quantitative. 3M EnvironmentalLaboratory 3M Environmental Laboratory Page 66 3M Medical Department Study: T-6316.1 Amendment Approval Analytical Report: FACT-TOX-003 Protocol FACT TOX-0L0R3N-U2104 Amendment No. 2 V T/& Makin T. Case, D.V.M., Ph.D., Sponsor Representative / J L &/ Date c John L. Butenhofi Ph.D., Study Director Date 3M Environmental Laboratory 3M Environmental Laboratory Page 67 3M Medical Department Study: T-6316.1 Record of Deviation Analytical Report: FACT-TOX-003 LRN-U2104 1. Identification Study I Pr?. Ha3 Deviation Type I (Checkone) r-Required Procedure/process: -a SOP _ - CJProtocol 11. - m e t h o d 0Equipment Procedure CI Other: Date(s) of occurrence; /n 4?9-5F uescrrprron: -0 - --- Ill. Actions Taken: (such as amendment issued, SOP revision, etc.) 1 Authorized By . - - . -- -- -($tudyDzrector / Project Lead) Date- Stud;j Diitcb'* Td, fl&,&& 3M Environmental Laboratory Form ETS-4-8.0 S p i o r Rlqrurdct;.t: Mu< Care ~ ~ o ver;,v Ti a t k , n ~ (assigned by Study Director or Project Lead at 3M Environmental Laboratory Page 68 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 Record of Deviation Study / Project No. Deviation Type (Check one) Document Number .......................................... _....... -- - I. Identification a - r- - _.- C o v ~ i c e- d2-24-228 SOP @Method D Eq~u- ipment Procedure ClProtocol 0 Other: 1 Date(s) of occu .............. _......................I ... ................. ........... I 7-/q 7-ye 11. Description: ... ................................. .- ..Requ-ired Proc_ edu_re/_ pro-cess: -_.____.____..___.___ . - - I _ _ - ~--~~~--~~.~~ .. ___ . ..................................... _c__---_- ........ -................................... __ ....... Actual Procedure/process: ............. . .................... __ .... - ................. .... . - .- --_ ............ ................................ ....... Ill. Actions Taken: ent issued, SOP revision, etc.) -- . .2-/59C& . ...... .. ............ __ ........ - -. __-I__ .- - - - - - ___ - --_ - (StudyDirector /Project Lead) - __ -- . - Form ETS-4-8.0 3M Environmental Laboratory (assigned by Study Director or Project Lead at the end of study or project) Page 69 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 Record of Deviation 1. Identification Study / Project No. FACT-TOX-003 Covance 6329-228 Deviation Type (Check one) 0 SOP UProtocol x Method 0Equipment Procedure 0 Other: Document Number(s): FACT-M-4.0, Date(s) of occurrence: 07/13/98,04/2 1/00, ETS-8-5.1 04/24/00,08/15/00,08/28/00,08/30/00 /I. Description: Reauired I P_r_o_ c_e_ d-u--re/Drocess: -__ __ - __ - - - I - ~ ~ Section 10.1.1 and- -10.1-.2 _st_at_e-s: -``Analyzea method blank-an-d a -ma&x blank prior toeach - Actual Procedure/process: On several occGions,-&aa&- at all for a .. were either a) analyzed after the c.ali.brati-on curveor b) not a.na-lyzed Wk4, Serum).' __ . . . . . . . . . . . . . . . -.-....... - _._ . . ._ . . ._ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ill. Actions Taken: (such as amen.dm-e-nt_i_s.s.-ue-d,-S.- OP revision, etc.) . . This deviation was written. - . . . . . . . __- Recorded By - Date . osI O - J l O , IV. Impact on-S k y _ ( Project . -. - - - - . ..... All blanks were ana..ly-zed-a-t l-ea-st--o- n.c.e- during the course of the study. When bl&kwere analyzed . _ after the calib.r.a.ti.on cur.ve, all con.trol-..o..f-bia.s.p..r.a..c.t.i.c.e..s..w..e.r.e...f.o.l.l.o..w. ed. In one..a.n.alysi-s,.bla_"nks were ___I not analyzed since these b_la_n~ k.s had been runpreviously.and.did not require reanalysis. In this particular ana.ly..s.i.s..(.0..8./.30/00, Wk4, Serum)-i. n_ stI ru_ m- ..e.n..t blan-ks w.e.r.e..u.s.e.d - t_ o.d_e_t_e.r_mI_i.n_ -^ e_limi.t.s.o.f qu_a_ n...tita-tion. This deviationhas no-a.dverse_ af_ fec.t on these study data. -__- Authorized By (Study Director /Project Lead) Date 3M Environmental Laboratory F o ET~S-4-8.0 3M Environmental Laboratory Deviation No. 3 (assigned by Study Director or Project Lead at the end of study or project) Page 70 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 Record of Deviation 2 1. Identification --I__ Study / Project No. __----__- ___- &[+-7-@xDeviation Type 3 W G f l u- d32s-2x2 --- 0 SOP Lia Method 0Eq~u-ipmeint Procedure (Check one) ClProtocol 0 Other: Document Number F A o L % A0 J E7X-J-~ s) of occurrence 11. Description: Ill. Actions Taken: - A<,/*> (such as amendment issued, SOP revision, etc.) ./LJ ADZ, Recorded By IV. Impact on Study/ Project I Date - Authorized By (StudyDirector /Project Lead) f -_ - I. 9//J -/ou JLd sh\ OklhOL: kLhN 9 3M Env ronmental Latomtory Form Em-4-8.0 a$'. Ip(lSor kOJ#C C i L Deviation No. Y (assigned by Study Director or Rojcct Lead at the end of study or project) 'c 3M Environmental Laboratory Page 71 3M Medical Department Study: T-6316.1 Record of Deviation Analytical Report: FACT-TOX-003 LRN-U2104 1. Identification Study / Project No.toxOO3 Deviation Type (Check one) 0 SOP o Protocol 4Method Other: Equipment I3rocedure I Document Number:F ACT-M-2.0 Date(s) of occurrence: 08/07/1998 11. Description: Required Procedure/process: - _ -- I Section 10.2.1 Analyze a matrix spike-andmatrix spike duplicatewith each analysis. With a ................................................... .................. ............. . - ~ ................. ................................................... .m.. i..n...i.m__u..m of 2 _s-p.ikes p-.-e-.-r.I._b-- atch. .. _-.-..- . .-.. - .. -... -- .._- I1, . . . ..... ..... . _ _ .. _ _. ._ _ _ , .-.. ........ ..... .. ~ . ... ___ . ..- ... I . . . . . . . . . _ _ . . ........ ...... .. ....... ... . . . . ................. ._ . . ..- .... Actual Procedure/process: The matrix spike and duplicates were analyzed in an earlier run and were _ not.r_ e-ru.n_ w- it_ h th.i_ s an.a_ lys.is_. - - . d . .l. . ,- ____ ............ _ .- _ _ . " .- . _ -- ........... _ .- . ~ _. . 1 ......................... ...... .. ..... - ...... ............ ........... ................. . .... .. ... ............ ._ .... . . -. . . . . . . . . . . . . .... ._ II - .. .. . _. Ill. Actions Taken: (such as amenhent issue_ d,.SOP revision, etc.) -Th- is- dev-iatio-i-@o-m.th-e-sta-ted.---me-t-h-od-is no_t-ed.- - - - -- - .- - - - - - t ........................................................................................................................ t __ ___ _ _ . - '- .-- . _-_ . ~ . .- - .................. -... ..... .. .. _-..._................... Recorded By: Harold Johnson I - &LL&L- S 4 4 ( IV. l m w t on Study/ Project 1 This willhave no impact on thestudy. - . _ .. I - -. - _ . ........... .. .. Authorized 'By (Stu(ir Direct& /Project Lead) __ __ .................. Date May 4,2001 Date Form ETS-4-80 3M Environmental Laboratory (assigned by Study Director or Projtxt Lead at the end of study or project) Page 72 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 Record of Deviation 1. Idenfification ........................................ Study / Project No. .. ...... ..... Deviation Type (Check one) ......................... ~c cTLIDY.:.~O?_-...... O SOP OProtocol . ,. ............ ......... ..... ................................... .,.. . ., ...... , . C... Q?Gn.L& -- d-3. a s -....---- 328 O Method CI Equipment :Procedure ~. Other: . ~~--___- Document Number 21 CFR sS. 120 (GI @I ___.-. ._ r ._-._-.(__.. .. 1 Date(s) of occurrence ! 11. Description: os/22lqcl .,. . . . . . . . . . . ......................... ............ ,........ Recorded By Date &&A . Authorized By -/Project 3M EnvironmentalLaboratory Form ETS-4-8.0 ____ Lead) Date _ _ _ _ _ _ _ ~....... ?/2-S/45 h&.~., 6 SpNOf f i N c n t c b . nffiv CaK. T& g-? Deviation No. (assigned by tudy Directoror Project Lead at the end of s t u d p a project) 3M Environmental Laboratory Page 73 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 Record of Deviation I Study / Project No. IDeviation Type (Check one) 1. Identification FACT-TOX-003 Covance 6329-228 0 SOP UProtocol X Method 0 Equipment Procedure 0 Other: Document Number(s): ETS-8-7.0 Date(s) of occurrence: 04/14/00,04/17/00 11. Description: IRequired Proce-d.ure/p.r-o.- ce--ss: -- - - - . - -__ _.- -- Section 14.311 st_a__tes, "The_l_fv_al-ue-fo-r the calibration curve must be 0.980 or better." S- -ec-tion 14.3.6 states, "A valid curve must c0nt-_h_a_t l_e% five activepoints."- - I __I__ -I__ _I _ Actual Procedure/process: T h e E @ O S E - O f i ~ b_&_ o- ~ &- ? e- fo-riheweek 104 livzr-ge_n_ecakd on 04/14/00 had an r;! value of0.9687. A four pI o.h__t c_u_r_v_ ewk usedto plo~ t_ _th_ e_ _E_ t_FOSE-OH c-a.lib- r-ati-o-n c-u- rv.e for the Week 53 liver,,generated on 04/17/00. . . . . . . . . . . . . . . . - ... -. ....... ...... ................ . . . . . . . . . . . . . . . ..- ...................................... .............................. ..................................................... ... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . -. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ill. Actions Taken: (s-u-ch-as amendme-n-t issued, _S_OP- revision, e-tc-.)- --- - _-__ -.. These data were flagged in the raw data and this deviation was written. -- - . __--_l_-----l_----.---.- Recorded By Date................ IV. Impact on Study / Project EtFOSE-OH data is flagged as qualitative, so these data willnot be adversely affected. Authorized By (Study Director /Project Lead) Date 9-L 3M Environmental Laboratory Page 74 3M Medical Department Study: T-6316.1 Record of Deviation Analytical Report: FACT-TOX-003 LRN-U2104 I. Identification I _ _____ Study / Project No. - --.-I- - - - - - _ _ _ __ __ - - - Fad- - / 228 7 G + - m 3 C O V m C C 0329 Deviation Type 0 SOP 0 Method &'Equipment Procedure CJProtocol D Other: ' . I.... . - ... I..... ___ ___- .. ......... __ Form ETS-4-8.0 3M Environmental Laboratory (assigned by Study Directoror Project Lead at the end of study or project) Page 75 3M Medical Department Study: T-6316.1 Record of Deviation Analytical Report: FACT-TOX-003 LRN-U2104 1. Identification Study / Project No. FACT-TOX-003 Covance 6329-228 Deviation Type (Check one) 0 SOP 0 Protocol X Method 0 Equipment ProcedAe 0 Other: Document Number(s): ETS-8-5.1 Date(s) of occurrence: 08/30/00 11. Description: Required Pro- ce-d-ure/pro-c-ess:- __ - __ -- A fivepo-int_c_urve or b_e_ t-ter is u- sed__for plo-t_t_ing the calibrat-ion cu_r__v_e.- I . . . . . . . . . . . . . . . . . . _...... .... .- ..... - . - .- ................... _. .. ........................... ....... .................. Actual Procedure/process: _ _ _ _ __ - - - _ _ -- A four p o h c G e w G used for plotting the -P F- O S U cali-brat-io-n curve for theweek 4 . . serum curve, .ge.ner-ated on 08/3O/OO. - -. . . . . . . . . .I. . . . ........................... ........... - . ...................................... -_............................................. .............. .......... ._............ .- - 111. Actions Taken: (suc.h a_s.amen-d-m. e.n--t".iIs..s_u._e__d, SO...P.....revision, etc_.)_ __ ._.^--..-I__,..I--.... ___I. .. .- __ These data were flagged in the raw data and this deviation was written. .. -. . IV. Impact on Study / Project These data will not be adversely affected. fzde a7L/.L Q C p a P t L f / I L c f + L JLWm<. Authorized By (Study Director /Project Lead) LYh @5/GY/Uf Date ita48 SpOnsDf QirectPr-, John GuknhJc Rtprts&\Id-b~~;M U v C M s ba/\n7, & 3M EnvironmentaI Laboratory Deviation NO. 9 Form ETS-4-8.0 (assigned by Study Director or Project Lead at the end of study or project) 3M Environmental Laboratory Page 76 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 3M Medical Department Study: T-6316.1 Analytical Report: FACT TOX-003 LRN-U2104 Appendix C: Extraction and Analytical Methods This appendix includes the following methods: Preparatory Methods FACT-M-1.O. Extraction of Potassium Perfluorooctanesulfonateor Other Anionic Fluorochemical Surfactants from Liver for Analysis Using HPLC-Electrospray/Mass Spectrometry, (8 pages) FACT-M-3.0, Extraction of Potassium Perfluorooctanesulfonateor Other Anionic Fluorochemical Surfactants from Serum for Analysis using HPLC-Electrospray/Mass Spectrometry, (8 pages) ETS-8-4.1, Extraction of Potassium Perfluorooctanesulfonateor other Fluorochemical Compounds from Serum for Analysis using HPLC-Electrosprayr'MassSpectrometry, (14 pages) ETS-8-6.0, Extraction of Potassium Perfluorooctanesulfonate or other Fluorochemical Compounds from Liver for Analysis using HPLC-Electrospray/MassSpectrometry, (14 pages) Analflical Methods FACT-M-2.0, Analysis of Liver Extracts for Fluorochemicals Using HPLCElectrospray/Mass Spectrometry, (8 pages) FACT-M-4.0, Analysis of Fluorochemicals in Serum Extracts Using HPLCElectrospray/MassSpectrometry, (8 pages) ETS-8-5.1, Analysis of Potassium Perfluorooctanesulfonateor other Fluorochemicals in Serum Extracts Using HPLC-Electrospray/MassSpectrometry, (9 pages) ETS-8-7.0, Analysis of Potassium Perfluorooctanesulfonate or other Fluorochemicalsin Liver Extracts Using HPLC-Electrospray/Mass Spectrometry, (10 pages) 3M Environmental Laboratory Page 77 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 3M ENVIRONMENTAL LABORATORY METHOD EX"RACTION OF POTASSIUM PEXFLU0ROOCTANESON44TEOR OTHER ANIONIC FLUOROCHEMXCALSURFACTANTSFROM LIVER FOE! &ALYSIS USING HPLC-ELECTROSPRAYMASPSESCTROIMETRY Method Number: FACT-M-1.0' ' .. Author: Lisa Clemen A,doptionDate: .5/2L/q 3 R.evision Date: @/,f Group Leader r L Tevchnical Reviewer 3 / t &/Pi? Date sl27/1Y Date 1.0 SCOPE ANDAPPLICATION -) I.IScope: This method is for the extraction of Potassium Perfluorooctanesulfonate (PFOS)or < other fluorochemical surfactants from liver. 3 1.2 Applicable Compounds: Fluorochemkal surfactants or other fluorinated compounds. 1.3 Matrices: Rabbit, rat, bovine, and monkey livers or other livers as designated in the 3 vdiciation report. -2 . a> Microsoft 7.0.1/95 FACT-M-1.0 Page 1 of 8 Extraction of PFOS fiom Liver 3M Environmental Laboratory Page 78 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 2.0 SUMMARY OF METHOD 2.1 This method describes howto extract potassium perfluorooctanesulfonate(PFOS) or other fluorochemical surfactants h m liver using ion pairing reagent and 5.0 m L s of ethyl acetate. An ion pairing reagent is added to each sample and partitioned into ethyl acetate. Four mLs of extract is removed to a centrifugetube and put onto a nitrogenevaporator u t i 1dry. Each extract is reconstituted in 1.O mL methanol then filtered througha 3 cc plastic syringe attached to a 0.2 p filter into glass autovials. 3.0 DEFINITIONS 3.1 None. 4.0 WARNINGS AM) CAUTIONS 4.1 Health and Safety Warnings: 4.1.1 Use universal precautions when handling animal livers, they may contain pathogens. . 5.0 INTERFERENCES 5.1 There are no knowninterferencesat thist h e . d 6.0 EQUIPMENT 6.1 The following equipment isused while carrying out this method. Equivalent equipment is acceptable. 6.1.1 Ultra-Turrax T25 Grinder for grinding liver samples 6.13 Vortex mixer, VWR, Vortex Genie 2 6.1.3 Centrifuge, Mistral 1000 or IEC 6.1.4 Shaker, Eberbach or VWR 6.1.5 Nitrogen Evaporator, Organomation 6.1.6 Balance 7.0 SUPPLXES AND MATFXUALS 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 Glass,type A, volumetric flasks 7.6 40 mL glass I-CHEM Vials 7.7 Plastic sampule vials, Wheaton, 6 mL 7.8 Polypropylene centrifuge tubes, 15 mL 7.9 Labels FACT-M- 1 .O Extraction of PFOS from Liver Page 2 of 8 3M Environmental Laboratory Page 79 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 7.10 Syringes, capable of measuring 10 pL to 50 pL 7.11 Glass,type A, volumetric pipettes 7.12 Graduated pipettes 7.13 Electronic pipettor, Eppendorf or equivalent 7.14 Timer 7.15 Disposable plastic 3 cc syringes 7.16 Filters, nylon syringe filters, 0.2 pm,25 mm 7.17 Crimp cap autovials Note: Prior to using glassware and bottles, rinse 3 times with rnetlmiol and 3 times with Milli- Qm water. Rinse syringesa hlinimum of 9 times withmethanol, 3 rinses fiom 3 separate vials. 8.0 REAGENTS AND STANDARDS 8.1 Reagents 8.1.1 SodiumHydroxide (J.T Baker or equivalent), (NaOH) 1ON:weigh approximatel 200 grams NaOH. Pour into a 1000mL beaker containing 500 liters (L)Miili-Qxi water, mix until all solids are dissolved. Storein a 1L nalgene bottle, 8.1.2 Sodium Hydroxide (J.T Baker or equivalent), (NaOH) 1N. Dilute 1ON 1:10. Measure 10mL of the 1ONNaOH solutioninto a 100K& volumetric flaskand I dilute to volume using Milli-Qm water. Store in a 125 mL nalgene bottle. 8.1.3 TetrabuQlammoniumhydrogen sulfate(Kodak or equivalent), (TBA) 0.5M W e i g approximately 169 grams of TBA into a 1 L volumetric; containing 500 L Milli-Q water. Adjust to pH 10using approximately 64mL 1014NaOH and dilute to volume with M W Q m water. Add NaOH slowly while adding the last 1I& of NaOH because the pH changes abruptly. Storein a 1Llnalgenebottle. 8.13.1 TBA requires a check prior to each use to ensire pH = 10. Adjust as needed using 1N NaOH solution. 8.1.4 Sodium carbonate/SodiumBicarbonateBuffer (J.T.Baker or equivalent), (Na&O$NaHC03) 0.25MWeigh approximately26.5 g of sodium carbonate (Na$OJ and 21.O g of sodium bicarbonate (NaHCOJ into a 1L volumetric flask and diluteto volume with Milli-Qm water. Store in a 1 L nalgene bottle. 8.1.5 PFOS (3MSpecialty Chemical Division), molecular weight = 538. 8.1.6 Ethyl Acetate, Omnisolv, glass distilled or HPLC grade. 8.1.7 Methanol, Omnisolv, glass distilled or HPLC grade. 8.1.8 Liver and control liver, received fkozen fiom testing laboratory. 8.1.9 Milli-Qm water, all water used in this method should be Milli-QTMwater and may be provided by a Milli-Q TOC Plus system. 8.2 Standards 8.2.1 Prepare PFOS standards for the standard curve. FACT-M-1 .O Extraction of PFOS from Liver Page 3 of 8 3M Environmental Laboratory Page 80 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 8.2.2 Weigh approximately 100 mg of PFOS into a 100 mC volumetric flask and record the actual weight. 8.2.3 Bring to volume with methanol for a stock standard of iipproximately 1000ppm (crg/mL). 8.2.4 Dilute the stock solution with methanol for a working standard 1 solution of approximately 50 ppm. 8.2.5 Dilute the stock solution with methanol for a working standard 2 solution of approx. 5.0 ppm, 8.2.6 Dilute the stock solution with methanol for a working standard 3 solution of approx. 0.50 ppm. 9.0 S A M P L E ~ L I N G 9.1 All livers are received'frozenand must be kept frozen until the extractionis performed. 10.0 QUALITCYONTROL 10.1 Matrix Spikes 10.1.1 Prepare and analyze matrix spike and matrix spike duplicate samples to determine the accuracy of the extraction. 10.1.2 Prepare each spike using liver chosen by the analyst, usually a control liver. 10.1.3 Expected concentrations will fall in the mid-range of the initial calibration curve. 10.2 Continuing Calibration Checks 10.2.1 Prepare and analyze continuing calibration check samples to determine the continued linearity of the initial calibration curve. 10.2.2 One check is prepared per group of ten samples. For example, if a sample set = 34,C four checks are prepared and extracted. 10.23 Prepare each continuingcalibration check fiom the same liver homogenateused to prep the initial curve. 10.2.4 The expected concentrationwill fall within the mid-ranj3e of the initialcalibration curve. 11.0 CALIBRATION AND STANDARDIZATION 11.1 Prepare Liver Homogenate to Use for Standards 11.1.1 Weigh approximately40 g of liver into a 250 mL Nalgene bottle containing 200 mLs Milii-QTMwater. Grind to a homogeneous solution. 11.'1.2 If 40 g is not available, use appropriate amounts of liver and water in keeping with a 15 ratio. 11.1.3 See section 13..0to calculate the actual density of Liver. FACT-M- 1.O Extraction of PFOS from Liver Page 4 of 8 3M Environmental Laboratory Page 81 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 11.1.4 Add 1 mL of homogeneous solution to a 15 mL centrifLge tube. Re-suspend homogeneous solution by shaking between aliquots while preparing a total of sixteen 1 mL aliquots of homogeneous solution in 15 mL centrifuge tubes. 11.1.5 Two 1 mL aliquots serve as matrix blanks. Use the standard concentrations and spiking amountslisted in table 1to spike, in duplicate, two standard curves for a total of fourteen samples. J 11.1.1 See section 13.0to calculate actual concentrations of PFOS in calibration standards. 11.2 Extrgct spiked liver homogenates following 12.14-12.24 of this method. Use these standards to establish each initial curve on the mass spectrometer. 12.0 PROCEDURES 12.1 0 b . a frozen liver samples. In spent tissue, note that the liver has not been packaged with other tissues. 12.2 cut appro&tely 1g of liver using a dissecting scalpel. 123 Weigh the sample directly into a tared plastic sampule vial. 12.4 Record the liver weight in the study notebook. 12.5 Label the sampule vial with the study number, weight, liver ID, date and analyst initials. 12.6 Add 2.5 mLs of water to sampule vial. 12.7 Grind the sample. Put the grinder probe in the sample and grind for about 2 minutes, or until the sample is homogeneous. 12.8 Rinse the probe into the sample with 2.5 d s water using a pipette. 12.9 Take the grinder apart and clean it With methanol after each sample. Follow AMDT-EP-22. 12.10 Cap the sample and vortex for 15 seconds. 3M Environmental Laboratory FACT-M-1.O Extraction of PFOS from Liver Page 5 of 8 Page- 82 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 12.11Pipette 1 mL homogenate into a 15 mL polypropylene centrifugetube. Label the centrifige tube with the identical information as the sampule vial. (See Worksheet for documenting the remaining steps.) 12.12 Spike liver homogenatcs with the appropriate amount of PFOS standard as described in section 11.1 or Table 1. 12.13Pipette two 1 mL aliquots of Milli-QTMwater to c e n a g e tubes. These will serve as instrument blanks. 12.14 Add 1 mL 0.5 M TBA and 2 mL of the 0.25 M sodium Carboniite/sodium bicarbonate buffer. 12.15 Using a volumetric pipette, add 5 m L s ethyl acetate. 12.16 Cap each sample and put on the shaker for 20 minutes. 12.17 Centrikge for 20 to 25 minutes, until layers are well separated,Set power on the centrifuge to approximately 3500 rpm. 12.18 Remove 4mLs of organic layer, using a 5 mL graduatedglass pipette, to a clean 15mL centrifige tube.Label this fie& tube with the same infomtioii as in 12.5. 12.19 Put each sample on the analyticalnitrogen evaporator until dry,approximately 2 to 3 hours. 1230Add 1.0mL of methanol to each centrifugetube using a graduated pipette., 12.21 Vortex mix for 30 seconds. 12.22 Attach a 0.2 juri nylon mesh filter to a 3 cc syringe and transfer the sample to this syringe. Filter into a 1.5 mL glass autovial. 12.23Label the autovial withthe study number, animal number and gender, sample timepoint, matrix,finalsolvent, extractiondate, and analyst(s) who performed the extraction. 12.24 Cap and hold for electrospray mass spectrometry analysis. 1235Complete the worksheet and tape to page of study notebook. 13.0 DATAANALYSIS AND CALCULATIONS 13.1 Calculations: 13.1.1 Calculate the density of liver (mg) in 1.OmLhomogenate using the following equation: g of Liver x Average weight of ten 1mLaliquots (ma (g of Liver + g of Water) 3M Environmental Laboratory FACT-M-1 .O Extraction of PFOS from Liver Page 6 of 8 Page 83 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 13.1.2 Calculate actual concentrations of PFOS in calibration standards using the following equation: pL of Standard x Concentration ( ~ /gmL) =Final Concentration (pg/g or m a g ) mg Liver'/ 1mL homogenate of PFOS in Liver "Average weight of liver in solution as determined in 13.1.1, by weighing ten 1 mL homogenates of approxkately 40 mg liver in 200 mL of Milli-Q water. 14.0 METHODPERFORMANCE 14.1` The method detection limit is equal.tq half the lowest standard in the calibration curve. 15.0. POLLU"I0N PREVENTIONAND WASTE MANAGEMENT 15.1 Samplewaste is disposedin biohazard containers, flammable solvent waste is disposed in high BTU containers,and used glass pipette waste is disposed in broken glass containers located in the laboratory. 16.0 RECORDS .16.1 Complete the extractionworksheet and tape into the study notebook. 17.0 TABLESD, IAGRAMSF,LOWCHARTS,AND VALIDATION DATA 17.1 The validation report associated with this method is FACT-M-1.O & 2.0-V-1. 18.0 REFERENCES 18.1 AMDT-EP-22, "Routine Maintenance of Ultra-Turrax T-25" 19.0 AFFECTEDDOCUMENTS 19.1 FACT-M-2, "Analysis of Liver Extractsfor Fluorochemicalsizsing HPLC-Electrospray Mass Spectrometry" 20.0 REVISIONS Revision Number. Reason For Revision - Revision Date 3M Environmental Laboratory FACT-M- 1.O Extraction of PFOS from Liver Page 7 of 8 Page 84 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 Extraction Worksheet for FACT-M-1 1Date and Initids for Std. t- e. Date & Initials ppm. MSfMSD used sample . 2for a final concentration of Cont. Checks used same homogenate as for std curve. FACT-M-1 .O Extraction of PFOS from Liver Page 8 of 8 3M Environmental Laboratory Page 85 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 3M ENVIRONMENTLAALBORATORY EXTRACTION OF POTASSIUMPERFLUOROOCTANESULFONATEOR OTHER ANIONIC ' F'LUOROCHEMICAL SURFACTANTSFROM SERUMFOR ANALYSIS USING ' HPLC-ELEXTROSPRAYWS SPECTROMETRY Method Number: FACT-M-3.0 Author: Lisa Clemen .Appro dBy: >)-qV$- 1 Laboratory Mahger Adoption Date: y t z l q 8 Revision Date: Date Lk g&)I Technical Reviewer lflzt1'73 Date 1 301.0 SCOPE ANDAPPLICATION 1.1 Scope: This method is for the extraction of potassium perfluorooctanesulfonate(PFOS) or other fluorochemical surfactants from serum. 1.2 Applicable Compounds: Fluorochemicd surfactantsor other fluorinatedcompounds. '3 1.3 Matrices: Rabbit, rat, and bovine serum or other sera as designated in the validation report. -. Microsoft 7.0.1195 FACT-M-3.0 Extraction of PFOS from S e m Page 1 of 8 3M Environmental Laboratory Page 86 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 2.0 SUMMARY OF METHOD 2.1 This method describes how to extract potassium perfluorooctaccesulfonate (PFOS) or other anionic fluorochemical surfactants fiom serum using an ion pairing reagent and 5 .O mL of ethyl acetate. An ion pairing reagent is added to the sample and the analyte ion pair is partitioned into ethyl acetate. Four mL of extract are removed and put onto a nitrogen . evaporator until dry. Each extract is reconstituted in 1.O mL of methanol, then filtered through a 3 cc plastic syringe attached to a 0.2 pm nylon filter into glass autovials. 3.0 DEFINITIONS 3.1 None. 4.0 WARNINGASND CAUTIONS 4.1 Health and Safety Warnings: 4.1.1 Use universal precautions, especially laboratory coats, goggles, and gloves when handling animal serum, it may contain pathogens, 5.0 INTERFERENCES 5.1 There are no known interferences at this time. 6.0 EQUIPMENT 6.1 The following equipment isused while carrying out thismethod. Equivalent equipment is. acceptable. 6.1.1 . Vortex mixer, VWR, Vortex Genie 2 6.1.2 Centrifbge, Mistral 1000 or IEC 6.1.3 Shaker, Eberbach or V W R 6.1.4 Nitrogen evaporator, Organomation 6.1.5 Balance, (k0.100 gm) 7.0 SUPPLIES AND MATERIALS 7.1 Gloves 7.2 Eppendorf or disposable pipettes 7.3 Nalgene bottles, capable of holding 250 mL and 1L 7.4 Glass, type A, volumetric flasks 7.5 40 mL glass I-CHEM vials 7.6 Polypropylene centrifuge tubes, 15 mL 7.7 Labels 7.8 Syringes, capable of measuring 10 pL to 50 pL 7.9 Glass, type A, volumetric pipettes 7.10 Graduated pipettes FACT-M-3 .O Extraction of PFOS from Serum Page 2 of 8 3M Environmental Laboratory Page 87 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 7.11 Electronic pipettor, Eppendorf or equivalent 7.12 Timer 7.13' Disposable plastic 3 cc syringes 7.14 Filters, nylon syringe fdters, 0.2 pm, 25 mm 7.15 Crimp cap autovials Note: Prior to using glassware and bottles, rinse 3 times with methanol and 3 times with MilliQm water. Rinse syringes a minimum of 9 times with methanol, 3 rinses from 3 separate Vials. 8.0 REAGENTS AND STANDARDS 8.1 Reagents 8.1.1 x, Sodium hydroxide (J-TBaker or equivalent), (NaOH) 1(ONweigh approximatel 200 grams NaOH. Pour into a 1000 mL beaker containing 500 liters (L) Milli-Q water, mix until all solids are dissolved Store in a 1 L Iqalgene bottle. 8.1.2 Sodium hydroxide (J.T Baker or equivalent), (NaOH) 1". Dilute 1ON 1:lo. Measure 10 mL of 1ONNaOH solution into a 100mL volumetric flask and dilute to volume using Milli-QTMwater. Store in a 125 mL NzJgene bottle. 8.13 e Tetrabutylammonhmhydrogen sulfate (Kodak or equivalent), (TBA) O S M . Wei approximately 169 grams of TBA into a 1L volumetric containing 500 L Milli-Q r' water. Adjust to pH 10using approximately 64mL of 1ON NaOH and dilute to volume with Milli-QW water. Add NaOH slowly while adding the last mL of NaOH because the pH changes abruptly. Store in a 1 L Nalgene bottle. 8.1.3.1 TBA requires a check prior to each use to ensure pH = 10. Adjust as needed using 1N NaOH solution, 8.1.4 Sodium CarbomWsodium bicarbonate buffer (J..T. Baker or equivalent), (Na&O,MaHCO,) 0.25M:Weigh approximately 26.5 e; of sodium carbonate (N+CO,) and 21.O g of sodium bicarbonate (NaHCO,) into a 1L volumetric flask and bring to volume with I ~ f 3 l i - Qw~ater. Store in a 1 L nalgene bottle. 8.1.5 PFOS (3MSpecialty Chemical Division), molecular weight = 538. 8.1.6 Other fluorochemicals, as appropriate. 8.1.7 Ethyl Acetate, Omnisolv, glass distilled or HPLC grade, 8.1.8 Methanol, Omnisolv, glass distilled or PLCgrade. 8.1.9 Serum, frozen liquid Erom Sigma. 8.1.10 Control sepun received with each sample set. 8.1.11 Milli-QTMwater, all water used inthis method should be: Milli-Qm water and may be provided by a Milli-Q TOC Plus system. 3M Environmental Laboratory FACT-M-3 .O Extraction of PFOS from Serum Page 3 of 8 Page 88 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 8.2 Standards 8.2.1 Prepare PFOS standards for the standard curve. 8.2.2 Prepare other fluorochemicalstandards, as appropriate. 8.2.3 Weigh approximately 100mg of PFOS into a 100mL volumetric flaskand record the actual weight. 8.2.4 Bring to volume with methanol for a stock standard of approximately 1000 ppm (I.1.g/mL). 8.2.5 Dilute the stock solution with methanol for a working standard 1solution of approximately 50 ppm. 8.2.6 Dilute the stock solution with methanol for a working standard 2 solution of . approx. 5.0 ppm. 8.2.7 Dilute the stock solution with methanol for a working standard 3 solutian of approx. 0.50 ppm. 9.0 SAMPLEHANDLING 9.1 All sera are received frozen and must be kept frozen until the extraction is performed. *10.0 O u mCONTROL 10.1 Matrix Blanks and Method Blanks 10.1.1 Two 1.O mL aliquots of the serum are extracted followingthisprocedure and used as matrix blanks. See section 11.1.2. 10.1.2 Two 1.0 mL aliquots of Milli-Qm water are extracted fisllowing this procedure and used as method blanks. 10.2 Matrixspikes 10.2.1 Prepare and analyze malxix spike and m a e spike duplicate samples to determine the accuracy of the extraction. 10.2.2 Prepare each spike using serum chosen by the analyst, ilsually control serum received with each sample set. 10.2.3 Expected concentrations will fall in the mid-range of the initial calibration curve. Additional spikes may be included and may fall in the low-range of the initial calibration curve. 10.3 Continuing Calibration Checks 10.3.1 Prepare and analyze continuing calibration check samples to determine the continued linearity of the initial calibration curve. 10.3.2 One check is prepared per group of ten samples. For e:cample, if a sample set = 34, four checks are prepared and extracted. 3M Environmental Laboratory FACT-M-3.O Extraction of PFOS from Serum Page 4 of 8 Page 89 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 103.3 Prepare each continuing calibration check from the same serum used to prep the initial curve. 10.3.4 The expected concentration will fall within the mid-range of the initial calibration curve. 11.0 CALXBRATAIONDNSTANDARDIZATION 11.1 Prepare Serum Standards 11.1.1 Transfer 1 mL of serum to a 15 mL centrifuge tube. 11.1.2 Ifthe majority ofserum samplevolumes are less than 1.0 mL, extract standards using serum volumes in the standards equal to the semn volumes in samples. Do not extract below 0.50 mL of serum. Record the serum volume on the extraction sheet. 11.1.3 Mix or shake between diquots while preparing a totalcd sixteen aliquots of s e m in 15 mL centrifugetubes. . 11.1.4 Two 1 mL or appropriate aliquots serve as matrix blanks. Typically use the standard concentrations and spiking amounts listed in table 1 to spike, in duplicate, two standard curves for a total of fourteen samples. 11.1.5 Refer to the validation report FACT-M-3.0-V-1 and FACT-M-4.0-V-1 which lists 4 the working ranges for calibration curves. Table 1 Approximate SpikingAmounts for Standards andl Spikes - Using 1.0 mL of Serum Working Standad (Approx-. Conc.) 0.500 ppm 5.00 ppm 5.00 ppm 5.00 ppm 50.0 ppm 50.0 ppm 50.0 ppm ~~ PL Approx. final conc. of PFOS ini serum .. Blank 20 0.0 10ppm 5 0.025 ppm 10 0.050 ppm 20 0.100 ppm 5 0.250ppm 10 0.500 ppm 15 0.750 ppm 11.1.4 See section 13.0 to calculate actual concentrations of PFOS in calibration standards. 11.2 Extract spiked serum standards following 12.6-12.16 of this rnlzthod Use these standards to establish each initial curve on the mass spectrometer. 3M Environmental Laboratory FACT-M-3.0 Extraction of PFOS from Serum Page 5 of 8 Page 90 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 12.0 PROCEDURES 12.1 Obtain frozen serum samples and allow to thaw. 12.2 Vortex mix for 15 seconds then remove 1.O mL or appropriate volume to a 15 mL polypropylene centrifuge tube. 1 2 3 Return serum samples to freezer after extraction amount has been removed. 12.4 Record the serum volume on the extraction worksheet. The final methanol volume will equal the initial serum volume. 12.5 Label the tube with the study number, serum ID, date and analyst initials. See attached worksheet for documenting the remaining steps. 12.6 Spike serum withthe appropriate amount of PFOS standard as described in section 11.1 or Table I for the calibration curve standards. Also spike matrix spikes and continuing calibration standards. 12.7 Vortex mix the standard curve samples, matrix spike samples, ,and continuing calibration samples for 15 seconds. 12.8 Add 1 mL 0.5 M TBA and 2 mL of the 0.25 M sodium carboaitdsodium bicarbonate buffer. 12.9 Using a volumetric pipette, add 5 mL ethyl acetate. 12.10 Cap each sample and put on the shaker for 20 minutes. 12.11 Centrifuge for 20 to 25 minutes, until layers are well separated. Set power on the centrifuge to approximately 3500 rpm. 12.12 Transfer 4 mL of organic layer, using a 5 mL graduated glass pipette, to a clean 15 mL centrifuge tube: Label this fresh tube with the same information as in 12.5. 12.13 Put each sample on the analyticalnitrogen evaporator until dry,approxhately 2 to 3 hours. 12.14Add 1.O mL or appropriate volume ofmethanol to each centrifugetube using a graduated pipette. (This volume equals the initial volume of serum used for the extraction.) 12.15 Vortex mix for 30 seconds. 12.16Attach a 0.2 pm nylon mesh fdter to a 3 cc syringe and transfer the sample to this syringe. Filter into a 1.5 mL glass autovial. 12.17Label the autovial with the study number, animal number and gender, sample timepoint, matrix, final solvent, extraction date, and analyst(s) who performed the extraction. 12.18 Cap and hold for HPLC-electrospray/mass spectrometry analysis. Extracts may be stored at 4" C until analysis. 12.19 Complete the extraction worksheet, attached to this document, and tape to page of study notebook. 3M Environmental Laboratory FACT-M-3 .O Extraction of PFOS from Serum Page 6 of 8 Page 91 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 13.0 DATAANALYSIASND CALCULATIONS 13.1 Calculations: 13.1.1 Calculate actual concentrations of PFOS,or other appropriatefluorochemical, in calibrationstan&& ushg the following equation: mL of Standard x Concentration lug /mL) = Final Concentration (pglmL) m L of Standard +Initial Serum Volume (mL) of I?FOSin Serum 14.0 METHOD PERFORMANCE 14.1 The-methoddetection limit is equal to halfthe lowest standard in the calibration curve. 15.0 POLLUTIOPNREVENTIOANND W m MANAGEMENT 15.1 Sample waste is disposed in biohazard containers, flammable solvent waste is disposed in high BTU Containers,and used glasspipette waste is disposed in broknglass containers located in the laboratory. 16.0 RECORDS 16.1 Complete the extractionworksheet attached to thismethod, and tape into the study notebook. 17.0 TABLESD,IAGRAMFSL. OWCHARTS.AND VALIDATIONDATA 17.1 The validation report associated with this method is FACT-M-3.0 & 4.0-V-1. 18.0 REFERENCES 18.1 None 19.0 AFFECTEDDOCUMENTS 19.1 FACT-M-4, "Analysis ofSerum Fxtmcts for Fluomchemicalsusing HPLC-Electrospray Mass Spectrometry" 20.0 REVISIONS Revision Number. Reason For Revision Revision - Date 3M Environmental Laboratory . FACT-M3.0 Extraction of PFOS from Serum Page 7 of 8 - .. _-. __ Page 92 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 Extraction Worksheet for FACT-M-3 rStudy # Sample Number set # H,O Blank Serum Blank PFOS approx. 0.5 ppm actual ppm #W PFOS approx. 5 ppm actual ppm #W - PFOS approx. 50 ppm actual ppm #W - Date and Initials for Std. or Comments I I --- I -- I I MS/MSD/- Cont. Checks: Spiked ' pprn. M S N S D used sample uL of a ppm std ( ) for a final concentration of . Cont. Checks used same serum as for std curve. FACT-M-3 .O Extractionof PFOS &om Serum Page 8 of 8 3M Environmental Laboratory Page 93 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 3M ENVIRONMENTALLABORATORY METHOD EXTRACTION OF POTASSIUM PJ~X~?LUOROOCT~SULFONAOTREOTHER ~~'I.,UOROCHEMICACLOMPOUNDS FROM SERUM FOR ANALYSISUSING HPLC- ELECTROSPRAY/MASSPS ECTROMETRY Method Number: ETS-8-4.1 Adoption Date: 03/01/99 Author: Lisa Clemen, Glenn Langenburg Revision Date: 412 7/49 Approved By: r) Laboratory Manager .& t &.A Group Leader Technical Reviewer Date Wzb/Yg Date C,li/aL/4 4 Date 1.0 SCOPEANDAPPLICATION 0 1.1 Scope: This method is for the extraction of potassium perflucirooctanesullbnate(PFOS) or other ffuorochemical compounds fiom senun. 1.2 Applicable compounds: Fluomchemical surfactants or other fluorinatedcompounds. 1.3 Matrices: Rabbit, rat, bovine, monkey,and human serum or other fluids as designated in the validation report. -2. . 3 9, Word 6/95 ETS-8-4.1 Extraction ofPFOS from Strum Page 1 of 14 3M Environmental Laboratory Page 94 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 2.0 SUMMARY OF METHOD 2.1 This method describes the procedure for extracting potassium perfluorooctanesulfonate (PFOS)or other fluorochemicalsurfactants fiom serum, or other fluids,using 8n ion pairing reagent and methyl-tert-butyl ether (MBE). In this method, seven fluorochemicalswere extracted: PFOS,PFOSA,PFOSAA, EtFOSE-OH, PFOSEA, M556, and surrogate standard (see 3.0 Definitions). An ion pairing reagent is added to the sample and the analyte ion pair is partitioned into MtBE. The MtBE extract is removed and put onto a nitrogenevaporator until dry. Each extract is reconstituted in 1.O mL of methanol, then filtered through a 3 cc plastic syringe aiached to a 0.2 pnnylon fdter into glass autovials. 2.2 These sample extracts are analyzed following method ETS-8-5.1 or othex appropriate methods. 3.0 DEFINITIONS 3.1 PFOS:perfluorooctanesule (anionof potassium salt) C,F,,SO,' 3.2 PFOSA perfluorooctane sulfonylamide CJ?l$OzNH, 3.3 PFOSAA:perfluomoctane sulfonylamido (ethy1)acetate C$,,S0,N(CHzCH3)CHzC0,' 3.4 EtFOSE-OH: 2(N-ethylperfluorooctane suEonamido)-ethyl alcohol I c8F,,s0ZN(~~~3)~~~~0H 3.5 PFOSEA:perfluoxooctanesulfonyl ethyhide C,F, 7S0,N(C132CH3)H 3.6 M556: C8F,$02NCH)(CH2COOH) 3.7 Surrogate standard lH-lH-2H-2H perfluorooctane sulfonic acid 4.0 WARNINGS AND CAUTIONS 4.1 Health and safety warnings 4.1.1 Use universal precautions, especially laboratory coats, goggles, and gloves when handling animal tissue, which may contain pathogens. 5.0 INTERFERENCES 5.1 There are no interferencesknown at thistime. 6.0 EOUIPMENT 6.1 The following equipment is used while performing this method. Equivalent equipment is acceptable. 6.1.1 Vortex mixer, VWR, Vortex Genie 2 6.1.2 Centrifuge, Mistral 1000 or IEC 6.1.3 Shaker, Eberbach or VWR ETS-8-4.1 Extraction of PFOS from Serum Page 2 of 14 3M Environmental Laboratory Page 95 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 6.1.4 Nitrogen evaporator, Organomation 6.1.5 Balance (k 0.100 g) 7.0 SUPPLIES AND MATERIALS 7.1 Gloves 7.2 Eppendorf or disposable pipettes 7.3 Nalgene bottles, capable of holding 250 I& and 1L 7.4 Volumetric flasks,glass, type A 7.5 I-CHEMVials,glass, 40 mL glass 7.6 Centrifuge tubes,polypropylene, 15mL 7.7 Labels 7.8 Oxford Dispenser-3.0 to 10.0mL 7.9 Syringes, capable of measuring 5 pL to 50 pL 7.10 Graduated pipettes 7.11 Syringes, disposableplastic, 3 cc 7.12 Syringe filters,nylon, 0.2 pm, 25 mm 1.13 Timer 7.14 Crimp cap autovials and caps 7.15 Crimpers Note: Prior to using glasswareand bottles, rinse 3 times with methanol and 3 times with Milli-Qm water. Rinse syringes a minimum of 9 times with niethanol, 3 rinses h m 3 separate vials. 8.0 REAGENTS AND STANDARDS 8.1 Type I rea ent grade water, Milli-Qm or equivalent; all water used in this method should be Milli-Qf M water and may be provided by a m i - QTOC PliusTMsystem 8.2 Sodium hydroxide (NaOH), J.T Baker or equivalent 8.3 Tetrabutylammoniumhydrogen sulfate(TBA), Kodak or equivalent 8.4 Sodium carbonate (Na.$03), J.T. Baker or equivalent 8.5 Sodiumbicarbonate (NaHCO,), J.T.Baker or equivalent 8.6 Methyl-T-Butyl Ether, Omnisolv, glass distilled or HPLC grade 8.7 Methanol, Omnisolv, glass distilled or HPLC grade 8.8 Serum or blood, fiozen 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 96 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 8.93 PFOSAA (3MSpecialty Chemical Division), molecular weight = 585 8.9.4 EtFOSE-OH (3MSpecialty Chemical Division), molecular weight = 570 8.9.5 PFOSEA (3M Specialty Chemical Division), moleculiu weight = 527 8.9.6 M556 (3MSpecialty ChemicalDivision), molecular weight =557 8.9.7 Surrogate standard: 4-H, perfluorooctanesulfonic acid (1-HJ-H, 2-H,2-H C,F,,SO,H) molecular weight = 428 8.9.8 Other fluorochemicals, as appropriate 8.10 Reagent preparation NOTE: When preparing larger volumes thanlisted in reagemi,standard, or surrogate preparation, adjust accordingly. 8.10.1 10N sodiumhydroxide (NaOH): Weigh approximately 200 gNaOH. Pour into a 1000mL beaker containing 500 mL Milli-QTMwater, inix until all solids are dissolved. Store in a 1L Nalgene bottle. 8.10.2 1 N sodium hydroxide (NaOH): Dilute 10NNaOH 1 10. Measure 10mL of 10 N NaOH solution into a 100mL volumetric flaskand (diluteto volume using Milli-Qm water. Store in a 125 mL Nalgene bottle. 8.10.3 0.5M tetrabutylammoniumhydrogen sulfate (TBA): Wei of TBA into a 1L volumetric containing500 mLMilEi-Q@l waaptperro. xAimdjautsetltyo1p6H9g d ` 10using approximately 44 to 54 mL of 10N NaOH (While adding the last mL of NaOH, add slowly because the pH changes abruptly). Dilute to volume with Milli-Qm water. Store in a 1L Nalgene bottle. 8.103.1 TBA requires a check prior to each use to emure pH = 10. Adjust as ' needed using 1N NaOH solution. 8.10.4 0.25 M sodium carbonatdsodiumbicarbonatebuffer (Na$OJNaHCO,): Weigh approximately 26.5 g of sodium carbonate (NqCO,) and 21.Og of sodium bicarbonate (NaHCO,) into a 1L volumetric flask and bring to volume with MilliQm water. Store in a 1 L Nalgene bottle. 8.11 Standards preparation 8.11.1 Prepare PFOS standards for the standard curve. 8.11.2 Prepare other fluorochemicalstandards, as appropriate. Multicomponent fluorochemical standards are acceptable (for example, one working standard solution containing 1.00 ppm PFOS,1.02ppm PFOSA, 0.987 ppm PFOSAA,and 1.10 ppm EtFOSE-OH.) 8.11.3 Weigh approximately 100mg of PFOS into a 100mL volumetric flask and record the actual weight. 8.11.4 Bring to volume with methanol for a stock standard of'approximately 1000 ppm (Pg/rnL)- 8.1 1.5 Dilute the stock solution with methanol for a working standard 1solution 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 Serum Page 4 of 14 3M Environmental Laboratory Page 97 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 I 8.12 8.11.7 Dilute working standard 1 with methanol for a working standard 3 solution of approx. 0.50 ppm. Surrogate stock standard preparation 8.12.1 Weigh approximately 50-60 mg of surrogatestandard 1-HJ-H, 2-H,2-H, C$,,SO,H into a 50 mL volumetric flaskand record the actual weight. 8.12.2 8.12.3 Bring to volume with methanol for a smogate stock of approximately 1000-1200 PP** Prepare a surrogateworking standard. Transfer approximately 1mL of surrogate stock to a 10mLvolumetric flask and bring to volumt: withmethanol for a working standard of 100ppm. Record the actual volume transferred. 9.0 SAMPLEHANDLING 9.1 All samples are received frozenand must be kept frozen until the extraction is performed. 9.2 Allow samples to thaw to room temperature prior to extraction. 10.0 OUALITYCONTROL 10.1 Solvent Blanks, Method blanks and matrix blanks 10.1.1 An aIiquot of 1.0 mL methanol is used as a solvent blsmk C I 10.1.2 Extract two 1.0 mL aliquots of Milg-QTMwater following this procedure and use as method blanks. 10.1.3 Extract two 1.0 mL aliquots of the serum following this procedure and use as matrix blanks. See 11.1.4. 10.2 Matrix spikes 10.2.1 Prepare and analyze matrix spike and matrix spike duplicate samples to determine the accuracy of the extraction. 10.2.2 Prepare each spike using a sample chosen by the analyst, usuallythe control matrix received with each sample set. 10.23 Expected concentrations will fall in the mid-range of the initial calibration curve. Additional spikesmay be included and may fallin the low-range ofthe initial calibration curve. 10.2.4 Prepare one matrix spike and matrix spike duplicate per 40 samples, with a minimum of 2 matrix spikes per batch. 10.3 Continuing calibration checks 10.3.1 Prepare continuingcalibration check samples to ensure the accuracy of the initial calibration curve. 10.3.2 Prepare, at a minimum,one continuing check per group of 10 samples. For example, if a sample set = 34, four checks are prepared and extracted. 10.3.3 Prepare each continuing calibration check from the sane matrix used to prepare the initial curve. 3M Environmental Laboratory ETS-8-4.1 Extraction ofPFOS from S a m Page 5 of 14 Page 98 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 10.3.4 The expected concentrations will fall within the mid-range of the initial calibration curve. Additional spikes may be included that fall in the low-range of the initial calibration curve. This is necessary if the analyst must quantitate using only the low end of the calibration curve (for example, 5 ppb - 100ppb, rather than 5 ppb - 1000 ppb). 11.o CALIBRATION AND STANDARDIZATION 11.1 Prepare matrix calibration standards 11.1.1 Transfer I mL of serum to a 15 mL centrifuge tube. 11.12 Ifmost samplevolumes are less than 1.0 mL, extract standardswithmatrix volumes equal to the sample volumes. Do not extract less than 0.50 mL of . matrix. Record each sample volume on the extraction sheet 11.1.3 While preparing a total of twenty aliquotsin 15mL c e n m g e tubes, mix or shake between aliquots. 11.1.4 Two 1mL aliquots, or other appropri'ate volume, sem: as matrix blanks. Typically use the standard concentrations and spiking amounts listed in Table 1, at the end of this section,to spike, in duplicate, two standard curves, for a total of eighteen standards, two matrix blanks, and two methoti blanks. 11.1.5 Refer to validation report ETS-8-4.O & ETS-8-5.0-V-1, which lists the working I ranges and the Linear CalibrationRange (LCR) for calibration curves. 11.1.6 Use Attachment D as an aid in calculatingthe concentrations of the working standards. See Section 13.0 to calculate actualconcentrationsof PFOS in calibration standards. 11.2 To each standard, blank, or continuingcheck, add appropriate amount of surrogate working standard for the concentration to fallwithin the calibmtion curve range 5 ppb - 1000 ppb. 11.3 Extract spiked matrix standardsfollowing 12.6-12.16 of this method. Use these standards to establish each initial curve on the mass spectrometer. 3M Environmental Laboratory ETS-8-4.1 Extraction of PFOS from Serum Page 6 of 14 Page 99 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 , Approximate spiking Table 1 amounts for standards 1 and spikes 12.0 PROCEDURE 124 Obtain h z e n samples and allow to thaw at room temperatureor in a lukewarm watexbath. 12.2 Vortex mix for 15 seconds, then transfer 1.0 mL or other appropriate volume to a 15 mL polypropylene centrifbgetube. 12.3 Return unused samples to freezerafter extraction.amounts have been removed. 12.4 Record the initial volume on the extraction worksheet. 12.5 Labelthe 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 s.tandards, ready for e.Ktractionwith surrogate standard as described in 11.2. 12.7 Spike each matrix with the appropriateamount ofstandard as described in 11.1, or Table 1in that section, for the calibration curve standards. Also prqme matrix spikes and contiwing calibration standards. I 12.8 Vortex mix the standard curve samples, matrix spike samples, and continuing calibration samples for 15 seconds. 1 12.9 Check to ensure the 0.5 M TBA reagent is at pH 10. If not, acljust accordingly. 12.10 To each sample, add 1mL 0.5 M TBA and 2 mL of 0.25M so13iumcarbonatelsodium 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 300rpm,for 20 minutes. 12.13 Centrifuge for 20 to 25 minutes at a setting of 3500 rpm,or until layers are well separated. ETS-8-4.1 ExtractionofPFOS from Scnun Page 7 of I4 3M Environmental Laboratory Page 100 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 12.14 12.15 12.16 12.17 12.18 12.19 . 12.20 12.21 12.22 Label a fresh 15 mL centrifuge tube with the same information as in 12.5. Remove 4.0 mL of the organic layer to this clean 15 mL centrifugetube. Put each sample on the analytical nitrogen evaporator until dry, approximately 1to 2 hours. Add 1.OmL of methanol to each centrifugetube using a graduatedpipette. Vortex mix for 30 seconds. Attach a 0.2 pm nylon mesh filter to a 3 cc syringe and transfixthe sample to this syringe. Filter into a 1.5 mL glass autovial or low-volume autovial when necessary. Label the autovial with the study number,animal number and gender, sample timepoint, matrix,finalsolvent, extraction date, and analyst(s) performing the extraction. Cap and store extracts at room temperature or at approximately 4 "C until analysis. Complete the extraction worksheet, attached to this document, and tape in the study notebook or include in study binder, as appropriate. 13.0 DATAANALYSIASN D CALCULATIONS 13.1 Calculations 13.1.1 Calculate actual concentrations of PFOS, or othex applicable fluorochemical, in calibration standards using the following equation: I mL of standard x concentration of standard fw/dl - . mL of standard +mL of surrogate standard +initial matrixvolume (mL) Final Concentration (pg/mL) of PFOS in matrix 14.0 METHOPDERFORMANCE 14.1 The metbod detection limit (MDL) is analyte and matrix specific. Refer to MDL report for specific MDL and limit of quantitation (LOQ values (see Attachments B and C). 14.2 The following quality controlsamples are extractedwith each batch of samples to evaluate the quality of the extraction and analysis. 14.2.1 Method blanks and matrix blanks. 14.2.2 Matrix spike and matrix spike duplicate samples to determine accuracy and precision of the extraction. 14.2.3 Continuing calibration check samplesto determinethe continued accuracy of the initial calibration curve. 14.3 Refer to section 14 of ETS-8-5.1 for method performance criteria. 15.0 POLLUTION PREVENTION AND WASTE MANAGEMENT 15.1 Sample waste is disposed in biohazard containers, flammable solvent waste is disposed in high BTU containers, and used glass pipette waste is disposed in broken glass containers located in the laboratory. ETS-8-4.1 Extraction of PFOS &om Serum Page 8 of 14 3M Environmental Laboratory Page 101 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 16.0 RECORDS 16.1 Complete the extraction worksheet attached to this method, and tape in &e 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, MDULOQ 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-1. 18.2 FACT-M-3.1, "Analysis of Serum or Other Fluid Extracts for Fluomchemicals using HPLC-Electrospray Mass Spectrometry" 19.1 ETS-8-5.1, "Analysis of Serum or Other Fluid Extracts for Fluorochemicals using HPLC-Electrospray Mass Spectrometry" 20.0 REVISIONS . Revision Number 1 Reason For Revision Section 12.21 Changedto include sample storage at room t e m p h e . 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 04/02/99 3M Environmental Laboratory ETS-8-4.1 Extraction of PFOS from Serum Page 9 of 14 Page 102 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 Study # Matrix Box # rr - Extraction Worksheet ETS-8-4.1 1 SurrogateStd approx. ppm actual ppm FC-Mix approx. 0.5 pm actual ppm FC-M~Xapprox. 5 ppm actual ppm FC-Mix approx. 50 ppm actual ppm # Comments ccv I I MS I I I I 1 I I I - I - I I I Blank Std ## Serum Extraction Method voltex 15 sec. Pipette Matrix Pipette 1 mL of 0.5 M TBA, pH 10. pH = amount = voiume mL Std. # Pipette 2 mL of 0.25 Na2C03/0.25M NaHCO3 buffer Std. # Dispense 5 mL of methyl-t-butyl ether - Shake 20 min. CenmfuKe 20-25 min. TN-AShaker weed Centrifuge meed Remove a 4 mL aliquot of organic layer Put on Nitrogen Evaporator to dryness Add methanol Volume Temperature: mL TN-A- Vortex 30 sec. Filter using a 3cc B-D swinge with a 0.2um filter into a 1.5 rnL autosample vial Cont. Cal. Verifications used same matrix as for std curve. 3mL I Date & Initials Attachment A 3M Environmental Laboratory ETS-84.1 Extraction of PFOS from Serum Page 10 of 14 Page 103 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 MDLLOQ values for rabbit serum Compound MDL LOQ Linear Calibration Range PFOS PFOSA PFOSAA EtFOSE-OH M556 PFOSEA @pb) 1.74 1.51 3.46 11.4 6.03 5.71 @pb) 5.55 4.79 20.5 36.2 19.2 18.2 Approximate concentrations to be used for preparing the Standard Calibration Curve - 5 ppb 1000ppb - 5 ppb 1000 ppb - 5 ppb 1000ppb - 5 ppb 1000ppb 5 ppb - 1000 ppb - 5 ppb lOOOppb determined. Two CUNCS in each ofthtse matric~w~ere urtracted and analyzedwith the rabbit serum curves to determineequivalence. Responsesin the rat, bovine,monkey,and human were equivalentto the rabbit responses, therefore, their MDL and LOQ will be the same values as determined in rabbit SenUn. please see LOQ Summary and MDL study in ETS-84.0 & 5.0-V-1 for fhrther information. Attachment B:MDULOQ Summary ---. 3M Environ.me.n. tal Laboratory ETS-8-4.1 Extraction ofPFOS from Serum Page 11of 14 Page 104 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 Rabbit Serum Full Range Prepared range of standards (PPb) (ndW - 0.995 970 LCR from curve Ob) (ng/mL) 24.8 - 978 LOW Curie - 4.94 248 4.94 - 248 Sigh curve - 97.8 978 97.8 - 978 I 1fi I 0.995 -978 I 4.94-978 % Recovery Range 83-108 85-104 85-106 1 94-111 I R:ID Range - 4.67-1 1.0 4.60-10.5 I Rabbit Serum Prepared range of standards (PPb) (ng/mL) FullRange Low curve High curve 1/X . 0.993-976 4.93 - 97.6 24.8 - 976 0.993 - 976 LCB fkom curve bb) (ng/mL) 4.93 - 976 4.93 - 97.6 24.8 - 978 - 4.93 976 % Recovery Range 88-103 . . 87-105 93-102 94-103 -- 5.10-14.7 -- 9.85-14.7 -- 5.08-13.9 - 5.10- .*14.5 Full Range 0.991 - 974 24.7 - 974 81-111 4.18-10.6 I I I I I . I I I I I rHighcune I I I 1 I ~~ 4.92-247 49.2-974 ' 0.991 - 974 9.74-247 97.4-974 9.74-974 97-107 85-108 95-115 6.38-21.8 4.33-12.5 ~~ 4.11-23.2 Attachment B:MDULOQ Summary ETS-8-4.1 Extraction of PFOS from Serum 3M Environmental Laboratory . Page 12 of 14 Page 105 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 Rabbit Serum Prepared range of standards O b ) (ng/mL) Full Range 0.993 - 976 1 Low Curve I 4.93-97.6 High curve 49.3 - 976 1/X - 0.993 493 LCR from curve Ob) (ng/mL) , 49.3-976 I 9.76-97.6 97.6 - 976 9.76 - 976 % Recovery Range ' 77-110 I 97-107 I 90-109 86-111 RSD Rmge - 11.;!-25.5 14.1.-21.3 - 11.5-19.6 1 1.1-2 1.2 Rabbit Serum Preparedrange of standards 0(ng/mL) Full Range 0.993 - 976 Low Curve - 4.93 248 I I I Highcurve I 1 r1E 49.3-976 0.993-976 LCRfiom curve @PV (ndmu 24c8 - 976 9.76 - 248 49.3- 976 9.76-976 %Recovery Range 96-106 91-110 I I 86-106 I 1 95-117 10.1-16.2 11.8-19.5 10.2-18.2-1 lO.l-l9F] . Rabbit Serum Prepared range of standards (PPb) (ng/mL) LCR from cwe (PPb) % Recovery Range Full Range - 0.993 976 24.8 - 976 88-106 Low Cwve 4.93 - 97.6 9.76- 97.6 100-105 High curve 97.6 - 976 97.6 - 976 81-111 I m 1 0.993 - 976 I 9.76-976 I 97-110 1 R3Fi Range - 4.82.17.9 - 5.9548.2 5.11 -9.74 4.77-19.5 I Attachment B:MDLiLOQ Summary ETS-8-4.1 Extraction of PFOS from Serum 3M Environmental Laboratory Page 13 of 14 Page 106 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 Ion Prep date@): Analyte(s): Sample matrix: Methodhevision: Target analyte(s): FC mix std approx. 0.500 ppm: FC mix std approx. 5.00 ppm: FC mix std approx. 50.0ppm: Surrogatestd approx 100ppm: Pair Standard Curves - Fluidis Standard number: Equipment number: Final solvent and TN: Blank fluididentifler: Actual concentrationsof standards in the FC mix Final vol I Calculated concentrations of standardsin the sample matrix PFOS PFOSA PFOSAA EtFOSE PFOSEA M556 Final conc Final conc Final conc Final conc Final conc Final conc Std ubnc AmY mikd Attachment C:Ion Pair Standard Curves ETS-84.1 Extraction of PFOS from Senun 3M Environmental Laboratory Page 14 of 14 Page 107 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 3M ENVIRONEMNTAL LABORATORY METHOD EXTRACTION OF POTASSIUMPERFLUOROOCTANESULFONATEOR OTHER FLUOROCEEMICAL COMPOUNDS FROM LIVER FOR ANALYSIS USING HPLC- ELECTROSPRAYMSAPSESCTROMETRY Method Number: ETS-8-6.0 Author: Lisa Clemen, Robert Wynne d l Approved By: Revision Date: $ Laboratory hkana&r U Date Technical Reviewer c 7/, q 109 Date 7Q A `2 (3 2 .-,1.0 SCOPE AND APPLICATION , 1.1 Scope: This method is for the extraction of potassium perfluorooctanesulfonate(PFOS) or 1 \ -2 other fluorochemical compounds fiom liver. .- 3 1.2 Applicable Compounds: Fluorochemical surfactants or other fluorinated compounds. < p) 1.3 Matrices: Rabbit, rat, bovine, and monkey livers or other tissues as designated in the Q :2. - `-2 validation report. -.-1 -6 Word 6.0195 ETS-8-6.0 Page 1of 14 Extraction of PFOS fiom Liver 3M Environmental Laboratory Page 108 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 2.0 SUMMARY OF METHOD 2.1 This method describes the procedure for extracting potassium peduorooctanesulfonate (PFOS) or other fluorochemical surfactants from liver, or other lissues, using an ion pairing reagent and methyl-tert-butyl ether (MtBE). In thismethod, sewn fluorochemicalscan be extracted: PFOS, PFOSA, PFOSAA, EtFOSE-OH,.PFOSEA, M556,and surrogate standard. An ion pairing reagent is added to the sample and the imalyteion pair is partitioned into MtBE. The MtBE extract is transferred to a centrifugetube and put onto a nitrogen evaporatoruntil dry. Each extract is reconstituted in 1.O mL methanol then filtered through a 3 cc plastic syringe attached to a 0.2 p n nylon filter into glass autovials. 2.2 These sample extracts are analyzed following method ETS-8-7.0 or other appropriate methods. 3.0 DEFINITIONS 3.1 PFOS: perfluorooctanesulfonate (anion of potassium salt) C8F,,!30, 3.2 PFOSA perfluorooctane sulfonylamide C8F,+302NH2 3.3 PFOSAA: pcrfluorooctane sulfonylamido (ethy1)acetate C,F,7SOzN(CHzCH,)CJ32COz 3.4 EtFOSE-OH:2(N-ethylperfluorooctane sulfonamid0)-ethyl alccthol ' C$ ,,SO,N(CH.$H,)CH,~OH 3.5 PFOSEA: perfluorooctane sulfonyl ethylamide C8F,,SO2N(C&CH,)H 3.6 M556: CJ?,7S0,N(H)(CH,COOH) 3.7 Surrogate standard: lH-lH-2H-2H perfluorooctane sulfonic acid 4.0 WARNINGS AND CAUTIONS 4.1 Health and Safety Warnings: 4.1.1 Use universal precautions, especially laboratory coats, goggles, and gloves when handling animal tissue, which may contain pathogens. 5.0 INTERFERENCES 5.1 There are no interferences known at this time. 6.0 EOUIPMENT 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 V W R ETS-8-6.0 Extraction of PFOS from Liver Page 2 of 14 3M Environmental Laboratory Page 109 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 6.1.5 Nitrogen Evaporator, Organomation 6.1.6 Balance (sensitivityto 0.100 g) 7.0 SUPPLIES AND MATERIALS 7.1 Gloves 7.2 Dissecting scalpels 7.3 Eppendorf or disposablepipettes 7.4 Nalgene bottles, capable of holding 250 mL and 1L 7.5 Volumetric flasks, glass, type A 7.6 I-CHEM vials, 40 mL glass 7.7 Plastic sampde vials, Wheaton, 6 mL (orappropriatesize) 7.8 Centrifuge tubes, polypropylene, 15 mL 7.9 Labels 7.10 Oxford Dispensor -3.Oto 10.0 ml 7.11 Syringes, capableof measuring 5 J.Lto 50 pL 7.12 Graduated pipettes 7.13 Syringes, disposable plastic, 3 cc 7.14 Syringe filters, nylon, 0.2 p,25 mm 7.15 Timer 7.16 Crimp cap autovials and caps 7.17 crimpers Note: Prior to using glassware and bottles, rinse 3 times Withmethanol and 3 timeswith MilliQm water. Rinse syringes a minimumof 9 times withmethanol, 3 rinses fiom 3 separate Vials. 8.0 REAGENTS AND STANDARDS 8.1 Type I reagent grade water, MW-Qm or equivalent; all water used in thismethod should be Milli-QTMwater and be providedby a Mali-Q TOC Plusm system 8.2 Sodium hydroxide (NaOH), J.TBaker or equivalent 8 3 Tetrabutylammoniumhydrogen sulfate(TJ3A), Kodak or equivalent 8.4 Sodium carbonate (Na$OJ, J.T.Baker or equivalent 8.5 Sodium bicarbonate (NaHCO,), J.T.Baker or equivalent 8.6 Methyl-tert-butyl ether, Omnkolv, glass distilled or HPLC grade 8.7 Methanol, Omnisolv, glass distilled or HPLC grade 8.8 Liver, frozen from supplier 8.9 Dry ice fi-om supplier 8.10 Fluorochemical standards 8.10.1 PFOS (3M Specialty Chemical Division), molecular weight = 538 ETS-8-6.0 Extraction ofPFOS from Liver Page 3 of 14 3M Environmental Laboratory Page 110 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 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 (3MSpecialty Chemical Division), molecular weight = 557 8.10.7 S m g a t e standard: 4-H, perfluorooctane sulfonic acid (1-H, 1-H, 2-H, 2-H C,F,,SO,H) molecular weight = 428 8.10.8 Other fluorochemicals, as appropriate 8.11 Reagent preparation NOTE: When preparing larger volumes than listed in reagent, standard, or surrogate preparation, adjust accordingly. 8.11.1. 10N sodium hydroxide (NaOH): Weigh approximately200 g NaOH. Pour into a 1000 mL beaker containing 500 mL Mi%-Q" water, mix until all solids are dissolved. Store in a 1L Nalgene bottle. 8.11.2 1N sodium hydroxide (NaOH): Dilute 10N NaOH 1:10. Measure 10I& of 10N NaOH solutioninto a 100mL volumetric flask and dilute to volume using Milli-Q" water. Store in a 125 mL Nalgene bottle. 8.1 1.3 0.5 M tetrabutylammonium hydrogen sulfate (TBA): Weigh approximately 169 g J of TBA into a 1L volumetric containing 500 mLMilli-Qm water. Adjust to pH 10using approximately44to 54 mL of 10N NaOH (While adding the last mL of NaOH, add slowly because the pH changes abruptly). Dilute to volume with Milli-Qm water. Store in a 1 L Nalgene bottle. 8.11.3.1 TBA requires a check prior to each use to ensure pH = 10. Adjust as needed using 1 N NaOH solution. 8.11.4 0.25 M sodium carbonatdsodiumbicarbonatebuffer @k&03/N&C03): Weigh approximately 26.5 g of sodium carbonate (Na.$OJ and 21.0 g of sodium bicarbonate (NaHC03) into a 1 L volumetric flask and bring to volume with Mi1l.i- Q'" water. Store in a 1 L Nalgene bottle. 8.12 Standards preparation 8.12.1. Prepare PFOS standards for the standard curve. 8.12.2 Prepare other fluorochemical standards, as appropriate. Multicomponent fluorochemical standards are acceptable (for example, one working standard solution containing 1.OO ppm PFOS, 1.02ppm PFOSA, 0.987 ppm PFOSAA, and 1.10 ppm EtFOSE-OH.) 8.12.3 Weigh approximately 100 mg of PFOS into a 100 mL volumetric flask and record the actual weight. 8.12.4 Bring to volume with methanol for a stock standard of approximately 1000ppm (PLg/mL). 8.12.5 Dilute the stock solution with methanol for a working standard 1 solution of approximately 50 ppm. ETS-8-6.0 Extraction of PFOS from Liver Page 4 of 14 3M Environmental Laboratory Page 111 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 8.12.6 Dilute the stock solution with methanol for a working standard 2 solution of approx. 5.0 ppm. 8.12.7 Dilute the stock solution with methanol for a working standard 3 solution of approx. 0.50 ppm. 8.13 Surrogate stock standard preparation 8.13.1 Weigh approximately 50-60mg of surrogate standard 1-H,1-H, 2-H,2-H, C$,,SO,H into a 50 ml voIumetric flask and record the actual weight, 8.13.2 8.13.3 Bring to volume with methanol for a surrogate stock of approximately 1000-1200 PP** Prepare a surrogate working standard. Transfer approximately 1.0 ml of surrogate stock to a 10mlvolumetric flask and bring to vo1um.ewithmethanol for a working standard of 10-20 ppm. Record the actual volume transferred. 9.0 SAMPLE HANDLING 9.1 All samples are received ftozen and must be kept fiozen until the extraction is performed. 10.0 O U A L I T ~CONTROL 10.1 Matrix blanks and method blanks 1 10.1.1 An aliquot of 1.0 mLmethanol is used as a solvent blank 10.1.2 Extract two 1.0mL aliquots of Milli-QTMwater following this procedure and use as method blanks. 10.1.3 Extract two 1.0mL aliquots of liver homogenate followingthis procedure and use as matrix blanks. Refer to 11.1.6. -. 10.2 Matrix spikes 10.2.1 Prepare and analyze matrix spike and matrix spike duplicate samples to determine the accuracy of the extraction. 10.2.2 Prepare each spike using a sample chosen by the analyst, usually a control liver received with each sample set. 10.23 Expected concentrationswill fall'inthe mid-range of tlie initial calibration curve. Additional spikes may be included and may fall in the low-range of the initial calibration curve. 10.2.4 Prepare one matrix spike and matrix spike duplicate pcr 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. 103.2 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 112 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 10.3.3 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 - 100ppb, rather than 5 ppb - 1000 ppb). 11.0 CALIBRATION AND STAND-IZATION 11.1 Prepare matrix calibration standards 11.1.1 Weigh approximately 40 g of liver into a 250 mL Nalgese bottle containing 200 m L s Milli-Q""'water. Grind to a homogeneous solution. 11.1.2 If 40g is not available,use appropriate amountsof liver and water to ensure a 1 5 ratio. 11.13 Refer to 13.0 to calculate the actual density of liver homogenate and the concentrationof solid liver tissue dispersed in 1.O mL of homogenate solution. 11.1.5 Add 1 mL of homogenateto a 15 mL centrihge tube. Re-suspendsolutionby shaking between aliquots while preparing a total of eighteen 1 mL aliquots of homogeneous solution in 15mL centrifugetubes. I 11.1.6 Two 1 mL aliquots, or other appropriate volume, serve as matrix blanks. ' 11.1.7 Typicafly 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 bianks. 11.1.8 Refer to validation reports ETS-84.0 and ETS-8-7.0-V-1or Attachment B, which lists the working ranges and the Linear Calibratica Range (LCR) for calibration curves. 11.1.9 Use Attachment C as an aid in calculating the concentrations of the working standards. .Referto 13.0 to calculate actual concentrationsof PFOS in calibration standards. 11.2 To each working standard,blank, or continuing verification, add appropriate amount of surrogate working standard for the concentration to fall within the calibration curve range 5 ppb - 1OOOppb. 3M Environmental Laboratory ETS-8-6.0 Extraction of PFOS fiom Liver Page 6 of 14 Page 113 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 11.3 Extract spiked liver homogenates following 12.14-12.25 of this method. Use these standardsto estabIish each initial curve on the mass spectrometer. 1 Table 1 Approximate Spiking Amounts for Calibration Standards Working Standard Pl - (Approx. Conc.) - .0.50m m A* 0.50 ppm 5.0 ppm 5.0 Durn 0.025 ppm 20 0.050 w m 40 10 20 0.500 Dum I 50 PPm 4 12.0 PROCEDURE 12.1 Obtain fiozen 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 tared plastic sampule vial. 12.4 Record the liver weight in'the study notebook 12.5 Return unused liver portions to fkeezer. 12.6 Add 2.5 mLs of water to sampule vial. 12.7 Grind the sample. Put the grinder probe in the sample and grind for about 2 minutes, or until the sampleis homogeneous. 12.8 Rinse the probe into the samplewith 2.5mLs water using a pipette. 12.9 Take the grinder apart and clean it with methanol after each sample. Refer to W T - E P 22. 12.10 Cap the sample and vortex for 15 seconds. Label the sampule vial with the study number, weight, liver ID, date and analyst initials. 3M Environmental Laboratory ETS-8-6.0 Extraction ofPFOS from Liver Page 7 of 14 Page 114 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 12.11 Pipette 1.0 mL, or other appropriate volume, of homogenate into a 15 mL polypropylene centrifuge tube. Label the centrifuge tube with the identical idolmation as the sampuie vial. Refer to attached worksheet for documenting the remaining,steps, 12.12 Pipette two 1 mL aliquots of Milli-Q" water to centrifugetubes. These will s a v e as method blanks. 12.13 Spike all samples, including blanks and standards ready for extraction with surrogate standard as described in section 11.2. 12.14 Spike each m a h with the appropriateamount of standard as described in 11.1, or Table 1 of that section, for the 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. If not, adjust ciccordingly. 12.17 To each sample, add 1niL 0.5 M TBA and 2 mL of the 0.25 M sodium carbonatdsodium bicarbonate buffer. 12.18 Using an Oxford Dispenser, add 5 mL methyl-tert-butyl ether. 12.19 Cap each sample and put on the shaker at a setting of 300 rpm, for 20 minutes. 12.20 Centrifuge for 20to 25 minutes at a setting of 3500 rpm,or until layers are well separated. 12.21 Label a fiesh 15mL centn'fugetube with the same information as in 12.10. 12.22 Remove 4.0mZ,of the organic layer to the fresh 15mL centrifuge tube. . 12.23 Put each sample on the analytical nitrogen evaporatoruntil dry,approximately 1to 2 hours. 12.24 Add 1.O mL to each centrifuge tube using a graduatedpipette. 12.25 Vortex mix for 30 seconds. 12.26 Attach a 0.2pnylon mesh filter to a 3 cc syringe and transfer the sample to this syringe. Filter into a 1.5 mL glass autovial or low-volume autovial when necessary. 12.27 Label the autovial with the study number, animal number and i:ender, sample timepoint, matrix, final solvent, extraction date, and analyst(s) perfoming the extraction. 12.28 Cap and store extracts at room temperature or at approximately 4 "C until analysis. 12.29 Complete the extraction worksheet., attached to this document, and tape in study notebook or include in study binder, as appropriate. 3M Environmental Laboratory ETS-8-6.0 . Extraction of PFOS from Liver Page 8 of 14 Page 115 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 13.0 DATAANALYSIASND CALCULATIONS 13.1 Calculations: 13.1.1 Calculate the average density of the liver homogenate b y recording each mass of ten separate 1.O mL aliquots of homogenate. Average density (mg/mL) =Average mass f m d of the a.liauots 1.0 mL aliquot 13.1.2 Calculate the amount of liver (mg) per 1.0 mLhomogenate (or concentrationof dispersed solid tissue per mL of homogenate suspension.) using the following equation: g of Liver x Average d a s h * ofhomomate (mdndLJ (g of Liver + g of Water) * refer to 13.1.1 for details. 13.1.3 Calculate actual concentralions of PFOS and other fluorochemicals in calibration standards using the following equation: J' JJLof Standardx Concentrationfun /mL) =FinalConcentration (pg/g or mgkg) mg Liver/ 1mL homogenate* of PFOS in Liver *refer to 13.1.2 for details. 14.0 bfETHOD PERFORMANCE 14.1 The method detection limit (MDL)is analyte and matrix specific. Refer to MDL report for specificMDL and limit of quantitation (LOQ)values (refer to Attachments B and C). 14.2 The followihg quality control samples are extractedwith each batch of samples to evaluate the quality of the extraction and analysis. 14.2.1 Method blanks and matrix blanks. 14.2.2 Matrix spike and matrix spike duplicate samples to determine accuracy and . precision of the extraction. 14.2.3 Continuing calibration verification samples to determine the continued accuracy of the initial calibration curve. 14.3 Refer to section 14 of ETS-8-7.0 for method performance criteria. 15.0 POLLUTIOPNREVENTIONAND WASTEMANAGEMENT 15.1 Sample waste is disposed in biohazard containers, flammable solvent waste is disposed in high BTU containers, and used glass pipette waste is disposedi n broken glass containers located in the laborkory. 3M Environmental Laboratory ETS-8-6.0 ExtractionofPFOS from Liver Page 9 of 14 Page 116 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 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 `TABLEDSI, AGRAMFSL.OWCHARTASN.D VALIDATION DATA 17.1 Attachment A, Extraction worksheet 17.2 Attachment B, MDLJLOQ values and sumxiary 17.3 Attachment C, Calibration standardcalculationand concentrationworksheet 18.0 REFERENCES 18.1 The validation report associatedWith this method is ETS-8-6.0 ,&7.0-V-1. 18.2 AMDT-EP-22, ``Routhe Maintenance of Ultra-Turrax T-25" 18.3 FACT-M-1.1, "Extractionof PFOS or Other Anionic FluorochemicalSurfactantsfrom Liver for Analysis Using HPLC-Electrospray/Mas Spectrometty" 19.0 AFFECTED DOCUMENTS 19.1 ETS-8-7.0, "Analysis of Liver Extracts for Fluorochemicalsusing HPLC-Electrospray Mass Spectrometry'' 20.0 RJCWSIONS Revision Number. .. Reason For Revision Revision 3M Environmental Laboratory ETS-8-6.0 Extraction ofPFOS from Liver Page 10 of 14 Page 117 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 Study # Matrix Box # may Date SpikedAnalyst ccv MS MSD Surrogate Std approx. pprn actual ppm # FC Mix Std approx. 0.5 ppm actual ppm # FCMixStd I FC Mix Std I Comments I approx. 5 ppin approx. 50 ppm actual pprn actual ppm # - # - I I R Date & Initials Vortex 30 sec. Filter usins a 3cc B-Dsyringe with a 0.2um SRI filterinto autosamplevial Cont. Cal. Verifications used the same matrix as for the standard curve. Attachment B: MDYLOQ Values ETS-8-6.0 Extraction of PFOS from Liver 3M Environmental Laboratory Page 1 1 of 16 Page 118 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 Compound PFOS PFOSA PFOSAA EtFOSE-OH M556 PFOSEA MDL (ppb) 8.45 3.50 24.6 108 82.3 33.9 LOQ (ppb) 26.9 11.1 78.3 345 262 108 Linear Calibration Range (LCR) Approximate concentrations to lie used for preparing the Standard Calibration Curve 30 ppb - 1200 ppb 12ppb - 1200ppb 30 ppb - 1200ppb 60PPI, - 900mb* 60ppb - 1200ppb 30ppb- 1200ppb MDL,/LOQ values in rat, bovine, and monkey liver were not statisticrtlly determined. Two curves in each of these matrices were extracted and analyzedwith the: rabbit liver curves to determine equivalence. Responses in the rat, bovine, and monkey liver curves were equivalent to the rabbit responses, therefore, their MDL and LOQ will be assumed to be equivalent to those values as determined for the rabbit liver. Refer to LOQ'Summary and MDL study in ETS-84.0 & 7.0-V-1 for finrthtr information * EtFOSE-OH estimates only for h4DL and LOQ. Did not meet criteria fcr validation. Compound: PFOS Compound: PFOSA Liver matrix Rabbit Prepared range of standards ( P P ~ )(ng/mL) 6.16 - 1232 Rangeof average curve (PP~)tndmL) 12 - 1200 LGlZ&m.? ave curve (ppb) (ndW 30 - 1200 Rangeof low std curve (ppb) (nglmL) 30 - 900 -60 9ao high std Attachment B:MDULOQ Values 3M Environmental Laboratory ETS-8-6.0 Extraction of PFOS from Liver Page 12 of 16 Page 119 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 Liver matrix Rabbit Prepared range of standards - (ppb) (n%nL) 6.17 1235 Rangeof average We (Ppb) (ndrnI-1 31 - 900 LCRfimm ave curve (Ppb) (ng/mL) -31 900 Rangeof low std CUNC @Pb) ( n d W NIA low std NIA high std high std Attachment C:Standard Calculations 3M Environmental Laboratory ETS-8-6.0 Extraction of PFOS from Liver Page 13 of 14 Page 120 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 Ion Pair Standard Curves - Tissue Prep date@): Analyte(s): Sample matrix: Methodrevision: Target analyte(s): FC mix std approx. 0.500ppm: FC mix std approx. 5.00ppm: FC mix std approx. 50.0 ppm: Surrogate std approx. 100 ppm: Standard number: Equipment number:' Final solvent and TN: Blank livedidentifier: Actual concentrationsof standardsin the FC mix Calculated concentrations of standards in the sample matrix Liver Rabbit Bovine Rat Monkey PFOS 1 PFOSA I 5-1000ppb I 5-1000 ppb I Estimates only, use rabbit values. Estimates only, use rabbit values. Estimates only, use rabbit values. PFOSAA 5-1000 ppb 1 EtFOSE-OH I 5-1000 ppb Attachment C Standard Calculations ETS-8-6.0 Extraction of PFOS from Liver 3M Environmental Laboratory Page 14 of 14 Page 121 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 3M ENVIRONMENTLAALBORATORY .. METHOD ANALYSIS OF kUOROCHEMICALS IN LIVER EXTRACTS USING HPLC-ELECTROSPRAYMASS SPECTROMETRY Method Number: FACT-M-2.0 Author: Lisa Clemen Approved By: 7 - r, /. Laboratory Manager Adoption Date: 5/& 198 Rtrvision Date: jA Date /hA Technical Reviewer 5-t27/96 Date -. I_ 1.0 SCOPE AND APPLICATION .: I.IScope: This method is for the analysis of extracts of liver or other tissues for fluorochemical sudactants using HPLC-electrospray/mas spectrometry. * 1.2 Applicable Compounds: Potassium perfluorooctanesulfonate, anionic fluorochemical surfactants, or other ionizable compounds. 1.3 Matrices: Rabbit, rat, bovine, and mobkey livers or other livers eis designated in the x m lirlatinn r m n n r ) -. -.J -LI) Word 7.0.1/95 FACT-M-2.0 Analysis of Liver Extract Using ESMS Page 1 of 8 3M Environmental Laboratory Page 122 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 2.0 SUMMARY OF METHOD 2.1 This method describes the analysis of fluorochemical surfactanti extracted from liver using HPLC-electrospray/mass spectrometry. The analysis is performed by monitoring a single ion characteristic of a particular fluorochemical, such as the potassium perfluorooctanesulfonate(PFOS) anion, M/Z= 499. Samples may also be screened to verify compound identification. 3.0' DEFINITIONS 3.1 None. 4.0 WARNINGS AND CAUTIONS 4.1 Health and Safety Warnings: 4.1.1 Use caution with the voltage cable for the probe. When the voltage cable is plugged into the probe DO NOT TOUCH THE PROBE,there is risk of electrical shock. 4.2 Cautions: 4.2.1 Do not runsolvent pumps above capacity of 400 bar (58100 psi). If pressure goes over 400 bar, the HP 1100will initiate automatic shutdown. 1 4.2.2 Do not run solvent pumps to dryness. 5.0 INTERFERENCES 5.1 Teflon should not be used for sample storage or any part of instrumentationthat comes in contact with the sample or extract. 6.0 EQUIPMENT 6.1 Equipment listed below may be changed in order to optimize the system. 6.1.1 Micromass Electrospray Mass Spectrometer 6.1.2 HP1100 low pulse solvent pumping system and autosampler. 7.0 SUPPLIESAND MATERIALS 7.1 Supplies 7.1.1 Nitrogen gas,refrigerated liquid, regulated to approximately 100psi. 7.1.2 HPLC column, specifics to be determined by the analyst. 7.1.3 Capped autovials or capped 15 mL centrifuge tubes. 8.0 REAGENTASND STANDARDS 8.1 Reagents 8.1.1 Methanol, HPLC grade or equivalent. Word 7.0.1195 FACT-M-2.0 Analysis of Liver Extract Using ESMS Page 2 of 8 3M Environmental Laboratory Page 123 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 8.1.2 Milli-QM water, all water used in this method should be Milli-Qm water and may be provided by a Milli-Q TOC Plus system. 8.1.3 Ammonium acetate, HPLC grade or equivalent. 8.2 Standards 8.2.1 Typically one H,O blank, one live.+blank, and seven liver standardsare prepared during the extraction procedure. See FACT-M-1. 9.0 SAMPLHEANDLING 9.1 Fresh liver standards are prepared with each analysis. Extracted standards and samples are stored in capped autovials or capped 15 mL centrifuge tubes until analysis. 9.2 If analysis will be delayed, extracted standards and samples may be refiigerated until analysis can be performed. 10.0 QUALITY CONTROL 10.1 Matrix Blanks and Method Blanks 10.1.1 Analyze a method blank and matfix blank prior to each calibration curve. 10.2 Matrix Spikes ' 10.2.1 Analyze a matrix spike and matrix spike duplicate with each analysis. 10.2.2 Expected concentrations will fall in the mid-range of the initial caIibration curve. Additional spike concentrations may fall in the low-range of the initial calibration curve. 10.2.3 See section 13 to calculate percent recovery. 10.3 Continuing Calibration Checks 103.1 Analyze a mid-range calibration standard after every tenth sample. If a significant change (.t30Yo) in peak area occurs, relative to the initial standard curve, stop the ruu.Only those samples analyzed before the last acceptable calibration standard will be used. The remaining samples must be reanalyzed. 1033 See section 13 to calculate percent difference. 10.4 System Suitability 10.4.1 System suitability (e.g. peak area, retention time and peak shape, etc.) will be assessed for each run. 11.0 CALIBRATIONAND STANDARDIZATION 11.1 Analyze the extracted liver standards prior to and following each set of extracts. The mean of two standard values, at each standard concentration,will be plotted by linear regression for the calibration curve using MassLynx or other suitable sofhvare. 3M Environmental Laboratory FACT-M-2 .O Analysis of Liver Extract Using E S N S Page 3 of 8 Page 124 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 11.2 The ? value for the data should be 0.98 or greater. Lower values may be acceptable at the discretion of the analyst. 11.3 Ifthe curve does not meet requirements, perform routine mainte:nmceor reextract the standard curve (if necessary) and reanalyze. 12.0 PROCEDURES 12.1 Acquisition Set up 12.1.1 Click on start button in the Acquisition Control Panel. Set up a sample list. Assign a filename using letter-MO-DAY-last digit of year-sample number, assign a method (MS) for acquiring, and type in sample descriptions. 12.1.2 To create a method click on scan button in the Acquisition control panel and select SIR. Set Ionization Mode as appropriateand mass to 499 or other appropriate masses.<A scan is usually collected along with the SIRs Save method. 12.13 Typically the sample list begins with the first set of liver standards and ends with the second set of standards. 12.1.4 Samples are analyzed with a continuing calibration check injected after every tenth sample. Solvent blanks should be analyzed periodically to monitor possible analyte carryover and are not considered samples but may be included as such. $2.2 Using the Autosampler 12.2.1 Set up sampletray accordingto the sample list prepared in section 12.1.l. Time 0.00 min. t 7.5 min. 11.0min.. I MeOH 45% 90% 90% Ammonium ricetate I 10% 1 10% Note: In this instrument configuration, the run must be set up on the electrospray s o h a r e with a "Waiting for inlet start" message tlefore the "Start'' button is pressed on the HP Workstation. 12.2.2.5 Press the "Start" button. FACT-M-2.0 Analysis of Liver Extract Using ES/MS Page 4 of 8 3M Environmental Laboratory Page 125 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 12.3 Instrument Sep-up 12.3.1 Refer to AMDT-EP-31 for more details. 123.2 Check the solvent level in reservoirs and refill if necessary. 12.33 Check the stainless steel capillary at the end of the proba. Use an eye piece to check the tip. The tip should be flat with no jagged edges. If the tip is found to be unsatisfactory, disassemblethe probe and replace the stainlesssteel capillary. - 123.4 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. 123.5 Turn on the nitrogen. A fine mist should be expelled with no nitrogen leaking around the tip of the probe. 12.3.6 The hstrument 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 12.3.63 ESI nebulizing gas 10-15 literdhow 123.63 LC constant flow mode flow rate 10- 500 uLhnin 12.3.6.4 Pressure4 0 0 bar (This parameter is not set, it is a guide to ensure the instrument is operating correctly.) 1 12.3.7 Carefully guide the probe into the opening. Insert probe until it will not go any M e r . Connect the voltage cables to the probe. 12.3.8 Record tune parameters in the instrument log. 123.9 Using the cross-flowcounter electrode in the E S N S source is recommended for the analysis of biological matrices. 123.10 Click on start button in the Acquisition Control Panel. Press the start button at top of sample list. Ensure start and end samplenumber .includesall samples to be analy2ed. 13.0 DATAANALYSIS AND CALCULATIONS 13.1 Calculations: - 13.1.1 Calculate matrix spike percent recoveries using the following equation: YORecovery = ObservedResult Background Result x 100 Expected Result 13.1.2 Calculate percent difference using the following equation: % Difference = Expected Conc. - Calculated Conc. x 100 Expected Conc. 3M Environmental Laboratory FACT-M-2.0 Analysis of Liver Extract Using ESMS Page 5 of 8 Page 126 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 13.1.3 Calculate actuai concentration of PFOS anion in total liver (mg): (ug PFOS anion~alch. r n std m e ) 14.0 METHOPDERFORMANCE 14.1 The method detection limit is equal to at least three times the baseline noise in the matrix blank. 14.2 The practical quantitation limit is equal to the lowest standard i nthe calibrationcurve. 15.0 POLLUTION PREVENTION AND WASTE MANAGEMENT 15.1 Sample waste is disposed in biohazard containers, flammable solvent waste is disposedin high BTU containers, and glass pipette waste is disposed in broken glass containers. All containers are located in the laboratory. 16.0 RECORDS 16.1 Store chromatogramsin the study folder. Each chromatogram should have the following ' information included either in the header or hand written on the chromatogram: study number, sample name, extraction date, and dilution factor (if applicable). 16.2 Plot calibration curve by linear regression and store in the study folder. 16.3 Print sample list fiom MassLynx and tape into the instrument nmlog. 4 16.4 Print data integration summary from MassLynx and tape into the instrument d o g . 16.5 Copy instrument runlog pages, including instrument parameters and sample results, and tape into appropriate study notebook. 16.6 Summarize data using suitable software and store in the study folder. 16.7 Back up electronic data to appropriate media Record in study notebook the file name and location of backup electronic data, 17.0 TABLESDI,AGRAMFSL,OWCHARTS,AND VALIDATIONDATA 17.1 Attachment A: FACT-M-2 Data reporting spreadsheet 17.2 The validation report associated with this method is FACT:M-1.0 & 2.0-V-1. 18.0 REFERENCES 18.1 AMDT-EP-31, "Operation of VG Platform Electrospray Mass Spectrometer" 3M Environmental Laboratory FACT-M-2.0 Analysis of Liver Extract Using ES/MS Page 6 of 8 Page 127 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 19.0 AFFECTEDDOCUMENTS 19.1 FACT-M-1.O, ``Extraction of PotassiumPeffluorooctanesulfonatefrom Liver for Analysis Using HPLC-Eltctrospray/MassSpectrometry" 20.0 REVISIONS Revision Number. Reason For Revision Revision - Date 3M Environmental Laboratory FACT-M-2.0 Analysis of Liver ExtractUsing ESMS Page 7 of 8 Page 128 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 Laboratory Study # Study: Test Material: M a M i n a l Solvent MethodRevision: Analytical Equipment SystemN u m k . Instrument SoftwareNersion: Filename: R-Squared Value: Slope: Y Intercept: Date of Extraction/Analyst: Date of Analysis/Analyst: Group I S a m p l d I Concentration Slope: Taken from linear regression equation. Grouprnose: Taken fiom the study folder. Sample# Taken from the study folder. Concentration (ug/mL):, Taken from the MassLynx integration summary. Initial Volume (mL): Taken from the study folder. Dilution Factor: Taken from the study folder. Final Conc. (ug/mL): Calculated by dividingthe initial volume from the concentration 3M Environmental Laboratory FACT-M-2.0 Analysis ofLiver Extract Using ESMS Page 8 of 8 Page 129 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 I 3M ENVIRONMENTALLABORATORY I . ANALYSIS OF FLUOROCHEMCALSIN SERUMEXTRACTUSSING HPLC-ELECTROSPRAYMASS SPECTRCMETRY Method Number: FACT-M-4.0 Author: Lisa Clemen q App_ro.ved By: Jr L -4. Laboratory M m e r Itevision Date: ' Ml/Q Date dk r . TechnicalReviewer Y/d98 Date L.6 ==.I !-? -r-. 1.0 SCOPE AND APPLICATION c , 5% c: (--(VI 1.1 Scope: This method is for the analysis of extracts of serum or tissue for fluorochemical surfactants using HPLC-electrospray/massspectrometry. 1.2 Applicable Compounds: Potassium perfluorooctane&onate, anionic flwrochemical dactants, or other ionizable compounds. 1.3 Matrices: Rabbit, rat,and bovine serum or othez seraas designated in the validationreport. Word 7.0.1/95 FACT-M-4.0 Anaiysis of Serum Extract Using ESMS Page 1 of 8 3M Environmental Laboratory Page 130 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 2.0 SUMMARY OF METHOD 2.1 This method describes the analysis of fluorochemical s&actants extracted fiom serum using HPLC-electrospray/mas spectrometry. The analysis is performed by monitoring a single ion characteristic of a particular fluorochemical,such as the potassium perfluorooctanesulfonate (PFOS)anion,M/Z=499. Samples may also be screened to veri& compound identification. 3.0 DEFINITIONS 3.1 None. 4.0 WARNINGS AND CAUTIONS 4.1 Health and Safety Warnings: 4.1.1 Use caution with the voltage cable for the probe. When the voltage cable is plugged into the probe DO NOT TOUCHTHEPROBE,there is risk of electrical shock. 4.2 Cautions: 4.2.1 Do not run solvent pumps above capacity of 400 bar (5800 psi). If pressure goes over 400 bar, the HP1100will initiate automatic shutdown. 4.2.2 Do not runsolventpumps to dryness. d 5.0 INTERFERENCES 5.1 Teflon should not be used for sample storage or any part of instnunentation that comes in contact with the sample or extract. 6.0 EOUIPMENT 6.1 Equipment listed below may be changed in order to optimize the system. 6.1.1 Micromass Electrospray Mass Spectrometer 6.1.2 HPllOO low pulse solvent pumping system and autosampler. 7.0 SUPPLIES AND MATERIALS 7.1 Supplies 7.1.1 Nitrogen gas, reegerated liquid, regulated to approximati:ly 100psi. 7.1.2 HPLC column, specificsto be determined by the analyst, 7.1.3 Capped autovials or capped 15 mL centrifuge tubes. 8.0 REAGENTS AND STANDARDS 8.1 Reagents 8.1.1 Methanol, HPLC grade or equivalent. FACT-M-4 .O Analysis of Serum Extract Using ESMS Page 2 of 8 3M Environmental Laboratory Page 131 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 8.1.2 Milli-Qm water, all water used in this method should be Milli-Qm water and may be provided by a Milli-Q TOC Plus system. 8.1.3 Ammonium acetate, HPLC grade or equivalent. 8.2 Standards 8.2.1 Typically one H20blank, one serum blank, and seven serum standards are prepared during the extraction procedure. See FACT-M-3. 9.0 SAMPLHEANDLING 9.1 Fresh serum standards are prepared with each analysis. Extracted standards and samples are stored in capped autovials or capped 15mL c e n ~ gtuebes until analysis. 9.2 If analysis will be delayed, extracted standards and samples may be refiigerated at 4 O C until analysis can be performed. 10.0 O u ~ ~ rCrOvNTROL 10.1 Matrix Blanks and Method Blanks 10.1.1 Analyze a method blank and a matrix blank prior to each calibration curve. 10.2 MatrixSpikes 1 10.2.1 Analyze a matrix spike and matrix spike duplicate with each analysis. 10.2.2 Expected concentrations will fall in the mid-range of the initial calibration curve. Additional spike concentrationsmay fall in the low-range of the initial calibration curve. 10.2.3 See section 13 to calculate percent recovery. 10.3 Continuing Calibration Checks 10.3.1 Analyze a mid-range calibration standard after every tenth sample. If a significant change (.t30%) in peak area occurs, relative to the initial:standard curve, stop the run.Only those samples analyzedbefore the last acceptablecalibration standard will be used. The remaining samples must be reanalyzed. 103.2 See section 13to calculatepercent difference. 10.4 System Suitability 10.4.1 System suitability (e.g., peak area, retention time, peak shape, etc.) will be assessed for each run. 11.0 CALIBRATIONAND STANDARDIZATION 11.1 Analyze the extracted serum standards prior to and following each set of extracts. The mean of two standard values, at each standard concentration, will 1Seplotted by linear regression for the calibration curve using MassLynx or other suitable software. 3M Environmental Laboratory FACT-M-4 .O Analysis of Serum Extract Using ES/MS Page 3 of 8 Page 132 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 11.2 The 3 value for the data should be 0.98 or greater. Lower values may be acceptable at the discretion of the analyst. 11.3 If the curve does not meet requirements, perform routine maintenance or reextract the standard curve (ifnecessary) and reanalyze. 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 filenameusing letter-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). Set Ionization Mode as appropriate and mass to 499 or other appropriate masses. A scan is usually collected along with the SIRS. Save method. 12.13 Typically the sample list begins withthe firstset of s e m i standards and ends with the second set of standards. 12.1.4 Samples are analyzed with a continuing calibration check.injected after every tenth sample. Solvent blanks should be analyzed periodically lo monitor possible analyte carryover and are not considered samples but may be included as such. 12.2 Using the Autosampler 12.2.1 Set up sample tray according to the sample list prepared i n section 12.1.1. 122.2 Set-up the HP1lOO/autosampler at the following conditi0.wor at conditions the analyst considers appropriate for optimal response. Record actual conditions in the instrument logbook 12.2.2.1 Sample size= 10 pL injectionwith a samplewash 12.2.2.2 Inject/sample = 1 12.2.23 Cycle time= 15 minutes 12.2.2.4 Sol Time 0.00 min. 7.5 min. 11.O min. 11.5 min. MeOH 45% 90% 90% 45% Ammonium acetate 55% 10% 10% 55% Note: In this instrument configuration,the runmust be set up on the electrospray software with a "Waiting for inlet start" message before the "Start"button is pressed on the HP Workstation. 12.2.2.5 Press the "Start" button. FACT-M-4.0 Analysis of Serum Extract Using ESMS Page 4 of 8 3M Environmental Laboratory Page 133 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 12.3 Instrument Set-up 12.3.1 Refer to Ah4DT-EP-3 1 for more details. 12.3.2 Check the solvent level in reservoirs and refill if necessary. 12.3.3 12.3.4 Check the stainless steel capillary at the end of the probe:. Use an eye piece to check the tip. The tip should be flat with no jagged edge:s. If the tip is found to be unsatisfactory, disassemble the probe and replace the stainless steel capillary. 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 10minutes. 123.5 Turn on the nitrogen. A fine mist should be expelled wiih no nitrogen leaking around the tip of the probe. 123.6 The instrumentuses these parameters at the following settings. These settingsmay change in order to optimize the response: 12.3.6.1 Drying gas 250-400 literslhour 12.3.6.2 ESI nebulizing gas 10-15 litedhour 12.3.63 HPLC constant flow mode flow rate 10- 500 p:L/min 12.3.6.4 Pressure -400bar (This parameter is not set, it is a guide to enswe the HPLC is operating correctly.) r( 123.7 carefully guide the probe into the opening. Insert probe .until it will not go any further. Connect the voltage cables to the prabe. 12.3.8 Record tune parameters in the instrument log. 12.3.9 Using the cross-flow counter electrode in the ES/MS sotme is recommended for the analysis of biological matrices. 12.3.1OClick on start button in the Acquisition Control Panel. Press the start button at top of sample list. Ensure start and end sample number includes all samples to be analyzed. 13.0 DATAANALYSIASND CALCULATIONS 13.1 Calculations: 13.1.4 Calculate matrix spike percent recoveries using the following equation: YORecovery = Observed Result - Background Resuli; x 100 Expected Result 13.1.5 Calculate percent difference using the following equation: YODifference = Emected Conc. - Calculated Conc. x 100 Expected Conc. 3M Environmental Laboratory FACT-M-4.0 Analysis of Serum Extract Using ESMS Page 5 of 8 Page 134 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 13.1.6 Calculate actual concentration of PFOS, or other fluorochemical, anion in serum (I.Lg/mL): ) x DilutionFactor x FinalVolume(mL) Initial Volume of senun (mL) 14.0 METHOPDERFORMANCE 14.1 The method detection limit is equal to halfthe lowest standard in the calibration curve. 14.2 The practical quantitation limit is equal to the lowest standard in1 the calibration curve. 15.0 POLLUTION PREVENTIONAND WASTE MANAGEMENT 15.1 Sample extract waste and flammable solvent is disposed in high BTU containers, and glass pipette waste is disposed in broken glass containers located in the laboratory. 16.0 RECORDS 16.1 Store chromatograms in the study folder. Each chromatogram must have the following information included either in the header or hand written on the Ichromatogram: study number, sample name, extraction date, and dilution factor (if applicable). 16.2 Plot calibration curve by linear regression and store in the study folder. 16.3 Print sample list fiom MassLynx and tape into the instrument runlog. 16.4 Print data integration summary fiom MassLynx and tape into thc:instrumentrunlog. 16.5 Copy instrument runlog pages, including instrument parameters and sample results, and tape into appropriate study notebook. 16.6 Summarize data using suitable software and store in the study folder. 16.7 Back up electronic data to appropriate medium. Record in study notebook the file name and location of backup electronic data. 17.0 TABLESD,IAGRAMS, FLOWCHARTANSD. VALIDATION DATA 17.1 Attachment A: FACT-M-4 Data reporting spreadsheet 17.2 The validation report associatedwiththis method is FACT-M-3.O & 4.0-V-1.' 18.0 REFERENCES 18.1 AMDT-EP-3 1,"Operation of VG Platform Electrospray Mass Slpectrometer" 19.0 AFFECTEDDOCUMENTS 19.1 FACT-M-3.0, "Extraction of Fluorochemical Anions fiom Semn for Analysis Using HPLC-Electrospray/Mas Spectrometry" 3M Environmental Laboratory FACT-M-4.O Analysis of Serum Extract Using ESMS Page 6 of 8 Page 135 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 Revision Number. Reason For Revision - Revision Date 3M Environmental Laboratory FACT-M-4.O Analysis of Serum Extract Using ESMS Page 7 of 8 Page 136 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 Laboratory Study # Study: Test Material: Matrix/Final Solvent: MethodRevision: Analytical Equipment System Number: Instrument SoftwarcNersion: Filename: R-Squared Value: Slope: Y Intercept: Date of ExtraCtiodAnalyst: Date of AnalysWhalyst: Group Dose Sample# Concentration og/mL Initial VoL mL Dilution Factor Final Conc. Slope: Taken fiom linear regression equation. GroupLDose: Taken frorn the study folder. Sample##:Taken fiom the study folder. Concentration (ug/mL): Taken from the MassLynx integratio , summary. Initial Volume (mL): Taken fiom the study folder. Dilution Factor: Taken from the study folder. Final Conc. (ug/mL): Calculated by dividing the initial volum from the concentration 3M Environmental Laboratory FACT-M-4.O Analysis of Serum ExtractUsing ESMS Page 8 of 8 Page 137 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 3M ENVIRONMENTLAALBORATORY A N A L Y S I S OF POTASSIUMPERFLUOROOCTANESULFCbNATE OR OTHER F L ~ ~ R ~ ~ H E M I CIANSLESRUMEXTRACTUSSING HPLC=ELECTROSPFUY/MSPAESCTSROMETRY Method Number: ETS-8-5.1 Adloption Date: 03/01/99 Author: Lisa Clemen, Robert Wynne Re vision Date: 9 Approved By: Laboratory Manager Group Leader c Technical Reviewer 4/=, *Date 4/w /99 Date ou/llt/ss Date rt .g1.o SCOPE AND APPLICATION <' 1.1 Scope: This method describes the analysis of serum extracts for flucrochemical surfactants using HPLC-electrospray/mass spectrometry. 3 1.2 Applicable Compounds: Fluorochemical surfactants or other fluorinated compounds, or other ionizabte compounds. --.. -. 3 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 ESMS Page I of9 3M Environmental Laboratory Page 138 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 2.0 SUMMAROFYMETHOD 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 characteristicof a particular fluorochemical,such as the perfluorooctanesulfonate(PFOS)anion, d z = 499. Additionally, samples may be analyzed using a tandem mass spc:ctrometer to firther 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 Q u a m 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 atmosphericpressure (Le., not under a vacuum). 3.2 Electrospray Ionization (ES,ESI): a method of ionizationperfonned at atmospheric pressure, whereby ions in solution are transferred to the gas phase via tiny charged droplets. These charged droplets are produced by the applicationof a strong ~dectricaflield. 3.3 Mass Spectrometry, Mass Spectrometer(MS), TandemMass Spectrometer( M S M S ) : The API Quattro II triple quadrupole systems are equipped With quitdrupole mass selective detectors. Ions are selectivelydiscriminated by mass to charge ratio ( d z )and subsequently ' detected. A single MS may be employed for ion detection or a serir:s( M S M S ) for more specific fragmentation information. 3.4 Conventional vs. %spray probe interface: The latest models of n/licromassQuattro 11 triple quadrupole systems (post 1998) utilize a "Z-spray" conformation. The spray emitted from a probe is orthogonalto the cone aperture. In the conventional conformationit is aimed directly at the cone aperture, after passing through a tortuous pathway in the counter electrode. Though the configurationis different, the methods of operation, cleaning, and maintenance are the same. However, 2-spraycomponents and conventional components are not compatible with one another, but only with similar systems (i.e., 2-spraycomponents are compatible with some other Z-spray systems, etc.) 3.5 Mass Lynx Software: System softwaredesigned 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 I1 triple quadrupole MassLynx or MassLynx N1` 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 W h e n handling samples or solvents wear appropriateprotective gloves, eyewear, and clothing. ETS-8-5.1 Analysis of Serum Extract Using ES/MS Page 2 of 9 3M Environmental Laboratory Page 139 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 4.2 Cautions: 4.2.1 Do not operate solvent pumps above capacity of400 bar (5800 psi) back pressure. If the back pressure exceeds 400 bar, the HP1100 will initiate automatic shutdown. 4.2.2 Do not run solvent pumps to dryness. 5.0 INTERFERENCES 5.1 To minimize interferences when analyzing samples, teflon should not be used for sample storage or any part of instrumentationthat comes in contact with the sample or extract. 6.0 EOUIPMENT 6.1 Equipment listed below may be modified in order to optimizethc: 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 degaiiser, column compartment, and autosampler 7.0 SUPPLIES AND MATERIALS 7.1 Supplies J 7.1.1 High purity grade nitrogen gas regulated to approximately 100psi (House air system) 7.1.2 HPLC analyticalcolumn, specificsto be determinedby tht: analyst and documented in the raw data 7.1.3 Capped autovials or capped 15 mL centrifuge tubes 8.0 mAGENTS AND STANDARDS 8.1 Reagents 8.1.1 Methanol, HPLC grade or equivalent 8.1.2 Milli-QW water, all water used in this method should be Milli-QM 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 eightcen matrix standards are prepared during the extraction procedure. See ETS-8-4.1. 9.0 SAMPLHEANDLING 9.1 Fresh matrix standards are prepared with each analysis. Extracted standards and samples are stored in capped autovials or capped 15mL centrifugetubes until analysis. ETS-8-5.1 Analysis of Serum Extract Using ESMS Page 3 of9 3M Environmental Laboratory Page 140 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 9.2 If analysis will be delayed, extracted standards and samples car1 be refrigerated at approximately 4" C, or at room temperature, until analysis can be performed. 10.0 OUALITCYONTROL 10.1 Solvent Blanks, Method Blanks and Matrix Blanks 10.1.1 Solvent blanks, method blanks and matrix blanks are prepared and analyzed with each batch to determine contamination or carryover. 10.1.2 Analyze a method blank and a matrix blank prior to each calibration curve. 10.2 Matrix Spikes 10.2.1 Matrix spikes are prepared and analyzed to determine tht: matrix effect on the recovery efficiency. 10.2.2 Matrix spike duplicates are prepared and analyzed to m a m e the precision and the recovery for each analyte. 10.2.3 Analyze a matrix spike and matrix spike duplicate per foity samples, with a minimum of2 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. ld.3 Continuing Calibration Verifications 10.3.1 Continuing calibration verificationsare analyzed to verify the continued accuracy of the calibration curve. 10.3.2 Analyze a mid-range calibration standard after every tentb sample, with a minimum of one per batch. 11.o CALIBRATIONA N D STANDARDIZATION 11.1 Analyze the extracted matrix standards prior to and following each set of extracts. The average of two standard curves will be plotted by linear regression (y = my + b), weighted l/x, not forced through zero, using MassLynx or other suitable soitware. 11.2 If the curve does not meet requirements,perform routine maintenance or reextract the standard curve (if necessary) and reanalyze. 11.3 For purposes of accuracy when quantitating low levels of analyte, it may be necessary to use the low end of the calibration curve rather than the 111range of the standard curve. Example: when attempting to quantitate approximately I O ppb of analyte, generate a calibration curve consisting of the standards from 5 ppb to 100ppb rather than the fill range of the curve (5 ppb to 1000ppb). This will reduce inaccuracy attributed to linear regression weighting of high concentration standards. 3M Environmental Laboratory ETS-8-5.1 Analysis of Serum Extract Using ES/MS Page 4 of 9 Page 141 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 12.0 PROCEDURES 12.1 Acquisition Set up 12.1.1 Click on start button in the Acquisition Control Panel. Set up a sampIe list. Assign a filename using MO-DAY-last digit of year-sample number, assign a method ( M S ) for acquiring, and type in sample descriptions. 12.1.2 To create a method click on scan button in the Acquisitiim control panel and select SIR (Single Ion Recording) or MRM. Set Ionization Mcde as appropriateand m a s to 499 or other appropriatemasses. A full scan is usually collected along with the SIRS. Save acquisition method. If MSNS instruments are employed, additional product ion flagmentation 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 endswith a set of extracted matrix standards. 12.1.4 Samples are analyzed with a continuingcalibration check injected after every tenth sample. Solvent blanks should be analyzed periodically to monitor possible analyte carryover and are not considered samples but may be included as such. 12.2 Using the AutosampIer 12.2.1 Set up sample tray according to the sample list prepared hi Section 12.1.1. I '12.2.2 Set-up the HP1lOO/autosamplerat the following conditions or at conditions the analyst considers appropriate for optimal response. Record actual conditions in the instrument logbook: 12.2.2.1 Sample size = 10 pL injection 12.2.2.2 Inject/sample = 1 12.2.2.3 Cycle time = 13.5 minutes 12.2.2.4 Solvent ramp = 1I Time I I 1 I 0.00min. I 8.50 min. 11.O min. 12.0 min. MeOH 40% 90% 90% 40% 1- I 2.0 a-I I Ammonium acetate I I I 60% 10% 10% 60% 12.2.2.5 Press the "Start"button. 12.3 Instrument Set-up 12.3.1 Refer to ETS-9-24.0for more details. 12.3.2 Check the solvent level in reservoirs and refill if necessary. 3M Environmental Laboratory ETS-8-5. I Analysis of Serum Extract Using ESMS Page 5 of 9 Page 142 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 12.3.3 12.3.4 Check the stainless steel capillary at the end of the probe. Use an eyepiece to check the tip. The tip should be flat with no jagged edges. If the tip is found to be unsatisfactory, disassemble the probe and replace the stainless steel capillary. Set HPLC pump to "On". Set the flow to 10 - 500 uWmin or as appropriate. Observe droplets coming out of the tip of the probe. Allow to equilibrate for approximately 10minutes. 12.3.5 Turn on the nitrogen. A fine mist should be expelled Wilth no nitrogen leaking around the tip of the probe. Readjust the tip of the probe if no mist is obsmed. 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 12.3.6.2 12.3.6.3 12.3.6.4 Drying gas 250-400 literskour ESI nebulizing-gas 10-15 literslhour HPLC constant flow mode, flow rate 10- 500 pL/min Pressure4 0 0 bar (Thisparameter is not set, it is a guide to ensure the HPLC is operating correctly.) 123.7 Carefully guide the probe into the opening. Insert probe imtil it will not go any further. Connect the voltage cables to the probe. 12.3.8 Print the tune page, with its parameters, and store it in the study binder with a copy taped into the instrument log. ' 12.3.9 Using the cross-flow counter electrode in the ESMS sour1;e is recommended for the analysis of biological matrices. 12.3.10Click on start button in the Acquisition ControlPanel (thismay v q a m o n g MassLynx versions, see appropriateMassLynx USERS GUIDE). Press the start button. Ensure start and end samplenumber includes all samples to be analyzed. 13,O DATAANALYSISAND CALCULATIONS 13.1 Calculations: 13.1.4 Calculate matrix spike percent recoveriesusing the following equation: % Recovery = Observed Result - Backmound Result x 100 Expected Result 13.1.5 CalcuIate percent difference using the following equation: % Difference = ExDected Conc. - Calculated Conc. x 100 Expected Conc. 13.1.6 CalcuIate actual concentration of PFOS,or other fluorochanical, in matrix (vg/mL): (ne:of PFOS calc. from std. Curve x Dilution F a c t d x 1 UB. JInitiaI Volume of matrix (mL) + mL of Surrogate Standard) 1000ng Final Volume (mL) ETS-8-5.1 Analysis of Serum Extract Using ESMS Page 6 of9 3M Environmental Laboratory Page 143 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 14.0 METHOPDERFORMANCE 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 3 value for the calibration curve must be 0.980 or better. 14.4 Matrix Spikes 14.4.1 Matrix spikepercent recoveries are must be withink30% ofthespiked concentration. 14.5 Continuing Calibration Verifications 14.5.1 Continuing calibration verification percent recoveries must be f 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 r' analyst. Document dl actions in the appropriate logbook. 14.7 If data are to be reported when performance criteria have not been met, the data must be footnoted on tables and discussed in the text of the report. 15.0 POLLUTION PREVENTION AND WASTE MANAGEMENT 15.1 Sample extract waste and flammablesolvent is disposed in high BTU contai'ners, and glass pipette waste is disposed in broken glass containers located in the laboratory. 16.0 RECORDS 16.1 Each page generated for a study must have the following information included either in the header or hand written on the page: study or project number, acquisitionmethod, integration method, sample name, extraction date, dilution factor (I f 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 calibrationcurve by linear regression, weighted l/x,then print these graphs and store in the study folder. 16.4 Print data integration summary, integration method, and chromatograms, f?om MassLynx, and store in the study folder. 3M Environmental Laboratory ETS-8-5.1 Analysis of Senun Extract Using E S N S Page 7 of9 Page 144 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 16.5 Summarize data using suitable soflware (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 stud/ notebook the file name and location of backup electronic data. 17.0 TABLESD.IAGRAMFSL. OWCHARTS, AND VALIDATION DATA 17.1 Attachment A: ETS-8-5.1 Data summary spreadsheet. 18.0 REFERENCES 18.1 FACT-M4.1, "Extractionof PotassiumPerfluorooctauesulfonatlsor Other Fluorochemical compounds &om Serum for AnalysisUshg HPLC-Electrospray/MassSpectrometry 18.2 ETS-9-24.0, "Operation and Maintenance of the Micromass Atmospheric Pressure lonization/Mass SpectrometerQuattro II 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-84.1, "Extraction of Potassium Perfluorooctanesdfonate or Other Fluorochemical Compounds from Serum for AnaIysis Using HPLC-Electrospray/Mas Spectrometry" 20,o REVISIONS Revision Number. 1 Reason For Revision Section 6.1.2 Clarification of HP1100 system components. Section 11.1 Average of two curves, not standard values, art: used for plotting linear regression and added the llx weighting of tht: curve. . Section 12.2.2.4 Clarification of solvent ramp. Section 17.1 Changed fiom attachment B to A. Revision 04/02/99 3M Environmental Laboratory ETS-8-5.1 Analysis of Serum Extract Using ESMS Page 8 of 9 Page 145 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 Laboratory Study # Group Dose Sample# Concentration Ug/mL Initfat VoL mI, Dilution Factor Find Conc, Ugld r' Slope: Taken fiom linear regression equation. Attachment A: Summary Spreadsheet ETS-8-5. I Analysis of Serum Extract Usgg ESMS 3M Environmental Laboratory Page 9 of 9 Page 146 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 3M ENVIRONMENTLAALBORATORY METHOD ANALYSIOSF POTASSIUMPERFLUOROOCTANESULIO~ROONTAHTEER F'LUOROCFfEMICALS IN LIVER EXT'k4CX.SUSING HPLC-ELECI'ROSPR.AY/'MASSSPECTROMETRY Method Number: ETS-8-7.0 Author: Lisa Clemen, Glenn Langenburg Approved By: Revision Date: N& I&--- Group Leader L. Technical Reviewer 3 /.I4133 Date Date ' 1.0 SCOPEAND APPLICATION \.i 1.1 Scope: This method is for the analysis of liver extracts for fluomlchemical surfactants using 3 HPLC-electrospray/mas spectrometry. 1.2 Applicable Compounds: Fluorochemical surfactants or other fluorinated compounds, or '3 other ionizable compounds. 2. 1.3 Matrices: Rabbit, rat, bovine, monkey h e r , or other tissues as designated in the validation 3 report. Word 6/95 ETS-8-7.0 Analysis of Liver Extract Using ESMS Page 1 of 10 3M Environmental Laboratory Page 147 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 2.0 SUMMARY O F METHOD 2.1 This method describes the analysis of fluorochemical surfactarlts extracted from liver using HPLC-electrospray/mass spectrometry, or similar system as appropriate. The analysis is performed by monitoring a single ion characteristic of a particiilar fluorochemical, such as the perfluorooctanesulfonate(PFOS)anion, d z = 499. Additionally, samples may be analyzed using a tandem mass spectrometer to further verify the identity of a compound by detecting daughter ions of the selected parent ion. 3.0 DEFINITIONS 3.1 Atmospheric Pressure Ionization (API): The Micromass Quattro IItriple quadrupole systems allow for various methods of ionization by utilizing varilms 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 perft,md at atmospheric pressure, whereby ions in solution are transfenred to the gas phase via tiny charged droplets. These charged droplets are produced by the application of a strong electrical field. 3.3 Mass Spectrometry, Mass Spectrometer (MS),TandemMass Spectrometer ( M S M S ) : The API Quattro II triple quadrupole mass spectrometeris equipped with two quadrupole , mass selective detectors and a collisioncell. Ions are selectivelydiscriminatedby mass to charge ratio ( d z )and subsequently detected. A single MS may be employed for ion detection or an ionmay be selected in the first quadrupole, hgmented in the collision cell, and these hgments may be analyzed in the second quadrupole. 3.4 Conventional vs. Zspray probe interface: The latest models of Micromass Quattro 11 triple quadrupole (post 1998) utilize a "Z-spray" conformation. The spray emitted from a probe is orthogonal to the cone aperture. In the conventional conformationit 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 componentsand cc~nventiondcomponents are not compatiblewithone another, but only with similar systems (Le. 2-spraycomponents are compatible with other Z-spray systems, etc.) 3.5 Mass Lynx Software: System software designed for the specific: operation of these Quattro II triple quadrupole systems. Cunrently MassLynx has Windows 95 and WindowsNT 4.0 versions. All vmions are similar. For more details refer to the nianual specific to the instrument (MicromassQuattro II triple quadrupole MassLynx 01MassLynx NT User's Guide). 4.0 WARNINGS A N D 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 A ~ l y s iosfLiver ExtractUsing ES/MS Page 2 of 10 3M Environmental Laboratory Page 148 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 4.1.2 When handling samples or solvents wear appropriate Flrotective gIoves, eyewear, and clothing. 4.2 Cautions: 4.2.1 Operate the solvent pumps below a back pressure of 4(lO bar (5800 psi). If the back pressure exceeds 400bar, the HP1100 will initiateautomatic shutdown. 4.2.2 Do not run solvent pumps to dryness. 5.0 INTERFERENCES 5.1 To minimize interferences when analyzingsamples,Teflon shill not be used for'sample storage or any part of instrumentation that comes in contact with the sample or extract. 6.0 EOUJPMENT 6.1 Equipment listed below may be modifiedin order to optimizethe system. Document any . modifications in the raw data as method deviations. 6.1.1 Micromass Quattro 11triple quadrupoleMass Spectrometerequipped with an electrospray ionization source. 6.1.2 HP1100 low pulse solvent pumping system, solventdepsser, column compartment, and autosampler 7.0 SUPPLIES AND MATERIALS 7.1 Supplies 7.1.1 High purity grade air regulated to approximately 100psi (house air system) 7.1.2 HPLC analytical column, specificsto be determinedby the analyst and documented in the raw data 7.1.3 Capped autovialsor capped 15ml centrifuge tubes 8.0 -A GENTS AND STANDARDS 8.1 Reagents 8.1.1 Methanol, HPLC grade or equivalent . 8.1.2 Milli-QTMwater (ASTM type I), all water used in this method should be ATSM type I, or equivalent, and be provided by a Milli-Q TO(: 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 approximately0.300 g ammonium acetate. Pour into a 2000 mL volumetric container containing 2000 mL Milli-Qm water, mix until all solids are dissolved. Store at room temperature. ETS-8-7.0 Analysis ofLiver Extract Using ESMS Page 3 of 10 3M Environmental Laboratory Page 149 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 8.2 Standards 8.2.1 Typically two method blanks, two matrix blanks, and aighteen matrix standards are prepared during the extraction procedure. Refer to ETS-8-6.0. 9.0 SAMPLE HANDLING 9.1 Fresh matrix standardsare prepared with each analysis. Extrac;ted standarb and samples are stored in capped autovials or capped 15 ml centrifuge tubes until analysis. 9.2 If analysiswill be delayed, extracted standards and samplesm(kybe stored at room temperature, or refiigerated at approximately 4' C, until analysis can be performed. 10.0 OUALlrV CONTROL 10.1 Method Blanks and Matrix Blanks 10.1.1 Solvent blanks, method blanks, and ma& blanks are Freparedand analyzed with each batch to determine contarninition 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 I recovery efficiency. ' 10.2.2 Matrix spike duplicates are prepared and analyzed to merecovery for each d y t e . the precision and the 10.2.3 Analyze a matrix spike and matrix spike duplicate per forty sampiep. With a minimum of 2 spikes per batch. 10.2.4 Matrix spike and matrix spike duplicate concentrations wilI fall in the mid-range of the initial calibrationcurve. Additional spike concentrationsmay fall in the low- range of the initial calibration curve. 10.3 Continuing Calibration Checks 10.3.1 Continuing calibration verificationsare analyzed to verify the continued accuracy of the calibration curve. 10.3.2 Analyze a mid-range calibration standard every tenth sample, with a minimum of one per batch. 11.0 CALIBRATION AND STANDARDIZATION 11.1 Analyze the extracted matrix standardsprior to and following each set of sample extracts. The average of two standard curves will be plotted by linear repession (y=mx +b), weighted l/x, not forced through the origin, using MassLynx 01' other suitable software. 11.2 Ifthe curve does not meet requirements perform routine maintenance or reextract the standard curve (if necessary) and reanalyze. 3M Environmental Laboratory ETS-8-7 .O Analysis of Liver Extract Using ES/MS Page 4 of 10 Page 150 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 11.3 For purposes of accuracy when quantitating low levels of analyte, it may be necessary to use the low end of the calibration curve rather than the full range of the standard e w e . Example: when attempting to quantitate approximately 10 pph of analyte, generate a calibration curve consisting of the standards from 5 ppb to 100ppb rather than the full range of the curve (5 ppb to 1000ppb). This will reduce inaccuracy attributed to linear regression weighting of high concentration standards. 12.0 PROCEDURES 12.1 Acquisition Set up 12.1.1 Set up the sample list. 12.1.1.1 Assign a sample list filename using MO-DAY-last digit of year-increasing letter of the alphabet starting with a 12.1.1.2 Assign a method (MS file) for acquiring 12.1.1.3 Assign an HPLC pro&= (Inlet file) 12.1.1.4 Type in sample descriptions and vial position numbers 12.1.2 To create a method click on method inthe Acquisition control panel thenmass spectrometerheadings and select SIR (SingleIon Recoiding) or MRM (Multiple Reaction Monitoring). Set Ionization Mode as appropn ate and mass to 499 or other appropriatemasses. A fill scan is usually collected alongwiththe SIRS. Save acquisition method. IfMS/MS htruments are employed, additional product ion fragmentation information may be collected. Refer to nrlicromass MassLynx GUIDE TO DATA ACQUISITION for additional information and MRM. 12.1.3 Typically the analytical batch runsequencebegins and ends with a set of extracted matrix standards. 12.1.4 Samples are analyzed with a continuing calibration verification injected standard after every tenth sample. Solvent blanks should be analyzed periodically to monitor possible analyte carryover and are not considered samples but may be included as such. 12.2 Using t h e Autosampler 12.2.1 Set up sample tray according to the sample list prepared in Section 12.1.1. 12.2.2 Set-up the HP1lOO/autosamplerat the following conditions or at conditionsthe analyst considers appropriatefor optimal response. Record actual conditionsin the instrument logbook ' 12.2.2.1 Sample size = 10 pL injection 12.2.2.2 InjecVsample = 1 12.2.2.3 Cycle time = 9 minutes , 3M Environmental Laboratory ETS-8-7.0 Analysis of Liver Extract Using ES/MS Page 5 of 10 Page 151 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 12.2.2.4 Solvent ramp conditions Time MeOH 0.00 min. 40% 1.0 min. 40% -4.5 min. 95% 6.5 min. 95% 7.0min. 40% 9.0 mi. 40% Ammonium acetate 60% . 12.2.2.5 Press the "Start" button. 12.3 Instrument Set-up 12.3.1 Refer to ETS-9-24.0, "Operation and Maintenance of the Micromass Quattro II Triple Quadrupole Mass Spectrometer Fitted with an Atmospheric Pressure Ionization Source," for more details. 12.3.2 `Checkthe solvent level inreservoirsand refill if necessary. 123.3 Check the stainless steel capillary at the end of the probe. Use an eyepiece to check the tip. The tip should be flat with no jagged edges. If the tip is found to be I! unsatisfhctory, disassemble the probe and replace the stihless steel capillary. 123.4 Turn on the nitrogen. 12.3.5 Open the tune page. Clicks on operate to initiate source block and`desolvation heaters. 12.3.6 Open the Inlet Editor. 12.3.6.1 12.3.6.2 12.3.6.3 12.3.6.4 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. 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 Allow to equilibrate for approximately 10 minutes. 12.3.7 The instrument uses these parameters at the following settings. These settings may change in order to optimize the response: 12.3.7.1 Drying gas 250-400litemhour 12.3.7.2 ESI nebulizing gas 10-15 liters/hour 123.7.3 HPLC constant flow mode flow rate 10-500 p.Wmin 12.3.7.4 Pressure 2400 bar (This parameter is not set, it is a guide to ensure the HPLC is operating correctly.) 12.3.7.5 Source block temperature 150' 12.3.7.6 Desolvation temperature 250" ETS-8-7.0 Analysis of Liver Extract Using ESMS Page 6 of 10 3M Environmental Laboratory Page 152 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 12.3.8 Print the tune page, with its parameters, and store it in the study binder with a Copy taped into the instrument log. 123.9 Click on start button in the Acquisition Control Panel (this may vary among MassLynx versions, refer to appropriate MassLynx User's Guide). Ensure start and end sample number includes all samples to be analyzed. 13.0 DATAANALYSIASND CALCULATIONS 13.1 Calculations: 13.1.4 Calculatematrix spikepercent recoveries using the following equation: Expected Result 13.1.5 Calculatepercent differenceushg the following equation: - % Difference = Exuected Conc. Calculated Conc. x 100 Expected Conc. 13.1.6 Calculate actual concentrations in matrix (pg/g): In. of PFOS calc. fromstd. Curve x Dilutioq Factor) x 1ug (Initial Wei&t of Liver (E) 1000 ng I Final Volume (A) 14.0 METHODPERFOF~MANCE 14.1 Method Detection Limit (MDL) and Limit of Quantitation (LOW are mehod, malyte,and . matrix specific. Refer to ETS-8-6.0,Attachment B for a listing of current validated MDL and LOQ values. 14.2 Solvent Blanks, Method Blanks and Matrix Blanks 14.2.1 Solvent blanks, method blanks, and matrix blanks must be below the lowest standard in the calibration curve. 14.3 Calibration Curves 14.3.1 The 3 value for the calibration must be 0.980 or better, 14.4 Matrix Spikes 14.4.1 Matrix spike percent recoveries must be within ik 30%of the spiked concentration. 14.5 Continuing Calibration Verification 14.5.1 Continuing calibration verification percent recoveries must be within k 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. 3M Environmental Laboratory ETS-8-7.0 Analysis of Liver Extract Using ESMS Page 7 of 10 Page 153 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 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 POLLUTIONPREVENTJAONND WASTMEANAGEMENT 15.1 Sample extract waste and flammable solvent is disposed in hi4;h BTU containers, and.glass pipette waste is disposed in broken glass containers located in the laboratory. 16.0 RECORDS . 16.1 Each page generated for a study must have the following information included either in the header or hand written on the page: study or project number, slcquisitionmethod, integration method, sample name, extraction date, dilution factor (if applicable), and analyst. 16.2 Print the tune page, sample list, and acquisition method from AdassLynx to include in the appropriate study folder. Copy these pages and tape into the instrument runlog. 16.3 Plot the calibration curve by linear regression, weighted l/x, then print these p p h s and store in the study folder. 16.4 h i n t data integration summary, integration method, and c h r o m . a t o f~rom MassLynx and store in the study folder. ,16.5 Summarize data using suitable sofhvare (Excel 5.W) and store in the study folder, refer to Attachment A for an example of a summaryqreadsheet. 16.6 Back up electronic data to appropriatemedium. Record in studbynotebook the filename and location of backup electronic data. 17.0 TABLESD,IAGRAMSF.LOWCHARTA NSD. VALIDATION DATA 17.1 Attachment A ETS-8-7.0 Data summary spreadsheet 18.0 I REFERENCES 18.1 FACT-M-2.1, "Extraction ofPotassium Perfluorooctanesulfonateor Other Fluorochemical Compounds from Liver for Analysis Using HPLC-Electrospray/Mass Spectrometry" 18.2 ETS-9-24.0, "Operation and Maintenance of the Micromass Atmospheric Pressure Ionization/Mass SpectrometerQuattro IItriple quadrupole Systm s " 18.3 The validation report associated with thismethod is ETS-8-6.0 & 7.0-V-1 19.0 AFFEC~EDDOCUMENTS 19.1 ETS-8-6.0, "Extraction of PotassiumPerfluorooctanesulfonateor Other Fluorochemical Compounds torn Liver or Fluid for Analysis Using HPLC-ElectrosprayMass Spectrometry" 3M Environmental Laboratory ETS-8-7.0 Analysis of Liver.Extract Using E&MS Page 8 of 10 Page 154 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 20.0 REVISIONS Revision Number Reason For Revision Revision - Date I 1 3M Environmental Laboratory ETS-8-7.0 Analysis of Liver Extract Using ES/MS Page 9 of 10 Page 155 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 Laboratory Study # Study: Test Material: MatrixlFinal Solvent: MethodRen'sion: Analytical Equipment System Number: Instrument Softwarflersion: Filename: R-Squared Value: Slope: Y Intercept: Date of ExtractiodAnalyst . Date of AnalysidAnalyst nope: Taken trom linear regression equatlon. Attachment A: Summary Spreadsheet ETS-8-7.0 Analysis of Liver Extract Using ES/MS 3M Environmental Laboratory Page 10 of 10 Page 156 3M Medical Department Study: T-6316.1 3M Medical Department Study: T-6316.1 Appendix D: Data Summary Tables Analytical Report: FACT TOX-003 LRN-U2104 Analytical Report: FACT-TOX-003 LRN-U2104 Table 9. FACT TOX-003 Data Summary for PFOS, PFOSA, PFOSAA, EtFOSE, M556 and PFOSEA-Rat Timept Group PFOS Avg t SD PFOSA Aug f SD PFOSAA Avg SD EtFOSE" Avg fSD Serum (-U_ -g/mL) M556 Avg t SD PFOSEA* Avg * SD M <LO@ CLOQ~ 0.00460 f 0.00158" NA NA NA Group 8 (n=5) (n=5) (n=5) 0.0 ppm F CLOQ~ <LOQ~ 0.0103 f 0.00375' NA NA NA Week 4 (n=5) (n=5) (n=5) M 0.174 f 0.0510 0.00500 f 0.000125 0.247 f 0.195' NA NA NA Group 9 (n=5) (n=5) (n=5) 1.0 ppm F 0.270 f 0.0687 0.00778 f 0.00254 0.294 f0.111' NA NA NA (n4) (n3) (n=5) M 0.0393 f 0.0109 cLOQC CLOQ~ NA NA NA Group 8 (n=5) (n=5) (n=5) 0.0 ppm Week F 0.126 f 0.0159 cLOQC CLOQ~ NA NA NA (n=5) (n=5) (n=5) ~ ~ 14 M 2.1 9 f 0.733 0.0110 f 0.000716 0.497 f 0.618 NA NA NA Group 9 (n=5) (n=5) (n=5) 1.0 ppm F 3.26 f 0.629 0.0164 f 0.00354 0.578 f 0.425 NA NA NA (n=6) (n=6) (n=6) Week Cr) "V Group 8 0.0 ppm Group 9 1.O ppm M 0.0490 f 0.0352 (n=5) <LO@ (n=5) F 0.0727 f 0.0565 (n=5) cLOQ' (n=5) M 1.35 f 0.452 0.00672 f 0.00251 (n=5) (n=5) F 3.02 f 1.54 0.0102 f 0.00252 CLOQ' (n=5) CLOQ' (n=5) 0.297 f 0.194 (n=5) 0.273 kO.110 <LOQ~ (n=5) CLOQ' (n=5) cLOQ' (n=5) <LOQ~ (n=5) I <LOO' h=S\ ! cLOQ' (n=q I <LOQ~ (n=5) 0.120 f 0.0289 (n=5) cLOQ' (n=5) <LOQ~ 0.202 f 0.0723 <LOQ' -Not amlici 3M Environmental Laboratory Page 157 3M Medical Department Study: T-6316.1 3M Medical Department Study: T-6316.1 Analytical Report: FACT TOX-003 LRN-U2104 Analytical Report: FACT-TOX-003 LRN-U2104 Timept Week 105 Group M Group 8 0.0 ppm F M Group 9 1.O ppm F PFOS Avg i SD 0.0137 * 0.0181 (n=lO) 0.0256 f 0.0145 (n=lO) 1.12 i 1.10 (n=lO) 1.61 i 0.631 (n=lO) PFOSA Avg t SD cLOQ' (n=lO) <LO@ (n=lO) 0.00720 i 0.00330 (n=lO) 0.00797 f 0.00198 (n=lO) PFOSAA Avg t SD clod (n=lO) CLOd (n=lO) 0.252 f 0.160 (n=lO) 0.259 i 0.156 @=lo) EtFOSE" Avg t SD CLOQ~ (n=lO) CLOQ~ (n=lO) CLOQ~ (n=lO) CLOQ~ (n=lO) M556 Avg t SD ' OL<CI (n=lO) CLOQ' (n=lO) 0.0645 i 0.0445 (n=lO) 0.0868 i 0.0294 (n=lO) PFOSEA" Avg t SD cLOQ" (n=lO) <LOQ~ (n=lO) <LOO" (n=lO) cLOQ" @=lo) NOTE: Results are expressedas group/genderaverages i the standard deviationassociatedwith that group/gender. NOTE: AI1 PFOSA, PFOSAA, PFOSEA, M556, and PFOS Week 4 results are not corrected for purity of the reference standard material. It Is not possible to verify true recoveryof endogenous analytefrom tissues without radio-labeledreference material.The only measurementof accuracyavailable at this time, matrix spike studies, indicate that, with the exceptionof PFOSEA and EtFOSE-OH,the sera and liver datacan be consideredto be accurate to within one standard deviationof the averagefortified samples recovery. 3M Environmental Laboratory Page 158 3M Medical Department Study: T-6316.1 3M Medical Department Study: T-6316.1 Analytical Report: FACT TOX-003 LRN-U2104 Analytical Report: FACT-TOX-003 LRN-U2104 Table 10. FACT TOX-003 Data Summary for PFOS, PFOSA, PFOSAA,EtFOSE, M556 and PFOSEkRat Liver (pg/g) Timept Group PFOS Avg t SD PFOSA Avg 2 SD PFOSAA * Avg SD EtFOSE* Avg t SD M556 Avg t SD PFOSEA* Avg t SD M 0.1 98 f 0.0822 cLOQa CLOQ~ NR NA NA Group 8 (n=5) (1-4) (n=5) 0.0 pprn F 0.0891 f0.0315 <LO@ CLOQ~ NR NA NA Week 4 (n=5) (n=5) (n=5) M 6.53 f 0.628 0.187 f 0.0401 0.683 f 0.613 NR NA NA Group 9 (n=5) (n=5) (n=5) 1.O ppm F 2.97 f 0.937 0.222 f 0.1 04 0.582 f 0.193 NR NA NA (n=5) (n=5) (n=5) M 2.96 f 4.83 0.0822 f 0.156 0.330 f 0.656 NR NA NA Group 8 (n=5) (n=5) (n=5) 0.0 ppm Week F 0.41 1 f 0.0625 0.01 30 f 0.000872 0.0411 f 0.0121 NR NA NA (n=5) (n=5) (n=5) 14 M 12.4 f 6.99 0.366 f 0.221 2.89 f 1.88 NR NA NA Group 9 (n=5) (n=5) (175) 1.O pprn F 9.1(9nf=51).53 0.580(nf=50).239 2.61(nf=52).49 NR NA NA M 1.44f 1.18 Group 8 (n=5) 0.0 ppm F 0.178 f 0.1 15 Week (n=5) 53 M 37.1 f 10.9 Group 9 (n=5) 1.0 pprn F 25.2 f 13.9 (n=5) 'EtFOSE and PFOSEA results are qualitative :LOO-Limit <LOQC (n=5) CLOQ~ (n5) cLOQe (n=5) CLOQ' (n=5) <LO' CI (n=5) 0.00647 f 0.0000786 (n=5) <LOQ~ (n=5) <LO@ (n=5) CLOQ' (n=5) cLOQg (n5) 0.204 f 0.0769 (n=5) 0.906 f 0.605 (n-5) <LO@ I--=\ ,a ,-e, 0.336 f 0.0682 (n=Sj <LOQ~ (n=5) 0.242 f 0.0999 0.527 f 0.131 <LOCI* 0.267 f 0.0660 cLOQg (n=5) (n=5) (n=5) (n=5) (175) of Quantitation = 0.0121 pg/g dLOO-Limit of Quantitation = 0.0125 pgg g ~ ~ ~ - ~ oifmQuiatntitation= 0.0308 pg/g 3M Environmental Laboratory Page 159 3M Medical Department Study: T-6316.1 3M Medical Department Study: T-6316.1 Analytical Report: FACT TOX-003 LRN-U2104 Analytical Report: FACT-TOX-003 LRN-U2104 Table 10. (Continued) FACT TOX-003 Data Summary for PFOS, PFOSk PFOSAA, EtFOSE, M556 and PFOSEkRat Liver (pglg) Timept Group PFOS * Avg SD PFQSA Avg i SD PFOSAA Avg i SD EtFOSE* Avg i SD M556 Avg i SD PFOSEA* Avg % SD Week 105 Group 8 0.0 ppm Group 9 1.0 ppm M 0.0653 f 0.0923 (n=lO) F 0.0872 f 0.0522 (n=lo) M 6.21 f 3.80 (n=lO) 9.63 i 4.35 F @=lo) <LOQ~ (n=10) <LOCIh (n=10) 0.371 * 0.187 (n=10) 0.550f0.112 (n=lO) eLOQ' (n=l0) <LOQ' (n=lO) 0.736 f 0.551 (n=lo) 0.771 f 0.411 (n=10) cLOQe (n=lO) eLOQe (n=lO) cLOQe (n=lO) <LOQe (n=lO) CLOQ' @=lo) CLOQ' (n=10) 0.250 i 0.0865 @=lo) 0.336 i 0.0748 @=lo) cLOQg (n=lO) CLOQ~ (n=10) <LO@ (n=lO) <LOCI' (n=lO) 3M Environmental Laboratory Page 160 3M Medical Department Study: T-6316.1 3 M Medical Department Study: T-6316.1 Appendix E: Data Spreadsheets Analytical Report: FACT-TOX-003 LRN-U2104 Analytical Report: FACT TOX-003 LRN-U2104 3M Environmental Laboratory Page 161 3M Medical Department Study: T-6316.1 . Analytical Report: FACT-TOX-003 LRN-U2104 3M Environmental Laboratory Page 162 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 3M Environmental Laboratory Page 163 3M Medical Department Study: T-6316.1 3M Environmental Laboratory Analytical Report: FACT-TOX-003 LRN-U2104 +- Page 164 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 3M Environmental Laboratory Page 165 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 3M Environmental Laboratory Page 166 3M Medical Department Study: T-6316.1 ,"I- NA *A NA NH A* NA 3M Environmental Laboratory Analytical Report: FACT-TOX-003 LRN-U2104 Page 167 3M Medical Department Study: T-6316.1 NA NA NA NA NA I NA N* NA s. WR 097m .. . OYR 09m om Om Analytical Report: FACT-TOX-003 LRN-U2104 3M Environmental Laboratory Page 168 3M Medical Department Study: T-6316.1 -jNA NA HA NA Analytical Report: FACT-TOX-003 LRN-U2104 M QPR- 3M Environmental Laboratory Page 169 3M Medical Department Study: T-6316.1 AMDTY 050598.2 CovanceY 6329-228 Analytical Report: FACT-TOX-003 LRN-U2104 !ai 193 PFOS Pvrig COWUlh FVlOl UhW UhW URLNr. UnLaoW NA NA NA NA NA NA NA NA NA NA UhW UhW UntmWl UhW PFOS cor dZ 172 0 73 I4 5 5.53 3% 368 413 4% NA NA Y65 1061 NA NA 353 w 173 15s 142 85.8 8S.9 151 S3.0 109 141 160 1% 126 PFOS Duulh F-UW 1 I 1 I I I I I NA NA 1 I NA NA I I I I I I I I I I 54 M 50 54 PFOS C.lc.Car. dt 160 Ofd 133 s 47 199 113 I58 200 NA NA em 710 NA NA 326 233 19 I44 I27 n3 75 8 138 48.1 98.4 6788 71S2 6978 57M 3835 360 I658 x4 2322 Y5S 132% 81% M If40 M 1% NA 3rd NA 3rd NA 1 0% 119% 11- NAM HA M NA 0316 0233 0 158 0 144 0 I27 oo m 00159 0191 0 1% c top( a m WI) OLE84 Owl 679 7 15 691 5 71 6 03 6S3 354 331 3s4 166 232 2 35% 246 NA ISS NA 41 6 OM22 3s 4 0031) 965 0628 31s 0931 FACTMJ 0 uY7 3M Environmental Laboratory Page 170 3M Medical Department Study: T-6316.1 - .. crap 8 CUIUVI c Y 2 1 W Ms C92IS.N US N2IS.NMSU NA N 2 1 W US NA N2IYMI MSD NA CWlY?J.I MS NA cY2IYIM MSI) NA I N2l*?J.I Ms NLIYBIMSD I NA NA NZIYZM Ubro Ubra _I_ I _I_ 84 4 I - _ _ hU7 I 382 I 152 I __ N A NA NA NA om I ow I I I I ~ I I 1 - I I I I 1 I _I_ I I AMDT# 050598.2 Covancdf 6329-228 Analytical Report: FACT-TOX-003 LRN-U2104 101617 <Lop 0181 c222 NA NA 215 OOIol 472 OIOI 05382 05x2 05382 05382 05382 05W 05382 OJW 05382 05382 05W m . 05382 05382 s Pufluam-uu-w UhW Unhpw UhW UhW Ub.a UbW Unhpw UntmW UbWn Ub*n Uohpwi UbW UbW PFOSAA Cak C o . . .A om om om om 674 I% 225 251 NA NA 60 75Y NA NA 000 om om om 000 Om om om I Om om I 000 Om 1 om ia 5 428 132 5 342 w 5 592 111 5 233 615 5 I ia 248 5 641 as 5 613 172 5 u6 133 5 155 124 5 8-56 N R - N a R s id .blr r- MM3ax)) Moba99y MM3ax)s MlbyR136 M(KyR937 E 0613 0.446 0355 OS56 106113 0582 1__* 0 IY3 3M Environmental Laboratory Page 171 3M Medical Department Study: T-6316.1 AMDTW 050598.2 Covance# 6329-228 Analytical Report: FACT-TOX-003 LRN-U2104 3M Environmental Laboratory Page 172 3M Medical Department Study: T-6316.1 AMDTX 050598.2 Covnncd 6329-228 YEEK I4 RAT LIVER REWORK c-w Smpk I DUY _ _ .,lid \VL I __ lOaa lW 40 I3 40 I 3 IW 3 I W3 - ImU3 I W3 IW 3 IOIIS 0 Yim Io m OW 10116 0yYY I m1s - luoul 0yY3 OW11 0 wB3 own IUM IW ~ IO161 OWiJ Iu U 3 - l O l l 4 1- Tub1 h1.u - P>UbSld "IU*.r :"rr.sUu IF.clor NA OYLJS _ _ _ NA OYIJS NA OYLlS NA UYZJS NA NA NA NA - NA NA NA NA NA OYLlS NA OYLJS NA OYZlS NA OYZJS _ _ NA OY2lS NA 09271 NA OY1JS NA OY21S - NA NA NA UY2JS OYLlS OY215 NA OY2JS NA OYLJS NA OY2JS NA 0 Y21S NA OY2lS NA UY2lS NA OY21S NA 0 Y275 NA oyns \=NolApl X2ll.rrml' lol IY3 PFUSPurlq Co.ralb. Fsctor uhw ubw uhm Uhw NA NA NA NA Ubw Uhrn Uhr Uhw u- UrAmW Uhw Uhw Uhrn UrsnaO uhw ubm UhM Uhw Uhm UrAmm Uh4 Uhm Unlmr Vatnow d PFUS cor. a111 om om om om lMl iim NA NA 963 u3 10s 674 m w 45a 4Yl 362 311 131 zy IJl M 159 4as un 3a 342 431 WOS Wvlw F..lor I I I I I I NA NA I 54 1 I I I I I 1 I I 80 80 80 E4l 73 u 25 u 7.5 - M.. PFOS 9 9 - 89% -NA -2 % - 0411 18.8 13.2 - I S 0 14.6 124 11.1 9.44 711 6.30 10.1 -Y 19 Analytical Report: FACT-TOX-003 LRN-U2104 - PPOSA EO.& -3%- 3.82 4.99 390 161 111 19 96.0 9.m 366 8 14 8 21 73 112 8.M 8.6s 126 I14 12.3 I13 13.4 I34 Y4S 1 9.44 14.3 14.4 1.10 7.16 5Yl S* 4% 43 4661 4% sm 318 314 528 I 49l 53 - 3% tmi FACT-M-2 0 Eu.IYJ 3M Environmental Laboratory Page 173 3M Medical Department Study: T-6316.1 R n c IS- I NA NA 1 10 21.7 1 31 0 I 12 5 I I 10 1 I 406 I 53.8 I 1 I m 10 10 10 IO 10 5 5 20 kd w- .blr om sioim 1137 SIOI2WM6 254 SIOIZ\hu(7 NA NA NA NA 111 SIOIN9014 1% SlOlXHlW 111 SIOINYII5 164 SIOI~I6 39 I SIOIN9017 616 SlOlXHllK 545 s101299Ou 218 SlOlzWDu 289 SlOlXHlZ4 374 SIOI~ 431 SI0129926 x)52 A10259X62 BY9 s101m2 2458 S101XHl32 1979 SIOIXHl33 6083 SlOlrraY mi siotrrxlu 380 sIolz99o1o 275 s101299011 u163 AIO25YJJ63 Analytical Report: FACT-TOX-003 LRN-U2104 lot 936 EIFOSE C J o rI NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR Nil NR NR NR NR NR NR NR NR NR WOSE cFnrhllolbr. I 1 I I I I 1 1 I I I I I I I 1 I I I 1 I I I I I I I I EtFOSE C.*..C&OI1 n NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR UR NR NR NR 3M Environmental Laboratory Page 174 3M Medical Department Study: T-6316.1 AMDTY 050598.2 CovsnerY 6329-228 Analytical Report: FACT-TOX-003 LRN-U2104 0.9949 09949 0.9949 0.9949 0.9949 0.9919 0 99.9 0.9919 0.9949 0.9919 0 9949 0.99.9 1 I I 1;\ :% ::! 0 8640 41111 0,1640 411 IW 3114s 01640 172 JOl4I 0 164 410 33120 0 1640 139 LW lC9M 0.164 362 IW 300lJ OW m IW 36916 0.1640 372 100 3W7 01640 313 lW 27346 01640 Ill6 I 1211 rms. P~I-WUC.*~ 164 0.710 1.16 1c6 3M Environmental Laboratory Page 175 3M Medical Department Study: T-6316.1 AMDTY OSOSyS.2 Cuvsocel6329-228 Analytical Report: FACT-TOX-003 LRN-U2104 mr-70 Gd 97 3M Environmental Laboratory Page 176 3M Medical Department Study: T-6316.1 AMDTI OM598.2 Covaac& 6319.228 Analytical Report: FACT-TOX-003 LRN-U2104 3M Environmental Laboratory Page 177 3M Medical Department Study: T-6316.1 I b.3 OW I "0.3 1 b.. OW I OW lib.. OW I OW L'b.. om I om AMDTY 050598.2 Cuvancd 6329.128 WFEK 53 RAT LWEI SY* * Mabod B l t wmi100-mo8it-i wwi100-muonit-a wLml4mhruBlt-l RBLMl4m-Lr.rBlt-1 C91197!MffiI-MS.I C91I9lMffil-MSD.1 91195IMKiI-MS-I NlI1JIMXil-MSD-I RBLMIW-MS-I RBLMIIO-WD-I C91195M C91199M CPIIJM C92UIM C91146M cP134IF C92116F C91111F C91316F C91390F C91IlM C9ll74M oiia7~ C91UIM C91310M OUWF C914llF C~I~IIF cP14YF C914lIF llvl"Z Lun*od " d m m Wb dI o.m o.m 0.m 4.17 116 224 401 114 111 161 IM llJ5 611 in 1s11 OW Im I89 191 111 41JlJ ima JOl48 31110 lW IOWS IWSI 10647 17146 1111 Analytical Report: FACT-TOX-003 LRN-U2104 3M Environmental Laboratory Page 178 3M Medical Department Study: T-6316.1 AMDTI 09598.2 CovsnceU 6329-228 Analytical Report: FACT-TOX-003 LRN-U2104 RBLMI4W MID-! CWl91M C912OJM NllllM C91UIM C911JlM C91119M C911JlM Oll31F 01334F C9134IF L92JJ4F LPIJJIF CP2360F L'91361F CP2,WF OXl4F OlJ83F C92161M COllddM CPlllOM V91111M C9111IM C9111JM C91JVPM C911II.U CYI3I.M CPlIIIM OUIOF LPl416F 01419F 0141JF Ol43OF 01131F CPU3PF LY1443F CPl45IF CPI4JlF M M hA NA NA NA Ibb N u C d n d l l l d kd<'mfiL".d NA NA NA NA NA NA M M NA M NA NA M NA NA NA NA NA NA NA NA hA NA NA NA N* NA NA NA HA M NA NA hA NA hA NA NA NA ll@ N m C d o d h* __ IdUd HI. a ___ I UXMJ 1 (XI* __ l u x x l Iw x 1 1,1451 I UIJJ I0141 I u141 lmoU I u)(xI I 04JJ IU l l l IOld0 I0160 UPI~J IWlI I WJ6 09916 IW J I 09YIO 09971 I WIY I UJ45 Iwul 09168 IM9I I014J ,0113 I 01Ul I 0811 "09961 YVV) 1 U07b 1W l l IW J l I0181 09811 0 9811 0 9963 09981 1 ow4 09916 10141 "9980 0 9PJ6 lull3 I Wl 0YI9U 0 PYl1 0 9911 R-NuRq A=LiUApl D;).ln* *A HA N.4 NA NA NA NA NA NA NA NA NA NA NA NA NA N4 NA NA NA NA N* NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA N* NA NA NA NA NA NA NA NA NA d UC Y.BuIu0. 101 171 mas M1 ha, CY.F-r 09949 0 9949 09949 09949 0 9949 09949 09949 0 9949 0 9949 0 9949 0 9949 0 9949 0 9949 0 9949 0 9949 0 9949 0 9949 0 9919 0 9949 0.9919 ow 0.9949 0.9949 0.9949 0.9949 0.9949 0 9949 0.9949 0 9949 0 9949 0.9949 0.9949 0.9949 0.9949 0 9949 0 5949 0 9949 0 9949 0.9949 0.9949 0.9949 0.9949 0.9949 0 9949 0 9949 0 9949 0.9949 0 9949 0.9919 0.9949 EiiGzT crr*ru.. FX,.. 0 8640 0 8640 0.8640 0 8640 0.mo 0.8640 0.1640 0 8640 0 1640 0 8640 u.aw 0 11640 0 a640 0 1640 o a640 0 8640 o.aw 0.1640 om a 0.1640 0.8640 0 1640 o a640 0.8640 0 8640 0.1640 0 1640 0.8640 0 a640 0.8610 0.1640 0.8640 0 1w 0 1640 0.8640 O.IMO 0.8640 0.1640 0 8640 0.8640 0.a640 0 I640 0 8640 0 1640 0.9640 0.1640 0.8640 0.8640 0.8640 0 1640 1191 1112 231 161 J3.1 3.44 9.43 0.M 13 0 186 I69 1.19 109 31.3 140 IJ8 I11 189 419 131 111 110 616 643 III 491 294 I I 1 10 10 1J15 DOJI5m011 7 JI 10 11701 WJIJOI9 111 IO 3111 WJlJWlO 371 10 20 10 10 1351 6118 9900 16395 11614 11968 Le WJlJm036 DOIlJmOlJ WllJm017 WJI5mOlI DOJlJmOlI WJlJmOl3 WJIJOOOU 6 I1 990 lJ.0 lotL.IJ109 OUI Wk.. sum* V.rlrd WOSA Old h r W C...rUr WlMSDIPD Frlw NA NA 09110 NA NA 09JIO NA NA 09510 NA NA NA 09J10 09JIO I% NA 09110 NA 11% NA 09510 09JLO NA 0.9110 11% NA 09510 NA 0.9JlO NA 09110 NA 0.9JlO NA OJJlO NA 09110 NA 0.9J10 NA 0.9J10 NA 09J10 141 NA o.mn NA NA 09510 09510 0.9J10 NA 0.9510 NA 0 9J10 NA 09J10 NA 0.9J10 NA 09JIO HA 09110 NA 09110 59.9 NA O.OJ11 NA 0.9510 09510 j 1 : NA 09110 tu 09110 NA 09110 NA 09510 0.9110 09110 09110 09JIO 09JIO OPJIO 0.9110 09110 09510 09110 O9JlO NA 09110 09510 09JIO 45.1 0.9510 4.31 09JlO *- mS* crr OW im "W - 139 161 - 191 196 -113 160 0.m0 O.W OW Q 11 0.W Om0 Urn0 OW - O W om Om0 0 37 009 0.m u ua 0 16 0J l Om -0 41 0 33 UXI 164 19J ,ai 13J I41 169 iJJ - 1 J I 141 166 729 .94 J92 380 &bJ J11 611 - 46A 411 - pFD6* DhUr Frlr, I I ~ I I ~ I _I _ I I ~ - I I I I I I 1 I I I - I I 1 , I I I I I I - 1 I I I I I I I I I I I I I I I 3 I I - I I + m. Wcur om 110 ow 114 246 iw 181 181 1J9 ow 000 Om 0 14 om "W OW ow 00 OW OW 0 31 ow om 009 0 16 0 J1 OW 046 0 31 4la 110 119 JUJ 146 351 180 JlO t6J xa 146 11) 511 PI 401 UI JII 61 I .w 446 3M Environmental Laboratory Lrr W.* IM TOX-Un-lnml8-P Page 179 3M Medical Department Study: T-6316.1 0.110 0 5w os12 0 b14 oa 4 0.55, 0 490 OW 10371 0 550 AMDTY 05059112 C u r a n d 6329.128 PFOSM Cde OW OW ow ow 11. 26(1 251 154 211 266 0.w 0.w 0.00 0.w ow 0.w 0.00 0.00 000 000 0.w 0.w ow 0.W OW 0.w om ow ow 0.00 574 113 111 761 m 527 4v7 6911 404 753 162 771 PfLUM DYmU. Fr(w I I I I I I I I I I I I I 1 1 1 I I 1 I I 1 I I 1 I 1 I I I I I 10 I I I I mM wr C L -3%0.w 0.00 OW 115 149 240 - 141 211 166 0.m 000 0.w OW 0.w 0.w 0.w 0.w 0.w ow ow ow 0.w ow 0.w 0.w - 0.W ow ow 0.70 591 196 1219 771 612 511 511 565 496 591 545 1811 414 OJ a96 707 414 110 11s 796 Analytical Report: FACT-TOX-003 LRN-U2104 m suI*r. M%M' SD WD NA NA NA NA 15% 1 IS NA NA NA NA 74 9 0551 s.ma.l. Vmwd 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 M NA M NA NA NA NA NA NA NA hSWI3 -ad h* crnruu F.Cl. 0 9770 09770 0 9770 09770 0 9770 0 9770 o9no 09770 0 9770 0 9770 0 9770 0 9770 09770 0 9770 o 9no 0 9770 0 9770 o9no 09770 0 9770 0 9770 0 9710 0.9770 09770 0 9770 09770 0 9770 0 9710 0 9770 0 9770 0 9770 09770 0 9770 0 9770 0 9770 0 9770 0 9770 0 9170 0 9770 0 9770 wno 0 9770 09770 0 9770 0 9770 0 9770 0 9770 0 5770 09770 0 9770 + WMP E.r OW oar OW in 231 472 4JP \R \x OW OW ow OW OW OW ow OW uw "W OW am ow OW ow ow OW ow ow am ow OW ow ow ow OW OW OW 000 OW 000 OW OW 601 ow ow OW ow ow ow 3M Environmental Laboratory Page 180 3M Medical Department Study: T-6316.1 AMDTY ososw.2 Cavaocd 6329-128 LPD YI a,. MSlMSDM NA NA NA NA 11% 3% NR NA NA NA NA NA NA NA NA Analytical Report: FACT-TOX-003 LRN-U2104 3M Environmental Laboratory Page 181 3M Medical Department Study: T-6316.1 s r p, RBl.MlunIu~~Ilt C~~IJJIM C911JJM C91139M C91lJlM OlJlJF - MI54 Crr. 3%- 0.m Om om - J61 310 - JI6 J3I - 156 217 -om om 0.m om 0.m o.m 0.m om 0.m 0.m _. Om 0.m 0.m o.m 0.00 o.m 0.m 003 - 0.m o.m 1JO JI4 401 I13 I94 JM I4 111 140 _ 101 . 434 .l6 311 10 14J 161 339 JO1 -110 1SS A M D T X 050s9a2 Cuvancd 6329-21 Analytical Report: FACT-TOX-003 LRN-U2104 on0 0.336 m su I*.. MSIMSDW D NA NA NA NA 1.418 4 JI'& JZ NA NA NA J4.7 0 0165 11.2 omia S.rr.ute V*W 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 NA NA NA NA NA M NA NA NA NA NA NA NA NA NA NA NA lo( 119 ~~~h~ CunN.. VW.. 0 9930 0.9910 0 WJO OWJO 09930 0 9930 0.99JO 0 WJO 0.991 0 9930 0.9930 0991 0.99JO 0.9930 099JO 09930 099JO 0.99JO 0.99JO 0.99910 0.WJO OWJO 09930 0 9930 OW30 O.WJ0 0.9930 0.9910 0.9PJO 0 99JO 0.9930 0.9930 0 9910 09930 0.99JO 0.9930 0 99JO 09930 0.99JO 0.9910 0.WJO O.WJ0 O.WJ0 0.99JO 0 9930 O.WJ0 0 9930 441 1J3 JJI J9J 1 0.w om om 0.m Om Om 0.m om 1 om om I om 0.m om 1 o.m om om 0.W Om 1 0.00 I ow 3M Environmental Laboratory Page 182 3M Medical Department Study: T-6316.1 AMDTlO50598.2 Cuvsocd 6329-lul Analytical Report: FACT-TOX-003 LRN-U2104 + PFOS cdc. cur 0 ,m "a) 4 77 226 214 11% 401 117% 124 110% IJI 760 1bI urn 44 I 0.M41 c L a ) (0.0107WIJ :WQcool07 uyu c WQ(0.01m WIJ 00712 CPIIJJM C9113YM C91151M CPl31JF OlJ3.F CYlJ48F 'LW(OOl07Wl) < LwcoolmWI) om ~LoQ~ool07"ylJ I15 0.211 146 0.146 im DlW 91 0 O.Wl0 I53 0 LJJ CYlI54F CYll5.F O13MF IJI 00131 6 42 rL~(oolm"y1~ I: 4 00114 C9136lF 29 9 0 0299 c9lJWF Ill 0112 CPlJ74F 111 0.111 CVlJ1JF 1 JO 0 I50 c91l6l.U 50J7 J.M CIllMM 7515 7.51 CYll7OM 117Q2 I2 7 r~iiia~ 1711 1.71 CV111IM 4bw 4.66 CYllIJLI 10141 101 CYlJWY 161 0.561 CYIJIIM l"1. 2 01 CVllllM 1671 1.68 $1.2 ('YlIlIM 6149 6 15 3 10 Ol4IOF 10717 10.7 CY1416F lWll 10 9 CYUI9F 1177 2.11 C9141lF 5114 511 CYl4JOF C914J IF C9143Y F 1351 6171 YYm 8-15 6 I1 9 90 CtlUlF IbJ9J 16.4 C9115IF 11614 11.6 4J.l C92WF 1068 15.0 435 R*Nah$.d A=WAF@ld& x ) - barldQuunu* 0.110 0 371 0 165 0.1J1 0.116 0.773 O.Jl1 06M 0.40~ 0 414 055l 0671 490 0.490 416 0 446 PFOS I Peal"PWSA =P d u d M- PFCSAA PP) WDn wmc ur C- d. hvIIMsDpID dr om 4.w NA om om NA om 165 uI1 IJ I 111 a51 11% 1% 4J9 I I Nn 9% 166 17% Im 11% NII om ~LnQ(0.0J14uy1) om om om OW om om Om om NA om 4.w NA om om om om I 9: I ::::0611 20 1 0111 I 496 707 710 IIJ 796 OJ91 1.111 0.414 0.135 0.496 0.707 0 710 0 115 0 7% 0.771 0.411 .. U .U >* U U U M mr.7 0 E . 4 91 3M Environmental Laboratory Page 183 ~~~ ~~ 3M Medical Department Study: T-6316.1 3M Medical Department Study: T-6316.1 Appendix F: Example Calculations Analytical Report: FACT-TOX-003 LRN-U2104 Analytical Report: FACT TOX-003 LRN-U2104 Formula Used for Sera Analyses in Study FACT TOX-003 AR (ng/mL) x DF x PC2 x FV (mL) x 1.0 1.18 = Reported Concentration (pg/mL) PC1 EV(mL) 1OOOng Calculation Used for Group 9, Week 105, Animal ID C92430/F 199ng/mLx 1 0 ~ 0 . 8 6 4 0x 1 mL x 1.Opg = 1.73pgImL 0.9949 1 mL 1OOOng AR- Analytical result from MassLynx summary DF- Dilution factor PC 1-Purity Correction-1 PC2-Purity Correction-2 FV-Final extract volume (1.O mL unless otherwise noted) EVLVolume of sera extracted Formula Used for Liver Analyses in Study FACT TO:K-003 a AR (ng/g>x curve (') x sc x DF x 1.0 pg = Reported Concentration (pg/g) a sample 1000 ng ('I a curve is assumed to be: 1 g liver 5 mL H 2 0 Calculation Used for Group 9, Week 53, Animal ID C92267M 497 ng/g x 1 g/ 5 mL x 0.8640 x 100 x 1.0 pg = 42.6 pg/g 1.0137g/ 5mL 0.9949 1000 ng aAR- Analytical result from MassLynx summary curve-Density of the liver standard curve, assumed to be lg liver/ 5 ml water 3 sample-Density of the liver sample (g sample/ 5 mL H20) PC 1-Purity Correction-1 PC2-Purity Correction-2 DF- Dilution factor 3M Environmental Laboratory Page 184 3M Medical Department Study: T-6316.1 3M Medical Department Study: T-6316.1 Appendix F: Example Calculations Analytical Report: FACT-TOX-003 LRN-U2104 Analytical Report: FACT TOX-003 LRN-U2104 Formula Used for Sera Analyses in Study FACT TOX-003 AR (ng/mL) x DF x PC2 x FV (mL) x 1.0 pg = Reported Concentration (pg/mL) PCl EV (mL) 1000 ng Calculation Used for Group 9, Week 105, Animal ID C92430/F 199 nglmLx 10 ~ 0 . 8 6 4 0x 1 mL x 1.Opg = 1.73pg/mL, 0.9949 1 mL 1OOOng AR- Analytical result from MassLynx summary DF- Dilution factor PC 1-Purity Correction-1 PC2-Purity Correction-2 FV7Final extract volume (1.O mL unless otherwise noted) EV-Volume of sera extracted Formula Used for Liver Analyses in Study FACT TOX-003 a AR (ng/g) x curve (I) x SC x DF x 1.0 pg = Reported Concentration (pg/g) a sample 1000 ng (I) 3 curve is assumed to be: 1 g liver 5 mL H20 Calculation Used for Group 9, Week 53, Animal ID C92267M 497 ng/g x 1 g/ 5 mL x 0.8640 x 100 x 1.0 pg = 42.6 pg/g 1.0137 g/ 5mL 0.9949 1000 ng AR- Analytical result from MassLynx summary 3 curve-Density of the liver standard curve, assumed to be l g liver/ 5 inl water 3 sample-Density of the liver sample (g sample/ 5 mL H20) PC 1-Purity Correction-1 PC2-Pur ity Correction-2 DF- Dilution factor 3M Environmental Laboratory Page 185 3M Medical Department Study: T-6316.1 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 Analytical Report: FACT TOX-003 LRN-U2104 Appendix G: Interim Certificates of Analysis 3M Environmental Laboratory i Page 186 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 Test Name Purity' 4ppearance [dentification NMR Metals (ICPMS) 1. Calcium 2. Magnesium 3. Sodium 4. Potassium* 5. Nickel 6. Iron 7. Manganese rota1 YOImpurity (NMR) rota1 % Impurity [LCMS) Total % Impurity (GCMS) Related Compounds - POAA Residual Solvents (TGA) Purity by DSC Inorganic Anions (IC) 1. Chloride 2. Fluoride 3. Bromide 4. Nitrate 5 . Nitrite 6. Phosphate 7. Sulfate4 Organic Acids ' (IC) 1. TFA 2. PFPA 3. HFBA 4. "PA Elemental Analysis? 1. Carbon 2. Hydrogen 3. Nitrogen 4. Sulfur 5. Fluorine COA023-018B 3M Environmental Laboratory Specifications White Crystalline Powder 1. Theoretical Value = 17.8% 2. Theoretical Value = 0% 3. Theoretical Value = 0% 4. Theoretical Value = 5.95% 5. Theoretical Value = 60% Result 86.4% Conforms Positive 1. 0.017 wt./wt.% 2. 0.007 wtJwt.% 3. 1.355 wt./wt.% 4. 6.552 wt./wt.% 5 . 0.003 wt./wt.% 6. 0.004 wt./wt.% 10.60 wt./wt.% None Detected 0.30 wtJwt.% None Detected Not Applicable3 1. CO.0 15 wt./wt.% 2. 0.27 wt./wt.% 3. <0.040 wt./wt.% 4. <0.009 wt./wt.% 5 . C0.006wt./wt.% 6. C0.007 wt./wt.Yo 7. 8.82 wt./wt.% 1. CO.1 wt./wt.% 2. <0.1 wt./wt.% 3. <0.1 wt./wt.% 4. <0.25 wt./wt.% 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.% Page 1 o f 3 Page 187 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 L 3048 Research Drive State College, PA 16801 Phone: (814)231-8032 Fax: (814)231-1253or (814)231-1580 INTERM CERTIFICATE OF ANA.LYSIS Centre Analytical Laboratories COA Reference #: 023-018B 1 Date of Last Analysis: 0813 1/00 Expiration Date: 08131/01 Storage Conditions: Frozen 5-10C Re-assessment Date: 08/31/01 'Purity = 100% - (sum of metal impurities, 1.39%+LC/MS hnpurjties, 10.60%+InorganicFluoride, 0.27%+NMR impurities, 1.OO%+ PO& 0.30%) Total impurity from all tests = 13.56% Purity = 100% - 13.56% = 86.4% *Potassiumis expected in this salt form and is therefore not considered an impurity. 3Purityby DSC is generally not applicable to materials of low purily. No endotherm was observed for this sample. 4Sulfurin the sample appears to be converted to SO4 and 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 so4 is not considered an impurity. 'TFA HFBA NFPA PFPA Trifluoroacetic acid Heptafluorobutyric acid Nonofluoropentanoic acid Pentafluoropropanoic acid 6Theoreticalvalue calculations based on the empirical formula, C8F17S03Xt (Mw=538) This work was conducted under EPA Good Laboratory Practice Stzidards (40 CFR 160). COA023-018B 3M Environmental Laboratory Page 2 of 3 Page 188 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 CEntrtz Analytical Laboratories, Inc. 3048 Research Drive Phone: (814) 231-8032 State College, PA 16801 Fax: (814) 231-1253 or (814) 231-1580 INTERIM CERTIFICATE OF ANALYSIS Centre Analytical Laboratories COA Reference #: 023-018B t LCMS Purity Profile: Impurity c c4 c5 C6 c7 Total Note: The C4 and C6 values were calculated'using the C4 and C6 s'tandardcalibration curves, respectively. The C5 value was calculated using the averag: response factors fiom the C4 and C6 standard curves. Likewise, the C7 value was calculated using the average response factors from the C6 and C8 standard curves. Prepared By: K~A# PA/*& Dawd S. Bell Date lytical Laboratories - ?h. Date anager, Centre Analytical Laboratories COA023-0 18B 3M Environmental Laboratory Page 3 of3 Page 189 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 Centre Analytical Laboratories, Inc. , /1 3048 Research Drive Phone: (814) 231-8032 State College, PA 16801 Fax:(814) 231-1253 or (814)231-1580 INTERIM CERTIFICATE OF ANAL YSIS Centre Analytical Laboratories COA Reference #: 023-022-2 3M Product: EtFOSE-OH Test Control Reference #: TCR-00017-52 Purity: 97.4% (1 3. Sodium 4. Potassium 5. Nickel 1. Chloride 2. Fluoride 3. Bromide 4. Nitrate 5 . Nitrite 2. PFPA 1. Carbon 2. Hydrogen 3. Nitrogen COA023-022-2 3M Environmental Laboratory 1. <0.001 wt./wt.% 2. <0.001 wt./wt.% 3. <0.001 wt./wt.% 4. <0.001 wt./wt.% 1. . <0.015 wt./wt.% 2. <0.005 wt./wt.% 3 . <0.040 wt./wt.% 4. 0.009 wt./wt.% 5 . c0.006 wt./wt.% 1. <o. 1 wt./wt.% 2. <0.1 wt./wt.% 1. Theoretical Value = 25.2% 2. Theoretical Value = 1.75% 3. Theoretical Value = 2.45% 1. 25.04 wt./wt.% 2. 1.69 wt./wt.% 3. 2.61 wt./wt.% Page 1 of 3 Page 190 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 Lrn 3048 Research Drive Phone:(814) 231-8032 State College, PA 16801 Fax: (814) 231-1253 or (814) 231-1580 INTERIM CERTIFICATE OF ANAL KSIS Centre Analytical Laboratories COA Reference #: 023-022-2 3M Product: EtFOSE-OH Test Control Reference #: TCR-00017-52 Date of Last Analysis: 11/26/00 Expiration Date: 11/26/01 Storage Conditions: <-lo "C Re-assessment Date: 11/26/01 'Purity = 100%- (total NMR impurities, 1.26%+ GCMS impurities, 1.29 + POAA, 0.10%) Total impurity fiom all tests = 2.65% Purity = 100%- 2.65%= 97.4% * 'TFA Trifluoroacetic acid fl HFBA Heptafluorobutpc acid NFPA Nonafluoropentanoic acid PFPA Pentafluoropropanoic acid 3Theoreticalvalue calculations based on the empirical formula, C ~ ~ H ~ O F ~ ~ N O ~ S (MW=571) COA023-022-2 3M Environmental Laboratory Page 2 Of3 Page 191 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 CEntrE Analytical Laboratories, Inc. 3048 Research Drive State College, PA 16801 f" Phone: (814) 231-8032 Fax: (814) 231-1253 or (814) 231-1580 INTERIM CERTIFICATE OF ANAL KSIS Centre Analytical Laboratories COA Reference #: 023-022-2 3M Product: EtFOSE-OH Test Control Reference #: TCR-00017-52 GCMS Purity Profile Peak # 1 2 Retention Time (fin) 13.934 17.307 I Identity PFOSDEA c7 O,b Impwity 0.36 0.93 This work was conducted under EPA Good Laboratory Practice Standards (40CFR 160). Prepared By: Scientist Centre Analytical Laboratories COAO23-022-2 3M Environmental Laboratory Page 3 of3 Page 192 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 CEntrE Analytical Laboiratories, Inc. L m 3048 Research Drive Phone: (814) 231-8032 State College, PA 16801 Fax: (814) 231-1253 or (814) 231-1580 INTENM CERTIFICATE OFANAL YSIS Centre Analytical Laboratories COA Reference #: 023-022-1 3M Product: EtFOSE-OH Test Control Reference #: SD-013 Purity: 88.9% 4. Potassium 1. <0.001 wt./wt.% 2. <0.001 wt./wt.% 3. <0.001 wt./wt.% 4. 0.002 wt.lwt% 5. <0.001 wt.lwt.% 6 . <0.001 wt./wt.% COA023-022- I 3M Environmental Laboratory 2. <0.005wt./wt.% 3. <0.040 wt./wt.% 4. <0.009wt.lwt.% 1. <0.1 wt./wt.% 1. Theoretical Value = 25.2% 2. Theoretical Value = 1.75% 1. 24.42 wt./wt.% 2. 1.78 wt./wt.% 3. 2.72 wt.lwt.% 4. 9.34 wt.lwt.% Page 1 Of 3 Page 193 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 L m 3048 Research Drive Phone: (814) 231-8032 State College, PA 16801 Fax: (814) 231.1253 or (814) 231-1580 INTERIM CERTIFICATE OF ANALIYSIS Centre Anaiytical Laboratories COA Reference #: 023-022-1 3M Product: EtFOSE-OH Test Control Reference #: SD-013 Date of Last Analysis: 11/26/00 Expiration Date: 11/26/01 Storage Conditions: <-lo "C Re-assessment Date: 11/26/01 'purity = 100% - (total metal impurities, 0.002% + totalNMEZimpurities, 0.90% + GCMS impurities, 10.21 + POAA, 0.03%) Total impurity fiom all tests = 11.14% Purity = 100% - 11.14%=88.9% a 'TFA Trifluoroacetic acid f? HFBA Heptafluorobutyric acid NFPA Nonafluoropentanoic acid PFPA Pentafluoropropanoic acid 3Theoreticalvalue calculationsbased on the empirical formula, C12H1817N03S , (MW=571) COA023-022-1 3M Environmental Laboratory Page 2 of 3 Page 194 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 CEntrG Analytical Laboratories, Inc. 3048 Research Drive State College, PA 16801 Phone: (814) 231-8032 Fax: (814) 231-1253 or (814) 231-1580 fl INTERIM CERTIFICATE OF ANALYSIS Centre Analytical Laboratories COA Reference #: 023-022-1 3M Product: EtFOSE-OH Test Control Reference #: SD-013 GUMS Purity Profile %Impurity . 0.12 0.23 0.51 0.21 0.34 0.62 0.11 0.11 1.11 1.55 1.07 3.30 0.93 10.21 This work was conducted under EPA Good Laboratory Practice Staridards (40 CFR 160). Prepared By: Scientist ratories 'Laboratory Manager Centre Analytical Laboratories f- COA023-022-1 3M Environmental Laboratory Page 3 Of3 Page 195 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 W J V: ANALYTICAL REPORT STUDY TITLE P W Y DETERMINATIONOF SAMPLE LOTS OF PFOS Sample # TCR-00065-022 DATA REOUIREMENTS Test Article Characterization STUDY DIRECTOR "THISISAN EXACTCOW OF 7HE ORIGINAL DOCUMENT" Kevin LIoyd ANALYTICAL REPORT COMPLETION DATE October 25,2000 PERFORMING LABORATORIE / TESTING FACILITIES Centre Analytical Laboratories, Inc. (Centrz) 3048 Research Drive State College, PA 16801 Phone: 814-231-8032 STUDY SPONSOR 3M Environmental Technology and Safety Services Building 2-3E-09 PO Box 33331 St. Paul, MN 55133-3331 PRO.I''ECT IDENTIFICATION Centre Study Number: 023-045 Total Pages: 15 3M Environmental Laboratory Page 196 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 Centre Study No.: 023-045 W GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT Centre Study Number 023-045, entitled "Punty Determination of Sample Lots of PFOS, Sample # TCR-00065-022" conducted for 3M Environmental Laboratory, was performed in compliance with US EPA Good LaboratoryPractice Standards)(40 CFR Part 160)by Centre Analytical Laboratories, Inc. with the following exceptions: The automated data collection systems used in this study were not fully compliant with 21 CFR 58.130 (e). Kevin Lloyd Date Study Director ' ' Centre Analytical Laboratories, Inc. w Date 3M Environmental Technology and Safety Services Centre Analytical Laboratories, I ~ c . 3M Environmental Laboratory Page 2 of 15 Page 197 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 Centre Study No.: 023-045 V QUALITY ASSURANCE STATEMEm Centre Study Number 023-045, entitled "Purity Determination of Sample Lots of PFOS, Sample # TCR-00065-022"was reviewed by Centre Analytical Laboratories' Quality Assurance Unit. All reviewed phases were reviewed for conduct ;iccording to Centre Analytical Laboratories' Standard Operating Procedures, the Stud:/ Protocol, and all applicable Good Laboratory Practice Standards. All findings were reported to the Study Director and to management. - Phase 1. Protocol Review Date Insoected 1o/ 19/00 Date Reported to Study Director and m e Management 10/25/00 Date Reported to Sponsor Manarrement 10/25/00 2. Raw Data Review 10/19/00 A W 3. Report Review 10/24/OO 10/25/OO 10/25/00 10/25/00 10/25/00 wiliiam spare Quality Assurance Officer /o 2 5 0 - Date "THIS ISAN EXACT COPY OF THE ORIGINAL DOCUMENT" BYSTt-mm2m Centre Analytical Laboratories, Inc. 3M Environmental Laboratory Page 3 of 15 Page 198 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 Centre Study No.: 023-045 V CERTIFICATION OF AUTHENTICITY This report, for Centre Study Number 023-045, is a true and complete representation of the raw data for the study. Submitted by: Centre Analytical Laboratories, Inc. 3048 Research Drive State College, PA 16801 (8 14) 23 1-8032 Study Director, Centre: Kevin Lloyd Date Study Director Centre Analytical Laboratories,Inc. 1 W Centre Analytical Laboratories, Inc. Facility Management: / RichardA. drazzi Date President Centre Analytical Laboratories, Inc. Centre Analytical Laboratories,Inc. 3M Environmental Laboratory Page 4 of 15 Page 199 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 Centre Study No.: 023-045 STUDY IDENTIFICATION PURITY DETERh4INATIONOF SAMPLE LOTS OF PFOS Sample # TCR-00065-022 TYPE OF STUDY: TEST SYSTEM: TEST ARTICLE: SPONSOR: I W STUDY DIRECTOR: TESTING FAClLlTES: ANALYTICAL PHASE TIMETABLE: V Characterization Not Applicable PFOS, Test Control Reference # TCR-00065-022 3M Environmental Technology and Safety Services Building 2-3E-09 PO Box 33331 St. Paul, MN 55133-3331 Kevin Woyd Center Analyticai Laboratories, Inc. Phone: (814) 231-8032 CentreAnalytical Laboratories,Inc. (Centre) 3048 &search Drive State College, PA 16801 Phone: 814-231-8032 Study Initiation Date: Analytical Start Date: Analytical Termination Date: 09/28/00 10/05/00 10/24/00 `THIS IS AN EXACT COPY OF THE ORICiINAL DOCUMENT" BY X-DATE .E&&& Centre Analytical Laboratories. Inc. 3M Environmental Laboratory Page 5 of 15 Page 200 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 Centre Study No.: 023-045 PROJECT PERSONNEL The Study Director for this project was Kevin Lloyd at Centre Analytical Laboratories, Inc. The following personnel from Centre Analytical Laboratories, Inc., were associated with various phases of the study: Name Gerry Shero David S. Bell Emily R. Stauffer Mark Ammerman Title Scientist Scientist Scientist Sample Custodian 'THIS IS AN EXACT COPY OF THE OR'lGlNALDOCUMENT" Centre Analytical Laboratories, Inc. 3M Environmental Laboratory Page 6 of 15 Page 201 3M Medical Department Study: T-6316.1 . . Analytical Report: FACT-TOX-003 Centre Study N o . . LRN-U2104 023-045 TABLE OF CONTENTS &gg TITLEPAGE ...................................................................................................................... 1 GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT ............................. 2 QUALITY ASSURANCE STATEMENT.......................................................................... 3 CERTIFICATIONOF AUTHENTICITY........................................................................... 4 STUDY IDENTlFICATION ............................................................................................... 5 PROJECTPERSONNEL.................................................................................................... 6 TABLE OF CONTENTS.................................................................................................7... 1.0 SUMMARY............................................................................................................... 8 2.0 INTRODUCTION.......................................................................................................8.. 3.0 TEST SYSTEM ............................................................................................................ 8 4.0 TEST ARTICE............................................................................................................ 8 5.0 EXPERIMENTALPROCEDURES ............................................................................9. 6.0RESULTS AND DISCUSSION.................................................................................. 10 8.0 CIRCUMSTANCESTHAT MAY HAVE AFFECTED TI% DATA....................10 9.0 RETENTION OFDATA AND SAMPLES ............................................................ IO a Table I. Results From LC/MS forTCR-00065-022........................................... 11 U- Figure 1.PFOS Calibration Standard (C100SOO-4)at 269pg/L .............................. 12 Figure 2.PFOS Calibration Standard (ClOO500-2) at 539pg/L .............................. 13 Figure 3. PFOS (TCR-00065-022) at 250pg/L. ESI Negative 1011Mode ...................... 14 . Appendix A: Study Protocol OOP-023-045:PURITYDETERlWNATION OF SAMPLELOTS OF PFOS. Sample # TCR.00065.022 Including Amendment 1 .........15 `THIS IS AN EXACT COPY OF THE ORIGINAL DOCUMENT" Centre Analytical Laboratories. Inc. 3M Environmental Laboratory Page 7 of 15 Page 202 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 Centre Study No.: 023-045 1.0 SUMMARY The punty of PFOS from sample # TCR-00065-022, was detemlined from the LCMS test data to be 88.0% pure as compared with the PFOS control ('TCR-00017-046,97.9% purity). 2.0 INTRODUCTION - This report details the results of the analysis of PFOS, Test Control Reference# TCR- 00065-022.The test was analysisby liquid chromatography mass spectrometry (LCMS). The L W S was conducted at Centre. The study was initiated on September 28,2000 when the Study Director signed protocol number OOP-023-045. The analytical start date was October 5,2000, and the experimental termination date was October 9,2000. 3.0 TEST SYSTEM There is no test system associated with this characterization study, therefore the G U requirement for test system description,justification, and identification do not apply. The route of administration, levels and frequency of administration also do not apply to characterization studies. ' 9 1 s IS AN EXACT COPY OF 1HE ORIGINAL DOCUMENT" 4.0 TEST ARTICLE BV4 f - f DATE1 0 The test article was PFOS, Test Control Reference# TCR-00065-022. 3M Environmental Laboratory supplied the test article and it was logged at Centre Analytical Laboratories, Inc. as follows: Compound PFOS TCR-00065-022 Lot or Nl3 Number 193 Centre Control No. 00-023-068 Punty TBD Date jieceived 10/05/00 The sample received was a white, crystalline solid. It was received under frozen conditions and was stored in a temperature-monitored freezer kept at <-1O"C. Centre Analytical Laboratories. Inc. 3M Environmental Laboratory Page 8 of 15 Page 203 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 Centre Study No.: 023-045 The analyticalkontrol standard PFOS was: Control Article PFOS Lot Number TCR-00017-046 Centre Control No. 00-023-042 Punty 97.9% Expiration _Da_te 8/3 1/2001 The chemical and physical data for PFOS is as follows. Common Name: Molecular weight: CAS Number: Structure: PFOS 499 ( C S F ~ ~ S-)O ~ 2795-39-3 I 0 1I c817s 0 4 -K+ 0 Note: the neutral molecule and standard fom that the PFOS (anion) is derived from is potassium perfluorooctane sulfonate ( C ~ F I ~ S O ~mKo)le,cular we:ight538. 5.0 EXPERIMENTAL PROCEDUItES LCNS Spectral Analysis PFOS sample TCR-00065-022was analyzed for purity using liquid chromatography/mass spectrometry(LCMS). The sample was analyzed accordingto the following procedure. The sample was prepared at about 250 pg/mL in methanol and analyzed by LCMS using a Hewlett-Packard 1100WLC system interfaced with a Hewlett-Packard 1100mass selective detector. The mass spectrometer was run in negative ionization mode using electrosprayionization (ESI) using Selective Ionization Monitoring for ion m/z 499. The liquid chromatography system was operated in reversed-phase mode using a C18 silica-based column. Each resulting chromatogram was calculated against the analyticsil control article (TCR00017-046) calibration curve. V Centre Analytical Laboratories,Inc. 3M Environmental Laboratory Page 9 of 15 Page 204 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 Centre Study No.:023-045 6.0 RESULTS AND DISCUSSION LCMS Spectral Analysis Responses were observed for the PFOS sample in negative ion mode using an ESI interface. Quantitation was performed using SIh4 mode, m/z 4951, and PFOS was detected at 7.8 minutes. TH-PFOSwas used as the internal stantlard, m/z 427, and was detected at 7.5 minutes. The total percent purity, calculated against the analytical control article (TCR-00017-046c)alibration curve was determined to be 88.0% from the average of three replicate analyses. 8.0 CIRCUMSTANCES THAT MAY HAVE AFFECTED THE DATA Electronic records are not fully compliant with 21 CFR 11, "Electronic records; Electronic Signature." However, approved SOPSwere in place and all instrumentation used in this study was fully calibrated and operational. All orighal raw data were printed as hard copies and fully audited by quality assurance. Verified ey.act copies and the electronic data will be stored in the archives at Centre Analytical Laboratories. Original d raw data will be returned to the Sponsor. 9.0 RETENTION OF DATA AND SAMPLES When the final report is final, all original paper data generated by Centre Analytical Laboratories, Inc. will be shipped to the sponsor, This does not include facility-specific raw data such as instrument logs, however exact copies of tempex-atmlogs will be submitted. Exact copies of all raw data, as well as a signed copy of the final analytical report and all original facility-specific raw data, will be retained i n the Centre Analytical Laboratories, Inc. archives for the period of time specified in 40 ClFR Part 160. Retained samples of reference substances are archived by the sponsor. `THIS IS AN EXACT COW OF THE ORIGINAL DOCUMENT" Centre Analytical Laboratories, Inc. 3M Environmental Laboratory Page 10 of 15 Page 205 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 Centre Study No.:023-045 Table I. Results From LC/MS Of TCR-00065-022E,SI Negative mode 1 3M ID I Retention I Mass 1 Value 1 Determined I % RecGerd 7e = 88.0 r * * 110 93 94 105 104 103 112 103 Standards Average = 103 Std Dev = 7 W * First two injections during instrument warm up, not reported. CCV = standard in run used to check calibration (Cl005004at 269pg/L) Centre Analytical Laboratories, Inc. 3M Environmental Laboratory Page 11 of 15 Page 206 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 Centre Study No.: 023-045 - Ir Figure 1. PFOS Calibration Standard (Cl00500-4)1at 269pgL Sample Name:C100500-4 ' Sample Info: ... ___p 1 1 Data file :C:\iPCHEM\1\~TA\100600\10060022.D Inetrument I Dilution ' : In8trument, 1 :1 Inj. NO. : I n j . Vol. : .`1 s $1 U 1ns:rurnent 1 Mon, 23. Oct. 2000 00:12:06 pm _- Paga 1 c.rf .1. I...-..*..(.;...;. _- .--aAp "THIS IS AN EXACT COPY OF THE ORIGINALDOCUMENT" I . ..-.*... .--. .. -. -.-`- >. . .. Job</& BY, Centre Analytical Laboratories, Inc. 3M Environmental Laboratory Page 12 of 15 Page 207 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 Centre Study No.:023-045 Figure 2. PFOS Calibration Standard (ClOOSOO-3) at 539pg/L Sample Name:C100500-3 Sample Info: Data f i l e :C:\HPCHEM\1\~TA\100600\10060015.D --11=111111111111111------*-1-------------M------------------~------B*---- Injection Date P ~ c oqperator Inetw e n t Dilution : 10/6/2000 : OS 6s w t - : Inetrument 1 :I 1:13:21 PM Seq Line : V i a l No. : Inj. No. : Inj. Vol. : A c q . Method I PF0S.M 01:45:47 pm (modified after loading) Analyeio for PFOS . msvi UT. .. ., 1 13 1s 1 5 fi1 $1 Instrument 1 Mon, 9. O c t . 2000 01:46:00 pm W Centre Analytical Laboratories, Inc. 3M Environmental Laboratory Page 1 of 1 Page 13 of 15 Page 208 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 Centre Study No.: 023-045 Figure 3. PFOS (TCR-00065-022)at 250pg/L, ESI Negative Ion Mode - -- Sample Name :L29092 1 rSample Info:C100500-8 -> 1 Data file :C:\HPCHEM\1\DATA\100600\10060019.D 1~111999999~9~~199~,~~~9~9,9999~999~~999~999~~9-~-~~---~-.-~9~~ Injection Date r A c q operator Instrument Dilution : 10/6/2000 : GS LI WU-U : Instrument 1 :1 2:20:49 PM Seq Line : Vial No. : Inj. No. : Inj. Vol. : 17 21 1 5 Irl Acq. Method : PF0S.M Analysis Method : C:\WPCHEM\1\METHODS\lOO6OO.M i Last Changed : h 0 / 2 3 / 2 0 0 0 Analysie for PFOS i 11:44:26 am U i :I .. "' 4t. 11 Instrument 1 Mon, 23. Oct. 2000 00:10:19 pm Page 1 at 1 '7HIS ISAN EXACT COPY OF I THE ORIGINAL DOCUMENT" Centre Analytical Laboratories, Inc. 3M Environmental Laboratory Page 140f 15 Page 209 3M Medical Department Study: T-6316.1 Analytical Report: FACT-TOX-003 LRN-U2104 Centre Study No.:023-045 APPENDIX A Study Protocol Purity Determination of Sample Lots of PFOS Including Amendment 1. 'THIS IS AN EXACT COPY OF THE ORIGINAL DOCUMENT" Centre Analytical Laboratories, Inc. 3M Environmental Laboratory Page 15 of 15 Page 210 3M Medical Department Study: T-6316.1 3M Medical Department Study: T-6316.1 Appendix H: Report Signature Page Analytical Report: FACT-TOX-003 LRN-U2104 Analytical ISeport: FACT TOX-003 LRN42104 John L. Butenhoff, Ph.D., Ph.D., Study Director Date h 0A f-L Marvin T. Case, D.V.M., Ph.D., Sponsor Representative k f I 4 Date , Ph.D., Principal Analytical Investigator Date R//L- William K. Reagen, Ph.D., Laboratory Manager drL?y6/7 Date 3M Environmental Laboratory Page 211