Document mmqb65xg2vrmqgyonj9z0XY9O

3M Medical Department Study: T-6316.5 3M Medical Department Study: T-6316.5 Analytical Study: FACT-TOX-013 LRN-U2095 Analytical Report: FACT TO X -013 LRN-U2095 Study Title Analytical Study 2(N-Ethylperfluorooctane sulfonamido)-ethanol in Two Generation Rat Reproduction Amended Analytical Laboratory Report Determination of the Presence and Concentration of PFOS, M556, PFOSAA, and PFOSA in the Liver and PFOS, M556, PFOSAA, PFOSA, and EtFOSE-OH in the Sera of Crl:CDBR VAF/Plus Rats Exposed to EtFOSE-OH Data Requirement Not Applicable Author 3M Environmental Laboratory Study Completion Date May 31, 2001 Performing Laboratories Sera Analyses Liver Analyses 3M Environmental Laboratory Building 2-3E-09, 935 Bush Avenue St. Paul, MN 55106 Battelle Memorial Institute 505 King Avenue Columbus, OH 43201-2693 Project Identification 3M Medical Department Study: T-6316.5 Argus In-Life Study: 418-009 Analytical Report: FACT TOX-013 3M Laboratory Request No. U2095 Total Number of Pages 143 3M Environmental Laboratory 3MEnvironmental Laboratory Page 1 Page 1 3M Medical Department Study: T-6316.5 R 3M Medical Department Study: T6316.5 Analytical Study: FACT-TOX-013 LRN-U2095 Analytical Report: FACT TOX-013 LEN-U2095 This page has been reserved for specific country requirements. ' 3M Environmental Laboratory 3MEnvironmental Laboratory Page 2 Page 2 3M Medical Department Study: T-6316.5 3M Medical Department Study: T-6316.5 GLP Compliance Statement Analytical Study: FACT-TOX-013 LRN-U2095 Analytical Study: FACT TOX-013 LRN-U2095 Analytical Laboratory Report Title: Determination of the Presence and Concentration of PFOS, M556, PFOSAA, and PFOSA in the Liver and PFOS, M556, PFOSAA, PFOSA, and EtFOSE-OH in the Sera of Crl:CDBR VAF/Plus Rats Exposed to EtFOSE-OH Study Identification Number: T-6316.5, FACT TOX-013, LRN-U2095 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. All raw data, protocol, analytical report and samples for this study are retained in archives at the 3M Environmental Laboratory and will be retained for a period of at least ten years. The analytical phase completed at the 3M Environmental Laboratory was performed in accordance with 3M ET&SS 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 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 the technical performance of each phase was entirely separate, no effect is expected from this exception. Some changes made in the standard preparation logs obscured the original entry, did not document the reason for the change and/or were not initialed and dated by the person making the change. The samples that were analyzed on 3/16/00 utilized standards that had an expiration date of 2/00. Liver values generated at contract laboratories were corrected by 3M Environmental Laboratory to reflect the official purity values from the COA. Revised final reports will be solicited from the contract laboratory and will be added as a report amendment at a later date. Expiration dates on some reagents and solutions were missing. The analytical report from Battelle is not signed or dated by the Principal Analytical Investigator or laboratory management. The Quality Assurance Statement in the Battelle analytical report does not include the dates of the QA inspection activities or the dates reported to the Study Director and laboratory management. The Quality Assurance Statement is not signed. The Argus and Battelle analytical reports do not include the names of all the contributing 3MEnvironmental Laboratory Page 3 3M Medical Department Study: T-6316.5 Analytical Study: FACT-TOX-013 LRN-U2095 3M Medical Department Study: T-6316.5 Analytical Study: FACT TOX-013 LRN-U2095 GLP Study-- Quality Assurance Statement Analytical Laboratory Report Title: Determination of the Presence and Concentration of PFOS, M556, PFOSAA, and PFOSA in the Liver and PFOS, M556, PFOSAA, PFOSA, and EtFOSE-OH in the Sera of Crl:CDBR VAF/Plus Rats Exposed to EtFOSE-OH Study Identification Number: T-6316.5, FACT TOX-013, LRN-U2095 This study has been inspected by the 3M Environmental Laboratory Quality Assurance Unit (QAU) as indicated in the following table. The findings were reported to the study director and laboratory management. Inspection Dates Phase Date Reported to Management Study Director 10/12/99 Extraction 10/26/99 10/26/99 6/5/00-6/14/00 Data 6/16/00 6/16/00 9/11/00-9/13/00 Draft report 9/14/00 9/14/00 5/14/01 Amended report 5/14/01 5/14/01 QAU Representative 3 O'-n- t l Date 2oQ 1 3M Environmental Laboratory 3MEnvironmental Laboratory Page 4 Page 4 3M Medical Department Study: T-6316.5 3M Medical Department Study: T-6316.5 Table of Contents Analytical Study: FACT-TOX-013 LRN-U2095 Analytical Study: FA C TTO X -013 LRN-U2095 GLP Compliance Statement............................................................................................... 3 GLP Study--Quality Assurance Statement....................................................................... 4 Study Personnel and Contributors......................................................................................7 Introduction and Purpose................................................................................................... 8 Test System.................................................................................................................... 8 Specimen Collection and Analysis.................................................................................9 Specimen Receipt and Maintenance..................................................................................9 Chemical Characterization.................................................................................................... 10 Dose Confirmation Analyses............................................................................................10 Method Summaries............................................................................................................... 11 3M Environmental Laboratory..........................................................................................11 Preparatory Method..................................................................................................... 11 Analytical Method........................................................................................................ 11 Analytical Equipment................................................................................................... 11 Deviations......................................................................................................................... 12 Data Quality Objectives and Data Integrity.......................................................................... 12 Data Summary, Analyses, and Results............................................................................... 13 Summary of Quality Control Analyses Results................................................................13 Summary of Sample Results............................................................................................14 Statistical Methods and Calculations....................................................................................14 Statement of Conclusion...................................................................................................... 14 Appendix A: Chemical Characterization, Control Matrices and Dose Confirmation Analyses................................................................................................................................ 15 Appendix B: Protocol............................................................................................................ 18 Appendix C: Extraction and Analytical Methods..................................................................37 ETS-8-4.1, Extraction of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds from Serum for Analysis Using HPLC-Electrospray/Mass Spectrometry, (14 pages).............................................................................................................................38 ETS-8-5.1, Analysis of Potassium Perfluorooctanesulfonate or Other Fluorochemlcals in Serum Extracts Using HPLC-Electrospray/Mass Spectrometry, (9 pages).................... 52 Appendix D: Data Summary Tables.................................................................................... 61 Appendix E: Data Spreadsheets......................................................................................... 64 Appendix F: Example Calculations......................................................................................70 Appendix G: Contract Lab Report..................................................................................... 71 . Appendix H: Interim Certificate of Analysis........................................................................ 137 Appendix I: Report Signature Page....................................................................................141 3M Environmental Laboratory 3MEnvironmental Laboratory Page 5 3M Medical Department Study: T-6316.5 Analytical Study: FACT-TOX-013 LRN-U2095 3M Medical Department Study: T-6316.5 Analytical Study: FACT TO X-013 LRN-U2095 Appendix J: Amendment 1 to FACT TOX-013 Final Report 142 List of Tables Table 1. Test System Population Demographics and Dosage Levels for Study (418-009)....................................................................................................... 8 Table 2. Characterization of the Test Article in Study FACT TOX-013 ...............................10 Table 3. Negative Ions Monitored in 3M Laboratory Analyses........................................... 12 Table 4. Deviation Summary for FACT TOX-013...............................................................12 Table 5. Determinations of the LOQ in the Analyses of Serum Extracts............................13 Table 6. Characterization of the Control Matrices Used for Sera Analyses in Study FACT TOX-013 .............................................................................................15 Table 7. Characterization of the Control Matrices Used for Liver Analyses in Study FACT TOX-013 ............................................................................................. 15 Table 8. Characterization of the Analytical Reference Materials Used for Sera Analyses in Study FACT TOX-013.........................................................................................16 Table 9. Characterization of the Analytical Reference Materials Used for Liver Analyses in Study FACT TOX-013.........................................................................................16 Table 10. Tween Dosing Confirmation for Study In-life #418-009...................................... 17 Table 11. Tween Dosing Confirmation-- Matrix Spikes for Study In-life #418-009............17 Table 12. Reported Fluorochemical Levels in Sera Analyses in Study FACT TOX-013... 61 Table 12. Reported Fluorochemical Levels in Sera Analyses in Study FACT TOX-013 (continued).............................................................................................................62 Table 13. Reported Fluorochemical Levels in Liver Analyses in Study FACT TOX-013...62 Table 13. Reported Fluorochemical Levels in Liver Analyses in Study FACT TOX-013 (continued)............................................................................................................ 63 3M Environmental Laboratory 3MEnvironmental Laboratory Page 6 Page 6 3M Medical Department Study: T-6316.5 3M Medical Department Study: T6316.5 Analytical Study: FACT-TOX-013 LRN-U2095 Analytical Report: PACT TOX-013 LRN-U2095 Study Personnel and Contributors Study Director Marvin T. Case, D.V.M., Ph.D, Study Director 3M Corporate Toxicology - Medical Department 3M Center, Building 220-2E-02 ^ St. Paul, MN, 55144-1000 651-733-5180 r - Sponsor John L. Butenhoff, Ph.D., Sponsor Representative 3M Corporate Toxicology - Medical Department 3M Center, Building 220-2E-02 St. Paul, MN 55144-1000 ^ Analytical Chemistry Laboratories Sera Analyses 3M Environmental Laboratory (3M Lab) ^ Kristen J. Hansen Ph.D., Analytical Investigator Liver Analyses Battelle Memorial Institute Jon C. Andre, Ph.D., Analytical Investigator 3M Lab Contributing Personnel David R. Bamidge. Ph.D. Lisa A. Clemen Lisa Dick, Ph.D. Kelly J. Dorweiler Mark E. Ellefson Sara E. Estes Barb A. Gramenz Sarah A. Heimdal Cari S. Hewitt Marlene M. Heying Harold O. Johnson Kelly J. Kuehlwein Sally A. Linda Joseph C. Pilon Scott R. Post Ian A. Smith Kathy M. Stock Anh-Dao Vo Bob W . Wynne 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. Control sera and liver will be maintained at the contract lab along with the test substance. 3M Environmental Laboratory 3MEnvironmental Laboratory Page 7 Page 7 3M Medical Department Study: T-6316.5 3M Medical Department Study: T6316.5 Analytical Study: FACT-TOX-013 LRN-U2095 Analytical Report: FACT TOX-013 LRN-U2095 Introduction and Purpose The purpose of the study is to determine the presence and concentration of PFOS, PFOSA, PFOSAA, and M556 in liver samples and PFOS, PFOSA, PFOSAA, EtFOS&OH, and M556 in sera samples collected from rats exposed to EtFOSE-OH. This study was initiated on 1 October 1998. T e s t System Five groups of FO generation male and female rats and 3 groups of F1 generation male and female rats were used as the test system. Table 1 outlines the rat population demographics and dosage levels for study 418-009. On day 4 of lactation, litters were culled to four male and four female pups, where possible. On day 21 of lactation, 25 male and 25 female pups in Groups I, II, and III were selected for continued evaluation. F1 generation male and female rats were given appropriate dosages of the test article via gavage beginning on day 22 of lactation or postpartum through the day before sacrifice. The test system species and strain selected was the CitCCPBR VAF/Plus* (Sprague-Dawley) rat received from Charles River Laboratories, Inc., and assigned temporary numbers until assigned to the study. Rats were permanently identified using MoneF self-piercing ear tags when assigned to the study. FO generation rats were identified with ear tags. Pups were not identified during lactation, as parameters were evaluated in terms of the litter. At weaning, each F1 generation rat selected for continued observation was identified with a Monelseif-piercing ear tag. FO female rats were approximately 65 days of age and weighed approximately 1 7 9 -2 2 9 g when received. FO male rats were approximately 5 8 -6 7 days of age and weighed approximately 223-331 g when received. Weight data are included in Argus Research Laboratories, Inc. final report (study number 418-009). Table 1. Test System Population Demographics and Dosage Levels for Study (418-009) Population Number of F0 Generation Rats per Sex Number of F1 Generation Rats per Sex Dosage (mgfkg/day) Dosage Group I (Control) Dosage Group II Dosage Group III Dosage Group IV Dosage GroupV 35 35 35 35 35 25 0 (vehicle) 25 1 25 5 -- 10 -- 15 3M Environmental Laboratory 3MEnvironmental Laboratory Page 8 Page 8 3M Medical Department Study: T-6316.5 3M Medical Department Study: T6316.5 Analytical Study: FACT-TOX-013 LRN-U2095 Analytical Report: PACT TOX-013 LRN-U2095 Spoolm en C ollection and A nalysis Sample specimens were collected by Argus (study 418-009) and sent to the 3M Environmental Laboratory for analysis. Liver and sera specimens were collected from F0 male rats at the completion of the cohabitation period and F0 female rats on day 21 postpartum. Liver specimens were collected from F0 generation litters, and stomach content specimens were collected from the F0 and F2 generation litters. The analysis of the stomach contents were not part of the scope of analysis determined by the study director. The number and type of specimens collected for analyses in the analytical phase of this study are presented below. Specimens Collected from Study Groups I through V (through 11/30/98): Serum Specim ens-- 45 specimens Liver Specimens-- 65 specimens Blood specimens were centrifuged after collection. Serum was then harvested and immediately frozen on dry ice and maintained frozen at -70C until shipped to the 3M Environmental Laboratory. Liver specimens collected from each animal were frozen and retained at -70C until shipped to the 3M Environmental Laboratory. Stomach content specimens were frozen at -20C until shipped to the 3M Environmental Laboratory. Liver, sera, and stomach content specimens were shipped to the 3M Environmental Laboratory frozen and on dry ice. Sera and liver samples were extracted beginning on October 1 1 ,1 9 9 9 using an ion pairing reagent and methyl-ferf-butyl ether (MtBE) for the sera and ethyl acetate for the liver samples. Liver samples were homogenized prior to the extraction procedure. Sample extracts were analyzed using high-performance liquid chromatography-etectrospray/tandem mass spectrometry (HPLC-ESM SM S) in the multiple response monitoring mode. PFOS, PFOSA, PFOSAA, EtFOSE-OH, and M556 levels were quantitated by external calibration. PFOSEA was not analyzed due to inconsistent analysis and failed QC. Analytical details are included in this report. Specimen Receipt and Maintenance The 3M Environmental Laboratory received from Argus, serum, liver and stomach content specimens collected at predetermined time points during and at the end of thein-life phase of Argus study 418-009 on 8 -4 -9 8,1 0-1-98 and 1-29-99. All specimens were received frozen on dry ice and were immediately transferred to storage at -20C 10C. Specimens that were analyzed at Battelle were shipped frozen on dry ice. Control matrices used in liver and sera analyses were obtained from commercial sources and are presented in Table 6 and 7. 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 10C. 3M Environmental Laboratory 3MEnvironmental Laboratory Page 9 Page 9 3M Medical Department Study: T-6316.5 3M Medical Department Study: T6316.5 Analytical Study: FACT-TOX-013 LRN-U2095 Analytical Report: FACT TOX-013 LRN-U2095 Chemical Characterization EtFOSE-OH CAS Number: 1691-99-2 Chemical Formula: CgF17S 0 2N(CH2CH3)CH2CH20 H Molecular Weight: 571.0 Chemical characterization information on the test article is presented in tabular form below. Chemical characterization information on the analytical reference materials used in this study is presented in tabular form in Appendix A (see Tables 8 and 9) and the interim Certificate of Analysis available in Appendix I. Table 2. Characterization of the Test Article in Study FACT TOX-013 Test Article Chemical Name Source EtFOSE-OH FM-3929 2(N-Ethyfperfluorooctane sulfbnamido)-ethanol 3M Expiration Date 05/2000 Storage Conditions Ambient temperature Chemical Lot# Physical Description 30035, 30037, 30039 Waxy Solid Purify To be determined* * The purity of the test article determined nominally by NMR analysis. Subsequent chemical characterization is occurring and this analytical reportwill be amended to indicate the purity when a certificate ofanalysis is issued. Doss C onfirm ation A nalyses The dose confirmation data were collected according to a method that was not fully validated. Dose confirmation analyses were performed on test article samples taken at the start of dosage, at 6 weeks, and at the end of dosage during the in-life phase of the study. Dose confirmation analyses were performed on 3 dose levels collected during the in-life phase of the study: the results are presented in Appendix A (see Tables 10 and 11). Dose confirmation was performed by diluting the Tween dose samples with Miili-Q water into the linear range of the instrument. For each sample, a matrix spike was prepared (at approximately 5 0-100% of the expected dose level). In all cases, samples were analyzed versus an unextracted curve using HPLC-ES/MS/MS. The instrumental parameters and analytical conditions described in ETS-8-5.1 were used for dose solution analyses. 3M Environmental Laboratory 3MEnvironmental Laboratory Page 10 Page 10 3M Medical Department Study: T-6316.5 3M Medical Department Study: T6316.5 Analytical Study: FACT-TOX-013 LRN-U2095 Analytical Report: FACT TOX-013 LRN-U2 095 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 are located in Appendix C. The methods and analytical equipment settings used by Battelle are presented in the Battelle final report (see Appendix G). 3IM Environm ental Laboratory Preparatory Method ETS-8-4.1, "Extraction of Potassium Perfluorooctanesulfonate or Other Fluorochemicai Compounds from Serum for Analysis using HPLC-Electrospray/Mass Spectrometry" Sera samples were extracted using an ion-pairing extraction procedure. An ion pairing reagent was added to the sample and the analyte ion-pair was partitioned into MtBE. The MtBE extract was transferred to a centrifuge tube and put onto a nitrogen evaporator until dry. Each extract was reconstituted in 1.0 mL of methanol, then filtered through a 3cc plastic syringe attached to a 0.2pm nylon filter into a glass autoviai. Analytical Method ETS-8-5.1, "Analysis of Potassium Perfluorooctanesulfonate or Other Fluorochemicals in Serum Extracts Using HPLC-Electrospray/Mass Spectrometry" The analyses were performed by monitoring one or more product ions selected from a Single primary ion characteristic of a particular fluorochemicai using HPLC-ESMSMS. For example, molecular ion 499, selected as the primary-ion for PFOS (Q F 17S 0 3-) analysis, was fragmented further to produce ion 99 (FSQ,-). The characteristic product-ion 99 was monitored for quantitative analysis. Analytical Equipment The following equipment and parameters are representative of those used during the analytical phase of this study. Liquid Chrom atograph: Hewlett-Packard* Series 1100 Liquid Chromatograph system Analytical column: Keystone* BetasilTM C182x50 mm (5 pm) Column temperature: Ambient Mobile phase components: Component A: 2mM aqueous ammonium acetate Component B: methanol Flow rate: 300 pL/min Injection volume: 10 pL Solvent Gradient: 10 minutes Start at 40%B Hold at 40% B for 1 minute Increase to 95%B over 3.5 minutes 3M Environmental Laboratory 3MEnvironmental Laboratory Page 11 Page 11 3M Medical Department Study: T-6316.5 3M Medical Department Study: T6316.5 Analytical Study: FACT-TOX-013 LRN-U2095 Analytical Report: FACT TOX-013 LRN-U2095 Hold at 95% B for 2 minutes Return to 4 0 % 8 over 0.5 minutes Hold at 40% B for 3 minutes Mass Spectrom eter: Micromass* API/Mass Spectrometer Quattro 11TMTriple Quadrupole system Software: Mass Lynx" 3.2 Cone Voltage: 2 0-60 V Collision Energy: 2 5 -4 5 eV Mode: Electrospray Negative Source Block Temperature: 150C 10C Z-spray source Analysis Type: Multiple Reaction Monitoring (MRM) Table 3. Negative Ions Monitored in 3M Laboratory Analyses Target Analyte Primary Ion (AMU) Product Ion (a m u ) PFOS 499.0 99.0 PFOSA PFOSAA EtFOSE-OH 498.0 584.0 630.0 78.0 169.0 59.0 M556 THPFOS 556.0 427.0 78.0, 169,0 80.0 D eviations Deviations from the original protocol and methods are documented in the table below: Table 4. Deviation Summary for FACT TOX-013 Deviation 0ate(8) of Occurrence Impact on Study Pipette was used instead Oxford dispenser 0.2-1 .OmL of sample was used for extraction instead of 1.OmL. Milk curd samples were not analyzed. 10/12/99 10/12/99 Entire study Standards and samples were prepared identically. No adverse impact on study. Current work indicates that volumes 0.5 mL provide results equivalent to 1 mL extraction volumes. Results of sample volumes <0.5 mL have not been validated and wiK be marked in the data table. No milk curd data is available for the final report Data Quality Objectives and Data Integrity The following data quality objectives (DQOs) were indicated in the method performance section of ETS-8-5.1, "Analysis of Potassium Perfluorooctanesulfonate or Other Fluorochemicals in Serum Extracts Using HPLC-Electrospray/Mass Spectrometry": 3M Environmental Laboratory 3MEnvironmental Laboratory Page 12 Page 12 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 Linearity: The coefficient of determination (r2) equal to or greater than 0.980 Lim its o f Quantitation (LO Q ): The LOQ for PFOS is 5.55 ppb, PFOSA is 4.79 ppb, PFOSAA is 20.5 ppb, EtFOSE-OH is 36.2 ppb, and M556 is 19.2 ppb. Acceptable Spike Recoveries: 70-130% Data Summary, Analyses, and Results With the exceptions noted in this report, data quality objectives for the analytical phase of this study outlined in the 3M Environmental Laboratory method ETS-8-5.1 (see Appendix C) and the Battelle final report (see Appendix G) were met. Although extraction and analysis were initiated in September 1998, the study was reprioritized and put on hold. Upon restarting the study, the decision was made to reextract and analyze the specimens. No data from the original analysis are included in this report. The data in this report reflect only that obtained from specimens extracted on, or after October 11,1999. Sum m ary o f Q u ality C ontrol A nalyses R esults Linearity: The coefficient of determination (r2) of the standard curves were0.980. Calibration Standards: Quantitation of the target analytes was based on linear regression analysis (1/x weighted) of two extracted matrix curves bracketing each group of samples. High or low points on the curve may have been deactivated to provide a better linear fit over the concentration range most appropriate to the data. Ail active curve points are accurate to within 70% of theoretical value. 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 outlier curve point may have been deactivated. Quantitation of each analyte was based on the response of one or more specific product ion(s) using the multiple response-monitoring mode of the instrument (see Appendix C). Lim its o f Quantitation (LO Q ): The LOQ is equal to the lowest accepted standard in the calibration curve (defined as a standard with a concentration that is within 30% of the theoretical value, and which has at least two times the analyte peak area detected in the extraction blanks). Table 5. Determinations of the LOQ in the Analyses o f Serum Extracts Analyte Method LOQ FFOS PFOSA PFOSAA EtFOSE-OH M556 5.55 ppb 4.79 ppb 20.5 ppb 36.2 ppb 24.9 ppb 3M Environmental Laboratory Page 13 3M Medical Department Study: T-6316.5 3M Medical Department Study: T6316.5 Analytical Study: FACT-TOX-013 LRN-U2095 Analytical Report: PACT TOX-013 LRN-U2 095 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, rabbit sera was selected as a suitable surrogate matrix for standard curves. Precision: Precision was determined by analysis of M S/M SD and was reproducible to within 10%. M atrix Spikes: Matrix spikes and matrix spike duplicates were extracted with each set of samples and analyzed during analytical runs. With the exception of M556, all sera matrix spikes were within 30% of the theoretical concentration. Both matrix spikes showed a recovery of 69% for the M556. These results were verified. Data quality objectives will be adjusted to reflect this recovery. Surrogates: The surrogate (THPFOS) was added to all samples and standards. THPFO S was not used for quantitation, but was used to monitor for gross instrument failure. The surrogate response of each analytical run was verified to determine that it did not vary more than 50% from the mean within each analytical run. Assuming spike recovery studies form a suitable indication of endogenous analyte recovery, sera data are quantitative to 30% for all analysis but M556; M 556 data is quantitated to 31% . The validity of this assumption has not been verified by other techniques. Sum m ary o f Sam ple R esults Sam ples from Control Anim als: Low levels of PFOS, PFOSA, PFOSAA, EtFOSE-OH, and M 556 were often detected in the sera and liver of the control animals. These levels were significantly lower than those found in the low dose test animals. Sam ples from Dosed Anim als: In general, PFOS, PFOSA, PFOSAA, EtFOSE-OH, and M556 levels found in the sera and liver of the test animals increased with 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-013. Statement of Conclusion v Under the conditions of the present studies, PFOS, PFOSA, PFOSAA, EtFOSE-OH, and M556 were observed in the sera and liver of rats dosed with EtFOSE-OH during the in-life phase of the study. 3M Environmental Laboratory 3MEnvironmental Laboratory Page 14 Page 14 3M Medical Department Study: T-6316.5 3M Medical Department Study: T6316.5 Analytical Study: FACT-TOX-013 LRN-U2095 Analytical Report: PACT TOX-013 LRN-U2095 Appendix A: Chemical Characterization, Control Matrices and Dose Confirmation Analyses Table 6. Characterization o f the Control Matrices Used for Sera Analyses in Study FACT TOX-013 Location 3M Lab Control Matrix Rat Serum (TN-A-2001) Rabbit Serum (TN-A-2573) Source Expiration Date Storage Conditions Chemical Lot # Physical Description N/R--not recorded Sigma 2010 Ambient 17H9306 Rat Serum Sigma 2010 Ambient 118H8418 Rabbit Serum Table 7. Characterization o f the Control Matrices Used for Liver Analyses in Study FACT TOX-013 Location Battello Memorial Institute Control Matrix Rat Liver Source Expiration Date Storage Conditions Chemical Lot # Physical Description N/R--not recorded Harlan N/R N/R N/R R a tU v e r 3M Environmental Laboratory 3MEnvironmental Laboratory Page 15 Page 15 3M Medical Department Study: T-6316.5 3M Medical Department Study: T-6316.5 Analytical Study: FACT-TOX-013 LRN-U2095 Analytical Study: FACT TOX-013 LRN-U2095 Table 8. Characterization of the Analytical Reference Materials Used for Sera Analyses in Study FACT T O X -013 Location i5M Lab Materials PFOS C aF^SO * PFOSA CBF17S 0 2NH2 PFOSAA CbF17S 0 2N((CH2CH3) (CHjCOOH)) Source Expiration Date 3 M Specialty C hem icals 08/3 1/01 N /R 0 1/0 1/20 1 0 N /R 0 1/0 1/20 1 0 Storage Conditions Chemical Lot Number Physical Description A m bient tem perature A m bient tem perature 171 L -15709 W h ite crystalline powder Light yellow w axy solid A m bient tem perature NB 112999-99 Tan w axy solid Purity 8 6.4% TBD TBD ` Surrogate standard-- 1H,1 H,2H,2H-Tetrahydroperfluorooctanesulfonic acid N/R-- not recorded TBD--to be determined NA-- not applicable EtFOSE-OH CbF17S 02N(CH2CH3) c h 2c h 2o h 3M IC P /P C P Division 0 1/0 1/20 1 0 A m bient tem perature 936 A m b er w axy solid TBD M556 CbF17S 02N ((H)(CHjCOOH)) 3M 0 1 /0 1 /2 0 1 0 A m bient tem perature THPFOS* c bh4f13s o 3h IC N B iom edicals 0 1/2 01 0 A m bient tem perature N B 113047-80 53406 W h ite pow der TBD Brown w axy solid NA Table 9. Characterization of the Analytical Reference Materials Used fo r Liver Analyses in Study FACT TOX-013 Location Battelle Memorial Institute Materials PFOS M556 PFOSAA PFOSA THPFOS* Source 3M 3M 3M 3M Expiration Date Storage Conditions Chemical Lot Number Physical Description 0 8 /3 1 /0 1 0 1/0 1/20 1 0 A m bient tem perature A m bient tem perature 171 NB 113047-80 /Vhite crystalline powder W h ite pow der 2010 A m bient tem perature 617 N /R 0 1 /0 1 /2 0 1 0 A m bient tem perature L -1 5 7 0 9 Light yellow w axy solid Purity 8 6.4% TBD TBD TBD ` Surrogate standard-- 1H,1H,2H,2H-Tetrahydroperfluorooctanesulfbnic add N/R--not recorded TBD--to be determined NA-- not applicable IC N N /R A m bient tem perature 59909 N /R NA 3MEnvironmental Laboratory Page 16 3M Medical Department Study: T-6316.5 3M Medical Department Study: T6316.5 Analytical Study: FACT-TOX-013 LRN-U2095 Analytical Report: FACT TOX-013 LRN-U2095 Table 10. Tween Dosing Confirmation for Study In-life #418-009 Group Dose Sample Number Expected Cone. Measured Cone. EtFOSE (ng/mLJ EtFOSE (ng/mL) Group 1-- Control 0 mg/mL Group 2--0.2 mg/mL Group 3-- 1.0 mg/mL Group 4-- 2.0 mg/mL Group 5--3.0 mg/mL B-418-009-A, 06/08/98 B-418-009-A, 07/15/98 B-418-009-B, 06/08/98 B-418-009-B, 07/15/98 B-418-009-C, 06/08/98 B-418-009-C, 07/15/98 B-418-Q09-D, 06/08/98 B-418-009-D, 07/15/98 B-418-009-E, 06/08/98 B-418-009-E, 07/15/98 0.00 NA 200000 200000 1000000 100000 2000000 2000000 3000000 3000000 0.00 NA NA NA 1020000 942000 2190000 2750000 3060000 3640000 Homogeneity Samples-- 3.0 mg/mL B-418-009-A, 05/08/98 1 0f6T B-418-009-A, 06/08/98 3of6M B-418-009-A, 06/08/98 5of6B 3000000 3000000 3000000 3250000 3690000 3790000 NA = Not applicable EtFOSE % Recovery Accuracy NA NA NA NA 102 94 110 138 102 121 108 123 126 Table 11. Tween Dosing Confirmation--Matrix Spikes for Study In-life #418-009 Sample Number Expected Cone. EtFOSE (ng/mL) Measured Cone. EtFOSE (ng/mL) EtFOSE % MS Recovery Accuracy B-418-009-B, 06/08/98-MS B-418-009-B, 07/15/98-MS B-418-009-C, 06/08/98-MS B-418-009-C, 07/15/98-MS B-418-009-D, 06/08/98-MS B-418-009-0,07/15/98-M S B-418-009-E. 06/08/98-MS B-418-009-E, 07/15/98-MS 1200 1200 900 900 900 900 1100 1100 NA NA 818 826 910 733 973 1089 NA NA 91 92 101 81 88 99 B-418-009-A, 05/08/98 1 of6T-MS B-418-009-A, 06/08/98 3of6M-MS B-418-009-A, 06/08/98 5 Of 6 B-MS 1100 1100 1100 949 1053 944 86 96 86 NA Not applicable 3M Environmental Laboratory 3MEnvironmental Laboratory Page 17 Page 17 3M Medical Department Study: T-6316.5 ss*m 3M Medical Department Study: T6316.5 Appendix B: Protocol Analytical Study: FACT-TOX-013 LRN-U2095 Analytical Report: FACT TOX-013 LRN-U2095 3M Environmental Laboratory 3M Environmental Laboratory Page 18 Page 18 3M Medical Department Study: T-6316.5 3M Medical Department Study: T6316.5 Analytical Study: FACT-TOX-013 ' Analytical A 3M Environmental Laboratory____________ P ro to c o l - A n a lytical Stu d y 2(N-Ethylperfluorooctanesulfonamido)-ethanol in Two Generation Rat Reproduction In-vivo study reference num ber: Argus 418-009 Study num ber: FACT 060998.1 T est substance: 2(N-Ethylperfluorooctanesulfonamido)-ethanol (N-EtFOSE-OH) Name and address of Sponsor: Marvin Case 3M Toxicology Services 3M Center Building 220-2E-02 St. Paul, MN 55144 - Nam e and address o f testing facility: 3M Environmental Technology and Services 935 Bush Avenue, Building 2-3E-09 * St. Paul, MN 55106 Experim ental start date: Expected term ination date: December 31,1998 M ethod num bers and revisions: FA C T-M -1.0, Extraction o f Potassium Perfluorooctanesulfonate or Other A nionic Surfactants from Liver for Analysis Using HPLC-Electrospray/Mass Spectrometry FA C T-M -2.0, Analysis o f Fluorochemicals in Liver Extracts U sing HPLCElectrospray/Mass Spectrometry FA C T-M -3.0, Extraction o f Potassium Perfluorooctanesulfonate or Other Anionic Surfactants from Serum for Analysis U sing HPLC-Electrospray/Mass Spectrometry FA C T-M -4,0, Analysis o f Fluorochemicals in Serum Extracts U sing HPLCElectrospray/Mass Spectrometry Author: Lisa Clemen ------- ------------------ Kris Hansen Date Study Director Marvin Case Sponsor Representative D ate 3M-^Environmental Laboratory 3MEnvironmental Laboratory 1 Page 19 Page 19 3M Medical Department Study: T-6316.5 : 3M Medical Department Study: T6316.5 Analytical Study: FACT-TOX-013 Analytical Report: FACT T O ^ ftK 'U 2095 LRN-U2095 1.0 Purpose _________________________________________________________ The analytical portion o f this dosing study is designed evaluate the levels o f perfluorooctane sulfonate (PFOS), or another metabolite o f 2(N-ethylperfluorooctanesulfonamido)-ethanol (NEtFOSE-OH) designated by the study director, in the liver o f the parent and subsequent generations o f the test system, or in die serum as necessary. The in life portion o f this study was conducted at Argus Research Laboratories. 2.0 Regulatory Compliance_________________________________________________ ____ This study is conducted in com pliance with the Food and Drug Administration Good Laboratory Practices regulation as stated in 21 CFR 58. Any exceptions w ill be noted in the final report. 3.0 Test Materials_________________________________________________ _______________ 3.1 Test, control, and reference substances and matrices 3.1.1 Analytical reference substance: Potassium perfluorooctanesulfonate (PFOS), lot #217 3.1.2 Analytical reference substance matrix: Rat liver and serum 3.1.3 Analytical control substance: None 3.1.4 Analytical control substance matrix: Rat liver and serum 3.2 Source o f materials 3.2.1 Analytical reference substance: 3M Specialty Chemical D ivision; traceability information w ill be included in the final report 3.2.2 Analytical reference substance matrix: Argus Research Laboratories; traceability information w ill be included in the final report 3.2.3 Analytical control matrix: 3 .2 3 .1 Rat liver - Argus Research Laboratories; traceability information w ill be included in the final report; or Rabbit liver - Covance Laboratories; traceability information w ill be included in the final report 3.2.3.2 Rat serum - Sigm a Chemical Company; traceability information w ill be included in the final report 3.3 Number o f test and control sam ples. Liver samples for testing were received from 40 test animals and 10 control animals. Serum samples w ill be tested at the discretion o f the Study Director. 3.4 Identification o f test and control samples: The samples are identified using the Argus Research Laboratories identifiers, which consist o f a letter follow ed by the Argus project number, the animal number, the group designation, and the draw date. 3M Environmental Laboratory 3MEnvironmental Laboratory Page 20 Page 20 3M Medical Department Study: T-6316.5 3M Medical Department Study: T6316.5 Analytical Study: FACT-TOX-013 Analytical Report: FACT TOXLR ' U2095 LRN-U2095 3.5 Purity and strength o f m aterials: Characterization o f the purity and identity o f the reference material is the responsibility o f the Sponsor. 3.6 S tability o f test m aterial: Characterization o f the stability o f the test material is the responsibility o f the Sponsor. 3.7 Storage conditions for test m aterials: Test materials are stored at room temperature. Samples are stored at --20 10 C. 3.8 D isposition o f test and/or control substances: Biological tissues and fluids are retained per GLP regulation. 3.9 Safety precautions: Refer to the material safety data sheets o f chemicals used. Wear appropriate laboratory attire, and follow adequate precautions for handling biological materials and preparing samples for analysis. 4.0 Experimental - Overview___________________________ I_________________________ Tissues from animals dosed as described in Argus Research Laboratories Protocol #418-009 are received for analysis o f fluorine compounds. At the discretion o f the Study Director, a series o f analytical tests w ill be performed on select tissues. Initially, all liver samples w ill be analyzed for PFOS by electrospray/mass spectrometry (ES/M S). On the basis o f findings from these analyses, additional sample matrices may be evaluated or other metabolites may be targeted. If additional analysis is performed, a protocol amendment w ill be written. 5.0 Experimental - A nalytical M ethods 5.1 FA C T-M -1.0, Extraction o f Potassium Perfluorooctanesulfonate or Other Anionic Surfactants from Liver for Analysis Using HPLC-Electrospray/Mass Spectrometry 5.2 FA C T-M -2.0, Analysis o f Fluorochemicals in Liver Extracts Using HPLCElectrospray/Mass Spectrometry 5.3 FA C T-M -3.0, Extraction o f Potassium Perfluorooctanesulfonate or Other Anionic pmm. Surfactants from Seram for Analysis U sing HPLC-Electrospray/Mass Spectrometry 5.4 FA C T-M -4.0, Analysis o f Fluorochemicals in Serum Extracts U sing HPLCElectrospray/Mass Spectrometry JP*"* 6.0 Data Analysis 6.1 D ata transform ations and analysis: Data w ill be reported as the concentration (weight/weight) o f fluoride per tissue or sample, or o f PFOS per unit o f tissue or fluid. 6.2 Statistical analysis: Statistics used may include regression analysis o f the serum concentrations over time, and standard deviations calculated for the concentrations within each dose group. If necessary, sim ple statistical tests, such as Student's t test, may be applied to evaluate statistical difference. 3M Environmental Laboratory 3MEnvironmental Laboratory 3 Page 21 Page 21 3M Medical Department Study: T-6316.5 3M Medical Department Study: T6316.5 Analytical Study: FACT-TOX-013 Analytical Report: FACT TOXLRN' U2095 LRN-U2095 7.0 Maintenance of Raw Data and Records / V 7.1 The follow ing taw data and records w ill be retained in the study folder in the archives fissa, according to AMDT-S-8: 7.1.1 Approved protocol and amendments 7.1.2 Study correspondence 7.1.3 Shipping records 7.1.4 Raw data 7.1.5 Electronic copies o f data 7.2 Supporting records to be retained separately from the study folder in the archives m m according to AMDT-S-8 w ill include at least the following: 7.2.1 Training records ' r* 7.2.2 Calibration records 7.2.3 Instrument maintenance logs 7.2.4 Standard Operating Procedures, Equipment Procedures, and M ethods 7.2.5 Appropriate specimens. 8.0 References 8.1 3M Environmental Laboratory Quality System Chapters 1 ,5 and 6 8.2 Other applicable 3M Environmental Laboratory Quality System Standard Operating Procedures r* 9.0 Attachments 9.1 FA C T-M -1.0, Extraction o f Potassium Perfluorooctanesulfonate or Other A nionic Surfactants from Liver for Analysis Using HPLC-Electrospray/Mass Spectrometry 9.2 FA C T-M -2.0, Analysis o f Fluorochemicals in Liver Extracts U sing HPLCElectrospray/Mass Spectrometry SSHOES 9.3 FA C T-M -3.0, Extraction of Potassium Perfluorooctanesulfonate or Other Anionic Surfactants from Serum for Analysis Using HPLC-Electrospray/Mass Spectrometry pm * 9.4 FA C T-M -4.0, Analysis o f Fluorochemicals in Seram Extracts U sing HPLCElectrospray/Mass Spectrometry 3M Environmental Laboratory 3MEnvironmental Laboratory 4 Page 22 Page 22 3M Medical Department Study: T-6316.5 ws 3M Medical Department Study: T6316.5 Analytical Study: FACT-TOX-013 Analytical Report: FACT TOX^ ^ 'U 2095 LRN-U2 095 Study Title Combined Oral (Gavage) Fertility Developm ent and'Perinatal/Postnatal Reproduction Toxicity Study o f N-EtFOSE in Rats PROTOCOL AMENDMENT NO. 1 Amendment Date: July 2 8 ,1 9 9 9 Performing Laboratory 3M Environmental Technology & Safety Services 3M Environmental Laboratory 935 Bush Avenue S t Paul, M N 55106 Laboratory Project Identification ET&SS FACT-TOX-013 L IR N U 2095 3M Environmental Laboratory 3M Environmental Laboratory 3MEnvironmental Laboratory Page 23 Page 23 3M Medical Department Study: T-6316.5 3M Medical Department Study: T6316.5 Analytical Study: FACT-TOX-013 Analytical Report: FACT T O ^ M 'U 2095 LRN-U2095 Protocol FACT-TOX-013 Amendment 1 T h is am endm ent m o d ifies the fo llo w in g p o rtio n (s) o f th e p rotocol: 1. PROTOCOL reads: The proposed study com pletion date is listed as 12/31/98. AMEND TOread: The proposed study com pletion data is 6/30/00. REASON: The proposed com pletion date w as changed to allow tim e for analyzing all matrices o f interest. Amendment Approval Marvin Case Ph.D., Sponsor Representative / T A - - J . Kris J. Hansen Ph.D., Study Director g/ -2-)5 D ate 3M Environmental Laboratory 3M Environmental Laboratory 3MEnvironmental Laboratory Page 24 Page 24 3M Medical Department Study: T-6316.5 3M Medical Department Study: T6316.5 Analytical Study: FACT-TOX-013 Analytical Report: FACT TOXLRN-U2095 LRN-U2095 Study Title Combined Oral (Gavage) Fertility Development and Perinatal/Postnatal Reproduction Toxicity Study o f N-EtFOSE in Rats PROTOCOL AMENDMENT NO. 2 Amendment Date: September 10,1999 Performing Laboratory 3M Environmental Technology & Safety Services 3M Environmental Laboratory 935 Bush Avenue St. Paul, M N 55106 Laboratory Project Identification ET&SS FACT-TOX-013 L IR N U 2095 3M Environmental Laboratory 3M Environmental Laboratory 3MEnvironmental Laboratory Page 25 Page 25 3M Medical Department Study: T-6316.5 3M Medical Department Study: T6316.5 Analytical Study: FACT-TOX-013 Analytical Report: FACT TOX-^ ^ 'U 2095 LRN-U2095 Protocol FACT-TO X-013 Amendment 2 T h is am endm ent m odifies th e fo llo w in g p o rtio n (s) o f th e pro to co l: 1. PROTOCOL reads: The protocol states that liver w ill be extracted and analyzed at the 3M Environmental Laboratory. AMEND to read: The liver specimens w ill be extracted and analyzed at Battelle M em orial Institute, 505 King Avenue, Columbus, Ohio 43201-2693. REASON: The liver specim ens w ill be sent to Battelle M emorial Institute for extraction and analysis due to time constraints in the 3M Environmental Laboratory. 2. PROTOCOL reads: The protocol states that serum specim ens w ill be extracted and analyzed follow ing methods: FACT-M -3.0, "Extraction o f Potassium Perfluorooctanesulfonate or Other A nionic Surfactants from Serum for Analysis U sing HPLC-Electrospray/Mass Spectrometry" FA CT-M -4.0, "A nalysis o f Fluorochemicals in Serum Extracts U sing HPLCElectrospray/Mass Spectrometry" AMEND to read: The serum specimens w ill be extracted and analyzed follow ing methods: ETS-8-4.1, "Extraction o f Potassium Perfluorooctanesulfonate or Other Fluorochem ical Compounds from Serum for A nalysis U sing HPLC-Electrospray M ass Spectrometry" ETS-8-5.1, "A nalysis o f Potassium Perfluorooctanesulfonate or Other Fluorochem ical Compounds in Serum Extracts HPLC-Electrospray M ass Spectrometry" REASON: The extraction and analytical methods FACT-M -3.0 and FACT-M -4.0, respectively, were updated on 04/27/99 to ETS-8-4.1 and ETS-8-5.1. !--* 3M Environmental Laboratory 3M Environmental Laboratory 3M Environmental Laboratory Page 26 Page 26 3M Medical Department Study: T-6316.5 3M Medical Department Study: T6316.5 m " Analytical Study: FACT-TOX-013 ' Analytical Report: FACT TOX^ f t ^ U 2095 LRN-U2095 Protocol FACT-TOX-013 Amendment 2 3. Protocol reads: The protocol states that liver specim ens w ill be extracted and analyzed " follow ing methods: FA C T -M -1.0, "Extraction o f Potassium Perfluorooctanesulfonate or Other A nionic - surfactants from Liver for analysis U sing HPLC-Electrospray/Mas Spectrometry" FA C T -M -2.0, "A nalysis o f Frluorochemicals in Liver Extracts U sing HPLC- . Electrospray/M ass Spectrometry" AMEND to read: The liver specim ens w ill be extracted and analyzed follow ing method: M ethod for A nalysis o f Perfluorooctane Sulfonate (PFOS) in Rat liver by LC/M S/M S, V ersion 1.0 REASON; Since the liver extraction and analysis was sub-contracted to Battelle M emorial Institute, this amendment w as written to include their liver methods and titles. Amendment Approval Marvin Case Ph.D ., Sponsor Representative k i-- ___________ Kristen J. Hansen Ph.D ., Study Director 3M Environmental Laboratory 3M Environmental Laboratory 3M Environmental Laboratory D ate D ate Page 27 Page 27 3M Medical Department Study: T-6316.5 3M Medical Department Study: T6316.5 Analytical Study: FACT-TOX-013 Analytical Report: FACT TOX^ f t ^ 'U 2095 LRN-U2095 Study Title Analytical Study 2(N-Ethylperfluorooctanesulfonamido)-ethanol in Two Generation Rat Reproduction PROTOCOL AMENDMENT NO. 3 Amendment Date: October 4 ,1 9 9 9 Performing Laboratory 3M Environmental Technology & Safety Services 3M Environmental Laboratory 935 Bush Avenue St. Paul, MN 55106 Laboratory Project Identification ET&SS FACT-TOX-013 L IR N U 2095 _ 3M Environmental Laboratory 3M Environmental Laboratory 3MEnvironmental Laboratory Page 28 Page 28 3M Medical Department Study: T-6316.5 3M Medical Department Study: T6316.5 Analytical Study: FACT-TOX-013 Analytical Report: FACT TOXLR ' U2095 LRN-U2095 Protocol FACT Tox-013 Amendment Number 3 T h is am endm ent m o d ifies th e fo llo w in g p o rtio n (s) o f th e pro to co l: 1. Protocol Reads: Kristen J. Hansen, Ph.D. is the Study Director. Amend to Read: James K. Lundberg, Ph.D. is the Study Director. Reason: Original study design has changed due to availability o f resources and James K. Lundberg w ill begin serving as the study director for FACT-TOX-013 as o f 4 October 1999. 2. Protocol Reads: - Section 7.1 states that the follow ing raw data and records w ill be retained in the study folder in the archives according to AM DT-S-8: Approved protocol and amendments; study correspondence; shipping records; raw data; and electronic copies o f data. Additionally, Section 7.2 states that supporting records to be retained separately from the study folder in the archives according to AM DT-S-8 w ill include at least the following: Training records; calibration records; instrument maintenance logs; Standard Operating Procedures, Equipment Procedures, and Methods; and appropriate specimens. Amend to Read: Section 7 states: "The original data, or copies thereof, w ill be available at the 3M Environmental Laboratory to facilitate audits o f the study during its progress and before acceptance o f the final report. When the final report is com pleted, all original paper data, including: approved protocol and amendments, study correspondence, dripping records, raw data, approved final report, and electronic copies o f data w ill be retained in the archives o f the 3M Environmental Laboratory. A ll corresponding training records, calibration records, instrument maintenance logs, standard operating procedures, equipment procedures, and methods w ill be retained in the archives o f the facility performing each analysis. Reason: To direct subcontract laboratories in the disposition o f the item s listed above. 3M Environmental Laboratory 3M Environmental Laboratory 3MEnvironmental Laboratory Page 29 Page 29 3M Medical Department Study: T-6316.5 8BW 3M Medical Department Study: T6316.5 Analytical Study: FACT-TOX-013 Analytical Report: FACT TOXLRN' U2095 LRN-U2095 Protocol FACT Tox-013 Amendment Number 3 3. Protocol reads: D isposition o f test and control substances: B iological tissues and fluids are retained per GLP regulation. Amend to read: Specim ens w ill be maintained in the 3M Environmental Laboratory specim en archives. A ll specim ens sent to sub-contract laboratories w ill be returned to the 3M Environmental Laboratory upon com pletion o f analysis and subm ission o f the sub-contract laboratory(s) final report. The specimens w ill be returned with the follow ing documentation: the signed original chain o f custody and records o f storage conditions w hile at the sub-contract facility. Reason: To define in detail the appropriate disposition o f specim ens analyzed at subcontract laboratories. - Amendment Approval Marv Case, D .V .M ., Ph.D., Sponsor Representative D ate " lb Kristen J. Hansen, Ph.D., Previous Study Director D ate Dale L. Bacon, PhrBT 3M Environmental Laboratory Management 3M Environmentsl Laboratory 3M Environmental Laboratory 3MEnvironmental Laboratory Page 30 Page 30 3M Medical Department Study: T-6316.5 3M Medical Department Study: T6316.5 Analytical Study: FACT-TOX-013 A n a ly tic a l R eport: FACT TOXLR ' U2095 LRN-U2095 Study Title Analytical Study o f 2(N-Ethylperfluorooctanesulfonamido)-ethanol in Two Generation Rat Reproduction PROTOCOL AMENDMENT NO. 4 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-U2095 FACT TOX-013 Argus Study: 418-009 3M M edical Department Study: T-6316.5 3M Environmental Laboratory 3M Environmental Laboratory 3MEnvironmental Laboratory Page 31 Page 31 3M Medical Department Study: T-6316.5 3M Medical Department Study: T6316.5 Analytical Study: FACT-TOX-013 Analytical Report: FACT TOXLR ' U2095 LRN-U2095 Protocol LRN-U2095 Amendment Number 4 T h is am endm ent m o d ifies th e follow ing portio n(s) o f the protocol: 1. Protocol reads: The study director for the present study was identified in the protocol as James K. Lundburg, Ph.D. A mend to read: The role of study director for the present study was reassigned to Marvin T. Case, D .V.M ., Ph.D., as of 20 January 2000. The previous study director, James K. Lundburg, 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). 2. Protocol reads: The sponsor for the present study was identified as Marvin T. Case, D .V.M ., Ph.D. Amend to read: The role of sponsor for the present study was reassigned to John L. Butenhoff, Ph.D., as of 20 January 2000. Reason: To ensure that the study director does not also carry the duties of study sponsor, the sponsor role was reassigned. In this manner, personnel responsibilities and workload are more evenly balanced. 3M Environmental Laboratory 3M Environmental Laboratory 3MEnvironmental Laboratory Paqe 32 Page 32 3M Medical Department Study: T-6316.5 _ 3M Medical Department Study: T6316.5 ae Analytical Study: FACT-TOX-013 LRN-U2095 Analytical Report: FACT TOX-013 LRN-U2095 Protocol LRN-U2095 Amendment Number 4 Amendment Approval John L ButenhoffP h D ., Sponsor Representative D ate 3 tk I M arvin T. Case, D. V.M., P h D ., Incoming Study Director JO .-- eh^L Date & 3M Environmental Laboratory 3M Environmental Laboratory 3MEnvironmental Laboratory Page 33 Page 33 3M Medical Department Study: T-6316.5 3M Medical Department Study: T6316.5 Analytical Study: FACT-TOX-013 Analytical Report: FACT TOX L R N 'U2095 LRN-U2095 Study Title Analytical Study o f 2(N-Ethylperfluorooctanesulfonamido)-ethanol in Two Generation Rat Reproduction PROTOCOL AMENDMENT NO. 5 Amendment Date: August 31, 2000 Performing Laboratory 3M Environmental Technology & Safety Services 3M Environmental Laboratory 935 Bush Avenue St. Paul, MN 55106 Laboratory Project Identification FACT -T O X -013 ET&SS LRN U2095 Argus Study: 418-009 3M Medical Department Study: T6316.5 r* 3M Environmental Laboratory 3M Environmental Laboratory 3M Environmental Laboratory Page 34 Page 34 3M Medical Department Study: T-6316.5 3M Medical Department Study: T6316.5 " Analytical Study: FACT-TOX-013 Analytical Report: FACT TOxLfftM 'U2095 LRN-U2 095 Protocol FA C T TO X-013 Amendment No. 5 T h is am endm ent m odifies th e fo llo w in g po rtio n (s) o f th e protocol: - 1. PROTOCOL READS: The Principle Analytical Investigator for the present study was identified as James K. Lundberg, Ph.D. 2. AMEND TO read: The role o f Principle Analytical Investigator for the present study was reassigned to Kristen J. Hansen Ph.D. REASON: The role of Principle Analytical Investigator was reassigned due to availability of resources. 3M Environmental Laboratory 3M Environmental Laboratory 3MEnvironmental Laboratory Page 35 Page 35 3M Medical Department Study: T-6316.5 3M Medical Department Study: T6316.5 Analytical Study: FACT-TOX-013 LRN-U2095 Analytical Report: FACT TOX-013 LRN-U2095 Protocol F A C T TOX-013 Amendment No. 5 Amendment Approval John L. Butenhoff, Ph.D., Sponsor Representative Date Marvin T. Case, D. V.M., Ph.D., Study Director ^ O^ht-b Date 3M Environmental Laboratory 3M Environmental Laboratory 3MEnvironmental Laboratory Page 36 Page 36 3M Medical Department Study: T-6316.5 3M Medical Department Study: T6316.5 Analytical Study: FACT-TOX-013 LRN-U2095 Analytical Report: FACT TOX-013 LRN-U2095 Appendix C: Extraction and Analytical Methods This appendix includes the following methods: ETS-8-4.1, Extraction of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds from Serum for Analysis Using HPLC-Electrospray/Mass Spectrometry, (14 pages) ETS-8-5.1, Analysis of Potassium Perfluorooctanesulfonate or Other Fluorochemicals in Serum Extracts Using HPLC-Electrospray/Mass Spectrometry, (9 pages) 3M Environmental Laboratory 3MEnvironmental Laboratory Page 37 Page 37 3M Medical Department Study: T-6316.5 m 3M Medical Department Study: T6316.5 x Analytical Study: FACT-TOX-013 Analytical Report: FACT TOXLR ' U2095 T.DM _TTOftQC 3M Environmental Laboratory. 1 I T I !f 1 n * " r 1 ! I v l " C m J L J *--/r x J L / V f l v t l . X V/X% M . pm M ethod p m E x t r a c t io n ,o f P o t a s s iu m P e r f l u o r o o c t a n e s u l f o n a t e o r O t h e r HPLC-F l u o r o c h e m ic a l c o m p o u n d s f r o m S e r u m f o r A n a l y s is U s in g E l e c t r o sp r a y /M a ss Sp e c t r o m e t r y (m m M ethod N um ber: ETS-8-4.1 Adoption Date: 03/01/99 Author: Lisa Clemen, Glenn Langenburg R evision Date: Approved By: - 0 1 1 S-- - Laboratory Manager /m m , fy i* (J tT ^ v - -- -------------------- Group Leader pm C/iM, A Technical Reviewer Date Date ... o v / W s ? Date pm 1.0 Scope and A pplication 1.1 Scope: This method is for the extraction o f potassium perfluorooctanesulfonate (PFOS) or other fluorochemical compounds from serum. 1.2 A pplicable com pounds: Fluorochemical surfactants or other fluorinated compounds. 1.3 M atrices: Rabbit, rat, bovine, monkey, and human serum or other fluids as designated in the validation report. Word 6/95 fmm 3M Environmental Laboratory 3MEnvironmental Laboratory ETS-8-4.1 Extraction o f PFOS from Serum Page 1of 14 Page 38 Page 38 3M Medical Department Study: T-6316.5 3M Medical Department Study: T6316.5 Analytical Study: FACT-TOX-013 A n a ly tic a l R eport: FACT TO J^^K '^2095 LRN-U2095 2.0 Summary of M ethod___________________________________________________________ 2.1 This method describes the procedure for extracting potassium perfluorooctanesulfonate (PFOS) or other fluorochemical surfactants from serum, or other fluids, using an ion pairing reagent and methyl-iert-butyl ether (MtBE). In this method, seven fluorochem icals were extracted: PFOS, PFOSA, PFOSAA, EtFOSE-OH, PFOSEA, M 556, and surrogate standard (see 3.0 Definitions), An ion pairing reagent is added to the sample and the analyte ion pair is partitioned into MtBE. The MtBE extract is removed and put onto a nitrogen evaporator until dry. Each extract is reconstituted in 1.0 m i. o f methanol, then filtered through a 3 cc plastic syringe attached to a 0.2 jum nylon filter into glass autovials. 2.2 These sample extracts are analyzed follow ing method ETS-8-5.1 or other appropriate m ethods. 3.0 Definitions___________________________________________________________________ 3.1 PFOS: perfluorooctanesulfonate (anion o f potassium salt) C8F17S 0 3` 3.2 PFOSA: perfluorooctane sulfonylam ide C8F17S 0 2NH2 3.3 PFOSAA: perfluorooctane sulfonylamido (ethyl)acetate C8F17S 0 2N(CH2CH3)CH2CO2' 3.4 EtFOSE-OH: 2(N-ethylperfluorooctane sulfonamido)-ethyl alcohol C8F17S 0 2N(CH2CH3)CH2CH20H 3.5 PFOSEA: perfluorooctane sulfonyl ethylamide C8FI7S 0 2N(CH2CH3)H 3.6 M 556: C8FnS 0 2N(H)(CH2C 0 0 H ) 3.7 Surrogate standard: 1H-1H-2H-2H perfluorooctane sulfonic acid 4.0 Warnings and Cautions_________________________________________________________ 4.1 H ealth and safety w arnings 4.1.1 U se universal precautions, especially laboratory coats, goggles, and gloves when handling animal tissue, which may contain pathogens. 5.0 Interferences_______________________________________________________________ 5.1 There are no interferences known at this time. 6.0 Equipment___________________________________________________________________ 6.1 The follow ing equipment is used w hile performing this method. Equivalent equipment is acceptable. 6.1.1 Vortex mixer, VWR, Vortex Genie 2 6.1.2 Centrifuge, Mistral 1000 or IEC 6.1.3 Shaker, Eberbach or VWR 3M Environmental Laboratory 3MEnvironmental Laboratory ETS-8-4.1 Extraction of PFOS from Serum Page 2 o f 14 Paqe 39 Page 39 3M Medical Department Study: T-6316.5 3M Medical Department Study: T6316.5 Analytical Study: FACT-TOX-013 Analytical Report: FACT TOX^ ^ ' ^ 2095 LRN-U2095 6.1.4 Nitrogen evaporator, Organomation 6.1.5 Balance ( 0.100 g) 7.0 Supplies and Materials________________________________________________________ 7.1 Gloves 7.2 Eppendorf or disposable pipettes 7.3 N algene bottles, capable o f holding 250 mL and 1 L 7.4 Volumetric flasks, glass, type A 7.5 I-CHEM vials, glass, 40 mL glass 7.6 Centrifuge tubes, polypropylene, 15 mL 7.7 Labels 7.8 Oxford Dispenser - 3.0 to 10.0 mL 7.9 Syringes, capable o f measuring 5 pL to 50 pL 7.10 Graduated pipettes 7.11 Syringes, disposable plastic, 3 cc 7.12 Syringe filters, nylon, 0.2 pm, 25 mm 7.13 Timer 7.14 Crimp cap autovials and caps 7.15 Crimpers Note: Prior to using glassware and bottles, rinse 3 tim es with methanol and 3 tim es w ith M illi-QTM water. Rinse syringes a minimum o f 9 times with methanol, 3 rinses from 3 separate vials. 8.0 Reagents and Standards______________________________________________________ 8.1 Type I reagent grade water, M illi-QTM or equivalent; all water used in this method should be M illi-QTM water and may be provided by a M illi-Q TOC PlusTM system 8.2 Sodium hydroxide (NaOH), J.T Baker or equivalent 8.3 Tetrabutylammonium hydrogen sulfate(TBA ), Kodak or equivalent 8.4 Sodium carbonate (NajCOj), J.T. Baker or equivalent 8.5 Sodium bicarbonate (N aH C 03), J.T. Baker or equivalent 8.6 M ethyl-T-Butyl Ether, Omnisolv, glass distilled or HPLC grade 8.7 Methanol, Omnisolv, glass distilled or HPLC grade 8.8 Serum or blood, frozen from supplier 8.9 Fluorochem ical standards 8.9.1 PFOS (3M Specialty Chemical D ivision), molecular weight = 538 8.9.2 PFOSA (3M Specialty Chemical D ivision), molecular weight = 499 ETS-8-4.1 Extraction of PFOS from Serum Page 3 o f 14 3M Environmental Laboratory 3MEnvironmental Laboratory Paoe 40 Page 40 3M Medical Department Study: T-6316.5 3M Medical Department Study: T6316.5 Analytical Study: FACT-TOX-013 LRN-U2095 Analytical Report: FACT TOX-013 LRN-U2095 8.9.3 PFOSAA (3M Specialty Chemical D ivision), molecular w eight - 585 8.9.4 EtFOSE-OH (3M Specialty Chemical D ivision), molecular weight = 570 8.9.5 PFOSEA (3M Specialty Chemical D ivision), molecular weight = 527 8.9.6 M 556 (3M Specialty Chemical D ivision), molecular w eight = 557 8.9.7 Surrogate standard: 4-H, periluorooctane sulfonic acid (l-H .l-H , 2-H, 2-H C8F13S 0 3H) molecular weight = 428 8.9.8 Other fluorochemicals, as appropriate 8.10 R eagent preparation NOTE: When preparing larger volumes than listed in reagent, standard, or surrogate preparation, adjust accordingly. 8.10.1 10 N sodium hydroxide (NaOH): W eigh approximately 200 g NaOH. Pour into a 1000 mL beaker containing 500 mL Milli-QTM water, m ix until all solids are dissolved. Store in a 1 L Nalgene bottle. 8.10.2 1 N sodium hydroxide (NaOH): Dilute 10 N NaOH 1:10. Measure 10 mL o f 10 N NaOH solution into a 100 mL volumetric flask and dilute to volum e using Milli-QTM water. Store in a 125 mL N algene bottle. 8.10.3 0.5 M tetrabutylammonium hydrogen sulfate (TBA): W eigh approximately 169 g o f TBA into a 1 L volumetric containing 500 mL Milli-QTM water. Adjust to pH 10 using approximately 44 to 54 mL o f 10 N NaOH (W hile adding the last mL o f NaOH, add slow ly because the pH changes abruptly). Dilute to volum e with Milli-QTM water. Store in a 1 L Nalgene bottle. 8.10.3.1 TBA requires a check prior to each use to ensure pH = 10. Adjust as needed using 1 N NaOH solution. 8.10.4 0.25 M sodium carbonate/sodium bicarbonate buffer (Na2C03/N aH C 03): W eigh approximately 26.5 g o f sodium carbonate (N a^ O j) and 21.0 g o f sodium bicarbonate (NaH C03) into a 1 L volumetric flask and bring to volum e with M illiQTM water. Store in a 1 L Nalgene bottle. 8.11 Standards preparation 8.11.1 Prepare PFOS standards for the standard curve. 8.11.2 Prepare other fluorochemical standards, as appropriate. Multicomponent fluorochemical standards are acceptable (for example, one working standard solution containing 1.00 ppm PFOS, 1.02 ppm PFOSA , 0.987 ppm PFOSAA, and 1.10 ppm EtFOSE-OH.) 8.11.3 W eigh approximately 100 mg o f PFOS into a 100 mL volumetric flask and record the actual weight. 8.11.4 Bring to volum e with methanol for a stock standard o f approximately 1000 ppm (ug/m L ). 8.11.5 D ilute the stock solution with methanol for a working standard 1 solution o f approximately 50 ppm. 8.11.6 D ilute working standard 1 w ith methanol for a working standard 2 solution o f approx. 5.0 ppm. ETS-8-4.1 Extraction of PFOS from Serum Page 4 o f 14 3M Environmental Laboratory 3M Environmental Laboratory Page 41 Page 41 3M Medical Department Study: T-6316.5 3M Medical Department Study: T6316.5 Analytical Study: FACT-TOX-013 LRN-U2095 Analytical Report: FACT TOX-013 L R N -U 2095 8.11.7 D ilute working standard 1 with methanol for a working standard 3 solution o f approx. 0.50 ppm. 8.12 Surrogate stock standard preparation 8.12.1 W eigh approximately 50-60 mg o f surrogate standard 1-H,1-H, 2-H, 2-H, C8F,3S 0 3H into a 50 mL volumetric flask and record the actual weight. 8.12.2 Bring to volum e with methanol for a surrogate stock o f approximately 1000-1200 ppm. " 8.12.3 Prepare a surrogate working standard. Transfer approximately 1 mL o f surrogate stock to a 10 mL volumetric flask and bring to volum e with methanol for a working standard o f 100 ppm. Record the actual volum e transferred. 9.0 Sample Handling_________________________________________ ^____________________ 9.1 A ll samples are received frozen and must be kept frozen until the extraction is performed. 9.2 A llow samples to thaw to room temperature prior to extraction. 10.0 Quality Control ____________________________________ 10.1 Solvent B lanks, M ethod blanks and m atrix blanks 10.1.2 Extract two 1.0 mL aliquots o f Milli-QTM water follow ing this procedure and use as method blanks. 10.1.3 Extract two 1.0 mL aliquots o f the serum follow ing this procedure and use as matrix blanks. See 11.1.4. 9.2 M atrix spikes 10.2.1 Prepare and analyze matrix spike and matrix spike duplicate samples to determine the accuracy o f the extraction. 10.2.2 Prepare each spike using a sample chosen by the analyst, usually the control matrix received with each sample set. 10.2.3 Expected concentrations w ill fall in the mid-range o f the initial calibration curve. Additional spikes may be included and may fall in the low-range o f the initial calibration curve. 10.2.4 Prepare one matrix spike and matrix spike duplicate per 40 samples, w ith a minimum o f 2 matrix spikes per batch. 0.3 C ontinuing calibration checks 10.3.1 Prepare continuing calibration check samples to ensure the accuracy o f the initial calibration curve. 10.3.2 Prepare, at a minimum, one continuing check per group o f 10 samples. For example, if a sample set - 34, four checks are prepared and extracted. 10.3.3 Prepare each continuing calibration check from the same matrix used to prepare the initial curve. II li ETS-8-4.1 Extraction of PFOS from Serum ronmental Laboratory 3MEnvironmental Laboratory Page 5 o f 14 Paqe 42 3M Envii Page 42 3M Medical Department Study: T-6316.5 3M Medical Department Study: T6316.5 Analytical Study: FACT-TOX-013 LRN-U2095 Analytical Report: FACT TOX-013 LRN-U2095 10.3.4 The expected concentrations w ill fall within the mid-range o f the initial calibration curve. Additional spikes may be included that fall in the low-range o f the initial calibration curve. This is necessary if the analyst must quantitate using only the low end o f the calibration curve (for exam ple, 5 ppb --100 ppb, rather than 5 ppb - 1000 ppb). 11.0 Calibration and Standardization ___________________________________________ 11.1 Prepare m atrix calibration standards 11.1.1 Transfer 1 mL o f serum to a 15 mL centrifuge tube. 11.1.2 If m ost sample volum es are less than 1.0 mL, extract standards with matrix volum es equal to the sample volum es. D o not extract less than 0.50 mL o f matrix. Record each sample volum e on the extraction sheet. 11.1.3 W hile preparing a total o f twenty aliquots in 15 mL centrifuge tubes, m ix or shake between aliquots. 11.1.4 Two 1 mL aliquots, or other appropriate volum e, serve as matrix blanks. Typically use the standard concentrations and spiking amounts listed in Table 1, at the end o f this section, to spike, in duplicate, two standard curves, for a total o f eighteen standards, two matrix blanks, and two method blanks. 11.1.5 Refer to validation report ETS-8-4.0 & ETS-8-5.0-V -1, which lists the working ranges and the Linear Calibration Range (LCR) for calibration curves. 11.1.6 U se Attachment D as an aid in calculating the concentrations o f the working standards. See Section 13.0 to calculate actual concentrations o f PFOS in calibration standards. 11.2 To each standard, blank, or continuing check, add appropriate amount o f surrogate working standard for the concentration to fall within the calibration curve range 5 ppb 1000 ppb. 11.3 Extract spiked matrix standards follow ing 12.6-12.16 o f this method. U se these standards to establish each initial curve on the mass spectrometer. 3M Environmental Laboratory 3MEnvironmental Laboratory ETS-8-4.1 Extraction o f PFOS from Serum Page 6 o f 14 Page 43 Page 43 3M Medical Department Study: T-6316.5 3M Medical Department Study: T6316.5 Analytical Study: FACT-TOX-013 LRN-U2095 Analytical Report: FACT TOX-013 LRN-U2095 Table 1 Approxim ate spiking am ounts for standards and spikes U sing 1.0 mL o f m atrix Working standard pL Approx, final cone, o f (approx, cone.) analyte in matrix -- - Blank 0.500 ppm 10 0.005 ppm 0.500 ppm 20 0.010 ppm 5.00 ppm 5 0.025 ppm 5.00 ppm 10 0.050 ppm 5.00 ppm 20 0.100 ppm 50.0 ppm 5 0.250 ppm 50.0 ppm 10 0.500 ppm 50.0 ppm 15 0.750 ppm 50.0 ppm 20 . 1.00 ppm 12.0 Procedure___________________________________________________________________ 12.1 Obtain frozen samples and allow to thaw at room temperature or in a lukewarm waterbath. 12.2 Vortex m ix for 15 seconds, then transfer 1.0 mL or other appropriate volum e to a 15 mL polypropylene centrifuge tube. 12.3 Return unused samples to freezer after extraction amounts have been removed. 12.4 Record the initial volume on the extraction worksheet. 12.5 Label the tube with the study number, sample ID, date and analyst initials. See attached worksheet for documenting the remaining steps. 12.6 Spike all sam ples, including blanks and standards, ready for extraction w ith surrogate standard as described in 11.2. 12.7 Spike each matrix with the appropriate amount o f standard as described in 11.1, or T able 1 in that section, for the calibration curve standards. A lso prepare matrix spikes and continuing calibration standards. 12.8 Vortex m ix the standard curve samples, matrix spike samples, and continuing calibration sam ples for 15 seconds. 12.9 Check to ensure the 0.5 M TBA reagent is at pH 10. I f not, adjust accordingly. 12.10 To each sample, add 1 mL 0.5 M TBA and 2 mL o f 0.25M sodium carbonate/sodium bicarbonate buffer. 12.11 U sing an Oxford Dispenser, add 5 mL methyl-terf-butyl ether. 12.12 Cap each sample and put on the shaker at a setting o f 300 rpm, for 20 minutes. 12.13 Centrifuge for 20 to 25 minutes at a setting o f 3500 rpm, or until layers are w ell separated. ETS-8-4.1 Extraction o f PFOS from Serum Page 7 o f 14 3M Environmental Laboratory Page 44 3MEnvironmental Laboratory Page 44 3M Medical Department Study: T-6316.5 (M M * 3M Medical Department Study: T6316.5 Analytical Study: FACT-TOX-013 LRN-U2095 Analytical Report: FACT TOX-013 LRN-U2095 12.14 Label a fresh 15 mL centrifuge tube with the same information as in 12.5. 12.15 Rem ove 4.0 mL o f the organic layer to this clean 15 mL centrifuge tube. 12.16 Put each sample on the analytical nitrogen evaporator until dry, approximately 1 to 2 hours. 12.17 Add 1.0 mL o f methanol to each centrifuge tube using a graduated pipette. 12.18 Vortex m ix for 30 seconds. . 12.19 Attach a 0.2 pm nylon mesh filter to a 3 cc syringe and transfer the sample to this syringe. Filter into a 1.5 mL glass autovial or low-volum e autovial when necessary. 12.20 Label the autovial with the study number, animal number and gender, sample tim epoint, matrix, final solvent, extraction date, and analyst(s) performing the extraction. 12.21 Cap and store extracts at room temperature or at approximately 4 C until analysis. 12.22 Complete the extraction worksheet, attached to this document, and tape in the study notebook or include in study binder, as appropriate. 13.0 Data Analysis and Calculations_____ ;________________________________________ 13.1 C alculations 13.1.1 Calculate actual concentrations o f PFOS, or other applicable fluorochem ical, in calibration standards using the follow ing equation: mL o f standard x concentration o f standard fug /m D ___________________ = mL o f standard + mL o f surrogate standard + initial matrix volum e (mL) Final Concentration (pg/mL) o f PFOS in matrix 14.0 Method Performance__________________________________________________________ 14.1 The method detection lim it (MDL) is analyte and matrix specific. Refer to M DL report for specific MDL and lim it o f quantitation (LOQ) values (see A ttachm ents B and C). 14.2 The follow ing quality control samples are extracted with each batch o f sam ples to evaluate the quality o f 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 o f the extraction. 14.2.3 Continuing calibration check samples to determine the continued accuracy o f the initial calibration curve. 14.3 Refer to section 14 o f ETS-8-5.1 for method performance criteria. 15.0 Pollution Prevention and W aste Management_________________________ 15.1 Sample waste is disposed in biohazard containers, flammable solvent w aste is disposed in high BTU containers, and used glass pipette waste is disposed in broken glass containers located in the laboratory. ETS-8-4. Extraction o f PFOS from Serum Page 8 o f 14 3M Environmental Laboratory is m 3MEnvironmental Laboratory Page 45 Page 45 3M Medical Department Study: T-6316.5 3M Medical Department Study: T6316.5 Analytical Study: FACT-TOX-013 LRN-U2095 Analytical Report: FACT TOX-013 LRN-U2095 16.0 Records ______________________________________________________ _ 16.1 Complete the extraction worksheet attached to this method, and tape in the study notebook or include in the 3-ring study binder, as appropriate. 17.0 Attachments_________________________________________________________________ 17.1 Attachment A , Extraction worksheet 17.2 Attachment B , MDL/LOQ values and summaty 17.3 Attachment C, Calibration standard concentration worksheet 18.0 References__________________________________________________________________ 18.1 The validation report associated with this method is E T S-8-4.0 & 5.0-V -l. 18.2 FACT-M -3.1, "Analysis o f Serum or Other Fluid Extracts for Fluorochem icals using HPLC-Electrospray M ass Spectrometry" 19.0 Affected Documents________ ;________________________________________________ 19.1 ETS-8-5.1, "Analysis o f Serum or Other Fluid Extracts for Fluorochemicals using HPLC-Electrospray M ass Spectrometry" 20.0 Revisions__________________ :________________________________________________ R evision Number 1 Reason For R evision Section 12.21 Changed to include sample storage at room temperature. Section 12.13 Added the shaker speed. Section 12.17 Final volum e is 1.0 mL; not adjusted for initial volum es less than 1,0 mL. R evision D ate 04/02/99 3M Environmental Laboratory 3MEnvironmental Laboratory ETS-8-4.1 Extraction of PFOS from Serum Page 9 o f 14 Page 46 Page 46 3M Medical Department Study: T-6316.5 <8S3BWi 3M Medical Department Study: T6316.5 Analytical Study: FACT-TOX-013 LRN-U2095 Analytical Report: FACT TOX-013 LRN-U2095 Extraction Worksheet ETS-8-4.1 Study# Matrix Box# Wk/Day DateSpiked/Analyst CCV MS MSD Surrogate Std approx, ppm actual ppm # FC-Mix approx. 0.5 pm actual ppm # FC-Mix approx. 5 ppm actual ppm # FC-Mix approx. 50 ppm actual ppm # Comments --. * ---- --- - -- -- -- -- -- -- -- Blank Std # amount = Serum Extraction Method : Vortex 15 sec. Pipette Matrix Volume mL Pipette 1 mL of 0.5 M TBA, pH 10. pH = Std. # Pipette 2 mL of 0.25 Na?COV0.25M NaHCCh buffer Std. # Dispense 5 mL of methyl-t-butyl ether TN-A- Shake 20 min. Shaker speed: Centrifuge 20-25 min. Centrifuge speed: Remove a 4 mL aliauot of organic layer Put on Nitrogen Evaporator to drvness Temperature: Add methanol Volume mL TN-A- Vortex 30 sec. Filter using a 3cc B-D svringe with a 0.2um filter into a 1.5 mL autosample vial Cont. Cal. Verifications used same matrix as for std curve. - - - - - - - - - - - - - mL Date & Initials i""* Attachment A 3M Environmental Laboratory 3MEnvironmental Laboratory ETS-8-4.1 Extraction o f PFOS from Serum Page 10 o f 14 Page 47 Page 47 3M Medical Department Study: T-6316.5 3M Medical Department Study: T6316.5 Analytical Study: FACT-TOX-013 LRN-U2095 Analytical Report: FACT TOX-013 LRN-U2 095 MDL/LOQ values for rabbit serum Com pound MDL LOQ Linear C alibration R ange (LCR) (PPb) (PPb) A pproxim ate concentrations to be used for preparing the Standard C alibration Curve PFOS 1.74 5.55 5 p p b - 1000ppb PFOSA 1.51 4.79 5 ppb - 1000 ppb PFOSAA 3.46 20.5 5 ppb - 1000 ppb EtFOSE-OH 11.4 36.2 5 ppb - 1000 ppb M556 6.03 19.2 5 ppb - 1000 ppb PFOSEA 5.71 18.2 5 ppb - 1000 ppb MDL/LOQ values in rat, bovine, monkey, and human serum, and monkey plasma were not statistically determined. Two curves in each o f these matrices were extracted and analyzed with the rabbit serum curves to determine equivalence. Responses in the rat, bovine, monkey, and human were equivalent to the rabbit responses, therefore, their MDL and LOQ will be the same values as determined in rabbit serum. Please see LOQ Summary and MDL study in ETS-8-4.0 & 5.0-V-l for further information. Attachment B: MDL/LOQ Summary 3M Environmental Laboratory 3MEnvironmental Laboratory ETS-8-4.1 Extraction o f PFOS from Serum Page 11 o f 14 Page 48 Page 48 3M Medical Department Study: T-6316.5 PRB4 3M Medical Department Study: T631S.5 Analytical Study: FACT-TOX-013 LRN-U2095 Analytical Report: FACT TOX-013 LRN-U2095 p"** SBW r* i r 1 (mm Compound: PFOS Prepared range Rabbit Serum o f standards (ppb) (ng/mL) Full Range Low Curve High curve 1/X 0.995 - 978 4.94 - 248 97.8 - 978 0.995 - 978 Compound: PFOSA Prepared range Rabbit Serum of standards (ppb) (ng/mL) Full Range Low Curve High curve 1/X 0.993 - 976 4.93 - 97.6 24.8 - 976 0.993 - 976 Compound: PFOSAA Prepared range Rabbit Serum of standards (ppb) (ng/mL) Full Range Low Curve High curve 1/X 0.991-974 4.92 - 247 49.2 - 974 0.991 - 974 LCR from curve (PPb) (ng/mL) 24.8 - 978 4.94 - 248 97.8-978 4.94 - 978 % Recovery Range 83-108 85-104 85-106 94-111 LCR from curve (PPb) (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 LCR from curve (PPb) (ng/mL) 24.7 - 974 9.74 - 247 97.4 - 974 9.74 - 974 % Recovery Range 81-111 97-107 85-108 95-115 RSD Range 4.67-11.0 5.34-12.0 4.84-9.80 4.60-10.5 RSD Range 5.10-14.7 9.85-14.7 5.08-13.9 5.10-14.5 RSD Range 4.18-10.6 6.38-21.8 4.33-12.5 4.11-23.2 ^ Attachment B: MDL/LOQ Summary 3M Environmental Laboratory 3MEnvironmental Laboratory ETS-8-4.1 Extraction o f PFOS from Serum Page 12 o f 14 Prr#=> AQ Page 49 3M Medical Department Study: T-6316.5 3M Medical Department Study: T6316.5 Analytical Study: FACT-TOX-013 Analytical Report: FACT TOL R N "U2 0 9 5 L R N -U 2095 Compound: EtFOSE-OH Prepared range Rabbit Serum o f standards (ppb) (ng/mL) Full Range Low Curve High curve 1/X 0.993 - 976 4.93 - 97.6 49.3 - 976 0.993 - 493 LCR from curve (ppb) (ng/mL) 49.3 - 976 9.76 - 97.6 97.6 - 976 9.76 - 976 % Recovery Range 77-110 97-107 90-109 86-111 Com pound: PFOSEA Prepared range Rabbit Serum o f standards (ppb) (ng/mL) Full Range Low Curve High curve 1/X 0.993 - 976 4.93 - 248 49.3 - 976 0.993 - 976 LCR from curve (PPb) (ng/m L ) 24.8 - 976 9.76 - 248 49.3-976 9.76 - 976 % Recovery Range 96-106 91-110 86-106 95-117 Compound: M556 Prepared range Rabbit Serum o f standards (ppb) (ng/mL) Full Range Low Curve High curve 1/X 0.993 - 976 4.93 - 97.6 97.6 - 976 0.993 - 976 LCR from curve (Ppb) (ng/m L ) 24.8 - 976 9 .7 6 -9 7 .6 97.6 - 976 9.76 - 976 % Recovery Range 88-106 100-105 81-111 97-110 RSD Range 11.2-25.5 14.1-21.3 11.5-19.6 11.1-21.2 RSD Range 10.1-16.2 11.8-19.5 10.2-18.2 10.1-19.1 RSD Range 4.82-17.9 5.95-18.2 5.11-9.74 4.77-19.5 Attachment B: MDL/LOQ Summary 3M Environmental Laboratory 3MEnvironmental Laboratory ETS-8-4.1 Extraction o f PFOS from Serum Page 13 o f 14 Pad 50 Page 50 JM Medical Department Study: T-6316.5 3M Medical Department Study: T6316.5 Analytical Study: FACT-TOX-013 Analytical Report: FACT TOL R N 'U2 0 9 5 LRN-U2095 Ion Pair Standard Curves - Fluids Prep date(s): Standard number: Analyte(s): Equipment number: Sample matrix: Final solvent and TN: Blank fluid/identifier: M ethod/revision: Target analyte(s): _ FC mix std approx. 0.500 ppm: FC mix std approx. 5.00 ppm: FC mix std approx. 50.0 ppm: Surrogate std approx. 100 ppm: Actual concentrations of standards in the FC mix PFOS PFOSA PFOSAA EtFOSE PFOSEA Std cone Std cone Std cone Std cone Std cone ug/mL ug/mL ug/mL ug/mL ug/mL 0.500 0.507 0.532 0.501 0.521 0.500 0.507 0.532 0.501 0.521 5.00 5.07 5.32 5.0! 5.21 5.00 5.07 5.32 5.01 5.21 5.00 5.07 5.32 5.01 5.21 50.0 50.1 53.2 50.1 52.1 50.0 50.1 53.2 50.1 52.1 50.0 50.1 53.2 50.1 52.1 50.0 50.1 53.2 50.1 52.1 M556 Std cone ug/mL 0.501 0.501 5.01 5.01 5.01 50.1 50.1 50.1 50.1 All Amt spiked mL 0.010 0.020 0.005 0.010 0.020 0.005 0.010 0.015 0.020 All Final vol mL 1.015 1.025 1.010 1.015 1.025 1.010 1.015 1.020 1.025 C alculated concentrations of standards in the sam ple m atrix PFOS PFOSA PFOSAA EtFOSE PFOSEA M556 Surrogate Final cone Final cone Final cone Final cone Final cone Final cone Std cone ng/mL ng/mL ng/mL ng/mL ng/mL ng/mL ng/mL 4.93 5.00 5.24 4.94 5.01 5.13 100 9.76 9.89 10.4 9.78 9.93 10.2 24.8 25.1 26.3 24.8 25.2 25.8 Surrogate 49.3 50.0 52.4 49.4 50.1 51.3 Final cone 97.6 98.9 104 97.8 99.3 102 ng/mL 248 251 263 248 252 258 500 493 500 524 494 501 513 735 746 782 737 749 766 976 989 1038 978 993 1017 All Ain't spiked mL 0.005 Validated ranges - approximate concentrations Serum PFOS PFOSA PFOSAA EtFOSE-OH PFOSEA M556 Rabbit 5.00-1000 | 5.00-1000 | 5.00-1000 | 5.00-1000 | 5.00-1000 | 5.00-1000 Bovine Estimates only. Use values for rabbit. Rat Estimates only. Use values for rabbit. Monkey & Plasma Estimates only. Use values for rabbit. Human Estimates only. Use values for rabbit. Attachment C: Ion Pair Standard Curves ETS-8-4.1 Extraction o f PFOS from Serum 3M Environmental Laboratory 3MEnvironmental Laboratory Page 14 o f 14 Pacre 51 Page 51 3M Medical Department Study: T-6316.5 3M Medical Department Study: T6316.5 Analytical Study: FACT-TOX-013 LRN-U2095 Analytical Report: FACT TOX-013 LRN-U2095 3M Environmental Laboratory M ethod A n a l y sis o f P o t a ssiu m P e r fl u o r o o c t a n e su l fo n a t e o r O t h e r F l u o r o c h e m ic a l s in Ser u m E x t r a c t s U sin g H P L C -E l e c t r o spr a y /M a ss S pe c t r o m e t r y - M ethod Num ber: ETS-8-5.1 Author: Lisa Clemen, Robert Wynne Approved By: ) X i l i -- W* Laboratory Manager Group Leader AL . pm* A CIawc-u __ Technical Reviewer A doption D ate: 03/01/99 R evision Date: H / ^ b P ^ ^ y 2. c f D ate D ate dh/ u / m D ate 1.0 Scope and Application_________________________________________________________ 1.1 Scope: This method describes the analysis o f serum extracts for fluorochem ical surfactants using HPLC-electrospray/mass spectrometry. 1.2 A pplicable C om pounds: Fluorochemical surfactants or other fluorinated compounds, or other ionizable compounds. 1.3 M atrices: Rabbit, rat, bovine, monkey, and human serum, or other fluids as designated in the validation report. Word 6/95 ETS-8-5.1 Analysis o f Serum Extract Using ES/MS Page 1 o f 9 3M Environmental Laboratory 3MEnvironmental Laboratory Page 52 Page 52 3M Medical Department Study: T-6316.5 9 tm 1 J 3M Medical Department Study: T6316.5 Analytical Study: FACT-TOX-013 Analytical Report: FACT TO:LR N "U2095 LRN-U2 095 2.0 Summary of Method____________________________________________ _______________ 2.1 This method describes the analysis o f fluorochemical surfactants extracted from serum or other fluids, using HPLC-electrospray/mass spectrometry, or similar system as appropriate. The analysis is performed by monitoring a single ion characteristic o f a particular fluorochem ical, such as the perfluorooctanesulfonate (PFOS) anion, m /z= 499. Additionally, samples may be analyzed using a tandem mass spectrometer to further verify the identity o f a compound by detecting daughter ions o f the parent ion. 3.0 Definitions _______________________________________________________________ 3.1 A tm ospheric Pressure Ionization (API): The Micromass Quattro II triple quadrupole system s allow for various methods o f ionization by utilizing various sources, probes, and interfaces. These include but are not limited to: Electrospray Ionization (ESI), Atmospheric Pressure chem ical Ionization (APcI), Thermospray, etc. The ionization process in these techniques occurs at atmospheric pressure (i.e., not under a vacuum). 3.2 E lectrospray Ionization (E S, ESI): a method o f ionization performed at atmospheric pressure, whereby ions in solution are transferred to the gas phase via tiny charged droplets. These charged droplets are produced by the application o f a strong electrical field. 3.3 M ass Spectrom etry, M ass Spectrom eter (M S), Tandem M ass Spectrom eter (M S/M S): The API Quattro II triple quadrupole system s are equipped with quadrupole mass selective detectors. Ions are selectively discriminated by mass to charge ratio (m /z) and subsequently detected. A single MS may be employed for ion detection or a series (M S/M S) for more specific fragmentation information. 3.4 C onventional vs. Z-spray probe interface: The latest m odels o f Micromass Quattro II triple quadrupole systems (post 1998) utilize a "Z-spray" conformation. The spray emitted from a probe is orthogonal to the cone aperture. In the conventional conformation it is aimed directly at the cone aperture, after passing through a tortuous pathway in the counter electrode. Though the configuration is different, the methods o f operation, cleaning, and maintenance are the same. However, Z-spray components and conventional components are not compatible with one another, but only with similar system s (i.e., Z-spray components are compatible with some other Z-spray system s, etc.) 3.5 M ass L ynx Software: System software designed for the specific operation o f these Quattro II triple quadrupole systems. Currently MassLynx has W indows 95 and W indowsNT 4.0 versions. A ll versions are similar. For more details see the manual specific to the instrument (Micromass Quattro II triple quadrupole M assLynx or MassLynx NT User's Guide). 4.0 Warnings and Cautions_____________________________________________________ 4.1 H ealth and Safety W arnings: 4.1.1 U se caution with the voltage cables for the probe. When engaged, the probe em ploys a voltage o f approximately 5000 Volts. 4.1.2 When handling samples or solvents wear appropriate protective gloves, eyewear, and clothing. 3M Environmental Laboratory 3MEnvironmental Laboratory ETS-8-5.1 Analysis of Serum Extract Using ES/MS Page 2 of 9 Pace 53 Page 53 3M Medical Department Study: T-6316.5 3M Medical Department Study: T6316.5 Analytical Study: FACT-TOX-013 Analytical Report: FACT TO/ -U2095 LRN-U2095 4.2 Cautions: 4.2.1 Do not operate solvent pumps above capacity o f 400 bar (5800 psi) back pressure. If the back pressure exceeds 400 bar, the HP 1100 w ill initiate automatic shutdown. 4.2.2 D o not run solvent pumps to dryness. 5.0 Interferences________________________________________________________________ 5.1 To m inim ize interferences when analyzing samples, teflon should not be used for sample storage or any part o f instrumentation that comes in contact w ith the sample or extract. 6.0 Equipment____________________________________________________________________ 6.1 Equipment listed below may be m odified in order to optim ize the system . Docum ent any m odifications in the raw data as method deviations. 6.1.1 6.1.2 Micromass Quattro II triple quadrupole Mass Spectrometer equipped w ith an electrospray ionization source HP 1100 low pulse solvent pumping system, solvent degasser, column compartment, and autosampler 7.0 Supplies and Materials________________________________ :_______________________ 7.1 Supplies 7.1.1 High purity grade nitrogen gas regulated to approximately 100 psi (H ouse air system ) 7.1.2 HPLC analytical column, specifics to be determined by the analyst and documented in the raw data. 7.1.3 Capped autovials or capped 15 mL centrifuge tubes 8.0 Reagents and Standards___________________________________ ;_____________________ 8.1 Reagents 8.1.1 Methanol, HPLC grade or equivalent 8.1.2 Milli-QTM water, all water used in this method should be Milli-QTM water or equivalent, and may be provided by a M illi-Q TOC Plus system or other vendor 8.1.3 Ammonium acetate, reagent grade or equivalent 8.2 Standards 8.2.1 Typically two method blanks, two matrix blanks, and eighteen matrix standards are prepared during the extraction procedure. See ETS-8-4.1. 9.0 Sample Handling___________________ '__________________ ,_______________________ 9.1 Fresh matrix standards are prepared with each analysis. Extracted standards and samples are stored in capped autovials or capped 15 mL centrifuge tubes until analysis. ^ 3M Environmental Laboratory 3MEnvironmental Laboratory ETS-8-5.1 Analysis o f Serum Extract Using ES/MS Page 3 of 9 Pacre 54 Page 54 3M Medical Department Study: T-6316.5 3M Medical Department Study: T6316.5 Analytical Study: FACT-TOX-013 Analytical Report: FACT TO^-^ S ' ^ 2095 LRN-U2095 9.2 I f analysis w ill be delayed, extracted standards and samples can be refrigerated at approximately 4 C, or at room temperature, until analysis can be performed. 10.0 Quality Control____________________________________________________________ 10.1 Solvent Blanks, Method Blanks and Matrix Blanks 10.1.1 Solvent blanks, method blanks and matrix blanks are prepared and analyzed with each batch to determine contamination or carryover. 10.1.2 Analyze a method blank and a matrix blank prior to each calibration curve. 10.2 Matrix Spikes 10.2.1 Matrix spikes are prepared and analyzed to determine the matrix effect on the recovery efficiency. 10.2.2 Matrix spike duplicates are prepared and analyzed to measure the precision and the recovery for each analyte. 10.2.3 Analyze a matrix spike and matrix spike duplicate per forty samples, w ith a minimum o f 2 spikes per batch. 10.2.4 Matrix spike and matrix spike duplicate concentrations w ill fall in the mid-range o f the initial calibration curve. Additional spike concentrations may fall in the low range o f the initial calibration curve. 10.3 Continuing Calibration Verifications 10.3.1 Continuing calibration verifications are analyzed to verify the continued accuracy o f the calibration curve. 10.3.2 Analyze a mid-range calibration standard after every tenth sample, with a minimum o f one per batch. 11.0 Calibration and Standardization___________________________________________ 11.1 Analyze the extracted matrix standards prior to and follow ing each set o f extracts. The average o f two standard curves w ill be plotted by linear regression (y = my + b), weighted 1/x, not forced through zero, using M assLynx or other suitable software. 11.2 I f the curve does not meet requirements, perform routine maintenance or reextract the standard curve (if necessary) and reanalyze. 11.3 For purposes o f accuracy when quantitating low levels o f analyte, it may be necessary to use the low end o f the calibration curve rather than the full range o f the standard curve. Example: when attempting to quantitate approximately 10 ppb o f analyte, generate a calibration curve consisting o f the standards from 5 ppb to 100 ppb rather than the full range o f the curve (5 ppb to 1000 ppb). This w ill reduce inaccuracy attributed to linear regression weighting o f high concentration standards. 3M Environmental Laboratory 3MEnvironmental Laboratory ETS-8-5.1 Analysis of Serum Extract Using ES/MS Page 4 of 9 Paqe 55 Page 55 3M Medical Department Study: T-6316.5 3M Medical Department Study: T6316.5 Analytical Study: FACT-TOX-013 Analytical Report: FACT T O ^ ^ K '^ 2095 LRN-U2 095 12.0 PROCEDURES_________________________________________________________________________ 12.1 A cquisition Set up 12.1.1 Click on start button in the Acquisition Control Panel. Set up a sample list. A ssign a filename using M O-DAY-last digit o f 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 Acquisition control panel and select SIR (Single Ion Recording) or MRM. Set Ionization Mode as appropriate and mass to 499 or other appropriate m asses. A full scan is usually collected along w ith the SIRs. Save acquisition method. If MS/MS instruments are em ployed, additional product ion fragmentation information may be collected. See Micromass M assLynx 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 o f extracted matrix standards and ends with a set o f extracted matrix standards. 12.1.4 Samples are analyzed with a continuing calibration check injected after every tenth sample. Solvent blanks should be analyzed periodically to monitor possible analyte carryover and are not considered samples but may be included as such. 12.2 U sing th e A utosam pler 12.2.1 Set up sample tray according to the sample list prepared in Section 12.1.1. 12.2.2 Set-up the HP1100/autosampler at the following conditions or at conditions the analyst considers appropriate for optimal response. Record actual conditions in the instrument logbook: 12.2.2.1 Sample size = 10 pL injection 12.2.2.2 Inject/sample = 1 12.2.2.3 Cycle time = 13.5 minutes 12.2.2.4 Solvent ramp = T im e 0.00 min. 8.50 min. 11.0 min. 12.0 min. MeOH 40% 90% 90% 40% 2.0 mM Ammonium acetate 60% 10% 10% 60% 12.2.2.5 Press the "Start" button. 12.3 Instrument Set-up 12.3.1 Refer to ETS-9-24.0 for more details. 12.3.2 Check the solvent level in reservoirs and refill if necessary. 3M Environmental Laboratory 3MEnvironmental Laboratory ETS-8-5.1 Analysis of Serum Extract Using ES/MS Page 5 of 9 Page 56 Page 56 3M Medical Department Study: T-6316.5 3M Medical Department Study: T6316.5 Analytical Study: FACT-TOX-013 Analytical Report: FACT TOS^ ^ K '^ 2095 LRN-U2095 12.3.3 Check the stainless steel capillary at the end o f the probe. U se an eyepiece to check the tip. The tip should be flat with no jagged edges. I f the tip is found to be unsatisfactory, disassem ble the probe and replace the stainless steel capillary. 12.3.4 Set HPLC pump to "On". Set the flow to 10 - 500 uL/min or as appropriate. Observe droplets com ing out o f the tip o f the probe. A llow to equilibrate for approximately 10 minutes. 12.3.5 Turn orr the nitrogen. A fine mist should be expelled with no nitrogen leaking around the tip o f the probe. Readjust the tip o f the probe if no m ist is observed. 12.3.6 The instrument uses these parameters at the follow ing settings. These settings may change in order to optimize the response: 12.3.6.1 Drying gas 250-400 liters/hour 12.3.6.2 ESI nebulizing gas 10-15 liters/hour 12.3.6.3 HPLC constant flow mode, flow rate 10 - 500 pL/min 12.3.6.4 Pressure <400 bar (This parameter is not set, it is a guide to ensure the HPLC is operating correctly.) 12.3.7 Carefully guide the probe into the opening. Insert probe until it w ill 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 w ith a copy taped into the instrument log. 12.3.9 U sing the cross-flow counter electrode in the ES/MS source is recommended for the analysis o f biological matrices. 12.3.10C lick on start button in the Acquisition Control Panel (this may vary among M assLynx versions, see appropriate MassLynx USER'S GUIDE). Press the start button. Ensure start and end sample number includes all samples to be analyzed. 13.0 Data Analysis and Calculations____________;_____________________________ 13.1 Calculations: 13.1.4 Calculate matrix spike percent recoveries using the follow ing equation: % Recovery = Observed Result - Background Result x 100 Expected Result 13.1.5 Calculate percent difference using the follow ing equation: % D ifference = Expected Cone. - Calculated Cone, x 100 Expected Cone. 13.1.6 Calculate actual concentration o f PFOS, or other fluorochemical, in matrix (pg/m L ): fag o f PFOS calc, from std. Curve x Dilution Factor) x 1 pg (Initial Volum e o f matrix (mLl + mL o f Surrogate Standards 1000 ng Final Volum e (mL) 3M Environmental Laboratory 3MEnvironmental Laboratory ETS-8-5.1 Analysis of Serum Extract Using ES/MS Page of 9 Page 57 Page 57 3M Medical Department Study: T-6316.5 3M Medical Department Study: T6316.5 Analytical Study: FACT-TOX-013 Analytical Report: FACT T O ^ ^ S ' ^ 2095 LRN-U2 095 14.0 Method Performance________________________________________________________ 14.1 M ethod Detection Limit (MDL) and Limit o f Quantitation (LOQ) are method, analyte, and matrix specific. Please see ETS-8-4.1, A ttachm ent B, for a listing o f 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 low est standard in the calibration curve 14.3 Calibration Curves 14.3.1 The r2value for the calibration curve must be 0.980 or better. 14.4 Matrix Spikes 14.4.1 Matrix spike percent recoveries are must be within 30% o f the spiked concentration. 14.5 Continuing Calibration Verifications , 14.5.1 Continuing calibration verification percent recoveries must be 30% o f the spiked concentration. 14.6 I f criteria listed in this method performance section isn't met, maintenance may be performed on the system and samples reanalyzed or other actions as determined by the analyst. Document all actions in the appropriate logbook. 14.7 I f data are to be reported when performance criteria have not been met, the data must be footnoted on tables and discussed in the text o f the report. 15.0 Pollution Prevention and Waste Management_______________________________ 15.1 Sample extract waste and flammable solvent is disposed in high BTU containers, and glass pipette waste is disposed in broken glass containers located in the laboratory. 16.0 R e c o r d s ____________________________________________________________________________ 16.1 Each page generated for a study must have the follow ing information included either in the header or hand written on the page: study or project number, acquisition method, integration method, sample name, extraction date, dilution factor (if applicable), and analyst. 16.2 Print the tune page, sample list, and acquisition method from MassLynx to include in the appropriate study folder. Copy these pages and tape into the instrument runlog. 16.3 Plot the calibration curve by linear regression, weighted 1/x, then print these graphs and store in the study folder. 16.4 Print data integration summary, integration method, and chromatograms, from M assLynx, and store in the study folder. 3M Environmental Laboratory 3MEnvironmental Laboratory ETS-8-S.1 Analysis of Serum Extract Using ES/MS Page 7 o f 9 Page 58 Page 58 3M Medical Department Study: T-6316.5 psws 1J 3M Medical Department Study: T6316.5 Analytical Study: FACT-TOX-013 Analytical Report: FACT TO^-^ f i ' ^ 2095 LRN-U2 095 16.5 Summarize data using suitable software (Excel 5.0) and store in the study folder, see A ttachm ent A for an example o f a summary spreadsheet. 16.6 Back up electronic data to appropriate medium. Record in study notebook the file name and location o f backup electronic data. 17.0 Tables. Diagrams. Flowcharts, and Validation Data______________________ 17.1 Attachment A: ETS-8-5.1 Data summary spreadsheet. 18.0 References__________________________________________________________________ 18.1 FACT-M -4.1, "Extraction o f Potassium Perfluorooctanesulfonate or Other Fluorochem ical compounds from Serum for Analysis Using HPLC-Electrospray/Mass Spectrometry 18.2 ETS-9-24.0, "Operation and Maintenance o f the Micromass Atmospheric Pressure Ionization/M ass Spectrometer Quattro II triple quadrupole System s" 18.3 The validation report associated with this method is ETS-8-4.0 & 5 .0 -V -l. 19.0 Affected Documents_________________________________________________________ 19.1 ETS-8-4.1, "Extraction o f Potassium Perfluorooctanesulfonate or Other Fluorochem ical Compounds from Serum for Analysis Using HPLC-Electrospray/Mass Spectrometry" 20.0 Revisions___________________ ________ '_________________________________________ R evision Number. 1 Reason For R evision Section 6.1.2 Clarification o f HP1100 system components. Section 11.1 Average o f two curves, not standard values, are used for plotting linear regression and added the 1/x weighting o f the curve. Section 12.2.2.4 Clarification o f solvent ramp. Section 17.1 Changed from attachment B to A. R evision D ate 04/02/99 3M Environmental Laboratory 3MEnvironmental Laboratory ETS-8-5.1 Analysis of Serum Extract Using ES/MS Page 8 of 9 Page 59 Page 59 3M Medical Department Study: T-6316.5 3M Medical Department Study: T6316.5 f Analytical Study: FACT-TOX-013 Analytical Report: FACT TOX^ ^ S ' ^ 2095 LRN-U2 095 Laboratory Study # Study: Test Material: Matrix/Final Solvent: Method/Revision: - Analytical Equipment System Number: Instrument Software/Version: Filename: R-Squared Value: Slope: Y Intercept: Date o f Extraction/Analyst: Date o f Analysis/Analyst: Group Dose Sample# Concentration ug/mL Initial Vol. mL Dilution Factor Final Cone. ug/mL Slope: Taken from linear regression equation. Group/Dose: Taken from the study folder. Sample#: Taken from the study folder. Concentration (ug/mL): Taken from the MassLynx integration summary. Initial Volume (mL): Taken from the study folder. Dilution Factor: Taken from the study folder. Final Cone. (ug/mL): Calculated by dividing the initial volume from the concentration Attachment A: Summary Spreadsheet ETS-8-5.1 Analysis of Serum Extract Using ES/MS 3M Environmental Laboratory 3MEnvironmental Laboratory Page 9 o f 9 Page 60 Page 60 3M Medical Department Study: T-6316.5 , 3M Medical Department Study: T6316.5 Analytical Study: FACT-TOX-013 LRN-U2095 Analytical Report: FACT TOX-013 LRN-U2095 Appendix D: Data Summary Tables Table 12. Reported Fluorochemical Levels in Sera Analyses in Study FACT TOX-013 Dosage Group Specimen ID PFOS (pg/mL) 1 10097F 0.0394 1 10105F 0.0181 1 10106F 0.0258 1 10107F 0.0343 1 10108F 0.0253 1 9922M* 0.0115 1 9930M* 0.0134 1 9931M 0.00725 1 9932M 0.0162 i 9933M* 0.0156 II 10121F 9.62 II 10126F 19.8 II 10136F 5.96 II 10140F 6.27 II 10142F 13.1 II 9961M * 34.8 II 9964M 30.4 II 9965M * 74.9 II 9967M 25.1 II 9970M * 38.9 III 10155F* 87.8 III 10156F 76.1 III 10164F 49.6 III 10172F 68.4 III 10177F 42.2 III 9997M 108 III 9999M * 178 III 10001M 94.9 III 10002M 113 III 10004M 130 IV 10187F 89.5 IV 10194F 73.4 IV 10203F 126 IV 10211F 99.7 IV 10214F 98.3 IV 10019M 302 IV 10024M* 477 IV 10029M* 296 IV 10033M 272 IV 10034M 249 * = Tentative values, initial volum e w as <0.5 mL PFOSA (pg/mL) <LOQ (4.79 ppb) <LOQ (4.79 ppb) <LOQ (4.79 ppb) <LOQ (4.79 ppb) <LQ (4.79 ppb) <LOQ (4.79 ppb) <LOQ (4.79 ppb) <LOQ (4.79 ppb) <LOQ (4.79 ppb) <LOQ (4.79 ppb) 0.0682 0.112 0.0663 0.0507 0.0665 0.0962 0.188 0.114 0.147 0.165 0.328 0.352 0.265 0.325 0.335 0.574 0.579 0.480 0.393 0.465 0.461 0.576 0.651 0.670 0.569 0.613 0.553 0.610 0.804 0.637 PFOSAA (pg/mL) <LOQ (20.5 ppb) <LOQ (20.5 ppb) <LOQ (20.5 ppb) <LOQ (20.5 ppb) <LOQ (20.5 ppb) <LOQ (20.5 ppb) <LOQ (20.5 ppb) <LOQ (20.5 ppb) <LOQ (20.5 ppb) <LOQ (20.5 ppb) 1.59 4.55 1.18 0.690 2.09 1.40 5.86 1.86 1.26 5.55 9.9 6.91 4.66 8.17 4.58 11.8 18.7 12.1 10.4 14.9 8.00 10.6 19.0 10.2 12.3 22.5 40.5 28.8 24.5 31.4 EtFOSE-OH (pg/mL) M566 (pg/mL) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ <36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (24.9 ppb) <LOQ (24.9 ppb) <LOQ (24.9 ppb) <LOQ (24.9 ppb) <LOQ (24.9 ppb) <LOQ (24.9 ppb) <LOQ (24.9 ppb) <LOQ (24.9 ppb) <LOQ (24.9 ppb) <LOQ (24.9 ppb) 1.86 4.19 0.952 1.15 2.45 4.69 5.18 4.54 3.44 6.11 43.3 22.3 18.0 17.0 17.9 29.1 73.6 25.1 38.1 37.8 39.0 25.6 39.6 28.8 33.8 71.3 102 94.9 90.9 56.7 3M Environmental Laboratory 3M Environmental Laboratory Page 61 Page 61 3M Medical Department Study: T-6316.5 3M Medical Department Study: T6316.5 Analytical Study: FACT-TOX-013 -U2095 Analytical Report: FACT TOX-013 LRN-U2095 Table 12. Reported Fluorochemical Levels in Sera Analyses in Study FACT TOX-013 (continued) Dosage Group Specimen ID PFOS (pg/mL) V NR NR V NR NR V NR NR V NR NR V NR NR V 10042M 238 V 10044M 235 V 1004SM 326 V 10051M 162 V 10054M 182 NR * Sample not received or reported * = Tentative values, initial volume was <0.5 mL PFOSA (pg/mL) NR NR NR NR NR 0.791 0.972 0.897 0.574 0.669 PFOSAA (pg/mL.) NR NR NR NR NR 25.2 20.7 26.8 14.0 15.8 EtFOSE-OH (pgftnL) NR NR NR NR NR <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) M656 (pg/mL) NR NR NR NR NR 62.4 55.6 93.8 30.7 55.5 Table 13. Reported Fluorochemical Levels in Liver Analyses in Study FACT TOX-013 Dosage Group I I 1 1 1 1 1 1 I 1 1 1 i 1 1 II il il il il II il II II 11 II (I It II II Specimen ID 10097F 10105F 10106F 10107F 10108F 9922M 9930M 9931M 9932M 9933M 10097M 10105M 10106M 10107M 10108M 10121F 10126F 10136F 10140F 10142F 9961M 9964M 9965M 9967M 9970M 10121M 10126M 10136M 10140M 10142M PFOS (pg/g) 0.149 <LOQ 0.121 <LOQ <LOQ 0.585 0.816 0.836 1.04 1.01 0.281 0.242 0.226 0.221 0.251 25.1 22.9 39.6 23.7 22.1 116 102 89.9 80.7 87.3 54.7 67.8 53.7 28.0 71.5 PFOSA (pg/g) <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <1.00 <LOQ <10Q <LOQ <LOQ <LOQ <LOQ <LOQ 0.514 0.708 1.40 0.601 0.508 4.49 4.10 2.88 3.88 5.42 1.90 2.65 2.35 1.22 2.06 PFOSAA (pg/g) <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <UOQ <LOQ <LOQ <LOQ <LOQ 2.68 4.34 5.01 2.67 2.67 11.0 15.6 9.79 6.62 10.4 5.87 14.6 9.44 2.82 6.55 M556(pg/g) <LOQ <LOQ <UOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ 1.19 1.75 2.86 1.55 1.41 12.8 10.2 11.6 8.95 11.6 5.39 7.75 4.64 3.27 4.58 3M Environmental Laboratory 3MEnvironmental Laboratory Page 62 Page 62 3M Medical Department Study: T-6316.5 : 3M Medical Department Study: T6316.5 Analytical Study: FACT-TOX-013 LRN-U2095 Analytical Report: FACT TOX-013 LRN-U2095 Table 13. Reported Fluorochemical Levels In Liver Analyses in Study FACT TOX-013 (continued) Doug* Group III III III III III III III III III III III III III III III IV IV IV IV IV IV IV IV IV IV IV IV IV IV IV V V V V V Spadnw n ID 10155F 10156F 10164F 10172F 10177F 9997M 9999M 10001M 10002M 10004M 10155M 10156M 10184M 10172M 10177M 10187F 10194F 10203F 10211F 10214F 10019M 10024M 10029M 10033M 10034M 10187M 10194M 10203M 10211M 10214M 10042M 10044M 1004SM 10051M 10054M p f o s (pg/g) 102 130 179 119 105 415 234 498 257 386 89.0 219 203 188 164 240 344 255 264 831 791 556 781 556 226 277 448 457 344 1218 1356 1132 1063 1054 PFOSA (pg/g) 2.22 2.24 1.94 2.17 2.88 10.8 9.41 8.67 8.29 8.41 5.11 6.14 6.26 6.20 6.91 2.80 4.06 3.14 3.39 4.56 12.8 11.0 11.2 12.6 11.0 6.36 9.96 9.93 11.4 8.11 16.4 13.0 10.9 9.80 | 10.8 PFOSAAQjg/g) 15.6 20.2 22.7 11.9 20.0 73.5 28.6 85.2 34.2 64.9 12.0 27.3 29.4 33.7 17.1 31.1 51.5 49.5 46.6 51.4 122 148 86.2 129 135 27.4 45.5 76.0 56.2 60.0 188 206 150 157 165 M 5S6(p(ft) 7.17 7.64 8.41 5.87 8.16 63.5 39.3 66.4 38.0 54.6 11.5 33.3 43.1 29.9 31.4 19.8 26.8 26.6 27.5 29.3 84.5 97.5 78.2 117 82.2 39.9 39.5 67.8 65.4 64.5 128 152 133 118 161 3M Environmental Laboratory 3MEnvironmental Laboratory Page 63 Page 63 3M Medical Department Study: T-6316.5 3M Medical Department Study: T6316.5 Appendix E: Data Spreadsheets Analytical Study: FACT-TOX-013 LRN-U2095 Analytical Report: FACT TOX-013 LRN-U2095 3M Environmental Laboratory 3MEnvironmental Laboratory Page 64 Page 64 $MMedica| Department Sturdy: .T-6316.5 w 3 M3 C (3o333(IHD-T1coiC)r 0tn> I0(Q*t)v) <Ti in 3MEnvironmental Laboratory F A C T -T O X -013 A rus#418-009 Argus 418-009, Two-Geaeralioo Reproductif Study of EtFOSE-OH in Produci NumbcrfTest Substance): ErFOSE-OH (T-6316.5) Matrix: R a Seront BTS-8-4.1& ETS-8-5.1 Auletica! E Davey 070799, Soup 020199 Instrument Software/Verekm: Mu$Lyox3J flau ste R-Squared Vaiue: See Haiag to theright SccAjtachmests , Slope: See Attachments Y-loMcepC Date* of Extraction/Aialyst: See Attachments (QT12/99 RWW Dates of Analysb/AaalysL UY14/99, 100099,10/22/99.03/16/00 MMH/1AS DaeofDasaRedoctton/Analya: m m , 10/21/99,10/25/99,03/17/30.03/23M0 MMH/IAS Sample Data RAT SERA n Group Dose Sumpia# l-M -l Val I, PFOS Croc. sfaL Crocantrutfro o f PFOS 0ta L e r % R e c M ero PFOS uasteL RSD S tr L D tv . MS/M SDRPD Method Bile IOI29-H20Bik-l 101294120 Blk-2 1 l 0.00 <LOQ< 2 4 5 R b) 0.00 <LOQ <LOO NA Matrix Bik RRS10l29-SoaBik-l l o.oo <LOQ< 24.8 ppb) RBS10129-SoaBtk-2 1 0.00 <LOQ QC-l00ppb RBSI0129-MS-1 1 23 i 93 <UX) NA RBS 10129-MSD-l 1 213 86 90 8 G ivapl 10097F 0.70 34.4 0.0394 Control 10105F 0.70 1S.8 O.OmgAgAday 10106F O.S0 16.1 0.0181 0.0258 Omg/mL 10107F 0.60 25.7 10108F 0.60 19.0 0.0343 0.0253 0.0286 29.2 0.00834 9922M** 9930M** 9931M 0.40 0.40 050 5.74 6.71 4.69 0.0115 0.0134 O.O752 9932M 9933M " 0.50 0.40 10.1 7.78 0.0162 0.0156 0.0129 27.4 0.00352 Group 2 10121F 0.70 336 9.62 1 mg/kgAlay 10126F 0.50 495 02tng/mL 10136F 1.00 297 19.8 5.96 10140F i.no 313 I0142F 0.60 394 627 13.1 52.4 10.9 5.74 9961M** 9964M 9965M** 0.40 0.50 0.20 347 379 374 34.8 30.4 74.9 9967M 9970M** 050 0.40 313 389 25.1 38.9 48.4 4 05 19.7 G ro u p ? 10155F** 0.40 434 87.8 5mg/kg/day 10156F 0.60 572 l.Omg/ml. J0J64F 0.70 433 10172P 0.60 515 76.1 49.6 68.4 28.9 10177F 0.70 369 9997M 0.70 379 42.2 108 64.8 18.8 9999M** 10001M 100Q2M 10004M 0.30 0.70 0.60 O.SO 259 331 340 324 178 94.9 113 130 25.8 125 32.2 Group 4 10I87F 0.60 686 89.5 UMhtif/kgAky 10194F 0.80 7 73.4 2.0mg/mL 10203F* 0.70 1100 126 102HF* 0.80 995 99.7 19.6 10214F 0.70 842 98.3 97.4 19.1 I0019M 0.60 446 302 10024M** ioo2m ** 0.30 0.40 360 296 477 296 10033M 10034M 0.50 0.50 340 311 272 249 28.4 319 90.6 Group? NR NR NR NR 15.0mgAgAi*y NR NR NR NR 3.mg/roL NR NR NR NR NR NR NR NR M l NR NR NR NR NR NR 10042M 10044M 1045M 10051M 1054M 0.80 0.80 0.50 1.00 0.90 475 469 405 401 404 238 235 326 162 182 27.8 229 63.7 Limit of Onantation (LOQ): PFOS * 5 55 ng/mL, PFOSA =4.79Rib. PF5AA - 2 0 5 ppb.ElF O SE -362 ppb.PFOSEA 18.2 ppb,M 556-19 2 ppb Correction factors not applicable for MS/MSD QC data Tentative value, PFOS concentration w js am within the ringe o f the c u m . it's approximately 15 above tbc highest standard. LAC 03/24/00 D ae Entend/By. 0VI1/00,03/23/00,03/24/00 LAC Date Verified/ By. Purity Eucred/VcriOcd: 09/13/00 LAC, hoj 9/13/00 ** Tentative values, initial voiumcbdowO-SrnL. LAC08/31/00 Analytical Study: FACT-TOX-013 11 1 l Rn -U2095 OJ 3! faKD HOP M *0(0D) ft 3fPrl>t nw % G>-3\ O<HJJ\ 01 &> M *< OfH&>t i--1 >(oHDt0 rt hr] FH>o dI xO1-3 IO ! oo KO H (J1 OJ Page 65 3M^Medical^Department Study: T-6316.5 w 3 M P< hl-i1- o 3 3 (D 3 rr i) V ItDr o H (D ft o *< 0) IQ (D (Ti O'. ers-84.1 3M Environmental Laboratory I ' " 1 1 1 .....! ' 1 ' I FACT-TOX-OI3 Argus# 418-409 Study: Argtu 418-009. Two-GererailoflReproduction Study of EtFOSE-OH in Rau Prodart Namb(Test Sabstaoce): EtFOSB-H (T-63165) Matrix: Methodfitevisfac RatSemm &TS-4-4.I&ETS-8-5.1 Analytical Equipment SystemNumber l>avey0TO799. Soup 020199 Instrument SoftwareA'ertion: Filename: M tssLytu33 Seelisting to the right R'SquxcdV ato: See Attachment* Slope S Attachment* Y-Iatocept SeeAnactmmu Datesof Extraokw/Analyst UY12A9 RWW Dales o f AndyotfAnalyst: V W .10/20/99.1(V22/.03/LM MMHAAS L hieoiD aa Reductioo'AndySt 10/15/9*. m m , 10/25/99, 03/176)0.03/2300 MMH/lAS SaapleDat* RATSERA F#____________ Gmy Deae Method Blk Matrix B k QC-IOOr * SM fkt 10129-H2O Bk-1 10129-H2O Bflt-2 RBS10129>SeraBfc-l RBSW129-Sn Bk-2 RBS10129-MS-1 RBS10I29-MSD-1 laWal VaL L 1 111 11 PFOSA Cm c 0r.W00* 1.57 OOO 122507 215 C a s s a ta tien f PFOSA a jtte L o r % Rc <LOQ <LOO <OQ <LOO 89% 87 M a PFOS ML <LOO <LOO 88% RSD SM. Der. M S/M S D R P D NA NA 3 G m pl 10097F 0.70 1.46 <LOQ Conard 0QOTngtfcg&y mgAnf. 10105F 10106F 10107F 10108F 9922M* 993<W- 0.70 0.50 00.6600 040 040 128 1.15 1.44 1.14 2.41 1.89 <LOQ tLOQ LOQ <LOO <LQ <LOQ <LOO NA NA 9931M 050 1.44 <LOQ 9932M 050 1.41 <LQ NA 9933M 040 133 <LOQ <LOQ NA G raop 1mg&gftxy 0. 2 n t j t o i - 10121F 10126F 10136F 10140F 10142F 9961M** 9964M 9965M" 9967M 9970M* 0.70 011.5.00000 060 040 002500 050 0.40 47.7 S6.0 66.3 50.7 39.9 38.5 94.2 22.7 7636..62 00..0161822 0.0663 0.0507 0.0665 03)962 0.188 0.114 0147 0165 0.0727 0.142 31.7 0.0231 26.4 0.0376 G reap3 Smg/kgAtoy I.OtngAnLr 10155F 10156F 10164F 10172F 10177F 9997M 9999M I0001M 0.40 0.60 0.70 0.60 0.70 070 030 070 211311 185 195 235 402 174 336 0328 0352 0.265 0325 0.335 0574 0579 0.480 0521 103 0.0331 10002M 10004M 060 0.50 236 233 0393 0.465 0.498 15.8 0.0786 Group 4 2.010X1ag/kgtfay ogfaL 10187F 10194F 10203F IG ltlF I0214P 10019M 10024M** 10029M** 10033M 10034M 060 080 070 0.80 070 .6Q 050 0.40 050 050 276 461 456 536 398 368 166 244 402 318 0461 0.576 0651 0670 0569 0613 0553 0610 0804 0.637 0565 0.643 14.1 0.0826 14.8 0.0951 Groop S 15.0n/fcK/dsy NR NR NR NR NR NR NR NR 3.0mgAoL NR NR NR NR NR NR 1042M I0044M NR NR oso 080 NR NR 633 778 NR NR 0.791 0972 NR NR NR 1004M 10051M 10054M 015.000 090 448 57* 602 0897 0.374 0669 0.780 208 O.J63 Limit of Quantitilioo (LOQ): PFOS = S55 nj/mL, PFOSA e 4.79 ppt>. PFOSAA * 205 ppb, EtFOSE 36.2 ppb, PlNR > Sample am received oof rpond. Correction facto not ppticafcie far MS/MSDQC data Tentativevalue,cwweotraiioowas4witliiBlbet)feoflltecotve,it's^imini*ieiylS**ove(Jkhighen sondanl- LAC 03/2400 faeEntsetf&y: QA1*, 05/23/00,05/14LAC Date Verified/Uy: *Tentative vafces, Initial volume below0.5 aL . LAC08/31A Analytical Study: FACT-TOX-013 ....1 LRN-U295 LU s s QCD* H O 0> M O fl) d 0) Krt 3 CD Prr CO rr ch `C hCT3t UJ H(Ti (Tl & 0) H Krr H O P> H d CD O K rr *3 > O F 1-3 HI cil O X O ovo en Page 66 ov oo xB H2 O- M < Ph __ -o 3 GO cd- o: c <T 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 vo <tuo fin I* ;8?a 4I 1 I. =aJ W l S I l I l 'i I * .sfJz!ililf l"^*' 4 5 S SS SS If! 1i 8 5 ! fi IS# fl Ufi!mu11 * M3 ^ 4 II fts fin "1 R rJ SS 5S i s Sd s Q s s Si i 4I S s f^ ^ *s*jg g23 2 ^ S3 5 O 2 2 2 2 m 2 3 3^ 33 3S2h ggsgg N pjoo<5oe S S S S 2 si |!88o'88 ss s i i s i I ! 13 2 a s s i s 5 S S 3 S .>* 8RS p*.opi--.<=*:"i S ? ?S-'3 S X H5$ S 3v g g g g g S R K8 do* I ( V* -- -- -- e e s s s 5 ? ^?? S ^ 8--;8--;S '' 8 8 383 8 8 ^8>8 i S i i g g g g g il I B ! ; JS a 11il Itili lii II1SI11 Ta*aa? SS jL|Lhb s i i l i s 3 s S cl elb,(k I3 I2 ii S l| I 2 S S S i s * s ? 2 2 2 2 2 P p 2S22 fsci&sfe:S:& 3oiSS ggggg ill Mm g1%s lifdt il! il! 2 if! 'i | i g! I l> ili ir> vo co*8* VO ITM GO +-> o <D 9*"* t<s3 ' O<Dh Q 'co5TM * <3D 3M Environmental Laboratory Page 67 b s 2 o <3 G <1 3 1R3 s R .Ok "3 3M Medical Department Study: T-6316.5 3M Medical Department Study: T6316.5 Analytical Study: FACT-TOX-013 LRN-U2095 Analytical Report: FACT TOX-013 LRN-U2095 pa* ~! ss 1 1 $5 NA HA 55 i i Z S i ss gg 111111 11g iJ 1!ISH3it| ih ! J s111 r}5 Pi II II U mu mu mu mu mu mu mu mu ggggg mu Ii sI "9 a I is 1 IpI In s! 3 H1 8 33 8 8 a s 8 89 $-- OS 8 s $ 3$ m u s^ g^ sg^s 2 8 8 8 ^ SJ 'rnl<! 3 3 = = ! = = g g g g g r.-^ o*3^ rm- l l i| iLsjjpil m i misfit -- -- -- 9 0 0 0 3 3 33 3 o 3 3 2 3 2 !" t3 3 8 2 82 eoe ggggg || 3 | S illiUfiiiiis i Is- El a| 11 tal 11IS i |iii iilil Ilj1* 2 CaOtao at to u,a.& th c Vi e V si iSMolr;2 2 2 2 31 2 giiSi m ! 1 ! 1 1 piss k L u>b>b siili iliii ggggg ftp Ball gill u if! if! l}!" !I ij>|l,i- JPt!liii flllHlWllliS 111! a t8 i e> if! n " lllff illii 3M Environmental Laboratory 3MEnvironmental Laboratory P^rr# 6ft Page 68 3M Medical Department Study: T-6316.5 , 3M Medical Department Study: T6316.5 Analytical Study: FACT-TOX-013 LRN-U2095 Analytical Report: FACT TOX-013 LRN-U2095 t 3M Environmental Laboratory 3MEnvironmental Laboratory Page 69 Page 69 3M Medical Department Study: T-6316.5 imw : 3M Medical Department Study: T6316.5 Analytical Study: FACT-TOX-013 LRN-U2095 Analytical Report: FACT TOX-013 LRN-U2095 Appendix F: Example Calculations Formula Used for Sera Analyses in Study FACT TOX-013 AR (ng/mL) x DF x SC x FV (mL) x 1.0 ng x PC = Reported Concentration (pg/mL) EV (m L) lOOIFng Calculation Used for Group 4, Anim al ID 10033M 340 ng/mL x 500 x 0.9275 x 1 mL x 1.0 pg x 0.864 = 272 pg/mL 0.5'mU 1000"Sg~ AR-- Analytical result from MassLynx summary DF-- Dilution factor SC-- PFOS salt correction constant (0.9275) FV-- Final extract volume (1.0 mL unless otherwise noted) EV-- Volume o f sera extracted PC-- PFOS purity correction factor (86.4%) 3M Environmental Laboratory 3MEnvironmental Laboratory Page 70 Page 70 3M Medical Department Study: T-6316.5 3M Medical Department Study: T-6316.5 Analytical Study: FACT-TOX-013 LRN-U2095 Analytical Study: FACT TOX-013 LRN-U2095 Appendix G: Contract Lab Report This appendix includes the following contract laboratory report: B a tte lle M e m o r ia l In s titu te , Study Number: N003604-D, 2 (N-Ethylfluorooctanesulfonamido)-ethano! in Two Generation Rat Reproduction, (65 pages) 3M Environmental Laboratory 3MEnvironmental Laboratory Page 135 Page 71 3M Medical Department Study: T-6316.5 Analytical Study: FACT-TOX-013 LRN-U2095 BIOLOGICAL SAMPLE ANALYSIS Battelle Study Number: N003604-D 3M Environmental Laboratory Study Number: FACT 060998.1 ... P u t t i n g T e c h n o l o g y T o W o r k FINAL REPORT 2 (N-Ethylfluorooctanesulfonamido)-ethanol in Two Generation Rat Reproduction SPONSOR 3M Toxicology Services 3M Center Building 220-2E-02 St. Paul, MN 55144 Testing Facility Battelle Memorial Institute 505 King Avenue Columbus, Ohio 43201-2693 Prepared Bv Patrick L. South, B.S. 3MEnvironmental Laboratory Page 72 3M Medical Department Study: T-6316.5 Analytical Study: FACT-TOX-013 LRN-U2095 Battelle Study Number: N003604-D 3M Environmental Laboratoiy Study Number: FACT 060998.1 FINAL REPORT 2 (N-EthylfluorooctanesuIfonamido)-ethanoI in Two Generation Rat Reproduction CV Joi/a. Andre, Ph.D. Battelle Principal Investigator Battelle Senior Program Director 'Lac ( 3MEnvironmental Laboratory it Page 73 3M Medical Department Study: T-6316.5 Analytical Study: FACT-TOX-013 LRN-U2095 Battelle Study Number: N003604-D 3M Environmental Laboratory Study Number: FACT 060998.1 2 (N-Ethylfluorooctanesulfonamido)-ethanol in Two Generation Rat Reproduction EXECUTIVE SUMMARY Rat liver samples sent to Battelle by 3M Environmental Technology and Services were analyzed by the previously validated method "Method for Analysis of Potassium Perfluorooctanesulfonate (PFOS) in Rat Liver by LC/MS/MS". Samples were extracted and analyzed by High-Performance Liquid Chromatography Mass Spectroscopy (LC/MS/MS) for PFOS, M-556, PFOSAA, and PFOSA content only. Related fluorochemicals mentioned in the analytical method were not investigated. The results for the concentration determinations in the liver samples from this study are attached as appendices to this report. Concentrations are reported as mass of analyte (fig) per gram of liver tissue extracted. 3MEnvironmental Laboratory m Page 74 3M Medical Department Study: T-6316.5 Analytical Study: FACT-TOX-013 LRN-U2095 Battelle Study Number: N003604-D 3M Environmental Laboratory Study Number: FACT 060998.1 QUALITY ASSURANCE STATEMENT This study was inspected by the Quality Assurance Unit and reports were submitted to the task leader, study director, and associated management as follows: Date Reported to Battelle Task Phase Inspected Inspection Date Leader/ Battelle ___________________________________________________ M anagem ent Date Reported to Offsite Study Director/ Management_______ Sample weights Sample homogenization Extraction Sample analysis Audit study file Audit final report Audit study file Audit final report 10/12/1999 10/12/1999 10/13/1999 10/13/1999 12/9/1999 12/9/1999 2/21/2001 2/21/2001 11/1/1999 11/1/1999 11/1/1999 11/1/1999 12/9/1999 12/9/1999 2/21/2001 2/21/2001 3/30/01 3/30/01 3/30/01 3/30/01 3/30/01 3/30/01 3/30/01 3/30/01 Hality Assurance Unit ^ attelle Memorial Institute Date 3MEnvironmental Laboratory IV Page 75 3M Medical Department Study: T-6316.5 Analytical Study: FACT-TOX-013 LRN-U2095 Battelle Study Number: N003604-D 3M Environmental Laboratory Study Number: FACT 060998.1 GOOD LABORATORY PRACTICES COMPLIANCE STATEMENT Study Title. 2 (N-Ethylfluorooctanesulfonamido)-ethanol in Two Generation Rat Reproduction This study was conducted in compliance with the Food and Drug Administration's Good Laboratory Practice Regulations (21 CFR 58), with the exception that the mass spectrometry data for the liver samples was collected and processed with the MassLynx software system (version 3.1), which was not fully validated. The study was listed on Battelle's Master List o f regulated studies. Battelle Principal Investigator Marvin T. Case, DVM, Ph.D. Study Director 3MEnvironmental Laboratory V Page 76 3M Medical Department Study: T-6316.5 Analytical Study: FACT-TOX-013 LRN-U2095 Battelle Study Number: N003604-D 3M Environmental Laboratory Study Number: FACT 060998.1 Table of Contents Page Executive Summary................................................................................................................................................iii Quality Assurance Statement................................................................................................................................ iv Compliance Statement........................................................................................................................................... v Table of Contents...................................................................................................................................................vi 1.0 Introduction............................................................................................................................................... 1 2.0 Reference Substances............................................................................................................................... 1 3.0 Receipt of Samples................................................................................................................................... 1 4.0 Analysis of Samples................................................................................................................................. 1 4.1 Summary of Method.................................................................................................................... 1 4.2 Results......................................................................................................................................... 3 4.2.1 Quality Control..............................................................................................................3 4.2.2 Sample Results............................................................................................................. 3 5.0 Conclusions................................................................................................................................................3 6.0 Acknowledgements................................................................................................................................... 4 7.0 Specimen Storage and Record Archives.................................................................................................. 4 List of Tables Table 1. Example of Instrument Parameters Used to Analyze Samples.............................................................2 Appendix A (Results) Summary Results for Rat liver Sample Analysis................................................................................................. A-l Appendix B (Daily Acceptance Criteria Summary) Daily Acceptance Criteria Summary....................................................................................................................B-l Appendix C (Sample Inventory List) Sample Inventory List............................................................................................................................................C-l Appendix D (Chromatograms) Representative Chromatograms............................................................................................................................D -l Appendix E (Protocol, Amendments, and Deviations) Protocol, Amendments, and Deviations.................................................................................................................E-l Appendix F (PFOS Purity Report) PFOS Purity Report...............................................................................................................................................F-l 3MEnvironmental Laboratory VI Page 77 3M Medical Department Study: T-6316.5 Analytical Study: FACT-TOX-013 LRN-U2095 Battelle Study Number: N003604-D 3M Environmental Laboratory Study Number: FACT 060998.1 1.0 Introduction This report presents a description of the method used to analyze PFOS, M-556, PFOSAA, and PFOSA in rat liver samples from 3M Study Number FACT 060998.1 (TOX-013) and the results from this analysis. See Appendix E for a copy of the study protocol, amendments, and protocol deviation reports). 2.0 Reference Substances The analytical reference substances for this study were supplied by 3M. The following lot number or tracking number designations apply: PFOS (lot 171), M-556 (TN-A-2203), PFOSAA (TN-A-1283) and PFOSA (L-15709). Note that based on information supplied to Battelle from 3M, PFOS has two equivalent names. The name appearing on the Material Safety Data Sheet and bottle label is potassium perfluoroalkyl sulfonate. The name more commonly used by 3M in analytical methods and correspondence is potassium perfluorooctanesulfonate. The latter name will be used in this report. See Appendix F for purity data supplied by 3M to Battelle. The reference substances were stored at room temperature. The surrogate standard was 1H,1H,2H,2H-Perfluorooctane sulfonic acid, lot number 59909, supplied by ICN. The surrogate standard was stored at room temperature. 3.0 Receipt of Samples Samples were received frozen and intact at Battelle, from 3M Environmental Technology and Services, in one batch on October 6,1999. Samples were generated by Argus Research under protocol number 418-009. See Appendix C for a copy of the inventory list. The samples were stored at approximately -20C. 4.0 Analysis of Samples 4.1 Summary of Method Samples were analyzed by a previously validated method (Battelle study number N003604-A). The current version of the method is attached to this report in Appendix E. Samples were analyzed by LC/MS/MS, and an example of the instrument parameters is listed in Table 1. Note that only PFOS, M-556, PFOSAA, and PFOSA (and the surrogate) were quantitated. The other related fluorochemicals, although present in the stock solutions, were not monitored. Quadratic regressions weighted 1/x were used to construct the calibration curves. 3MEnvironmental Laboratory Page 78 3M Medical Department Study: T-6316.5 Analytical Study: FACT-TOX-013 LRN-U2095 Battelle Study Number: N003604-D 3M Environmental Laboratory Study Number: FACT 060998.1 Table 1. Example of Instrument Parameters Used to Analyze Samples LC/MS/MS System Autosampler HPLC pumps VIass spectrometer Analytical column Mobile phase components Gradient profile Injection volume Flow Column temp HPLC pressure MS source Dcsolvation as Nebulizer as Source block temp Desolvation temp Cone voltage Collision energy Collision gas Multiplier Resolution Ions monitored Total run time Approximate retention times: Make: Gilson Model: 234 Make: Gilson Models: 305 and 306 Make: Micromass______ Model: Quattro LC with Z-spray source Keystone Betasil C 18,5 urn, 2 x 50 mm, Part No. 055-701 -2 Component A: Ammonium acetate(2mM):methanol, 60:40, v:v Component B: Ammonium acetate(2mM):methanol, 5:95, v:v Time, min %B Flow. mL/min 0 0 0.3 1 0 0.3 4.5 100 0.3 6 100 0.3 6.1 100 0.6 8.5 100 0.6 9 0 0.3 11 0 0.3 10 UL______________________________________ LC column flow at start split to 20 pL/min into the MS Ambient_______________________________________ Approximately 840 psi at gradient start_____________ Electrospray, Negative Ion_______________________ Nitrogen at ~575 L/hr___________________________ Nitrogen at ~80 L/hr_____________________________ 140C__________________________________________ 250C__________________________________________ 70 V for SS, PFOS 20 V for M-556, PFOSAA, PFOSA________________ 40 eV__________________ _______________________ Argon, gas cell, at ~2.5xl0'3 mb 650 V__________________________________________ 12,0 for MSI; 10,0 for MS2_______________________ 427> 81 MRM transition for SS 499>99 MRM transition for PFOS 556>78 MRM transition for M-556 584> 169 MRM transition for PFOSAA 498>78 MRM transition for PFOSA_____ _________ 11 minutes_____________________________________ SS: 4 min PFOS: 4.2 min M-556: 4.4 PFOSAA: 4.5 PFOSA: 5 2 3MEnvironmental Laboratory Page 79 3M, Medical Department Study: T-6316.5 Analytical Study: FACT-TOX-013 LRN-U2095 4.2 Results Battelle Study Number: N003604-D 3M Environmental Laboratory Study Number: FACT 060998.1 4.2.1 Quality Control System suitability acceptance criteria were established during the method validation and are included in Appendix E, Section IX Acceptance Criteria. Relevant statistics from each sample set are provided in Appendix B. Representative chromatograms are given in Appendix D. 4.2.2 Sample Results The results of the sample analyses as well as a method detection limit determination are presented in Appendix A. All liver samples were initially extracted as undiluted homogenates. After the data were reviewed, dilutions of the homogenates were performed in order to bring analyte concentrations within the calibration range. The first analysis that provided acceptable data for an analyte was used in reporting. Four extraction sets were required to provide data for each analyte. The limit of quantitation is defined as the concentration of the lowest standard which meets acceptance criteria for accuracy (25% RE; see Appendix E for definition). The notation BLOQ denotes "Below Limit of Quantitation" for samples that had concentrations lower than the theoretical concentration for the 0.13 pg/g calibration standard. The notation ALOQ denotes "Above Limit of Quantitation" for samples that had concentrations higher than the theoretical concentration for the 13 pg/g calibration standard. Samples that were initially ALOQ were diluted with blank liver homogenate and re-extracted. Samples that were expected to be ALOQ were first diluted with blank liver homogenate before extraction. The "Corrected PFOS Cone" presented in the results tables is the concentration found for the diluted sample multiplied by its dilution factor (final volume + sample homogenate volume). The method detection limit (MDL) of PFOS was calculated in Battelle study N003296F to be0.0173 pg/g from the analysis of 7 replicate preparations of 0.13 pg/g calibration standard. The MDL was calculated by multiplying the standard deviation of the found concentrations of the 7 reps by 3.143; the Signal-to-Noise (S/N) ratio was calculated by dividing the mean found concentration of the 7 reps by their standard deviation. The method of MDL determination was provided by the Sponsor. 5.0 Conclusions All analyses met acceptance criteria unless otherwise noted. 3MEnvironmental Laboratory 3 Page 80 3M Medical Department Study: T-6316.5 Analytical Study: FACT-TOX-013 LRN-U2095 Battelle Study Number: N003604-D 3M Environmental Laboratory Study Number: FACT 060998.1 6.0 Acknowledgements Acknowledgement of principal contributors participating in the performance of this study at Battelle is presented in the following list. Participant Title Jon C. Andre, Ph.D. Richard W. Slauter, Ph.D., D.A.B.T. Patrick L. South, B.S. Gerke H. van der Zwaag, M.S. Battelle Principal Investigator Senior Program Director Mass spectroscopist Sample preparation chemist 7.0 Specimen Storage and Record Archives See Appendix E, protocol amendment 3 for records archival information. All residual liver samples, extracts, and unused test article will be disposed of or returned to the Sponsor as directed by the Sponsor. 3MEnvironmental Laboratory 4 Page 81 3M Medical Department Study: -6316.5 Analytical Study: FACT-TOX-013 LRN-U2095 Battelle Study Number: N003604-D 3M Environmental Laboratory Study Number: FACT 060998.1 APPENDIX A -RESULTS 3MEnvironmental Laboratory Page 82 3M Medical Department Study: T-6316.5 Analytical Study: FACT-TOX-013 LRN-U2095 Battelle Study Number: N003604-D 3M Environmental Laboratory Study Number: FACT 060998.1 PFOS, M-S56, PFOSAA, PFOSA IN RAT LIVER BATTELLE STUDY: N003604-D SPREADSHEET SOFTWARE: DATA ENTERED: MANUALLY EXCEL 97 Sample Dose Group Animal Number 1 1 9922 2 1 9930 3 1 9931 4 1 9932 5 1 9933 6 2 9961 7 2 9964 8 2 9965 9 2 9967 10 2 9970 11 3 9997 12 3 9999 13 3 10001 14 3 10002 15 3 10004 16 4 10019 17 4 10024 18 4 10029 19 4 10033 20 4 10034 21 5 10042 22 5 10044 23 5 10045 24 5 10051 25 5 10054 26 1 10097 27 1 10105 28 1 10106 29 1 10107 30 1 10108 31 2 10136 32 2 10140 33 2 10142 34 2 10121 35 2 10126 36 3 10177 37 3 10155 38 3 10156 39 3 10164 40 3 10172 41 4 10187 42 4 10194 43 4 10203 44 4 10211 45 4 10214 46 1 10097 47 1 10105 48 1 10106 49 1 10107 50 1 10108 51 2 10136 52 2 10140 53 2 10142 54 2 10121 55 2 10126 56 3 10177 57 3 10155 58 3 10156 59 3 10164 60 3 10172 61 4 10187 62 4 10194 63 4 10203 64 4 10211 65 4 10214 BLOQ = BELOW LIMIT O F QUANTITATION Analysis date key: Sample Type Maternal Maternal Maternal Maternal Maternal Maternal Maternal Maternal Maternal Maternal Maternal Maternal Maternal Maternal Maternal Maternal Maternal Maternal Maternal Maternal Maternal Maternal Maternal Maternal Maternal Fetal Fetal Fetal Fetal Fetal Fetal Fetal Fetal Fetal Fetal Fetal Fetal Fetal Fetal Fetal Fetal Fetal Fetal Fetal Fetal Maternal Maternal Maternal Maternai Maternal Maternal Maternal Maternal Maternal Maternal Maternal Maternal Maternal Maternal Maternal Maternal Maternal Maternal Maternal Maternal PFOS Cone, iig/g 6.77E-01 9.44E-01 9.68E-01 1.20E+00 1.17E+00 1.34E+02 1.18E+02 1.04E+02 9.34E+01 1.01E+02 4.80E+02 2.71 E+02 5.76E+02 2.97E+02 4.47E+02 9.62E+02 9.15E+02 6.43E+02 9.04E+02 S.43E+02 1.41E+03 1.57E+03 1.31E+03 1.23E+03 1.22E+3 1.72E-01 BLOQ 1.40E-01 BLOQ BLOQ 4.61 E+01 2.74E+01 2.56E+01 2.906+01 2.65E+01 1.21 E+02 1.18E+02 1.50E+02 2.07E+02 1.38E+02 1.90E+02 2.78E+02 3.98E+02 2.95E+02 3.06E+02 3.25E-01 2.80E-01 2.61E-01 2.56E-Q1 2.90E-01 6.21 E+01 3.24E+01 8.28E+01 6.33E+01 7.85E+01 1.77E+02 1.03E+02 2.53E+02 2.35E+02 2.18E+02 2.S2E+02 3.21 E+02 5.19E+02 5.29E+02 3.98E+02 M-556 Cone, pg/g BLOQ BLOQ BLOQ BLOQ BLOQ 1.28E+01 1.02E+01 1.16E+01 8.95E+00 1.16E+01 6.35E+01 3.93E+01 6.64E+01 3.80E+1 5.46E+01 8.45E+01 9.7SE+01 7.82E+01 1.17E+02 8.22E+01 1.28E+02 1.52E+02 1.33E+02 1.186+02 1.616+02 BLOQ BLOQ BLOQ BLOQ BLOQ 2.86E+00 1.55E+00 1.41F+00 M .19E+00 1.75E+00 8.16E+00 7.17E+00 7.64E+00 8.41E+00 5.87E+00 1.98E+01 2.68E+01 2.66E+01 2.75E+01 2.93E+01 BLOQ BLOQ BLOQ BLOQ BLOQ 4.64E+00 3.27E+00 4.56E+00 5.39E+00 7.75E+00 3.14E+01 1.15E+01 3.33E+01 4.31 E+01 2.99E+01 3.99E+01 3.96E+01 6.78E+01 6.54E+01 6.45E+01 PFOSAA Cone, pg/g BLOQ BLOQ BLOQ BLOQ BLOQ 1.10E+01 1.56E+01 9.79E+00 6.62E+00 1.04E+01 7.35E+01 2.86E+01 8.52E+01 3.42E+01 6.49E+01 1.22E+02 1.48E+02 8.62E+01 1.29E+02 1.35E+02 1.88E+02 2.06E+02 1.50E+02 1.57E+02 1.656+02 BLOQ BLOQ BLOQ BLOQ BLOQ 5.01 E+00 2.67E+00 2.67E+00 2.68E+00 4.34E+00 2.00E+01 1.56E+01 2.02E+01 2.27E+01 1.19E+01 3.11 E+01 5.15E+01 4.95E+01 4.66E+01 5.14E+01 BLOQ BLOQ BLOQ BLOQ BLOQ 9.44E+00 2.82E+00 6.55E+00 5.87E+00 1.46E+01 1.71 E+01 1.20E+01 2.73E+01 2.94E+01 3.37E+01 2.74E+01 4.55E+01 7.60E+01 5.62E+01 6.00E+01 PFOSA Cone, ua/a BLOQ BLOQ BLOQ BLOQ BLOQ 4.49E+00 4.10E+00 2.88E+00 3.86E+00 5.42E+ 1.08E+01 9.41 E+00 8.67E+00 8.29E+00 8.41 E+00 1.28E+01 1.10E+01 1.12E+01 1.26E+01 1.10E+01 1.64E+01 1.30E+01 1.09E+01 9.80E+00 1.08E+01 BLOQ BLOQ BLOQ BLOQ BLOQ 1.40E+00 6.01 E-01 5.08E-01 5.14E-01 7.08E-01 2.88E+00 2.22E+00 2.24E+00 1.94E+O0 2.17E+00 2.80E+00 4.06E+00 3.14E+00 3.39E+00 4.56E+00 BLOQ BLOQ BLOQ BLOQ BLOQ 2.35E+00 1.22E+00 2.06E+00 1.90E+00 2.65E+00 6.91 E+00 5.11 E+00 6.14E+00 6.26E+00 6.20E+00 6.36E+00 9.96E+00 9.93E+00 1.14E+01 8.11 E+00 Normal font = October 13,1999 Underline = October 16,1999 Bold - October 18,1999 Bold Underline - October 20,1999 All sam ples undiluted All sam ples undiluted All sam ples diluted All sam ples diluted 3MEnvironmental Laboratory A -l Page 83 3M Medical Department Study: T-6316.5 Analytical Study: FACT-TOX-013 LRN-U2095 Battelle Study Number: N003604-D 3M Environmental Laboratory Study Number: FACT 060998.1 METHOD DETECTION LIMIT (MDL) RESULTS STUDY: N003604-D ANALYSIS DATE AND INSTRUMENT ID: DATA ENTERED: SPREADSHEET SOFTWARE: 130ct99; 9053 Electronically and manually Excel 97 All cones in (ig/g Calculated Concentration o f Replicate 1 Calculated Concentration o f Replicate 2 Calculated Concentration o f Replicate 3 Calculated Concentration o f Replicate 4 Calculated Concentration o f Replicate 5 Calculated Concentration o f Replicate 6 Calculated Concentration o f Replicate 7 Mean Concentration Std. Dev. M -556 0.1269 0.1220 0.1433 0.1368 0.1670 0.1225 0.1190 0.1339 0.0170 Spike Level 0.1331 MDL determined S/N 0.05345 7.88 Valid Yes LOQ (det. from 10 x std.dev. "noise") LOQ (det. from cal curve low std.) 0.17005 0.1331 Curve Coeff of Determination 0.9978 Date analyzed Method 130ct99 LC/MS/MS Key 1 - Spike Level too high; Spike Level must be < lOx MDL 2 - Spike Level too low; Spike Level must be > MDL 3 - S/N too low; S/N must be > 5 4 - Coeff o f Det o f calibration curve unacceptable PFO SA A 0.1484 0.1281 0.1484 0.1605 0.1661 0.1513 0.1385 0.1488 0.0128 0.1334 0 04011 11.66 Yes 0.12763 0.1334 0.9937 130ct99 LC/MS/MS PFO SA 0.1167 0.1325 0.1521 0.1441 0.1490 0.1382 0.1413 0.1391 0.0119 0.1315 0.03725 11.74 Yes 0.11853 0.1315 0.9891 130ct99 LC/MS/MS 3MEnvironmental Laboratory A-2 Page 84 3M Medical Department Study: T-6316.5 Analytical Study: FACT-TOX-013 LRN-U2095 Battelle Study Number: N003604-D 3M Environmental Laboratory Study Number: FACT 060998.1 APPENDIX B-DAELY ACCEPTANCE CRITERIA SUM M ARY 3MEnvironmental Laboratory Page 85 3M Medical Department Study: T-6316.5 Analytical Study: FACT-TOX-013 LRN-U2095 Battelle Study Number: N003604-D 3M Environmental Laboratory Study Number: FACT 060998.1 PFOS et al IN RAT LIVER STUDY NUMBER: N003604-D DATA ENTERED: MANUALLY SOFTWARE: EXCEL 97 REGRESSION PARAMETERS Analysis Date October 1 3 ,1999 October 1 6 ,1999 October 1 3 ,1999 October 20 ,1 9 9 9 Analyte PFOS M-556 PFOSAA PFOSA PFOS M-556 PFOSAA PFOSA PFOS M-556 PFOSAA PFOSA PFOS PFOSA X2Coeff -0.0164 0.00646 0.00232 -155 -0.00389 8.88E-06 0.00360 -250 0.00393 0.0221 0.00370 -71.9 0.00615 -1 5 3 X Coeff 1.88 0.627 0.0970 1.50E+04 1.82 0.578 0.0931 1.73E+04 2.17 0.312 0.0853 1.23E+04 2.07 1 .68E + 04 Intercept -0.0772 -0.0213 -0.00398 -872 -0.00920 -0.00807 -0.00321 -986 -0.0905 -0.00269 -0.000603 -722 -0.0381 -1 0 2 P + 0 3 Coeff of Determination 0.984 0.996 0.994 0.989 0.999 0.998 0.998 0.990 0.985 0.984 0.993 0.996 0.997 ft SQQ Comments/ Deviations One rep of Cal pt 1 excluded One rep of Cal pt 1 excluded One rep of cal pt 7 excluded One rep of cal pt 5 excluded One rep of cal pt 7 excluded One rep of pts 1, 3, 5 excluded One rep of pts 4, 7 excluded 3MEnvironmental Laboratory B-l Page 86 3M Medical Department Study: T-6316.5 Analytical Study: FACT-TOX-013 LRN-U2095 _ Battelle Study Number: N003604-D 3M Environmental Laboratory Study Number: FACT 060998.1 PFOS et al IN RAT LIVER STUDY NUMBER: N003604-D DATA ENTERED: MANUALLY SOFTWARE: EXCEL 97 QC Results Analysis Date October 13, 1999 Analyte PFOS M-556 PFOSAA PFOSA October 16,1999 PFOS M-556 PFOSAA PFOSA October 18, 1999 PFOS M-556 PFOSAA PFOSA October 2 0 ,199 9 PFOS PFOSA QC Level, ng/m L 10 3.3 0.7 0.16 10 3.3 0.7 0.16 10 3.3 0.7 0.16 10 3.3 0.7 0.16 10 3.3 0.7 0.16 10 3.3 0.7 0.16 10 3.3 0.7 0.16 10 3.3 0.7 0.16 10 3.3 0.7 0.16 10 3.3 0.7 0.16 10 3.3 0.7 0.16 10 3.3 0.7 0.16 10 3.3 0.7 0.16 10 3.3 0.7 0.16 %RSD 6.6 3.8 7.9 3.1 4.7 11.7 3.0 11.5 6.5 5.1 8.7 14.5 16.9 9.2 5.8 4.6 5.5 4.5 8.3 9.7 3.4 2.7 8.0 17.7 4.3 2.1 7.4 17.7 11.6 6.1 8.0 8.6 8.7 11.7 7.0 15.1 14.7 17.0 21.2 28.8 14.7 11.4 15.1 21.3 16.7 11.5 12.6 6.4 2.4 2.0 3.7 5.3 2.3 4.0 2.3 3.4 %RE -4.0 -4.6 -6.4 -12.2 -2.4 2.5 -6.6 -5.1 -1.7 -10.4 -13.0 8.2 -4.3 -2.1 1.3 3.9 14.8 15.5 8.8 -2.7 -2.8 5.7 -3.7 4.5 -0.8 -2.9 -7.0 15.8 -3.9 5.4 -4.4 13.7 -4.5 -1.7 -20.3 -6.1 -2.9 23.4 11.6 15.4 0.8 -1.8 -20.6 -18.5 0.0 13.7 0.0 16.0 -8.0 -12.0 -21.7 -16.0 -1.6 6.5 1.9 11.5 3MEnvironmental Laboratory R-? Page 87 3M Medical Department Study: T-6316.5 Analytical Study: FACT-TOX-013 LRN-U2095 Battelle Study Number: N003604-D 3M Environmental Laboratory Study Number: FACT 060998.1 APPENDIX C-SAMPLE INVENTORY LIST 3MEnvironmental Laboratory Page 88 3M Medical .Department Study: T-6316.5 Analytical Study: FACT-TOX-013 LRN-U2095 _ Battelle Study Number: N003604-D 3M Environmental Laboratory Study Number: FACT 060998.1 Study TOX-013, Argus 418-009. Sample Information for shipment to Battelle Sample 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 Sample Description F0-9922-orpl-M FO-9930-grpl-M F0-9931-grpl-M F0-9932-grpl-M FO-9933-gipl-M F0-9961-grpll-M F0-9964-grpll-M F0-9965-grpll-M F0-9967-grpll-M F0-9970-grpll-M F0-9997-grplll-M F0-9999-grplll-M FO-10001-grplll-M F0-10002-grplll-M F0-100O4-qrplll-M F0-10019-grplV-M F0-10024-grplV-M FO-10029-grplV-M F0-10033-grplV-M F0-10034-grplV-M F0-10042-grpV-M F0-10044-grpV-M FO-10045-grpV-M F0-10051-grpV-M F0-10054-grpV-M FO-10097-flrpl-F F0-10105-grpl-F F0-10106-grpl-F F0-10107-grpl-F F0-10108-grpl-F F0-10136-grpll-F F0-10140-grpll-F FO-10142-grpll-F FO-10121-grpH-F FO-10126-prpH-F F0-10177-grpUI-F F0-10155-grplll-F F0-10156-grplll-F F0-10164-grplll-F F0-10172-grplil-F F0-10187-grplV-F F0-10194-prplV-F FO-10203-arplV-F FO-10211-grplV-F F0-10214-grplV-F FO-10097-grpl-F F0-10105-grpl-F FO-10106-grpl-F FO-10107-grpl-F FO-10108-grpi-F FO-10136-grpll-F FO-10140-prpll-F FO-10142-grpH-F F0-10121-grpll-F FO-10126-grpH-F FO-10177-grplH-F F0-10155-qrplll-F F0-10156-grplll-F FO-10164-grplH-F FO-10172-grplll-F F0-10187-grplV-F F0-10194-grplV-F F0-10203-grplV-F FO-10211-grplV-F F0-10214-grplV-F Sample Type Maternal Rat Liver Maternal Rat Liver Maternal Rat Liver Maternal Rat Liver Maternal Rat Liver Maternal Rat Liver Maternal Rat Liver Maternal Rat Uver Maternal Rat Liver Maternal Rat Liver Maternal Rat Liver Maternal Rat Uver Maternal Rat Uver Maternal Rat Uver Maternal Rat Uver Maternal Rat Uver Maternal Rat Uver Maternal Rat Uver Maternal Rat Uver Maternal Rat Uver Maternal Rat Uver Maternal Rat Uver Maternal Rat Uver Maternal Rat Uver Maternal Rat Liver Fetal Uver Fetal Liver Fetal Uver Fetal Uver Fetal Uver Fetal Uver Fetal Uver Fetal Uver Fetal Uver Fetal Uver Fetal Uver Fetal Uver Fetal Uver Fetal Liver Fetal Liver Fetal Uver Fetal Uver Fetal Uver Fetal Uver Fetal Liver Maternal Rat Uver Maternal Rat Liver Maternal Rat Uver Maternal Rat Uver Maternal Rat Uver Maternal Rat Liver Maternal Rat Uver Maternal Rat Uver Maternal Rat Uver Maternal Rat Uver Maternal Rat Uver Maternal Rat Uver Maternal Rat Uver Maternal Rat Uver Maternal Rat Uver Maternal Rat Uver Maternal Rat Uver Maternal Rat Uver Maternal Rat Liver Maternal Rat Uver 3MEnvironmental Laboratory C -l Page 89 3M Medical Department Study: T-6316.5 Analytical Study: FACT-TOX-013 LRN-U2095 Battelle Study Number: N003604-D 3M Environmental Laboratory Study Number: FACT 060998.1 APPENDIX D-REPRESENTATIVE CHROMATOGRAMS 3MEnvironmental Laboratory Page 90 3M Medical Department Study: T-6316.5 Analytical Study: FACT-TOX-013 LRN-U2095 Battelle Study Number: N 003604-D 3M Environmental Laboratory Study Number: FACT 060998.1 3MEnvironmental Laboratory D-l Page 91 3M Medical Department Study: T-6316.5 Analytical Study: FACT-TOX-013 LRN-U2095 _ Battelle Study Number: N003604-D 3M Environmental Laboratory Study Number: FACT 060998.1 3MEnvironmental Laboratory n -2 Page 92 3M M edical D epartm ent Study: T-6316.5 Analytical Study: FACT-TOX-013 L R N -U 2095 Battelle Study Number: N003604-D 3M Environmental Laboratory Study Number: FACT 060998.1 3MEnvironmental Laboratory rvi Page 93 3M Medical Department Study: T-6316.5 Analytical Study: FACT-TOX-013 LRN-U2095 _ Battelle Study Number: N003604-D 3M Environmental Laboratory Study Number: FACT 060998.1 3MEnvironmental Laboratory r\ a Page 94 3M Medical Department Study: T-6316.5 Analytical Study: FACT-TOX-013 LRN-U2095 Battelle Study Number: N003604-D 3M Environmental Laboratory Study Number: FACT 060998.1 3MEnvironmental Laboratory D-5 Page 95 3M Medical Department Study: T-6316.5 Analytical Study: FACT-TOX-013 LRN-U2095 Battelle Study Number: N003604-D 3M Environmental Laboratory Study Number: FACT 060998.1 APPENDIX E-PROTOCOL, AMENDMENTS, AND DEVIATIONS 3MEnvironmental Laboratory Page 96 3M M edical D epartm ent Study: T-6316.5 A nalytical Study: FA CT-TO X-013 L R N -U 2095 _ Battelle Study Number: N003604-D 3M Environmental Laboratory Study Number: FACT 060998.1 fir r c . ' TOA - l)/~$ 3M Environmental Laboratory P r o t o c o l - A n a l y t ic a l S t u d y 2(N-Ethylperfluorooctanesulfonamido)-ethanol in Two Generation Rat Reproduction In-vivo study reference number: Argus 418-009 Study number: FACT 060998.1 Test substance: 2(N-Ethylperfluorooctanesulfonamido)-ethanol (N-EtFOSE-OH) Name and address of Sponsor: Marvin Case 3M Toxicology Services 3M Center Building 220-2E-02 St. Paul, MN 55144 Name and address of testing facility: 3M Environmental Technology and Services 935 Bush Avenue, Building 2-3E-09 St. Paul, MN 55106 Experimental start date: Expected termination date: December 31,1998 Method numbers and revisions: FACT-M-1.0, Extraction of Potassium Perfluorooctanesulfonate or Other Anionic Surfactants from Liver for Analysis Using HPLC-Electrospray/Mass Spectrometry FACT-M-2.0, Analysis of Fluorochemicals in Liver Extracts Using HPLC- Electrospray/Mass Spectrometry ' FACT-M-3.0, Extraction of Potassium Perfluorooctanesulfonate or Other Anionic Surfactants from Serum for Analysis Using HPLC-Electrospray/Mass Spectrometry FACT-M-4.0, Analysis of Fluorochemicals in Serum Extracts Using HPLCElectrospray/Mass Spectrometry Author: Lisa Clemen Kris Hansen Study Director Date Marvin Case Date Sponsor Representative 3MEnvironmental Laboratory E-l Page 97 3M M edical D epartm ent Study: T-6316.5 Analytical Study: FACT-TOX-013 L R N -U 2095 _ Battelle Study Number: N003604-D 3M Environmental Laboratory Study Number: FACT 060998.1 1.0 Purpose ____________________________________________________________ The analytical portion of this dosing study is designed evaluate the levels of perfluorooctane sulfonate (PFOS), or another metabolite of 2(N-ethylperfluorooctanesulfonamido)-ethanol (NEtFOSE-OH) designated by the study director, in the liver of the parent and subsequent generations of the test system, or in the serum as necessary. The in life portion of this study was conducted at Argus Research Laboratories. 2.0 Regulatory Compliance________________________________________________ This study is conducted in compliance with the Food and Drug Administration Good Laboratory Practices regulation as stated in 21 CFR 58. Any exceptions will be noted in the final report. 3.0 Test Materials_________________________________________________________ 3.1 Test, control, and reference substances and matrices 3.1.1 Analytical reference substance: Potassium perfluorooctanesulfonate (PFOS), lot #217 3.1.2 Analytical reference substance matrix: Rat liver and serum 3.1.3 Analytical control substance: None 3.1.4 Analytical control substance matrix: Rat liver and serum 3.2 Source of materials 3.2.1 Analytical reference substance: 3M Specialty Chemical Division; traceability information will be included in the final report 3.2.2 Analytical reference substance matrix: Argus Research Laboratories; traceability information will be included in the final report 3.2.3 Analytical control matrix: 3.2.3.1 Rat liver - Argus Research Laboratories; traceability information will be included in the final report; or Rabbit liver - Covance Laboratories; traceability information will be included in the final report 3.23.2 Rat serum - Sigma Chemical Company; traceability information will be included in the final report 3 3 Number of test and control samples. Liver samples for testing were received from 40 test animals and 10 control animals. Serum samples will be tested at the discretion of the Study Director. 3.4 Identification of test and control samples: The samples are identified using the Argus Research Laboratories identifiers, which consist of a letter followed by the Argus project number, the animal number, the group designation, and the draw date. 2 3MEnvironmental Laboratory Page 98 3M M edical D epartm ent Study: T-6316.5 A nalytical Study: FACT-TO X-013 L R N -U 2095 Battelle Study Number: N003604-D 3M Environmental Laboratory Study Number: FACT 060998.1 3.5 Purity and strength of materials: Characterization of the purity and identity of the reference material is the responsibility of the Sponsor. 3.6 Stability of test material: Characterization of the stability of the test material is the responsibility of the Sponsor. 3.7 Storage conditions for test materials: Test materials are stored at room temperature. Samples are stored at -20 10 C. 3.8 Disposition of test and/or control substances: Biological tissues and fluids are retained per GLP regulation. 3.9 Safety precautions: Refer to the material safety data sheets of chemicals used. Wear appropriate laboratory attire, and follow adequate precautions for handling biological materials and preparing samples for analysis. 4.0 Experimental - Overview___________________________________________________ Tissues from animals dosed as described in Argus Research Laboratories Protocol #418-009 are received for analysis of fluorine compounds. At the discretion of the Study Director, a series of analytical tests will be performed on select tissues. Initially, all liver samples will be analyzed for PFOS by electrospray/mass spectrometry (ES/MS). On the basis of findings from these analyses, additional sample matrices may be evaluated or other metabolites may be targeted. If additional analysis is performed, a protocol amendment will be written. 5.0 Experimental - Analytical Methods__________________________________________ 5.1 FACT-M-1.0, Extraction of Potassium Perfluorooctanesulfonate or Other Anionic Surfactants from Liver for Analysis Using HPLC-Electrospray/Mass Spectrometry 5.2 FACT-M-2.0, Analysis of Fluorochemicals in liver Extracts Using HPLCElectrospray/Mass Spectrometry 5.3 FACT-M-3.0, Extraction of Potassium Perfluorooctanesulfonate or Other Anionic Surfactants from Serum for Analysis Using HPLC-Electrospray/Mass Spectrometry 5.4 FACT-M-4.0, Analysis of Fluorochemicals in Serum Extracts Using HPLCElectrospray/Mass Spectrometry 6.0 Data Analysis____________________________ ,_____________________________ 6.1 Data transformations and analysis: Data will be reported as the concentration (weight/weight) of fluoride per tissue or sample, or of PFOS per unit of tissue or fluid. 6.2 Statistical analysis: Statistics used may include regression analysis of the serum . concentrations over time, and standard deviations calculated for the concentrations within each dose group. If necessary, simple statistical tests, such as Student's t test, may be applied to evaluate statistical difference. 3MEnvironmental Laboratory F.-3 3 Page 99 3M- Medical Department Study: T-6316.5 Analytical Study: FACT-TOX-013 LRN-U2095 _ B attelle Study Num ber: N 0 0 3 6 0 4 -D 3M Environm ental Laboratory Study Num ber: F A C T 060998.1 7 .0 Maintenance of Raw Data and Records 7 .1 T h e fo llo w in g raw data and records w ill b e retained in the study fold er in the arch ives according to A M D T -S -8: 7.1.1 A pproved protocol and am endm ents 7 .1 .2 Study correspondence 7 .1 .3 Shipping records 7 .1 .4 R aw data 7 .1 .5 E lectronic copies o f data 7 .2 Supporting records to b e retained separately from the study fold er in the arch ives according to A M D T -S -8 w ill inclu de at least the follow in g: 7 .2 .1 T raining records 7 .2 .2 C alibration records 7 .2 .3 Instrum ent m aintenance logs 7 .2 .4 Standard O perating P rocedures, E quipm ent P rocedures, and M eth od s 7 .2 .5 A ppropriate sp ecim en s. 8 .0 References _ 8.1 3 M E n v ir o n m e n ta l L a b o r a to r y Q u a lity S y s t e m C h a p te r s 1 , 5 a n d 6 8.2 O th e r a p p lic a b le 3 M E n v ir o n m e n ta l L a b o r a to r y Q u a lity S y s t e m S ta n d a r d O p e r a tin g Procedures 9 .0 Attachments 9 .1 F A C T -M -1 .0 , Extraction of Potassium Perfluorooctanesulfonate o r O th er A n io n ic Surfactants from L iver for A n alysis U sin g H PL C -E lectrospray/M ass Spectrom etry 9 .2 F A C T -M -2 .0 , A n alysis o f F lu orochem icals in L iver E xtracts U sin g H PL C E lectrospray/M ass Spectrom etry 9 .3 F A C T -M -3 .0 , E xtraction o f P otassiu m P erfluorooctanesulfonate or O ther A n io n ic Surfactants from Serum for A n alysis U sin g H PL C -E lectrospray/M ass Spectrom etry 9 .4 F A C T -M -4 .0 , A nalysis o f R u oroch em icals in Serum E xtracts U sin g H PL C E lectrospray/M ass Spectrom etry | ! 3MEnvironmental Laboratory ca 4 Page 100 3M M edical D epartm ent Study: T-6316.5 A nalytical Study: FACT-TO X-013 L R N -U 2095 _ Battelle Study Number: N003604-D 3M Environmental Laboratory Study Number: FACT 060998.1 METHOD FORANALYSIS OFPOTASSIUM PERFLUOROOCTANESULFONATE (PFOS) INRATLIVERBYLC/MS/MS Version 1.0 . ' Study N o.!_________________________ A nalyst/Sate:___________________________ R evisions to the method D ru e o l'R e v is io n R e v ised bv A pprov cd bv IBI^B^^BIB^BHBB BIB^Bk^^^Bh^^^Bb b b b ^ b b b b h b b b b B B B ^ B B B i BBBBBH^HB b b b b b b b BflHBHHB^BHBHBB iB B B B B B b b b b b b b b b W ritten by: Patrick L. South _________Date: /ip s 3 iv > 9 9 * Approved by: C< ____________ Date: JJcdpt/lCc . Andre., P h D . ger, B ionalydcal Chem istry Gm j j Jt <9 f. f W > 3MEnvironmental Laboratory Page 1 of 16 cc P age 101 3M M edical D epartm ent Study: T-6316.5 A nalytical Study: FACT-TO X-013 L R N -U 2095 _ Battelle Study Number: N003604-D 3M Environmental Laboratory Study Number: FACT 060998.1 METHOD FORANALYSIS OFPOTASSIUM PERFLUOROOCTANESULFONATE (PFOS) IN RATLIVERBY LC/MS/MS V ersion L0 Study No.: Analyst/D ate: _ L Summary The extraction and analysis o f potassium perfluorooctanesulfonate and related fluorochemicals in rat liver is performed. Calibration standards are prepared by spiking blank liver homogenate w ith solvent standards from two independently-prepared stocks. The calibration standards are fortified w ith surrogate standard, buffered, and extracted with ethyl acetate. The organic phases are evaporated to dryness and reconstituted in methanol for analysis by LC/MS/MS. EL P u r po se To extract and analyze potassium perfluorooctanesulfonate and related fluorochemical compounds found in Sprague-Dawley rat liver. in . Samples See Chain o f Custody records if applicable. IV . G e n e r a l I n st r u c t io n s Calibrate all required balances according to the SOP on balance usage. Make equivalent dilutions when the volume needed varies from the volume stated in the method. Label all standard and reagent solutions as specified in the appropriate SOP. I f you intend tn reuse a solution for future tasks, be sure tire label includes the preparation date and study number for which the solution was initially prepared. Sign on the final page a fth is m ethod to signify that you have followed the method as written, all materials and reagents are current, and all equipment has been properly calibrated. Ifyou deviate from the method, document the change, and obtain the approval o f the unit manager, study director, or task leader as soon as possible. Initial and date all data entries on the page on which they were made. If only one person enters all data on a single day, the documentation may be made in a single location on that page.. I f multiple staff make entries, the additional entries must be initialed and dated by the person malting the entry. Line-outs or NA denotes "Not Applicable". The method is w ritten in general chronological order, but the sequence o f steps may be altered if the analyst deems it appropriate, unless the order for certain activities is specified. Stocks w ill be used fo r the duration o f the study unless consumed or unless stability is considered suspect No correction will be made for purity o r salt content o f any test article but PFOSAA. Use glass volumetric, Eppendorf repeater, or positive-displacement pipets for dispensing methanolic solutions. Contact with Teflon by the test article should be minimized. . V. Materials See Table 1 for all required chemicals, reagents, and solvents. Use Table 1 fo r documentation. Check all labels carefhlly to ensure that all materials are not expired and that they are the proper purity o r grade. Page 2 of 16 3MEnvironmental Laboratory CA Page 102 3M M edical D epartm ent Study: T-6316.5 A nalytical Study: FA CT-TO X-013 . LRN-U2095 Battelle Study Number: N003604-D 3M Environmental Laboratory Study Number: FACT 060998.1 . METHOD FORANALYSIS OFPOTASSIUM PERFLUOROOCTANESULFONATE (PFOS) INRATLIVERBYLC/MS/MS . Version 1.0 Study N<w_________________________ Analyjt/D ate:___________________________ M aterials U se Potassium Perfluorooctanesulfbnate (PFOS) lH ,nU K t2H Perfluorooctane Sulphonic A dd M-536 . MS70 PFOSAA PFOSA PFOSEA R at Liver A nalytical Standard Surrogate Standard A nalytical Standard A nalytical Standard A nalytical Standard A nalytical Standard A nalytical Standard M atrix Ammonium Acetate, N ILCJLO , Sodium Hydroxide, NaOH Tetrabutylammonium Hydrogensulfate (TBA), iCHsfCHALNiHSCM Sodium Carbonate, NiteCO, Sodium Bicarbonate, NaHCOi Ethyl Acetate M obile Phase Reagent Prep E xtra Prep E x tra Prep E x tra Prep E xtra Prep T a b let. M aterials S u p p lier G rade o r P u ri tv 3M ICN 3M 3M 3M 3M 3M H arlan SpragueDawley S to rag e Tem o Room Temp Room T em p Room Temp Room Ternn Room Temp Room Temp Room Temp --20C RT RT RT Lot o r ID RT RT RT M ethanol M illi-Q Water pH 7 Buffer pH 10 Buffer M obile Phase, Stocks, WS Reagent Prep, M obile Phase pH m eter calibration pH m eter calibration M illipore RTASTM Typel RT RT RT I R.T means Room Temperature. V L E q u ipm en t See Table 2 for all required major pieces o f equipm ent Use the table to document the actual piece (e.g. make, model) o f equipm ent Check calibration ofall equipment requiring calibration (e.g. balances) to ensure i t is current Page 3 of 16 3MEnvironmental Laboratory F.-7 P age 103 3M M edical D epartm ent Study: T-6316.5 Analytical Study: FACT-TOX-013 L R N -U 2095 _ Battelle Study Number: N003604-D 3M Environmental Laboratory Study Number: FACT 060998.1 METHOD FORANALYSIS OFPOTASSIUM PERFLUOROOCTANESULFONATE (PFOS) INRATLIVERBYLC/MS/MS - - Version 1.0 Study N o.:_________________________ Analyst/D ale:___________________________ E q u ip m en t Analytical Balance W eight Set U se W eigh Standards or R eagents Calibrate Balance Table X Equipment M an u factu rer M odel. X Lm -SN 1 Pipettor Pipet Samples Pipettor Pipet Samples Pipettor Pipettor Pipet EtOAc extraction phase Pipet Reagents, WIS Eppendorf - Repeater Vortexer- M ix Samples Freezer (-20C) R efrig erato r a -9 Q Centrifuge Store QCs, Blank L iv e r Store Buffer, Stocks Phase separation T est Tubes Centrifuge Tubes T est Tubes Transport tubes M agnetic stirrer O rbital Shaker E v a p o ra to r Liver sample hom ogenization Extract Samples Evaporate Extracts Store QCs Stir matrix Extract Samples Evaporate E xtracts Stockwell Scientific Blue Falcon Blue Falcon Elkay Polypropylene, 15 mL Polypropylene, 15 mL Polypropylene, 12 x75 mm 5 mL polypropylene SW8599 2096 . 2002 127-T160-56P Zymark Turbovap L V Syringe Filters Filter Extract H om oeenizer pH meter Electrode Volumetric Flasks, Class A Volumetric Pipets, G ass A Transfer Pipets. Plastic Grind liver Determine Buffer pH Determine Buffer pH M ake Volumetric D ilutions M ake Volumetric D ilutions Transfer Extracts to Centrifuge Filters and LC Inserts NA NA Samco NA NA NA NA Page 4 of 16 3MEnvironmental Laboratory C_Q Page 104 3M M edical D epartm ent Study: T-6316.5 A nalytical Study: FA CT-TO X-013 L R N -U 2095 _ Battelle Study Number: N003604-D 3M Environmental Laboratory Study Number: FACT 060998.1 J METHOD FOR ANALYSIS OF POTASSIUM PERFLUOROOCTANESULFONATE (PFOS) IN RAT LTVER BY LC/MS/MS V enan 1.0 Study No.: Analyst/Date: _ VEL PROCEDURE A. Preparation of2 mMAmmoniumAcetate W eigh 0.1500 j; 0.0020 g o f ammonium acetate and transfer to a 1000-mL volumetric flask. Dissolve the solid in w ater and dilute to volume with water. Solution may be used fo r one month stored a t room temperature. Actual mass o f ammonium acetate:__________ Actual final volum e:_________ Date o f preparation:____________________ Study No: ____________ B. Preparation of~29% SodiumHydroxide Solution W eigh 200 2 g of sodium hydroxide into a beaker. Add 500 mL o fMOli-Q w ater and m ix to dissolve. Cool and transfer to a polypropylene bottle for storage. Solution may be stored for 6 months a t room temperature. Actual m a o f sodium hydroxide: _ _ _ _ _ _ Actual volume M illi-Q w a te r_________ Date o f preparation:____________________ Study N o :_______________ C. Preparation of--2.9%SodiumHydroxide Solution ' Add 10 mL o f--29% Sodium Hydroxide Solution to a 100-mL volumetric flask and dilute to volume w ith M illi-Q water. Transfer to a polypropylene bottle for storage. Solution may be stored fo r 6 months at room temperature. Actual volume o f-29% NaOH so lu tio n :__________ Actual final volume: _________ D ate o f preparation: ____ Study N o:_______________ ' D. Preparation ofTetrabutylammoniumHydrogensulfate (TBA) Solution, 0.5M, (pH 10) pH M eter Calibration pH buffer 7 pH buffer 10 pH reading:________ pH reading:________ Add 169* IgafT B A to-S O O m L afM illi-Q w aterinabeaker. Adjust the pH to 10.00 t 0.02 using -55-60 mL o f 29% Sodium Hydroxide Solution, dilute to 1000 mL wife . Milli-Q water, and mix. Adjust the pH to 10.00 * 0.02 using -2.9% NaOH and mix. Transfer to a polypropylene bottle for storage. Solution may be used for one month Stored at room temperature, but the pH m ust be checked n rio r to each use. Adjust to pH 10.0 i 0.02 with 2.9% Sodium Hydroxide Solution as necessary. Page 5 of 16 3MEnvironmental Laboratory r? a Page 105 3M M edical D epartm ent Study: T-6316.5 Analytical Study: FACT-TOX-013 L R N -U 2095 _ Battelle Study Number: N003604-D 3M Environmental Laboratory Study Number: FACT 060998.1 METHODFORANALYSIS OFPOTASSIUM PERFLUOROOCTANESULFONATE (PFOS) INRATLIVERBYLC/MS/MS ' Version 1.0 Study No.: ` A n alyit/D ate:___________________________ Actual m ass o f T B A :__________ Actual final volum e:_________ . Actual final p H :__________ Date of preparation:____________________ Study N o :_______________ pH after techedring and/or readjusting:_________ E . P re p a ra tio n o f 0 .2 5 M C a rb o n a te B u ffe r Weigh 2 6J 0.1 g o f sodium carbonate and 21.0 * 0.1 g o f sodium bicarbonate and transfer to the same 1000-mL volumetric flask. Dissolve the materials in M illi-Q water, dilute to volume w ith M illi-Q water, mix, and transfer to a polypropylene bottle for storage. Solution may be used fo r 1 month when stored refiigerated. Actual mass o f sodium carbonate: __________ Actual mass of sodium bicarbonate:__________ Actual final volume: _________ Date o f preparation:____________________ Study N o:_______________ F . P re p a ra tio n o f M o b ile P h a se Component A: M ix together 600 mL of 2 mM ammonium acetate and 400 mL of methanoL Solution may be used fo r 1 month when stored at room temperature. Actual volume of 2 mM ammonium acetate:________ mL Actual volume o f methanol: ________mL Date ofpreparation:____________________ Study N o:_______________ Component B: M ix together SO mL of 2 mM ammonium acetate and 950 mL of methanoL Solution may be used for 1 month when stored at room temperature. Actual volume o f 2 niM ammonium acetate: Actual volume of methanol: m l. Date ofpreparation:____________________ Study N o :_______________ mL G . P re p a ra tio n o f S to ck S u rro g a te S ta n d a rd a n d W o rk in g S u rro g a te S ta n d a rd (W SS) 1. Stock Surrogate Standard (250,000 ng/mL): W eigh 25 t 2 mg o f 1H, 1H, 2H, 2H,-pcrfluorooctane sulphonic ad d and transfer to a 100-mL volumetric flask. Dissolve in methanoL dilute to volume with m ethanol, and mix. Store refiigerated, protected fiom UV light Actual W eight:________________ Actual Dilution Volum e:___________________ Date ofPreparation:__________________ Study No: _________________ Page 6 o f16 3MEnvironmental Laboratory F .in Page 106 3M M edical D epartm ent Study: T-6316.5 A nalytical Study: FACT-TO X-013 L R N -U 2095 Battelle Study Number: N003604-D 3M Environmental Laboratory Study Number: FACT 060998.1 METHOD FORANALYSIS OFPOTASSIUM PERFLUOROOCTANESULFONATE (PFOS) INRATLIVERBYLC/MS/MS V ersion 1.0 Study N o.:____________ ;____________ A nalyst/D ate:___________________________ 2. WSS (1000 ng/mL): D ilute 100 /JL ctfstock, surrogate standard to 25 mL with m ethanol and m ix.. Actual Volume of Stock Internal Standard:___________ Actual D ilution V olum e:___________________ Date ofP rep aratio n :__________________ H. Preparation ofCalibration Solvent Stocks andWorking Standards 1. Solvent Stocks: For each analyte weigh the specified amount of standard (independently weighed as A and B replicates) listed in Table 3 and transfer into separate volum etric flasks. Dissolve in methanol, dilute to volume w ith methanol, and m ix well. Store refrigerated, protected from UV light. 2. M ixed Solvent Stocks: . P ipetthe specified amount of each analytical standard Replicate A as listed in Table 3 and transfis' into a single volumetric flask. Dissolve in methanol, dilute to volume w ith methanol, and mix well. Store refrigerated, protected from UV lig h t Repeat the process with Replicate B stocks. The mixed solvent stocks are used to prepare Ike working standards. Date ofpreparation:____________________ Study N o :_______________ > 3. W orking Standards (WS): D ilute the m ixed stocks and working standards with m ethanol as specified in Table 3 and m ix well. 4 Date of preparation:_______________________________ S tan d a rd Stodc 1A Stodc IB Stock 2A Stodc 2B Stock 3A Stock 3B Stock 4A Stock 4B Stodc SA Stodc SB Stodc 6A S o u rce PFOS PFOS M-SS6 M -556 M 570 M 570 FFOSAA PFOSAA PFOSA PFOSA PFOSEA Table 3. Calibration Solvent Stocks and W orking Standards T arget A ctu al A m ount T arget A ctu al A m ount A n aly tical S td . F in al V ul S tu d i, o r W S im L ) SO X 1 mg 10 25 0.S mg - me- 10 SO X 1 mg H H B f f i S B 10 25 X0.5 mg 10 50 X 1 mg mg* I 10 25 X0.5 mg MB* 1 10 93 X 1 mg L > ME* 10 46 X0.5 mg mg* 10 50 X 1 mg 10 25 X 0.5 mg "> ' > m g* 10 50 X 1 mg ______________ M L 10 F in a l V ol. (mL) ' - ,, -P s. '** Kn::: ' n.... - * 4 N om inal C one (n/m L > 5,000,000 2^00,000 5,000,000 2J00.000 5,000,000 2,500,000 5.000,000 2.500,000 5,000.000 2,500,000 5.000,000 Page 7 of Id 3MEnvironmental Laboratory F.-l 1 Page 107 3M M edical D epartm ent Study: T-6316.5 Analytical Study: FACT-TOX-013 L R N -U 2095 Battelle Study Number: N003604-D 3M Environmental Laboratory Study Number: FACT 060998.1 METHOD FOR ANALYSIS OF POTASSIUM PERFLUOROOCTANESULFONATE (PFOS) IN RAT LIVER BY LC/MS/MS V ersion 1.0 - Study N o.:__________ A nalyst/D ate:___________________________ Stock 6B FFOSEA 25 0.5 mg m tm 10 rr'wmi ....w....irvr j 2.500.000 Mixed Stocks 1 thru Stock A 6 Rep A 5 mL each** -----------"L each j. > i* !>k 50 9o*J* d8L> ~} }. 500,000 Mixed Stocks I thru Stock B 6RepB 5 mL each** 7 50 250,000 - I vt* <* * WS 1 Mixed Stock A 1 mL** . mL stm r Xi 25 bgs.sa::.t;.w.s::::.::::::?-::.::*:TM:-::: :;>:x:j. 20,000 W S2 M ixed Stock B 1 mL** m L 25 10,000 ^........: . ' ' ' ' W S3 WS 1 2 mL** mTt- 10 * a y >. 4000 WS 4 W S2 2 mL** m L 10 " 2000 WS 5 WS 3 2.5 mL** mL 1 10 1000 W S6 W S4 2.5 mL** m L 10 500 WS 7 W S5 2 mL** 10 ::;ti**r; 200 * Weigh all analytical standards to a t least the nearest 0.01 mg. ** Use volum etric or positive-displacement pipet(s). L Preparation of Calibration Standards and Blanks l . Liver homogenate Prepare blank liver homogenate in bulk by weighing approximately 40 g o f blank liver into a 500 mL Nalgene bottle containing 200 mL of Milli-Q water. Grind to a homogeneous suspension. Aliquot into approx 30 mL portions for frozen (approx -2 0 Q storage. Actual M ass o fLiven____________ Actual volume o f w ater___________ Date o fp re p :___________________ Study:_____________________ Determine density o f calibradon/QC matrix: MIX HOMOGENATE THOROUGHLY and determine the mass in milligrams o f 10 replicate weighings o f 1 mL portions of the THOROUGHLY MIXED homogenate. M K HOMOGENATE IMMEDIATELY PRIOR TO EACH ALIQUOT REMOVAL. R eplicate# Mass (inai a 2. Liver Calibration Standards Prepare each Ever calibration standard by adding 0.45 mL o f undiluted liver homogenate (STIR HOMOGENATE WHILE ALIQUOTING) into a 15 mL extraction tube and adding 50 pL o f WS or MeOH. Prepare triplicate cal standards and 6 blanks. See Table 5 for volumes. The diluted liver density is assumed to be approximately 150 mgfatL. M ix well. Page 8 of 16 3MEnvironmental Laboratory cn Page 108 3M M edical D epartm ent Study: T-6316.5 Analytical Study: FACT-TOX-013 L R N -U 2095 _ Battelle Study Number: N003604-D 3M Environmental Laboratory Study Number: FACT 060998.1 METHOD FOR ANALYSIS OF POTASSIUM PERFLUOROOCTANESULFONATE (PFOS) IN RAT LIVER BY LCM S/M S V ernon 1.0 Study No.: A nalyst/D ate: _ C al S tii/B lan k 1 2 3 4 5 6 7 Blank S ource W Sl WS 2 WS 3 W S4 WS 5 WS 6 WS 7 MeOH T a ra e t Voi G aL) 50 50 50 50 50 50 50 50 T ab les. C alibration Standard and B lanks A ctual V oi T arget A ctual N om inal G aL ) F in al Voi F inal V oi C one ' - . *> *i 1...........' ' ' : (m L ) 0.5 0.5 0.5 0.5 0.5 0.5 (m L ) 8 8 6S i8S & :tynxK*S::K::>fc::? : * *'- 1 v ' 1;, *J '/ i> >> , (n s/m U 2000 1000 400 200 100 50 i:!i: ! 0.5 20 1 0.5 iililllli 0 N o m in al C one (u a /a ) 13 6.6 2.6 1J 0.66 0.33 0.13 0 Date o f preparation o f cal stds/blank:____________________________ J. Preparation of Quality Control Liver Samples (QCs) 1. Quality Control Working Standards D ilute the following source volumes m ethanol in volumetric flask.! and mix w ell. Prepare fresh when used. Actual volumes are in parentheses. S o in ID OC WS 1 QCW S2 QC WS 3 QCW S4 Z S ource M ixed Stock A M ixed Stock B QC WS 1 QCW S2 T abled. QC WS P reparation V oi S o u rc e . mL F in al V oi. mL I 3 .(........... i i f i l l i to ffc ' ~ * r ` l i > 50 f - 7.5 ( 100 f 1 1 sg& m sm -> . 1 50( ) Preparation o f Quality Control Liver Samples C one, n a/m L 15.000 5000 1125 250 Prepare each QC in bulk by filling the volumetric flask approximately h alffull w ith u n diluted liv e r homogenate (S T IR HO M O G EN ATE W H ILE ALIQUOTING), adding the appropriate QC WS, mixing, and diluting to volume w ith undiluted Uver homogenate (SU R HOMOGENATE WHILE ALIQUOTING). MIX THOROUGHLY and dispense Z5-mL aliquots into polypropylene tubes and store at approximately -2 0 C Table 7. Q C P reparation QC S ource Voi S ource. m L F in al V oi. m L C one. na/m L C o n e , lui/ n 1 OCWS 1 25 C i 1500 1 10 2 QC WS 2 3 QCW S 3 Z5C . . 2* t i 500 112.5 3.3 0.7 4 OCWS 4 Z5 ) 25 0.16 Date ofQC prep:___________________ Study._____________________ K. Preparation of MS Check Standard for System Suitability Pipet 250 mL of WS 2 a t--10,000 ng(mL and Z 5 mL of WSS at -1000 ng/mL in methanol into the same 50-mL volumetric flask. Dilute to volume with MeOH and mix. Page 9 of 16 3MEnvironmental Laboratory Page 109 3M M edical D epartm ent Study: T-6316.5 A nalytical Study: FACT-TO X-013 L R N -U 2095 Battelle Study Number: N003604-D 3M Environmental Laboratory Study Number: FACT 060998.1 METHOD FOR ANALYSIS OF POTASSIUM PERFLUOROOCTANESULFONATE (PFOS) IN RAT LIVER BY LC/MS/MS V ersion L0 . Study N o.:____________________________ A nalyst/D ate:______________________________ L. Preparation of homogenizer recovery liver samples . M. To determine the recovery from the homogenization process, nnhom ogeaized blank liver w ill be fortified in duplicate a t 3 concentration levels and homogenized as follows. This needs to be done every day that homogenization of study samples is performed. 1. Place approximately 0.3 g o f unbomogenized blank liver into each o f 6 ,1 5 mL polypropylene centrifuge tubes. Record weights o f liver. X Add 100 pL o f WS 1 ,3 , and 4 (one WS per duplicate tubes) to prepare - fortifications a t approximately 4, 0.8, and 0.4 pg/g. 3. M ultiply the mass ofliver in g by 2.5 and add this manymL ofw ater. 4. Homogenize each liver sample, and rinse homogenizer probe with another volume o f w ater used in step 3, adding rinse to homogenized sample. . 5. Clean homogenizer w ith MeOH between samples. 6. Cap and vortex homogenate for use in extraction. Preparation of Dilation Check Sample 1. Place 2.95 mL o f undiluted liver homogenate (STIR HOMOGENATE WHILE ALIQUOTING) into a 15 mL extraction tube and add 50 pL o f M ixed Stock A. X D ilute 50 pL of step 1 solution (VORTEX SOLUTION WHILE ALIQUOTING) with 0.45 mL of undiluted liver homogenate (STIR. HOMOGENATE WHOLE ALIQUOTING) in 3,1 5 mL extraction tubes. 3. This sample should be prepared for extraction only on days when study samples w ill be diluted and extracted. .- N. Homogenization of study samples 1. Place approximately 0.5 g of unhomogenized study sample liver into a 15 mL polypropylene tube. Record weights ofliver. X M ultiply the mass o f liver in g by X5 and add this manymL of water. 3. Homogenize each liver sample, and rinse homogenizer probe with another volume o f water used in step X adding rinse to homogenized sample. 4. C lean hom ogenizerw ith M eO H between samples. 5. Cap and vortex homogenate for use in extraction. O. Analysis Standards, Blanks, QCs, and Samples 1. M IX LIVER HOMOGENATES THOROUGHLY BEFORE ALIQUOTING and p ip 500 /jL of each QC (4 replicates per level), and other samples being extracted into 15-mL polypropylene extraction tubes. The cal stds an d blanks are already aliquoted. X To the B lanks-IS (3 reps), add 100 jL o f MeOH and vortex. 3. To the Blanks + IS (3 reps) and to the remaining samples, add 100 iL WSS and vortex. 4. Add 0.5 mL o f 0.5 M TBA (pH 10) to all tubes and vortex briefly. 5. Add 1 mL o f 0X5 M carbonate buffer and vortex briefly. 6. Add 2 J mL of ethyl acetate. Place the tubes sideways on the orbital shaker at a ' setting o f300 for -2 0 minutes. 7. Centrifuge tubes at a setting o f3500 rpm for -2 0 minutes to separate layers. 8. Transfer 2 mL of the top organic layer to a dean polypropylene tube. Page 10 of 16 3MEnvironmental Laboratory E-14 Page 110 3M M edical D epartm ent Study: T-6316.5 Analytical Study: FACT-TOX-013 L R N -U 2095 Battelle Study Number: N003604-D 3M Environmental Laboratory Study Number: FACT 060998.1 METHOD FOR ANALYSIS OF POTASSIUM PERFLUOROOCTANESULFONATE (PFOS) IN RAT LIVER BY LC/MS/MS Version 1.0 Study N o.:___________________________ Analyjt/Date: ______________________________ 9. Evaporate to dryness tm der nitrogen at a setting of 30*C fo r-6 0 minutes. 10. Reconstitute the residues in 500 mL o f methanol with vortexing. 11. Syringe-filter extracts into antosnmplcr vials for analysis. Store vials refrigerated (up to 1 month) if LC/MS/MS will not be performed the same day. Since 3-day room temperature extract stability was demonstrated during validation, the - extracts o f the cal stds, blanks, and QCs may be reused for up to 3 days after ' their initial preparation if held at room temperature (recap the vials if reusing). Date of cal std/blank extract prep:________________________ Date of QC extract prep:_______________________________ P. LC/MS/MS Analysis . 8 . 1. Use the system conditions specified in Table y f. The conditions which are designated may be modified by the analyst to produce acceptable peak shape. L C /M S /M S S ystem Table 8 - LC/M S/M S Conditions A u to sam p fcr H PLC Pum ps M ass S pectrom eter M ak e:____ Make: ____ M ake: M odel: M odel: M odel: ID: ID: ID: A n a ly t ic a l c o lu m n Keystone Betasil C18, 5m. 2 x 50 mm. Part No. 055-701-2, S/N: :L oc M o b ile P h ase C o m p o n en ts Component A Ammonium acetatem ethanol, 60:40, v x Comoonent B: Ammonium acetate:methanol. 5:95. v x ' G ra d ie n t p rofile Tim e, min 0 % B Flow. mL/min 0 0.3 1 0 0.3 4.5 100 0.3 6 100 0.3 6.1 100 0.6 8.5 100 0.6 9 0 0.3 ' 11 0 0.3 In je ctio n volum e d im split 10 uL ( ______________________________:_______________________ LC column flow split to *30 ML/min ( uL/m inl into the MS at run start C o lu m n T em p Ambient lU 'L C P ressu re 1000 psi at gradient start ( osi) ' M S Source Electrosnrav. Negative Ion U esolvatinn as N eb u lizer as Nitrogen at 575 L/hr ( Nitrogen at 80 L/hr ( L/hr) L/hr) S o u r c e B in d s T e m p *140C( o 1)cmiI\ a t i o n T e m p *250C f a C o n e voltage *70 V (___ *20 v r V) P F O S , - V) PFOSA, PFOSAA PFOSEA, M-556, M570 C ollision energy *40 eV (___ eV) PFOS, I t PFOSA PFOSAA, M-556, M570 *30 eV ( eV) PFOSEA C ollisio n as 1 Argon at *2.5 x HT* mb gas cell ( mb) M u ltip lie r R estitution I 650 V ( ___ Y)___ ____ ._________________________________ ___________ 1 1 *12.0 for MSI ( ): *10.0 for M S2 ) JV/ouAi ACft " S f" } Page 11 of 16 3MEnvironmental Laboratory C-1 * P age 111 3M M edical D epartm ent Study: T-6316.5 A nalytical Study: FACT-TO X-013 L R N -U 2095 _ Battelle Study Number: N003604-D 3M Environmental Laboratory Study Number: FACT 060998.1 METHOD FOR ANALYSIS OF POTASSIUM PERFLUOROOCTANESULFONATE (PFOS) IN RAT LIVER BY LC/MS/MS V enion 1.0 Study No.: Analyst/Date: _ Ions m onitored Total run time A pproxim ate retention times: 427>81 MRM transition for Surrogate Standard (SS) 1 499>99 MRM transition for PFOS 1 556>78 MRM transition for M-556 570169 MRM transition for M570 1 584>169 MRM transition for PFOSAA 1 498>78 MRM transition for PFOSA 526>169 MRM transition for PFOSEA *11 minutes ( min) SS: 4 min ( min) PFOS: 4.3 min ( ___min) M-556: 4 a min ( ____min) M 570:4.6 min ( __ min) PFOSAA: 4.7 m in e min) PFOSA: 5.1 min (______min) PFOSEA: 5.7 min 1 min) * Parameters that may be changed by the analyst Actual values in ( ). 2. The above conditions should be suitable for the Micromass Quattro LC (S/N 9053). Modifications may be necessary if another Micromass Quattro Series spectrometer is used. Split the flow post-column via a Keystone BIO-tee or sim ilar device. 3. Calibrate the mass spectrometer using a suitable reference compound, o r verity that the calibration is suitable by visual inspection (on the tune page) that a suitable mobile phase ion is still accurately determined. Resolution may need to be higher than that used for analyzing samples. -' 4. To check the proper performance o f the instrument, inject the instrument check standard. The results should be comparable to a recent injection if available. 5. Use an automated chromatography integration software system to collect the output from the analysis. 6. Loading O rder See the loading report from the automated chromatography integration software system. 7. Make single injections of each cal standard, QC, study sample, or blank. Make at least 4 injections of the instrument check standard. 8. Run set sizes should typ ica lly no t exceed 80 injections due to instrum ent response roll-off considerations. Longer runs may be performed, but they pose a risk o f yielding unacceptable curve results. VUL CALCULATIONS 1. Spreadsheet Software: ___________________ V ersion________ 2. MS Analysis Software:_____________________ V ersion_______ 3. Calculate the average density of the liver homogenate (10 reps) in mg/mL. 4. Using the average density of the homogenate, calculate its liver density (mg of liver per mL of diluted homogenate): Undiluted liver density (mg/mL) - (g o f liver x average density o f homogenate)/(g o f liver + g of water) where g of liver and g o f water are masses used to prepare bulk homogenate; density of water is assumed to b e l g/mL. Diluted liver density (mg/mL) " Undiluted density + D iln Factor Page 12 of 16 3MEnvironmental Laboratory F.-16 Page 112 3M M edical D epartm ent Study: T-6316.5 A nalytical Study: FACT-TO X-013 L R N -U 2095 Battelle Study Number: N003604-D 3M Environmental Laboratory Study Number: FACT 060998.1 METHOD FOR ANALYSIS OF POTASSIUM PERFLUOROOCTANESULFONATE (PFOS) IN RAT LIVER BY LC/MS/MS ' V ersion 1.0 Study N o.:___________________________ A n alyit/D ate:_____________________________ W here diin factor " 2/1.8 m 1.1111 to accountfor 10% rtfln ofliver homogenate in cal std and QC matrices. 5. Calculate the actual concentration (ng/mL) ofPFOS and other flnorochemicals in the suspensions of calibration standards and QCs by using the mass of analytes and dilution factors only (no liver density collection). Use purity correction for FFOSAA only. 6 . fa lm tn ti the actual concentration o f PFOS and other fln n m rhe m ira lt In liw r fnr rim calibration standards and QCs as follows: Conc(|ig/g) " Cone (ng/mL) * Diluted Liver density (mg/mL) x 1000 mg/g x 10 pg/ng 7 . Assure that the integrations o f the peak areas of the test article and surrogate standard are correct Flag manual integrations where performed. Calculate the exact concentration of each .- liver standard. 8. Calculate the regression equation relating the peak response ratio (test article/SS) o f each calibration standard (y-axis) to test article concentration in liver (x-axis) for PFOS, M -556, M570, and FFOSAA. Calculate the regression equation relating the peak area of r h calibration standard to test article concentration in liver for PFOSA and PFOSEA. PFOS, M 356, M570, and FFOSAA are quantitated by using the surrogate standard as an internal standard: PFOSA and PFOSEA are quantitated without reference to the surrogate fexternal standard calibration curve). Use a quadratic regression weighted 1 /x , origin excluded, fo r all analytes. 9. Calculate a determined concentration for each injection of calibration standard, QC, and ' sample using the regression parameters and the peak response ratios or areas. 10. rnlm lati the relative error, average relative error, standard deviation, and relative standard deviation for all QCs. Calculate foe relative error for each injection of calibration standard. 11. rn lm la tK the average recovery fo r die hom ngenrar nera veiy fnTtifiratifin e 12. Calculate the relative standard deviation for foe PFOS to SS peak area ratio o f the replicate * o f the standard. IX. a c c e p t a n c e C r it e r ia A. MS Check Standard (System Suitability) At least 3 injections of foe MS Check Standard must provide a %RSD of 10% or less for the PFOS to SS peak area ratio. B. Calibration Standards The percent relative errors for the concentration-level averages of the calibration standards should m eet the following limits: Page 13 of 16 3MEnvironmental Laboratory E-17 P age 113 3M M edical D epartm ent Study: T-6316.5 Analytical Study: FACT-TOX-013 L R N -U 2095 _ Battelle Study Number: N003604-D 3M Environmental Laboratory Study Number: FACT 060998.1 METHOD FOR ANALYSIS OF POTASSIUM PERFLUOROOCTANESULFONATE (PFOS) IN RAT LIVER BY LC/MS/MS V ersion L0 Study No.: Analyst/Date: _ Table 9. Calibration Standard Acceptance lim its ANALYTE % Error PFOS 20 05% at LOO) M -556 20 05% at LOO) M570 20 05% at LOO) PFOSAA 20 05% at LOO) PFOSA 20 05% at LOO) PFOSEA 25f30% atL 001 Up to 5 calibration standard injections may be excluded from the curve, provided that one injection remains per level. Removal o f an entire level may be done if approval is obtained. If an entire level is-.removed, the samples bracketed by the remaining calibration range w ill be - considered acceptable. The calibration curve should have a coefficient of determination o f 0.97 or better. C. QCj The concentration-level average percent relative errors and percent relative standard deviations ofthe QCs should meet the following limits: Table 10. QC Acceptance limits ANALYTE % PFOS M -556 M570 20 20 20 PFOSAA 20 PFOSA PFOSEA 20 25 Removal of individual values from the QC calculations may be done if accompanied by a reasonable explanation (e.g_, instrument m alfunction or Dixon's Q test results). I f d ie average determ ined concentration fo r any Q C level exceeds the acceptance lim it, the tad : leader or study director should be notified. The nin may be repeated or a portion of the run may be considered acceptable. F or example, if the low QC fails the stated requirements, samples may be accepted that have concentrations bracketed by the highest calibration standard and a mid-level QC concentration. D. Homogenizer Recovery and Dilution Check Samples The average recovery across the 3 levels o f homogenizer recovery samples as well as that o f the dilution check samples should fill w ithin the range o f70-130% inclusive. Removal of individual outliers fiom the calculations may be done if accompanied by a reasonable explanation. E. Sensitivity (LOQs) ' The validated lim its o f quantitation are nominally 0.13 pg/g each for PFOS, M -556, M570, and PFOSAA. Page 14 of 16 3MEnvironmental Laboratory C 1Q Page 114 3M M edical D epartm ent Study: T-6316.5 A nalytical Study: FA CT-TO X-013 L R N -U 2095 _ Battelle Study Number: N003604-D 3M Environmental Laboratory Study Number: FACT 060998.1 METHOD FOR ANALYSIS OF POTASSIUM PERFLUOROOCTANESULFONATE (PFOS) IN RAT LIVER BY LC/MS/MS VenioB 1.0 Study N o.:___________________________ . ' Anaiyrt/Date: _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ For PFOSA and PFOSEA, the validated LOQs are nominally 0.33 pg/g each. Due to the nature of die preparation of die calibration standards, lower concentrations afPFO SA and PFOSEA w ill be carried through the extraction. These lower concentration values w ill be evaluated with each run set, and may be included in the regressions if they m eet acceptance criteria. If they are included, study samples which are quantitated to have concentrations below the validated level (nominally 0.33 pg/g) w ill be appropriately flagged. F. Specificity The method suffers from endogeneous m atrix interferences a t levels sometimes exceeding 20% o fLOQ. The intercept o f die calibration curve appears to offer some correction fo r any - effect on quantitations. Acceptai performance (error) o f the lowest used standard, therefore, w ill be considered sufficient evidence that bracketed study samples are quantified property. G. General The above acceptance criteria indicate that this method is capable o f producing occasional errors outside the normal acceptance criteria of a validated method (15% normally). Where indicated, replicate analyses lessen die impact o f these occasional outliers. X . RESULTS See attached hard copy o f spreadsheet or see file on network drive. XL C o m m e n t s 3MEnvironmental Laboratory Page 15 of 16 T? t n Page 115 3M M edical D epartm ent Study: T-6316.5 A nalytical Study: FA CT-TO X-013 L R N -U 2095 _ Battelle Study Number: N003604-D 3M Environmental Laboratory Study Number: FACT 060998.1 METHOD FORANALYSIS OFPOTASSIUM PERFLUOROOCTANESULFONATE (PFOS) INRATLIVER BYLC/MS/MS Verjion U> Study No.: - Analyst/D ate:____ X3L C o n c l u sio n s XHL S ig n a t u r e s Analysts _____________________________________________ . - - _____________________________________ _____________________________________________ _____________________________________________ TechnicalReview QCReview D ate: ' Date: D ate: D ate: Date: D ate: D ate: Date: 3MEnvironmental Laboratory Page 16 of 16 Page 116 3M M edical D epartm ent Study: T-6316.5 A nalytical Study: FA CT-TO X-013 L R N -U 2095 _ Battelle Study Number: N003604-D 3M Environmental Laboratory Study Number: FACT 060998.1 Study Title C om b in ed Oral (G avage) F ertility D ev elo p m en t and Perinatal/Postnatal R eproduction T oxicity Study o f N -E tF O SE in R ats PROTOCOL AMENDMENT NO. 1 Amendment Date: July 2 8 ,1 9 9 9 Performing Laboratory 3M E nvironm ental T ech n ology & Safety Services 3M E nvironm ental Laboratory 935 Bush A venue S t Paul, M N 551 0 6 Laboratory Project Identification ET& SS F A C T -T O X -013 L IR N U 2095 3M Environmental Laboratory 3MEnvironmental Laboratory E-21 Page 117 3M M edical D epartm ent Study: T-6316.5 Analytical Study: FACT-TOX-013 L R N -U 2095 _ Battelle Study Number: N003604-D 3M Environmental Laboratory Study Number: FACT 060998.1 Protocol FACT-TOX-013 Amendment 1 This amendment modifies the following portion(s) of the protocol: 1. PROTOCOL READS: The proposed study completion date is listed as 12/31/98. A m end TO READ: The proposed study completion data is 6/30/00. Reason: The proposed completion date was changed to allow time for analyzing all matrices of interest. Amendment Approval r Marvin Case Ph.D., Sponsor Representative Kris J. Ik Ph.D., Study Director g /2 -/^ 5 Date 3M Environmental Laboratory 3MEnvironmental Laboratory T"' ^ f l Page 118 3M M edical D epartm ent Study: T-6316.5 Analytical Study: FACT-TOX-013 L R N -U 2095 _ Battelle Study Number: N003604-D 3M Environmental Laboratory Study Number: FACT 060998.1 Study Title Combined Oral (Gavage) Fertility Development and Perinatal/Postnatal Reproduction Toxicity Study of N-EtFOSE in Rats PROTOCOL AMENDMENT NO. 2 Amendment Date: September 10,1999 Performing Laboratory 3M Environmental Technology & Safety Services 3M Environmental Laboratory 935 Bush Avenue St. Paul, MN 55106 Laboratory Project Identification ET&SS FACT-TOX-013 LIRNU2095 i 3M Environmental Laboratory 3MEnvironmental Laboratory t: 0-2 Page 119 3M M edical D epartm ent Study: T-6316.5 Analytical Study: FACT-TOX-013 L R N -U 2095 _ Battelle Study Number: N003604-D 3M Environmental Laboratory Study Number: FACT 060998.1 Protocol FACT-TOX-013 Amendment 2 This am endm ent m odifies the follow ing portion(s) o f the protocol: 1. PROTOCOL reads: The protocol states that liver will be extracted and analyzed at the 3M Environmental Laboratory. AMEND TO read: The liver specimens will be extracted and analyzed at Battelle Memorial Institute, 505 King Avenue, Columbus, Ohio 43201-2693. REASON: The liver specimens will be sent to Battelle Memorial Institute for extraction and analysis due to time constraints in the 3M Environmental Laboratory. 2. PROTOCOL reads: The protocol states that serum specimens will be extracted and analyzed following methods: FACT-M-3.0, "Extraction of Potassium Perfluorooctanesulfonate or Other Anionic Surfactants from Serum for Analysis Using HPLC-Electrospray/Mass Spectrometry" FACT-M-4.0, "Analysis o f Fluorochemicals in Serum Extracts Using HPLCElectrospray/Mass Spectrometry" AMEND to read: The serum specimens will be extracted and analyzed following methods: ETS-8-4.1, "Extraction o f Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds from Serum for Analysis Using HPLC-Electrospray Mass Spectrometry" ETS-8-5.1, "Analysis o f Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds in Serum Extracts HPLC-Electrospray Mass Spectrometry" REASON: The extraction and analytical methods FACT-M-3.0 and FACT-M-4.0, respectively, were updated on 04/27/99 to ETS-8-4.1 and ETS-8-5.1. 3M Environmental Laboratory 3MEnvironmental Laboratory t: 'a Page 120 3M M edical D epartm ent Study: T-6316.5 Analytical Study: FACT-TOX-013 L R N -U 2095 Battelle Study Number: N003604-D 3M Environmental Laboratory Study Number: FACT 060998.1 Protocol FACT-TOX-013 Amendment 2 3. PROTOCOL reads: The protocol states that liver specimens will be extracted and analyzed following methods: FACT-M-1.0, "Extraction of Potassium Perfluorooctanesulfonate or Other Anionic surfactants from Liver for analysis Using HPLC-Electrospray/Mas Spectrometry" FACT-M-2.0, "Analysis o f Frluorochemicals in Liver Extracts Using HPLC- . Electrospray/Mass Spectrometry" AMEND TO read: The liver specimens will be extracted and analyzed following method: Method for Analysis of Perfluorooctane Sulfonate (PFOS) in Rat liver by LC/MS/MS, Version 1.0 REASON: Since the liver extraction and analysis was sub-contracted to Battelle Memorial Institute, this amendment was written to include their liver methods and titles. Amendment Approval * _ ________ Marvin case Ph.D.-, Sponsor Representative h p f .l is p . f o b hk Kristen J. Hansen Ph.D., Study Director 3M Environmental Laboratory 3MEnvironmental Laboratory r nr Date P age 121 3M M edical D epartm ent Study: T-6316.5 A nalytical Study: FACT-TO X-013 L R N -U 2095 _ Battelle Study Number: N003604-D 3M Environmental Laboratory Study Number: FACT 060998.1 Study Title Analytical Study 2(N-Ethylperfluorooctanesulfonamido)-ethanol in Two Generation Rat Reproduction PROTOCOL AMENDMENT NO. 3 Amendment Date: October 4,1999 Performing Laboratory 3M Environmental Technology & Safety Services 3M Environmental Laboratory 935 Bush Avenue St. Paul, M N 55106 Laboratory Project identifcation ET&SS FACT-TOX-013 LIRNU2095 3MEnvironmental Laboratory 3MEnvironmental Laboratory E-26 Page 122 3M M edical D epartm ent Study: T-6316.5 Analytical Study: FACT-TOX-013 L R N -U 2095 _ Battelle Study Number: N003604-D 3M Environmental Laboratory Study Number: FACT 060998.1 * j. Protocol FA C T Tox-013 Amendment Number 3 This amendment modifies the following portion(s) of the protocol: 1. P ro to co l Re a d s : Kristen J. Hansen, Ph.D. is the Study Director. A mend to Read: James K. Lundberg, Ph.D. is the Study Director. Reason: Original study design has changed due to availability o f resources and James K. Lundberg will begin serving as the study director for'FACT-TOX-013 as o f 4 October 1999. 2. Protocol Reads: Section 7.1 states that the following raw data and records will be retained in the study folder in the archives according to AMDT-S-8: Approved protocol and amendments; study correspondence; shipping records; raw data; and electronic copies o f data. Additionally, Section 7.2 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: Training records; calibration records; instrument maintenance logs; Standard Operating Procedures, Equipment Procedures, and Methods; and appropriate specimens. A mend to Read: Section 7 states: "The original data, or copies thereof, will be available at the 3M Environmental Laboratory to facilitate audits o f the study during its progress and before acceptance o f the final report. When the final report is completed, all original paper data, including: approved protocol and amendments, study correspondence, shipping records, raw data, approved final report, and electronic copies o f data will be retained in the archives of the 3M Environmental Laboratory. All corresponding training records, calibration records, instrument maintenance logs, standard operating procedures, equipment procedures, and methods will be retained in the archives o f the facility performing each analysis. Reason: To direct subcontract laboratories in the disposition of the items listed above. 3M Environmental Laboratory 3MEnvironmental Laboratory P age 123 3M M edical D epartm ent Study: T-6316.5 Analytical Study: FACT-TOX-013 L R N -U 2095 _ Battelle Study Number: N003604-D 3M Environmental Laboratory Study Number: FACT 060998.1 Protocol FACT Tox-013 Amendment Number 3 3. Protocol reads: Disposition o f test and control substances: Biological tissues and fluids are retained per GLP regulation. Amend to read: Specimens will be maintained in the 3M Environmental Laboratory specimen archives. All specimens sent to sub-contract laboratories will be returned to the 3M Environmental Laboratory upon completion o f analysis and submission of the sub-contract laboratory(s) final report The specimens 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 o f specimens analyzed at subcontract laboratories. Amendment Approval Marv Case, D.V.M., Ph.D., Sponsor Representative H (^ r Date f t L & ----------- ,__________^ ________ ;______________________ t O / Z f U Kristen J. Hansen, Ph.D., Previous Study Director Date \ Dale L. Bacon, PteT 3M Environmental Laboratory Management 3M Environmental Laboratory 3MEnvironmental Laboratory c oo Page 124 3M M edical D epartm ent Study: T-6316.5 A nalytical Study: FA CT-TO X-013 L R N -U 2095 Study Title A nalytical Study o f 2(N -Ethylperfluorooctanesulfonam ido)-ethanol in Tw o Generation Rat Reproduction PROTOCOL AMENDMENT NO. 4 Amendment Date: 20 January 2000 Performing Laboratory 3M Environmental Technology & Safety Services 3M Environmental Laboratory 935 Bush Avenue St. Paul, M N 55106 Laboratory Project Identification ET&SS LRN-U2095 FACT TOX-013 Argus Study: 418-009 3M M edical Department Study: T -6316.5 3M Environmental Laboratory 3MEnvironmental Laboratory E-29 Page 125 3M M edical D epartm ent Study: T-6316.5 Analytical Study: FACT-TOX-013 L R N -U 2095 Protocol L R N -U 2095 Amendment Number 4 This amendment modifies the following portion(s) of the protocol: 1. Protocol reads: The study director for the present study was identified in the protocol as James K. Lundburg, Ph.D. A mend to read: The role of study director for the present study was reassigned to Marvin T. Case, D.V.M ., Ph.D., as of 20 January 2000. The previous study director, James K. Lundburg, 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 C FR Part 58). 2. Protocol reads: The sponsor for the present study was identified as Marvin T. Case, D.V.M ., Ph.D. A mend to read: The role of sponsor for the present study was reassigned to John L. Butenhoff, Ph.D., as of 20 January 2000. Reason: To ensure that the study director does not also carry the duties of study sponsor, the sponsor role was reassigned. In this manner, personnel responsibilities and workload are more evenly balanced. m j r* _____________ i _ i i _ l 3MEnvironmental Laboratory E-30 Page 126 3M M edical D epartm ent Study: T-6316.5 Analytical Study: FACT-TOX-013 L R N -U 2095 Protocol LRN-U2095 Amendment Number 4 Amendment Approval John L B utenhoffP h D ., Sponsor Representative to , D ate 3 h t A M a rv in T. Case, D . V.M ., P h D ., Incom ing Study Director J .P __ R -U aju Date V Q ^tr 3 M Environmental I ahnratnrv 3MEnvironmental Laboratory E-31 Page 127 3M M edical D epartm ent Study: T-6316.5 Analytical Study: FACT-TOX-013 L R N -U 2095 Study Title Analytical Study o f 2(N-Ethylperfluorooctanesulfonamido)-ethanol in Two Generation Rat Reproduction PROTOCOL AMENDMENT NO. 5 Amendment Date: August 31,2000 Performing Laboratory 3M Environmental Technology & Safety Services 3M Environmental Laboratory 935 Bush Avenue St. Paul, MN 55106 Laboratory Project identification FACT-TOX-013 ET&SS LRN U2095 Argus Study: 418-009 3M Medical Department Study: T6316.5 3MEnvironmental Laboratory Page 128 3M M edical D epartm ent Study: T-6316.5 A nalytical Study: FA CT-TO X-013 L R N -U 2095 - Protocol FA C T TO X-013 Amendment No. 5 This am endm ent m odifies th e follow ing portion(s) of the protocol: 1. PROTOCOL r e a d s : The Principle Analytical Investigator for the present study was identified as James K. Lundberg, Ph.D. 2 . AMEND TO r e a d : The role o f Principle Analytical Investigator for the present study was reassigned to Kristen J. Hansen Ph.D. REASON: The role o f Principle Analytical Investigator was reassigned due to availability of resources. 3 M Fnvironm antal Laboratorif 3MEnvironmental Laboratory E-33 Page 129 3M M edical D epartm ent Study: T-6316.5 ' - Amendment Approval Analytical Study: FACT-TOX-013 L R N -U 2095 Protocol F A C T TO X-013 Amendment No. 5 John L. Butenhoff, Ph.D., Sponsor Representative ; rc ^ M arvin T. Case, D .V .M ., Ph.D., Study Director Date Date Fnwirnnmontal I ahnm tnn/ 3M Environmental Laboratory E-34 Page 130 3M M edical D epartm ent Study: T-6316.5 Analytical Study: FACT-TOX-013 L R N -U 2095 _ Battelle Study Number: N003604-D 3M Environmental Laboratory Study Number: FACT 060998.1 DEVIATION REPORT B attelle Study N um ber: N 003 6 0 4 -D 3 M E nvironm ental T ech n ology and S ervices Study N um ber: F A C T 0 6 0 9 9 8 .1 2 (N -E th ylp erflu orooctan esu lfon am id o)-eth an ol in T w o G eneration R at R eproduction TYPE OF DEVIATIONS: PROTOCOL DATES O F DEVIATIONS: October 18, 1999 NA TU RE O F DEVIATIONS: Some o f the analytical method acceptance criteria were not met for the LC/M S/M S analysis conducted 180ct99 at Battelle. These deviations relate to protocol amendment 2. See below for summary. A nalvte A cceptance criterio n n o t m et PFOS M -556 M -556 M -556 M -556 PFOSAA PFOSAA QC3 exceeded 20 error (-20.3 actual! QC2 exceeded 20% error (23.4 actual) ( QC3 exceeded 20% RSD (21.2 actual) QC4 exceeded 20% RSD (28.8 actual) Dilution recovery exceeded 130% (131.5 actual with 21.6 RSD) QC3 exceeded 20 error (-20.6% actual) QC4 exceeded 20 RSD (21.3 actual) CAUSE O F DEVIATIONS: Sample preparation and/or LC/M S/M S variabilities over the course o f the sample set may have contributed to the deviations. IM PA C T O F DEVIATIONS O N T H E STUDY: The errant QC values w ere bracketed by acceptable QC concentration levels w hich demonstrates that the calibration curves generally provided good accuracy over tho tested range. The dilution recovery for M-556 was not considered to be exceedingly high enough, at only approximately 1.5 above the normal acceptance level, to have significantly impacted the data. CORRECTIVE ACTION :This protocol deviation summary was prepared for inclusion in the final report. APPROVED BY: Cv Jon dyA ndre, Ph.D. Battelle Principal Investigator Z . - Z . l - ' C 'i Date k u v/W k J~~C xsij-- Jam es-K . Lwndbarg , PhrD . Study D irector f r s t>V*\ f t p J / fi'yuu Q-4- ( Date N003604-D Protocol Deviation 1, Page 1 of 1 3MEnvironmental Laboratory E-35 P age 131 3M M edical D epartm ent Study: T-6316.5 A nalytical Study: FA CT-TO X-013 L R N -U 2095 _ Battelle Study Number: N003604-D 3M Environmental Laboratory Study Number: FACT 060998.1 DEVIATION REPORT B attelle Study N um ber: N 0 0 3 6 0 4 -D 3M E nvironm ental T ech n ology and S ervices Study N um ber: F A C T 0 6 0 9 9 8 .1 2 (N -E th ylp eifluorooctan esulfonam ido)-eth anol in T w o G eneration R at R eproduction TYPE OF DEVIATIONS: PROTOCOL DATE O F DEVIATIONS: October 20, 1999 NA TU RE O F DEVIATIONS: PFO S QC3 exceeded the 20% RE method requirement (actual -2 1 .7 % ). The dilution recovery check standard did not meet the 70-130% recovery requirement (actuals 5.0% for PFOS and 4.6% for PFOSA). These method deviations relate to amendment 2 o f the study protocol. CAUSE O F DEVIATIONS: Sample preparation and/or LC/M S/M S variabilities over the course o f the sample set may have contributed to the QC deviation. Sample preparation error appears to have been the cause for the dilution recovery results. IM PA CT OF DEVIATIONS ON T H E STUDY: The errant QC value level w as bracketed by acceptable QC concentration levels which demonstrates that the calibration curves generally provided good accuracy for study samples over the tested range. A comparison o f the results obtained for the diluted study samples from 20Oct99 and previous results that w ere Slightly LO Q (130ct99 and 180ct99) demonstrated good agreem ent betw een the 2 determinations. This w ould indicate that the dilution o f the study samples was perform ed correctly 20Oct99 so that no impact on the quantitations occurred. CORRECTIVE ACTION: This protocol deviation summary was prepared for inclusion in the final report. APPROVED BY: Battelle Principal Investigator o D ate Study Director Date j N003604-D Protocol Deviation 2, Page 1 of 1 E-36 3MEnvironmental Laboratory Page 132 3M M edical D epartm ent Study: T-6316.5 A nalytical Study: FACT-TO X-013 L R N -U 2095 - Battelle Study Number: N003604-D 3M Environmental Laboratory Study Number: FACT 060998.1 DEVIATION REPORT B attelle Study N um ber: N 00 3 6 0 4 -D 3M E nvironm ental T ech n ology and S ervices Study N um ber: F A C T 0 6 0 9 9 8 .1 2 (N -E thylperfluorooctanesulfonam ido)-ethanol in T w o G eneration R at R eproduction T Y P E O F D EV IA T IO N S: PR O TO C O L D A T E S O F D E V IA T IO N S: O ctober 12, 1999 through O ctober 2 0 ,1 9 9 9 N A T U R E O F D E V IA T IO N S: T he lot num ber o f PFO S used w as not 2 1 7 as per section 3 .1 .1 o f th e p rotocol. T h e sou rce o f reference substance m atrix w as not A rgu s R esearch L aboratories as sp ecified in section 3 .2 .2 o f the protocol. In itial an alyses o f liver did not exclu sively target P F O S as p er section 4 .0 o f p rotocol. C A U S E O F D E V IA T IO N S : O nly P F O S lo t num ber 171 w as available at B attelle. H arlan w as th e supplier o f control rat livers used to prepare blanks, standards, and Q C s fo r th e analytical portion o f th e study. A ll 4 analytes o f interest (P F O S, M -5 5 6 , P F O S A A , and P F O S A ) w ere m onitored during each analysis. IM P A C T O F D E V IA T IO N S O N T H E S T U D Y : PFO S lot num ber 171 and H arlansupplied liv er w ere both u sed fo r B attelle's valid ation o f the analytical m eth od (B attelle study num ber N 0 0 3 6 0 4 -A ). T h ese m aterials a llo w ed achievem en t o f th e reported m ethod acceptance criteria so that there is no im pact on the study. T he concurrent analysis o f P F O S and m etab olites w as an efficien cy im provem ent. C O R R E C T IV E A C T IO N : T h is protocol d eviation report w as prepared. APPROVED BY: C Cud'_L_ Jon(G . A ndre, P h .D . B attelle P rincipal Investigator z- zz^ D ate r _______ Jam es K . Lundberg, P h .B . Study D irector N003604-D Protocol Deviation 3, Page 1 of 1 E-37 3MEnvironmental Laboratory __ D ate P age 133 3M M edical D epartm ent Study: -6316.5 A nalytical Study: FA CT-TO X-013 L R N -U 2095 Battelle Study Number: N003604-D 3M Environmental Laboratory Study Number: FACT 060998.1 APPENDIX F - PFOS PURITY REPORT 3MEnvironmental Laboratory Page 134 3M M edical D epartm ent Study: T-6316.5 ETSS 2 3W A nalytical Study: FACT-TO X-013 L R N -U 2095 _ Battelle Study Number: N003604-D 3M Environmental Laboratory Study Number: FACT 060998.1 ^651 778 4226 04/26/99 09:56 0 :02/03 NO:341 I 2I From: Subject: D a te: 3M SPECIALTY ADHESIVES & CHEMICALS ANALYTICAL LABORATORY L isa Clem en - (8-5568) - ET& SS- 2-03-09 Request # 57830 T om K estner - (3 -5 6 3 3 ) - SA & C Analytical Lab 236-2B -11 Chemical Characterization ofPOSF-BasedFluorochemieals by `H-NMR &1}F-NMR Spectroscopy M atch 2 4 ,1 9 9 9 : Prelim inary report for FC -95 (PFO S), lot 171 SAMPLE DESCRIPTION F C -9 5 , lo t 171 (P F O S ), TN-A-0834; N o m in a l product = C F ir S 0 3(-) K (+ ) (w h ite p o w d er) INTRODUCTION: T h is sa m p le w a s s u b je c te d to 'H -N M R and 1SF -N M R spectral a n alyses to determ in e the p u rity o f th e n o m in a l product and to characterize as m any impurity com ponents as possible. A portion o f the sam ple w as accurately w eighed, spiked with a known amount o f 1,4-bis(tiifluorom eth yl)b en zene (p-H F X ), and then totally dissolved in DM SO-d for subsequent analysis by N M R . A 400 M H z 'H -N M R sp ectru m ( # h 5 7 8 3 0 .4 0 1 ) and a 3 7 6 M H z t9F -N M R spectrum ( # (5 7 8 3 0 .4 0 1 ) w e re acquired u s in g a V a r ia n U N IT Y p lu s 4 0 0 F T -N M R spectrom eter. U se o f the p-H FX internal standard w as intended to perm it the determ ination o f the absolute w eigh t percent concentrations o f the assigned com ponents w ithout n ecessarily needing to identify or quantify all the com ponents in the sam ple mixture. RESULTS: T he com bined N M R spectral data w ere used to assign all o f the major and m ost o f the minor com ponents in this sam p le as r ec eiv ed . T h e qu alitative and quantitative com positional results that w e re derived fro m the sin g le trial N M R internal standardization an alyses are summarized in T A B L E -1 on the follow in g page. 1 have reported both relative and absolute w eig h t percent concentrations. One possible reason that the absolute wt.% values add up to m ore than 100% m ay be due to the fact that I assum ed all o f the com ponents contained 8 carbons. If there w ere any shorter chain hom ologs present (i.e., 7, 6, S, ere. carbons), then the average com pound m olecular w eigh ts w o u ld h a v e b e e n so m e w h a t le s s than th ose used in the calcu lation s. In general, the f*F -N M R te ch n iq u e is n o t particularly w ell suited for identifyin g or quantifying sm all amounts o f various fluorochem ical h o m o lo g impurity com ponents u n less the chains are very short. A more com plete characterization o f any other fluorochem ical h om ologs w ould require analysis b y electrospray M S or a sim ilar technique. A d dition al w ork w ou ld b e required in an effort to positively verify the tentatively assigned com p on en ts listed in T A B L E -1 (den oted by p o ssib le). S m all amounts o f other unidentified im purities are also detected in the N M R spectra, but additional w ork w ou ld b e required in an effort to identify or quantify th ese other m aterials. C op ies o f the N M R spectra w ill be provided for you at a later date. If you have any questions about the results in this initial report for FC -95, lot 171. please let m e know. I apologize for the delay in com pleting this initial work. T om K estner c: Rick Payfer - SAAC Analytical L ab -2 3 6 -2 B -l! Fik iUfcrcncs: LC37S30CC061 3MEnvironmental Laboratory Pat- t P -1 Page 135 3M M edical D epartm ent Study: T-6316.5 ETSS 2 3W March 2 4 ,1 9 9 9 . Analytical Study: FACT-TOX-013 L R N -U 2095 _ Battelle Study Number: N 003604-D 3M Environmental Laboratory Study Number: FACT 060998.1 ^ 651 778 4226 04/26/99 09:56 0 :0 3 /0 3 NO:341 SA & C A nalytical Lab Request # 37830 Initial R eaort fo r F C -93. lo t 171 " TABLE-1 Sam ple: FC -95, lot 171 (PFOS), TN -A -0834 O v er a ll Q u an titative C o m p o sitio n a l R e su lts b y 'H /`*F-N M R Internal S tan d ard ization A n a ly s e s Structural A ssig n m e n ts C F 3( C F ^ - S 0 3(-) K (+ ) (N orm al chain; assum e x =7 for calculation purposes) NM R A bsolute W eight% C on cen tration s (initie trial m easurem ent) 7 0 .3 % NM R R ela tiv e W eight* C oncentrations (tin a ie trial measurement) 6 8 .6 % C F ^ C F J i -CFCCFjK C F jJj-S O jM K (+ ) (Internal m onom ethyl branch; assum e x+ y 5 , a * 0 , A y * 0, for calculation purposes) 17.7% 173% (C F j) jC F -(C F j)*-SO j( - ) K (+ ) (Isopropyl branch; assum e x =3 for calculation purposes) QFji-CF(CFi)-SOi(-) K M (A lpha branch; assum e x * 6 for calculation purposes) P o ssib le F -S F ^ -C J V S O jL ) K (+) (assum e x 3 for calculation purposes) C F jK C F ^ -C C C F jJ H C F ^ -S O jH K (+ ) (Internal gem -dim ethyl branch: assum e x+y - 4 and x * 0 for calculation purposes) 03P o s s ib le CFj-S F *-C |F u -S (-) K (+ ) (assum e x * 7 for calculation purposes) P ro b a b le C iH j r S O j(") K (+ ) (H ydrocarbon sulfonate salt; assum e x a 3 for calculation purposes) (C F 3) 3C -<C Fj) x-S 0 3 (-) K (+ ) (t-butyl branch; assum e x * 4 for calculation purposes) 10.3% 3.3% 0 .3 7 % 0 .1 6 % . 0.11% 0 .0 3 1 % 0 .0 2 7 % 10.2% 3 .2 % 036% 0 .1 6 % 0 .1 0 % 0 .0 3 0 % 0 .0 2 6 % 3MEnvironmental Laboratory Pip.2F-2 Page 136 3M M edical D epartm ent Study: T-6316.5 3M Medical Department Study: T6316.5 A nalytical Study: FA CT-TO X-013 LRN-U2095 Analytical Report: FACT TOX-013 LRN-U2095 Appendix H: Interim Certificate of Analysis 3M Environmental Laboratory 3MEnvironmental Laboratory Page 131 Page 137 3M M edical D epartm ent Study: T-6316.5 proB) 3M M e d i c a l D e p a r t m e n t S t u d y : T 6 3 1 6 . 5 Analytical Study: FACT-TOX-013 LRN-U2095 A n a l y t i c a l R e p o r t : FACT T O X -013 L R N -U 2095 INTERIM CERTIFICATE OF ANALYSIS Revision 1(9/7/00) Centre Analytical Laboratories COA Reference #: 023-018B 3M Product: P F O S ,L otl71 Reference#: SD-009 _____________________ Purity: 86.4% Test Name Specifications PurityJ Result 86.4% Appearance Identification NMR Metals (ICP/MS) 1. Calcium 2. Magnesium 3. Sodium 4. Potassium2 5. Nickel 6 . Iron 7. Manganese Total % Impurity (NMR) Total % Impurity (LC/MS) Total % Impurity (GC/MS) 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 A cids5 (IC) 1. TFA 2. PFPA 3. HFBA 4. NFPA Elemental Analysis6: 1. Carbon 2. Hydrogen 3. Nitrogen 4. Sulfur 5. Fluorine 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% Conforms Positive 1. 0.017 wt./wt.% 2. 0.007 wt/wt.% 3. 1.355 wt/wt.% 4. 6.552 wt./wt.% 5. 0.003 wt./wt.% 6 . 0.004 wt./wt.% 7. <0.001 wt./wt.% 1 .0 0 wt./wt.% 10.60 wt./wt.% None Detected 0.30 wt./wt.% None Detected Not Applicable4 1 . <0.015 wt/wt.% 2. 0.27 wt./wt.% 3. <0.040 wt./wt.% 4. <0.009 wt/wt.% 5. <0.006 wt/wt.% 6 . <0.007 wt/wt.% 7. 8.82 wt/wt.% 1 . <0 .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.% COA023-018B 3M E n v i r o n m e n t a l L a b o r a t o r y 3MEnvironmental Laboratory Page 1 o f 3 Page 132 Page 138 3M M edical D epartm ent Study: T-6316.5 : 3M Medical Department Study: T6316.5 A nalytical Study: FACT-TO X-013 LRN-U2095 Analytical Report: FACT TOX-013 LRN-U2 095 INTERIM CERTIFICATE OF ANALYSIS Centre Analytical Laboratories COA Reference #: 023-018B Date o f Last Analysis: 08/31 /0Q Expiration Date: 08/31/01 Storage Conditions: Frozen <-10C Re-assessment Date: 08/31/01 Purity = 100% - (sum o f metal impurities, 1.39% +LC/MS impurities, 10.60%+Inorganic Fluoride, 0.27%+NMR impurities, 1.00%+ POAA, 0.30%) Total impurity from all tests = 13.56% Purity = 100% -13.56% = 86.4% 2Potassium is expected in this salt form and is therefore not considered an impurity. 3Purity by DSC is generally not applicable to materials o f low purity. N o endotherm was observed for this sample. 4Sulfur in the sample appears to be converted to SO4 and hence detected using the inorganic anion method conditions. The anion result agrees w ell with the sulfur determination in the elemental analysis, lending confidence to this interpretation. Based on the results, the SO4 is not considered an impurity. 5TFA HFBA NFPA PFPA Trifluoroacetic acid Heptafluorobutyric acid Nonofluoropentanoic acid Pentafluoropropanoic acid ^Theoretical value calculations based on the empirical formula, CsFi7S03*K+ (MW=538) This work was conducted under EPA Good Laboratory Practice Standards (40 CFR 160). COA023-018B M-- i 3M Environmental Laboratory -- .1-- -- . -- - ... --11 ... 1- 3M Environmental Laboratory Page 2 o f 3 Page 133 Page 139 3M M edical D epartm ent Study: T-6316.5 3M Medical Department Study: T6316.5 A nalytical Study: FACT-TO X-013 LRN-U2095 Analytical Report: FACT TOX-013 LRN-U2095 INTERIM CERTIFICATE OF ANALYSIS Centre Analytical Laboratories COA Reference #: 023-018R LC/MS Purity Profile: Impurity C4 C5 C6 C7 Total wt./wt. % 1.03 1.56 6.38 1.63 10.60 Note: The C4 and C6 values were calculated using the C4 and C6 standard calibration curves, respectively. The C5 value was calculated using the average response factors from the C4 and C6 standard curves. Likewise, the C7 value was calculated using the average response factors from the C6 and C8 standard curves. Prepared By: ________________________________ ____ David S. Bell Date Scientist, Centre Analytical Laboratories Reviewed B y :__________________________ ;_____ ____ John Flaherty Date Laboratory Manager, Centre Analytical Laboratories COA023-018B 3M Environmental Laboratory 3MEnvironmental Laboratory Page 3 o f 3 Page 134 Page 140 ,3M M edical D epartm ent Study: T-6316.5 , 3M Medical Department Study: T6316.5 Analytical Study: FACT-TOX-013 LRN-U2095 Analytical Report: FACT TOX-013 LRN-U2095 Appendix I: Report Signature Page T ^- Marvin T. Case D.V.M., Ph.D., Study Director Date John L. Butenhoff, Ph.D., Sponsor Representative Date ^ fCm'S FA! 3M Environmental Laboratory 3MEnvironmental Laboratory Page 135 P age 141 3M M edical D epartm ent Study: T-6316.5 3M Medical Department Study: T-6316.5 A nalytical Study: FA CT-TO X-013 L R N -U 2095 Analytical Study: FACT TO X -013 LRN-U2095 Appendix J: Amendment 1 to FACT TOX-013 Final Report TO X-013 Final Study Report Am endm ent 1 Study number: TOX 013 Study title: Analytical Study 2(N-Ethylperfluorooctane sulfonamido)-ethanol in Two Generation Rat Reproduction Study Director: Marvin T. Case, D.V.M., Ph.D. Amendment date: May 7, 2001 Amendment number: 1 This amendment modifies the following portion of the final report: A final signed report from Battelle Memorial Institute, presenting the results for PFOS, PFOSAA, PFOSA, and M-556 levels in rat liver specimens, replaces the draft Battelle report in Appendix G. Liver results in this report are identical to those presented in the original TOX-013 report (Table 13, pages 22-23). As in the original liver data, the PFOS values reported in the Battelle report were corrected by 3M for purity of the reference standard material. The final Battelle report differs from the draft report in the following ways: All signature pages are signed and dated. The Quality Assurance Statement page has four additional audit dates added. Table of Contents page numbers were corrected. Two Battelle participants were eliminated from page 4, `Acknowledgements.' T h e s to ra g e a n d arch iv e instructions (p a g e 4 ) a re n o w fo u n d in th e 3 M T O X - 0 1 3 protocol amendment 3. Inclusion of 3M TOX-013 protocol amendments 4 and 5, thus changing the total number of pages. Minor wording changes. Other changes to the TOX-013 report include: The cover page was updated to reflect the total number of pages and the title was changed to say "Amended Final Report." The Table of Contents was updated to reflect the added amendment. The additional audit date of the Amended Analytical Laboratory Report TOX013 was added to the Quality Assurance Statement. 3M Environmental Laboratory 3MEnvironmental Laboratory Page 135 Page 142 3M M edical D epartm ent Study: T-6316.5 3M Medical Department Study: T-6316.5 Approved by: A nalytical Study: FA CT-TO X-013 L R N -U 2095 Analytical Study: FACT TO X-013 LRN-U2095 Kristen H. Hansen, Ph.D., Principal Analytical Investigator 0 S -/3 O /O I Date Marvin T. Case, D.V.M., Ph.D., S tu d y D ire c to r Bill Reagan, Ph.D., Environmental Laboratory Manager Date O J /J y /o / Date 3M Environmental Laboratory 3MEnvironmental Laboratory Page 135 P age 143