Document NEXZLKxzYQ3z3D6bvkO8znN7w

3M Medical Department Study: T-6316.5 BACK TOMAIN 3M Medical Department Study: T-6316.5 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 3M Medical Department Study: T6316.5 BACKTOMAIN Analytical Report: FACT TOX-013 LRN-U2095 BfflW 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 BACKTOMAIN 3M Medical Department Study: T-6316.5 GLP Compliance Statement 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 BACK TOMAIO 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 , 3 o Q i Date 3M Environmental Laboratory 3MEnvironmental Laboratory Page 4 Page 4 3M Medical Department Study: T-6316.5 BACK TO MAIN 3M Medical Department Study: T-6316.5 Table of Contents Analytical Study: FACT TOX-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 BACK TOMAIN 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 BACKTOMAIN 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 B A C K TO MAIN 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 ost 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 Monel*seif-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) P op u latio n Num ber of F0 G en eratio n R ats per Sex N um ber o f F1 G en eratio n R ats per Sex Dosage (mg/kg/day) Dosage Group I (Control) Dosage Group II Dosage Group III Dosage Group IV Dosage Group V 35 35 35 35 35 25 0 (vehicle) 25 1 25 5 -- 10 _ 15 pm* 3M Environmental Laboratory 3MEnvironmental Laboratory Page 8 Page 8 J M Medical Department Study: T-6316.5 3M Medical Department Study: T6316.5 BACKTO MAIN Analytical Report: PACT TOX-013 LRN-U2095 Specim 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 BACKTOICAIN 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 Expiration Date EtFOSE-OH FM -3929 2(N-Ethyfperfluorooctane sulfbnam ido)-ethanol 3M 05/2 0 0 0 Storage Conditions Am bient tem perature Chemical Lot# Physical Description 30035, 30037, 30039 W axy Solid Purify To be determ ined* * The purity o f the test article determ ined nominally by N M R analysis. Subsequent chem ical characterization is occurring and this analytical report w ill be am ended to indicate the purity when a certificate of analysis is issued. D oss Confirmation A nalyses T h e d o s e c o n firm a tio n d a ta w e re c o lle c te d a c c o rd in g to a m e th o d th a t w a s n o t fully v a lid a te d . 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 BACKTOICAIN 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 E n viro n m e n ta l L a b o rato ry 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 autovial. 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 BACK TO CAIN 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) o f 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. Summary of Quality Control 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 A nalyte M ethod LO Q FFOS PFOSA PFOSAA EtFO SE-O H M 556 5.55 ppb 4 .7 9 ppb 2 0 .5 ppb 36.2 ppb 2 4 .9 ppb 3M Environmental Laboratory Page 13 3M Medical Department Study: T-6316.5 3M Medical Department Study: T6316.5 BACKTOICAIN 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. Summary 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 B A C K TO MAIN 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 L ocatio n 3M Lab C o n tro l M atrix R at Serum (T N -A -2001) R a b b it S erum (T N -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 fo r Liver Analyses in Study FACT TOX-013 L o catio n B attello M em o rial In s titu te C o n tro l M atrix R a t L iv e r 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 BACK TO CAIN 3M Medical Department Study: T-6316.5 Analytical Study: FACT TOX-013 LRN-U2095 Table 8. Characterization of the Analytical Reference Materials Used for Sera Analyses in Study FACT TOX-013 L o catio n 45M L ab M a te ria ls PFOS C ,, F 17S 0 3 . PFOSA c bf 17s o 2n h 2 PFOSAA CbF17S 02N((CH2CH3) (C H 2C O O H )) 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 0 2N(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 M 556 c bf 17s o 2n ((H)(CHjCOOH)) 3M 0 1 /0 1 /2 0 1 0 A m bient tem perature THPFOS* c bh 4f 13s 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 TO X-013 L o c a tio n B a tte lle M em o rial In s titu te M a te ria ls PFOS M 556 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 BACKTOMAIN 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/mL) 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 Homogeneity Samples-- 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-418-009-D, 06/08/98 B-418 -0 0 9 -0 ,0 7 /1 5 /9 8 B-418-009-E, 06/08/98 B-418-009-E, 07/15/98 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 0.00 NA 200000 200000 1000000 100000 2000000 2000000 3000000 3000000 3000000 3000000 3000000 0.00 NA NA NA 1020000 942000 2190000 2750000 3060000 3640000 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-M S 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 , 3M Medical Department Study: T6316.5 Appendix B: Protocol BACKTOMAIN Analytical Report: FACT TOX-013 LRN-U2095 3M Environmental Laboratory 3MEnvironmental Laboratory Page 18 Page 18 3M Medical Department Study: T-6316.5 3M Medical Department Study: T6316.5 Analyt i c m BACKTOICAIN )/^ 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: M arvin 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 Experim ental sta rt date: Expected term ination date: December 31,1998 M ethod num bers and revisions: FA CT-M -1.0, Extraction o f Potassium Perfluorooctanesulfonate or O ther Anionic Surfactants from Liver for Analysis Using HPLC-Electrospray/M ass Spectrom etry FA CT-M -2.0, Analysis o f Fluorochemicals in Liver Extracts Using HPLCElectrospray/M ass Spectrometry FA CT-M -3.0, Extraction of Potassium Perfluorooctanesulfonate or O ther Anionic Surfactants from Serum for Analysis Using HPLC-Electrospray/M ass Spectrom etry FA CT-M -4,0, Analysis o f Fluorochemicals in Serum Extracts Using HPLCElectrospray/M ass Spectrometry Author: Lisa Clemen --------- ----------------------5_l% Kris Hansen D ate Study D irector M arvin 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 B A C K TO MAIN AnalytrcdX K B pui l : xwa- uxj LRN-U2095 1.0 Purpose _________________________________________________________ The analytical portion of this dosing study is designed evaluate the levels of perfluorooctane sulfonate (PFOS), or another m etabolite o f 2(N-ethylperfluorooctanesulfonamido)-ethanol (NEtFOSE-OH) designated by the study director, in the liver of the parent and subsequent generations of the test system, o r in die 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 o f 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: 3.23.1 Rat liver - Argus Research Laboratories; traceability information w ill be included in the final report; or Rabbit liver - Covance Laboratories; traceability information will be included in the final report 3.2.3.2 Rat serum - Sigm a Chemical Company; traceability information will be included in the final report 3.3 Number o f 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 o f 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. 3M Environmental Laboratory 3MEnvironmental Laboratory Page 20 Page 20 3M Medical Department Study: T-6316.5 3M Medical Department c Study: u T6316.5 BACKTOICAIN ' Analyticar Repuxu: i u a -v j T .T D T > ,T ,,T T O nQ C : 3.5 P u rity an d strength of m aterials: Characterization of the purity and identity of the reference material is the responsibility of the Sponsor. 3.6 S tability o f te st m aterial: Characterization of the stability o f the test m aterial is the responsibility of the Sponsor. 3.7 Storage conditions fo r test m aterials: Test materials are stored at room temperature. Samples are stored at --20 10 C. 3.8 D isposition o f test an d /o r 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. W ear 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 of fluorine compounds. At the discretion o f the Study Director, a series of analytical tests w ill be performed on select tissues. Initially, all liver samples will be analyzed for PFOS by electrospray/mass spectrometry (ES/M S). 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 w ill be written. 5.0 Experimental - A nalytical M ethods 5.1 FA CT-M -1.0, Extraction of Potassium Perfluorooctanesulfonate or Other Anionic Surfactants from Liver for Analysis Using HPLC-Electrospray/Mass Spectrometry 5.2 FA CT-M -2.0, Analysis of Fluorochemicals in Liver Extracts Using HPLCElectrospray/M ass Spectrometry 5.3 FA CT-M -3.0, Extraction of Potassium Perfluorooctanesulfonate or Other Anionic Surfactants from Seram for Analysis Using HPLC-Electrospray/Mass Spectrometry 5.4 FA CT-M -4.0, Analysis of Fluorochemicals in Serum Extracts Using HPLCElectrospray/M ass Spectrometry 6.0 Data Analysis pm* 6.1 D ata transform ations and analysis: D ata w ill be reported as the concentration (weight/weight) of fluoride per tissue or sample, or o f PFOS per unit o f tissue or fluid. 6.2 S tatistical 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. 3M Environmental Laboratory 3MEnvironmental Laboratory 3 Page 21 Page 21 3M Medical Department Study: T-6316.5 3M Medical Department Study: T6316.5 BACKTOICAIN Analytrcax Kepun.: iu a - u u LRN-U2095 fSWSHl . -- r* &*< Simse (mm. 7/.0V Maintenance of Raw Data and Records 7.1 The following raw data and records will be retained in the study folder in the archives 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 of data 7.2 Supporting records to be retained separately from the study folder in the archives according to AMDT-S-8 will include at least the following: 7.2.1 Training records ' 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 9.0 Attachments 9.1 FA CT-M -1.0, Extraction of Potassium Perfluorooctanesulfonate or Other Anionic Surfactants from Liver for Analysis Using HPLC-Electrospray/Mass Spectrometry 9.2 FA CT-M -2.0, Analysis o f Fluorochemicals in Liver Extracts Using HPLCElectrospray/M ass Spectrometry 9.3 FA CT-M -3.0, Extraction of Potassium Perfluorooctanesulfonate or Other Anionic Surfactants from Serum for Analysis Using HPLC-Electrospray/M ass Spectrometry 9.4 FA CT-M -4.0, Analysis o f Fluorochemicals in Seram Extracts Using HPLCElectrospray/M ass Spectrometry 3M Environmental Laboratory 3MEnvironmental Laboratory 4 Page 22 Page 22 fsmm3.M Medical Department Study: T-6316.5 3M Medical Department Study: T6316.5 B A C K TO MAIN ' Analytical tepore: AUX IUA-U1J LRN-U2 095 Study Title Combined Oral (Gavage) Fertility Development and'Perinatal/Postnatal Reproduction Toxicity Study o f N-EtFOSE in Rats PROTOCOL AMENDMENT NO. 1 Amendment Date: July 28,1999 Performing Laboratory 3M Environm ental Technology & Safety Services 3M Environm ental Laboratory 935 Bush Avenue S t 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 23 Page 23 3M Medical Department Study: T-6316.5 3M Medical Department Study: T6316.5 BACK TO CAIN Analytxcax Keporc : a l i i u a -u u 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 r e a d s : The proposed study completion date is listed as 12/31/98. AMEND TOr e a d : The proposed study com pletion data is 6/30/00. REASO N: The proposed com pletion date was changed to allow tim e for analyzing all m atrices o f interest. Amendment Approval M arvin Case Ph.D ., Sponsor Representative Kris J. a Study D irector J o ^ t U !4 < ? f Dat/ D ate 3M Environm ental Laboratory 3M Environmental Laboratory 3MEnvironmental Laboratory Page 24 Page 24 3M Medical Department Study: T-6316.5 3M Medical Department Study: T6316.5 B A C K TO MAIN A n a ly tic a l tvcyui u, l'nui x\uxx wj--LRN-U2 095 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, MN 55106 Laboratory Project Identification ET&SS FACT-TOX-013 L IR N U 2095 * - 3M Environm ental Laboratory 3M Environmental Laboratory 3MEnvironmental Laboratory Page 25 Page 25 3M Medical Department Study: T-6316.5 3M Medical Department Study: T6316.5 BACKT OMAIN LRN-U2095 Protocol FA CT-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 B attelle M em orial Institute, 505 King Avenue, Columbus, Ohio 43201-2693. REASON: The liver specimens 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 specimens w ill be extracted and analyzed following methods: FACT-M -3.0, "Extraction o f Potassium Perfluorooctanesulfonate or Other Anionic Surfactants from Serum for Analysis Using HPLC-Electrospray/M ass Spectrom etry" FACT-M -4.0, "Analysis o f Fluorochemicals in Serum Extracts U sing HPLCElectrospray/M ass Spectrometry" AMEND to read: The serum specimens w ill be extracted and analyzed following m ethods: ETS-8-4.1, "Extraction o f Potassium Perfluorooctanesulfonate or Other Fluorochem ical Compounds from Serum for Analysis Using HPLC-Electrospray M ass Spectrometry" ETS-8-5.1, "Analysis 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 BACKT OMAIN Analytical Repul i; irtuX x u x j LRN-U2095 Protocol FACT-TOX-013 Amendment 2 3 . P r o t o c o l r e a d s : The protocol states that liver specimens w ill be extracted and analyzed following methods: FA CT-M -1.0, "Extraction o f Potassium Perfluorooctanesulfonate or O ther Anionic surfactants from Liver for analysis Using HPLC-Electrospray/M as Spectrometry" FA C T-M -2.0, "A nalysis o f Frluorochemicals in Liver Extracts Using HPLCElectrospray/M ass Spectrometry" A M E N D t o r e a d : The liver specimens w ill be extracted and analyzed following m ethod: M ethod for A nalysis o f Perfluorooctane Sulfonate (PFOS) in R at liver by LC/M S/M S, V ersion 1.0 REASON; Since the liver extraction and analysis was sub-contracted to B attelle M em orial Institute, this am endm ent w as w ritten to include their liver methods and titles. Amendment Approval A fo \. M arvin Case Ph.D ., Sponsor Representative b it-- ._____________ K risten J. Hansen Ph.D ., Study Director _ 3 M Environm ental Laboratory 3M Environmental Laboratory 3M Environmental Laboratory D ate Page 27 Page 27 3M Medical Department Study: T-6316.5 3M Medical Department Study: T6316.5 BACKTOMAIN Analytical Report: r a u i v a -u u LRN-U2095 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, 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 BACKTOMAIN Analytical Keport : f a u t m - u u 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 Jam es K. Lundberg . w ill begin serving as the study director for FACT-TOX-013 as o f 4 O ctober 1999. 2. Protocol Reads: - Section 7.1 states that the following raw data and records w ill 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 w ill include at least the following: Training records; calibration records; instrument maintenance logs; Standard Operating Procedures, Equipm ent 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 completed, 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, instrum ent maintenance logs, standard operating procedures, equipment procedures, and methods w ill be retained in the archives o f the facility perform ing 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 3M Medical Department Study: T6316.5 BACKTOMAIN Analytical Report: jj'a u t t u a -uj.3 LRN-U2095 Protocol FACT Tox-013 Amendment Number 3 3. Protocol reads: D isposition o f test and control substances: Biological tissues and fluids are retained per GLP regulation. Amend to read: Specimens w ill be maintained in the 3M Environmental Laboratory specim en archives. A ll specimens sent to sub-contract laboratories w ill be returned to the 3M Environm ental Laboratory upon completion o f analysis and subm ission o f the sub-contract laboratory(s) final report. The specimens w ill be returned w ith the following docum entation: 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 specimens analyzed at subcontract laboratories. - Amendment Approval M arv Case, D.V.M ., Ph.D., Sponsor Representative D ate -ti* -- ______________________________________________ t o / s m Kristen J. Hansen, Ph.D., Previous Study D irector D ate Dale L. Bacon, PhrBT 3M Environmental Laboratory M anagement 3M Environm ental Laboratory 3M Environmental Laboratory 3MEnvironmental Laboratory Page 30 Page 30 3M Medical Department Study: T-6316.5 3M Medical Department Study: T6316.5 BACKTOMAIN ' Analyticctx ncpuit: r LKN-U2095 Study Title A nalytical Study o f 2(N-Ethylperfluorooctanesulfpnamido)-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 Environm ental Laboratory 3M Environmental Laboratory 3MEnvironmental Laboratory Page 31 Page 31 3M Medical Department Study: T-6316.5 3M Medical Department Study: T6316.5 . BACKTOMAIN ' Analytical Keport: r a w t u a -uxj LRN-U2095 Protocol LR N -U 2095 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: T h e study director for th e present study w as identified in the protocol as Jam es K. Lundburg, Ph.D. A mend to read: T h e role of study director for the present study w as reassigned to Marvin T. Case, D .V .M ., Ph.D ., as of 20 January 2000. T h e previous study director, Jam es K. Lundburg, has been reassigned to the role o f Principle Analytical Investigator. Reason: T h e role o f study director w as reassigned in an effort to ensure com pliance with Good Laboratory Practice Standards that outline study personnel requirements (refer to 21 C FR Part 58). 2. Protocol reads: T h e sponsor for the present study was identified as Marvin T. Case, D .V .M ., Ph.D. Amend to read: T h e role of sponsor for the present study w as reassigned to John L. Butenhoff, Ph.D., as of 20 January 2000. Reason: T o ensure that the study director does not also carry the duties o f study sponsor, the sponsor role w as reassigned. In this m anner, personnel responsibilities and workload are more evenly balanced. 3 M Environm ental Laboratory 3M Environmental Laboratory 3MEnvironmental Laboratory Paqe 32 Page 32 3M Medical Department Study: T-6316.5 3M Medical Department Study: T6316.5 BACKTO MAIN Analytical Report: FACT TOX-013 LRN-U2095 Protocol LRN-U2095 Amendment Number 4 mm, ' Amendment Approval John L B utenhoffP hD ., Sponsor Representative Date I M arvin T. Case, D. V.M., P h D ., Incoming Study Director J O .-- eh$rL 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 BACKT OMAIN Analytical Report: f a c t t u i -u u LRN-U2095 Study Title Analytical Study of 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 FA CT -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 Analytiuai BACKTOMAIN LRN-U2 095 " Protocol FA C T TOX-013 Amendment No. 5 mt This am endm ent m odifies the following portion(s) of the protocol: - 1. PROTOCOL READS: The Principle Analytical Investigator for the present study was identified as James K. Lundberg, Ph.D. 2 . AM END TO r e a d : The role of 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 B A C K T O MAIN Analytical Report: FACT TOX-013 LRN-U2095 Protocol FA C T TOX-013 Amendment No. 5 Amendment Approval John L. Butenhoff, Ph.D., Sponsor Representative a' Date rc ^ M arvin T. Case, D. V.M., Ph.D., Study Director 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 BACKTOMAIN 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 3M Medical Department Study: T6316.5 BACKTOMAIN Analyticax Kepuu-: iu a - u u LRN-U2095 3M E Lnvironmental aboratory131^ I | I ' j I J A l r l l I T I I v i ^ JL V / JL. A. jw*a M ethod pm 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 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 HPLC- E lectro spra y /M ass Spec tr o m etr y fmm M ethod N um ber: ETS-8-4.1 Adoption Date: 03/01/99 Author: Lisa Clemen, Glenn Langenburg Revision Date: Approved By: -0 Laboratory Manager ,****, 1 i_ Date U t-- ---------------- Group Leader WS Date A Technical Reviewer .... dv/ W w Date 1.0 Scope and Application 1.1 Scope: This method is for the extraction o f potassium perfluorooctanesulfonate (PFOS) or other fluorochemical compounds from serum. 1.2 Applicable compounds: 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 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 BACK TO CAIN Analytical K e p u n : rii i u a -uio 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 fluorochemicals were extracted: PFOS, PFOSA, PFOSAA, EtFOSE-OH, PFOSEA, M556, and surrogate standard (see 3.0 D e finitio ns), 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 following method ETS-8-5.1 or other appropriate methods. 3.0 Definitions____________________________________________________________________ 3.1 PFOS: perfluorooctanesulfonate (anion o f potassium salt) C8F17S 0 3` 3.2 PFOSA: perfluorooctane sulfonylamide 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 C8F 17S 0 2N(CH2CH3)CH2CH20 H 3.5 PFOSEA: perfluorooctane sulfonyl ethylamide C8FI7S 0 2N(CH2CH3)H 3.6 M556: 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 Use universal precautions, especially laboratory coats, goggles, and gloves when handling animal tissue, which may contain pathogens. 5.0 Interferences_________________________________________________________________ 5.1 There are no interferences known at this time. 6.0 Equipment_____________________________________________________________________ 6.1 The following equipment is used while performing this method. Equivalent equipment is acceptable. 6.1.1 Vortex mixer, VWR, Vortex Genie 2 6.1.2 Centrifuge, Mistral 1000 or IEC 6.1.3 Shaker, Eberbach or VWR 3M Environmental Laboratory 3MEnvironmental Laboratory ETS-8-4.1 Extraction o f 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 BACKTOICAIN Analytical Keport: f a c t t u a -u u 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 Nalgene bottles, capable of holding 250 mL and 1 L 7.4 Volumetric flasks, glass, type A 7.5 I-CHEM vials, glass, 40 mL glass 7.6 Centrifuge tubes, polypropylene, 15 mL 7.7 Labels 7.8 Oxford Dispenser - 3.0 to 10.0 mL 7.9 Syringes, capable 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 times with methanol and 3 times with Milli-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, Milli-QTM or equivalent; all water used in this method should be Milli-QTM water and may be provided by a Milli-Q TOC PlusTM system 8.2 Sodium hydroxide (NaOH), J.T Baker or equivalent 8.3 Tetrabutylammonium hydrogen sulfate(TBA), Kodak or equivalent 8.4 Sodium carbonate (NajCOj), J.T. Baker or equivalent 8.5 Sodium bicarbonate (NaHC03), J.T. Baker or equivalent 8.6 Methyl-T-Butyl Ether, Omnisolv, glass distilled or HPLC grade 8.7 Methanol, Omnisolv, glass distilled or HPLC grade 8.8 Serum or blood, frozen from supplier 8.9 Fluorochem ical standards 8.9.1 PFOS (3M Specialty Chemical Division), molecular weight = 538 8.9.2 PFOSA (3M Specialty Chemical Division), molecular weight = 499 ETS-8-4.1 Extraction o f PFOS from Serum Page 3 o f 14 3M Environmental Laboratory 3MEnvironmental Laboratory Paoe 40 Page 40 3M Medical Department Study: N-6316.5 3M Medical Department Study: T6316.5 BACKT OMAIN Analytical Report: FACT TOX-013 LRN-U2095 8.9.3 PFOSAA (3M Specialty Chemical Division), molecular weight - 585 8.9.4 EtFOSE-OH (3M Specialty Chemical Division), molecular weight = 570 8.9.5 PFOSEA (3M Specialty Chemical Division), molecular weight = 527 8.9.6 M556 (3M Specialty Chemical Division), molecular weight = 557 8.9.7 Surrogate standard: 4-H, periluorooctane sulfonic acid (l-H .l-H , 2-H, 2-H C8F 13S 0 3H) molecular weight = 428 8.9.8 Other fluorochemicals, as appropriate 8.10 Reagent preparation NOTE: When preparing larger volumes than listed in reagent, standard, or surrogate preparation, adjust accordingly. 8.10.1 10 N sodium hydroxide (NaOH): Weigh approximately 200 g NaOH. Pour into a 1000 mL beaker containing 500 mL Milli-QTM water, mix until all solids are dissolved. Store in a 1 L Nalgene bottle. 8.10.2 1 N sodium hydroxide (NaOH): Dilute 10 N NaOH 1:10. Measure 10 mL o f 10 N NaOH solution into a 100 mL volumetric flask and dilute to volume using Milli-QTM water. Store in a 125 mL Nalgene bottle. 8.10.3 0.5 M tetrabutylammonium hydrogen sulfate (TBA): Weigh approximately 169 g of TBA into a 1 L volumetric containing 500 mL Milli-QTM water. Adjust to pH 10 using approximately 44 to 54 mL of 10 N NaOH (While adding the last mL o f NaOH, add slowly because the pH changes abruptly). Dilute to volume 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): Weigh approximately 26.5 g of sodium carbonate (N a^O j) and 21.0 g o f sodium bicarbonate (NaHC03) into a 1 L volumetric flask and bring to volume with MilliQTM 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 Weigh approximately 100 mg o f PFOS into a 100 mL volumetric flask and record the actual weight. 8.11.4 Bring to volume with methanol for a stock standard o f approximately 1000 ppm (ug/mL). 8.11.5 Dilute the stock solution with methanol for a working standard 1 solution o f approximately 50 ppm. 8.11.6 Dilute working standard 1 with methanol for a working standard 2 solution of approx. 5.0 ppm. ETS-8-4.1 Extraction o f 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 BACKTOMAIN Analytical Report: FACT TOX-013 LRN-U2095 8.11.7 Dilute working standard 1 with methanol for a working standard 3 solution of approx. 0.50 ppm. 8.12 Surrogate stock standard preparation 8.12.1 Weigh approximately 50-60 mg 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 volume 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 volume with methanol for a working standard o f 100 ppm. Record the actual volume transferred. 9.0 Sample Handling 9.1 All samples are received frozen and must be kept frozen until the extraction is performed. 9.2 Allow samples to thaw to room temperature prior to extraction. 10.0 Quality Control 10.1 Solvent Blanks, M ethod blanks and m atrix blanks .a- --poJLt 10.1.2 Extract two 1.0 mL aliquots of Milli-QTM water following this procedure and use as method blanks. 10.1.3 Extract two 1.0 mL aliquots o f the serum following 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 of 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 will fall in the mid-range o f the initial calibration curve. Additional spikes may be included and may fall in the low-range of the initial calibration curve. 10.2.4 Prepare one matrix spike and matrix spike duplicate per 40 samples, with a minimum o f 2 matrix spikes per batch. 0.3 Continuing 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. It ETS-8-4.1 Extraction o f PFOS from Serum emmental 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 B A C K T O MAIN Analytical Report: FACT TOX-013 LRN-U2095 10.3.4 The expected concentrations will fall within the mid-range o f the initial calibration curve. Additional spikes may be included that fall in the low-range of the initial calibration curve. This is necessary if the analyst must quantitate using only the low end o f the calibration curve (for example, 5 ppb --100 ppb, rather than 5 ppb - 1000 ppb). 11.0 Calibration and Standardization ___________________________________________ 11.1 P repare m atrix calibration standards 11.1.1 Transfer 1 mL o f serum to a 15 mL centrifuge tube. 11.1.2 If most sample volumes are less than 1.0 mL, extract standards with matrix volumes equal to the sample volumes. Do not extract less than 0.50 mL of matrix. Record each sample volume on the extraction sheet. 11.1.3 While preparing a total o f twenty aliquots in 15 mL centrifuge tubes, mix or shake between aliquots. 11.1.4 Two 1 mL aliquots, or other appropriate volume, serve as matrix blanks. Typically use the standard concentrations and spiking amounts listed in Table 1, at 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 Use 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 following 12.6-12.16 of this method. Use 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 BACK TOMAIN Analytical Report: FACT TOX-013 LRN-U2095 Table 1 Approximate spiking amounts for standards and spikes Using 1.0 mL of m atrix Working standard pL Approx, final cone, of (approx, cone.) analyte in matrix -- - Blank 0.500 ppm 10 0.005 ppm 0.500 ppm 20 0.010 ppm 5.00 ppm 5 0.025 ppm 5.00 ppm 10 0.050 ppm 5.00 ppm 20 0.100 ppm 50.0 ppm 5 0.250 ppm 50.0 ppm 10 0.500 ppm 50.0 ppm 15 0.750 ppm 50.0 ppm 20 . 1.00 ppm 12.0 Procedure___________________________________________________________________ 12.1 Obtain frozen samples and allow to thaw at room temperature or in a lukewarm waterbath. 12.2 Vortex mix for 15 seconds, then transfer 1.0 mL or other appropriate volume to a 15 mL polypropylene centrifuge tube. 12.3 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 samples, including blanks and standards, ready for extraction with surrogate standard as described in 11.2. 12.7 Spike each matrix with the appropriate amount o f standard as described in 11.1, or Table 1 in that section, for the calibration curve standards. Also prepare matrix spikes and continuing calibration standards. 12.8 Vortex mix the standard curve samples, matrix spike samples, and continuing calibration samples for 15 seconds. 12.9 Check to ensure the 0.5 M TBA reagent is at pH 10. If 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 Using an Oxford Dispenser, add 5 mL methyl-terf-butyl ether. 12.12 Cap each sample and put on the shaker at a setting of 300 rpm, for 20 minutes. 12.13 Centrifuge for 20 to 25 minutes at a setting o f 3500 rpm, or until layers are well 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 3M Medical Department Study: T6316.5 BACKTOMAIN 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 Remove 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 mix 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-volume autovial when necessary. 12.20 Label the autovial with the study number, animal number and gender, sample timepoint, matrix, final solvent, extraction date, and 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 Calculations 13.1.1 Calculate actual concentrations o f PFOS, or other applicable fluorochemical, in calibration standards using the following equation: mL o f standard x concentration o f standard fug /m D __________________ = mL o f standard + mL o f surrogate standard + initial matrix volume (mL) Final Concentration (pg/mL) o f PFOS in matrix 14.0 Method Performance_______________________________________________________ 14.1 The method detection limit (MDL) is analyte and matrix specific. Refer to M DL report for specific MDL and limit o f quantitation (LOQ) values (see Attachm ents B and C). 14.2 The following quality control samples are extracted with each batch o f samples to evaluate the quality of the extraction and analysis. 14.2.1 Method blanks and matrix blanks. 14.2.2 Matrix spike and matrix spike duplicate samples to determine accuracy and precision of the extraction. 14.2.3 Continuing calibration check 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 P o l l u t io n P r e v e n t io n a n d W a s t e M a n a g e m e n t ______________________________ 15.1 Sample waste is disposed in biohazard containers, flammable solvent waste is disposed in high BTU containers, and used glass pipette waste is disposed in broken glass containers located in the laboratory. ETS-8-4.1 Extraction o f PFOS from Serum Page 8 o f 14 3M Environmental Laboratory HWW 3MEnvironmental Laboratory Page 45 Page 45 3M Medical Department Study: T-6316.5 3M Medical Department Study: T6316.5 B A C K T O MAIN Analytical Report: FACT TOX-013 LRN-U2095 16.0 R e c o r d s __________________________________________________________________ _ 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 A t t a c h m e n t s _______________________________________________________________________________ 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 ETS-8-4.0 & 5.0-V -l. 18.2 FACT-M-3.1, "Analysis o f Serum or Other Fluid Extracts for Fluorochemicals using HPLC-Electrospray Mass Spectrometry" 19.0 A f f e c t e d D o c u m e n t s __________ ;___________________________________________________________ 19.1 ETS-8-5.1, "Analysis of Serum or Other Fluid Extracts for Fluorochemicals using HPLC-Electrospray Mass Spectrometry" 20.0 Revisions___________________ :________________________________________________ Revision Number 1 Reason For Revision Section 12.21 Changed to include sample storage at room temperature. Section 12.13 Added the shaker speed. Section 12.17 Final volume is 1.0 mL; not adjusted for initial volumes less than 1,0 mL. Revision Date 04/02/99 3M Environmental Laboratory 3MEnvironmental Laboratory ETS-8-4.1 Extraction o f PFOS from Serum Page 9 o f 14 Page 46 Page 46 3M Medical Department Study: T-6316.5 B A C K T O MAIN 3M Medical Department Study: T6316.5 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 s Vortex 15 sec. Pinette 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 soeed: 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 svrinee with a 0.2um filter into a 1.5 mL autosamole vial Cont. Cal. Verifications used same matrix as for std curve. - - - - - - - - - - - - - mL Date & Initials 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 B A C K T O MAIN Analytical Report: FACT TOX-013 LRN-U2 095 MDL/LOQ values for rabbit serum Compound MDL LOQ Linear Calibration Range (LCR) (PPb) (PPb) Approximate concentrations to be used for preparing the Standard Calibration Curve PFOS 1.74 5.55 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 of 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 3M Medical Department Study: T6316.5 BACKTOMAIN Analytical Report: FACT TOX-013 LRN-U2095 Compound: PFOS Prepared range Rabbit Serum of standards (ppb) (ng/mL) Full Range Low Curve High curve 1/X 0.995 - 978 4.94 - 248 97.8 - 978 0.995 - 978 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 RSD Range 4.67-11.0 5.34-12.0 4.84-9.80 4.60-10.5 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 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 RSD Range 5.10-14.7 9.85-14.7 5.08-13.9 5.10-14.5 Compound: PFOSAA Prepared range Rabbit Serum of standards (ppb) (ng/mL) Full Range 0.991-974 LCR from curve (PPb) (ng/mL) 24.7 - 974 % Recovery Range 81-111 RSD Range 4.18-10.6 Low Curve 4.92 - 247 9.74 - 247 97-107 6.38-21.8 High curve 49.2 - 974 97.4 - 974 85-108 4.33-12.5 pm* 1/X 0.991 - 974 9.74 - 974 95-115 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 P^rr<a AQ Page 49 3M Medical Department Study: T-6316.5 3M Medical Department Study: T6316.5 BACK TO CAIN Analytical Keport: F A U T T U A - U H LRN-U2095 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 Compound: P FOSEA 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 Compound: M556 Prepared range Rabbit Serum o f standards (ppb) (ng/mL) Full Range Low Curve High curve 1/X 0.993 - 976 4.93 - 97.6 97.6 - 976 0.993 - 976 LCR from curve (ppb) (ng/mL) 49.3 - 976 9.76 - 97.6 97.6 - 976 9.76 - 976 LCR from curve (PPb) (ng/m L ) 24.8 - 976 9.76 - 248 49.3-976 9.76 - 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 77-110 97-107 90-109 86-111 % Recovery Range 96-106 91-110 86-106 95-117 % 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 B A C K TO MAIN Analyuxuax Kepuru: Iftli 1UA-UX-3 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: Method/revision: Target analyte(s): _ FC mix std approx. 0.500 ppm: FC mix std approx. 5.00 ppm: FC mix std approx. 50.0 ppm: Surrogate std approx. 100 ppm: Actual concentrations of standards in the FC mix 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 5.01 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 Calculated concentrations of standards in the sample matrix 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 BACKTOMAIN Analytical Report: FACT TOX-013 LRN-U2095 3M Environmental Laboratory M ethod A n a ly sis o f P o ta ssiu m P er flu o r o o c ta n esu lfo n a te o r O t h e r F lu o r o c h em ic a ls in Seru m E x tra cts U sin g H P L C -E lectrospray/M ass Spectro m etry - M ethod N um ber: ETS-8-5.1 Author: Lisa Clemen, Robert Wynne Approved By: l it J ! -- Laboratory Manager Group Leader A L. .... S* A ' Technical Reviewer Adoption Date: 03/01/99 Revision Date: ^ y 2. c f Date Date oh/u /m Date 1.0 Scope and Application_______________________________________________________ 1.1 Scope: This method describes the analysis o f serum extracts for fluorochemical surfactants using HPLC-electrospray/mass spectrometry. 1.2 Applicable 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 3MkwW Medical DepX artment StudyJ : T-6316.5 3M Medical Department Study: T6316.5 BACK TO CAIN Analytical Report: r a u t t u -uis LRN-U2 095 2.0 S u m m a r y o f M e t h o d ______________________________________________________ __________________ 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 of a particular fluorochemical, such as the perfluorooctanesulfonate (PFOS) anion, m/z= 499. Additionally, samples may be analyzed using a tandem mass spectrometer to further verify the identity o f a compound by detecting daughter ions o f the parent ion. 3.0 D e f in it io n s _____________________________________________________________________________ 3.1 A tm ospheric Pressure Ionization (API): The Micromass Quattro II triple quadrupole systems 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 chemical Ionization (APcI), Thermospray, etc. The ionization process in these techniques occurs at atmospheric pressure (i.e., not under a vacuum). 3.2 Electrospray Ionization (ES, ESI): a method 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 Mass Spectrometry, Mass Spectrom eter (MS), Tandem Mass Spectrom eter (MS/MS): The API Quattro II triple quadrupole systems are equipped with quadrupole mass selective detectors. Ions are selectively discriminated by mass to charge ratio (m/z) and subsequently detected. A single MS may be employed for ion detection or a series (MS/MS) for more specific fragmentation information. 3.4 Conventional vs. Z-spray probe interface: The latest models of Micromass Quattro II triple quadrupole systems (post 1998) utilize a "Z-spray" conformation. The spray emitted from a probe is orthogonal to the cone aperture. In the conventional conformation it is aimed directly at the cone aperture, after passing through a tortuous pathway in the counter electrode. Though the configuration is different, the methods 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 systems (i.e., Z-spray components are compatible with some other Z-spray systems, etc.) 3.5 M ass Lynx Software: System software designed for the specific operation of these Quattro II triple quadrupole systems. Currently MassLynx has Windows 95 and WindowsNT 4.0 versions. All versions are similar. For more details see the manual specific to the instrument (Micromass Quattro II triple quadrupole MassLynx or MassLynx NT User's Guide). 4.0 W a r n in g s a n d C a u t io n s _________________________________________________________________ 4.1 H ealth and Safety W arnings: 4.1.1 Use caution with the voltage cables for the probe. When engaged, the probe employs 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 o f Serum Extract Using ES/MS Page 2 o f 9 Pace 53 Page 53 3M Medical Department Study: T-6316.5 3M Medical Department Study: T6316.5 BACK TO CAIN Analytical Report: f a c t t o x -u i 3 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 will initiate automatic shutdown. 4.2.2 Do not run solvent pumps to dryness. 5.0 I n t e r f e r e n c e s _______________________________________________________________________________ 5.1 To minimize interferences when analyzing samples, teflon should not be used for sample storage or any part o f instrumentation that comes in contact with the sample or extract. 6.0 E q u ip m e n t ____________________________________________________________________________________ 6.1 Equipment listed below may be modified in order to optimize the system. Document any modifications in the raw data as method deviations. 6.1.1 6.1.2 Micromass Quattro II triple quadrupole Mass Spectrometer equipped with an electrospray ionization source HP 1100 low pulse solvent pumping system, solvent degasser, column compartment, and autosampler 7.0 S u p p l ie s a n d M a t e r ia l s ________________________________________ :____________________________ 7.1 Supplies 7.1.1 High purity grade nitrogen gas regulated to approximately 100 psi (House air system) 7.1.2 HPLC analytical column, specifics to be determined by the analyst and documented in the raw data. 7.1.3 Capped autovials or capped 15 mL centrifuge tubes 8.0 R e a g e n t s a n d S t a n d a r d s _________________________________________ ;_________________________ 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 Milli-Q TOC Plus system or other vendor 8.1.3 Ammonium acetate, reagent grade or equivalent 8.2 Standards 8.2.1 Typically two method blanks, two matrix blanks, and eighteen matrix standards are prepared during the extraction procedure. See ETS-8-4.1. 9.0 S a m p l e H a n d l in g ________________________'_______________________,____________________________ 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 B A C K TO MAIN Analytical Keport: f a u t t u a -u u LRN-U2095 9.2 I f analysis will be delayed, extracted standards and samples can be refrigerated at approximately 4 C, or at room temperature, until analysis can be performed. 10.0 Q u a l it y C o n t r o l __________________________________________________________________________ 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, with a minimum o f 2 spikes per batch. 10.2.4 Matrix spike and matrix spike duplicate concentrations will fall in the mid-range o f the initial calibration curve. Additional spike concentrations may fall in the lowrange 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 C a l ib r a t io n a n d S t a n d a r d iz a t io n _____________________________________________________ 11.1 Analyze the extracted matrix standards prior to and following each set o f extracts. The average o f two standard curves will be plotted by linear regression (y = my + b), weighted 1/x, not forced through zero, using MassLynx or other suitable software. 11.2 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 of analyte, generate a calibration curve consisting of the standards from 5 ppb to 100 ppb rather than the full range o f the curve (5 ppb to 1000 ppb). This will reduce inaccuracy attributed to linear regression weighting o f high concentration standards. 3M Environmental Laboratory 3MEnvironmental Laboratory ETS-8-5.1 Analysis o f 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 B A C K TO MAIN Analyticax Keporc: c x x u a - u u LRN-U2 095 12.0 PROCEDURES_________________________________________________________________________________ 12.1 Acquisition Set up 12.1.1 Click on start button in the Acquisition Control Panel. Set up a sample list. Assign a filename using MO-DAY-last digit o f year-sample number, assign a method (MS) for acquiring, and type in sample descriptions. 12.1.2 To create a method click on scan button in the Acquisition control panel and select SIR (Single Ion Recording) or MRM. Set Ionization Mode as appropriate and mass to 499 or other appropriate masses. A full scan is usually collected along with the SIRs. Save acquisition method. If MS/MS instruments are employed, additional product ion fragmentation information may be collected. See Micromass MassLynx GUIDE TO DATA ACQUISITION for additional information and MRM (Multiple Reaction Monitoring). 12.1.3 Typically the analytical batch run sequence begins with a set 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 Using the Autosam 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 = Time 0.00 min. 8.50 min. 11.0 min. 12.0 min. MeOH 40% 90% 90% 40% 2.0 mM Ammonium acetate 60% 10% 10% 60% 12.2.2.5 Press the "Start" button. 12.3 Instrument Set-up 12.3.1 Refer to ETS-9-24.0 for more details. 12.3.2 Check the solvent level in reservoirs and refill if necessary. 3M Environmental Laboratory 3MEnvironmental Laboratory ETS-8-5.1 Analysis o f 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 BACKTOICAIN Analytrcax Keporc: i u a -u u LRN-U2095 12.3.3 Check the stainless steel capillary at the end o f the probe. Use an eyepiece to check the tip. The tip should be flat with no jagged edges. If the tip is found to be unsatisfactory, disassemble the probe and replace the stainless steel capillary. 12.3.4 Set HPLC pump to "On". Set the flow to 10 - 500 uL/min or as appropriate. Observe droplets coming out of the tip o f the probe. Allow 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 mist is observed. 12.3.6 The instrument uses these parameters at the following settings. These settings may change in order to optimize the response: 12.3.6.1 Drying gas 250-400 liters/hour 12.3.6.2 ESI nebulizing gas 10-15 liters/hour 12.3.6.3 HPLC constant flow mode, flow rate 10 - 500 pL/min 12.3.6.4 Pressure <400 bar (This parameter is not set, it is a guide to ensure the HPLC is operating correctly.) 12.3.7 Carefully guide the probe into the opening. Insert probe until it will not go any further. Connect the voltage cables to the probe. 12.3.8 Print the tune page, with its parameters, and store it in the study binder with a copy taped into the instrument log. 12.3.9 Using the cross-flow counter electrode in the ES/MS source is recommended for the analysis of biological matrices. 12.3.10Click on start button in the Acquisition Control Panel (this may vary among MassLynx versions, see appropriate MassLynx USER'S GUIDE). Press the start button. Ensure start and end sample number includes all samples to be analyzed. 13.0 D a t a A n a l y s is a n d C a l c u l a t io n s ______________ ;___________________________________ 13.1 Calculations: 13.1.4 Calculate matrix spike percent recoveries using the following equation: % Recovery = Observed Result - Background Result x 100 Expected Result 13.1.5 Calculate percent difference using the following equation: % Difference = Expected Cone. - Calculated Cone, x 100 Expected Cone. 13.1.6 Calculate actual concentration o f PFOS, or other fluorochemical, in matrix (pg/mL): fag o f PFOS calc, from std. Curve x Dilution Factor) x 1 pg (Initial Volume o f matrix (mL>) + mL o f Surrogate Standards 1000 ng Final Volume (mL) 3M Environmental Laboratory 3MEnvironmental Laboratory ETS-8-5.1 Analysis o f 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 BACK TO CAIN Analycicax Report: ihui iua-u u LRN-U2 095 14.0 M e t h o d P e r f o r m a n c e _____________________________________________________________________ 14.1 Method Detection Limit (MDL) and Limit 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 lowest standard in the calibration curve 14.3 Calibration Curves 14.3.1 The r2value for the calibration curve must be 0.980 or better. 14.4 Matrix Spikes 14.4.1 Matrix spike percent recoveries are must be 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 If data are to be reported when performance criteria have not been met, the data must be footnoted on tables and discussed in the text o f the report. 15.0 P o l l u t i o n P r e v e n t io n a n d W a s t e M a n a g e m e n t ______________________________________ 15.1 Sample extract waste and flammable solvent is disposed in high BTU containers, and glass pipette waste is disposed in broken glass containers located in the laboratory. 16.0 Records_____________________________________________________________________ 16.1 Each page generated for a study must have the following information included either in the header or hand written on the page: study or project number, acquisition method, integration method, sample name, extraction date, dilution factor (if applicable), and analyst. 16.2 Print the tune page, sample list, and acquisition method from MassLynx to include in the appropriate study folder. Copy these pages and tape into the instrument runlog. 16.3 Plot the calibration curve by linear regression, weighted 1/x, then print these graphs and store in the study folder. 16.4 Print data integration summary, integration method, and chromatograms, from MassLynx, and store in the study folder. 3M Environmental Laboratory 3MEnvironmental Laboratory ETS-8-S.1 Analysis o f Serum Extract Using ES/MS Page 7 o f 9 Page 58 Page 58 3M Medical Department Study: T-6316.5 3M Medical Department Study: T6316.5 B A C K TCi MAIN Analytical Keporc: (wr tua-u u LRN-U2 095 16.5 Summarize data using suitable software (Excel 5.0) and store in the study folder, see Attachment 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 R e f e r e n c e s _________________________________________________________________________________ 18.1 FACT-M-4.1, "Extraction o f Potassium Perfluorooctanesulfonate or Other Fluorochemical 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/Mass Spectrometer Quattro II triple quadrupole Systems" 18.3 The validation report associated with this method is ETS-8-4.0 & 5.0-V -l. 19.0 A ffected Documents 19.1 ETS-8-4.1, "Extraction o f Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds from Serum for Analysis Using HPLC-Electrospray/Mass Spectrometry" 20.0 Revisions Revision Number. 1 Reason For Revision Section 6.1.2 Clarification of 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. Revision Date 04/02/99 3M Environmental Laboratory 3MEnvironmental Laboratory ETS-8-5.1 Analysis o f 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 BACKTOMAIN Analytical Keport: c a u i t u a -u u 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 o f 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 BACK TO CAIN Analytical Report: FACT TOX-013 LRN-U2095 Appendix D: Data Summary Tables Table 12. Reported Fluorochem ical Levels in Sera Analyses in Study FACT TO X-013 Dosage Group Specimen ID PFO S (pg/mL) PFOSA (pg/mL) PFOSAA (pg/mL) EtFOSE-OH (pg/mL) M566 (pg/mL) 1 10097F 0.0394 1 10105F 0.0181 1 10106F 0.0258 1 10107F 1 10108F 0.0343 0.0253 1 9922M* 1 9930M* 0.0115 0.0134 1 9931M 0.00725 1 9932M 0.0162 i 9933M* 0.0156 II 10121F 9.62 II 10126F 19.8 II 10 136F 5.96 II 10140F II 10 142F II 9 9 6 1 M * 6.27 13.1 34.8 II 9 9 6 4 M 30.4 II 9 9 6 5 M * 74.9 II 9 9 6 7 M 25.1 II 9 9 7 0 M * 38.9 III 10155F * 87.8 III 10156F 76.1 III 10 164F 49.6 III 10 172F 68.4 III 10177F 42.2 III 9 9 9 7 M 108 III 9 9 9 9 M * III 10 001M 178 94.9 III 10 002M 113 III 10004M 130 IV 10187F IV 10194F 89.5 73.4 IV 10203F 126 IV 1 0 2 1 1F 99.7 IV 10214F 98.3 IV 10019M 302 IV 10024M * 477 IV 10029M * IV 10033M IV 10034M 296 272 249 * = Tentative values, initial volume was <0.5 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 <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 .5 5 1.18 0.690 2.09 1.40 5.86 1.86 1.26 5.55 9.9 6.91 4 .6 6 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 <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LO Q (36.2 ppb) <LO Q (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LO Q (36.2 ppb) <LO Q (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LO Q (36.2 ppb) <LO Q (36.2 ppb) <LO Q (36.2 ppb) <LOQ (36.2 ppb) <LO Q (36.2 ppb) <LOQ (36.2 ppb) <LO Q (36.2 ppb) <LOQ (36.2 ppb) <LO Q (36.2 ppb) <LO Q (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LO Q (36.2 ppb) <LO Q (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) <LO Q (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) <LO Q (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 4 3 .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 3MEnvironmental Laboratory Page 61 Page 61 3M M edical D epartm ent Study: T-6316.5 3M Medical Department Study: T6316.5 BACK TO CAIN Analytical Report: FACT TOX-013 LRN-U2095 Table 12. Reported Fluorochem ical Levels in Sera Analyses in Study FACT TO X-013 (continued) Dosage G roup Specim en 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 N R * 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 P FO S A A (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 <LO Q (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LO Q (36.2 ppb) M 556 (pg/mL) NR NR NR NR NR 62.4 55.6 93.8 30.7 55.5 Table 13. Reported Fluorochem ical Levels in Liver Analyses in Study FACT TOX-013 Dosage Group I I 1 1 1 1 l 1 I l 1 l i 1 l II il il il il ll il II ll ll ll ll ll II II Specim en 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 PFO S(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 .0 0 <LOQ <10Q <LOQ <LOQ <LOQ <LOQ <LOQ 0.514 0.708 1.40 0.601 0.508 4 .4 9 4.10 2.88 3.88 5.42 1.90 2.65 2.35 1.22 2.06 PFO S A A <pg/g) <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <UOQ <LOQ <LOQ <LOQ <LOQ 2.68 4 .3 4 5.01 2.67 2.67 11.0 15.6 9.79 6 .6 2 10.4 5.87 14.6 9.44 2.82 6.55 M 5 5 6 (p g /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 .5 8 .aaas 3M Environmental Laboratory 3MEnvironmental Laboratory Page 62 Page 62 3M Medical Department Study: T-6316.5 i 3M Medical Department Study: T6316.5 B A C K TO MAIN Analytical Report: FACT TOX-013 LRN-U2095 Table 13. Reported Fluorochem ical Levels In Liver Analyses in Study FACT TO X-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 S padnw 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 PFO SA (pg/g) PFOSAA (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 .0 8 1 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 I 10.8 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(P04) 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 past 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 BACKTOMAIN Analytical Report: FACT TOX-013 LRN-U2095 : 3M Environmental Laboratory 3MEnvironmental Laboratory Page 64 Page 64 O.Xt- o<N UN ^ < Ph GBO' 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 <S3uO CP S 2< a| 27.4 0.00352 52.4 5.74 48.4 19.7 28.9 18.8 25.8 32.2 27.8 63.7 2<Cm2t ^ sss gg t * O <I CQ 'B ! B m i sos a s"i 3 v2\ * a i &a ' ! < i 33 5 | Vjjjij $ m 11 1 a| OS 5sSKsiRs25a.ga*S ||g3 00s?a<*s<s9toroj."Cjjto*gfsSBjSS v.<*ftn g* g| ggggg sssss i HV iW {-i !si M I Soo83o.So. SS f2o-arsCjviP-d22^r^P n5jt2| S5SS SSSS3 gg ISSSS ggggg S$9f 5 * is 1 i fiiii al|J|il! fillliii i** -- -- -- Sdfo5SccSicS??O5?o5c?$cigS 8-<*8->S`SSOSOSoS sssciseetSPStSio tSt8P tSt8O.tSt5tSRtggggg St8tt-8*o Pi *I8 *7<S ptl i,,lf!lllg Sfi3i i| fj i oeoono%on?!tIeVn)IOt5531S2I2?|8R SS lSilios liiil ss ** lilOiOI*1^s lg>ogoggi|a!i 1 fek fei i ! i i 2 |g ggggg g i 1SS 5 m ii ijii h o si 1S s? fij ifJM na 5 U S lili 1i j <3!*r c> jff 53 |!i | til i j i f l Jl !lili SI lllillilltl lilil 3M Environmental Laboratory Page 65 b a g <o3 <1 4R RO .p. 3M^M edical^Department Study: T-6316.5 OJ S td 0 < H H O 2 3 (D 3 ffut ir f0cofui)r fot *< 1f1u5 fl) cors KTS-8-5.1 3MEnvironmental Laboratory BACK TO MAIN FACT-TOX-013 Argus# 418-009 Sfltdy: Atgtu 418-009. Two-Generation Reproduction Study of ElFOSE-QH in Rats Prod*U N i mbef(Tesl Substance): Matrix: McKxSiRcviofl: Analytical Equipment System Number Instrument SoitwareA'enioo: Filename: R-StfJMvdViltG- EtFOSB-OH (T-63165) Rat Seram ETS-8-4.I 4ETS-8-5.1 Davey 070799. Soup 020199 MassLytu 3 J Sec lifting to the right SeeAttachments Slope Y-Intocept See Attachment* SeeAtttctanecii Dates nTExtraction/Analyst 1/12/W RWW Dales of Amtyiii/Analyin: 10/14/99,10/20/99.10/22/99,03/16/TO MMHAAS Date of Data Radaclkm/Analyst: 10/1*99,10/21/99, KV25/99,03/17/00.03/23A MMH/lAS Sample Dal RATSERA F6____________ Gcwp Dok Method Elk Matrix Bk QC-lOOfpb Crewpl Cannot O.Omg/fcgiiay 0rog/mL GrMip2 1 mg/kg/ti*y 0.Ir tty r L Snraple 10129-H2O Blk-1 10129-H2O BQl-2 RBS10129-Sen BA-1 RBS10129-Sen Bk-2 RBS 10129-MS-1 RBS10129-MSD-1 10097F m o st* 10106F 10107F 10I08F 9922M** 993<W-* 9931M 9932M 9933M10121F 10126F 10136F 1014QF 10142F 9961M** 9964M 996SU *' 9967M 9970M- Initial Voi. wL I 1 1 1 l 1 0.70 0.7 0.50 0.60 .60 0.40 0.40 oso 050 0.40 0.70 0 so 1.00 1.00 0.60 0.40 0.50 0.20 0.50 0.40 prosa Caw. HW* 0.00 1.57 0.00 1J7 220 215 1.46 1.28 1.15 1.44 1.14 2.41 1.89 144 1.41 133 47.7 56.0 66.3 50.7 39.9 38.5 94.2 217 73.6 66.2 Cam tati itjun *f PFOSA agteLor %Rc cLOQ <LOO cLOQ <UX) 89* 87* cLOQ <LQQ -cLOQ cLOQ cLOQ <LQQ cLOQ <LOQ cLOQ <LOQ 0.0682 0.112 0.0663 0.0507 0.0665 03)962 0.188 0.114 0.147 ai6S Mra FFOSA *WL <LOO <LOO 88% <LOQ <LOQ 0.0727 0.142 USD SM -D cr. MS/MSD RD NA NA 3* NA NA NA NA 31.7 0.0231 26.4 0.0376 GranpS 5 mgAgAUy l.Qmg/mL Group 4 (0.0 mgAg/day 2.0mg/mL G toupj 15.0 mg/fcjidxy 3.0mg/mL 10155F** 10I56F 10164F 10172F 10I77F 9997M 9999MIG001M 10002M 10004M 10I87F 0194F 10203F 102UF I02MP 10019M 10024M** 10029M* 10033M 10034M NR NR NR 0.40 0.60 0.70 0.60 0.70 0.70 0J0 0.70 0.60 0.50 0.60 0.80 0.70 0.80 0.70 0.60 OJO 0.40 0.50 0.50 NR NR NR 131 211 185 195 235 402 174 336 236 233 276 461 456 536 398 368 166 244 402 318 NR NR NR 0328 0.352 0.265 0J25 0.335 0574 0579 0.480 0J93 0.465 0.461 0.576 0.651 0.670 0569 0.613 0553 0.610 0.804 0.637 NR NR NR 0J21 0.498 0585 0.643 10.3 0.0331 15.8 0.0786 14.1 0.0826 14.8 0.0951 NR NR 1042M I0044M NR NR 0.80 080 NR NR 633 778 NR NR 0.791 0.972 NR NR NR 1004M 10051M 10054M OJO TOO 0.90 448 574 602 0.897 0.374 0.669 0.780 208 0.163 Luot of Qucitation (LQQ): PFOS * $55 nj/mL, PFOSA = 4.79 ppb, PPOSAA 205 ppb, EtFOSE=36.2 ppb, PINK Sample not received nor repaced. Correctionf*cwtnotappcateforMS/MSDQCdata Tentative valse, eonwotraioo was nut wittrin the tinge of the Curve, it's approximately 15* above liar togbeii standarrl LAC 03/24* Date EntswSBy: G3/II*, OVZ3AW,03/14/06 LAC Date Verified/By: "* Tentative varies, InitljJ volume below 0.5 oL. LAC 08/31/00 nalytical Study: FACT-TOX-013 LRN-U295 IsP sCD Q* Hn o> M o (D O P> rt 3 CD 3rt cn rr C Pt *< CTi OJ H cr in p H-1 < fHOt fU J--1 TC3D O H ft > F HO cHXOI tof oKOHo aioj Page 66 O oOv >< 3 H2 < p H __ -a a CO co*r '- t. 13 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 tv"o (0L0) fin < e o 1 Z S SS SS s *n ft (3 K h 35 23 S s s Si I - I* ! o* 4 Icfc s S1 1 f s < ! i s S d g Q s s s CD ffi 5* NNfO ft 1 f l II m u mu -- -- d gj ? * 3 3 2S2 S5g v 35 o 2 <e o 2 3 2 2 *3-]3 3 3 2 Si riIs.. O oq 8 S22 M I* Si g5al I4!S-S# t i l l JIIllf lfHl l s i i f I J lfil sa 3J 1n 88 oo 88 oo S S slial 11123 assis 5SS3S *i -i ."1 t SR2S? ?-ss S S S S 8 $ JS | ; a1 U -- -- -- o o655 d8 8 o8 o? oS 5o$ 8 ^ o 88? o --'-- o 8. R 8 8 8 odd 8 o 8686 o ci d o $6 ooo 85; oo S O 86 8 6 oddd i S S i S i S f B S JS i avan t*aa? 11 U. - U. [r. i l gs3l s i i l i g s 1 s * s ? l i l S o I t i l iI I s SSS 3i S 5 CL Et. b. Cu s3s Si 23S S2P p 2 2 2 2 fse lfesfe-fefe s s is s i i SSSS I l l II !1 g %s 1 l i fdt ^jfd il! i lo !~ if ! ill 'sI fI!l> ili in VO COww* VO 1co" +to- s~ tt Ooh Q If*8* O '-o3 . 3M Environmental Laboratory Page 67 t a g <oP3S R 3u .SROk 5^ "3 3M M edical D epartm ent Study: T-6316.5 3M Medical Department Study: T6316.5 B A C K T O IC A IN Analytical Report: FACT TOX-013 LRN-U2095 FACT-TOX-013 Argus#418-009 NNAA Tentativevalue*, initial votone below0.3 mL. LAC08/3I/00 t 5656 Si ZZ 5K S t t iJ gg !S1 fl i sn i jt 1 1 1 1 1 1 1 1 1 1 g 1i it tiuIha i! | j I I I I 11 m u m u m u m u m u m u m u m u ggggg m u Pi i S 1 it H| S3 8o8o3a 8o8*S--5e8So2| JS$$ m u s^gosOg* s a*aisri 8r#i O2tE;TrjIOi ==12333 ggggg "ioom; !| iLtnpil llliliflilili 1 1| tit !* i af -- -- -- 0*000ooooo11 11 3o3o3o3o?o3oo33o3ofoS11111 o3Sco8oSooooooggggg i l i i i t El la| mu* ft il ilmocmaositaoWoJ2M313rri2l,a2t2h2o,C5it1i1 sal **8 oomooo!i!118pi3smslili ggtt.tt.tfcg* siili i l ii i ggggg ini glIiIal]lil1 m! ! .| dii i[a !i a S t8 i l! o l! filli o" if! i}! lili 3M Environmental Laboratory 3MEnvironmental Laboratory Page 68 3M M edical D epartm ent Study: T-6316.5 3M Medical Department Study: T6316.5 B A C K TO MAIN Analytical Report: FACT TOX-013 LRN-U2095 Tentative vahiet, initial vfltamebe1Ow0.5 m L. LA C 08/31/00 3M Environmental Laboratory 3MEnvironmental Laboratory Page 69 Page 69 3M M edical D epartm ent Study: T-6316.5 3M Medical Department Study: T6316.5 BACKTOMAIN 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 pg x pc = R eported C oncentration (pg/mL) EV (m L) 100"ng 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 10(J0"Hg~ 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 of sera extracted PC--PFOS purity correction factor (86.4%) 3M Environmental Laboratory 3MEnvironmental Laboratory Page 70 Page 70 3M M edical D epartm ent Study: T-6316.5 3M Medical Department Study: T-6316.5 BACK FOM AIN Analytical Study: FACT TOX-013 LRN-U2095 Appendix G: Contract Lab Report This appendix includes the following contract laboratory report: Battelle Memorial Institute, 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 M edical D epartm ent Study: T-6316.5 BACK TOMAIN BIOLOGICAL SAMPLE ANALYSIS Battelle Study Number: N003604-D 3M Environmental Laboratory Study Number: FACT 060998.1 C lB 3 ll6 ll6 Puttins Technology To Work 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 M edical D epartm ent Study: T-6316.5 BACK TOTIAIN Battelle Study Number: N003604-D 3M Environmental Laboratoiy Study Number: FACT 060998.1 FINAL REPORT 2 (N-EthylfluorooctanesuIfonamido)-ethanoI in Two Generation Rat Reproduction Battelle Senior Program Director 3MEnvironmental Laboratory ii Page 73 3M M edical D epartm ent Study: T-6316.5 BACK TOM AIN 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 iii Page 74 3M M edical D epartm ent Study: T-6316.5 BACK TOMAIN Battelle Study Number: N003604-D 3M Environmental Laboratory Study Number: FACT 060998. t 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: Phase Inspected Inspection Date Date Reported to Battelle Task Leader/ Battelle Management 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 Date 3MEnvironmental Laboratory iv Page 75 3M M edical D epartm ent Study: T-6316.5 BACK TOMAIN 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 M aster List o f regulated studies. Battelle Principal Investigator Marvin T. Case, DVM, Ph.D. Study Director 3MEnvironmental Laboratory V Page 76 3M M edical D epartm ent Study: T-6316.5 BACK TOM AIN 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 M edical D epartm ent Study: T-6316.5 BACK TOM AIN 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 1 Page 78 3M M edical D epartm ent Study: T-6316.5 BACK TOTIAIN 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 Svstem Autosampler HPLC pumps VIass spectrometer Analytical column Mobile phase components Gradient profile Injection volume Flow Column temp HPLC pressure MS source Desob ation as Nebulizer gas 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 pun, 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 .5 x l0 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, M edical D epartm ent Study: T-6316.5 BACK TOMAIN 4.2 Results Battelle Study Number: N 003604-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 M edical D epartm ent Study: T-6316.5 BACK TOM AIN Battelle Study Number: N 003604-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 P age 81 3M M edical D epartm ent Study: T-6316.5 BACK TOM AIN Battelle Study Number: N003604-D 3M Environmental Laboratory Study Number: FACT 060998.1 APPENDIX A -RESULTS 3MEnvironmental Laboratory Page 82 3M M edical D epartm ent Study: T-6316.5 BACK TOM AIO Battelle Study Number: N 003604-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 a 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 28 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, pg/fl 6.77E-01 9.44E-01 9.68E-01 1.20E+00 1.17E+00 1.34E+02 1.18E+02 1.04E+02 9.34 E+01 1.01 E+02 4.80E+02 2.71 E+02 5.76E+02 2.97E+02 4.47 E+02 9.62E+02 9.15E+02 6.43 E+02 9.04E+02 6.43 E+02 1.41E+03 1.57E+03 1.31 E+03 1.23E+03 1.22E+03 1.72E-01 BLOQ 1.40E-01 BLOQ BLOQ 4.61 E+01 2.74E+01 2.56E+01 2.90E+01 2.65E+01 1.21 E+02 1.18E+02 1.50E+02 2.07E+02 1.38 E+02 1.90E+02 2.78E+02 3.98E+02 2.95E+02 3.06E+02 3.25E-01 2.80E-01 2.61 E-01 2.56E-01 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 2.35 E+02 2.18E+02 2.62E+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+Q1 8.95E+00 1.16E+01 6.35E+01 3.93E+01 6.64E+01 3.80E+01 5.46 E+01 8.45 E+01 9.75E+01 7.82E+01 1.17E+02 8.22E+01 1.28 E+02 1.52E+02 1.33E+02 1.186+02 1.616+02 BLOQ BLOQ BLOQ BLOQ BLOQ 2.86E+00 1.55E+00 1.41E+00 f 1.19E+00 1.75E+00 8.16E+00 7.17E+00 7.64E+00 8.41E+Q0 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 3.95E+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 2.0SE+02 1.50E+02 1.57E+02 1.65E+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 2.94 E+01 3.37E+01 2.74E+01 4.55E+01 7.60E+01 5.62E+01 6.00E+01 PFOSA Cone, pg/g BLOQ BLOQ BLOQ BLOQ BLOQ 4.49E+00 4.10E+00 2.88E+00 3.86E+00 5.42E+00 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 1.40 E+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+00 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 2.65 E+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 P age 83 3M M edical D epartm ent Study: T-6316.5 BACK TOM AIN 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 M-556 0.1269 0.1220 0.1433 0.1368 0.1670 0.1225 0.1190 Mean Concentration Std. Dev. 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 PFOSAA 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 PFOSA 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 BACK T O MA IN A Battelle Study Number: N003604-D 3M Environmental Laboratory Study Number: FACT 060998.1 APPENDIX B-DAELY ACCEPTANCE CRITERIA SUM MARY 3MEnvironmental Laboratory Page 85 3M M edical D epartm ent Study: T-6316.5 BACK TOM AIN 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 13, 1999 October 16, 1999 October 18, 1999 October 20, 1999 Analyte PFOS M-556 PFOSAA PFOSA PFOS M-556 PFOSAA PFOSA PFOS M-556 PFOSAA PFOSA PFOS PFOSA X ' Coeff -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 -153 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 -1.02E+03 Coeff of Determination 0.984 0.998 0.994 0.989 0.999 0.998 0.998 0.990 0.985 0.984 0.993 0.996 0.997 0.999 Comments/ Deviations One rep of Cal pt 1 excluded One rep of Cal pt 1 excluded One rep of cal p t7 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 M edical D epartm ent Study: T-6316.5 BACK TOM AIN _ Battelle Study Number: N 003604-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 M edical D epartm ent Study: T-6316.5 BACK TOM AIN Battelle Study Number: N003604-D 3M Environmental Laboratoiy Study Number: FACT 060998.1 APPENDIX C-SAMPLE INVENTORY LIST 3MEnvironmental Laboratory Page 88 3M M edical .D ep artm en t Study: T-6316.5 BACK TOM AIN _ Battelle Study Number: N 003604-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 FCM0203-grplV-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 F0-10177-grplll-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 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 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 FetalUver 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 BACK TO M AIN Battelle Study Number: N003604-D ' 3M Environmental Laboratory Study Number: FACT 060998.1 APPENDIX D-REPRESENTATIVE CHROMATOGRAMS 3MEnvironmental Laboratory Page 90 3M M edical D epartm ent Study: T-6316.5 BACK TOM AIN Battelle Study Number: N 003604-D 3M Environmental Laboratory Study Number: FACT 060998.1 3MEnvironmental Laboratory D -l P age 91 3M M edical D epartm ent Study: T-6316.5 BACK TOM AIN _ Battelle Study Number: N 003604-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 BACK TOM AIN Battelle Study Number: N 003604-D 3M Environmental Laboratory Study Number: FACT 060998.1 3MEnvironmental Laboratory rvi Page 93 3M M edical D epartm ent Study: T-6316.5 BACK TOM AIN _ Battelle Study Number: N 003604-D 3M Environmental Laboratory Study Number: FACT 060998.1 3MEnvironmental Laboratory T\ A Page 94 3M M edical D epartm ent Study: T-6316.5 BACK TOM AIN Battelle Study Number: N 003604-D 3M Environmental Laboratory Study Number: FACT 060998.1 3MEnvironmental Laboratory D-5 Page 95 3M M edical D epartm ent Study: T-6316.5 BACK TOM AIN 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 B A C K T O C IA IO _ Battelle Study Number: N 003604-D 3M Environmental Laboratory Study Number: FACT 060998.1 fir r c . - 7-/t - 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/M ass Spectrom etry ' 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 BACK TOM AIN _ Battelle Study Number: N 003604-D 3M Environmental Laboratory Study Number: FACT 060998.1 1.0 P urpo se _________________________________________________________________________ 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 Reg ulato ry C o m pliance___________________________________________________________ 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 Te st M aterials_____________________________________________________________________ 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 BACK TOMAIN Battelle Study Number: N 003604-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 E xperim ental - 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 E xperim ental - 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 D ata 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- M edical D ep artm en t Study: T-6316.5 BACK TOM AIN _ Battelle Study Number: N 003604-D 3M Environmental Laboratory Study Number: FACT 060998.1 7.0 M a in ten a n c e o f R aw D a ta a n d Rec o r d s_______________________________________ 7.1 The following raw data and records will be retained in the study folder in the archives 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 of data 7.2 Supporting records to be retained separately from the study folder in the archives according to AMDT-S-8 will include at least the following: 7.2.1 Training records 7.2.2 Calibration records 7.2.3 Instrument maintenance logs 7.2.4 Standard Operating Procedures, Equipment Procedures, and Methods 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 9.0 A ttachm ents___________________________________________________________________ 9.1 F A C T -M -l.O , Extraction o f Potassium Perfluorooctanesulfonate or Other A nionic Surfactants from Liver for Analysis Using HPLC-Electrospray/M ass Spectrom etry 9.2 FACT-M-2.0, Analysis of Fluorochemicals in Liver Extracts Using HPLC- Electrospray/Mass Spectrometry 9.3 FACT-M-3.0, Extraction of Potassium Perfluorooctanesulfonate or Other Anionic Surfactants from Serum for Analysis Using HPLC-Electrospray/Mass Spectrometry 9.4 FACT-M-4.0, Analysis of Ruorochemicals in Serum Extracts Using HPLC- Electrospray/Mass Spectrometry 3MEnvironmental Laboratory t:.a 4 Page 100 3M M edical D epartm ent Study: T-6316.5 BACK TOM AIN _ Battelle Study Number: N 003604-D 3M Environmental Laboratory Study Number: FACT 060998.1 METHOD FOR ANALYSIS OF POTASSIUM PERFLUOROOCTANESULFONATE (PFOS) IN RAT LIVER BY LC/MS/MS V ersio n 1.0 ' Study No.s A nalyst/S ate: _ Revisions 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 : P atrick L . S outh _________ D ate: ' A pproved by : C< J d t l c . A n d re ., P h D . _____________ D ate: Gm j j J t f O> ;er, B io n a ly d c a l C h e m istry 3MEnvironmental Laboratory Page 1 of 16 cc P age 101 3M M edical D epartm ent Study: T-6316.5 BACK TOM AIN _ Battelle Study Number: N 003604-D 3M Environmental Laboratory Study Number: FACT 060998.1 1 METHOD FOR ANALYSIS OF POTASSIUM PERFLUOROOCTANESULFONATE (PFOS) IN RAT LIVER BY LC/MS/MS V ersion L0 S tudy N o.: A nalyst/D ate: _ L Su m m ary T he extraction and analysis o f potassium perfluorooctanesulfonate and related fluorochem icals in ra t liv er is perform ed. C alibration standards are prepared by spiking blank liver hom ogenate w ith solvent standards tram tw o independently-prepared stocks. The calibration standards are fortified w ith surrogate standard, buffered, an d extracted w ith ethyl acetate. The organic phases are evaporated to dryness and reconstituted in m ethanol fo r analysis by LC/M S/M S. EL P u r p o se T o extract and analyze potassium perfluorooctanesulfonate and related fiuorochem ical com pounds found in Sprague-D aw ley ra t liver. in . Samples See C hain o f Custody records i f applicable. IV. General Instructions C alibrate all required balances according to the SO P on balance usage. M ake equivalent dilutions w hen th e volum e needed varies from the volum e stated in the m ethod. L abel a il standard and reagent solutions as specified in the appropriate SO P. I f you intend to reuse a solution fo r fu tu re tasks, be sure tire label includes the preparation date and study num ber for w hich the solution w as initially prepared. Sign on the final page a fth is m ethod to signify th a t you have follow ed th e m ethod as w ritten, all m aterials and reagents are current, an d all equipm ent has been properly calibrated. If you deviate from the m ethod, docum ent the change, and obtain the approval o f the unit manager, study director, o r task leader as soon as possible. Initial and date all data entries on the page on w hich they were m ade. I f only one person enters a ll data on a single day, the docum entation m ay be marfe in a single location on that page.. I f m ultiple sta ff m ake entries, the additional entries m ust be initialed and dated by the person m alting tire entry. L ine-outs o r NA denotes "N ot A pplicable". T he m ethod is w ritten in general chronological order, b u t the sequence ctf steps m ay be altered i f the analyst deem s i t appropriate, unless the order fo r certain activities is specified. Stocks w ill be used fo r th e duration o f th e study unless consum ed o r unless stability is considered suspect No correction w ill be m ade fo r purity o r salt content o f any test article b u t PFOSAA. a U se glass volum etric, E ppendorf repeater, or positive-displacem ent pipets fo r dispensing m ethanolic solutions. a C o n tact w ith T eflon by th e te s t a rtic le should b e m inim ized. . V. Materials See T able 1 for all required chem icals, reagents, and solvents. Use T able 1 fo r docum entation. C heck a ll labels carefolly to ensure that all m aterials are not expired and that they are the proper purity o r grade. Page 2 of 16 3MEnvironmental Laboratory c e. Page 102 3M M edical D epartm ent Study: T-6316.5 BACK TAM AIN 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 S tudy N<w___________________________ A n a ly it/D a te :_____________________________ M aterials U se P o ta ssiu m P e rflu o ro o ctan e sulfbnate (PFOS) lH ,n U K t2 H P e rflu o ro o ctan e Sulphonic A d d M -536 . MS70 PFOSAA PFOSA PFOSEA R at Liver A nalytical S ta n d a rd S u rro g a te S ta n d a rd A nalytical S ta n d ard A nalytical S ta n d a rd A nalytical S ta n d ard A nalytical S ta n d ard A nalytical S ta n d ard M atrix Amm onium Acetate, N IL C O , Sodium Hydroxide, N aO H Tetrabutylam m onium H ydrogensulfate (T B A ), rC H ,(C H ,),L N < H S C M Sodium Carbonate, N ajC O j Sodium B icarbonate, NaHCOi E thyl Acetate M obile Phase R eagent Prep E x tra Prep E x tra Prep E x tra Prep E x tra Prep T a b le t. M ateriali S upplier G rade o r P u r i tv 3M IC N 3M 3M 3M 3M 3M H arlan SpragueD aw ley S to ra g e Tem p Room Tem p Room Tem p Room Tem p Room T ern o Room Tem p Room T em p Room Tem p --20C RT RT RT Lot o r ID RT RT RT M ethanol M illi-Q W ater pH 7 Buffer pH 10 Buffer M obile Phase, Stocks, WS R eagent Prep, M obile Phase pH m eter c a lib ra tio n pH m eter c a lib ra tio n M illipore A STM Typel RTRT RT RT I R T m eans Room Tem perature. V L E q u ip m e n t See Table 2 for all required m ajor pieces o f equipm ent U se the table to docum ent the actual piece (e.g. m ake, m odel) o f equipm ent Check calibration ofa ll equipm ent requiring calibration (e.g. balances) to ensure i t is cu rren t Page 3 of 16 3MEnvironmental Laboratory F .-7 P age 103 3M M edical D epartm ent Study: T-6316.5 BACK TOM AIN _ 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 S tudy N o .:___________________________ Analyst/Daie:_______________________ E quipm ent A nalytical B alance W eight Set Use W eigh Standards or R eagents C alibrate Balance T ab le 2. E quipm ent M anufacturer M odel. XLm-SN 1 P ipettor P ipet Sam ples P ipettor P ipet Sam ples P ipettor P ipettor P ipet E tO Ac extraction phase P ip et R eagents, W IS E ppendorf - R epeater V ortexer- M ix Sam ples Freezer (-20C) R efrigerator a-9 Q C en trifu g e S tore QCs, B lank L iver S tore B uffer, Stocks P hase separation T est Tubes Centrifuge Tubes T est Tubes T ransport tubes M agnetic stirrer O rbital Shaker E v a p o ra to r L iver sam ple hom ogenization E xtract Sam ples E vaporate E xtracts Store QCs S tir m atrix E xtract Sam ples E vaporate E xtracts S tockw ell S c ie n tific Blue Falcon B lue Falcon E lkay Polypropylene, 15 mL P o ly p ro p y len e , 15 mL Polypropylene, 12 x 7 5 mm 5 mL polypropylene SW 8599 2096 . 2002 1 2 7 -T 1 6 0 -5 6 P Zyxnark Turbovap L V Syringe Filters F ilter E xtract H om ogenizer pH m eter E le ctro d e V olum etric Flasks, C lass A V olum etric Pipets, G ass A T ransfer Pipets. P lastic G rind liver D eterm ine B uffer pH D eterm ine B uffer pH M ake V olum etric D ilutions M ake V olum etric D ilutions T ransfer E xtracts to C entrifuge F ilters and LC Inserts NA NA Sam co NA NA NA NA Page 4 of 16 3MEnvironmental Laboratory CQ Page 104 3M M edical D epartm ent Study: T-6316.5 BACK TOM AIN _ Battelle Study Number: N003604-D 3M Environmental Laboratory Study Number: FACT 060998.1 METHOD FOR ANALYSIS OF POTASSIUM PERFLUOROOCTANESULFONATE (PFOS) IN RAT LTVER BY LC/MS/MS V e n a n 1.0 S tudy N o.: A nalyjt/D ate: V EL PROCEDURE A. Preparation of 2 mM Ammonium Acetate W eigh 0.1300 + 0.0020 g o f am m onium acetate and transfer to a 1000-m L volum etric flask. D issolve the solid in w ater and dilute to volum e w ith w ater. Solution m ay be used fo r one m onth stored a t room tem perature. A ctual m ass o f am m onium a c e ta te :___________ A ctual fin al v o lu m e :__________ D ate o f p re p ara tio n :______________________ Study N o: ____________ B. Preparation of~29% Sodium Hydroxide Solution W eigh 200 2 g o f sodium hydroxide into a beaker. A dd 500 m L o f M Oli-Q w ater and m ix to dissolve. Cool and transfer to a polypropylene bottle fo r storage. Solution may be stored fo r 6 m onths a t room tem perature. A ctual m a o f sodium hydroxide: _ _ _ _ _ _ A ctual volum e M illi-Q w a te r __________ D ate o f p re p ara tio n :______________________ Study N o :________________ C. Preparation of --2.9% Sodium Hydroxide Solution ' A dd 10 mL o f--29% Sodium H ydroxide Solution to a 100-m L volum etric flask and dilute to volum e w ith M illi-Q w ater. T ransfer to a polypropylene bottle for storage. Solution m ay be stored fo r 6 m onths a t room tem perature. A ctual volum e o f ~29% NaOH so lu tio n :___________ Actual final volum e: __________ D ate o f preparation : _______________________ _ Study N o :________________ ' D. Preparation of Tetrabutylammonium Hydrogensulfate (TBA) Solution, 0.5 M, (pH 10) pH M eter C alibration pH buffer 7 pH b u ffer 10 pH reading:________ pH read in g :________ A dd 1 6 9 i Ig a fT B A to -S O O m L a fM illi-Q w a te rin a b e a k e r. A djust the pH to 10.00 t 0.02 using -5 5 -6 0 m L o f 29% Sodium H ydroxide Solution, dilute to 1000 m L wife . M illi-Q w ater, and m ix. A djust the pH to 10.00 * 0.02 using -2 .9 % NaOH and m ix. T ransfer to a polypropylene bottle fo r storage. Solution m ay be used fo r one m onth stored a t room tem perature, b u t th e pH m ust be checked n rio r to each use. A djust to pH 10.0 i 0.02 w ith 2.9% Sodium H ydroxide Solution as necessary. Page 5 of 16 3MEnvironmental Laboratory it n Page 105 3M M edical D epartm ent Study: T-6316.5 BACK TOM AIN _ 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 aly st/D ate:_____________________________ A ctual m ass o f T B A :___________ A ctual fin al v o lu m e :_________ . A ctual fin al p H :___________ D ate o f p re p ara tio n :_____________________ 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 W eigh 26.5 1 0.1 g o f sodium caihonate and 21.0 * 0.1 g o f sodium bicarbonate and transfer to the sam e 1000-mL volum etric flask. Dissolve the m aterials in M illi-Q w ater, dilute to volum e w ith M illi-Q w ater, m ix, and transfer to a polypropylene bottle fo r storage. Solution m ay be used fo r 1 m onth when stored refiigerated. A ctual m ass o f sodium carbonate: __________ A ctual m ass o f sodium bicarbonate:__________ A ctual fin al volum e: __________ D ate o f p re p ara tio n :_____________________ Study N o :________________ F . P re p a ra tio n o f M o b ile P h a se C om ponent A: M ix together 600 mL o f 2 m M ammonium acetate and 400 m L o f methanoL Solution m ay be used fo r 1 m onth when stored a t room tem perature. A ctual volum e o f 2 m M amm onium a c e ta te :________ mL A ctual volum e o f m ethanol: ________ mL D ate o f p re p ara tio n :_____________________ Study N o :________________ C om ponent B : M ix together 50 mL o f 2 m M ammonium acetate and 950 mL o f m ethanoL Solution m ay be used fo r 1 m onth when stored a t room tem perature. A ctual volum e o f 2 niM ammonium acetate: A ctual volum e o f m ethanol: m l. D ate o f p re p ara tio n :_____________________ Study N o :________________ mL G . P re p a ra tio n o f S to c k 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 S S ) 1. Stock Surrogate Standard (250,000 ng/mL): W eigh 25 a 2 m g o f 1H, 1H, 2H, 2H ,-pcrfluorooctane sulphonic a d d and transfer to a 100-mL volum etric flask. D issolve in methanoL dilute to volum e w ith m ethanol, and m ix. Store refiigerated, protected fiom U V lig h t A ctual W eig h t:_________________ A ctual D ilution V olum e:____________________ D ate o fP rep aratio n :___________________ Study No: __________________ Page 6 of16 3MEnvironmental Laboratory F.in Page 106 3M M edical D epartm ent Study: T-6316.5 BACK TOMAIN 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 S tudy N o .:____________ ;______________ A n aiy st/D ate:_____________________________ 2 . W SS (1000 ng/m L): D ilu te 100 /JL ctf stock, surrogate standard to 25 mL w ith m ethanol and m ix.. A ctual V olum e o f Stock Internal S tan d ard :____________ A ctual D ilution V olum e:____________________ D ate o fP re p a ra tio n :___________________ H . Preparation of Calibration Solvent Stocks and Working Standards S tandard Stodc 1A Stodc IB Stock 2A Stodc 2B Stock 3A Stock 3B Stock 4A Stock 4B Stodc SA Stodc SB Stodc 6A 1. Solvent Stocks: F o r each analyte w eigh the specified am ount o f standard (independently w eighed as A and B replicates) listed in Table 3 and transfer into separate volum etric flasks. D issolve in m ethanol, dilute to volum e w ith m ethanol, and m ix w ell. S tore refrigerated, protected from UV light. 2. M ixed Solvent Stocks: . P ip e tth e specified am ount o f each analytical standard R eplicate A as listed in T able 3 and transfer into a single volum etric flask. D issolve in m ethanol, dilute to volum e w ith m ethanol, and m ix w ell. Store refrigerated, protected from UV lig h t R epeat the process w ith R eplicate B stocks. Themixedsolventstocksare - - usedtopreparethe working standards. D ate o f p re p a ra tio n :_____________________ Study N o :________________ 3. W orking Standards (W S): D ilute th e m ixed stocks and w orking standards w ith m ethanol as specified in T able 3 an d m ix w ell. D ate o f p rep aratio n :__________________________________ 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 ctual A m ount T arg et A ctual Am ount A nalytical Std. S tudi, o r W S 50 x 1 mg 25 0.S m g i t . : : SO x 1 m g g g a S issS sS m K S 25 t 0.5 m g i r>i Esc:i f H'l p ;j| 50 x 1 mg * .m g * 25 * 0.5 mg 93 x 1 m g fell0 : 46 x 0.5 mg 50 x 1 mg B:i3wSf mg 25 x 0.5 mg mg* 50 x 1 m g ________________ S S _ Final Vul im L) 10 10 10 10 10 10 10 10 10 10 10 F inal Vol. (m L) ' 1 .cs?r. 't-'SS j :Vii:i: ^5Hs: :? N om inal Conc (n/m L> 5,000,000 2,500,000 5,000,000 2 J 0 0 .0 0 0 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 BACK TOTIAIN 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 - S tudy N o .:__________ A n aly st/D ate:_____________________________ Stock 6B M ixed Stock A M ixed Stock B WS I W S2 W S3 WS 4 WS 5 W S6 WS 7 FFOSEA Stocks 1 thru 6 Rep A Stocks I thru 6R epB M ixed Stock A M ixed Stock B WS 1 W S2 WS 3 W S4 W S5 25 0.5 m g 5 mL each** 5 mL each** 1 mL** -------.n iL e a c h ty [ip.: 7> mL _ 1 mL** mL 2 mL** 2 mL** 2.5 m L** 2.5 mL** 2 mL** ~ mL mL l 10 *Ks:r.: tin 2.500.000 50 SS:luf!:::: ':H?ltj:Vtur.t 500,000 50 ~>*wftJ?!i:f.H:: 250,000 >M>;2 52* 25 :xtrssHx?:::;i:-: i5'y.>i 20,000 fincans:::s:::s::;: 25 SUHilwSttttRl 10,000 Bt::trPrj; :H:;H:*::iT: 10 4000 10 g S S I s S 2000 10 :t"HJt!: 1000 10 500 10 ~j*&**!;Ifi * 200 * W eigh all analytical standards to a t least the nearest 0.01 m g. ** Use volum etric o r positive-displacem ent p ip ers). L Preparation of Calibration Standards and Blanks l . L iver hom ogenate P repare blank liv er hom ogenate in bulk by w eighing approxim ately 40 g o f blank liv er into a 500 m L Nalgene bottle containing 200 mL of M illi-Q w ater. G rind to a hom ogeneous suspension. A liquot into approx 30 mL portions fo r frozen (approx -2 0 Q storage. A ctual M ass o f L iven_____________ A ctual volum e o f w ater____________ D ate o f p re p :____________________ S tudy:______________________ D eterm ine density o f calibradon/Q C m atrix: M IX HOM OGENATE THOROUGHLY and determ ine the m ass in m illigram s o f 10 replicate w eighings o f 1 mL portions o f th e THOROUGHLY MIXED hom ogenate. M K HOM OGENATE IM M EDIATELY PRIOR TO EACH ALIQUOT REM OVAL. R eplicate# M ass (inai a 2. L iver C alibration Standards Prepare each Ever calibration standard by adding 0.45 mL o f undiluted liver hom ogenate (STIR HOMOGENATE WHILE ALIQUOTING) into a 15 mL extraction tube and adding 50 pL o f WS or M eOH. Prepare triplicate cal standards and 6 blanks. See Table 5 fo r volum es. The diluted liv er density is assum ed to be approxim ately 150 m glm L. M ix w ell. Page 8 of 16 3MEnvironmental Laboratory cn Page 108 3M M edical D epartm ent Study: T-6316.5 BACK TOM AIN _ 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 Vernon 1.0 S tudy N o.: A nxlyst/D ate: _ Cal S td/B lank 1 2 3 4 5 6 7 B lank S o u rc e W Sl WS 2 WS 3 W S4 WS 5 WS 6 WS 7 M eO H T a b le s . C a lib ra tio n S tan d ard s and Blanks T ara et Voi A ctual Vol T arg et A ctual N om inal 0-L) (aL ) F inal Vol Final Vol C one (m L) (m L) (ns/m U 50 0.5 2000 50 n s g g p i 0.5 50 xri: 50 tgri |t 0.5 0 .5 50 1k^SSH:*3 rt: 0 .5 1000 ;:Sb: :hi 400 200 100 50 [X " i-j *st1' " 0.5 50 50 0.5 20 50 1 0.5 <: < 0 N om inal Cone (u a /u ) 13 6.6 2.6 1J 0 .6 6 0.33 0.13 0 D ate o f preparation o f cal stds/blank:______________________________ J. Preparation of Quality Control Liver Samples (QCs) 1. Q uality C ontrol W orking Standards D ilute the follow ing source volum es m ethanol in volum etric flasks and m ix w ell. P repare fresh w hen used. A ctual volum es are in parentheses. Soin ID OC WS 1 QCW S2 QC WS 3 QCW S4 Z Source M ixed Stock A M ixed Stock B QC WS 1 QCW S2 w. -W T ab led . Q C W S P rep aratio n Vol Source. mL Final Vol. m L mm IC --? . 50 ( ) Preparation o f Q uality C ontrol L iver Sam ples Cone, mi/mL 15,000 5000 1125 250 P repare each QC in bulk by fillin g the volum etric flask approxim ately h a lf fu ll with undiluted liver homogenate (STIR HOMOGENATE WHILE ALIQUOTING), adding the appropriate QC W S, m ixing, and diluting to volum e w ith undiluted Uver hom ogenate (STOR. HOMOGENATE W HILE ALIQUOTING). M IX THOROUGHLY and dispense Z5-m L aliquots into polypropylene tubes and store a t approxim ately -2 0 C T able 7. Q C P rep aratio n QC Source Vol Source. mL F inal V ol. m L Cone. n*a/mL Cone. U!T2 1 OCWS 1 llS H t S iiiM iS i 1500 1 10 2 QC WS 2 500 3.3 3 QCWS 3 Z5f ` 112.5 0.7 4 OCWS 4 Z5f ) 25 0.16 D ate o f QC p rep :____________________ S tudy.______________________ K. Preparation of MS Check Standard for System Suitability P ipet 250 itL o f WS 2 a t--10,000 ng(mL and Z 5 mL o f WSS a t -1000 ng/mL in m ethanol into the sam e 50-mL volum etric flask. D ilute to volum e w ith M eOH and m ix. Page 9 of 16 3MEnvironmental Laboratory f_i a Page 109 3M M edical D epartm ent Study: T-6316.5 BACK TOMAIN Battelle Study Number: N 003604-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 . S tudy N o .:______________________________ A n aly st/D ate:________________________________ L. Preparation of homogenizer recovery liver samples . M. To determ ine the recovery from the hom ogenization process, nnhom ogeaized blank liv er w ill be fortified in duplicate a t 3 concentration levels and hom ogenized as follow s. T his needs to be done every day th at hom ogenization o f study sam ples is perform ed. 1. Place approxim ately 0.3 g o f unbom ogenized blank liver into each o f 6 ,1 5 mL polypropylene centrifuge tubes. Record w eights o f liver. X A dd 100 pL o f WS 1 ,3 , and 4 (one WS p er duplicate tubes) to prepare - fortifications a t approxim ately 4, 0.8, and 0.4 pg/g. 3. M ultiply th e m ass o fliv e r in g by 2.5 and add this m anym L ofw ater. 4. H om ogenize each liv er sam ple, and rinse hom ogenizer probe w ith another volum e o f w ater used in step 3, adding rinse to hom ogenized sam ple. . 5. C lean hom ogenizer w ith MeOH betw een sam ples. 6. C ap and vortex hom ogenate for use in extraction. Preparation of Dilation Check Sample 1. Place 2.95 mL o f undiluted liver hom ogenate (STIR HOM OGENATE W HILE ALIQUOTING) into a 15 mL extraction tube and add 50 pL o f M ixed Stock A. X D ilute 50 pL o f step 1 solution (VORTEX SOLUTION W HILE ALIQUOTING) w ith 0.45 mL o f undiluted liver hom ogenate (STIR. HOM OGENATE WHOLE ALIQUOTING) in 3 ,1 5 mL extraction tubes. 3. T his sam ple should be prepared for extraction only on days when study sam ples w ill be diluted and extracted. .- N. Homogenization of study samples 1. Place approxim ately 0.5 g o f unhom ogenized study sam ple liver into a 15 mL polypropylene tube. R ecord w eights o fliv er. X M ultiply the m ass o f liver in g by X5 and add this m anym L o f w ater. 3. H om ogenize each liver sam ple, and rinse hom ogenizer probe w ith another volum e o f w ater used in step X adding rinse to hom ogenized sam ple. 4. Clean homogenizer with MeOH between samples. 5. Cap and vortex hom ogenate for use in extraction. O . Analysis Standards, Blanks, QCs, and Samples 1. M IX LIVER HOMOGENATES THOROUGHLY BEFORE ALIQUOTING an d p ip et 500 /jL o f each QC (4 replicates per level), and other sam ples being extracted into 15-mL polypropylene extraction tubes. T he c al std s a n d blan k s a re already aliquoted. X To the B la n k s-IS (3 reps), add 100 jL o f M eOH and vortex. 3. T o the B lanks + IS (3 reps) and to the rem aining sam ples, add 100 jL W SS and v o rte x . 4. A dd 0.5 mL o f 0.5 M T B A (pH 10) to all tubes and vortex briefly. 5. A dd 1 mL o f 0X 5 M carbonate buffer and vortex briefly. 6. A dd 2 J mL o f ethyl acetate. Place the tubes sidew ays on the orbital shaker a t a ' setting o f300 fo r -2 0 m inutes. 7. C entrifuge tubes a t a setting o f3500 rpm for -2 0 m inutes to separate layers. 8. T ransfer 2 mL o f the top organic layer to a d ean polypropylene tube. Page 10 of 16 3MEnvironmental Laboratory E-14 Page 110 3M M edical D epartm ent Study: T-6316.5 BACK TOTIAIN Battelle Study Number: N 003604-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.:___________________________ Anaiyjt/Date: ______________________________ 9. Evaporate to dryness tm der nitrogen a t a setting o f 30*C fo r-6 0 m inutes. 10. R econstitute the residues in 500 mL o f m ethanol w ith vortexing. 11. Syringe-filter extracts into antosnm plcr vials fo r analysis. Store vials refrigerated (up to 1 m onth) if LC/M S/M S w ill not be perform ed the sam e day. Since 3-day room tem perature extract stability w as dem onstrated during validation, the - extracts o f the cal stds, blanks, and QCs may be reused fo r up to 3 days after ' th eir initial preparation i f held a t room tem perature (recap the vials i f reusing). D ate o f cal std/blank extract p rep :_________________________ D ate o f QC extract p re p :_________________________________ P. LC/MS/MS Analysis . tfPyfafo 8 . 1. U se the system conditions specified in Table ys. The conditions w hich are designated m ay be m odified by the analyst to produce acceptable peak shape. L C /M S /M S System T able 8 - LC /M S/M S C onditions A utosam pfcr H PLC Pum ps M ass S pectrom eter A nalytical colum n M obile P hase C om ponents G ra d ie n t profile Injection volum e Plirn split M a k e :_____ M odel: ID : M ake: _____ M odel: ID: M ake: M odel: ID : K eystone B etasil C18, 5m. 2 x 50 m m . P art No. 055-701-2, S /N : :L oc Com ponent A Ammonium acetate m ethanol, 60:40, vrt Com oonent B: Am m onium acetate:m ethanol. 5:95. vy ' T im e, m in %B Flow . m L/m in 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 colum n flow sp lit to *30 ML/min ( uL /m inl into the M S a t run start C olum n Tem p Am bient lU 'L C P ressure 1000 psi a t gradient s ta rti osi) ' M S Source Electrosnrav. N egative Ion D esolvation as N ebulizer as Nitrogen at 575 L /hr ( N itrogen a t 80 L /hr ( L /h r) L /hr) S o u r c e B l o c k T e m p *140C ( o D c m i Is 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, PFO SA A PFO SEA M -556, M 570 C ollision energy *40 eV (___ eV ) PFO S, I t PFOSA PFOSAA, M -556, M 570 C ollision as *30 eV ( eV) PFOSEA 1 Argon a t *2.5 x HT* mb gas cell ( mb) M ultiplier R e stitu tio n I 650 V ( ___ Y)___ ____ .____________________________________ ____________ 1 1 *12.0 for M SI ( ): *10.0 for M S 2 ) jv/ouaj kcto "sr"; * feeffirlVf Page 11 of 16 3MEnvironmental Laboratory T7_1* P age 111 3M M edical D epartm ent Study: T-6316.5 BACK TOM AIO _ Battelle Study Number: N 003604-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 S tudy N o .:___________________________ Analyst/Date:_______________________ Ions m onitored Total run tim e A pproxim ate reten tio n tim es: 427>81 MRM transition fo r Surrogate Standard (SS) 1 499>99 MRM transition fo r PFOS 1 556>78 MRM transition fo r M -556 5 7 0 1 6 9 MRM transition for M 570 1 584>169 MRM transition fo r PFOSAA 1 498>78 MRM transition fo r PFOSA 526>169 MRM transition fo r PFOSEA *11 m inutes ( SS: 4 m in ( PFOS: 4.3 m in ( m in) m in) ___m in) M -556: 4 a m in ( ____m in) M 570:4.6 m in ( __ m in) PFOSAA: 4.7 m in e m in) PFOSA: 5.1 m in (______ m in) PFOSEA: 5.7 m in 1 m in) * Param eters that m ay be changed by the an aly st A ctual values in ( ). 2. The above conditions should be suitable for the M icrom ass Q uattro L C (S/N 9053). M odifications m ay be necessary if another M icrom ass Q uattro Series spectrom eter is used. Split the flow post-colum n via a K eystone B IO -tee or sim ilar device. 3. C alibrate the m ass spectrom eter using a suitable reference com pound, o r verity that the calibration is suitable by visual inspection (on the tune page) th at a suitable m obile phase ion is still accurately determ ined. R esolution m ay need to be higher than that used for analyzing sam ples. -' 4. To check the proper perform ance o f th e instrum ent, inject the instrum ent check standard. The results should be com parable to a recent injection if available. 5. U se an autom ated chrom atography integration softw are system to collect the output from the analysis. 6. Loading O rder See the loading report from the autom ated chrom atography integration softw are system . 7. M ake single injections o f each cal standard, QC, study sam ple, or blank. M ake a t least 4 injections o f the instrum ent check standard. 8. R un set sizes should typically not exceed 80 injections due to instrum ent response roll-off considerations. L onger runs m ay be perform ed, b u t they pose a risk o f yielding unacceptable curve results. VUL CALCULATIONS 1. Spreadsheet Software: ____________________ V e rsio n ________ 2. M S A nalysis Softw are:_______________________V ersio n ________ 3. C alculate the average density of th e liver hom ogenate (10 reps) in m g/mL. 4. U sing the average density o f the hom ogenate, calculate its liver density (m g o f liver per mL o f diluted hom ogenate): U ndiluted liver density (m g/m L) - (g o f liv er x average density o f hom ogenate)/(g o f liver + g of w ater) w here g o f liv er and g o f w ater are m asses used to prepare b u lk hom ogenate; density of w ater is assum ed to b e l g/mL. D iluted liver density (m g/m L) " U ndiluted density + D iln Factor Page 12 of 16 3MEnvironmental Laboratory F.-16 Page 112 3M M edical D epartm ent Study: T-6316.5 BACK TOMAIN Battelle Study Number: N 003604-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 a ly st/S ate :_______________________________ W here diln fa c to r--2/1.8 * 1.1111 to account fo r 10% diln o f liv e r hom ogenate in cal std and Q C m atrices. 5 . C alculate the actual concentration (ng/m L) o fPFOS and other flnorochem icals in th e suspensions o f calibration standards and QCs by using the m ass o f analytes and dilution factors only (no liver density correction). Use purity correction for FFOSAA only. 6. falmtnti the actual concentration of PFOS and other flnnmriiMniraR hi liw r fn r rim calibration standards and Q Cs as follow s: C onc(|ig/g) " C one (ng/m L) * D iluted Liver density (mg/mL) x 1000 m g/g x 1 0 pg/ng 7 . A ssure th at the integrations o f the peak areas o f the test article and surrogate standard are correct F lag m anual integrations w here perform ed. Calculate the exact concentration o f each .- liver standard. 8 . C alculate the regression equation relating the peak response ratio (test ardde/S S ) o f each calibration standard (y-axis) to test article concentration in liv e r (x-axis) fo r PFO S, M -556, M 570, and FFOSAA. C alculate the regression equation relating the peak area o f r h calibration standard to test article concentration in liver fo r PFOSA and PFOSEA. PFO S, M 356, M 570, and FFOSAA are quantitated by using the surrogate standard as an internal standard: PFOSA and PFO SEA are quantitated w ithout reference to the surrogate fexternal standard calibration curve). U se a q u a d ratic regression w eighted V x, orig in excluded, fo r all analytes. 9. C alculate a determ ined concentration fo r each injection o f calibration standard, QC, and ' sam ple using the regression param eters and the peak response ratios o r areas. 10. Oatentare the relative error, average relative error, standard deviation, and relative standard deviation fo r all QCs. C alculate die relative error for each injection o f calibration standard. 11. C alculate the average recovery fo r die hnnuigenm r recovery fnTtifirarirma 12. C alculate the relative standard deviation for the PFOS to SS peak area ratio o f the replicate * in jection s o f the check, standard. IX. a c c e p t a n c e C r it e r ia A. MS Check Standard (System SuitabDity) A t least 3 injections o f the M S Check Standard m ust provide a %RSD o f 10% o r less fo r the PFOS to SS peak area ratio. B. Calibration Standards T he percent relative errors fo r th e concentration-level averages of the calibration standards should m eet die follow ing lim its: Page 13 of 16 3MEnvironmental Laboratory E-17 P age 113 3M M edical D epartm ent Study: T-6316.5 BACK TOTIAIN _ 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 L 0 Study No.: Analyst/Date: _ T able 9. C alibration Standard Acceptance lim its ANALYTE % Error PFOS 20 05% at LOO) M -556 20 05% at LOO) M 570 20 05% at LOO) PFOSAA 20 05% at LOO) PFOSA 20 05% at LOO) PFOSEA 25 i30% atL 001 Up to 5 calibration standard injections m ay be excluded from the curve, provided th at one injection rem ains p er level. Rem oval o f an entire level may be done if approval is obtained. I f an entire level is-.removed, the sam ples bracketed by the rem aining calibration range w ill be - considered acceptable. T he calibration curve should have a coefficient o f determ ination o f 0.97 o r better. C. QCs The concentration-level average percent relative errors and percent relative standard deviations o f th e QCs should m eet the follow ing lim its: T able 10. QC A cceptance lim its ANALYTE % PFOS 20 M -556 20 M 570 20 PFOSAA 20 PFOSA 20 PFOSEA 25 Rem oval o f individual values from the QC calculations may be done if accom panied by a reasonable explanation (e.g_, instrum ent m alfunction or D ixon's Q test results). If die average determined concentration for any QC level exceeds the acceptance limit, the task leader or study director should be n otified . T he ran may be repeated o r a portion o f the run m ay be considered acceptable. F or exam ple, if the low QC fails the stated requirem ents, sam ples may be accepted th at have concentrations bracketed by die highest calibration standard and a m id-level QC concentration. D. Homogenizer Recovery and Dilution Check Samples The avenge recovery across the 3 levels o f hom ogenizer recovery sam ples as w ell as that o f the dilution check sam ples should f ill w ithin the range o f 70-130% inclusive. Rem oval o f individual outliers from the calculations m ay be done if accom panied by a reasonable explanation. E. Sensitivity (LOQs) The validated lim its o f quantitation are nom inally 0.13 pg/g each for PFOS, M -556, M 570, and PFOSAA. Page 14 of 16 3MEnvironmental Laboratory C 1Q Page 114 3M M edical D epartm ent Study: T-6316.5 BACK TOM AIN _ Battelle Study Number: N 003604-D 3M Environmental Laboratory Study Number: FACT 060998.1 METHOD FOR ANALYSIS OF POTASSIUM PERFLUOROOCTANESULFONATE (PFOS) IN RAT LIVER BY LC/MS/MS V enioB 1.0 S tudy N o .:_____________________________ . ' A naiyrt/D ate: _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ F o r PFOSA and PFOSEA , the validated LOQs are nom inally 0.33 pg/g each. Due to the nature o f die preparation o f die calibration standards, low er concentrations o f PFO SA and PFOSEA w ill be carried through th e extraction. These low er concentration values w ill be evaluated w ith each ru n set, and m ay be included in the rgressions if they m eet acceptance criteria. I f they are included, study sam ples w hich are quantitated to have concentrations below the validated level (nom inally 0.33 pg/g) w ill be appropriately flagged. F. Specificity T he m ethod suffers from endogeneous m atrix interferences a t levels som etim es exceeding 20% o f LOQ. T he intercept o f d ie calibration curve appears to offer som e correction fo r any - effect on quantitations. A cceptai perform ance (error) o f the low est used standard, therefore, w ill be considered sufficient evidence that bracketed study sam ples are quantified properly. G. General T he above acceptance criteria indicate that th is m ethod is capable o f producing occasional errors outside the norm al acceptance criteria o f a validated m ethod (15% norm ally). W here indicated, replicate analyses lessen die im pact o f these occasional outliers. X. RESULTS See attached hard copy o f spreadsheet o r see file on netw ork drive. XL C o m m e n t s 3MEnvironmental Laboratory Page 15 of 16 T?tn Page 115 3M M edical D epartm ent Study: T-6316.5 BACK TATIAIN _ Battelle Study Number: N 003604-D 3M Environmental Laboratory Study Number: FACT 060998.1 METHOD FOR ANALYSIS OF POTASSIUM PERFLUOROOCTANESULFONATE (PFOS) IN RAT LIVER BY LGMS/MS Version U> Study No.: - A nalyit/D ate___ X3L C onclusions XHL S ig n a t o r e s A nalysts ________________________________________________ . - - _______________________________________ ________________________________________________ ________________________________________________ Technical Review ________________________________________________ '_________________________________________ QC Review D ate: ' D ate D ate D ate D ate D ate D ate Date 3MEnvironmental Laboratory Page 16 af 16 Page 116 3M M edical D epartm ent Study: T-6316.5 BACKTOM AIN _ 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 ofN-EtFOSE in Rats PROTOCOL AMENDMENT NO. 1 Amendment Date: July 28,1999 Performing Laboratory 3M Environmental Technology & Safety Services 3M Environmental Laboratory 935 Bush Avenue S t Paul, MN 55106 Laboratory Project Identification ET&SS FACT-TOX-013 LIRNU2095 3M Environmental Laboratory 3MEnvironmental Laboratory E-21 Page 117 3M M edical D epartm ent Study: T-6316.5 BACK TOTIAIN _ Battelle Study Number: N 003604-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 mend TOREAD: 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 Marvin Case Ph.D., Sponsor Representative Kris J. Ph.D., Study Director g /2-/^5 Date 3M EnvironmentalLaboratory 3MEnvironmental Laboratory r? Page 118 3M M edical D epartm ent Study: T-6316.5 BACK TOM AIN _ Battelle Study Number: N 003604-D 3M Environmental Laboratory Study Number: FACT 060998.1 Study Title Combined Oral (Gavage) Fertility Development and Perinatal/Postnatal Reproduction Toxicity Study ofN-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 r? 0*2 Page 119 3M M edical D epartm ent Study: T-6316.5 BACK TOM AIN _ Battelle Study Number: N 003604-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 TOread: 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 of 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 of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds from Serum for Analysis Using HPLC-Electrospray Mass Spectrometry" ETS-8-5.1, "Analysis of 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, w ere updated on 04/27/99 to ETS-8-4.1 and E T S-8-5.1. 3M Environmental Laboratory 3MEnvironmental Laboratory t: D/i Page 120 3M M edical D epartm ent Study: T-6316.5 BACK TOTIAIN Battelle Study Number: N 003604-D 3M Environmental Laboratory Study Number: FACT 060998.1 Protocol FACT-TO X-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 ofFrluorochemicals in Liver Extracts Using HPLC- . Electrospray/Mass Spectrometry" AMEND TOread: 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 fc k Kristen J. Hansen Ph.D., Study Director 3M EnvironmentalLaboratory 3MEnvironmental Laboratory T? O C Date P age 121 3M M edical D epartm ent Study: T-6316.5 BACK TOMAIN _ 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 Environm ental T echnology & Safety Services 3M Environm ental Laboratory 935 Bush Avenue St. Paul, M N 55106 Laboratory Project identlfcation ET&SS FACT-TO X-013 LIR N U 2095 3MEnvironmental Laboratory 3MEnvironmental Laboratory E-26 Page 122 3M M edical D epartm ent Study: T-6316.5 BACK TOM AIN _ Battelle Study Number: N 003604-D 3M Environmental Laboratory Study Number: FACT 060998.1 * i* Protocol FACT Tox-013 Amendment Number 3 This am endm ent m odifies the follow ing portion(s) o f the protocol: 1. Protocol Reads: 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 of resources and James K. Lundberg will begin serving as the study director for'FACT-TOX-013 as of 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 of 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 of the study during its progress and before acceptance of the final report. When the final report is completed, all original paper data, including: approved protocol and amendments, study correspondence, shipping records, raw data, approved final report, and electronic copies of data will be retained in the archives of the 3M Environmental Laboratory. All corresponding training records, calibration records, instrument m aintenance logs, standard operating procedures, equipm ent procedures, and methods will be retained in the archives of the facility performing each analysis. Re a s o n : To direct subcontract laboratories in the disposition of the items listed above. 3M Environmental Laboratory 3MEnvironmental Laboratory T*? P age 123 3M M edical D epartm ent Study: T-6316.5 BACK TOTIAIN _ Battelle Study Number: N 003604-D 3M Environmental Laboratory Study Number: FACT 060998.1 Protocol FACT Tox-013 Amendment Number 3 3. Protocol reads: Disposition of test and control substances: Biological tissues and fluids are retained per GLP regulation. A mend 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 of 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 of specimens analyzed at subcontract laboratories. Amendment Approval TG toL Marv Case, D.V.M., Ph.D., Sponsor Representative Date & L J k ---- --------------- ^ ________;_____________________ t O / Z f U Kristen J. Hansen, Ph.D., Previous Study Director Date 1 Dale L. Bacon, PfcB., 3M Environmental Laboratory Management !f fs h 1 Date 3M Environmental Laboratory 3MEnvironmental Laboratory T ? TO Page 124 3M M edical D epartm ent Study: T-6316.5 BACK TOM AIN Study Title Analytical Study o f 2(N-Ethylperfluorooctanesulfonamido)-ethanol in Two Generation Rat Reproduction PROTOCOL AMENDMENT NO. 4 Am endm ent Date: 20 January 2000 Perform ing Laboratory 3M Environmental Technology & Safety Services 3M Environmental Laboratory 935 Bush Avenue St. Paul, MN 55106 Laboratory P roject Identification ET&SS LRN-U2095 FACT TOX-013 Argus Study: 418-009 3M Medical Department Study: T-6316.5 3M Environmental Laboratory 3MEnvironmental Laboratory E-29 Page 125 3M M edical D epartm ent Study: T-6316.5 BACK TOM AIN Protocol LRN-U2095 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 CFR 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. o i l r--______ __ ___I $ _ L ______ i __ _ ,, 3MEnvironmental Laboratory E-30 Page 126 3M M edical D epartm ent Study: T-6316.5 Amendment Approval BACK TOTIAIN Protocol LRN-U2095 Amendment Number 4 John L B utenhoffP h D ., Sponsor Representative Fste*t r y to , Date fh k A /M A M a rv in T. Case, D. V.M., P h D ., Incoming Study Director J.P_R-Uaju (h^r Date V 3 M Environmental I ahnratnrv 3MEnvironmental Laboratory E-31 Page 127 3M M edical D epartm ent Study: T-6316.5 BACK TOM AIN Study Title Analytical Study o f 2(N-Ethylperfluorooctanesulfonamido)-ethanol in Two Generation Rat Reproduction PROTOCOL AMENDMENT NO. 5 Am endm ent Date: August 31,2000 Perform ing Laboratory 3M Environmental Technology & Safety Services 3M Environmental Laboratory 935 Bush Avenue St. Paul, MN 55106 Laboratory P roject 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 BACK TOM AIN - Protocol FA C T TOX-013 Amendment No. 5 This am endm ent m odifies th e follow ing portion(s) of the 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. REASO N: The role of Principle Analytical Investigator was reassigned due to availability of resources. 3 M Fm /ironm antal Laboratorif 3MEnvironmental Laboratory E-33 Page 129 3M M edical D epartm ent Study: T-6316.5 Amendment Approval BACK TOM AIN Protocol FA C T TOX-013 Amendment No. 5 John L. Butenhoff, Ph.D ., Sponsor Representative ; T C *u M a rvin T. Case, D .V.M ., Ph.D., Study Director Date Date 3 M Fnwirnnmontal I ahnratnrv 3MEnvironmental Laboratory E-34 Page 130 3M M edical D epartm ent Study: T-6316.5 BACK TOM AIO _ Battelle Study Number: N 003604-D 3M Environmental Laboratory Study Number: FACT 060998.1 DEVIATION REPORT Battelle Study Number: N003604-D 3M Environmental Technology and Services Study Number: FACT 060998.1 2 (N-Ethylperfluorooctanesulfonamido)-ethanol in Two Generation Rat Reproduction TY PE O F D EVIA TION S: PR O TO C O L DA TES O F DEVIATIONS: O ctober 18, 1999 N A T U R E O F D EV IA TIO N S: Som e o f the analytical m ethod acceptance criteria w ere not m et for the L C /M S/M S analysis conducted 180ct99 at B attelle. These deviations relate to protocol am endm ent 2. See below for sum m ary. A nalvte PFOS M -556 M -556 M -556 M -556 PFOSAA PFOSAA A cceptance criterio n n o t m et Q C 3 e x ce ed e d 2096 e r ro r (-20.3% a ctu al) Q C2 exceeded 2096 erro r (23.4% actual) ( QC3 exceeded 20% R SD (21.2% actual) Q C4 exceeded 20% R SD (28.8% actual) D ilution recovery exceeded 130% (131.5% actual w ith 21.6% RSD) QC3 exceeded 20% erro r (-20.6% actual) Q C4 exceeded 20% R SD (21.3% actual) C A U SE O F D EV IA TIO N S: Sam ple preparation and/or L C /M S/M S variabilities over the course o f the sam ple set m ay have contributed to the deviations. IM P A C T O F D EV IA TIO N S O N T H E STU D Y : T he errant Q C values w ere bracketed by acceptable QC concentration levels w hich dem onstrates that the calibration curves generally provided good accuracy over tho tested range. The dilution recovery for M -556 w as not considered to be exceedingly high enough, a t only approxim ately 1.5% above the norm al acceptance level, to have significantly im pacted the data. C O R R E C T IV E A C T IO N :T his protocol deviation sum m ary w as prepared for inclusion in the final report. APPROVED BY: Cv Jon ( tj A ndre, Ph.D . B attelle Principal Investigator Z.- Z.l-'Oi D ate f a d . V/Wk Jam ea-K . Lw ndbar g , P h rD . Study D irector f r w r U K T . f r s t>V*\ f t p J I /fatS .I k Q*- f D ate N 003604-D Protocol Deviation 1, Page 1 o f 1 3MEnvironmental Laboratory E-35 P age 131 3M M edical D epartm ent Study: T-6316.5 BACK TATIAIN jk _ Battelle Study Number: N 003604-D 3M Environmental Laboratory Study Number: FACT 060998.1 DEVIATION REPORT Battelle Study Number: N003604-D 3M Environmental Technology and Services Study Number: FACT 060998.1 2 (N-Ethylpeifluorooctanesulfonamido)-ethanol in Two Generation Rat Reproduction TY PE O F DEVIA TION S: PR O TO C O L D A TE O F DEVIA TION S: O ctober 20, 1999 N A TU R E O F D EV IA TIO N S: P F O S QC3 exceeded the 20% R E m ethod requirem ent (actual -2 1 .7 % ). T he dilution recovery check standard did not m eet the 70-130% recovery requirem ent (actuals 5.0% for PFO S and 4.6% for PFO SA ). T hese m ethod deviations relate to am endm ent 2 o f the study protocol. C A U SE O F D EV IA TIO N S: Sam ple preparation and/or L C /M S/M S variabilities over the course o f the sam ple set m ay have contributed to the QC deviation. Sam ple preparation erro r appears to have been the cause for the dilution recovery results. IM P A C T O F D E V IA TIO N S O N T H E STUD Y: T he errant Q C value level w as bracketed by acceptable Q C concentration levels w hich dem onstrates that the calibration curves generally provided good accuracy fo r study sam ples over the tested range. A com parison o f the results obtained fo r the diluted study sam ples from 20O ct99 and previous results th at w ere Slightly L O Q (1 3 0 c t9 9 and 180ct99) dem onstrated good agreem ent betw een the 2 determ inations. T his w ould indicate that the dilution o f the study sam ples w as perform ed correctly 20O ct99 so that no im pact on the quantitations occurred. C O R R E C T IV E A C TIO N : T his protocol deviation sum m ary w as prepared for inclusion in the final report. APPROVED BY: B attelle Principal Investigator t-T /h o l D ate Study D irector j Z holiii D ate N003604-D Protocol Deviation 2 , Page 1 o f 1 E-36 3MEnvironmental Laboratory Page 132 3M M edical D epartm ent Study: T-6316.5 BACK TOMAIN - Battelle Study Number: N003604-D 3M Environmental Laboratory Study Number: FACT 060998.1 DEVIATION REPORT Battelle Study Number: N003604-D 3M Environmental Technology and Services Study Number: FACT 060998.1 2 (N-Ethylperfluorooctanesulfonamido)-ethanol in Two Generation Rat Reproduction TYPE OF DEVIATIONS: PROTOCOL DATES OF DEVIATIONS: October 12, 1999 through October 20,1999 NATURE OF DEVIATIONS: The lot number of PFOS used was not 217 as per section 3.1.1 of the protocol. The source of reference substance matrix was not Argus Research Laboratories as specified in section 3.2.2 of the protocol. Initial analyses of liver did not exclusively target PFOS as per section 4.0 of protocol. CAUSE OF DEVIATIONS: Only PFOS lot number 171 was available at Battelle. Harlan was the supplier of control rat livers used to prepare blanks, standards, and QCs for the analytical portion of the study. All 4 analytes of interest (PFOS, M-556, PFOSAA, and PFOSA) were monitored during each analysis. IMPACT OF DEVIATIONS ON THE STUDY: PFOS lot number 171 and Harlansupplied liver were both used for Battelle's validation of the analytical method (Battelle study number N003604-A). These materials allowed achievement of the reported method acceptance criteria so that there is no impact on the study. The concurrent analysis of PFOS and metabolites was an efficiency improvement. CORRECTIVE ACTION: This protocol deviation report was prepared. APPROVED BY: C C LhI Jon(G. Andre, Ph.D. Battelle Principal Investigator z- zz^ Date James K. Lundberg, Ph.B. Study Director N003604-D Protocol Deviation 3, Page 1 o f 1 E-37 3MEnvironmental Laboratory AvuU 0^6.j Date P age 133 3M M edical D epartm ent Study: T-6316.5 BACK TOM AIN 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 BACK TAM AIN _ Battelle Study Number: N 003604-D 3M Environmental Laboratory Study Number: FACT 060998.1 1^651 778 4226 04/26/99 09:56 0 : 02/03 NO:341 I 2I From: Subject: Date: 3M SPECIA LTY ADHESIVES & CHEM ICALS ANALYTICAL LABORATORY Lisa Clemen - (8-5568) - ET&SS- 2-03-09 Request # 57830 Tom K estner - (3-5633) - SA&C Analytical Lab 236-2B-11 Chtm ical Characterization o fPOSF-Based Fluorochemicals by `H -N M R & lfF -N M R Spectroscopy M atch 2 4 ,1 9 9 9 : Prelim inary report for FC-95 (PFOS), lot 171 SAM PLE DESCRIPTION FC-95, lot 171 (PFO S), T N -A -0834; N om inal product = C F irS 0 3(-) K(+) (white pow der) IN T R O D U C T IO N : T his sam ple was su b jected to 'H -N M R and 1SF-N M R spectral analyses to determ ine the purity o f the nom inal product and to characterize as m any impurity components as possible. 'AL: A portion o f the sam ple was accurately weighed, spiked with a known amount o f 1,4-bis(tiifluorom ethyl)benzene (p-HFX), and then totally dissolved in DMSO-d for subsequent analysis by NMR. A 400 M H z 'H -N M R spectrum (# h57830.401) and a 376 M H z t9F-NM R spectrum (# (57830.401) were acquired u sin g a V arian UNITYplus 400 FT-NM R spectrometer. Use o f the p-HFX internal standard was intended to perm it the determination o f the absolute weight percent concentrations of the assigned components without necessarily needing to identify or quantify all the components in the sample mixture. RESULTS: The combined N M R spectral data were used to assign all of the major and m ost o f the minor com ponents in this sample as received. The qualitative and quantitative compositional results that were derived from the single trial NM R internal standardization analyses are summarized in TABLE-1 on the following page. 1 have reported both relative and absolute w eight percent concentrations. One possible reason that the absolute wt.% values add up to more than 100% may be due to the fact that I assumed all of the components contained 8 carbons. If there were any sh o rter ch ain h o m o l o g s p r e s e n t ( i . e . , 7, 6, S, e re . c a r b o n s ) , th e n th e a v e r a g e c o m p o u n d m o l e c u l a r w e i g h t s w ould h ave been som ew hat less than those used in the calculations. In general, the f*F-NMR technique is not particularly well suited for identifying or quantifying small amounts of various fluorochemical hom olog impurity components unless the chains are very short. A more complete characterization of any other fluorochem ical homologs would require analysis by electrospray MS or a similar technique. A dditional w ork would be required in an effort to positively verify the tentatively assigned com ponents listed in T A B L E -1 (denoted by possible). Sm all amounts o f other unidentified impurities are also detected in the N M R spectra, but additional w ork would be required in an effort to identify or quantify these other m aterials. Copies o f the N M R spectra will 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 me know. I apologize for the delay in completing this initial work. Tom Kestner e: Rick Payfer - SAAC Analytical L a b -2 3 6 -2 B -l! Fila iUfcrcncs: LC37S30CC061 3MEnvironmental Laboratory Pat- 1P -1 Page 135 3M M edical D epartm ent Study: T-6316.5 BACK TOM AIN ETSS 2 3W March 24,1999 _ Battelle Study Number: N003604-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 Analytical Lab Request # 37830 Initial R e a o rt fo r FC -95. lo t 171 " TABLE-1 S am p le: FC-95, lot 171 (PFOS), TN-A-0834 O verall Q uantitative C om positional R esults by 'H /`*F-NMR Internal Standardization A nalyses S tru ctu ral Assignments C F3( C F ^ - S 0 3(-) K(+) (Normal chain; assume x =7 for calculation purposes) NMR Absolute Weight% Concentradona (initie trial measurement) 70.3% NMR Relative Weight* Concentrations (staile trial measurement) 68.6% C F ^ C F J i -CFCCFjK C F jJj-SO jM K(+) (Internal mooomethyl branch; assume x+y 5, a * 0, A y * 0, for calculation purposes) 17.7% 173% (C Fj)jCF-(C Fj)*-SOj(-) X(+) (Isopropyl branch; assume x =3 for calculation purposes) 10.3% 10.2% Q Fji-CF(CFi)-SOi(-) K M (Alpha branch; assume x * 6 for calculation purposes) Possible F-SFi-C JV S O jM K(+) (assume x * 3 for calculation purposes) C F jK C F^-C C C FjJ H C F ^ - S O jH K(+) (Internal gem-dimethyl branch: assume x+y - 4 and x * 0 for calculation purposes) 3.3% 0.37% 0.16% 3.2% 036% 0.16% P ossible C F j-S F rC Jv -S O ji-) K(+) (assume x * 7 for calculation purposes) Probable C iH j rS O j(") K(+) (Hydrocarbon sulfonate salt; assume x a 3 for calculation purposes) (CF3)3C-<CFj)x-S03(-) K(+) (t-butyl branch; assume x * 4 for calculation purposes) . 0.11% 0.031% 0.027% 0.10% 0.030% 0.026% 3MEnvironmental Laboratory P ip .2F-2 Page 136 3M M edical D epartm ent Study: T-6316.5 3M Medical Department Study: T6316.5 BACKTOMA IN 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 3M Medical Department Study: T6316.5 BACKTO MAIN Analytical Report: FACT TOX-013 LRN-U2095 INTERIM CERTIFICATE OF ANALYSIS Revision 1(9/7/00) C entre Analytical Laboratories COA Reference #: 023-018B 3M Product: PFO 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.% C O A 023-018B 3M Environmental Laboratory 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 B A C K T O MAIN 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 /00 Expiration Date: 08/31/01 Storage Conditions: Frozen <-10C Re-assessment Date: 08/31/01 P urity = 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. No 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 well with the sulfur determination in the elemental analysis, lending confidence to this interpretation. Based on the results, the SO4 is not considered an impurity. 5TFA HFBA NFPA PFPA Trifluoroacetic acid Heptafluorobutyric acid Nonofluoropentanoic acid Pentafluoropropanoic acid ^Theoretical value calculations based on the empirical formula, CsFi7S0 3 *K+ (MW=538) This work was conducted under EPA Good Laboratory Practice Standards (40 CFR 160). COA023-018B 3M Environmental Laboratory (*W i -- - --- 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 BACKTOM AIN Analytical Report: FACT TOX-013 LRN-U2095 INTERIM CERTIFICATE OF ANALYSIS Centre Analytical Laboratories COA Reference #: 023-018R LC/MS Purity Profile: Im purity 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 BACKTOMAIN Analytical Report: FACT TOX-013 LRN-U2095 Appendix I: Report Signature Page % /yuy^ Marvin T. Case D.V.M., Ph.D., Study Director Date John L. Butenhoff, Ph.D., Sponsor Representative Date ^ far i s IM /" Ffil 3M Environmental Laboratory 3MEnvironmental Laboratory Page 135 P age 141 3M M edical D epartm ent Study: T-6316.5 BACK TOM AIN 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 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: BACK TAM AIN Analytical Study: FACT TO X-013 LRN-U2095 Kristen H. H ansen, P h.D ., Principal Analytical Investigator O S-/30 /0 1 D a te M arvin T . C a s e , D .V .M ., P h .D ., Study Director D a te Bill R e a g a n , P h .D ., E n v iro n m e n ta l L a b o ra to ry M a n a g e r D a te 3M Environmental Laboratory 3MEnvironmental Laboratory Page 135 P age 143