Document Rpaw5BOxZ1ORxp4LnM604pVJk

3M Medical Department Study: T6316.5 '*T f ORIGINAL Analytical Report: FACT TOX-013 LRN-U2095 sat-oW Study Title Analytical Study 2(N-Ethylperfluorooctane sulfonamido)-ethanol in Two Generation Rat Reproduction 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 CrLCD^BR VAF/Plus* Rats Exposed to EtFOSE-OH Data Requirement Not Applicable Author 3M Environmental Laboratory Study Completion Date At signing 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 S 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 0^0 -o p i -o o --'.rn co C~i pC~iJ) vO ro ro Total Number o f Pages 135 CVJ'P.e\ '.i} NO CBIr\rl-iTlti 3M Environmental Laboratory MX Page 1 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 This page has been reserved for specific country requirements. 3M Environmental Laboratory 2*3 Page 2 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 GLP Compliance 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 CrbCD^BR 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. \f J LV- I Study Director Date Sponsor Representative Date 3M Environmental Laboratory Page 3 3M Medical Department Study: T6316.5 Analytical Report: 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:CD*BR VAF/PIus* 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 QAU Representative 3M Environmental Laboratory Page 4 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 Table of Contents 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 Fluorochemlcal Compounds from Serum for Analysis Using HPLC-Electrospray/Mass Spectrometry, (14 pages)....................................................................................................................38 ETS-8-5.1, Analysis of Potassium Perfluorooctanesulfonate or Other Fluorochemicals 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...................................................................... 131 3M Environmental Laboratory Page 5 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 Appendix I: Report Signature Page................................................................................ 135 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 Fiuorochemical Levels in Sera Analyses in Study FACT TOX-013..... 61 Table 12. Reported Fiuorochemical Levels in Sera Analyses in Study FACT TOX-013 (continued)...................................................................................................... 62 Table 13. Reported Fiuorochemical Levels in Uver Analyses in Study FACT TOX-013.....62 Table 13. Reported Fiuorochemical Levels in Liver Analyses in Study FACT TOX-013 (continued)................................................................................................... 63 3M Environmental Laboratory Page 6 3M Medical Department Study: T6316.5 Analytical Report: FACT 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 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 Can S. Hewitt Marlene M. Heying Harold O. Johnson Kelly J. Kuehlwein Sally A. Linda Joseph C. Pilon Scott R. Post lan 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 aM Page 7 3M Medical Department Study: T6316.5 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, EtFOSE-OH, and M556 in sera samples collected from rats exposed to EtFOSE-OH. This study was initiated on 1 October 1998. Test 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 CrfrCCfBR 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 MoneP 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*self-piercing ear tag. FO female rats were approximately 65 days of age and weighed approximately 179-229g when received. FO male rats were approximately 58-67 days of age and weighed approximately 223-331 g when received. Weight data are included in Argus Research Laboratories, Inc. final report (study number 418-009). Table 1. Test System Population Demographics and Dosage Levels for Study (418-009) Population Number of F0 Generation Rats per Sex Number of F1 Generation Rats per Sex Dosage (mgfeg/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 3M Environmental Laboratory Page 8 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 Specim en Collection and Analysis 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 Specimens--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 -70*C 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 -20'C 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 11, 1999 using an ion pairing reagent and methyl-tert-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-electrospray/tandem mass spectrometry (HPLC-ESMSMS) 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 84-98,10-1-98 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 -20*C 10*C. 3M Environmental Laboratory Page 9 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 Chemical Characterization EtFOSE-OH CAS Number: 1691-99-2 Chemical Formula: CF17S 0 2N(CH2CH3)CHjCH20H 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-Q13 Test Article Chemical Name Source Expiration Date Storage Conditions Chemical Lot # Physical Description Purity EtFOSE-OH FM-3929 2(N-Ethylperfluorooctane sulfbnamido)-ethanol 3M 05/2000 Ambient temperature 30035, 30037, 30039 Waxy Solid To be determined* * The purity of the test article determ ined nominally by N M R analysis. Subsequent chem ical characterization is occurring and this analytical report w ll be am ended to indicate the purity when a certificata of analysis is issued. Dose Confirmation Analyses The dose confirmation data were collected according to a method that was not fully validated. Dose confirmation analyses were performed on test article samples taken at the start of dosage, at 6 weeks, and at the end of dosage during the in-life phase of the study. Dose confirmation analyses were performed on 3 dose levels collected during the in-life phase of the study: the results are presented in Appendix A (see Tables 10 and 11). Dose confirmation was performed by diluting the Tween dose samples with Milli-Q water into the linear range of the instrument. For each sample, a matrix spike was prepared (at approximately 50-100% 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 Page 10 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 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). 3M Environmental Laboratory Preparatory Method ETS-8-4.1, "Extraction of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds from Serum for Analysis using HPLC-Electrospray/Mass Spectrometry" Sera samples were extracted using an ion-pairing extraction procedure. An ion pairing reagent was added to the sample and the analyte ion-pair was partitioned into MtBE. The MtBE extract was transferred to a centrifuge tube and put onto a nitrogen evaporator until dry. Each extract was reconstituted in 1.0 mL of methanol, then Filtered through a 3cc plastic syringe attached to a 0.2pm nylon filter into a glass autoviai. Analytical Method ETS-8-5.1, "Analysis of Potassium Perfluorooctanesulfonate or Other Fluorochemicals in Serum Extracts Using HPLC-Electrospray/Mass Spectrometry' The analyses were performed by monitoring one or more product ions selected from a Single primary ion characteristic of a particular fluorochemical using HPLC-ESMSMS. For example, molecular ion 499, selected as the primary-ion for PFOS (QF17S 0 3-) analysis, was fragmented further to produce ion 99 (FSOj-). 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 Chromatograph: Hewlett-Packard* Series 1100 Liquid Chromatograph system Analytical column: Keystone* BetasilTM C1#2x50 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 S.92. Page 11 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2 095 Hold at 95%B for 2 minutes Return to 40%B over 0.5 minutes Hold at 40%B for 3 minutes Mass Spectrometer; Micromass* APl/Mass Spectrometer Quattro irT rip le Q uadruple system Software: Mass Lynx" 3.2 Cone Voltage: 20-60 V Collision Energy: 25-45 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 (am u ) Product Ion (am u) PFOS 499.0 99.0 PFOSA 498.0 78.0 PFOSAA 584.0 169.0 EtFOSE-OH 630.0 59.0 M556 556.0 78.0, 169.0 THPFOS 427.0 80.0 Deviations Deviations from the original protocol and methods are documented in the table below: Table 4. Deviation Summary fo r FACT TOX-013 D eviation Date(s) of Occurrence Impact on Study Pipette was used instead Oxford dispenser 0.2-1 .OmL of sample was used for extraction instead of 1.0mL. 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 will be marked in the data table. No milk curd data 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 3.93 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 (LOQ): 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 Analyses R esults Linearity: The coefficient of determination (r2) of the standard curves were^0.980. 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. All 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 (LOQ): The LOQ is equal to the lowest accepted standard in the calibration curve (defined as a standard with a concentration that is within 30% of the theoretical value, and which has at least two times the analyte peak area detected in the extraction blanks). Table 5. Determinations of the LOQ in the Analyses o f Serum Extracts Analyte Method LOQ PFOS PFOSA PFOSAA EtFOSE-OH M556 5.55 ppb 4.79 ppb 20.5 ppb 36.2 ppb 24.9 ppb 3M Environmental Laboratory 29*/ Page 13 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 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 MS/MSD and was reproducible to within 10% . Matrix 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. THPFOS 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; M556 data is quantitated to 31%. The validity of this assumption has not been verified by other techniques. Summary of Sample R esults Samples from Control Anim als: Low levels of PFOS, PFOSA, PFOSAA, EtFOSEOH, and M556 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. Samples from Dosed Animals: 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 Page 14 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 Appendix A: Chemical Characterization, Control Matrices and Dose Confirmation Analyses Table 6. Characterization of the Control Matrices Used for Sera Analyses in Study FACT TOX-013 Location 3M Lab Control Matrix Rat Serum (TN-A-2001) Rabbit Serum (TN-A-2573) Source Expiration Date Storage Conditions Chemical Lot # Physical Description N/R-- not recorded Sigma 2010 Ambient 17H9306 Rat Serum Sigma 2010 Ambient 118H841B Rabbit Serum Table 7. Characterization of the Control Matrices Used for Liver Analyses in Study FACT TOX-013 Location Battello Memorial Institute Control Matrix Rat Liver Source Expiration Date Storage Conditions Chemical Lot # Physical Description N/R-- not recorded Harlan N/R N/R N/R Rat Liver 3M Environmental Laboratory Page 15 3M Medical Department Study: T6316.S Analytical Report: FACT TOX-013 LRN-U2095 Table 8. Characterization of the Analytical Reference Materials Used for Sera Analyses In Study FACT TOX- Location IM Lab Materials PFOS e * 1fso* PFOSA C fF n S O jN H , PFOSAA C jF ijS O jH ftC M ^ H ) (C H jC O O H )) S o u rc e SM Specialty Chemicals N /R N /R E xp iratio n D ate S torage C o n d itio n s C hem ical Lo t Num ber P h ysical D escrip tio n 0 8 /3 1 /0 1 A m b ie n t te m p e ra tin e 0 1 /0 1 /2 0 1 0 A m b ie n t te m p e ra tu re 171 VIMIe c ry a ta lin e pow der L -1 5 7 0 9 L ig h t yetow r w ax y so W 0 1 /0 1 /2 0 1 0 A m b ie n t te m p e ra tu re N B 1 1 2 *9 9 -9 9 T a n w axy io M P urity 8 8 .4 % TBD TBD standard--*Sarrogate 1H ,1H ,2H ,2H-Tetrahydropertluorooctanesulfonlcacd N/R-- not recorded TBD-- to be determ ined NA-- not applicable EtFOSE-OH C ,F ,,S O ,N {C H iC H J CHjC H jO H 3 M IC P /P C P Dbnaion 0 1 /0 1 /2 0 1 0 A m b ie n t ta m p e re tire 936 A m ber waxy a d d TBD M556 C.FnSOAKW (CH.CHJ) 3M 0 1 /0 1 /2 0 1 0 A m taiem te m p e ra tu re N B 1 1 3 0 4 7 -8 0 \M ib e pow der TBD THPFOS* C iH ^ n N O ^ IC N B lo m e d lc a ls 0 1 /2 0 1 0 A m b ie n t te m p e ra tu re 53406 B row n waxy a o td NA Table 9. Characterization of the Analytical Reference Materials Used for Liver Analyses in Study FACT TOX-013 Location Battalia Memorial Institute Materials PFOS M556 PFOSAA PFOSA THPFOS* S o u rc e 3M 3M SM 3M E xpiration D ate S torage C on ditions C hem ical L o t Num ber P hysical D escrip tio n 0 8 /3 1 /0 1 01A 31/2010 A m b ie n t te m p e ra tu re 171 A m b ie n t te m p e ra tu re N B 1 1 3 0 4 7 -8 0 W h ite crys ta llin e pow der W h ite p o w d e r 2010 A m b ie n t te m p e ra tu re 617 N /R 0 1 /0 1 /2 0 1 0 A m b ie n t te m p e ra tu ra L -1 5 7 0 9 L ig h t y e to w w a x y a c id P u rity 8 8 .4 % TBD TBD TBD Surrogate standard-- 1H , 1H,2H.2H-Tetrahydroperfluorooctaresu!fonic acid N /R -- not recorded TBD-- to be determ ined NA-- not appScable IC N N /R A m b ie n t te m p e ra tu re 59909 N /R NA 3M Environmental Laboratory Z97 Page 16 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 Table 10. Tween Dosing Confirmation for Study In-life #418-009 Group Dose 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 NA = Not applicable Sample Number B-418-009-A, 06/08/98 B-418-009-A, 07/15/98 B-418-009-B, 06/08/98 B-418-009-B, 07/15/98 B-418-009-C, 06/08/98 B-418-009-C, 07/15/98 B-418-Q09-D, 06/08/98 B-418-009-0, 07/15/98 B-418-009-E, 06/08/98 B-418-009-E, 07/15/98 B-418-009-A, 05/08/98 1 of6T B-418-009-A, 06/08/98 3 of 6 M B-418-009-A, 06/08/98 5 of 6 B Expected Cone. EtFOSE (ng/mL) 0.00 NA 200000 200000 1000000 100000 2000000 2000000 3000000 3000000 3000000 3000000 3000000 Measured Cone. EtFOSE (ng/mL) 0.00 NA NA NA 1020000 942000 2190000 2750000 3060000 3640000 3250000 3690000 3790000 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-llfe #418-009 Semple Number Expected Cone. EtFOSE (ng/mL) Measured Cone. EtFOSE (ng/m L) 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-O, 07/15/98-MS B-418-009-E, 06/08/98-MS B-418-009-E, 07/15/98-MS B-418-009-A, 05/08/98 1 of 6 T-MS B-418-009-A, 06/08/98 3 of 6 M-MS B-418-009-A, 06/08/98 5 of 6 B-MS NA Not applicable 1200 1200 900 900 900 900 1100 1100 1100 1100 1100 NA NA 818 826 910 733 973 1089 949 1053 944 NA NA 91 92 101 81 88 99 86 96 86 3M Environmental Laboratory Z 9 Page 17 3M Medical Department Study: T6316.5 Appendix B: Protocol Analytical Report: FACT TOX-013 LRN-U2095 3M Environmental Laboratory JL9 Page 18 m in 3M Medical Department Study: T6316.5 A 3 ^ * } y 3M Environmental Laboratory______________ P rotocol - Analytical Study 2(N-EthyIperfluorooctanesulfonamido)-ethanol in Two Generation Rat Reproduction In-vivo study reference number: Argus 418-009 Study number: FACT 060998.1 Test substance: 2(N-EthyIperfluorooctanesulfonamido)-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 o f Potassium Perfluorooctanesulfonate or Other Anionic Surfactants from Liver for Analysis Using HPLC-Electrospray/Mass Spectrometry FACT-M -2.0, Analysis o f Fluorochemicals in Liver Extracts Using HPLCElectrospray/Mass Spectrometry FACT-M -3.0, Extraction of Potassium Perfluorooctanesulfonate or Other Anionic Surfactants from Serum for Analysis Using HPLC-Electrospray/Mass Spectrometry FACT-M -4.0, Analysis o f Fluorochemicals in Serum Extracts Using HPLCElectrospray/Mass Spectrometry Author: Lisa Clemen Kris Hansen Study Director ijis ln Date Marvin Case Sponsor Representative Date it 3M 'Environmental Laboratory 3oo 1 Page 19 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 1.0 Purpose________________________________________________ _______________ The analytical portion of this dosing study is designed evaluate the levels of perfluorooctane sulfonate (PFOS), or another metabolite 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, or in the serum as necessary. The in life portion of this study was conducted at Argus Research Laboratories. 2.0 Regulatory Compliance_________________________________________ _ This study is conducted in compliance with the Food and Drug Administration Good Laboratory Practices regulation as stated in 21 CFR 58. Any exceptions will be noted in the final report. 3.0 Test Materials__________________________________________________ _ 3.1 Test, control, and reference substances and matrices 3.1.1 Analytical reference substance: Potassium perfluorooctanesulfonate (PFOS), lot #217 3.1.2 Analytical reference substance matrix: Rat liver and serum 3.1.3 Analytical control substance: None 3.1.4 Analytical control substance matrix: Rat liver and serum 3.2 Source of materials 3.2.1 Analytical reference substance: 3M Specialty Chemical Division; traceability information will be included in the final report 3.2.2 Analytical reference substance matrix: Argus Research Laboratories; traceability information will be included in the final report 3.2.3 Analytical control matrix: 3.2.3.1 Rat liver - Argus Research Laboratories; traceability information will be included in the final report; or Rabbit liver - Covance Laboratories; traceability information w ill be included in the final report 3.2.3.2 Rat serum - Sigma Chemical Company; traceability information will be included in the final report 3.3 Num ber o f test and control sam ples. Liver samples for testing were received from 40 test animals and 10 control animals. Serum samples will be tested at the discretion o f the Study Director. 3.4 Identification of test and control sam ples: The samples are identified using the Argus Research Laboratories identifiers, which consist o f a letter followed by the Argus project number, the animal number, the group designation, and the draw date. 3M Environmental Laboratory al 2 Page 20 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 3.5 Purity and strength of materials: Characterization of the purity and identity o f the reference material is the responsibility of the Sponsor. 3.6 Stability o f 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 o f test and/or control substances: Biological tissues and fluids are retained per GLP regulation. 3.9 Safety precautions: Refer to the material safety data sheets of chemicals used. Wear appropriate laboratory attire, and follow adequate precautions for handling biological materials and preparing samples for analysis. 4.0 Experimental - Overview__________________________ "________________________ _ Tissues from animals dosed as described in Argus Research Laboratories Protocol #418-009 are received for analysis of fluorine compounds. At the discretion of the Study Director, a series o f analytical tests will be performed on select tissues. Initially, all liver samples will be analyzed for PFOS by electrospray/mass spectrometry (ES/MS). On the basis of findings from these analyses, additional sample matrices may be evaluated or other metabolites may be targeted. If additional analysis is performed, a protocol amendment will be written. 5.0 Experimental - Analytical Methods______________________________________ 5.1 FACT-M-1.0, Extraction of Potassium Perfluorooctanesulfonate or Other Anionic Surfactants from Liver for Analysis Using HPLC-Electrospray/Mass Spectrometry 5.2 FACT-M-2.0, Analysis of Fluorochemicals in Liver Extracts Using HPLCElectrospray/Mass Spectrometry 5.3 FACT-M-3.0, Extraction of Potassium Perfluorooctanesulfonate or Other Anionic Surfactants from Serum for Analysis Using HPLC-Electrospray/Mass Spectrometry 5.4 FACT-M-4.0, Analysis o f Fluorochemicals in Seram Extracts Using HPLCElectrospray/Mass Spectrometry 6.0 Data Analysis___________________________________________________________ 6.1 Data transformations and analysis: Data will be reported as the concentration (weight/weight) o f fluoride per tissue or sample, or o f PFOS per unit o f tissue or fluid. 6.2 Statistical analysis: Statistics used may include regression analysis 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 30 JL 3 Page 21 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U209S 7.0 Maintenance o f 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 3 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 3 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 FACT-M -1.0, Extraction of Potassium Perfluorooctanesulfonate or Other Anionic Surfactants from Liver for Analysis Using HPLC-Electrospray/Mass Spectrometry 9.2 FACT-M -2.0, Analysis of Fluorochemicals in Liver Extracts Using HPLCElectrospray/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 Fluorochemicals in Serum Extracts Using HPLCElectrospray/Mass Spectrometry 3M Environmental Laboratory 303 4 Page 22 3M Medical Department Study: T6316.5 ' Analytical Report: FACT TOX-013 LRN-U2095 Study Title Combined Oral (Gavage) Fertility Development and'Pcrinatal/Postnatal Reproduction Toxicity Study o f N-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, M N 55106 Laboratory Project identification ET&SS FACT-TOX-013 LERN U2095 3M Environmental Laboratory 3M Environmental Laboratory Page 23 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 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. AMEND TO read: The proposed study completion data is 6/30/00. REASON: The proposed completion date was changed to allow time for analyzing all matrices o f interest. Amendment Approval Marvin Case Ph.D., Sponsor Representative Kris J. Study Director Jo * I Z-/35 Date 3M Environmental Laboratory 3M Environmental Laboratory $S Page 24 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LEN-U2 095 Protocol FACT Tox-013 Amendment Number 3 3. Protocol reads: Disposition o f test and control substances: Biological tissues and fluids are retained per GLP regulation. Amend to read: Specimens will be maintained in the 3M Environmental Laboratory specimen archives. A ll specimens sent to sub-contract laboratories w ill be returned to the 3M Environmental Laboratory upon completion o f analysis and submission o f the sub-contract laboratory(s) final report The specimens will be returned with the following documentation: the signed original chain o f custody and records o f storage conditions while at the sub-contract facility. Re a s o n : To define in detail the appropriate disposition o f specimens analyzed at subcontract laboratories. Amendment Approval f y i A W * T G to JL Marv Case, D.V.M ., Ph.D., Sponsor Representative Date Kristen J. Hansen, Ph.D., Previous Study Director Dale L. Bacon, Ph.D., 3M Environmental Laboratory Management 3M Environmental Laboratory 3M Environmental Laboratory Date is h i Date Page 3, 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 Study Title Analytical 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 Medical Department Study: T-6316.5 3M Environmental Laboratory 3M Environmental Laboratory Page 31 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 Protocol LRN-U2095 Amendment Number 4 This am endm ent 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. Amend to read: The role of study director for the present study was reassigned to Marvin T. Case, D.V.M., Ph.D., as of 20 January 2000. The previous study director, James K. Lundburg, has been reassigned to the role of Principle Analytical Investigator. Reason: The role of study director was reassigned in an effort to ensure compliance with Good Laboratory Practice Standards that outline study personnel requirements (refer to 21 CFR Part 58). 2. Protocol reads: The sponsor for the present study was identified as Marvin T. Case, D.V.M., Ph.D. Amend to read: The role of sponsor for the present study was reassigned to John L. Butenhoff, Ph.D., as of 20 January 2000. Reason: To ensure that the study director does not also carry the duties of study sponsor, the sponsor role was reassigned. In this manner, personnel responsibilities and workload are more evenly balanced. 3M Environmental Laboratory 3M Environmental Laboratory Page 32 3M Medical Department Study: T6316.S Analytical Report: FACT TOX-013 LRN-U2095 Protocol LRN-U2095 Amendment Number 4 Amendment Approval John L ButenhoffPh.D., Sponsor Representative Date fj/lM-zV' T . & 4 Marvin T. Case, D. V.M., PhD ., Incoming Study Director LO.-- Q-b-e- Date & 3M Environmental Laboratory 3M Environmental Laboratory jt $ 6 f Page 33 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-O13 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 C T -T O X -0 13 ET&SS LRN U2095 Argus Study: 418-009 3M Medical Department Study: T 6 3 16.5 3M Environmental Laboratory 3M Environmental Laboratory / Page 34 3M Medical Department Study: T631S.5 Analytical Report: FACT TOX-013 LRN-U2 095 Protocol FACT TOX-013 Amendment No. 5 This am endm ent modifies 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. AMEND TOREAD: The role o f Principle Analytical Investigator for the present study was reassigned to Kristen J. Hansen Ph.D. REASON: The role of Principle Analytical Investigator was reassigned due to availability o f resources. 3M Environmental Laboratory 3M Environmental Laboratory Page 35 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LEN-U2095 Protocol FACT TOX-013 Amendment No. 5 Amendment Approval John L. Butenhoff, Ph.D., Sponsor Representative r c <aX_ Marvin T. Case, D.V.M., Ph.D., Study Director Date Date 3M Environmental Laboratory 3M Environmental Laboratory 312- Page 36 3M Medical Department Study: T6316.5 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 - * r s is Page 37 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 3M Environmental Laboratory M ethod E x t r a c t io n ,o f P o t a ssiu m P e r fl u o r o o c t a n e su l fo n a t e o r O t h e r Fluo ro ch em ical co m po unds from Serum fo r An a lysis U sin g H P L C - E lectr o spray/M ass Spectro m etry Method Number: ETS-8-4.1 Adoption Date: 03/01/99 Author: Lisa Clemen, Glenn Langenburg Revision Date: Approved By: 0 -/ / i -- Laboratory Manager Date fM ----------Group Leader Date Technical Reviewer Oh/ i U m 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 Matrices: Rabbit, rat, bovine, monkey, and human serum or other fluids as designated in the validation report. Word 6/95 3M Environmental Laboratory ETS-8-4.1 Extraction o f PFOS from Serum 323/ V Page 1 o f 14 Page 38 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 2.0 Summary of Method______________________________________ __________ _ 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 Definitions). An ion pairing reagent is added to the sample and the analyte ion pair is partitioned into MtBE. The MtBE extract is removed and put onto a nitrogen evaporator until dry. Each extract is reconstituted in 1.0 mL o f methanol, then filtered through a 3 cc plastic syringe attached to a 0.2 pm nylon filter into glass autovials. 2.2 These sample extracts are analyzed following method ETS-8-5.1 or other appropriate methods. 3.0 Definitions__________________________________________________ ___________ 3.1 PFOS: perfluorooctanesulfonate (anion o f potassium salt) C8F17S 0 3' 3.2 PFOSA: perfluorooctane sulfonylamide CgF,7S 0 2NH2 3.3 PFOSAA: perfluorooctane sulfonylamido (ethyl)acetate CgF17S 0 2N(CH2CH3)CH2CO2' 3.4 EtFOSE-OH: 2(N-ethylperfluorooctane sulfonamido)-ethyl alcohol C8F17S 0 2N(CH2CH3)CH2CH2OH 3.5 PFOSEA: perfluorooctane sulfonyl ethylamide CgF,7S 0 2N(CH2CH3)H 3.6 M556: C8F17S 0 2N(H)(CH2COOH) 3.7 Surrogate standard: 1H-1H-2H-2H perfluorooctane sulfonic acid 4.0 Warnings and Cautions_________________________________________________________ 4.1 Health and safety warnings 4.1.1 Use universal precautions, especially laboratory coats, goggles, and gloves when handling animal tissue, which may contain pathogens. 5.0 Interferences__________________________________________________________ 5.1 There are no interferences known at this time. 6.0 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 ETS-8-4.1 Extraction of PFOS from Serum Page 2 o f 14 Page 39 3M Medical Department Study-. T6316.5 Analytical Report: FACT TOX-013 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 o f holding 250 mL and 1 L 7.4 Volumetric flasks, glass, type A 7.5 I-CHEM vials, glass, 40 mL glass 7.6 Centrifuge tubes, polypropylene, 15 mL 7.7 Labels 7.8 Oxford Dispenser - 3.0 to 10.0 mL 7.9 Syringes, capable o f measuring 5 pL to 50 |iL 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 (N a^O j), 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 Fluorochemical standards 8.9.1 PFOS (3M Specialty Chemical Division), molecular weight = 538 8.9.2 PFOSA (3M Specialty Chemical Division), molecular weight = 499 ETS-8-4.1 Extraction o f PFOS from Serum Page 3 of 14 3M Environmental Laboratory ^ 3 / 4. Page 40 3M Medical Department Study: T6316.5 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 CgF,3S 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 ION sodium hydroxide (NaOH): Weigh approximately 200 g NaOH. Pour into a 1000 mL beaker containing 500 mL Milli-QTM water, mix until all solids are dissolved. Store in a 1 L Nalgene bottle. 8.10.2 1 N sodium hydroxide (NaOH): Dilute lO NN aO H 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 o f TBA into a 1 L volumetric containing 500 mL Milli-QTM water. Adjust to pH 10 using approximately 44 to 54 mL o f 10 N NaOH (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 o f sodium carbonate (Na^COj) 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 (pg/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 o f approx. 5.0 ppm. ETS-8-4.1 Extraction of PFOS from Serum Page 4 o f 14 3M Environmental Laboratory Page 41 3M Medical Department Study-. T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 8.11.7 Dilute working standard 1 with methanol for a working standard 3 solution o f approx. 0.50 ppm. 8.12 Surrogate stock standard preparation 8.12.1 Weigh approximately 50-60 mg o f surrogate standard l-H .l-H , 2-H, 2-H, CgF13S 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 matrix blanks 10.1.1 An aliquot o f 1.0 mL methanol is used as a solvent blank. 10.1.2 Extract two 1.0 mL aliquots o f 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. 10.2 Matrix spikes 10.2.1 Prepare and analyze matrix spike and matrix spike duplicate samples to determine the accuracy o f the extraction. 10.2.2 Prepare each spike using a sample chosen by the analyst, usually the control matrix received with each sample set. 10.2.3 Expected concentrations will fall in the mid-range o f the initial calibration curve. Additional spikes may be included and may fall in the low-range o f the initial calibration curve. 10.2.4 Prepare one matrix spike and matrix spike duplicate per 40 samples, with a minimum o f 2 matrix spikes per batch. 10.3 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. 3M Environmental Laboratory ETS-8-4.1 Extraction o f PFOS from Serum Page 5 o f 14 Page 42 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2 095 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 o f the initial calibration curve. This is necessary if the analyst must quantitate using only the low end o f the calibration curve (for example, 5 ppb - 100 ppb, rather than 5 ppb - 1000 ppb). 11.0 Calibration and Standardization ______________________________ _ 11.1 Prepare matrix calibration standards 11.1.1 Transfer 1 mL 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. D o not extract less than 0.50 mL o f 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, m ix 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 o f this method. U se these standards to establish each initial curve on the mass spectrometer. 3M Environmental Laboratory ETS-8-4.1 Extraction o f PFOS from Serum Page 6 of 14 Page 43 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 Table 1 Approximate spiking amounts for standards and spikes Using 1.0 mL of matrix Working standard pL Approx, final cone, o f (approx, cone.) analyte in matrix - Blank 0.500 ppm 10 0.005 ppm 0.500 ppm 20 0.010 ppm 5.00 ppm 5 0.025 ppm 5.00 ppm 10 0.050 ppm 5.00 ppm 20 0.100 ppm 50.0 ppm 5 0.250 ppm 50.0 ppm 10 0.500 ppm 50.0 ppm 15 0.750 ppm 50.0 ppm 20 1.00 ppm 12.0 Procedure____________________________________________________________ _ 12.1 Obtain frozen samples and allow to thaw at room temperature or in a lukewarm waterbath. 12.2 Vortex 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-ferr-butyl ether. 12.12 Cap each sample and put on the shaker at a setting o f 300 rpm, for 20 minutes. 12.13 Centrifuge for 20 to 25 minutes at a setting o f 3500 rpm, or until layers are w ell separated. ETS-8-4.1 Extraction of PFOS from Serum Page 7 of 14 3M Environmental Laboratory * 3 ZA Page 44 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 12.14 Label a fresh 15 mL centrifuge tube with the same information as in 12.S. 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 fixe /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 MDL report for specific MDL and limit o f quantitation (LOQ) values (see Attachments B and C). 14.2 The following quality control samples are extracted with each batch o f samples to evaluate the quality o f the extraction and analysis. 14.2.1 Method blanks and matrix blanks. 14.2.2 Matrix spike and matrix spike duplicate samples to determine accuracy and precision o f the extraction. 14.2.3 Continuing calibration check samples to determine the continued accuracy o f the initial calibration curve. 14.3 Refer to section 14 o f ETS-8-5.1 for method performance criteria. 15.0 Pollution Prevention and Waste Management_______________________ 15.1 Sample waste is disposed in biohazard containers, flammable solvent waste is disposed in high BTU containers, and used glass pipette waste is disposed in broken glass containers located in the laboratory. ETS-8-4.1 Extraction of PFOS from Serum Page 8 o f 14 3M Environmental Laboratory M tf 3A / Page 45 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 16.0 Records_______________________________________________ _____________ _ 16.1 Complete the extraction worksheet attached to this method, and tape in the study notebook or include in the 3-ring study binder, as appropriate. 17.0 Attachments_________________________________________________ _ 17.1 Attachment A, Extraction worksheet 17.2 Attachment B , MDL/LOQ values and summary 17.3 Attachment C, Calibration standard concentration worksheet 18.0 References__________________________ ,________________________________ 18.1 The validation report associated with this method is ETS-8-4.0 & 5.0 -V -l. 18.2 FACT-M-3.1, "Analysis o f Serum or Other Fluid Extracts for Fluorochemicals using HPLC-Electrospray Mass Spectrometry" 19.0 Affected Documents____________________________________________________ 19.1 ETS-8-5.1, "Analysis o f 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 ETS-8-4.1 Extraction of PFOS from Serum Page 9 o f 14 Page 46 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2 095 Extraction Worksheet ETS-8-4.1 Study # Matrix Box # Wk/Day DateSpiked/Analyst CCV MS MSD Surrogate Std approx, ppm actual ppm # FC-Mix approx. 0.5 pm actual ppm # FC-Mix approx. 5 ppm actual ppm # FC-Mix approx. 50 ppm actual ppm # Comments -- --- --- Blank Std# -- -------- amount" Serum Extraction Method Vortex 15 sec. Pipette Matrix Volume mL Pipette 1 mL of 0.5 M TBA, pH 10. pH = Std. # Pipette 2 mL of 0.25 Na?COy0.25M NaHCOt buffer Std. # Dispense 5 mL of methyl-t-butyl ether TN-A- Shake 20 min. Shaker speed: Centrifuee 20-25 min. Centrifuge speed: Remove a 4 mL aliauot of organic laver Put on Nitrogen Evaporator to dryness Temperature: Add methanol Volume mL TN-A- Vortex 30 sec. Filter using a 3cc B-D syringe with a 0.2um filter into a 1.5 mL autosamole vial C ont Cal. Verifications used same matrix as for std curve. - - - - - - - mL Date & Initials Attachment A 3M Environmental Laboratory ETS-8-4.1 Extraction o f PFOS from Serum Page 10 o f 14 Page 47 3M Medical Department Study: TG316.5 Analytical Report: FACT TOX-013 LRN-U2095 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 ppb - 1000 ppb 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 ETS-8-4.1 Extraction ofPFOS from Serum Page 11 o f 14 Page 48 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 Compound: PFOS Prepared range Rabbit Serum o f standards (ppb) (ng/mL) Full Range Low Curve High curve 1f X 0.995 - 978 4.94 - 248 97.8 - 978 0.995 - 978 Compound: PFOSA 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 24.8 - 976 0.993 - 976 Compound: PFOSAA Prepared range Rabbit Serum o f standards (ppb) (ng/mL) Full Range Low Curve High curve 1/X 0.991 - 974 4.92 - 247 49.2 - 974 0.991 - 974 LCR from curve (PPb) (ng/m L ) 24.8 - 978 4.94 - 248 97.8 - 978 4.94 - 978 LCR from curve (Ppb) (ng/m L ) 4.93 - 976 4.93 - 97.6 24.8 - 978 4.93 - 976 LCR from curve (ppb) (ng/m L ) 24.7 - 974 9.74 - 247 97.4 - 974 9.74 - 974 % Recovery Range 83-108 85-104 85-106 94-111 % Recovery Range 88-103 87-105 93-102 94-103 % Recovery Range 81-111 97-107 85-108 95-115 RSD Range 4.67-11.0 5.34-12.0 4.84-9.80 4.60-10.5 RSD Range 5.10-14.7 9.85-14.7 5.08-13.9 5.10-14.5 RSD Range 4.18-10.6 6.38-21.8 4.33-12.5 4.11-23.2 Attachment B: MDL/LOQ Summary 3M Environmental Laboratory ETS-8-4.1 Extraction of PFOS from Serum Page 12 o f 14 Page 49 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 Compound: EtFOSE-OH Prepared range R abbit Scrum o f standards (ppb) (ng/mL) Full Range Low Curve High curve 1/X 0.993 - 976 4 .9 3 -9 7 .6 49.3 - 976 0.993 - 493 Compound: PFOSEA Prepared range R abbit 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 R ange Low Curve High curve 1/X 0.993 - 976 4.93 - 97.6 97.6 - 976 0.993 - 976 LCR from curve (PPb) (ng/m L ) 49.3 - 976 9 .7 6 -9 7 .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 ETS-8-4.1 Extraction o f PFOS from Serum Page 13 o f 14 Page 50 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 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 5312 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 Am't 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 o f 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 Am't spiked mL 0.005 Validated ranges - approximate concentrations Serum PFOS PFOSA PFOSAA Rabbit 5.00-1000 1 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 EtFOSE-OH | 5.00-1000 | PFOSEA 5.00-1000 M556 [ 5.00-1000 Attachment C: Ion Pair Standard Curves ETS-8-4.1 Extraction o f PFOS from Serum 3M Environmental Laboratory -& 3 Z 7 Page 14 o f 14 Page 51 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 3M Environmental Laboratory Method Analysis qf Potassium Perfluorooctanesulfonate or Other Fluorochemicals in Serum E xtracts Using H PLC-Electrospray/Mass Spectrometry Method Number: ETS-8-5.1 Author: Lisa Clemen, Robert Wynne Approved By: 'c H Laboratory Manager -- Adoption Date: 03/01/99 Revision Date: ti/zc, Date Group Leader __ Cfris* A ~____________________________ Technical Reviewer Date ohIm / 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 Compounds: Fluorochemical surfactants or other fluorinated compounds, or other ionizable compounds. 1.3 Matrices: Rabbit, rat, bovine, monkey, and human serum, or other fluids as designated in the validation report. Word 6/95 ETS-8-5.1 Analysis of Serum Extract Using ES/MS Page 1 of 9 3M Environmental Laboratory T S 2 lr Page 52 3M Medical Department Study. T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 2.0 Summary of Method__________________________________ 2.1 This method describes the analysis o f fluorochemical surfactants extracted from serum or other fluids, using HPLC-electrospray/mass spectrometry, or similar system as appropriate. The analysis is performed by monitoring a single ion characteristic o f a particular fluorochemical, such as the perfluorooctanesulfonate (PFOS) anion, m lz - 499. Additionally, samples may be analyzed using a tandem mass spectrometer to further verify the identity o f a compound by detecting daughter ions o f the parent ion. 3.0 Definitions__________________________________________________ 3.1 Atm 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 Spectrometer (MS), Tandem Mass Spectrometer (MS/MS): The API Quattro II triple quadrupole systems are equipped with quadrupole mass selective detectors. Ions are selectively discriminated by mass to charge ratio (m/z) and subsequently detected. A single MS may be employed for ion detection or a series (MS/MS) for more specific fragmentation information. 3.4 Conventional vs. Z-spray probe interface: The latest models o f Micromass Quattro II triple quadrupole systems (post 1998) utilize a "Z-spray" conformation. The spray emitted from a probe is orthogonal to the cone aperture. In the conventional conformation it is aimed directly at the cone aperture, after passing through a tortuous pathway in the counter electrode. Though the configuration is different, the methods o f operation, cleaning, and maintenance are the same. However, Z-spray components and conventional components are not compatible with one another, but only with similar 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 o f 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 Warnings and Cautions_________________________________________________ 4.1 Health and Safety Warnings: 4.1.1 Use caution with the voltage cables for the probe. When engaged, the probe employs a voltage o f approximately 5000 Volts. 4.1.2 When handling samples or solvents wear appropriate protective gloves, eyewear, and clothing. 3M Environmental Laboratory ETS-8-5.1 Analysis of Serum Extract Using ES/MS 4^ 32? Page 2 of 9 Page 53 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 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 Interferences_______________________________________________ ___________ _ 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 Equipment________________________________________________________ 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 HP1100 low pulse solvent pumping system, solvent degasser, column compartment, and autosampler 7.0 Supplies and Materials___________________________________________________ 7.1 Supplies 7.1.1 High purity grade 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 Reagents and Standards_______________________________ ;__________________ 8.1 Reagents 8.1.1 Methanol, HPLC grade or equivalent 8.1.2 Milli-QTM water, all water used in this method should be Milli-QTM water or equivalent, and may be provided by a Milli-Q TOC Plus system or other vendor 8.1.3 Ammonium acetate, reagent grade or equivalent 8.2 Standards 8.2.1 Typically two method blanks, two matrix blanks, and eighteen matrix standards are prepared during the extraction procedure. See ETS-8-4.1. 9.0 Sample Handling_________________________________________________________ 9.1 Fresh matrix standards are prepared with each analysis. Extracted standards and samples are stored in capped autovials or capped 15 mL centrifuge tubes until analysis. 3M Environmental Laboratory ETS-8-5.1 Analysis o f Serum Extract Using ES/MS 330 Page 3 of 9 Page 54 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 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 Quality Control_____________________________________________ 10.1 Solvent Blanks, Method Blanks and Matrix Blanks 10.1.1 Solvent blanks, method blanks and matrix blanks are prepared and analyzed with each batch to determine contamination or carryover. 10.1.2 Analyze a method blank and a matrix blank prior to each calibration curve. 10.2 M atrix 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 C ontinuing calibration verifications are analyzed to verify th e co n tin u ed accuracy o f the calibration curve. 10.3.2 Analyze a mid-range calibration standard after every tenth sample, with a minimum o f one per batch. 11.0 Calibration and Standardization________________________________________ 11.1 Analyze the extracted matrix standards prior to and 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 o f the standards from 5 ppb to 100 ppb rather than the fall 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 ETS-8-5.1 Analysis ofSeramExtract Using ES/MS Page 4 of 9 Page 55 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 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 Autosampler 12.2.1 Set up sample tray according to the sample list prepared in Section 12.1.1. 12.2.2 Set-up the HP1100/autosampler at the following conditions or at conditions the analyst considers appropriate for optimal response. Record actual conditions in the instrument logbook: 12.2.2.1 Sample size = 10 pL injection 12.2.2.2 Inject/sample = 1 12.2.2.3 Cycle time = 13.5 minutes 12.2.2.4 Solvent ramp = Time 0.00 min. 8.50 min. 11.0 min. 12.0 min. MeOH 40% 90% 90% 40% 2.0 mM Ammonium acetate 60% 10% 10% 60% 12.2.2.5 Press the "Start" button. 12.3 Instrument Set-up 12.3.1 Refer to ETS-9-24.0 for more details. 12 3 .2 Check the solvent level in reservoirs and refill if necessary. 3M Environmental Laboratory ETS-8-5.1 Analysis of Serum Extract Using ES/MS J& T33Z. Page 5of9 Page 56 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 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. I f 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 o f the tip o f the probe. Allow to equilibrate for approximately 10 minutes. 12.3.5 Turn on 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 i f 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 jiL/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 o f biological matrices. 12.3.1 OClick 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 Data Analysis and Calculations_________________________________________ 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): fne o f PFOS calc, from std. Curve x Dilution Factor! x 1 u e (Initial Volume o f matrix (mLl + mL o f Surrogate Standard) 1000 ng Final Volume (mL) 3M Environmental Laboratory ETS-8-5.1 Analysis of Serum Extract Using ES/MS $3 % Page 6 of 9 Page 57 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 14.0 Method Performance_________________________________________ _ 14.1 Method Detection Limit (MDL) and Limit o f Quantitation (LOQ) are method, analyte, and matrix specific. Please see ETS-8-4.1, Attachment B, for a listing o f current validated M DL 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 M atrix 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 If criteria listed in this method performance section isn't met, maintenance may be performed on the system and samples reanalyzed or other actions as determined by the analyst. Document all actions in the appropriate logbook. ... 14.7 If data are to be reported when performance criteria have not been met, the data must be footnoted on tables and discussed in the text o f the report. 15.0 Pollution Prevention and Waste Management____________________________ 15.1 Sample extract waste and flammable solvent is disposed in high BTU containers, and glass pipette waste is disposed in broken glass containers located in the laboratory. 16.0 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 ETS-8-5.1 Analysis of Serum Extract Using ES/MS Page 7 o f9 Page 58 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 16.5 Summarize data using suitable software (Excel 5.0) and store in the study folder, see Attachm ent A for an example o f a summary spreadsheet. 16.6 Back up electronic data to appropriate medium. Record in study notebook the file name and location o f backup electronic data. 17.0 T ables. Diagrams. Flowcharts, and Validation Data______________________ _ 17.1 Attachment A: ETS-8-5.1 Data summary spreadsheet. 18.0 References________________________________________________________ 18.1 FACT-M-4.1, "Extraction o f Potassium Perfluorooctanesulfonate or Other 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 Affected 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 o f HP1100 system components. Section 11.1 Average o f two curves, not standard values, are used for plotting linear regression and added the 1/x weighting o f the curve. Section 12.2.2.4 Clarification o f solvent ramp. Section 17.1 Changed from attachment B to A. Revision Date 04/02/99 3M Environmental Laboratory ETS-8-5.1 Analysis of Serum Extract Using ES/MS Page 8 of 9 Page 59 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 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 of Extraction/Analyst: Date of Analysis/Analyst Group Dose Sample# Concentration ug/mL Initial Vol. mL Dilution Factor Final Cone. ug/m L Slope: Taken from linear regression equation. G roup/Dose: Taken from the study folder. Sam ple#: Taken from the study folder. Concentration (ug/mL): Taken from the MassLynx integration summary. Initial Volume (mL): Taken from the study folder. Dilution F actor: Taken from the study folder. Final Cone. (ug/mL): Calculated by dividing the initial volume from the concentration Attachment A: Summary Spreadsheet ETS-8-5.1 Analysis of Serum Extract Using ES/MS 3M Environmental Laboratory Page 9 of 9 Page 60 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 Appendix D: Data Summary Tables Table 12. Reported Fluorochemical Levels in Sera Analyses in Study FACT TOX-013 Dosage Group S pecim en 10 PFOS (pg/mL) PFOSA (pg/mL) PFOSAA (pgfmL) EtFOSE-OH (pg/mL) M 566 (pg/m L) 1 10097F 0.0394 1 10105F 1 10106F t 10107F 1 10108F 1 9822M* 1 9930M* 0.0181 0.0258 0.0343 0.0253 0.0115 0 .0 1 3 4 1 9931M 0 .0 0 7 2 5 1 9932M 0.0162 1 9933M* 0.0156 II 10121F II 10126F 9.62 19.8 II 10136F 5.96 II 10140F 6.27 II 10142F 13.1 II 99 61M * 34.8 II 9964M 30.4 II 99 6 5 M * 7 4 .9 II 9 9 67M 25.1 II 99 70M * 38.9 III 1 0 1 5 5 F ' 87.8 III 10156F 76.1 III 10164F 4 9 .6 III 1172F 6 8 .4 III 10177F 42.2 III 9997M 108 III 99 99M * 178 ill 10001M 94.9 III 10002M 113 III 10004M 130 IV 10187F IV 10194F 6 9 .5 73.4 IV 10203F 126 IV 10211F 99.7 IV 10214F 98.3 IV 10019M 302 IV 10024M* 477 IV 10029M* 296 IV 10033M 272 IV 10034M 249 * Tentativo value*, initial volume waa <0.5 m t < 1 0 0 (4.79 ppb) < 10 Q (4.79 ppb) <LOQ (4.79 ppb) <LOQ (4.79 ppb) <LOQ (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) < tO Q (20.5 ppb) <LOQ (20.5 ppb) <LOQ (20.5 ppb) <LOQ (20.5 ppb) <LOQ (20.5 ppb) <LOQ (20.5 ppb) <LOQ (20.5 ppb) <LOQ (20.5 ppb) <LOQ (20.5 ppb) 1.59 4.55 1.18 0.690 2.09 1.40 5.86 1.86 1.26 5.55 9.9 6.91 4.66 8.17 4.56 11.8 18.7 12.1 10.4 14.9 8.00 10.6 19.0 10.2 12.3 22.5 40.5 28.6 24.5 31.4 <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LO Q (36.2 ppb) <UOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (38.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (24.9 ppb) <LO Q (24.9 ppb) <LO Q (24.9 ppb) <LOQ (24.9 ppb) <LO Q (24.9 pob) <LOQ (24.9 ppb) <LOQ (24.9 ppb) <LOQ (24.9 ppb) <LOQ (24.9 ppb) <LOQ (24.9 ppb) 1.86 4 .1 9 0.952 1.15 2.45 4.69 5.18 4 .5 4 3 .4 4 6.11 4 3 .3 2 2 .3 18.0 17.0 17.9 29.1 7 3 .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 Page 61 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 Table 12. Reported Fiuorochemical Levels in Sera Analyses in Study FACT TOX-013 (continued) Doeage Group Specim en 10 PFOS (pglmL) PFOSA (pg/mL) PFOSAA (pg/mL) EtFOSE-OH (pg/mL) M5S6 (pg/mL) V NR V NR NR NR V NR NR V NR NR V NR NR V 10042M 238 V 10044M 235 V 10045M 326 V 10051M 162 V 10054M 182 N R " Sample not received or reported * * Tentative values, initial volume vas <0.5 m i. NR NR NR NR NR 0.791 0.972 0.897 0.574 0.669 NR NR NR NR NR 2S.2 20.7 26.8 14.0 15.8 NR NR NR NR NR LOQ (36.2 ppb) <LO Q (3621 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) NR NR NR NR NR 62.4 55.6 93.8 30.7 55.5 Table 13. Reported Fiuorochemical Levels in Liver Analyses In Study FACT TOX-013 Doeage Group 1 1 1 1 1 1 1 1 1 1 1 1 ( 1 1 11 II II 11 II II II II II II II It II It II S pecim en ID 10097F 10105F 10106F 10107F 10108F 9922M 9930M 9 9 3 1M 9932M 9933M 10097M 10105M 10106M 10107M 10108M 10121F 10126F 10136F 10140F 10142F 9961M 9964M 9965M 9967M 9970M 10121M 10126M 10136M 10140M 10142M PFOS (pgig) 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 39.8 23.7 22.1 116 102 89.9 80.7 87.3 54.7 67.8 53.7 28.0 71.5 PFOSA(pgZg) <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ 0.514 0.708 1.40 0.601 0.506 4.49 4.10 2.88 3.88 5.42 1.90 2.6S 2.35 1.22 2.06 P FO S A A <pg/g) <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ 2.68 4.34 5.01 2.67 2.87 11.0 15.6 9.79 6.62 10.4 5.67 14.6 9.44 2.82 6.55 ms {Mia) <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <L0Q <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 4.84 3.27 4.58 3M Environmental Laboratory 8T 4S* Page 62 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 Table 13. Reported Fluorochemical Levels in Liver Analyses In Study FACT TOX-013 (continued) Do m o * Group III III III III III III III III III III III III III III ill IV IV IV IV IV IV IV IV IV IV IV IV IV IV IV V V V V V S pecim en ID 10155F 10156F 10164F 10172F 10177F 9997M 999M 10001M 10002M 10004M 10155M 10156M 10164M 10172M 10177M 10187F 10194F 10203F 10211F 10214F 10019M 10024M 10029M 10033M 10034M 10187M 10194M 10203M 10211M 10214M 10042M 10044M 10045M 10051M 10054M PFOS (pg/g) 102 130 179 119 105 415 234 498 257 386 89.0 219 203 188 153 164 240 344 255 264 831 791 556 781 556 226 277 448 457 344 1218 1356 1132 1063 1054 PFOSA (pg/g) 2.22 2.24 1.94 2.17 2.88 10.8 9.41 8.67 8.29 8.41 5.11 6.14 6.26 6.20 6.91 2.80 4.06 3.14 3.39 4.56 12.8 11.0 11.2 12.6 11.0 6.36 9.96 9.93 11.4 8.11 16.4 13.0 10.9 9.80 10.8 PFOSAA (pg/g) 15.8 20.2 22.7 11.9 20.0 73.5 28.6 65.2 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 6 S 6 (|jg rg ) 7.17 7.64 8.41 5.87 8.16 6 3 .5 39.3 66.4 38.0 54.6 11.5 33.3 43.1 29.9 31.4 19.8 26.8 26.6 27.5 29.3 84.5 97.5 78.2 117 82.2 39.9 39.5 67.8 65.4 64.5 128 152 133 118 161 3M Environmental Laboratory Page 63 3M Medical Department Study: T6316.5 Appendix E: Data Spreadsheets Analytical Report: FACT TOX-013 LRN-U2095 3M Environmental Laboratory Page 64 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 > l| <z * ,,a S 3o 27.4 0.00352 -- 8 S i = R5 3 S 51 S9 t JJ 3 im ! fts 8o eci 1 P 3 5 = S !f \ =3 { S3 1i 11 ! u"8 i 1 s a5 * s Ss e8 ss e e8 <e? po g oq o S s 2 5 5 s ? a s U 2 1 5 5r * 5 * = S S 5 3 5 5 g * P s ggs t S i a VH i 5 VULO1 2*" i ii ] II jji i L f | | | | i l m 8 8 8o S s s s i S f t S S i E n S 5S SS55 S lis S S*S = SSSS s i i s i i I *? I. s la -- % f: I -- s joe ao s e e i e g ea 8- S--8o OS 8 fot 8o 5o 58 od fi S JE | ddc S g |8 8 3o Re So 2 a8 Sd 9 8e 8e S S S 8' S ~8i 8o P 1 59 r? -If-- maaa ja ) XnI5 IS aa y $ a 5 $ a 5 2 s 5 : 5? s S 3 m 83 u 53 5 k ilii l a a a |s eo i I s i i RS* *S 1*1*1 Um z i XS Z l! |l| k k 5 5 5 5 SS ! | i l i g 2 S? I l 333 i i i l f li s ili 1 1 !; 1 ] I i|i a1 1 - I n i ! i l l! ! ! } ! l i ! ti i \1 1 Oi wj h o o Il S fl *u e i* 2l i fe l i} S is li ! 1? i l! 1 1 1 1 1 11 3M Environmental Laboratory Page 65 3M Medical Department Study: T6316.5 Analytical Report FACT TOX-013 LRN-U2095 A fi 4tMXn. TwOoienM iproduboe Study o EiFOSE-Ot! is iUU EiFf)S&OH(T-4JliJ) OWlMODYl ' T 0 * W W W I I P M , W * > 1 >fcu^u(TcH SabrtMccfc 3M Environmental Laboratory e r3 i Page 66 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 1 %< i 2 i i ai f S3 5 2 5 H 32 55 32 te 32 5 f <e a ifi i =3 foi e fi Iji SS m u i m i 32 = l S M g 2 ? j 2 ; = 3 5 1 3 I S S 2 f i f i ? set t e fi fi fi 2 a n 1 1 In -be-o9x 94 ooood fia25 8 IH*s1 3S^i>ig*Sa*f,g 11 SI =sJ l l l l l i i ' 88 8O80 = r 3 8 8 2 8 d8 8O8O9<A8 = S = S3 S S E 5 3 S 5 S 5 5 2 5 2 2 3 *s"1--i Oi 0l i*13 3 3 3 3 t e t t e 3 1 3 3 3 i n - - - - e g s i s ? 1 1 S 1 bfi ae 8- -88 ?d i0adao d9 1 gfi 9 g fai o8 fi e* o8 8d so d e dfi 1 2 3 3 2 t e t t e I 3 2 ss JcIJjl jjuiais --r* 1 ii i i l i 1 l m ssJSMa :loMfa8a!5 g e S 5 iSii*ii*ls sms oo5oe *M*1i*lli il l II Hill eSsji s8sS 1|11 sssss i m i ii mm Mm ft 1 1 9 ill! Hi i|! i{5 jl I ill 3M Environmental Laboratory 4ft U S Qi Page 67 3M Medical Department Study: T6316.5 3M Environmental Laboratory NBtei<Tc S lU U M j: A s|w 4lt-009, To43oimk fcfatfK tioaSiudyofEtRUS&OHs R m Ei=OSfi-OHfT-A31i-5> Analytical Report FACT TOX-013 LRN-U2095 t 3VV & Page 68 Toui(ivtvaJws,ialiblv(ivracbdottQ.5mL LACNO MB 3M Medical Department Study: T6316.5 Analytical Report: PACT TOX-013 LRN-U2095 Tontivaiaci,tortaivnfemtelo*OJ rL. LACOWIA i >i| ii 2 sS 3S 2T A A-- 2fl 25 s s S 3 IIm 1 1 S ! ! 4 5 3 3 5 3 i S Sc n UI? iati *} iai5 i l jS b Ji Ji IuJ JJ s 3 n II] r il il SS Si 1s 31 51 13 5! mS Q.si sls 3 =5 =3 3 =33 = - 5 ? S -s f*1iaT s-i RA s a s a =2 ; * s s s a s 2 3 S3 S I f f i i I f f f l m SS 1 1 S 1 1 1 1 1 5 5 1 2 1 SSS =? s i s 3 5 a s * * 3 a S 3 s S a a s a s s s a s 8 5 3 S ! K iil s e S Si s eee $ a 9 d s e a o a o 9 o p A as fl as --A 2 2 2 2 1 9 o S e R9 a S a 2 2 2 1 2 S o S a P fl a e P fl 1 2 2 2 2 SSt SB2 2 2 1 1 j :j I2 II IMI mui Afl Afl M A i | ie lo o 5 35 i -S o AM Sa |S ceceo ai 1s *** s S am S Sa e im i lili! i lifli sasss IH Ili _s i! i liiis U l f l1 m A 1: 8 1 1 3 !! il! r fol H3 if! j !l l 3M Environmental Laboratory J & rS H S Page 69 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 Appendix F: Example Calculations Formula Used for Sera Analyses in Study FACT TOX-013 AR (ng/mL) * DF * SC * FV (mL) * 1.0 pg * PC = Reported Concentration (jig/mL) EV (m L) iOOO ng Calculation Used for Group 4, Anim al ID 10033M 340 ng/mL * 500 x 0.9275 * 1 mL x 1.0 pg x 0.864 = 272 pg/mL 0.5'mL rOOng" 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 Page 70 3M Medical Department Study: T6316.5 Analytical Report: 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)-ethanol in Two Generation Rat Reproduction, (59 pages) 3M Environmental Laboratory Page 71 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 BIOLOGICAL SAMPLE ANALYSIS Battelle Study Number N003604-D 3M Environmental Laboratory Study Number: FACT 060998.1 I l Baitene . . . Putting Technology To lAtork FINAL REPORT 2 (N-EthylfluorooctanesuIfonamido)-ethanoI in Two Generation R at Reproduction SPONSOR 3MToxicology Services 3M Center Building 220-2E-02' St Paul, MN 55144 StudyInitiation: September IS, 199S Testing Facility Battelle Memorial Institute SOSKing Avenue Columbus, Ohio 43201-2693 Prepared Bv Patrick L. South, B.S. 3M Environmental Laboratory e rs i* Page 72 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX- 0 1 3 LRN-U2095 Batidle StudyNumber N0036C4-D 3MEnvironmental Laboratory StudyNumber FACT 060998.1 FINAL REPORT 2 (N-Ethylfluorooctanesulfonam ido)-ethanol in Two Generation R at Reproduction Joa C. Andre, PhD. Batidle PrincipalInvestigator Richard W . Slanter, P h D ., D A B .T . B atidle Senior Program D irector Date D ate 3M Environmental, Laboratory ii & 3 if Page 73 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2Q95 Battelle StudyNumber. N003604-D 3MEnvironmentalLaboratory StudyNumber: FACT 060998.1 2 (N-Ethylfluorooctanesulfonam ido)-ethanol in Two Generation R at 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 ChromatographyMass 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 (jig) per gramofliver tissue extracted. 3M Environmental Laboratory iii Page 74 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 Baitelle StudyNumber.N003604-D 3MEnvironmentalLaboratoryStudyNumber FACT 060998.1 QUALITY ASSURANCE STATEMENT This study was inspected by the Quality Assurance Unit and reports were submitted to the task leader, study director, and associated management as follows: 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 10/12/1999 10/12/1999 10/13/1999 10/13/1999 Quality Assurance Unit Battelle Memorial Institute Date 3M Environmental Laboratory IV Page 75 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 Batidle StudyNumber N003604-D 3MEnvironmentalLaboratoiy StudyNumber FACT060998.1 GOOD LABORATORY PRACTICES COMPLIANCE STATEMENT Study Title: 2 (N-EthyIfluorooctanesulfonamido)-ethanol in Two Generation R at R ep rod u ction This study was conducted in compliance with the Food and Drug Administration's Good Laboratoiy Practice Regulations (21 CFR 58), with the exception that the mass spectrometry data for the liver samples was collected and processed with the MassLynx software system (version 3.1), which was not fully validated. The study was listed on Battelle's Master List o f regulated studies. Jon C. Andre, PhJD. Battelle Principal Investigator Kris Hansen, PhJ ). Study Director D ate D ate 3M Environmental.Laboratory V Hr Page 76 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 Battelle StudyNumber N003604-D 3MEnvironmentalLaboratory StudyNumber FACT 060998.1 Table of Contents Executive Summary........ ..... Quality Assurance Statement. Compliance Statement....... Table o f Contents................................................ . 1.0 Introduction........................................... . 2.0 Reference Substances............................ , 3.0 Receipt of Samples ............................ 4.0 Analysis o f Samples _............................ 4.1 Summary o f Method................ 4.2 Results..... ................................ 4.2.1 Quality Control.... ....... 4.2.2 Sample Results_____ 5.0 Conclusions........................................... 6.0 Acknowledgements................................ 7.0 Specimen Storage and Record Archives. S ists iii rv .V vi ,1 ..1 ..1 ..1 ..1 ..2 ..2 ..3 ..3 ...4 ,.4 L ist o f Tables Table i. Example of InstrumentParameters Used to Analyze Samples......................... 2 Appendix A (Results) Summary Results for Rat liver Sample Analysis................. ......................................... .A-I Appendix B (D aily 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 (Protocol, Amendm ents, and Deviations) Protocol, Amendments, andDeviations...........................................................................................................-E-l Appendix F (PFOS Purity Report) PFOS Purity Report......................................................................................................................................-E"! 3M Environmental Laboratory VI Page 77 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 Battelle Study Number ND03604-D 3M EnvironmentalLaboratoryStudyNumber 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 studyprotocol, amendments, andprotocol deviation reports (signed deviation reports "werenot available for inclusion here). 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 Got 171), M-556 (TN-A-2203), PFOSAA (TN-A-1283) and PFOSA (L-15709). Note thatbased on information supplied to Battelle from 3M, PFOS has two equivalent names. The name appearing on the Material Safety Data Sheet andbottle label is potassium perfLuaroalkyl sulfonate. The name more commonly used by 3M in analytical methods andcorrespondence is potassium perQuorooctancsulfonate. The latter name will be used in this report See Appendix F far purity data supplied by 3M to Battelle. The reference substances were stored at room temperature. The surrogate standard was 1H,lF^2H,2H-Perfluorooctane sulfonic acid, lot number59909, supplied by ICN. The surrogate standard was stored at room temperature. 3.0 Receipt of Sam ples 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 underprotocol number 418-009. See Appendix C for a copy of the inventory list The samples were stored at approximately-208C. 4.0 Analysis of Sam ples 4.1 Summary of Method Samples were analyzed by a previously validated method (Battelle study numberN003604-A). The current version of the method is attached to this report in Appendix E. Samples were analyzedby 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 L/x wereused to construct the calibration curves. 3M Environmental Laboratory Page 78 3M Medical Department; Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 Battee StudyNumber. NQ03604-D 3M Environmental Laboratory StudyNumber FACT 060998.1 Table 1. Example of Instrument Parameters Used to Analyze Samples L C /M S/M S S> stem A u lu saiii|ili'r HPLC pumps M ass spectrom eter A nalytical colum n M itbili- phase com ponents G rad ien t profile In jection voliiine Flow C olum n tem p H PLC pressure M S source D esnlvatiun as N ebulizer as S ource hlocL tem p D esaltatio n tem p C one voltage C ollision c n c r t' C ollision ".is M ultiplier R esolution Ions m onitored T o tal ru n time A pproxim ate reten tio n tim es: Make: G ilson M odel: 234 Make: G ilson M odels: 30S and 306 Make: M icrom ass Model: Quattro LC wife Z-spray source Keystone Betasil C l 8, Sum , 2 x 5 0 mm. P art No. 055-701-2 Component A: Ammonium acetate(2mM )dnethanol, 60:40, v:v Component B: Ammonium acetate(2mM )3nefean0l, 5 :95, vrv Time, min %B Flow. mlAnln 0 0 0J 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 pL LC column flow at start split to 20 uLAnin into the M S A m bient Approximately 840 psi at gradient start Electrospray. N egative Ion N itrogen at ~575 L/hr N itrogen at ~80 L/hr 140*C 250*C 70 V for SS, PFOS 20 V fbrM -556. PFOSAA. PFOSA 40 eV Argon, gas cell, at -2.5x1 O'3 mb 650 V 12.0 for M S I: 10.0 fo r M S2 427>81 MRM transition for SS 499>99 M RM transition for PFOS 556>78 MRM transition for M -556 584>169 M RM transition for PFOSAA 498>78 MRM transition for PFOSA 11 m inutes SS: 4 m in PFOS: 4.2 m in M -556: 4.4 PFOSAA: 4.5 PFOSA: 5 2 3M E: Laboratory W 3S A Page 79 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 4.2 Results Battclle StudyNumber N003604-D 3MEnvironmentalLaboratory StudyNumber FACT060998.1 4.2.1 Quality Control System suitability acceptance criteria were established during the method validation and are included in Appendix C, Section EXAcceptance Criteria. Relevant statistics horn each sample set are provided in Appendix B. Representative chromatograms are given in Appendix D. 4.2.2 Sample Results The results o f 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 foe lowest standard which meets acceptance criteria for accuracy (25% RE). 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 thathad concentrations higher than foe theoretical concentration for foe 13 pg/g calibration standard. Samples that were initially ALOQ were diluted with blank liver homogenate and reextracted Samples that were expected to be ALOQ were first diluted with blank liver homogenate before extraction. The "Corrected PFOS Cone" presented in foe results tables is foe concentration found for foe diluted sample multiplied by its dilution factor (final volume + sample homogenate volume). The method detection Umit (MDL) of PFOS was calculated to be 0.0173 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 foe 7 reps by 3.143; foe Signal-toNoise (S/N) ratio was calculated by dividing foe mean found concentration of the 7 reps by their standard deviation. The method of MDL determination was providedby foe Sponsor. 5.0 Conclusions All analyses met acceptance criteria unless otherwise noted 3M Environmental Laboratory 3 Page 80 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 Baelle Study Number N003604-D 3MEnvironmentalLaboratory StudyNumber FACT 060998.1 6.0 Acknowledgem ents Acknowledgement o f principal contributors participating in die performance o f this study at Battclle is presented in the following list Participant Title Jon C. Andre, PhJD. Richard W. Slauter, PLD., DAJB.T. Patrick L. South, B.S. Gerke H. van der Zwaag, M.S. Bobby C. Braswell, B.A. Cynthia J. Ryan, B.S. Battclle Principal Investigator Senior Program Director Mass spcctroscopist ' Sample preparation chemist Quality assurance auditor Lead quality assurance auditor 7.0 Specimen Storage and Record Archives All original paper as well as electronic copies o f data, will be forwarded to 3M for archival Copies o fboth the final report and all original paper data generated in conjunction with this study will be maintained by Battclle. All residual liver samples, extracts, and unused test article will be disposed o f or returned to the Sponsor as directedby the Sponsor. 3M Environmental Laboratory 4 SS7 Page 81 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 Battelle StudyNumber N003604-D 3M EnvironmentalLaboratoryStudyNumber. FACT060998.1 APPENDIX A -RESULTS 3M Environmental Laboratory rrzs* Page 82 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 Battello Study Number N003604-D 3M Environmental Laboratory Study Number: FACT 060998 1 PFOO, M 4M , PfOaAA, P fO M w hat u v n MTTaxI STUDY: SPMAD3H6ST SOFTWARE DATA B fm iD ! MANUALLY BXC8LIT IPI D m Aitami >--_Nu2bjr 1 a 4s 1 1 9022 9830 1 9831 1 9832 7 1 9833 a 9931 a 9984 a 9909 a 9987 1110 t2 1134 1133 17 13 13 X a 9970 3 9997 3 9999 3 10001 3 10002 3 4 10004 10019 4 10024 4 10039 4 4 10033 10034 21 8 10042 2232 34 23 23 27 8 10044 8 10048 8 10081 8 10084 1 10097 1 10108 3att 30 31 32 33 34 33 33 3337 3430 41 4432 44 43 1 10109 1 10107 t 10109 a 10138 a 10140 a 10142 a 10121 2 10128 3 10177 3 10156 3 10159 3 10184 3 4 10172 10197 44 10194 10203 4 4 10211 10214 4437 1 10097 1 10109 1 10109 1 10107 30 1 10108 31 aS3 34 SB 2 10139 2 10140 2 10142 2 10121 10129 S573 33 10177 10188 10188 30 31 3323 10184 10172 10187 10194 10203 10211 as 10214 3UOQ BELOW UMT OF QUANTITATION Analysisdatehay: Materni MMaawtenitael tMoratlaimmai!l MMMMaaaattteuturtrnreaaai!i Materni Matterai Matti IMtaattaenrntti MMMaaatttltitinneaaii MMMMaaaattteeetrrrtnnneaaa!!! Materna! MMMaaattteeerrrnnnaaa!!! Fatai FFFFFFaaaaaattttttaatttteeee!! FFFFaaaattttattaeeii FFaattttee FFFaaatttttteee Fatte FFFaaatttttteee MMaattaermnaaf! MMMMMMMaaaaaaatttttteteeeeearrrrrnnnnnnnaaaaaa!!!!!l MMMaaattteeerrrnnnaaa!!! MMaatteerrnnaa!! MMaatteerrnnaa!! Material MMaatteerrnnaa!! Materna! PF03 Cone, MAS* Cana, FFCOaBmA,A ffoba 1.248708476666* 111iu...311j487e886****o99222 9.348*01 1.019*92 4J0e*92 2.71* 7*6*82 2J7B+92 299..441279C8**+*9922 8438*92 994413488**9922 1418*91 1.878*91 1418*03 1238*93 112.72286*9.1 BLOQ 1.BB4LL0OO6QQ41 4.816*01 222575940888+**904111 214JM88E*+9012 1.136*02 1.508*92 214419788**9922 21..7910E6*X92 32.498966**9922 33..0299E8*-0912 2806*01 241E-01 82240510888*-60911 3448*91 9498*91 t9..3m3c8*+9o1i 1.778*92 l21iB.403f3r68H**99I22J 2198*92 23942188**9922 3J.?5* a3.9.s9e8**9a2 aBLuOsQa BBLUOMQ 11.4B082U66M**0011 ufl.9sfleg**0Qn u114*Ms40sa866e****t00a11 4*e*oi 23744888**0011 7A2S*01 1112-.212*7666***0013 1428*92 11.413988**0022 1.B91L8O*Q92 BBLUOMQ BLOQ BLOQ 218.59366**00 u31..17i5e&6s*S0S0 4.108*00 uretaa 1416* 31J4Z96E**091S 222497945888***000111 2338*01 BBLLOOQQ 4.BBB5LLU4OO6MQQ* 3.278*00 4.586* U72.174I48M8**0010t u0231a36e8**t90a11i 12539*86+*0011 22976988++0011 2548+01 245E+01 BLOQ BBBBLLLLOOOQQQn 11..13Q4G#** B15.72S66** 1.046* 7.338* IMS* 1823* 1*14-242236861* 1436* 11U*2S66**M 11.43*866**0022 2M3* 11.J57066** 1.B9L48O*Q08 BLOQ 2180BBB71LLL6EOOO*QQQ*00 2*76 24.*34866*00 2i211j0.201t072e*8666t*4*o01i i415t..11a51s4We6**+1 1146* BBLLOOQQ BLOQ BBLUOMQ 124*4266** 2586* 11141.7871666***01 1.208*01 12974186**01 2378*01 12574486**01 7.806* 22902088**0011 MOOQG lati4n1Qq 221M08C*10000 IMirtfl IMBIBO 5192...9440H720868B***ftf00ilQQ10 22sg*gg 814n1aggQoQf I.i08*4ft 1.126*01 111..391B408^^**78011 10E*4n 1O9TI0I 91..099068**40?0 BBLLOOQQ BLOQ BLOQ BLOQ 10.4160*1 TM148 7086 222828M6*0 21.29448E**4 2178*4 24.300086**4 2148*4 1386* 4386* BBLUOMQ BBMLLjOOOQQQ 21.J2S286**4 2086* 1.B0E* 2366* 1166** U20.22i9s06*8**f44ia 386* 9.998*4 81..P14566** 2118*4 Namtaltan ta Otant . i l 1333 te tU B fe tO n i" O ta te * 23.1333 AH samplaa undiluted A il samplaa undiluted Alt aam plm diluted Ait sam plm diluted 3M Environmental Laboratory A-l Page 83 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 Battelle Study N um ber N 003604-D 3M Environmental Laboratory StudyN um ber 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 A ll cones In iig/g C alculated C oncentration o f R eplicate 1 Calculated C oncentration o f R eplicate 2 C alculated C oncentration o f R eplicate 3 C alculated Concentration o f R eplicate 4 C alculated C oncentration o f R eplicate 3 C alculated C oncentration o f R eplicate 6 C alculated Concentration o f R eplicate 7 M -556 0.1269 0.1220 0.1433 0.1368 0.1670 0.1225 0.1190 PFOSAA 0.1484 0.1281 0.1484 0.1605 0.1681 0.1513 0.1385 PFOSA 0.1167 0.1325 0.1521 0.1441 0.1490 0.1382 0.1413 M ean C oncentration Std. Dev. 0.1339 0.0170 0.1488 0.0128 0.1391 0 .0 1 1 9 Ip i 1 jn ^ Spike Level S/N 0.1331 0.1334 0.1315 illillllllil B -- 7.88 11.66 11.74 LOQ (d e t from 10 x std.dev. "noise") LO Q (d e t from cal curve low std.) Curve Coeff o f D eterm ination D ate analyzed M ethod 0.17005 0.1331 0.9978 1 3 0 c t9 9 LC/M S/M S 0.12763 0.1334 0.9937 130ct99 LC/M S/M S W BBSSSBSBSi 0.11853 0.1315 0.9891 1 3 0 c t9 9 LC/M S/M S Key 1 - Spike Level too high; Spike L evel m ust be < lOx MDL 2 - Spike Level too low , Spike Level m ust be > MDL 3 - S/N too low , S/N m ust be > 5 4 - Coeff o f D et o f calibration curve unacceptable 3M Environmental Laboratory A-2 3to Page 84 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 Batidle StudyNumber. N003604-D 3MEnvironmental Laboratory StudyNumber FACT 060998.1 APPENDIX B-DAILY ACCEPTANCE CRITERIA SUMMARY 3M Environmental Laboratory . -^80 3 / Page 85 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 B atidle Study N um b er N 003604-D 3M Environm ental Laboratory Study N u m b er FA C T 060998.1 P F O S t a l tN RATUVER STUDY NUMBER: N003604-D DATA ENTERED: MANUALLY SOFTWARE: EXCEL 97 RESRESSION PAfM M C TO S Analvalp O a tt O rtn h f 13.1888 Ootttosr 16. 1800 OdDbai IS , 190 O dobaf 20,1800 A n a lvts pFoe H affft PFOSAA Pf=OSA PFOS M-586 PFOSAJK PFOSA PFOS M-9SS PfO SAA PFOSA PFOS PFOSA 1 ? C a *t 0.0164 4X00646 000232 196 4X00388 9.99E49 aooseo 390 a00383 0.0231 OOC3TQ -71.9 ao o eia .153 X Com ff 1JI6 aaz7 0.0070 1.906+04 1-83 0.578 00831 1.736+04 2.17 0312 0.0853 1.336+04 2.07 1.686+04 fnterespt 0.0773 4X0313 4X00306 872 -0.0002) 4.00007 4.00321 886 -Q.0009 4.00290 4X000603 -722 4X0361 -1.02E-33 C oaff o f P F w w in rto n 0L864 0886 0904 0968 0988 0886 0886 0880 0966 O JM 0903 0.906 0987 0908 Cm u * M 6 OvvM q m O n . d p i a# CM |* 1 antudad Ona rae at Cal M1 aadudad Ona rap of cal pt7 -anudad O n rap o f cal pi 5 aariudad Ona rap of cal pt 7 tacfeidad O r* rap o f pea 1 ,9 ,9 n d u d a tf Ona rap a l pta 4 .7 ataludad 3M Environmental Laboratory B -l -8 + Page 86 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 Baitelle StudyNumber N003604-D 3M Environmental Laboratory StudyNumber FACT 060998!l PFOS et a! IN RAT LIVER STUDY NUMBER: N003604-D DATA ENTERED: MANUALLY SOFTWARE: EXCEL 97 . QC Result A nalysis D ate October 13. 1969 A nalyte PFOS M -558 PFOSAA PFOSA October 1 8 ,1 8 6 6 October 18,1989 PFOS u-sse PFOSAA PFOSA PFOS M -558 PFOSAA PFOSA October 2 0 ,1 6 8 8 PFOS PFOSA QC Level, na/mL, ~%RSD 10 33 0 .7 0 .1 8 10 M 0 .7 0 .1 8 10 3 .3 0.7 0 .1 8 10 3 .3 0 .7 0 .1 8 8 .6 33 73 3.1 4 .7 1 1 .7 3 .0 113 83 3.1 8 .7 143 1 8 .8 8 .2 5 .8 4 .8 10 3 .3 0 .7 0 .1 8 10 3 .3 0 .7 0 .1 8 10 3 .3 0.7 0 .1 6 10 3.3 0 .7 0 .1 8 53 43 8 .3 8 .7 3 .4 2 .7 8 .0 1 7 .7 4 .3 2.1 7 .4 1 7 .7 1 1 .8 8.1 6 .0 8 .8 10 3 .3 0 ,7 0 .1 8 10 33 0 .7 0 .1 8 10 3 .3 0 .7 0.18 10 3 .3 0 .7 0 .1 8 8 .7 1 1 .7 7 .0 15.1 1 4 .7 1 7 .0 213 2 8 .8 1 4 .7 1 1 .4 15.1 2 1 .3 1 8 .7 113 12.8 6 .4 10 2.4 3.3 2.0 0.7 3 .7 0 .1 8 10 53 2 .3 3 3 4 .0 0.7 2.3 0 .1 6 3 ,4 %RE 4 .0 -4 .6 6 .4 -1 2 3 -2 .4 23 -8.6 8.1 -1 .7 -1 0 .4 -1 3 .0 83 4 3 -2.1 1 .3 3 .9 143 153 8 .8 2 J -2 .8 5 .7 3 .7 0 .8 -2 .9 -7 .0 is .a -3 .9 5 .4 4.4 13 .7 -4 3 -1 .7 -2 0 3 -4 .1 -2 .9 2 3 .4 1 1 .6 1 5 .4 0 .8 1 3 -2 0 .6 -1 8 3 0.0 13 .7 0.0 1 8 .0 -8 .0 -12.0 -2 1 .7 -1 8 .0 -1.6 63 13 1 1 .5 3M Environmental Laboratory B-2 U > S (,3 Page 87 3 M .Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 B attelle Study N u m b er N 003604-D 3M Environm ental Laboratory Study N um ber FACT 060998.1 APPENDIX C-SAMPLE INVENTORY LIST .3M Environmental Laboratory S* 3 . Page 88. 3M Medical Department Study. T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 Batefle StudyNumber N003604-D 3M Environmental Laboratory Study Number FACT 060998.1 S tu d y T O X - 0 1 3 . A r s u * 1 8 - 0 0 8 . S a m p le Infonm a B c n fo r te ifc m e n t >9 O l i t e l a S am p le 1 2 a 4 3 a 7 a 9 10 11 12 13 14 IS 10 17 18 18 20 21 22 23 24 23 28 27 28 29 30 31 32 33 34 38 36 37 38 39 40 41 42 43 44 *5 *a *7 48 49 so 31 52 53 54 55 58 57 58 59 80 81 82 63 84 I- M S a m p le D e sc rip tio n FO -9922-orpl-M FO -9930-orpt-M F 0 -9 83 1-9P M 4 F O -9932-flfpl-M F 0-9933-flrp l-M F O -9981-grpll-M F0-9S84-orp)M 4 FO-O SSS-ofpll-M FO -9987-otpH -M F0-9970-0fpH -M F 0 -B 9 97 -s rp lll-M F O -8 99 9 -o rp lll-M F O -io o o i-a m U M 4 F O -1 00 0 2-trp lll-M F O -t0004-erp M 44 FO -10019-O TPlV-M F0-10024-om tV -M FO -10029-tX P lV -M F0-10033-o rp tV -M FO -10034-orptV -M F 0-10042-0H 3V -M F O -10044-flfpV -M F O -10045-prpV-M F 0 -1 0 0 5 1-prpV -M F O -10034-ffp V -M F 0-10097-aip t-F FO -10105-on>t-F FO -10108-O T l-F FO -10107-grpt-F FO -10108-o rp l-F FO -10136-orpH -F F 0-1014O -<jrpll-F FO -10142-orpH -F F O -1 01 2 1-o rp ll-F FO -1012B -orpll-F F O -io m -o n jiiK F FO -10135-flrpltt-F FO-10 1 3 6-g rp H t-F F O -10184-flrp lH -F F 0 -10172-o rp lll-F FO -10187-flfplV -F F O -1 01 9 4-flrp lV -F F O -1 02 0 3-o rp lV -F FO-10 2 1 1 -c rp iV -F F O -1 02 1 4-flrp lV -F FO -1 00 8 7-o rp l-F FO -10105-srpl-F FO -10106-Q rpl-F FO-10 1 0 7-p rp l-F FQ -10108-tjrp l-F F O -10138-o rp lF F F O -1 01 4 0-o rp ll-F F 0-10142-o rp lt-F F O -1 01 2 1-o rp lF F FO -10128-O iplLF F O -1 01 7 7-o rp lII-F FO -10155-grpH t-F FO-10158-OTTiUI-F F O -10184-orptll-F FO -10172-orptlt-F FO-10 1 8 7 -o rp lV -F F O -1 01 9 4-o rp lV -F F0-10 2 0 3-o rp fV -F I FO-10 2 1 1-grpJV-F I FO -10214-flrptV -F Sam ple T y p e 4aternai R at U var Aatem al R a t U v er tatem al R a t U v a r 4atem al R at L M r Itatem al R a t U v a r rtatam al R a tU v a r ktatamaJ R a t U v a r M aternal R a t U v a r M aternal R a t U v a r M aternal R a t U v a r M aternal R a t U v a r M aternal R a t U v a r M aternal R a t U v a r M aternal R a t U v a r M aternal R at U v a r M aternal R a t U v ar M aternal R a t U v ar M aternal R at U v ar M aternal R at U var M aternal R at U v ar M aternal R at U v ar M aternal R at U v ar M aternal R a t U v ar M aternal R at U v a r M aternal R a t U v a r F atal U v ar Fatal U v ar Fatal U var Fate) U v ar Fatal U var Fatal U v ar Fatal U var Fatal U v ar Fatal U v ar Fatal U v ar Fatal U var Fatal U var Fatal U var F atal U var Fetal U var F atal U var Fatal U var Fatal U v ar Fatal U var Fatal U var M aternal R a t U v a r M atam al R a t U var M aternal R a t U v a r M aternal R a t U v a r M aternal R a t U v a r M aternal R a t U v a r M aternal R a t U v a r M aternal R at U v ar M aternal R at U v a r M aternal R at U v ar M aternal R at U v ar M atam tf R at U var M aternal R a t U v ar M aternal R at U v ar M aternal R a t U v a r M aternal R a t U v a r M aternal R at U v a r M aternal R a t U v a r M aternal R a t U v a r {M aternal R at U v a r 3M Environmental Laboratory C-l Page 89 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 Batidle StudyNumber N003604-D 3MEnvironmental Laboratory StudyNumber FACT060998.1 APPENDIX D-REPKESENTAUVE CHROMATOGRAMS 3M Environmental Laboratory Page 90 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 Battelle Study Number N003604-D 3M Environmental Laboratory StudyNumber. FACT 060998.1 3M Environmental Laboratory D -l # b 3 7 Page 91 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 ^ LRN-U2095 ' . Battello Study N um ber. N 003604-D 3M Environm ental Laboratory Study N u m b er FA C T 060998.1 3M Environmental .Laboratory D-2 Page 92 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 Battelle Study Number. N0Q3604-D 3M Environmental Laboratory StudyNumber FACT 060998.1 3M Environmental Laboratory D-3 Page 93 3M Medical Department Study: T6316.5 'Analytical Report: FACT TOX-013 LRN-U2095 BatteUe StudyNumber N003604-D 3MEnvironmentalLaboratory StudyNumber FACT 060998.1 3M Environmental Laboratory D-4 T Page 94 3M Medical Department Study. T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 Battelle Study Number. N003604-D 3M Environmental Laboratory Study Number. FACT 060998.1 3M Environmental Laboratory D-5 Page 95 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 Batidle StudyNumber. N003604-D 3M Environmental Laboratory Study Number FACT 060998.1 APPENDIX E-PROTOCOL, AMENDMENTS, AND DEVIATIONS 3M Environmental-Laboratory Page 96 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2 095 Battelle StudyNumber N003604-D 3M Environmental Laboratory StudyNumber FACT 060998.1 7 ~ 7~C4 - j/-^ 3 M E n v ir o n m e n t a l L a b o r a t o r y _________________ _____ Protocol -Analytical Study 2(N-EthyIperfluorooctanesuIfonainido)-etiianol in Two Generation Rat Reproduction In-vivo study reference number; Argus 418-009 Study number; FACT 060998.1 Test substance: 2(N-Ethylpexfluorooctanesulfonanudo)-ethanol (N-EtFOSE-OH) Name and address of Sponsor: Marvin Case 3M Toxicology Services 3M Center Building 220-2E-02 S t Paul, MN 55144 Name and address of testing facility: 3M Environmental Technology and Services 93S Bush Avenue, Building 2-3E-09 S t Paul, MN 55106 Experimental start date: Expected termination date: December 31,1998 Method numbers and revisions: FACT-M-1.0, Extraction of Potassium Perflnorooctanesulfonate or OtherAnionic Surfactants from Liver for Analysis Using HFLC-Electrospray/Masa Spectrometry FACT-M-2.0, Analysis of Fluorochemicals in Liver Extracts Using KPLCElectruspray/Mass Spectrometry FACT-M-3.0, Extraction of Potassium Perfluorooctanesulfonate or OtherAnionic Surfactants from Serum for Analysis Using HFLC-Electrospny/Maas Spectrometry FACT-M-4.0, Analysis of Fluorochemicals in Serum Extracts Using HFLCElectrospray/Mass Spectrometry Author Lisa Qemen ------- ----------------- i / i s h x Kris Hansen Date Study Director 7G < *. Marvin Case Sponsor Representative / Date f 3M Environmental Laboratory E-l 1 Page 97 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 BatteUe Study N um ber. N 003604-D 3M Environm ental Laboratory Study N um ber FA CT 060998.1 ______________________________________________________ _ The analytical portion of this dosing study is designed evaluate the levels of perfluorooctane sulfonate (PFOS), or another metabolite of 2(N-ethylpeifluorooctanesulfonimido)-ethanol (NEtFOSE-OH) designated by the study director, in the liver of the parent and subsequent generations of die test system, or in the serum as necessary. The in life portion of this study was conducted at Argus Research Laboratories. 2.0 Regulatory Compliance________________________________________ _ This study is conducted in compliance with die Food and Drag Administration GoodLaboratory Practices regulation as stated in 21 CFR 58. Any exceptions will be noted in the fin al report. 3.0 TestMaterials_______________________________________ _ 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: Ratliver and scram 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 3.221 Analytical reference substance matrix: Argus ResearchLaboratories; traceability information will be Included in the final report 3 3 3 Analytical control matrix: 3 3 3 .1 .Rat liver-Argus Research Laboratories; traceability information will be included in the final report; or Rabbit liver - Covance Laboratories; traceability information winbe included in the final report 3 3 3 3 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. Seram 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 aletter followed by the Argus project number, the animal number, the group designation, and the draw date. 2 3M Environmental Laboratory E-2 VT37 Page '98 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 Battelle Study Number: N003604-D 3MEnvironmental 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 die Sponsor. 3.6 Stability of test material: Characterization of the stability of die test material is die 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 5 Safety precautions: Refer to the material safety data sheets of chemicals used. Wear appropriatelaboratory attire, andfollow adequate precautions for h an d lin g biological materials and preparing samples for analysis. 4.0 E xperimental - Overview______________________________________ Tissues bom 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 (ES7MS). 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. M J ENTAL- Analytical Methods 5.1 FACT-M-1.0, Extraction of Potassium Pcrfluorooctancsulfonatc or OtherAnionic Surfactants from liver for Analysis Using HPLC-Elcctrospray/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 Seram Extracts Using HFLCElectrospray/Mass Spectrometry 6.0 Data Analysis_____________________________ ;______________________________ 6.1 Data transformations and analysis: Data will be reported as the concentration (weigbt/weight) of fluoride per tissue or sample, or of PFOS per unit of tissue or fluid. 63 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. 3 3M Environmental Laboratory E-3 SM Z 7S Page 99 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 Battefle StudyNumber N003604-D 3M Environmental Laboratory StudyNumber FACT 060998.1 7.0 Maintenance of Raw Data ami Recoups__________________________ 7.1 The following raw data and records will be retained in the study folder in die archives according to AMDT-S-8: 7X 1 Approved protocol and amendments 7.1.2 Stcufy correspondence 7X 3 Shipping records 7X 4 Raw data 7X 5 Electronic copies of data 7.2 Supporting records tobe retained separately from the study folder in die archives according to AMDT-S-8 will include at least the following: 7X 1 Training records 7X 2 Calibration records 7X 3 Instrument maintenance logs 7X 4 Standard Operating Procedures, Equipment Procedures, and Methods 7X 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 9.1 FACT-M-1D, Extraction of Potassium Perfluorooctanesulfonate or Other Anionic Surfactants from Liver for Analysis Using HPLC-Electrospray/Msss Spectrometry 9.2 FACT-M-2D, Analysis of Fluorochemicals in liv er Extracts Using HPLCElectrospray/Mass Spectrometry 9 3 FACT-M-3.0, Extraction of Potassium Perflnorooctanesulfonate or Other Anionic Surfactants from Serumfor Analysis Using HPLC-EIectrospray/Mass Spectrometry 9.4 FACT-M-4.0, Analysis of Fluorochemicals in Serum Extracts Using HPLCElectrospray/Mass Spectrometry 3M Environmental Laboratory E-4 Z7 ( . 4 Page 100 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 B attelle Study N u m b er N 003604-D 3M Environm ental Laboratory Study N u m b er FA CT 060998.1 METHODFOB.ANALYSIS OFPOTASSIUM PXBFL'UOROOCTANXSTJLFONATX C*FOS)lNBATLIVZRBV LOMSMS Vanias U> ShirWiki Amtbmttomtm l)U '1 : i- *i *)Lsvisioosto tho|-- I'.ii'.'.i'iv 1'. | - 'Writtenbv: PatrickL. Sooth ______ Date; App roved by: C ________ P A a d n ,P K D . r, B io o a iy d a d C h e m iitry 9*^ f P!i i f t f f , 3M Environmental Laboratory PaislrfW C -l E-5 277 Page 101 3M Medical Department Study: T6316.5 Analytical Report;: FACT TOX-013 LRN-U2095 BatteUe StudyNumber:N003604-D 3M Environmental Laboratory StudyNumber: FACT 060998.1 M E T H O P F O R A N A LY SIS O F PO TA SSIU M FX R FL T JO R O O C T A N ISU LFO N A X B fP F O S ) IN R A T T.TVTTBB Y L O M S /M 3 V e n ta U atadvMej A a a h ra t/P a te i X Soutfuty floandandallT la SE&actbn and n a ir ii* o fp o ta ln a paflaaro octB M B iS ata n d a b M d tk a t liver bpedbnned. Calibeada aauidaida aeapMptw d by ^fld agb lnelt M m to a e i-- vd dt advent etmdarda ftom two bidtpm rinaly-prBpaiad aocM. T b e c a U b fH ta a n d a id ita lM M M l with pgTofaa agmdaid. bettered, and aaiiaiap l w ith athyi acetate. Tba organic phaata a a - MTrratwi m dry-- mid icrrmditnaid i miehennl a nelyl by TPAWU IL POTTOS* T o ooract ta d aealyiapocairfginpB gncrriric a nnnmaBaie and related flaornchrm lralcanpognda ftamd la S pnfa*D am lay ta l Jtafc IT T, S ra m .-M Soo d a la atCustodylecorda tfa ppUcabh . IV . G zrosA L ltisrsxrcnoN a Calibrate i l l reqoiiedhainraaccardfan a the SOP ca halmncaonge. M ake eqaivaknt dnudaaa when tba volume needed verb * flrs a the volume Matadbi the BOAthotL L ib d sUamdbud and reagent a o h s k o n s ^ a d fie d lm h a appropriate SOP. H > e a b ta d reune a aolndm fe r Arturo ta rio . be a n a tbn label indadca die pR pendm data aad atndy m in iW fry liK -- twirfafly p y y w y f Sign a n te final page c f tide method to aigaiQrlhat yon have Iblkiwed the method a e v rd tta , aU a a m ia ln a d itaeents * e en ota*. and aU eqolpnaaaM i been prepedy edB bm at K yee deviate fa ta tbs method, docomoa* the cbeage, and obtain (he approval o f the neb aaaasac, amdy (flitetot. or auk leader a t toon a * p a n b le . In itial aad data iH data antriee o ath s pace oawfcLdithey w en nmda. I f onty am paraoa eaten aU data on a dnaJa day, the doenm enatta maybe made la a d n jle locadan an fta t p p I f m ultiplemmS ttvo id d U cn slen txlaiZ B B fttb sin lriilcd tfd d rtrilQ fA ft wwpw wmH m tba cutiy, Linw ooa atH A denote* "N ot AppHrahUT. The aw bodi writum la genet! chronological order,butthe aeqtaaca efrtepa so y h a tb a n d I f Iba anaiyitdeema b appropriate , anlaaa the etder fcrcatafaiactM tlee la ipnrfflad. Stoda w illb e oacdlbrthedntattoa o f the nndyenlcaa rra a m in i ocacleaa v a b fflly k wwMimrf fmt^1--^ H o eonecticow fll be made Ib r peirity o n a lt content o f any tern a r id * but FPOSAA. Uaaglaaa volumetric, E pp aidta f repeats; or poattivo-dbplacam qtplpeaftr iBgpand a t t C fta tirt w itb Teflon by the tea artic le t a M be laiariaed. V. M aterials Sea Table ltb r an reunited chearicala. taagean, and en tran t. Uae Table 1 bar doemaentadaa. Check an labela carefully to eeaaie d m aU materials a n ant expired aad feat they a n the pnper purity or pad *. Paa2 o f Id 3M Environmental Laboratory C -2 E-6 S?Z7* Page 102 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-I12095 BaJtelle StudyNumber N003604-D 3M Environmental Laboratory Study Number FACT 060998.1 M ETH O D F O R ANALYSES O F PO TA SSIU M P E B IL U O R O O C T A N E SU L FO N A T I (PFO S) D f B A T L IV E R BY L O M S /M 5 Vantoa L9 te*rKti aatntPtB Xl.llVtt.llN INe Pntaiahiaa h jftn w m afr soffocare (PROSI SuMhooi A dd 14rSM , USTO FFOSAA rpo&A FFQSEA Sal Liver Analytical Standard Smrocata Standard Analytical Sts&dtfd Analytical Standard Analytical Stasrd Analytical Stmdsrd Analytical StBUSSRi Matrix NHXAO Sadism Hydroxide, NaOH ffytiUftiuul COEA1. .ICHjfCBiUKHSOO ftvHiim C steStt^ NnCO, Soritnm BicvbasuUB, NaHCO, E&yi Acema Mobile Phase Reagent Prep ExtractPrep ExtractPrep Extract Prep Extract Prep T aM at Materiale Mmhvr lei .ttK'or I'mill 3M 1CN 3M 3M 3M 3M 3M Rarian SpatneDawfarr 11 **<Ut"C IVttlf* Kaom Tassp Room Tamp Tessa Pa^i--I Tama Roam Tema Roam Tassa --30*C RT RT KT 1 1 * " RT RT KT KCctbsool KfilU-Q Water pH TBotter pH 10 Buffa- Mobile Pham, Scoda. WS BnajcttPrcpt Mobile F hase calibration pH meter MUUpars RTASTMTypel RX RX RX R X m oas Room Temperatura. V L X q u it v o c n t SeoTib* 2 t a all roquiitd majer pisca e f aiaipacoL U ee tbe able to doaimoit Use ttS m i pleca (a.j. malr^ mnrtcQ <tcqgipmenl. Cberb ealibradca o*fl equipuseat rcqoiriaf cantados (ajp bolmcGg) (g emuraitUasrosL Pa*o S e f li 3M'Environmental Laboratory C-3 E-7 17? Page 103 3M Medical Department Study: T6316.5 Analytical Report:' FACT TOX-013 LRN-U2 095 Battelle Study Number N003604-D 3MEnvironmental Laboratory StudyNumber: FACT 060998.1 METHODTORANALYSISOFPOTASSIUM FXKFLUOROOCTANXSULFONATX(TTOS)INBATLIVERBYLOMS/MS VoafcraUl Anbnatew ______ TsMs2. h A w l ________ F|ii|Mncnf_________ I K f_____ Mumitacnucr____ Minici A a ly tk a l B > lm W dgb Standard or R o a e n ta W dfbcSac Ci ISb u b B ^ kdci \ ,i vN 1 Plpednr P tpetS im plg ***** Pipetear PSpcssr V anear- Freear (-1 0 *0 R cflig s a c a r 0 -9 *Q _ Certtrifago fip ttS n p k i P ipaE lO A c Pipet Reagenti, W IS X )QKfffiw p lfl Store QCa, Blaak U ver Stara B o tte , Stocka psw SBpHtiOB Pjajg iiArwf RfipCSttf Tac Toba U ver am ple SUdcwoU Potypaopytcnc, SWSS99 ' Centnflig T a b a T a tT a b a T n n ip o it M i cuaje sane Ckbitsl S b ib r E v ip o ra n r Extract Samplea Evaporata Extracta Store QCa Stlrisatrbc S m p ict Evaporan Extracta Bios Falena Bine Falena EU ajr botyprepyl-- i. 13 mL Polypropylene, 1 2 x 7 3 urn 1 3 mL polvwocvlene Zysunk TaxtxrvapLV 209 . 2002 137-T160-3P S /itagoF U ta eH aan Electred* Volaanstria F lo ta . a*M a C lan A T r e n te Pipeta, F ia d lo PBtor JBxunct Grind livor Determine B o tte oH Determina B o tte oH Mah Volnnutri DOndost M ake Volumetrie Dadnni Ite r a te Extracta to Csntrifiigg FUtsn and L C Inaero NA NA * MA NA . NA NA 3M Environmental Laboratory P*4D i l i C-4 E-8 23 z o Page 104 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 Battello StudyNimben N003604-D 3M Environmental Laboratory StudyNumber FACT 060998.1 ; M ETH O D F O R A N A LY SIS O F PO TA SSIU M PXB7LUOROOCTANESULFONATX (PFO S) IN RA T L IV E R BY LO M & M S Venirne U StudTWa MhaffittB VU PnoczsnoBX A . P re p a ra tio n o f 2 m M A m nionhnn A cetate T n llT ili 1TTTI _*_1tTTTT | rdimmrail irm entit mil tniuftr W i IQfifliiiT illfiiiiilihi fiaafc. D tao lvatb eao lld in water aoddBua to v o ta artifem ec. Solution a n y ka m ad f i r ana ameth atorad i Actual naaa cdemmrtrim Beatalo; Actual fin al v o te s D ate odpreparado c . Study No: B . P re p a ra tio n o f ~29% S odium H y d ro x id e S olution Weigh 200 a 2 gad atxfinmfcydraddebeo abealeer.Add3 0 0 m L .o flfllU -Q w aterand m ix te dlnolvm. Cool n d m a a firto a poiyptupytaa beala f i r maga. SotsnSoamay bo atorad f i r 6 month a t room tam pm enra. Actual maas o f sofluta lydroaidet _ _ _ _ _ Actual volume M H H -Q a a to : _ _ _ _ _ Date o f preparation: _ _ _ _ _ _ _ _ _ _ _ _ Study N o :________________ C P re p a ra tio n o f ~ 2 J % S odium H y d ro x id e S olution Add 10 mL of~2S% Sodium Hydroxldo Solution to a lO O -aL volumetric fladcaad d llu tt ta volume w ith M B 2-Q malar. T tm a fir to a yolypnpyiane bottle f i r Stoopa Soiudoa may be atorad f i r 6 montisi at room tempemma. A ftm l velam a ad--2S K N aO H abbaio _ _ _ _ _ _ A ctm l fin al votame: _ _ _ _ _ _ Data odpreparation: S ln rtrN b s D . P rep aratio n o f T etrab u ty lam m o n iu m H y d ro y n iasifata (IB A ) S o latio n , (L5 M , (p H 10) pH M ater CaUhnrion pH baffler 7 pH bafflac 10 pff lesflo^ pHleeting* Add 169 s 1 god T B A to~3O0 m L arM U JL-Q w irar ta tb a aker. A fls n tfia p H 10.00 a 0.02ualng--35-60 n L ad29% Sodium Hydroxide Safadiaa rifiutato 1000 m L w ifi MOB-Q water, and m ix. A ttu a ti pH to 10.00 a 0 m a d o g *4 J % N a O H a a d te tn ta ta d tr to a polypropylene b o o k f i r atonga. Sotado may be uteri f i r ana m nafi atorad at room tom peate, b it tfea bH t iW lh t tfw tlted a a W 'te e a e h a m Adfatoto pH 10.0 a 0.02.witis 2-9% SodtenHydraodd* Solution 1 Paga 3 e d iti 3M Environmental Laboratory C -3 E-9 3*1 Page 105 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 Battelle Study Number. N003604-D 3M Environmental Laboratory StudyNumber FACT 00998.1 M ETH O D F O R A N A LY SIS O F FO TA SSIO M FHXFLU O R O O C TA N ESU LFO N A IX (P F O S )JN S A T LTVZR B Y L C /M S M 3 Venia* LO BtmfrfNoj Aaabufl)* A c m ln a o f lB A : __________ A c fn lflo tlv o b iB ic i_________ Acinaifin al pH: _ _ _ _ _ D a** a f perpoUiac _ _ _ _ _ _ _ _ _ _ _ _ Study No: _ _ _ _ _ _ _ _ _ _ pH fierrecbecm and/ortea<$u*ting:________ X . P re p a ra tio n o f 0.25 M C a rb o n a ta B u ffer W eigh 26.5 a 0 .1 1 o f jodium c a ta s te to d IL O * 0 .1 1 o f aodhna bicatbcuata a d taxmlato the a m lOOO-cnL -volumetric flaak. M acche the m tcriala teM Iltt-Q w etB ^ flhue to 'volons w ith M IIU -Q water. m ia. od taneftr to a paiypnpyhaa bolt]* to r Borage. Soluriac may b ued for1 month wbea unrad refrigeraceli. Actual n u c f K xfiun ewbnnaWC A ffla i t ta o f aodinm bicarbonate ; Actual fin i, votane: _ _ _ _ _ _ ' Date cfjrrtpatadoa: _ _ _ _ _ _ _ _ _ _ _ _ : Stupito:___________ F . p re p a ra tio n o f M obil P h u t Component A: M x together 600 inL of 1 jbM ammcnbun acato and 400 toL td Saturimi may ha uacd f t r 1 m th *w h am n d m o m tampiiraliira. A c a ti volume o f 1 tn M emmonhini ac etate________ L A stati votam i a f methanol: ________ m L Data a f prepam loa: _ _ _ _ _ _ _ _ _ _ _ _ _ Study No: _ _ _ _ _ _ _ _ ComponentB: M ix together 30 m L e f3 n M ammonium acetati i d >30 L a t Solution may ba un d t o r i month when M a n d a tm an temperatura. Actual volume o f 2 o U ammcuiusi acetate________ m L ' ActualvahnM o f methanol; _ _ _ _ _ _ m L '* Date ef an ralliai: Study No: _ _ _ _ _ _ _ _ G . P re p a ra tio n o f S tock S u rro g ate S ta n d a rd a n d W orkfatf S u rro g ata S ta n d a rd (W SS) 1. Slade Surrogata Standard 30.000 njhuL): W eigh 15 2 rag o f IH . IH . 2H . ZB.-yafjnorooctane artphreric ad d and craax&r to a lOO^uX.volumetric fliric , D iiao lra la methanol. (Stale to rolsaaa with m rrhanal. and adx. Stum n ftig eala d . protected t o n W llg b t. Actual A rim i DOudea Votum Pate ofPrcpaiatioei:___________________ Study No: - PasaSoflfi '3M Environmental Laboratory C-6 E-10 -W T 3 -2 Page 106 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX^013 LRN-U2095 Battelle StudyNumber. N003604-D 3M Environmental Laboratory Study Number FACT 060998.1 MZTHOD FO R ANALYSIS OF POTASSIUM PEBFLUOROOCTANESULFONATX (PFOS) IN RAT LXVXXBY LOMS/MS V erdea LO ' Stady Ne A a e tre t/P B c 3. WSS (1000 njftnL): LDOate 100 j Ofdock arroga itaadenl 23 m L w U i aetfcaael redafac. f*M l Votam e r f Stock Tmm a l Stenderti: A stati D fln flra V o lo n s P e a atPrapoiz dnn: H . P re p a ra tio n o f C a lib ra tio n S olvent Stocks a n d W o rk in g S tan d ard s L Solvent Stades For eecb anelate w d g h 6 a t a d a d anuat o f rouderd adapcadaritr w d g h e d u A endB repIlcatm ) U aed in T d ile 3 and to n a te tota e s en ta vnlnm arie f lu iti m n lv In m u t i m i .Mut, tu wjwm mmA tnixm CL Store n f ilg a i tBd, p ro a c ttd i t o UV ttght. 2. Mbttd Solvent Stades 1 Pjpff amAHirfOfCOCl iutasi| A,qg ^ Tabla 3 n id to rn ite tata s dngle volumetrie S u k . D laotve la m edm d, dDatt ta voltane w iih frvtrtamnl. ad taix-wen. Stare refligented, protactad f i n tJV light. Repexttas jro oe n w ltb P e p ilo Brndr. n w h r i > 'i n t a i t i e . . . m id U p rtp a rm th a rn riM n y tt a* A itd e Date o f prepaadOK Stndr Ko; 3 . W oddag Stenderti (W S): D ilata tha tatxed ito c k i m d woriogg a m d ir ii vrith rertkennl 1l prcifiod la Table 3 and m ix e e fl. Pet q f preperartotv >f.nnf.ti (t 'mil tc FPOS Stalk IB EPOS Stadt XA M-1M Stack 2B M-JM Stock 3A. M370 MJ70 Stack 4A 1 PFOSAA Stadt 4B ! FFOSAA Stadt JA PFOSA Stadt 5B PFOSA Stack SA PFOSEA T etto 3.'CeObraBoa SatvmdSiedeed'WortdeaSteederSs V(imI Ammut V lIM IIIlt \ ( l. llM iL .lt S it i. V ttl.ll V n l S ih if,, u r\\s> uni. 30 * 1 tac ISBMBMlTMritiil 23 * 0-3 m i ffiPm h im S ) 10 io 30 e 1 ma B9HBBiBttfll 23 * 0.3 BZ lqOyH rSvral 10 io 30 * 1 mz 10 1 23 * 0J mz M M u f l *10 93 * 1 m< p a r H M iB oe ia 10 44 s 0.3 m WtaWbdhitttitSaklHBl 10 1 1 IU fl 1 1 I | 50 R 1 Oll H (H m | IfffMUittiiSiiM 10 I LO 1 50 t 1 n i 10 1 Page 7 cf 16 3M Environmental Laboratory C-7 E -ll Jftfc i t s Page' 107 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 Battelle Study Number N003604-D 3M Environmental Laboratory Study Number FACT 060998.1 'M E T H O D F O R A NA LY SIS O F PO TA SSIU M TX RFLUO ROO CTA NX SULFO NA TE (PFO S) IN R A T LIV ER. BY L O M S /M S Vanhm LO Study Nclc A nalyit/D ate: _ Stock SB S m A Stock A Uberi StockB WS 1 WS2 WS 3 WS 4 WSJ WS WS 7 PFOSEA Storta 1 tora Seo A Storta 1 tora XcoB Mixed Stock A U b ai Stock B WS I WS2 WS 3 WS* WS 3 23 ajm * bsfflnMUHKMOt 3 mL eech** IrowmMHMBfli 3 aL BiWtSlBl&ifm each HUSlM HUM H 1 mL* ImL* g S | s g 3 mL** 2mL** l_5mL-- 1 1 ml_~ 2mL m B to w a MWBIHIHiiilflfaeil UlClfflmSBB&Bi biSa9neH>2ft|Q| fetB W B B 10 1pm im nifflt 1300.000 30 300.000 30 S H 230,000 23 20,000 23 jn s g g g g g 10.000 10 ItnlMWftiliitf *000 10 iiiBMIIm M I 2000 10 HBimawmi 1000 10 mniwromwl soo 10 StatwaaaB 100 Wdgh J analytical andante ta at Iravtho acareetO.Ol mg. -- TTw limili 11Ir nr i lltvi infiltrim i ni jilin 1(1) L P re p a ra tio n of C a lib ra tio n S ta n d a rd s and BLanlcs 1. liver hamssam e Prepere blank Uver homogenete b bulle by weighing approximately 40 g o f blank liver into a 300 mLNmlgene botri* conndniag 200 m L c fM m -Q water. Grind to a homogmeoni nnponrion, Ahqoot has appena 30 m Lpoitioosfer . frozen (approx -2 0 *C ) etarage. nfA c tn a lM m o fliv e r ____________ A lt! VPhim w ater Due ofprep: '_______ Stodr Decornine dcarity ofcaUbtadeai'QC matrix : M IX H Q M O G E H A IB TH O R O U G H LY and determina the mam In mHUstamn of 10 replicata weigbinp of 1 n i pardon ofdtoTHOROUGHLYMIXED homogenate. MIXHOMOGENATEIMMEDIATELYPRIORTOEACH ALIQUOTREMOVAL R eplicale* M ao Iom'i I U v e r CaUbmdon Standards P repare each 9ver calibrarte Vendant by adding 0.A3 m L of nndUxneAIher homogenate (STIR HOM O G ENATE WEULB A U Q U O TIN G ) tnm a 13 L oxgarritm tube and adding 30 pL o fW S a t M cCB. Prepare triplicala cal Sftandards and bienIre. Sea Table 3 Cbrvolunxa. Ih * dQutad Uvea draaby la nenmncd to be approximately 130 mgftaL. MfatweU. Pige lo f 1 3M Environmental Laboratory C -* E-12 Page 108 3M Medical Department Study : T6316.5 Analytical Report'-. FACT TOX-013 LRN-U2095 Battelle Study N u m b er N 003604-D 3M Environm ental Laboratory Study N u m ber FACT 060998.1 M ETH O D FO R ANALYSTS O F PO TA SSIU M X R F L U O R O O C T A N S T IL F O N A T I (PFO S) IN R A T L IV E R B Y L O M S /M S V erd e 1-0 Stm trW od ' A a a ln t/D tte i tr.ii S dLU l.m l* i 2 3 4 3 < 7 Blank Tabla S. Caffbredan Stsndardi and X a k a Sun CC Tu nict V iil AUU.il V'-til T .iru c t \U iijl 1 L l ...L l i t u u l Vul tiii.il V1 ClilK H ill.) <itil i il" m! i WS 1 ____ _____ a I WS 2 50 IB3fNtys&Bi 1 0J 0J . C_ - - - - 1 2000' 1000 WS 3 WS 4 jo RntfllT********* 50 Ie h h e & o s 0.5 ffflfttfMKffffiffi 400 o j W o tim 200 WS 3 1 WS 6 I WS 7 *> so - 1 . msmaasm 30 ftfl q j U H B E r S 100 QJ5 p iM r ^ fr ir ii so 0.5 U U i U l j i 20 M eOH 1 30 0.5 0 m 5 ~ "8 Z ...... u 9M 0J3 0.13 0 D ale r f atroatadoa atcal gda/btadc J . P re p a ra tio n o f Q uality C o n tro l li v e r Sam ples (Q C l) 1. Quality Control W orking Standanl P flttte the lbllowmg cures volume m rtftannl to volumetric C id a m d m ix w ell. Ptepare fro h whennaed. Actual vohonea are In pnothcK a. Still! I l l QC WS 1 OCW S2 OCW S3 OCW S4 S im rce M ixed Stock A M ixed Stock B QCW S1 QC W S I T a b le d . Q C W S Preparado ,.JV *il S uurcc.m l. B S H R a H S ffltiiih tiB fit tin .il V u l. m l. .,,`-'WLj ',11 _ p m mriiimrnnii wunnmii feag^r^ "3^IEUglilrKgffi em anm 13.000 9000 1133 230 3 . - Prepararon o f Qoallty Controlliv e r Sample* Prepara each QC In bolle by fillin g the volumetric flaak approadmataty hal f fill! w ith undOvaed liver bcmogenaa STTRBOM QGENATB W HU JS A LIQ U O TIN G ), adding the appropriate QC W S, mhrtng. aadrtltnrtng to volume with undiluted Bver bamogemea (S TIR B D M D G EN A TS WH3LB AL1QUOTING). M IX TH O R O U G H LY etui iflapenea 1 3 -e d . alUpxXi b n polypropylene tabea and tore at approximately -IO *C T ab le 7. Q O n m n a la i QC Source Vtl Source. m L Final V o l. 111L Cone. 1 oc ws i S S i l l 1300 oc w si2 p38gS^igMM3BSTaWBCI5hw W W B B K I 300 3 OC W S3 4 QC WS 4 ife iisi 112J 23 10 3J 0.7 0 .M Data a f O C crept S tu d y ._____ K . P re p a ra tio n of M S CTierk S ta n d a rd fo r S ystem S u itab ility . Pipet 230 uL o fWS 2 at-10,000 ngfaL t a d l i o l ofWSS at-1000 ag/mL la metbanol into die aune 50-m L valnmcerlc flaalc. Dilute ta volume w ith M eCH and m ix. Page 9 o f 1$ 3M Environmental Laboratory C -9 E-13 m- Page'10 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 BatteUe Study Number. N003604-D 3M Environmental Laboratory StudyNumber FACT 060998.1 M ETH O D F O R A N A LY SIS O F PO TA SSIU M FXRFLTJOBOOCTANXS1ILFONATX (PFO S) IN R A T LTVZR B Y L C /M ^ M S V ertio U ) B tedr Ha.! Anabaa/Petet ~ L P rep aratio n o f ho m o feo ix er recovery fiver u x ap les To determine tbe recovery from the honmprrir a r to procci. entow in|TeiTidhlaefcUv<r la witrorimi lM te o rftm M m .il . TUm " *w ha Jana mmryifay thee lv..n/>fm lTrine n /*n rfy mwplM t. L Flaceapprtadnately 0.3 lafeah eaen B b sd b lB iilcB verh iao caeb afS , U a L polypropylene co d iiftige tnbca. Record-iwl|tn-i o f Over. X Add 100 pL o f W S l , 3 , aod 4 (oca W S p * <hp&ae tnfaes) is proper* thrtiflattiom at sppradnmiety 4 , 0.X a d 0.4 (t|fg . X M ultiply tbs xcaaa aflivex in j by X J andadd tb it meny m L o f water. 4. Homes e a l a t o liver n m ^ e , ad rfaje h cm o je n in r probe w ith aatshbr . o 'ln rM >y w rw n^l l n w f l , ih ia V T T 1* 5. Q u a boxoDgcsisrw ith M eCH berw cm iu& plaa. 4. Capandvcroaxhom osem ateta'otilnocoaalon. M . P re p a ra tio n o f D Q stion Check. S am ple X Place ZR3 mL afcndUnted Bvcrhopogaani jS I1 K BQMOGENA.TB ~WBILH AUQUOTTNG) turn a 13 mL axsacdaa tub* and add 30 |iL cfbOaad S lack A. X SQmc30 pL o f step 1 aatatbm (V O R TE X S O LU TIO N 'W H ILH ALIQUOTTNG) w ith 0.45 mL o fundiluted liver hemogeeata C H U t HO M OG ENATE W H IL E A U Q U O n N G ) la X 13 mL extractJoa tome. 3. T h li n m p b ibculd be prepared ih f c crracdoa only an dayi when Batty aaaplae oriS be dnmed and eauancd. N . H om ogenization o f stu d y sam ples L X1 3. 4. 5. Place approximately 0,3 g of aahatanst nh i d ttndy am p le liver to o a U m L polypropylene tuba. Record weights o f liver. M ultiply the m en o f liver In g b y X J xndtdd ttili many sL o fwater, Homogenize each liver am ple, endrinse homogeaizzrprobe w ithenodnr vqIxubm t f m tc ru fc d i& m p 2 a td d h |iiD M (d bomofasised s s f l i dean bem ogaiizsr w ith M eO H between m nplea. Cap ezzlvortex hotnogesam ib r see la extcacdoo. O . A n a ly s is S ta n d a rd s , B la n k s , Q C s , a n d S am p les 1. M IX L IV E R HOM OG ENATES TH O R O UG HLYBEFO KB AUQUCTHNO and pipet 300 ><L o f each QC (4 RpUcaaaa per level), aad other am p le * M a g extracted into 13-caL polypropylene ex to rtio n tube* T h e cal atdi sod blank are already aliqooted. X T a IheBlanks-1 5 (3 reps), add 100 jX c fM e O H s a d v e rte x 3. To tie Blank! + IS (3 rope) end to the renalntng om plat. add 100 *iL ViSS sad vortex 4. Add Od m L a fO J M T B A (pH 10) >a all tabes m d vertex bdtoy. 3. A d d Im LafO jaM catbonuebcG B bxm dvortoxbdcQ r. 6 . Add U m L of ethyl acetate. Place the tnbea sideinqn an tbaorfaitaidaeltBrst a te ttin j o f300 fc r-2 0 niman, 7 . Ceamfage tu b a at a eettfag c f 3300 rpm tor~20 rrhrotee to eepentolayen. X T n n d e rlm L afth e top orpude layer m a dean polypropylene tdbe. F a te 10 o f IS 3M Environmental Laboratory C-10 E-14 J f i if Page 110 3M Medical Department Study. T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 Battelle Study Number: N003604-D 3M Environmental Laboratory Study Number. FACT 060998.1 M ETH O D Y O U A N A LY SIS O F PO TA SSIU M PERFLU O RO O CTA N ESTJLFO N A TX (PFO S) IN H A T L IV E R BY L O M S /M S V endra L0 SaadyNaj A nilrat/Tlalr 9. Evaporata lo tbynes ander nitrogen at a attling o f30*C tb r~ 6 0 adm an, JO. Benw trirma tba leaMuea In 300yLo f n e ttano! wbbvocimdag. 11. Syria ge^ U te1am e t trun m tr m p laf t f a li fc r m M i Stnr'riU (up to 1 am arti) If LC M SrW S w in act be pcrihrroart tba su m day. S b a M f roos k s ^ c r s D n ttn c tilib Q i^ iM d tn o iM M id flu exorna o f tbs a l a fe , blanks, and Q C t nay be naacd lotap 3 daga r f t e tfaeir initial pregando t f held atnnatranp eam re (wop Ita v ia li g ra n a ta ^ Darned cal atlA laalt enactpreac D x n r fO C n rtrartoren: P . L C /M S/M S A nalysis yCU ic th a ijrseaa condition pocifled fa Tafcla Tba mnrtkiooa vrbkb a n dcalgnated pay be modified by the amdyata protteaafnapeahla peak kbipc LCVM V M S >>'lem T ab la -L O M S /M 3 CoadHI--a l l l 'I.C M .i'S S|iolIhimiiUT y iW M a te _____ M o d el: Mbda|: ID ; ID : M a t e _____ M odel: ID : K a y * B a tte i C lt,3 > J, 1 % 30 m a i PartN o. 033-701-0, S/N: : LoC M o llile 1` U.inv: Component A : Ammncinm accm agncflim nl. <0:40, vat Comooneot B : Am m onirai atm iB -m gbraoi. 3:93. Try Ci.ittk'iir jirntili* Thna. m ia S iif i TH w iW lU n rfa 0 0 03 1 0 03 4 3 100 0 3 6 100 0 3 6.1 100 0.6 U 10O 0.6 . 0 0J 11 0 0 3 ltiL \(iiiii animile 10 uL (___ HL)_________________ i l'HV 1 Tr . mhirrm flow snUf to 30 uLAda f id A n in l fag> tbs M S at ran ftatt 1 iliiruu Kini> lll'I.C liiL '-iin : M > ViUtLC H.tS 1 Ambient 1 1000 H i * raflent art ( 1 B orn m o t -Netatvo Ion 1 Nitroten at 573 Uba { pai) U h i) * N cliu li/cv H.lN 1 Nitroreo at 10 L/hr ( 'io H itc lit n i. Id illi 1 4 0 *C ( C l L/hrt U c'iiU .H M it funi 230*C o *70 V (___ vip p o a. - *20 V f V lP FO S A .FF O S A A .T TO S E A . M -336.M J70 1 n lh 'in ii 4 0 aV (__ ____ eV ) PTOS. S LF FO S A , PFOSAA, M -336, M 370 30 V f eV>FFOSEAv l ollisHin J.i'a Arven i t x 10* mb t u ceQ < mbl Multiplier *630 V f V) KcsilllllitMl *1X 0 ib rM S I f >.10.0 tor M S 2 f 1 1 1 i Os^outA Atn, * * icsrjbrlvt Pigs a an C -ll 3M Environmental Laboratory E-15 0 3 *7 Page 111 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 Battelle Study Number. N003604-D 3M Environmental Laboratory Study Number FACT 060998.1 METHOD FOR ANALYSIS OF POTASSIUM FXRFXUOROOCTANZSULFONATX (PFOS) IN RAT LIVER BY LC/M SM S Ventea L0 Study y u A aah nt/D itc; 4 Z 7 > tl M R M transition t e Surrogate Standard (SS) I'm-* tinm il.itctl 49g>99 M R M tnmabina ftg PROS 534>71M R M tesnsidoalbrM JS 6 1 5 7 0 1 4 9 M RM tansbonferM STO 5S4>1$9 M R M transition ftrF P O S A A * 4 9 0 7 1 M R M transitVta ft PFOSA 5 3 0 1 4 9 M R M transition ftir PFOSBA |..t it i 'in 'm i, \|ij'i tic *11 mfanan ( SS: 4 reia ( dal m ini sli ilium Imu': - PROS: 4J m in i M -33& 4 3 edu ( M 3 70:4 . m in t m ini aM total PFOSAA: 4.7 m in (______nrin) . FFOSA: 3.1 m b (______ m b ) - IFO S E A : 3.7 m b f m ini *?izim eositlatznaybechsafB dbytiusare!yat. Actaal vahes l a ( ). 2. T ie shove coaditcxa shoold be ruitabk ib r the M kroaass Q eanm LC (JVN 9033). ModMlrarlrm* nay ha necessary i f anotherVOcromare Qoaaiu Sariaa speorpmerer I* tried. Split tha flow pnstH-nfrtrrm via t Keystone B IO tea ar sim ilar device. 3 . Calibrate fee masa spectrometer Bring a referible refacace componed. a rv e rift fla t tfaa caKbration Is n im b i* by v ia l hopeadas (cmthe tasa page) fla t a nfyMs wyiKfla p)stn kM il lirtf l b higher tbaa that traed br analyzing --."pt-- OHQTM ( l tB 4. flf tb4 h * 111* 1* ffWT ftTTT^yf standard. Use remits abenld ba companbla to a recant injection If evnBribin. 3. Usa no sammmad dan iiLaingfsphy totngiadan software syetasn as coDact fen output from tha analysts. 6. Loading O rder Sea the loadla* report than (ba tntnmalad chromatography Integration software system. 7. MUce b^ccsLooi o f o d i t t l M u da d , QC itody -- qtU i s I l V t t i g. Ran sa sfaga should typically apt amend SO Injections doe to lustrum p^P^nfqi t Bf y Vi* p ^ ^ ^ i . V t ^ k t y r t t ft risk o fyielding tmncceptnble c a m nanks. VHL CALCULATIONS L Spreadsheet Software: ____________________V e rd n ________ 1 M S Anahrdj Software: _ Verdna 3. Calculate lbs avenga denary of the Uvas homogenise (10 reps) la sig b iL . 4. Using the avenge density g f ft homognea, calrnhre In liver density (mg o f liver per mL o f dthrted hnrnngrnala): UndUnted 2ver density (m g/m L) - (g ofliv e r z average dm lily otfhcmogtnatayCgaf liv e r+ g ofwamr) where g o Liver and g e f water are miares m ed to prepare bu lk h e b o fe a re ; density of water is assumed to be lg M L DtliSed Livar density (m g/taL) - UndSated density * D tta Factor ' Fags 12 of IS 3M Environmental Laboratory C-12 E-16 ^ 3 S-Sr . Page 112 ~~ 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 BatteUe Study Number NQ03604-D 3M Environmental Laboratory Study'Number FACT 060998.1 M E T H O D F O R A NA LY SIS O F FOTASSHXM PZR FLU O R O O C TA N X SU LFO M A TZ (PFO S) IN R A T UCVKR B Y LC /M S/M S Vocio U Stadvlfo^ Aaahat/Pat; Wbere dlla ficc ar-2 /L - LU11 lo ccauitftrlO%dBa attivarbonotaana la cal tdod QC ostrica. 5. dmetnletMeamurinw rfTPDWfut I. Wa. i|ii m inai nf nlftintlnfi nnrtnnli irai f|f* itrjM e liti im a n f inaljlaa in o lia fica n nly (no Svcr dcasfy comedo). Un pdqr comedo ftrPPOSAAoriy. 6. Calmiate hecaal oooraiirnitlori ofPFOS ad fa floonxtankal I Brerte tto caEbtatton andanti ad OCasfiUcwc Coac(p^'{) " Cane C&*AnL) VSated U va ieu d tj (fctffaL) x 1000asg'g x UT1pftag gl7 . A s a ri d a l ito b te g ra tia a ia ftto pede arcai ths to t M ic ia and azregRa M o to ri R e c o m a . 1 | m arn ai hneyraKnw t m p e r lh m n l f 1m h m B e t h m Ii. - r -- <| < Uva andari. '. *. Cabriate Ito regnalo aqoadoa Riattai ita peto reapeaattrio te * *RfclaS)afdb t erilbrettaa andari fr-*ds) tote arida coacaaireitaBl Bear Qwada)t e PPaHAMA M370, aadPFOSAA. Calcolata ito regresttaa quatta retadag diapale m d a d i alS ndoa andarito to t arida cnaceamieB taB verte WC8A ndPPOSEA. 1TOS,Vf336. MITO, adPFOSAA are qiranfllafcri by la i Iharrotata aaatort s a Internai *t*~**"<,PFlC?s * mrA VFCS& A tre onanritated ritfaootrefeg a eg te tfaa rnm wte Immtrmi aadsd criftmttancorvo).Pia <redrtlcregimiti wdghted IH ,righeaactadod. tur a llia a ly ta . 9, n^ww?iiiil frf i^atlWttal*lll<!hnT MMl- atsple orini da ngrcslos parnaso and tto peak lapooaa odo* or ama. 10. Calcolate tbo relativa cnor. svenga reladve orar, andari devhttae, odtatti an d a ri deriatianlbr all QCa. CainiInni tto reladve to rte eh njcaioo afcalibriti(m to ri. IL Cabalate tto venga recovery for da hoaiogBifaarrecoverytetlflcalltm. IX Clrw1l*"derive .tjrvl.rrf rfjn/tlrtnn h r th W n t In SS prmfrm rrtywVrtw.mylU-*a byecdota of Ibe check andari. I IX . ACCSeTANCX CStiTXlUA A . M S C h eck S ta n d a rd (Syftcx S tritabU ity) Ai lcaa 3 tqjcctlcna oftto MS Ctoch Standard moatprovida a KRSD of 10%orkw t e Iha PFOS k>S3 peak arcando. B . C alli)radon S tan d ard Tba perecat relative enon ftrr tbe concenaarion4cvel aveags ritto eallbrrtae andarti tfatmld o c a ibe following limita: 3M Environmental Laboratory P *H D o t 16 C-13 E-17 -tpr 3 * ? Page 113 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 BatteUe StudyNumber. N003604-D 3M Environmental Laboratory StudyNumber FACT 060998.1 METHOD FOR ANALYSIS O F POTASSIUM PERFLUOROOCTANESULFONATI (FFOS) IN RAT LIV ER BY LC/M3/MS V enloaL l to ifltiL i A n a h rat/D atg T tb lft 9. CaHbacica AN. UA li-. pros iM X M370 ' FTO6AA PPOSA PTOSEA S o d ti ".I,,.. i 20 05 % atLO O l 1 20 03% at LO O) 1 20 03% atLOO) 1 20 03% atLO O ) 20 0 3 % at LOCH ___ ______________ mxfUp is 3 o U tn d o a sm daid injection* b * exdnded flu e dhecarve,provided Unit ooe offe^ctJoeram ainiperleveL Removal an enrfro level m aybe dona I f approval hi obtained, f f n cadis lsvd IRBaored. dM am plcs bndeetad'bjr dw r a c ia iD f a llfa a tia a iBa i|B b com ldcied acceptable. The (nirbrarinncE rw ahnoldhnvai coefficient of dcterm inniioeaf -0 J 7 or better. ! C. QO The cooceatiatioa4evct average p n a t relative a ra n and percent idaitve standard dovisdenaaf the'QCi Aooid n a a d a tbUowina Hanha: T ith lftO C A m p m n lln jl ANALVTE pros 20 M -jje M370 20 20 PFOSAA 20 PPOSA 20 PTOSEA 23 I Removal of indMchnl value from the Q C clcniationati*y be don a if accompaniedt j a neonabla rrplanarton ( e j , bucnnDea TnaWmctinn or D ixon'I Q feat raaitteX i f the area^o determi ned noncratratlon for any Q C lc l exceedsthe aerrytincS B a it. fra task lo u ts or study d n a o r Sbodd to notified. Th era saaybt re p e a te d * po tio n c fthe l a n r be coaidcaed acceptable. fo r sxampie, i f the lo w Q C & O i the anted laquir -- ante, am ple may be accepted flu*, tavo oaocenmuioB hrariirtrd hy a blghrat raHhradcn nandard aad a nrid-lovei QC coocataniion. D . H om o g eab cr R ecovery a a d D ilatio n C heck S am ples The n e a fs w o w ij acre** the 3 level* o f fcamoicnbnr recovery aasplee a* w all as th e a f the dQndan check aanpleadiaald& ll w ithin the ta afae f? (j-U O % iD cfc ^ *ai Removai o f fcafrvUbaal outliers from thacalcnlalianf may bo done i f accompanied by a reaaonabie . Bplanetlon, X . S ensitivity (LO Q s) The validated Bnriu o f quantitation are nom inilly 0.13 B dPFO SA A each fcr IF O S , 1 * 3 3 6 ,145TH Page 14 o f 1$ 3M Environmental Laboratory C -1 4 E-18 je f 3?< 3 Page 114 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 Battelle Study Number N003604-D 3M Environmental Laboratory StudyNumber. FACT 060998.1 MTTHOD FO R ANALYSIS O F POTASSIUM PERILUOROOCTAN1SIJLFONATX (PFOS) IN RAT UVK R BY LOM S/M S VanhaaUO StdyN < u_________________ A natm /D am Par ROSA andROSEA, Sia validatedLOQa am aoniBanjr 0.33 pcfgaadu D m to b e n aan ofibeprcpanclaa of tbecaBbcadoa stendardi. l0w crracaaiidanaarRO 3A .m d PTOSEA01 ba caxriadthrough thaeanaciion. Thaaalower areceotasnav a in a r il ba avvinatetiwith neb nm act. ai mar ha tadndadln the marminna If criteria. IT they ire Inchatnl. je ity samples which sm qoasdtstadlBltava ersacentatlaaB M aw tfcevaildstod lavai (nw riaafly 0.33 p<fg) vdUba m im prinaly flagged. F . S p e c ific ity 2SttefLOQ. Tbabacreapt oftbacalttaidoncor* apeara to oflfcraomaoonoctlaafcrmgr eflfcct on gaanrttariae. Aacaptaba pasjfanam ca (erroO fthe lowest mart sttndasd.them fer i. tn h . M i il i f a r i if lld e M miAi-mm lh a t tn w rfa rw l m d y ampi-- -- < T ~ <p tp y if r . G . G eneral Tba above acceptance criteria Indicata that this metbod !a capable o f prododng occadooal aeran cratslda * a normal acceptance a lla ria o fa validated method (13% nanaaQy). W ham iah caad. replican saaiyau lessen the Im paci o f th e renirinnal octUcn. X . BX3CLT3 See attached hard copy o f spreadsheet or see fila on network driva. X L C om m ents 3M Environmental Laboratory P ic a IS o f 16 C -1 5 E-19 & f/ Page 115 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 Battelle Study N um ber. N 003604-D 3M Environm ental Laboratory Study N um ber; FA CT 060998.1 M ETH O D F O R A N A LY SIS O F PO TA SSIU M PXRyLUO R O O C T A N IS tT L F O N A T I (T O S ) IN R A T U V Z R B Y L C /M S/M S V in li U Stm iy Kau_;________________ X U . CoNcxcaroNB XJSL SictuiUBXS AatpU Technical R rriew QC Review - Du Sete D ate 3M Environmental Laboratory IiliU tfM C-16 E -2 0 JUT 3 ? i - Page 116 3M Medical Department Study: T6316.5 Analytical .Report: FACT -TOX-013 LRN-U2095 Battelle StudyNumber. N003604-D 3M Environmental Laboratory StudyNumber. FACT 060998.1 Study Tide Combined Oral (Gavage) Fertility Development andPerinatal/Postnatal Reproduction Toxicity Study o fN-EtFOSE in Rata 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 LERNU2095 f 3 U E nvironm ental Labo ratory 3M Environmental Laboratory E-21 li> Page 117 3M Medical Department Study: T6316.5 ' Analytical Report: FACT TOX-013 LRN-U2095 Battelle Study Number N003604-D 3M Environmental Laboratory StudyNumber FACT 060998.1 Protocol FA CT-TO X -013 Amendment 1 This amendment modifies the following portlon(s) of the protocol: 1. PROTOCOLBEADS: The proposed stody completion date is Batedas 12/31/98. AMENDTOREAD: The proposed study completion data,is 6/30/00. REASON:Theproposed completion date was changed to allow time foranalyzing all matrices of interest Amendment Approval MarvinCase PhDn SponsorRepresentative J o HD ai# l $ f . KrisJ. L PhD.., StudyDirector Date 3 M E nvironm ental Laboratory 3M Environmental Laboratory >22 Page 118 3M Medical Department Study: T6316.5 Analytical Report:. PACT TOX-013 .LRN-U2095 Batidle Study Number. N003604-D 3M Environmental Laboratory StudyNumber: FACT 060998.1 ' Study Title C om bined O ral (G arage) F ertility D evelopm ent and Fcrinatal/P oatnatal R eproduction T o x icity Study o f N -E tFO SE in R ats PROTOCOL AMENDMENT NO. 2 Amendment Date: Septem ber 1 0 ,1 9 9 9 Performing Laboratory 3M E nvironm ental T echnology & S afety Services 3M E nvironm ental Laboratory 935 Bush Avenue S t. P aul, M N 55106 Laboratory Project identification ET& SS FA CT-TO X -013 U R N U 2095 x 3 M E nvironm ental Labo ratory 3M Environmental Laboratory E -2 3 Page 119 3M Medical Department Study: T6316.5 'Analytical Report': FACT TOX-013 LRN-U2095 BiitteUe StudyNumber. NQ03604-D 3M Environmental Laboratory Study Number: FACT 060998.1 Protocol FA CT-TQ X -013 Amendment 2 This amendment modifies the following portlon(s) of the protocol: 1. PROTOCOLreads: The protocol states that liverwill 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. R E A S O N : The liver specimens will be sent to Battelle Memorial Institute for extraction and analysis due to time constraints in the 3M Environmental Laboratory. 2. PROTOCOLreads: The protocol states that serum specimens will be extracted and analyzed .following methods: FACT-M-3.0, "Extraction of Potassium Perfluorooctanesulfonate or OtherAnionic Surfactants from Serum for Analysis Using HPLC-Electroapray/Mass Spectrometry" FACT-M-4.0, "Analysis o fFluorochemicals in Serum Extracts Using HPLCElectrospray/Mass Spectrometry" AMENDTOread: The serum specimens will be extracted and analyzed following methods: ETS-8-4.1t "Extraction o f Potassium Perfluorooctanesulfonate or OtherFluorochcmical Compounds from Serum for Analysa Using HPLC-ElectrosprayMass Spectrometry" ETS-8-5.1, "Analysis o f Potassium Perfluorooctanesulfonate or OtherFluorochemical Compounds in Serum Extracts HPLC-ElectrosprayMass Spectrometry" R E A S O N : The extraction and analytical methods FACT-M-3.0 andFACT-M-4.0, respectively, were updated on 047/99 to ETS-8-4.1 and ETS-8-5.1. 3M E nvironm ental L aboratory 3M Environmental Laboratory E-24 V rt 3 9 6 Page 120 3M Medical Department Study: T6316.5 "_ Analytical Report: FACT TOX-013 LRN-U2095 Battelle StudyNumber N003604-D 3M Environmental Laboratory Study Number FACT 060998.1 Protocol FA CT-TO X-013 Amendment 2 3. PROTOCOLreads: The protocol states that liver specimens will be extracted and analyzed following methods: FACT-M-1.0, "Extraction ofPotassium Perfluorooctaaesulfonate or OtherAnionic surfactants fromLiver for analysis Using HPLC-Electrospray/Mas Spectrometry" FACT-M-2.0, "Analysis ofFriuorochemicals in Liver ExtractsUsing HPLCElectrospray/Mass Spectrometry** AMEND to r e a d : The liver specimens will be extracted andanalyzed following method: Method for Analysis o fPerfluorooctane Sulfonate (PFOS) in Ratliverby LC/MS/MS, Vernon 1.0 Re a s o n : Since die Ever extraction and analysis was sub-contracted to Battdle Memorial Institute, this amendment was written to include theirlivermethods andtitles. - Amendment Approval ;M[arvin2 k Case Phi).', Sponsor Representative (ftp Kristen J. Hansen PhJD., Study Director \ 3 M E nvironm ental Lebo ratory 3M Environmental Laboratory E-2S Date Page 121 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 -' LRN-U2 09.5 Battette StudyNumber N003604-D 3M Environmental Laboratory Stu<fyNumber FACT 060998.1 Stu dy Title A nalytical Study 2(N -E thylperfluorooctanesulfbnam ido)-ethanol in Tw o G eneration B a t R eproduction PROTOCOL AMENDMENT NO. 3 Am endm ent Date; O ctober 4 ,1 9 9 9 P erform ing Laboratory 3M Environm ental T echnology A Safety Services 3M E nvironm ental Laboratory 933 B ush Avenue S t P aul, M N 55106 Laboratory P ro je ct Id e n tifica tio n ET& SS FA CT-TO X -013 U X N U 2095 3MEmbonmantalLaboratory 3M Environmental Laboratory E-26 Page 122 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 B atidle Study N um ber N 003604-D 3M Environm ental Laboratory Study N um ber. FA CT 060998.1 Protocol FACT Tox-013 Am endm ent N um ber3 This amendment modifies the following portlon(s) of the protocol: 1. Protocol Reads: Kristen J. Hansen, PhJD. is the Study Director. Amend toRead: James ELLundberg, PhD. is die 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: i 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 13. states that supporting records to be retained separately from foe study 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. Amend to Read: Section 7 states: "The original data, or copies thereof, will be available at foe 3M t Environmental Laboratory to facilitate audits of the study during its progress andbefore acceptance of the final report 'When the final report is completed, all original paper data,' including: approved protocol and amendments, study correspondence, shipping records, raw data, approved fin a l report, and electronic copies of datawill he retained in foe archives of the 3M Environmental Laboratory. All corresponding training records, calibration records, instrument maintenance logs, standard operating procedures, equipmentprocedures, and methods will be retained in the archives of the facility performing each analysis. Reason: To direct subcontractlaboratories in foe disposition o f the items listed above. 3MEnvironmental Laboratory 3M Environmental Laboratory E-27 Page 123 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 " LRN-U2095 Battelle Study Number N003604-D 3M Environmental Laboratory StudyNumber FACT 060998.1 Protocol FACT Tax-013 Am endm ent N um ber 3 3. Protocol heads: Disposition of test andcontrol substances: Biological tissues and fluid arc retainedper GLP regulation. A m e n d t o read; Specimens will be maintained in the 3M Environmentallaboratory specimen rrhfw specimens sent to sub-contract laboratories will be returnedto the 3MEnvirosmsatal Laboratory upon completion of analysis andsubmissionof the sub-contract laboritory(s) final report. The specimens will be returnedwiththe following documentation- fheaigned original chain ofeustody andrecords of storage conditions while atthe sub-contract Utility. - Reason: To define in detail t1ieppropriate disposition ofspecimetrfanalysed mt lhmntmo laboratories. Amendment Approval T C tg jL Marv Case, D.V.M., PhJX, Sponsor Representative H doii& dS h L fL Date 41a ------KristenJ. Hansen, PhJD., Previous StudyDirector Date </ / p -- _____________ __________ 1 * 1 i s f n l Dale L. Bacon, PfcST 3M Environmental LaboratoryManagement Date 3M Environmental Laboratory 3M Environmental Laboratory E-28 W r HOO Page 124 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 Battelle Study Number N003604-D 3M Environmental Laboratory Study Number. FACT 060998.1 D EV IA TIO N REPO RT BatteJle Study Number: N003604-D 3M Environmental Technology and Services Study Number. FACT 060998.1 2 (N-Ethy]perfhiorooctanesulfonamido)-ethanol in Two Generation Rat Reproduction TYPE OF DEVIATIONS; PROTOCOL DATES OP DEVIATIONS: October 18,1999 NATURE OP DEVIATIONS: Some o f the analytical method acceptance criteria were not met to t the LC/MS/MS analytic conducted 18Qct99 at BatteHe. Theee deviationa relate toprotocol ameodineetZ. See below for tummary. AiulMi* A .w p M n .L * a n .1*1h 'u i:*t t , i FFOS M-556 M-556 M-556 M-556 PFOSAA FFOSAA QC3 exceeded 2 0 * error (-20.3 * actnal) QC2 exceeded 2 0 * error (23.4* actual! OC3 exceeded 2 0 * RSD (21.2* actual) OC4 exceeded 2 0 RSD 0 8 .8 actual) Dilution recovery exceeded 130* (131.5% actnal w ith 2 1 .6 * RSD) OC3 exceeded 2 0 error (-20.6* actnal) OC4 exceeded 2 0 * RSD (21.3* actual) CAUSE OF DEVIATIONS: Sample preparation and/or LC/MS/MS variabilities over the eoene of the am ple set may have contributed to die deviationa. . IMPACT OF DEVIATIONS ON THE STUDY: The errant QC value* were bracketed by acceptable QC concentration levels which demonstrates that the calibration curve* generally provided good accuracy over the tested range. The dilution recovery for M-3S6 was not considered to be exceedingly Ugh enough, at only approximately 1.3 above the normal acceptance level, to have significantly impacted the data. CORRECTIVE ACTION:Thli protocol deviation summary was prepared for Inclusion in the final report. APPROVED BY: Jan C. Andre, Ph-D. Battelle Principal Investigator Date Kristen I. Hansen, Ph.D . Study Director N00364-D Protocol Deviation 1, Page 1 o f 1 Date 3M Environmental Laboratory E -2 9 X& H0 \ Page 125 3M Medical Department- Study: T6316.5 Analytical Report: PACT TOX-013 LRN-U2095 Batce StudyNumber. N003604-D 3M Environmental Laboratory StudyNumber FACT 060998.1 DEVIATION REPORJ Battcllf. Study Number N003604-D 3M Environmental Technology and Services Study Number FACT 060998.1 2 (N-EthyIperfluorooctanesulfonamido)-ethanol in Two Generation Fat Reproduction TY PE O F DEVIATIONS: PROTOCOL DATE OP DEVIATIONS: October 20. 1999 NATURE OF DEVIATIONS: PFOS QC3 exceeded (he 2 9 * RE method requirement (actnal -2 1 .7 } . The dilution recovery check rtandaxd did not meet the 70*130 recovery requirement (actual* 3 .0 for PFOS and 4.6% for PFOSA). These method deviations relate to amendment 2 o f foe study protocol. CAUSE OF DEVIATIONS: Sample preparation and/or LC/MS/MS variabilities over the cornea o f the temple set may have contributed to the QC deviation. Sample preparation error appear* to have been foe cause for the dilution recovery resulta. IMPACT OF DEVIATIONS ON THE STUDY: D ie errant QC value level waa bracketed fay acceptable QC concentration levels which demonstrates that the calibration curve* generally provided good for study sample* over the tested range. A comparison of the results obtained for the diluted study samples from 20Oct99 and previous resulti that were slightly ALOQ (130ct99 and 180ct99) demonstrated good agreement between the 2 determination. This would indicate that the dilution of foe study samples was performed correctly 20Oct99 so foal no impact on the quantitations occurred. CORRECTIVE ACTION:Thia protocol deviation aummary was prepared for inclusion In the final report. APPROVED BY: Jon C. Andre, Ph.D. Battalia Principal Investigator Date Kristen I. Hansen, Fh.D . Study Director D ate V. ` N003604-D Protocol Deviation 2, Page 1 o f 1 3M Environmental Laboratory E-30 Page 126 3M Medical Department Study. T6316.S Analytical Report: FACT TOX-013 - LRN-U2 095 Battelle StudyNumber. N003604-D 3M Environmental Laboratory Study Number FACT 060998.1 DELATION REPORT Battelle Study Number N003604-D 3M Environmental Technology and Services Study Number FACT 060998.1 2 (N-Etbylpcrfluorooctanesulfonamido)-ethaxiol in Two Generation Bat Reproduction TYPE OF DEVIATIONS: PROTOCOL DATES OF DEVIATIONS: October 12, 1999 through October 20,1999 NATURE OF DEVIATIONS: The lot number of FFOS 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 FFOS 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 die 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 andHarlansupplied liver were both used for Battello'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: Jon C. Andre, PhD . Battelle Principal Investigator Date Kristen I. Hansen, PhD . Study Director N003604-D Protocol Deviation 3, Page 1 o f 1 3M Environmental Laboratory E-31 S Dale HO 3 Page 127 3MMedical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 Battdle StudyNumber. N003604-D 3M EnvironmentalLaboratory StudyNumber. FACT 060998.1 APPENDIX F - PFOS PURITY REPORT 3M Environmental Laboratory H o Ll Page 128 3M Medical Department Study: T6316.5 ETSS 2 3U Analytical Report: FACT TOX-013 LRN-U2095 - Battellc Study N u m ber N 003604-D 3M Environm ental Laboratory Study N u m b er FA CT 060998.1 ^ 651 773 4226 04./26/99 09:56 f? :02/Q3 NO; X From: Sublet: D ate; 3M SPECIALTY ADHESIVES * CHEMICALS ANALYTICAL LABORATORY L iu Oeroen - (8-3368) - ET&SS- 2-03-09 Repast #5713* Tom Xesmer-(3-3633)-SAAC Analytical Lab-236-2B-II OumlcaJ Qunctarization afTOST-Baird Fluondumicsh by 'S-NMR A aF-ffMS Sprctrotcoyy M arch 24,1999: Prelim inary report forFC-93 (PFOS), lot 171 SAMPLEDEStixlh 1LION FC-95, la 171 (PFCS), TN-A-0834;Nominal productC.FirSQj(-) K(+) (whimpowder) T his sam ple was su b jecte d IH-NM R and t*F-NMR spectral analyses co dcan ain e the purity o f the nom inal product and tn characterize sa many im purity components as possible. A portion o f the sample was accurately weighed, spiked with a known amount of l,4-bis(triflaoreinethyI)be& zeae (p-H FX ), and then totally dissolved in DMSO-d, for subsequent snalyais by NMR. A 400 M H z 'H-NM R spectrum (# h57830.401) sod a 376 M Hz '*F*NMR spectrum (# 07830.401) were acquired using aV arian UNITYplus SCO FT-NMR spec trom eter. Use of the p-HFX Internal standard was intended to perm it the determ ination of the absolute weight percent concentrations of the assigned components w ithout necessarily needing to identify or quantify all the components in the sample mixture. RESULTS; The combined NMR spectral data were used to assign all o f the major and most o f die minor com ponents in this sam ple as received. The qualitative and quantitative compositional results that went derived from the Single trial NMR internal standardization analyses are summarized in TABLE-1 on the following page. I 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 1assumed all of the components contained 8 carbons. If there w a s any shorter chain hom olop present (i.e^ 7 ,6 ,3 , etc. carbons), then the average compound m olecular w eight! would have been somewhat 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 clog im purity com ponents unless the chains are very short. A m ore compLets characterization of any other fiuorochandcal h o tao lo p would require analysis by electrospray M S or a sim ilar technique. A dditional w ork would be required in an effort to positively verify the tentatively assigned components Bated ba TABLE-1 (denoted by possible). Sm all amounts o f ether unidentified impurities a n also detected is the'N M R spectra, but additional work would be required in an effort to identify or quantify these other marrials. Copies o f the NMR spectra w ill be provided for you at a later date. If you have any questions about the results in this initial report for FC-93, lot 171. please let mo know . I apologize for the delay in completing this in itial work. T om X estner Wek Payta - 3A*C Analytical Lsb - J34-JB-11 1Uala ta a o : LCSUJOOOOtt 3M Environmental Laboratory fact t F -l m ho* Page 129 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095. Battelle Study Number N003604-D 3M Environmental Laboratory StudyNumber FACT 060998.1 ETSS 2 3W g 651 77S 4226 04/26/99 09:56 0 :Q3/03 N0:34t March 24,1999 &AACAnalytical LabRequest#57830 - Initial Renert far lot 1Tt TABLE-1 Semple: FC-95. lot 171 P K Si, N-A-C834 O verall Quantitarive Compositional Results by 'W a F-NMR Internal S tan d u d latio o Analyses Structural Aerignmenta CFrfCFjVSO*) K<+) {Normal chain;Menus x7 tar calculados purposes) NMRAhBOl Wright* fllntie BUI waaaarmiMnt^ 103% NM RliittB Weights (rifltie ttiel nuniMM| 693% CF^CFaJrCFCCFjHCF,)^^.) K(+) CtetenuUncnocntffayl branch entona x+yJ, m*<0,A y* 0,forcakuladnaporpora) 17.7* 175* . (CF^CF-CCFjX-SOC-IICO) (Isopropyl bnaeh; acamex *5 fer cilcularina porpora) CJ'^rCFCCFjJ^On-) XJL+) (Alpha branch; m b tm x 6 fc r cilcularton porpora*) Feasible F-SFrCJ'wSO^) K(+) (aune x 1for calculadospurposes) CFHCFjJ^CFjlrCFjlj-SOj-) K+) (Internal iwa-dlnvediyl breach: w ane r+y 4 tad x <*0 for caktrisrinn purposes) Possible CFrSFrCJFsrSOi K(+) (awuaaex 7 fbrcalculadospurpose) Probable C^Hto.l-SOJ(-} KM (Hydrocarbon rulfcomic n it; w u m r * t tor calculation purpoara) (CFjhC-CCFilrSO, K/+) (t-butyi branch; rame x 4 fer calculden purpoima) 103% 33 % 031% 0.16* an* 0.031* 0027* 102* 35* 056% 016% OIQ* 0530* 0526* 3M Environmental Laboratory F-2 Page 130 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 Appendix H: Interim Certificate of Analysis 3M Environmental Laboratory ^ 07 Page 131 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-TJ2095 INTERIM CERTIFICATE OF ANALYSIS Revision 1(9/7/00) Centre A nalytical Laboratories COA Reference #: 023-018B 3M Product: P F O S ,L o tl7 1 R eference #: SD-009 ______________________Purity: 86.4% Test Name Specifications Purity4 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 (1C) 1. Chloride 2. Fluoride 3. Bromide 4. Nitrate 5. Nitrite 6. Phosphate 7. Sulfate4 Organic Acids s (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./vrt.% 3. 1.355 wt/wt.% 4. 6.552 wt/wt.% 5. 0.003 wt.Avt.% 6. 0.004 wt/wt.% 7. <0.001 wt/wt.% 1.00 wt/wt.% 10.60 wt/wt.% None Detected 0.30 wt/wt.% None Detected Not Applicable4 1. <0.015 wt/wt.% 2. 0.27 Wt/wt.% 3. <0.040 wt/wt.% 4. <0.009 wt/wt.% 5. <0.006 wt/wt.% 6. <0.007 wt/wt.% 7. 8.82 wt/wt.% 1. <0.1 wt./wt.% 2. <0.1 wt/wt.% 3. <0.1 wt/wt.% 4. <0.25 wt/wt.% 1. 12.08 wt/wt.% 2. 0.794 wt/wt.% 3. 1.61 wt/wt.% 4. 10.1 wt/wt.% 5. 50.4 wt/wt.% COA023-018B 3M Environmental Laboratory m VST Page 1 o f3 Page 132 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 INTERIM CERTIFICATE OF ANALYSIS Centre A nalytical Laboratories COA Reference #: 023-018B Date o f Last Analysis: 08/31/00 Expiration Date: 08/31/01 Storage Conditions: Frozen <-10C Re-assessm ent Date: 08/31/01 'Purity = 100% - (sum o f metal impurities, 1.39% +LC/MS impurities, 10.60%+Inorganic Fluoride, 0.27%+NMR impurities, 1.00% +POAlA, 0.30%) Total impurity from all tests = 13.56% Purity = 100% - 13.56% = 86.4% 2Potassium is expected in this salt form and is therefore not considered an impurity. 3Purity by DSC is generally not applicable to materials o f low purity. N o endotherm was observed for this sample. 4Sulfur in the sample appears to be converted to SO4 and hence detected using the inorganic anion method conditions. The anion result agrees w ell with the sulfur determination in the elemental analysis, lending confidence to this interpretation. Based on the results, the SO4 is not considered an impurity. 5TFA HFBA NFPA PFPA Trifluoroacetic acid Heptafluorobutyric acid Nonofluoropentanoic acid Pentafluoropropanoic acid 6Theoretical value calculations based on the empirical formula, CgFnSCTfK* (M W =538) This work was conducted under EPA Good Laboratory Practice Standards (40 CFR 160). C O A 023-018B 3M Environmental Laboratory Page 2 o f 3 Page 133 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 INTERIM CERTIFICATE OF ANALYSIS Centre Analytical Laboratories COA Reference #: 023-018B LC/MS Purity Profile: Impurity C4 C5 C6 Cl Total wtVwt. % 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 C l 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 4 Hio Page 3of3 Page 134 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 Appendix I: Report Signature Page -C lA yl^ ry Marvin T. Case D.V.M., Ph.D,,S tudy Director Date <^cL^ Z. John L. Butenhoff, Ph.D., Sponsor Representative c Date u^ / i n 6 Mr'S*4*, FA l 3M Environmental Laboratory Page 135