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<gHQ - oy-06. 373 {oz>o g/l7g,a z ) _________________ ________________________________________________ &o A R Z O fc -a W Analytical Laboratory Report ON THE Determination of the Presence and Concentration of Perfluorooctanesulfonate (PFOS) (CAS Number: 2759-39-3) in the Serum of Sprague-Dawley Rats Exposed to Potassium Perfluorooctanesulfonate via Gavage Laboratory Report No. U2779 Requester Project No. 3M Tox 6295.13 Study Dates Study Initiation: 10 June 1999 Sam ple Analysis Initiation: 22 June 1999 Sam ple Analysis Completion: 28 June 1999 Study Completion: At signing 01172 Perfluorooctanesulfonate CAS Num ber-2759-39-3 3M Environmental Laboratory Report No. FACT Tox-108 Laboratory Request Number (LRNHJ2779 Table of Contents Study Personnel and Contributors.............................................................................................. 1 Statement of Compliance.......................................................................................................... 2 GLP Study................................................................................................................................3 Quality Assurance Statement.................................................................................................... 3 Introduction.............................................................................................................................. 4 Purpose................................................................................................................................4 Test System......................................................................................................................... 4 Sample Collection andAnalysis.............................................................................................. 5 Sample Receipt........................................................................................................................5 Materials and Methods..............................................................................................................6 Chemical Characterization..................................................................................................... 6 Method Summaries............................................................................................................... 7 Protocol Deviations................................................................................................................8 Data Summary, Analyses, and Results....................................................................................... 8 Summary of Quality ControlAnalyses Results......................................................................... 8 Summary of Sample Results..................................................................................................8 Statistical Methods.................................................................................................................... 9 Data Quality Objectives and Data Integrity..................................................................................9 Statement of Conclusion........................................................................................................... 9 References............................................................................................................................. 10 Attachments......................................................................................................................... 10 OCllTS Pagei PerfluorooctanesuKbnate CAS Num ber-2759-39-3 Stu d y P ersonnel and C ontributors Analytical Chemistry Laboratories: Servm Analyses Advanced BioAnalytical Services, Inc. 15 Catherwood Road Ithaca, NY 14850 Kristen J. Hansen, Ph.D., StudyDirector John R. Perkins, Ph.D., Assistant Scientific Director In-life Testing Laboratory Argus Research Laboratory, Inc. 905 Sheehy Drive, Building A Horsham, PA 19044 Raymond G. York, Ph.D. DABT, StudyDirector Sponsor 3M Toxicology Services - Medical Department 3M Center, Building 220-2E-02 St Paul, MN 55144-1000 Marvin T. Case, D.V.M., Ph.D., SponsorRepresentative 3M Environmental Laboratory Report No. FA C TTox-108 Laboratory Request Number (LRN)-U2779 001174 Page 1 of 10 Perfluorooctanesuifonate CAS Num ber-2759-39-3 3M Environmental Laboratory Report No. FACT Tox-108 Laboratory Request Number (LRN)-U2779 Statem ent of C ompliance Study Title: Analytical Laboratory Report on the Determ ination of the Presence and Concentration of Perfluorooctanesuifonate (P FO S ) in the Serum o f Sprague-Dawley Rats Exposed to Potassium Perfluorooctanesuifonate via G avage Study Identification N u m b er FACT Tox-108 This study w as conducted in com pliance with Food and Drug Administration Good Laboratory Practice (G LP ) Regulations for Noncfinical Laboratory Studies [Data R equirem ents): 21 C FR ( Part 58)], with the exceptions in the bulleted list below . In addition, the present study has been audited retrospectively by an independent quality assurance unit. Audit procedures and findings for audits perform ed at the 3M Environm ental Laboratory and at participating contract laboratories are housed with docum entation pertinent to this study in archives at the 3M laboratory and will b e retained for a period not to exceed ten years. T he analytical portion completed at the 3M Environm ental Lab w as perform ed in accordance with 3M Environm ental Technology and Safety Services Standard O perating Procedures. Exceptions to G LP compliance: Tw o separate study directors w ere assigned to the in-life phase and the analytical phase of this study T h e ABS final report does not have a Statem ent o f Com pliance T h e Q AU statem ent in the ABS final report indicates com pliance with EPA 40 C FR Part 792, rather than FD A 21 C FR Part 58 D ose confirm ation analyses w ere not conducted according to the G LP regulations; analytical method w as not fully validated Not all raw data w ere verified by the group leader or designee / Study Director _______________ U - P - t i - o o Date Sponsor Representative 001175 Page 2 of 10 Parfluorooctanesulfonate CAS Num ber-2759-39-3 3M Environmental Laboratory Report No. FACT Tox-108 Laboratory Request Number (LRN)-U2779 GLP Study Q uality A ssurance Statement Study Title: Analytical Laboratory Report on the Determ ination o f the Presence and Concentration of Perfluorooctanesulfbnate (P FO S ) in the Serum of Sprague-Dawley Rats Exposed to Potassium Perfluorooctanesuifonate via G avage Study Identification N um ber FACT Tox-108 This study has been inspected by the 3M Environm ental Laboratory Q uality Assurance Unit (Q A U ) as indicated in the following tab le. T h e findings w ere reported to the study director and m anagem ent In s p e c t io n D a t e s From 12-30-99 I To 01-06-00 Phase I Draft Final Report Da te R eported to I Management | StudyDirector 0 1 -1 1 -0 0 01-07-00 2 - I l-O O Date 001176 Perfluorooctanesulfonate CAS Num ber-2759-39-3 3M Environmental Laboratory Report No. FACTTox-108 Laboratory Request Number (LRN)-U2779 In t r o d u c t io n o I C3F17------- S --------O I o Perfluorooctanesulfonate (PFOS) CAS N um ber 2759-39-3 C hem ical Form ula: CgF-| 7S O 3- M olecular W eight: 4 9 9 Purpose T h e purpose o f the study is to determ ine the presence and concentration of PFO S (C g F i7S0 3 ` ) in rat serum sam ples collected during the study of F1 offspring o f FO generation fem ale rats exposed to potassium perfluorooctanesulfonate via g a v a g e .. D ata quality objectives for this analytical study (outlined in the 3M Environm ental Technology and Safety Services protocol for F A C T T o x -108 ) w ere met. Test System In the present study, two groups o f fem ale rats w ere used as the test system. Group I consisted o f control FO fem ale rats that w ere adm inistered only Tw een 80 (vehide). Group II FO fem ale rats w ere adm inistered 1.6 mg of potassium perfluorooctanesulfonate per kg of body w eight/day in 0.5% Tw een 80 (test artid e). T h e dose corresponds to a concentration o f 0.3 2 mg/mL. Table 1 outlines the FO generation fem ale rat population dem ographics for study T -6 2 9 5 .13. Group II FOfemale rats were treated with the test artid e 4 2 days prior to cohabitation, and through day 14 (pharm acokinetic subgroup) or day 21 postpartum (lactation and delivery). F 1 m ale and fem ale rat pups w ere not directly adm inistered the test artid e, but w ere exposed to the chem ical through m aternal gestation (in utero exposure) and during lactation. F1 pups w ere identified as being from Group I or II. Cross fostering w as random ized; half of the pups from each group w ere cross fostered with G roup I or II fem ales creating four F1 anim al subgroups (A-D ). T he identification and cross-fostering procedure is docum ented in the in-life study report (Final Report 41 8 -0 1 4 for study T -629 5.13 page 11-10). Various physical and biological param eters w ere monitored in test animals. Table 1. F0 Generation Rat Population Demographics for Study T-6295.13 P o p u l a t io n In itia l Q u a n tity A cclim ated Control (G roup 1) Exposed or Treated (G roup II) Pharm acokinetic Sam ple Subgroup T o tal 86 virgin fem ale rats 42 fem ale rats 36 fem ale rats 10 fem ale rats Selected for Study 78 virgin fem ale rats 42 m ated fm ale rats 36 m ated fem ale rats 10 m ated fem ales (8 control, 2 treated rats) S elected for C r o s s F o s t e r in g -- 25 m ated fem ale rats 25 m ated fem ale rats None Page 4 of 10 001177 Perfluorooctanesutfonate CAS Num ber-2759-39-3 3M Environmental Laboratory Report No. FACT Tox-108 Laboratory Request Number (LRN)-U2779 T he test system species and strain selected w as the Cri:CDBR VAF/Plus (Sprague-Daw ley) rat received from C harles River Laboratories, In c , perm anently identified using a Monel self-piercing ear tag. F0 generation rats w ere identified with e a r tags. F1 generation rats w ere identified as part o f either Group I or II litters, but w ere not individually tagged, and all param eters w ere evaluated in term s o f the litter (dam and litter numbers are in A ttachm ent E , T ab le 4). F0 virgin fem ale rats w ere at least 60 days o f age and weighed approxim ately 2 0 0 -2 2 5 g when received (W eight data are included in Argus Research Laboratories, Inc., final report for study T -6 2 9 5 .1 3 on file at the 3M archives). M ale rats of the sam e source and strain w ere used only as breeders and w ere not adm inistered the test article or considered part o f the test system. Sample Collection and Analysis FO and F1 anim als assigned to the control pool w ere culled on day 1 postpartum. On day 14 postpartum, sam ples w ere collected from the FO m ated fem ale rats and F1 pups assigned to the pharm acokinetic subgroup. On day 22 postpartum , sam ples w ere collected from the rem aining anim als in the test system. All collected sam ples w ere im m ediately frozen on dry ice and maintained frozen (-7 0 "C or below) until shipped to the 3M Environm ental Laboratories. In th e analytical study reported here, sera sam ples collected from the population of exposed anim als (generation FO) and their offspring (generation F1) w ere analyzed for the presence of PFO S. S era sam ples w ere extracted by a liquid-liquid extraction procedure using an ion pairing agent and ethyl acetate. T he extracts w ere quantitatively analyzed using turbo ion spray liquid chrom atography/m ass spectrom etry (L C /M S ) in selected ion monitoring (S IM ) mode, and PFO S levels w ere evaluated against extracted standards. Analytical details are included in this report; further details are available in the study binder m aintained by the 3M Fluorine A nalytical Chem istry Team (FA C T); the binder is located in the 3M archives. A nalyses assessing the presence and concentration o f PFO S in the sera of Sprague-Daw ley rats w ere conducted in com pliance with Good Laboratory Practice Regulations (21 C FR 58). Validated methods and standard operating procedures w ere followed during the preparation and analysis of the samples associated with this study. S am ple R eceipt Sam ples w ere received from Argus Research Laboratories sporadically, during and at the end o f the in-life phase of study, from Novem ber 1998, through February, 1999. Sam ples received w ere packaged in dry ice. Specim ens w ere registered with the 3M Environm ental Laboratory and transferred to a freezer for storage. Sam ples o f body tissues and fluids other than rat serum w ere collected and received from Argus Research Laboratories (Protocol 41 8-0 14, Study T -629 5.13 ), but w ere not part of the scope of analysis in the protocol determ ined by the study director. Specim ens sent to Advanced BioAnalytical Services, Inc., (ABS) for analysis w ere received and tracked according to the Sam ple Tracking System Standard Operating Procedure. Specim ens sent to ABS will be returned to the 3M Environm ental Laboratory upon completion o f analysis and submission of the subcontract laboratory(s) final report. Specim ens will be m aintained in the 3M Environm ental Laboratory specimen archives. Sam ple receipt, identification, storage, and chain o f custody protocols and data are located in the study binder for this report (FA C T T ox-108); th e binder is located in the 3M archives. Page 5 of 10 001178 Perfluorooctanesulfonate CAS Num ber-2759-39-3 3M Environmental Laboratory Report No. FACT Tox-108 Laboratory Request Number (L R N H I2779 Materials and M ethods Chemical Characterization T ab le 2 presents information regarding characterization o f the test, control, and reference substances used in the in-life phase o f this study. Table 2. Characterization and Treatment of the Test Substance, Control, and Reference Materials in Study FACT Tox-108 1 Test Substance Source Preparation Maximum Storage Time Storage Conditions Chemical Lot Number Physical Description and Identity Analyses Purity Stability Control Material Source Preparation Maximum Storage Time Storage Conditions Chemical Lot Number Physical Description and Identity Analyses Purity Stability A nalyses Performed a t AB S Potassium Perfluorooctanesulfonate 3M Specialty Chemicals Daily--Records maintained in the raw data Expiration May 2000 Room temperature 217 FC-95, White powder 99.28% 48-hr data of prepared formulations on file with the sponsor Deionized Water J.T. Baker, Phiilipsburg NJ Suspensions were prepared daily Unknown Room temperature Tween80--M03H05, L06662, AND M29477 0.5% Tween80 in Reverse Osmosis Deionized Water There are no known contaminants in the diet that would interfere with this study. 48-hr data of prepared formulations on file with the sponsor Page 6 of 10 001179 Perfluorooctanesulfonate CAS Num ber-2759-39-3 3M Environmental Laboratory Report No. FACT Tox-108 Laboratory Request Number (LRN)-U2779 Table 2. Characterization and Treatment of the Test Substance, Control, and Reference Materials in Study FACT Tox-108 (continued) Analytical Reference Material Source Preparation Maximum Storage Time Storage Conditions Chemical Lot Number Physical Description and Identity Analyses Purity Stability ABSA n a l y s e s P e r f o r m e d a t PFOS (Perfluorooctanesulfonate) 3M Specialty Chemicals Analytical procedure in the ABS final report (Attachment F) Unused reference material will be retained for use by the 3M Environmental Laboratory and will be discarded when the quality of preparation no longer affords evaluation Room temperature 171 Light-colored powder 99.49% Undetermined Dose analyses w ere perform ed on test substance sam ples taken at various tim es during the In-life phase o f the study: data are located in the study binder for this report (F A C T T o x -1 08 ) and are presented in Attachm ent D. R eserve sam ples o f control m aterials from the 3M Environm ental Laboratory analyses will be stored at the 3M Environm ental Laboratory for a period not to exceed 10 years following the effective date o f the final test rule, or until the quality o f the preparation no longer affords evaluation, as will any reserve sam ples o f test substance returned from the in-life phase o f the study. R eserve samples of control m aterial from ABS will be stored at the subcontract laboratories in such a m anner that the control m aterials will be preserved for use in ensuing studies. Method Summaries Following is a brief description o f the methods used during this analytical study by AB S. D etailed descriptions of these m ethods are presented in Attachm ent C. ABS Preparatory and Analytical Method: Method Validation for the Quantitation o f Perfluorooctanesulfonate (P F O S ) in R at Serum by Turbo Ion Spray LC/M S. Analytical standard stock solution, standard spiking solutions. Internal standard stock solutions, internal standard working solutions, control blanks, quality control stock solutions, quality control sam ples, and study sam ples are prepared in dilutions with appropriate volum es of methanol or purified w ater in preparation for extraction. Tetrabutyl am m onium hydrogen sulfate (1m L of 0.5 m olar solution) is added to all quality control and study sam ples, along with 2m L o f 0 .2 5 molar sodium carbonate/ 0.25 m olar sodium bicarbonate. W ater (500pL) is added to the control blank, and 500pL of the internal standard working solution is added to all other tubes. Five m L of ethyl acetate is then added to each tube, and the tubes are placed on a reciprocal shaker at m edium speed for 20 m inutes. T he tubes are then centrifuged, and the organic layer is transferred to a clean polypropylene tube. T h e organic layer is evaporated to dryness in a TurboVapTM at about 2 0 *C under nitrogen. T h e dried extracts are then reconstituted with acetonitrile and w ater, and 200pL o f the extract is transferred to an autosam pler tube for analysis. Page 7 of 10 001180 Perfluorooctanesulfonate CAS Num ber-2759-39-3 3M Environmental Laboratory Report No. FACTTox-1Q8 Laboratory Request Number (LRNHJ2779 Equipment used at ABS: LC/MS/MS System: A u to sam pler W aters 717plus M ass sp ectro m eter PE SC IE X API 365 with TurbolonSprayTM interface A nalytical colum n: Keystone BetasilTM Cis, 2.1 x50 mm (5pm ) M obile phase components: Com ponent A -- 10:90 methanol:2 mM am m onium acetate Com ponent B-- 90:10 m ethanol:2 mM am m onium acetate Injection volum e: 3-5pL Colum n tem perature: Am bient Curtain gas: U H P nitrogen N ebulizer gas: U H P nitrogen Ion spray voltage: -3 0 0 0 V D edustering potential: -35 V N egative ions monitored: m /z= 499 .0 for P FO S [M -K]m /z= 427.0 for tetra-H -PFO S [M -H ]Total run tim e: 8 .0 minutes Protocol Deviations T here w ere no deviations from the original protocol.* Data Summary, A nalyses, and Results Summary of Quality Control Analyses Results (ABS) T he inter-assay precision (relative standard d eviatio n-R S D ) data from daily quality control sam ples ranged from 1 .5 7 -4 .0 1 % for PFO S, and the inter-assay accuracy (relative erro r-R E ) ranged from -7 .3 2 -1 0 .1 % . Both are acceptable ranges. T h e inter-assay precision (R S D ) of standards ranged from 1 .1 1 -5 .8 3 % for PFO S. The inter-assay accuracy (R E ) from standards ranged from -5 .8 6 -1 1 .0 % . All are acceptable ranges. Coefficients o f determ ination w ere 0 .995 4. R efer to the ABS final report (Attachm ent F, T ab le 2) for precision and accuracy data on the analyses of quality control partial volum e (one-to-ten dilutions). Summary of Sample Results (ABS) Following is inform ation regarding sam ple analyses in the present study: T h e sam ples in analytical run num bers 1 and 3 w ere reassayed because the m easured concentrations exceeded the calibration range and upper lim it o f quantitation (ULQ ) (refer to T able 6 in attachm ent F). Sm aller volum es w ere reextracted from the sam ples and reanalyzed. Reassayed sam ples resulted in m easured concentrations within the calibration range, and the data w ere reported (refer to Table 6 in attachm ent F). O riginal data or copies thereof are available a t the 3M Environm ental Laboratory. Page 8 of 10 001181 Perfluorooctanesutfonate CAS Num ber-2759-39-3 3M Environmental Laboratory Report No. FA C TTox-108 Laboratory Request Number (LRN)-U2779 Statistical Methods A t A B S, d ata from peak areas w ere integrated by the PE S C IE X program M acQ uanTM , version 1.4, on a M acintosh com puter. Following peak area integration, M acQ uan results data w ere converted to text files, m oved to the AB S file server, and analyzed with the W atsonTM software package (version 5.3.1.01 from P S S , Inc., W ayne, PA). T h e W atson analysis applied a w eighted (1 ty2) quadratic regression to the data, and calculations w ere perform ed on unrounded num bers; however, standard and quality control data w ere rounded to three significant figures prior to reporting the data. Data Q uality O bjectives and Data Integrity No circum stances existed during the present study that would have affected the quality or integrity of the data. T he following d ata quality objectives (DQ O s) w ere followed during the study: Linearity-- T he coefficient of determ ination (r2) of the standard curve was equal to or greater than 0 .9 8 Limits o f Quantitation (LOQ )-- Equal to the lowest acceptable standard in the calibration curve D uplicate acceptable precision-- <30% Spike acceptable recoveries-- 70% to 130% U se o f confirm atory methods-- no confirmatory methods w ere used Dem onstration o f specificity-- specificity w as dem onstrated by chromatographic retention tim e, m ass spectral [M -K] product ion characterization Assum ing th at spike recovery studies form a suitable indication o f endogenous analyte recovery, data are quantitative to 30% . The validity of this assumption has not been verified by other techniques. In serum analyses, th e limit o f detection (LO D ) for PFO S is approxim ately 1.75ng/m L and the limit of quantitation (LO Q ) is 0.05pg/m L P FO S . Sta tem ent of C onclusion Statem ent of Conclusion: Under the conditions of the present studies, PFO S w as observed in the sera of F0 fem ale rats exposed during the in-life phase o f the study. Additionally, PFO S w as observed in sera tissues taken from F1 generation pups from fem ale rats exposed to the test substance, and in F1 generation pups exposed to the test substance via lactation, but not exposed in utero. Page 9 of 10 001182 Perfluorooctanesulfonate CAS Num ber-2759-39-3 R eferences None. 3M Environmental Laboratory Report No. FA C TTox-108 Laboratory Request Number (LRNHJ2779 A ttachments Attachm ent A: Sam ple R eceipt and Chain o f Custody Docum entation Attachm ent B: Protocol Attachm ent C: Extraction and Analytical Methods Attachm ent D: Dose Confirm ation Analyses Attachm ent E: D ata Sum m ary and Tables Attachm ent F: Analytical Reports from Contributing Laboratories Attachm ent G: Report Signature Page Page 10 o f 10 001183 Perfluorooctanesulfonate CAS Num ber-2759-39-3 3M Environmental Laboratory Report No. FACT Tox-108 Laboratory Request Number (LRN)-U2779 Attachment A Sam ple Receipt and C hain of C ustody Documentation Sam ple receipt and chain of custody information is docum ented in the binder for this report (FA C T T ox-108); the binder is located in the 3M archives. 001184 PerfluorooctanesuHbnate CAS Num ber-2759-39-3 Attachment B Protocol 3M Environmental Laboratory Report No. FACT Tox-108 Laboratory Request Number (LRN)-U2779 001185 3M Environmental Technology and Services PO Box 33331 St. Paul. M N 55133-3331 612 778 6442 Protocol #FACT-TOX-108 Study Title Oral (Gavage) Cross-Fostering Study of PFOS In Rats PROTOCOL Author Lisa Clemen Date: June 8,1999 Performing Laboratory 3M Environmental Technology & Safety Services 3M Environmental Laboratory 935 Bush Avenue St. Paul, MN 55106 Laboratory Project Identification FACT-TOX-108 U2779 3M Environmental Laboratory Page 1 of 10 0 0 1 1 8 6 Protocol #FACT-TOX-108 Study Identification Oral (Gavage) Cross-Fostering Study of PFOS in Rats Test Material Sponsor Sponsor Representative Study Director Study Location(s) In vivo Testing Facility Analytical Testing Laboratory Perfluorooctane sulfonic acid potassium salt (T-6295) 3M Toxicology Services - Medical Department 3M Center, Building 220-2E-02 St. Paul, MN 55144-1000 Marvin T. Case, D.V.M., Ph.D. 3M Toxicology Services Telephone: 651-733-5180 Facsimile: 651-733-1773 Kristen J. Hansen, Ph.D. 3M Environmental Technology and Safety Services Building 2-3E-09 651-778-6018 Argus Research Laboratories, Inc. 905 Sheehy Drive, Building A Horsham, PA 19044 3M Environmental Laboratory Building 2-3E-09 935 Bush Avenue St. Paul, MN 55106 3M Environmental Laboratory Pase2of1 001187 Sub-Contract Laboratory Proposed Study Timetable Study Initiation Date Study Completion Date Protocol #FACT-TOX-108 Advanced Bioanalytical Services, Inc. 15 Catherwood Road Ithaca, NY 14850 Battelle Memorial Institute 505 King Avenue Columbus, Ohio 43201-2693 June 08,1999 June 08,2000 1. Study Oral (gavage) cross-fostering study of potassium perfluorooctane sulfonic acid (PFOS) in rats. 2. Purpose This analytical study is designed to determine levels of potassium perfluorooctanesulfonate (PFOS) in specimens of liver and serum of rats. The in-life portion of this study was conducted at Argus Research Laboratories, study #418-014. All serum samples will be extracted and analyzed at Advanced Bioanalytical Services, Inc. and all liver samples extracted and analyzed at Battelle Memorial Institute. Additional analyses may be performed at the 3M Environmental Laboratory as methods are developed and validated. If additional analyses are performed an amendment to this protocol will be written. 3. Regulatory Compliance This study will be conducted in accordance with the United States Food and Drug Administration, Good Laboratory Practices Standards, Final Rule 21 CFR 58, with the exception that analysis of the test material mixture for concentration, solubility, homogeneity, and stability will not be conducted, and is the responsibility of the Sponsor. 4. Quality Assurance The 3M Environmental Laboratory Quality Assurance Unit will review the protocol and audit study conduct, data, and final report to determine compliance with Good Laboratory Practice Standards and with 3M Environmental Laboratory Standard Operating Procedures. The QA Unit at the sub-contract laboratory will audit their study conduct, data, and results report prior to submitting to the 3M Environmental Laboratory. 3M Environmental Laboratory Page 3 o f 10 001188 Protocol UFACT-T0X-1Q8 5. Test Material 5.1 Refer to Argus Research Laboratory protocol for study #418-014. 6 . Control Matrices 6.1 Identification Rat liver and serum and/or rabbit liver and serum, traceability numbers will be recorded in the raw data and included in the final report 6.2 Source Argus Research and/or Sigma Chemical 6.3 P hysical Description Rat liver and serum and/or rabbit liver and serum 6.4 P urity and Stability Not applicable 6.5 Storage Conditions Frozen at -20 C 10 C or -50 C 10 C 6.6 Reserve Matrix A portion of the control matrix will be retained in the 3M archives for as long as the quality of the preparation affords evaluation, but not longer than ten years following the effective date of the final test rule (if applicable). 6.7 Disposition Matrices will be retained at the 3M Environmental Laboratory per GLP regulation. Certain matrices (feces, urine, and blood) may be disposed after QAU verification. 6.8 Safety Precautions Refer to MSDS for chemicals used. Wear appropriate laboratory attire, and follow adequate precautions for handling biological materials and preparing samples for analysis. 7. Reference Material 7.1 Identification Potassium perfluorooctanesulfonate (PFOS), lot #s 171, 215, or 217 (equivalent lots) 7.2 Source 3M Specialty Chemicals 7.3 Physical Description White powder 7.4 Purity and Stability Purity of PFOS is 99% or greater. Stability has not been determined. 7.5 Storage Conditions Room temperature 7.6 Reserve Material A reserve sample from each batch of PFOS used in this study will be retained as long as the quality of the preparation affords evaluation, but not longer than ten years following the effective date of the final test rule (if applicable). 7.7 Disposition Unused reference material will be retained for use by the 3M Environmental Laboratory and will be discarded when the quality of preparation no longer affords evaluation. 3M Environmental Laboratory Page 4 of 10 001189 Protocol #F A C T -T 0X -108 7.8 S afety Precautions Refer to MSDS for chemicals used. Wear appropriate laboratory attire, and follow adequate precautions for handling biological materials and preparing samples for analysis. 8. Test System Female rats were used as the test system and were maintained and dosed as described in Argus Research protocol #418-014. Group 1 control animals did not receive the test substance while the group 2 treated animals received the test substance at a concentration of 1.6 mg/kg/day for 6 weeks. Refer to Argus Research protocol #418-014 for tabular presentation of data. Dosing of the female animals in group 2 was continued during mating, gestation, and lactation. Crossfostering o f the pups bom to the control dams were randomized, half of the pups were placed on control dams while the other half were placed on the PFOS dosed dams. Likewise, the same was done with the group 2 pups. Each of these pups will be marked to identify whether they are from control or PFOS dosed dams. 9. Specimen and Sample Receipt The 3M Environmental Laboratory will receive homogeneity samples and specimens of the following body tissues and fluids from the indicated points in the study. All specimens will be packed on dry ice for shipping. Body tissue/fluid Serum - Dam and Pup animals Urine and feces - Dam animals Liver - Dam and Pup animals Milk Secreting Glands - Dam animals Milk - Dam animals Collected At lactation day 14 and termination of the study After termination of the study At lactation day 14 and termination of the study At lactation day 14 and termination of the study At lactation day 14 Expected # of specimens 60 Dam and 34 Pup 48 Urine and 48 Feces 60 Dam and 34 Pup 58 10 Total number of expected specimens: 352 Total number of test animals: 36 Total number of control animals: 42 Specimens sent to 3M Environmental Laboratories will be received and tracked according to applicable Standard Operating Procedures. 3M Environmental Laboratory Page 5 o f 10 001190 Protocol XFACT-TOX-108 10. Preparatory Methods 10.1 FACT-M-1.1, Extraction of Potassium Perfluorooctanesulfonate or Other Anionic Fluorochemical Surfactant from Liver for Analysis Using HPLC-Electrospray/Mass Spectrometry 10.2 ETS-8-4.1, Extraction of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds from Serum or Other Fluid for Analysis Using HPLCElectrospray/Mass Spectrometry 10.3 If preparatory methods other than those listed above are used, an amendment to this protocol will be written. Any deviations from these methods will be documented and included with the study data. 10.4 If analyses are sub-contracted to other laboratories, an amendment will be written to include their methods and copies of each method will be attached to this protocol. 11. Analytical Methods 11.1 FACT-M-2.1, Analysis of Fluorochemicals in Liver Extracts Using HPLCElectrospray/Mass Spectrometry 11.2 ETS-8-5.1, Analysis of Potassium Perfluorooctanesulfonate or Other Fluorochemicals in Serum or Other Fluid Extracts Using HPLC-Electrospray/Mass Spectrometry 11.3 If analytical methods other than those listed above are used, an amendment to this protocol will be written. Any deviations from these methods will be documented and included with the study data. 11.4 If analyses are sub-contracted to other laboratories, an amendment will be written to include their methods and copies of each method will be attached to this protocol. 12. Da ta Quality Objectives The number of spikes/duplicates, use of surrogates, and information on other data quality indicators are included in the analytical methods. In addition, the following criteria will be met: 12.1 Linearity r > 0.98 12.2 Limits o f detection / quantitation 12.2.1 Method Detection Limit (MDL) for PFOS a) Serum: 1.75 ppb b) Liver: 15 ppb 12.2.2 Limit of Quantitation (LOQ) - Equal to the lowest acceptable standard in the calibration curve 3M Environmental Laboratory Page 6 of 10 001191 Protocol UFACT-TOX-108 J 12.3 Duplicate acceptable precision < 30% for the method 12.4 Spike acceptable recoveries 70% -130% 12.5 Use o f confirm atory m ethods Indeterminate samples will be re-analyzed using a confirmatory method. If a confirmatory method is used, an amendment to this protocol will be written. 12.6 Dem onstration o f specificity Chromatographic retention time, mass spectral daughter ion characterization. 13. Sub-Contracted Analysis 13.1 All analyses as detailed in this protocol will be performed at 3M Environmental Laboratories, Building 2-3E-09, 935 Bush Avenue, St. Paul, MN 55106, at Advanced Bioanalytical Services, Inc., 15 Catherwood Road, Ithaca, NY 14850, or at Battelle Memorial Institute, 505 King Avenue, Columbus, Ohio 43201-2693. 13.2 An amendment to this protocol will be written if analyses are performed at laboratories other than 3M Environmental Laboratories, Advanced Bioanalytical Services, Inc., or Battelle Memorial Institute. 14. Statistical Analysis Averages and standard deviations will be calculated. The statistical methods that will be used are described below: 14.1 Data transformations and analysis Data will be reported as the concentration (weight/weight or weight/vol) of PFOS or metabolite per tissue or fluid. 14.2 Statistical analysis Statistics used may include regression analysis of 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. 15. Report A report containing all the results of the study will be prepared by the 3M Environmental Laboratory. If analyses are sub-contracted to other laboratories, each laboratory will prepare a report and submit it to the 3M Environmental Laboratory for inclusion in the 3M Environmental Laboratory report. Each report will include, but not be limited to, the following, when applicable: 15.1 Name and address o f the facility performing the study 15.2 Dates upon which the study was initiated and completed 15.3 A statement of compliance by the Study Director addressing any exceptions to Good Laboratory Practice Standards 3M Environmental Laboratory Page 7 of 10 Protocol #FACT-TOX-108 15.4 Objectives and procedures as stated in the approved protocol, including any changes in the original protocol 15.5 The test substance identification by name, chemical abstracts number or code number, strength, purity, and composition or other appropriate characteristics, if provided by the Sponsor 15.6 Stability and the solubility of the test substances under the conditions of administration, if provided by the Sponsor 15.7 A description of the methods used to conduct the test(s) 15.8 A description of the test system 15.9 A description of any circumstances that may have affected the quality or the integrity o f the data 15.10 The name of the Study Director and the names of other scientists, professionals, and supervisory personnel involved in the study 15.11 A description of the transformations, calculations, or operations performed on the data, a summary and analysis of the analytical chemistry data, and a statement of the conclusions drawn from the analyses 15.12 Statistical methods used to evaluate the data, if applicable 15.13 The signed and dated reports of each of the individual scientists or other professionals involved in the study, if applicable 15.14 The location where raw data and the final report are to be stored 15.15 A statement prepared by the Quality Assurance Unit listing the dates that study inspections and audits were made, and the dates of any findings reported to the Study Director and Management If it is necessary to make corrections or additions to a report after it has been accepted, the changes will be made in the form of an amendment issued by the Study Director. The amendment will clearly identify the part of the report that is being amended, the reasons for the amendment, and will be signed by the Study Director. 16. Location of Raw Data, Records, and Final Report Original data, or copies thereof, will be available at the 3M Environmental Laboratory to facilitate audits of the study during its progress and before acceptance of the final report. When the final report is completed, all original paper data, including those items listed below, will be retained in the archives of 3M Environmental Laboratory for at least a period of time as specified by regulation, and as established by 3M Environmental Laboratory Standard Operating Procedures. 3M Environmental Laboratory Page 8 o f 10 001193 Protocol #FACT-TOX-108 16.1 The following raw data and records will be retained in the study folder in the study/project archives according to 3M Environmental Laboratory Standard Operating Procedures: 16.1.1 Approved protocol and amendments 16.1.2 Study correspondence 16.1.3 Shipping records 16.1.4 Raw data 16.1.5 Approved final report (original signed copy) 16.1.6 Electronic copies of data 16.2 The following supporting records will be retained separately from the study folder in the archives according to 3M Environmental Laboratory Standard Operating Procedures: 16.2.1 Training records 16.2.2 Calibration records 16.2.3 Instrument maintenance logs 16.2.4 Standard Operating Procedures, Equipment Procedures, and Methods 17. Specimen Retention Specimens will be maintained in the 3M Environmental Laboratory specimen archives for a period o f time as specified by regulation or as long as the quality of the preparation affords evaluation, but not longer than ten years following the effective date of the final test rule (if applicable), and as established by 3M Environmental Laboratory Standard Operating Procedures. 18. Protocol Amendments and deviations Planned changes to the protocol will be in the form of written amendments signed by the Study Director and the Sponsor's Representative. Amendments will be considered as part of the protocol and will be attached to the final protocol. All changes to the protocol will be indicated in the final report. Any other changes will be in the form of written deviations, signed by the Study Director and filed with the raw data. 3M Environmental Laboratory 001194 Page 9 of 10 19. A ttachments 19.1 Attachment A Preparatory and analytical methods 20. Signatures Protocol HFACT-TQX-108 Kristen J. Hansen, Ph.D., 3M Environmental Laboratory Study Director Date 3M Environmental Laboratory Page 10 o f 10 001195 Study Title Oral (Gavage) Cross-Fostering Study of PFOS in Rats PROTOCOL AMENDMENT NO. 1 Amendment Date: August 12, 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-TOX108 LIRNU2779 3M Environmental Laboratory 001196 Protocol FACT-TOX108 Amendment No. 1 This amendment modifies the following portion(s) of the protocol: 1. PROTOCOL reads: Section 2.0 states that all liver samples will be extracted and analyzed at Battelle Memorial Institute. AMEND TOread: All liver samples will be extracted and analyzed at the 3M Environmental Laboratory. REASON: Due to time constraints, the liver extraction and analysis will be performed at the 3M Environmental Laboratory. 2. PROTOCOL reads: Section 10.0 and 11.0 list the following methods to use for extraction and analysis: FACT-M-1.1 "Extraction of Potassium Perfluorooctanesulfonate or Other Anionic Fluorochemical Surfactant from Liver for Analysis Using HPLC-Electrospray/Mass Spectrometry" FACT-M-2.1 "Analysis of Fluorochemicals in Liver Extracts Using HPLC-Electrospray/Mass Spectrometry" AMEND to read: The extraction and analytical methods to follow at the 3M Environmental Laboratory are: ETS-8-6.0 "Extraction of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds from Liver for Analysis Using HPLC-Electrospray/Mass Spectrometry" ETS-8-7.0 "Analysis of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds in Liver Extracts Using HPLC-Electrospray/Mass Spectrometry" REASON: The extraction and analytical methods FACT-M-1.1 and FACT-M-2.1-, respectively, were updated on 07/22/99 to ETS-8-6.0 and ETS-8-7.0. These methods were updated to replace the extraction solvent ethyl acetate with a different extraction solvent MTBE (methyl tert butyl ether), POAA and Monoester were removed from the standard mix, and M556 was added to the standard mix. The analytical method was updated to include linear regression with 1/x weighting and a few minor changes in the HPLC 1100 instrument parameters. 3M Environmental Laboratory 00119V Protocol FACT-TOX108 Amendment No. 1 3. PROTOCOL reads: Section 10.4 and 11.4 state that if analyses are sub-contracted to other laboratories an amendment will be written to include their methods. AMEND TOREAD: The extraction and analytical methods to follow at Advanced Bioanalytical Services will be attached to the protocol. REASON: The analytical methods at the sub-contract laboratory were not included in the original protocol. 4. PROTOCOL reads: Section 12.2.1 b) lists the liver method detection limit as 15 ppb. AMEND TOread: The liver method detection limit is 8.50 ppb (ng/g). REASON: The validation supporting methods ETS-8-6.0 and ETS-8-7.0 includes a lower method detection limit for PFOS. Amendment Approval 1/Ua/ vh-v^ T & Marvin Case, D.V.M., Ph.D., Sponsor Representative 1 -AMjcJ- !<??& Date ___________________ ----------------------------- Kris J. Hansen, Ph.D., Study Director Date 3M Environmental Laboratory 001198 Study Title Oral (Gavage) Cross-Fostering Study of PFOS in Rats PROTOCOL AMENDMENT NO. 2 Amendment Date: September 30,1999 Performing Laboratory 3M Environmental Technology & Safety Services 3M Environmental Laboratory 935 Bush Avenue St. Paul, MN 55106 Laboratory Project Identification ET&SS FACT-TOX108 LIRNU2779 3M Environmental Laboratory 001139 Protocol FACT-TOX108 Amendment No. 2 This amendment modifies the foliowing portion(s) of the protocol: 1. Protocol reads: Section 2.0 states that all liver samples will be extracted and analyzed at the 3M Environmental Laboratory. Amend to read: No liver samples will be extracted and analyzed. Reason: Liver results are no longer required. 2. Protocol reads: Section 16 states that the original data, or copies thereof, will be available at the 3M Environmental Laboratory to facilitate audits of the study during its progress and before acceptance of the final report. When the final report is completed, all original paper data, including: approved protocol and amendments, study correspondence, shipping records, raw data, approved final report, electronic copies of data, training records, calibration records, instrument maintenance logs and standard operating procedures, equipment procedures, and methods will be retained in the archives of the 3M Environmental Laboratory. Amend to read: Section 16 states that the original data, or copies thereof, will be available at the 3M Environmental Laboratory to facilitate audits of the study during its progress and before acceptance of the final report. When the final report is completed, all original paper data, including: approved protocol and amendments, study correspondence, shipping records, raw data, approved final report, and electronic copies of data will be retained in the archives of the 3M Environmental Laboratory. All corresponding training records, calibration records, instrument maintenance logs, standard operating procedures, equipment procedures, and methods will be retained in the archives of the facility performing each analysis. Reason: Clarification of the disposition of archived records if analyses are performed at a sub-contract laboratory. 3M Environmental Laboratory 001200 Protocol FACT-TOX108 Amendment No. 2 3. Protocol reads: Section 17 states that specimens will be maintained in the 3M Environmental Laboratory specimen archives. Amend to read: Specimens will be maintained in the 3M Environmental Laboratory specimen archives. All specimens sent to sub-contract laboratories will be returned to the 3M Environmental Laboratory upon completion of analysis and submission of the sub-contract laboratory(s) final report. The specimens will be returned with the following documentation: the signed original chain of custody and records of storage conditions while at the sub-contract facility. Reason: Clarification o f the disposition of specimens and documentation for analyses performed by a sub-contract laboratory. Amendment Approval Marvin Case, D.V.M., Ph.D., Sponsor Representative j J ' Date f J 2------------------------------ Kristen J. Hansen, Ph.D., Study Director lo ls lW Date 3M Environmental Laboratory 001201 Perfluorooctanesulfbnate CAS Number-2759-39-3 Attachments Extraction and A nalytical M ethods 3M Environmental Laboratory Report No. FACTTox-108 Laboratory Request Number (LR N H I2779 Proprietary and Confidential 001202 nA D V A N C E D BIOANALYTICAL i S E R V IC E S . INC. METHOD VALIDATION REPORT TITLE: METHOD VALIDATION FOR THE QUANTITATION OF PERFLUOROOCTANESULFONATE (PFOS) IN RAT SERUM BY TURBO ION SPRAY LC/MS DATE: 26 August 1999 REPORT: 99VDJA01.MI.DOC AUTHORS: David J. Anderson, M.S. Amie J. Prince, B.S. Holly D. Ross, M.S. PREPARED FOR: 3M Environmental Technology and Safety Services St. Paul, MN 55133-3331 NUMBER OF PAGES: 50 001203 15 Catherwood Road Ith aca. New York 14850 ( 6 0 7) 2 6 6 - 0 6 6 5 Fax (6 07) 2 6 6 - 0 7 4 9 AUTHORS: FOR: DATE: TITLE: David J. Anderson, M.S. Amie J. Prince, B.S. Holly D. Ross, M-S. 3M Environmental Technology and Safety Services 26 August 1999 METHOD VALIDATION FOR THE QUANTITATION OF PERFLUOROOCTANESULFONATE (PFOS) IN RAT SERUM BY TURBO ION SPRAY LC/MS ABSTRACT A sensitive, specific, accurate, and reproducible analytical method was developed by Advanced BioAnalytical Services, Inc., Ithaca, New York to quantitate perfluorooctanesulfonate (PFOS) in rat serum samples. Serum samples (50 pL) were extracted by a liquid-liquid extraction procedure to isolate the analyte from rat serum. Sample extracts were reduced to dryness, reconstituted, and analyzed by turbo ion spray liquid chromatography/mass spectrometry (LC/MS) in the negative ion mode. The assay demonstrated a lower limit of quantitation (LLQ) of 0.05 pg/mL using 50-pL sample aliquots. The calibration curves were fit from 0.05 pg/mL to 20 pg/mL for PFOS by a weighted (1/y2) quadratic equation. The coefficients of determination of the calibration curves ranged from 0.9962 to 0.9965. Precision and accuracy quality control (QC) samples were prepared at concentrations of 0.2, 6, and 18 pg/mL PFOS. Quality control (QC) samples were prepared at a concentration of 100 pg/mL PFOS for partial volume analysis. The intra- and inter assay precision (RSD) results calculated from all QC samples ranged from 1.92% to 4.87% for PFOS. The intra- and inter-assay accuracies (RE) calculated from QC samples ranged from -3.58% to 5.58% for PFOS. The mean extraction recoveries were from 87.6% to 100% for PFOS and 89.9% for the internal standard (IS). PFOS was measured as stable in rat serum for up to 24 hours at ambient temperature. PFOS was measured as stable in rat serum at -20 C, currently for up to 33 days, and ADVANCED B IO A N A L Y T IC A L S E H V IC E S . IN C . Page 2 o f 50 99VD JA O I.M I.D O C after three freeze/thaw cycles. Reliable results were obtained for sample extracts reinjected 27 hours after initial reconstitution. ADVANCED B IO A N A L Y T IC A L S E R V IC E S . IN C . Page 3 o f 50 001205 99V D JA O I.M I.D O C QAU STATEMENT Periodic inspections of the method validation for the quantitation of PFOS in rat serum were conducted by the Quality Assurance Unit of Advanced BioAnalytical Services (ABS) for compliance with EPA GLP regulations (40 CFR Part 792). The study was inspected on the following dates: 13,21 May 1999; 7, 8, 14, 15 June 1999; 1, 2 July 1999; 3, 4 August 1999. Results of the inspections were reported to ABS Management on: 13, 21 May 1999; 7, 8,14,15 June 1999; 1,2 July 1999; 3, 4 August 1999. Results of the inspections were reported to the Study Director on 5 August 1999. Based on the inspections and the data reviewed, this report is a complete and accurate representation of the data. Quality Auditor ADVANCED B IO A N A L Y T IC A L S E R V IC E S . IN C . Page 4 o f 50 001206 99VD JA 0I.M I.D C X: SIGNATURE PAGE TITLE: METHOD VALIDATION FOR THE QUANTITATION OF PERFLUOROOCTANESULFONATE (PFOS) IN RAT SERUM BY TURBO ION SPRAY LC/MS Report Number: 99VDJIAOL.MI.DQ Reported by: David J. Ande^on, M.S. Research Scientist 2 Date Reviewed by: <^(pddJjULh &unaJL__ Kathleen Cormack, B.S. Associate Auditor 7.(ouaQc Date Authorized for Release by: John RJ Perkins, Ph.D. Assistant Scientific Director Qjp Date 'IT*7^ ADVANCED B IO A N A L Y T IC A L S E R V IC E S . IN C . Page 5 o f 50 00120*7 99VD JA O i.M I.D O C TABLE OF CONTENTS ABSTRACT............................................................................................................................ QAU STATEMENT............................................................................................................. .. SIGNATURE PAGE............................................................................................................ .. TABLE OF CONTENTS..................................................................................................... .. LIST OF TABLES................................................................................................................8 LIST OF FIGURES.............................................................................................................. 9 1. INTRODUCTION.......................................................................................................10 2. EXPERIMENTAL................................................................................ ......................10 2.1. Chemicals and Materials............................................................................... 10 2.2. LC/MS Instrumentation.................................................................................. 10 2.3. Sample Preparation and Extraction procedure..................................... 11 2.4. Rat Serum Validation Data........................................................................... 11 2.5. Assay Evaluation..............................................................................................12 2.5.1. Intra- and Inter-Assay Accuracy....................................................................12 2.5.2. Intra- and Inter-Assay Precision.................................................................... 12 2.5.3. Partial Volume Analysis.................................................................................12 2.5.4. Lower Limit of Quantitation (LLQ).............................................................. 13 2.5.5. Selectivity....................................................................................................... 13 2.5.6. Carryover Evaluation......................................................................................13 2.6. STABILITY OF PFOS IN QUALITY CONTROL SAMPLES........................................13 2.6.1. Ambient-Temperature Stability of PFOS in Rat Serum................................13 2.6.2. Freezer Stability of PFOS in Rat Serum at -20 C........................................14 2.6.3. Freeze/Thaw Stability in Rat Serum.............................................................. 14 2.7. REPRODUCIBILITYOFREINJECTINGEXTRACTED SAMPLES................................14 2.8. Extraction recovery....................................................................................... 14 3. RESULTS AND DISCUSSION................................................................................. 15 3.1. Assay Evaluation Results............................................................................ 16 3.1.1. Intra- and Inter-Assay Accuracy................................................................... 16 ADVANCED B IO A N A L Y T IC A L S E R V IC E S . IN C . Page 6 of 50 99VD JA O I.M I.D O C 001Z08 3.1.2. Intra- and Inter-Assay Precision..................................................................... g 3.1.3. Linearity.......................................................................................................... . 3.1.4. Partial Volume Analysis........................................................ ig 3.1.5. Lower Limit of Quantitation (LLQ).............................................................. . 3.1.6. Selectivity....................................................................................................... . 3.1.7. Carryover Evaluation......................................................................................... 3.2. Stability of PFOS in Quality Control Samples........................................17 3.2.1. Ambient Temperature Stability of PFOS in Rat Serum ............................... 17 3.2.2. Freezer Stability of PFOS in Rat Serum at -20 C.......................................18 3.2.3. Freeze/Thaw Stability of PFOS in Rat Serum................................................18 3.3. REPRODUCIBILITY OF REINJECTING EXTRACTED SAMPLES...............................18 3.4. EXTRACTION RECOVERY...................................................................................... 18 4. CONCLUSIONS............................ 19 5. DATA RETRIEVAL.................................................................................................... 19 6 . REFERENCES............................................................................................................. 19 7. TABLES....................................................................................................................... 20 8 . FIGURES..................................................................................................................... 31 9. APPENDIX A: QUANTITATION OF PERFLUOROOCTANESULFONATE (PFOS) IN RAT SERUM BY TURBO ION SPRAY LC/M S.................................. 37 ADVANCED B ID A N A L Y T IC A L S E R V IC E S . IN C . Page 7 o f 50 99VD JA O I.M I.D O C 001209 LIST OF TABLES Table 1: Intra-Assay Accuracy and Precision of the PFOS Assay in Rat Serum for QC Samples........................................................................................................20 Table 2: Inter-Assay Accuracy and Precision of the PFOS Assay in Rat Serum for QC Samples from Three Validation R uns........................................................ 21 Table 3: Accuracy and Precision of the PFOS Assay in Rat Serum for Calibration Standards from Three Validation Runs..............................................................22 Table 4: Calibration Curve Parameters for PFOS in Rat Serum .................................... 23 Table 5: Partial Volume Analysis of PFOS in Rat Serum.............................................. 24 Table 6 : Lower Limit of Quantitation of PFOS in Rat Serum........................................ 25 Table 7: Ambient-Temperature Stability of PFOS in Rat SerumAfter 24 Hours.......... 26 Table 8 : Freezer Stability of PFOS in Rat Serum at -20 C ...........................................27 Table 9: Stability of PFOS in Rat Serum After Three Freeze/Thaw Cycles................. 28 Table 10: Reproducibility of Reinjecting Extracted Samples Containing PFOS after 27 Hours in Reconstitution Solution..................................................................29 Table 11: Extraction Recovery of PFOS from Rat Serum................................................. 30 ADVANCED B IO A N A L Y T IC A L S E R V IC E S , IN C . Page 8 o f 50 001210 99V D JA 01.M I.D O C LIST OF FIGURES Figure 1: Full-Scan Single MS Mass Spectrum o f PFOS................................................31 Figure 2: Full-Scan Single MS Mass Spectrum of Tetra-H-PFOS..................................32 Figure 3: Mass Chromatograms of PFOS and its Internal Standard in Control Blank Rat Serum Extract............................................................................................. 33 Figure 4: Mass Chromatograms of PFOS in a Rat Serum Extract Sample Containing Internal Standard Only (Zero Sample)............................................................. 34 Figure 5: Mass Chromatograms of Calibration Standard 1 in Rat Serum Extract Containing PFOS (0.05 pg/mL) and the Internal Standard............................. 35 Figure 6 : Mass Chromatograms of Calibration Standard 10 in Rat Serum Extract Containing PFOS (20 pg/mL) and the Internal Standard................................ 36 ADVANCED B fO A N A L Y T IC A L S E R V IC E S . IN C . Page 9 o f 50 001211 99VD JA 01.M I.D O C 1. INTRODUCTION The purpose of this report is to describe the method validation for the quantitative determination of perfluorooctanesulfonate (PFOS) in rat serum samples after modification of a previous method (1). The objectives of the method validation were to validate a simple extraction procedure, determine the lower limit of quantitation for routine analysis, determine the extraction recoveries of the analyte and internal standard (IS), determine the analyte stability in rat serum at ambient temperature, long-term freezer storage, and over three freeze/thaw cycles, and to provide specific, accurate, and reproducible quantitative results by turbo ion spray liquid chromatography/mass spectrometry (LC/MS). 2. EXPERIMENTAL 2.1. Chemicals and Materials Perfluorooctanesulfonate (PFOS, Lot# 171) was obtained from 3M, Inc. The internal standard (IS) for PFOS was lH,lH ,2 H,2 H-perfluorooctane sulfonic acid (Tetra-HPFOS). The internal standard (Lot# 59909) was obtained from 3M, Inc. A detailed list of chemicals and materials is found in Appendix A. Stock and working solutions which were vised to prepare the calibration curves for the analytes were prepared as described in Appendix A. Stock solutions used in the preparation of quality control (QC) samples were prepared separately from those used in preparation of the calibration curves. Preparation of all solutions used during extraction and analysis are described in Appendix A. 2.2. LC/MS INSTRUMENTATION The liquid chromatography/mass spectrometry system consisted of two LC-10AD pumps (Shimadzu, Columbia, MD 21046), a SCL-10A pump controller (Shimadzu, m ADVANCED B IO A N A L V T IC A L S E R V IC E S . IN C . Page 10 o f 50 001212 99VD JA 01.M I.D O C Columbia, MD 21046), a WISP 717plus autosampler (Waters Associates, Millipore Corporation, Milford, MA 01757), a Betasil C1S(2 x 50 mm, 5 pm) column (Keystone Scientific, Inc., Bellefonte, PA 16823), and a PE SCIEX API 365 mass spectrometer (PE SCIEX, Concord, Ontario). A detailed list of the instrumentation and instrument conditions is found in Appendix A. 2.3. Sample Preparation and Extraction Procedure For each analytical run (tray), duplicate 50-pL aliquots of the calibration curve samples were prepared as described in Appendix A. The nominal (theoretical) concentrations of PFOS in the calibration curves were 0.05, 0.1, 0.25, 0.5,1, 2, 4 , 8, 16, and 20 pg/mL. Rat serum quality control samples were prepared in advance of the validation study at nominal (theoretical) concentrations of 0.2, 6 , and 18 pg/mL for QC1, QC2, and QC3, respectively, as detailed in Appendix A. A dilution QC (QC4, 100 pg/mL) was prepared at a concentration exceeding the upper limit of the calibration curve range (20 pg/mL), and was assayed using a 10-fold dilution for partial volume analysis. Calibration standards and QC samples were extracted by the procedure detailed in Appendix A. 2.4. Rat Serum Validation Data The data were collected using selected ion monitoring (SIM) turbo ion spray LC/MS in the negative ion mode. Peak areas were integrated by the PE SCIEX program MacQuan, version 1.4, residing on a Macintosh computer. Following peak area integration, the results tables from MacQuan were saved as text files and uploaded to the Advanced BioAnalytical Services (ABS) file server where a weighted (1/y2) quadratic regression was performed using the software package Watson v 5.3.1.01 (PSS, Inc., Wayne, PA 19087). All data were rounded to no less than three significant figures by ABS prior to reporting in Tables 1 through 11. The data for the rat serum validation are stored in ABS Notebook 2304. 001213 ADVANCED B IO A N A L Y T IC A L S E R V IC E S . IN C . Page I I o f 50 99VD JA 01.M I.D O C All calculations were based on the peak area ratio of the analyte to the internal standard. Concentrations of each analyte in quality control samples were determined by inverse prediction from the calibration curve. 2.5. ASSAY EVALUATION 2.5.1. Intra- and Inter-Assay Accuracy The intra- and inter-assay accuracy of the method was assessed by determining the relative error observed in the analysis of quality control samples. The mean concentration for each quality control level was divided by the theoretical concentration. One was subtracted from the result, converted to percent and expressed as RE. QC1 through QC3 were assayed in replicates of five. In addition, the RE was reported for standards at all levels over three runs. 2.5.2. Intra- and Inter-Assay Precision The intra- and inter-assay precision of the method was assessed by determining the Relative Standard Deviation (RSD) observed for quality control sample data. The mean concentration and RSD were calculated for the first run and over three runs for intra-assay and inter-assay precision, respectively. QC1 through QC3 were assayed in replicates of five. In addition, the RSD was reported for standards at all levels over three runs. 2.5.3. Partial Volume Analysis The effect of dilution on the analysis of PFOS in rat serum was determined by partial volume analysis. QC4 (100 pg/mL) was prepared containing PFOS at approximately five times the upper limit of quantitation (ULQ). Five replicates of QC4 were diluted ten-fold and analyzed. The RSD and RE were reported. ADVANCED B IO A N A L Y T IC A L S E H V IC E S . IN C . Page 12 o f 50 001214 99VD JA O I.M I.D O C 2.5.4. Lower Limit of Quantitation (LLQ) Rat serum control samples from six separate individuals were spiked with PFOS at a concentration of 0.05 pg/mL. The LLQ was determined by obtaining the backcalculated concentrations from these control samples. The overall mean, RSD, and RE were also calculated. 2.5.5. Selectivity The selectivity of the assay was determined by LC/MS. To monitor for interference from the biological matrix, rat serum samples containing neither the analyte nor the internal standard (control blank) were assayed with all experiments. 2.5.6. Carryover Evaluation The carryover of analyte from one injection' to the next was assessed by analyzing a control blank injected immediately after a high calibration standard (STD 10, 20 pg/mL). 2.6. Stability of PFOS in Quality Control Samples QCl through QC3 were used to determine the stability of the analyte during sample storage, extraction, and analysis. 2.6.1. Ambient-TemperatureStability of PFOS in Rat Serum The stability of PFOS was evaluated by storing QC samples at each concentration level at ambient temperature (ca. 25 C) for nominal timepoints o f 0, 2.5, 6 , and 24 hours after thawing. All QCs were assayed in replicates of five. ADVANCED B IO A N A L Y T 1 C A L S E R V IC E S . IN C . Page 13 o f 50 99VD JA 01.M I.D O C 2.6.2. Freezer Stability of PFOS in Rat Serum at -20 C The freezer stability of PFOS was evaluated by storing QC samples at each concentration level at -20 C for 7, 13, and 33 days. Additional freezer stability timepoints will be reported in an addendum to this report. All QCs were assayed in replicates of four. 2.6.3. Freeze/Thaw Stability in Rat Serum The stability of PFOS was evaluated after three freeze/thaw cycles. The mean concentrations of the QC samples after three freeze/thaw cycles (i.e., at least 2 hours frozen storage at the nominal temperature o f -20 C followed by thawing at ambient temperature for 30 minutes) were compared to the mean concentrations of freshly thawed QC samples. All QCs were assayed in replicates of five. \ 2.7. REPRODUCIBILITY OF REINJECTING EXTRACTED SAMPLES The reproducibility o f reinjecting reconstituted serum extracts was investigated by reinjecting a set of previously-assayed standards and QC samples which had been stored after injection at approximately 25 C for 27 hours. The RE of the QC samples was used to assess processed sample stability in the reconstitution solution. All QCs were assayed in replicates of five. i, 2.8. Extraction Recovery The extraction recovery of PFOS from rat serum was determined by comparing the peak area ratio (PAR) of samples (0.25,4, and 16 pg/mL) spiked after extraction (post-extract) with the PAR of samples spiked before extraction (pre-extract). The internal standard was spiked post-extraction for all samples. The recovery of the internal standard (4 pg/mL) was assessed following a similar approach using PFOS as the reference. The extraction recovery (% Recovery) was determined by dividing the pre-extract PAR by the post-extract PAR and expressing the result as a percentage. Five replicates were used at each concentration level. ADVANCED B IO A N A L Y T IC A L S E R V IC E S . IN C . Page 14 o f 50 99V D JA 0I.M I.D O C 001216 3. RESULTS AND DISCUSSION A quantitative analytical procedure using turbo ion spray LC/MS in the negative ion mode was developed to meet the high sensitivity, specificity, and reproducibility requirements for the determination of PFOS in rat serum. The full-scan single MS mass spectrum of PFOS showed an abundant [M-K]' ion at m/z = 499 (Figure 1). The full-scan single MS mass spectrum of Tetra-H-PFOS showed an abundant [M-H]' ion at m/z = 427 (Figure 2). The following selected ion monitoring (SIM) was used to quantify the analytes in rat serum: PFOS Tetra-H-PFOS (IS) m/z = 499.0 m/z = 427.0 The peak labeling of full-scan data does not accurately represent the performance of the instrument in SIM mode. The SIM ions were derived from separate experiments using narrow-range scanning, which more accurately depicts the operation of the instrument in the SIM mode. The mass chromatograms of a representative control blank rat serum extract are shown iri Figure 3. Mass chromatograms from a representative control rat serum extract containing only the internal standard (zero sample), are shown in Figure 4. Figures 5 and 6 are mass chromatograms of representative extracts from calibration standards 1 and 10, respectively. ADVANCED B IO A N A L Y T IC A L S E R V IC E S . IN C . Page 15 o f 50 001Z 7 99VD JA 01.M I.D O C 3.l . assay Evaluation Results 3.1.1. Intra- and Inter-Assay Accuracy The intra-assay accuracy (RE) data from QC samples ranged from -2.35 to 5.58% for PFOS (Table 1). The inter-assay accuracy ranged from -3.58 to 4.95% for PFOS (Table 2). These data indicate acceptable intra- and inter-assay accuracy for the determination of PFOS in rat serum. 3.1.2. Intra- and Inter-Assay Precision The intra-assay precision data (RSD) ranged from 1.99 to 3.28% for PFOS at all QC concentration levels (Table 1). The inter-assay precision results from QC samples ranged from 1.92 to 4.87% for PFOS (Table 2). The RSD ranged from 0.613 to 4.38% for calibration standards at all levels (Table 3). These data indicate acceptable intra-and inter-assay precision for the determination of PFOS in rat serum. 3.1.3. Linearity The calibration curves were fit by a weighted (1/y2) quadratic regression. Coefficients of determination (r2) were >0.9962 for PFOS in rat serum. The calibration curve statistics are shown in Table 4. 3.1.4. Partial Volume Analysis The results of partial volume analysis of QC4 is shown in Table 5. The precision (RSD) of QC4 samples diluted 1 in 10 was 2.71%. The accuracy was 5.89%. These data indicate acceptable accuracy and precision for partial volume analysis for the determination of PFOS in rat serum. iII 1' lillil'T"" l1liili il li ~ li ii'f t if iir iir i ADVANCED B IO A N A L Y T IC A L S E R V IC E S . IN C . Page 16 o f 50 001218 99VD JA O l.M l.D O C 3.1.5. Lower Limit of Quantitation (LLQ) Table 6 shows the lower limit of quantitation (LLQ) data. The serum LLQ experiment demonstrated that 0.05 pg/mL is an acceptable LLQ for PFOS. The precision (RSD) was 4.95% for PFOS. The RE was 4.57%. 3.1.6. Selectivity The assay was specific for PFOS. No chromatographic interferences were observed in any of the control serum samples analyzed (Figure 3). The control blanks and zero samples did show some evidence of small chromatographic peaks at the retention times of the analyte. These peaks were not quantifiable as they were below the lower limit of quantitation (LLQ). 3.1.7. Carryover Evaluation Carryover of PFOS and the IS was evaluated by injection of an extracted rat serum control blank following an injection of an extracted high standard (STD 10). The response for the small chromatographic peak at the retention time for PFOS was comparable to the response of a rat serum control blank injected before the high standard. Thus, carryover is negligible for PFOS. There was no evidence of carryover for the internal standard. 3.2. Stability of PFOS in Quality Control Samples 3.2.1. Ambient Temperature Stability of PFOS in Rat Serum The results of the ambient stability of PFOS in QC samples are shown in Table 7. The mean predicted concentrations deviated from -4.45 to 10.5% from the 0-hour values for all QC levels and time points. Based on these data, PFOS was stable in rat serum for up to 24 hours at ambient temperature. ADVANCED B ID A N A L Y T IC A L R F R V IC E S . IN C . Page 17 o f 50 001219 99VD JA 01.M I.D O C 3.2.2. Freezer Stability of PFOS in Rat Serum at -20 C The results of the long-term stability of PFOS in QC samples stored at -20 C are presented in Table 8. The PFOS concentrations deviated from 0-hour values from -2.76 to 12.3% for all QC levels stored up to 33 days. Results of freezer storage after two, four, and eight months will be reported as an addendum to this report. 3.2.3. Freeze/Thaw Stability of PFOS in Rat Serum The results of the freeze/thaw stability of PFOS in QC samples after three freeze/thaw cycles is shown in Table 9. PFOS was stable after three freeze/thaw cycles with deviations ranging from -1.38% to 10.1% from the 0-cycle samples for all QC levels (Table 9). 3.3. Reproducibility of reinjecting Extracted Samples The results of reinjecting reconstituted samples containing PFOS after storage for 27 hours at approximately 25 C in reconstitution solution are shown in Table 10. Reinjecting processed samples after 27 hours in reconstitution solution was appropriate with RE values ranging from -6.33 to 4.59% at 27 hours for all QC levels. 3.4. Extraction Recovery The mean recoveries of PFOS and Tetra-H-PFOS are shown in Table 11. The mean recoveries ranged from 87.6 to 100% for PFOS. The mean recovery for Tetra-HPFOS was 89.9%. ADVANCED B ID A N A L Y T IC A L S E R V IC E S . IN C . Page 18 o f 50 001220 99V D JA O I.M I.D O C 4. CONCLUSIONS The turbo ion spray LC/MS assay procedure for the determination of PFOS in rat serum has proven to be sensitive, specific, accurate, and reproducible. Its high sensitivity allows reliable and reproducible quantitation of PFOS down to a level of 0.05 pg/mL in rat serum based on 50-pL samples. 5. DATA RETRIEVAL The data for the rat serum validation are stored in ABS Notebook 2304 and in the ABS Archives. 6. REFERENCES 1. Advanced BioAnalytical Services, Inc. Report 98AGKP02.MMM. Analytical Report for the Determination of Perfluorooctanoate and Perfluorooctanesulfonate in Human Serum by LC/MS. Grace K. Poon, Ph.D., David Hardwick, B.S., and Ellen Pace, M.A.T., 6 February 1998. ADVANCED B IO A N A L Y T IC A L S E R V IC E S . IN C . Page 19 o f 50 001221 99VD JA 01.M I.D O C 7. TABLES Table 1: Intra-Assay Accuracy and Precision of the PFOS Assay in Rat Serum for QC Samples PFOS Concentration (jig/mL) QC1 QC2 QC3 Theoretical Cone. 0.2 6 18 Run 9 0.210 6.52 17.9 0.202 6.31 18.0 0.206 6.17 16.7 0.205 6.32 18.0 0.194 6.35 17.3 Mean RSD (%) RE (%) 0.204 6.34 17.6 2.93 1.99 3.28 1.77 5.58 -2.35 RSD = (SD/Mean) x 100 RE = [(Mean/Theoretical)-l] x 100 ADVANCED B IO A N A L Y T 1 C A L S E R V IC E S . IN C . Page 20 o f 50 001222 99V D JA O I.M I.D O C Table 2: Inter-Assay Accuracy and Precision of the PFOS Assay in Rat Serum for QC Samples from Three Validation Runs PFOS Concentration (jig/mL) Theoretical Cone. Run 9 QC1 0.2 0.210 0.202 0.206 0.205 0.194 QC2 6 6.52 6.31 6.17 6.32 6.35 QC3 18 17.9 18.0 16.7 18.0 17.3 Run 10 Run 11 0.187 0.186 0.182 0.189 0.182 0.194 0.197 0.184 0.192 0.184 6.44 6.44 6.32 6.19 6.31 6.17 6.13 6.41 6.15 6.24 16.9 17.3 17.5 18.0 17.4 18.2 17.1 17.6 17.7 17.0 Mean 0.193 6.30 17.5 RSD (%) 4.87 1.92 2.66 RE (%) -3.58 4.95 -2.73 RSD = (SD/Mean) x 100 RE = [(Mean/Theoretical)-l] x 100 ADVANCED B IO A N A L Y T 1 C A L S E R V IC E S . IN C . Page 21 o f 50 001223 99VD JA 01.M I.D O C Table 3 Accuracy and Precision of the PFOS Assay in Rat Serum for Calibration Standards from Three Validation Runs PFOS Calibration Standard Concentration (p.g/mL) Theoretical STD1 STD2 STD3 STD4 STD5 STD6 STD7 STD8 Cone. 0.05 0.1 0.25 0.5 1 2 4 8 Run 9 0.0483 0.0972 0.251 0.480 0.942 2.10 4.48 7.61 0.0530 0.101 0.252 0.483 0.938 2.13 4.55 7.70 Run 10 0.0491 0.0951 0.239 0.469 0.954 2.16 4.58 8.05 0.0542 0.101 0.251 0.479 0.942 2.11 4.43 7.87 Run 11 0.0510 0.0960 0.251 0.469 0.945 2.21 4.40 7.64 0.0510 0.0995 0.252 0.477 0.948 2.13 4.53 7.78 Mean 0.0511 0.0983 0.249 0.476 0.945 2.14 4.50 7.78 RSD (%) 4.38 2.65 2.07 1.15 0.613 1.90 1.62 2.13 RE (%) 2.20 -1.67 -0.207 -4.78 -5.53 7.05 12.4 -2.80 RSD = (SD/ Vlean) x 100 RE = [(Mean/Theoretical)-l] x 100 STD9 STD10 16 20 15.8 20.0 16.2 19.9 15.9 19.6 15.7 20.0 15.8 20.3 15.6 20.0 15.8 20.0 1.22 1.08 -1.00 -0.0764 I I ADVANCED B IO A N A L Y T IC A L S E R V IC E S . IN C . Page 22 o f 50 001224 99VD JA 01.M I.D O C Table 4: Calibration Curve Parameters for PFOS in Rat Serum Run # Quadratic Coefficient Coefficient (A)a Linear (B) 9 -0.0010769 0.13730 10 -0.0010516 0.14535 11 -0.0012359 0.15173 Mean -0.0011215 0.14479 a: y = Ax2 +Bx + C, weighted 1/y2 Intercept Coefficient of (C) Determination (r) 0.00042009 0.9965 0.0014910 0.9962 0.00088515 0.9964 0.00093206 0.9964 ADVANCED B ID A N A L V T IC A L S E R V IC E S . IN C . Page 23 o f 50 001225 99VDJA01.M 1.DOC Table 5: Partial Volume Analysis of PFOS in Rat Serum PFOS Cone. (ug/mL) Theoretical Cone. QC4 100 0 in 10 dilution) Run 9 111 105 105 104 104 Mean RSD (%) RE (%) 106 2.71 5.89 RSD = (SD/Mean) x 100 RE = [(Mean/Theoretical)-l] x 100 i: ? ADVANCED B ID A N A L Y T IC A L S E R V IC E S . IN C . Page 24 o f 50 001226 99VD JA 01.M I.D O C Table 6: Lower Limit of Quantitation of PFOS in Rat Serum Run 11 PFOS Theoretical Concentration Cone. Found (jig/mL) 0.0548 LLQ (0.05 ng/mL) 0.0489 0.0499 0.0554 0.0527 0.0520 Mean RSD (%) RE (%) 0.0523 4.95 4.57 RSD = (SD/Mean) x 100 RE = [(Mean/Theoretical)-l] x 100 ADVANCED B IO A N A L V T IC A L S E R V IC E S . IN C . Page 25 o f 50 001227 99V D JA 01.M I.D O C Table 7: Ambient-Temperature Stability of PFOS in Rat Serum After 24 Hours Run 11 QC (Theoretical Cone.) QC 1 (0.2 pg/mL) Mean RSD (%) %DEV from 0 Hour 0 Hour 0.194 0.197 0.184 0.192 0.184 0.190 3.03 NA PFOS (pg/mL) 2.5 Hour 6 Hour 0.187 0.177 0.181 0.179 0.184 0.180 0.180 0.183 0.178 0.189 0.182 0.182 1.91 2.52 -4.20 -4.45 24 Hour 0.190 0.187 0.191 0.182 0.190 0.188 2.01 -1.16 QC 2 (6 pg/mL) Mean RSD (%) %DEV from 0 Hour QC 3 (18 pg/mL) Mean RSD (%) %DEV from 0 Hour 6.17 6.17 6.36 6.13 6.30 6.38 6.41 6.26 6.35 6.15 6.28 6.22 6.24 6.24 6.11 6.22 6.25 6.28 1.82 0.797 1.86 NA 0.525 1.05 18.2 18.0 18.1 17.1 18.0 18.0 17.6 17.7 17.6 17.7 17.9 18.2 17.0 18.0 18.0 17.5 17.9 18.0 2.84 0.719 1.26 NA 2.33 2.39 6.55 6.53 6.85 6.66 6.81 6.68 2.16 7.42 19.1 19.4 19.2 19.4 19.7 19.4 1.19 10.5 NA: Not Applicable RSD = (SD/Mean) x 100 %DEV from 0 Hour = [(X Hour Cone. - 0 Hour Conc.)/0 Hour Cone.] x 100 T ADVANCED B IO A N A L Y T IC A L S E R V IC E S . IN C . Page 26 o f 50 001228 99VD JA 01.M I.D O C Table 8: Freezer Stability of PFOS in Rat Serum at -20 C QC Nominal Concentration 0 Hour (Run 2)* 0.213 PFOS (ug/mL) 7 Days 13 Days (Run 6)* (Run 3)** 0.225 0.211 QC 1 (0.2 ug/mL) 0.209 0.208 0.208 0.222 0.218 0.219 0.210 0.217 0.214 0.207 Mean RSD (%) %DEV from 0 Hour 0.209 1.24 NA 0.221 1.32 5.72 0.213 1.55 1.84 6.02 6.22 5.97 QC 2 5.71 6.15 5.84 (6 ug/mL) 6.00 6.39 6.07 6.06 6.27 5.99 5.69 Mean 5.89 6.26 5.97 RSD (%) 3.08 1.62 1.58 %DEV from 0 Hour NA 6.19 1.20 20.2 19.1 18.8 QC 3 18.6 19.5 18.2 (18 ug/mL) 18.9 19.8 18.8 18.2 19.3 17.9 Mean RSD (%) %DEV from 0 Hour 18.9 18.9 3.96 NA 19.4 18.4 1.37 2.42 2.52 -2.76 NA: Not Applicable RSD = (SD/Mean) x 100 %DEV from 0 Hour = [(Cone. - 0 Hour Conc.)/0 Hour Cone.] x 100 *From Protocol FACT-TOX-110 **From Protocol FACT-TOX-111 33 Days (Run 16) 0.245 0.234 0.228 0.232 0.235 3.17 12.3 6.45 6.13 6.58 6.37 6.38 2.97 8.28 18.6 18.8 18.7 18.7 18.7 0.395 -1.30 ADVANCED B IO A N A L Y T IC A L S E R V IC E S . IN C . Page 27 o f 50 Q01Z23 99V D JA 01.M I.D O C Table 9: Stability of PFOS in Rat Serum After Three Freeze/Thaw Cycles Run 10 PFOS (pg/mL) QC (Theoretical Cone.) QC 1 (0.2 pg/mL) Mean RSD (%) %DEV from 0 Cycle 0 Cycle 0.187 0.186 0.182 0.189 0.182 0.185 1.70 NA 3 Cycles 0.177 0.177 0.185 0.185 0.189 0.182 3.12 -1.38 QC 2 (6 pg/mL) Mean RSD (%) %DEV from 0 Cycle 6.44 6.44 6.32 6.19 6.31 6.34 1.66 NA 6.59 6.61 6.44 6.39 6.40 6.49 1.63 2.35 QC 3 (18 pg/mL) Mean RSD (%) %DEV from 0 Cycle 16.9 17.3 17.5 18.0 17.4 17.4 2.31 NA 19.2 19.1 19.2 19.6 18.7 19.2 1.80 10.1 NA: Not Applicable RSD = (SD/Mean) x 100 %DEV from 0 Cycle = [(3 Cycle - 0 Cycle)/0 Cycle] x 100 ADVANCED B IO A N A L Y T IG A L S E R V IC E S . IN C . Page 28 o f 50 001230 99VD JA O I.M I.D O C Table 10: Reproducibility of Reinjecting Extracted Samples Containing PFOS after 27 Hours in Reconstitution Solution QC1 (0.2 jig/mL) Mean RSD (%) RE (%) QC2 (6 |ug/mL) Mean RSD (%) RE (%) QC 3 (18 (ig/mL) Mean RSD (%) RE (%) NA: Not Applicable RSD= (SD/Mean) x 100 RE= [(Mean/Theoretical)-1] x 100 PFOS (ug/mL) t=0 Hours t=27 Hours (Run 10) (Run 12) 0.187 0.186 0.182 0.189 0.182 0.185 1.70 -7.50 0.187 0.182 0.188 0.188 0.190 0.187 1.61 -6.33 6.44 6.39 6.44 6.02 6.32 6.19 6.19 6.55 6.31 6.22 6.34 6.28 1.66 3.23 5.62 4.59 16.9 17.3 17.3 17.5 18.0 17.4 17.4 2.31 -3.29 18.2 18.1 17.3 17.6 17.7 2.40 -1.73 1 , | 1 ! ! ADVANCED B IO A N A L Y T 1 C A L S E R V IC E S . IN C . Page 29 o f 50 001231 99VD JA 01.M I.D O C Table 11: Extraction Recovery of PFOS from Rat Serum Theoretical Cone. Pre-Extraction Post-Extraction in serum (pg/mL) Response (Area Ratio) Response (Area Ratio) % Recovery* PFOS 0.03160 0.02880 110 Spike 1 0.02850 0.03010 94.7 (0.25 pg/mL) 0.02930 0.02950 99.3 Rim 14 0.02790 0.02930 95.2 0.03050 0.02960 103 Mean 0.02956 0.02946 100 PFOS Spike 2 (4 pg/mL) Run 14 Mean 0.4597 0.4751 0.4776 0.4724 0.4772 0.4724 0.5270 0.5304 0.5335 0.5298 0.5392 0.5320 87.2 89.6 89.5 89.2 88.5 88.8 PFOS Spike 3 (16 pg/mL) Run 14 Mean 1.649 1.679 1.719 1.535 1.612 1.639 1.874 1.857 1.869 1.887 1.864 1.870 88.0 90.4 92.0 81.4 86.5 87.6 Tetra-H-PFOS Spike IS (4 pg/mL) Run 13 Mean 0.4068 0.3884 0.4043 0.3921 0.4078 0.3999 0.4460 0.4423 0.4445 0.4512 0.4412 0.4450 91.2 87.8 91.0 86.9 92.4 89.9 ^(Individual Response for Pre-Ext./Individual Response for Post-Ext.) x 100 ADVANCED B IO A N A L Y T IC A L S E R V IC E S . IN C . Page 30 o f 50 001232 99VD JA 01.M I.D O C 8. FIGURES Figure I : Full-Scan Single MS Mass Spectrum of PFOS Spectrum from PFOS QI 3.19e7 cps Relative Ion Abundance (% ) ADVANCED B ID A N A L Y T IC A L SERVICES. INC. Page 31 o f 50 001233 99VD JA 01.M I.D O C Figure 2 Full-Scan Single MS Mass Spectrum of Tetra-H-PFOS Spectrum from Tetra-H-PFOS QI I.94e7cps Relative Ion Abundance (% ) ADVANCED B IO A N A L V T IC A L S E R V IC E S . IN C . Page 32 o f 50 001234 99VD JA 01.M I.D O C Figure 3: Mass Chromatograms of PFOS and its Internal Standard in Control Blank Rat Serum Extract m/z - 427.0 1,07e3 cps Time, min All quantitation results and figures were prep ared from non-sm oothed d ata. Analyte PFOS Tetra-H-PFOS (IS) Ion m/z = 499.0 m/z = 427.0 Retention Time 3.1 2.9 001235 ADVANCED B IO A N A L Y T 1 C A L S E R V IC E S . IN C . Page 33 o f 50 99VD JA 01.M I.D O C Figure 4: Mass Chromatograms of PFOS in a Rat Serum Extract Sample Containing Internal Standard Only (Zero Sample) m/z -4 2 7 .0 2.34c5 cps 90 80 i 70 60 50 ^ 40 30 H 20 10 oue 3 < Co m/z = 499.0 Time, min 3.78e2 cps Time, min All quantitation results and figures were prepared from non-smoothed data. Anajy te PFOS Tetra-H-PFOS (IS) Ion m/z - 499.0 m/z = 427.0 Retention Time 3.1 2.9 001236 ADVANCED B IO A N A L Y T IC A L S E R V IC E S . IN C . Page 34 o f 50 99VD JA 01.M I.D O C Figure 5: Mass Chromatograms of Calibration Standard 1 in Rat Serum Extract Containing PFOS (0.05 pg/mL) and the Internal Standard m/z -4 2 7 .0 3.82e5 cps ae3 J<3 All quantitation results and figures were prepared from non-smoothed data. Analvte PFOS Tetra-H-PFOS (IS) Ion m /z: 499.0 m/z = 427.0 Retention Time 3.1 2.9 001237 ADVANCED B IO A N A L Y T IC A L S E R V IC E S . IN C . Page 35 o f 50 99VDJA01.M 1.DO C Figure 6: Mass Chromatograms of Calibration Standard 10 in Rat Serum Extract Containing PFOS (20 ng/mL) and the Internal Standard m/z " 427.0 3.52e5 cps All quantitation results and figures were prepared from non-smoothed data. Analvte PFOS Tetra-H-PFOS (IS) IflQ m/z = 499.0 m/z = 427.0 Retention Time 3.1 2.9 001208 ADVANCED BIO A N A LY TIC A L ccQ\/ireq imp Page 36 o f 50 99V D JA 01.M I.D O C 9. APPENDIX A: QUANTITATION OF PERFLUOROOCTANESULFONATE (PFOS) IN RAT SERUM BY TURBO ION SPRAY LC/MS AUTHORS: David J. Anderson, M.S. Amie J. Prince, B.S. Holly D. Ross, M.S. TABLE OF CONTENTS A. 1.0 Chemical Structure(s)........................................................................................... 38 A.2.0 Specimen(s).......................................................................................................... 38 A.3.0 Assay Principle..................................................................................................... 38 A.4.0 Compounds........................................................................................................... 38 A.5.0 Chemicals............................................................................................................. 38 A.6.0 Materials and Equipment......................................................................................39 A.7.0 Preparation of Solutions.......................................................................................42 A.8.0 Preparation of Quality Control Samples.............................................................. 45 A.9.0 Liquid-liquid Extraction Procedure..................................................................... 46 A. 10.0 Instrument Conditions..........................................................................................47 A. 11.0 Calculations.......................................................................................................... 49 ADVANCED B IO A N A L Y T IC A L S E R V IC E S . IN C . Page 37 o f 50 001239 99VD JA O I.M I.D O C A.1.0 CHEMICAL STRUCTURE(S) O CgF|7----- S----- 0'K + 0 Potassium Perfluorooctanesulfonate MW = 538 O II C6F |3-----C-----C----- S----- OH H2 H2 || o 1H, 1H, 2H, 2H-Perfluorooctane Sulfonic Acid MW = 428, Internal Standard A.2.0 SPECIMEN(S) This assay uses 50-pL aliquots of rat serum. Rat serum samples are stored at -20 C. A.3.0 ASSAY PRINCIPLE PFOS and its internal standard, Tetra-H-PFOS, are extracted from rat serum samples (50 pL) using a liquid-liquid extraction procedure. The organic layer is evaporated to dryness and then reconstituted. An aliquot is then analyzed by turbo ion spray liquid chromatography/mass spectrometry (LC/MS) in the negative ion mode. A.4.0 COMPOUNDS PFOS: Lot# 171, 99.976% purity, 3M, Inc., Minneapolis, MN. Tetra-H-PFOS: Lot# 59909, 90% purity, 3M, Inc., Minneapolis, MN. A.5.0 CHEMICALS All chemicals may be substituted with that of an equivalent manufacturer and grade of chemical. 001240 ADVANCED B IO A N A L Y T IC A L ^ P H V IC E S , IN C . Page 38 o f 50 99VD JA 01.M I.D O C Acetonitrile Ammonium Acetate Ethyl Acetate Methanol Rhodapex mass calibration solution Sodium Bicarbonate Sodium Carbonate, Annhydrous Tetrabutylammonium Hydrogen Sulfate Water Rat Serum Rat Serum Cat# 015-4, Burdick & Jackson, Muskegon, MI 49442 Cat# 24,019-2, Aldrich, Milwaukee, WI 53201 Cat# 100-4, Burdick & Jackson, Muskegon, MI 49442 Cat# 230-4, Burdick & Jackson, Muskegon, MI 49442 A mixture of 0.11 mM trifluoroacetic acid (J.T. Baker, Phillipsburg, NJ 08865), 0.52 mM Rhodapex CO-436, 1 mM ABEX EP110 and 1 mM ABEX EP-120 (all three supplied by Rhone-Poulenc, Inc., Cranbury, NJ 08512) in 1:1 methanolrwater. Cat# 3509-01, J.T. Baker, Phillipsburg, NJ 08865 Cat# 3604-01, J.T. Baker, Phillipsburg, NJ 08865 HPLC Grade, Cat# J360-07, J.T. Baker, Phillipsburg, NJ 08865 High Purity (NANOpure), Bamstead Model D7331 Ultrapure Water System, Dubuque, LA 52001 Lampire Biological Laboratories, Piperville, PA 18947 Harlan Bioproducts for Science, Inc., Indianapolis, IN 46229 A.6.0 MATERIALS AND EQUIPMENT Mass Spectrometer PE SCIEX API 365 atmospheric pressure ionization tandem triple quadrupole mass spectrometer equipped with TurboIonSprayTM interface, PE SCIEX, Concord, Ontario L4K 4V8 Data system API Standard Software, MacQuan v 1.4, LC2Tune v 1.3, MacDAD v 1.3, Bundler v 1.3, Multiview v 1.3, 001241 ADVANCED B IO A N A L Y T IC A L S E R V IC E S . IN C . Page 39 o f 50 99VD JA O I.M I.D O C and Sample Control v 1.3, on a Power Macintosh, PE SCIEX, Concord, Ontario L4K 4V8 HPLC Pump Shimadzu LC-10AD pump, Shimadzu Co., Columbia, MD 21046 LC Pump Controller Shimadzu SCL-10A, Shimadzu Co., Columbia, MD 21046 Autosampler Waters 717plus, Waters Associates, Millipore Corporation, Milford, MA 01757 HPLC Column Betasil C,,, 5 pm particle size, 2.1 x 50 mm, Cat #852055-701, Keystone Scientific, Inc., Bellefonte, PA 16823 Harvard syringe pump 11 Harvard Apparatus Inc., South Natick, MA 01760 Multi-tube vortexer Cat# 58816-115, VWR Scientific, West Chester, PA 19380 Balance Model FX-300, AND Ltd., Tokyo, Japan pH Meter Model 340, Coming Inc., Coming, NY 14830 Mechanical shaker Cat# 6000, Eberbach Corp., Ann Arbor, MI 48106 NANOpure-UV water purification system Bamstead, Dubuque, IA 52001 Microbalance Model MT-5, Mettler-Toledo Inc., Hightstown, NJ 08520 Micro weigh boats Cat# 0219-0041, Perkin-Elmer Corp., Norwalk, CT 06859 Weigh boats Cat# 12577-025, VWR Scientific, West Chester, PA 19380 Sorvall RT-6000D refrigerated centrifuge Cat# 83071, DuPont Co., Wilmington, DE 19898 Beckman GS6KR Refrigerated centrifuge Beckman, Palo Alto, CA 94304 001242 ADVANCED B IO A N A L Y T IC A L S E P V IC E S . IN C . Page 40 o f 50 99VD JA 01.M I.D O C TurboVap LV Evaporator Pipettes Pipette tips Polypropylene tubes Screw-capped Polypropylene vials Screw-capped Polypropylene vials Plug Tite Multipurpose Caps Pyrex volumetric flask Solvent Filtration Apparatus Nylon Titan Membrane Filters PEEK tubing Liquid Nitrogen Gastight Glass Syringes ' Cat# 43750, Zymark Instruments, Hopkinton, MA 01748 Cat# P-5000, P-1000, P-200, P-100, Rainin Instrument Co., Woburn, MA 01888 RT-20, RT-200, C-5000, CP-25, CP-50, CP-250, Rainin Instrument Co., Woburn, MA 01888 13 mm x 100 mm, Cat# 15070-574, VWR Scientific, West Chester, PA 19380 16.5 x 57 mm, Cat# 60.542, Sarstedt, Inc., Newton, SC 28658 16.5 x 101 mm, Cat# 60.541, Sarstedt, Inc., Newton, SC 28658 Cat# 78-127-0019-100, Elkay Products, Inc., Worcester, MA 01607 Cat# 28014P-10, 28014P-25, 28014P-100, 28014P1000, Kimble/Kontes, Vineland, NJ 08360 Cat# 953781-0000, 953751-0000, 953753-0000, 953826-0000, 953827-0000, Kimble/Kontes, Vineland, NJ 08360 0.45 pm Scientific Resources Inc., Cat# 74547-NN, Eatontown, NJ 07724 0.005" i.d. x 1/16" o.d., Cat# 1535, Upchurch Scientific Inc., Oak Harbor, WA 98277 Supplied in-house from bulk tank, BOC Gasses, Buffalo, NY 14210-2005 Cat# 1750, Hamilton Company, Reno, NV 89510 Other general laboratory glassware and supplies were used. ADVANCED BIO A N A LV TIC A L Page 41 o f 50 001243 99VD JA O I.M I.D O C A.7.0 PREPARATION OF SOLUTIONS NANOpure water (18 megaohm-cm.) or equivalent should be used wherever water is called for. Mix all solutions well. A.7.1 Preparation of Analytical Standard Stock Solutions Analytical Standard Stock Solution, PFOS (1 mg/mL): Weigh approximately 5 mg of PFOS (after correction for purity) on a microbalance and transfer it to a polypropylene vial. Dilute with the appropriate volume o f methanol to yield a 1 mg/mL solution. Prepare a fresh solution every three months. Store the solution at 4 C and bring to ambient temperature before use. Standard Spiking Solutions Prepare Standard Working Solutions A through J in 5-mL class "A" volumetric flasks according to the following dilution scheme. Dilute to the 5-mL mark with water to yield the final concentration. Prepare fresh solutions every three months. Store the solutions at 4 C and bring to ambient temperature before use. Standard Working A B C D> E F G H I J Volume Spiked Solution Used (pL) 1000 Stock 800 Stock 400 Stock 200 Stock 100 Stock 250 200 pg/mL (A) 125 200 pg/mL (A) 62.5 200 pg/mL (A) 500 10 pg/mL (F) 250 10 pg/mL (F) PFOS Final Cone. (pg/mL) 200 160 80 40 20 10 5 2.5 1.0 0.5 ADVANCED B IO A N A L Y T IC A L S E R V IC E S . IN C . Page 42 o f 50 001244 99VD JA 01.M I.D O C A.7.2 Preparation of Internal Standard Stock Solutions Internal Standard Stock Solution, Tetra-H-PFOS (1 mg/mL): Weigh approximately 5 mg of Tetra-H-PFOS on a microbalance and transfer it to a polypropylene vial. Dilute with an appropriate volume of methanol to yield a 1 mg/mL solution. Prepare a fresh solution every three months. Store the solution at 4 C and bring to ambient temperature before use. A.7.3 Preparation of Internal Standard W orking Solution Internal Standard W orking Solution (4 pg/mL Tetra-H-PFOS): Add 400 pL of Internal Standard Stock Solution to a 100-mL class "A" volumetric flask. Dilute to the mark with water to give a 4 pg/mL Tetra-H-PFOS solution. Store the solution at 4 C and bring to ambient temperature before use. Prepare fresh solution as needed. A.7.4 Preparation of Standard Curve and Control Blank Prepare fresh calibration standards for each analytical run by combining 360 pL of rat control serum and 40 pL of the analytical standard working solution (see table below) in labeled 1.7-mL microtubes. Prepare control blanks and zero samples by combining 360 pL o f rat control serum with 40 pL of water. Vortex each for 60 seconds. mmm AOVANCED BIO A N A LY TICA L S E R V IC E S . INC. Page 43 o f 50 001245 99VD JA 01.M l.D O C Analytical Standard Dilution Table: Standard # 10 9 8 7 6 5 4 3 2 1 Volume Spiked (pL) 40 40 40 40 40 40 40 40 40 40 Solution to use A B C D E F G H I J pL control serum 360 360 360 360 360 360 360 360 360 360 Final Cone. (pg/mL) 20 16 8 4 2 1 0.5 0.25 0.1 0.05 A.7.5 Preparation of Other Solutions 0.25 M Sodium Carbonate/0.25 M Sodium Bicarbonate: Weigh 26.5 g of sodium carbonate and 21 g of sodium bicarbonate and dissolve in approximately 900 mL of HPLC-grade water. Transfer the solution to a 1000-mL volumetric flask and dilute to the mark with HPLC-grade water. Mix the solution thoroughly. Store at ambient temperature. Prepare a fresh solution every three months. 1 M Ammonium Acetate: Add 7.7 g of ammonium acetate to a 100-mL volumetric flask, dissolve in NANOpure water, and stir until completely dissolved. Dilute to the mark with NANOpure water. Store at ambient temperature. Prepare a fresh solution every three months. 2 mM Ammonium Acetate: Combine 2 mL of 1M ammonium acetate and 900 mL NANOpure water in a 1000-mL volumetric flask; stir until completely mixed. Dilute to the mark with NANOpure water. Store at ambient temperature. Prepare a fresh solution every three months. 001246 ADVANCED BIO A N A LY TICA L SE R V IC E S. INC Page 44 o f 50 99VDJA01.M J.DO C 10 M Sodium Hydroxide: Add 40 g of sodium hydroxide to a 100-mL volumetric flask, dissolve in NANOpure water, and stir until completely dissolved. Dilute to the mark with NANOpure water. Store at ambient temperature. Prepare a fresh solution every three months. 1 M Sodium Hydroxide: Add 10 mL of 10 M sodium hydroxide to a 100-mL volumetric flask. Dilute to the mark with NANOpure water; stir until completely mixed. Store at ambient temperature. Prepare a fresh solution every three months. 0.5 M Tetrabutylammonium Hydrogen Sulfate, pH 10: Weigh 16.98 g of tetrabutylammonium hydrogen sulfate and dissolve in 40 mL of NANOpure water. Adjusted the pH to 10.0 with 10 M and 1 M sodium hydroxide. Transfer the solution to a 100-mL volumetric flask and dilute to the mark with NANOpure water. 10:90 MethanoI:2 raM Ammonium Acetate (Mobile Phase Eluent A): Add 100 mL of methanol and 900 mL of 2 mM ammonium acetate to a 1000-mL Pyrex bottle. Mix the solution thoroughly and filter the solution through a 0.45 pM filter with a vacuum flask. Store at ambient temperature. Prepare a fresh solution every three months. 90:10 Methanol:2 mM Ammonium Acetate (Mobile Phase Eluent B): Add 900 mL of methanol and 100 mL of 2 mM ammonium acetate to a 1000-mL Pyrex bottle. Mix the solution thoroughly and filter the solution through a 0.45 pM filter with a vacuum flask. Store at ambient temperature. Prepare a fresh solution every three months. A.8.0 PREPARATION OF QUALITY CONTROL SAMPLES A.8.1 Preparation of Quality Control Stock Solutions QC Stock Solution, PFOS (1 mg/mL): Weigh approximately 5 mg of PFOS (after correction for purity) on a microbalance and transfer it to polypropylene vial. Dilute 0 0 1 2 4 7 ADVANCED BIO A N A LY TICA L S E R V IC E S . INC. Page 45 o f 50 99V D M O I.M I.D O C with the appropriate volume of methanol to yield a 1 mg/mL solution. Store the solution at 4 C and bring to ambient temperature before use. Prepare a fresh solution every three months. A.8.2 Preparation of Serum QC Samples QC4 (Dilution QC, 100 pg/mL PFOS): Add 1000 pL QC Stock Solution to a 10mL class "A" volumetric flask to yield a 100-pg/mL solution. Dilute to the mark with control serum, cap and mix. Aliquots of approximately 0.2 mL are placed in polypropylene vials and stored frozen at -20 C. QC3 (18 pg/mL PFOS): Add 450 pL QC Stock Solution to a 25-mL class "A" volumetric flask to yield a 18-pg/mL solution. Dilute to the mark with control serum, cap and mix. Aliquots of approximately 0.5 mL are placed in polypropylene vials and stored frozen at -20 C. QC2 (6 pg/mL PFOS): Add 150 pL of QC Stock Solution to a 25-mL class "A" volumetric flask to yield a 6-pg/mL solution. Dilute to the mark with control serum, cap and mix. Aliquots of approximately 0.5 mL are placed in polypropylene vials and stored frozen at -20 C. QC1 (0.2 pg/mL PFOS): Add 111.1 pL of QC3 to a 10-mL class "A" volumetric flask to yield a 0.2-pg/mL solution. Dilute to the mark with control serum, cap and mix. Aliquots of approximately 0.5 mL are placed in polypropylene vials and stored frozen at -20 C. A.9.0 LIQUID-LIQUID EXTRACTION PROCEDURE 1. Prepare fresh calibration standards for each run according to Section A.7.4. 2. Thaw QC samples. 3. Prepare QC4 samples (diluted 1 in 10) in two steps: a) Add 180 pL of control serum and 20 pL of QC4 into 1.7-tnL microtubes and 001248 ADVANCED BIO A N A LV TIC A L e icro v/ ir-cre : iM r- Page 46 o f 50 99VD JA 01.M I.D O C vortex. b) Aliquot 50 pL of diluted QC4 into labeled 16 x 100 mm polypropylene tubes. 4. Aliquot 50 pL of each control blank, zero sample, calibration standard, and quality control samples 1-3 into labeled 16 x 100 mm polypropylene tubes. Standards and blanks are analyzed in duplicate. All QC samples are analyzed in replicates of five. 5. Add 1 mL of 0.5 M tetrabutylammonium hydrogen sulfate, pH 10 to each tube. 6 . Add 2 mL of 0.25 M sodium carbonate/0.25 M sodium bicarbonate to each tube. 7. Add 500 pL of the Internal Standard Working Solution to each tube (except control blank, add 500 pL water). 8. Add 5 mL ethyl acetate to each tube. 9. Shake the tubes on a reciprocal shaker at medium speed for 20 minutes. 10. Centrifuge the tubes at 3000 rpm for 20 minutes at 20 C. 11. Freeze the aqueous phase in an acetone/dry ice bath and keep the tubes in the bath an additional 5 minutes. 12. Transfer the ethyl acetate layer to a fresh, labeled 13 x 100 mm polypropylene tube. 13. Evaporate the ethyl acetate layer to dryness in a TurboVapTM at approximately 20 C under nitrogen. 14. Reconstitute the dried extracts in two steps: Add 500 pL acetonitrile and vortex for 60 seconds. Then add 500 pL water and vortex for 60 seconds. 15. Transfer 200 pL of the extract to a labeled polypropylene autosampler vial. A. 10.0 INSTRUMENT CONDITIONS HPLC Conditions: Eluent (gradient) WWW ADVANCED B IO A N A L Y T IC A L S E R V IC E S . IN C . Mobile Phase A: 10:90 methanol:2 mM ammonium acetate (v/v) Mobile Phase B: 90:10 methanol:2 mM ammonium acetate (v/v) 001249 Page 47 o f 50 99V D M 01.M J.D O C Eluent Gradient Conditions: Time (min) 0 1.0 5 5.5 8 Flow Rate Autosampler Autosampler Temperature Injection Volume HPLC Column HPLC Column Temperature Typical Initial Column Pressure Autosampler Run Time %B 45 100 100 45% Stop 300 pL/min. Waters 717plus Ambient 3 to 5 pL Keystone Betasil C,,, 5 pm particle size, 2.1 mm x 50 mm Ambient 90 bar 8.0 minutes Analyte Representative Retention Time (minutes) PFOS Tetra-H-PFOS (IS) 3.1 2.9 Mass Spectrometer Conditions: Curtain Gas Nebulizer Gas TurboIonSprayTM Temperature TurboIonSprayTM Auxiliary Gas UHP Nitrogen UHP Nitrogen 400 C UHP Nitrogen at 8 L/min ADVANCED B ID A N A L Y T IC A L S E R V IC E S . IN C . Page 48 o f 50 001250 99VDJAO I.M J.DO C Ions Monitored: Analvte PFOS Tetra-H-PFOS (IS) Ionization Mode Ion Spray Voltage Declustering Potential MS Acquisition Time Pause Time Ion Monitored m/z = 499.0 m/z = 427.0 Negative Ion -3000 V -35 V 5 minutes 2 ms Dwell Time 500 ms 500 ms Calibrate the mass axis of the instrument by infusion of Rhodapex mass calibration solution (Section A.5.0) at a flow rate of 10 pL/min. Optimize the sensitivity of the instrument using an infusion of a 10 pg/mL solution of the analyte at 10 pL/min into a flow o f 190 pL/min of mobile phase using the gradient condition at which the analyte elutes from the LC column (100% eluent B). Mass spectral peak widths should be approximately 0.6 amu at half-height. One set of calibration standards was injected at the beginning and one set of calibration standards was injected at the end of each analytical run (tray). A. 11.0 CALCULATIONS Calculated concentrations are based on peak area ratios of PFOS to Tetra-H-PFOS. The peak area for the analyte ion is divided by the peak area for the corresponding internal standard ion. Data generated from samples in this study were acquired and integrated using PE SCIEX software applications Sample Control (v. 1.3) and MacQuan (v. 1.4). The validation data were formatted in the ABS laboratory information management system (Watson, v.5.3.1.01). All data were rounded to no less than three significant figures by ABS prior to reporting. 001251 ADVANCED B IO A N A L Y T IC A L S E R V IC E S . IN C . Page 49 o f 50 99V D M O I.M I.D O C Acceptance Criteria for Sample Analysis Calibration standards: The coefficient of determination (r2) must be >0.98. At least three-fourths of the individual calibration standard replicates will have deviations within 15% from their nominal concentrations. LLQ acceptance criteria: At least one replicate at the LLQ must exhibit a deviation within 20% from its nominal concentration. If this criterion is not met, both replicates are rejected and the standards at the next higher level are subjected to the same test. Quality control samples: At least two-thirds of the individual QC sample replicates will have deviations within 15% from their nominal concentrations, with at least one replicate at each QC concentration meeting this criterion. ADVANCED B IO A N A L Y T IC A L S E R V IC E S . IN C . Page 50 o f 50 001252 99VD JA O I.M I.D O C Perfluorooctanesutfonate CAS Number-2759-39-3 Attachment D Dose C onfirmation A nalyses 3M Environmental Laboratory Report No. FACTTox-108 Laboratory Request Number (LRNHJ2779 Proprietary and Confidential 001253 Argus 418-014 Study: Product Number(Test Substance): Matrix: Method/Revision: Analytical Equipment System Number: Instrument Softwarc/Vcrsion: Date of Exlraclion/Analyst: Date of Analysis/Analyst: Date of Data Reduction/Analyst: Argus 418-014, Oral (Gavage) Cross-Fostering Study of PFOS in Rats PFOS (T-6295.9) Tween Dosing Vehicle See list to right ETS-8-4.1 & ETS-8-5.1 See Attachments Madeline04l098 MassLynx 3.2 9/13/99 IAS See Attachments Sec Attachments 9/15/99 IAS 9/16/99 IAS Sample Data 0 0 0 Grp I Tween Dosing Confirmation Group Dose Sample # QC Group 1 Control 0.0 mg/kg/day Group 2 1.6 mg/kg/day 0.32 mg/ml B-418-014-B, 11/1/98 (302ppb MS) B-418-014-B, 1/27/99 (302ppb MS) B-418-0I4-A (11/01/98) Diluted 1/1 B-418-014-A (1/27/99) Diluted 1/1 B-418-014-B (11/01/98) Diluted 1/808 B-418-014-B (1/27/99) Diluted 1/808 Limit of Quantitation Limit (LOQ) = PFOS = 30 ng/g Method Detection Limit (MDL): PFOS = 15 ng/g PFOS = Perfluorooctanesulfonate Expected Cone. PFOS ng/ml 201 201 0 0 320000 320000 PFOS Cone. ng/ml 199 141 0 0 252516 276069 PFOS % Recovery Accuracy 99% 70% <LOD <LOD 79% 86% NR = Sample not received nor reported. NA = Not Applicable Date Entered/Analyst: Date Verifted/Analyst: 9/27/99 GM L 001ZS4 ETS-8-4.1 Excel 97 Version SR2 Attachment D FACT Tox-108 dose confirm.xls 1/ 11/00 4:19 PM Perfluorooctanesu lionate CAS Number-2759-39-3 Attachment E Data S ummary Tables 3M Environmental Laboratory Report No. FACTTox-108 Laboratory Request Number (LRNHJ2779 Proprietary and Confidential 001-255 Perfluorooctanesulfbnate CAS Number-2759-39-3 3M Environmental Laboratory Report No. FACT Tox-108 Laboratory Request Number (LRN)-U2779 Table 1. Concentrations of PFOS in Rat Serum Samples from Study FACT Tox-108 < L L a Less than the Lower Limit of Quantitation (0.05pg/mL) 1 Rat N umber 1 "P"( R a t N u m b e r w i t h a 1 In d ic a t e s a P u p S a m p l e ) 18901 18901P 18903 18903P 18904 18906 18907 18907P 18908 18909 18910 1891OP 18911 18911P 18912 18913 18913P 18915 18915P 18917 18917P 18918 18918P 18919 18920 18922 18922P 18923 18923P 18924 18924P 18926 18926P 18927 18927P PFOS C o n c e n t r a t io n (pg/mL) 0.0507 <LLQ[0.0323]<(0.05) 4.80 35.7 <LLQ[0.0462]<(0.05) <LLQ[0.0329]<(0.05) 1.35 33.5 2.41 1.06 <LLQ[0.0316]<(0.05) <LLQ[0.0382]<(0.05) 0.0798 0.123 <LLQ[0.0376]<(0.05) <LLQ[0.0341]<(0.05) <LLQ[0.0398]<(0.05) <LLQ[0.0221]<(0.05) <LLQ[0.0242]<(0.05) <LLQ[0.0355]<(0.05) <LLQ[0.0221]<(0.05) <LLQ[0.0323]<(0.05) <LLQ(0.0247]<(0.05) <LLQ[0.0340]<(0.05) 1.96 <LLQ[0.0412]<(0.05) <LLQ[0.0367]<(0.05) <LLQ(0.0291]<(0.05) <LLQ[0.0331]<(0.05) 2.72 30.7 <LLQ[0.0323]<{0.05) <LLQ[0.0229]<(0.05) <LLQ[0.0305]<(0.05) <LLQ[0.0318]<(0.05) Proprietary and Confidential 001256 Perfluorooctanesuifbnate CAS Number-2759-39-3 3M Environmental Laboratory Report No. FACT Tox-108 Laboratory Request Number (LRNHJ2779 Table 1. Concentrations of PFOS In Rat Serum Samples from Study FACT Tox-108 (continued) <LLQ: Less than the Lower Limit of Quantitation (0.05|jg/mL) I Ra t N um ber 1 (R a t N u m b e r w it h a " P " 1 In d ic a t e s a P u p S a m p l e ) 18929 18931 18931P 18932 18934 18934P 18935 18935P 18936 18936P 18937 18938 18939 18939P 18940 18940P 18941 18942 18943 18943P 18946 18947 18948 18950 18951 18951P 18952 18952P 18953 18953P 18956 18956P 18957 18957P 18958 18958P PFOS C o n c e n t r a t io n (pg/mL) 2.40 <LLQ[0.0382]<(0.05) <LLQ[0.0196]<(0.05) <LLQ[0.0399]<(0.05) 1.25 34.1 <LLQ[0.0316]<(0.05) <LLQ[0.0206]<(0.05) <LLQ[0.0302]<(0.05) <LLQ[0.0230]<(0.05) 1.80 <LLQ[0.0359]<(0.05) <LLQ[0.0311 ]<(0.05) <LLQ[0.0308]<(0.05) <LLQ[0.0330]<(0.05) <LLQ(0.0231]<{0.05) 5.34 1.12 65.1 82.2 157 86.9 96.1 66.4 72.4 59.2 90.8 48.3 67.0 53.8 97.5 89.3 69.1 93.2 68.3 56.2 Proprietary and Confidential 001257 Perfluorooctanesutfbnate CAS Number-2759-39-3 3M Environmental Laboratory Report No. FACT Tox-108 Laboratory Request Number (LRN)-U2779 Table 1. Concentrations of PFOS in Rat Serum Samples from Study FACT Tox-108 (continued) <LLQ: Less than the Lower Limit of Quantitation (0.05pg/mL) Ra t N umber (Ra t N u m b e r w it h a "P " In d ic a t e s a P u p S a m p l e ) 18959 18960 18960P 18961 18961P 18963 18964 18964P 18965 18967 18968 18969 18970 18970P 18971 18972 18972P 18973 18973P 18974 18976 18977 PFOS C o n c e n t r a t io n (pg/mL) 59.2 78.0 93.6 69.1 47.6 83.6 85.6 92.9 75.9 74.2 80.9 78.6 99.3 96.9 218 66.9 58.2 81.9 79.5 172 77.9 124 Proprietary and Confidential 001258 Perfluorooctanesutfonate CA5 Number-2759-39-3 3M Environmental Laboratory Report No. FACTTox-108 Laboratory Request Number (LRNHJ2779 Table 2. Repeat Analysis of Rat Serum Samples from Study FACT Tox-108 <ULQ: Greater than the Upper Limit of Quantitation (20.0pg/mL) Ra t N u m b er (Rat Numberwrma "P" Indicatesa Pup Sample) 18903P 18907P 18924P 18934P 18971 TREATMENT T im e 0 Day 14 0 Day 14 0 Day 14 0 Day 14 1 Day 14 O r ig in a l C o n c e n t r a t io n Uig/mL| >ULQ-28.5284>(20) >ULQ-30.8172>(20) >ULQ-27.5496>(20) >ULQ-36.8737>(20) >ULQ-219.5172>(200) O r ig in a l C urve Num ber 3 3 3 3 1 Reason for R eassay 1 1 1 1 2 Reassay CONC. (ugimL) 35.7 33.5 30.7 34.1 218 Reassay C urve N um ber 5 5 5 5 3 Reasons for Reassay: Reasons for Reported Cone: 1) Run 3 concentration exceeded calibration range 2) Run 1 concentration exceeded calibration range 1) Run 5 concentration within calibration range 2) Run 3 concentration within calibration range R epo rted Conc. (ug/mL) 35.7 33.5 30.7 34.1 218 R easo nfo r Repo r ted Conc. 1 1 1 1 2 Table 3. Pharmacokinetic Subgroup--Concentrations of PFOS in Dam and Litter Serum Samples from Study FACT Tox-108 <LLQ: Less than the Lower Limit of Quantitation (0.05pg/mL) D osage G roup I Dam Number 18910 PFOS C o n c e n t r a t io n (pg/mL) <LLQ (0.0316]<(0.05) L it t e r N u m b e r 18910P PFOS C o n c e n t r a t io n (p g /m L ) <LLQ [0.0382]<(0.05) I 18911 0.0798 18911P 0 .1 2 3 I 18913 <LLQ [0.0341]<(0.05) 18913P <LLQ [0.0398]<(0.05) I 18915 <LLQ (0.0221]<(0.05) 18915P <LLQ [0.0242]<(0.05) 18926 <LLQ (0.0323]<(0.05) 18926P <LLQ (0.0229]<(0.05) I 18927 <IL Q [0.0305]<(0.05) 18927P <LLQ (0.0318]<(0.05) I 18939 <LLQ [0.0311]<(0.05) 18939P <LLQ (0.0308]<(0.05) I 18940 <LLQ (0.0330]<(0.05) 18940P <LLQ [0.0231 ]<(0.05) II 1 8 9 5 6 97.5 18956P 89.3 II 18971 218 18971P NA N A -S a m p le not assayed. Insufficient sam ple volum e for analysis. Proprietary and Confidential 001259 Perfluorooctanesulfonate CAS Number-2759-39-3 3M Environmental Laboratory Report No. FACTTox-108 Laboratory Request Number (LRN)-U2779 Table 4. PFOS Concentrations in Dam and Cross-Fostered Litter Serum Samples by Dosage Group <LLQ: Less than the Lower Limit of Quantitation (0.05pg/mL) -- : Sample not received I Dosage I Group Dam Number PFOS C o n c e n t r a t io n (pg/mL) S ubset Num ber C ross Foster Lit t e r N u m b e r PFOS C o n c e n t r a t io n (pg/mL) 1 1 Untreated Dams i 18903 i 18907 i 18908 i 18909 i 18918 i 18920 i 18924 i 18929 i 18934 i 18936 i 18937 i 18941 i 18942 I 1 Untreated Dams i 18901 i 18904 i 18906 i 18912 i 18917 i 18919 i 18922 i 18923 i 18931 i 18932 i 18935 i 18938 I II Treated Dam s h 18943 h 18947 h 18948 h 18957 h 18960 il 18964 h 18967 h 18968 h 18969 h 18970 h 18973 h 18974 II Treated Dam s h 18946 h 18950 it 18951 h 18952 h 18953 h 18958 h 18959 h 18961 it 18963 h 18965 h 18972 h 18976 h 18977 4.80 1.35 2.41 1.06 <LLQ[0.0323]<(0.05) 1.96 2.72 2.40 1.25 <LLQ[0.0302]<(0.05) 1.80 5.34 1.12 0.0507 <LLQ[0.0462]<(0.05) <LLQ[0.0329]<(0.05) <LLQ[0.03761<(0.05) <LLQ[0.0355]<(0.05) <LLQ[0.0340]<(0.05) <LLQ[0.Q412]<(0.05) <LLQ[0.0291 ]<(0.05) <LLQ[0.0382]<(0.05) <LLQ(0.0399]<(0.05) <LLQ[0.0316]<(0.05) <LLQ[0.0359]<(0.05) 65.1 86.9 96.1 69.1 78.0 85.6 74.2 80.9 78.6 99.3 81.9 172 157 66.4 72.4 90.8 67.0 68.3 59.2 69.1 83.6 75.9 66.9 77.9 124 A Treated Litters A A A A A A A A A A A A AI 18976P 18963P 18953P 18961P 18946P 18952P 18972P 18950P 18959P 18977P 18958P 18951P 18965P B Untreated Litters B 18931P B 18932P B 18938P B 18935P B 18922P B 18923P B 18917P B 18919P B 18901P B 18904P B 18912P B 18906P C Treated Litters C 18968P c 18973P c 18970P c 18969P c 18974P c 18967P c 18964P c 18943P c 18957P c 18948P c 18947P c 18960P Untreated Litters D 18918P D 18929P D 18941P D 18920P D 18908P D 18937P D 18934P D 18909P D 18907P D 18942P D 18924P D 18903P D 18936P -- -- 53.8 47.6 -- 48.3 58.2 -- -- -- 56.2 59.2 -- <LLQ[0.01961<(0.05) -- -- <LLQ[0.0206]<(0.05) <LLQ[0.0367]<(0.05) <LLQ[0.0331 ]<(0.05) <LLQ[0.02211<(0.05) -- <LLQ[0.0323]<(0.05) -- -- -- -- 79.5 96.9 -- -- -- 92.9 82.2 93.2 -- -- 93.6 <LLQ[0.0247]<(0.05) -- -- -- -- -- 34.1 -- 33.5 -- 30.7 35.7 <LLQ[0.0230]<(0.05) Proprietary and Confidential 001260 Perfluorooctanesulfonate CAS Number-2759-39-3 3M Environmental Laboratory Report No. FACT Tox-108 Laboratory Request Number (LRN)-U2779 Attachment F A nalytical Reports from C ontributing Laboratories Proprietary and Confidential 001261 ADVANCED BIOANALYTICAL S E R V IC E S . INC. BIOANALYTICAL REPORT Title: Quantitative Determination of Perfluorooctanesulfonate (PFOS) in Rat Serum from Study #FACT-TOX-108 Using Turbo Ion Spray LC/MS Date: Authors: 27 August 1999 David J. Anderson, M.S. Amie J. Prince, B.S. Holly D. Ross, M.S. Prepared For: 3M Environmental Technology and Safety Services St. Paul, MN 55133-3331 ABS Report No.: Study Protocol: 99ADJA01 .MI.DOC FACT-TOX-108 Number of Pages: 16 Summary and Conclusions The concentration of perfluorooctanesulfonate (PFOS) was determined in rat serum samples collected during the study #FACT-TOX-108 using a sensitive, specific, accurate, and reproducible analytical method developed by Advanced BioAnalytical Services, Inc., Ithaca, NY. Rat serum samples (50 pL) were extracted by a liquid-liquid extraction procedure to isolate PFOS and the internal standard, lH,lH,2H,2H-perfluorooctane sulfonic acid (Tetra-H-PFOS). Following evaporation and reconstitution, sample extracts were analyzed by turbo ion spray liquid chromatography/mass spectrometry (LC/MS) in the negative ion mode. All samples were successfully analyzed within four runs. The lower limit of quantitation was 0.05 pg/mL for PFOS. The precision of this assay (RSD) as determined from the analysis of quality control samples for PFOS was <4.01%. The precision of this assay (RSD), as determined from the calibration standards for PFOS was <5.83%. The relative error (RE) of the assay, as determined from the analysis of the quality control samples ranged from -7.32 to 10.1%. The RE of the assay, as determined from the analysis of calibration standards ranged from -5.86 to 11.0%. 001262 15 Catherwood Road Ithaca, New York 14850 (607) 266-0665 Fax (607) 266-0749 Title: ADVANCED BIOANALYTICAL SERVICES SIGNATURE PAGE Quantitative Determination of Perfluorooctanesulfonate (PFOS) in Rat Serum from Study #FACT-TOX-108 Using Turbo Ion Spray LC/MS Reviewed by: Authorized for Release by: Kthleen Cormack, B.S. Associate Auditor 7 John IL. Perkins, Ph.D. Assistant Scientific Director Z9Atnn I W Date Date Date ADVANCED B IO A N A L Y T IC A L S E R V IC E S , IN C . Page 2 o f 16 001263 99AD JA O I.M I.D O C QAU STATEMENT Periodic inspections of the bioanalytical portion of study #FACT-TOX-108 were conducted by the Quality Assurance Unit of Advanced BioAnalytical Services (ABS) for compliance with EPA GLP regulations (40 CFR Part 792). The study was inspected on the following dates: 22 June 1999; 1, 2, 7, July 1999; 12 August 1999. Results of the inspection were reported to ABS Management and Project Team Leader on: 22 June 1999; 1, 2, 7, July 1999; 12 August 1999. Results of the inspections were reported to the Study Director on 12 August 1999. Based on the inspections and the data reviewed, this report is a complete and accurate representation of the data. Kathleen Corinack, B.S. Associate Auditor Date ADVANCED B IO A N A L Y T IC A L S E R V IC E S . IN C . Page 3 o f 16 001264 99ADJA01.M I.DO C TABLE OF CONTENTS SIGNATURE PAGE.............................................................................................................2 QAU STATEMENT..............................................................................................................3 TABLE OF CONTENTS.......................................................................................................4 LIST OF TABLES.................................................................................................................5 1. INTRODUCTION............................... 6 1.1. Study Description and Objective..............................................................................6 2. METHODS....................................................................................................................6 2.1. Analytical Procedure(s)....................................................... 6 2.2. Assay Site...................................................................................................................7 2.3. Data Processing..........................................................................................................7 3. RESULTS...................................................................................................................... 7 3.1. Assay Performance....................................................................................................7 3.2. Analytical Results for Study #FACT-TOX-108...................................................... 8 4. SUMMARY AND CONCLUSIONS...........................................................................8 5. DATA RETRIEVAL............................................ 8 6 . REFERENCES..............................:..............................................................................8 7. TABLES........................................................................................................................9 ADVANCED B IO A N A L Y T IC A L S E R V IC E S . IN C . Page 4 o f ! 6 001265 99ADJA01.M I.DO C LIST OF TABLES Table 1: Summary of Sample and Assay Informationfor Protocol FACT-TOX-108..... 9 Table 2: Inter-Assay Precision and Accuracy for PFOS Quality Control Samples in Rat Serum for Study #FACT-TOX-108................................................................. 10 Table 3: Inter-Assay Precision and Accuracy for PFOS Calibration Standards in Rat Serum for Study #FACT-TOX-108................................................................. 11 Table 4: Calibration Curve Statistics for the Determination of PFOS in Rat Serum for Study #FACT-TOX-108.....................................................................................12 Table 5: Concentrations of PFOS in Rat Serum Samples from Study #FACT-TOX-108 ............................................................................................................................. 13 Table 6: Repeat Analysis of Rat Serum SamplesfromStudy #FACT-TOX-108..........16 ADVANCED . B IO A N A L Y T IC A L S E R V IC E S . IN C . Page 5 o f 16 001266 99ADJAOI.MI.DOC 1. INTRODUCTION 1.1. Study Description and Objective The objective of this study was to determine the concentrations of perfluorooctanesulfonate (PFOS) in rat serum samples collected during the study #FACTTOX-108, "Oral (Gavage) Cross-Fostering Study of PFOS in Rats." Rat serum samples were analyzed using a liquid-liquid extraction and turbo ion spray liquid chromatography/mass spectrometry (LC/MS) assay. 2. METHODS 2.1. Analytical Procedure(s) PFOS concentrations were determined in rat serum according to the validated LC/MS method (1). Serum samples (50 pL) were extracted by a liquid-liquid extraction procedure using ethyl acetate to isolate PFOS and the internal standard (IS), lH,lH,2H,2H-perfluorooctane sulfonic acid (Tetra-H-PFOS) from rat serum. Following evaporation to dryness and subsequent reconstitution, sample extracts were separated by reversed-phase chromatography on a 2.1 x 50 mm (5 pm) BetasilTM Clg column (Keystone Scientific, Inc., Bellefonte, PA) with an initial mobile phase of 55% Eluent A (10:90 methanol:2 mM ammonium acetate) and 45% Eluent B (90:10 methanol:2 mM ammonium acetate). PFOS concentrations were determined by turbo ion spray liquid chromatography/mass spectrometry (LC/MS) in selected ion monitoring (SIM) mode. The negative ions monitored were: m/z 499.0 for PFOS [M-K]' m/z = 427.0 for Tetra-H-PFOS [M-H]' Study sample concentrations were determined from a weighted (i/y2), quadratic regression of peak area ratios (peak area of PFOS/peak area of Tetra-H-PFOS) versus nominal concentration of ten calibration standards. The nominal concentrations of the calibration standards were 0.05, 0.1, 0.25, 0.5, 1, 2,4, 8, 16, and 20 pg/mL PFOS. The lower limit of quantitation (LLQ) for this assay was determined to be 0.05 pg/mL PFOS. Quality control (QC) serum samples at three different concentrations (0.2, 6, and 18 pg/mL PFOS) were analyzed with each assay batch in replicates of four. Dilution QC samples (QC4, 100 pg/mL) were prepared at each dilution that was used during a particular assay and were analyzed with each assay batch in replicates of four as needed. The acceptance criteria for calibration standards stipulated that the back-calculated concentrations of at least three-fourths of the individual calibration standard replicates must not deviate more than 15% from their nominal concentration (except at the LLQ). At least one duplicate at the LLQ must exhibit a deviation within 20% of the nom in^jQ 1 2 6 V ADVANCED B IO A N A L Y T IC A L S E R V IC E S . IN C . P a g e 6o fl6 99ADJA01.MI.DOC concentration . The coefficient of determination (r) must be 0.98. The acceptance criteria followed are in accordance with ABS Standard Operating Procedures. The acceptance criteria for the quality control samples stipulated that at least two-thirds of the individual QC sample replicates must not deviate more than 15% from their nominal concentrations. At least one replicate at each QC concentration must exhibit a deviation within 15%. A summary of the sample and assay information for study #FACT-TOX-108 is given in Table 1. 2.2. Assay Site All samples were analyzed at Advanced BioAnalytical Services, Inc., Ithaca, NY. 2.3. Data Processing The data were collected using turbo ion spray LC/MS selected ion monitoring (SIM) in the negative ion mode. Peak areas were integrated by the PE SCIEX program MacQuan, version 1.4, residing on a Macintosh computer. Following peak area integration, the results tables from MacQuan were saved as text files and uploaded to the ABS file server where a weighted (l/y2) quadratic regression was performed using the software package Watson (v 5.3.1.01 PSS, Inc., Wayne, PA 19087). Calculations were performed on unrounded numbers. All calibration standard and QC results were rounded to no less than three significant figures before reporting. 3. RESULTS 3.1. Assay Performance The performance of the assay for PFOS as determined from the analysis of daily quality control samples is documented in Table 2. The inter-assay precision (RSD) of quality control samples ranged from 1.57 to 4.01% for PFOS. There was no marked inaccuracy in the results from these quality control samples; the relative error (RE) ranged from -7.32 to 10.1%. The performance of daily calibration curves is documented in Table 3. The inter-assay precision (RSD) of the standards ranged from 1.11 to 5.83% for PFOS. There was no marked inaccuracy in the results from these standards; the relative error (RE) ranged between -5.86 to 11.0%. 001268 ADVANCED B IO A N A L Y T IC A L S E R V IC E S . IN C . Page 7 o f 16 99ADJA01.MI.DOC The slope, y-intercept, and coefficient of determination (r2) for each analytical run are presented in Table 4. The r2values ranged from 0.9954 to 0.9982 for PFOS in rat serum. All ABS personnel assigned to analyze samples for this project were required to successfully extract quality control (QC) samples and calibration standards. Results of the extraction were reviewed by a trained analyst against the pre-defined acceptance criteria for this assay. Results are maintained in the study records of ABS. 3.2. Analytical Results for Study #FACT-TOX-108 PFOS concentrations in rat serum from Study #FACT-TOX-108 are presented in Table 5. All data were rounded to no less than three significant figures before reporting in Table 5. Study samples requiring repeat analysis for Study #FACT-TOX-108 are presented in Table 6 . 4. SUMMARY AND CONCLUSIONS The concentration of PFOS was determined in rat serum samples collected during the study #FACT-TOX-108. PFOS was isolated from serum by a liquid-liquid extraction procedure and determined by LC/MS. The quality of the determinations was satisfactory throughout. The LLQ was 0.05 )ig/mL PFOS. The precision of the assay, as determined from the analysis of quality control samples was <4.01% for PFOS. 5. DATA RETRIEVAL The calculated concentration data from the analysis of rat serum samples for Study #FACT-TOX-108 are maintained on file in the archives of the Advanced BioAnalytical Services, Inc., Ithaca, NY in ABS Notebook 2319. 6. REFERENCES 1. Advanced BioAnalytical Services Validation Report 99VDJA01 .MI.DOC. Method Validation for the Quantitation of Perfluorooctanesulfonate (PFOS) in Rat Serum by Turbo Ion Spray LC/MS. David J. Anderson, M.S. Amie J. Prince, B.S., and Holly D. Ross, M.S. 26 August 1999. 001269 ADVANCED B IO A N A L Y T IC A L S E R V IC E S . IN C . Page 8 o f 16 99ADJA01.MI.DOC 7. TABLES Table 1: Summary of Sample and Assay Information for Protocol FACT-TOX108 Species/Matrix: Rat/Serum Sample Collection and Storage Information: Anticoagulant/Stabilizer: Reported Sample Collection Dates: Dates Received at ABS: Storage Temperature at ABS: None 7 January 1999 to 29 January 1999 16 June 1999 -20 C Assay Information: Assay Period: 22 June 1999 to 28 June 1999 Analyte: Potassium perfluorooctanesulfonate (PFOS) Analytical Standard: Lot No: Source: PFOS 171 3M Internal Standard: Lot No: Source: 1H,1H,2H,2Hperfluorooctane sulfonic acid (Tetra-H-PFOS) 59909 3M Calibration Range: Regression Method: Weighting Factor: Lower Limit of Quantitation: Upper Limit of Quantitation: i/yquadratic 2 0.05 pg/mL 20 pg/mL ADVANCED B ID A N A L Y T IC A L S E R V IC E S . IN C . Page 9 o f 16 001270 99ADJA01.MI.DOC Table 2: Inter-Assay Precision and Accuracy for PFOS Quality Control Samples in Rat Serum for Study #FACT-TOX-108 PFOS Concentration (ug/mL) Run No. QC1 QC2 QC3 QC4 QC4 0.2 6 18 100 100 (1 in 10 Dilution) (1 in 20 Dilution) 1 0.187 6.19 17.5 0.192 6.22 17.3 0.191 6.22 17.5 0.182 6.14 17.3 2 0.191 6.13 16.9 0.191 5.91 16.8 0.190 6.20 16.5 0.192 6.19 16.7 3 0.172 6.02 17.0 0.174 6.22 16.7 0.175 5.94 17.0 0.177 6.08 17.1 5 0.184 6.37 17.3 0.187 6.36 17.5 0.197 6.39 17.5 0.183 6.33 17.1 Mean 0.185 6.18 17.1 RSD (%) 4.01 2.33 1.92 RE -7.32 3.04 -5.00 110 109 110 108 NA NA NA NA 114 111 110 109 109 113 111 109 110 1.57 10.1 NA NA NA NA NA NA NA NA 107 106 100 103 NA NA NA NA 104 2.92 3.97 RSD = (SD/Mean) x 100% RE = [(Mean-Nominal)/Nominal] x 100% NA: Not Applicable. ADVANCED B IO A N A L Y T IC A L S E R V IC E S . IN C . Page 10 o f 16 001271 99ADJA01.MI.DOC HD > m 9 3] n< \\ m zn nui d 0 r> Table 3: Inter-Assay Precision and Accuracy for PFOS Calibration Standards in Rat Serum for Study #FACT-TOX-108 Run No. STD1 0.05 STD2 0.1 STD3 0.25 1 0.0479 0.105 0.240 0.0490 0.112 0.258 2 0.0508 0.102 0.245 0.0503 0.0985 0.240 3 0.0474 0.105 0.237 0.0513 0.109 0.245 5 0.0498 0.0936 0.231 0.0553 0.0982 0.244 Mean 0.0502 0.103 RSD (%) 4.90 5.83 0.242 3.20 RE 0.450 2.82 -3.04 RSD = (SD/Mean) x 100% RE = [(Mean-Nominal)/Nominal] x 100% PFOS Concentration (ng/mL) STD4 STD5 STD6 STD7 0.5 1 2 4 0.488 0.891 2.03 4.24 0.509 0.935 2.12 4.51 0.488 0.973 2.07 4.37 0.483 0.970 2.05 4.41 0.476 0.944 2.12 4.52 0.479 0.934 2.07 4.54 0.483 0.952 2.19 4.45 0.507 0.933 2.21 4.47 0.489 0.941 2.11 4.44 2.54 2.71 3.09 2.23 -2.21 -5.86 5.34 11.0 STD8 8 7.64 8.03 7.97 7.93 7.91 7.84 7.79 7.89 7.88 1.52 -1.55 STD9 16 15.4 16.1 15.4 15.9 15.6 15.8 15.8 15.6 15.7 1.62 - 1.86 STD 10 20 20.5 20.0 19.8 20.4 20.1 20.0 19.9 20.1 20.1 1.11 0.446 O Page 11 of 16 99ADJAOI.MI.DOC Table 4: Calibration Curve Statistics for the Determination of PFOS in Rat Serum for Study #FACT-TOX-108 Run No. Quadratic Linear Coefficient (A) Coefficient (B) 1 -0.0007277 0.1225 2 -0.0005896 0.1392 3 -0.0006758 0.1401 5 -0.0004928 0.1345 Mean -0.0006215 0.1341 y = Ax2 + Bx + C, weighted 1/y2 Intercept (C) 0.001121 0.0009484 0.001454 0.001190 0.001178 Coefficient of Determination (r2) 0.9959 0.9982 0.9959 0.9954 0.9964 ADVANCED B IO A N A L Y T IC A L S E R V IC E S . IN C . Page 12 o f 16 001273 99ADJA01.MI.DOC Table 5: Concentrations of PFOS in Rat Serum Samples from Study #FACTTOX-108 Rat PFOS Concentration Number* (pg/mL) 18901 0.0507 18901P <LLQ[0.0323]<(0.05) 18903 4.80 18903P 35.7 18904 <LLQ[0.0462]<(0.05) 18906 <LLQ[0.0329]<(0.05) 18907 1.35 18907P 33.5 18908 2.41 18909 1.06 18910 <LLQ[0.0316]<(0.05) 18910P <LLQ[0.0382]<(0.05) 18911 0.0798 1891IP 0.123 18912 <LLQ[0.0376]<(0.05) 18913 <LLQ[0.0341 ]<(0.05) 18913P <LLQ[0.0398]<(0.05) 18915 <LLQ[0.0221]<(0.05) 18915P <LLQ[0.0242]<(0.05) 18917 <LLQ[0.0355]<(0.05) 18917P <LLQ[0.0221]<(0.05) 18918 <LLQ[0.0323]<(0.05) 18918P <LLQ[0.0247]<(0.05) 18919 <LLQ[0.0340]<(0.05) 18920 1.96 18922 <LLQ[0.0412]<(0.05) 18922P <LLQ[0.0367]<(0.05) 18923 <LLQ[0.0291 ]<(0.05) 18923P <LLQ[0.0331]<(0.05) 18924 2.72 18924P 30.7 18926 <LLQ(0.0323]<(0.05) 18926P <LLQ[0.0229]<(0.05) 18927 <LLQ[0.0305]<(0.05) 18927P <LLQ[0.0318]<(0.05) 18929 2.40 *Rat Number with a "P" indicates a pup sample. <LLQ: Less than the Lower Limit of Quantitation (0.05 pg/mL) ADVANCED B ID A N A L Y T IC A L S E R V IC E S . IN C . Page 13 o f 16 001274 99ADJA01.MI.DOC Table 5: (Continued) Concentrations of PFOS in Rat Serum Samples from Study #FACT-TOX-108 Rat PFOS Concentration Number* (pg/mL) 18931 <LLQ[0.0382]<(0.05) 1893IP <LLQ[0.0196]<(0.05) 18932 <LLQ[0.0399]<(0.05) 18934 1.25 18934P 34.1 18935 <LLQ[0.0316]<(0.05) 18935P <LLQ[0.0206]<(0.05) 18936 <LLQ[0.0302]<(0.05) 18936P <LLQ[0.0230]<(0.05) 18937 1.80 18938 <LLQ[0.0359]<(0.05) 18939 <LLQ[0.0311]<(0.05) 18939P <LLQ[0.0308]<(0.05) 18940 <LLQ[0.0330]<(0.05) 18940P <LLQ[0.0231]<(0.05) 18941 5.34 18942 1.12 18943 65.1 18943P 82.2 18946 157 18947 86.9 18948 96.1 18950 66.4 18951 72.4 1895 IP 59.2 18952 90.8 18952P 48.3 18953 67.0 18953P 53.8 18956 97.5 18956P 89.3 18957 69.1 18957P 93.2 18958 68.3 18958P 56.2 *Rat Number with a "P" indicates a pup sample. <LLQ: Less than the Lower Limit of Quantitation (0.05 pg/mL) 001275 ADVANCED B IO A N A L Y T IC A L S E R V IC E S . IN C . Page 14 o f 16 99ADJA01.MI.DOC Table 5: (Continued) Concentrations of PFOS in Rat Serum Samples from Study #FACT-TOX-108 Rat PFOS Concentration Number* (ug/mL) 18959 59.2 18960 78.0 18960P 93.6 18961 69.1 18961P 47.6 18963 83.6 18964 85.6 18964P 92.9 18965 75.9 18967 74.2 18968 80.9 18969 78.6 18970 99.3 18970P 96.9 18971 218 18972 66.9 18972P 58.2 18973 81.9 18973P 79.5 18974 172 18976 77.9 18977 124 *Rat Number with a "P" indicates a pup sample. ADVANCED B IO A N A L Y T IC A L S E R V IC E S . IN C . Page IS of 16 0012V6 99ADJA01.MI.DOC (mi) m > z -< m H Table 6: Repeat Analysis of Rat Serum Samples from Study t/FACT-TOX-108 Rat Treatment Time Number* Original Cone. ug/ml 18903P 18907P 18924P 18934P 18971 0 Day 14 >ULQ-28.5284>(20) 0 Day 14 >ULQ-30.8172>(20) 0 Day 14 >ULQ-27.5496>(20) 0 Day 14 >ULQ-36.8737>(20) 1 Day 14 >ULQ-219.5172>(200) >ULQ: Greater than the Upper Limit of Quantitation. *Rat Number with a "P" indicates a pup sample. Original Curve Number 3 3 3 3 1 Reason for Reassay 1 1 1 1 2 Reassay Cone. ug/ml 35.7 33.5 30.7 34.1 218 Reassay Curve Number 5 5 5 5 3 Reported Cone. ug/ml 35.7 33.5 30.7 34.1 218 REASONS FOR REASSAY: 1) . Run 3 concentration exceeded calibration range. 2) . Run 1 concentration exceeded calibration range. REASONS FOR REPORTED CONC: 1) . Run 5 concentration within calibration range. 2) . Run 3 concentration within calibration range. Reason for Reported Cone. 1 1 1 1 2 Oo N Page 16 o f 16 vi 99ADJAO I.M I.DO C ADVANCED BIOANALYTICAL S E R V IC E S . INC. ADDENDUM TO THE BIOANALYTICAL REPORT Title: Addendum - Quantitative Determination of Perfluorooctanesulfonate (PFOS) in Rat Serum from Study #FACT-TOX-108 Using Turbo Ion Spray LC/MS Date: 13 October 1999 Addendum Report: 99ADJAOLMI.ADD.DOC Original Report: 99ADJA01 .MI.DOC Authors: David J. Anderson, M.S. Amie J. Prince, B.S. Holly D. Ross, M.S. Prepared For: 3M Environmental Technology and Safety Services St. Paul, MN 55133-3331 Number of Pages: 3 001278 15 Catherwood Road Ithaca. New York 14850 (607) 266-0665 Fax (607) 266-0749 ADVANCED BIOANALYTICAL SERVICES Title: SIGNATURE PAGE Addendum - Quantitative Determination of Perfluorooctanesulfonate (PFOS) in Rat Serum from Study #FACT-TOX-108 Using Turbo Ion Spray LC/MS ABS Report No.: 99ADM. ai.Mi.i SD.DOC Reported by: __________ David J. Anddj/son, MS'. Research Scientist 13 gc r Date Reviewed by: Authorized for Release by: John Rj Perkins, Ph.D. Assistant Scientific Director Accepted by: Kns Hanson, Ph.D. 3M Study Director Date JS-aiT.fo Date h! Pot Date ADVANCED B IO A N A L Y T IC A L S E R V IC E S , IN C . Page 2 o f 3 001279 99ADJAOI.MI.ADD.DOC ADDENDUM SUMMARY ADDENDUM Addition of Study Director signature on the Signature Page. The signature page of this addendum indicates acceptance of the original report. REASON FOR ADDENDUM Original GLP report requires the signature of the Study Director. ADVANCED B ID A N A L Y T IC A L S E R V IC E S . IN C . Page 3 o f 3 001280 99ADJA01 .M I.A D D .D O C Perfluorooctanesulfbnate CAS Number-2759-39-3 Attachment G Report S ignature Page 3M Environmental Laboratory Report No. FACTTox-108 Laboratory Request Number (LRNHJ2779 Date Vt (fp-u* Marvin T. Case, D.V.M., Ph.D., Sponsor Representative ate Dale L Bacon, Laboratory Manager " V f <r / o <? Date Proprietary and Confidential 001281 Study Title Analytical Laboratory Report on the Determination of the Presence and Concentration of Perfluorooctanesulfonate (PFOS) in the Serum of Sprague-Dawley Rats Exposed to Potassium Perfluorooctanesulfonate via Gavage REPORT AMENDMENT NO. 1 Amendment Date: 19 April 2000 Performing Laboratory 3M Environmental Technology & Safety Services 3M Environmental Laboratory 935 Bush Avenue St. Paul, MN 55106 Laboratory Project Identification ET&SS LRN-U2779 ET&SS FACT Tox-108 3M Environmental Laboratory 001282 FACT Tox-108 Report Amendment No. 1 This amendment modifies the following portion(s) of the final report: 1. Final Report: Make the following addition to the final report text. Add: After the final report was issued (signed and archived), it was discovered that the purity values stated for the analytical reference material and test substance were inaccurate. These values were based only on NMR analyses. Subsequent chemical characterization is occurring to determine the actual purity of theses substances (PFOS Lot 171 and 217). The final report will be amended to detail the analytical results using the purity values from the certificates of analysis when they are issued for PFOS Lot 171 and 217. Reason: To describe the changes that will be made to the final report when the certificate of analysis is issued for PFOS Lot 171 and 217. 2 . Final Report: Make the following addition to the exceptions list in the Statement of Compliance section. Amend to Read: The purity and stability of the test substance and analytical reference material are unknown and was not determined prior to the initiation of this study. Reason: The Statement of Compliance section was incomplete. 3. Final Report: The purity of the test substance is 99.28% (Lot 217), and analytical reference material is 99.49% (Lot 171) as listed in Table 2. Amend to read: The purity of the test substance and analytical reference material listed in Table 2 is unknown. Reason: The purity values stated for the test substance and analytical reference material listed in Table 2 are incorrect. 3M Environmental Laboratory 001283 Amendment Approval FA C T Tox-108 Report Amendment No. 1 Marvin T. Case, D. V.M., Ph.D., Sponsor Representative t C h <*- f h ---------Kristen J. Hansen, Ph.D., Study Director ' Date 6 V / 9/C Date 3M Environmental Laboratory 001284