Document K6e0DVkwKDJgB04p1D5Ja5yBQ

X/TPi P 11 I 000272 /M W * - EXPOSURE ASSESSMENT PROTOCOL W ork R eq u est N o.: 15076 S ervice C o d e N o.: 1361 P erfo rm in g In s titu tio n : Haskell Laboratory for H ealth and Environm ental Sciences Study T itle : E xp o su re A ssessm en t o f M easu red S erum P erflu o rin ated O ctan o ic A cid A n ion in th e M a n u fa c tu re o f A m m onium P erflu o ro o ctan o a te (A P FO ) a t F a ye ttev ille , NC S tu d y D irecto r: Joseph F. Viskocil, Jr. C IH S enior Industrial Hygienist Haskell Laboratory for H ealth and Environm ental Sciences 1 09 0 Elkton Road Newark, DE 191714 ~\ , C o -in vestig ato rs: Robin C . Leonard, Ph.D . Principal Epidem iologist Haskell Laboratory for Health and Environm ental Sciences DuPont Company Elkton Road, P.O . Box 50 Newark, DE 19714 M ary A. Kaiser, Ph.D . Research Fellow Corporate C enter for Analytical Sciences S . M ark Kennedy, P h.D . Haskell Laboratory for H ealth and Environm ental Sciences 1090 Elkton Road Newark, DE 191714 Company SM.ni2ed. Does M l <=">*i" TSCACBI 000273 Jennifer Locklear Industrial Hygienist Fayetteville W orks DuPont Company 2 2 8 2 8 N C Highway 8 7 W est Fayetteville, NC 2 83 0 6 Bonnie G . W ilson, RN Fayetteville Site Nurse Fayetteville W orks DuPont Company 2_2 8 2 8.. N C... Hi-g--h--w--a--y-- 8 7 W est Study Sponsor: David M . Rurak SHE Manager D uP ont Fluoroproduets BMP GRP 711-2216-C Company Sanitized. D oes not contain TSCA CBI 2 000274 To: David M. Rurak SHE Manager Fluoroproducts AUTHORIZATION FOR SERVICES From: Joseph F. Viskocil, Jr. CIH Senior Industrial Hygienist Haskell Laboratory Requested By: Date: December 12,2003 T itle: Exposure Assessment of Measured Serum Perfluorinated Octanoic Acid Anion in the Manufacture of Ammonium Perfluorooctanoate (APFO) at Fayetteville, NC Authority is requested to undertake the study identified above and described as fo llo w s: The primary purpose of this project is to measure worker exposure In the manufacture APFO in Fayetteville, NC. for assessment of the effectiveness of the industrial hygiene controls measures in place to minimize exposure to APFO. The secondary purpose of this occupational exposure project is to characterize APFO exposure in workers engaged in the manufacture of APFO and to determine 1) whether the major route of exposure is via inhalation or dermal, 2) the length of time required for the compound to reach steady-state, and 3) if there is a sufficient population, determine the length of time for serum levels to return to baseline following cessation of exposure. This will be accomplished by longitudinal sampling and analysis of serum PFOA anion levels according to a repeated samples design, air monitoring, and estimating dermal and oral contributions to overall exposure. Correlations between external and internal measures of exposure will be calculated. The data will be examined for within- and between-subject variation associated with specific tasks on the process line. AUTHORIZATION FOR SERVICES Additional Support Required from the Plant: Air sampling equipment including pumps, calibrators, battery elim inators,..... Personnel time - additional IH resources to conduct air sampling and investigate process upsets, data anomalies INITIATION DATE: Immediately upon ESTIMATED COMPLETION: approval- Estim ated approval date December 31,2009 December 15,2003 ESTIMATED COST: December 15, 2003 to December 31,2004 cost GENERAL LEDGER/SUBACCOUNT: 935Q -C V T 91601002-8980880Q Company Sanitized. D oes not contain TSC CBI 000275 If this request meets with your approval, enter the general ledger and subaccount, sign below, and return. AUTHORIZED APPROVALf V ^ M ^ X p A T E : >2\ j t s / o \ 00276 Table of Contents 0 Page No, I. R eview and A p p ro v a l............................................. ................ .......... ,.................. 6 Principal investigator and C o-investigators............. ......................... .......... 6 II. In tro d u c tio n ........... ............................ .............. . . . . -- ........, .............. ..................7 A bstract........... .............................................. ........................................... ............7 Definitions O b jectives........................ ..................................................................................... 7 B ackground............-- ... ........ ................................. .........................................7 ill. S u rv e illa n c e M e th o d s ....... ............................................ .......................... -- ...... 9 Participant Selection................. 9 D ata Collection___ _________ 9 D ata A n alysis........................................... ........................... ......... .......... . 11 Exposure A s s es s m e n t................ ............... ...................... .............................11 Factors That M ay Influence the Surveillance Design j and Analytical M e th o d s ...,.............. ....................... ..................... ..................12 Hum an Subject P rotection ...................................... 12 Protocol Am endm ents and D eviations.......... ............ 12 Q uality A ssu ran ce........... .................................. 12 IV . R e p o rtin g R e s u lts ------- ---- 13 V . R e q u ired R e s o u rce s ................. 13 V I. A rc h ivin g o f S tu d y D o c u m e n ts ..................... 13 V II. B ib lio g ra p h y ....... .......... 13 Exygen Blood Method Clayton Air M ethod W ipe Test Validation 4 000277 \ VIII. Appendix Appendix 1: Standard O perating Procedure: Blood Collection Appendix 2: Standard Operating Procedure: Process for Analysis of Blood Biomonitoring Sam ples Appendix 3: Standard Operating Procedure: Study-Specific Standard O perating Procedure: Docum entation, Accountability, and Procedures for Consent Form s, and Collection & Processing of Serum Sam ples Appendix 4: Standard O perating Procedure: Docum entation, Accountability, and S am ple Chain of Custody Requirem ents for the Shipping of Serum Sam ples Appendix 5: Blood Collection Checklist Appendix 6: Air Sam pling Strategy Appendix 7: Standard O perating Procedure: Q uality Assurance Procedures: Industrial Hygiene Laboratory and Field W ork Appendix 8: Standard O perating Procedure: Sam ple Handling, Chain-ofC u s to d y , Preservation and Shipping --Air Sam ples Appendix 9: Analytical M ethod for Air Sam ples Appendix 10: Air Monitoring D ata Form Appendix 11 : Perfluorooctanoic Acid W ipe T est Analysis 5 000278 REVIEW AND APPRO VAL STUDY DIRECTOR AND COINVESTIGATORS I attest that, to the best of my knowledge, this protocol is scientifically sound, describes the study and procedures to b e used with appropriate regulatory requirem ents, Good Industrial Hygiene Practices, th e research protocol and any am endm ents thereof. Study Director Approved: Operations Manager, Haskell Laboratory P ate: 6 000279 IN T R O D U C T IO N Abstract This occupational exposure study will characterize A P FO PFOA anion in workers engaged in the m anufacture of A PFO and to determ ine 1) w hether the m ajor route of exposure is via inhalation or derm al, 2) the length of tim e required for the com pound to reach steady-state, and 3 ) if there is a sufficient population, determ ine the length of tim e for serum levels to return to baseline following cessation of exposure. This will be accom plished by sam pling and analysis of serum P FO A anion levels according to a repeated sam ples design, air monitoring, and estim ating derm al and oral contributions to overall exposure. Correlations betw een external and internal m easures of exposure will be calculated. The d a ta will be exam ined for within- and betw een-subject variation associated with specific tasks on the process line. Definitions: A P F O - Ammonium Perfluorooctanoate Biomonitoring -Industrial Hygiene serum sam ples analyzed for PFO A anion used as an indication of occupational exposure LG - Liquid Chrom atography M S - M ass Spectrom etry P FO A - Perfluorooctanoic Acid P FO A Anion -perfluorooctanoate anion, the analyte of both PFO A and A P F O by LC /M S /M S Objective: T h e objective of this study is to m onitor serum levels of P FO A anion levels in m anufacturing and m anufacturing support w orkers of A P FO as a m easure the effectiveness of industrial hygiene controls. BACKGROUND Despite the absence of any persuasive scientific evidence establishing a causal association between exposure to PFO A and specific health effects, there continues to be 7 0002S0 questions raised on the health affects of PFO A. Epidem iological studies on the health effects of PFO A exposure have focused on occupational populations. A currently validated analytical method (with a lim it of quantification as low as 0 .5 ppb) allows increased understanding of background levels. In Fayetteville A P F O production began in the fall of 2 00 3 . Prior to start-up baseline blood serun sam ples w ere taken. W ith this background, w e have a unique opportunity to monitor exposure as a function of process tim e.This PFO A anion in serum levels will be m easured asroutinely an indication of the effectiveness of industrial hygiene controls. It is our goal to m inim ize em ployee exposure to A P FO in the A F P O m anufacturing process and to assure com pliance with the DuPont Acceptable Exposure Limit (AEL) of 0.01 m g/m 3. 8 000281 SURVEILLANCE M ETHODS PARTICIPANT SELECTION Biological m onitoring evaluates the absorption of P FO A /A P FO by m easuring the chem ical or it$ m etabolites in body fluids. Serum sam pling is the preferred m ethod for biological monitoring because there is a validated analytical m ethod for m easuring PFO A anion, rather than a m etabolite or the fluoride ion in this m edium . W orkers in the A P FO m anufacturing process are chosen because they are likely to have highest exposures a t the Fayetteville W orks, w e w ere able to get a pre-start up baseline, and w e can track serum levels with tim e from the process start-up. D u P o n fs goal is to m inim ize exposure. W orkers em ployed in the new A P FO production unit at Fayetteville W orks and for whom a baseline level of serum P FO A anion w as determ ined before beginning work in the unit will be included in this surveillance study. Participants will be all A P FO w orkers at the DuPont Fayetteville W orks site who volunteer for participation in this study, and sign a consent form. DATA COLLECTION Every em ployee assigned to the A P F O m anufacturing area will asked to participate in the PFO A anion blood m onitoring program . The em ployee will be asked to sign a consent form and a baseline blood sam ple will be taken before the em ployee begins work in the A P FO m anufacturing area. A fter the baseline sam ple is collected, th e em ployee will participate in the norm al 6-m onth sam pling program . Once in the blood m onitoring program , the em ployee is expected to have blood drawn and analyzed every 6-m onths for the duration of the study w hether or not they rem ain in the A P FO m anufacturing area. For each participant, date of birth and date of hire will be collected. For each task with potential for exposure to APFO , job title, work area and begin and end dates will be recorded. 9 000282 #) A total of 9 job titles and 5 work areas have been identified as having potential for exposure to AP.FO at the Fayetteville Plant. The num ber of unique task/w ork area com binations with potential for A P FO exposure will be determ ined. Blood Sampling Standard O perating Procedure: Collecting and Processing Blood Sam ples Appendix -1 Standard Operating Procedure: Process for Analysis of Blood Biomonitoring Sam ples -A ppendix - 2 Standard O perating Procedure: Study-Specific Standard O perating Procedure: Docum entation, Accountability, and Procedures for Consent Form s, and Collection & Processing of Serum Sam ples - Appendix - 3 Standard O perating Procedure: Docum entation, Accountability, and S am ple Chain of Custody Requirem ents for the Shipping of Serum Sam ples - Appendix - 4 Blood Collection Checklist - Appendix - 5 A ir S am pling A ir Sam pling Strategy - Appendix - 6 Standard O perating Procedure: Q uality Assurance Procedures: Industrial Hygiene Laboratory and Field W ork - Appendix - 7 Standard O perating Procedure: S am ple Handling, C hain-of-C ustody, Preservation and Shipping - Air Sam ples - Appendix - 8 Analytical M ethod for Air Sam ples - Appendix - 9 A ir Monitoring D ata Form - Appendix - 1 0 W ip e T est Perfluorooctanoic Acid W ipe Test Analysis - Appendix -1 1 10 000283 DATA ANALYSIS These exposure data will be analyzed as follows: 1. The general statistical procedures described below will be used in the analysis of the data. Analytical results of the regular sam ples will be analyzed using appropriate statistical m ethods. 2 . Descriptive statistics will be generated on each exposure m etric with all m easurem ents pooled. 3. W here there is sufficient data, regression of serum P FO A anion for each individual will be done on gender, age, m ost frequent ta s k , 4 . Analysis of variance for each tim e point m easurem ent of serum PFO A anion will determ ine the betw een- and within-individual variation, and 5. W here there is sufficient data, estim ate blood clearance rates of PFO A anion in w orkers by continued surveillance following cessation o f exposure EXPOSURE ASSESSMENT Exposure will be m easured by the following param eters: 1. Serum level of P FO A anion, prior to beginning work in th e AP'FO Manufacturing A rea and m easured every six m onths (per the unit schedule) 2 . Personal air monitoring of each participant, selected to cover all tasks and all shifts with potential for exposure to A P FO 3. A rea air monitoring for the laboratories, the m anufacturing lines, and the packing and shipping of A P FO 4. Estim ate the derm al contribution to the biomonitoring result fo r each worker, and 5. If necessary estim ate the contribution of other routes of exposure. Th e investigators will com pile a docum ent for each a rea that will sum m arize data related to A P FO exposure: (1) descriptions of processes w here exposure to A P F O m ay occur, including nature of and dates of engineering controls, changes in operating procedures and potential exposure points, (2) specific tasks perform ed in work areas w here exposure to A P FO m ay occur (3) w ork a re a nam es, job titles and tim e periods with potential for exposure, (4) docum entation of personal protective equipm ent use, (5) a ir sam pling d ata and analytical m ethods em ployed, (6) plant production records and (7) details on work conditions and practices gained from interviews and observations. h 002S4 Because derm al contact m ay be a significant route of exposure, estim ates of the am ount and frequency of skin contact will be used to estim ate a derm al dose using the surface area of the body covered, frequency of contact, length of contact and results of the surface wipe tests. Personal air monitoring data will be used as a direct m easure of inhalation exposure. FACTORS THAT MAY INFLUENCE THE SURVEILLANCE DESIGN AND ANALYTIC METHODS Analytic methods are new and recently validated. O nly one laboratory is G LP certified to do the blood analysis and only one laboratory is currently A iH A accredited for the air sam ple analysis. Timing of results could becom e an issue. The size of the surveillance population will not be large thus limiting the statistical power. The w ipe test analysis is com plex. Sam ple turn around tim e will not allow wipe tests to be used as a quick check for contam ination or the effectiveness of a decontam ination procedure. HUMAN SUBJECT PROTECTION H askell Q uality D irective Q D 007-C -001 requires com pliance with the U .S . Federal regulation known as the Health Insurance Probability and Accountability A ct of 2 00 3 (H IP A A ) and is supported by standard operating procedures that specify privacy and confidentiality requirem ents. PROTOCOL AMENDMENTS AND DEVIATIONS Intended (planned) changes to the protocol will be m ade by protocol am endm ent. Unintended or unexpected divergence from this protocol will be docum ented in the field raw d ata as a deviation. The Study D irector will be notified of all protocol am endm ents and deviations as soon as possible. All changes in or revisions of the approved protocol and the reasons for the changes will be form ally prepared by the Study Director, signed and dated by the Study D irector and Sponsor, and m aintained with the protocol. QUALITY CONTROL Field staff will record observed exposure data on standardized forms and send the observed d ata to Haskell Laboratory within seven days of collection. H askell staff will review form s and reconcile missing or discrepant information w here appropriate. Sam pled workers will be contacted if necessary to resolve discrepant, missing, or inconsistent data. Data will be transm itted electronically along with selected hardcopy data to the Study Director. 12 0Z8S REPORTING OF RESULTS Status reports will be review ed by the Study Director following the receipt of th e quarterly air monitoring results and upon receipt of blood results, final sum m ary report w ill be issued at the conclusion of the study. A communication of the final results will be to the site industrial hygienist who will com m unicate them to the study participants. RESOURCES TO CONDUCT THE STUDY Sam pling Equipm ent - 10 A ir Sampling Pum ps, chargers, battery elim inator, calibrator, and m aintenance log Personal Assistance - An experienced industrial hygienist to conduct air sam pling, provide field observations, ensure adherence to this protocol and investigate deviations from the protocol. S ee Air Sam pling Strategy - Appendix 6 ARCHIVING OF STUDY DOCUMENTS T he protocol, am endm ents to the protocol, raw data, consent form s, analytical results, com puter printouts, and final report will be archived through the standard procedures of the Haskell Laboratory Inform ation Services Group. BIBLIOGRAPHY R eference Exygen Blood Method R eference Clayton A ir M ethod W ipe Test Validation 13 0 0 0 2 8 6 Appendix-1 Standard Operating Procedure: Blood Collection 1. BLOOD COLLECTION SCHEDULE: Personal communications will be maintained with each study participant to confirm the dates to begin sampling. Blood samples will be drawn every 6-months. To cover all shifts, blood samples will be collected within a 10-day period. Baseline samples will be collected prior to an employee working in the area. The employee will then join the normal 6-month rotation. 1.1. Field Laboratory for Blood Collection Blood samples will be collected at Fayetteville Site Medical by the site nurse. 1.2. Aborted Blood Collection: If an anticipated sampling is canceled, workers will be notified promptly and sampling will be re-scheduled as-soon as-possible after the originally scheduled day. If there are medical or physical reasons that contraindicate the drawing of a blood sample on a scheduled day, then that study subject's sampling will be rescheduled to occur on the next day that the same or similar tasks will be performed. 2. PROCESSING BLOOD SAMPLES: At the field laboratory, blood samples will not sit ambient for more than 2 hours prior to serum separation. 3. TRAVEL BLANKS: iC. Travel Blanks will be an unused polypropylene tube supplied by Exygen. This balak will be shipped with collected blood samples to insure that the samples were not contaminated during shipping. 4. SAMPLE IDENTIFICATION: The site nurse/industrial hygienist will create a sampling identification system that identifies the site and person sampled. The chain-of-custody form will record the shipping date. 6. PROCESSING SAMPLES FOR FROZEN STORAGE AND SHIPMENT: 6.1. SAMPLE STORAGE: Appendix 3- Standard Operating Procedure: Documentation, Accountability, and Procedures for Consent Forms, and Collection & Processing of Serum Samples 000287 6.2. SAMPLE SHIPMENT: Appendix 4: Standard Operating Procedure: Documentation, Accountability, and Sample Chain of Custody Requirements for the Shipping of Seram Samples 7. SAMPLE ANALYSIS: The detailed procedures for analysis of study samples for PFOA airepresented in Attachment 3. 8. DATA ANALYSIS AND STATISTICAL METHODS: Analytical results of the regular samples will be analyzed using appropriate statistical methods. 9. CONTROL OF BIAS: Bias within the selection process for study participants and in the collection of blood samples should only arise from the voluntary nature of participation. Comparisons of the demographics, work histories, and usual tasks performed of the volunteers and the abstentions will be made in order to identify any systematic differences in the two groups. 10. REQUIRED FIELD DATA RECORDS: All field notes and data sheets must be filled out promptly, accurately, and in indelible ink by study personnel. All pages must be signed. If more than one individual records data on a page, it must be clear which individual recorded specific data. All entries must be dated on the day of entry and signed or initialed by the parson entering the data. The field staff will maintain original documents or legible verified copies of the following information: 1. A description of the tasks performed by the sampled participant, including personal protective equipment used. 2. A record of serum sample handling including time from collection to frozen storage. 3. A copy of the freezer temperature log for the period the serum samples were stored. 4. Personal work habits of each worker during his/her shift such as hand washing, sweating, accidental contamination, etc. Any observation that would provide an understanding of a potential route of exposure should be recorded. 11. PROTOCOL AMENDMENTS AND DEVIATIONS: Intended (planned) changes to the protocol will be made by protocol amendment. Unintended or unexpected divergence from this protocol will be documented in the field raw data as a deviation. The Study Director will be notified of all protocol amendments and deviations as C00288 soon as possible. All changes in or revisions of the approved protocol and the reasons for the changes will be formally prepared by the Study Director, signed and dated by the Study Director and maintained with the protocol. 12. REPORTING OF DATA: Upon completion of the analytical and field portions of the study, analytical and field reports will be prepared by the Principal Investigators. Upon completion of these reports, a draft final report will be prepared for review and approval by the Study Director. Upon approval, a final report will be issued. The report will contain narrative descriptions of the environmental conditions, work practices, worker background and experience, and other pertinent information collected during each replicate (campaign) of the study. In addition, the final report will include: 1. Name and address of the facilities performing the study and the data on which the study was initiated and completed 2. Objectives and procedures stated in the approved protocol, including any deviations and/or amendments in the original protocol 3. Identification and description of the test and reference substance(s) 4. A description of all methods used 5. A summary of the data generated while conducting the study and a description of all transformation, calculations, statistics, or other operations performed on the data 6. The name and dated signature of the Study Director 7. A description of all conclusions drawn from analysis of the samples 8. Complete description of the test system 9. Location of raw data, final report, and all specimens 10. A verified copy of the study protocol including protocol deviations and amendments 11. Location of the test site Upon acceptance o fthe final report, all study specific original raw data will be submitted to the Sponsor along with exact copies of non-study specific support data. Original documents will be the property of the Sponsor. Subsequent support records shaU be copied, certified as exact copies, and included with the original raw data and submitted to the Sponsor for archival. 13. QUALITY ASSURANCE: The field phase of this protocol will be conducted and documented as completely as possible to ensure the quality and integrity of the data generated for this portion of the study. 000289 ) The analytical portion of this study will be performed in compliance with the Good Laboratory Practice (GLP) as specified in 40 CFR 160. The analytical portion and the final report of (bis study will be inspected. The findings of these inspections will be made available to the Study Director and Test Facilities Management. 14. RECORDS RETENTION: Reports, raw data, and records pertaining to this study will be archived by Haskell Information Sciences, 000290 Appendix-2 Process for Analysis o f Blood Biom onltoring Sam ples Preparation Phase; 1. Plant site handle appropriate communications, scheduling of phlebotomy, and all other pre-sampling arrangements 2. Plant site requests supplies from Exygen at least 4 weeks in advance; estimated number of samples is needed at this time 3. Exygen arranges for supplies to be shipped and schedules samples for analysis 4. Supplies are shipped to plant site with instructions on processing and shipment Sam pling Phase; 5. Blood is sampled and processed into serum at the plant site 6. Serum samples are shipped directly from the plant site to Exygen laboratory with a sample listing and Chain-of-Custody records. The plant will cc S. Mark Kennedy on the sample listing. Analysis and A nalytical Reporting Phase: 7. Exygen analyzes the samples and prepares a draft report to review. The draft report and copies of the raw data are sent to S. Mark Kennedy for review 8. S. M ark Kennedy reviews the analytical data, makes any corrections/upgrades to the draft, and gives the contract lab permission to finalize the analytical report 9. The contract lab finalizes the analytical report and sends it to S. Mark Kennedy. S, Mark Kennedy sends a copy of the report to Study Director Joseph F. Viskocil who archives the original report at Haskell and informs the plant site industrial hygienist of the results Completion Phase: 10. The site handles appropriate communications at the site 11. Upon receipt o f the invoice from the contract lab, S. Mark Kennedy will provide the appropriate (plant-site-specifie) charge code to have the bill paid against a standing purchase order 12. Samples will be retained for 12 months per the method validation. Samples will be discarded after 12 months upon the Study Director's approval. 000291 ) Appendix-3 Specific Standard O perating Procedure: Docum entation, A ccountability, and Procedures for Consent Form s, and Collection & Processing o f Serum Sam ples 1. SCOPE AND APPLICABILITY This SOP governs the documentation, accountability, and procedures for consent forms, and collection & processing of serum samples. 2. REPONSIBIIJTIES 2.1 The Study Director is responsible for conducting the necessary training covering proper documentation, accountability, and procedures for consent forms, and collection & processing of serum samples. . 2.2 The Phlebotomist who performs the blood collection is responsible for ensuring that the Consent Form is completely and correctly filled out by each participant prior to venipuncture. 2.3 The Phlebotomist & Site Industrial Hygienist are responsible for ensuring that forms and the samples are properly collected and labeled. 3. Names Study Director: Joseph F. Viskocil, Jr CEH Phlebotomist: Bonnie Wilson, RN- Site Nurse Fayetteville Site Industrial Hygienist : Jennifer Locklear 4. MATERIALS AND EQUIPMENT 4.1. Consent Forms (duplicate or triplicate) 4.2. Serum Separator tubes (Red top) -(Supplied by Exygen) 4.3. Vacutainers, Needle holders and Needles-(Supp3ied by Exygen) 4.4. Centrifuges 4.5. Transfer Pipettes and Polypropylene Tubes-(Supplied by Exygen) 000292 5. PROCEDURE 5.1. Have the participant complete the Consent form prior to initiation of venipuncture. 5.2. Ensure that the consent form completed by each participant receives the same code number as the blood sample from that participant. 5.3. Original consent for each participait will be sent to Study Director for storage. 5.4. Access to consent forms to be decided 5.5. Collect blood specimen using usual venipuncture technique. Fill tube completely. 5.6. Gently invert the serum separator tube 5 times to mix. 5.7. Allow blood to clot a minimum of 30 minutes but no longer than 1 hour. 5.8. Centrifuge for 15 minutes at 2500 to 3500 rpm 5.9. Remove from centrifuge 5.10. Serum portion o f the collected sample in the SS tube is now ready to be pipetted into the specially provided polypropylene; tubes. 5.11. Seal the polypropylene tubes 5.12. The polypropylene tubes (labeled with specimen 1QDnumber) are now ready for transport to the lab. 5.13. Please see Appendix 2 for details on how to complete the chain of custody form and prepare the sample for shipping. 5.14. Samples will need to be frozen immediately at -20C and stored frozen. 5.15. Samples will also need to be shipped frozen. See Appenxix-4 6. REFERENCES - Appendix-4 7. FIGURES Figure 1. Consent form 000293 CONSENT TO PARTICIPATE IN PFOA EXPOSURE ASSESSMENT PROGRAM Study title: Exposure Assessment of Measured Serum Perfluorinated Octanoic Acid Anion in the Manufacture of Ammonium Perfluorooctanoate (APFO) at Fayetteville, NC Introduction: You a re being asked to p articip ate in a research study. Y our participation is voluntary. Your decision whether or not to participate will have no effect on your employment or the services you receive through DuPont's Occupational Health Program or Integrated Health Services, or Benefits Administration. Please ask questions of your site's OH Specialist or Nurse if there is anything you do not understand. What is the purpose o f this study? The purpose of the program is to learn about the relationship between specific tasks and the exposure level of PFOA that may occur in workers at this plant site, and to identify ways to reduce this exposure to as low a level as possible. Are there any benefitsfrom participating in this study? The benefit of participating in this study is that you may gain important knowledge about how exposures may vary depending on the ways certain tasks are carried out. This knowledge will help our OH Specialists, industrial hygienists, and engineers find ways to reduce these potential exposures. What does this study involve? Your participation in this study may last up to 5 years- A blood sample will be drawn every 6-months and analyzed for the serum level o f PFOA. No other blood levels w ill be determined. Will the study be affected if you do notparticipate? The more people that participate in this study, the more accurate and representative the results will be. In addition, we may be able to use these results to control exposures at other sites if we have enough participants to provide a good sample size. What are the risks involved with being enrolled in this study? The only risk o f oarticiaatine in this study wouldbe the small risk o fa hematoma (bruise) where the blood is drawn. Other important items you should know: W ithdraw al from the study: You may choose to stop your participation in 000294 this study at any time. Your decision to stop your participation will have no effect on your employment or the services you receive through DuPont's Occupational Health Program or Integrated Health Services, or Benefits Administration. New inform ation: To the best of our ability, any significant new findings during this research study will be made known to you. Confidentiality: Every effort will be taken to protect the names of the participants in this study. However, there is no guarantee that the information cannot be obtained by legal process or court order. Funding: Haskell Laboratory has been provided funding to conduct this study by the DuPont Fluoroproducts business. Number o f participants: We expect Approximately 30 participants. Who should you call with questions about this study? Questions about this study may be directed to the site OH Specialist, Jennifer Locklear; or the site Nurse, Bonnie Wilson, RN. You may also contact the Study Director, Joe Viskocil, at Haskell Laboratory: during normal business hours. Willyou be paid to participate in this study? No. Blood samples will be drawn during the work shift. CONSENT I have read the above information about (name of study) and have been given an opportunity to ask questions. I agree to participate in this study and I have been given a copy of this consent document for my own records. Print name:______________ _ _ ______ Pass Number: ________ _ Your signature: _______ ____________ ______ Date:, W itness:______________ ______________________ _ _ Date: Company Sanitized- D oes nol contain TSCACW 000295 Appendix-4 TITLE PAGE Standard Operating Procedure: Documentation, Accountability., and Sample Chain o f Custody Reonirem ents for the Shinning o f Seram Sam ples A nalytical Facility: Exygen Research 3058 Research Drive Stat " " '" '.0 3 Analytical Facility M anagement: John Flaherty, Vice President, Operations Company Sanitized. Does not contain TSCACBf Page 1 o f 7 000296 1. SCOPE AND APPLICABILITY This SOP governs the documentation, accountability, and sample chain o f custody requirements for the shipment o f serum samples to the analytical facility. 2. REPONS1BELITIES 2.1 The phlebotomist who performs the blood collection is responsible for ensuring that the Chain o f Custody/Analysis Request Form (COC) (Figure 1) is completely and correctly filled out and signed by staff members who package and ship the serum samples. 2.2 The Principal Investigator at the analytical facility is responsible for ensuring that the chain o f custody remains intact after the samples are received by the analytical facility. 3. DEFINITIONS Analytical Facility Management: John Flaherty, Vice President o f Operations, Exygen Research 3058 Research Drive, State College, PA 16801 Principal Investigator: Emily Decker, Scientist Exygen Research, 3058 Research Drive, State College, PA 168011 Company Sanitized. Does not contain TSCA CM Page 2 o f 7 000297 4. MATERIALS AND EQUIPMENT 4.1 Chain of Custody/Analysis Request Forms, triplicate design. 4.2. A readily available source o f dry ice pellets. Also, necessary personal protective equipment appropriate for handling dry ice. 4.3 Coolers numbered and coded with a unique identification assigned for this project. They must be purchased from Uline Shipping Supplies Specialists. The Uline part number for the required coolers is S-7888. (This part number corresponds to a package which includes one 19" x 12" x 12.5" cooler and one shipping box). 4.4 Plastic bags for use as secondary sample packaging prior to shipping. These will also be obtained from Uline, part number S-1707 (3" x 5", 4 mil reclosable poly bags, 1000 per carton). 5. PROCEDURE 5.1 .Before collecting any blood, the completed and signed consent form must be verified. When each sample is collected a unique alphanumeric identification code will be assigned to each sample. This number will be entered into the "Sample Identification" column o f the COC sheet. Likewise, the date and time o f each blood sample collection will be recorded, and the date and time Y . that the serum sample preparation is completed will be entered into the appropriate column on the COC sheet. 5.2. Each sample cooler will be assigned a unique alphanumeric identification number, which will be written on the cooler. 5.3. Before packaging, the primary serum sample containers must be placed individually into small plastic bags as secondary packaging. The secondary packaging must have the unique sample code written on it in permanent black ink. 5.4. Before the samples are packed in dry ice, a person other than the packager will verify that the samples documented on the COC form match exactly with those being packed in the cooler. The verifier then initials and dates the "Cooler Contents Verified" prompt. 5.5.Samples are to be dispersed in a cooler full o f dry ice pellets, arranged in such a w ay as to minimize the possibility o f impact, and such that they are completely covered with the pellets. The cooler must be completely filled with dry ice pellets. No more than 20 samples are to be placed in one cooler. The cooler will be covered promptly after packing with dry ice pellets to minimize loss of dry ice to sublimation. Page 3 o f 7 00Z98 5.6. The time between the completion o f serum preparation and packaging for shipment must be able to be accounted for on the COC page. There m ust not be any unexplained gap in the chain of custody for the samples. 5.7. After the serum samples are packaged, the Cooler ID# prompt will be completed. Section 2 ("Personnel Involved in Shipment o f Samples") o f the COC form also will be completed at this time, if it has not been previously. The printed name, signature, initials and date o f signature need to be recorded for each person who entered information anywhere on the form. The page number prompts also need to be filled in. 5.8. The person who packaged the samples then completes the first line in the "Relinquished by" section, and verifies that any unused prompts are lined out. 5.9. The COC form is then faxed to Exygen Research Sample Receiving using the fax number at the top o f the form. After it is faxed, the pink copy is removed and becomes part o f the client's records. The wMte original and yellow copies are to be placed in an envelope, which is then placed into the shipping carton with the cooler that contains the samples to which the COC form corresponds. 5.10. The serum samples are required to be shipped to Exygen Research Sample Receiving at the address given at the top o f the COC form by FedEx overnight delivery. This is the only earner and method o f shipment that is acceptable. 5.11. Upon receipt o f the samples by Exygen Research, the chain o f custody procedure will follow Exygen SOP V401, "Contract Research Sample Handling" . After Exygen Sample Receiving completes the "Received by" section, a copy o f the COC will be faxed back to the client, 5.12. Any problems with delivery or condition o f samples upon arrival at Exygen will be documented. The Exygen Principal Investigator will notify the client o f any problems immediately. 5.13. Correction and Clarification o f Raw Data 5.13.1. As soon as any information has been entered on the COC form, it becomes raw data. At this point, this form is not to be discarded for any reason. If it becomes damaged, information may be transcribed onto another sheet, but the transcription process will be documented via footnote on the new page The original damaged sheet must be attached to the transcribed page. 5.13.2. I f errors are made in handwritten entries, or if clarifications are to } be made, the errant information will be crossed out with a single line, so as not to obscure the original entry. Then, depending upon Page 4 o f 7 000299 the nature of the error, an appropriate error code is chosen (see Figure 2), and the error code is initialed and dated. If the error is more complex than what is covered by the error code list, a detailed explanation will be provided by footnote on the COC page. 6. REFERENCES Exygen Research SOP V401, "Contract Research Sample Handling" 7. FIGURES Figure 1. Chain o f Custody/Analysis Request Form Figure 2. List o f Error Codes 8. SIGNATURE Analytical Facility Management Date Page 5 o f 7 000300 Figure 1. Chain o f Custodv/Aaalvsis Request Form IT CHAIN OF CUSTODY RESEARCH i Research Sam ple Receiving 3 0 4 8 Research Drive M ate C ollege, PA 16801 I COLLECTION SITE INFORMATION Collection Site (name &address): STAFF INVOLVED IN SAMPLE SHIPMENT 1. (PrintedName) Signature 2 ......... ,.................. ..................... (Printed Name} initials/Date Phone: Fax: SSpaWre 3. (Printed Name) -- iwfcais/oate Email: ?r imtiah/Ditfe IMPORTANT SpecifyAMorPMfortime entries, Indudemonth, day. andyearfordales. Contents of package inusi.beverified by someone other than the packager, and thisverification must be attested to by initialingand dating the prompt* Thoroughly documentthe reason forany time gap betweenthe time t he serum preparation IscompleteS andthe time packaged by adding a footnote to the bottom of the page. Eachperson who enters information on this form must add their name, signature, initials, and date above. Lineout unused prompts. S a m p le id e n tific a tio n S e ru m S am p le P rep. C o m p le te d D a te & Tim e PLEASE RAXTHE COMPLETED FQRM to&ygen Sample Reosmg Cooler Contents Verified , poorId stuping samples _________Initial &Date" KEEPTHEPINKCOPY andricijde the d t a two with am ple shp,nent C oaler ID # : Relinquished by Date I Time (R eceived by Date it Note WtsensanpiesanveatF^^.dTsin-cf-cesiQdYpocsdures folow fxyssrr SOPV4QX`CaitractReseai'ch Samplenaudmgr Page I Time of Company Sanitized. Does not contain TSCA CBI Page 6 o f 7 000301 Figure 2. List of Error Codes EXPLANATION OF ERROR CODING Correction o f raw data is made bv drawing a single line through the erroneous data and/or write-over, and filling in the correct data. Care m ust be taken not to obscure the original entry. The correction is initialed and dated by the individnai making the correction, and an explanation is given for the correction. The following error codes shall be used to explain corrections: (wl) (cc) (wo) (sp) (ir) (ce) (re) (ne) (ee) (te) (wd) (sm) (le) (tl) Inadvertently recorded in Wrong Location Changed for greater Clarity Write Over (Technician inadvertently wrote over the error instead o f drawing a line through the error) Spelling error Inadvertently not Recorded at time of initial observation Calculation Error Recording Error Numerical transcription Error Entry not initialed and dated at time o f entry Transcription Error Wrong Date entry Stray Marks appearing on data with no other feasible explanation Late Entry Technical error Codes, other than those defined above may be used, if explained or defined by footnote on the COC form. Errors that cannot be explained using an error code m ust be fully detailed at the time of correction. Error codes are to be circled and written above or close to the correction. I f this is not feasible, the error should be footnoted with an asterisk or other symbol, and explained at I the bottom o f the page. I } Page 7 o f 7 000302 Example COC Sheet and Use of Error Codes E 3 ^1 CHAIN OF CUSTODY RESEARCH en Research Sam ple R eceiving 3048 Research p riv * S tate C ollege. PA 16801 CLINIC INFORMATION STAFF INVOLVED IN SAMPLE SHIPMENT C linic (n am e & a d d re ss): Hftme P Clinic- h dA <`<$ $ _ 'S tetl f.A Cffy..5ikk jap odi. C linic Id en tifica tio n C ode: P h o n e: (f tr'e a r W c f 7 -cfcart- # Fax: ' i - A f f r . f - Em ail: e tm ilffrc lin l'C .C D m 1. RC-MunC (Printed Name) Signature 7 TYoa^ P. <PrintedyName> J& , Signature 3. TT Stn'itL (Printed tome) S -fJ-A cN ih i Initials/Date T p s Y<57, i*A<* IniUals/Oate CTS 09-I8-Q3 toitiais/Qate IMPORTANT SpecifyAMor PMfortime entries. Indtidemonth, day, and yearfordates. Contents of package mustbe verified by someone other than the packager, and this verification must be attested to by initialing arid dating the prompt* Thoroughly document the reason for any time gap between the time the serum preparation is Completed ana the time packaged by adding a footnote to the bottom of the page. Each person who enters information on this form mist add their name, signature, initials, and date above, line out unused prompts. t C lin ic S a m p le Id e n tif ic a tio n B lo c d S a m p le C o lle c tio n D a t e -St T i m e Piep.I S e r u m S a m p l e C o m p le te d D a te & T im e Am i O liti) 1 1 3 AW tnhilm 3'm fn fit 00 2 - ................................................................. v-simo v l / d q ________1 oihtloj '<0PM A /iO 3 ! onusla ix M m oilid'3 Harm f\ ^ T A ^ (^ m < r i a a i k 'sPm!3 ________t a miria 'a> Ptn P U iA S E lA X T H E C Q M P I H H 3 F O W W t o 6 q m S am p le fie o a w n g [ C o o l e r C o n t e n t s V e r i f i e d u a t - p rio rto sN p p in g sa m p fe s. L In itia i& D a te * ^ IAKEff*THEPINKCOPY and jndudethe other tw o with sample shipment C ooler ID # ; --- p s if i- m ' 15 3 O C f J 07/llf&3 Relinquished by j-... (^arnf>JtRA :0 a ^ . Noia: WhensampfesaniueatExygeachairwif-custodyprocedures follow ExygenSOPV401.'Contractte o re ti Sample HaHing." Company Sanitized. D oes not contain TSCA CBI 000303 -'-N j A p p en d ix -5 BLOOD COLLECTING CHECKLIST Employees Name Employees Job Title Date and Tim e B lood Sample taken Collecting Blood Sample Red Capped Collection Tube Completely Filled Filled Tube inverted 5 times Centrifuge for 15 minutes @ 2500-3500 rpm Yes No Yes No . Start Stop (military time) " N Pipet Serum into Polypropylene tube with specimen ID Specimen ID Number Complete Chain-of-Custody (COC) Form Y es No There m ust not be any unexplained gap in the C O C for the sam ples. ^^/Serum Placed in - 2 0 PC Freezer at i "r ' . . (muttary time) A / o' Serum Sam ples m ust be shipped frozen - Sample is frozen --v -.j -j.-cj Sample Shipped to: ><?' .... 4 ` Exygen Research 3058 Research Drive State College, PA 16801 YES NO Shinned F ed E x overnight O N LY , (per Standard Operating Procedure: Documentation, Accountability, and Sample Chain of Custody Requirements for the Shipping of Serum Samples - Appendix - 4) Phlebotomist (Site N urse) Nam e IPrintl __________ Signature . ________ _ .Date________ _ _____________;_________ __________________________________ Please Send the Original Blood Collecting Checklist to Joseph F- V isk ocil, Jr. CIH Stine-Haskell Research Center PO Box 5 0 ,1 0 9 0 Elkton Road Newark, DE 19714-0050 Company Sanitized. D oes not contain TSCACBt 000304 Appendix- 6 A IR S A M P LIN G STR A TE G Y T h e goal of exposure assessm ent Is to recognize all exposures and to m inim ize all exposures. W orkers m ay be exposed w hile perform ing tasks, by incidental contact from background contam ination, or because of nearby tasks perform ed by others. Although the highest exposures usually result from a w orker's own task, exposures significant can occur from the other tw o sources. Serum levels of PFO A anion are an indication of overall exposure. A ir sam pling results are an indication of airborne exposure. Th e significance of exposure by incidental contact cannot be assessed until the airborne exposure is controlled. Since the num ber of job assignm ents at Fayetteville is sm all, w e will sam ple each job assignm ent quarterly. W here job assignm ents extend into evenings and w eekends, accom m odations will be m ade to m onitor w orkers at these tim es. Tw o A P FO Field/C C R Technicians are on duty fo r each 12-hour shift. O ne technician monitors the Control Room and the other technician does th e fieldwork. Since the fieldwork technician is m ore likely to have an exposure, the m ajority o f A P F O /C C R technician sam ples in the field. Proposed A ir Sampling R an for the first 12-M onths / C ollect six (6) personal samples Time-W eighted-Average (TW A) quarterly for each 12-hour shift schedule (APFO Field/CCR technician) 24 samples C ollect six (6) personal samples Time-W eighted-Average (TW A) quarterly for 8-hour shift schedule (APFO Lab and M echanical Technician) 1.2 samples C ollect three (3) personal samples Time-W eighted-Average (TW A) quarterly for 8-hour shift schedule for APFO Supervisors 3 samples C ollect three (3) personal samples Time-W eighted-Average (TWA) quarterly for 8-hour shift schedule for APFO FLS 3 samples C ollect six (6) personal samples (duration o f task) quarterly for technical/professional personnel (chem ist and engineers) 6 samples 000305 Total 48 regularly scheduled personal sam ples per quarter Sampling Strategy to be re-assessed after 2 -quarters Sampling w ill be scheduled to ensure that off-shifts and weekends will be proportionally represented For an 8-hour shift; a minimum o f 6- hours sam pling is required to qualify as a 8-hour TWA For a 12-hour shift; a minimum of 10- hours sam pling is required to qualify as a 12-hour TWA 000306 APPENDIX? QUALiTYASSURANCE PROCEDURES INDUSTRIAL HYGIENE LABORATORY AND FIELD WORK This procedure specifies quality assurance procedures for industrial hygiene field w ork. Laboratory Standard Operating Procedures To m inim ize the possibility of contam ination, external surfaces of adapters, tubing and the air sam pling pum ps should be w iped down with a 5 0 /5 0 solution of isopropyl alcohol. T he wipe should be assum ed to be contam inated and disposed of properly. Minim um personal protective equipm ent should include safety glasses with sid e shields and nitrile gloves. This procedure m ust b e follow ed to assure equipm ent calibration and m aintenance is done properly. R elevant quality control procedures are incorporated into this procedure. Sample Numbering System Each field sam ple m ust be assigned a unique num ber. T h e industrial hygienist will assign a num ber for each survey. Th e hygienist will add a unique sequential num ber for each sam ple taken on a survey. E xam ple: 82-24-01 82 Y e a r of Survey 1 24 Survey num ber ! 01 Sequential sam ple num ber Field Sampling S eal the O V S cassettes or other sim ilar sam pling devices in plastic bags for transport to and from use site. C alibrate sam ple pumps before and a fte r each sam ple. C alibrate sam ple pum ps with counters, or rotom eters a t least before and after each survey. Before and afte r flow rates should be within 10% . Instruct test subjects not to tam per with personal sam plers and observe for com pliance. Void any sam ples which are suspect. ) j Place sam pler inlet in downward orientation to prevent contam ination. Im m ediately after a sam ple is taken, place caps into filter cassettes. 000307 Pressure and tem perature corrections to 2 5 C and 7 6 0 m m Hg m ust be m ade to actual air sam ple volum es to calculate ppb concentrations of gases and vapors, for com parison with exposure limits. Pressure and tem perature corrections are not m ade, but actual a ir sam ple volum es are used to calculate m g/m 3 of particulates for com parison with exposure limits. Control, Known and Duplicate Samples Control, known and duplicate sam ples m ust be subm itted to the analytical laboratory, along with air sam ples. Control sam ples are collection devices which have been opened a t the site, im m ediately closed and sealed, and assigned a sequential num ber. T h ey provide a check on the analytical zero. Known sam ples are collection devices, which have been spiked with a known quantity of a chem ical at Haskell Laboratory (or at the site under the hygienist's direction), carried on the survey, and assigned a sequential num ber. They provide a check on analytical accuracy. In som e cases, it m ay not be feasible to prepare reliable know n sam ples and they m ay b e om itted upon concurrence of the Study Director. D uplicate sam ples are a re a air sam ples taken from six to tw elve inches apart and assigned sequential num bers. They provide a check on sam pling and analytical precision. Results of control, known and duplicate sam ples m ust be evaluated with the chem ist in view o f the specific analytical m ethod used to determ ine adequacy. G enerally, known sam ple results should be within 25% of the spiked am ount and duplicate sam ple values should be 2 5 % or less apart. Reasons for deviations should be determ ined and corrective actions taken. 000308 Control results should be less than 5 % of the exposure lim it dose on the collection device at the average survey sam ple volum e or non-detectable (w hichever is less stringent). S am ple results should be corrected for control sam ple results. Reasons for deviations should be determ ined and corrective actions taken. Minim um required num bers of control, known and duplicate sam ples which m ust b e subm itted, are given in the following tab le. Number o f Samples F ie ld Control Known D uplicate Pairs <10 1 2* 0 11-20 3 2 1 2 1 -4 0 3 4** 2 *** >41 4 y| *** Tw o at one level ** Tw o at each of two levels *** O ne pair at each of tw o levels **** Additional pairs m ay be needed if a wide range of concentrations is expected. INDUSTRIAL HYGIENE SURVEY RECORDS - Quantitative Survey Data ! A ir Sam pling D ata - All air sam pling data must be entered on Air Monitoring D ata Form which must be sent to the Study Director for perm anent retention. O th er Q uantitative S urvey D ata - O ther data, such as ventilation, room and equipm ent dim ensions, or other data which will be used in a quantitative m anner for calculations, m ust be entered on the A ir M onitoring D ata Form. Survey Field N otes (other than quantitative data) Th ese notes are taken only as an inform al key to m em ory. Significant inform ation will be sum m arized in the survey report. These field notes m ay be discarded when no longer needed. How ever, if in the industrial hygienist's judgm ent, original non-quantitative notes need to be retained, the inform ation should be attached to Air M onitoring D ata Form for perm anent retention. 000009 Analysis and Analytical Reporting Phase: Clayton analyzes the sam ples and prepares a draft report to review . The draft report and copies of the raw data are sent to M ary A . K aiser, P h.D . for review. 1. M ary A. K aiser, PhD reviews the analytical data, m akes any corrections/upgrades to th e draft, and gives the contract lab perm ission to finalize th e analytical report 2 . Th e contract lab finalizes the analytical report and sends it to M ary A. Kaiser, PhD 3 . M ary A. Kaiser, PhD sends a copy of the report to S tudy Director, Joseph F. Viskocil w ho archives th e original report a t Haskell and informs the plant industrial hygienist of th e results 000310 Appendix - 8 SAMPLE HANDLING, CHAIN-OF-CUSTODY, PRESERVATION and SHIPPING AIR SAMPLES The person collecting the sample should wear nitrile disposable lab or exam gloves and should limit his/her contact with the samples. Sample tubes (OVS) will be provided by the contracted laboratory and should be stored in a clean, dry location. Sample tubes and collected samples are not required to be chilled, however sample tubes and collected samples should not be subjected to temperature extremes. In order to minimize the possibility of introducing APFO contamination into samples, the following protocol should be followed: Avoid polytetxafluoroethylene (PTFE). Avoid aluminum foil. Do not use self-stick memo notes. Avoid blue ice (the OVS tubes are shipped at ambient temperature). Avoid pre-wrapped foods or snacks. O Wear C lothing that has b een w a sh ed at lea st s ix tim es. Use only containers supplied by contract laboratory. Following sample collection, the OVS tubes are capped and placed in a zip-loc bag. Seal the zip-loc bag. Complete the sample label and chain-of custody (COC) form, including the volume of air sampled through the tube. The completed sample label is attached to the outside of the zip-loc bag. Rack the samples in a small box or cooler with bubble wrap. Include the completed COC form. Complete a mailing label and airbill and ship by overnight carrier to: Clayton Group Services 22345 Roethel Drive Novi, M I 46375 Contact at the laboratory is Karen Coonan Samples should be stored and shipped refrigerated to the laboratory. Company Sanitized. D oes not contain T5C ACBI 000011 A p p en d ix - 9 December 4, 2003 M ary A . Kaiser, Ph.D. Research Fellow Corporate Center for Analytical Services Dupont Central Research & D evelopm ent DUPONT Experimental Station P.O. B ox 80302 W ilmington, DE 19880-0302 Clayton Work Order N o. 0301008Q Clayton Proposal N o. 03DETLAB015.REV1 Dear Dr. Kaiser: W e are pleased to provide the follow in g D ra ft report on the sam pling and analytical m ethod conditions follow ed for the method validation for This draft report is based on our proposal dated July 18,2003. The sam pling parameters m ay change as w e progress with the m ethod validation.- N ot all o f the parameters outlined in this report have been fully validated. If you have any questions, please call m e at (248) 344-1770. Sincerely, Kem Charron Program M anager-Special Projects Laboratory Services KC Enclosure 000312 22345 Roethel Drive Novi, Ml 48375 248.344.1770 Fax 248.344.2654 Clayton Group Services, Inc. Perfluorooctanoic Acid (PFOA) Empirical Formula: Molecular W eight CAS Number: CF3(CF2) 6C 0 2H 414.06 335-67-1 Clayton W ork Order N o.: 03070834 Draft Report Date: February 9 ,2 0 0 4 OSHA: None Properties NIOSH: None Appearance: W rite Powder ACGIH: None M elting Point: 55 -56 C AEL: 0.56 ppb (9 .4 8 ug/rn3)_________ ,_________ _ __________ ,, ____________ _________ Synonyms: Peutadecafluorooctanoic acid Sampler: Sampling and Storage O SH A Versatile Sampler, SKC Cat. N o. 2 2 6 -3 0 -1 6 Flow Rate: A rea or personal monitoring: 1.0 L/xnin for up to 4 8 0 minutes for a sample volum e o f 4 8 0 L. F en ce line monitoring: 15 L/min for up to 4 8 0 minutes for a sam ple volum e o f 7 2 0 0 L. Storage: Refrigerated or ambient temperatures Shipping: Samples can be shipped at ambient temperature Sample Stability: 6 0 days at refrigerated or ambient temperatures_____________._________ ^ E quipm ent 1. SKC OSHA Versatile Sampler (O V S) C a t # 226-30-16 ~ ^ 2. HPLC System: LC pump with automated gradient controller, autosaxnpler, optional tunable U V absorbance detector 3. M ass Spectrometer: Triple quadrupole mass spectrometer 2 - 1000 amu m ass range minimum, A PI interface with Electrospray probe, negative and positive ionization m ode capability, and data acquisition/reduction software 4. Column: A gilen t Hypersil ODS, 2.1 x 100 mm, 3 pm particle size, Cat. # .7 9 9 1 6 0 D -3 5 2 , or equivalent 5. 16 mL amber glass vial with screw cap and polytetrafluoretbylene/rubber liner. The polytetrafluorethylene side o f the liner is positioned in the cap towards the sample 6. 4 mL amber glass vial w ith screw cap and polytetrafluoretbylene/rubber liner. The polytetrafluorethylene side o f the liner is positioned in the cap towards the sample 7. Waters total recovery glass autosampler vials with polytetrafluoretbylene/rubber liner. The polytetrafluorethylene side o f the liner is positioned in the cap towards the sam ple 8. .Syringe: 10, 25, 50, & 100 pL 9. Disposable glass test tube, 16 x 125 m in w ith caps 02/09/04 DRAFT lof6 www.daytongrp.com Environmental Services Occupational Health and Safety Laboratory Services 0 0 0 3 1 3Laboratory Services Clayton Work Order: 03070834 Perfluorooctanoic Acid /V Clayton GRO UP 5ERVJC6S Reagents and Standards 1. Perfluorooctanoic A cid (PFOA), Aldrich 171468-5G , L ot # 04702M A 2 . 1,2-di-13C-Perfluorooctanoic A cid ( 13C-PFOA), Internal Standard, Dupont, date o f synthesis January 2 ,2 0 0 3 3. Water (H20 ) , Direct-Q 4. M ethanol (M eOH), HPLC grade, B&J Cat. # 230-4 5. Am m onium Acetate, Reagent grade, J.T. Baker Cat. # 0599-08 6. 2 m M A m m onium Acetate: dissolve 0.1388 g A m m onium A cetate in 900 mL H20 7. Extraction Solvent: 100% MeOH 8. Stock A nalytical Standard Solution (SAS): D issolve 56.9 m g 0.1 m g o f PFOA (corrected for material purity) in l OmL o f MeOH Concentration: ~ 5690 pg/mL 9. Calibration Standards: Dilutions o f SAS in MeOH resulting in PFOA concentrations o f 1 1 .4 ,2 8 .4 ,4 5 .5 ,5 6 .9 ,1 1 4 , and 227 ng/mL 10. Stock Spike Standard Solution (SSS): D issolve 56.8 mg 0.1 mg o f PFOA (corrected for material purity) in 10 m L o f MeOH Concentration: ~ 5680 pg/mL 11. Spiking Standard Solutions: Dilutions o f SSS in MeOH resulting in PFOA concentrations o f 568 and 56.8 pg/mL 12. Stock Internal Standard Solution: D issolve 7.2 m g 0 . 1 m g l,2 -d i-13C-Perfluorooctanoic A cid (corrected for material purity) in 10 mL o f MeOH Concentration: ~ 720 pg/mL 13. Internal Standard Solution: Added to calibration standards and sam ples prior to analysis. D ilution o f die Stock Internal Standard Solution in M eO H resulting in a 13C-PFOA concentration o f 1.4 pg/mL 02/09/04 DRA FT 2 of6 Laboratoty.Services 000314 Clayton Work Order: 03070834 Perfluorooctanoic Acid Clayton c r o u p services M easurem ent Technique: H igh Performance Liquid Chromatography (EDPLC) coupled w ith Tandem M ass Spectrometry Detectors: Triple Quadrupole M ass Spectrometer (Micromass Quattro LC, or equivalent) Extraction: 4 mL o f extraction solvent with shaking for 60 minutes on a flatbed mechanical shaker Instrum ent Conditions HPLC Conditions M ass Spectrom eter Conditions Column: A gilent H ypersilO D S, 2.1 x 100 mm, 3 pm particle size Ionization M ode: Electrospray Negative Column Temp: F low Rate: Injection Volume: RunTime: Retention Time: 30 C 0.3 mL/minute 5 pL 14 minutes 6.4 minutes M obile Phase: Time (min) 0 5 9 10 14 Gradient %A %B 90 10 10 90 10 90 90 10 90 10 Curve - A = 2 mM Ammonium Acetate B = Methanol Wavelength (UV): N ot Applicable Nebulizer Gas (L/hr): D esolvation Gas (L/hr): Source B lock Temperature (C): D esolvation Tempera ture (C): Collision Gas Pressure (mBar): Multiplier: 96 690 120 350 3.1e-3 650 Source Settings Capillary Voltage (kV): Extractor (V): RF Lens (V): Analyzer Settings M SI LM&HM Resolution: MS I Ion Energy (V); MS2 LM&HM Resolution: MS2 Ion Energy (V): Entrance (V): Exit (V): ES -Ion 1.5 1 0.5 T4 0.5 15 1.5 0 0 M ass Spectrometer M ethod M S Function: Multiple Reaction Monitoring (MRM) ESP- Inter Channel D elay for MRM (s): 0.1 Span (Daltons): 0.5 Start time: 0 m in E nd time: 14 m in Analyte Specific Settings: Transition (amu) D w ell 1st PFOA 412.9 - 3 6 8 .9 (0 ) 0.5 Internal Standard ( 13C-PFOA) 4 1 4 .9 - 3 6 9 .9 (Q) 0.5 (Q) = Transition used for quantitation Cone Voltage fV) 10 10 C ollision Enerev CeVi 10 10 02/09/04 D R A FT 3 of 6 Laboratory Services 000315 Clayton Work Order: 03070834 Perfluorooctanoic Acid in GROUP SERVICES Sam pling 1. Samplers (SK C 226-30-16) are received from Hie manufacturer (SKC). 2. Calibrate each sam pling pump with a representative collection d evice in line. 3. Depending on the application, there are tw o flo w rate options: (1) For area or personal monitoring, sample at a flow rate o f 1.0 L/min for up to 480 minutes to obtain a sample volum e o f 480 L. (2) For fen ce line monitoring, sample at a flow rate o f 15 L/m in for up to 480 minutes to obtain a sample volume o f 7200 L. 4. Label the sam ple w ith appropriate sample identification. 5. A t the com pletion o f sampling, re-calibrate each pump w ith a representative collection device in line, 6. Cap (he ends o f each sampler. 7. Samples are stored at ambient temperature. 8. A chain o f custody form is filled out and sept w ith the samplers under ambient conditions to the Laboratory for analysis. Sample Preparation Note: Samples are extracted just prior to analysis. 1. Extract each OVS sampling tube in three separate sections. P lace each section in a separate disposable glass test tube. Section 1: GFF Filter Section 2: X AD -2 Front portion o f media plus foam plug Section 3: X A D -2 Back portion o f media 2. A dd 4 m L o f M ethanol to each test tube and shake for 60 m inutes o n a m echanical flatbed shaker set at ~2 cycles per second. 3. Transfer approximately 3.5 mL o f each extract to a 4 mL amber glass vial by filtering through a Gelman 13-m m GHP Acrodisc. Extracts should be stored at refrigerated temperatures (2 to 6 C). 4. For LC/M S/M S analysis, transfer 200 pL each sample extract to a glass autosampler viaL A dd 10 pL o f a 1.4 pg/m L internal standard solution (l,2 -d i-BC-Perfluorooc.tanoic Acid). 02/09/04 DRAFT 4 of6 Laboratori'Services 000316 Clayton W ork Order: 03070834 \ Perfluorooctanoic Acid Clayton c r o u p services Calibration and Quality Control 1. Calibrate daily with six working standards over the range o f 11.4 to 227 ng/m L for PFOA. a) Calibration standards are prepared b y transferring 200 pL each PFOA standard solution to a glass autosampler vial. Add 10 pL o f a 1.4 pg/m L internal standard solution (1 ,2 -d i-l3C-Perfluorooctanoic Acid). b) A n alyze standards together with sam ples and blanks. A calibratiort curve is analyzed at the beginning and end o f each run, and Continuing Calibration Verification (CCV) standards are analyzed a minimum o f every ten samples. Acceptance criteria for a CCV is 20%. c) Generate an internal standard, linear calibration curve from the observed p eak area response factors and standard concentrations, applying 1/X weighting. The correlation coefficient (R) for the calibration curve must be > 0.992 (R2 > 0.985). 2. Determine the Extraction Efficiency (EE) at least once with each lot o f samplers. Prepare at least one level in duplicate within the expected sampling range. Include one media blank per lo t a) Inject pL amounts o f PFOA spiking standard solution onto the GFF portion o f an OVS tube. . b) A llo w the tube to air-dry at ambient temperature. c) Cap the tube and store at refrigerated temperatures (2 to 6 C) until extraction. d) Extract the PFOA-spiked and blank tubes a s described in Sample Preparation. A nalyze an aliquot from each sample along with standards. Transfer 200 pL each extract solution to a glass autosampler rial. Add 10 pL o f a 1.4 pg/m L Internal Standard solution (l,2 - d i- l3C-Perfluorooctanoic Acid). e) Calculate the ratio for the ng o f PFOA found on the media spike to the theoretical amount spiked. The concentrations found in each o f die three sections m ust be sum m ed to obtain the total amount o f PFOA found. The ratio reflects the recovery o f PFOA from the media. The mean recovery o f the replicates equals the EE for PFOA. M easurement 1. S et the liquid chromatograph and mass spectrometer conditions in accordance w ith those specified on page 3, or w ith suitable adjustments to optimize chromatography and sensitivity. 2. Inject the sp ecified aliquot o f each standard and sample. 3. Record the peak area response factor for PFOA and Internal Standard peaks. The observed retention tim e is 6.4 minutes for both PFOA and I3C-PFOA. 02/09/04 D R A FT 5 of 6 Laboratory Services 000317 Clayton Work Order: 03070834 Perfluorooctanoic Acid #%\Claytori CROUP SERVICES C alcu lation s L Calculate the response factor (RF) for PFOA. RF = (PFOA A r e a / 13C-PFOA Area) 2. Determine the mass (ng) o f PFOA collected using a first order linear regression curve plotting RF versus PFOA concentration. a) Analyte found: ng = (n g /m L x F V ) Where: ng/mL -- obtained b y interpolation o f the PFOA peak RF on die calibration curve F V =5= the Extraction Final volum e (mL) b ) Concentration (mg/m3) = (p gSaraplc- pgu^*) /V Where: (rgSimpfe = xg amount found in the sam ple (n g found / 1000 ng/pg) pgBIank = pg amount found in the blank sam ple (ng found / 1000 ng/pg) V = air sam ple volum e (L) This report prepared by: Supervisor, Method Validations Laboratory Services This report reviewed by: Senior V ice President Director, Laboratory Services February 9,2004 02/09/04 DRAFT 6 of 6 Laboratory Services 000318 Clayton Group Services, Inc Empirical Formula: Molecular Weight: CAS Number: OSHA: None NIOSH: None ACGIH: None AEL: 0 .5 6 ppb Synonyms: Pentadecafluorooctanoic acid Clayton Work Order No.: 03070834 Draft Report Date: December 4,2003 Properties Appearance: Odor: Melting Point: White Powder HHHI Sampling and Storage Sampler: OSHA Versatile Sampler SKC Cat. No. 226-30-16 Storage: To be determined Shipping: Samples should be shipped at refrigerated temperatures (2-6 C) Sample Stability: To be determined Equipment 1. SKC OSHA Versatile Sampler (OVS) C at No. 226-30-16 2. HPLC System: LC pump with automated gradient controller, autosampler, optional tunable UV absorbance detector 6. Mass Spectrometer: Triple quadrupole mass spectrometer 2 - 1000 amu mass range minimum, API interface with Electrospray probe, negative and positive ionization mode capability, and data acquisition/reductipn software 7. Column: Agilent Hypersil ODS, 2.1 x 100 mm, 3 pm particle size, Cat. # 799160D -352 or equivalent 8. 16 mL amber glass vial with screw cap and polytetrafluorethylene/rubber liner. The polytetrafluorethylene side o f the liner is positioned in the cap towards the sample 9. 4 mL amber glass vial with screw cap and polytetrafluorethylene/rubber liner. The polytetrafluorethylene side o f the liner is positioned in the cap towards the sample 10. Waters total recovery glass autosampler vials with polytetrafluorethylene/rubber liner. The polytetrafluorethylene side o f the liner is positioned in the cap towards the sample 12. Syringe: 10, 25, 50, & 100 pL 13. Disposable glass test tube, 16 x 125 mm with caps 12/04/003 DRAFT 1of 4 Laboratory Services 000319 Clayton Work Order: 03070834 Measurement Technique: High Performance Liquid Chromatography (HPLC) coupled with Tandem Mass Spectrometry Detectors: Triple Quadrupole Mass Spectrometer (Micromass Quattro LC, or equivalent) Extraction: 4 mL o f extraction solvent with shaking for 60 minutes on a flatbed mechanical shaker Instrument Conditions: HPLC Conditions Mass Spectrometer Conditions Column: Agilent Hypersil ODS, 2.1 x 100 mm, 3 pm particle size Ionization Mode: Electrospray Negative Column Temp: 3 0 C Flow Rate: 0.3 mL/minute Injection Volume: 5 pL Run Time: 14 minutes Retention Time: 6.4 minutes Mobile Phase: Time (min) 0 5 9 10 14 Gradient %A %B 90 10 10 90 10 90 90 10 90 10 Curve A = 2 mM Ammonium Acetate B = Methanol Wavelength (UV): N ot Applicable Nebulizer Gas (L/hr): 96 Desolvation Gas (L/hr): 690 Source Block Temperature (C): 120 Desolvation Temperature (C):350 Collision Gas Pressure (mBar):3,le-3 Multiplier: 650 Source Settings Capillary Voltage (kV): Extractor (V): RF Lens (V): Analyzer Settings MSI LM&HM Resolution: MSI Ion Energy (V): MS2 LM&HM Resolution: MS2 Ion Energy (V): Entrance (V): Exit (V): ES -Ion 1.5 1 0.5 14 0.5 15 1.5 0 0 Mass Spectrometer Method MS Function: Multiple Reaction Monitoring (MRM) BSP- Inter Channel D elay for MRM (s); 0.1 Span (Daltons): 0.5 Start time: 0 min End time: 14 min Analyte Specific Settings: Transition /annri Dwell fsi 0.5 Internal Standard ( 0.5 (Q) = Transition used for quantitation Cone Voltage (V) 10 10 Collision Energy IeVI 10 10 12/04/003 DRAFT 2 of4 Laboratory Services 000320 Clayton Work Order: 03070834 Sampling 1. Samplers (SKC 226-30-16) are received from the manufacturer (SKC). 2. Calibrate each sampling pump with a representative collection device in line. 3. Depending on the application, there are two flow rate options: (1) For area or personal monitoring, sample at a flow rate o f 1.0 L/min for up to 480 minutes to obtain a sample volume o f 960 L. (2) For fence line monitoring, sample at a flow rate o f 15 L/min for up to 1440 minutes to obtain a sample volume o f 21600 L. 4. Label the sample with appropriate sample identification. 5. A t the completion o f sampling, re-calibrate each pump with a representative collection device in line. 6. Cap the ends o f each sampler. 7. Samples are stored at refrigerated temperatures (2 to 6 C). 8. A chain o f custody form is filled out and sent with the samplers under refrigerated conditions to the Laboratory for analysis Sample Preparation Note: Samples are extracted just prior to analysis. 1. Extract each OVS sampling tube in three separate sections. Place each section in a separate disposable glass test tube. Section 1: GFF Filter Section 2: XAD-2 Front portion o f media plus foam plug Section 3: XAD-2 Back portion o f media 2. Add 4 mL o f Methanol to each test tube and shake for 60 minutes on a mechanical flatbed shaker set at ~2 cycles per second, 3. Transfer approximately 3.5 mL o f each extract to a 4 mL amber glass vial by filtering through a Gelman 13-mm GHP Acrodisc, Extracts should be stored refrigerated temperatures (2 to 6 C). 4. For LC/MS/MS analysis, transfer 200 pL each sample extract to a glass autosampler vial. Add 10 pL o f a 1.4 pg/mL Internal Standard solution Calibration and Quality Control 1 . Calibrate daily with six working standards over the range o f 11,4 to 227 ng/mL for a) Calibration standards are prepared by transferring 200 pL each standard solution to a glass autosampler vial. Add 10 pL o f a 1.4 pg/mL Internal Standard solution ( ( !) 12/04/03 DRAFT 3 of4 Laboratory Services 000321 Clayton Work Order: 03070834 b) Analyze standards together with samples and blanks. A calibration curve is analyzed at the beginning and end o f each run, and Continuing Calibration Verification (CCV) standards are analyzed a minimum o f every ten samples. Acceptance criteria for a CCV is 20%. c) Generate an internal standard, linear calibration curve from the observed peak area response factors and standard concentrations. 2. Determine the Extraction Efficiency (EE) at least once with each lot o f samplers. Prepare at least one level in duplicate within the expected sampling range. Include one media blank per lot. a) Inject pL amounts o fB B M sp ik in g standard solution onto the GFF portion o f an OVS tube. b) A llow the tube to air-dry at ambient temperature. c) Cap the tube and store at refrigerated temperatures (2 to 6 C) until extraction. d) Extract the PFOA-spiked and blank tubes as described in Sample Preparation. Analyze an aliquot from each sample along with standards. Transfer 200 pL each extract solution to a glass autosampler vial. Add 10 pL o f a 1.4 pg/mL Internal Standard solution ( | e) Calculate the ratio for the ng o f m ^ f o u n d on the media spike to the theoretical amount spiked. The concentrations found in each o f the three sections must be summed to obtain the total amount o f f l U found. The ratio reflects the recovery o f | equals the EE f o r f l H l 1from the media. The mean recovery o f the replicates M easurem ent 1. Set the liquid chromatograph and mass spectrometer conditions in accordance with those specified on page 2, or with suitable adjustments to optimize chromatography and sensitivity. 2. Inject the specified aliquot o f each standard and sample. 3. Record the peak area response factor for S H and Internal Standard peaks. The observed retention time is 6.4 minutes for both t H f l l and Internal Standard. 12/04/003 DRAFT 4 of4 L aboratory Services 000322 Appendix-10 Air Monitoring Data Form Sample Number Date Monitoring Type __ Personal Area Chemical Ageut(s) Sample Type ___Personal __ Source Bulk Sample Duration TWA STEL ...Ceiling PPE* - Respirator Air Supplied ,Full Face Air Purifying w /ov/acid gas/high Efficiency Particulate Filter, none - Full Acid Suit: nitrile or laytex gloves; other Type of Control ___Closed System ___ Local Exhaust Name -- Mechanical Exhaust ___Natural Ventilation Employee Infonntion - i r - '< \ SSN/Employee Number Unit Area Jo b Name Shift Supervisor B u ild in g Sample Information Floor Area Description of Location Tasks- See Personal Monitoring Log Pump Manufacturer Start Time Initial Flow Kate Sample Media Temperature (F) "Vvi:5 Agent -f'V Sam ple Collection Inform ation Pump Model Pump Number Stop Time Total fin ie (min) Final Flow Rate Average Flow Rate Sample Volume Lot Number Environmental Data Humidity (%) Wind Direction Wind Speed (mph) ;VtK cs Analytical Result Final Result .V Method Sample Analyzed By Limit of Detection Sample Collected By * Standard PPE includes Safety Glasses, Hard Hat, Steel Toe Acid Suit Boots 000323 Date: Employee Name: W ork Area: Sample Number: Pump Number: lim e INFORMATION WORKSHEET Comments 000324 A p p e n d ix -11 Wipe Test Procedure M aterials: W Y P AI.T. L40 disposable w ipers; K im berly-Clark #05701, VWR #21908-000 Cheesecloth wipes; VWR PK-200 4X4 #21910-107 - Cotton Pads; VWR PK-100 4X4 #21902-985 Tem plates for W ipe Sam pling 10 X 10cm paper; SKC Inc. #225-2415 Isopropyl alcohol Omnisolv 4L 99.9% ; VWR #EM-PX1834-1 W ater Omnisolv 4L; VWR #EM -W X0004-1 Scintillation vials, 20 mL, disposable, glass w /polyethylene cap VW R #66020-326 S a fe ty : The minimum PPE for this procedure is safety eyew ear w ith sideshields and nitrile gloves. All generated w aste is to be disposed in an appropriate manner. Procedure: 1. M ake 1 L o f mopping solvent (M S) by m ixing 500 m L o f isopropyl alcohol (EPA) with 500 mL of water (M3LLI-Q or other w ater purification system w ater may be substituted for EM Omnisolv w ater). MS is stored in a 1 L Nalgene volum etric flask. 2. Identify the area to be w ipe tested, Using the 10 X 10 cm Tem plate for W ipe Sampling to define the 100 square centim eter area to be tested. 3. Fill a transfer pipette w ith MS from the 1 min. scintillation vial. Evenly drip half of the contents (-0.75 mL) on a folded cheesecloth w ipe and return the balance o f the MS to the vial. Remove the paper tem plate and mop the test area w ith the wipe. Insert w ipe into the vial, gently push the wipe to the bottom w ith the transfer pipette and close the cap tightly. Dispose of the pipette in the w aste jug. 4. In order to m inim ize the possibility o f introducing APFO contam ination into samples, the following protocol should be followed: Avoid polytetrafluoroethylene (F IF E ). Avoid aluminum foil. Do not use self-stick memo notes. Avoid blue ice (the sam ples axe shipped at am bient tem perature). A void pre-Wrapped foods or snacks. W ear clothing that has been washed at least six times. 5. Follow ing sample collection, the vials are capped and placed in a zip-loc bag. Seal the zip-loc bag. 6 . Com plete the sam ple la b e l. The com pleted sample label is attached to the outside o f the zip-loc bag. 7. Pack the sam ples in a small box or cooler with bubble wrap. 000325 TITLE Method o f Analysis for the Determination o f Perfluorooctanoic A cid (PFOA) in Serum and Plasma by LC/MS/MS AUTHORS Emily Decker and Paul Connolly DATE ISSUED Decem bers, 2003 SPONSORS DuPont Haskell Laboratory for Health & Environmental Sciences Newark, DE DuPont Study Number: DuPont-12445 3M Environmental Laboratory ' St Paul, MN 3M Study Number: E03-0188 PERFORMING LABORATORY Exygen Research 3058 Research Drive State College, PA 16801 METHOD NUMBER ExM-008-211 revision 1 TOTAL NUMBER OF PAGES 27 ; 000326 MANAGEMENT APPROVAL Q Jv Paul Connolly Technical Leader, LC-MS Exygen Research 12-12-1 OS (j Date (2/L/w /-io h n Flaherty Vice President Exygen Research x fyi/k Date Dr. S. Mark Kennedy Sponsor Representative DuPont Dr. W illiam K. Reagen Sponsor Representative 3M Date Date Exygen Research _ Page 2 of 27 0 0 3 2 7 } TABLE OF CONTENTS TITLE.............. ............................. .............. .......... ..... - .............-V" ..................... .................... 1 MANAGEMENT APPROVAL.................................. .....................- ...................................... 2 TABLE OF CONTENTS............................... ....... ............................ ....................................... . 3 LEST OF TA B LES............ .............................................. .......................................................... 4 LIST OF FIG U RES............... ......................... ................... ...... .........................,........................4 1. SUM M ARY........................................... ......... .................. ....... .................. ......... ,...... .......5 2. EXPERIM ENTAL COM POUNDS........... ............................................. .......... ......... ...... 6 3. CHEMICALS AND SUPPLIES.............. ........................................... .......... .................... 7 3.1. Chemicals............................ ................... .................. ......... ................. ...................... 7 3.2. Standards..... ................................. .................. ..................... ........ ................. ......... 7 3.3. E quipment and Supplies................. ............... ............................................... .......... 8 3.4. Solutions............................................................................................ ........................9 3.5. P reparation of Standards and F orheication Solutions.......................... . 9 3.5JL Stock solution..... .............................. ...... ...........................-- ................. 9 3.5.2. Fortehcation Solutions............ ......... ....................................... ................9 3.5.3. Calibration Standards............ .......... ................................ ...... .......... . l l 4. M ETHOD............................................................ ....... ....................................... ...............H 4.1. Flow Diagram.............................. -- ....................................................................11 4.2. Sampleprocessing............... ................... .................................................................11 4.3. Sample Preparation............................ ............................................................ . 12 4.4. Extraction.............. .......... ................................. ........................... ......................... 12 4.5. Quantitation............... .............................. ........................... ........ ...........................12 4.5.1. LC/MS/MS System and Operating Conditions ................................... 12 4.5.2. Tune File Parameters.,......,.........,......................................... ......... ...........14 4.5.3. Calibration Procedures............ ......... ....... .................. ...... ................. . 14 4.5.4. Sample Analysis ........... ,........................................................ ................ ......14 4.6. Acceptance Criteria.......................................................... .................... ............... . 15 4.7. Performance Criteria........................... ......... ........ ............................................ . 16 4.8. Time Required for Analyses.................................................... ........... ........ ......... 16 5. CALCULATIONS..................... .......... .......... ........ ........ .......... ...... .................. 16 6. SAFETY............................ ......... .................................................................................. ...... 17 Page 3.of 27 0003Z8 LISTOFTABLES Table I. Recovery of PFOA from Fortifications In R abbit Serum ............ ............... 18 Table H Recovery of PFOA from Fortifications In Rabbit Plasma-...................... ........... 19 LIST OF FIGURES Figure 1 . Calibration curve for PFOA in Control Rabbit S erum ................. ................. ..... 20 Figure 2. Representative Chromatogram o f a Control R abbit Serum Sam ple for PFOA w ith - 1 ng/mL of 13C-PFOA ....................... ..................... ....... .................... ........ ......... 21 Figure 3. Representative Chromatogram o f a 0.5 ppb Standard for PFO A in Control Rabbit Serum w ith - 1 ng/m L o f 13C~PFOA........................ ............................. ........ . 22 Figure 4. Representative Chromatogram of Control R abbit Serum Fortified at 0.5 ppb with PFOA and w ith - 1 ng/m L o f 13C-PFOA ............................................... ................23 Figure 5. Calibration curve for PFOA in Control Rabbit P lasm a...................... .............. 24 Figure 6 . Representative Chromatogram o f a Control R abbit Plasm a Sam ple for PFOA w ith 1 ng/mL o f 13C-PFO A ............ .................................... ............................................. 25 Figure 7. Representative Chromatogram o f a 0.5 ppb Standard for PFOA in Control Rabbit Plasm a with - 1 ng/mL o f 13C~PFOA................................................. ....... ........ 26 Figure 8. Representative Chromatogram o f Control R abbit Plasm a Fortified at 0.5 ppb w ith PFOA and w ith ~ 1 ng/mL o f l3C-PFOA ........... ........ ....... ........ ,................. ....... 27 000329 ) 1. SUMMARY This report details a method of analysis fo r perfluorooctanoic acid (PFOA) in serum and plasma. PFOA is extracted from serum and plasm a by protein precipitation in acetonitrile by m eans o f a robotic liquid sam ple handler. Quantification o f PFOA is accom plished by liquid chrom atography/tandem m ass spectrom etry (LC/MS/M S) analysis using selected reaction m onitoring (SRM ). The chem ical form ula of PFOA is given in section 2 o f this method. The low er lim it o f quantitation (LLOQ) for this m ethod is 0.5 ppb fo r PFOA in serum and plasma. Q uantification is performed using extracted calibration standards containing an internal standard. This m ethod was developed using rabbit serum and rabbit plasm a. The overall percent recovery standard deviation for PFO A in rabbit serum at 0 .5 ,3 , and 40 ppb was 95% 8.5% (Table I). The overall percent recovery standard deviation for PFOA in rabbit plasm a at 0.5, 3, and 40 ppb was 99% 6.3% (T able H). A representative calibration curve for PFOA in rabbit serum is shown in F ig u re 1. R epresentative chromatograms for PFOA in rabbit serum are show n in F ig u res 2 to 4. A representative calibration curve fo r PFOA in rat plasm a is shown in F ig u re 5. Representative chromatograms fo r PFOA in rat plasm a are shown in F igures 6 to 8. Exygen Research 00033 Page 5 o f27 2. e x p e r im e n t a l c o m p o u n d s The structure of peifluorooctanoic acid (PK>A) and tire internal standard are given below. Chemical Name = M olecular weight - perfluorapctanoic acid 414 13C - PFO A Chemical Name M olecular weight perfluorooctanoic acid, [1,2-di- C] 416 O1! OH Exygen Research Page 6 of 27 0003Q/ 3. CHEMICALS AND SUPPLIES 3.1. Chemicals C hem ical M ethanol (MeOH) A cetonitrile (ACN) Ammonium Acetate OmniSoIv W ater G rade HPLC HPLC Reagent HPLC Source ~ EM Science EM Science S igm a-A ldrich EM Science C atalog No. JT 9 0 9 3 -2 AXO145-1 ' A-7330 W X 0004-1 3.2. Standards S ta n d a rd Lot Number perOuorooctanoic acid (PFOA) perfluorooctanoic acid, [l,2 -d i-13C] 21002 NA P u rity (% ) 97 96.35 S o u rce Oakwood Products DuPont Kxveen Research 000332 Page 7 o f27 3.3. Equipment and Supplies Equipm ent S upplier Balance, analytical (display at least 0.0001 g) . M ettler Disposable m icropipets (50-100UL, 100-200uL)' D rum m ond - - (VW R) Class A pipets and volum etric flasks various suppliers Hypercarb drop-in guard column (4 mm) K eystone (part #844017-400) Stand-alone drop-in guard cartridge holder - K eystone 125-m LLDPE narrow-m outh bottles N algene 2 xnL clear HPLC vial Mt VWR Standard lab equipm ent (graduated cylinders, various suppliers disposable tubes etc.) M ulitprobe II Ex, Robotic liquid handling system Packard M BP D isposable Conductive Tips (1 mL) - Packard (part #6000655) Array protein precipitation columns, 2 mL (part A rgonaut #120-2020-T) Isolute array collection plate, 1 m L (part #121-5202) A rgonaut 12x75 mm disposable culture tubes VWR LC/MS/MS and HPLC systems A s described in section 4.5. N ote: Equivalent m aterials m ay be substituted for those specified in this method if they can be shown to produce satisfactory results. N otes: 1. In order to avoid contam ination, the use o f disposable labw are is highly recommended (tubes, pipets, etc.). 2. PTFE o r PTFE lined containers or equipm ent, including PTFE-lined HPLC vials for the HPLC autosam pler m ust got be used. 3. It is necessary to check the -solvents (m ethanol) for the presence of contaminants by LC/M S/M S before use. Certain lo t num bers have been found to be unsuitable for use. 4. Use disposable m icropipets or pipets to aliquot standard solutions to make calibration standards and sample fortifications, 5. The hypercarb cartridges should be changed when the system contaminants (elevated baseline) move to w ithin 1 m inute from the elution ofP F O A . Exygen Research 333 Page 8 o f27 3.4. Solutions (1) 2 mM ammonium acetate solution is prepared by weighing 0.15 g of ammonium acetate and dissolving in *1L o f water. N ote: The aforementioned exam ple is provided for guidance, alternative volumes may be prepared as long as the ratios o f the solvent to solute are maintained. 3,5. P reparation of Standards and Fortification Solutions A nalytical standards are used for three purposes: 1. C alibration Standards - These standards are prepared in control rabbit serum o r rabbit plasma and are used to calibrate the response o f the detector used in the analysis. 2. Laboratory Control Spikes - These fortifications are usually prepared at concentrations corresponding to the LLOQ and lOx LLOQ and are used to determ ine analytical recovery. Laboratory control spikes are prepared in control rabbit serum or rabbit plasm a. 3. M atrix Spikes - These fortifications are prepared by spiking into the field sam ples at known concentrations. M atrix spikes are used to evaluate the effect o f the sample m atrix on analytical recovery. The absolute volum es o f the standards m ay be varied by the analyst as long as the correct proportions o f solute to solvent are m aintained. 3.5.1. Stock solution Prepare individual stock solutions o f 100 pg/m L o f PFOA and 13C-PFOA by w eighing out 10 m g o f analytical standard (corrected for purity) and dilute to 100 m L with m ethanol in a 100-rnL volum etric flask. Each stock solution (in 125-mL LDPE bottles) is to be stored in a refrigerator at 2C to 6C and is stable for a m axim um period o f 1 year from the date o f preparation. 3.5.2. Fortification Solutions a. Prepare a fortification standard o f 1.0 pg/m L (1000 ng/inL) for PFOA by diluting 1.0 m L o f the fortification solution described in 3.5.1 to 100 m L with acetonitrile in a volum etric flask. b. Prepare a fortification standard o f 1.0 pgfrnL for 13C-PFOA by diluting 1.0 mL o f die 13C-PFOA solution described in 3.5.1 to 100 m L w ith acetonitrile in a volum etric flask. c. Prepare a fortification standard o f 0.1 pg/m L (100 ng/mL) for PFOA by diluting 10.0 mL of the fortification solution described in 3.5.2.a to 100 m L w ith acetonitrile in a volum etric flask. d. Prepare a fortification standard o f 0.005 pg/m L for PFOA containing 0.001 pg/m L of 13C-PFOA by diluting 5.0 mL o f the PFOA solution Exygen Research 000334 Page 9 o f 27 described in 3.5.2.C and 0.1 mL o f the 13C-PFOA solution described in 3.5.2. b to 100 mL with acetonitrile in a volum etric flask. e. Prepare a fortification standard of 0.002 pg/m L for PFOA containing 0.001 pg/m L of 13C-PFOA by diluting 2.0 m L o f the PFO A solution described in 3.5.2.C and 0.1 ro L o f the *3C-PFOA solution described in 3.5.2. b to 100 mL with acetonitrile in a volum efric flask. f. Prepare a fortification standard o f 0.001 pg/m L for PFOA containing 0.001 pg/m L of 13C-PFOA by diluting 1.0 m L o f the PFOA solution described in 3.5.2.C and 0.1 mL of the *3C-PFOA solution described in 3.5.2. b to 100 mL w ith acetonitrile in a volum etric flask. g. Prepare a fortification standard o f 0.0005 pg/m L for PFOA containing 0.001 pg/m L of 13G-PFQA by diluting 0.5 m L o f the PFOA solution described in 3.5.2.C and 0.1 mL of the 33C-PFOA solution described in 3.5.2. b to 100 mL with acetonitrile in a volum etric flask. h. Prepare a fortification standard o f 0.0001 pg/m L fo r PFOA containing 0.001 pg/m L o f 13C-PFOA by diluting 0 .1 m L o f the PFOA solution described in 3.5.2.c and 0.1 m L o f th eT3C-PFOA solution described in 3.5.2. b to 100 mL w ith acetonitrile in a volum etric flask. i. Prepare a fortification standard o f 0,00005 pg/m L for PFOA containing 0.001 pg/m L of 13C-PFOA by diluting 0.05 m L o f the PFOA solution described in 3.5.2.C and 0.1 mL of the 13C-PFOA solution described in 3.5.2.b to 100 m L w ith acetonitrile in a volum etric flask. j. Prepare a fortification standard o f 0.001 pg/m L of 13C-PFOA by diluting 0 .1 mL o f the 13C-PFOA solution described in 3.5.2.b to 100 mL w ith acetonitrile in a volum etric flask. Store all fortification standard solutions in a refrigerator (in 125-mL LDPE bottles) at 2C to 6C for a m axim um period o f 1 year from the date of preparation, after which tim e it is necessary to make new standards using the stock solution. Exygen Research 000335 Page lO o f 27 3.5.3. Calibration Standards The calibration standards are processed through the extraction procedure, identical to the samples. The fortification of the standards before extraction is done according to the follow ing table: Cone. Of M ixed Fortification Solution (ng/m L ) 0.05 0.1 0.5 1.0 2.0 5.0 Fortification Volume (pL) 500 500 500 500 500 500 Volum e o f C o n tro l Sample (mL) 0.05 0.05 0.05 0.05 0.05 0.05 Cone, of E x tracte d C alib ratio n Standard (Ppb) 0.5 1 5 10 20 50 4. METHOD 4.1. Flow Diagram The flow diagram of the m ethod is given below , followed by a detailed description o f each step. M ethod Flow D iagram M easure 0.05 mL of sample (using disposable m icropipette) into protein precipitation colum n i Protein precipitation in acetonitrile using M ultiprobe A l LC/MS/MS analysis 4.2. Sam ple Processing N o sample processing is needed for serum o r plasm a samples. However,, frozen sam ples m ust be allowed to com pletely thaw , un-aided, at room temperature. Sam ples stored refrigerated should also be allow ed to equilibrate to room tem perature. All samples m ust be thoroughly m ixed before being sam pled for extraction. Exygen Research 000336 Page II o f 27 4.3. S a m p l e P r e p a r a t i o n a. Each batch o f samples analyzed (typically 35 or less) m ust include at least one reagent control (acetonitrile blank), one m atrix control (m ethod blank), two m atrix controls fortified at known concentrations to verify procedural recovery for the batch. b. A t least one sample per batch should be extracted in duplicate c. A t least one sample extracted should be separately fortified at a known concentration and carried through, the procedure to verify recovery. Additional matrix spikes may be perform ed at the sponsor's request. 4.4. Extraction a. Configure the M ultiprobe deck w ith the follow ing labware: 1.1000 pL disposable tips 2 .1 2 x 75 mm test tube rack 3. SPE m anifold with protein precipitation columns and 1 m L collection tray (plug any unused holes in the SPE m anifold) 4. Tip chute with disposal bag. b. Load 0.05 mL o f sample into the appropriate protein precipitation column. c. Place at least 1 m L of A cetonitrile containing the appropriate concentration of analyte and internal standard into a disposable 12 x 75 mm test tube. d. U sing the M ultiprobe's control softw are, W inprep, create a program that performs the following steps in sequence: 1. G et clean pipette tip(s). 2. Go to the appropriate 12 x 75 mm test tube(s) and aspirate an air plug followed by 500 pL o f ACN solution follow ed by a transport air plug. 3. Go to the appropriate protein precipitation colum n(s) on the SPE m anifold and dispense the ACN solution. 4. Go to the tipchute and drop the tip(s). 5. Repeat M ultiprobe procedure steps 1-4 until ACN solutions have been added to all required protein precipitation columns, 6 . Activate the SPE m anifold vacuum system in 10 to 20 second cycles until all ACN solution has been drawn through the protein precipitation column (usually 10 to 15 cycles). Note: Additional vacuum cycles can be initiated by opening the vacuum node o fthe W inprep m ethod and selecting the vacuum step "Preview. " e. Transfer ~ 400pL to an autosam pler vial w ith a pipette for analysis. 4.5. Quantitation 4.5J . LC/M S/M S System and Operating Conditions Instrum ent: PE SCIEX API 4000 Biom oleeular M ass A nalyzer SCIEX Turbo Ion Spray Liquid Introduction Interface C om puter: Dell O ptiPlex G X 110 000337 Exygen Research Page 12 of 27 Software: PE Sciex Analyst 1.2 HPLC Equipment: Hewlett Packard (HP) Series 1100 Quatpump G1311A Vacuum Degasser G1322A Autoinjector G1313A Column Compartment G1316A N ote: Two 4 x 10 mm hypercarb drop-in guard cartridge (K eystone, part # 844017-400) are attached in-line after the purge valve and before the sample injector port to trap any residue contaminants that m ay b e in the m obile phase and/or HPLC system. HPLC Column: Genesis Cs (JonesChrom atography), 2.1 mm x 50 mm, 4p Column Temperature: 30 C Injection Volume: 5 pL M obile Phase (A): 2 mM Ammonium A cetate in W ater M obile Phase (B): M ethanol T im e 0 .0 3 3 .5 3 .7 7 7 .5 9 9 .5 12 %A 40 40 0 0 0 40 40 40 40 %B 60 60 100 100 100 60 60 60 60 F lo w fm L /m in ) 0 .3 0 .3 0 .3 0 .5 0 .5 0 .5 0 .5 0 .3 0 .3 It m ay be necessary to adjust the H P IC gradient in order to optim ize instrum ent perform ance. Ions m onitored: A nalyte PFOA 13C -P F O A M ode N egative N egative Transition M onitored 413 -> 369 4 1 5 -> 3 7 0 A pproxim ate Retention Time ~2.8 min. ~2,8 min. The retention tim es may vary, on a day-to-day basis, depending on the column, batch o f m obile phase, etc. D rift in retention tim es is acceptable within an analytical run, as long as the drift continues through the entire analysis and the standards are interspersed throughout the analytical run. N ote: An alternative LC/MS/MS system m ay be used once, dem onstrated to be equivalent. Exvgen Research 1/00338 Page 13 o f 27 4.5.2. Tune File Parameters The m ass spectrom eter is tuned for each analyte by infusing a ~ 1 jig/m L standard solution of PFOA (at 10 pL/min, using an infusion pump) via a "T" into a stream of m obile phase containing 60% m ethanol and 40% '2m M am monium acetate in w ater at 0.3 mT/m in flow rate. The analyte is initially iu n ed fo r th e parent ion and then tuned for the product ion. Once the instrum enffs tuned, the optim ized param eters are saved as a tune file. This tune file is then used during routine analysis. 4.5.3. Calibration Procedures a. Inject the same aliquot (5 pL) o f each calibration standard into the LC/M S/M S. b. U se linear standard curves for quantitation. lin e a r standard curves are generated for each analyte by linear regression using 1 /x w eighting o f the ratio analyte peak area/intem ai standard peak area versus the ratio o f the concentration of analyte/concentration o f internal standard using A nalyst 1.2 (or equivalent) software system . Any calibration standard found to be a statistical outlier by using the appropriate outlier test (e.g. H uge O utlier Test), may be excluded from the calibration curve. However, the total num ber of calibration standards that could be excluded m ust not exceed 20 % o f the total num ber o f standards injected and at least one calibration standard at the LLOQ m ust be retained c. The correlation coefficient (R) for calibration curves generated m ust be >0.9925 (R2 >0.985). If calibration results fall outside these lim its, then appropriate steps must be taken to adjust instrum ent operation, and the standards or the relevant set o f samples should be reanalyzed. 4.5.4. Sample Analysis a. Inject the same aliquot (5 pL ) o f each standard, sample, recovery, control, etc. into the LC/MS/MS system. b. Standards corresponding to at least five or m ore concentration levels m ust be included in an analytical set. c. A n entire set o f calibration standards should be injected at the beginning o f a set follow ed by calibration standards interspersed every 5-10 samples (to account fo r a second set o f standards). As an alternative, an en tire'set of calibration standards may be included at the beginning and at the end o f a sam ple set. In either case, calibration standards m ust be the first and last injection in a sample set. d. The concentration of each sam ple/fortification/control is determ ined from the standard curve, based on the peak area o f each analyte. The standard responses should bracket responses o f the residue found in each sample set. Exygen Research Page 14 o f 27 000339 If necessary, dilute the samples in 50:50 m ethanol: w ater containing 1 ng/mL of 13C-PFOA to give a response w ithin the standard curve range r e. Fortification recoveries falling w ithin 85 to 115% (80 to 12 0 % fo r levels at the LLOQ) are considered acceptable. f. Extracted samples must be stored refrigerated betw een 2C to 6C until analysis. g. Samples in which no peaks are detected (i.e. s ig n a l: noise ratio < 3:1) at the corresponding analyte retention tim es will be reported as ND (not detected). Samples in w hich peaks are detected at the corresponding analyte retention times but are less than the low est concentration of the calibration standards (0.5 ppb where LLOQ = 0.5 ppb) w ill be reported as < 0.5 ppb. 4.6. Acceptance Criteria The following criteria must be m et to ensure the presence o f PFOA and 13CPFOA: 1. The chromatograms m ust show the follow ing peaks: PFOA: a daughter ion at 369 amu from a parent o f 413 amu 13C-PFOA: a daughter ion at 370 am u from a parent of 415 amu 2. A ny analyte present in m ethod blanks m ust be at least 3-fold low er than the LLOQ. Any analyte present in the reagent blank m ust be at least 5fold low er than the LLOQ. 3. Recoveries o f lab control spikes and m atrix spikes m ust be between .85115% (80-120% for levels at the LLOQ) o f their known values. Any m ethod fortification (lab control spike) falling outside the acceptable lim its warrants fe-extraction o f the entire analytical set. Any m atrix spike outside the acceptable range should be evaluated by the analyst to determ ine if re-extraction is warranted. 4. Any calibration standard found to be a statistical outlier by using an appropriate outlier test, may be excluded from the curve. However, the total num ber of calibration standards that could be excluded m ust not exceed 2 0 % of the total num ber of standards injected and at least one calibration standard at the LLOQ m ust be retained. 5. The correlation coefficient (R) for calibration curves generated m ust be >0.9925 (R2 >0.985). If calibration results fall outside these lim its, then appropriate steps m ust be taken to adjust instrum ent operation, and the standards or the relevant set o f samples should be reanalyzed. 000340 Exygen Research Page 15 o f 27 4.7. P e r f o r m a n c e C r i t e r i a The following two criteria m ust be perform ed as a system suitability test, before the com mencement o f analysis, when using an instrum entation set-up that has not been used for this method. First Criterion: Run a standard solution on LC/MS/MS corresponding to the estim ated LLOQ and obtain a signal-to-noise ratio o f at least 9:1, com pared to a reagent blank. If this criterion cannot be met, optim ize and change instrum ent operating param eters. Second Criterion: Run a set of standards of five or m ore concentration levels, spanning a range starting at or below the LLOQ up to the highest concentration level to be included in the analysis. Generate a calibration curve for the analyte and obtain a linear regression with a coefficient of determ ination (R2) o f at least 0.985. Once this criterion is m et, samples may be analyzed w ith standards interspersed. 4.8. Tim e R equired for Analysis A set o f 35 sam ples (1 reagent control, 1 m atrix control, 1 laboratory spike, 2 laboratory control spikes, and 30 sam ples) can be taken through the extraction procedure in approxim ately 2 hours by one person. The LC/M S/M S analysis (standards and 35 samples) will take approxim ately 9 hours. 5. CALCULATIONS a. U se Equation 1 to calculate the am ount o f analyte found (in ng/mL, based on peak area) using the standard curve (linear regression param eters) generated by the M ass Lynx software program. Equation 1: A nalyte found (ppb) = (A nalyte Peak area/TS peak area) - intercept)) x IS cone, (ppb) slope b. F or sam ples fortified with known am ounts o f analyte prior to extraction, use Equation 2 to calculate the percent recovery. Equation 2 : Recovery (%) = Analvte found (ppb) - avg. Analvte found in the corresponding sample (ppbl x 100% Amount Analyte added (ppb) Exygen Research 000341 Page 16 o f 27 6 . SAFETY The analyst should read the m aterial safety data sheets for all standards and reagents before perform ing this method. U se universal precautions when handling standards and reagents, including working in fum e hoods and wearing laboratory coats, safety glasses, and gloves. U se blood-borne pathogen handling precautions when handing serum and plasm a. Fxvren Research 000342 Page 17 o f 27 Table I Summary o f PFOA Recoveries for 0.5 ppb Fortification in R abbit Serum Exygen ID 0300808 Fort. 1 0300808 Fort. 2 0300808 Fort 3 Sponsor ID Lot 99H8400 L ot99H 8400 L ot99H 8400 E x tra ctio n A nalysis Fort. L ev el D ate D ate (ppb) 0 5 /2 2 /0 3 0 5 /2 3 /0 3 0 .5 0 5 /2 2 /0 3 0 5 /2 3 /0 3 0 .5 0 5 /2 2 /0 3 0 5 /2 3 /0 3 0 .5 AVERAGE: STAN DA RD DEVIATION: RELATIVE STAN DA RD DEVIATIO N: % Recovery 95 85 88 89 5.1 5 .7 Sum mary o f PFOA Recoveries for 3 ppb Fortification in R abbit Serum E xygen ID 0300808 Fort 4 0300808 Fort 5 0300808 Fort. 6 Sponsor ID Lot 99H8400 L ot99H 8400 Lot 99H8400 E xtraction A nalysis Fort. L evel D ate D ate (p p b ) 0 5 /2 2 /0 3 0 5 /2 3 /0 3 3 0 5 /2 2 /0 3 0 5 /2 2 /0 3 0 5 /2 3 /0 3 0 5 /2 3 /0 3 3 3 AVERAGE: STANDARD DEVIATIO N: RELATIVE STAN DA RD DEVIATIO N: % Recovery 108 102 107 106 3 .2 3 .0 Sum mary o f PFOA Recoveries for 40 ppb Fortification in R abbit Serum Exygen ID 0300808 F ort 7 0300808 Fort 8 0300808 Fort. 9 Sponsor ID Lot 99H8400 Lot 99H8400 Lot 99H8400 E xtraction A nalysis F ort Level D ate D ate (ppb) 0 5 /2 2 /0 3 0 5 /2 2 /0 3 0 5 /2 2 /0 3 0 5 /2 3 /0 3 0 5 /2 3 /0 3 0 5 /2 3 /0 3 40 40 40 AVERAGE: STAN DA RD DEVIATION: RELATIVE ST A N D A R D D EV IA TIO N : % Recovery 90 91 90 90 0 .6 0 .6 OVERALL AVERAGE: OVERALL STANDARD D EV IA TIO N : O VERALL RELATIVE STAN DA RD D EV IA T IO N : 95 8 .5 8 .9 Exygen Research 000343 Page 18 o f 27 Table H. Recovery of PFOA from Fortifications In Rabbit Plasma Sum mary of PFOA Recoveries for 0.5 ppb Fortification in Rabbit Plasm a Bcygen ID 0300922 F o r ti 0300922 Fort. 2 0300922 Fort. 3 Sponsor ID Lot 40K7400 L ot40K 7400 L ot40K 7400 E x tra ctio n A nalysis Fort. L evel- D ate D ate (ppb) 0 5 /2 2 /0 3 0 5 /2 3 /0 3 0 .5 0 5 /2 2 /0 3 0 5 /2 3 /0 3 0 .5 0 5 /2 2 /0 3 0 5 /2 3 /0 3 0 .5 AVERAGE: STAN DA RD DEVIATION: RELATIVE STANDARD DEVIATION: * % Recovery 99 100 100 100 0 .6 0 .6 Summary o f PFOA Recoveries for 3 ppb Fortification in Rabbit Plasm a Exygen ID 0300922 Fort. 4 0300922 Fort. 5 0300922 Fort. 6 Sponsor ID L ot40K 7400 L ot40K 7400 L ot40K 7400 E xtraction A nalysis Fort Level D ate D ate (ppb) 0 5 /2 2 /0 3 0 5 /2 3 /0 3 3 0 5 /2 2 /0 3 0 5 /2 3 /0 3 3 0 5 /2 2 /0 3 0 5 /2 3 /0 3 3 AVERAGE: STA N D A R D DEVIATION: RELATIVE STAN DA RD DEVIATION: % Recovery 107 106 105 106 1 .0 0 .9 Sum m ary o f PFOA Recoveries for 40 ppb Fortification in R abbit Plasm a Exygen ID 0300922 Fort. 7 0300922 Fort. 8 0300922 Fort 9 Sponsor ID L ot 40K74Q0 L ot40K 7400 L ot40K 7400 E xtraction A nalysis Fort. L evel D ate D ate (PPb) 0 5 /2 2 /0 3 0 5 /2 3 /0 3 40 0 5 /2 2 /0 3 0 5 /2 3 /0 3 40 0 5 /2 2 /0 3 0 5 /2 3 /0 3 40 AVERAGE: STAN DA RD DEVIATION: RELATIVE ST A N D A R D DEVIATION: % Recovery 91 92 92 92 0 .6 0 .6 OVERALL AVERAGE: O VERALL STAN DA RD D EV IA TIO N : OVERALL RELATIVE STA N D A R D D EV IA TIO N : 99 6 .3 6 .3 Exygen Research 000344 Page 19 o f 27 Figure 1. Calibration curve for PFOA in Control Rabbit Serum Serum (or method.rdb (PFOA): lin e a r" Regression f t /X" weighting): y =0.126 x +0.0131 ( r =0.9996) Exygen Research 000345 Page 20 o f 27 Figure 2. Representative Chromatogram of a Control Rabbit Serum Sample for PFOA with ~ 1 ng/mL of l3C-PFOA Rxveen Research Page 21 of 27 000346 R gure 3- jsate Exygen Research Page 22 of 27 000347 Wanre A Representative Chromatogram of Control Rabbit Serum FlgUre4' Fortified at 0.5 ppb with PFOA and with - 1 ngtaL of C- PFOA F.xveen Research Page 23 of 27 000348 Figure 5. Calibration curve for PFOA in Control Rabbit Plasma 1 Rssrifa-rrtfnkbpO^T^'FtgCTCnHW'wd1ir:y==Q1SB)c+QOIC0(r=QSBi) Exygen Research Page 24 of 27 000349 Figure 6 . Representative Chromatogram of a Control Rabbit Plasma Sample for PFOA with 1 ng/mL of 13C-PFOA i*i^II aRtitoettifaftmasid atiMWBMwanirf 11 Exygen Research Page 25 o f 27 0003So Figure 7. Representative Chromatogram of a 0.5 ppb Standard for PFOA in Control Rabbit Plasma with 1 ng/mL of CPFOA .1 Exygen Research Page 27 o f 27 000352 December 4,2003 M ary A. Kaiser, Ph.D. Research Fellow Corporate Center for Analytical Services Dupont Central Research & Development DUPONT Experimental Station P.O. Box 80302 W ilmington, DE 19880-0302 Clayton Work Order No. 03010080 Clayton Proposal No. 03DETLAB015.REV1 Dear Dr. Kaiser: We are pleased to provide the follow ing D raft report on the sampling and analytical method conditions follow ed for the method validation for This draft report is based on our proposal Sated July 18,2003. The sampling parameters may change as w e progress with the method validation. N ot all o f the parameters outlined in this report have been fully validated. I f you have any questions, please call m e at (248) 344-1770. Sincerely, Kem Charron Program Manager-Special Projects Laboratory Services K Enclosure 0 0 3 sa Clayton Group Services, Inc, Empirical Formula: M olecular W eight: CAS Number: OSHA: N one NIOSH: N one ACGIH: None AEL: 0 ,5 6 ppb Synonyms: Pentadecafluorooctanoic acid Clayton Work Order N o.: 03070834 Draft Report Date: Decem ber 4 ,2 0 0 3 Properties Appearance: Odor: M elting Point: W hite Powder Sampling and Storage Sampler: Osha V ersatile Sampler SKC C at No. 226-30-16 Storage: To be determ ined Shipping: Sam ples should be shipped at refrigerated temperatures Sample Stability: T o be determined Equipm ent 1. SKC OSHA V ersatile Sampler (O VS) C atN o. 226-30-16 2. HPLC System : LC pump with automated gradient controller, autosampler, optional tunable U V absorbance detector 6. M ass Spectrom eter: Triple quadrupole mass spectrometer 2 - 1000 amu m ass range minimum, API interface with Electrospray probe, negative and positive ionization m ode capability, and data acquisition/reduction software 7. Column: A gilen t H ypersilO D S, 2.1 x 100 mm, 3 pm particle size, Cat. # 79 9 1 6 0 0 -3 5 2 or equivalent 8. 16 mL amber glass vial with screw cap and Teflon/rubber liner 9. 4 mL amber glass v ial with screw cap and Teflon/rubber liner 10. Waters total recovery glass autosampler vials with Teflon/rubber liner 12. Syringe: 10, 2 5 ,5 0 , & 100 pL 13. D isposable glass test tube, 16 x 125 mm with caps | 12/04/003 DRAFT 2of4 Laboratory Services 000354 Clayton Work Order: 03070834 P erfluorooctanoic A cid Measurement Technique: H igh Performance Liquid Chromatography (HPLC) coupled with Tandem M ass Spectrom etry Detectors: Triple Quadrupole Mass Spectrometer (M icrom ass Quattro LC, or equivalent) Extraction: 4 mL o f extraction solvent for 60 minutes Instrum ent C onditions: H PL C C onditions M ass Spectrom eter C onditions Column: A gilent Hypersil ODS, 2.1 X 100 mm, 3 pm particle size Ionization M ode: Electrospray N egative Column Temp: 30 C Flow Rate: 0.3 mL/minute Injection Volum e: 5 pL Run Time: {4 minutes Retention Time: 6.4 minutes M obile Phase: Tim e (mtn) 0 5 9 10 14 Gradient %A %B 90 10 10 90 10 90 90 10 90 10 Curve - - - A = 2 mM Ammonium Acetate B = Methanol W avelength (UV): N ot Applicable N ebulizer Gas (L/hr); 96 D esolvation Gas (L/hr): 690 Source B lock Temperature (C ):120 D esolvation Temperature (C ):350 C ollision Gas Pressure (mBar):3 .1e-3 M ultiplier: 650 Source Settings Capillary V oltage (kV): Extractor (V ): RF Lens (V): A nalyzer Settings M SI LM&HM Resolution: M SI Ion Energy (V ): M S2 LM&HM Resolution: M S2 Ion Energy (V): Entrance (V ): E xit (V): ES -Ion 1.5 1 0.5 14 0.5 15 1.5 0 0 M ass Spectrom eter M ethod M S Function: M ultiple Reaction M onitoring (MRM) ESP- Inter Channel D elay for MRM (s): 0.1 Span (D altons): 0.5 Start time: 0 m in End time: 14 min A nalyte Specific Settings: Transition famu) D w ell fsi 9 4 1 2 ,9 - 368.9 (Q ) 0.5 Internal Standard H 4 1 4 .9 - 369.9 (Q ) 0.5 (Q ) = Transition used for quantitation Cone V oltage (V) 10 10 C ollision Energy feV l 10 10 1.2/04/003 DRAFT 2 of 4 Laboratory Services 003SS Clayton Work Order: 03070834 P erfluorooctanoic A cid Sampling 1. Samplers (SK C 226-30-16) are received from the manufacturer (SK C). 2 . Calibrate each sam pling pump with a representative collection device in line. 3 . Depending on the application, there are two flow rate options: (1) For area or personal m onitoring, sample at a flow rate o f 1.0 L/min for up to 48 0 m inutes to obtain a sam ple volum e o f 960 L. (2) For fen ce lin e m onitoring, sam ple at a flow rate o f 15 L/min for up to 1440 m inutes to obtain a sam ple volum e o f 21600 L. 4 . Label the sam ple with appropriate sample identification. 5 . A t the com pletion o f sam pling, re-calibrate each pump w ith a representative collection device in line. 6 . Cap the ends o f each sampler. 7 . Samples are stored at refrigerated temperatures. 8. A chain o f custody form is filled out and sent w ith the sam plers under refrigerated conditions to the Laboratory for analysis Sample P reparation Note: Samples are extracted just prior to analysis. Samples are stable for X days at X temperatures. 1. Extract each OVS sam pling tube in three separate sections. Place each section in a separate disposable glass test tube. Section 1: GFF Filter Section 2: X AD -2 Front portion o f media plus foam plug Section 3: X A D -2 Back portion o f media 2. Add 4 rtiL o f M ethanol to each test tube and shake for 60 m inutes on a m echanical shaker. 3. Transfer approxim ately 3 .5 mL o f each extract to a 4 mL amber glass vial by filtering through a Gelman 13-tnm GHP A crodisc. Extracts should be stored refrigerated. 4. For LC/M S/M S analysis, transfer 200 pL each sam ple extract to a j pL o f a 1.4 pg/m L Internal Standard solution ( J Add 10 Calibration and Quality Control 1. Calibrate daily w ith six working standards over the range o f 11.4 to 227 ng/m L fo r 'P H l. a) Calibration standards are prepared by transferring 200 pL each f H | | standard solution to a glass autosampler vial. Add 10 pL o f a 1.4 pg/m L Internal Standard solution | 12/04/03 DRAFT 3 of4 Labora tory Services 00356 Clayton Work Order: 03070834 Perfluorooctanoic Acid b) A nalyze standards together with sam ples and blanks, A calibration curve is analyzed at the beginning and end o f each run, and Continuing Calibration V erification (CCV) standards are analyzed a minimum o f every ten sam ples. Acceptance criteria for a CCV is 20%, c) Generate an internal standard, linear calibration curve from the observed peak area response factors and standard concentrations. 2. Determine the Extraction E fficiency (EE) at least once with each lot o f sam plers. Prepare at least one level in duplicate within the expected sam pling range. Include one media blank per lot. a) Inject pL amounts o f f l B spiking standard solution onto the GFF portion o f an OVS tube. b) A llow the tube to air-dry at ambient temperature. c) Cap the tube and store at refrigerated temperatures until extraction. d) Extract the fH H -sp ik ed and blank tubes as described in Sample Preparation. A nalyze an aliquot from each sam ple along with standards. Transfer 200 pL each extract solution to a glass autosampler vial. Add 10 p L o fa 1.4 pg/m L Internal Standard solution ( e) Calculate the ratio for the ng o f j B B found on the media spike to the theoretical amount spiked. The concentrations found in each o f the three sections m ust be sum med to obtain the total amount o fA B B found. The ratio reflects the recovery o f M i from the m edia. The mean recovery o f the replicates equals the EE for BA. Measurement 1. Set the liquid chromatograph and mass spectrometer conditions in accordance w ith those specified on page 2, or with suitable adjustments to optim ize chromatography and sensitivity. 2 . Inject the specified aliquot o f each standard and sample. 3 . Record die peak area response factor for A B B and Internal Standard peaks. The observed retention tim e is 6.4 m inutes for both B B sod Internal Standard. 12/04/0Q3 DRAFT 4 of4 Laboratory Services 00.357