Document V8e7920gMxoD7krvKM41z3g4

AR i . 33/3 - 495 M M 6 - r t l D uP ont-11763 Supplement Number 1 TRADE SECRET Study Title H-25509: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats Supplement Number 1 Test Guidelines: U.S. EPA Health Effects Test Guidelines OPPTS 870.3200 (1998) OECD Guidelines for Testing o f Chemicals Section 4: Health Effects, Number 410 (1981) Author: Carol Finlay, B.A. Study Completed on: August 4,2003 Supplement Number 1 Completed on: January 14,2004 Performing Laboratory: E.I. du Pont de Nemours and Company Haskell Laboratory for Health and Environmental Sciences Elkton Road, P.O. Box 50 Newark, Delaware 19714-0050 Laboratory Project ID: DuPont-11763 Company Sanitized. Does not <* #*wi Page 1 o f 81 Company Sanitized. Does not contain TSCA CBI H-25509: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats_____________ D u P o n t-11763 Supplement Nmnber 1 QUALITY ASSURANCE STATEM ENT Haskell Sample Number(s): 25509 Dates o f Inspections: Supplement Number 1: December 12,15, 2003 Dates Findings Reported to: Study Director: December 15,17, 2003 Management: December 15,17, 2003 Reported by: iX W rifr 1< l l t q _________ Wonda KftKelly Quality Assurance Auditor ^ (1 i l i Date K&mpany Sanitized. D oes not contain TSCA CBI H-25509: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats_____________ DuPont-11763 Supplement Number 1 C E R T IF IC A T IO N We, the undersigned, declare that this report provides an accurate evaluation of data obtained from this study. Approved by: ^ Scott E. Loveless, PhJD. Management Issued by Study Director: f A ^ Cfo t n i _ Carol Finlay, B.A.J Staff Toxicologist I3 " 5 V W '2 0 0 ^ Date v Date jP g iW y Sanitized. Does not contain TSCA CBS H-25509: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats_____________ DuPont-11763 Supplement Number 1 TABLE OF CONTENTS Page Q U A LITY ASSURANCE STA TEM EN T...........................................................................................2 C E R T IF IC A T IO N .................................................................................................................................. 3 STUDY IN FO R M A TIO N .......................................................................................................................5 R EA SO N F O R SU PPLEM EN T NUM BER 1....................................................................................6 SUM M A RY ........... ................ 7 A PPEN D IX A ANALYTICAL R E P O R T .......................................................................................... 9 JggfMpapff Sanitized. Does not contain TSCA CBJ -4 - H-25509: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats __________ STUDY INFORM ATION Substance Tested: Svnonvms/Codes : Submitter's Notebook Numberts) Haskell Number: 25509 DuPont-11763 Supplement Number 1 Sponsor: E.I. du Pont de Nemours and Company Wilmington, Delaware 19898 U.S.. Study Initiated/Completed: 22-Nov-2002 / (see report cover page) Itgjnpanjr Sanitized. Does not contain TSCA CB -5 - H-25509: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats ______ DuPont-11763 Supplement Number 1 REASON FOR SUPPLEMENT NUM BER 1 This supplement contains the results o f the analyses o f plasma samples collected at terminal sacrifice and analyzed for the presence o ff JSgWpalfi? Sanitized. D oes Hot contain TSCA CBI H-25509: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats_____________ DuPont-11763 Supplement Number 1 SUM M ARY A 28-day dermal toxicology study was conducted with male Crl:CD(SD)IGS BR rats. Ten rats per group were exposed dermally for 6 hours/day for a total o f 28 consecutive days to dosages o f 0, 5, 50, or 500 mg/kg/day H-25509. The ground test material was mixed with mineral oil, applied to the shaved, intact skin o f the rats, and covered with occlusive dressings for the duration of the daily exposure. All rats were sacrificed at the end of the exposure period. Under the conditions o f the study, the NOEL for toxicity was 50 mg/kg/day, based on decreased cholesterol at 500 mg/kg/day. In addition to assessments for toxicology endpoints, blood was collected for analyses at the end o f the exposure period. Whole blood was collected on study day 27 and retained for analysis of total fluorine. The whole blood was analyzed by use o f a Wickbold torch combustion method and an ion selective electrode. The limit o f detection (LOD) for the method is 0.5 ppm (500 ppb). At the 28-day sacrifice, blood was collected, centrifuged, and plasma retained for ana^ys^ 4 B H was extracted from plasma by protein precipitation in acetonitrile. The compound was quantified by liquid chromatography/tandem mass spectrometry (LC/MS/MS) analysis using selected reaction monitoring (SRM). Quantification was performed using extracted calibration standards containing an internal standard. The lower limit o f quantitation (LLOQ) for this method is 10 ppb. Results o f these analyses are presented in this supplemental report. One sample each o f the stock mineral oil and stock dressing material used to occlude the test sample application was also analyzed for the presence o f total fluorine. The Wickbold torch combustion method was used for this analysis. T h e ^ U f e v e l s in plasma were 61.4 ppb, 155.5 ppb, 1315.9 ppb, and 14176 ppb for dosages o f 0, 5, 50, and 500 mg/kg/day, respectively. The total fluorine levels in blood were 1075 ppb, 1470 ppb, and 9316 ppb for dosages o f 0,50, 500 mg/kg/day, respectively. All total fluorine levels at 5 mg/kg/day were below the limit o f detection. There was 500 ppb fluorine in the stock mineral oil and 1760 ppb fluorine in the stock bandage material. offH^jareThe results of the analysis summarized in Table 1 (based on Appendix A, Table HI o f Exygen report). The results o f the analysis o f total fluorine in the mineral oil dressing material are in Table 1. The analyses o f total fluorine were in the original report and have been included in Table 1 for comparative purposes. The details o f the analyses which were conducted by Exygen Research, are reported in Appendix A. Sanitizes. V m 9 m i contain T tsri& c*n -7 - H-25509: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats_____________ D u P o n t-11763 Supplement Number 1 TABLE 1 MEASURED CONCENTRATION (ppb) ___________ Blood Analyses___________ Dosage (mg/kg/day) WHOLE BLOOD FLUORINE8 PLASMA MEAN S.D. n MEAN S.D. n 0 1075 5 <500* 50 1470 500 9316 N=number o f samples S.D. = Standard deviation * All values below limit o f detection, a LOD = 500 ppb b L L O Q - 10 ppb________________ 700.0 0.0 308.6 5742.4 5 5 5 5 61.4 155.5 1315.9 14176 27.9 25.5 482.3 4534.0 10 10 10 10 SAMPLE ANALYSIS SAMPLE TOTAL FLUORINE tnnbl Mineral Oil 500 Bandage Material__________________________ 1760___________ JPompany Sanitized. D oes noi contain TSCA CBf H-25509: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats_____________ DuPont-11763 Supplement Number 1 APPENDIX A Analytical Report Sanitized. Dows not contain TSCA CBI H-25509: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats DuPont-11763 Supplement Number 1 STUDY TITLE H-25509: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats DATA REQUIREMENTS Analytical Method Requirements STUDY DIRECTOR Carol Finlay ANALYTICAL PHASE COM PLETED ON September 23, 2003 PERFORMING LABORATORY / TESTING FACILITY Exygen Research 3058 Research Drive State College, PA 16801 Phone: 814-272-1039 STUDY SPONSOR E. L du Pont de Nemours and Company (DuPont) P.O. Box 50 Newark, DE 9714-0050 USA Sanitised. D oes not contain TSCA CBi H-25509: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats_____________ DuPont-11763 Supplement Number 1 GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT milled "H-25509: Repeated-Dose Dermal Toxicity 28Jay Study in M ale Rats," conducted for DuPont, was performed in compliance with US EPA TSCA (40 CFR part 792) by Exygen Research. SilyR. Decker Principal Investigator Exygen Research _____ i d A & L Carol Finlay Study Director DuPont ' j ^ 5 '. ............ S. Mark Kennedy * Sponsor Representative DuPont f a i ft Date M -b e ,t " U t? Date Date Exygen Research Page 2 of 72 li!*}? Sanitized. Does lot contain TSCA CBI -11 - H-25509: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats_____________ DuPont-11763 Supplement Number 1 QUALITY ASSURANCE STATEMENT Exygen Research's Quality Assurance Unit reviewe_)_______________ entitled, "H-25509: Repeated-Dose Dermal Toxicity 253!>ay Study m Male Rats". All phases were reviewed for conduct according to Exygen Research's Standard Operating Procedures, the Study Protocol, and all applicable Good Laboratory Practice Standards. All findings were repotted to the Study Director and to management. Phase Date lhsbected Date Reported Date Reported to to Exygen Principal Management Investigator Date Reported to Study Director and SDonsor Management 1. Fortification and Extraction 07/23/03 07/29/03 08/13/03 08/19/03 2. Raw Data & Draft 08/11,12/03 Report Review 08/14/03 08/16/03 08/19/03 3. Raw Data &Final Analytical Report Review 09/22/03 09/22/03 09/23/03 09/23/03 ^ d k ^ J d j.u ) ___________ Lydia JyShaffer(/ (J Quality Assurance Auditor _ iik Date Exygen Research Page 3 of 72 P& ty SnlHz<&Does not contain TSCA CBI - 12- H-25509: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats____________ DuPont-11763 Supplement Number 1 CERTIFICATION OF AUTHENTICITY This report, for the raw data for te study. is a true and complete representation of Submitted by: Exygen Research 3058 Research Drive State College, PA 16801 (814) 272-1039 Principal Investigator, Exygen: cker Scientist/ftincipal Investigator Exygen Research ... Date* Exygen Research Facility Management: / J o h n M. Flaherty / Vice President Exygen Research Study Director, DuPont: Date Carol Finlay DuPont ( 0 Date Sponsor Representative, DuPont SS. Mark Kennedy DuPont _____________________ ' Exygen Research 9 - 0<sr Date Page 4 o f 72 Company Sanitized. Odes not contain TSCA CBl -13 - H-25509: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats_____________ DuPont-11763 Supplement Number 1 H-25509: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats SPONSOR STUDY NUMBER: DuPont-11763 TYPE OF STUDY: SAMPLE MATRIX: TEST SUBSTANCE: SPONSOR: STUDY DIRECTOR: Residue Rat Plasma E L du Pont de Nemours and Company P.O. Box 50 Newark, DE 19714-0050 Phone: (302) 366-5255 Carol Finlay DuPont TESTING FACILITY: ANALYTICAL PHASE TIMETABLE: Exygen Research 3058 Research Drive State College, PA 16801 Study Initiation Date: 11/22/02 Analytical Start Date: 07/22/03 Analytical Termination Date: 07/31/03 Analytical Report Completion Date: xx/xx/xx Exygen Research Page 5 o f 72 Company Sanitized. Does not contain TSC CB1 -14- H-25509: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats_____________ DuPont-11763 Supplement Number 1 PROJECT PERSONNEL The Study Director for this project at DuPont was Carol Finlay. The following personnel from Exygen Research were associated with various phases of the study. Name Paul Connolly Emily Decker Mark Neeley Mark Ammerman Tide Technical Lead-LC/MS Scientist Scientist Sample Custodian Exygen Research Page 6 of 72 jjg jg liX -15- Soes BelfconfalnTSCA CBI H-25509: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats __________ DuPont-11763 Supplement Number 1 /****"* TABLE OF CONTENTS TITLE PAGE.................................................................................................. GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT. QUALITY ASSURANCE STATEMENT................................................. CERTIFICATION OF AUTHENTICITY.................................................. STUDY IDENTIFICATION............................................................. .......... PROJECT PERSONNEL......................................................................... TABLE OF CONTENTS......................................................................... LIST O F TABLES...................................................................................... LIST OF FIGURES................................................................................... LIST OF APPENDICES........................................................................... 1.0 SUMMARY ........................................................................................ 2.0 OBJECTIVE........................................................................................ 3.0 INTRODUCTION............ ................................................................. 4.0 TEST SYSTEM.................................................................................. 5.0 REFERENCE MATERIAL............................................................... 6.0 DESCRIPTION OF ANALYTICAL M ETH O D ........................... 6.1 Extraction Procedure............................................................... . 6.2 Preparation of Standards and Fortification Solutions 6.3 Chromatography............................................................. 6.4 instrument Sensitivity..... ............................................. 6.5 Description o f Instrument and Operating Conditions 6.6 Quantitation and Example Calculation....................... 7.0 EXPERIMENTAL DESIGN................................................................................... 8.0 RESULTS................................................................................ ................................. 9.0 CIRCUMSTANCES THAT MAY HAVE AFFECTED THE QUALITY OR INTEGRITY OF THE D A TA ................................................................................ 10.0 CONCLUSIONS.................................................. 11.0 RETENTION OF DATA AND SAMPLES...... ..I ..2 ..3 ..4 ..5 ..6 ..7 ..7 ..8 ..8 ..9 ..9 ..9 ..9 10 11 11 11 12 12 12 13 14 15 15 15 15 LIST OF TABLES Table I. Summary o fi^ p R l (ng/mL) in Rat Plasma Control Samples.......................... 17 Table U Summary of Recoveries o f Q j( p f t% ) in Rat Plasma Sam ples....................... IS Table BL Summary of Residue Found for m P g / m L ) in R at Plasma Samples......19 Table IV. Summary of Residue Found fo (ng/mL) in Re-Extracted Rat Plasma Samples................. ....................................................................................20 Exygen Research Page 7 of 72 .Sanitized. Does not contain TSCA CM H-25509: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats________ DuPont-11763 Supplement Number 1 LIST O F FIG U R ES Figure 1. Typical Calibration Curve for .........................................................-..... 22 Figure 2. Chromatogram Representing a Calibration Standard 10 ppb with - 5 ng/mL o f 13C 4 ^ B t ...............................................................................23 Figure 3. Chromatogram Representing Control Rat Plasm a fop of with - 5 ng/mL ..........24 F ig u re d Chromatogram Representing Control R at Plasma Fortified at 10 ppb with 5 ng/mL of 13C | Figure 4. Chromatogram Representing Rat Plasma Sample with ~ 5 ng/mL LIST O F APPEN D IC ES A ppendix .4 Study Protocol DuPont-117631 Amendments and Deviation...... Appendix B LC/MS/MS" . Page Iin Serum by 52 Exygsn Research Page 8 o f 72 ggflipany Sanitized. Does no! confalo TSCA CBI -17- H-25509: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats DuPont-11763 Supplement Number 1 1.0 SU M M A RY Exygen Research (Exygen) analyzed samples of rat plasma for residues of f l i according to protocol DuPont-11763 (Appendix A) using the analytical method Appendix B). The limit of quantitation for this method is 10 ng/mL, which was established at Exygen in the method validation stu d y M H H H I Residues o :N H f e in the rat plasma samples ranged from 19.3 ng/mL to 26,000 ng/mL. The average recovery standard deviation forflBSBrin fortified rat plasma samples was 101% 4.6% . , 2.0 OBJECTIVE The objective of this analytical phase was to analyze rat plasma samples received at Exygen using the method entitled "M ethod of Analysis for the Determination Serum by LC/MS/MS". 3.0 INTRODUCTION This report details die results of fee analysis of rat plasma samples for analytical method ( ising the The study DuPont-11763 was initiated on November 22, 2002, when the study director signed protocol number 1012. The analytical start date was M y 22, 2003, and the analytical termination date was M y 31,2003. 4.0 TEST SYSTEM The control rat plasma used for control samples and laboratory control fortified samples was received from DuPont. Forty rat plasma samples were received at Exygen on M y 22,2003, logged in by Exygen personnel and placed in frozen storage (< -20C). Sample login and chain of custody information can be found in the raw data package associated with this study. Storage records will be kept at Exygen Research and a true copy of the storage records will be furnished upon request. Exygen Research Page 9 of 72 @pan; Sanitized Does not contain TSCA CBi -18- H-25509: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats_____________ DuPont-11763 Supplement Number 1 5.0 REFERENCE MATERIAL The analytical standard received at Exygen on October 9, 2002 f rom Oakwood Products, Inc. The control article (internal standard) received at Exygen on February 20, 2003 from DuPont The available information for die reference material is listed below. The analytical standard was stored dessicated in a refrigerator and the control article was stored at room temperature. Oomnoi Exvaen Inventory No. Lot No. P uritvf% i ExDiration Date The structure of below. and the internal standard are given Exygen Research Page 10 of 72 gmpari Sanitized. D oes 1 contain TSC A CBI -19- H-25509: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats DuPont-11763 Supplement Number 1 6.0 DESCRIPTION OF ANALYTICAL METHOD 6.1 Extraction Procedure 1. Measure 0.1 mL of sample into 2 mL eppendorf centrifuge tubes (fortify with analyte as needed, close lid, and vortex ~ 10 seconds). 2. Add enough acetonitrile containing internal standard at 0.005 pg/mL (accounting for fortification volume) to make extraction volume 500 pL and vortex for ~ 10 seconds. 3. Centrifuge for ~ 10 minutes at - 14,000 rpm. 4. Analyze samples using electrospray LC/MS/MS. 6.2 Preparation o f Standards and Fortification Solutions The stock standard solution o i W w a s prepared on May 21, 2003, at a concentration of approximately 100 pg/mL by dissolving approximately 10 mg of the standard (corrected for purity) in methanol. H ie stock standard solution o f was prepared on March 17, 2003, at a concentration of approximately 123 pg/mL by dissolving approxim atelylO m g of the standard (corrected for purity) in methanol. A fortification solution o f ^ H - a t 10 pg/mL was prepared by taking 10 mL of the stock standard solution and b rin g in g tfi^ o lu m e up to 100 in acetonitrile. A 0.98 pg/mL fortification solution of f l M v w a s prepared by taking 0.8 mL of the stock and bringing the volume up to 100 mL with acetonitrile. A set of standards containing internal staxum d^vas prepared by dilution of the 10.0 pg/mL d i s s o l u t i o n and the 0.98 pg/mL V flH H M o lu tio n in the following manner. These solutions were used to fortify the samples and the extracted calibration standards. Initial Cone. Volume (mL) Diluted to (mL) Final Cone. (Pg/mL)1 (pg/mL)1 10.0 10.0 1002 1.0 10.0 1.0 1002 0.1 10.0 0.1 1002 0.01 1of H M b n l v ___ 0.5 mL of the 0.98 pg/mL solution was addeibjrto^CMnaking final volume to give a concentration of 0.005 pg/mL of S H M P f c n each solution. The calibration standards are processed through the extraction procedure, identical to the samples. The extracted standards are assigned a two-week expiration date from the date o f extraction based on refrigerator stability data obtained during the method validation Exygen Research Page 11 of 72 j99)|ag>Sanitized. Does notcontain TgtCAOBI -20- H-25509: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats________ DuPont-11763 Supplement Number 1 study The fortification of the standards before extraction is done according to the following table; Cone. Of Fortification Solution (pg/mL) 0.01 0.01 0.1 0.1 0.1 Fortification Volume (pL) 100 200 50 100 200 Volume of Control Sample (mL) 0.1 0.1 0.1 0.1 0.1 Cone, of Extracted Calibration Standard <PPb) 10 20 50 100 200 The extraction solution was prepared by taking 0.S tnL o f die 0.98 p g /m L V H II ^ M solution and bringing the volume up to 100 mL with acetonitrile. This solution was also used for the dilution solvent. The stock standard solution and all fortiQcation/calibration standard solutions were stored in a refrigerator (4 2C) when not in use. Documentation o f standard preparation can be found in the raw data associated with this report. 6.3 Chromatography Quantification accomplished by analysis using electrospray LC-MS/MS. The retention tim e a fw H fe w a s - 2.Q min., with no significant interfering peaks (< 20% of the LOQ standard) in the control samples corresponding to die analyte retention times. 6.4 Instrument Sensitivity The smallest amount o ^ W b j e c t e d during the chromatographic run was equivalent to 10 ng/mL. 6.5 Description of Instrument and Operating Conditions A PE Sciex LC-MS/MS coupled to a Hewlett Packard HPLC system was used. Data acquisition and processing w o e performed using Analyst 1.2 software. Detailed operating conditions are listed below: Mass Spec: Interface: C om puter Software: PE SCIEX API 4000 Biomolecular Mass Analyzer SCIEX Turbo Ion Spray Liquid Introduction Interface Turbo Ion Spray temperature = 350 C with N2 at -7 L/min Dell Optiplex G X 110 PE Sciex Analyst: Version 1.2 Exygen Research rage 12 of 72 -21 - jCpmpany Sanitized. Does not contain TSCA CB! H-25509: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats DuPont-11763 Supplement Number 1 HPLC: Hewlett Packard (HP) Series 1100 HP Quat Pump HP Vacuum Degasser HP Autoinjector HP Column Compartment HPLC Column; Genesis Cg (Jones Chromatograhpy), 2.1 mm x 50 mm, 4p Column Temp.: 30 C Mobile Phase (A): 2 mM Ammonium Acetate in water Mobile Phase (B): Methanol Time 0 3.0 3.5 3.7 7.0 7.5 9.0 9.5 12.0 Injection VoL: 5 pL Ions monitored: %A 40 40 0 0 0 40 40 40 40 % B H ow OnL/min) 60 0 3 60 0.3 100 0.3 100 0.5 100 0.5 60 0.5 60 0.5 60 0.3 60 0.3 Mode Transition Monitored Negative 413 - 369 Negative 415->370 6.6 Quantitation and Example Calculation Five microliters of sample or calibration standard were injected into the LC-MS/MS. The peak area was measured and the standard curve was generated by linear regression using Vn weighting of the ratio analyte peak area/intemal standard peak area versus the ratio of the concentration of analyte/concentraticm of internal standard using Analyst 1.2 (or equivalent) software system. The residue concentration was determined from the following equations: Equation 1 was used to calculate the amount of analyte found (in ppb or ng/mL, based on ratio of analyte peak area to internal standard peak area) using the standard curve (linear regression parameters) generated by the Analyst software program. Equation 1: Analyte found (ppb) = ftAnalvte Peak area/IS peak areal - intercept!) XIS cone. (ng/mL) x DF slope Exygen Research Page 13 of 72 Sgmpany Sanitized. Does not contain TSCA CBI -22- H-25509: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats_____________ DuPont-11763 Supplement Number 1 For samples fortified with known amounts of analyte prior to extraction. Equation 2 was used to calculate the percent recovery. Equation 2: Recovery (%) = Analvte found (ppb) - ave. Analvte found in control (ppbt x 100 Amount Analyte added (ppb) An example of a calculation using an actual sample follows: Rat plasm a sample Exygen fortified w ithflHM } Where: analyte peak area = 79538 IS peak area = 172676 IS cone. (ng/mL) = 4.167 ng/mL intercept = 0.0344438 slope = 0.179697 dilution factor (DF) = l ppb added (fort level) = 10 amt found in control = < 10 (Not quantifiable) F ran equation 1: Analyte found (ppb) = f09538/1726761 - 0,03444381 x 4.167 x 1 0.179697 = 9.88 ppb F ran equation 2: % Recovery = (9.88 ppb - 01 x 100 10 ppb = 99% Note: This example calculation was done using rounded numbers, and therefore may be slightly different from the values shown in the RAW DATA. 7.0 EXPERIMENTAL DESIGN Each analytical set was extracted according to method j m U B f l f c n d consisted of a reagent blank (except for one matrix control, two laboratory matrix Exygen Research Page 14 of 72 Company Sanitized. Does not contain TSCA CBi -23- H-25509: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats__________ DuPont-11763 Supplement Number 1 controls fortified at known concentrations, one field sample fortified at a known concentration, ten to twenty field samples, and one field sample extracted in duplicate. 8.0 RESULTS T h d ^ H B tb u n d in the control rat plasma samples are listed in Table I. Individual recoveries f o 4 f l 0 l in the laboratory fortified rat plasma samples are detailed in Table II. The average percent recoveries standard deviation for f l H F i n the rat plasma samples was 101% 4.6% . Individual results for the f i B R in the rat plasma samples are given in Table H I. At the requestof the sponsor, ten rat plasma samples were re-extracted to confirm the residue detected. The results for the re-extracted samples are given in Table IV. Residues of m m in the rat plasma samples ranged from 19.3 ng/mL to 26,000 ng/mL. A typical calibration curve for M B and representative chromatograms are given in Figures 1-5. 9.0 ORCUMSTANCES THAT MAY HAVE AFFECTED THE QUALITY OR INTEGRITY OF THE DATA There are no known circumstances that have affected the quality or integrity of the data. 10.0 CONCLUSIONS The rat plasma samples were successfully analyzed according to metho 11.0 RETENTION OF DATA AND SAMPLES When the final report is complete, all original paper data generated by Exygen Research will be shipped to die sponsor. This does not include facility-specific raw data such as instrument logs. Exact copies of all raw data, as well as a signed copy of the final analytical report and all original facility-specific raw data, will be retained in fee Exygen Research's archives for the period of time specified in 40 CFR Part 792. Retained samples of reference substances are archived by the sponsor. Exygen Research Page 15 o f 72 JSpmpan^ Sanitized. Does not contain TSCA CB1 -24- H-25509: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats________ _____ DuPont-11763 Supplement Number 1 TABLES Exygers Research Page 16 of 72 jpmpany Sanitized. Does not contain TSCA CBt -25- H-25509: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats_____________ DuPont-11763 Supplement Number 1 Table I. Summary t lng/mL) In Rat Plasma Control Samples Sponsor _______ ID_______ #17 Control 07/22/03 #17 Control 07/23/03 #18 Control 07/31/03 (n g foli-). < 10.0 < 10.0 < 10.0 Exygen Research Page 17 o f 72 St&mpanx Sanitized. Does not contain TSCA CB1 -26- H-25509: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats ________ DuPont-11763 Supplement Number 1 Table n . Summary of Recoveries Samples in Rat Plasma Sponsor ID #17 (LCS A) 07/22/03 #17 {LCS B) 07/22/03 667165 (Matrix Spk) #17 (LCS A) 07/23/03 #17 (LCS B) 07/23/03 667185 (MtfixSpk) #18 (LCS A) 07/31/03 #18 (LCS B) 07/31/03 667166 (Matrix Spk) (na/mL) 9.88 1050 1220 10.2 1000 2970 10.3 97.0 184 Amount Fortlfied (ng/mL) 10.0 1000 1000 10.0 1000 1000 10 100 100 AVERAGE: STD DEV; RSD: Recovery (%) 99 105 109 102 100 95 103 97 96 101 4.6 4.5 Exygen Research -27- Page 18 of 72 , Does hot contain TSCA CBI H-25509: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats__________ DuPont-11763 Supplement Number 1 Table III. Summary of Residue Found for flH B (n g/m L ) in Rat Plasma Samples Sponsor ID 667165 667165 Dup 667166 667167 667168 667169 667170 667171 667172 667173 667174 667175 667176 667177 667178 667179 6671B0 667181 667182 667163 667184 667185 667185 Dup 667166 667187 667188 667169 6671SO 667191 667192 667193 667194 667195 667196 667197 667198 667199 667200 667201 667202 667203 667204 PFOA (ng/mL) 132 118 79.7 65.6 75-3 40.4 37.8 36.4 38.5 eoe 407 155 165 111 179 197 161 132 134 175 146 2020 2050 999 958 1220 973 1940 1050 2040 965 979 12500 16100 11300 9660 26000 13600 13000 13300 14600 11500 Exygen Research Page 19 of 72 JBgfflpany Sanitized. Does noi contain TSCA C ii H-25509: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats_____________ DuPont-11763 Supplement Number 1 Table IV. Summary of Residue Found fcr^ B H ^ n g/m L ) in ReExtracted Rat Plasma Samples Sponsor ID____________ I'ng/mL) 667165 94.7 667166 88.6 667166 Dup 86.8 667187 64.9 667166 73.9 667169 40.6 667170 37.3 667171 19.3 667172 38.2 667173 64.3 667174 47.4 Exyger. Research -29- Page 20 of 72 Does not contain TSCA OBI H-25509: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats_____________ DuPont-13763 Supplement Number 1 FIGURES Exygen Research Page 21 of 72 -30- J ^ n p a n y Sanitized. D oes not contain TSCA CBl H-25509: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats_____________ DuPont-11763 Supplement Number 1 Figure 1. Typical Calibration Curve for 072203* URM!* RWWMion f l 7*aigtitoi): y m 0.I788S7 x 0A3M43S (r . 0.9997133) Exygen Research Page 22 of 72 -31 - m p m % gan iiized. D oes net contain TSCA CBi H-25509: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats_____________ ! DuPont-11763 Supplement Number 1 Exygen Research Page 23 o f 72 iPsmpany Sanitized. Does not contain TSCA OBI -32- H-25509: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats_____________ DuPont-11763 Supplement Number 1 Figure 3. Chromatogram Representing Control Rat Plasma for pith ~ 5 ng/mL of ,3C4 Exygen Research Page 24 of 72 g&flipany Sanitized. D oes not contain TSCA CEN 33 H-25509: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats___________ DuPont-11763 Supplement Number 1 Figure 4. Chromatogram Representing Control Rat Plasma Fortified at 10 ppb with Exygen Research Page 25 o f 72 Company Sanitized. D oes not contain TSCA CBi 34 H-25509: Repeated-Dose Dermal Toxicity DuPont-11763 28-Day Study in Male Rats________________________________________________________ Supplement Number 1 (/***'\ Figure 5. Chromatogram Representing Rat Plasma Sample for( with - 5 ng/mL of -g - T^SlT s: B - 'i f = ar Exygen Research TT" Page 26 of 72 Company Sanitized. D oes not contain TSCA CB! H-25509: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats_____________ DuPont-11763 Supplement Number 1 APPENDIX A Study Protocol DuPont-11763 and Amendments and Deviation /"""N Exygen Research Page 27 of 72 |?9itipany Sanitized. Does not contain TSCA CBt -36- H-25509: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats_____________ DuPont-11763 Supplement Number 1 Jill 23.2003 6 :17AM HASKELL 1 / UM H-2S509: Repeitod-DowDm*I Toxichy 28-OtyStudyin M ile R i HO. 6266 f. 2/23 DaPont-inO Protocol HariteU A nim al W elfare Comnrittoe Ni RCCEIVED TOC JUL.E3. 6:S6Wt Exygen Research -I- PRINT TOE JU ..2 3 . GS40flH Page 28 o f 72 tSSfrPQnySanitized. Does not contain TSCA CBI -37- H-25509: Repeated-Dose Dermal Toxicity DuPont-11763 28-Day Study in Male Rats________________________________________________________ Supplement Number 1 Jill. 23.2003 6:17AM HASKELL ! / 1300 B-2509: Repada&DotoDamnlTtHfcfy 28-Pur5w4yla MllnTUa Ha 6286 P. 3/23 Dupom-l 1763 TABLE OF CONTENTS Page n-m tODucnoN. --3 OBJECTIVE, -3 SPONSOR.AND TESTFACILITY ...3 REGULATORY COMPLIANCE... -.3 STUDY DESIGN.,. MATERIALS AND METHODS. NtHNWMMMIMfM ....... 4 __ 4 A. TestSt&atence,.TM.- 4' B. Test Species____ _ ___4 C. AnimalHo*bsdry. D. Pretest Period.* L ppytfng Rpw^y F. AtaignmectloGroupi.- ___S ... ...6 _6 G. AdamrijtntkmofTcstSufciiaiict.., .._ 7 H. Body Wdgjrts------------- ------------- L Food CoanmylKm and FoodEfficiency. 3. CUmcalObwrvalionsend Mortality. X. Total Fluorine and FerflaorowtanotcAdd Level Evaluations,----- __ 7 -.TM...88 L. Clinical Pattology Evaluation._________ . . . _______ ,,,,____ 8 t, Hematology_____ ___ 2. d im o i OMroistry....... .9 mnwtmHiumwwmnee9 3. Urinalysis. 9 M. AnatomicalPathology.. ____10 1. Pretort- ----- 10 2, Dosing P bajc. ----- 10 STATISTICAL ANALYSES.. ___ .13 SAFETYAND HOUSEKEEPING., RECORDS AND SAMPLE STORAGE-.KtriMMakH) ____13 ----- 14 REFERENCES__ ___________ ------14 PROTOCOL APPENDDC. SIGNATURES________ ..... . ____15 nmwu.m_.__16 REOEIVED TDC JU..23. 626AM Exygen Research 2PRINT TIME JUL.23. 6!40fiM Page 29 of 72 Company Sanitized. D oes not contain TSCA CBI -38- H-25509: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats DuPont-11763 Supplement Number 1 JUL. 23, 2005 6:17*M HASKELL 1 / 1300 H-2J509; BcpcMadDue DmmlToxldtj' IS-Pay Saidria MileRun NO. 6286 P. 4/23 - textei. The<to*agasofthetestaobatanco were selected on the baril of a range finding study. OBJECTIVE The ohfcctive ofthis study is to evaluatethe subehmme toxicitypotential o fH-25509 administered deanaltyta malaratfor28 days. ThedemolimdeofadauabtcadoB was selected because it is a potential toute ofboron exposure. SPONSORAND TEST FACEiTY This studyis sponsored by EX da Pot* doNcmoroindGMipinKWUmntgtoii, Delaware. The sponsort approvalwas efieetivethedatetho sponsorauthorizedduewodecmHieWaric Authorization Fonn. Thestudy will be conductedat HukeO libaratotyfcr Healthand EnvironmentalSciences, EX du Pont doNaturai* sod Company, Nowstit^ D dawts- REGULATORY COMPLIANCE This study willbe oonductedin coaqjfiaace with U.S. EPA TSCA (40 CFRpart 792) Good Labmstoty Plastics Staadaids, which ate consistentwith &e OECDPrindplet ofGood Laboratory Practice(as revised in 1997)pdbHshodin BNWMOCHEMWn.11 Thestudy design is baaed on ILS. EnvironmentalProtection Agency(EPA) OfficeofPrevention, Pestidda.and Toxic Substances (OPPTS0,w Organizationfat Economic Cooperation and Devetopmeat (OECD),(4)test gmdeBnet. A rm o fnoncon^tuncewfflbcdocmiuaateduithe final report RECEIVED T lfE JLL .23. S:E6f)M Exygen Research PRINT TIME JUL.Z3. 6!3SWM Page 30 of 72 jGflPpanjf Sanitized. Does not contain TSCA CBI -39- H-25509: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats_____________ DuPont-11763 Supplement Number 1 Bl.23.2W 3 6:11AM HASKELL 1 / 1 3 0 4 HUJSOft IUpand-DcuDermalTaeHy . 2Pg3ft4lnia>toi 0 .6286 F. 5/23 __________________ DdPm-W63 ; STUDYDESIGN ! T lw rtadydadgoiaasfblloiw t: ' Groan I m V VH NumherfGraun 10 10 10 10 Dosage(mgfeg) 0 (Control) 5 50 500 StudyParameters Frequency Body Weight Test day 0 and at 3-4 dayinterval* thereafter FoodConsumptiaa Testday0 andweekly thereafter Crimea! Observation* Daily Obsarrattooa for dermal eSccta Daily MorHii(iMotibradityC3da Twice daily Bloodcollection fin-Total Fluorine Analysis Chimed Pathology Teatdays 0,7,14,27 Week4 H H P o o d aaly ris Wedk4 Necropsy Weak 4 MATERIALS ANDMETHODS A. Test Substance The test substance was rep lied by thesponsorand waj assigned theunique HasfcellLaboratory Number 25509. B. Test Special Mele CrL-CD*(SD)IGSBR.nt> siilibe obtained from CharlesRiver Laboratori*, Ine. Il locton 0f th rapplier(ctty'itato) wfllbe documentaiin th smdyrecoids ad finiitepori. The CrLCD(SD)IGS BR rathai becodocted eu th b u a ofestensive erperience with t e stm a et Hiekell Uboratory ani ite emtabflitywithitspeetto hanSineas, tangevity, aentvty, and low incidenix of apcatanecmidUettes, '( f k RECEIVED TIME JU ..23. 6!26fW Exygen Research 4 PRINT TITE JU ..E3. 6i39flM Page 31 of 72 'Cpmpany Sanitized Does not contain TSCA CBI -40- H-25509: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats_____________ DuPont-11763 Supplement Number 1 JUL.23. 2003 6: 13AM HASKELL 1 / 1300 NO. 6286 f. 6/23 K-23509; Bipated-DowPezmal Toxtdty IM hjySttfrfaM altfcm _____ Dupont-inJ C. AnimalHrabandry M reUw iU bebooaedm itsinle Reel, wire-reefe cage* soapcoded above cage boards. Animal nxm aw fflbe maintainedatatemperetureof223^iriaidativehw fedity of5020%. Animal reams will bo artificially nominated (fluorescentlight)on an approximate 12-hour light/dank cyde. All ri wifibe provided tap waterad Ufum. AS rats wifi1 fed PMI* Nutrition Intenutioiml, IXC Certified RodentLtfeDiel* 5002 a i m u m . As specified in 0 Hasten Lahomwryanimal health Badenvironmentalmonitoringprogram, fee followingprocedure* are pcxfenned periodicallyto are that.contaminant levds am below thosethat would be expectedto impactthe scientificintegrity ofthe Rudy. Water aonplessresndyiedfcr totalbacterial runts, arrifee prromce ofcxiliibrmj, lead, ' ' and othercontsmmanta. * Feed sampler are analyzed far total bacterial, spore, and fungal aranti Semples &omfteiHywiahed cages and cage racks ate anatyxed to enrereadequate sanitationby the cagewaihert. Certified animal feed it seed, guaranteed by themaoucrartr to meet specifiednutritional quiKancats endnotto exccad stated imnrinmm tpncentnfliciBofkey contaminanti, induding specifiedheavymetals, aflatoxin, dilmiaetedhydrocarbons, and organophoaphaiea. The presence ofthere contanrinaitfi belowfee nasinnnn concentration stated by the manufiictCTer would nothe expected to inqpact fee integrityo ffee study. TTieanimal healfe and environmentrimoMtoringprogram ii ednriniataedbyfee attending laboratory animal veterinarian. 1 ^ arenuintalnM separately from study records and may be included in fee fetaltepori atthe discretionoffee studydirector. D. Pretest Period Upon rival atHaricdl Laboratory, iH rets will be houst?i oneper cagein quarantine. The rats willbe: quarantinedibr a nfefenua o f 5day& identified temporarilyby cage identificatiaa. weighed at b ait 3 times during quarantine, observed wife reaped to weightgain and any gross signs ofdisease orixgury. The tats willbe released from quaresimeby fee laboratory animai veterinarian ordesignee on fee beans o fbody weights andclinical signs. I BECEIVED TIME JUL.23. 6126fit) -5- FttlMT TIME JUL.23. 6 :39AM . ii i f t Exygen Research Page 32 o f 72 -41 - P pm pany Sanitized. D oes not contain TSCA CIBI H-25509: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats D uPont-n /t>3 Supplement Number 1 / 0m,\ JUL. 23. 2003 6: tBAM HASKELL 1 / 1300 - NO. 628i f, 1/23 H-ZJ30S: Rrpc*tahOo*eDennlTIaiy 28-Day Stndyh MaleBats DuFm1-11763 Rate that me accidentally killedor removed fiom study daring thepretestperiod will be discarded withoutnecropsy. Rati tost re founddead or sacrificed in extremis during thepretest period wiMbe lent to fathology and given a gyosiexamiiurfiopto died: fiir the presence of disease. Dependent up<mthere fin d ig , fartherdiagnostic jmKcdma may be employed at the discrctfonoftlm study director, a pathologist, or toclabaratoryarimal veterinarian. The results will notbe repotted in the final reportwale eonaiiteed significantto the evaluationoftoe study. E. Range Finding Study A range finding study was conducted to aid in dosageselection. Thetest substance, moistened with mmeraLdl, wasappliedtotheahaved, intact sldnofgnH tpsoftw om aleraaatadosageof 10,100,orl000jng/lcg&ra6-hwrtajjureperiodbrfourc<msecutivedffys. Thoamount . cadi rat received was determinedftom thebodyweighton each dayofdosing. Two malerats were dosed withmineral oil and savod as controls. Allrati were observed for clinical signs and dermal Shots on each day ofdoaiogaSer the S-hourexposure period. Tho day alterthe lastdose, approximately 0.6-1 tnL ofblood was collected into EDTA tubes firan die orbital sinus o feachra t Therats were sacrificed by carbon dioxide asphyxiation. PlasmawaspepB edm ^tom ^oB m at approximately -20'C until analyzed tor concentration ! F. Assignment to Groups The rats, selected on toe bases ofadequate body waight gain and freedom from anydinical signs i ofdisease or injury, will be distributedby computerized, stratified randomizationinto study i groups as designated in the Study Design, so diet them are no statistically significant differences among group body weightmeans. Theweightvariation ofselectednits willnot exceed 20% of the mesa weight. Afiar asrigamem to poops, each rat win bokwsed individually. Each ratwill be assigned a unique 6-digitHukeHanimalnumberand an individual cage identificationnumber. Prior to assignmentto groups, each ratwillbe temporarilyitalified b y cage idsafifiostian. Afiei sigDuumtto groups, the last 3 digits ofthe Haskell animalnumberwill be tattooed on the tail o feach,ra t The Haskell animalcumber and cageidentificationnumberwill both be included on the cage labeL Atstudy start (test day 0) the rats willbe 8 weeks old. On test day 0, when possible, rats with body weights that are notwithin20y. ofthemeanwillbe removed fixnn study and rcplsced with rats havingbody weighQ within that range (subject to the same selection criteria as the original rats). B ^ th rt haven been assigned to atert group or whichhavebeen,removed form study on test i dayO, for ont-of-iangc bodyweight, will bereleased tor other laboratorypurposes, orbs sacrificedby carixm dioxide asphyxiation anil discarded withoutpathological evaluation, at the discretion o f the study director. - f i- ! RECEIVED TIME JUL.23. 6.26RM PRINT TIME JUL.23. SS39RM ; L I Exygen Research Page 33 o f 72 Rstmpany Sanitized. Do* not contain TSCA CBt 42 H-25509: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats_____________ DuPont-1 l / b3 Supplement Number 1 JL . 23.2003 6:18AM HASKELL 1 / 1300 - NO. 6286 P. 8/23 . B-2JJ09: Kfpcaicd-DoccDensn Toxicity 28-DrStudyinMiloBaa DuPgnt-U7iJ Q. Administration ofTestSidutance .Approaamstaly24hour*prim to tiw firsttreatment, &e fin ofeachrat will be closely shavedto eaq the sk a from the back and trank, The targetareato be treatedis 5 crux 7.4 cm 37 cm1. The 37 cm3treatmeid area* approximately ctpal to 10%ofthe total body surface area for rati m the 200 to 300 g range ofbody weights. T ie test rabstwicewinbe moistened with minoralofl to form a thickpaste tad appliedit: thin andnoifoim layerto coveras much surface o fthe target a rea possibie. For somedoses,the volume oftestsubstance maybe too small to cover (he entire target ares.Theqgm ahnatearntotecouem dw U lbem idrodcm thebadeofeashnt with a waterinsoluble marker andAe percentage ofthe masked area coveredwill be documented in the studyrecords. T1mtimsBbstance,witlbeoovertdwitha2-plyganzepadfi>Bawedby siKstossivetayeraofmetdigau and self-adhesivebandage. Use controltats will receive tnmeraloilattfceiame vohimeas tbehigh-dose r a ttlh e ratswiUbefittedwithplasticcoaan daring the exposureperiod to prevent ingestion oftheteatsubstance and disruption ofthe - - wrappings. Groups oflO nude rata willbo treated at dosages of0 (control), 5,50, nr SOOmgo f test substance per kg body weight TherelswiH be treated for 28 consecutive days. The amount o f test sdbstenee neededto treat eachratwillbebased on themost recently determinedbody weight measurement The exposurepedod wifi be approximatelyChours. After As exposureperiod, the collars and wrappings willbe removed andexcess testsubstancewflj bewashedoff with soap and warm tap water. The testsite o feach rat will (ken gently be pitted dry andthe retreturned to its cage. Control snimais wilt receive the same washingtechnique as the muted animals. The animalswiUbeleshtwed daringthe studyto facilitatethe evaluation ofdermal effects. Tbe tire area thatwas originally shaved (including the untreated cental skin) will bo reshaved. The animals willhereahaved only after an evaluation. a BodyWeights The rats will be weighed at 3-4 dayintervals duringthe study. _ . L Food Conaoirqption and Food Efficiency The amoomt offood consumed by eachrat willbe detennined weeklyby w eiring each feeder at dbegmmng and end ofthe weekand subtrading the finalweightand tire amount o fspillage fem foe feederduringfire w t^ fromthe initialweight Fromthesemeasurements,mean daily food consumption overthe week willbe determined. From the ftod consumption and body weight data, the mean daily food efficiencywillbe calculated. i f g | | | i | ' f I ! f ? 1 j : ! RECEIVED Tire JJL.aa. 6 :2E1 Exygen Research PRINT T O E JU-.23. 6 =39fln Page 34 of 72 I&mpany Sanitized. Does not contain TSCA CBf -43 - H-25509: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats DuPont-11763 Supplement Number 1 JUl.23.2O03 6: 19AM KASKELL 1 / 1300 - NO. 6206 F. 9/23 H-2JS09T lUpntcd-DawDcaailTinieiQr 1-Day3twlyaiMaleR a t . ________________ DB?oa-11763 1 Clinical Observations andMortality Therats wig be observedfor dim cil signs and dermal eBects afterremoval of thetest substance, The rats will dso be (fosoved for climcd signs* each weighing. TheDraiae ScalewiE be used to score skin irritation. Adjacent areas ofuntreated tide wfflbe used for comparison. Rets will be checked twice daily for mortality and for sign offllnes*.fojmy, orabnormalbehavior. . K. Total Fluorine and PerfluorooctanoicAcid Level Evaluation Blood (approximately0.6-1 mL) wiDbe cdlocted'into EDTA tubes frmn the orbital sinus ofall rats on test days 0,7,14,21, and 27. Thebleed willbe collected the animals approximately one hots after removalo ffoe last substance. The blood wffl be refrigerated The total fiuorina contentoffoe bloodsamplet maybe determinedbyutmg a WickboM torch . . combustionmethod fbBowed by analysis with a fluoride ion selective electrode. Initially, only thelrigh-dosesamplei dHbe analyzed. AdditiandanalysmwM tem pested by foe stody directorbased on the msatta ofthehigh-dose analysis. AdditionalMoodwill be collected from foe vena cavaof all rats at necropsy into a tube containingEDTA. Piasttu will betqw edm dgged frozen at-80*C to-20Crmtg analyzed for concentration Plasma will be evaluated from control and high-dose rats. The aecSsoSuo^sH now - andintermediate-dose groups will be based on. results in the high-dosegroup. Plasma samplet will be extractedby organic solventproteinpredpitetion using a protein precipitation column, and then analyzed f o ^ H p ty IC-MS-MS. L. ClinicalPathologyEvaluation A cHsical pathology evaluationwiQbe conductedon ail rat*foeday afier foe last dose. The day before collection ofblood samples forfoe dim eil pathologyevaluation, the animals will be jdaced in sutaboliam cages. These animals wfllbefostedoveroi^it (approximately 16hours) and mine will he collected from each animal Blood samples forhematologyand dim ed chemistrymeasuremente wig bo collected from foe ariritd stem ofeach animd while foe animal is underfight carbon dioxide anesthesia. Blood samples forplestnnfluoride an d M V w ilt be _ . collected at sacrifice from the abdominalvena cava ofeach animal while the animal is under carbon dioxide anofocaa. AMtiooalblood collected fosn.foe vena eavawUlbe placed in a serum toteprocessed to serum, and frozen at -S0*Cto .20*C Bone marrow smears will be prepared at foe final sacrifice from all surviving animals andwillbe evaluated Ifwarranted by experimental findings. At foe discretion o fthe todydirector or clinicalpathologist, additional samples for selected dinied pathologyteatswig be collected from animals showingdim ed evidenceo ftoxicity or sacrificed in extremis. RECEIVED TIME JU ..23. SiaSRM Exygen Research PRIKT TUE JU-.Z3- 6:39fiM Pag 35 o f 72 C&npiuiK Sanitized. Does not contain TSCA CBJ H-25509: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats ____________ DuPont-11763 Supplement Number 1 JUL 23. 2003 6:19AM HASXEll 1 / 1300 H-IM09: RtpfflleiCcieDaniilTajadiy a-BnyStainMalUsa t. Hematology NO. 6286 P. 10/23" DuPont-HTO Blood samp) win be evaluatedforquality byviiuriexonMaticmprfortoandyHi. I t e M owing hematologyparamele affli be dataaniaed: red blood cell count red cell diftabuliaa width hemoglobin abaolite reticulocyte const hematocrit platelet count mettcotpascnlar votane whitebloodcdl count meni cwptucnWbemoglobin tffaeclitl whiteblood cali count met crepuscularhemoglobin concentration microteopieblood smearexamination.. prothrombin time *etfvted partial thromboplastin time fe additesi,blood meats, am te untonew nrethykne-blue, willbe prepared from all hematologysamples a il will be evaluated, ifrequired, to substantiateor d eify the results of tenutolo findings. 2. Clinical Quaniatiy The following serum asportate aminotransferase alenine aannotransfeaie total bilirnbm jocose total protean albumin globulin creatiiiino cbolestvol triglyceride* sottace potassium chloride fluoride* 3. Urinalysis The following u rm slyn l pTm riiTl w ill h r- rh * * m i-n * A - quality color clarity volume osmolality SjK mucose betono hfljrubin Uaod urobilinogen fluoride* . protein microscopicurine sediment examination ----------------------------------------------------- -- ----------------------------------& RECEIVED TIME JUL.23. 6iE6PM PRIfTT TIME JLA..23 6*33*) If | ; Exygen Research Page 36 of 72 Bttes f f o t etrniain TSCA CBI H-25509: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats_____________ DuPont-11763 Supplement Number 1 Jlil.23.2M 3 6:19AM HASKELL 1 / 1300 - 0.6286 P. 11/23 - H-I509: X*pMt*d-DMtDndTiefcy tt-DiySmarinMitoRni Pigtat-lH O M. Aaatonc*l PMhoogy 1. Prtest SceMsteri&li and Mthode, SectionD. Prtt Perod. 2, DosingPhase ^ rata foundded.aedmallylcined, sa c rifi fa or aaarificcdby design will ta d e ^ a g im id im a M a ^ m tla itia a . A H t*l*rsm ovB dfitftady(eic^fco(B t-af- nwge body wreightm test dy0) wffl be sent to Paihologyfor gross valuationaad collection of thaes. R tam fllbeouftimgtiicd by carbcmifogMteancsdiaiaaad cxiangutaatioti, Rai Sfiedby<iengnw aiU fiw todfter3|ua,ontheaftaDo^bfiw l^*dicdutadictifice. A ^s aiflce^b ep erfh tind on m jr iv ^i t.fcU o 1elbe&uactmiclphology '" cvafcution. lioonierofsscrifice for icheduleddeeftawillberindom smon* dltrestm aat groups. Blood singles for cotgulstimpsramsteaMdpouflbfofatoeBnslysiswilibecollectocl *acrfflccfodm tboabdonnnidw ow oi^^eontiiuiidtrcibi(liondeoisfhtxa. P lian t w fflbeprepafM & am foannl^raiCTedforpO M iblBiniiyjii/inilfteplasm aw nibejtoredat apprajdmatdy-S0*Cto-20`>C(iaeSoctioiiK.), i 1 I ! j s i j; | I | i ``I !I f RECEIVED TOE JUU.E3. 6:a6RM Exygen Research - 10PRINT TIME 1U-.Z3. S'.39RM t?- I ! Page 37 o f 72 pSftfp&HySanitized, Does hot contain TSCACB? H-25509: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats DuFont-i i /OJ Supplement Number 1 Jill. 23. 2003 6:19AM HASKELL 1 / 1300 HO, 286 P. 12/23 K-25509: Rq>ottd4>ewDanllriciQr 2MhySadyiaMil)UI JDofom-11763 th e following tissueswlbe kaed to rn M t wind an found dead or incidentally killed (tissue integritypermitting), sacrificed in extremis, removed firm study ( rap t tnrt-of-rungc body w eight test dtyO), or aatrificed by dcrign. liver MVflAnh"I&" 0fll"tt duodenum jqumim iletmt pppnri cokm rectum safivsry gUndb pancreas irrjrmprg'YlPtn kidneys urinary blidder heart aorta HematoooieticSvstem spleen tbyioiit nu u ^n iitr lymphnode mesentericlymph node bonenuarow* EwdftgyingRvhfrtti p M tuy^nd d^m idtfand ptrs&yimd gltacb adrenal gland* fenwtfkneajoint BciHo^active Svstcm Male ' testet epffidymides prostate seminal vesicles Muceflanaous skm eyes (includingoptic gross obMrviaoQjr hmg* teethes. nasal tissue larynx pharynx Nsi'vom Svttcs^L M ntoenM ooataw cerebellar eortex, meduQa^pons) spinal cord (cervical, md-thoraede, lumbar) sciatic nerve iwUhteftamm (e ^ fiaidtjidBcdfir, lanoufeptftO 01fannl^rrate* AHtissaa wifi be placedin thaajpiojaite fixative. Rats sacrificed by deagnvffl havefltefcfiowiag organs weighed; liver, kidneys, adrenal glands, thymus,boun, spleen, heart, testes, and epididymides. Relativeorgan weights (percent offinal W y weight; ratio to teste weight) will be ealculsied. Finalbodyweighti determinedjust prior to necnqHywillbe used in file assessment ofotgsn weightchange*. Organs fiorn rate fcund deed, ssedfleed in ertnentij, or eddentsllykilled maybe w d#ad Mbediscretion ofthe pathologist orstudy director. Bone marrow smears willbe prepared s tthe finalsacrifice fiom 11surviving tats end will be evaluated ifweremed by ca^erisnsalalfindings. AHtissues coBeoted fiern rati in die high-dosage tad control groups, and fiom rats that ate found dead or aooidentilly killed (tissue mtegritypermitting), or ere sacrificed in extremis, will be fimhet processed to Slide*, staioed with hematoxylin andeodn, and examinednrictoaoepically. - 11- RCCEIVED T ltt JUL.23. 6'2GflM PRINT TIME 3U ..23. 6i39M Exygen Research Page 38 of 72 ^ m p m y Sahltized. D oes not contain TSCA CBi -47- H-25509: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats DuPont-11763 Supplement Number 1 JU l.23.20B 6:20AM HASKill 1 / 1300 - H&62B6 P, 13/23 B-2SSW: SqpMleM)BMDatnITaxKitr 2i-DySarfynM>etU M feaHPB MostgrossJaiioatand any additions!target organs Bxm rats in Aslow- sad intennediite- dta*gegroi *w fllbe^atW m kro*copioilty. Selected grow obreavattoatferwhich a ifeCK>icopfediig[K^vrouldnotbeeddjtiv*(a(astaoarferiti3,pododeimalitli, chronic tail m astitis, calottes, anddefonnfeesoffeetoefe toe, tail, or earpinna) willbe hived, but will generallynotbe processed fornricroacopic evaluation. Tissues ftom nta removed fiwn study (forotherthen ofe-of-r*agobody weighton test dyC),wfll notbeproeesaed formicroscopic evalustion ante considered necessaryby the srndydirectororpathologiat Additionalproceduresto identify and/or clarifyhistologic featuresoflesionsm ay be performed at fee discretion offeepaibologiit and wffl be documentedin the final report. : r \ RECEIVED TIME JLL.23. BSESflM Exygen Research PRINT TIME JU.,23'. 6 !39P)M Page 39 o f 72 -48- Sanitized. S e e s M l eonfaln TSCA CBI H-25S09: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats_____________ DuPont-11763 Supplement Number 1 Z**'V JUL.3.003 6:20AM HASKELL t / 1300 H-25509; Kqxatod-DtucDermalTcodcfty H-JarStudrinMtieKati - C.6266 P. H/3 - PoPem-insS ! sta tistic a l a n a ly ses Significance wfl! bejudged atp < 0.05. Method o f Statideal Analysis Parameter PnUmmarvTest Kprefijainarytastisnot Ifptriiminary testis significant significant BodyW riest Body WeightGain Ton for lack of tendi** Sequential application'*1 oftiteJoackheereTeroatr*trend testTM Preliminarytests for pairwise comparison Food Consumption Food Efficiency Organ Lotto's testfor bamogowity^ tad ShijOTfr-Wilkte*^ fornom titir^ OR* One-wayanalysts of variance'1"1fijUowad witbDamctfs tu*0 Rraalcal-WaUis test'" 1 followedwith Dunn's test"51 Leveaft's test for One-wayanalysisof Kniabd-Waliia test?11 Clinical Pathology0 Shapiro-Wilklest''1 fijinottnalitv* variant*1followed with DtmnCtfa tesi111 followedwith Dorm's W* Survival Incidence ofClinical Observation None Codnan-Armitage test fo r tread'14 Incidence o fDermal i Effects i k Jhdtwiiccoe npirisflisodoeisbedpfBltoa^teeairBodyeonducledlfzattitfocl^koftPBixiis b B toSfcaF ta*W ifetM biga)fietebeitam geb teafed g8ifietai;sniisvnim afD u uisttietw ai W Im afodhidnlobcrwtleabiagdedM bdBtkH thanc e tra vdu,c)rMlttTMate pcrfcnMdnn talffcetw oidad vaine. ?orm iele, ilhffimSiiBnrnparted <0.1, C.QSlru*ed faoyMlcubdonj i i U ^ b > e m it n ^ p t t S a ^ h a i s m ^ h d c o ia t x a a 4 , a T ^ , S M U , f ',> v /I A t Othermethodsw fflteuaeit ifappropriate, atthetime ofaanlyas.Tho statistical methods used willbe described a thefinal report SAFETY AND HOUSEKEEPING Good houMkeepinsproeedsta wilibeFsctissd to avoid cosSsBiiaBioa ofthe test substance and to avoid potential healthhazards. Do voidskin contact, gloveswill beworn whenhandling fitstest subatanoe, Annual carcasses and feces wfflbe indnosted. i ! RECEIVED THE JU ..23. 6:2SfW Exygen Research PRINT THE JU..23. 6!39flH ! Page 40 o f 72 eHiparty Sanlfteel. Se* iot contain t ca e! -49- H-25509: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats_____________ DuPont-11763 Supplement Number 1 JDl.23.2D03 6:20AM HASKELL 1 / 1300 H-25509: lUpc.d.Eto*rteaTcry 2M 9yStud?iaM ileRt8 RECORDS AND SAMPLESTORAGE HO. 6286 P. 15/23 . Daftoet-mQ All data and recordsforanalytical chm dehaiians conductedby the sponsorwill be ardbivad by foe sponsor. A sample ofChetest substancewillbe collected for archivepurposes and retained at Haskdl Laboratory, Newark, Delaware, Specimens(if applicable), raw data, and the final report will be retained at Haskell Laboratory, Newark, Delaware, at Iron MountainRecords Management, Wilmington, Delaware. REFERENCES 1. EPA/TSCA Good LaboratoryPractice Standards (40 CFR 792). (1989). 2. OECD Principles o fGood LaboratoryPractice(arevised in 1997, publishedin ENV/MQCHEM(98)17 (OCDE/GD(92)32). 3. United States EavuramerttalPicRectlouAgency (EPA), OfficeofPrevention, Peaticidea, and Toxic Substances (OPPTS) HealthEffects Test Guidelines, OPPTS 870.3200,21/28-Day Dermal Toxicity (AUG-1998). 4. Orgmisation for EconomicCooperation and Development, Guidelines forTesting of Chemicals, Seotiran4 (Part410): HealthEffects (1981). 5. Draper, N it. and Smith, t t (1981). Applied RegressionAnalysis, 2"1*1edition, pp 256-273. Wiley, New York. 6. Selwyn,MLR, (1995). Theuse oftrend tests to determine a no-obrervablc-eSect level in animal safety studies. Journal o ftheAmerican Collegi o fToxicology 14(2), 158-168. 7. Jonckheere, A it (1954). A distribudoa-freeKL-aampletest againstordered alternatives. Siam ctrikaAl, 133-145. 8. Levane, H. (I960). Robust testfor equalityofvariances. Contributions to Probability and Statistics (J. Olkio, e t) , pp 278-292. Stanford UniversityPress, Palo Alta. 9. Shapiro, S.S. and W ilt, M.B, (1965). An analystso fvariance test fornormality (complete samples). Siomctriha 52,591-611, 10. Snodecor, G.W. and Cochran, W.G. (1967). StatisticalMethods, 6* edition, pp 246-248 and ^ . , 349-352. The Iovra State UniversityPress, Iowa. 11. Durateti, C.W. (1955). A multiple comparisonprocedure for comparing several treatments with t contrai. J. Amer. Statist Assoc. 50,1096-1121. 12. Kxuskal, WJH. and Wallis,W A. (1952). Useo franks Inone-criterion malysls ofvariance. J. Amer. Statist Assoc. 47,583-621. 13. Durm, O J. (1964). Multiple contrasts using rank sums. Technomttrict6,241-252. 14. Fisher, RA. (1985). StatisticalMethodsfo r Research Workers, 1301edition, Hsffiter, New York, RECEIVED TIME JUL.23. 6:2SAM Exygen Research PRINT TDC JUL.23. 6:3SM Page 41 of 72 .Company Sanitized. D oes not contain TSCA CBI H-25509: Repeated-Dose Dennal Toxicity 28-Day Study in Male Rats DuPont-11763 Supplement Number 1 JUL 33. 2003 :21AM HASKELL I / 1300 EaSSO: RipeitatDau& aaulToxctir 2HIaatBdyinM alato____________ HO. 6286 P. 16/23 DsPaaJI7S3 Study Function PROTOCOLAPPEND Study Director Clinica] PariwlogyEvaluation Per&ioiaoctaJtoicAcid LevelEvaluations Carol Finlay StaffTssicologijt Nancy B. Everds Principal Rcaeardt ClinicalPathologist and Manager Shunt A. Gamico Anatomic Pathology Evaluation JahflF . wpwi Principal ReacarchPathologiat Initiation ofTest Subataaoe Administration November 25,2002 Collection o fBlood forFluorineAnalysb November 25,2002 December2,2002 December9,2002 December 16,2002 December 22,2002 Clinical PathologyEvaluation December23,2002 AnatomicPathology Evaluation Scheduled Sacrifice December 23,2002 December 23,2002 RECEIVED Tire: JUL.23. 6t26f*1 Exygea Research IS - PRINT m e JU ..23. 6:30AM Page 42 of 72 .Company Sanitized. Does not contain TSCA cts -51 - H-25509: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats____________ _ DuPont-11763 Supplement Number 1 JUL. 23.2003 6:21AM HASKELL 1 7 13M - KO. S2SS P. 17/23 - H-25509; Rqwta&DmDmulTaiiatjr 20-D trtojrlnK ileR m _________________________________ SIGNATURES s r L N kcyB .** W M ad etb c f c - f e w T i L. ^M nR K m ea AmujcM ialatfii SeottB. L en k a KaaRb M aaw aod Dbcctar (GmUW # -^ .m asp 2 > - Du ................ | f i Da Dmc Dfl0 2 .-.Q ei/.-T x ;& t< Cm ; 1 r1 \1 1 ! j 1E RECEIVED THE JUL.23. 6S2SAH Exygen Research - 16. PRINT T O E JU L .2 3 . S;3SFK ! i Page 43 o f 72 SanitlzedL Does not contain TSCA CBi -52- H-25509: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats DuPont-11763 Supplement Number 1 JUL 23. 2003 6:21AU HASKELL 1 / 130(1 &35S09: R quaaU JoK ttasal Toting ' W<ARequestNumber 13951 ServiceCod 1012 Protocol AmendmentNo. 1 NO. 6286 P. 18/23 DcPott-UTCS The protocol is emended follows (change bolded): 1. Pag*4, StudyDesign, add testday21 to Frequency far Blood CollectionforTotal EToorine Analysis. 2- Page 8,SccticmL. C3imclPalogyEyahattoivreplK S*sentau*wi&'Blood sin^toto*5)llaoa,pljsm fioorid^ao4PFO A w m becorectodtjB crificefejnithe ebdourind vena eovaofeschsmmalwlijU the somaliundercsAoa dioxideanesthesia."- ; - ReBoaelei These than* were iosdvotently omitted from theoriginalprotocol i ilI. | I Approvedbjs StudySbeetai Bub i f RECEIVED TUE JUL.23. 6:2SM1 Exygen Research PRINT TIME JUL.23. 6:30PM Page 44 o f 72 -53- Company Sanitized. D oes Wot contain tssca CT H-25509: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats_____________ DuPont-11763 Supplement Number 1 1/* N . JUL 23.2003 6:21AM HASXEU ! / 1 0 KQ. 6266 P. 19/23 - H-2J509: ItqM BtaW oM D aaairm fy ^-PrrSm tyiaifakRM Ptfmat.11783 1 Protocol Amendment2 The protocol is emended u follows: 1. Assign Aattomic Pathologut respansiWlitie to PeterC, Mann, Veterinary Pe&ologist j_ RatMMlff StBdypotonnalreassigned due to wgmiattloMl change. ***"*+r----------------------------------------------- Ja-^dJL .- V 3 StoifD iatax * M*rCMass a im a | RECEIVED TIME JUL.23. 6:26flM Exygen Research PRINT TIME JUL.23. 6:3WW Page 45 o f 72 -54- gawHIzaig. Dee not eonlain TSCA CBi H-25509: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats DuPont-11763 Supplement Number 1 JUL 33. 2603 HASKELL 1 / 130 H-23IW: RipMtaPDwcDerail Taricbj . P g Hsu - 0.6286 P. 20/23 . PaD-oa-ino Protocol Agsca&nent 3 The protocolis (mended >follows: I m gK ite ' second to laa tsanttmenm the fr i t pjui^ ijih fo ra 'faitisllv',. onlythe KgfrAviejiampIt^arftlV analyzed." to; 'Ttaitiilly, only dte day27 asoqiletfiom the fixitfive mis ofcash group wQlheanaJyxed." SeMMi: The flaorins level*msyheeompwxd to fiuorinetevdi fia-otherstadial conducted with this test substance. MNmSltrt M tCjptU^. NvVMwkr J J sJ l 1 RECEIVED TIME JUL.23. 6 :2 6 * Exygen Research PRINT TDC JIJL.E3, 6 :3 8 * I Page 46 o f 72 Does notcontainTSCA CBI -55- H-25509: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats DuPont-11763 Supplement Number 1 JUL. 13.2003 (:21AM HASKELL 1 / 1360 0.286 P. 21/23 . H-2350; XipMUd-DaRDoaoilToxidt/ 21-IHyStailytoMelcRiag Dufoai.I17i3 Protocol Amendment4 The protocol ii m ended u Mow: ProtocolAmendment 5,dd the folloymgwateaco: "Fluorineblood Anslyr will beporfamed stJsdtoen laboratory, Deepwater,NX 08023". Reason: To add the name and addnmoftbe flamme bloodanalysis. I l!AAtCVM tftfa a id fa u a L '^ 3 ti [ RECEIVED TWE ILL.23. 6S26H1 Exygen Research \ PRINT TINE JUL.23. 6:39RM 47 72 'I -56- H-25509: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats_____________ DuPont-11763 Supplement Number 1 S '* ': JUL 2 3 .2M3 6:22AM HASKEll 1 / 1300 - NO. 628 f. 22/23 . H-25309: RtpcnofrQaicnoaiMlTajdeiiy U-D*ys**rkUMb** ________________________________________ nac,um U Protocol A m radm eatJ The protocol is amended u follows: 1. Protocol Amendment4, delete Aitem. Reason: The item tiddetod becausethesentenceneed* to Veadded to the protocol rather than to theprotocol mendmait. Thii dungs also addRssea the incorrectprotocol 2. Protocol, Pago 8, Section ILFluoriiu BloodA n a ljm tn d S B M H H M M lfL c re l Eviiaationi add 0 felkwriogsentence ^ u o rin e U o o d iB a ^ m S bagecfiS adat JidotonLabontoy, Dcqwater.NJ. 08023". Reason: To add the nameto addicts ofthe laboratorypeHmmng the foiorineHood analytic, M rlteki * ! Fi 1f i I: .............. ................ RECEXVED TIME JUL.23. :26flM Exygea Research |i t f .... - t - PRINT TOE JUL.23. S:38flM Page 48 of 72 Company Sanitized. Does no! contain TSCA CBI H-25509: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats DuPont-11763 Supplement Number 1 JL,23.2003 6:22AM SASKEU t / 1300 H-25S09: B^noM)4nDesnlT<inclV a-CySW|riMJ.Bil .................... - -* 0.6286 P. 23/23 DaW H7<3 Protocol Aowadmenl6 tile protocol is nended fo llo w . Theresults o fthdSBHfcplMtn*mtyr willbe presented in t supplemental report. B au m T hsH H M xim * *nUjr metliodhi notbeen fcUyviKdKed. Amonibr-------------1 ty u L ,^ <UM tj.................... StoOfDim** i V .1rc RECEIVED I I I JUL.E3. !ESRM Exygen Research PRINT TIME DJL.23. 6:38fti i Page 49 o f 72 Company Sanitized. Does not contain TSCA CBl -58- H-25509: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats_____________ DuPont-11763 Supplement Number 1 H-2SS0?! Reptaled-DoseDenial Toncily 2B-DiySluJy'm M a lc R n t f _____________ J3uPom-H7t3 Page S, Materials and Methods, Section K. Total Fluorine a tx ^ M B ^ jp ( p ^ p L e v e l Evaluations, third paragraph, change to read: "Plasma gantplM w tjlbeSractcd by organic solvent proton precipitation and then analyzed io r fg tg h y an appropriatemethod at Exygen Research (3058 Research Drive, State College, PA 16801).* Reason: To add the name andaddress of the laboratory performing tbeH H N nalyria. /tr m d br-_______tA JO L SkatyttncttY Exygen Research 1 f it -l- f Page SO of 72 Company Sanitized. Does not contain tsc. cbi -59- H-25509: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats______________ DuPont-11763 Supplement Number 1 Exyeen ^^RESEARCH mprecsc taewch, \ Frown Reute. 'P an 1 ' of 1 PROTOCOL DEVIATION Deviation Numb: t V Date of Occurrence: <0/17/03.07/22/03 Protocol N um ber D uPont-11763 1. Section 12. Analytical Itethod Section standard, used alternate volumes for fortification preparation, and prepared additional concentrations! 2. Section 12. Analytical Method Section 1.3.a-dld not prepare a reagent blankwith Set C72203A. \ \ \ fr ammde-iMleUTiaIPMWilmgmJAf;SKOgPlla tti, te. 1. Protocol Deviation Issued and wttl revise the method to account for variable concentrations and preparattons of standards as long as the concentrations are accurately recorded. 2. Protocol Deviation issued. \ Racon*,<i mm.onsura Date: 1. No negative Impact because stSndard'preparation was accurately documented and the actual concentrations prepared were used fpr calculation purposes. 2. No negative Impact because all other matrix controls and QCsamples meet the acceptance criteria for that set. ManagementSignature (Sponsor! USFMMWROTOCOk.OEVIATIONhtoo Exygen Research O ats B a c a ti QAU Review \ .LOS OsJm Ig m scatm 16501, U5A Y fTkc,:ft'aBo14JS37I2J.Z101199 ejcygerwom Page 51 o f 72 Company Sanitized. Does not contain TSCA OEH -60- H-25509: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats_____________ DuPont-11763 Supplement Number 1 APPENDIX B Analytical Method V H B H H B P "Method of Analysis for the Determination of Serum by LC/MS/MS" in Exygen Research Page 52 o f 72 -61 - Company Sanitized. Does not contain TSCA C8t H-25509: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats___________ DuPont-11763 Supplement Number 1 n _ "RESEARCH f f f lk \ iT riJS Method ofAnalysis for the Determinati byU C /h/M S tin Serum \ \ \ \\ \ APTHOR Bmily Decker DATE ISSUED ` June 9,2003 \ SPONSOR DuPont BasiceliLaboratory forHealth & Environmental Sciences \ P.O.Box. 50 Newark, DE 19714-0050 USA D altet Study Number DuPoat-13090 PERFORMING LABORATORY \ Exygen Research 3058 Research Drive State CoUege.pA 16801 Exygen Research TOTAL NUMBER OP PAGES \ 20 tSSEpt u I bOI, USA fi BM jn.1019 Page 53 of 72 Company Sanitized. Does not contain TSCA CBl -62- H-25509: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats DuPont-11763 Supplement Number 1 ^ Exygen Study No.: 008-317^ n _ RESEARCH TITLE Mt&od ofAnalysis for the Determinattion t(Ff PPeerrBBuucoioactanoic Acid (PFCOWAj|Jiin Serum rvam m s AUTHOR Emily Decker PATE ISSUED June 9,2003 \ SPONSOR \ DuPont HaskellLaboratory forHealth &Environmental Sciences \ P.O. Bon 50 Newark, DE 19714-0050 USA DuPont todyNumber: DuPont-13090 PERFORMING LABORATORY Exygen Research 3058 Reseath Drive State College.PA 16801 ExM-008-276 TOTAL NUMBER OF PAGES 20 Exygen Research M 028IJX 19 m f i 6M.272.1019 igeMoni Page 53 of 72 .Company Sanitized. Does not contain TSCA CW H-25509: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats DuPont-11763 Supplement Number 1 management Approval , $ fR . e tite '' Scientist1' Exygen Research /'John Flaherty Vice President Exygen Research ' bale Dato S. M art Kennedy / SponsorRepresentative DuPont Date i I K I . F!- ! i Exygra Research Exygen Research" PSc2oC20 Page 54 o f 72 Company Sanitized. D oes not contain TSCA CB H-25509: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats_____________ DuPont-11763 Supplement Number 1 TABLE OF CONTENTS TITLE.... ................................... MANAGEMENT APPROVAL..,. TABLE OFCONTENTS--------- LEST OF TABUES---------- ------ UST OFHOUSES.......... .......... 1. SUMMARY____________ 2, EXPERIMENTALCOMPOUNDS 3. CHEMICALS AND SUPPLIES 3.1. Chemicals ,,-- ---------- 3.2. STANDARDS---------------------------------------- .... 3 3 . Equipment and supplies-------------------------------------------- 3.4. S o l u t io n s.......---------------------------------- ------- --- ----------3 3 . Preparation of Standards and Fotm ncA iioN Solutions,, 33.1. Stock solution____ __ ________ .....------------------ 3 3 3 . FORTPlCATKaiSCSOTJONS............ -- .................,, ........ 3 3 3 . CaubraticnStandasds....-------------------------- ----- 4. METHOD----------------- ---- -------- ------ - ----------------------------- 4.1. FIgw Diacram .............---------------- ---------------------------- 4 3 . SAMPLEPBOCESSWa---------- ...... 4 3 . SamplePreparation-------- ----------- -- 4.4. E x t r a c t io n ....... ............ ..... 4 3 . Quantitation---------------- ------- ,,...------------------------- 4 3 .1. LCTMS/MS System and Operating C ondititm ----------- 4 .53. TuneR lb Parameters........ .................... ................... . 4 3 3 . CaukrationProcedures-- 4 3 .4 . Sample Analysis-- ,,........... 4.6. AcceptanceCrttesua.................. ..............,,.. 4.7. PH8WRMANCBCRITERIA.-- ...... ................... 4 3 . TafflRBQUKEDFCRANALYSIS-.................... 5. CALCULATIONS------------------- --------------- 6. SA FETY - ,,..... 1 ___ 2 ------3 ' ------4 ___ 4 ------5 ___ 6 ....7 ....8 _______ 8 ________ 9 ................ 9 ................ 9 _______10 ...........,, 1 0 ___ ___ 10 _______ 10 .............. 11 _______11 _______11 .............. 11 ........... ,,.1 2 .............. 12 ... ........... 13 ..... ......... 14 ... ...... 14 _______ IS ____ _ 15 _______ 15 Bxygu Research Exygen Research Page 3 of20 Page 55 o f 72 -64- [Company Sanitized. D oes not contain TSCA CBf H-25509: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats_____________ DuPont-11763 Supplement Number 1 LIST O F TABLES j Table 1 Recovery Fortifications In Monkey Scrum. .--IS j i LIST O P FIGURES Hgure 1. Calibration carve fo tfM P in Control Monkey Serum. 17 ..IS " Hgure 3. Representative Chromatogram of a 10 ppb Standard for Monkey Serum with - 5ng/m Lof UC | lir. Control .19 Figure 4. Chromatogram of Control Monkey Serum Fortified at 10 ppb and with - 5 ngfmL of UC 4H B B -....... -- -- ___ ,,20 Exygea Research Exygen Research l t i. Page4 o f20 L I Page 56 o f 72 .Company Sanitized. D oes not contain TSCA C8I H-25509: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats________ _ DuPont-11763 Supplement Number 1 1. SUMMARY This report details a method of analysis for serum. I' PFOA is extracted from serum by protein precipitation in acetonitrile. Quantification of accomplished by liquid chromatography/ttiidein spectrometry (I/MS1MS) analysis using selected reaction monitoring (SEM). The chemicalformulaefR R B ta given in section 2 of this method. The lower limit of quantitation (LLOQ) for this method is 10 ppb for ( H P in scrum. ' _. Quantification is performed using extracted calibration standards containing an internal standard. t This method was developed using monkey serum. The overall percept recovery standard deviation fo rH B ^ 111tnonkey serum at 10,100, end 1000 ppb was 94% 6.9% (Table I). A representative calibration curve fcnRRR^bmonlrey serum is shown in Figure 1. Representative chromatograms f o r H R P t a monkey serum .tie shown in Figures 2 to 4. l Screen Research Exygen Research t I rI i i PageS of20 ! Page 57 o f 72 r'^Company Sanitized- Does not cental T*A -66- H-25509: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats ______ DuPont-11763 Supplement Number 1 Exygen Research Exygen Research Pago6 of20 Page 58 o f 72 jpjMnpanjf Sanitized. D oes not contain t s c a c w -67- H-25509: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats______________ DuPont-11763 Supplement Number 1 3. CHEM ICALS AND SUPPLIES . 3 0 . Chkmkals Chemical Methanol (MeOH) Acetonitrile (ACN) OmniSolv Water Grade EEOC HELC Reagent E P IC Source EM Science EM Science Sigma-Aldrich EM Science Catalog No. I9093-2 AXD145- A-330 'WX0004-1 3 2 . STANDABDS Standard Lot Number Purity - -S o u rce. 1 I 1 \ i I j. Exygenesrarch Bxygen Research Page7 of20 Page 59 of 72 jCsmpany Sanitized. Doe no! contain TSCA cm -68- H-25509: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats_____________ DuPont-11763 Supplement Number 1 3 3 . EQWTMENTAM)SUPPLIES Balance, analytical (display at least0.0001 g) ' Mettlec Centrifuge Bppendorf 2 mL eppendcrf centrifuge tubes VWR Disposable micropipets <S0-t06uL, 100-200uL) Drummond (VWR) ii Class A pipes andvolumetric flasks Hyperearb drop-inguard column (4 sun) various suppliers Keystone (part#'844017-400) Stand-alone drop-in guard cartridgebolder Keystone 125-rriLLDPE narrow-mouth bottles Nadgene 2 mL clear BPLC viallit (cat # 5181-3400) HP Standard lab equipment (graduated cylinders, various suppliers disposable tubes etc.) i LOMS/MS and HPLC systems As described in section A S, Note: Equivalent materials may be substituted for those specified in this method 1 if they can be fihownto produce satisfactory an!ts. Notes: 1. In order to avoid contamination, tile use of disposable labware is highly recommended(tubes, pipets, etc.). 2. PIPE or FERE lined containers or equipment, including PTFE-lined HPLC vials forthe HPLC autosampler mustnot be used. 3. It is necessary to co ed the solvents (methanol) for the presence of contaminants by LC/MS/MS before use. Certain lot numbers have been found to be unsuitable foruse. 4. Use disposable micropipets or pipets to aliquot standard solutions to make calibration standards and sample fortifications. 5. The hyperearb cartridges should be changed when the system contaminants (elevatedbaseline) move to within 1 minute from the elution 3.4. Solutions (1) 2 mM ammonium acetate solution is prepared by weighing B.15 g of ananoiiium acetate and dissolving in 1 L of QnmiSoIv Water. Note: The aforementionedexample is provided for guidance, alternative volumes may be prepared;as long as the ratios ofthe solvent to solute ate maintained. . Bxygp*S n atch Plgc8 ot20 | j i. j :I; t Exygen Research Page 60 o f 72 SfjflppanySanitized. D oes not contain TftftA '" -69- H-25509: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats_____________ DuPont-11763 Supplement Number 1 3.5. PKEPR.TKJNOPStaNOXRDEANCFORTinCTON SOLUTIONS Analytical standards me usedfor three purposes: 1. Calibration Standards - These standards are prepared in control monkey serum and are used to calibrate the response of the detector used in the analysis. 2. Laboratory Control Spikes - These fortifications are usually prepared at concentrations corresponding to the IXOQ and lOx IXQQ and are used to determine analytical recovery. Laboratory control prices are prepared in control monkey serum. 3. Matrix Spikes - These fortificalions are prepared by spiking into the field samples at known concentrations. Matrix spikes are used to evaluate the effect of die sample matrix on analytical recovery. ' '~ > j f R I -; 'I The absolute volumes of the standards maybe varied by the analyst as long as the correct proportions of solute to solvent are maintained. ! 35.1. Stock solution Prepare individual stock solutions of 100 jig/zhL ofM B k and 13G H P by weighing out 10 mg of analytical standard (corrected for purity) and dilute to 100 xoL with methanol in a 100-mL volumetric flask. Each stock solution fin 125-mL U5PE bottles) is to be stored in a refrigerator at 2C to 6C end is stable far a maximum period of 1 year from foe date of preparation. j i 3.5.2. Fortification Solutions I a. Prepare a fortification standard of 10.0 pgfaiL forflJ)J0l by diluting 10.0 mL of the (M ferd u tio n described in 35.1 to 100 mL with acetonitrilein a volumetric flask. ____ t b. Prepare a fortification standard of 1.0 pg/rhL for n C ^ | by j diluting 1.0 mL of the u C|HB>>olution described in 3.5.1 to 100 mL with acetonitrile in a volumetric flasic. c. Prepare a fortification standard of 1.0 pg/mL. fo rflH R o n tain tcg k 0.005 (ig/mL o f DcB H H b y diluting lO .t^ n ^ f th eM V so lu tio n described m 35.2.a and 05 mL of the 13cB W M blution described in 35.2.b to 100 mL with acetonitrile in a volumetricflask. | i d. Prepare a fortification standard of 0.1 pg/mL farflflB R ontaining l i 0.005 pg/mL of I3 4 H M b y diluting I D m Lof foelH K so lution described in 355. ead C5 nriL,of foe^'CQBBfoludQn described m 3.55.b to 100 mL with acetonitrile in a volumetric flask. ; e. Prepare a fortification standard of 0.01 pgtaL forflH kcontalning f 1 0.005 pg/mL of diluting 0.1 mL of thdM W B olution described in 355.a and 0.5 mL of th e`3C -JM H P!ution described in 3.5.2.b to 100 mL with acetonitrile in a volumetric flask. ! Rxygeo Research Exgen Research Page 9 of20 Page 61 of 72 l i g g p .Simlifeea, VomWormnMn T8CA CBI -70- H-25509: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats ____________ DuPont-11763 Supplement Number 1 i i : Store aE fortification standard solutions in a refrigerator (in 125-mLIi>FB j bottleB) at 2 ^ to 6"C for m am nun period of 1 year ftcan the dale of -j 1 preparation, after which time It is necessary to make new standards using 1 i the tooksolution. | ] i.J .i. Catibratwn Standards Jj ] The calibration standards are processed through the extraction procedure, j i identical to the camples. The fortification of fire standards before p extraction i>dooe accordingto the following table: '[ Cone. Of Mixed Fortification Solution (ue/m Q 0.01 0j01 0.1 0.1 0.1 Fortification Volume (pL) 100 200 50 100 200 Volume of Control Sample (mL) 0.1 0.1 0.1 0.1 0.1 Coac. of Extracted Calibration Standard (ppb) 10 20 50 100 200 4. METHOD 4.L Flow Diagram The flow diagram of the method is given below, followed by a detailed description ofeach step. M EteflB avdaitBOT Measure 0.1 mLof sample (using disposable micropipette) (fortify samples when designated) i- .4 Protein precipitation in scetonitrile 'l 4- \ LC/MS/MS analysis I 1 2 . Sa m ple P rocessing ] No sample processing is needed for serum samples. However, frozen samples t most be allowed to completely thaw, tin-aided, at room temperature. Samples stored refrigerated should also be allowed to equilibrate to room temperature. All ! samples must be thoroughly mixed before being sampled for extraction. l ExygeaReaeoth Page 10 of20 Exygen Research Page 62 of 72 ^ m x ' ^ n i z M Doe not contain TSCA c r H-25509: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats_____________ DuPont-11763 Supplement Number 1 j 4 3 , Sample Preparation t- f a. Each batch of samples extracted (typically 30 or less) must include at least one reagent control (acetonitrile blank), one matrix control (method blank), and two matrix controls fortified at known concentrations to verify procedural recovery for thebatch. b. At least one ampleper batch should be extracted in duplicate o. At least one sample extracted should be separately fortified at a known concentration and earned through the procedure to verify recovery. Additional matrix Bpikeamay be performed at the sponsor's request 4A. E xtraction X. Measure O.I mL of sample into 2 mL eppeadorf centrifuge tubes (fortify with analyte as-needed, close lid, and vortex ~ 10 seconds). Z Add enough acetonitrile containing internal standard at 0.005 pg/mL j (accounting for fortification volume) to make extraction volume 500 pL and vortex for -1 0 xeconds, 3. Centrifugefor-1 0 minutes at ~ 14,000ipm. 4. Analyze samples using electrospr&y LC/MS/MS. j- 4 3 . Quantitation l ! 4.S.I. LC M SM S System and Operating Conditions Instrument PE SC3EXA H 4000 BiomolecularMass Analyzer SCIEX Ttabo Ion Spray liquid Introduction Interface Computer DeUOptiPlexaXUO Software: PE Sdex Analyst 13 HPLC Equipment: HewlettPackard(HP) Series 1100 Quatpump G1311A Vacuum DegasserQ1322A Antoinjector 01313A Column CompartmentG1316A Note: Two 4 X 10 mm hypsteaxb drop-in guard cartridges (Keystone, part #. 844017-400) are attached in-line after the purge valve and before the sample injector port to trap any residue contaminants a t may be in the mobile phase and/orHPLC system. HPLC Column; Genesis Cs (Tones Chromatography), Z I mm x 50 mm, 4u. f ExygeaResearch Page II of20 Exygen Research Page 63 o f 72 Company Sanitized. Does not contain Tfsr* -72- S a n t o ! 1,.D oes fR>!contain TSCA CB i H-25509: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats________ _ DuPont-11763 Supplement Number 1 Column Temperature:30* C i Injection Volume: 5pL MobilePhase (A); 2 mM AmmoniumAcetEte in OmnlSolv Water MobilePhase (B): Methanol ' Tune % 2 Hftff (nytrAgin) 0.0 40 . 60 03 ' 3 ' 40 1 1 03 il 3.5 0 3.7 0 100 100 0.3 0.5 7 0 100 03 j 7.5 40 60 0.5 I 9 40 60 03 J 9.5 40 60 0.3 12 40 60 0.3 It may be necessary to adjust the HFLC gradient in older to optimize instrument perfonnance. i Ions monitored: j Approximate Mods Transition Monitored Retention Time Negative 413 -> 369 -2.8 min. t i k i Negative 415->370 -2.8 min. The retention times may vary, on a day-to-day bams, depending on the batch of mobile phase etc. Drift in retention times is acceptable within an analytical run, as long as the drift continues through the entire analysis and the standards sic interspersed throughoutthe analytical run. Note; An alternative LC/MS/MS system may be used once demonstrated to be equivalent. 4_1.2. Time file Panmeters The mass spectrometer is tuned for the analyte by infusing s - 1 pg/mL standard solution a flp M fc t 10 pL/min, using an infusion pump) via a 'T " into a stream of mobile phase containing 60% methanol sad 40% 2mM ammonium acetate at 0.3 ml/min flow rate. The analyte is initially timed for the parent ion and then tunedfbrthe product ion. Once the instrument is tuned,the optimized parameter: 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) of each calibration standard into the LC/MS/MS. i r i I ! i Exyjtn Research Exygen. Research Page 12 of20 ( Page 64 of 72 Company Sanitized. Does not contain TSCA c w -73- H-25509: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats_____________ DuPont-11763 Supplement Number 1 b. Use Unesr standard corves for quantitation. linear standard curves are generated for each analyte bylinear regression using 1/x weighting of the ratio analyte peak area/intemal standard peak area verms the ratio of the concentration of arudyte/conceutration of Internal standard using Analyst 1.2 (or equivalent) software system. Any calibration standard found to be a statistical outlierby using die appropriate outlier test (e.g. Huge Outlier Test), may be excluded from the calibration curve. However, tire total number of calibration standards that could be excluded must not exceed.20% of the total number of standards injected and at least one calibration standard at the LLQQmustberetained. c. 71 correlation coefficient (r) for calibration curves generated must be 20.9925 (t* 20.985). If calibration results fall outside these limits, jhen appropriate steps must be taken to adjust instrument operation, and die standards or the relevantactofsamples shouldbe reanalyzed. 4.5.4. SampleAnalysis a. Inject the same aliquot (5 pL) of each standard, sample, recovery, control, etc. into the LG/MS/M5 system. b. Standards corresponding to at least five or more concentration levels must be included in an analytical set. c. An entire set of calibration standards should be injected at the beginning of a l set followed by calibration standards intetspessed every 5-10 samples (to account for a second set of standards). As an alternative, an entire set of calibration standards may be included at (be beginning and at the end of a sample set In either case, calibration standards must be the first and last injection in a sample set d. The concentration of each aample/fortification/cantrol is determined from the standard curve, based on foe peak area of each, analyte. The standard responsesshould bracketresponses o fthe residue found in each sample nit. E necessary, dilute the samples in acetonitrile to give a response within the standard curverange. e. Fortification recoveries falling within 85 to 115% (80 to 120% for levels at the UX1Q) are considered acceptable. f. Extracted saasptss must be stored refrigerated between 2*C to 6C until analysis. .i ; g. Samples in which no peaks are detected (he, signal: noiso ratio < 3;1) at the corresponding analyte retention times will be reported as ND (not detected). Samples in which peaks ant detected at the corresponding analyte retention f Exygcn Rejecrcii PSO13of20 Exygen Research Page 65 o f 72 &3ttfpany Similized. Doss no! contain t s c <''**! -74- H-25509: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats_____________ DuPont-11763 Supplement Number 1 i times but arc less than the lowest concentration of the calibration standards (10 ppb where LLOQ = 10ppb) will be reported as <10ppb. 4,6, Acceptance Ckhbbm. | th e following criteria must be met to ensure the presence ofj| ! 1. The chromatogramsmust show tire following peaks; t W I a daughterion at369 amufroma parent of413 amn adanghterionat370 rnnuftoma parartof4i5am u 2. Any analyte present in method blanks must be at least 5-fbldlowcr than the LLOQ. Any analyte present in tire reagent blank must be at least 5- ' fold lower than the ILO Q. 3. Recoveries of lab control spikes and matrix spikes mast be between 85- 115% (80-120% for levels at the LLOQ) of their known values. Any method fortification (lab control' spike) falling outside the acceptable limits warrants re-extraction of the eatire analytical set Any matrix spike outride 1he acceptable range' should be evaluated by the analyst to determine if nxsttnsctian. is warranted. i t 4. Any calibration standard found to be a statistical outlier by using an appropriate outlier test, may be excluded form the curve. However, tire ! total number of calibration standards that could be excluded must not exceed 20% of tire total number of standards injected and at least one calibration Standard at die LLOQ most be retained. 5. The correlation coefficient (r) for calibration curves generated must be 20.0925 (i* 20.985). If calibration results fall outside these limits, then appropriate steps must be taken to adjust instrument operation, and the standards orthe relevantset of samples should be reanalyzed. 4.7. Performance Criteria The following two criteria must be performed as a system suitability test, before tin commencement of analysis when tiring an instrumentation set-up that has not been used for this method. F&griteri<m: . Run a standard solution an LC/MS/M5 corresponding to the estimated LLOQ and obtain a rignri-to-noise ratio of at least 9:1, compared to a reagent blank. If this criterion cannot be met, optimize and change instrument operating parameters. } \ Exygta Research Exygen Research Page 14of20 j Page 66 of 72 f&fflpSriy Sanitized. D ees wot contain TSCA CBi H-25509: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats_____________ DuPont-11763 Supplement Number 1 i i , Run a set of standards uf five or more concentration levels, spanning a range ! starting at orbelow the LLOQ up to the highest concentration Ipvel to be included in the analysis, Generate a calibration curve far each analyte and obtain a linear regression with a coefficient of determination (r*) of at least 0.985. Once this criterion is met, samples may he analyzed with standards interspersed. 4.8. Time Requeus fo* Attalybis A set of 35 samples <1 reagent control, l matrix control, 1 laboratory spite, 2 laboratory control spites, andSOsamples) can tre taken thronji the extraction t procedure in approximately 8 boors by one person. The LC/MS/MS analysis ' (standards and 35 samples) will take approximately 9 hours. I 5. CALCULATIONS tu Use Equation 1to calculate the amount of analyte found (in ng/nnL, based on peak area) using the standard curve (linear regression parameters) generated by the Mass Lynx software program. '> E quation 1; Analyte found (ppb) = fAnalytePckuti/lS peakmrt- intaccptVlx IScone. (njM.) lope b. For samples fortified with known amounts of analyte prior to extraction, nse Equation 2 to calculate foepercentrecovery. Equation 2: Recovery (96) = Analytefound fonbV- a m Analyte found in control fppb) x 100% Amount Analyte added(ppb) .6 SAFETY The analyst should read die material safety data sheets for all standards and reagents before performing this method. Use universal precautions when I J handling standards and reagents, mcindmg working in fume hoods and wearing laboratory coats, safety glasses, mid gloves. Use blood-borne pathogen handling precautions whenhandlingserum. i ! Bxygea Research Exygen Research Page 15 o f 20 Page 67 of 72 .Company Sanitized. Does notcontainTSca cal -76- H-25509: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats_____________ DuPont-11763 Supplement Number 1 Table L Recovery otSBBM rom Fortifications In Monkey Serum Recoveriestor 10ppb FortificationIn MimkeySeram Bryom ID m om m a SSOOIMSpkB m o o m sik c Spouor Enaction Aaalyj F a t Level m Sato Lot403400 m o m Dea ... ftp 05/3003 . 10 Lot40X3400 CS30TO MflMO 10 LM-WCM00 <wwm OSOMB 10 AVERAGE: STANDARDDEVIATION: RELA-nVBSTANBARDDEVIATION: Recovery 95 04 93 90 2a 23 Sum m ary Rncovcriea fo r 100 p pb F ortiflcaflon in M onkey Seram Bxyeea ID 03008 3{flcD ' (Booeos spies 0300S08 SjdcP Sponsor Btractu A ulpU F at.Level ID tktB Duto 14X40X0400 esm m 010009 Lot40X7400 asnam , 0*30/03 .. fti*> 100 100 Lot40X7400 osoam 09904 100 AVERAGE: STANDARDDEVIATION: RELATIVE STANDARDDEVIATION: fc lte o v e y 92 90 93 n %& 1.7 Sum m ary o f R P B B eco v erie* fo r 1000p p b FortiB catra In M onkey S en n a &W ID .03008 SpkO 0300*06 SpltH 0300808 Spkt Sponsor Srtnctkm AaelysU F a t Level ID Date Data ( Lot40X7400 ostv m Lot40X7400 m o m m om 093003 1900 1000 Lot40X7400 m a m 093003 1000 AVERAGE: STANDARDDEVIATION: RELATIVESTANDARDDEVIATION: %Recovery i 93 SI 94 13.0 13J0 OVERALLAVERAGE: OVERALLSTANDARDDEVIATION; OVERALL RELATIVE STANDARDDEVIATION: 94 6S 73 Bcygra Rerch Exygen Research Page 16 o f 20 Page 68 o f 72 `iS ^ p in y 8 a M IM . Upes wot contain Tf?rA w -77- H-25509: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats __________ DuPont-11763 Supplement Number 1 Exygaa Research Exygen Research ' Page 37 of20 Page 69 of 72 Company Sanitized. Does ffof contain TSCA CBt -78- H-25509: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats_____________ DuPont-11763 Supplement Number 1 / Rgure 2. Representative Chromatogram of a Control Monkey Serum Sample for M B fen th - 5 ug/mL o fuC d * B P /"""N "i Exygcn Research Exygea Research Pngel8ot20 f } I Page 70 of 72 Ppinpany Sanitized Does not contain TSCA CBS -79- H-25509: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats_____________ DuPont-11763 Supplement Number 1 Figure 3. Representative Chromatogram of a 10 ppb Standard for i Control Monkey Serum with ~ 5 ng/mL of UC- It j i [ i j I i 1 / Bicygsn Research Exygen R esearch ! i t. Page 19 of20 Page 71 o f 72 .Company Sanitized. Does not eontain t s c a cp -80- H-25509: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats_____________ DuPont-11763 Supplement Number 1 Figure 4. Representative Chromatogram of Control Monkey Serum Fortified at 10 ppb with flH B l and with ~ 5 ng/mL of UC- i 1 ! | ! ExygeB Research Bxygen Research' IS Page 20 o f 20 ! Page 72 o f 72 .Company Sanitized. Does not contain TSCA OBI -81-