Document o9zBrQ368LB7GvLdnDLMgEGJo

3M EnvironmentalLaboratory FinalReport-AnalyticaSltudy Single-DosIentravenouPsharmacokinetiSctudyofT-6067inRabbits In-VivoStudyReferencNeumber: HWI#6329-151 StudyNumber: AMDT-120694.1 TestSubstanceL:-13492(T-6067) Name and AddressofSponsor: 3M SCD Division 367 Grove Street St.Paul,NfN 55106 Name and Address of TestingFacility: 3M EnvirorunentaTlechnology & Services 935 Bush Avenue St.Paul,MN 55106 Method Numbers AMDT-M-1-0, AMDT-M-2-0, AMDT-M-4-0, AMDT-M-5-0, AMDT-M-8-0, AMDT-M-14-0, and Revisions: Thermal Extractionof Fluorideby Means of a Modified Dohnnann DX2000 OrganicHalideAnalyzer-Liver FluorideMeasurement by.Means of an Orion EA940 Expandable Ion Analyzer Extractionof Fluorochemicalsfrom RabbitLiver Analysisof RabbitLiverExtractforFluorochemicalsUsing ElectrosprayMass Spectrometry Analysisof FluorideUsing theSkalarSegmented Flow Analyzer with Ion SelectiveElectrode Thermal Extractionof Fluorideby Means of a Modified Dohnnann DX2000 OrganicHalideAnalyzer-Serum InitiatioDnate: See attachedprotocol Author:James D. Johnson Approved By: D. @*son Study Dil@oor Compietion Date -rna,Itj 13-caL 11)2.14c LX - ooocoi 1.0 SUMMARY Tissuaendserumsampleswereanalyzefdortotaolrganifcluorianfetesringle intravenousdosesto individuarlabbitsinordertocollectinformationinsupportof a dermal absorptionstudy. Serum samples were analyzedat2,4,6,8,12,24,and 48 hourspost dose. There is a rapiddecreaseinserum levelof totalorganicfluorinewith time. The biological half-lifieson theorderof 4 hours. There isvery littlteotalorganicfluorinepresentinliveror serum at48 hourspost intravenousdose of L-13492 (T-6067).The pharmacokineticstudysuggeststhat interpretatioofndermal absorptionof L-13492 (tobe done and reportedina separatestudy)willbe more difficultI.tisunlikelythattheserum levelsat 48 hours and theliversamples at28 days willcontainany detectableorganic fluorinewhatevertheextentofdermal absorption. 2.0 INTRODUCTION This studywas designedtoprovideinformationas towhether L-13492 when administeredas a singleintravenousdose resultsintotalorganicfluorideincreasein whole liverat48 hourspostdose or inserum at2,4,8, 12,24, or 48 hours. This informationistobe acquiredinordertointerpreat subsequentdermal absorption studywiththesame compound (HWI#6329-152, AMDT-011095.1). Thedermal absorptionstudywillbe reportedseparately. 3.0 TEST MATERIALS 3.1 Test,Control,and Reference Substances and Matrices 3.1.1AnalyticalReference Substance: FC-95, lot161 or 171. They are equivalent. 3.1.2AnalyticalReference Matrix: Bovine liverand bovine serum 3.1.3AnalyticalControl Substance: None 3.1.4AnalyticalControl Matrix: Bovine liverand bovine serum 3.2 Source of Materials:3M ICP/PCP DivisionforFC-95, bovine liverfrom grocerystoreb,ovineserum from Sigma ChemicalCompany 3.3.Purityand StrengthofReferenceSubstance:Responsibiliotfy Sponsor. 3.4 Stabilityof Reference Substance: To be determined by Sponsor. 2 0()000;! 3.5StorageConditionfsorTestMaterialsR:oom temperatufroerFC-95.For biologicalsamples thestorageis-20100 C. 3.6Dispositionof Test and Control Substances: Biologicaltissuesand fluidswill be retainedper GLP Regulationforthetimeperiodrequiredforstudieslongerthan 28 days. This studyisinparallewlitha 28 day absorptionstudy,so alltissueswill be retained. 4.0 EXPERIMENTAL -Overview Serum and tissuesfrom animalsdosed as described(HWI#6329-15 1)were available foranalysisby combustion with subsequentanalysisby selectivieon electrodeor by electrospramyass spectrometry.The tissueand serum samples were analyzedfor totalorganicfluorinetocollectinformationinorderto supporta dermal absorption study.The extentof analysiswillbe atthediscretioonf the Study Director.The samplesarisefrom a previousstudy(HWI#6329-151) inwhich singlerabbitswere dosed intravenouslwyithL-13492 atdoses ranging4 to24 mg/kg. (Seeattached protocolfordosing,tissuecollectione,tc.)In thefinalreportfrom HWI on this studythe dosingisfrom 4 to24 mg/kg. In the originalprotocolt,he dosingisto 160 mg/kg. A rabbitdosedat40 mg/kg died5 minutesafterinjectionN.o tissues were saved. 5.0 EXPERIMENTAL -METHODS 5.1AMDT-M-1-0, Tbennal Extractionof Fluorideby Means of a Modified Dohrmann DX2000 OrganicHalideAnalyzer-Liver 5.2AMDT-M-2-0, FluorideMeasurement by Means of an Orion EA940 Expandable Ion Analyzer 5.3AMDT-M-4-0, Extractionof Fluorochemicalsfrom RabbitLiver 5.4AMDT-M-5-0, Analysisof RabbitLiverExtractforFluorochemicalsUsing ElectrosprayMass Spectrometry 5.5AMDT-M-8-0, AnalysisofFluorideUsing the SkalarSegmented Flow Analyzer with Ion SelectiveElectrode 5.6AMDT-M-14-0, Thermal Extractionof Fluorideby Means of a Modified Dohrmann DX2000 OrganicHalideAnalyzer-Serum 3 000003 6.0DATA ANALYSIS Thedataareattachefdortotaolrganifcluoriannealysiosfliveart48 hourspost dose. The totalorganicfluorineforwhole liverforcontrolanimals,4 mg/kg, 16 mg/kg, and 24 mg/kg intravenousdosesof a 45% solutionare20 ug, 43 ug, 66 ug,and 54 ug. Each datapointrepresentsone animal.An animal dosed with 40 mg/kg died 5 minutesafterinjection. The dataareattachedforserum concentrationosf totalorganicfluorineat2,4, 6,8, 12,24,and 48 hourspostdose. What isimmediatelyapparentisthatthe concentrationdsecreaserapidlywith time and arenon-detectablaet48 hours. The biologicahlalf-lifaeppearstobe about4 hours(seepages 84-86). Itappearslikelythatthissaltof perfluorooetanoatiesrapidlyeliminatedfrom femalerabbits.Itisnot known ifthereisa sex differencienthe eliminatioonf the perfluorooctanoatienrabbitsasisobservedwith rats. Other datawas collectedusingelectrospramyass spectrometryand Skalar segmented flow analyzerwith ionselectivelectrode(seeappendices).Thisdata, althoughsupportivei,ntheopinionof theStudy Directorisnot requiredtoreachthe conclusionstatedhere and thereforeisnotdiscussedin detail. 6.1 Circumstances that May Affectthe Quality of the Data: The problem with thisanalysisisthatthereisnot a good biologicamlarker and thusthe pharmacokineticstudydoes not providea gciodavenue fortheinterpretatiofn a dermal absorptionstudy 7.0 CONCLUSION There isvery littlteotalorganicfluorinperesentinliverat48 hourspostintravenous dose of L-13492. The pharmacokineticstudysuggeststhatinterpretatioofndermal absorptionof L-13492 (tobe done and reportedina separatestudy)willbe very difficult. 8.0 MAINTENANCE OF RAW DATA AND RECORDS 8.1 Raw Data and Data: Raw data,approvedprotocola,pprovedfinalreport, appropriatsepecimens,and electronidcatawillbe maintainedintheAMDT archives. 0()()()044 9.0APPENDICES 9.1Protocoalnd Amendments 9.1.P1rotocoalndFinalReportH:WI#6329-151"Single-DoIsnetravenous PharmacokineticStudy of T-6067 in Rabbits" (Protocoltype TP8084.PK for dosingof animals,tissuecollectione,tc.) 9.1.2 AnalyticalprotocolAMDT-120694.1 9.2 Signed Reports from IndividualScientistsN:one 9.3 QualityAssurance Unit Statement:See attached 9.4 Key PersonnelInvolvedin the Study: See attached 9.5 Materialsand Equipment: See methods 9.6 Solutions,Reagents,and Standards:See methods 9.7 Sample Preparation:See methods 9.8 QualityControl Practices:See methods 9.9 Test Methods: See ProtocolAMDT-120694.1 9.10 Instrument Settings:See methods 9.11 Data: See attached. 9.11.1Summary and raw data;ug F-inwhole liveras determinedby thermal extractiofnollowedby analysisusingOrion ion analyzer. 9.11.2Summary and raw data;ppm F- inserum as determinedby thermal extractiofnollowedby analysisusingOrion ion analyzer. 9.11.3Summary and raw data;ppm F-inserum as determinedby thermal extractiofnollowedby analysisusingSkalarsegmented flow analyzerwith ion selectivelectrode. 9.11.4Summary and raw data;analysisof liverextractsusing electrospray mass spectrometry. 000()()S 9.1.1Protocoland FinalReport:HWI#6329-151 "Single-DoseIntravenousPharmacokineticStudy of T-6067 inRabbits"(ProtocotlypeTP8084.PK for dosingof animals,tissuecollectione,tc.) 000006 HAZLF.OCCN W IS C 0 N S IN P (J f 0 P F I ( i 6 r) 'K .4 '1 Sponso 3M St. Paul, Minnesota CCMNING FINAL REPORT Study Title: Single-Dose Intravenous Pharmacokinetic Study of T-6067 in Rabbits Author: Steven M. Glaza Study Completion Date: February 1, 1995 Performing Laboratory: Hazleton Wisconsin, Inc. 3301 Kinsman Boulevard Madison, Wisconsin 53704 Laboratory Pro,iectIdentification: HWI 6329-151 P h o n e 6 0 S 2 -1 4 4 7 1 P R E c-S 'Yl D :@@L i v E R Page I of 28 r3 I K i @I 1 8 L @@1) MA 60 r) @i 24 72 2 7 53 704 OOOOQ7 Page 2 of 28 QUALITY ASSURANCE STATEMENT HWI 6329-151 This report has been reviewed by the Quality Assurance Unit of Hazleton Wisconsin, Inc., in accordancewith-the Food and Drug Administration (FDA) Good LaboratoryPracticeRegulations,21 CFR 58.35 (b) (6) (7). The following inspections were conducted and findings reported to the Study Director and management. Written status reports of inspections and findings are issued to Hazleton management monthly according to standard operating procedures. Inspection Dates From To - Phase Date Reported to Date to Study Director Management 11/27/94 12/09/94 12/28/94 01/17/95 01/30/95 11/27/94 12/09/94 12/28/94 01/19/95 01/30/95 Protocol Review Sample Collection Protocol Amendment Data/Report Review Report Rereview 11/27/94 12/09/94 12/28/94 01/19/95 01/30/95 12/10/94 01/10/95 01/10/95 02/10/95 02/10/95 te&llia M. Danner Date Representative, Quality Assurance Unit 000008 Page 3 of 28 STUDY IDENTIFICATION Single-Dose Intravenous-Pharmacokinetic Study of T-6067 in Rabbits HWI 6329-151 Test Material Sponsor Sponsor's Representative Study Director Study Location Study Timetable Experimental Start Date Experimental Termination Date T-6067 3M Toxicology Services 220-2E-02 3M Center St. Paul, MN 55144 John L. Butenhoff, PhD 3M Toxicology Services 220-2E-02 3M Center St. Paul, MN 55144 (612) 733-1962 Steven M. Glaza Hazleton Wisconsin, Inc. P.O. Box 7545 Madison, Wl 53707-7545 (608) 241-7292 Ha*zletonWisconsin, Inc. Building No. 3 3802 Packers Avenue Madison, WI 53704 December 9, 1994 December 11, 1994 000009 Page 4 of 28 HWI 6329-151 KEY PERSONNEL Acute Toxicolo Steven M. Glaza Study Director Manager Francis (Bud) W. McDonald Study Coordinator Patricia Padgham In-life Supervisor Rose M. Bridge Report Supervisor Ouality Assurance Sherry R. W. Petsel Manager Laboratory Animal Medicine Cindy J. Cary, DVM -Diplomate, ACLAM Supervisor Anatomical Patholoqy Thomas E. Palmer, PhD Anatomical Pathologist Jack Serfort/ Deborah L. Pirkel Supervisors Necropsy Anne Mosher Supervisor Pathology Data OOOOJLO Page 5 of 28 CONTENTS Quality Assurance Statement Study Identification Key Personnel Summary Objective Regulatory Compliance Test and Control Materials Test System Procedures Results Discussion Signature Reference Pathology Report Table I IndividualBody Weights (g) 2 IndividualClinicalSigns Appendix A Protocol Deviation Protocol TP8084.PK Protocol Amendment No. 1 HWI 6329-151 Paqe 2 3 4 6 7 7 7 8 9 11 11 11 11 12 13 14 15 16 17 27 000()ILJL Page 6 of 28 HWI 6329-151 SUMMARY This study was done to assess the level of systemic exposure of T-6067 when administeredby intravenousinjectionto rabbits. Female Hra:(NZW)SPF rabbits were assigned at random to five groups (one/group). On Day 0, the animals received a single intravenousinjectionof the vehicle (sterilewater for injection)or 4, 16, 24, or 40 mg of T-6067/kg of body weight at a dose volume of 0.5 mL/kg. Clinical observations were conducted at approximately0.5, 2, 4, 24, and 48 hours after intravenousinjection. Body weights were determined just before test material administration(Day 0). A blood sample (approximately 4 mL) was collected from an auricularartery or marginalear vein of the survivinganimalsat 2-, 4-, 6-, 8-, 12-, 24-hourspost-injection. In addition,at the time of experimentaltermination(48-hourspost-injection), approximately20 mL of blood was obtained from each surviving animal. All samples were centrifuged, separated into serum and cellular fractions, and sent to the Sponsor. The one animal (40 mg/kg) found dead during the study was necropsied but tissues were not saved. Animals surviving to the end of the 48-hour post-injectionperiod were anesthetizedwith sodium pentobarbital, bled via the posterior vena cava, and exsanguinated. An abbreviatedgross necropsy examination was not done, however, tissues were collected. The whole liver, bile, and both kidneys from each animal surviving to terminationwere collectedand sent frozen to the Sponsor after terminationof the in-life phase. The animals treated at 0, 4, 16, and 24 mg/kg appeared normal throughout the study. The animal treated at the 40 mg/kg dose level died within 5 minutes of dosing. At necropsy,the lungs in this animalwere diffuselydark red. This change was attributed to acute death and postmortem change. 0()O():L2 Page 7 of 28 HWI 6329-151 OBJECTIVE The objective of this study was to assess"the level of systemic exposure to the test material, T-6067, when administeredas a single intravenousinjection to rabbits. REGULATORY COMPLIANCE This study was conducted in accordance with the U.S. Food and Drug Administration'sGood LaboratoryPractice Regulations for Nonclinical LaboratoryStudies, 21 CFR 58, with the exception that analysis of the test mixtures for concentration,homogeneity/solubility,and stability was not conducted. All procedures used in this study were in compliance with the Animal Welfare Act Regulations. In the opinion of the Sponsor and study director, the study did not unnecessarilyduplicate any previous work. TEST AND CONTROL MATERIALS Identification The test material was identifiedas T-6067 and described as a clear, colorless liquid. The control materialwas SterileWater for Injection,USP (Abbott Laboratories, Lot No. 86-748-DM-02; Exp. March 1, 1996), and was described as a clear, colorlessliquid. Purity and Stability The Sponsor assumes responsibilityfor test material purity and stability determinations(includingunder test conditions). A sample of the test material/vehiclemixtures for concentration,solubility,homogeneity,and stability analyses was not taken before administrationas this was not requested by the Sponsor. The purity and stability of the USP grade control material were considered to be adequate for the purposes of this study. Storage and Retention The test material was stored at room temperature. The control material was stored refrigerated. Any unused test material was returned to the Sponsor after completionof all testing accordingto Hazleton Wisconsin (HWI) Standard Operating Procedure (SOP). Any remaining vehicle may be used for other testing and will not be discardedafter issuanceof the final report. 00001.13 Page 8 of 28 HWI 6329-151 Safety Precautions The test and control material handling procedures were according to HWI SOPs and policies. TEST SYSTEM Test Animal Adult albino rabbits of the Hra:(NZW)SPF strain were received from HRP, Inc., Kalamazoo, Michigan on November 16, 1994 and maintained at the Hazleton Wisconsin facility at 3802 Packers Avenue, Madison, Wisconsin. Housing After receipt, the animals were acclimated for a period of at least 7 days. During acclimation and throughout the study, the animals were individually housed in screen-bottom stainless steel cages in temperature- and humiditycontrolledquarters. Environmentalcontrols for the animal room were set to maintain a temperature of 19* to 230C, a relative humidity of 50% 20%, and a 12-hour light/12-hourdark lighting cycle. In cases where variationsfrom the required temperature and humidity conditions existed, they were documented and considered to have had no adverse effect on the study outcome. Animal husbandry and housing at HWI complied with standards outlined in the "Guide for the Care and Use of Laboratory Animals".1 Animal Diet The animals were provided access to water ad 7ibitum and a measured amount of Laboratory Rabbit Diet HF #5326, PMI Feeds, Inc. The feed is routinely analyzed by the manufacturer for nutritional components and environmental contaminants. Samples of the water are periodicallyanalyzed by HWI. There were no known contaminants in the feed or water at levels that would have interferedwith or affected the resultsof the study. Selection of Test Animals The animals were identified by animal number and corresponding ear tag and were selected at random based on health and body weight requirements. 0000IL4 Page 9 of 28 HWI 6329-151 Study Design Female animals weighing from 2,511 to 2,781 g at initiation of treatment were placed into the following study groups: Grour) Dose Level Dose Volume Number Treatment (mg T-6067/kql (mL/kq) of Animals 1 (Control) 0 2 T-6067 4 3 T-6067 16 4 T-6067 40 5 T-6067 24 0.5 1 0.5 1 0.5 1 0.5 1 0.5 1 Sterile Water for Injection,USP. Justificationfor Species Selection Historically, the New Zealand White albino rabbit has been the animal of choice because of the large amount of background information on this species. PROCEDURES Dose Preparation and Administration The test materialwas diluted with Sterile Water for Injectionto achieve a specificconcentrationfor each dose level. An individualdose of each respective test solution or control was calculatedfor each animal based on its body weight on the day of treatment. The respectivetest solution was administered by intravenousinjection into a marginal ear vein. The dose was given as a slow push (approximately30 to 60 seconds in duration). The prepared test solutionswere stored at room temperature until administered. After administration,any remaining test solutionswere discarded. Reason for Route of Administration Intravenous injection is an acceptable route to assess systemic exposure. Observations of Animals Clinical observations were conducted at approximately0.5, 2, 4, 24 and 48 hours after intravenousinjection. Body weights were determinedjust before test material administration(Day 0). 00001.5 Page 10 of 28 SamipleCollection HWI 6329-151 Blood samples (approximately4 mL) were collected from either ear via the catheterizationof the auricular artery or'from the marginal ear vein of all survivinganimals at 2, 4, 6, 8, 12, and 24 hours post-injection. At the time of necropsy (approximately48-hours post-injection)approximately20 mL of blood was obtained from the posterior vena cava of each surviving animal. All samples were stored at room temperature until centrifuged and separated into serum and cellular fractions. The blood samples were then stored in a freezer set to maintain a temperatureof -20*C 10*C until shipped to the Sponsor. Pathology The animal found dead immediately after dosing (40 mg/kg) was subjected to an abbreviated gross necropsy examination and any abnormalities were recorded. No tissues were collected from this animal. At terminationof the experimental phase, surviving animals were anesthetizedwith sodium pentobarbital and exsanguinated without necropsy however tissues were collected. The whole liver, bile, and both kidneys from each animal surviving to terminationwere collected and immediatelyplaced on dry ice, then frozen by placing in a freezer set to maintain a temperatureof -20*C 10*C. After tissue/bilecollection,the animals were discarded. Shipment of Tissues After completionof the in-life phase the blood samples,livers, bile, and kidneys were sent frozen (on dry ice) to the Sponsor (James D. Johnson, 3M E.E. & P.C., Bldg. 2-3E-09, 935 Bush Avenue, St. Paul, MN, 55106). The Sponsor is responsiblefor the retention and dispositionof the samples. HWI does not accept any responsibilityfor the analysis of the samples collected in this study nor are these results presentedin this report. StatisticalAnalyses No statisticalanalyses were required by the protocol. Location of-Raw Data, Records, and Final Report The raw data, records, and an original signed copy of the final report will be retained in the archives of HWI in accordance with HWI SOP. C)000:LG Page 11 of 28 RESULTS Body Weights Individualbody weights are in Table 1. HWI 6329-151 Clinical Observations Individualclinical signs are in Table 2. The animalstreated at 0, 4, 16, and 24 mg/kg appeared normal throughout the study. The animal treated at 40 mg/kg died within 5 minutes of dosing. Pathology The animals treated at 0, 4, 16, and 24 mg/kg survived to terminationof the experimental phase and were not examined grossly when sacrificed. The animal treated at 40 mg/kg died on the day of treatment and was necropsied. The lungs in this animal were diffuselydark red. This change was attributedto acute death and postmortem change. . DISCUSSION The level of systemic exposure of T-6067 was evaluated in female albino rabbits when administeredas a single intravenousinjectionat levels of 0, 4, 16, 24, and 40 mg/kg. The animals treated at 0, 4, 16, and 24 mg/kg appeared normal throughoutthe study. Administrationof this material at 40 mg/kg resulted in the death of the animal. SIGNATURE Steven M. Glaza Date Study Director Acute Toxicology REFERENCE 1. NIH PublicationNo. 86-23 (revised1985). OOOA017 Page 12 of 28 HWI 6329-151 PATHOLOGY REPORT There was one Group-4 female rabbit treated at 40 mg/kg that died on Day 0 and was necropsied. At necropsy, the lungs in this animal were diffusely dark red. This change was attributed to acute death and postmortem change. The liver, bile, and both kidneys from this animal were inadvertentlynot collected as required by protocol. After necropsy, the animal was discarded. ThomWE.'Palmer, PhD---' Date Pathologist (6329-ISI.slh) 122294 oooo:Ls Page 13 of 28 GrouD 1 2 3 4 5 Table I IndividualBody Weights (g) Dose Level (mg/kq) Sex Animal Number 0 Female F52762 4 Female F52763 16 Female F52768 40 Female F52769 24 Female F52770 Day 0 2,781 2,770 2,735 2,511 2,778 HWI 6329-151 Page 14 of 28 Dose Level Group (mg/kg) Sex 1 0 Female 2 4 Female 3 16 Female 4 40 Female 5 24 Female Table 2 IndividualClinical Signs Animal Number F52762 F52763 F52768 F52769 F52770 Observation 0.5 Appeared normal Appeared normal Appeared normal Dead (=5 minutes post-injection) Appeared normal Indicates condition exists. Not applicable. HWI 6329-151 Hour 24 48 .1 000020 Page 15 of 28 HWI 6329-151 APPENDIX A Protocol Deviation Protocol TP8084.PK Protocol Amendment No. I 00002:L Page 16 of 28 Protocol Deviation HWI 6329-151 -- Protocol Page 8, 7. Experimental Design, E. Termination,(2) Scheduled Sacrifice,(a) Sample Collection. The whole liver and bile from each animal dying during the study, sacrificein a moribund condition, or survivingto terminationwill be collected. Both kidneys from each animal will also be collected. Actual Procedure The liver, bile, and kidneys from the animal that died on test (Group 4, No. F52769) were inadvertentlynot collected at necropsy. This deviation is not considered to have had an adverse effect on the outcome of the study. oooo;42 HAZLr;ECC:N W W IS C 0 N S IN POST OFf:iCF BOX /545 MAOISON Wl @3107 1545 Page 17 of 28 CORNING Sponsor: 3M St. Paul, Minnesota PROTOCOL TP8084.PK Study Title: Single-Dose IntravenousPharmacokineticStudy of T-6067 in Rabbits Date: December 6, 1994 Performing Laboratory: Hazleton Wisconsin, Inc. 3301 Kinsman Boulevard Madison, Wisconsin 53704 LaboratoryProject Identification: HWI 6329-151 000023 Page 18 of 28 STUDY IDENTIFICATION TP8084.PK Page 2 Single-Dose IntravenousPharmacokineticStudy of T-6067 in Rabbits HWI No. Test Material Sponsor Sponsor's Representative Study Director Study Location Proposed Study Timetable Experimental Start Date Experimental Termination Date Draft Report Date 6329-151 T-6067 3M Toxicology Services 220-2E-02 3M Center St. Paul, MN 55144 John L. Butenhoff, PhD 3M Toxicology Services 220-2E-02 3M Center St. Paul, MN 55144 (612) 733-1962 Steven M. Glaza Hazleton Wisconsin, Inc. P.O. Box 7545 Madison, Wl 53707-7545 (608) 241-7292 Hazleton Wisconsin, Inc. Building No. 3 3802 Packers Avenue Madison, Wi 53704 December 9, 1994 December 11, 1994 January 13, 1995 0000,%4 Page 19 of 28 TP8084.PK Page 3 1. Study Single-Dose IntravenousPhamacokinetic Study in Rabbits 2. Purpose To assess the level of systemic exposurewhen the test material is administeredas a single intravenousinjectionto rabbits 3. Regulatory Compliance This study will be conducted in accordancewith the followingGood Laboratory Practice Regulations/Standards/Guidelines with the exceptionthat analysisof the test materialmixturesfor concentration,solubility,homogeneity,and stabilitywill not be conducted: [ ] Conduct as a Nonregulated Study [X] 21 CFR 58 (FDA) 40 CFR 160 (EPA-FIFRA) 40 CFR 792 (EPA-TSCA) C(81)30(Final)(OECD) 59 Nohsan No. 3850 (Japanese MAFF) NotificationNo. 313 (Japanese MOHW) All proceduresin this protocol are in compliancewith the Animal Welfare Act Regulations. In the opinion of the Sponsor and study director, the study does not unnece,ssarildyuplicateany previous work. 4. Ouality Assurance The protocol,study conduct,and the final reportwill be audited by the Quality Assurance Unit in accordancewith Hazleton Wisconsin (HWI) StandardOperating Procedures (SOPS) and policies. 5. Test Material A. Identification T-6067 B. PhysicalDescription (To be documented in the raw data) C. Purity and Stability The Sponsorassumes responsibilityfor purity and stability determinations(includingunder test conditions). Samplesof testmaterial/vehiclmeixture(s)for concentration,solubility, homogeneity,and stabilityanalyseswill be taken before administrationif requestedby the Sponsor. These samples (if taken)will be sent to the Sponsor after experimental terminationfor possibleanalysis. oooozs Page 20 of 28 D. Storage Room temperature TP8084.PK Page 4 E. Reserve Samples Reservesampleswill not be required for this study. F. Reteqtion Any unused test materialwill be returnedto the Sponsor after completionof the in-lifephase.ofthe study. G. Safety Precautions As required by HWI SOPs and policies 6. Control Material A. Identification Sterilewater for injection B. PhysicalDescription Clear, colorlessliquid C. Purity and Stability The purity and stabilityof this USP grade material is consideredto be adequate for the purposes of this study. D. Storage Refrigerated E. Reserve SaMDles See Section, 5. E. Reserve Samples F. Retention Any remainingcontrol material may be used for other testing and will not be discardedafter issuanceof the final report. G. Safety Precautions As required by HWI SOPs and policies 7. ExperimentalDesign A. Animals (1) Species Rabbit (2) Strain/Source Hra:(NZW)SPF/HRP, Inc. (3) Age at Initiation Adult 0000%6 Page 21 of 28 TP8084.PK Page 5 (4) Weight at Initiation 2.5 to 3.5 kg (5) Number and Sex 5 females (6) Identification Individualnumbered ear tag (7) Husbandry (a) Housing Individually,in screen-bottomstainlesssteel cages (heavy gauge) (b) Food A measured amount of Laboratory Rabbit Diet HF #5326 (PMI Feeds,Inc.). The food is routinelyanalyzedby the manufacturerfor nutritionalcomponents and environmentalcontaminants. (c)Water Ad libitum from an automatic system. Samples of the water are analyzedby HWI for total dissolvedsolids, hardness,and specified microbiologicalcontent and for selectedelements, heavy metals, organophosphates,and chlorinated hydrocarbons. (d) Contaminants There are no known contaminants in the food or water thatwould interferewith this study. (e) Environment Environmentalcontrols for the animal room will be set to maintain a temperatureof 19*C to 23*C, a relativehumidity of 50% +20%, and a 12-hour light/12-hourdark cycle. (f) Acclimation At least 7 days (8) Selection of Test Animals Based on health and body weight according to HWI SOPS. An adequate number of extra animals will be purchased so that no animal in obviouslypoor health is placed on test. (9)Justificationfor Species Selection Historically,the New Zealand White albino rabbit has been the animal of choice because of the large amount of It background informationon this species. Page 22 of 28 TP8084.PK Page 6 B. Dose Administration (1) Test Groups Group Dose Level (mg/kqla- Number of Females 1 0 (Control) 1 2 4 1 3 16 1 4 40 1 5 160 1 a The dose volume will be 0.5 mL/kg of body weight. C. Dosing Procedures (1) Dosing Route Intravenousinjectioninto a marginalear vein over approximately30 to 60 seconds. (2) Reason for-Dosing Route Intravenousinjectionis an acceptableroute to assess systemic exposure. (3) Dosing Duration Single dose (4) Dose Preparation The testmaterialwill be dilutedwith sterilewater for injectionto achievea specificconcentrationfor each dose level. Individualdoses will be calculatedbased on the animal'sbody weight taken just before test material administration.The preparedtest mixtureswill be stored at room temperatureuntil administration. D. Observationof Animals (1) ClinicalObservations The animalswill be observed for clinicalsigns of toxicityat approximately0.5, 2.0, 4.0, 24, and 48 hours after treatment. oooozs Page 23 of 28 TP8084.PK Page 7 (2) Body Weights Just beforetest material administration. (3) Sample Collections (a) Frequency 2, 4, 6, 8, 12, 24, and 48 hours post-injection (b) Number of Animals All (c)Method of Collection Blood samples (approximately4 ML) will be collected from eitherear via the catheterizationof the auriculararteryor from the marginalear vein at 2, 4, 6, 8, 12, and 24 hours post-injection. Approximately20 mL of blood (actualvolume to be documentedin the raw data) will be obtained from the posteriorvena cava of each animal at the time of necropsy(48 hours post-injection).Approximately 20 mL of blood will be collected from moribund animalsduring the study, also, if possible. The sampleswill be stored at room temperature and then centrifuged,and the separate serum and cellular fractionsstored in 4 freezerset to maintaina temperatureof -20*C 10*C. The separated serum and cellularfractionswill be sent frozento the Sponsor after experimentaltermination. Sampleswill be shipped to: James D. Johnson 3M E.E. & P.C. Bldg. 2-3E-09 935 Bush Avenue St. Paul, MN 55106 James D. Johnsonor his alternatewill be notifiedby telephoneat (612) 778-5294prior to the shipmentof the samples. E. Termination (1) Unscheduled Sacrifices and Deaths Any animaldying during the study or sacrificedin a moribundcondition,will be subjectedto an abbreviated gross necropsy examinationand all abnormalitieswill be recorded. Animals in a moribundconditionwill be anesthetizedwith sodium pentobarbital(via injectionin the marginalear vein), bled via the vena cava, and exsanguinated. 000029 Page 24 of 28 TP8084.PK Page 8 (2) Scheduled Sacrifice At approximately48 hours post-injection,animals survivingto terminationwill be anesthetizedwith sodium pentobarbital(viainjectionin the marginalear vein), bled via the vena cava, and exsanguinated. An abbreviated gross necropsy examination will not be done, however, tissueswill be collected. (a) Sample Collection The whole liver and bile from each animal dying during the study, sacrificedin a moribund condition, or survivingto terminationwill be collected. Both kidneysfrom each animal will also be collected. The tissueswill be placed on dry ice immediatelyafter collectionand then placed in a freezer set to maintain a temperature of -20*C 10*C. The tissues(liver,bile, kidneys)will be sent frozen on dry ice to the Sponsor after experimental termination. The sampleswill be shippedto the personlistedin Section7.D.(3).(c).The Sponsoris responsiblefor the retentionand dispositionof the samples. F. StatisticalAnalyses @o statisticalanalysesare required. 8. ReRort A final report includingthose items listed below will be submitted. Descriptionof the test and controlmaterials Descriptionof the test system Procedures Dates of experimentalinitiationand termination Descriptionof any toxic effects Gross pathologyfindings(if applicable) Gross pathologyreport (if applicableand requestedby the Study Director) 000030 Page 25 of 28 TP8084.PK Page 9 9. Location of Raw Data. Records, and Final Regort Originaldata, or copies thereof,will be availableat HWI to facilitateauditingthe studyduring its progressand before acceptanceof the final report. When the final report is completed, all originalpaper data, includingthose item listed below will be retained in the archives of HWI according to HWI SOP. Protocol and protocol amendments Dose preparation records In-liferecords Body weights Dose administration Observations Sample collectionrecords Shipping records Pathology Records Study correspondence Finalreport (originalsignedcopy) The followingsupportingrecordswill be retainedat HWI but will not be archivedwith the study data. Animal receipt/acclimationrecords Water analysis records Animal room temperature and humidity records Refrigeratorand freezer temperaturerecords Instrumentcalibration and maintenance records 00003:L Page 26 of 28 TP8084.PK Page 10 PROTOCOL APPROVAL z g@@ John L. Butenhoff, PhD Sponsor's Representative 3M Steven M. Glaza Study Director Acute Toxicology Hazleton Wisconsin, Inc. ,Kkpresentative Quality Assurance Unit Hazleton Wisconsin, Inc. (6329-151.protdskl) 12 Date iz--,clq Date Date oooo3z Page 27 of 28 HAZLIZ'C(ZN W IS C 0 N S IN POST OFFICE BOX 1545 MADISON. Wi 13707-7545 i.CORNING co",Cklny PROTOCOL TP8084.PK Single-Dose IntravenousPharmacokineticStudy of T-6067 in Rabbits NWI 6329-151 SRonsor 3M Toxicology Services 220-2E-02 3M Center St. Paul, MN 55144 Sponsor's Representative John L. Butenhoff, PhD Contractor Hazleton Wisconsin, Inc 3301 Kinsman Boulevard Madison, Wl 53704 Study Director Steven M. Glaza Amendment No. I This amendmentmodifies the followingportion of the protocol: Effective December 9, 1094 1. Page 6- 7. ExperimentalDesign, B. Dose Administration:(1) Test Groups. Due to the death of the Group 4 animal (40 mg/kg), modify the dose level for the Group 5 animal as indicatedby the followingunderlinedchange: Group Dose Level (mg/kqla- Number of Females 1 0 (Control) 1 2 4 1 3 16 1 4 40 1 5 24 1 a The dose volume will be 0.5 mL/kg of body weight p tI o n E" 6 Ll 2 t @,clR E '@ , N@l 1 li iiL '/I-, J 2 .1 ;Ili 0000,3.3 Amendment No. I Page 28 of 28 PROTOCOL APPROVAL HWI 6329-151 Page 2 John L. Butenhoff, PhD Sponsor's Representative 3M Steven M. Glaza Study Director Acute Toxicology Hazleton Wisconsin, Inc. @2 /,;- t--@ Oipr-esentative Quality Assurance Unit Hazleton Wisconsin, Inc. (6329-151.Aml.dsk2) ae 5ate )Y--) Date 000034 9.1.2 AnalyticalprotocolAMDT-120694.1 000035 3M EnvironmentalLaboratory Protoco-lAnalyticaSltudy Single-DosIentravenouPsharmacokinetiSctudyofT-6067inRabbits In-VivoStudyReferencNeumber: HWI#6329-151 StudyNumber: ANfDT-120694.1 TestSubstance:L-13492(T-6067) Name and AddressofSponsor: 3M SCD Division 367 Grove Street St.Paul,MN 55106 Name and Address of TestingFacility: 3M EnvironmentalTechnology and Services 935 Bush Avenue St.Paul,MN 55106 Proposed InitiatioDnate: July25, 1995 Proposed Completion Date: August 25, 1995 Method Numbers AMDT-M-1-0, AMDT-M-2-0, AMDT-M-4-0, AMDT-M-5-0, AMDT-M-8-0, AMDT-M-14-0, and Revisions: Then-nalExtractionofFluorideby Means of a Modified Dohnnann DX2000 OrganicHalideAnalyzer-Liver FluorideMeasurement by Means ofan Orion EA940 Expandable Ion Analyzer Extractionof Fluorochemicalsfrom RabbitLiver AnalysisofRabbitLiverExtractforFluorochmicalsUsing ElectrosprayMass Spectrometry AnalysisofFluorideUsing the SkalarSegmented Flow Analyzer with Ion SelectiveElectrode Therman Extractionof Fluorideby Means of a Modified Dohnnann DX2000 OrganicHalideAnalyzer-Serum Author:James D. Johnson Approved By: /a@SatemuedssylDiI/@Anson Stmudy ?@r/ector Date John Butenhoff,PhD Date Sponsor Representative 1.0 PURPOSE To assestsheleveolfsystemiecxposurwehen thetesmtateriaLl-13492(T-6067i)s administeredasa singleintravenousinjectiotnorabbits. 2.0 TEST MATERIALS 2.1 Test,Control,and Reference Substances and Matrices 2.1.1AnalyticalReference Substance: FC-95, lot161 or 171. They are equivalent. 2.1.2AnalyticalReference Matrix: Bovine liverand bovine serum 2.1.3AnalyticalControl Substance: None 2.1.4AnalyticalControl Matrix: Bovine liverand bovine serum 2.2 Source of Materials:3M ICP/PCP Division(2.1.1),grocerystore(2.1.2,2.1.4liver)S,igma Chemical Company (2.1.22,.1.4-scrum) 2.3Number of Testand ControlSamples: Liverand serum from 3 testanimals and I controlanimal. One animaldiedafterinjectioonf a 40 mg/kg dose,and was notreplaced.Other biologicatlissues(kidney,bile,cellulafrractionw)illbe availableforanalysisifdeemed appropriatbey the Study Director. 2.4 Identifleatioonf Test and Control Samples: The samples areidentifieudsing theHWI animal identificatinounmber which consistsof a lettearnd fivedigit number, plusthetissueidentitaynd day identit(yserum). 2.5Purity and Strength of Reference Substance: To be determinedby Sponsor. 2.6 Stabilityof Reference Substance: To be determinedby Sponsor. 2.7Storage Conditions for Test Materials:Room temperature(2.1.1), -20 10-C (2.1.22,.1.4).Testand Controlsampleswillbe receivedaccordingto AMDT-S-10-0. 2.8Dispositionof Specimens: Biologicatlissuesand fluidswillbe retainedper GLP Regulationforthetime periodrequiredforstudieslongerthan 28 days. This studyisinparallewlitha 28 day dermal absorptionstudyso alltissueswillbe retained. 2.9 Safety Precautions: Refer to appropriateMSDS. Wear appropriatelaboratory attireU.se cautionwhen handlingknivesforcuttingthesamples. 2 000oa7 3.0EXPERIMENTAL -Overview Thetissufersomanimaldsosedasdescrib(eHdWI#6329-151a)r,eavailabfloer analysifsorfluorinceompounds. At thediscretioonf theStudy Directora, seriesof analyticatlestscan be performed.The screeningforfluoridien liverviacombustion (SeeMethods--nextSection)istheappropriataenalysitsopresentdefinitivdeatafor fluorinientheliver. 4.0 EXPERIMENTAL -Methods 4.1Liver and Serum screeningmethods: (attached) 4.1.1AMDT-M-1-0,, Thermal ExtractioonfFluorideby Means ofa Modified Dohrmann DX2000 OrganicHalideAnalyzer-Liver 4.1.2AMDT-M-2-0, FluorideMeasurement by Means ofan Orion EA940 Expandable Ion Analyzer 4.1.3AMDT-M-4-0, Extractionof Fluorochemicalsfrom RabbitLiver 4.1.4AMDT-M-5-0, Analysisof RabbitLiverExtractforFluorochmicals Using ElectrospraMyass Spectrometry 4.1.5AMDT-M-8-0, Analysisof FluorideUsing theSkalarSegmented Flow AnalyzerwithIon SelectivEelectrode 4.1.6AMDT-M-14-0, ThermalExtractioonfFluorideby Means of a ModifiedDohrmann DX2000 OrganicHalideAnalyzer-Serum 5.QDATA ANALYSIS 5.1Data Reporting: Data willbe reportedasa concentratio(nweight/weighto)f fluoridpeer tissueor fluido,r as FC- 143 (electrospramyass spectrometryp)erunit oftissueor fluid. 6.0 MAINTENANCE OF RAW DATA AND RECORDS 6.1 Raw Data and Records: Raw data,approvedprotocola,ppropriatsepecimens, approvedfinalreporta,nd electronidcatawillbe maintainedintheAMDT archives. 3 oooozis 7,0 REFERENCES 7.1 AMDT-S-10-0, Sample TrackingSystem 8.0 ATTACHMENTS 8.1AMDT-M-1-0, Thermal Extractionof Fluorideby Means of a Modified Dohrmann DX2000 OrganicHalideAnalyzer-Liver 8.2AMDT-M-2-0, FluorideMeasurement by Means of an OrionEA940 Expandable Ion Analyzer 8.3AMIDT-M4-0, Extractioonf Fluorochemicalsfrom RabbitLiver 8.4AMDT-M-5-0, Analysisof RabbitLiverExtractforFluorochmicalsUsing ElectrosprayMass Spectrometry 8.5 AMDT-M-8-0, Analysisof FluorideUsing the SkalarSegmented Flow Analyzer with Ion SelectiveElectrode 8.6AMDT-M-14-0, Thermal Extractionof Fluorideby Means of a Modified Dohrmann DX2000 OrganicHalideAnalyzer-Serum 4 ()000,3.4) 3M EnvironmentaLlaboratory Method TherrnalExtractionof Fluorideby Means of a Modified Dohrmann DX2000 OrganicHalideAnalyzer- Liver Method IdentificatiNounmber: AMDT-M-1 RevisionNumber: 0 AdoptionDate: /10 RevisionDate: None Author RichYoungblom Approved by: roupLea74/ OUP Le /0IZ3 Daie @@u-alitAyssurance Date Software:MS Word 5.la AffectedDocuments: AMDT-M-2 FluoridMeeasurementby Means ofan OrionEA940 ExpandableIonAnalyzer AMDT-EP-3 RoutineMa@itenanceofa ModifiedDohrmanTiDX2000 OrganicHalideAnalyzer 000040 1.0 SCOPE, APPLICABLE COMPOUNDS, AND MATRICES 1.1Scope:Thismethod isfortheoperationofa Dohrmann DX2000 when itisused toextract fluoridferom variousmatrices.The fluoridiestypicallcyollecteidnTISAB solutionforanalysis withan ionselectiveelectrode. 1.2ApplicableCompounds: Fluorocheniicalosrotherfluorinatecdompounds, 1.3Matrices:Biologicatlissuesp,articularlliyver. 2.0 KEYWORDS - 2.1Fluoride,fluorinee,xtractiopny,rolysisi,onizatioino,nselectiveelectrodeD,ohnnann, halide, DX2000, fluorocherr@cals. 3.0 PRECAUTIONS 3.1Glassware and exhaustgasescan be extremelyhot. 3.2Glasswareisfragileb,rokenglassmay causeinjuries. 3.3Pressurizedgases,propercompressedgas handlingpracticersequired. 3.4Solventbasedsamplesmay flashm,ay need toallowthem todrydown beforestartinrgun. 3.5Potentiabliohazardsdue tothebiologicamlatrices.Use appropriatpeersonalprotective equipment. 4.0 SUPPLIES AND MATERIALS 4.1Compressed Oxygen, Hydrocarbon free,regulatedto30 PSI. 4.2Compressed Helium, High PurityGrade,regulatedto45 PSI. 4.3Quartz glasssample boat withTeflonTMtubing,Dohrmann 890-097 or equivalent. 4.4Quartzglasscombustiontube,RelianceGlass G-9405-012 orequivalent. 4.5Orion940999 TotalIonicStrengthAdjustmentBuffer(TISAB II)or equivalent. 4.6Sample collectiovnialsH,DPE. 4.7Milli-QTIwlater 4.8Polystyrenepipettes. 4.9ActivatedCharcoal,E. Merck 2005 orequivalent. 4.10Hamilton Syringeorequivalent. 4.11Miscellaneouslaboratorgylassware 5.0 EQUIPMENT- 5.1Rosemount DohrTnannDX2000 OrganicHalideAnalyzer,modifiedforfluoridextraction. 5.2IBM compatible386 or 486 computer. 5.3DX2000 softwarev,ersion1.00,modifiedforfluoridextraction. 5.4Excel Spreadsheet,version5.0orgreater 6.0 INTERFERENCES 6.1 Sample sizeislimitedtoapproximately150mg, dependingon samplemoisturecontent.This may varyfrommatrixtomatrix. 2 000041 7.0 SAMPLE HANDLING 7.1Samples arenot tobe handled with barehands. Fluoridemay leachfrom theskinto the sample. Use forcepsor probe to transfertissues. 7.2Samples of liverarecutfrom frozenliverand placedina taredand labeledweigh boat.Use a cleanscalpeland cuttingboard.The cuttingboardand scalpelshouldbe cleanedwith water, methanol,or methanol-watersolutionaftereach liveriscut. 8.0 --CALIBRATION AND STANDARDIZATION 8.1 PreparatioonfCalibratioSntandards 8.1.1The standardsrequiredforeach projectwillneed to be appropriatfeorthatindividualproject. Refer toprotocolforthatproject. 8.1.2Typically50-500 ppm FC-95 inmethanol standardsareused. 8.1.3For rabbitliverstudiesu,se beefliverasthematrix.Cut a pieceofftozenbeefliver(100150 mg) and weigh itin a labeledand taredweigh boat. 8.2 Calibration- Overview The normal calibratioinsthefluoridceurve(AMDT-M-2). However, ifan optionalspikedliver curveisrequiredtheprocedurelistedbelow isused. 8.2.1 A calibratiocnurve fortheDX2000 isgeneratedby spikingsamples withknown standards and combusting them using thesame methods and matrixtype as thesamples to be tested. 8.2.2Typically,threereplicateosf each standardand fiveconcentrationosf standardswillbe spiked. 8.2.3Standardcur%,ewillbe plottedas Mass Spiked F (ug)on thex-axisand StandardMass Recovered F (ug)on they-axis.Generatea regressioncurveand calculatteheequationfortheline and ther2 value. 8.2.4Mass Spiked F (ug)= (Amount spikedinmL) x ( Conc.of standardinppm) x (0.6004)* *FC-95 is60.04% F therefore0.6004 isthefactorused toconvertFC-95 toF 8.2.5StandardMass Recovered F (ug)= (TISAB volume inmL) x (Orionreadinginppm) 8.3Calibration- Procedure 8.3.1StartUp 8.3.1.1Run 2 or more Clean Cycleswhen startinignstrumenetachday. More cleancyclesmay be used iftheprevioussamples containedhighconcentrationosf fluoride. 8.3.2Blanks 8.3.2.1Preparesample usingthesame methods and typeof matrixas thetestsample. 8.3-2.2For rabbitstudies,use beefliveras thematrix.Prepareatleast3 samples of beef liver (100 - 150 mg) forblanks. 8.3.2.3Put sample inDohrmann boat.Combust each sample as describedin section9.0and analyzesample accordingto method AMDT-N,1-2 forthe]onselectivelectrodeanalysis. 3 ()00042 8.3-2.4For rabbitstudiest,hemeter readingfora blanksample should be 0.03 ppm or lower beforeproceedingwiththecalibrationB.um samplesuntilthislimitisreached,or untilinthe judgement of theoperatorthereadingisstablewith respecttohistoricarleadings(previous48 hours). 8.3.2.5For non-rabbitstudies,theblankreadingsshouldreacha predeterminedionconcentration beforeproceedingwiththecalibration. 8.3.2.6Itmay be necessaryto mix approximately50 mg of charcoalwith thesample to aid combustion. 8.3.3 Standard Curve 8.3.3.1Weigh out atleast15 matrixsamples (5 standardswith3 replicateesach)in taredand labeledweigh boats.For rabbitstudies,weigh 100-150 mg beefliversamples. Record weightsin studydata. Storethematrixsamples on dry iceor icepacks tokeep them frozenuntilused. 8.3.3.2 Placeweighed beefliversample inDohrmann sample boat. 8.3.3.3StartwiththeloweststandardconcentrationU.sing a Hamilton syringe,ejecta fixed quantityof thestandardon or inthematrix. For rabbitstudiesu,se 4 uL of standardand ejectiton or inthebeefliver. 8.3.3.4At least3 replicatesshouldbe used forthelowest standardconcentrationm;ore replicates may be used atthediscretioonf theanalyst. 8.3.3.5Combust the sample asdescribedinsection9.3and analyzeaccordingto AMDT-M-2. 8.3.3.6Run all15 standards.Ifone replicatiessignificantdliyfferenftrom theothertwo replicatensm, anothersample forthatstandard.Indicateindatathatthenew replicatreeplacesthe oldreplicataend thatthenew replicatweillbe used to calculatteheregressioncurve. 8.3.3.7When allstandardshave been run,calculattehe r2. r2must be atleast0.95. Ifitisnot at least0.95,consultwith supervisor. 8.3.3.8A new standardcurveshouldbe run when thecombustion tubeor sample matrixis changed. New standardcurvemay alsobe run atthediscretioonf theanalyst. 8.4 Storage Conditions for Standards 8.4.1Storagerequirementsforstandardsaredependenton theindividualstandardsused. Typically,standardsarestoredatroom temperatureinplasticscrew topbottles. 8.4.2 New FC-95 standardsshouldbe preparedatleastonce a month. 9.0 PROCEDURES 9.1TypicalOperating Conditions: 9.1.1Combustion tubetemperature= 950'C. 9.1.2Oxygen and Helium flow 50 cc/rninute. 9.1.3Vaporization/Dryintgime 240 seconds. 9.1.4Bake time = 300 seconds. 9.2StartUp Procedure: 9.2.1Iftheprogram isnot starteds,tarttheEOX program on thePC. 9.2.2Open the SYSTEM SETUP window. 9.2.3Put thefurnacemodule and thecellintheREADY mode. 9.2.4Close the SYSTEM SETUP window. 4 000043 9.2.5When theoven has reachedtheREADY temperature,run theCLEAN BOAT program found intheCELL CHECK menu. 9.2.6 See AMDT-EP-3 fordetailosf the Dohrmann software. 9.3Sample ExtractionProcedure: 9.3.1Open theSAMPLE HATCH and placethesample intheBOAT. Itmay be necessaryto n-dxapproximately50 mg ofcharcoalwiththesample toaidcombustion. Ifthisisdone,charcoal shouldalsobe mixed in while establishintghebaselineand when generatingthestandardcurve. 9.3.2Close SAMPLE HATCH. 9.3.3Add appropriatveolume ofTISAB solutioonr 1:1 TISAB:Milli-QTIlwatermixturetoa labeledsample collectiovnial.Typically0.6mL to 15 mL areused. For rabbitstudiesu,se 1.0or 2.0mL of 1:1 TISAB:Milli-QTM watermixture. 9.3.4 Placethevialso thatthetipof theCOMBUSTION TUBE isintheTISAB atleast0.25 inches.Gases releasedduringpyrolysismust bubble throughtheTISAB. 9.3.5 Run theEOX-SOLIDS program found in theRUN menu. 9.3.6When theEOX program isfinishedr,emove thecollectiovnialfrom thecombustion tube. 9.3.7IfundilutedTISAB was used tocollecthesample,add an equalvolume of Milli-QT,w4ater totheTISAB tomake 1:1 TISAB:Milli-QTm. 9.3.8Rinse theend of the combustiontubewithMilli-QTMwaterand wipe with a KIMWIPE to remove any TISAB remainingon thetube. 9.3.9Open thesample hatchand remove any remainingash from theboat. Ash can be removed witha cottontippedapplicatoror vacuumed out. Itmay be necessarytoscrapparticleosffthe bottom witha spatulaor othersimilardevice.A drop ofMilli-QTMwater may be added tothe boatto aidintheClean Cycle. 9.3.10 Close thebatch. 9.3.11 Run the CLEAN BOAT program. 9.3.12Sample isready foranalysisby ion selectiveelectrode(ANMT-M-2). 9.4 Sample Calculations 9.4.1 Use thestandardcurve tocalculatethesample value. 9.4.2Sample Mass RecoveredF (ug)= (TISAB volinmL) x (Orionreadiniiznpl2m -intercel2t) (slope) 10.0 VALIDATION 10.1 QualityControl 10.1.1Daily StartUp Check Samples: Once thestandardcurveisestablishede,ach day of analysisisstartedby analyzingQC samples.The QC samples areto be the same as thelowest concentrationspikedsamples used togeneratethestandardcurve.Each concentrationmust be done intriplicautnelessthefirsttwo replicateasrewithin20% of thestandardcurve,thena third replicatiesnot necessary. 10.2 Precisionand Accuracy: See method development analysisand sample analysisin FluorideNotebooks 2,3,and 5. Precisionand accuracyvarieswhen analyzingsamples of different matricesand differenrteferencecompounds. 10.3 Other N'alidatioPnarameters: NA 5 000044 11.0 DATA ANALYSIS 11.1Calculations 11.1.F1orthestandacrudrveu,seregressainoanlysiinsExcelv,ersio5n.0orgreater. 11.1.2To calculattehefluoridceontractioinnthesample,seemethod AMDT-M-2. 11.2 Analyzing theData 11.2.1r2must be atleast0.95orgreater".Outliersm"ay be excludediftwo of thethreereplicates arewithin20% ofeach otherand theoutlieirsgreaterthan200% oftheaverageof thosetwo or lessthan50% oftheaverageofthosetwo. Any suchoutliersshouldbe pointedoutinthedataand notedintheFinalReportalongwiththereasonitwas consideredan outlier. 12.0 ATTACHMENTS None 13.0 REFERENCES 13.1Rosemount Dohrmann DX2000 OrganicHalideAnalyzerOperator'Msanual (Manual 915349,revisioBn, December 1993) 13.2 ANIDT-M-2 FluorideMeasurement by Means of an Orion EA940 ExpandableIon Analyzer 13.3ANOT-EP-3 RoutineMaintenanceofa ModifiedDohnnann DX2000 OrganicHalide Analyzer 14.0 REVISIONS Revision Numbe Reason forChanize Revision Date 6 0()004Zi 3M Environmental Laboratory Method FluorideMeasurement by Means ofan Orion EA940 Expandable Ion Analyzer MethodIdentirteaNtuimobner: AMDT-M-2 RevisionNumber: 0 AdoptionDate:10-4-fsRevisionDate: None Author:Rich Youngblom Approved By: G@, Leader Date Z4:@f QualitAyssurance -Lo-4,-,t Date Software:MS Word 5.la AffectedDocuments: AMDT-M-1 ThermalExtractioonfFluoridbey Means ofa Modified Dohrmann DX2000 OrganicHalideAnalyzer 000046 1.0 SCOPE, APPLICABLE COMPOUNDS. AND MATRICES 1.1SCOPE: This method isforthecalibratioannd operationof an Orion EA940 Expandable Ion Analyzer. 1.2 APPLICABLE COMPOUNDS: Fluoride. 1.3APPLICABLE pH of 6.0. MATRICES: Liquidsamples inan appropriatebuffersolution.Preferred 2.0 KEYWORDS 2.1Fluoride,fluorinei,onselectiveelectrode 3.0 PRECAUTIONS 3.1No hazardsidentifiewdiththismethod. 4.0 SUPPLIES AND MATERIALS 4.1Orion 940999 TotalIonicStrengthAdjustmentBufferH (TISABII)or equivalent. 4.2Orion Model 900001 electrodfeillinsgolution(AgCl)or equivalent. 4.3 Orion 940907 100 ppm fluoridestandardor equivalent. 4.4Milli-QTMwateror equivalent. 4.5Magnetic stirbars. 4.6Lab tissues. 4.7Sample collectiovnials. 4.8 Plastic100 mL volumetricflasks. 4.9 Polystyrenepipettes. 4.10 Miscellaneouslaboratoryglassware. 5.0 EQUIPMENT 5.1Orion Model EA940 Expandable Ion Analyzer orequivalent. 5.2Orion Model 960900 SolidStateCombination Fluorideelectrodeor equivalent. 5.3Magnetic StirPlate. 5.4IBM compatible386 or486 computer(onlyneeded ifusingOrion 3E software). 5.5Orion RS232 interfacecable(onlyneeded ifusingOrion 3E software). 5.6MicrosoftExcel 5.0(onlyneeded ifusingOrion 3E software). 6.0 INTERFERENCES 6.1Itisrecommended thatthepH be ator near6.0.A 1:1 mixtureofTISAB and sample/Milli- Q TI-I water will generally bring sample to pH of 6.0. 6.2 Sample temperature may effect fluoride measurement. Itis recommended that the sample be atroom temperatureas thestandardswere when themeter was calibrated. 6.3The ratethesamples arestirreadtshouldbe consistenwtith theratethestandardswere stirred. 2 000047 6.4AirbubbletsrappeudnderelectrocdaengiveerroneourseadingsM.ake surenoairistrapped under electrode. 7.0 SAMPLE HANDLING 7.1 No specialhandlingnecessary. 8.0 CALIBRATION AND STANDARDIZATION 8.1 Preparationof CalibrationStandards 8.1.1Measure 50 mL of TISAB 11into5 100 mL plastivcolumetrifclasks. 8.1.2Labeltheflaskass 0.05,0.1,0.5,1.0,and 1.5ppm F-,alongwiththedateand yourinitials. 8.1.3Pipett0e.05,0.1,0.5,1.0,and 1.5niL of 100ppm fluoridsetandaridntotheappropriately labeledflasks. 8.1.4Add approximatel3y0 mL ofMilli-QTMwatertoeachflask. 8.1.5Shake theflaskstorr@xthesolutions. 8.1.6Eliminateairbubblesfrom theflaskbsy tippintgheflaskosn theirsidesand rollintgheairin theflasksovertheairbubbles. 8.1.7Bringthevolume intheflasksup tothe100 mL mark withMiUi-QTM water. 8.1.8Invertand shaketheflaskfsorthefinamlixing. 8.1.9Record standardisnStandardsLog Book. 8.2 Calibration 8.2.1Ifnecessaryr,emove tapefromelectrodfeillinhgole. 8.2.2Invertprobetowet topseal. 8.2.3Ejecta few dropsof fillinsgolutiofnrom bottomofelectrodteowet lowerseal. 8.2.4Filltheelectrodweithfilhngsolution. 8.2.5The meterand theF- electrodaeretypicallcyalibratebdy direcmteasurementwithno blank correctiouns,ingstandardswithconcentratioonfs0.05,0.1,0.5,1.0,and 1.5ppm F-,following themanufacturer'isnstructions. 8.2.6Record theslopeintheappropriatleogbook. 8.2.7Cleantheelectrodbey rinsinwgithMilli-QTM waterand wipingthesidesdown withlab tissues. 8.3 StorageConditionsforStandards 8.3.1Calibratiosntandardsarestoredatroom temperature. 9.0 PROCEDURES 9.1Calibratioannd Measurement,Standardmethod: 9.1.1The sample tobe measuredneedstobe rru*xewdithTISAB usingtheproportions recommended by theTISAB manufacturer. 9.1.2Placea stirbar'inthesampleand placethesampleon thestiprlate. 9.1.3Allow thesampletomix fora few secondsbeforeinsertintgheelectrodeW.hen the electrodiesinsertedm,ake surethereareno airbubblestrappedundertheelectrode. 9.1.4The sampleshouldbe thesame temperaturaes thecalibratiosntandardsand stirreadtthe same rateas the calibratisotnandards. 9.1.5When thereadingshave stabilizerde,cordthereadingintheappropriatleogbook. 3 0()0048 9.2CalibratioAnd Nleasuremen4UsingOrion3E Software: 9.2.1Calibration: 9.2.1.1Follow steps8.2.1to 8.2.4. 9.2.1.2PressFunctionKey #8 (F8). 9.2.1.3The computer screenwillask you toconfirm thenumber of standardstobe used, concentratioonf thestandards,and whetheror nota blankistobe includedinthecalibration. Make any necessarychanges totheinformationpresentedand clickon CONTINLTE. 9.2.1.4Placetheelectrodeinthefirssttandardon thestirplateand clickon CONTINLTE. 9.2.1.5Observe thereadingson thegraphicdisplayon thecomputer. When thereadingshave stabilizedp,ressACCEPT READING. 9.2.1.6Repeat step9.2.1.4and 9.2.1.5forthe remair@ng standards. 9.2.1.7Afterthefinalstandard,thecomputer willdisplaytheslopeof thecurve,as well as the intercepatnd correlationR.ecord theslope,intercepta,nd correlatioinntheappropriatleog book and clickon CONTINLTE. The calibratiodnataisautomaticallcyopiedtoC:\Orion\Data\Calib.txt. 9.2.2Data Spreadsheet: 9.2.2.1SelecteitherNEW or OPEN from the FILE menu toopen a new or existingspreadsheet tostoredatain. 9.2.2.2Record thename of thespreadsheetused in theappropriatelog book. 9.2.3Fluoride Measurement: 9.2.3.1Follow steps 9.2.1 through 9.2.4 9.2.3.2Enter the name of the sample in the appropriate place on the screen. 9.2.3.3Click on the NEW SANTLE button 9.2.3.4When the readings have stabilizedc,lickon the RECORD appropriatelog book. button and wtite the resultin the 10.0 VALIDATION 10.1 QualityControl: 10.2 Precision and Accuracy 10.3 Other Validation Parameters According to Reference 13.2,the range of detectionis0.02 ppm fluorideup to a saturatedsolutionof fluotide. 11.0 DATA ANALYSIS 11.1 Calculations None necessary. 11.2 Analyzin(eyp the Data None necessary. 12.0 ATTACHNIENTS None 13.0 REFERENCES 4 ()()0043 13.1Orion Model EA940 Expandable Ion Analyzer InstructioMnanual, Orion Research Incorporated,1991. 13.2Orion Model 960900 SolidStateCombination FluorideElectrodeInstructioMnanual, Orion Research Incorporated,1991. 14.0 REVISIONS Revision Number Reason for Chanize Revision Date 5 0()0050 3M Environmental Laboratory- Method Extractionof Fluorochemicalsfrom Rabbit Livers SOP IdentiricatioNnumber: AMDT-M-4 Revision Number: 0 Adoption Date: /a RevisionDate: None Author: Dave Christenson/CynthiWaeber Approved By: r/uo7uLpeaadder Dar, QualityAssurance Dam Software:MS Word, 6.0 AffectedDocuments: M-5, AnalysisofRabbitExtractforFluorochemicalsUsing Electrospray Mass Spectroscopy. 0000511 1.0 SCOPE 1.1 Scope: This method isfortheextractioonf fluorocherriicfarlosm rabbitlivers. Ethylacetateisused to extractfluorochemicalfsrom theliversforanalysisby electrospraymass spectroscopy. 1.2 Applicable Compounds: Fluorochemicals or other fluorinatedcompounds. 1.3 Matrices: Rabbit Livers. 2.0 KEYWORDS 2.1 Fluorochemicals,rabbitliverse,lectrospramyass spectrometer,fluorinated compounds, extraction. 3-0-PRECAUTIONS 3.1 Use gloveswhen handlingtherabbitHvm, theymay containpathogens. 4.0 SUPPLIES AND MATERIALS 4.1 Supplies 4.1.1 Syringe,capableof measuring 100 gL 4.1.2 Eppendorf typeor disposablepipets 4.1.3 4.1.4 4.1.5 4.1.6 Gloves Plasticgrindingtubes Plasticentrifugetubes,15 mL Labels 4.1.7 Nitrogen 4.1.8 Timer 4.1.9 FiltersT,itannylon syringefilter0s.,2gm. 4.1.10 Analyticaplipets:glassvolumetricpipets. 4. 1. 11 Disposableplastic3 cc syringes. 4.1.12 Crimp cap autovials. 4.2 Reagents 4.2.1 Aqueous Ammonium Acetate(Aldrich)a,pprox.250 ppm: Preparea 2500 ppm aqueous solutionof ammonium acetateby adding 250 mg ammonium acetatetoa 100 mL volumetricflaskand diluteto volume with MiUi-Q water.Dilutethissolution1:10 fora 250 ppm solution. 4.2.2 Sodium carbonate/SodiumBicarbonateBuffer(J.T.Baker), (Na2CO,/NaHC03) 0.25M: Weigh 26.5g of sodium carbonate(N%C03) and 21.0g ofsodium bicarbonate(NaHC03) intoa I L volumetlicflaskand bringtovolume with NERI-Q water. 4.2.3 Diluteacetonitrisloelutiond,iluteacetonitri1l:e1 withMilli-Qwater. 4.2.4 EthylAcetate 4.2.S Methanol 4.2.6 Nfilli-wQater 4.2.7 1H,IH,2H,2H -perfluorooctanesulfonaiccid(Aldrich) 4.2.8 FC-95 (3M SpecialtCyhemicalDivision) 00005;! 2 5.0 EQUIPMENT 5.1 Ultra-TurrTa'x25Grindefrorgrindinlgivesramples. 5.2 Vortexmixer 5.3 Centrifuge 5.4 Shaker 5.5 AnalyticaElvaporator 6.0 INTERFERENCES 6.1 nere areno known interferenceastthistime. 7.0 5AMPLE HANDLING 7.1 'Me rabbitliversarereceivedfrozen,and must be keptfrozenuntiltheextractioins performed. 8.0 CALIBRATION AND STANDARDIZATION 8.1 Preparationof InternalStandards 8.1.1 Preparean internalstandardof approximately12 ppm 1H, IH,2H,2Hperfluorooctanesulphoradcidto be added to each liversample. 8.1.2 Weigh atleast0.1 g of IH,IH,2H,2H-perfluorooctanesulphoniaccidintoa 100 mL volumetricflask.Record theactualweight. 8.1.3 Bring itup tovolume withmethanol,thisisthestockstandard. 8.1.4 To a 250 mL volumenic flask,add 3 mLs ofthestockstandardand bringto volume with MIIH-Q water.Calculatetheactualconcentrationof the standard. actualmg perfluoroctane- sull2honiaccid X 0.1 L 3_rnL 250 mL actualconcentrationp,pm 8.2 Prepare FC-95 Anion Standards 8.2.1 PrepareFC-95 standardsforthestandardcurve. 8.2.2 Weigh approximately100 mg of FC-95 intoa 100 mL volumetricflask. Record theactualweight. 8.2.3 Bring up tovolume withdiluteacetoniaile. 8.2.4 Dilutethesolutionwithdiluteacetonitff1l:e10 fora solutionof approximately100 ppm. Dilutethissolution1:10 withdiluteacetonitrifloer a solutionof approx.10 ppm. 8.2.5 Use the10 ppm solutionto make working standardswith valuescloseto 5.0 ppm, 1.0ppm and 500 ppb. 8.3 Prepare Beef Liver Homogenate to Use for Standards 8.3. 1 Weigh 40 g ofBovine liverintoa 250 mL Nalgene bottlecontaining 200 mLs Milli-Qwater.Grind toa homogenous solution. 8.3.2 Add I mL of thesolutiontoa 15 mL centrifugteube.Preparea totalof eight I mL aliquotsofthesolutionin 15 mL centrifugteubes.Be suretoresuspend solutionby shaking itbetween aliquots. 000053 3 8.3.3 Spikesevenof the I mL aliquotwsiththefollowingamounts ofworking standardsinstep9.12of theprocedure.One I mL aliquotservesas the blank. Working Standard (ApproximateConc.) 500 ppb 500 ppb 500 ppb 500 ppb I ppm 5 ppm 5 ppm UL lw- 200 300 400 Soo 200 300 Approxirmtefinal concentratioonf FC-95 inliver Blank 0.292 pm 0.584 ppm 0.877 ppm 1.168 ppm 4 ppm 5.'848 ppm 8.772 ppm 8.4 Calculatetheactualvalueof thestandards: uL of standardx concentratio(ninpr-m-l= finacloncentratio(nppm) 171 mg liver/I ml homogenate ofFC -95 inliver *Average weight ofbovine liverinsolutionasdeterminedby weighing I mL homogenates of40 mg liverin200 mL ofNUUI-Q water.The amount of FC-95 isreportedas equivalentsof FC-95 potassium salt. 8.5 Calibration 8.5.1 Extractthespikedbeefliverhomogenate fohowing 9.13to9.23of this method. Use thesestandardsto establishyour curve on themass spectrometer. 8.5.2 Alternativelya,standardcurvemay be generatedusingratiosof responses of theperfluorooctansulfonaatneionand theinternalstandardanionversus concentrationof theperfluorooctanesulfonaatneion. 8.6 Storage Conditions for Standards 8.6. 1 New standardsarepreparedwith each analysisS.tandardsarestoredin covered plasticentrifugetubesuntiltheanalysison themass spectrometer isperformed. 8.7 Storage Conditions for Standards 8.7. 1 Beef liverhomogenates may be frozenafterpreparation. 9.0 PROCEDURES9.1 Obtain frozenliversamples.In spenttissuen,ote thattheliverhas not been packaged with othertissues. 9.2 Use a dissectinsgcalpeland cutoffapproximatelyI g of liver. 9.3 Weigh thesample directlyintoa Laredplastigcrindingtube. 9.4 Record theliverweightinthestudynotebook. 9.5 Put a labelon thevialwiththestudynumber, weight,rabbitID,dateand analyst initials. 000054 4 9.6 9.7 9.8 9.9 9.10 9.11 9.12 9.13 9.14 9.15 9.16 9.17 9.18 9.19 9.20 9.21 9.22 9.23 9.24 9.25 Add 2.5 mLs water. Grind the sample.Put thegrinderprobe inthesample and grindforabout 2 minutes,untilthesample isa homogeneous solutionwith no largechunks. Rinse theprobeoffintothesample with2.5mLs waterusinga pipet. Take thegrindearpartand cleanitwithmethanolafteerachsample.Follow AMDT-EP-22. Cap thesample and vortexfor15 seconds. PipetI mL intoa 15 mL centrifugteube.Label thecentrifugteubewiththeidentical infor-matioansthegrindingtube.(SeeAMDT-M-4 Worksheet fordocumenting the remainingsteps.) Spike thebeefliverhomogenates with theappropriataemount of FC-95 standard as describedin 8.3. Spike thesamples and beefliverhomogenates with 100 uL of intemal standard. Add 1 mL ofthesodium carbonate/sodiumbicarbonatbeufferand I mL ar=onium acetate. Using an analyticaplipet,add 5 mL ethylacetate. Cap thesample and vortex20 to 30 seconds. Put them in theshakerfor20 min. Centrifugefor20 to 25 minutes,untilthelayersar-ewellseparated.Setthepower on thecentrifugeto 25. Remove 4 mLs ofthetoporganiclayertoa fresh15 mL centrifugteubewith a 5 mL graduatedglasspipet.Transferthelabeltothefreshtube. Blow thesample down on theanalyticaelvaporatorto neardrynesswith nitrogen, approximately30 to40 minutes. Bring theremainingsample up in 1 niL diluteacetonitriwliethan analyticaplipet. Vortex 15 seconds. Transferthesample toa 3 mL syringe.Attacha 0.2gm nylon mesh filtera,nd filter thesample intoa freshcentrifugteubeor a autovialL.abel thetubeor vialwith the studynumber and animal number. Cap and hold foranalysisby electrospramyass spectroscopy. Complete AMDT-M-4 worksheetand attachtopage of studynotebook. 10.0 VALIDATION 10. 1 Quality Control- not applicable 10.2 Precisionand Accuracy- not applicable 10.3 Other ValidationParameters- not applicable 11.0 DATA ANALYSIS 11.1 None 12.0 ATTACHMENTS 12.1 WorksheetAMDT-M-4 13.0 REFERENCES 13.1 ANIDT-EP-22 RoutineMaintenanceofUltra-Tur-rTa-x25 14.0 REVISIONS Revision Number Reason r Change Revision Date 000055 5 Study Sample Number set Rlank I-iver Worksheet AMDT-M-4 FC-95 approx 0.5 ppm actual PPM #W 100 )00111, 100 u 400 11 FC-95 approx I ppm actual ppm #W 500 til@ - FC-95 approx. 5 ppm actual PPM #W Date and InitialfsorStd. 2()0uL 1(10lif, ,tudvni)rnhe'arxherethe nricrin:winlrk-,gheeit-.I;nrntt-pdnd ninrea cnnxFxtrnetinn Pir)etI rnT,nf LiverSoltitinn Pinet 100 uT-of 12 nnrn Intt-rnnSit-Ancl;trd Vnrtex I@ -,er Pir@etI mT, of 250 nnm ArnrnnniiimAretate ii Pinet I rnT,of 0-25 NaCO,/0-25M Pinet5 mT. nf F;.thyAlretnte, Nal4CO, Buffer vn"y 70-1()!Zt-r Sh;ike20 minCentrifnpt7-t)-?;min Remnve,a 4 mT. aliniinntf cirpiniIr;ive-r Bit)w iinwr to near ciryne@g(e<,O 2r,ml@) vath N@ AritiI m- r)f1-1 Acernnitrilf-/74.n Vnrte.-xIIq TNii I Filttriyqing ;iIrr B-D syrinpe with ;10 "iim qR I filterinto a I '@mL aningamnir, via) ooo6!;(; 6 3M Environmental Laboratory Method Analysisof Rabbit Liver Extract for Fluorochemicalsusing Electrospray Mass Spectroscopy SOP IdentiricatioNnumber: AMDT-M-5 RevisionNumber: 0 Adoption Date: RevisionDate: None Author: Dave Christenson/CynthiWaeber Approved By: @fu,proup Leeaaddeerr Date i5ualitAyssurance Date Software:MS Word, 6.0 AffectedDocuments: M-4, ExtractionofFluorochernicalfsrom RabbitLivers 1.0 SCOPE 1.1 Scope: This method isfortheanalysisofextractsofrabbitliveror othertissuesor fluidsforfluorochemicalussingthe electrospramyass spectrometerT.he analysis isperformed by singleionmonitoringofFC-95 anion,M/Z= 499,theinternal standardM/Z = 427, and otherappropriatemasses. 1.2 Applicable Compounds: Fluorochemicalsor otherfluorinatecdompounds. 1.3 Matrices:RabbitLivers(samples)B,eef Liver(smndards),othertissuesand fluids. 2.0 KEYWORDS 2.1 Fluorochemicals,fluorinatecdompounds, electrospramyass spectroscopy,mass spectrometer,rabbitlivers. 3.0 PRECAUTIONS 3.1 Use cautionwith thevoltagecablefortheprobe.When thevoltagecableisplugged intotheprobe DO NOT TOUCH THE PROBE, thereisriskof electricaslhock. 3.2 Do notrun thepump above it'scapacityof 4000 psi.Ifpressuregoes over 4000 psi stopand releasepressure.The peak tubingmay be plugged.Troubleshootback to findtheplug and replacetheplugged tubing.See AMDT-EP- 15 3.3 Do notrun thepump to dryness. 4.0 SUPPLIES AND MATERIALS 4.1 Supplies 4. 1. 1 Nitrogengasregulatedto 140 psi. 4.1.2 Fluofixcolumn orequivalent.'. -@ 4.1.3 100 uL or 250 uL flattipsyringeforsample injection. 4.2 Reagents 4.2.1 Diluteacetonitrimloebilephase,diluteacetonitri1l:e1 withMilli-Qwater. 4.2.2 Milli-Qwater,allwaterusedinthismethod shouldbe Milli-Qwater. 5.0 EQUIPMENT 5.1 VG Trio2000 ElectrosprMaayss Spectrometeorrequivalent. 5.2 ISCO SyringePump 5.3 SpectraphysicAsS300 Autosampler 5.4 100 uL Assembly 5.5 Autovialsor capped centrifugteubes. 6.0 INTERFERENCES 6.1 There areno known interferenceastthistime. 7.0 SAMPLE HANDLING 7.1 Keep theextractedsamples incapped 15 mL centrifugetubesor in capped autovials untilready foranalysis. 0()0058 2 8.0 CALIBRATION AND STANDARDIZATION 8.1 Preparationof CalibrationStandards 8. 1. 1 Seven beefliverstandardsand one blankbeefliverarepreparedduringthe extractiopnrocedure.(SeeAMDT-M-4, section8.0) 8.2 Calibration 8. 2. 1 Run theseven beefliverstandardstwice,startingwith theloweststandard to obtainthestandardcurve. 8. 2.2 Typicallyone standardisrun aftereach 5 to 7 samples.Choose a standard in the same range of concentrationas the samples. 8.3 Storage Conditions for Standards 8.3. 1 Freshstandardsarepreparedwitheach analysisS.tandardsarestoredin coveredplasticentrifugetubesuntiltheanalysison themass spectometeris performed.Samples and standardsareNOT refrigerated. 8.4 Storage Conditions for Beef Liver Homogenstes 8.4. 1 Beef liverhomogenates may be frozenafterpreparation. 9.0 PROCEDURE 9.1 InitialSet-up 9.1.1 Set softwareto"Operateon",Ion Mode ES'. 9.1.2 Record backingpressurein theinstrumentlog. 9.1.3 Fillthesolventcylinderwithmobile phase. 9.1.4 Setthepump to"Run". Settheflowto 1000 uL/rnin.Observes &oplets coming outof thetipof theprobe.The pressureshouldbe at 1700 to 1800 psi. 9.1.5 Check thefused silicattheend of theprobe.Use an eye piecetocheck for chips.The tipshould be flatwithno jagged edges.Ifany chipsarefound cutoffthetipofthesilicwaitha column cutterand pullthesilictahroughto theappropriatelength. 9.1.6 Check yournitrogensupply.Turn on thenitrogen.There shouldbe no nitrogenleakingaround thetipof theprobe.A finernistshouldbe coming out of thetip. 9.1.7 Carefullyguidetheprobeintotheopening.Insertituntilitwon't go any furtherC.onnect thevoltagecableto theprobe. 9.1.8 Go tothe"Editor"page,and setIonizatioMnode toES-,and the appropriatemasses to427 and 499. 9.1.9 Ifitisnotin singleionmc>dego to"Option" and setSIR. 9.1.IOStartAcquisitionA.ssigna filename, MO-DAY-YR + letterR.ecord itin the logbook. 9. 1. 11 Run thebeefliversamples firstr,unningeach standardtwiceatthe beginningof therun..Run a QC check by running one standardafterevery 5 to7 samples. 9.2 Manual Injection 9.2. 1 Draw 150 uL of sample intoa syringeI.njectthesample intotherheodyne injectiopnori.Injectslowly.Record thesample ID inthe logbook. 9.2.2 Turn thevalveto"On". 9.2.3 Wait two minutes,and injecthenextsample. 9.2.4 Record thescannumber foreach simple inthelogbook. 3 oooorj9 9.3 Using the Autosampler 9.3.1 SetupsampletrayA,B,orC. 9.3.2 Record thesamples and theirpositionsin theinstrumentlog book. Up to 17 vialsmay be ineach run. 9.3.3 Set-upthe sampler: 9.3.3.1 Push the sample button 9.3.3.2 Setsample loop size= 100 uL 9.3.3.3 Setinject/sampl=e 2 9.3.3.4 SetCycle time= 0 9.3.3.5 Name thefileL:ivers 9.3.3.6 Idenfifythetrayused 9.3.3.7 Add thesamples to Queue by pressing"Enter" 9.3.3.8 Press"Run" tostart 10.0 VALIDATION 10.1 Quality Control 10. 1.IRun a standardevery5 to7 samples.Ifa significancthange( 50%) in peak heightoccursstoptherun.Only thesamples beforethelastacceptable standardwillbe used.The remainingsamples willbe reanalyzed. 10.2 Precisionand Accuracy 10. 2. 1 See Method ValidationReportnumber AMDT-M-5.0.V 1 10.3 Other Validation Parameters 10.4 Referto Method ValidationReport Number ANMT-M-5.0.Vl 11.0 DATA ANALYSIS 11.1 11.2 Calculations Plotthestandardcurve,usingthemean of thetwo valuesobtainedforeach standard. 11. 2. lread peak heightsor areasforthesamplesfrom theprintoutU.se linear regressiontodeterminethesample concentrations. 11. 2.2 Calculatethemg of FC-95 anion,or otherfluorochen-dcailn thetotalrabbit liver-. mg FC-95 anioninthetotalrabbitliver mg FC-95 anionfrom std.curve gms of liverused foranalysis x Totalmass ofliverg,ms 11.3 11.4 Make a resulttsableand enteritinthestudybook. Printa chromatogram foreach sample,withthepeaks labeledwith thesample or standardDD.Writethestudynumber on theprintouti,nitiadla,te,and put itinthe study folder.Stapleallchromato@rramstogetherand number pages. 4 0000(io i 12.0 ATTACHMENTS None 13.0 REFERENCES 13.1 AMDT-EP- 17 14.0 REVISIONS Revision Numbe Reason for chang!: Revision Date ooooci 5 3M Environmental Laboratory Method Analysis of Fluoride Using the Skalar Segmented Flow Analyzer With Ion SelectiveElectrode Method IdentiricatioNnumber: AMDT-M-8 Adoption Date: RevisionNumber: 0 RevisionDate: None Author: Deb Wright/ CynthiaWeber Appmved By: /o/s/@ps Date QualityAssurance Date Software:IBM MS Word, 6.0 AffectedDocuments: AMDT-EP-26, Analyzer Operationand Maintenanceof theSkalarSegmented Flow oooo(;2 1.0 SCOPE 1. 1 This method isfortheanalysisforfluoridet,hermallyextractedfrom samples using theDohrmann DX2000 (AMDT-M- 1),and collecteidnTISA B foranalysiswithan Ion SelectiveElectrode(ISE).The analysisisperformedusingtheSkalar Segmented Flow Analyzer withISE. 1.2 Samples can be tissuess,erum, biologicamlaterialo,r othermaterialsextractedon theDohrmann. 2.0 KEYWORDS 2.1 Skalar,segmented flow,fluoride. 3.0 PRECAUTIONS 3.1 Followstandarldaboratorsyafetypractices. 4.0 SUPPLIES AND MATERIALS 4.1 Supplies 4.1.1 Sample cups,4 mL plasticups with caps 4.1.2 Autopipets,oxfordorequivalentwithplasncnps 4.1.3 Polypropylenevolumetricflasks,100 ML 4.1.4 Cartridgecomponents,referto theSkalarMethods forcomponents and part numbers. 4.1.5 Sample prefilterEsv,ergreen 4.2 Reagents 4.2.1 Brij35, 30% S.F.A.S.Detergent 4.2.2 TISAB 11buffersolutionP:urchaseTISAB 11from Orion.To I liteorf TISAB 11add 2.5mL or 100'pprhfluoridesolutionand I mL Brij. 4.2.3 Sampler rinsingsolutionD:iluteTISAB 111:1 withMilli-Qwater. 4.2.4 NitricacidsolutiofnordecontaminationI, N (labgrade):Slowly add 64 mLs concentratenditriaccid(HN03) to250 mLs of Milli-Qwater.Bring thevolume up toI L withMilli-Qwater. 4.3 Standards 4.3.1 Stock solution1,00 ppm F:purchasedfrom Orion. 4.3.2 Intermediatestandard,10 ppm: Dilute10 n-tlsofstocksolutionto 100 mLs with Milli-Qwater.Use polypropylenevolumetricflasks. 4.3.3 Working standard:Make up thefollowingworking standardsby adding the volumes of intermediatoer stockstandardindicatedon thetableu,sing oxfordor pumpmate pipeest,o50 nilsofTISAB and dilutingto 100 mLs withMilli-Qwater. Working Standard mLs of Stock Standard 0.015 ppm - 0.03 ppm - 0.06 ppm - 0.09 Ppm - 0.12 ppm - 0.15 ppm - 0.3 ppm 0.3 mLs of IntermediateStandard 0.15 ?F 3 0.6 0.9 1.2 1.5 - 0.6 ppm 0.6 2 1.2 ppm 1.2 L5 ppm 1.5 5.0 E!QUIPMENT 5.1 SkalarSegmented Flow Auto Analyzer Sans'" System equipped with ISE 6.0 INTERFERENCFS 6.1 High concentrationosfalkalinitcyh,loridep,hosphate,sulfatoer ironcan cause interferences. 7.0 SAMPLE HANDLING 7.1 Samples should be storedin polyethylenebottlesS.amples shouldbe analyzed within30 days. 8.0 CALTBRATION AND STANDARDIZATION 8.1 Preparation of CalibrationStandards 8. 1.1 Preparecalibratiosntandardsasinsection4.3. 8.2 Calibration 8.2. 1 The standardsareanalyzedat thebeginningof therun. 8.3 Storage Conditions for Standards 8.3. 1 Standardsarestoredincapped polypropylenevolumetricflasks.New standardsarepreparedat a minimum of every sixmonths, or as necessary. 9.0 PROCEDURE 9.1 Start Up Procedure 9.1.1 Clamp down thepumpdecks, airbarsand sampler-pump tubing. 9.1.2 Put thefluoridelectrodeisn theelectrodechamber. 9.1.3 Turn on thepower of thesampler,pumps, offsetpotentiometerand heafing bath. 9.1.4 Put thereagent-lineisn theappropriatebottles. 9.1.5 Turn on theinterfacec,omputer,displayand printerM.ake sure you turn on the interfacebeforethe computer. 9.1.6 Let thesystem stabilizfeorapproximately30 minutes. 9.2 Startinga Run 9.2.1 Createa sample tableby selectinFgILES, TABLE, and CREATE, typein thename of thefilea,nd pressENTER. 9.2.2 Printthesample table,insertedin thesystem tableby pushing ESC, PRINT, GROUP 1.This willprinttheentirerun. 9.2.3 Dialthesampler settingtso theappropriatneumber of samples,number of seconds forsample wash, and number of seconds forthesample. 9.2.4 Fillthesample traywith thestandards,samples,washes and driftsI.W and FW/RUNOUT cups on thesamplerdo not need tobe filled. 9.2.5 Set the baseline. 3 ()00064 9.2.S. ISelectGRAPHICS, REAL TIME. Ifyou cannotgetreal-timey,ou may be in theData Handling Panel.Switch totheAnalysisPanelby selectinCgONTROL PANEL and pushingF7. 9. 2.5. 2 Use thesmallscrewdriverfortheoffsetpotentiometertosetthe baseline.Adjustthebaselineuntilitisapproximately3/4 inchfrom thebottom of thescreen. 9. 2.5. 3 Check thehigheststandardand adjustthegain,ifnecessary,with theinterfacsecrew #3. 9.2.6 Go toCONTROL PANEL, and toanalysispanel.Deselecttheanalysisthat willnotbe run.(Selectordeselectanalysisby pressingENTER.) PressTab toreturntotheAnalysisPanel. 9.2.7 Press thespacebarto bringup thelocalmenu. 9.2.8 SelectSTART tostartheanalysis. 9.2.9 Type yourID (initialtsh)e,sampletablewhiphyou createdunder9.2.1(or pressENTER forchoices)c,hoose runningwith orwithoutthesystem table and selectSTART ANALYSIS. 9. 2. 10 Afterstartintghesoftware,starthesampler.Make surethatthesampler is settotherightnumber of sarnplesand thatthesample/wash/airtimes are OK. 9.2.1 IS electGRAPHICS, REAL TIME toview theprogressof theanalysis. 9.3 Loading and Printingthe Data-File 9.3.1 Go to CONTROL PANEL, pressthespacebartobringup thelocalmenu and selectLOAD. SelectAUTOCALCULATION and enterthefilename(or highlighthefiletobe printedand pressENTER). 9.3.2 To view thecalibratiocnurve,go toGRAPHICS, CALIBRATION CURVE. 9.3.3 To printhehighleveclurve,pushPRINT SCREEN. 9.3.4 To printthelow levelscreen,push ESC to getout of graphics.Select SETTINGS. Change themax y valuetoapproximately900.Go toCAL CURVE and pressESC, and Enter.PressPRINT SCREEN. 9.3.5 Return toSETNNOS and change themax valueback to4095, go toEDIT, pressENTER and PRINT SCREEN toprintsample peaks. 9.3.6 To printtheresultsgo toCONTROL PANEL, SPACEBAR, OUTPUT, OUTPUT. SelectPRINTER fortheEpson or PRN fortheLaser. 9.4 Shutdown 9.4.1 Put allthereagent-liniensMilli-Qwater. 9.4.2 Let thesystem rinseforapproximately30 minutes. 9.4.3 Afterthesystem hasrinsedcompletely,turnoffthesampler,pump and offsetpotentiometerT.urn offtheheatingbathon weekends. Leave liquidin the lines. 9.4.4 Take theelectrodeoutand soak in 100 ppm F overnight. 9.4.5 Release thepump-decks, airbarsand samplerPLIMP-tubing. 9.4.6 SelectFILES, pressALT F and selectQUIT toexittheprogram. 9.4.7 On Friday,turnoffthecomputer,displayand interfacfeortheweekend. 10.0 VALIDATION 10.1 Quality Control 10. 1.1 Run a standard(mid tohighconcentratione)very 10 samples.Ifa significancthange in peak heightoccurs,only thesamples beforethelast acceptablestandardwillbe used.The remainingsamples willbe reanalyzed. 4 ()00065 10.2 Precisionand Accuracy 10.7. 1See Method ValidationReport number AMDT-M-8.0.V 1 10.3 Other ValidationParameters 10.4 RefertoMethod ValidatioRneportNumber AMDT-M-8.0.V I 11.0 12ATA ANALYSTS 11.1 11.2 11.3 11.4 Calculations 11. 1. 1The standardcurve isplottedby theSkalarsoftware. 11. 1.2 Allcalculationasredone by theSkalarsoftware.r2 shouldbe 0.995or better. Preparespreadsheetsto summarize data.Includesample volume, weightsused etc. Write thestudynumber on theprintoutsi,nitiadla,tetheprintouta,nd bind together withallpackage documents and placein thestudyfolderM.ake a copy of the summary sheetand tapeintothestudynotebook.Back up alldataand spreadsheets onto studydisk and backup disks. ElectroniDcata 11.4.1GLP studiesE:lectronidcataiscopiedontotheStudyfloppydiskforeach study,and alsodataiscopiedontoa floppydiskthatisstoredinthelab. 11.4.2OtherstudiesA:lldataiscopiedontoa floppydiskthatisstoredinthelab. 12.0 ATTA(:HMENTS None 13.0 REFERENCES 13.1 AMDT-M-1, ThermalExtractioonfFluoridbey Means ofa ModifiedDohr-rnann DX2000 OrganicHalideAnalyzer-Liver 13.2 SkalarMethods,#335,SkalarMethodsManual 13.3 ANIDT-EP-26,OperationandMaintenanceoftheSkalarSegmentedFlow Analyzer 14.0 REVISIONS Revision Numbe Reason forchange Revision Date 0000,66 5 3M EnvironmentalLaboratory Method Thermal ExtractionofFluorideby Means of a ModifiedDohrmann DX2000 OrganicHalideAnalyzer- Serum Method IdentificatiNounmber: AMDT-M-14 RevisionNumber: 0 AdoptionDate: RevisionDate: None Author. Rich Youngblom Approved by: roup Leader Date p@L QualityAssurance Date Software: MS Word 5.1a AffectedDocuments: AMDT-M-2 FluorideMeasurement by Means of an Orion EA940 Expandable Ion Analyzer AMDT-EP-3 RoutineMaintenance ofa Modified Dohrmann DX2000 OrganicHalideAnalyzer 1.F- Le2 "129)q6 000067 1.0 SCOPE. APPLICABLE COMPOUNDS, AND MATRICES 1.1Scope: Thismethod isfortheoperationof a Dohrmann DX2000 when itisused toextract fluoridferom variousmatrices.The fluoridiestypicallcyollecteidnTISAB solutiofnoranalysis withan ionselectiveelectrode. 1.2Applicable Compounds: Fluorochernicalosrotherfluorinatecdompounds. 1.3Matrices:Biologicaflluidsp,articularsleyrum. 2.0 KEYWORDS 2.1Fluoridef,luorine,xtractiopny,rolysisi,onizatioino,nselectiveelectrodeD,ohrmann, halide, DX2000, fluorocheniicals. 3.0 PRECAUTIONS 3.1 Glasswareand exhaustgasescan be extremelyhot. 3.2Glasswareisfragileb,roken glassmay causeinjuries. 3.3 Pressurizedgases,propercompressed gas handlingpracticersequired. 3.4Solventbasedsamplesmay flashm,ay need toallowthem todry down beforestartinrgun. 3.5Potentiabliohazardsdue tothebiologicamlatrices.Use appropriatpeersonalprotective equipment. 4.0 SUPPLIES AND MATERIALS 4.1Compressed Oxygen, Hydrocarbon free,regulatedto 30 PSI. 4.2Compressed Helium, High PurityGrade,regulatedto.45 PSI. 4.3Quartzglasssample boatwithTeflonTMtubing,Dohrmann 890-097orequivalent. 4.4Quartzglasscombustiontube,RelianceGlass G-9405-012 or equivalent. 4.5Orion940999 TotalIonicStrengthAdjustmentBuffer(TISAB Il)or equivalent. 4.6Sample collectiovnialsH,DPE. 4.7Milh-QTM water 4.8Polystyrenepipettes. 4.9ActivatedCharcoal,E. Merck 2005 or equivalent. 4.10Han-@ltonSyringeor equivalent. 4.11Miscellaneouslaboratorgylassware 5.0 EQUIPMENT 5.1Rosemount Dohrmann DX2000 OrganicHalideAnalyzer,modifiedforfluoridextraction. 5.2MM compatible386 or 486 computer. 5.3DX2000 softwarev,ersion1.00,modifiedforfluorideextraction. 5.4Excel Spreadsheet,version5.0or greater 6.0 INTERFERENCES 6.1 Sample sizeislimitedtoapproximately100 41.Thismay varyfrom matrixto matrix. 2 000068 7.0 SAMPLE T4ANDLING 7.1Samples aretobe handledwithplastipcipettesA. new pipetteistobe used foreach sample. 8.0 CALIBRATION AND STANDARDIZATION 8.1PreparatioonfCalibratioSntandards 8.1.T1hestandarrdesquirefdoreachprojecwtilnleedtobeapproprifaotrethaitndividuparloject. Refertoprotocolforthatproject. 8.1.2Typically50-500 ppm FC-95 inmethanolstandardsareused. 8.1.3For rabbitserwn studiesu,sebeefserum asthematrix. 8.2 Calibration- Overview The normal calibratioinsthefluoridceurve(AMDT-M-2). However, ifan optionalspikedserum curveisrequiredtheprocedurelistebdelow isused. 8.2.1A calibratiocnurvefortheDX2000 isgeneratedby spikingsampleswithknown standards and combusting them usingthesame methods and matrixtypeasthesamplestobe tested, 8.2.2Typicallyt,hreereplicateosf eachstandardand fiveconcentrationosf standardswillbe spiked. 8.2.3Standardcurvewillbe plotteads Mass SpikedF (ug)on thex-axisand StandardMass RecoveredF (ug)on they-axis.Generatea regressiocnurveand calculatteheequationfortheline and ther2 value. 8.2.4Mass SpikedF (ug)= (Amount spikedinrnL)x (Conc.ofstandardinppm) x (0.6004)* *FC-95 is60.04% F therefor0e.6004isthefactorused to convertFC-95 toF 8.2.5StandardMass RecoveredF (ug)= (TISAB volumeinmL) x (Orionreadinginppm) 8.3Calibration-Procedure 8.3.1StartUp 8.3.1.1Run 2 ormore CleanCycleswhen startinignsu=ent eachday. More cleancyclesmay be usediftheprevioussamplescontainedhighconcentrationosffluoride. 8.3.2Blanks 8.3.2.1Preparesample usingthesame methods and typeof matrixas thetestsample. 8.3.2.2For rabbitstudiesu,sebeefserum asthematrix. 8.3.2.3Putserum blankin Dohrmann boat.Combust sample asdescribedinsection9.0and analyzesampleaccordingtomethod AMDT-M-2 fortheionselectiveelectrodaenalysis. 8.3.2.4For rabbitstudiest,hemeterreadingfora blanksample shouldbe 0.03ppm or lower beforeproceedingwith thecalibratioBnu.m samplesuntilthislin-@itsreached,or untilinthe judgement of theoperatorthereadingisstablewithrespecto1-@storicraeladings(previous48 hours). 8.3.2.5For non-rabbitstudiest,heblankreadin2sshouldreacha predeterminedionconcentration beforeproceeding@kiththecalibration. 8.3.2.6Itmay be necessarytomix approximately50 mg of charcoalwiththesampletoaid combustion. 3 oooo(;9 8.3.3StandardCurve 8.3.3.I1fbeefserwnisfrozent,hawatleasetnoughtocomplettehestandarcdurveanalysifsorthe day (=30 mL). 8.3.3.2PipettelOOgLof beefserum intoDohrmann sampleboat. 8.3.3.3StartwiththeloweststandarcdoncentratioUns.ing aHamiltonsyringee,jecta fixed quantityofthestandardon orinthematrix.For rabbitstudiesu,se 4 uL of standardand ejectiton or inthebeef senim. 8.3.3.4At least3 replicatesshouldbe used forthelowest standardconcentrationm;ore replicates may be used atthediscretioonf theanalyst. 8.3.3.5Combust thesample as describedin section9.3and analyzeaccordingto AMDT-M-2. 8.3.3.6Run all15 standards.Ifone replicatiessignificantldyifferenftrom theothertwo replicatersu,n anothersample forthatstandard.Indicateindatathatthenew replicatreeplacesthe oldreplicataend thatthenew replicatweillbe used to calculatteheregressioncurve. 8.3.3.7NVhen allstandardshave been run,calculattehe r2. r2 must be at least0.95.Ifitisnot at least0.95,consultwith supervisor. 8.3.3.8A new standardcurveshouldbe run when thecombustion tube or sample matrix is changed. New standardcurvemay alsobe run atthediscretioonf theanalyst. 8.4 StorageConditionsforStandards 8.4.1Storagereqtdrementsforstandardsaredependenton theindividualstandardsused. TypicaUy, standardsarestoredatroom temperatureinplasticscrew topbottles. 8.4.2New FC-95 standardsshouldbe preparedatleastonce a month. 9.0 PROCEDURES 9.1TypicalOperating Conditions: 9.1.1Combustion tubetemperature= 950'C. 9.1.2Oxygen and Helium flow= 50 cc/minute. 9.1.3Vaporization/Dryingtime= 240 seconds. 9.1.4Bake time = 300 seconds. 9.2 StartUp Procedure: 9.2.1Iftheprogram isnot starteds,tartheEOX program on thePC. 9.2.2Open the SYSTEM SETUP window. 9.2.3Put the furnacemodule and thecellin theR.EADY mode. 9.2.4Close the SYSTEM SETUP window. 9.2.5When theoven has reachedtheR-EADY temperature,run theCLEAN BOAT program foundin the CELL CHECK menu. 9.2.6 See AMDT-EP-3 fordetailosf theDohrmann software. 9.3Sample ExtractionProcedure: 9.3.1Open theSAMPLE HATCH and pipetteIOOGL of sample intothe BOAT, Itmay be necessarytomix approximately50 mg of charcoalwiththesample toaidcombustion. Ifthisis done,charcoalshould alsobe mixed 'Inwhile establishintghebaselineand when generatingthe standardcurve. 9.3.2 Close SAMPLE HATCH. 4 0()0070 9.3.3 Add appropriatevolume of TISAB solutionor 1:1 TISAB:Milli-QTM watermixture toa labeledsample collectiovnia].Typically0.6 mL to 15 mL areused. For rabbitstudiesu,se 1.0or 2.0 mL of 1:1 TISAB: Milli-QTMwater mixture. 9.3.4 Placethevialso thatthetipof theCOMBUSTION TUBE isinthe TISAB atleast0.25 inches.Gases releasedduringpyrolysismust bubble throughtheTISAB. 9.3.5 Run theEOX-WATER program found intheRLN menu. 9.3.6 When theEOX program isfinishedr,emove thecollectiovnia]from thecombustion tube. 9.3.7 IfundilutedTISAB was used tocollecthesample,add an equalvolume of Milli-QTMwater totheTISAB tomake 1:1 TISAB: Milli-QTM. 9.3.8Rinse theend ofthe combustion tubewithMilli-QTmwater and wipe witha KIMWIPE to remove any TISAB remainingon thetube. 9.3.9 Open thesample hatchand remove any remainingash from the boat. Ash can be removed with a cottonfippedapplicatorand/orvacuumed out. Itmay be necessaryto scrapparticleosffthe bottom witha spatulaor othersimilardevice.A drop ofMilli-QT"water may be added tothe boattoaidintheClean Cycle, 9.3.10 Close thehatch. 9.3.11 Run the CLEAN BOAT program. 9.3.12 Sample isreadyforanalysisby ion selectiveelectrode(AMDT-M-2). 9.4 Sample Calculations 9.4.1 Use thestandardcurve to calculattehesample value. 9.4.2Sample Mass Recovered F (ug)= (TISAB volinmL) x (Orionreadinpi-npl2m - intercel2t) (Slope) 10.0 VALIDATION 10.1QualityControl 10.1.D1ailyStartUp Check Samples:Oncethestandarcdurveisestablisheadc,hdayof analysisisstartedby analyzingQC samples. The QC samples aretobe the same as thelowest concentrationspikedsamples used to generatethestandardcurve. Each concentrationmust be done intriplicatuenlessthefirsttwo replicateasrewit@dn20% of thestandardcurve,then a third replicatiesnot necessary. 10.2 Precisionand Accuracy: See method development analysisand sample analysisin FluorideNotebooks 2,3,and 5. Precisionand accuracyvarieswhen analyzingsamples ofdifferent matricesand differenrteferencecompounds. 10.3 Other ValidationParameters: NA 11.0 DATA ANALYSIS 11.1 Calculations 11.1.1 For thestandardcurve,use regressionanalysisin Excel,version5.0 or greater. 11.1.2To calculattehe fluoridceontractioninthe sample,see method AMDT-M-2. ,5 000071 11.2AnalyzingtheData 11-2.1r2mustbeatleas0t.95orgreate"rO.utliermsa"y beexcludeidftwoofthethrereeplicates arewithin20% of each otherand theoutlierisgreaterthan200% of theaverage of thosetwo or lessthan 50% of theaverageof thosetwo. Any such outliershould be pointedout in thedataand notedin theFinalReport alongwith thereasonitwas consideredan outlier. 12.0 ATTACHMENTS None 13.0 REFERENCES 13.1Rosemount Dohnnann DX2000 Organic HalideAnalyzerOperator'sManual (Manual 915349,revisionB, December 1993) 13.2 AMDT-M-2 FluorideMeasurement by Means of an Orion EA940 Expandable Ion Analyzer 13.3AMDT-EP-3 Routine Maintenanceof a Modified DohrTnann DX2000 Organic Halide Analyzer 14.0 REVISIONS Revision Numbe Reason forChange Revision Date 6 000072 9.3 QualityAssurance Unit Statement 000073 AttachmentD GLP Study QualityAssurance Statement Study Title: Single-dose Intravenous Pharmacokinetic Study of T-6067 in Rabbits Study Number- AMDT- 120694.1 Name ofAuditor KariRambo Thisstudyhas been inspectedby theQualityAssurance Unit asindicatedin thefollowingtable. The findingswere reportedto thestudydirectorand management. InspectionDates EM T-Q 11-09-95 11-13-95 Phase FinalReport Date InspectionReported to M agcment StudyDirec 11-13-95 11-13-95 QAU Auditor Date 000074 9.4 Key Personnel Involvedin the Study 00007S 3M Environmental Laboratory Key Personnel Thermalextractiofnollowebdy analysiussingOrionionanalyzer: Jim Johnson Deb Wright Rich Youngblom Deann Plununer Analysisofliverextractussingelectrospramyass spectrometry: Jim Johnson Dave Christenson Thermal extractionfollowedby analysisusingSkalarsegmented flow analyzerwith ionselectiveelectrode: Jim Johnson Deb Wright Rich Youngbiom Deann Plununer Documentation and Reporting: Jim Johnson Rich Youngblom QualityAssurance Unit: Gale Van Buskirk CynthiaWeber KariRambo 0()0076 9.11 Data 000077 9.11.1Summary and raw data;ug F -inwhole liveras determinedby thermalextractiofnollowedby analysisusing Orion ion analyzer. 000078 Summary of Combustion Data - Liver AMDT-120694.1, HWI 6329-151 As Referenced inFinalReport section6.0DATA ANALYSIS Totalug FluorideinWhole Liver Mean per Dose Group ug ControlGroup 19.7 4.0mg/kg dose (T6067) 42.9 16 mg/kg dose (T6067) 66.4 24 mg/kg dose (T6067) 54.4 000079 RPT1 51L.XLS L13492 PK ID liverbik 1 liverblk2 liverblk3 liverspike 1 liverspike2 liverspike 3 F52762 - 1 F52762 - 2 F52762 - 3 F52763 - 1 F52763 - 2 F52763 - 3 F52768-1 F52768-2 F52768-3 F52770-1 F52770-2 F52770-3 liverspike4 liverspike5 Liverblank % rcvry 91% 94% 84% 96% 87% Actual ppm Finliver (W/W) 0.501 0.286 0.262 1.08 1.00 0.985 0.384 0.245 0.242 0.732 0.483 0.524 0.727 0.794 0.803 0.806 0.775 0.776 1.07 1.19 0.228 Average ppm Finliver (W/W) 0.290 0.580 0.775 0.786 liver burned (grams) 0.145 0.150 0.136 0.127 0.143 0.129 0.120 0.136 0.142 0.111 0.146 0.133 0.123 0.133 0.115 0.130 0.134 0.142 0.136 0.111 0.123 Whole liver weight (grams) 67.7 67.7 67.7 73.9 73.9 73.9 85.7 85.7 85.7 89.2 69.2 69.2 TotalFinwhole liver (@tg) 19.7 42.9 66.4 54.4 Dosage (mg/kg) 0.0 4.0 16 24 Page 1 000080 9.11.2Summary and raw data;ppm F- inserum as determinedby thermalextractiofnollowedby analysisusing Orion ion analyzer. oooosl HWI 6329-151 AMDT 120694.1 Dohrmann Serum Analysis AnalysisDates:07/27/95-07/31/95 All serum samples were thermallyextractedby a modifiedDohrmann DX2000 OrganicHalideAnalyzerand collectedina 1:1milliQ waterand TISAB solution.The samples were measured on an Orion EA940 expandableion analyzer.The Dohnnann was calibrateudsing 34ppm, 40ppm, 62ppm, 100ppm, 124ppm, 250ppm, and 500ppm FC-95 standards. The Orion was calibratebdy dir-ecmteasurrment withno blank correction using0.05ppm, O.Ippm, 0.5ppm, l.Oppm and 1.5ppm F standards.The slope,intercepta,nd correlatiownere recordedinthe appropriatelogbook. A summary tableisincluded,showing theppm F- ineach sample (seepage 2). 'Me valueof "ND" has been enteredforany sample withan Orion readingof below 0.05. An initiaclalibratiocnurvewithstandard deviation,%RSD, R' valueand equationofthelineison pages 3 and 4. Pages 5 and 6 show theexcelspreadsheetthatwas generatedwhen the samples were being analyzed. Pages 7 and 8 show thesame spreadsheet with "ND" insertedwhere the Orion readingisbelow 0.05. e@-h. Lt4l"*- a6" qo LL PageI ofB II 0()0082 Ll3492 PK Dosage: Omg/kg Dosage: 4 mg/kg Dosage: 16 mg/kg Dosage: 4fA)-mg/kg WL STUDY# 6329-151 SERUM HWI 6329-151 Fluorideconcentratioinnrabbitserum (ppm F-) Sample F52762 F52763 F52768 F52770 2.0hour 4.0hour 6.0 hour 8.0hour 12.0hour 24.0hour 48.0hour 1.25 5.84 ND 3.56 2.98 1.22 NO 4.09 3.25 2.27 1.89 2.01 1.41 ND 14.9 8.05 4.39 2.87 2.72 ND ND 41.0 19.4 12.7 8.08 4.40 ND ND 0()0083 151CHART.XLS Chart 12 10.00 4 mg/kg 0 U. cr0;_ 0 0 m 0 1.00 11 i- i i 0 5 10 15 20 time thourspost dose) c .rl .;@b tp Page 1 151CHART. XLS Chart 11 100.0 16 mg/kg F@@ LL 0 cc; 10.0 00 a 1.0 - 0ome@ 0 2 4 6 8 10 0 time(hourpsostdose) c xcli -I-- Page 1 151 CHART.XLS Chart 10 100.0 - - LL tc; 10.0 0 u 24 mg/kg 0 1.0 - 0L C) 2 4 6 8 10 a time (hourspostdose) (Z x c@- b Page 1 ....................................... 34.ppm.l 34-ppm-2 34-ppm-3 40-ppm-1 40-ppm-2 40-ppm-3 40-ppm-4 62-ppm-1 62-ppm-2 62-ppm-3 100-ppm-1 100-ppm-2 100-ppm-3 124-ppm-1 124-ppm-2 124-ppm-3 250-ppm-1 250-ppm-2 250-ppm-3 500-ppm-1 500-ppm-2 500-ppm-3 0.07175 0.1 0.05614 0.1 0.06462 0.1 0.08668 0.1 0.06728 0.1 0.05939 0.1 0.06385 0.1 0.07291 0.1 0.0753 0.1 0.07839 0.1 0.0902 0.1 0.1026 0.1 0.1126 0.1 0.1371 0.1 0.1451 0.1 0.1617 0.1 0.3217 0.1 O@2447 0.1 0.3078 0.1 0.4438 0.1 0.4584 0.1 0.4888 0.1 NORMAN SERUM CURVE I ML'@F-Cas:,: 2.0 0.004 34 2.0 0.004 34 2.0 0.004 34 2.0 0.004 40 2.0 0.004 40 2.0 0.004 40 2.0 0.004 40 2.0 O@004 62 2.0 0.004 62 2.0 0.004 62 2.0 0.004 100 2.0 0.004 100 2.0 0.004 100 2.0 0.004 124 2.0 0.004 124 2.0 0.004 124 2.0 0,004 250 2.0 0.004 250 2.0 0.004 250 2.0 0.004 500 2.0 0.004 500 2.0 0.004 500 Acitue ........... .... 176% 138% 158% 180% 140% 124% 133% 98% 101% 105% 75% 85% 94% 92% 97% 109% 107% 82% 103% 74% 76% 81% 1.4350 1.1228 1.2924 1,7336 1.3456 1,1878 1.2770 1.4582 1.5060 1.5678 1.8040 2.0520 2.2520 2.7420 2.9020 3.2340 6.4340 4.8940 6.1560 8.8760 9,1680 9.7760 to ................. 0.0817 O@0817 0.0817 0.0961 0.0961 0.0961 0.0961 0.1489 0.1489 0.1489 0.2402 0.2402 0.2402 0.2978 0.2978 0.2978 0.6004 0.6004 0.1435 0.11228 0.12924 0.17336 0.13456 0.11878 0.1277 0.14582 0.1506 0.15678 0.1804 0.2052 0.2252 0.2742 0.2902 0.3234 0.6434 0.4894 0.6004 1.2008 1,2008 1.2008 0.6156 0.8676 0.9168 0.9776 . ....... STOEV: %RSD: 0.015629 12 STDEV: %RSD: 0.024057 17 STOEV: %RSD: STOEV: %RSD: STDEV: %RSD: STOEV: %RSD: STOEV: %RSD: 0.005495 3.6 0.022443 II 0.025096 8.5 0.082072 14 0.045915 5.0 SERUM CURVE I NORMAN (07126196) 0.9-- 0.5-- 0.7-0.6 LU 0 A 0.4 0.3.- 0.2 0.1 0 O.DODD O@2000 y - 0.743x + 0.064 Rz 0.9722 0.4000 0.6000 0.8000 MASS SPIKED (ug) 1.0000 1.2000 1.4000 ooOos"@, -r.t w- it)-@gs 0.9 0.8 0,7 0.6 0.5 0.4 0.3 0.2 0.1 o O.DDOO SERUM CURVE I NORMAN (07125195) y 0.743x+ 0,064 R 2 0.9722 0.20DO 0.4000 0.6000 0.8000 MASS SPIKED (ug) 1.0000 1.2DOO STUDY# 6329-151 SERUM Sample ID BLANK-1 BLANK-2 BLANK-3 BLANK-4 BLANK-5 BLANK-6 62-PPM-1 62-PPM-2 62-PPM-3 62-PPM-4 62-PPM-5 62-PPM-6 250-PPM-1 250-PPM-2 250-PPM-3 F52762-2.OHR F52763-2.OHR F52768-2.OHR F52770-2.OHR F52762-4.OHR F52763-4,OHR F52768-4,OHR F52770-4.OHR 62-PPM-1 62-PPM-2 250-PPM-1 250-PPM-2 62-PPM-3 SERUM BLANK SERUM BLANK SERUM BLANK SERUM BLANK SERUM BLANK SPIKE 62-1 SPIKE 62-2 SPIKE 62-3 SPIKE 250-1 SPIKE 250-2 SPIKE 250-3 SERUM BLANK SERUM BLANK F52762-6HR F52763-6HR F52768-6HR F52770-6HR F52762-SHR F52763-8HR F52768-8HR Actual reading (pem F-) 0.0507 0.0355 0.03" 0.0308 0.0353 0.0290 0.0856 0.110 0.0835 0,0778 0.0746 0.0718 0.190 0.213 0.223 0.0626 0.205 0.746 2.05 0.292 0.163 0.402 0.971 0.189 0.135 0.221 0.246 0.130 0.0458 0.0352 0.0340 0.0358 0.0350 0.0628 0.0689 0.0715 0.155 0.261 0.217 0.0936 0.0289 0.0222 0.114 0.220 0.636 0.178 0.094 0.144 Sample Qty (mL org) 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 TISAB finalvat (mL) 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 mL FC95 Conc.FC95 spiked solution (ppm) 0.004 62 0.004 62 0.004 62 0.004 62 0.004 62 0.004 62 0.004 250 0.004 250 0.004 250 0.004 62 0.004 62 0.004 250 0.004 250 0,004 62 0.004 62 0.004 62 0.004 62 0.004 250 0.004 250 0.004 250 % Actual recovery ppm F- (ug/ug) insample 1.01 0.710 0.689 0.617 0.707 0.581 115% 1.71 148% 2.20 112% 1.67 104% 1.56 100% 1.49 96% 1.44 63% 3.80 71% 4.27 74% 4.47 1.25 4.09 14.9 41.0 5.84 3.25 8.05 19.4 254% 3.78 181% 2.70 74% 4.42 82% 4.92 174% 2.60 0.916 0.703 0.680 0.715 0.700 84% 1.26 92% 1.38 96% 1.43 52% 3.10 87% 5.22 72% 4.34 1.87 0.577 0.445 2.27 4.39 12.7 3.56 1.89 2.87 Mass spiked (ugF-) 0.15 0.15 0.15 0.15 0.15 0.15 0.60 0.60 0.60 0.15 0.15 0.60 0.60 0.15 0.15 0.15 0.15 0.60 0.60 0.60 Mass recovered (ugF-) 0.101 0.0710 0.0669 0.0617 0.0707 0.0581 0.171 0.220 0.167 0.156 0.149 0.144 0.380 0.427 0.447 0.125 0.409 1.49 4.10 o.5a4 0.325 0.805 1.94 0.378 0.270 0.442 0.492 o@260 0.0916 0.0703 0.0680 0.0715 0.0700 0.126 0.138 0.143 0.310 0.522 0.434 0.187 0.0577 0.0445 0.227 0.439 1.27 0.356 0.189 0.287 0()008@) 4 STUDY# 6329-151 SERUM Sample ID F52770-8HR F52762-12HR F52763-12HR SPIKE 62-1 SPIKE 250-1 SPIKE 250-2 F52768-12HR F52770-12HR F52762-24HR F52763-24HR F52768-24HR F52770-24HR F52762-48HR F52763-48HR F52768-48HR F52770-48HR BLANK-1 BLANK-2 SPIKE 62-1 SPIKE 62-2 SPIKE250-1 SPIKE250-2 SPIKE250-3 Actual reading (ppm F-) 0.404 0.149 0.101 0.0920 0.187 0.225 0.136 0.220 0.0609 0.0704 0.0442 0.0387 0.0282 0.0264 0.0260 0.0357 0.0830 0.0314 0.0635 0.0644 0.157 0.239 0.240 Sample oty (mL org) 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 TISAB finavlol (mL) 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 mL FC95 Conc. FC95 spiked soloon (ppm) 0.004 62 0.004 250 0.004 250 0.004 62 0,.004 62 0.004 250 0.004 250 0.004 250 % Actual recovery ppm F- (ug/ug)_in Sample 8.08 2.98 2.01 124% 1.84 62% 3.74 75% 4.51 2.72 4.40 1.22 1.41 0.883 0.775 0.564 0.527 0.520 0.715 1.66 0.627 85% 1.27 86% 1.29 52% 3.15 80% 4.78 80% 4.80 Mass spiked (ugF-) 0.15 0.60 0.60 0.15 0.15 0.60 0.60 0.60 Mass recovered (ugF-) 0.808 0.298 0.201 0.184 0.374 0.451 0.272 0.440 0.122 0.141 0.0883 0.0775 0.0564 0.0527 0.0520 0.0715 0.166 0.0627 0.127 0.129 0.315 0.478 0.480 0()00.90 -r.E. PW-L9 @ -4" STUDY# 6329-151 SERUM Sample ID BLANK-1 BLANK-2 BLANK-3 BLANK-4 BLANK-5 BLANK-6 62-PPM-1 62-PPM-2 62-PPM-3 62-PPM-4 62-PPM-5 62-PPM-6 250-PPM-1 250-PPM-2 250-PPM-3 F52762-2.OHR F52763-2.OHR F52768-2.OHR F52770-2.OHR F52762-4.OHR F52763-4.OHR F52768-4.OHR F52770-4.OHR 62-PPM-1 62-PPM-2 250-PPM-1 250-PPM-2 62-PPM-3 SERUM BLANK SERUM BLANK SERUM BLANK SERUM BLANK SERUM BLANK SPIKE 62-1 SPIKE 62-2 SPIKE 62-3 SPIKE 250-1 SPIKE 250-2 SPIKE 250-3 SERUM BLANK SERUM BLANK F52762-6HR F52763-6HR F52768-6HR F52770-6HR F52762-SHR F52763-8HR F52768-8HR Actual reading (ppm F-) 0.0507 0.0355 0.0344 0.0308 0.0353 0.0290 0.0856 0.110 0.0835 0.0778 0.0746 0.0718 0.190 0.213 0.223 0.0626 0.205 0.746 2.05 0.292 0.163 0.402 0.971 0.189 0.135 0.221 0.246 0.130 0.0458 0.0352 0.0340 0.03SS 0.0350 0.0628 0.0689 0.0715 0.155 0.261 0.217 0.0936 0.0289 0.0222 0.114 0.220 0.636 0.178 0.094 0.144 Sample Qty (mL org) 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 TISAB finalvol (mL) 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 mL FC95 spiked Cone. FC95 solution (ppm) 0.004 62 0.004 62 0.004 62 0.004 62 0.004 62 0.004 62 0.004 250 0.004 250 0.004 250 0.004 62 0.004 62 0.004 250 0.004 250 0.004 62 0.004 62 0.004 62 0.004 62 0.004 250 0.004 250 0.004 250 % recovery (ug/ug) 115% 148% 112% 104% 100% 96% 63% 71% 74% 254% 181% 74% 82% 174% 84% 92% 96% 52% 87% 72% Actual ppm Finsample 1.01 ND ND ND ND ND 1.71 2.20 1.67 1.56 1.49 1.44 3.80 4.27 4.47 1.25 4.09 14.9 41.0 5.84 3.25 8.05 19.4 3.78 2.70 4.42 4.92 2.60 ND ND ND ND NO 1.26 1.38 1.43 3.10 5.22 4.34 1.87 ND ND 2.27 4.39 12.7 3.56 1.89 2.87 Mass spiked (ugF-) 0.15 0.15 0.15 0.15 0.15 0.15 0.60 0.60 0.60 0.15 0.15 0.60 0.60 0.15 0.15 0.15 0.15 0.60 0.60 0.60 Mass recovered (ugF-) 0.101 ND ND ND ND ND 0.171 0.220 0.167 0.156 0.149 0.144 0.380 0.427 0.447 0.125 0.409 1.49 4.10 0.584 0.325 0.805 1.94 0.378 0.270 0.442 0.492 0.260 ND NO ND ND ND 0.126 0.138 0.143 0.310 0.522 0.434 0.187 ND ND 0.227 0.439 1.27 0.356 0.189 0.287 0()00!il 10 -x C\-Z. @t@8196 '@rE,. 31 STUDY# 6329-151 SERUM Sample ID F52770-BHR F52762-12HR F52763-12HR SPIKE 62-1 SPIKE 250-1 SPIKE 250-2 F52768-12HR F52770-12HR F52762-24HR F52763-24HR F52768-24HR F52770-24HR F52762-48HR F52763-48HR F52768-48HR F52770-48HR BLANK-1 BLANK-2 SPIKE 62-1 SPIKE 62-2 SPIKE250-1 SPIKE250-2 SPIKE250-3 Actual Sample reading Qty (ppm F-) (mL org) 0.404 0.1 0.149 0.1 0.101 0.1 0.0920 0.1 0.187 0.1 0.225 0.1 0.136 0.1 0.220 0.1 0.0609 0.1 0.0704 0.1 0.0442 0.1 0.0387 0.1 0.0282 0.1 0.0264 0.1 0.0260 0.1 0.0357 0.1 0.0830 0.1 0.0314 0.1 0.0635 0.1 0.0644 0.1 0.157 0.1 0.239 0.1 0.240 0.1 TISAB finalvol (mL) 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 mL FC95 Conc, FC95 spiked so(ution (ppm) 0.004 62 0.004 250 0.004 250 0.004 62 0.004 62 0.004 250 0.004 250 0.004 250 % Actual recovery ppm F- (ug/ug) insample 8.08 2.98 2.01 124% 1.84 62% 3.74 75% 4.51 2.72 4.40 1.22 1.41 ND ND NO ND ND ND 1.66 ND 85% 1.27 86% 1.29 52% 3.15 80% 4.78 80% 4.80 Mass spiked (ugF-) 0.15 0.60 0.60 0.15 0.15 0.60 0.60 0.60 Mass recovered (ugF-) 0.808 0.298 0.201 0.184 0.374 0.451 0.272 0.440 0.122 0.141 ND ND ND ND ND ND 0.166 ND 0.127 0.129 0.315 0.478 0.480 000092 -T @p@ @G. oc@b @) x 9.11.3Summary and raw data;ppm F-inserum as determinedby thermalextractiofnollowedby analysisusing Skalarsegmented flow analyzerwith ion selectivelectrode. 0000313 RE: 6329-151 SERUM SRMPLES RMDT 120694.1 Date of Rnalysis:8/2/95 Rnalyst: DOW The samples are burned in the Bohrman at 950 C using 0.10 mL of the serum. The gas is collectedin 2.0 mL of 1:1 TiSRB/Milli-O water. The samples are then analyzed on a Skalar Segmented Flow Rnalyzer using the Ion SpecificElectrode(ISE)Method. TISRB buffer isadded to each sample as itproceeds through the system. The sample then goes through a heated mixing coil before the potentialbetween the ion selectiveelectrodeand the reference electrodeismeasured. The signalisamplifiedand relatedto the fluorideconcentration. The instrument was calibratedin the ranges of 0.015 - 0.15 ppm and 0.15 - 1.50ppm fluoride.The standard curve for the high range was plottedusing the inverselogarithm option.The standard curve for the low range islinear.HIIstandards and samples were then calculatedby th-eSkalarsoftware using these curves. RIIresultsbelow 0.0001 ppm appear an the raw data as #.####. R qualitycontrolstandard was analyzed every 10 samples to check for accuracy and drift. Raw data istaken from the appropriatecalibratedrange of the Skalar printoutand summarized on an Excel spreadsheet. The finalresultsare adjusted for the collectionvolume and any subsequent dilutions. rD4i- 0()00911 SUMMARY OF 6329-151 SERUM SAMPLES AMDT 120694.1 Dose Level: 0 Dose Level: 4 mgtkg Dose Level: 16 mgfkg Dose Level: WMgfkg a.,L F52762 F52763 F52768 F52770 1.56 6.18 ND 3.96 3.44 1.43 0.55 4.10 3.44 2.49 2.24 2.51 1.60 0.46 15.6 8.08 4.72 3.36 2.94 0.96 0.57 32.9 20.2 14.8 9.26 4.62 0.80 0.82 T@ 6329151S.SUM 0()009!-j Page 1 SERUM CURVE I 7-31-95 NORMAN Sample ID Spk 34-1 Spk 34-2 Spk 34-3 Spk 40-1 Spk 40-2 Spk 40-3 Spk 62-1 Spk 62-2 Spk 62-3 Spk 100-1 Spk 100-2 Spk 100-3 Spk 124-1 Spk 124-2 Spk 124-3 Spk 250-1 Spk 250-2 Spk 250-3 Spk 500-1 Spk 500-2 Spk 500-3 Skalar DI:TISAB Result finalvol (ppm) (mL) mL FC 95 Conc SolutionFC 95 Soin Spiked (ppm) Mass Spiked (ugF-) Mass % Recovered RecovM (ugF-) 0.09 0.07 0.08 0.08 0.07 0.07 0.09 0.09 0.09 2.0 0.004 34.00 0.08 0.17 211% STANDARD DEVIATION: 2.0 0.004 34.00 0.08 0.13 163% 2.0 0.004 34.00 0.08 0.16 191% % RSD: 2.0 0.004 40.00 0.10 0.16 164% STANDARD DEVIATION: 2.0 0.004 40.00 0.10 0.14 147% % RSD: 2.0 0.004 40.00 0.10 0.15 154% 2.0 0.004 62.00 0.15 0.18 120% STANDARD DEVIATION: 2.0 0.004 62.00 0.15 0.18 119% % RSD: 2.0 0.004 62.00 0.15 0.18 124% 0.11 0.12 0.13 0.16 0.17 0.18 2.0 0.004 100.0 0.24 0.21 88% STANDARD DEVIATION: 2.0 0.004 100.0 0.24 0.24 100% % RSD: 2.0 0.004 100.0 0.24 0.27 110% 2.0 0.004 124.0 0.30 0.32 108% STANDARD DEVIATION: 2.0 0.004 124.0 0.30 0.34 114% % RSD: 2.0 0.004 124.0 0.30 0.37 124% 00..3236 22..00 00..000044 225500..00 00..6600 00..6572 18171%% STANDARD DEVIATION: 0.32 2.0 0.004 250.0 0.60 0.65 108% % RSD: 0.47 0.49 0.52 2.0 0.004 500.0 1.20 0.94 78% STANDARD DEVIATION: 2.0 0.004 500.0 1.20 0.99 82% % RSD: 2.0 0.004 500.0 1.20 ' 1.04 87% 0.2450 12.9998 0.0826 5.3307 0.0263 2.1670 0.1138 11.4530 0.0778 6.7516 0.1318 12.9196 0.0442 5.3672 SERUM CURVE I (NORMAN) 7/3110 y 0.7743x+ 0.086 R'- 0.9762 1.20 1.00 030 O.W 020 0001 000 020 040 ow 0+&0 1.00 2wo KIAU SPIKED (ug) BERCRVLN.SUM 0000f)(; Page 1 1995-08-0123:20 OutPut of: 950802B I Opera(or :DDW Date of theAnalysis:1995-08-0208:38 AnalysisFileNaine :C:\SKALARDATA@HWIDATA@SERUNR950802BI 6329151S.XLS I Tracer 1.50 1.48 98% 2 Drift t.50 1.48 99% 3 Wash ND 4 Standard 1 0.015 0.017 111% 5 Standard 2 0.03 0.03 92% 6 Standard 3 0.06 0.06 101% 7 Standard 4 0.09 0.09 101% 8 Standard 5 0.12 0.12 99% 9 Standard 6 0.15 0.15 100% 10 Standard 7 0.30 0.28 93% 11 Standard 8 0.60 0.62 103% 12 Standard 9 1.20 1.23 102% 13 Standard 10 1.50 1.47 98% 14 Dfift 1.50 1.49 990/0 15 Wash ND 16 SERUM BLK 1 0.07 17 SERUM BLK 2 0.05 2.0 o@ 10 1.39 2.0 0.10 0.97 18 SERUM BLK 3 0.05 2.0 0.10 0.96 19 SERUM BLK 4 0.04 2.0 0.10 0.80 20 SERUM BLK 5 0.05 2.0 0.10 0.96 21 SERUM BLK 6 0.04 2.0 0.10 0.78 22 SPK 62-1 0.11 2.0 0.10 2.17 0.004 62.00 0.15 23 SPK 62-2 0.12 2.0 0.10 2.45 O@004 62.00 0,15 24 SPK 62-3 0.10 2.0 0.10 l@94 0.004 62.00 0.15 oo% 25 SPK 62-4 0.09 2.0 0.10 1.89 0.004 62.00 0.15 26 Drift 1.50 1.47 98% 27 Wasit ND 28 SPK 62-5 0.09 2.0 0.10 1.84 0.004 62.00 0.15 29 SPK 62-6 0.09 2.0 0.10 1.74 0.004 62.00 0.15 30 SPK 250-1 0.20 2.0 0.10 3.96 0.004 250.00 0.60 Page 1 6329151S.XLS 31 SPK 250-2 0.22 2.0 0.10 4.40 0.004 250.00 0.60 32 SPK 250-3 0.23 2.0 0.10 4.54 0.004 250.00 0.60 33 F52762-2 0.08 2.0 0.10 1.56 34 F52763-2 0.21 2.0 0.10 4.10 35 F52768-2 0.78 2@O 0.10 15.58 36 F52770-2 1.65 2.0 0.10 32.94 37 F52762-4 0.31 2.0 0.10 6.18 38 Drift 1.50 1.47 98% 39 Wash ND 40 F527634 0.17 41 F527684 0.40 42 F52770-4 1.01 43 SPK 62-1 0.26 44 SPK 62-2 0.15 45 SPK 250-1 0.23 46 SPK 250-2 0.25 47 SPK 62-3 0.15 48 SERUM BLK 1 0,06 49 SERUM BLK 2 O@04 50 Drift 1.50 1.42 95% 2.0 0.10 3.44 2.0 0.10 8.09 2.0 0.10 20.18 2.0 0.10 5@18 0.004 62.00 0.15 2.o 0.10 2.97 0.004 62.00 0.15 2.0 0.10 4.52 0.004 250.00 0.60 2.0 0.10 5.08 0,004 250.00 0.60 2.0 0.10 2.93 O@004 62.00 0.15 2.0 0.10 1.20 2.0 0.10 0.90 51 Wash ND 52 SERUM BLK 3 0.05 2.0 0.10 0.92 53 SERUM BLK 4 0.04 2.0 0.10 0.88 54 SERUM BLK 5 0.04 2.0 0.10 0.80 55 SPK 62-1 0.07 2.0 0.10 1.47 0.004 62.00 0.15 56 SPK 62-2 O@08 2.0 0.10 1.62 0.004 62.00 0.15 57 SPK 62-3 0.08 2.0 0.10 1.65 0.004 62.00 0.15 58 SPK 250-1 0.17 2.0 0.10 3.38 0.004 250.00 0.60 59 SPK 250-2 0.28 2.0 0.10 5.64 0.004 250.00 0.60 60 SPK 250-3 0.23 2.0 0.10 4.62 0.004 250.00 0.60 Oak. 61 BLK 1 0.11 2.0 0.10 2.15 62 Drift 1.50 1.38 92% 63 Wash RD 64 BLK 2 0.04 2.0 0.10 0.74 65 BLK 3 0.02 2.0 0.10 0.47 66 F52762-6 ND 2@O 0.10 ND Page 2 6329151S.XLS 67 F52763-6 0.12 2.0 0.10 2.49 68 F52768-6 0.24 2.0 0.10 4.72 69 F52770-6 0.74 2.0 0.10 14.80 70 F52762-8 0.20 2.0 0.10 3.96 71 F52763-8 0.11 2.0 0.10 2.24 72 F52768-8 0.17 2.0 0.10 3.36 73 F52770-8 0.46 2.0 0.10 9.26 74 Drift 1.50 1.43 95% 75 Wash ND 76 F52762-12 0.17 2.0 0.10 3.44 77 F52763-12 0.13 2.0 0.10 2,51 78 SPK 62-1 0.12 2.0 0.10 2.48 0.004 62.00 0.15 79 SPK 250-1 0.21 2.0 o@ 10 4.18 0.004 250.00 0.60 80 SPK 250-2 0.24 2.0 0.10 4.80 0.004 250.00 0.60 81 F52768-12 0.15 2.0 0.10 2.94 82 F52770-12 0.23 2.0 0.10 4.62 83 F52762-24 o@07 2.0 0.10 1.43 84 F52763-24 0.08 2.0 0.10 1.60 85 F52768-24 0,05 2.0 0.10 0.96 86 Drift 1.50 1.39 93% 87 Wash ND 88 F52770-24 O@04 2.0 0.10 0.80 89 F5276248 0.03 2.0 0.10 0.55 90 F5276348 0.02 2.0 0.10 0.46 91 F52768-48 0.03 2.0 0.10 0.57 92 F52770-48 0.04 2.0 0.10 0.82 93 Drift 1.50 1.43 95% 94 Wash ND C) Page 3 1995-08-0213:20 Outputof : 95080211 Softwar:e versio6n.1 C1990,93 Operator DDV Bats Of tbt AAIIYSIS 1995-08-0208:38 Analysis file lass C:ISIALAR@DATA@IVIDATA@SIRON@950802BJ fluoride1.5 Calibratioonrder--InverseLogarithit Slop# I --/.I///, I - el Resultc 101 s i = correctedvalueof the sample cl : correctevdalueof thecoaceatratioIn s z Slope of the electrode &2 -0.06000 al 0.00070 al -1.23789 flaorideL Calibratioonrder= 2 Cor,relationr = 0.JYYU Result- a2 ilf alt i f aO &2 -0.00000 al 0.00025 ao -0.00103 Sampler Type Number SAIOOO :I Sample Tile 50 sec. Wash Till$ 120 see. Air file I sec. Take up Single special lone needleHeight 70 n. Diluter needleBeight 80 u dilutionFactor 10 dilutionVolute 2.5 al. lesample 1 Dilutionruns I Userfile ?IT Reproces No I-a-L 0()0100 1995-08-1213:20 Outht of 95080231 fluoride1.5 Path onabor signaltype Decolor systesfulber dilate lssisple dil Threshold diG output Viadov event 3 Debubblid Yes 0 lo lo 4095 0 Off 61 ST&Bdatd Ignore s2 s?andard Ignore s3 st&adard Ignore s4 s?aodard Ignore s5 slaidird Ignore s6 s?aodard 0,150 s7 s?aadard 68 slaadard 0.300 0.600 s9 standard 1.200 slO slazdard 1.500 Order. laverieLoqaritha DiiLinsion-.PPK startvalue 500 Do triggeLritit 1800 Soc Peak shape Pointed StArtignore io Sic old ignore 120 Bec measure window 75 1 filter No logeaeration 10 for&Ula : output li.ift fluorideL Path author Signaltype Decolor systesluaber diluto lesasple dil Threshold diG output Window event 0 Debubbled lo 0 lo lo 4095 0 Off 000101 1915-08-0213:20 Catht of 15080211 sl s?andard 0.015 s2 standard 0.030 s3 slandard 0,060 s4 s?andard 0.090 15 s?aadird 0.120 s6 s?andard 0.150 17 s?andard Ignore sS s?andard Ignore s9 standard Ignore slO sliitdard Ignore Order: 2 Disension startValue 500 DU triggetrilit 1800 sac Peak ships Pointed itkrtignore 60 SIC old ignore 120 Sec Noisure window 75 1 filter lo loginerition lo formula C4:--c3 output 1.1111 0()0102 1995-08-0213:20 Oltht of : 95080211 floorid1e.5 flaoridLe PM PM Pos Typ Ideat Cl loseltf line ck losilIt live vt iv IzitiiWlash 3 0.058 65 4 1.#### 0 1t Tracer 3 1.475 209 4 0.3996 0 2d Drift 3 1,481 385 4 0.3995 0 31 Wash 3 0.058 621 4 1.1101 0 4 61 Standard1 3 0.065 735 4 0.0167 0 5 12 Standard2 3 0.069 911 1 0.0275 0 6 53 Standard3 3 0.086 lo85 4 0.0606 0 7 64 Standard4 3 0.105 1262 1 0.0912 0 8 95 StandardS 3 0.126 1436 1 0.1187 0 9 s6 Standard 6 3 0.156 1612 4 0.1503 0 10 s7 Standard 7 3 0,280 1786 4 0.2336 0 11 sS Standard 8 3 0.618 1962 4 0.3348 0 12 s9 Standard9 3 1.228 2131 4 0.3gio 0 13 910 Standard10 3 1,469 2310 1 9.3997 0 14 d Drift 3 1.489 2486 1 0.3994 a 15 v Wish 3 0.058 2700 j 1,1101 0 16 2 $11ON BLI 1 3 0.091 2836 4 0.0656 0 17 u slium ILI 2 3 o.ogo 3011 1 0,0181 0 la u silak BLI 3 3 o.079 3187 4 0.0481 0 19 a SIIUK BLI 4 3 0,075 3363 4 0.0400 20 u SSIUK BLI 5 3 0.079 3533 4 0.0479 0 21 u SIRON ILI 6 3 0,075 3711 4 0.0388 D 22 u SPI 62-1 3 0.118 3887 4 O.ID87 0 23 u SPI 62-2 3 0.129 1063 4 0.1225 0 21 u SPI 62-3 3 0.109 4237 4 0.0972 0 25 u SPK 62-4 3 0.107 1411 4 0.0945 0 26 d Drift 3 1.474 1588 4 0.3996 0 27 v Wash 3 0.058 4829 1 1.1111 0 28 u SPI 62-5 3 0.106 4939 4 0,0921 0 29 u SPI 62-6 3 0.102 5113 4 0.0870 0 30 u S?l 250-1 3 0.198 5287 4 0.1849 0 31 u SPI 250-2 3 0.220 5465 1 O.Zeco 0 32 u SPI 250-3 3 0.227 5639 4 0.2048 0 33 u F52762-2 3 0.096 581c 4 o.07g2 0 34 u P52753-2 3 0.205 5989 4 0.1902 0 35 a 152768-2 3 0.779 6165 1 0.3598 0 36 u ?52770-2 3 1.647 6339 4 0.3919 0 37 a F52762-4 3 0.309 6515 4 0.2471 0 38 d Drift 3 1.474 6689 4 0.3996 0 39 v Va;b 3 0.058 6918 4 I.Ifff 0 io u F52763-4 3 0.172 7039 4 0.1647 0 41 u 152768-4 3 0.404 7214 4 0.2828 a 42 u F52770-4 3 1.009 7389 4 0.3833 - 0 43 u SPI 62-1 3 0.259 7567 4 0.2232 0 44 u SPI 62-2 3 0.154 7740 1 0.1183 a 15 u SPI 250-1 3 0.226 7914 4 0.2041 0- 46 u SPI 250-2 3 0.251 8090 1 0.2203 0 47 u SPI 62-3 3 0.152 8266 1 0.1467 0 48 u SIRUK ELI 1 3 o.oa6 8439 4 0.0599 0 49 u SIRUM BLI 2 3 0.078 8616 4 0.0448 a 50 d Drift 3 i.tio 8790 4 0.3999 0 51 v Wash 3 0.058 9ol6 4 I.Illj a 52 u SilUK BLI 3 3 0,078 9139 4 o.o462 0 53 u SilUK OLL 1 3 0.077 9315 4 O.Ci4l 0 PaloI of 2 0()0103 1995-08-0213:20 Outht of : 15080211 fluoride1.5 fluorideL PM ppi PosTTPIdelt Ck lessift Tiss Ck lostitf liat 51 u SZIUN ELI S 3 0.075 9491 1 0.0400 0 55 u SPE 62-1 3 0.053 9665 4 0,0731 0 51 u SPI 62-2 3 0.098 9841 1 0.0809 0 57 u SPI 62-3 3 0.099 10016 4 0.0825 0 So u BPI 250-1 3 0.169 10191 4 0.1121 0 59 a SPI 250-2 3 01282 10316 1 0.2347 0 60 a SPI 250-3 3 0.231 10539 4 0,2071 a 61 u BLI 1 3 0.117 10715 1 0.1071 0 iz 4 Drift 3 1.382 10890 4 0.3997 0 63 v Wash 3 0.058 11011 4 $.##If 0 14 u BLI 2 3 0.071 11212 4 0.0369 0 65 a BLI 3 3 0.068 Ilill1 0.0236 0 61 u 152762-6 3 0.063 11588 4 0.0122 0 67 a 152763-6 3 0.131 11766 4 0.1244 0 is a F52768-6 3 0.236 ll9iO 1 0.2099 0 19 1 152770-1 3 0,740 12116 4 0,3546 0 70 a 152762-9 3 0.198 12292 1 0.1849 0 71 u 152763-8 3 0.121 12166 4 0.1121 0 72 u 152768-8 3 0.168 12$40 4 0.1611 0 T3 a 152770-8 3 0.463 12817 4 0.3001 -.0 74 d Drift 3 1.428 12990 4 0.3999 0 75 v Mash 3 0.058 13230 1 1.#### 0 76 u 152762-12 3 0.172 13311 4 0.1647 a 77 u f52763-12 3 2.132 13517 4 011256 0 78 u BPI 62-1 3 0.130 13651 1 0.1238 a 79 u SPI 250-1 3 0.209 13861 4 0.1921 0 Be $?1 250-2 3 0.240 14043 4 O@2121 0 $1 152768-12 3 0.152 11217 1 0.1165 0 82 u F52770-12 3 0.231 11391 4 0.2072 0 83 u 152762-24 3 0.092 lisil 1 0.0711 0 84 u F52763-2i 3 0.098 14713 4 0.0802 0 85 u 152768-24 3 0,079 14918 1 0.0179 0 96 d Drift 3 1.388 15091 4 0.3998 0 87 1 Vash 3 0,058 15223 1 1.##1# 0 88 a 152770-21 3 0.075 15414 4 0.04DO 0 $I a P52762-48 3 0.070 15618 4 0.0277 0 90 u 152763-18 3 0.067 15790 1 0.0231 0 91 u 152768-48 3 0.070 15964 4 0.0285 0 92 u F52770-48 3 0.076 161(2 4 0.0412 0 53 4 Drift 3 1.432 16318 4 0.3999 0 94 v Wash 3 0.058 16si5 1 1.1111 0 wt ty lutoutWasb 3 0.058 16793 1 1.;### 0 Page 2 of 2 ooolo.I Calibration curve of 950802BI : Fluoride L Eli*-rrlraumIN;M!r4wr--r.B 6 Mg.7.7 r.MV-Til 0. OnO4g720rt?-TM 94 0.1940644- 0 -0.0010,11-6 0 Order Measured 900 r 0.99908 i.76835S8 Calibration curve of 9SO802Bt Fluoride 1.5 MM r-4 /+ / 0 Measured Order Inverse Logarithm 409S S 6068 0()010( Raw data of 9SO80281 : Fluoride I.S E;Flit7ire@@Er.7M 69 st@@ 147 EM[M64 -A to 0 0 Esc=Exii FI=Help Time Ot-ti-P=Editpeaks z,t,A-1@ I sooo 0()0101"fl Raw data of 95080281 : Fluoride I.S 4588 574 -6 409S 5 16 Io 45i6o Esc=Exit FI=Help Time erti-P=Edit peaks 9560 0()Olos Raw data of 950802BI Fluoride 1.5 9016 NIFFTM-M 4095 I fI ,I 0 893S Esc=Exit FI=Help Time Crtl-P=Edit peaks 1 11 935@ ooolo@) Raw data of 950802BI '.Fluoride 1.5 12466 WtAff -M 97-4) 4095 19, -1'6 gt 0 12435 Esc=Exit FI=Heip Time Crti-P=Edit peaks t7435 ooolto 9.11.4Summary and raw data;analysisof liverextracts using electrospramyass spectrometry. This data,althoughsupportivei,ntheopinionof theStudy Directorisnot requiredto reachtheconclusionstatedin FinalReport Section6.0,and thereforeisnot discussedin detail. ooollt A-k, tc- to-cl@- 14,0 Dxvk Si mtAg-.@ 74keLrc STVVY - File:08019SF LAD-BASE - The MS Data System Sample:HWI# 6329-151: L13492 (PH) 080195F ZFS- 3a 45 @I'D40 itjj tILn IL 513 3 5 CA Y.FS- 32 546 2!59 to ,Son 290 Awr--#= b-:@29 151 C,,e@ L 1-6@ File:080195E Sample: 08019SE LAB-BASE The MS Data Sgsten 0 23 Y.FS 2a 25 La Son 50 100 150 as@ 250 300 (Y) File:080195E Sample: LAD-BASE - The NS Data System 0 OEIO.95E 20 oz)-itsir)(;OMBINE (196 o 206 43 .90 TO ll")+(ZZ9 VFS 471.08 35566.90 40022..0000 / 33 9e66!.8884 - 436.00 L4 455@3A3.@.80 45 472.00 / 473.80 474.00 491.00 500 533.00 552.00 go" 551 578. J-I "WI File:080195E ample: 8019SE 262 is@. 6.98? LAB-BASE - The NS Data System 42 .00 OIL/ aI ZFS- 48 .88 406.gio \ 4-29--.0-6I/ 0 8 -- N/z a 450 500 -- File:080195E Sample: 080195E'324 (0.640) LAB-BASE - The NS Data Sgsten Ot/ COMBINE:(321 to 326)-(((284 to 302)+(369 1 zFS- 413.00 378.80 4144.1090. as 471 go 58 114/z 158 400 450 560 Aua 0 C32q lc,51 LI'344Z File:080195G Sample: 088195U 33 V.FS%D @I 100- r4 4) IL 45 LAB-BASE The NS Data System 0 (4 210 LL Y.FS- 0, scn 341 12 66 500 -11-6--6 1293 15--os 1580 21 2053 2@fee