Document wqY0Yr9QLRjXpJBL39E0Y43MD

3M EnvironmentaLlaboratory FinalReport-AnalyticSatludy Single-DoIsnetravenouPsharmacokinetSitcudyofT-6054inRabbits In-VivSotudyReferencNeumber:HWI#6329-138 StudyNumber: ANMT-122094.2 TestSubstanceF:C-129(T-6054) Name and AddressofSponsor: 3M SCD Division 367 Grove Street St.Paul,MN 55106 Name and Address ofTestingFacility: 3M EnvironmentalTechnology& Services 935 Bush Avenue St.Paul,NlN 55106 Method Numbers AMDT-M-1-0, AMDT-M-2-0, AMDT-M4-0, AMDT-M-5-0, AMDT-M-8-0, and Revisions: Thermal ExtractionofFluorideby Means of a Modified Dohrinann DX2000 OrganicHalideAnalyzer-Liver FluorideMeasurement by Means ofan Orion EA940 Expandable Ion Analyzer ExtractionofFluorochemicalsfrom RabbitLiver Analysisof RabbitLiverExtractforFluorochemicalsUsing ElectrospraMyass Spectrometry Analysisof FluorideUsing the SkalarSegmented Flow Analyzer with Ion SelectivEelectrode InitiatioDnate: See attachedprotocol Author:James D. Johnson A provedBy: X,mes i@:J*son i tudyDir(jefor Completion Date 1.0 SUMMARY Liversampleast48 hourspostintravenoduosseofFC-129(T-6054i)nrabbitwsere analyzedfortotalorganicfluorinaend perfluorooctanesulfonate. Afteran intravenousdose ofFC-129 inrabbitst,hereisa detectabliencreaseoftotal organicfluorineinliverat48 hourspostdose atdosesrangingfrom 0.128 to 12.8 mg/kg. A substantialmount ofthistotalorganicfluoriniesintheform of perfluorooctanesulfonatTeh.ispharrnacoldnetisctudyshows thatthereisa convenientmarker (perfluorooctanesulfonaftoer)assessingtheextentofdermal absorption. 2.0 INTRODUCTION This studywas performed in orderto providedatafortheassessmenta subsequent dermalabsorptionstudy(HWI#6329-133). Knowing thedispositioonfan intravenousdose of FC-129, facilitatiensterpretatioofna dermal absorptionstudy. Analysisof liverand serum samplesfrom rabbitsdosed intravenouslwyithFC-129 fortotalorganicfluorid(ecombustionanalysisa)nd specificcompounds . (electrospramyass spectrometryp)rovidedataastowhetherfluorinatecdompounds areinthe liverat48 hourspostdose and whether perfluorooctanesulfonaitse present.Perfluorooctanesulfonaitsea very good marker fora dermal studysince previouswork hasshown ittobe persistenitnliverand serum inrabbits(biological halflife>1.month). 3.0 TEST MATE - A S 3.1Test,Control,and ReferenceSubstancesand Matrices 3.1.1AnalyticaRleferenceSubstance:FC-95,lot161or 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 3.2Source ofMaterials:3M ICP/PCP DivisionforFC-95,bovineliverfrom grocerystoreb,ovineserum from Sigma Chemical Company. 3.3.Purity and StrengthofReference Substance:ResponsibilitoyfSponsor. 3.4Stabilityof ReferenceSubstance: To be determinedby Sponsor. 3.5StorageConditionfsorTestMaterialRso:om temperatufroerFC-95.For biologicalsamples thestor-agies-20+100 C. 3.6Dispositionof Specimens: Biologicaltissuesand fluidswillbe retainedper GLP Regulationforthetimeperiodrequiredforstudieslongerthan28 days. This studyisinparallewlith a 28 day absorptionstudy,soalltissuewsillbe retained. 4.0 EXPERIMENTAL -Overview Serum and tissuesfrom animalsdosed as described(HWI#6329-1.38),were available foranalysisfortotalorganicfluorineand fluorinatecdompounds. The samples were analyzedby combustionand/orelectrospmymass spectrometrytotheextent necessarytoprovidesufficiendtatafortheinterpretatioofna second studyon the extentof dermalabsorptionofFC-129 (HWI#6329-133). 5.0 EXPEREQENTAL -METHODS 5.1AMI[)T-M-1-0, ThermalExtractioonf Fluorideby Means ofa Modified Dohnnann DX2000 OrganicHalideAnalyzer-Liver 5.2AAIDT-M-2-0, FluorideMeasurement by Means of an Orion EA940 Expandable Ion Analyzer 5.3AMDT-M-4-0, ExtractioonfFluorochemicalsfrom RabbitLiver 5.4AMDT-M-5-0. AnalysisofRabbitLiverExtractforFluorochemicalUssing ElectrosprayMass Spectrometry 5.5AAIDT-M-8-0, AnalysisofFluorideUsing theSkalarSegmented Flow Analyzer with Ion SelectivEelectrode 6.0 DATA ANALYSIS The dataareattached.For combustionanalysisfortotalorganicfluorinet,herabbits dosed with 0,0.128,0.64,1.28,and 12.8mg/kg had 18 (below thelevelof practical quantitation6)1,, 118,164,and 2239 ug/whole liverr,espectivelyT.he body weight 3 ofthe 12.8mg/kg rabbit(F52752)was 2.7kg. Thus,thetotaldose giventhisrabbit was 34.6mg. The amount of totalorganicfluorinienthisdose is19 mg. The total organicfluorineinliveraftertheintravenousdose inrabbitF52752 represents 11.8% of thedose. The dataforan electrospramyass spectrometryanalysisisattached.There is detectablpeerfluorooctanesulfonaitnealltreatedrabbits.The high dose rabbit (F52752)had about335 ug perfluorooctanesulfonparteesentinliverat48 hours.If the 34.6mg dose isexpressedasFC-95, (thatis,assuming 100% biotransformation) the dose is31.8mg. The 335 ug presentinwhole liverinthisanimalrepresents 1.05% ofthedose. This representas substantiaalmount ofbiotransformatioonf FC-129 to perfluorooctanesulfonaatte48 hours. Other datawas collecteudsingSkalarsegmented flow analyzerwith ionselective electrode(seeappendices)T.his data,althoughsupportivei,ntheopinionofthe Study Directorisnot requiredtoreachtheconclusionstatedhereand thereforeisnot discussedin detail. 6.1 Circumstances that May Affectthe Quality of the Data: The problem with thisanalysisisthatina pharmacoldnetisctudyreportedseparatel(yHWI#6329-159), therewas a delayinperfluorooctanesulfonactoencentratioonbservedin serum that indicatedan unexplaineddistributipoanttern.The leveldropped dramaticallayt12 and 24 hoursbut returnedto a maximum at48 hourswitha subsequentdecay in serum levelsthatindicateadbiologicahlalf-lifoef> 1 month. Thisdelayin perfluorooctanesulfonalteevelsmay be shiftedtoa latertimewhen perfluorooctanesulfonaitsreesultinfgrom biotransformationT.here may actuallbye a higherlevelofperfluorooctanesulfonaitneliverata latertime periodand this studyonly went to48 hours.Thiswould indicattehatperfluorooctanesulfoniastaen even bettermarker fora dermal absorptionstudythanwould be indicatedby this data. 7.0 CONCLUSION After an intravenousdose of FC-129 inrabbitst,hereisa detectableincreaseof total organicfluorinienliverat48 hoursatdosesrangingfrom 0.64to 12.8mg/kg. A substantiaalmount of thistotalorganicfluorineisintheform of perfluorooctanesulfonatTeh.ispharmacokineticstudyshows thatthereisa convenientmarker forassessingtheextentof dermal absorption. 4 8.0MAINTENANCE OF RAW DATA AND RECORDS 8.1Raw Dataand Data:Raw dataa,pprovepdrotocoalp,provefdmalreport, appropriatsepecimens,and electronidcatawillbe maintainedintheAMDT archives. 9.0 APPENDICES 9.1 Protocol and Amendments 9.1.1Protocoland FinalReport:HWI#6329-138 "Single-DoseIntravenous PharmacokineticStudyof T-6054 inRabbits"(ProtocoltypeT?8084.PK for dosingof animals,tissuecollectione,tc.) 9.1.2 AnalyticalprotocolAMDT- 122094.2 9.2 Signed Reports from IndividualScientistsN:one 9.3 QualityAssurance Unit Statement:See attached 9.4 Key PersonnelInvolvedinthe 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 Quality ControlPractices:See methods 9.9 Test Methods: See ProtocolAMDT-122094.2 9.10 Instrument Settings:See methods 5 9.11Data:Seeattached. 9.11.1Summary and raw data;ug F-inwhole liveras determinedby thermal extractiofnollowedby analysisusingOrion ionanalyzer. 9.11.2Summary and raw data;analysisof liverextractussingelectrospray mass spectrometry. 9.11.3Summary and raw data;ug F'inwhole liveras determinedby thermal extractiofnollowedby analysisusingSkalarsegmented flow analyzerwithion selectiveelectrode. 6 9.1.1Protocoland FinalReport:HWI#6329-138 "SingleDose IntravenousPharrnacokinetiSctudy of T-6054 in Rabbits" (ProtocoltypeTP8084.PK fordosingof animals, tissuecollectione,tc.) HAZLrzatON W IS C 0 N S IN POST OFFICE BOX 7545 MADISO@j,Wl 53707 7545 Sponsor: 3M St. Paul, Minnesota FINAL REPORT j CORNING Coinviny CEI i(FEEB8 E7 Study Title: Single-Dose Intravenous Pharmacokinetic Study of T-6054 in Rabbits Author: Steven M. Glaza Study tomgletion Date: February ?4, 1995 Performing Laboratory: Hazleton Wisconsin, Inc. 3301 Kinsman Boulevard Madison, Wisconsin 53704 LaboratoryProject Identification: HWI 6329-138 Page 1 of 24 P h o n e 6 0 8 -2 4 1 -4 4 7 1 E X P R E S S - M A IL D E L IV E R Y 3,'Ol K ItlS MArl BLVD F a.x.. -60 B.- 2 4 11@--72 2 7 MAD[ SO N. vv I - 53704 Page 2 of 24 HWI 6329-138 QUALITY ASSURANCE STATEMENT This report has been reviewed by the Quality AssuranceUnit of Hazleton Wisconsin, Inc., in accordancewith the Food and Drug Administration (FDA)Good LaboratoryPracticeRegulations,21 CFR 58.35 (b) (6) (7). The following inspectionswere conductedand findingsreportedto 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 12/09/94 12/09/94 ProtocolReview 12/19/94 12/19/94 Animal Observation 02/02/95 02/02/95 Data/Report Review 12/09/94 12/19/94 02/02/95 01/10/95 01/10/95 03/10/95 egeccilia M. Danner Date Representative, Q3ua@iaty AssuvranceUnit Page 3 of 24 STUDY IDENTIFICATION Single-Dose Intravenous Pharmacokinetic Study of T-6054 in Rabbits HWI 6329-138 Test.Material Sponsor Sponsor.l.sRepresentative Study Director Study Location Study Timetable Experimental Start Date Experimental Termination Date T-6054 3M Toxicology Service MedicalDepartment 3M Center, Bldg. 220-2E-02 P.O. Box 33220 St. Paul, MN 55133-3220 John L. Butenhoff, PhD 3M Toxicology Service Medical Department 3M Center, Bldg. 220-2E-02 P.O. Box 33220 St. Paul, MN 55133-3220 (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, Wl 53704 December 17, 1994 December 19, 1994 Page 4 of 24 KEY PERSONNEL HWI 6329-138 Acute Toxicology Steven M. Glaza Study Director Manager Francis (Bud) W. McDonald Study Coordinator .Patricia Padgham In-life Supervisor Rose M. Bridge Report Supervisor Ouality Assurance Laboratory Animal Medicine Cindy J. Cary, DVM Diplomate, ACLAM Supervisor Anatomical Pathology Jack Serfort/ Deborah L. Pirkel Supervisors Necropsy Anne Mosher Supervisor Pathology Data Sherry R. W. Petsel Manager Page 5 of 24 CONTENTS Quality Assurance Statement Study Identification Key Personnel Summary Objective Regulatory Compliance Test and Control Materials Test System Procedures Results Discussion Signature Reference Table 1 IndividualBody Weights (g) 2 IndividualClinicalSigns Appendix A Protocol TP8084.PK HWI 6329-138 Paqe 2 3 4 6 7 7 7 8 9 11 11 11 11 12 13 14 15 Page 6 of 24 HWI 6329-138 SUMMARY This study was done to assess the level of systemic exposure of T-6054 when administered by intravenous injectionto rabbits. Female Hra:(NZW)SPF rabbits were assigned at random to five groups (one/group). On Day 0, the animals received a single intravenousinjection of the vehicle (sterilewater for injection)or 0.128, 0.64, 1.28, or 12.8 mg of T-6054/kg of body weight (Groups 1 through 5, respectively). The dose volume was 0.5 mL/kg for all groups. Clinical observations were conducted at approximately 0.5, 2, 4, 24, and 48 hours after intravenous injection. Body weights were determined just before test material administration(Day 0). A blood sample (approximately 4 mL) was collected from an auricular artery or marginal ear vein of the animals at 2-, 4-, 6-, 8-, 12-, and 24-hourspost-injection. In addition, at the time of experimental termination (48-hours post-injection),approximately 20 mL of blood was obtained from each animal. All samples were centrifuged, separated into serum and cellular fractions, and sent to the Sponsor. Approximately 48 hours post-injection,the animals were anesthetized with sodium pentobarbital, bled via the posterior vena cava, and exsanguinated. An abbreviated gross necropsy examination was not.done, however, tissues were collected. The whole liver, bile, and both kidneys from each animal were collected and sent frozen to the Sponsor after terminationof the in-life phase. All five animals appeared normal throughout the study. Page 7 of 24 HWI 6329-138 OBJECTIVE The objectiveof this study was to assess the level of systemicexposure to the test material,T-6054, when administeredas a single intravenousinjection to rabbits. REGULATORY COMPLIANCE This study was conducted in accordancewith the U.S. Food and Drug Administration'sGood LaboratoryPracticeRegulationsfor Nonclinical LaboratoryStudies,21 CFR 58, with the exceptionthat analysisof the test mixturesfor concentration,homogeneity/solubilitya,nd stabilitywas not conducted. All proceduresused in this study were in compliancewith the Animal Welfare Act Regulations. In the opinion of the Sponsor and study director,the study did not unnecessarilyduplicateany previouswork. TEST AND CONTROL MATERIALS Identification The test materialwas identifiedas T-6054 and described as an amber 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. Purityand Stability The Sponsor assumesresponsibilityfor test materialpurity and stability determinations(includingunder test conditions). A sample of the test material/vehiclemixturesfor concentration,solubility,homogeneity,and stabilityanalyseswas not taken before administrationas this was not requestedby the Sponsor. The purity and stabilityof the USP grade control material were consideredto be adequatefor the purposes of this study. Storage and Retention The test materialwas stored at room temperature. The control materialwas stored refrigerated. Any unused test materialwas returnedto the Sponsor after completionof all testing accordingto HazletonWisconsin(HWI) Standard Operating Procedure (SOP). Any remainingvehicle may be used for other testingand will not be discardedafter issuanceof the final report. Page 8 of 24 HWI 6329-138 Safety Precautions The test and control material handlingprocedureswere according to HWI Sops and policies. TEST SYSTEM Test Animal Adult albino rabbits of the Hra:(NZW)SPFstrain were receivedfrom HRP, Inc., Kalamazoo, Michigan on November 16, 1994 and maintained at the Hazleton Wisconsin facility at 3802 PackersAvenue, Madison, Wisconsin. Housing After receipt,the animalswere acclimatedfor a period of at least 7 days. During acclimationand throughoutthe study, the animalswere individually housed in screen-bottomstainlesssteel cages in temperature-and humiditycontrolledquarters. Environmentalcontrolsfor the animal room were set to maintain a temperatureof 19* to 23*C, a relativehumidityof 50% 20%, and a 12-hourlight/12-hourdark lightingcycle. 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 compliedwith standards outlined in the "Guide for the Care and Use of LaboratoryAnimals Animal Diet The animals were provided access to water ad 7ibitum and a measured amount of LaboratoryRabbit Diet HF #5326, PMI Feeds, Inc. The feed is routinely analyzed by the manufacturerfor nutritionalcomponentsand environmental contaminants. Samples of the water are periodicallyanalyzed by HWI. There were no known contaminantsin the feed or water at levels that would have interferedwith or affectedthe resultsof the study. Selection of Test Animals The animals were identified by animal number and correspondingear tag and were selected at random based on health and body weight requirements. Page 9 of 24 Study Design HWI 6329-138 Female animalsweighing from 2,702 to 2,891 g at initiationof treatmentwere placed into the followingstudy groups: Group Dose Level Dose Volume Treatment (mg T-6054/kg) (mL/kq) I (Control) 0 0.5 2 T-6054 0.128 0.5 3 T-6054 0.64 0.5 4 T-6054 1.28 0.5 5 T-6054 12.8 0.5 SterileWater for Injection,USP. Number of Animals 1 1 1 1 1 Justificationfor SDecies Selection Historically,the New Zealand White albino rabbit has been the animal of choice because of the large amount of background informationon this species. PROCEDURES Dose Preparation and Administration The test materialwas dilutedwith SterileWater for Injectionto achievea specificconcentrationfor each-dose level. An individualdose of each respectivetest solutionor controlwas calculated for each animal based on its body weight on the day of treatment. The.respectivetest solutionwas administeredby intravenousinjectioninto 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 temperatureuntil administered. After administration,any remainingtest solutionswere discarded. Reason for Route of Administration Intravenousinjectionis an acceptableroute to assess systemic exposure. Observations of Animals Clinical observationswere conductedat approximately-0.5,2, 4, 24, and 48 hours after intravenousinjection. Body weights were determinedjust before test material administration(Day 0). Page 10 of 24 Sami2leCollections HWI 6329-138 A blood sample (approximately4 mL) was collectedfrom either ear via the catheterizationof the auriculararteryor from the marginalear vein of all animalsat 2, 4, 6, 8, 12, and 24 hours post-injection.At the time of necropsy (approximately48-hourspost-injection),approximately20 mL of blood was obtained from the posteriorvena cava of each animal. All samples were stored at room temperatureuntil centrifugedand separatedinto serum and cellularfractions. The blood sampleswere then stored in a freezerset to maintain a temperatureof -20*C 10*C until shipped to the Sponsor. Pathology At terminationof the experimentalphase (approximately48-hours post-injection),animalswere anesthetizedwith sodium pentobarbital,bled via the posterior vena cava, and exsanguinated. An abbreviatedgross necropsy examinationwas not conducted, however, tissueswere collected. The whole liver, bile, and both kidneys from each animalwere collected and immediately placed on dry ice, then frozen by placing in a freezerset to maintaina temperatureof -20*C 10*C. After tissue/bilecollection,the animalswere discarded. Shipment of Tissues After completionof the in-lifephase the.bloodsamples,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 retentionand dispositionof the samples. HWI does not accept any responsibilityfor the analysisof the samples collectedin this study nor are these resultspresentedin this report. StatisticalAnalyses No statisticalanalyseswere requiredby the protocol. Location of Raw Data, Records, and Final Report The raw data, records,and an originalsignedcopy of the final reportwill be retained in the archives of HWI in accordancewith HWI SOP. Page 11 of 24 RESULTS Body Weights Individual body weights at initiation are in Table 1. HWI 6329-138 Clinical Observations Individualclinical signs are in Table 2. All five animals appeared normal throughout the study. Pathology All animals survived to termination of the experimental phase and were not examined grossly when sacrificed. DISCUSSION The level of systemic exposure of T-6054 was evaluated in female albino rabbits when administeredas a single intravenous injection at levels of 0, 0.128, 0.64, 1.28, and 12.8 mg/kg. All animals appeared normal throughout the study following administrationof this,material. SIGNATURE Steven M. Glaza Study Director Acute Toxicology Date REFERENCE 1. NIH PublicationNo. 86-23 (revised 1985). GroUR 1 2 3 4 5 Page 12 of 24 Table I IndividualBody Weights (g) Dose Level (mg/kg) Sex Animal Number 0 Female F52792 0.128 Female F52793 0.64 Female F52750 1.28 Female. F52751 12.8 Female F52752 Day 0 2,784 2,805 2,891 2,702 2,783 HWI 6329-138 Grou R .1 Dose Level (mg/kq) 0 Sex Female Page 13 of 24 Table 2 IndividualClinicalSigns Animal Number Observation 0.5 F52792 Appeared normal .1 .HWI 6329-138 Hour 2 4 24 48 2 0.128 Female F52793 Appeared normal t 3 0.64 Female F52750 Appeared normal 4 1.28 Female F52751 Appeared normal t t 5 12.8 Female F52752 Appeared normal t .1 Indicatesconditionexists. Page 14 of 24 APPENDIX A Protocol TP8084.PK HWI 6329-138 HwAIZsLEcN0TON NS I N POST OFC:I('E 13OX 1!@45 MADISO@L. Wl 53701 1545 Page 15 of 24 itCORNING CI)fllpalrf Sponsor: 3M St. Paul, Minnesota PROTOCOL TP8084.PK Study Title: Single-DoseIntravenousPhamacokinetic Study of 7-6054 in Rabbits Date: December 13, 1994 Performing Laboratory: Hazleton Wisconsin, Inc. 3301 Kinsman Boulevard Madison, Wisconsin 53704 Laboratory Project Identification: HWI 6329-138 P t) c. ,@ t30 li 2 14 1 Ni-\ , i,lI VERY 330 K I@l .@r.vlA N Bt V[) PvAl N Page 16 of 24 STUDY IDENTIFICATION TP8084.PK Page 2 Single-Dose Intravenous Phamacokinetic Study of T-6054 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 6329-138 T-6054 3N Toxicology Service Medical Department 3M Center, Bldg. 220-2E-02 P.O. Box 33220 St. Paul, MN 55133-3220 John L. Butenhoff, PhD 3M Toxicology Service Medical Department 3M Center, Bldg. 220-2E-02 P.O. Box 33220 St. Paul, MN 55133-3220 (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 17, 1994 December 19, 1994 January 23, 1995 Page 17 of 24 TP8084.PK Page 3 1. Study Single-Dose IntravenousPhamacokinetic Study in Rabbits 2. Purpose To assess the level of systemic exposure when the test material is administeredas a single intravenousinjectionto rabbits 3. Regulatory Compliance .Thisstudy will be conducted in accordance with the following Good Laboratory Practice Regulations/Standards/Guidelinewsith the exceptionthat analysisof the test materialmixturesfor concentration,solubility,homogeneity,and stabilitywill not be conducted: Conduct as a Nonregulated Study [XI 21 CFR 58 (FDA) 40 CFR 160 (EPA-FIFRA) 40 CFR 792 (EPA-TSCA) C(81)30 (Final)(O.ECO) 59 Nohsan No. 3850 (Japanese MAFF) Notification No. 313 (Japanese MOHW) All procedures in this protocol are in compliancewith the Animal Welfare Act Regulations. In the opinion of the Sponsor and study director, the study does not unnecessarilyduplicate any previous work. 4. Ouality Assurance The protocol, studyconduct, and the final report will be auditedby the Quality Assurance Unit in accordance with Hazleton Wisconsin (HWI) Standard Operating Procedures (SOPS) and policies. 5. Test Material A. Identification T-6054 B. Physical Description (To be documented in the raw data) C. Purity and-StabilitY The Sponsor assumesresponsibilityfor purity and stability determinations(includingunder test conditions). Samplesof test material/vehiclmeixture(s)for concentration,solubility, homogeneity,and stabilityanalyses will be taken before administrationif requestedby the Sponsor. These samples (if taken) will be sent to the Sponsor after experimental terminationfor possibleanalysis. Page 18 of 24 TP8084.PK Page 4 D. Storage Room temperature E. Reserve SamDles Reserve sampleswill not be requiredfor this study. F. Retention Any unused test material will be returnedto the Sponsor after completionof the in-life phase of the study. G. Safety Precautions As required by HWI SOPs and policies 6. ControlMaterial A. Identification Sterilewater for injection B. PhysicalDescription Clear, colorlessliquid C. Purityand-StabilitY The purity and stability of this USP grade material is considered to be adequate for the purposes of this study. 0. Storage Refrigerated E. Reserve SamRles 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 Page 19 of 24 TP8084.PK Page 5 (4) Weight at Initiation 2.5 to 3.5 kg (5) Number and-Sex 5 females (6) Identification Individual numbered ear tag (7) Husbandry (a) Housing Individually,in screen-bottomstainless steel cages (heavy gauge) (b) Food A measured amount of Laboratory Rabbit Diet HF #5326 (PMI Feeds, Inc.). The food is routinelyanalyzedby the manufacturer for nutritional components and environmental contaminants. (c) Water Ad 7ibitum 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 that would interferewith this study. (e) Environment Environmentalcontrols for the animal room will be set to maintain a temperature of 19*C to 23*C, a relative humidity of 50% +20%, and a 12-hour light/12-hourdark cycle.. (f)Acclimation At least 7 days (8) Selection of Test Animals Based on healt an 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 Sele@Z-t@ Historically,the New Zealand White albino rabbit has been the animal of choice because of the large amount of background informationon this species. Page 20 of 24 TP8084.PK Page 6 B. Dose Administration (1) Test GrouRs Group Dose Level (MCI/kq)' Number of Females 1 0 (Control) 2 0.128 3 0.64 4 1.28 5 12.8 a The dose volume will be 0.5 mL/kg of body weight. C. Dosing Procedures (1) Dosing Route intravenousinjectioninto a marginal ear vein over approximately 30 to 60 seconds. (2) Reason for Dosing Route Intravenousinjection is an acceptable route to assess systemic exposure. (3) Dosing Duration Single dose (4) Dose PreRaration The test materialwill be dilutedwith sterilewater for injectionto achieve a specificconcentrationfor each dose level. Individualdoses will be calculatedbased on the animal'sbody weight taken just before test material administration.The prepared test mixtures will be stored at room temperatureuntil administration. D. Observation of Animals . (1) ClinicalObservations - T e animals wi e observed for clinical signso toxicity at approximately0.5, 2.0, 4.0, 24, and 48 hours after treatment. (2) Body Weights Just before test material administration. (3) Sample Collections (a) Ergqu-e-n-cl 2, 4, 6, 8, 12, 24, and 48 hours post-injection Page 21 of 24 TP8084.PK Page 7 (b) Number of Animals All (c) Method of Collection Blood samples (approximately4 mL) will be collected from either ear via the catheterizationof the auricular arteryor from the marginal ear vein at 2, 4, 6, 8, 12, and 24 hours post-injection. Approximately20 mL of blood (actual volume to be documented in 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 samples will be stored at room temperature and then centrifuged,and the separate serum and cellular fractionsstored in a freezer set to maintaina temperatureof -20'C tlO*C. The separated serum and cellularfractionswill be sent frozenon dry ice to the Sponsor after experimentaltermination. Samples will be shipped to: James D. Johnson 3M E.E. & P.C. Bldg. 2-3E-09 935 Bush Avenue St. Paul, MN 55106 James D. Johnson or his alternatewill be notified by telephoneat (612) 778-5294prior to the shipmentof the samples. E. Termination (1) Unscheduled Sacrifices and Deaths Any animal dying during the study or sacrificedin a moribund condition,will be subjected to an abbreviated gross necropsy examinationand all abnormalitieswill be recorded. Animals in a moribund conditionwill be anesthetizedwith sodium pentobarbital(via injectionin the marginal ear vein), bled via the vena cava, and exsanguinated. Tissues, as described in section E. Termination,(3) Sample Collection,will be collected. Page 22 of 24 TP8084.PK Page 8 (2) Scheduled Sacrifice At approximately48 hours post-injection,animals survivingto terminationwill be anesthetizedwith sodium pentobarbita(lvia injectionin the marginalear vein), bled via the vena cava, and exsanguinated. An abbreviated gross necropsy examinationwill not be done, however, tissueswill be collected. (3) Sample Collection The whole liver and bile from each animaldying during the study,sacrificedin a moribundcondition,or survivingto terminationwill be collected. Both kidneysfrom each animalwill also be collected. The tissueswill be placed on dry ice immediatelyaftercollectionand then placed in a freezerset to maintaina temperatureof -20*C 10*C. The tissues(liver,bile,kidneys)will be sent frozenon dry ice to the Sponsorafterexperimentaltermination. The sampleswill be shippedto the person listed in Section7.0.(3).(c).The Sponsoris responsiblefor the retentionand dispositionof the samples. F. StatisicalAnalyses No statisticalanalysesare required. 8. Rep6rt A final reportincludingthose items listedbelowwill 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) Page 23 of 24 TP8084.PK Page 9 9. location of Raw Data, Records, and Final Report Originaldata, or copies thereof,will be availableat HWI to facilitateauditing the study during its progress and 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 collection records Shipping records Pathology Records Study correspondence Final report (originalsignedcopy) The followingsupportingrecords will be retained at HWI but-will not be archived with the study data. Animal receipt/acclimationrecords Water analysis records Animal room temperature and humidity records Refrigeratorand freezer temperaturerecords Instrumentcalibration and maintenance records Page 24 of 24 TP8084.PK Page 10 PROTOCOL APPROVAL John L. Butenhoff,PhD Sponsor's Representative 3M Toxicology Service Medical Department /,ZDate Steven M. Glaza Date Study Director Acute Toxicology HazletonWisconsin, Inc. Representatie Date QualityAssuance Unit Hazleton Wisconsin,Inc. (6329-138.protdsk2) 9.1.2 AnalyticalprotocolAMDT-122094.2 3M EnvironmentaLlaboratory Protoco-lAnalyticaSltudy Single-Dos-eIntravenouPsharmacokinetiSctudyofT-6054inRabbits In-VivoStudyReferenceNumber: HWI#6329-138 StudyNumber: AMDT-122094.2 TestSubstance:FC-129 (T-6054) 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 InitiationDate: 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, and Revisions: Thermal Extractionof Fluorideby Means of a Modified Dohrmann DX2000 Organic HalideAnalyzer-Liver FluorideMeasurement by Means of an Orion EA940 Expandable Ion Analyzer Extractionof Fluorochemicalsfrom Rabbit Liver Analysisof RabbitLiverExtractforFluorochemicalsUsing ElectrosprayMass Spectrometry Analysisof FluorideUsing the SkalarSegmented Flow Analyzer with Ion SelectiveElectrode Author: James D. Johnson Approved By: aSmatemu@edssy D 3-5-,Inson Study LWwee'c or Date John ButCnhoff,PhD Date Sponsor Representative 1.0 PURPOSE Thisstudyisperformedinordertoprovidienformatinoencessartyoassestshe extentof dermal absorptionof FC- 129 (T-6054)in a subsequentdermal absorption study,HWI#6329-133. The studyisdesignedto provideinformationas to whether FC-129 and its metabolitesaredetectableinliverand othertissuese,itheras totalorganicfluorineor as specificompounds when the FC-129 isadministeredas an intravenousdose;and to ascertainw,hether perfluorooctanatweillprovidea marker fordermal absorption. 2.0 TEST MATERIALS 2.1 Test,Contro 1,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:3 M ICP/PCP Division(2.1.1),grocery store(2.1.2,2.1.4liver)S,igma Chemical Company (2.1.22,.1.4-serum) 2.3Number of Test and Control Samples: Liverand serum from 4 testanimals and I controlanimal,otherbiologicatlissues(kidney,bile,cellulafrractionw)illbe availableforanalysisifdeemed appropriateby theStudy Director. 2.4Identificatioonf 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. 2.7 Storage Conditionsfor Test Materials:Room temperature(2.1.1), -20 IOOC (2.1.22,.1.4).Test and Controlsampleswillbe receivedaccordingto AMDT-S- 10-0. 2.8DispositioofnSpecimensB:iologictailssuaensdfluidwsilbleretainpeedr GLP Regulationforthetimeperiodrequiredforstudieslongerthan 28 days. This studyisinparallewlith a 28 day dermal absorptionstudyso alltissueswillbe retained. 2.9 Safety Precautions:Referto appropriateMSDS. Wear appropriatelaboratory attireU.se cautionwhen handlingknivesforcuttingthesamples. 3.0 EXPERTMENTAL -Overview The tissuesand serum from animalsdosed asdescribed(HWI#6329-138), are availablfeoranalysisforfluorinceompounds. At thediscretioonftheStudy Directora, seriesofanalyticatlestscan be performed.The screeningforfluoridein liverviacombustion(SeeMethods--nextSection)istheappropriataenalysisto presentdefinitivdeataforfluorinientheliver.For confirmatioonfthepresenceof specificompounds intissuesand serum,electrospramyass spectrometrycan be used. 4.0 EXPERIMENTAL -Methods 4.1 Liver and Serum screening methods: (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 of an Orion EA940 Expandable Ion Analyzer 4.1.3AMDT-M-4-0, Extractioonf Fluorochemicalfsrom RabbitLiver 4.1.4AMDT-M-5-0, AnalysisofRabbitLiverExtractforFluorochemicals Using ElectrosprayMass Spectrometry 4.1.5AMDT-M-8-0, Analysisof FluorideUsing theSkalarSegmented Flow Analyzerwith Ion SelectivEelectrode 3 5.0DATA ANALYSIS 5.1Data Reporting:Datawillbe reporteadsa concentrati(owneight/weigohft) fluorideper tissueor fluid,or as FC-95 (electrospramyass spectrometry)per unitof tissueor fluid. Statisticused,atthediscretioonf theStudy Director,may include averagesand standarddeviationsfrom differendtose groups.Ifnecessary,simple standardstatistictaelstssuch as the Student'sttestmay be appliedtodetermine statisticdailfference. 6.0 MAINTENANCE OF RAW DATA AND RECORDS 6.1 Raw Data and Records: Raw data,approved protocol,appropriatespecimens, approved finalreport,and electronidcatawillbe maintainedin theAMDT archives. 7.0 REFERENCES 7.1AMDT-S-10-0, Sample TrackingSystem 8.0 ATTACTMENTS 8.1AMDT-M-1-0, Thermal Extractionof Fluorideby Means of a Modified Dohrmann DX2000 OrganicHalideAnalyzer-Liver 8.2 AMDT-M-2-0, FluorideMeasurement by Means of an Orion EA940 Expandable Ion Analyzer 8.3 AMDT-M4-0, Extractionof Fluorochemicalsfrom RabbitLiver 8.4AMDT-M-5-0, Analysisof Rabbit LiverExtractforFluorochemicalsUsing ElectrosprayMass.Spectrometry I 8.5 AMDT-M-8-0, Analysisof FluorideUsing the SkalarSegme ntedFlow Analyzer with Ion SelectiveElectrode -4- 9 3M Environmental Laboratory Method Tbermal Extractionof Fluorideby Means of a ModifiedDohrmann DX2000 OrganicHalideAnalyzer- Liver Method IdentificatiNounmber: AMDT-M-1 RevisionNumber: 0 AdoptionDate: RevisionDate: None Author-RichYoungblom Approved by: roup Lead roUP Lt /,gDaie QualityAssurance Date Software:MS Word 5.1a AfrectedDocuments: AMDT-M-2 FluoridMeeasurementby Means ofanOrionEA940 ExpandableIonAnalyzer AMDT-EP-3 RoutineMaintenanceofa ModifiedDohnnann DX2000 OrganicHalideAnalyzer -1.0SCOPE. APPLICABLE COMPOUNDS, AND MATRICES 1.1Scope:Thismethod isfortheoperatioonfa DohrrnannDX2000 when itisusedtoextract fluoridferom variousmatrices.The fluoridiestypicallcyollecteidnTISAB solutiofnoranalysis withan ionselectiveelectrode. 1.2ApplicableCompounds: Fluorochemicalosrotherfluorinatecdompounds. 1.3Matrices:Biologicatlissuesp,articularlliyver. 2.0 KEYWORDS 261Fluoridef,luorinee,xtractiopny,rolysisi,or@zatioino,nselectiveelectrodeD,ohnnann, halide, DX2000, fluorochemicals. 3.0 PRECAUTIONS 3.1 Glasswareand exhaustgasescan be extremelyhot. 3.2Glasswareisfragileb,rokenglassmay causeinjuries. 3.3Pressurizedgases,propercompressedgas handlingpracticersequired. 3.4Solventbasedsamplesmay flashm,ay needtoallowthem todrydown beforestartinrgun. 3.5Potentiabliohazarddsue tothebiologicamlatrices.Use appropriatpeersonalprotective equipment. 4.0 SUPPLIES AND MATERIALS 4.1Compressed Oxygen, Hydrocarbonfree,regulatedto30 PSI. 4.2Compressed Helium,High PurityGrade,regulatetdo45 PSI. 4.3 Quartzglasssample boatwithTeflonl*'4tubiDnogh,rinann890-097or equivalent. 4.4Quartzglasscombustiontube,RelianceGlass G-9405-012 orequivalent. 4.5Orion940999 TotalIonicStrengtAhdjustmentBuffer(TISAB II)orequivalent. 4.6Sample collectiovnialsH,DPE. 4.7Nfilh-QTmwater 4.8Polystyrenpeipettes. 4.9ActivatedCharcoal,E.Merck 2005orequivalent. 4.10HamiltonSyringeor equivalent. 4.11Miscellaneouslaboratorgylassware 5.0 EQU PMENT 5.1Rosemount Dohr-inanDnX2000 OrganicHalideAnalyzer,modifiedforfluorideextraction. 5.2IBM compatible386 or486 computer. 5.3DX2000 softwarev,ersion1.00,modifiedforfluorideextraction. 5.4Excel Spreadshee4version5.0orgreater 6.0 INTERFERENCES 6.1 Sample sizeislimitetdoapproximatel1y50mg, dependingon samplemoisturecontent.This may varyfrom matrixtomatrix. 2 7.0 SAMPLE HANDLING 7.1Samples arenot to be handled withbarehands. Fluoridemay leachfrom theskintothe sample. Use forcepsor probe totransfertissues. 7.2Samples ofliverare cut from frozenliverand placedina taredand labeledweigh boat. Use a cleanscalpeland cuttingboard. The cuttingboard and scalpelshould be cleanedwithwater, methanol,or methanol-watersolutionaftereach liveriscut. 8.0 CALIBRATION AND STANDARDIZATION 8.1 PreparationofCalibrationStandards 8.1.1The standardrsequiredforeachprojecwtillneedtobe appropriatfeorthatindividuaplroject. Refer toprotocolforthatproject. 8.1.2Typically50-500 ppm FC-95 inmethanol standardsareused. 8.1.3For rabbitliverstudiesu,se beefliverasthematrix.Cut a pieceof frozenbeefliver(100 150 mg) and weigh itin a labeledand taredweigh boat. 8.2 Calibration- Overview The normal calibratioinsthe fluoridecurve (AMDT-M-2). However, ifan optionalspikedliver curve isrequiredthe procedurelistedbelow isused. 8.2.1 A calibratiocnurve forthe DX2000 isgeneratedby spikingsamples withknown standards and combusting them using the same methods and matrix typeas thesamples tobe tested. 8.2.2Typically,threereplicateosf each standardand fiveconcentrationosf standardswillbe spiked. 8.2.3 Standardcurve willbe plottedasMass Spiked F (ug)on thex-axisand StandardMass Recovered F (ug)on they-axis.Generatea regressioncurve and calculatteheequationfortheline and ther2 value. 8.2.4Mass SpikedF (ug)= (Amount spikedinmL) x ( Conc. of standardin ppm) x (0.6004)* *FC-95 is60.04% F therefore0.6004 isthe factorused toconvertFC-95 toF 8.2.5StandardMass Recovered F (ug)= (TISAB volume inmL) x (Orionreadingin ppm) 8.3Calibration- Procedure 8.3.1 Start Up 8.3.1.1Run 2 or more CleanCycleswhen startinignstrumenteachday. More cleancyclesmay be used ifthe previoussamples containedhighconcentrationosf fluoride. 8.3.2Blanks 8.3.2.1Prepare sample using thesame methods and typeof matrix asthe testsample. 8.3.2.2For rabbitstudies,use beefliveras thematrix.Prepare atleast3 samples ofbeef liver (100 - 150 mg) forblanks. 8.3.2.3Put sample in Dohrmann boat. Combust each sample as describedin section9.0 and analyze sample accordingtomethod AMDT-M-2 fortheion selectivelectrodeanalysis. 3 8.3.2.4Forrabbitstudiest,hemeter readingfora blanksample shouldbe 0.03ppm or lower beforeproceedingwiththecalibratioBnu.m samplesuntilthislimitisreached,or untilinthe judgement of theoperatorthereadingisstablewithrespecttohistoricraeladings(previous48 hours). 8.3.2.5For non-rabbitstudiest,heblankreadingsshouldreacha predeterminedionconcentration beforeproceedingwiththe calibration. 8.3.2.6Itmay be necessarytomix approximately50 mg of charcoalwiththesample toaid combustion. 8.3.3Standard Curve . 8.3.3.1Weigh outatleast15 matrixsamples (5 standardswith3 replicatesach)intaredand labeledweigh boats.For rabbitstudiesw,eigh 100-150 mg beefliversamples.Record weightsin studydata.Storethematrixsampleson dry iceor icepacks tokeep them frozenuntilused. 8.3.3.2Placeweighed beefliversample in Dohrmann sample boat. 8.3.3.3StartwiththeloweststandardconcentrationU.sing a Hamiltonsyringe,ejecta fixed quantityof thestandardon orin thematrix.For rabbitstudiesu,se4 uL ofstandardand ejectiton or inthebeefliver. 8.3.3.4At least3 replicatesshouldbe used forthelowest standardconcentrationm;ore replicates may be used atthediscretioonftheanalyst. 8.3.3.5Combust thesample asdescribedin section9.3and analyzeaccordingto AMDT-M-2. 8.3.3.6Run all15 standards.Ifone replicatiessignificantdliyfferenftrom theothertwo replicatersu,nanothersample forthatstandard.Indicateindatathatthenew replicatreeplacesthe oldreplicataend thatthenew replicatweillbe used tocalculatteheregressiocnurve. 8.3.3.7When allstandardshave beenrun,calculattehe r2. r2must be atleast0.95. Ifitisnotat least0.95,consultwith supervisor. 8.3.3.8A new standardcurveshouldbe run when thecombustiontubeor sample matrixis changed. New standardcurvemay alsobe run atthediscretioonf theanalyst. 8.4 StorageConditionsforStandards 8.4.1Storagerequirementsforstandardsaredependent on theindividuasltandardsused. Typically,standardsarestoredatroom temperatureinplastisccrew topbottles. .8-4.2New FC-95 standardsshouldbe preparedatleastonce a month. 9.0 PROCEDURES 9.1TypicalOperating Conditions: 9.1.1Combustion tubetemperatur=e 9500C. 9.1.2Oxygen and Helium flow= 50 cc/minute. 9.1.3Vaporization/Dryintgime= 240 seconds. 9.1.4Bake time = 300 seconds. 9.2StartUp Procedure: 9.2.1Iftheprogram isnot starteds,tartheEOX program on thePC. 9.2.2Open theSYSTEM SETUP window. 9.2.3Put thefurnacemodule and thecellintheREADY mode. 9.2.4Closethe SYSTEM SETUP window. 4 9.2.5When theoven has reached theREADY temperature,run theCLEAN found intheCELL CHECK menu. 9.2.6 See AMDT-EP-3 fordetailosf theDohrmann software. BOAT program 9.3Sample ExtractionProcedure: 9.3.1Open the SAMPLE HATCH and placethesample in theBOAT. Itmay be necessaryto mix approximately50 mg ofcharcoalwith thesample toaidcombustion. Ifthisisdone,charcoal should alsobe mixed inwhile establishintghebaselineand when generatingthestandardcurve. 9.3.2 Close SAMPLE HATCH. 9.3.3 Add appropriatevolume ofTISAB solutionor 1:1TISAB:Milli-QTIlwater mixtureto a labeledsample collectionvial.Typically0.6mL to 15 mL are used. For rabbitstudiesu,se 1.0or 2.0mL of 1:1 TISAB:Milli-QTm watermixture. 9.3.4 Place thevialso thatthetipof theCOMBUSTION TUBE isintheTISAB atleast0.25 inches.Gases releasedduringpyrolysismust bubble throughthe TISAB. 9.3.5 Run theEOX-SOLIDS program found intheRLTN menu. 9.3.6 When theEOX program isfinishedr,emove the collectiovnialfrom the combustion tube. 9.3.7 IfundilutedTISAB was used tocollecthe sample,add an equalvolume of Nfilli-wQaTtemr totheTISAB tomake 1:1 TISAB:Mlli-QTm. 9.3.8Rinse theend of the combustion tubewith Milli-QTmwaterand wipe with a KIMWIPE to remove any TISAB remaining on the tube. 9.3.9 Open the sample hatch and remove any remaining ash from theboat. Ash can be removed with a cottonfippedapplicatoror vacuumed out. Itmay be necessarytoscrapparticleosffthe bottom with a spatulaor othersimilardevice.A drop of Mlji-QTM water may be added tothe boatto aidin theClean Cycle. 9.3.10 Close thehatch. 9.3.11 Run the CLEAN BOAT program. 9.3.12 Sample isready foranalysisby ionselectiveelectrode(AMDT-M-2). 9.4 Sample Calculations 9.4.1 Use the standardcurve tocalculatethesample value. 9.4.2 Sample Mass Recovered F (ug) (TISAB volin mL-)x (OrionreadinginRpm - intercept) (Slope) 10.0 VALIDATION 10.1 QualityControl 10.1.1DailyStartUp Cbeck Samples:Once thestandardcurveisestablisheeda,chday of analysisisstartedby analyzingQC samples. The QC samples aretobe the same as the lowest concentrationspiked samples used togeneratethe standardcurve. Each concentrationmust be done intriplicatuenlessthe fimttwo replicateasre within20% of thestandardcurve,thena third replicateisnot necessary. 10.2 Precisionand Accuracy: See method development analysisand sample analysisin FluorideNotebooks 2,3,and 5. Precisionand accuracy varieswhen analyzingsamples of different matricesand differentreferencecompounds. 10.3 Other ValidationParameters: NA 5 11.0 DATA ANALYSIS 11.1Calculations 11.1.1For thestandardcurve,useregressioannalysiisnExcel,version5.0orgreater. 11.1.2To calculattehefluoridceontractioinnthesample,seemethod AMDT-M-2. 11.2 Analyzing theData ll,.2.r12must be atleast0.95orgreater".Outliersm"ay be excludediftwo ofthethreereplicates arewithin20% ofeachotherand theoutlieirsgreaterthan200% oftheaverageofthosetwo or lessthan50% oftheaverageofthosetwo. Any suchoutliersshouldbepointedoutinthedataand notedintheFinalReportalongwiththereasonitwas consideredan outlier. 12.0 ATTACHMENTS. None 13.0 REFERENCES 13.1Rosemount Dohrmann DX2000 OrganicHalideAnalyzerOperator'Msanual (Manual915349,revisioBn, December 1993) 13.2 ANMT-M-2 FluorideMeasurementby Means ofan OrionEA940 ExpandableIon Analyzer 13.3AMDT-EP-3 RoutineMaintenanceofa ModifiedDohrmann DX2000 OrganicHalide Analyzer 14.0 REVISIONS Revision Numbe Reason forChange Revision Date 6 3M Environmental Laboratory Method FluorideMeasurement by Means ofan Orion EA940, Expandable Ion Analyzer Method IdentificatiNounmber: AMDT-M-2 RevisionNumber: 0 AdoptionDate: 10-4-fsRevisionDate: None Author.Rich Youngblom Approved By: G@, Leader Date QualityAssurance Date Software:MSWord5.la AffectedDocuments: AMDT-M-1 ThermalExtractioonfFluorideby Means ofa Modified Dohrmann DX2000 OrganicHalideAnalyzer 1.0 SCOPE, APPLFCABLE COMPOUNDS, AND MATRFCES 1.1SCOPE: Thismethod isforthecalibratiaonnd operatioonfan OrionEA940 Expandable Ion Analyzer. 1.2APPLICABLE COMPOUNDS: Fluoride. 1.3APPLICABLE MATRICES: LiquidsamplesinanappropriabtueffersolutionP.referred pH of 6.0. 2.0 KEYWORDS 2.1 Fluoridef,luorinei,onselectiveelectrode 3.0 PRECAUTTONS 3.1No hazardisdentifwiietdhthimsethod. 4.0 SUPPLIES AND MATERIALS 4.1Orion940999 TotalIonicStrengtAhdjustmentBufferIl(TISABII)or equivalent. 4.2OrionModel 900001 electrodfeillinsgolutio(nAgCl)orequivalent. 4.3Orion 940907 100ppm fluoridsetandardorequivalent. 4.4Nfilli-QTwmateror equivalent. 4.5Magnetic stibrars. 4.6Lab tissues. 4.7Sample collectiovnials. 4.8Plastic100mL volumetrifclasks. 4.9Polystyrenepipettes. 4.10Miscellaneouslaboratorgylassware. 5.0 EQUTPMENT 5.1OrionModel EA940 ExpandableIonAnalyzeror equivalent. 5.2Orion Model 960900 SolidStateCombinationFluorideelectrodoerequivalent. 53 MagneticStirPlate. 5.4MM compatible386 or486 computer(onlyneededifusingOrion3E software). 5.5OrionRS232 interfacceable(onlyneededifusingOrion3E software). 5.6MicrosoftExcel5.0(ordyneededifusingOrion3E software). 6.0 INTERFERENCES 6.1ItisreconunendedthatthepH be atornear6.0.A 1:1 mixtureofTISAB and sample/MilliQTNIwaterwillgenerallbyringsampletopH of6.0. 6.2Sample temperaturemay effectfluoridmeeasurement. ftisrecommended thatthesample be atroom temperatureas thestandardswere when themeterwas cali'brated. 6-3The ratethesamplesarestirreadtshouldbeconsistenwtiththeratethestandardswere stirred. 2 6.4Air bubblestrappedunder electrodcean give erroneousreadings.Make sureno airistrapped under electrode. 7.0 SAMPLE HANDLING 7.1 No specialhandlingnecessary. 8.0 CALIBRATION AND STANDARDIZATION 8.1PreparatioonfCaUbrationStandards S.1.1Measure50riLofTISAB IIinto5 100mL plastivcolumetrfilcasks. 8.1.L2abeltheflaskass0.050,.1,0.5,1.0a,nd 1.5ppm F-,alongwiththedateandyourinitials. 8.1.P3ipett0e.050,.1,0.5,1.0a,nd1.5niLof100ppm fluorisdteandaridntotheappropriately labeledflasks. 8.1.4Add approximately30 mL of Milli-QThwiatertoeach flask. 8.1.5Shake theflaskstomix thesolutions. 8.1.6Eliminateairbubblesfrom theflasksby tippingtheflaskson theirsidesand rollingtheairin theflasksover theairbubbles. 8.1.7Bringthevolume intheflasksup tothe 100 mL mark withNfini-QT14Water. 8.1.8Invertand shake theflasksforthefinamlixing. 8.1.9Record standardsin StandardsLog Book. 8.2 Calibration 8.2.1Ifnecessary,remove tapefrom electrodfeillinhgole. 8.2.2Invertprobe to wet topseal. 8.2.3Ejecta few dropsof fillinsgolutionfrom bottom of electrodteo wet lowerseal. 8.2.4Filltheelectrodweithfillinsgolution. 8.2.5The meter and theF- electrodearetypicallcyalibratebdy directmeasurement with no blank correctionu,singstandardswith concentrationosf 0.05,0.1,0.5,1.0,and 1.5ppm F-,following themanufacturer'isnstructions. 8.2.6Record theslopeintheappropriatleogbook. 8.2.7Cleantheelectrodbey rinsingwithMilli-QTMwaterand wipingthesidesdown with lab tissues. 8.3 StorageConditionsforStandards 8.3.1Calibratiosntandardsare storedat'roomtemperature. 9.0 PROCEDURES 9.1Calibrationand Measurement, Standard method: 9.1.1The sample to be measured needs tobe mixed withTISAB usingtheproportions recommended by theTISAB manufacturer. 9.1.2Placea stirbarinthesample and placethesample on thestirplate. 9.1.3Allow thesample tomix fora few secondsbeforeinsertintgheelectrode.When the electrodeisinsertedm,ake surethereareno airbubblestrappedundertheelectrode. 9.1.4The sample shouldbe thesame temperatureasthecalibratiosntandardsand stirreadtthe same rateas thecalibratiosntandards. 9.1.5When thereadingshave stabilizerde,cordthereadingintheappropriatelogbook. 3 9.2CalibrationAnd Nleasurement,Using Orion 3E Software: 9.2.1Calibration: ,9.2.1.F1ollow steps8.2.1to 8.2.4. 9.2.1.2PressFunctionKey #8 (F8). 9.2.1.3The computer screenwillask you toconfirmthenumber of standardstobe used, concentratioonf thestandardsa,nd whetherornota blankistobe includedinthecalibration. Make any necessarychanges totheinformationpresentedand clickon CONTINUE. 9.2.1.4Placetheelectrodein thefirssttandardon thestirplateand clickon CONTRQLTE. 9.2.1.5Observe thereadingson thegraphicdisplayon thecomputer. When thereadingshave stabilizepdr,essACCEPT READING. 9.2.1.6Repeat step9.2.1.4and 9.2.1.5fortheremairdngstandards. 9.2.1.7Afterthefinalstandardt,hecomputerwilldisplaytheslopeof thecurve,as wellas the intercepatnd correlationR.ecord theslope,intercepta,nd correlatioinntheappropriatelogbook and clickon CONTINUE. The calibratidoantaisautomaticallcyopiedtoC:\Orion\Data\Calib.txt. 9.2.2Data Spreadsheet: 9.2.2.1SelecteitherNEW orOPEN from theFILE menu toopen a new orexistinsgpreadsheet tostoredatain. 9.2.2.2Record thename of thespreadsheetused intheappropriatleogbook. 9.2.3FluorideMeasurement: 9.2.3.1Follow steps9.2.1through9.2.4 9.2.3.2Enterthename ofthesample intheappropriatpelaceon thescreen. 9.2.3.3Clickon theNEW SANFPLE button 9.2.3.4When thereadingshave stabilizecdl,ickon theRECORD buttonand writetheresultinthe appropriatleogbook. 10.0 VALIDATION 10.1 QualityControl: 10.2 Precisionand Accuracy 10.3 Other ValidationParameters AccordingtoReference13.2,therangeof detectioins0.02 ppm fluoriduep toa saturatesdolutioonf fluoride. 11.0 DATA ANALYSIS 11.1 CalculationsNone necessary. 11.2 Analyzing the Data None necessary. 12.0 ATTACHMENTS None 13.0 REFERENCES 13.1Orion Model EA940 ExpandableIonAnalyzer InstructioMnanual,Orion Research Incorporated1,991. 13.2Orion Model 960900 SolidStateCombinationFluorideElectrodeInstructioMnanual, Orion Research Incorporated1,991. 14.0 REVISIONS Revision Numbe Reason-forChanp-e Revision Date 3 EnvironmentaLlaboratory Method Extractionof Fluorochemicalsfrom Rabbit Livers SOP IdentiricatioNnumber: AMDT-M-4 RevisionNumber: 0 Adoption Date: RevisionDate: None Author.Dave Christenson/CyndiWieaber Approved By: up Leader /0 - 31Dam Qualit@Assurance Date Software:MS Word, 6.0 AffectedDocuments: M-5, Analysisof RabbitExtractforFluorochemicalsUsing Electrospray Mass Spectroscopy. ,LO SCOPE 1.1 1.2 1.3 Scope: This method isfortheextractioonf fluorochemicalfsrom mbbit livers. Ethylacetateisusedtoextracftluorochemicalfsrom theliversforanalysisby electrospraymass spectroscopy. ApplicableCompounds: Fluorochemicalsor otherfluorinatecdompounds. Matrices:RabbitLivers. 2-0 KEYWORDS 2.1 Fluorochemicalsr,abbitliverse,lectrospramyass spectrometerf,luorinated compounds, extraction. M PRECAUTIONS 3.1 Use gloveswhen handlintgherabbiltiverst,heymay contaipnathogens. 4.0 SUPPLI]ES AND MATERIALS 4.1 Supplies 4.1.1 Syringe,capableofmeasuring 100 liL 4.1.2 Eppendorf typeordisposablepipets 4.1.3 Gloves 4.1.4 Plasticgrindingtubes 4.1.5 Plasticentrifugteubes,15 mL 4.1.6 Labels 4.1.7 Nitrogen 4.1.8 Timer 4.1.9 FiltersT,itannylonsyringefilter0s.,2gm. 4.1.10 Analyticaplipetsg:lassvolumetricpipets. 4. 1.11 Disposableplasti3c cc syringes. 4. 1.12 Crimp cap autovials. 4.2 Reagents 4.2.1 Aqueous Ammonium Acetate(Aldrich)a,pprox.250 ppm: Prepar-ae2500 ppm aqueoussolutionof ammonium acetateby adding250 mg annnonium acetatetoa 100mL volumenicflaskand dilutetovolume withMiffi-Q water.Dilutethissolution1:10 fora 250 ppm solution. 4.2.2 Sodium carbonate/SodiumBicarbonateBuffer(J.T.Baker), (N%CO3/NaHC03)0.25 M: Weigh 26.5g of sodium carbonate(Na2CO3) and 21.0g of sodium bicarbonat(eNAHCO@ intoa IL volumetricflaskand bringtovolume withNUI-Q water. 4.2.3 Diluteacetonitdlseolutiond,iluteacetonitri1l:e1 withMilli-Qwater. 4.2.4 EthylAcetate 4.2.5 Methanol 4.2.6 Milh-Q water 4.2.7 IH,IH,2H,2H -perfluorooctanesulfoanciicd(Aldrich) 4.2.8 FC-95 (3M SpecialtCyhemicalDivision) 2 5.0 EOUIPMENT 5.1 Ultra-Tun-aTx25 Grinderforgrindingliversamples. 5.2 Vortexmixer 5.3 Centrifuge 5.4 Shaker S.5 AnalyticaElvaporator 6.0 INTFRFERENCES 6.1 There areno known inteiferenceastthistime. 7.0 SAMPLE HANDLING 7.1 Ile rabbitliversarereceivedfrozen,and must be keptfrozenuntiltheextractioins performed. 8.0 CALIBRATION AND STANDARDIZATION 8.1 Preparationof InternalStandards 8.1.1 Preparean internasltandardofapproximately12 ppm IH,IH,2H,2Hperfluorooctanesulphonaiccidtobe added toeach liversample. 8.1.2 Weigh atleast0.1 g of 1H,IH,2H,2H-perfluorooctanesulphonaiccidintoa 100 mL volumetricflaskR.ecord theactualweighl 8.1.3 Bring itup tovolume withmethanol,thisisthestockstandard. 8.1.4 To a 250 mL volumetricflask,add 3 mLs of thestockstandardand bringto volume with Milli-Qwater.Calculatteheactualconcentratioonf the standard. actualmg perfluoroctane- sulphonicacid X 0.1 L 3 mL 250 mL actualconcentrationp,pm 8.2 Prepare FC-95 Anion Standards 8.2.1 PreparFeC-95 standardfsorthestandarcdurve. 8.2.2 Weigh approximately100 mg of FC-95 intoa 100 mL volumetricflask. Record theactualweight. 8.2.3 Bringup tovolume withdiluteacetonitrile. 8.2.4 Dilutethesolutiownithdiluteacetonitdl1e:10 fora solutioonf approximately100 ppm. Dilutethissolution1:10 withdiluteacetonitdlfeor a solutionof approx.10 ppm. 8.2.5 Use the10 ppm solutiontomake working standardswithvaluescloseto 5.0ppm, 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 Nalgenebottlceontaining 200 mLs Mlli-Q water.Grind toa homogenous solution. 8.3.2 Add Iffilof thesolutiontoa 15 mL centrifugteube.Preparea totalofeight I mL aliquotosf thesolutionin 15 mL i!entrifutguebes.Be suretoresuspendsolutionby shakingitbetween aliquots. 8.3.3 SpikesevenoftheI niL aliquotwsiththefollowingamounts ofworldng standardsin step9.12of theprocedure.One I mL aliquotservesas the blank. Worldng Ytanda-rd (ApproximateConc.) 500 ppb 500 pp 500 ppb 5M ppb 1 P,pm 5 ppm 5 pTm UL ioo2U5 300 400 500 200 300 Approximate final concentratioonf FC-95 inliver Blank 0.292 ppm 0.584 ppm ---v877 ppm 1.168 ppm 2.924 ppm 5.848 ppm 8,772 ppm 8.4 Calculatetheactualvalueofthestandards: uL ofstandardx concentratio(ninppm) 171 mg liver/Inilhomogenate finacloncentratio(nppm) ofFC -95inliver *Average weightof bovineliverinsolutionasdeterminedby weighing I mL homogenatesof40 mg liverin200 mL ofMiUi-Q water.Ile amount of FC-95 isreportedas equivalentosf FC-95 potassiumsalt. 8.5 Calibration 8.5.1 Extractthespikedbeefliverhomogenate following9.13 to9.23of this method.Use thesestandardstoestablisyhour curveon themass spectrometer. 8.5.2 Alternativelay,standardcurve may be generatedusingratiosofresponses of theperfluorooctansulfonaatneionand theinternasltandardanionversus concentratioonf theperfluorooctanesulfonaatneion. 8.6 Storage Conditionsfor Standards S.6.1 New standardasrepreparedwitheachanalysiSst.andardasrestoreidn coveredplasticentrifugteubesuntiltheanalysison themass spectrometer isperformed. 8.7 Storage Conditionsfor Standards 8.7. 1 Beef liverhomogenatesmay be frozenafterpreparation. 9.0 PROCEDURES 9.1 Obtainfrozenliversamples.Inspenttissuen,ote thattheliverhasnot been packaged withothertissues. 9.2 Use a dissectinsgcalpeland cutoffapproximaie-lIyg ofliver. 9.3 Weigh thesampledirectliyntoa taredplastigcrindingtube. 9.4 Record theliverweightinthestudynotebook. 9.5 Put a labelon thevialwiththestudynumber, weight,rabbitID,dateand analyst initials. 9.6 Add 2.5mLs water. 9.7 Grind thesample.Put thegrinderprobe inthesample and grindforabout2 minutes,untilthesample isa homogeneous solutionwithno largechunks. 9.8 Rinse theprobe offintothesample with2.5mLs waterusinga pipet. 9.9 ' Take thegrinderapartand cleanitwith methanolaftereach sample.Follow AMDT-EP-22. 9.10 Cap thesample and vortexfor15 seconds. 9.11 PipetI mL intoa 15 mL centrifugteube.Label thecentrifugteubewiththeidentical infonnatioans thegrindingtube.(SeeAMDT-M-4 Worksheet fordocumentingthe remainingsteps.) 9.12 Spikethebeefliverhomogenateswith theappropriataemount of FC-95 standard as describedin 8.3. 9.13 Spike thesamplesand beefliverhomogenates with 100 uL of intemalstandard. 9.14 Add 1 mL of thesodium carbonate/sodiumbicarbonatbeufferand I mL arm-nonium acetate. 9. 5 Using an analyticaplipet,add 5 mL ethylacetate. 9.16 Cap thesample and vortex20 to30 seconds. 9.17 Put them intheshakerfor20 min. 9.18 Centrifugefor20 to25 minutes,untilthelayersarewellseparatedS.etthepower on thecentrifugteo25. 9.19 Remove 4 n-ilosf thetoporganiclayertoa fresh15 mL centrifugteubewitha 5 mL graduatedglasspipet.Transferthelabeltothefreshtube. 9.20 Blow thesample down on theanalyticaelvaporatortoneardrynesswithnitrogen, approximately30 to40 minutes. 9.21 Bringtheremainingsample up in 1 mL diluteacetonitdlweithan analyticaplipet. 9.22 Vortex 15 seconds. 9.23 Transferthesample toa 3 mL syringe.Attacha 0.2pm nylon mesh filtera,nd filter thesampleintoa fr-eschentrifugteubeor a autovialL.abel thetubeor vialwith the studynumber and animalnumber. 9.24 Cap and hold foranalysisby electrospramyass spectroscopy. 9.25 Complete AAMT-M-4 worksheetand attachtopageof studynotebook. 10.0 VALIDATION 10.1 QualityControl- notapplicable 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 AMDT-EP-22 RoutineMaintenanceofUltra-TuiTaTx-25 14.0 REVISIONS Revision Number Reason forChanEe Revision Date Study -sample Number set _muk f,iver Worksheet AMDT-M-4 FC-95 approx0.5ppm actual ppm - 70t)iiT. 100 uL 4(10iit, FC-95 approx I ppm actual ppm #W 500111, FC-95 approx.5 ppm actual PPM Date and InitialfsorStd. 200111, qudy ntimher where tht-nrioin-awlorkqheet igfricatpdqpetnfprt-2 cnny. I I- I I -- T,iverP.Ytrae-fiPnmnr -"Q. PIWA I mT. of T,iverRnliitin?i Pinet100 iiLnf 12 nntnInternaqltgntinrd I;td#- Vnrtt-.x115qt-e. Pinet I MT. nf 750 nnm Ammoniurn Aretate Igtel# Pinet I mT. nf 0-25 NsLrO./O.25M N;THCO. Ruffer Pinet15mT. nf Pthyl Are"tL-- Vnrtt-Y20@30 -gec gb;ike20 min rt-13trifMi2l5m min- Remnyt-n 4 mT. ;tlioinifntnrpanirlavt-.r- Rlow dnwn tonegrdrynegg(<o 25 mL) withN. Add I m- nf 1-1Aef!tnnitrilt-/14.0 TN# T)atik- Initinl-q Filteurrinaa IcC R-T)gyrincrweitha 0-2um ';Rlfilteirntoa 1-5ml, attto-g:lmvniIaL], 6 3M Environmental Laboratory Method Analysisof Rabbit Liver Extractfor Fluorochemicalsusing Electrospray Mass Spectroscopy SOP IdentiricatiNounmber: AMDT.M.5 RevisionNumber: 0 Adoption Date: RevisionDate: None Author-Dave Christenson/CynthWieaber Approved By: roupLeader Date QualityAssurance Dam Software:MS Word, 6 0 AffectedDocuments:M-4, ExtractioonfFluorocberiiicfarlosm RabbitLivers 1.0 SCOPE 1. 1 1.2 1.3 Scope: Thismethod isfortheanalysisof extractosf rabbitliveror othertissuesor fluidsforfluorochemicalussingthe electrospramyass spectrometerT.he analysis isperformedby singleionmonitoringof FC-95 anion,M/Z= 499,theinternal standardM/Z = 427, and otherappropriatemasses. ApplicableCompounds: Fluorochemicalsor otherfluorinatecdompounds. Matrices:RabbitLivers(samples)B,eef Liver(standardso)t,hertissueasnd fluids. 2.0 KEYWORDS 2.1 Fluorochemicalsf,luorinatecdompounds, electrospramyass spectroscopym,ass spectrometerr,abbitlivers. 3.0 PRECAUTIONS 3.1 Use cautionwiththevoltagecablefortheprobe.When thevoltagecableisplugged intotheprobeDO NOT TOUCH THE PROBE, thereisriskof electricsahlock. 3.2 Do notrun thepump above it'scapacityof 4000 psi.Ifpressuregoes over4000 psi stopand releasepressuren.e peak tubingmay be pluggeclTroubleshootback to findtheplugand replacethepluggedtubing.See AMDT-EP-15 3.3 Do not run thepump todryness. 4.0 SUPPLIES AND MATERIALS 4.1 Supplies 4.1. 1 Nitrogengasregulatedto 140 psi. 4.1.2 Fluofixcolumn or equivalenl . 4.1.3 100 uLor250 uL flatipsyringeforsampleinjection. 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 T:do 2000 ElectrospraMyass Spectrometeror equivalent. 5.2 ISCO SyringePump 5.3 SpectraphysicAsS300 Autosampler 5.4 100 uL Assembly 5.5 Autovialsorcapped centrifugteubes. 6.0 INTERFERENCES 6.1 'Mere areno known interferenceastthistime. 7.0 SAMPLE HANDLING 7.1 Keep theextractedsamplesincapped 15 mL centrifugteubesor incapped autovials untilready foranalysis. 2 8.0 CALIBRATION AND STANDARDIZATIQN 8.1 Preparationof CalibrationStandards 8. 1. 1 Seven beefliverstandardsand one blankbeefliverarepreparedduringthe extractiopnrocedure(.SeeAMDT-M-4, section8.0) 8.2 Calibration 8.2. 1 Run thesevenbeefliverstandardstwice,startinwgiththeloweststandard toobtainthestandardcurve. 8.2.2 Typicallyone standardisrun aftereach 5 to7 samples.Choose a standard inthesame rangeof concentratioans thesamples. 8.3 Storage Conditionsfor Standards S.3.1 FreshstandardasrepreparedwitheachanalysiSst.andardsarestoredin coveredplasticentrifugteubesuntiltheanalysiosn themass spectometeris performed.Samples and standardsareNOT refrigerated. 8.4 Storage Conditionsfor Beef Liver Homogenates 8.4.1 Beeflivehromogenatemsay befrozenafteprreparation. 9.0 PROCEDURE 9.1 InitialSet-up 9.1.1 Set softwareto"Operateon",Ion Mode ES-. 9.1.2 Record backingpressureintheinstrumentlog. 9.1.3 Fillthesolventcylinderwithmobilephase. 9.1.4 Setthepump to"Run". Settheflowto 1000 ul@min.Observesdroplets coming outofthetipof theprobe.The pressureshouldbe at 1700 to 1800 psi. 9.1.5 Check thefusedsilicattheend of theprobe.Use an eye piecetocheck for chips.The tipshouldbe flatwithno jagged edges.Ifany chipsarefound cutoffthetipofthesilicwaitha column cutterand pullthesilictahroughto the appropriatleength. 9.1.6 Check your nitrogensupply.Turn on thenitrogenT.here shouldbe no nitrogenleakingaround thetipof theprobe.A finemistshouldbe coming outof thetip. 9.1.7 Carefullyguidetheprobeintotheopening.Inserittuntilitwon'tgo any furtherC.onnect thevoltagecabletotheprobe. 9.1.8 Go tothe"Editor"page,and setlonizadonMode toES-,and the appropriatemasses to427 and 499. 9.1.9 Ifitisnotinsingleionmode go to"Option"and setSIR. 9. 1.IOStartAcquisitionA.ssigna filename, MO-DAY-YR + letteRre.cord itin thelogbook. 9.1. 11 Run thebeefliversamplesfirstr,unningeach standardtwiceatthe beginningof therun..Run a QC check by runningone standardafterevery 15to7 samples. 9.2 Manual Injection 9.2. 1 Draw 150 uL of sampleintoa syringeI.njecthesample intotherheodyne injectiopnort.Injecstlowly.Record thesampleID inthelogbook. 9.2.2 Turn thevalveto"On". 9.2.3 Wait two minutes,and injecthenextsample. 9.2.4 Record thescannumber foreach sample inthelogbook. 9.3 Using the Autosampler 9.3.1 Set up sample trayA, B, or C. 9.3.2 Record thesamples and theirpositionsinthe instrumentlog book. Up to 17 vialsmay be ineach run. 9.3.3 Set-up thesampler: 9.3.3.1 Push thesample button 9.3.3.2 Set sample loop size= 100 uL 9.3.3.3 Setinject/sample= 2 9.3.3.4 Set Cycle time= 0 9.3.3.5 9.3.3.6 Name thefileL:ivers Identifythetrayused 9.3.3.7 9.3.3.8 Add thesamples toQueue by pressing"Enter" Pmss "Run" tostart 10.0 VALIDATION 10.1 Quality Control 10. 1.1 Run a standardevery5 to7 samples.Ifa significancthange( 50%) in peak heightoccursstoptherun.Only the samples beforethelastacceptable standardwillbe used.The remaining samples willbe reanalyzed. 10.2 Precision and Accuracy 10. 2. 1See Method ValidationReportnumber AMDT-M-5.0.V 1 10.3 Other ValidationParameters 10.4 RefertoMethod ValidatioRneportNumber AMDT-M-5.0.Vl 11.0 DATA -ANALYSIS 11.1 11.2 Calculations Plotthe standardcurve,usingthemean of thetwo valuesobtainedforeach standard. 11. 2. 1Read peak heightsor areasforthe samples from theprintout.Use linear regressionto determine thesample concentrations. 11. 2.2 Calculatethemg of PC-95 anion,or otherfluorochemicalinthe totalrabbit liver mg FC-95 anioninthetotalrabbitliver m g FC-9-5anion from-std.curvt, gms ofliverused foranalysis x Totalmass of liverg,ms 11. 3 11. 4 Make a resultstableand enteritinthe studybook. Printa chromatogram foreach sample,with thepeaks labeledwith thesample or standardD:).Write thestudynumber on the p-n'ntouitn,itiadla,te,and put itinthe studyfolder.Stapleallchromatograms togetherand number pages. 12.0 ATTACHMENTS None 13.0 R FERENCES - 13.1 AMDT-EP-17 14.0 REVISIONS - Revision Num Reason f-gr-gh-a-nm Revision Date 3M Environmental Laboratory Method Analysisof FluorideUsing the Skalar Segmented Flow Analyzer With Ion SelectiveElectrode Method IdentiricatiNounmber: AMDT-M-8 Adoption Date: RevisionNumber: 0 RevisionDate: None Author Deb Wright/CynthiaWeber ApprovedBy: up Leader Date QualityAssurance Dam Software:EBM MS Word, 6.0 AffectedDocuments: AMDT-EP-26, OperationandMaintenanceof theSkalarSegmentedFlow Analyzer 1.0 SCOPE 1. 1 1.2 Thismethod isfortheanalysisforfluoridet,hermalleyxtractefdrom s=ple s using theDohrmann DX2000 (AMDT-M-1), and collecteidnTISAB foranalysiswithan Ion SelectiveElectrode(ISE).The analysisisperformed usingtheSkalar Segmented Flow Analyzer with ISE. Samples can be tissuess,erum, biologicalmaterial,orothermaterialsextractedon theDohrmann. 2.0 KEYWORDS 2.1 Skalar,segmented flow,fluoride. 3.0 PRECAUTIONS 3.1 Follow standardlaboratorysafetypractices. 4.0 SUPPLIES AND MATERIALS- 4.1 Supplies 4.1.1 Sample cups,4 mL plasticups withcaps 4.1.2 Autopipets,oxfordor equivalentwithplastictips 4.1.3 Polypropylenevolumetricflasks,loo ML 4.1.4 Cartridgecomponents,refertotheSkalarMethods 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 IIbuffersolutionP:urchaseTISAB IIfrom Orion.To 1 liteorf TISAB 11add 2.5 mL or 100 ppm fluoridesolutionand 1 mL Brij. 4.2.3 Sampler rinsingsolutionD:iluteTIS AB 111:1 withNtlli-Qwater. 4.2.4 Nitriaccidsolutiofnordecontaminatio1nN, (labgrade):Slowlyadd 64 mLs concentratenditriaccid(HN03)to250mLs ofNfilliw-aQterB.ring thevolume up toIL withMilli-Qwater. 4.3 Standards 4.3.1 Stocksolution1,00 ppm F:purchasedfrom Orion. 4.3.2 Intermediatsetandard1,0 ppm: Dilute10 mLs ofstocksolutiotno 100mLs withNfilli-wQater.Use polypropylenveolumetrifclasks. 4.3.3 Working standardM:ake up thefollowingworkingstandardbsy addingthe volumes ofintermediatoerstockstandardindicateodn thetableu,sing oxfordorpumpmate pipetst,o50 mLs ofTISAB and dilutintgo100 mLs withMilli-Qwater. Working Standard 0.015ppm --- 0.03ppm 0.06ppm 0.09 ppm mLs ofStockStandard - MLS of IntermediatSetandard 0.15 0.3 0.6 0.9 0.12 ppm 1.2 0.15 ppm - 1.5 0.3 ppm 0.3 0.6 ppm 0.6 __j 2 1.2 ppm 1.2 1.5 ppm 1.5 5.0 EQUIPMENT 5.1 SkalarSegmented Flow Auto Analyzer Sanspl"System equipped with ISE 6.0 INTERFERENCES 6.1 High concentrationosf alkalinityc,hloride,phosphate,sulfateor ironcan cause interferences. 7.0 SAMPLE HANDLING 7.1 Samples should be storedinpolyethylenebottlesS.amples should be analyzed within30 days. 8.Q CALTBRATION AND STANDARDIZATION 8.1 Preparation of CalibrationStandards 8.1.1 Prepare calibratiosntandardsas in section4.3. 8.2 Calibration 8.2. 1 The standardsareanalyzed atthebeginningof therun. 8.3 Storage Conditions for Standards 8.3. 1 Standardsarestoredincapped polypropylenevolumetricflasks.New standardsarepreparedata minimum of ever-ysixmonths, 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 thefluoridelectrodesin theelectrodechamber. 9.1.3 Turn on thepower of thesampler,pumps, offsetpotentiometerand headng bath. 9.1.4 Put thereac,,ent-liinnestheappropriatebottles. 9.1.5 Turn on theinterfacec,omputer,displayand printerM.ake sure you turn on the interfacebefore the computer. 9.1.6 Letthesystemstabilifzoerapproximatel3y0 minutes. 9.2 Startinga Run 9.2.1 Createa sample tableby selectinFgILES, TABLE, and CREATE, type in thename of thefilea,nd pressENTER. 9.2.2 Printthesample table,insertedinthe system tableby pushing ESC, PRINT, GROUP 1.This willprinttheentirerun. 9.2.3 Dialthe samplersettingstotheappropriatenumber of samples,number of seconds forsample wash, and numtiirof seconds forthe sample. 9.2.4 Fillthesample traywith the standards,samples,washes and driftsI.W and FW/RUNOUT cups on thesampler do not need tobe filled. 9.2.5 Set thebaseline. 3 9.2.5. 1SelectGRAPHICS, REAL TIME. Ifyou cannotgetreal-timey,ou may be intheData HandlingPanel.Switch totheAnalysisPanel by selectinCgONTROL PANEL and pushingF7. 9. 2.5.2 Use thesmallscrewdriverfortheoffsetpotentiometetrosetthe baselineA.djustthebaselineuntilitisapproximately3/4 inchfrom thebottom of thescreen. 9.2.S.3 Check thehigheststandardand adjustthegain,ifnecessary,with theinterfacsecrew #3. 9.2.6 Go toCONTROL PANEL, and toanalysispanel.Deselecttheanalysisthat willnot be run.(Selector deselectanalysisby pressingENTER.) PressTab toreturntotheAnalysisPanel. 9.2.7 Pressthespacebartobringup thelocalmenu. 9.2.8 SelectSTART tostartheanalysis. 9.2.9 Type yourID (initialtsh)e,sampletablewhich you createdunder9.2.1(or pressENTER forchoices)c,hooserunningwithor withoutthesystemtable and selectSTART ANALYSIS. 9.2. 10 Afterstartintghesoftware,starthesampler.Make surethatthesampleris settotherightnumber of samplesand thatthesample/wash/aitrimesare OK. 9.2. 11 SelectGRAPHICS, REAL TIME toview theprogressof theanalysis. 9.3 Loading and Printing the Data-File 9.3.1 Go toCONTROL PANEL, pressthespacebartobrinaup thelocalmenu and selecLtOAD. SelectAUTOCALCULATION and enterthefilename(or highlightthefiletobe printedand pressENTER). 9.3.2 To view thecalibraticounrve,go toGRAPHICS, CALIBRATION CURVE. 9.3.3 To printthehighlevelcurve,push PRINT SCREEN. 9.3.4 To printthelow levelscreen,push ESC togetoutof graphicsS.elect SEIMNGS. Change themax y valuetoapproximately900.Go toCAL CURVE and pressESC, and Enter.PressPRINT SCREEN. -9.3.5 ReturntoSETTINGS and change themax valueback to4095, go toEDIT, pressENTER and PRINT SCREEN toprintsample peaks. 9.3.6 To printheresultgso toCONTROL PANEL, SPACEBAR, OUTPUT, OUTPUT. SelectPRINTER fortheEpson or PRN fortheLaser. 9.4 Shutdown 9.4.1 Putallthereagent-liniensMilli-Qwater. 9.4.2 ]Letthesystemrinseforapproximately30 minutes. 9.4.3 Afterthesystem hasrinsedcompletely,turnoffthesampler,pump and offsetpotentiometerT.urn offtheheatingbathon weekends.Leave liquidin thelines. 9.4.4 Take theelectrodeoutand soak in 100 ppm F overnight. 9.4.5 Releasethepump-decks, airbarsand samplerpump-tubing. 9.4.6 SelectFILES, pressALT F and selectQUIT toexittheproaram. 9.4.7 On Friday,turnoffthecomputer,displayand interfacfeortheweekend. 10.0 VALIDATION - 10. 1 Quality Control 10. 1.1Run a standard(mid tohighconcentratione)very 10 samples.Ifa significacnhtange inpeak hei0-htoccurs,onlythesamplesbeforethelast acceptablestandardwillbe used.The remainingsampleswillbe reanalyzed. 4 10.2 Precisioannd Accuracy 10.2.1SeeMethodValidatiRoenporntumberAMDT-M-8.0.V1 .10.3 Other ValidationParameters 10.4 Re.fetroMethod ValidatioRneportNumber AMDT-M-8.0.Vl 11.0 DATA 11.1 11.2 11.3 11.4 ANALYSIS Calculations 11. 1.1The standardcurveisplottedby theSkalarsoftware. 11. 1.2 Allcalculationasredone by theSkalarsoftwarer.2 shouldbe 0.995or better. Preparespreadsheetstosummarize data.Includesample volume, weightsused etc. Write thestudynumber on theprintoutsi,nitiadla,tetheprintouta,nd bind together with allpackage documents and placeinthestudyfolderM.ake a copy of the summary sheetand tapeintothestudynotebook.Back up alldataand spreadsheets onto studydiskand backup disks. ElectroniDcam 11.4. 1GLP studiesE:lectronidcam iscopiedonto theStudy floppydiskforeach study,and alsodataiscopiedontoa floppydiskthatisstoredin thelab. 11. 4.2 Other studiesA:lldataiscopiedontoa:floppydiskthatisstoredin thelab. 12.0 ATTACHMENTS None 13.0 REFERENCES 13.1 AMDT-M-1, ThermalExtractioonfFluozideby Means ofa ModifiedDohrmann DX2000 OrganicHalideAnalyzer-Liver 13.2 SkalarMethods,#335,SkalarMethods Manual 13.3 AMDT-EP-26, Operafionand Maintenanceof theSkalarSegmented Flow Analyzer 14.0 REVISION@ --- Revision Num Reason forchanae Revision Date 5 9.3 QualityAssurance Unit Statement Anch=nt D GLP Study QualityAssurance Statement StudyTitle:Single-doseIntravenousPharmacokineticStudy of T-6054 in Rabbits StudyNumber.- AMDT-122094.2 Name ofAuditor.KariRambo iiiistudyhas been inspectedby theQualityAssuranceUnitasindicateidnthefollowingtable. The findingswere reportedto thestudydirectoarnd management. InspectionDates E= 12 10/14/95 10/19/95 Phase FinalReport Date InspectioRneportedto Managcmt Smft Director 10/19/95 10/19/95 QAU Auditor Date 9.4 Key Personnel Involvedin the Study 3-M Environmental Laboratory Key Personnel Thermalextractiofnollowebdy analysiussingOrion Ionanalyzer: Jim Johnson Deb Wright RichYoungblom Deann Plummer Analysisofliverextractussingelectrospramyass spectrometry: Jim Johnson Dave Christenson Thermal extractiofnollowedby analysiussingSkslarsegmentedflow analyzerwithIonselectiveelectrode: Jim Johnson Deb Wright ;'RicYhoungblom Deann Plummer Documentationand Reporting: Jim Johnson RichYoungblom QualityAssuranceUnit: GaleVan Buskirk CynthiaWeber KariRambo 9-11 Data 9.11.1Summary and raw data;ug F-inwhole liveras determinedby thermalextractiofnollowedby analysisusing Orion ion analyzer. .. ........ Summary ofCombustion Data -Liver ANDT-122094.2, HWI 6329-138 As Referenced in FinalReport section6.0DATA ANALYSIS Total ug Fluoridein Whole Liver Mean per Dose Group ug ControlGroup 18.0 0.129 mg/kg dose (T6054) 60.5 0.64mgtkg dose (T6054) 118 1.28mglkg dose (T6054) 164 12.8mglkg dose (T6054) 2239 RPT138.XLS FC129 PK ID LiverBlank-I LiverBlank-2 LiverBlank-3 LiverSpike-I LiverSpike-2 F52750-1 F52750-2 F52750-3 F52793-1 F52793-2 F52793-3 F52792-1 F52792-2 F52792-3 F52751-1 F52751-2 F52751-3 F52752-1 F52752-2 F52752-3 LiverBlank-I LiverBlank-2 Liverspike63-1 Liverspike63-2 Liverspike126-1 Liverspike126-2 Liverspike126-3 % rcvry 97% 95% 97% 96% 95% 82% 83% Actual Average ppm F- ppm F- inliver inliver (WNW) (WNV) 1.11 0.371 0.253 1.04 0.860 1* 71 1.* 53 1.59 1.54 0.839 0.840 0.879 0.959 0.253 0.234 0.225 0.187 2.64 2.34 2.45 2.37 23.3 24.5 23.8 23.0 0.364 0.272 0.985 1.06 2.38 1.76 2.31 Liver burned (grams) 0.145 0.137 0.128 0.141 0.168 0.118 0.121 0.130 0.141 0.125 0.123 0.141 0.129 b.148 0.108 0.133 0.133 0.143 0.142 0.137 0.134 .0.109 0.148 0.137 0.121 0.142 0.109 Whole TotalFliver inwhole weight liver (grams) (pg) 73.9 lis 68.8 60.5 80.2 18.0 66.8 164 94.9 2239 Dosage (mg/kg) 0.64 0.128 0.0 1.28 12.8 Page I 9.11.2Summary and raw data;analysisof liverextracts using electrospraymass spectrometry. HWI# 6329-138 A-( A Sir4e-Dossintraver*uPsharmacokinedc Study: protocolNumb*r. TestMaterial: matrix: Flsquared VSILW: Response FactorAniount: TP8084.PK T-6054InRabbits(Fe-'2' Liver o.9826 1.92EOO DLC ArmlYst: Date: Method: 416/95 AL4DT-M-4 FlsonsVG 2000 ElectrOspramys llnsewnwt: 040695A LAB13ASE File: Group Dose Group 1: o mg/kg sompie i on count F-txer"a wt Ratio 9 F52792' 0.0245 1.2662 Iutdion c tion Total ofliver factor iLg/9 9 1 0.0299 80.1586 Totalamount of FC-96per liver nv o.002 Group 2: 1.3227 1 F52793 0.2346 0.128mg lkg 0.2722 68-8400 0.019 Group 3: o.64niglkg F52750 0.4983 1.2397 0.6169 73.9156 o.046 0.5998 66.7527 0.040 Group 4: 1.26mg*g Group 5'. 12.8nV/kg F52751 0.5016 1.2M i.iS52 1 F52752 2.7225 3.5264 94.8862 0.335 - RatioofU499 IonCount4,t427Ioncount ytritghe resultby 4/5.The 415 and rnuitipi 31rffgoflivewras used In **Theconcs ntradonw*s ,culatebdy usingthestandardcu1r8v.e4InMethod cikilscalcldatoInnapplyingformull AMDT-*a4r'e0d,wre1fore614lorgerthan they curve isobtained. factorIstheresultOf8 ml 17,1Ing.The concentratiorIwnthestandard by 415,thecorrectresult liadonratherthanthecalculatecdoncentratioInnthe standardcurve thiscalct shouldbe.By nwidplying A-Z HWI# 6329-138 Conc, 0.4 0.8 1.2 1.6 4 8 12 Sample F52792 F52793 F52750 F52751 F52752 499 Ion Count 9091 63720 117051 183223 342495 654597 774077 427 Ion Count 74303 112333 146156 145956 135711 157098 128461 W,499 I.C./W427I.C. 0.1224 0.5672 0.8009 1.2553 2.5237 4.1668 6.0258 n 427 Ion Count @k,499I.C./M@427I.C. 2399 12826 41969 41908 256267 97837 54664 84226 83555 94129 0.0245 0.2346 0.4983 0.5016 2.7225 6.IB258 A Method C:DLCLIVE1 Sample Operator Run date 07-26-1995 16:12:19 version: 35 Printed on 07-26-1995 AT 16:12:32 Straight Line Fit forced through origin. --------------------------------- Componen-t#:I 4.1668 2.537 + 1.2SS3 + .8009 .S672 .1224 .4 :1.*2 .8 1.6 4 8 AMOUNT Component 1= EXTERNAL STANDARD CALIBRATION LEVEL AMOUNT AREA ----------- --------- 1 0.4000 0 2 0.8000 1 3 1.2000 1 4 1.6000 1 OA: 5 4.0000 3 6 8.0000 4 7 12.0000 6 Y SLOPE X + INTERCEPT --------------------------------------------------- Area Amount = 5.2126E-01 1.9184E+00 Amount Area + O.OOOOE-+OO + O.OOOOE+00 R squared = 0.9826 A-,3 + 12 6-!, Fc ctI File:040695A SamP] @e:Hwl # 040695A Los V.rs 69 541 27 Of LAB-BASE - The MS Data 89stem DVIroll19- JL 4JL JL5 5 jLg 4 37 3383 28 7 2135 4243 4584 10246 1 13 9 too 734 7 2 3306@ 7 437 429 12 jL 4 77 -t2526 .,L5.15 3149 1.83223 2999 2669 JL36 300 Son 29919 4250 4000 4284 4594 LO Fiie:040695A 04 695A 97837 47 3 Lee LAB-BASE 'IA 54 64 4 The NS Data gusten low ) 8 26 83555 5 ZFS 989 ? 4458839 L 44? 5 \ 1434 5003 \ 939 148 \ 0 x3.9 co 6 4 as Lee Y.IFS r E 2399 47 1 Ito JL2826 4874 0 Son 47 la 4880 4969 LL 5000 5208 File:048695Ati Sample:HWI 040695A Igo LAB-BASE The MS Data S9stem jL632 04 71 2 q 38 JL64 9" JIL 3 ZFS Los Y.FS Son 95 26 2a 43299 4 7 iLL24 612 682 x4.9 623 857 Leal loL3 1+ 2 69 1 74 71 70 22 51 is 73 2411 392 4 7 27 a see 200 s 42 6 JLG r JLOOG File:04@695A Sample:Hl-ll 040695A lee V.Fg 74303 25 6 LAD-IJASE The 11S Da-ta S-gate 112333 26 6 146156 20 8 145936 29 a 135711 31 a 15709 33 5 2498 2799 11 289'301 lee 9091 20 -\4 gel 63720 26 9 @@ll 117051 28 9 \\d jL83223 29 9 342495 3149 65459 33 6 File:04069SA Sample:HUI # 640 95A 1.92 146506 5 JL &a"- LAIB-BASE X17jLL5 3 The MS Da-La Sgstem /415 JL365JL4 5958 6358 61 3 %FS .t586 4742 97 573 58 J60L193938% XFS .tZS625 563-t son13 560 57 0 Lg.S7255 50 3 F-5 00 -r 39 959 58 5-C)OO 600- 479481 6.t 3 6JLBG 9.11.3Summary and raw data; ug F- inwhole liveras determinedby thermalextractionfollowedby analysisusing Skalarsegmented flow analyzerwith ion selectivelectrode. This data,althoughsupportive,intheopinionof the Study Directorisnot requiredtoreach theconclusionstatedin FinalReport Section6.0,and thereforeisnot discussedin ,detail. RE: 6329-138 LIUER SRMPLES RMDT 1220,94.2 oateofRnalySis:4-20-95 Rnalyst: DOW The samples are burned in the Bohrman at 950 c using between 13.1and 0.2 grams of the liuer.The gas is collectedin 1.0 mL of 1:1TISR13/Milli-wOater then an additionalI mL of 1:1 allow for sufficientuolume for Skalar TISRB/Milli-Ois added to lyzed on a Skalar Segmented analysis.The samples are then ana de (ISE)Method. Flow Rnalyzer using the Ion SpecificElectro TISRB buffer isadded to each sample as itproceeds through the mple then goes through a heated mixing coil system. The sa tialbetween the ion selectiueelectrode and the before the poten asured. The signalisamplifiedand reference electrode isme relatedto the fluorideconcentration. The instrument was calibratedin the ranges of 0.015 - 8.15 ppm and 0.15 - 1.50 PPM fluoride.The standard curue for the high range was plottedusing the inuerse logarithm option.The standard curue for the low range islinear.Rilstandards and samples were then calculatedby the Skalarsoftware using these curues. Rilresultsbelow 0.0001 ppm appear on the raw data as #.####. ftqualitycontrolstandard was analyzed euerg 10 samples to check for accuracy and drift. Raw data istaken from the appropriate calibratedrange of the Skalar printoutand summarized on an Excel spreadsheet. The finalresultsare.adjustedforthe collectionuolume and any subsequent dilutions. Li SUMMARY OF 6329-138 LIVER SAMPLES AMDT 122094.2 GROUP 1 Dose Level: 0 F52792-1 0.02 F52792-2 ND F32792-3 ND GROUP2 F52793-1 0.07 Dose Level: 0.128mg/kg F52793-2 0.06 F52793-3 0.06 GROUP3 F52750-1 0.11 Dose level:0.64mgtkg F52750-2 0.11 F52750-3 0.11 GROUP4 F52751-1 0.16 Dose Level: 1.28mg/kg F52751-2 0.17 F52751-3 0.17 GROUP5 F52752-1 0.42 Dose Level: 12.8mgtkg F52752-2 0.76 F52752-3 0.73 ------------ ----------- 2.0 0.1409 0.27 80.1586 22 2.0 0.1287 ND ND 80.1586 ND 2.0 0.1481 ND 80.1586 ND 2.0 0.1410 1.00 2.0 0.1253 0.99 2.0 0.1229 1.01 68.8400 69 1.00 68.8400 68 68.8400 70 2.0 0.1176 1.95 2.0 0.1206 1.85 2.0 0.1303 1.73 73.9156 144 1.84 73.9156 137 73.9156 128 2.0 0.1078 3.02 2.0 0.1332 2.58 2.0 0.1328 2.62 66.7527 202 2.74 66.7527 172 66.7527 175 2.0 0,1429 5.91 94.8862 560 2.0 0.1419 10.67 9.05 94.8862 1012 2,0 0.1370 10.58 94.8862 1004 95-07-06 15:57 OutPut of :950420AI )erator :DDW iteoftheAnalysis 1995-04-2007:12 nalysiFsileName C:\SKALAR\DATA\HWID ATA\LIVERS%95042OAl 13BLIVER.XLS 'I@iwassomm I 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 '30 131 Tracer Drift Wash Standard1 Standard2 Standard 3 Standard4 Standard5 Standard6 Standard7 Standard8 Standard9 Standard10 Drift Wash BLK-1 BLK-2 SPK-1 SPK-2 50-1 50-2 50-3 93-1 93-2 93-3 Drift Wash 92-1 92-2 92-3 51-1 1.50 1.50 0.015 0.03 o.o6 0.09 0.12 0.15 0.30 0.60 1.20 1.50 1.50 1 1.50 1.46 1.47 ND 0.015 0.03 0.06 0.09 0.12 0.15 0.28 0.61 1.23 1.47 1.48 ND 0.10 0,03 0.09 0.09 0.11 0.11 0.11 0.07 0.06 0.06 1.47 ND 0.02 ND ND 0.16 97% 980/0 980/0 101% 101% 100% 99% 100% 94% 102% 103% 98% 99% 98% NONE= 2.0 0.1445 1.36 2.0 0.1365 0.48 0.004 63.00 2.0 0.1410 1.26 0.004 63.00 2.0 0.1677 1.02 2.0 0.1176 1.95 73.9156 144.06 2.0 0.1206 1.85 73.9156 136.68 2.0 0.1303 1.73 73.9156 128.20 2.0 0.1410 1.00 68.8400 68.55 2.0 0.1253 0.99 68.8400 68.46 2.0 0.1229 1.01 68.8400 69.57 2.0 0.1409 0.27 80.1586 21.73 2.0 0.1287 ND 80.1586 ND 2.0 0.1481 ND 80.1586 ND 2.0 0.1078 3.02 66.7527 201.87 Page 1 13BLIVER.XLS 32 51-2 33 51-3 34 52-1 35 52-2 36 52-3 37 BLK-1 0.17 0.17 0.42 0.76 0.73 0.04 2.0 0.1332 2.58 66,7527 172.39 2.0 0.1328 2.62 66.7527 174.92 2.0 0.1429 5.91 94.8862 560.42 2.0 0.1419 10.67 94.8862 1012.39 2.0 0.1370 10.58 94.8862 1004.27 2.0 0.1334 0.55 38 Drift 1.50 1.51 100% 39 Wash 40 BLK-2 41 SPK-63-1 42 SPK-63-2 43 SPK 126-1 44 SPK 126-2 45 SPK 126-3 ND 0.02 0.09 0.09 0.15 0.14 0.14 2.0 0.1092 0.44 2.0 0.1484 1.21 2.0 0.1370 1.32 2.0 0.1212 2.40 2.0 0.1439 1.91 2.0 0.1085 2.53 0.004 63.00 0. 0.004 63.00 0. 0.004 126.00 0 0.004 126.00 0 0.004 126.00 0 46 Drift 1.50 1.50 100% 47 Wash ND Page 2 1995-04-20 Software 09:36 version 6.1 OutPUt Of cl990,93 : 95042OAl Operator : DDW Date of the Analysis : 1995-04-20 07:12 Analysis F@le Name : C:\SKALAR\DATA\HWIDATA\LIVERS\95042OAl AKBT 17-zoci4.'L Fluoride 1.5 Calibration order = Inverse Logarithm Slope s = #.##### Result cl 101 x s I a2 = al = aO = -0.00000 0.00065 -1.24984 x = corrected value of the sample cl = corrected value of the concentration 1 s = Slope of the electrode Fluoride L Calibration order 2 Co.rrelation r = 0. 99991 Result a2 X2 + al * x + aO a2 = al = aO = 0.00000 0.00020 0.00010 Sampler Type Number Sample Time Wash Time Air Time Take up special needle Height : SAIOOO :1 : 50 sec. : 120 sec. : I see. : Single : None : 70 mm. Diluter needle Height : 80 mm dilution Factor : 10 dilution Volume : 2.5 mi. Resample :1 Dilution runs 1 User file : TXT Reproces : No 1995-04-20 09;36 OutPut of Fluoride 1.5 Path number Signal type Decolor system Number dilute Resample dil Threshold diG output Window event 3 : Debubbled : Yes :0 : No : No : 4095 :0 : Off sl standard : s2 standard : s3 standard : s4 standard : s5 standard ; s6 standard : s7 standard : ss standard : S9 standard : slO standard : .Order : Inverse Dimension : PPM start Value trigger Limit Peak shape start ignore eNd ignore Measure window Filter Regeneration formula output Ignore Ignore Ignore Ignore Ignore 0.150 0.300 0.600 1.200 1.500 Logarithm 500 DU 1800 Sec Pointed 60 Sec 120 Sec 75 No No Fluoride L Path number Signal type Decolor system Number dilute Resample dil Threshold diG output Window event 0 : Debubbled : No :0 : No : No : 4095 :0 : Off 95042OAl 1995-04-20 09:36 OutPut of sl standard : 0.015 s2 standard : 0.030 s3 standard : 0.060 s4 standard : 0.090 s5 standard : 0.120 S6 standard : 0.150 s7 standard : Ignore s8 standard : Ignore S9 standard : Ignore slO standard : Ignore Order : 2 Dimension : PPM start Value trigger Limit Peak shape start ignore : 500 DU : 1800 See : Pointed : 60 See eNd ignore : 120 See Measure window : 75 % Filter : No Regeneration : No formula C4:=c3 output 950420AI 1995-04-20 09:36 output of : 950420AI Fluoride 1.5 Fluoride L PPM Pos Typ Ident Ch Result F Time wt iw initial Wash 3 0.056 1t -Tracer 3 1.457 2d Drift 3 1.470 3w Wash 3 0.056 4 sl Standard 1 3 0.063 5 s2 Standard 2 3 0.070 6 s3 7 s4 Standard 3 3 0.087 Standard 4 3 0.106 8 s5 Standard 5 3 0.128 9 s6 10 s7 11 s8 Standard 6 3 0.155 Standard 7 3 0.282 Standard 8 3 0.614 12 s9 Standard 9 3 1.232 13 slO Standard 10 3 1.467 14 d Drift 3 1.483 15 w Wash 3 0.056 16 u BLK-1 3 0.112 17 u BLK-2 3 0.071 18 u 19 u SPK-1 SPK-2 3 0.105 3 0.103 20 u 21 u 50-1 3 0.124 50-2 3 0.122 22 u 23 u 50-3 3 0.123 93-1 3 0.093 24 u 93-2 3 0.088 25 u 93-3 3 0.088 26 d Drift 3 1.467 27 w Wash 3 0.056 28 u 92-1 3 0.065 29 u 92-2 3 0.063 30 u 92-3 3 0.061 31 u 51-1 3 0.163 32 u 51-2 3 0.172 33 u 51-3 3 0.174 34 u 52-1 3 0.422 35 u 52-2 3 0.757 36 u 52-3 3 0.725 37 u 38 d BLK-1 3 0.074 Drift 3 1.507 39 w Wash 3 0.056 40 u BLK-2 3 0.067 41 u SPK-63-1 3 0.106 42 u SPK-63-2 3 0.106 43 u SPK 126-1 3 0.151 44 u SPK 126-2 3 0.143 45 u 46 d SPK 126-3 3 0.143 Drift 3 1.495 47 w wt rw Wash RunOut Wash 3 0.056 3 0.056 65 210 384 626 733 907 1085 1261 1436 1611 1785 1959 2135 2311 2485 2727 2836 3011 3187 3363 3538 3714 3886 4060 4238 4414 4588 4740 4936 5111 5285 5463 5637 5813 5989 6163 6339 6515 6689 6930 7034 7214 7390 7566 7740 7914 8090 8324 8565 PPM Ch Result F Time 4 0.0001 0 4 0.7942 0 4 0.8006 0 4 0.0001 0 4 0.0147 0 4 0.0302 0 4 0.0603 0 4 0.0904 0 4 0.1190 0 4 0.1505 0 4 0.2602 0 4 0.4429 0 4 0.6965 0 4 0.7988 0 4 0.8066 0 4 0.0001 0 4 0.0986 0 4 0.0326 0 4 0.0889 0 4 0.0857 0 4 0.1146 0 4 0.1115 0 4 0.1130 0 4 0.0702 0 4 0.0623 0 4 0.0621 0 4 0.7988 0 4 0.0001 0 4 0.0191 0 4 0.0143 0 4 0.0117 0 4 0.1586 0 4 0.1686 0 4 0.1705 0 4 0.3468 0 4 0.5053 0 4 0.4918 0 4 0.0368 0 4 0.8183 0 4 0.0001 0 4 0.0238 0 4 0.0898 0 4 0.0902, 0 4 0.1456 0 4 0.1375 0 4 0.1371 0 4 0.812.3 0 4 0.0001 0 4 0.0001 0 Page 1 of 0. 1842330 Calibration curve of 9S0420AI : Fluoride L 0.0001048 0 Ordet- 2 Measured goo r 0.99991 1.72628841 Calibration curve of 9SO42OAt W,3 96 Fluoride t.5 0.0562S47 0 Order Measured Inverse Logarithm 4095 s 6205 Raw data of 950420AI : Fluoride I.S 240 64 c) 0 Esc=Exit FI=Help Time Crtl-P=Edit peaks 1 2625 1 40:9S Raw data of 9504'A"OA:I Fluor-idet-5 7'37-3 2C-4 go-"--t:-94 %t 0 1373 Time Esc=Exit FI=Help 1 Crtl-P=Edit Peaks 3998 Raw data of 950420AI : Fluoride 1.5 !P;Fli-7i,-rm,m-kW-M- qqR 262 F;4 P4 A Ap I @I @,l @J 10 1 3998 Time 1 Esc=Exit Fi=Help Crtl-P=Edit Peaks 13 Raw data of 9SO4'IIOAI Fluoride 1.5 Mow wmwf r E,62-q 409S i Rd P4 6923 Time 9248 Esc=Exit FI=Help Crtl-P=Edit peaks 1