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3M Environmental Laboratory FinalReport- AnalyticalStudy Single-DoseIntravenousPharmacokinetic Study ofT-6052 in Rabbits In-Vivo Study ReferenceNumber: HWI#6329-134 StudyNumber: ANMT-111694.1 TestSubstance:FC-120 (T-6052) Name and Address ofSponsor: 3M SCD Division 367 Grove Street St.Paul,MN 55106 Name and Address of TestingFacility: 3M EnvironmentalTechnology & Services 935 Bush Avenue St.Paul,N4N 55106 Method Numbers AMDT-M-1-0, AMI)T-M-2-0, AMDT-M4-0, AMDT-M-5-0, AMDT-M-8-0. AMDT-M-14-0, and Revisions: Thermal Extractionof Fluorideby Means of a Modified Dohnnann DX2000 Organic HalideAnalyzer-Liver FluorideMeasurement by Means of an Orion EA940 Expandable Ion Analyzer Extractionof Fluorochemicalsfrom RabbitLiver Analysisof RabbitLiverExtractforFluorochemicalsUsing ElectrosprayMass Spectrometry Analysisof FluorideUsing the SkalarSegmented Flow Analyzer With Ion SelectiveElectrode Thermal Extractionof Fluorideby Means of a Modified Dohrmann DX2000 Organic HalideAnalyzer-Serum InitiationDate: See attachedprotocol Author: James D. Johnson Approved By: ,@aSmaetumsed@sy DirJJd(oson StudyD irwor Compleiion Date 1.0SUMMARY The livesrampleast48 hoursaftesringlientravenoaudsministratoifFoCn-120 (T-6052)were analyzedby combustionfortotalorganicfluorineT.-6052 isa 0.02% solutionof FC-120. Only the200 mg/kg (10ug/kg)and 1000 mg/kg (50 ug/kg)sampleshad detectabloerganicfluorine4:8 and 106 ug/whole liver, respectivelyT.hese triviaalmounts oforganicfluorinearea reflectioonfthelow doses. There isa marker fordermal absorptionstudiesi,fthedosesarehigherthan used inthisstudy. 2.0 E*4TRODUCTION The liverand serum samplesfrom HWI#6329-134 were availableforanalysis.This compound isperfluorodecanesulfona(taenunonium salt).There isnotexpectedto be any biotransforinatioofnthiscompound and thepharinacokineticasnd disposition areexpectedtobe similartothatfound forperfluorooctanesulfonatTeh.e tissuesat 48 hours were analyzedby combustionfortotalorganicfluorineand by electrospray mass spectrometryforperfluorodecanesulfonaatneion.The datawere tobe analyzedtoprovidedatafortheassessmentof a subsequentden-nalabsorptionstudy. The highdose isjust50 ug/kg.T-6052 isa 0.02% solutionof FC-120,which is 25% solids. 3.0 TEST MATERIALS *3.1Test,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 3.2Source of Materials:3M ICP/PCP DivisionforFC-95, bovine liverfrom. grocerystoreb,ovineserum from Sigma Chemical Company 3.3.Purity and Strength ofReference Substance:Responsibilitoyf Sponsor. 3.4 Stabilityof Reference Substance: To be determinedby Sponsor. 3.5Storage ConditionsforTestMaterials:Room temperatureforFC-95.For biologicalsamplesthe storageis-20100 C. 2 3.6Dispositionof Specimens: Biologicaltissueasnd fluidswillbe retainedper GLP Regulationforthetimeperiodrequiredforstudieslongerthan28 days.This studyisinparallewlith a 28 day absorptionstudy,so alltissuewsillbe retained. 4.0 EXPERIMENTAL -Overview Serum and tissuesfrom animalsdosed as described(HWI#6329-134), were available foranalysisforfluorinecompounds. Sinceperfluorodecanesulfonaatneionisnot biotransformedt,heanalysiswas accomplishedwithcombustion and subsequent analysisforfluorine.The.fluorinedataarerelated irectltyo perfluorodecanesulfonateanionconcentrationA.dditionalanalysisofliversampleswith electrospray mass spectrometryprovidesevidencethattheperfluorodecanesulfonaatneionis present.Data from theseanalysiswillbe used toassesstheextentof dermal absorptionina subsequentstudy(HWI#6329-135). 5.0 EX]PEREWENTAL -METHODS 5.1AMDT-M-1-0, ThermalExtractionofFluorideby Means ofa Modified Dohnnann DX2000 OrganicHalideAnalyzer-Liver 5.2AM:DT-M-2-0, FluorideMeasurement by Means ofan Orion EA940 Expandable Ion Analyzer 5.3AMDT-M4-0, Extractioonf Fluorochemicalfsrom RabbitLiver 5.4AMDT-M-5-0, AnalysisofRabbitLiverExtractforFluorochemicalUssing ElectrospmyMass Spectrometry 5.5 AMDT-M-8-0, Analysisof FluorideUsing theSkalarSegmented Flow AnalyzerWith Ion SelectivEelectrode 5.6AM:DT-M-14-0, Tbermal ExtractioonfFluorideby Means ofa Modified Dohrmann DX2000 OrganicHalideAnalyzer-Senim 6.0 DATA ANALYSIS The data(Skalari)sattachedforcombustionanalysis.The totalorganicfluorinien liverat48 hours afteran intravenousdose of FC- 120 was nondetectedforthe control0,.1 uglkg,and 1.0uglkg groups.The totalorganicfluorinemeasured for 3 the 10 ug/kg and 50 ug/kg rabbitswere 48 and 106 ug/whole liverr,espectively. Electrospraymass spectrometry(seeattached)confirmed thepresenceof perfluorodecanesulfona(tme/z--599). Other datawas collectedusingtheDorhman organichalideanalyzer,Orion ion analyzer(liverand serum),Skalarsegmented flow analyzerwithion selective electrode(serum),and electrospramyass spectrometry(liver-) seeappendices.This data,althoughsupportivei,n theopinionof theStudy Directorisnot requiredto reach the conclusionstatedhere and thereforeisnot discussedindetail. 6.1 Circumstances that May Have Affected the Quality of the Data: The problem with thisanalysisisthatthereisnot nearlyenough fluorineinthe liverafter theseintravenousdoses because the doses are too low. 7.0 !CONCLUSION This phannacokinetic study isnot usefulin terms of providingdata forthe assessmentof a dermal absorptionstudy. The perfluorodecanesulfoniaccidanion expectedisnot observedinliverexceptfora traceatthehighestdose (50 uglkg).If the dermal absorptionstudydoses arehigh enough, thereisa marker. 8.0 MAINTENANCE OF RAW DATA AND RECORDS 8.1 Raw Data and Data: Raw data,approved protocol,approved fmal report, appropriatespecimens,and electronidcata willbe maintainedintheAMDT archives. 9.0 APPENDICES 9.1 Protocoland Amendments 9.1.1Protocoland FinalReport:HWI#6329-134, "Single-DoseIntravenous PhannacokineticStudy of T-6052 in Rabbits" (ProtocoltypeTP8084.PK for dosingof animals,tissuecollectione,tc.) 9.1.2 AnalyticalprotocolAMDT- 111694.1 9.2 Signed Reports from IndividualScientistsN:one 9.3 Quality Assurance Unit Statement: See attached 4 9.2SignedReportsfromIndividuSaclientisNtosn:e 9.3QualitAyssurancUenitStatementS:eeattached 9.4Key PersonneIlnvolveidntheStudy:Seeattached 9.5MaterialasndEquipment:Seemethods 9.6SolutionRse,agentsa,nd StandardsS:eemethods 9.7SamplePreparatioSne:emethods 9.8QualitCyontroPlracticeSse:emethods 9.9TestMethods:SeeProtocoAlMDT-1 11694.1 9.10InstrumenStettingSse:emethods 9.11Data:Seeattached. 9.11.S1ummaryandrawdata;ug F-inwholelivearsdeterminbeyd thermal extractiofnollowedby analysisusingOrion ion analyzer. 9.11.2Summary and raw data;analysisof liverextractussingelectrospray mass spectrometry. 9.11.3Summary and raw data;ug F- in whole liveras determinedby thermal extractiofnollowedby analysisusingSkalarsegmented flow analyzerwith ion selectivelectrode. 9.11.4Summary and raw data;ppm F-in serum as determinedby thermal extractiofnollowedby analysisusingOrion ionanalyzer. 9.11.5Summary and raw data;ppm F-inserum as determinedby thermal extractiofnollowedby analysisusingSkalarsegmented flow analyzerwith ion selectivelectrode. 5 9.1.1FinalReport:HWI#6329-134, "Single-Dose IntravenousPharmacokineticStudy of T-6052 in Rabbits"(Protocoltype TP8084.PK fordosingof animals,tissuecollectione,tc.) HAZLCECCN W IS C 0 N S IN Pos r OFFICE BOX 7545 MADI'@O@l. Wl 53707-7F)45 Sponsor: 3M St. Paul, Minnesota iCORNING Coiripiiny FINAL REPORT Study Title: Single-Dose Intravenous Pharmacokinetic Study of T-6052 in Rabbits Author: Steven M. Glaza Study Completion Date: February 1, 1995 Performing Laboratory: Hazleton Wisconsin, Inc. 3301 Kinsman Boulevard Madison, Wisconsin 53704 LaboratoryProject Identification: HWI 6329-134 Page 1 of 24 P h o n e 6 0 3 -2 4 1 -4 4 7 1 E x P R E S S - M A IL D E L I V E R Y 3301 K IN S M A N PLVD. F ax MADISON, 608 'vV 1 53704 Page 2 of 24 QUALITY ASSURANCE STATEMENT HWI 6329-134 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 inspectionswere conducted and findingsreported to the Study Directorand management. Written status reports of inspections and findings are issued to Hazleton management monthly according to standard operatingprocedures. Inspection Dates From To - Phase 11/05/94 11/05/94 Protocol Review 11/11/94 11/11/94 Animal Observation 01/10/95 01/10/95 Data/Report Review 01/30/95 01/30/95 Report Rereview Date Reported to Date to Study Director Management 11/08/94 11/11/94 01/10/95 01/30/95 12/10/94 12/10/94 02/10/95 02/10/95 M. Danner Date Representative,Quality Assurance Unit Page 3 of 24 STUDY IDENTIFICATION Single-Dose Intravenous Pharmacokinetic Study of T-6052 in Rabbits HWI 6329-134 Test Material Sponsor Sponsor's Representative Study Director Study Location Study Ti-metable Experimental Start Date Experimental Termination Date T-6052 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, WI 53707-7545 (608) 241-7292 Hazleton Wisconsin, Inc. Building No. 3 3802 Packers Avenue Madison, WI 53704 November 11, 1994 November 13, 1994 Page 4 of 24 KEY PERSONNEL HWI 6329-134 Acute Toxicology -LaboratoryAnimal Medicine Steven M. Glaza Study Director Manager Cindy J. Cary, DVM Diplomate, ACLAM Supervisor Francis (Bud) W. McDonald Study Coordinator Anatomical Pathology Patricia Padgham In-life Supervisor Rose M. Bridge Report Supervisor Ouality Assurance 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 6329-134 Paqe 2 3 4 6 7 7 7 8 9 11 11 11 .11 12 13 14 15 Page 6 of 24 SUMMARY HWI 6329-134 This study was done to assess the level of systemic exposure of T-6052 when administered by intravenous injection to 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 2, 20, 200, or 1,000mg of T-6052/kg of body weight (Groups I through 5, respectively). The dose volume was 0.5 mL/kg for Groups I through 4 and 1.02 mL/kg for Group 5. 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-hours post-injection. In addition,at the time of experimentaltermination (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 termination of the in-life phase. All five animals appeared normal throughout the study. Page 7 of 24 OBJECTIVE HWI 6329-134 The objective of this study was to assess the level of systemic exposure to the test material, T-6052, when administered as a single intravenous injection to rabbits. REGULATORY COMPLIANCE This study was conducted in accordance with the U.S. Food and DrugAdministration's Good Laboratory Practice Regulations for Nonclinical Laboratory Studies, 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 identified as T-6052 and described as clear, colorless liquid. The control material was Sterile Water for Injection,USP (Abbott Laboratories, Lot No. 86-748-DM-02; Exp. March 1, 1996), and was described as a clear, colorless liquid. 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 will be returned to the Sponsor after completion of all testing accordingto Hazleton Wisconsin (HWI) Standard Operating Procedure (SOP). Any remaining vehicle may be used for other testing and will not be discarded after issuance of the final report. Page 8 of 24 HWI 6329-134 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 October 5, 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 humidity,controlledquarters. Environmental controls for@the animal room were set to maintain a temperature of 19' to 23*C, 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 Animal Diet The animals were provided access to water ad libitum 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 periodically analyzed by HWI. There were no known contaminants in the feed or water at levels that would have interferedwith or affected the results of 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. Page 9 of 24 Study Design HWI 6329-134 Female animals weighing from 2,813 to 3,031 g at initiation of treatment were placed into the following study groups: Group Dose Level Dose Volume Treatment (mg T-6052/kq) (mL/kg) 1 (Control) 2 3 4 5 T-6052 T-6052 T-6052 T-6052 0 2 20 200 1,000 0.5 0.5 0.5 0.5 1.02 Sterile Water for Injection,USP. Number of Animals 1 1 Justification for Sgecies 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 Pre2aration and Administration The test material was diluted with Sterile Water for Injectionto achieve a specific concentration for each dose level in Groups 2 through 4. The test material was administered undiluted at the 1,000 mg/kg dose level, using the bulk density of 0.98 g/mL to determine the dose volume. An individual dose of each respective test solution or control was calculated for 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 solutions were stored at room temperature until administered. After administration, any remaining test solutions were discarded. Reason for Route of Administration Intravenous injection is an acceptable route to assess systemic exposure. Observations of Animals Clinical observations were conducted at approximately 0.5, 2) 41 24, and 48 hours after intravenousinjection. Body weights were determined just before test material administration(Day 0). Page 10 of 24 HWI 6329-134 Samgle Collection A blood sample (approximately4 mL) was collected from either ear via the catheterizationof the auricularartery or from the marginal ear vein of all animals at 2, 4, 6, 8, 12, and 24 hours post-injection. At the time of necropsy (approximately48-hours post-injection), approximately 20 mL of blood was obtained from the posterior vena cava of each 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 temperature of -20*C 10*C until shipped to the Sponsor. PatholM At termination of the experimental phase (approximately48-hours post-injection),animals were anesthetizedwith sodium pentobarbital,bled via the posterior vena cava, and exsanguinated. An abbreviated gross necropsy examination was not conducted, however, tissues were collected. The whole liver, bile, and both kidneys from each animal were collected and immediately placed on dry ice, then frozen by placing in a freezer set to maintain a temperature of -20*C 10*C. After tissue/bile collection, the animals were discarded. Shipment of Tissues After completion of 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 responsible for the retention and disposition of the samples. HWI does not accept any responsibility for the analysis of the samples collected in this study nor are these results presented in this report. Statistical Analyses No statistical analyses were required by the protocol. Location of Raw Data, Records, and Final Report The raw data, records, and an original signed'copyof the final report will be retained in the archives of HWI in accordance with HWI SOP. Page 11 of 24 RESULTS Body Weights Individualbody weightsat initiationare in Table 1. HWI 6329-134 Clinical Observations Individualclinicalsigns are in Table 2. All five animalsappearednormal throughout the study. Pathology All animals survived to terminationof the experimentalphase and were not examined grossly when sacrificed. DISCUSSION The level of systemic exposure of T-6052 was evaluated in female albino rabbits when administeredas a single intravenousinjectionat levels of 0, 2, 20, 200, and 1,000 mg/kg. All animals appeared normal throughoutthe study followingadministrationof this material. SIGNATURE Steven M. Glaza Study Director Acute Toxicology -OL:m Date REFERENCE 1. NIH PublicationNo. 86-23 (revised1985). GrouD 1 2 3 4 5 Page 12 of 24 Table 1 IndividualBody Weights (g) Dose Level (mg/kq) Sex Animal Number 0 Female F52548 2 Female F52549 20 Female F52559 200 Female F52566 1,000 Female F52567 Day 0 3,031 2,921 2,813 2,912 2,853 HWI 6329-134 GrouD Dose Level (mg/kq) 1 0 Sex Female Page 13 of 24 Table 2 IndividualClinical Signs HWI 6329-134 Animal Number F52548 Observation Appeared normal Hour 0.5- 2 4 24 48 1( 1( If 2 2 Female F52549 Appeared normal 1( .1 3 20 Female F52559 Appeared normal t 4 @00 Female F52566 Appeared normal .1 t 5 1,000 Female F52567 Appeared normal Indicates condition exists. Page 14 of 24 APPENDIX A Protocol TP8084.PK HWI 6329-134 HAZLR;-CCN W IS C 0 N S IN POST OFFL(:E BOX 7545 MAD[fi0t.I.Wi S*,1101 :54!, Page 15 of 24 CORNING Sponsor: 3M St. Paul, Minnesota PROTOCOL TP8084.PK Study Title: Single-DoseIntravenous PharmacokineticStudy of T-6052 in Rabbits Date: November 9, 1994 PerformingLaboratory: HazletonWisconsin, Inc. 3301 Kinsman Boulevard Madison, Wisconsin 53704 LaboratoryProJect Identification: HWI 6329-134 F'fl j,S Page 16 of 24 STUDY IDENTIFICATION TP8084.PK Page 2 Single-Dose intravenousPharmacokineticStudy of T-6052 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 6329-134 T-6052 3M ToxicologyServices 220-2E-02 3M Center St. Pau-1,MN 55144 John L. Butenhoff,PhD 3M ToxicologyServices 220-2E-02 3M Center St. Paul, MN 55144 (612) 733-1962 Steven M. Glaza HazletonWisconsin, Inc. P.O. Box 7545 Madison, WI 53707-7545 (608) 241-7292 Hazleton Wisconsin,Inc. BuildingNo. 3 3802 Packers Avenue Madison, Wl 53704- Week of November 7, 1994 Week of November 7, 1994 Week of December 12, 1994 Page 17 of 24 TP8084.PK Page 3 1. Study Single-Dose IntravenousPharmacokineticStudy in Rabbits 2. Purpose To assess the levelof systemicexposurewhen the test material is administeredas a singleintravenousinjectionto rabbits 3. Regulatory Comt)liance This study will be conductedin accordancewith the followingGood LaboratoryPracticeRegulations/Standards/Guidelinweisth the exceptionthat analysisof the test materialmixturesfor concentrations,olubility,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 (JapaneseMOHW) All proceduresin this protocolare in compliancewith the Animal WelfareAct Regulations. In the opinion of the Sponsor and study director,the studydoes not unnecessarilyduplicateany previous work. 4. Ouality Assurance The protocol,studyconduct,and the final reportwill.beauditedby the Quality AssuranceUnit in accordancewith HazletonWisconsin (HWI) StandardOperatingProcedures(SOPS) and policies. 5. Test Material A. Identificalion T-6052 B. Physical Dgscription (To be documentedin the raw data) C. purityand Stability The Sponsorassumesresponsibilityfor purity and stability determinations(includingunder test conditions).Samplesof test material/vehiclmeixture(s)for concentrations,olubility, homogeneity,and stabilityanalyseswill be taken before administrationif requestedby the Sponsor. These samples(if taken)will be sent to the Sponsorafterexperimental terminationfor possibleanalysis. Page 18 of 24 TP8084.PK Page 4 D. Storage Room temperature E. Reserve SamDles Reservesampleswill not be requiredfor.this study. F. Retention Any unusedtest materialwill be discardedafter issuanceof the finalreport,unlessdirectedotherwiseby the Sponsor. G. Safety Precautions As requiredby HWI SOPs and policies 6. ControlMaterial A. Identifigation Sterilewaterfor injection B. physicalDescription Clear,colorlessliquid C. Purityand StabilitY The purityand stabilityof this USP grade materialis consideredto be adequatefor the purposesof this study. D. Storage Refrigerated E. Reserve SamRIes tee Section,5. E. Reserve Samples F. Retention Any remainingcontrolmaterial may be used for other testing and will not be discardedafter issuanceof the final report. G. Safetv Precautions As requiredby HWI SOPs and policies Experimental Design 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) Weightat 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-bottomstainlesssteelcages (heavy gauge) (b) Food A measured amount of Laboratory Rabbit Diet HF #5326 (PMIFeeds,Inc.). The food is routinelyanalyzedby the manufacturer-fornutritionalcomponentsand environmentalcontaminants. (c)Water Ad libitum from an automatic system. Samples of the water are analyzed by HWI for total dissolved solids, hardness,and specifiedmicrobiologicalcontentand for selectedelements,heavy metals, organophosphates,and.chlorinatedhydrocarbons. (d)Contaminants There are no k6own contaminantsin the food or water thatwould interferewith this study. (e) Environment Environmentalcontrolsfor the animal room will be set to maintaina temperatureof 19*C to 230C, a relativehumidity of 50% 20%, and a 12-hour light/12-hourdark cycle. (f)6cclimation At least 7 days (8) 5electionof Test Animals Based on health and body weight according.toHWI SOPS. An adequatenumber of extra animals will be purchased so that no animalin obviouslypoor health is placed on test. (9)Justificationfor SpeciesSelection Historically,the New ZealandWhite albino rabbit has been the animalof choice because of the large amount of backgroundinformationon this species. Page 20 of 24 TP8084.PK Page 6 B. Dosg Administration (1) Test Groups Dose Level (mg/kg)* Number of Females 1 0 (Control) 1 2 2 3 20 4 200 5 1000 a The dose volumewill be 0.5 ml/kg for Groups 1-4 and approximately1.0 mL/kg of body weight (dependingon the bulk densityof the test material)for Group S. 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 in Groups 1-4. The testmaterialwill be administeredundilutedat the 1,000mg/kg dose level, using the bulk densityto determinethe dose volume. Individualdoses will be calculatedbased on the animal's body weight taken just before test material administration.The preparedtest mixtureswill be stored at room temperatureuntil administration. 0. Observationof Animals (1) ClinicalObservations The animalswill be observedfor clinicalsigns of toxicityat approximately0.5, 2.0, 4.0, 24, and 48 hours after treatment. Page 21 of 24 TP8084.PK Page 7 (2) BodyWeights Just beforetestmaterialadministration. (3) Sample Co lections (a) Frecuency 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 fromeitherear via the catheterizatioonf 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 obtainedfrom the posteriorvena cava of each animalat the time of necropsy(48 hours post-injection).Approximately 20 mL of blood will be collectedfrom moribund animalsduringthe study,also, if possible. The sampleswill be stored at room temperatureand then centrifuged,and the separateserum and cellular fractionsstoredin a freezerset to maintaina temperatureof -20*C tlO*C. The separatedserum and cellularfractionswill be sent frozento the Sponsor after experimentaltermination. Sampleswill be shipped to: James 0. Johnson 3M E.E. & P.C. Bldg. 2-3E-09 935 Bush Avenue St. Paul, MN 55106 James 0. Johnsonwill be notifiedby telephoneat (612)778-5294prior to the shipmentof the samples. E. Termination (1)UnstheduledSacrificesand Deaths Any animaldyingduring the study or sacrificedin a moribundcondition,will be subjectedto an abbreviated gross necropsyexaminationand all abnormalitieswill.be recorded. Animalsin a moribundconditionwill be anesthetizedwith sodium pentobarbital,bled via the vena cava, and exsanguinated. Page 22 of 24 TP8084.PK Page 8 (2) Scheduled crifice At approximately48 hours post-injection,animals survivingto terminationwill be anesthetizedwith sodium pentobarbital,bled via the vena cava, and exsanquinated. An abbreviatedgross necropsyexaminationwill not be done,however,tissueswill be collected. (a)Sample Collection The whole liver and bile from each animal dying during the study,sacrificedin a moribundcondition, or survivingto terminationwill be collected. Both kidneysfrom each animalwill also be collected. The tissueswill be placedon dry ice immediatelyafter collectionand then placed in a freezerset to maintain a temperatureof -20*C 10*C. The tissues(liver,bile,kidneys)will be sent frozenon dry ice to the Sponsor after experimental termination@ The sampleswill be shippedto the personlistedin Section7.D.(3).(c).The Sponeoris responsiblefor the retentionand dispositionof the samples. F. StatisticalAnalyses No statisticalanalysesare required. 8. Report A finalreportincludingthose itemslistedbelowwill be submitted. .Descriptioonf the test and controlmaterials Descriptionof the test system Procedures Dates of experimentalinitiationand termination Descriptionof any toxic effects Gross pathologyfindings/grospsathologyreport (if applicable) Page 23 of 24 TP8084.PK Page 9 9. Location of Raw Data, Records, and Final ReDort Originaldata, or-copiesthereof,will be availableat HWI to facilitateauditingthe studyduring its progressand before acceptanceof the final report. When the final reportis completed, all originalpaperdata, includingthose item listedbelowwill be retained in the archivesof NWI according to HWI SOP. Protocol and protocol amendments Dose preparationrecords In-liferecords Body weights Dose administration Observations Sample collectionrecords Pathology Records Study correspondence Final report(originalsignedcopy) The followingsupportingrecordswill be retainedat HWI but will .notbe archivedwith the study data. Animal receipt/acclimatiornecords Water analysisrecords Animal room temperatureand humidity records Refrigeratorand freezertemperaturerecords Instrumentcalibrationand maintenancerecords Page 24 of 24 PROTOCOL APPROVAL 2. john L. Butenhoff, PhD Sponsor's Representative 3M Steven M. -Glaza Study Director Acute Toxicology Hazleton Wisconsin,Inc. R6@resentative quality AssuranceUnit Hazleton Wisconsin, Inc. (6329-134.protdskl) Date Date Date TP8084.PK Page 10 9.1.2 AnalyticalprotocolAMDT-1 11694.1 3M EnvironmentaLlaboratory Protoco-lAnalyticaSltudy Single-DoseIntravenousPharmacokinetic Study ofT-6052 inRabbits In-Vivo Study Reference Number: HWI#6329-134 StudyNumber: AMDT-111694.1 Test Substance: FC-120 (T-6052) Name and Address of Sponsor: 3M SCD Division 367 Grove Street St.Paul,NfN 55106 Name and Address ofTestingFacility: 3M EnvironmentalTechnology and Services 935 Bush Avenue St.Paul,MN 55106 Proposed InitiationDate: July25, 19.95 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: Thermal Extractionof Fluorideby means of a Modified Dohrrnann DX2000 Organic HalideAnalyzer-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 Dohrmann DX2000 Organic HalideAnalyzer-Serum Author: James D. Johnson Approved By: Qvw"@ "es D -ig*son Date It"udyDirddtor John Butenhoff,PhD Date Sponsor Representative 1.0 PURPOSE Thisstudyisperformeidnordertoprovidpeharmacokinetdiactafortheassessment of a subsequentdermal absorptionstudy(HWI#6329-135). 2.0 TEST MATERIALS 2.1Test,Control,and ReferenceSubstancesand Matrices 2.1.1AnalyticalReferenceSubstance: 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)g,rocerystore(2.1.22,.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 the Study Director. 2.4 Identiflcatioonf Test and Control Samples: The samples areidentifieudsing theHWI animal identificatinounmber which consistsof a lettearnd fivedigit number, plusthetissueidentitya,nd day.identit(yserum). 2.5 Purity and Strength of-ReferenceSubstance: To be determinedby Sponsor. 2.6 Stabilityof Reference Substance:To be determinedby Sponsor. 2.7 Storage Conditions forTest Materials:Room temperature(2.1.1), -20 IOOC (2.1.22,.1.4).Testand Controlsamples willbe receivedaccordingto AMDT-S-10-0. 2.8-DispositionofSpecimens: Biologicatlissueasnd fluidswillbe retainepder GLP Regulationforthetime periodrequiredforstudieslongerthan 28 days. This study isinparallewlith a 28 day dermal absorptionstudyso alltissueswillbe retained. 2.9 Safety Precautions: Refer to appropriateMSDS. Wear appropriatelaboratory attire*U.se cautionwhen handlingknives forcuttingthe samples. 2 3.0 EXPERIMENTAL -Overview The tissuesfrom animalsdosed as described(HWI#6329-134), areavailablefor analysifsorfluorinceompounds. At thediscretioonftheStudy Directora, seriesof analyticatlestscan be performed.The screeningforfluoridienliverviacombustion (SeeMethods--nextSection)istheappropriataenalysistopresentdefinitivdeatafor fluorinientheliver.Electrospramyass spectrometrywillbe performedinorderto confirmthepresenceofspecifimcolecules.The materialbeingstudiedisa perfluorodecanesulfonaiccidsalt(Ammonium). This materialisnotexpectedtobe biotransformed.Ifthematerialissimilarto perfluorooctanesulfonaiccidanion,it willbe persistenitntheliver.Analysisof liverfortotalorganicfluorinweill provideinformationas to theextentof persistence. 4.0 EXPERIMENTAL -Methods 4.1Liverand Serum screeningmethods:(attached) 4.1.1AMDT-M-1-0, Thermal Extractioonf Fluorideby Means of a Modified Dohnnann DX2000 OrganicHalideAnalyzer-Liver 4.1.2AMDT-M-2-0, FluorideMeasurement by Means ofan Orion EA940 Expandable Ion Analyzer 4.1.3AMDT-M-4-0, Extractioonf Flubrochemicalfsrom RabbitLiver 4.1.4AMDT-M-5-0, Analysisof RabbitLiverExtractforFluorochemicals Using ElectrosprayMass Spectrometry 4.1.5AMDT-M-8-0, Analysisof FluorideUsing theSkalarSegmented Flow Analyzerwith Ion SelectiveElectrode 4.1.6AMDT-M-14-0,Thermal Extractioonf Fluorideby Means of a ModifiedDohrmann DX2000 OrganicHalideAnalyzer-Serum 5.0DATA ANALYSIS 5.1Data Reporting:Datawillbe reporteadsa concentrati(owneight/weigohft) fluorideper tissueor fluido,r as FC- 120 (electrospramyass spectrometry)per unit of tissueor fluid.Statistiucsed,atthediscretioonf the Study Director,may includeaveragesand standarddeviationsfrom differendtose groups.Ifnecessary, simplestatistictaelstssuch as the Student'st testmay be appliedto determine statisticdailfference. 6.0 MAINTENANCE OF RAW DATA AND RECORDS 6.1 Raw Data and Records: Raw data,approved protocol,appropriatespecimens, approved finalreport,and electronidcata willbe maintainedintheAMDT archives. 7.0 REFERENCES 7.1 AMDT-S-10-0, Sample TrackingSystem 8.0 ATTACHMENTS 8.1 AMDT-M-1-0, Thermal Extractionof Fluorideby Means of a Modified Dohrmann DX2000 OrganicHalideAnalyzer-Liver 8.2AMDT-M-2-0, FluorideMeasurement by Means of an Orion EA940 Expandable Ion Analyzer 8.3 AMDT-M-4-0, Extractionof Fluorochemicalsfrom RabbitLiver 8.4AMDT-M-5-0, Analysisof RabbitLiverExtractforFluorochemicalsUsing ElectrosprayMass Spectrometry 8.5 AMDT-M-8-0, Analysisof FluorideUsing the SkalarSegmented Flow Analyzer with Ion SelectiveElectrode 8.6AMDT-M-14-0,Thermal Extractioonf Fluorideby Means of a Modified Dohrmann DX2000 Organic HalideAnalyzer-Serum 4 3M Environmental Laboratory Method Thermal Extractionof Fluorideby Means of a Modified Dohrmann DX2000 OrganicHalideAnalyzer -Liver Method IdentificatiNounmber: AMDT-M-1 RevisionNumber: 0 AdoptionDate: RevisionDate: None Author-Rich Youngblom Approved by: roupLea7,/ ? r*oup L, /o/3 Dafe QualityAssurance Date Software:MS Word 5.la AffectedDocuments: ANMT-M-2 FluoridMeeasurementby Means ofan OrionEA940 ExpandableIonAnalyzer AMDT-EP-3 RoutineMaintenanceofa ModifiedDohrmann DX2000 OrganicHalideAnalyzer 1.0 SCOPE. APPLICABLE COMPOUNDS. AND MATRICES 1.1Scope:Thismethod isfortheoperatioonf a Dohrmann DX2000 when itisusedtoextract fluoridferom variousmatricesT.he fluoridiestypicallcyollecteidnTISAB solutiofnoranalysis withan ionselectiveelectrode. 1.2ApplicableCompounds: Fluorochemicalosrotherfluorinatceodmpounds. 13 Matrices:Biologicatlissuepsa,rticularlliyver. 2.0 KEYWORDS 2.1Fluoridef,luorinee,xtractiopny,rolysisi,onizatioino,nselectiveelectrodeD,ohrmann, halide, DX2000, fluorochemicals. 3.0 PRECAUTIONS 3.1Glasswareand exhaustgasescan be extremelyhot. 3.2Glasswareisfragileb,rokenglassmay causeinjuries. 3.3Pressurizegdases,propercompressedgas handlingpracticersequired. 3.4Solventbased samplesmay flashm,ay need toallowthem todrydown beforestartinrgun. 3.5Potentiabliohazardsdue tothebiologicamlatrices.Use appropriatpeersonalprotective equipment. 4.0 SUPPLIES AND MATERIALS 4.1Compressed Oxygen, Hydrocarbonfree,regulatetdo30 PSI. 4.2Compressed Helium,High PurityGrade,regulatetdo45 PSI. 4.3QuartzglasssampleboatwithTeflonrmtubing,Dohrmann 890-097 orequivalent. 4.4Quartzglasscombustiontube,RelianceGlass G-9405-012 orequivalent. 4.5Orion940999 TotalIonicStrengtAhdjustmentBuffer(TISAB Il)orequivalent. 4.6Sample collectiovnialsH,DPE. 4.7NElh-QTM water 4.8Polystyrenepipettes. 4.9ActivatedCharcoal,E.Merck 2005 orequivalent. 4.10HamiltonSyringeor equivalent. 4.11Miscellaneouslaboratorgylassware 5.0 EQUIPMENT 5.1Rosemount Dohrmann DX2000 OrganicHalideAnalyzer,modifiedforfluorideextraction. 5.2IBM compatible386 or486 computer. 5.3DX2000 softwarev,ersion1.00m,odifiedforfluorideextraction. 5.4Excel Spreadshee4version5.0orgreater 6.0 INTERFERENCES 6.1 Sample sizeislimitetdoapproximatel1y50 mg, dependingon samplemoisturecontent.This may varyfrom matrixtomatrix. 2 7.0 SAMPLE F4ANDLING 7.1Samples arenottobe handledwithbarehands. Fluoridemay leachfrom theskintothe sample. Use forcepsorprobetotransfetrissues. 7.2Samples ofliverarecutfromfrozenliverand placedina taredand labeledweigh boat.Use a cleanscalpeland cuttinbgoard.The cuttingboardand scalpeslhouldbecleanedwithwater, methanol,or methanol-watesrolutioanftereachliveriscut. 8.0 CALIBRATION AND STANDARDIZATION 8.1PreparationfCalibratiSotnandards 8.1.T1hestandarrdesquirfeodreachprojecwtilnleedtobeapproprifaotrethaitndividpuraolject Refertoprotocolforthatproject. 8.1'2Typically50-500ppm FC-95 inmethanolstandardsareused. 8.1.3For rabbiltiverstudiesu,sebeefliverasthematrix.Cut a pieceoffrozenbeefliver(100150 mg) and weigh itina labeledand taredweigh boat. 8.2 Calibratio-nOverview The normalcalibratiiosnthefluoridceurve(AMDT-M-2). However,ifanoptionaslpikedliver curveisrequiredtheprocedurelistebdelow isused. 8.2.1A calibraticounrvefortheDX2000 is-generatebdy spikingsampleswithknown standards and combustingthem usingthesame methods and matrixtypeasthesamplestobe tested. 8.2.2Typicallyt,hreereplicatoefseachstandardand fiveconcentratioonfsstandardwsillbe spiked. 8.2.3Standardcurvewillbe plotteadsMass SpikedF (ug)on thex-axisand StandardMass RecoveredF (ug)on they-axis.Generatea regressiocnurveand calculatteheequationfortheline and ther2 value. 8.2.4Mass SpikedF (ug)= (AmountspikedinniL)x (Conc.ofstandaridnppm) x (0.6004)* *FC-95 is60.04% F therefor0e.6004 isthefactorusedtoconvertFC-95 toF 8.2.5StandardMass RecoveredF (ug)= (TISAB volume inmL) x (Orionreadinginppm) 8.3Calibratio-nProcedure 8.3.1StartUp 8.3.1.1Run 2 ormore CleanCycleswhen startinignstrumenetachday. More cleancyclesmay be usediftheprevioussamplescontainedhighconcentrationosffluoride. 8.3.2Blanks 8.3.2.1Preparesample usingthesame methods and typeofmatrixasthetestsample. 8.3.2.2Forrabbitstudiesu,sebeefliverasthematrix.Prepareatleast3 samplesofbeefliver (100- 150 mg) forblanks. 8.3.2.3Putsample inDohrmann boat.Combust each sample asdescribedinsection9.0and analyzesample accordingtomethod AMDT-M-2 fortheionselectiveelectrodaenalysis. 3 8.3.2.4For rabbitstudiest,hemeter readingfora blank sample shouldbe 0.03ppm or lower beforeproceedingwiththecalibrationB.um sampleswitilthislimitisreached,or untilinthe judgement of theoperatorthereadingisstablewithrespecttohistoricarleadings(previous48 hours). 8.3.2.5For non-mbbitstudiest,heblankreadingsshouldreacha predeterminedionconcentration beforeproceedingwith thecalibration. 8.3.2.6Itmay be necessarytomix approximately50 mg of charcoalwiththesample toaid combustion. 8.3.3 Standard Curve 8.3.3.1Weigh outatleast15 matrixsamples(5 standardswith3 replicateesach)intaredand labeledweigh boats.For rabbitstudiesw,eigh 100-150 mg beefliversamples.Record weightsin studydata.Storethematrixsampleson dryiceor icepacks tokeep them frozeauntilused. 8.3.3.2Placeweighed beefliversampleinDohrmann sampleboat. 8.3.3.3StartwiththeloweststandardconcentrationU.sing a Hamilton syringe4,@ecta fixed quantityof thestandardon or inthematrix.For rabbitstudiesu,se 4 uL of standardand ejectiton or inthebeefliver. 8.3.3.4At least3 replicatesshouldbe usedforthelowest standardconcentrationm;ore replicates may be used atthediscretioonf theanalyst. 8.3.3.5Combust thesample as describedinsection9.3and analyzeaccordingtoAMDT-M-2. 8.3.3.6Run all15 standards.Ifone replicatiessignificantdliyfferenftrom theothertwo replicatersu,n anothersample forthatstandard.Indicatein datathatthenew replicatreeplacesthe oldreplicataend thatthenew replicatweillbe used tocalculatteheregressiocnurve. 8.3.3.7When allstandardshave beenrun,calculattehe r2. r2 must be atleast0.95.Ifitisnotat least0.95,consultwith supervisor. 8.3.3.8A new standardcurveshouldbe run when thecombustion tubeor sample matrixis changed.,New standardcurvemay alsobe runatthediscretioonftheanalyst. 8.4 StorageConditionsforStandards 8.4.1Storagerequirementsforstandardsaredependenton theindividuasltandardsused. Typically,standardsarestoredatroom temperatureinplastisccrew topbottles. 8.4.2 New FC-95 standardsshouldbe preparedatleastonce a month. 9.0 PROCEDURES 9.1TypicalOperating Conditions: 9.1.1Combustion tubetemperature= 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 the SYSTEM SETUP window. 9.2.3Put thefurnacemodule and thecellintheREADY mode. 9.2.4Close the SYSTEM SETUP window. 4 9.2.5When theoven has reachedtheREADY temperaturer,un theCLEAN BOAT program found intheCELL CHECK menu. 9.2.6See AMDT-EP-3 fordetailosfthe Dohrmann software. 9.3Sample ExtractionProcedure: 9.3.1Open theSAMPLE HATCH and placethesample intheBOAT. Itmay be necessaryto mix approximately50 mg of charcoalwiththesample toaidcombustion.Ifthisisdone,charcoal should alsobe mixed inwhileestablishintghebaselineand when generatingthestandardcurve. 9.3.2 Close SAMPLE HATCH. - 9.3.3Add appropriatveolume ofTISAB solutionor 1:1 TISAB:Milli-QTI-wlatermixturetoa labeledsample collectiovnial.Typically0.6n-iLto 15 mL areused. For rabbitstudiesu,se 1.0or 2.0mL of 1:1 TISAB:Milli-QT@,wlaterrrdxture. 9.3.4PlacethevialsothatthetipoftheCOMBUSTION TUBE isintheTISAB atleast0.25 inches.Gases releaseduringpyrolysimsust bubblethroughtheTISAB. 9.3.5Run theEOX-SOLIODS program found in theRUN menu. 9.3.6When theEOX program isfinishedr,emove thecollectiovnialfrom thecombustiontube. 9.3.7IfundilutedTISAB was usedtocollecthesample,add an equalvolume of Milli-QTbw4ater totheTISAB tomake 1:1TISAB:Milli-QTm. 9.3.8Rinsetheend of thecombustiontubewithMilli-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 cottontippedapplicatoorr vacuumed out.Itmay be necessarytoscrapparticleosffthe bottom witha spatulaorothersimilardevice.A drop of Milli-QTMwater may be added tothe boattoaidintheClean Cycle. 9.3.10Close thehatch. 9.3.11Run theCLEAN BOAT program. 9.3.12Sample isreadyforanalysibsy ion selectiveelectrod(eAMDT-M-2). 9.4 Sample Calculations 9.4.1Use thestandardcurvetocalculattehesamplevalue. 9.4.2Sample Mass RecoveredF (ug)= (TISAB volinmL) x (Orionreadinginppm -intercel2t) (Slope) 10.0 VALIDATION 10.1QualityControl 10.1.D1ailyStartUp CheckSamples:Oncethestandarcdurveisestablisheeadc,hdayof analysisisstartebdy analyzingQC samples.The QC samples aretobe thesame as thelowest concentratiosnpikedsamplesused togeneratethestandardcurve.Each concentratiomnust 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. Precisioannd accuracyvarieswhen analyzingsamplesof different matricesand differenrteferencecompounds. 10.3 Other ValidationParameters: NA 5 11.0 -DATA ANALYSIS 11.1 Calculations 11.1.F1orthestandarcdurveu,seregressiaonnalysinsExcelv,ersio5n.0orgreater. 11.1.2To calculattehefluoridceontractiointhesample,seemethod AMDT-M-2. 11.2 AnalyzingtheData 11.2.1r2 must beatleast0.95orgreater".Outliersm"ay be excludediftwo ofthethreereplicates arewithin20% ofeachotherandtheoutlieirsgreatetrhan200% oftheaverageofthosetwo or lessthan50% oftheaverageofthosetwo. Any suchoutliersshouldbe pointedoutinthedataand notedintheFinalReportalongwiththereasonitwas considereadn outlier. 12.0 ATTACHMENTS None 13.0 REFERENCES 13.1Rosemount DohrrnannDX2000 OrganicHalideAnalyzerOperator'Msanual (Ivfanua9l15349,revisioBn, December 1993) 13.2 ANMT-M-2 FluoridMeeasurementby Means ofan OrionEA940 ExpandableIon Analyzer 13.3AMDT-EP-3 RoutineMaintenanceofa ModifiedDohrmann DX2000 OrganicHalide Analyzer 14.0 REVISIONS Revision Number Reason forChange Revision Date 6 3M Environmental Laboratory Method FluorideMeasurement by Means of an Orion EA940 Expandable Ion Analyzer Method IdentiricatioNnumber: AMDT-M-2 RevisioNnumber:0 Adoption Date: Io-4-fs- RevisionDate: None Author Rich Youngblom Approved By: G@, Leader Date QualityAssurance Date Software: MS Word S.1a ArfectedDocuments: AMDT-M-1 Thermal Extractionof Fluorideby Means of a Modified Dohrmann DX2000 Organic HalideAnalyzer 1.0 SCOPE, APPLICABLE COMPOUNDS, AND MATRICES 1.1SCOPE: Thismethod isforthecalibratiaond operatioonf an OrionEA940 Expandable Ion Analyzer. 1.2APPLICABLE COMPOUNDS: Fluoride. 1.3APPLICABLE MATRICES: Liquidsamplesinan appropriabtueffersolutionP.referred pH of6.0. 2.0 KEYWORDS 2.1Fluoridef,luorinei,onselectiveelectrode 3.0 PRECAUTIONS 3.1No hazardsidentifiweidththismethod. 4.0 SUPPLIES AND MATERIALS 4.1Orion940999 TotalIonicStrengthAdjustmentBuffer11(TISABII)orequivalent. 4.2OrionModel 900001 electrodfeillinsgolutio(nAgCl)orequivalent. 4.3Orion940907 100 ppm fluoridsetandarodrequivalent. 4.4Mfili-QTmwateror equivalent. 4.5Magnetic stirbars. 4.6Lab tissues. 4.7Sample collectiovnials. .4.8Plastic100mL volurnetrifclasks. 4.9Polystyrenepipettes. 4.10Miscellaneouslaboratorgylassware. 5.0 EQUIPMENT 5.1OrionModel EA940 ExpandableIonAnalyzeror equivalent. 5.2Orion Model 960900 SolidStateCombinationFluorideelectrodoerequivalent. 5.3MagneticStirPlate. 5.4IBM compatible386 or486 computer(onlyneededifusingOrion3E software). 5.5OrionRS232 interfacceable(onlyneededifusingOrion3E software). 5.6MicrosoftExcel5.0(onlyneededifusingOrion 3E software). 6.0 INTERFERENCES 6.1Itisrecommended thatthepH be atornear6.0.A 1:I.mixturoefTISAB and sample/MlliQ TMwater willgenerallbyringsampletopH of 6.0. 6.2Sample temperaturmeay effecftluoridmeeasurement. Itisrecommended thatthesample be atroom temperatureasthestandardswere when themeterwas calibrated. 6.3The ratethesamplesarestirreadtshouldbe consistenwtiththeratethestandardswere stirred. .2 6.4Air bubblestrappedunder electrodcean giveerroneousreadings.Make sureno airistrapped under electrode. 7.0 SAMPLE HANDLING 7.1 No specialhandlingnecessary. 8.0 CALIBRATION AND STANDARDIZATTON 8.1 PreparationofCalibratioSntandards 8.1.1Measure50 n-lLofTISAB IIinto5 100niL plastivcolumetrifclasks. 8.1.2Labeltheflaskass0.05,0.1,0.5,1.0,and 1.5ppm F-,alongwiththedateand yourinitials. 8.1.3Pipett0e.05,0.1,0.5,1.0,and 1.5mL of 100 ppm fluoridsetandardintotheappropriatel" labeledflasks. 8.1.4Add approximately30 mL of Nfilli-QTw'a4tertoeach flask. 8.1.5Shake theflaskstomix thesolutions. 8.1.6Eliminateairbubblesfrom theflasksby tippingtheflaskson theirsidesand rollingtheairin theflasksover theairbubbles. 8.1.7Bring thevolume intheflasksup tothe100 mL mark with Nfini-QT,w4ater. 8.1.8Invertand shake theflasksforthefinalmixing. 8.1.9Record standardsinStandardsLog Book. 8.2 Calibration 8.2.1Ifnecessary,remove tapefrom electrodfeillinhgole. 8.2.2Invertprobe towet topseal. 8.2.3E ecta few dropsof fillinsgolutionfrom bottom of electrodteo wet lowerseal. 8.2.4Filltheelectrodweithfillinsgolution. 8.2.5The meter and theF- electrodaeretypicallcyalibratebdy directmeasurement withno blank correctionu,singstandardswithconcentrationosf 0.05,0.1,0.5,1.0,and 1.5ppm F-,following themanufacturer'sinstructions. 8.2.6Record theslopein theappropriatleogbook. 8.2.7Clean theelectrodbey rinsingwithMilli-QTMwaterand wipingthesidesdown withlab tissues. 8.3 StorageConditionsforStandards 8.3.1Calibratiosntandardsarestoredatroom temperature. 9.0 PROCEDURES 9.1Calibrationand Measurement, Standard method: 9.1.1The sample tobe measured needs to be mixed withTISAB usingtheproportions recommended by theTISAB manufacturer. 9.1.2Placea stirbarinthesampleand placethesample on thestirplate. 9.1.3Allow thesample tomix fora few secondsbeforeinsertintgheelectrode.When the electrodeisinsertedm,ake surethereareno airbubblestrappedundertheelectrode. 9.1.4The sample shouldbe thesame temperatureas thecalibratiosntandardsand stirreadtthe same rateas thecalibratiosntandards. 9.1.5@Vhen thereadingshave stabilizerde,cordthereadingintheappropriatelogbook. 3 9.2 CalibrationAnd Nleasurement,Using Orion 3E Software: 9.2.1Calibration: 9.2.1.1 Follow steps8.2.1to8.2.4. 9.2.1.2Press FunctionKey #8 (F8). 9.2.1.3The computer screenwillask you toconfirm thenumber of standardstobe used, concentrationof thestandards,and whether ornot a blank istobe includedinthe calibration. Make any necessarychanges to theinformationpresentedand clickon CONTINUE. 9.2.1.4Place theelectrodeinthe firssttandardon the stirplateand clickon CONTINUE. 9.2.1.5Observe thereadingson thegraphicdisplayon thecomputer. When the readingshave stabilizedp,ressACCEPT READING. 9.2.1.6Repeat step9.2.1.4and 9.2.1.5fortheremaining standards. 9.2.1.7Afterthe finalstandard,the computer willdisplaytheslopeof thecurve,as wellas the intercepatnd correlationR.ecord theslope,intercepta,nd correlatioinnthe appropriatelog book and clickon CONTINUE. 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 to storedata in. 9.2.2.2Record thename of thespreadsheetused in the appropriatelog book. 9.2.3Fluoride Measurement: 9.2.3.1Follow steps9.2.1through9.2.4 9.2.3.2Enter thename of thesample inthe appropriateplaceon the screen. 9.2.3.3Click on theNEW SANTLE button 9.2.3.4When thereadingshave stabilizedc,lickon theRECORD buttonand writetheresultinthe appropriatelog book. 10.0 VALIDATION 10.1 QualityControl: 10.2 Precisionand Accuracy 10.3 Other ValidationParameters AccordingtoReference13.2,therangeof detectioins0.02 ppm fluorideup toa saturatedsolutionof fluoride. 11.0 DATA ANALYSIS 11.1 CalculationsNonenecessary. 11.2 Analyzing the Data Nonenecessary. 12.0 ATTACHIMENTS None 13.0 REFERENCES 4 13.1Orion Model EA940 ExpandableIonAnalyzer InstructioMnanual,Orion Research Incorporated1,991. 13.2Orion Model 960900 SolidStateCombination FluorideElectrodeInstructioMnanual, Orion Research Incorporated1,991. 14.0 REVISIONS Revision Number Reason forChanize Revision Date 3M Environmental Laboratorl Method Extractionof Fluorochemicalsfrom Rabbit Livers SOP IdentiricatiNounmber: AMDT-M-4 RevisionNumber: 0 Adoption Date: RevisionDate: None Audior.Dave ChristensorVCynthWieaber Approved By: roupLeader /0 - 31Date QualityAssurance -3 5Date Software:MS Word, 6.0 AffectedDocuments: M-5, AnalysisofRabbitExtractforFluorochernicaUlssing Electrospray Mass Spectroscopy. 1.0 SCOPE 1. 1 Scope: Thismethod isfortheextractioonf fluorochemicalfsrom rabbitlivers. Ethyl acetateisused toextractfluorocherriicaflrsom theliversforanalysisby electrospraymass spectroscopy. 1.2 Applicable Compounds: Fluorochemicalsor otherfluorinatecdompounds. 1.3 Matrices: Rabbit Livers. 2.0 KEYWORDS 2.1 Fluorochemicals,rabbitliverse,lectrospraymass spectrometer,fluorinated compounds, extraction. 3.0 PRECAUTIONS 3.1 Use gloveswhen handlingtherabbitliverst,heymay containpathogens. 4.0 SUPPLIES AND MATERIALS 4.1 Supplies 4.1.1 -Syringec,apableofmeasuring100 pL 4.1.2 Eppendorf typeor disposablepipets 4.1.3 Gloves 4.1.4 Plasticgrindingtubes 4.1.5 Plasticentrifugetubes,15 mL 4.1.6 Ubels 4.1.7 Nitrogen 4.1.8 Timer 4.1.9 FiltersT,itannylon syringef:aters0,.2 4.1. 10 Analyticalpipets:glassvolumetdc pipets. 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 volumetdc flaskand dilutetovolume withMRH-Q water.Dilutethissolution1:10 fora 250 ppm solution. 4.2.2 Sodium carbonate/SodiumBicarbonateBuffer(J.T.Baker), (Na2CO3/NaHC03) 0.25 M: Weigh 26.5 g of sodium carbonate(N%COO and 21.0 g of sodium bicarbonate(NAHCO) intoa 1 L volumetricflaskand bringtovolume with NfiUi-Qwater. 4.2.3 Diluteacetonitrisloelutiond,iluteacetonitri1l:e1with Miui-Q water. 4.2.4 EthylAcetate 4.2.5 Methanol 4.2.6 MIIU-Q water 4.2.7 IH,IH,2H,2H -perfluorooctanesulfonaiccid(Aldrich) 4.2.8 FC-95 (3M SpecialtCyhemical Division) 2 5.0 EQUIPMENT 5.1 Ultra-Tun7-7a5xGrindefrorgrindilnigvesramples. 5.2 Vortexrrdxer 5.3 Centrifuge 5.4 Shaker 5.5 AnalyticaElvaporator 6.0 INTERFERENCE 6.1 Ilereareno known interferencaetsthistime. 7.0 SAMPLE -HANDLING 7.1 llierabbitliverasrereceivedfrozena,nd must be keptfrozenuntiltheextractioins performed. 8.0 CALIBRATION AND STANDARDIZATION 8.1 PreparatioonfInternaSltandards 8.1.1Preparean internasltandardofapproximatel1y2ppm IH,lH,2H,2Hperfluorooctanesulphoanciicdtobe added toeach liversample. 8.1.2 Weigh atleast0.1 g of IH,1H,2H,2H-periluorooctanesulphoanciicdintoa 100mL volumetrifclaskR.ecordtheactualweighl 8.1.3 Bringitup tovolume withmethanol,thisisthestockstandard. 8.1.4 To a 250 mL volumetricflaska,dd 3 mLs ofthestockstandardand bringto volume with MUli-Q'WaterC.alculatteheactualconcentrationfthe standard. actualmg perfluoroctane- sul2honicacid X linl 0.1L 250 mL actualconcentratiopnp,m 8.2 Prepare FC-95 Anion Standards 8.2.1 PrepareFC-9@ standardsforthestandardcurve. 8.2.2 Weigh approximatel1y00mg of FC-95 intoa 100 n:LLvolumetrifclask. Recordtheactualweighl 8.2.3 Bringup tovolume withdiluteacetomi@ffle. 8.2.4 Dilutethesolutiownithdilutaecetonitri1l:1e0 fora solutioonf approximatel1y00ppm. Dilutethissolutio1n:10withdilutaecetonitdlfeor a solutioonfapprox.10ppm. 8.2.5 Use the10ppm solutiotnomake.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 liveirntoa 250 mL Nalgenebottlceontaining 200 mLs NfiUi-Qwater.Grind toa homogenous solution. 8.3.2 Add 1mL ofthesolutiotnoa 15mL centrifugteube.Preparea totalofeight I mL aliquotosf thesolutioinn15 mL centrifugteubesB.e suretore- suspendsolutiobny shakingitbetweenaliquots. 3 S.3.3 SpikesevenoftheImL aliquowtisththefollowianmgountosfworking standardsin step9.12of theprocedure.One I mL aliquotservesas the blank. Worldng Standard UL (ApproximateConc.) - 5 ppb 100 500 pp 500 ppb 300 500 ppb 400 1 ppm 500 5 ppm 200 5 ppm 300 Approximate final concentratioonf FC-95 inliver Blank 0.292 ppm 0.584 ppm 0.877 ppm 1.168 ppm 2.924 pp 5.F4Tppm 8.772 ppm 8.4 Calculatetheactualvalueof thestandards: uL of standardx concentratio(ninppm) = finacloncentratio(nppm) 171 mg liver*I/ml homogenate ofFC -95inliver *Average weightof bovine.Uverinsolutionas determinedby weighing 1 mL homogenatesof 40 mg liverin200 mL of MiRi-Q water.The amount of FC-95 isreporte4as equivalentosf FC-95 potassiumsalt. 8.5 Calibration 8.5.1 Extractthespikedbeefliverhomogenate following9.13to9.23of this method.Use thesestandardstoestablisyhour curve on themass spectrometer. 8.5.2 Alternativelay,standardcurvemay be generatedusingratiosof responses of theperfluorooctansulfonaatneionand theinternasltandardanionversus concentratioonf theperfluorooctanesulfonatneion. 8.6 Storage Conditionsfor Standards 8.6.1 New standardasrepreparedwitheachanalysisS.tandardasrestoreidn coveredplasticentrifugteubesuntiltheanalysison themass spectrometer isperformed. 8.7 Storage Conditionsfor Standards 8.7. 1 Beef livehromogenates may be frozenafterpreparation. 9.0 PROCEDIJRFS 9.1 ObtainfrozenEver samples.In spenttissuen,ote thattheliverhasnotbeen packaged with othertissues. 9.2 Use a dissectinsgcalpeland cutoffapproximatelyIg of liver. 9.3 Weigh thesampledirectliyntoa taredplastigcrindingtube. 9.4 Record theliverweightin thestudynotebook. 9.5 Put a labelon thevialwiththestudynumber, weight,rabbitID,dateand analyst initials. 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. IL8 9.19 9.20 9.21 9.22 9.2 3 9.24 9.25 Add 2.5 mLs water. Grind thesample.Put thegrinderpr-obeinthesample and grindforabout 2 minutes,untilthesample isa homogeneous solutionwithno largechunks. Rinse theprobe offintothesample with2.5mLs water usinga pipet. Take thegrinderapartand cleanitwith methanol aftereach sample.Follow AMDT-EP-22. Cap thesample and vortexfor15 seconds. PipetI mL intoa 15 mL centrifugteube.Label thecentrifugteubewiththeidentical informationasthegrindingtube.(SeeAMDT-M-4 Worksheet fordocumentingthe remainingsteps.) Spike thebeefliverhomogenates withtheappropriataemount of FC-95 standard as describedin 8.3. Spike thesamplesand beefliverhomogenates with 100 uL of internasltandard. Add I mL ofthesodium carbonate/sodiumbicarbonatbeufferand 1 mL ammonium acetate. Using an analyticaplipet,add 5 mL ethylacetate. Cap thesample and vortex20 to30 seconds. Put them intheshakerfor20 min. Centrifugefor20 to25 minutes,untilthelayersarewellseparatedS.etthepower on thecentrifugteo25. Remove 4 mLs of thetoporganiclayertoa fresh15 mL centrifugteubewith a 5 mL graduatedglasspipet.Transferthelabeltothefreshtube. Blow thesample down on theanalyticaelvaporatortoneardrynesswithnitrogen, approximately30 to40 minutes. Bringtheremaininsgampleup m I mL dilutaecetonitfwfiltehan analyticpailpet. Vortex 15 seconds. Transferthesampletoa 3 mL syringe.Attacha 0.2gm nylonmesh filtearn,d filter thesample intoa freshcentrifugteubeora autovialL.abel thetubeor vialwiththe studynumber and animalnumber. Cap and hold foranalysisby electrospramyass spectroscopy. Complete AMDT-M-4 worksheetand attachtopage of studynotebook. 10.0 VALIDATION 10.1 QualityControl- 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 AMDT-EP-22 RoutineMaintenanceofUltra-TurraTx-25 14.0 REVISIONS Revision Number Reason forChange Revision Date 5 Study - Sample Number set# Rlan T iver Worksheet AMDT-M-4 FC-95 approx0.5ppm actual ppm 1ni)viT. 700 iiT. 100 uL 40()iiT. - FC-95 approx I ppm actual PPM #W.. - '500iiT, - FC-95 approx.5 ppm actual ppm #W Date and InitialfsorStd. - 200 iiT. 100 iiT, '%tudy number x@vht-rethe.-nrivinal worimheetiglomtm antin]Rfl$f1lcnnv_ T.iver'F.xtrac-Phrnnefl-qql PinetI mL nf T.iv&Ir;nliition Pimt 100 iiTn.f 12 nnm InternaSltsneiqrd I;td#- Vnrtex15 qpr Pint-It mT. nf 750 nnrn Ammnniiirn AcLtntp. Std # PinetI mT. nf 0-25N&rO./O@2,SM N;tHrO. lptuffer Pint-1t5mT. nf FthylAretqttVnrtt-7Y()-10ger@qhake20 min- rt-ntrifiia7r0-?15min- Rt-mnye ;t4 mT, :iiiniinnftorynnicI:% Rlnw dnwn tnnl-adrrvnt-q(q<O25 mT-)withN@ AM I m- nf I-IAcetnnitrilt-/14.0 TN# VnrtemIS czipFiltetrigina lee B-D gyrinpewithal 0 7iim 9RI filteirnto;t1 5 ml. ntitngamnlvei-;,il T)nttk- Tnitinl-q 6 3M EnvironmentalLaboratory Method Analysisof Rabbit Liver Extract for Fluorochemicalsusing Electrospray Mass Spectroscopy SOP IdentiricatiNounmber: AMDT-M-5 RevisionNumber: 0 Adoption Date: 4 --1 RevisionDate: None Author.Dave Christenson/CynthWieaber Approved By: ?;7aroduep r@ad@er Date QualityAssiirance Dam Software:MS Word, 6.0 AffectedDocuments:M-4, ExtractioonfFluorochemicalfsromRabbitLivers 1.0 SCOPE 1. 1 Scope: This method isfortheanalysisof extractsof rabbitliveror othertissuesor fluidsforfluorochemicalsusing the electrospraymass spectrometerT.he analysis isperformed by singleion monitoringofFC-95 anion,M/Z= 499, theinternal standardM/Z = 427, and otherappropriatemasses. 1.2 Applicable Compounds: Fluorochemicalsor otherfluorinatecdompounds. 1.3 Matrices: RabbitLivers(samples),Beef Liver-(standardso)t,hertissuesand fluids. 2.0 KEYWORDS 2.1 Fluorochemicals,fluorinatecdompounds, electrospraymass spectroscopy,mass spectrometer,rabbitlivers. 3.0 PRECAUTIONS 3.1 Use cautionwiththevoltagecablefortheprobe.When thevoltagecableisplugged intotheprobe DO NOT TOUCH THE PROBE, thereisriskof electricaslhock. 3.2 Do not run thepump above it'scapacityof 4000 psi.Ifpressuregoes over 4000 psi stopand releasepressure.The peak tubingmay be plugged.Troubleshoot back to findtheplug and replacetheplugged tubing.See AMDT-EP-15 3.3 Do notrun thepump todryness. 4.0 SUPPLIES AND MATERIALS 4.1 Supplies 4.1.1 Nitrogengas regulatedto 140 psi. 4. 1.2 Fluofixcolumn orequivalenl 4.1.3 100 uLor250 uL flatipsyringeforsample injection. 4.2 Reagents 4.2.1 Diluteacetonitrimloebilephase,diluteacetonitri1l:e1 withMilli-Qwater. 4.2.2 Milli-Qwater,allwaterused in thismethod shouldbe Milli-Qwater. 5.0 EQUIPMENT 5.1 VG Trio2000 ElectrospraMyass Spectrometeror equivalent. 5.2 ISCO SyringePump 5.3 SpectraphysicAsS300 Autosampler 5.4 100 uL Assembly 5.5 Autovialsorcapped centrifugetubes. 6.0 INTERFERENCES 6.1 There areno known interferenceastthistime. 7.0 SAMPLE HANDLING 7.1 Keep theextractedsamples incapped 15 mL centrifugetubesor incapped autovials untilready foranalysis. 2 8.0 CALIBRATION AND STANDARDIZATION 8.1 Preparation of CalibrationStandards 8.1. 1 Seven beefliverstandardsand one blank beefliverarepreparedduringthe extractiopnrocedure.(See AMDT-M-4, section8.0) 8.2 Calibration 8. 2. 1 Run the seven beef liverstandards twice,startingwith the lowest standard to obtainthe standardcurve. 8.2. 2 Typicallyone standardisrun aftereach 5 to7 samples.Choose a standard in thesame range of concentrationas thesamples. 8.3 Storage Conditions for Standards 8.3.1 Fresh standardsareprepared with each analysis.Standardsare storedin covered plasticcentrifugetubes untilthe analysison the mass spectometer is performed. Samples and standards are NOT refzigerated. 8.4 Storage Conditions for Beef Liver Homogenates 8. 4. 1 Beef liverhomogenates may be frozen afterpreparation. 9.0 PROCEDURE 9.1 InitialSet-up 9.1.1 Set software to "Operate on", Ion Mode ES*. 9.1.2 Record backing pressure in the instrument log. 9.1.3 Fillthe solventcylinder with mobile phase. 9.1.4 Set the pump to "Run". Set the flow to 1000 uL4r@n. Observes droplets coming out of the tipof the probe. The pressure should be at 1700 to 1800 psi. 9.1.5 Check the fused silicaat the end of the probe. Use an eye piece to check for chips.The tipshould be flatwith no jagge4 edges. Ifany.chips are found cut off the tipof the silicawith a column cutterand pullthe silicathrough to the appropriatelength. 9.1.6 Check your nitrogen supply. Turn on the nitrogen.There should be no nitrogen leaking around the tipof the probe. A fine mist should be coining out of the tip. 9.1.1 Carefullyguide the probe intothe opening. Insertituntilitwon't go any further.Connect the voltage cable to the probe. 9. 1. 8 Go tothe "Editor" page, and setIonizationMode toES-, and the appropriatemasses to 427 and 499. 9.1.9 If itisnot in singleion mode go to "Option" and setSIR. 9. 1. 10 StartAcquisition.Assign a filename, MO-DAY-YR + letterR.ecord itin the log book. 9. 1. 11 Run the beef liversamples firstr,unning each standard twice at the beginning of the run..Run a QC check by running one standard afterevery 5 to 7 samples. 9.2 Manual Injection 9.2. 1 Draw 150 uL of sample intoa syringe.Injectthe sample intothe rheodyne injectionport.Injectslowly. Record thesample ID in the log book. 9.2.2 Turn the valve to "On". 9.2.3 Wait two minutes, and injectthe next sample. 9.2.4 Record the scan number for each sample in the logbook. 9.3 Using the Autosampler 9.3.1 Set up sample trayA, B, or C. 9.3.2 Record thesamplesand theirpositionisn theinstrumentlogbook. Up to 17 vialsmay be ineach run. 9.3.3 Set-upthesampler: 9.3.3.1 Push thesample button 9.3.3.2 Setsample loopsize= 100 uL 9.3.3.3 Setinject/sampl=e2 9.3.3.4 SetCycle time= 0 9.3.3.5 Name thefileL:ivers 9.3.3.6 Identiftyhetrayused 9.3.3.7 Add thesamplestoQueue by pressing"Enter" 9.3.3.8 Press"Run" tostart 10.0 -VALIDATION 10.1 Quality Control. 10. 1.1 Run a standardevery5 to7 samples.Ifa significancthange(50%) in peak heightoccursstoptherun.Only thesamplesbeforethelastacceptable standardwillbe used.The remainingsamples 'Wilble reanalyzed. 10.2 Precisionand Accuracy 10. 2. 1See Method ValidatioRneportnumber AMDT-M-5.0.Vl 10.3 Other Validation Parameters 10.4 RefertoMethod ValidatioRneportNumber AMDT-M-5.0.Vl 11.0 DATA ANALYSIS 11.1 11.2 Calculations Plotthestandardcurve,usingthemean ofthetwo valuesobtainedforeach standard. 11.2. 1Read peak heightsor areasforthesamplesfrom theprintoutU.se linear regressiontodeterminethesample concentranons. 11. 2.2 Calculatethemg ofFC-95 anion,orotherfluorocherr@cailnthetotalrabbit liver - mg FC-95 anioninthetotalrabbitliver mg FC-95 anionfrom std.curve gms of liverusedforanalysis x Totalmass ofliverg,ms 11.3 Make a resulttsableand enteritinthestudybook. 11.4 Printa chromatogramforeach sample,withthepeakslabeledwiththesample or standardD:).Writethestudynumber on theprintouti,nitiadla,te,and putitinthe studyfolderS.tapleallchromatograms togetherand number pages. 4 12.0 ATTACHMENTS None 13.0 REFERENCES 13.1 ANDT-EP- 17 14.0 REVISIONS Revision Num Reason forchangc Revision Datr, 5 3M EnvironmientalLaboratory Method Analysisof Fluoride Using the Skalar Segmented Flow Analyzer With Ion SelectiveE.lectrode Method IdentiricatioNnumber: AMDT-M-8 Adoption Date: RevisionNumber: 0 RevisionDate: None Author:Deb Wright/CynthiaWeber Approved By: up Leader Date QualityAssurance Dam Software:IBM MS Word, 6.0 AffectedDocuments: AMDT-EP-26, Operationand Maintenanceof theSkalarSegmented Flow Analyzer 1.0 SCOPE 1. 1 1.2 This method isforthe analysisforfluoridet,hermallyextractedfrom samples using theDohrmann DX2000 (AMDT-M-1), and collecteidnTISAB foranalysiswith an Ion SelectiveElectrode(ISE).The analysisisperformed usingtheSkalar Segmented Flow Analyzer with fSE. Samples can be fissuess,erum, biologicalmaterial,or othermaterialsextractedon the Dohrmann. 2.0 KEYWORDS 2.1 Skalar,segmented flow,fluoride. 3.0 PRECAUTIONS 3.1 Follow standardlaboratorsyafetypractices. 4.0 SUPPLIES AND MATIERIALS 4.1 Supplies 4.1.1 Sample cups,4 mL plasticcups with caps 4.1.2 Autopipets,oxford or equivalentwith plasticdps 4.1.3 Polypropylenevolumetricflasks,100 mL 4.1.4 Cartridgecomponents, refertothe SkalarMethods 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 E from Orion.To I liteorf TISAB R add 2.5mL or 100 ppm fluoridesolutionand 1 mL Brij. 4.2.3 Sampler rinsingsolutionD:iluteTISAB 111:1 withNElli-Qwater. 4.2.4 Nitricacidsolutionfordecontamination,I N (labgrade):Slowly add 64 mLs concentratenditriaccid(HN03) to250 mLs of Milli-Qwater.Bring thevolume up to1 L with Milli-Qwater. 4.3 Standards 4.3.1 Stocksoludon, 100 ppm F: purchased from Orion. 4.3.2 Intermediatestandard,10 ppm: Dilute10 mLs of stocksolutionto 100 mLs with Milli-Qwater.Use polypropylenevolumetricflasks. 4.3.3 Working standard:Make up thefollowingworking standardsby adding the volumes of intermediateor stockstandardindicatedon thetable,using- oxfordorp umpmate pipets,to50 mLs of TISAB and dilutint-o 100 mLs withMilli-Qwater. Working Standard mLs of Stock Standard mLs of IntermediateStandard 0.015 ppm 0.15 0.03 ppm 0.3 0.06 ppm 0.6 0.09 ppm 0.9 0.12 ppm I.- 0.15 ppm 1.5 0.3 ppm 0.3 - 0.6 ppm 0.6 2 17 ppm 1.2 1.5ppm 1.5 5.0 EQUTPMENT 5.1 SkalarSegmented Flow Auto Analyzer Sansp'"S'ystem equipped with ISE 6.Q 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. 8.0 CALTBRATION AN]2 STAN12ARDIZATION 8.1 Preparation of Calibration Standards 8. 1.1 Preparecalibratiosntandardsas in section4.3. 8.2 Calibration 8.2. 1 The standardsare analyzedatthebeginningof therun. 8.3 Storage Conditions for Standards 8.3. 1 Standardsarestoredincapped polypropylenevolumetricflasks.New standardsareprepared ata 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 thefluoridelectrodesin theelectrodechamber. 9.1.3 Tum on thepower of the sampler,pumps, offsetpotentiometerand heating bath. 9.1.4 Put therea-ent-lineisntheappropriatebottles. 9.1.5 Turn on theinterfacec,omputer, displayand printer.Make sure you turn on the interfacebefore the computer. 9.1.6 Let thesystem stabilizfeorapproximately30 minutes. 9.2 Startinga Run 9.2.1 Createa sample tableby selectingFILES, TABLE, and CREATE, type in the name of thefilea,nd pressENTER. 9.2.2 Printthesample table,insertedin thesystem tableby pushing ESC, PRINT, GROUP 1.This willprinttheentirerun. 9.2.3 Dialthesampler settingstothe appropriatenumber of samples,number of seconds forsample wash, and number of seconds forthe sample. 9.2.4 Fill'thesample traywith thestandards,samples,washes and driftsR.V and FW/RUNOUT cups on the sampler do not need tobe filled.' 9.2.5 Set thebaseline. 3 9.2.5. 1SelectGRAPHICS, REAL TIME. Ifyou cannotgetreal-timey,ou may be intheData Handling Panel.Switch totheAnalysisPanel by selectingCONTROL PANEL and pushin-F7. 9.2.5.2Use thesmallscrewdriverfortheoffsetpotentiometertosetthe baseline.Adjustthebaselineuntilitisapproximately3/4 inchfrom the bottom of thescreen. 9.2.5.3 Check the higheststandardand adjustthegain,ifnecessary,with theinterfacescrew #3. 9.2.6 Go toCONTROL PANEL, and toanalysispanel.Deselectthe analysisthat willnot be run.(Selector deselectanalysisby pressingENTER.) PressTab to returnto theAnalysisPanel. 9.2.7 Pressthe spacebartobringup the localmenu. 9.2.8 SelectSTART tostartheanalysis. 9.2.9 Type your ID (initialst)h,esample tablewhich you createdunder 9.2.1(or pressENTER forchoices),choose runningwith or withoutthesystem table and selectSTART ANALYSIS. 9.2.10 Afterstartintghesoftware,startthesampler.Make surethatthe sampler is settotherightnumber of samples and thatthesample/wash/airtimes are OK. 9.2. 11 SelectGRAPHICS, REAL TIME toview theprogressof theanalysis. 9.3 Loading and Printing the Data-File 9.3.1 Go toCONTROL PANEL, pressthespacebartobringup thelocalmenu and selecLtOAD. SelectALITOCALCULATION and enterthefilename(or highlightthefiletobe printedand pressENTER). 9.3.2 To view thecalibratiocnurve,go toGRAPHICS, CALIBRATION CURVE. 9.3.3 To printthehi-h levelcurve,push PRINT SCREEN. 9.3.4 To printthelow levelscreen,push ESC toget out ofgraphics.Select SETTINGS. Change themax y valuetoapproximately900. Go toCAL CURVE and pressESC, and Enter.PressPRINT SCREEN. 9.3.5 Return toSETTINGS and change themax valueback to4095, go toEDIT, pressENTER and PRINT SCREEN toprintsample peaks. 9.3.6 To printtheresultgso to CONTROL PANF-L, SPACEBAR, OUTPUT, OUTPUT. SelectPRINTER for theEpson or PRN fortheLaser. 9.4 Shutdown 9.4.1 Put allthereagent-lineisnMilli-Qwater. 9.4.2 Let thesystemrinseforapproximately30 minutes. 9.4.3 Afterthesystem has rinsedcompletely,tum offthesampler,pump and offsetpotentiometerT.um offtheheatingbathon weekends. Leave liquidin the lines. 9.4.4 Take theelectrodeout and soak in 100 ppm F overnight. 9.4.5 Releasethepump-decks, airbus and sampler pump-tubing. 9.4.6 SelectFILES, pressALT F and selectQUIT toexittheprogram. 9.4.7 On Friday,turnoffthecomputer,displiyand interfaceforthe weekend. 10.0 VALIDATION 10.1 QualityControl 10.1.1Run a standar(dmidtohi0-hconcentratieovne)ry10samplesI.fa significancthange inpeak heightoccurs,only thesamples beforethe last acceptablestandardwillbe used.The remainin- samples willbe reanalyzed. 4 10.2 Precisionand Accuracy 10. 2. 1See Method ValidatioRneportnumber AMDT-M-8.0.V I 10.3 Other ValidationParameters 10.4 RefertoMethod ValidatioRneportNumber AMDT-M-8.0.Vt 11.0 DATA 11.1 11.2 11.3 11.4 ANALYSIS Calculations 11. 1.1The standardcurve isplottedby theSkalarsoftware. 11. 1.2Allcalculationasredone by theSkalarsoftware.r' shouldbe 0.995or better. Preparespreadsheettso summarize data.Includesample volume, weightsused etc. Write thestudynumber on theprintoutsi,nitiadla,tetheprintouta,nd bind together withallpackagedocuments and placein thestudyfolderM.ake a copy of the summary sheetand tapeintothestudynotebook.Back up alldataand spreadsheets onto studydiskand backup disks. ElectroniDcata 11. 4.IOLP studiesE:lectronidcataiscopiedonto theStudy floppydiskforeach study,and alsodataiscopied ontoa floppydiskthatisstoredin thelab. 11.4.2 Other studiesA:lldataiscopiedonto a floppydiskthatisstoredin thelab. 12.0 ATTACHMENTS None 13.0 REFERENCES 13.1 13.2 13.3 AMDT-M-1, TherrnaIExtractioonfFluorideby Means ofa ModifiedDohrmann DX2000 OrganicHalideAnalyzer-Liver SkalarMethods,#335,SkalarMethods Manual AMDT-EP-26, Operationand MaintenanceoftheSkalarSegmented Flow Analyzer 14.0 REVISIONS Revision Number, Reason forchange Revision Date 5 3M Environmental Laboratory Method Thermal ExtractionofFluorideby Means of a ModifiedDohrmann DX2000 Organic HalideAnalyzer-.Serum Method IdentlflcatiNounmber: AMDT-M-14 RevisionNumber: 0 AdoptionDate: lo -3 -.,r1 RevisionDate: None Author.RichYoungblom Approved by: u Leader 101.31 @,-g Date z QualityAssurance Date Software:MS Word 5.1a AffectedDocuments: AMDT-M-2 FluoridMeeasurementby Means ofanOrionEA940 ExpandableIonAnalyzer AMDT-EP-3 RoutineMaintenanceofa ModifiedDohrznannDX2000 OrganicHalideAnalyzer 1.0 SCOPE, APPLICABLE COMPOUNDS. AND MATRICES 1.1Scope:Thismethod isfortheoperatioonfa Dohrmann DX2000 when itisusedtoextract fluoridferom variousmatricesT.he fluoridiestypicallcyollecteidnTISAB solutiofnoranalysis withan ionselectiveelectrode. 1.2ApplicableCompounds: Fluorocherr@caolrsotherfluorinatecdompounds. 1.3Matrices:Biologicaflluidsp,articularsleyrum. 2.0 KEYWORDS 2.1Fluoridef,luorine,xtractiopny,rolysisi,onizatioino,nselectiveelectrodDeo,hrmann, halide, DX2000, fluorochemicals. 3.0 PRECAUTIONS 3.1 Glasswareand exhaustgasescan be extremelyhot. 3.2Glasswareisfragileb,rokenglassmay causeinjuries. 3.3Pressurizedgases,propercompressedgas handlingpracticersequired. 3.4Solventbased samplesmay flashm,ay need toallowthem todrydown beforestartinrgun. 3.5Potentiabliohazardsdue tothebiologicamlatrices.Use appropriatpeersonalprotective equipment. 4.0 SUPPLTES AND MATERTALS 4.1Compressed Oxygen, Hydrocarbonfree,regulatedto30 PSI. 4.2Compressed Helium,High PurityGrade,regulatetdo45 PSI. 4.3QuartzglasssampleboatwithTeflonTMtubingD,ohrmann 890-097orequivalent. 4.4Quartzglasscombustiontube,RelianceGlass G-9405-012 orequivalent. 4.5Orion940999TotalIonicStrengtAhdjustmentBuffer(TISAB Il)orequivalent. 4.6Sample collectiovnialsH,DPE. 4.7Nfilli-QTwmater .4.8Polystyrcnepipettes. 4.9ActivatedCharcoalE,.Merck 2005orequivalent. 4.10HamiltonSyringeor equivalent. 4.11Miscellaneouslaboratorgylassware 5.0 EQUTPMENT 5.1Rosemount Dohrmann DX2000 OrganicHalideAnalyzer,modifiedforfluorideextraction. 5.2MM compatible386 or486 computer. 5.3DX2000 softwarev,ersion1.00,modifiedforfluorideextraction. 5.4Excel Spreadsheetv,ersion5.0orgreater 6.0 INTERFERENCES 6.1 Sample sizeislimitedtoapproximatel1y00 gi.Thismay varyfrom matrixtomatrix. 2 7.0 SAMPLE HANDLING 7.1 Samples are tobe handled with plasticpipettes.A new pipetteistobe used foreach sample. 8.0 CALIBRATION AND STANDA]KDTZA 8.1 Preparationof CalibrationStandards TION 8.1.1The standardsrequiredforeach projectwillneed tobe appropriateforthatindividualproject. Refer toprotocolforthatproject. 8.1.2Typically50-500 ppm FC-95 inmethanol standardsareused. 8.1.3For rabbitserum studies,use beefserum as thematrix. 8.2 Calibration-Overview The normal calibratioinsthefluozidceurve (AMDT-M-2). However, ifan optionalspikedserum curve isrequiredtheprocedure listebdelow isused. 8.2.1A calibratiocnurvefortheDX2000 isgeneratedby spikingsamples with known standards and combusting them usingthe same methods and matrix type asthe samples tobe tested. 8.2.2Typically,threereplicateosf each standardand fiveconcentrationosf standardswillbe spiked. 8.2.3 Standardcurve willbe plottedas Mass Spiked F (ug)on the x-axisand Standard Mass Recovered F (ug)on they-axis.Generate a regressioncurve and calculattehe equationfortheline and ther2 value. 8.2.4Mass SpikedF (ug)= (Amount spikedinmL) x (Conc. of standardinppm) x (0.6004)* *FC-95 is60.04% F therefore0.6004 isthe factorused to convertFC-95 to F 8.2.5StandardMass Recovered F (ug)= (TISAB volume innlL)x (Orionreadinginppm) 8.3CaUbration - Procedure 8.3.1StartUp 8.3.1.1Run 2 ormore Clean Cycleswhen startinignstrumenteachday. More cleancyclesmay be used ifthe previoussamples containedhigh concentrationosf fluoride. 8.3.2Blanks 8.3.2.1Prepare sample using the same methods and type of matrixas the testsample. 8.3.2.2For rabbitstudiesu,se beefserum asthe matrix. 8.3.2.3Put serum blank-inDohrmann boat. Combust sample asdescribedinsection9.0 and analyzesample accordingtomethod AMDT-M-2 forthe ion selectivelectrodeanalysis. 8.3.2.4For rabbitstudiest,he meterreadingfora blanksample should be 0.03 ppm or lower beforeproceedingwith the calibrationB.um samples untilthislimitisreached,or untilinthe judgement of theoperatorthereadingisstablewith respecttohistoricarleadings(previous48 hours). 8.3.2.5For non-rabbitstudies,theblank readingsshouldreach a predeterminedion concentration beforeproceeding with thecalibration. 8.3.2.6Itmay be necessarytomix approximately50 mg of charcoalwith thesample to aid combustion. 3 8.3.3Standard Curve 8-3.3.1Ifbeefserum isfrozen,thaw atleastenough tocompletethestandardcurve analysisforthe day (=30mL). 8.3.3.2PipettelOOgLof beefserum intoDohrmann sample boat. 8.3.3.3StartwiththeloweststandardconcentrationU.sing a Hamilton syringe,(@ecta fixed quantityof thestandardon or inthematrix.For rabbitstudiesu,se4 uL of standardand ejectiton orin thebeefserwn. 8.3.3.4At least3 replicatesshouldbe used forthelowest standardconcentrationm;ore replicates may be used atthediscretioonf theanalyst. 8.3.3.5Combust thesampleasdescribedin section9.3and analyzeaccordingto AMDT-M-2. 8.3.3.6Run all15 standards.Ifone replicatiessignificantdliyfferenftrom theothertwo replicatersu,n anothersample forthatstandard.Indicateindatathatthenew replicatreeplacesthe oldreplicataend thatthenew replicatweillbe used to calculatteheregressiocnurve. 8.3.3.7)When allstandardshave been run,calculattehe r2. r2must be atleast0.95.Ifitisnot at 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 8.4.1Storagerequirementsforstandardsaredependenton theindividuasltandardsused. TypicaUy,standardsarestoredatroom temperatureinplastisccrew topbottles. 8.4.2New FC-95 standardsshouldbe preparedatleastonce a month. 9.0 PROCEDURES 9.1TypicalOperating Conditions: 9.1.1Combustiontubetemperatur=e 950'C. 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 isnotstarteds,tarttheEOX program on thePC. 9.2.2Open theSYSTEM SETUP window. 9.2.3Put thefurnacemodule and thecellintheREADY mode. 9.2.4Close the SYSTEM SETUP window. 9.2.5When theoven hasreachedtheREADY temperaturer,un theCLEAN BOAT program found intheCELL CHECK menu. 9.2.6See AMDT-EP-3 fordetailosftheDohrmann software. 9.3Sample ExtractionProcedure: .9.3.1Open theSAMPLE HATCH and pipetteI00jiLof sampleintotheBOAT. Itmay be necessarytomix approximately50 mg of charcoalwiththesample toaidcombustion.Ifthisis done,charcoalshouldalsobe mixed inwhile establishintghebaselineand when generatingthe standardcurve. 9.3.2Close SAMPLE HATCH. 4 9.3.3Add appropriatveolume ofTISAB solutionor 1:1TISAB:Milli-QTm watermixturetoa labeledsample collectiovnial.Typically0.6mL to 15 mL areused. For rabbitstudiesu,se 1.0or 2.0mL of 1:1 TISAB:Milli-QTM watermixture. 9.3.4Placethevialso thatthetipoftheCOMBUSTION TUBE isintheTISAB atleast0.25 inches.Gases releaseduringpyrolysimsust bubblethroughtheTISAB. 9.3.5Run theEOX@-WATER program found intheRUN menu. 9.3.6When theEOX program isfinishedr,emove thecollectiovnialfrom thecombustiontube. 9.3.7IfundilutedTISAB was used tocollecthesample,add an equalvolume of Milli-QTMwater totheTISAB tomake 1:1TISAB:Milli-QTM. 9.3.8Rinse theend of thecombustiontubewithMilli-Q"rwmaterand wipe witha KIMWIPE to remove any TISAB remainingon thetube. 93.9 Open thesample hatchand remove any remair@ngash from theboat.Ash can be removed witha cottontippedapplicatoarnd/orvacuumed out.Itmay be necessarytoscrapparticleosffthe bottom witha spatulaor othersimilardevice.A drop of Milli-QTmwater may be added tothe boat toaidintheClean Cycle. 9.3.10 Close thehatch. 9.3.11 Run the CLEAN BOAT program. 9.3.12Sample isreadyforanalysibsy ionselectiveelectrod(eAMDT-M-2). 9.4 Sample Calculations 9.4.1Use thestandardcurveto calculattehesample value. 9.4.2Sample Mass RecoveredF (ug)= (TISAB volinrnL)x (Orionreadingin12pm-intercel2t) (Slope) 10.0 VALIDATION 10.1 QualityControl 10.1.1DailyStartUp Check Samples: Once thestandardcurveisestablishede,ach day of analysisisstartedby analyzingQC samples.The QC samples aretobe thesame as thelowest concentratiosnpikedsamplesused togeneratethestandardcurve.Each concentratiomnust be done intriplicautnelessthefirsttwo replicateasrewithin20% ofthestandardcurve,thena third replicatiesnot necessary. 10.2 Precisionand Accuracy: See method development analysisand sample analysisin FluorideNotebooks 2,3,and 5. Precisionand accuracyvarieswhen analyzingsamplesof different matricesand differenrteferencecompounds. 10.3 Other ValidationParameters:NA 11.0 DATA ANALYSIS 11.1 Calculations 11.1.1For thestandardcurve,use regressionanalysisin Excel,version5.0or greater. 11.1.2To calculattehefluoridceontractioinnthesample,seemethod AMDT-M-2. 5 11.2AnalyzintgheData 11.2.r12mustbeatleas0t.95orgreate"rO.utliermsa"y beexcluedd iftwo of thethreereplicates arewithin20% of eachotherand theoutlieirsgreaterthan200% of theaverageof thosetwo or lessthan50% of theaverageof thosetwo. Any such outliersshouldbe pointedout inthedataand notedintheFinalReportalongwiththereasonitwas consideredan outlier. 12.0 ATTACHMENTS None 13.0 REFERENCES 13.1Rosemount Dohrmann DX2000 OrganicHalideAnalyzerOperator'Msanual (Manual 915349,revisionB, December 1993) 13.2 AMDT-M-2 FluorideMeasurement by Means of an OrionEA940 Expandable Ion Analyzer 13.3AMDT-EP-3 RoutineMaintenanceofa Modified Dohrmann DX2000 OrganicHalide Analyzer 14.0 REVISIONS Revision Number Reason forChange Revision Date 6 9.3 QualityAssurance Unit Statement AttachmentD GLP Study QualityAssurance Statement Study Title: Single-dose Intravenous Pharmacokinetic Study of T-6052 in Rabbits Study Number- AMDT- 111694.1 Name ofAuditor.K-ariRambo This studyhasbeen inspectedby theQualityAssuranceUnit asindicateidnthefollowingtable. The findingswere reportedtothestudydirectoarnd management. InspectioDnates EM :IQ 10/13/95 10/19/95 Phase FinalReport Date InspectioRneportedto Manag=nt StudyDire 10/19/95 10/19/95 l@7 @QAU Auditor Date . 9.4 Key PersonnelInvolved inthe Study 3M Environmentgl Laboratory Key Personnel Thermal extractiofnollowedby analysiussingOrionIonanalyzer: Jim Johnson Deb Wright RichYoungblom Deann Plummer Analysisofliverextractussingelectrospramyass spectrometry: Jim Johnson Dave Christenson Thermal extractiofnollowedby analysisusingSkalarsegmentedflow analyzerwithIonselectiveelectrode: Jim Johnson Deb Wright "RichYoungblom 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 analysis usingOrion ionanalyzer. Thisdata,althoughsupportivei,ntheopinionof the StudyDirectorisnot requiredtoreachthe conclusion statedinFinalReportSection6.0,and thereforiesnot discussedin detail. Summary ofCombustion Data -Liver AMDT-111694.1, HWI 6329-134 As ReferencedinFinalReport section6.0DATA ANALYSIS Totalug FluorideinWhole Liver Mean per Dose Group ControlGroup 2.0mg/kg dose(T6052) 20.0mg/kg dose(T6052) 200 mglkg dose(T6052) 1000 mg/kg dose(T6052) ug 26.3 17.9 17.0 35.1 77.7 RPT1 34L.XLS FC120 PK ID LiverBlank-I LiverBlank-2 LiverSpike-1 LiverSpike-2 LiverSpike-3 F52548-1 F52548-2 F52548-3 F52549-1 F52549-2 F52549-3 F52559-1 F52559-2 F52559-3 F52566-1 F52566-2 F52566-3 F52567-1 F52567-2 F52567-3 LiverBik-I LiverBik-2 LiverSpk-1 LiverSpk-2 LiverSpk-3 LiverSpk-4 LiverSpk-5 LiverSpk-6 % rcvry 103% 92% 84% 80% 90% 92% 94% 108% 94% Actual pprn Fin liver (WM) 0.355 0.181 1.16 1.34 1.24 0.328 0.337 0.207 0.179 0.159 0.264 0.277 0.222 0.211 0.298 0.372 0.333 0.853 0.98 0.772 0.133 0.108 1.10 0.980 1.06 2.72 3.24 2.43 Average ppm Fin liver (WM) 0.291 0.200 0.237 0.334 0.867 liver burned (grams) 0.109 0.140 0.135 0.105 0.102 0.106 0.127 0.116 0.101 0.131 0.124 0.107 0.138 0.125 0.143 0.139 0.133 0.137 0.101 0.108 0.112 0.124 0.110 0.139 0.132 0.105 0.101 0.117 Whole liver weight (grams) 90.3 90.3 90.3 89.3 89.3 89.3 71.9 71.9 71.9 105.1 105.1 105.1 89.7 89.7 89.7 TotalF- in whole liver (ug) 26.3 17.9 17.0 35.1 77.7 Dosage (mg/kg) 0.0 2.0 20.0 200 1000 Page 1 9.11.2Summary and raw data;analysisof liver extractsusing electrospramyass spectrometry. HWI # 6329-134 Study: ProtocolNumber: T*st Material: Matrix: R Squared Value: Response FactorAnwunt: Analyst: Data: Method: Instrument: LABBASE File: Single-DoseIntravenousPharmacoklnegc TPOO84.PK T-6052inRabbits(FC 120) Liver Screening N/A DLC 4/4/95 FisonsVG 2000 EkkctmsprayMS 040495C A- I CO- 31 - '?5 Group Dose Group 1: 0 mg/kg SterilWeater Group 2: 2 mg /kg Group 3: 20 rng/kg Group 4: 200 mg/kg Group 5: 1000 nV/kg Samplo F52548 IonCount Extractewdt Area N.D. 1.0036 Dilution Concentration Totalmass factor lwg ofliver 9 1 N.D. 90.344 Totalanwunt Of FC-95 per liver nV N.D. % of FC,95 F52549 N.D. 1.0055 1 F52559 N.D. 1.0026 1 F52566 $ 1 1 F52567 $ 1.0027 1 N.D. 89.284 N.D. N.D. 71.921 N.D. 105.089 89.664 S = PositiveresponseforIonmonitored. SIR ofI&598 & 599 The concentratiownas calculatebdy usingthestandardcurveand multiplyintgheresultby 4/5.The 4/5 factorIstheresultofa miscalculatioInnapplyingformula8.4InMethod AMDT-LI-4-0.137 mg ofliverwas used In thiscalculatiornatherthan 171mg. The concentrationIsnthestandardcurvearetherefor5e14largerthantiny shouldbe. By multiplyintghecalculatecdoncentrationInthestandardcurveby 4/5,thecorrectresultIsobtained. LAb BAM 14,v).t 134 Sariip,Ie 1 *1c I iv I)peratnr illc 1-i?.rji.1F3te 05 -08--1-9'F@. 0'?"34:@,6 Ver z.ion Prirtt-eialn 05-)G-t995 AT 09:36:14 Lirie Fit ft)i-ce-Ithi-oii@ihOrii..Iirl. r-%Tvn- "gnn A-Z l'2)1mi 91r6( 4 1.6 I-V E L 4 Y AMOIJI,4T Corfiponerit t EXTERNAL STANDARD CALIBRATlOt4 AREA 0.4000 0. @;()00 1.(5000 4.0000 0000 1 0 ()0 91001,-7 1371"1 107036 501406 10 ocr9il v INTER'k'.El:'I' 1?13,31 E 04 617 1.4E - ()5 x- 0 . 12 Arficijtr, + 0 . 0 0 0 0 E q e.5 1)0 ()0 C 0 0 File:040495C Sample:HWI # 6329-134; W4-0499-C- LAB-BASE - The MS Data System FC-120 (PX) RABBIT LIVER EXTRACTS V.FS. 27 3 24 2 6 5. 53 73 949 1 82 1349 9 11 8 12 7 looXFS- v.FS-' ISen 13 3 349 12 8 rD 27 20 8 2 r4 tw 6 6573 3 24 25 2 19.39 46 6 860 logo 1138 1208 1349 1480 File:040495C Sample:HWI # 6329-134; 048495CI@@- LAB-BASE - The MS Data Sgsten FC-120 CPX) RABBIT LIVER EXTRACTS 126I14S3 4 1 03 9 rLo4 11I1L388 v.FS LO r4 (0 I@_fpLpo1J2L42747@@ rVN6t4,L)-- kp N fo to tL 100- 14 976 13 6 3 v.FS [son'jliiii"iii@6ii ii-iiii ii-6ii 34043 1258 to File:040495C Sample:HWI # 6329-134; 040495C loo- 183 8 LAB-BASE - The MS Data Sgsten FC-120 (PX) RABBIT LIVER EXTRACTS .J1L"a v.FS JLJL2i111,37 1196 1248 \JL21 13-8 1349 1 L9 Lee. . ...... .................. 19 12 7 1349 ...... LeeZFSisenildso 3440454383 1258 JL44976 1363 File:040495C Sample:HWI 040495C 6329-134; LAB-BASE - The MS Data Sgsten FC-120 (PX) RABBIT LIVER EXTRACTS 9,18 ZFS,Son 82 27 65 24 25 2 3 73 26ii '14'88 600 age 9 JL247 logo 1200 t4ei4i 9.11.3Summary and raw data; ug F- in whole liveras determinedby thermalextractionfollowedby analysis using Skalarsegmented R'ow analyzerwith ion selectivelectrode. KA-IW RE: 6329-134 LIUER SRMPLES RMDT 111684.1 Date of Rnalysis:3-30-95 Rnalyst: ODW The samples are burned inthe Bohrman at 950 C using between 0.1 and 0.2 grams of the liver.The gas iscollectedin 1.0mL of 1:1TISHB/Milli-Owater then an additional2 mL of 1:1 TISHB/Milli-Oisadded to allow for sufficientvolume for Skalar analysis.The samples are then analyzed on a Skalar Segmented Flow nnalyzer using the Ion SpecificElectrode(ISE)Method. TISHB buffer isadded to each sample as itproceeds through the system. The sample then goes through a heated mihing coil before the potentialbetween the ion selectiveelectrode and the reference electrode ismeasured. 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 plotted using the inverse logarithm option.The standard curve for the low range islinear.filsltandards and samples were then calculatedby the Skalarsoftware using these curves.filrlesultsbelow 0.0001 ppm appear on the raw data as #.####. R qualitycontrolstandard was analyzed every 10 samples to check for accuracy and drift. Raw data istaken from the appropriate calibratedrange of the Skalar printoutand summarized on an EHcel spreadsheet. The finalresultsare adjusted for the collectionvolume and any subsequent dilutions. .......... SUMMARY OF 6329-134 LIVER SAMPLES AM DT 111694.1 T GROUP 1 Dose Level:0 GROUP2 Dose Level: 2 mgfkg GROUP3 Dose Level: 20 mgtkg GROUP4 Dose Level: 200 mgtkg GROUPS Dose Level: 1000 mgfkg F52548-1 F52548-3 F52549-1 F52549-2 F52549-3 F52559-1 F52559-2 F52559-3 F52566-1 F52566-2 F52567-1 F52567-2 F52567-3 ND ND 0,017 ND 0.016 ND 0.015 ND 0.019 0.02 0.05 0.04 0.04 3.0 0.1057 ND 3.0 0.1160 ND 3.0 0.1008 0.51 3.0 0.1309 ND 3.0 0.1239 0.39 3.0 0.1074 ND 3.0 0.1377 0.33 3.0 0.1246 ND 3.0 0.1426 0.39 3.0 0.1393 0.52 3.0 0.1374 1.14 3.0 0.1007 1.26 3.0 0.1084 1.17 ND 90.3439 ND 90.3439 ND 89.2836 45 ND 89.2836 ND 89.2836 35 71.9209 ND ND 71.9209 24 71.9209 ND 0.46 105.0891 41 105.0891 55 89.6636 102 1.19 89.6636 113 89.6636 105 134-L.SUM 134-L.XLS NAM, I Tracer 1.50 1.24 83% 2 Drift 1.50 1.27 85% 3 Wash 0.016 4 Sid 1 0.015 0.020 131% 5 Sid 2 0.03 0.03 85% 6 Sid 3 0.06 0.06 99% 7 Sid 4 0.09 0.09 98% 8 Sid 5 0.12 0.13 105% 9 Sid 6 0.15 0.15 980/@ 10 Sid 7 0.30 0.29 96% II Sid 8 0.60 0.61 101% 12 Sid 9 1.20 1.24 103% 13 Sid 10 1.50 1.47 98% 14 Drift 1.50 1.31 88% is Wash 0.016 16 Blk-IA 0.02 3.0 0.1087 0.61 17 Blk-IB ND 18 Bik-2A ND 3.0 0.1087 ND 3.0 0,1399 ND 19 Blk-2B ND 3.0 0.1399 ND 20 Spk-IA 21 Spk-IB 22 Spk -2 0.05 3.0 0.1347 1.18 0.05 3.0 0.1347 1.15 0.06 3.0 0.1047 1.66 23 Spk-3 0.06 3.0 0.1019 1.65 24 F52548-1 ND 3.0 0.1057 ND 90.3439 ND 25 F52548-3 ND 3.0 0.1160 ND 90.3439 ND 26 Drift 1.50 1.28 85% 27 Wash 0.016 28 F52549-1 29 F52549-2 30 F52549-3 31 F52559-1 32 F52559-2 33 F52559-3 34 F52566-1 35 F52566-2 36 F52567-1 0.017 ND 0.016 ND 0.015 ND 0.019 0.02 0.05 3.0 0.1008 0.51 89.2836 45 3.0 0.1309 ND 89.2836 ND 3.0 0.1239 0.38 89.2836 34 3.0 0.1074 ND 71.9209 ND 3.0 0.1377 0.33 71.9209 24 3.0 0.1246 ND 71.9209 ND 3.0 0.1426 0.39 105.0891 41 3.0 0.1393 0.52 105.0891 55 3.0 0.1374 1.14 89.6636 102 Page 1 0.004 0.004 0.004 0.004 63.00 63.00 63.00 63.00 134-L.XLS 37 F52567-2 0.04 38 Drift 1.50 1.26 84% 39 Wash 0.016 40 F52567-3 0.04 41 Drift 1.50 1.28 85% 42 Wash 0.016 3.0 0.1007 1.26 89.6636 113 3.0 0.1084 1.17 89.6636 105 Page 2 1995-03-30 13:29 OutPut of : 950330B1 Software version 6.1 cl990,93 Operator : ddw Date of the Analysis : 1995-03-30 11:18 Analysis File Name : C:\SKALAR\DATA\95033OB1 Fluoride 1.5 Calibration order = Inverse Logarithm Slope s Result x cl 0 &acid&& 0 = 10cl s i x = corrected value of the sample cl = corrected value of the concentration 1 s = Slope of the electrode a2 al aO = -0.00000 0.00085 -1.13248 Fluoride L Calibration order 2 Correlation r = 0.99665 Result = a2 x:o + al * x + aO a2 = al aO - 0.00000 0.00031 0.01615 Sampler Type Number Sample Time Wash Time Air Time Take up special needle Height : SA1000 :1 : 50 sec. : 120 sec. : 1 sec. : Single : None : 70 mm. Diluter needle Height : 80 mm dilution Factor : 10 dilution Volume : 2.5 ml. Resample Dilution runs I User file : TXT Reproces : No 1995-03-30 13:29 Fluoride 1.5 Path number Signal type Decolor system Number dilute Resample dil Threshold diG output Window event OutPut of 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 95033OB1 1995-03-30 13:29 OutPut of sl standard 0.015 s2 standard 0.030 s3 standard 0.060 s4 standard 0.090 ss standard 0.120 s6 standard 0.150 s7 standard Ignore ss standard Ignore S9 standard Ignore slO standard Ignore Order : 2 Dimension : PPM start Value : 500 DU trigger Limit : 1800 Sec Peak shape : Pointed start ignore : 60 Sec eNd ignore : 120 Sec Measure window : 75 Filter : No Regeneration : No formula c4:=c3 output 950330BI 1995-03-30 13:29 output of : 950330BI Fluoride 1.5 Fluoride L PPM PPM Pon Typ Ident Dil Weight Ch Result F Cor. Valu Time wt iw Initial Wash 1 1.000 3 0.074 4 0.0161 0 128 65 0 0 0 1t Tracer 1 1.000 3 1.242 4 0.8874 2167 2313 212 2167 0 0 2d Drift 1 1.000 3 1.274 4 0.9072 2207 2370 388 2207 0 0 3w Wash 1 1.000 3 0.074 4 0.0161 0 181 574 0 0 0 4 sl Standard 1. 1 1.000 3 0.075 4 0.0196 11 192 747 11 0 0 5 s2 Standard 2 1 1.000 3 0.078 4 0.0255 30 212 911 30 0 0 6 s3 Standard 3 1 1.000 3 0.096 4 0.0594 137 320 1086 137 0 0 7 s4 Standard 4 1 1.000 3 0.113 4 0.0883 226 410 1264 226 0 0 8 S5 Standard 5 1 1.000 3 0.138 4 0.1257 338 524 1437 338 0 0 9 s6 Standard 6 1 1.000 3 0.153 4 0.1465 399 586 1612 399 0 0 10 s7 11 s8 Standard 7 Standard 8 1 1.000 3 0.289 4 0.2901 1 1.000 3 0.605 4 0.5171 797 992 1787 797 0 0 1362 1568 1963 1362 0 0 12 s9 Standard 9 13 siO Standard 10 14 d Drift 15 w Wash 1 1.000 3 1.239 4 0.8854 1 1.000 3 1.466 4 1.0359 1 1.000 3 1.313 4 0.9317 1 1.000 3 0.074 4 0.0161 2163 2386 2137 2163 0 0 2460 2694 2312 2460 0 0 2256 2440 2487 2256 0 0 0 184 2668 0 0 0 Page 1 of 3 1995-03-30 13:29 OutPut of : 950330BI Fluoride 1.5 Fluoride L Pos Typ 16 u 17 u 18 u 19 u 20 u 21 u 22 u PPM PPM Ident Dil Weight Ch Result F Cor. Valu Time BLK 1-A 1 1.000 3 0.077 4 0.0220 19 200 2836 19 0 0 BLK 1-B 1 1.000 3 Absen A -39 1.38 3012 4 0.0041 -39 0 0 BLK 2-A 1 1.000 3 too 1 -> -27 148 3242 4 0.0078 -27 0 0 BLK 2-B 1 1.000 3 0.068 4 0.0029 -43 128 3360 -43 0 0 SPK 1-A 1 000 3 0.092 4 0.0530 117 288 3536 117 0 0 SPK 1-B 1 1.000 3 0.092 4 0.0517 113 280 3714 113 0 0 SPK 2 1 1.000 3 0.095 4 0.0581 133 298 3888 133 0 0 23 u 24 u SPK 3 F52548-1 1 1.000 3 0.094 4 0.0559 1 1-OdO 3 0.072 4 0.0121 126 288 4059 126 0 0 -13 145 4236 -13 0 0 25 u 26 d 27 w F52548-3 Drift Wash 1 1.000 3 Absen A -23 132 4412 4 0.0090 -23 0 0 1 1.000 3 1.282 4 0.9117 2216 2368 4588 2216 0 0 1 1.000 3 0.074 4 0.0161 0 149 4771 0 0 0 28 u F52549-1 1 1.000 3 0.074 4 0.0171 3 152 4904 3 0 0 29 u 30 u 31 u F52549-2 F52549-3 F52559-1 1 1.000 3 Absen A -22 128 5113 4 0.0094 -22 0 0 1 1.000 3 0.073 4 0.0155 -2 148 5285 -2 0 0 1 1.000 3 0.073 4 0.0140 -7 144 5461 -7 0 0 Page 2 of 3 1995-03-30 13:29 OutPut of : 95033OB1 Fluoride 1.5 Fluoride L PPM PPM Poo Typ Ident 32 u F52559-2 33 u F52559-3 Dil Weight Ch Result F Cor. Valu Time 1 1.000 3 0.073 4 0.0152 -3 148 5606 -3 0 0 1 1.000 3 Absen A -31 120 5813 4 0.0066 -31 0 0 34 u F52566-1 1 1.000 3 0.075 4 0.0186 8 160 5982 8 0 0 35 u F52566-2 1 1.000 3 0.078 4 0.0242 26 178 6163 26 0 0 36 u 37 u F52567-1 F52567-2 1 1.000 3 0.092 4 0.0520 1 1.000 3 0.087 4 0.0422 114 266 6338 114 0 0 83 236 6513 83 0 0 38 d 39 w Drift Wash 1 1.000 3 1.262 4 0.8998 1 1.000 3 0.074 4 0.0161 2192 2346 6689 2192 0 0 0 154 6871. 0 0 0 40 u 41 d F52567-3 Drift 1 1.000 3 0.087 4 0.0422 1 1.000 3 1.282 4 0.9122 83 236 7038 83 0 0 2217 2368 7213 2217 0 0 42 w Wash wt rw RunOut Wash 1 1.000 3 0.074 4 0.0161 1 1.000 3 0.074 4 0.0161 0 150 7450 0 0 0 0 152 7688 0 0 0 Page 3 of 3 IC) 4000 flow data of 9SO33081 : Fluoride I.S @4ool t %0 0 o =-Exit L I Fl-Help 4 %0 I Crtl-P-Edit peaks -TTirm I 437S 'rE 4000 Raw data of SS033081 Fluoride 1.5 0 2423 Ene-Exit I Fl-Help 19 19 I Crtl-P-Edit peaks Time I 4000 Raw data of SS033001 8796 MLEfft"@ : Fluoride I.S 309 KMIMM4 All o@ilo 0 6788 Eno-Exit : Fl-Help : Crtl-P-Edit peaks Ti" I QOL,)-T4- 11173 2.9365883- *rbC Calibration - curve of 950330131 : Fluoride L 0.018 MORPLULIS6M4 0.0161470 0 Order 2 Heatsured . r 0 ssess 4000 1.4315392- Calibration curve of 95033081 Fluoride I.S 0.0737255 Order Measured Inverse Logarithm 4cmo A Awl 9.11.4Summary and raw data;ppm F- in serum as determinedby thermalextractiofnollowedby analysis usingOrionion analyzer. This data,althoughsupportivei,nthe opinionofthe Study Directorisnot requiredtoreachtheconclusion statedinFinalReport Section6.0,and thereforiesnot discussedindetail. HWI 6329-134 AMDT 111694.1 Dohrmann Serum Analysis AnalysisDates:07/31/95- 08/2/95 Allserum samples were thermallyextractedby a modifiedDohnnann DX2000 Organic HalideAnalyzerand collecteidna 1:1 milliQ waterand TISAB solution.The sampleswere measured on an Orion EA940 expandableion analyzer.The Dohrmann was calibrateudsing34ppm, 40ppm, 62ppm, 100ppm, 124ppm, 250ppm, and 500ppm FC-95 standards. The Orion was calibratebdy directmeasurement with no blankcorrection using0.05ppm, O.lppm, 0.5ppm, l.Oppm and 1.5ppm F-standards.'Me slope,intercepta,nd correlatiownere recordedin theappropriatleogbook. A summary tableisincludeds,howing theppm F- ineach sample (seepage 2). The summary tablealsoshows theactualOrionreadings.An initial calibratiocnurvewith standarddeviation%,RSD, R' valueand equationof thelineison pages 3 and 4. Pages 5 and 6 show theexcelspreadsheetthatwas generatedwhen the samples were being analyzed. The Dohrmann FC95 calibratiocnurvewas not used to generatethedata. Page I of 6 STUDY # 6329-134 SERUM FC120 PK HM 63294U Fluorideconcentrationinrabbitserum (ppm F-) Dosage: Sample 0 mgtkg F52548 2 mg/kg F52549 20 mg/kg F52559 200 mg/kg F52566 1000 mg/kg F52567 2 hour 0.453 0.372 0.357 0.400 0.402 4 hour 6 hour 0.359 0.334 0.329 0.322 0.283' 0.532 0.282 0.539 0.367 0.427 8 hour 0.411 0.384 0.291 0.436 0.519 12 hour 24 hour 48 hour 0.383 1.70 0.678 0.353 0.68 0.642 0.290 0.584 0.594 0.296 0.564 0.718 0.327 0.684 0.560 ActualOrion Reading (ppm F-) Dosage: Sample 0 mg/kg F52548 2 mg/kg F52549 20 mg/kg F52559 200 mg/kg F52566 1000 mg/kg F52567 2 hour 0.0226 0.0186 0.0179 0.200 0.0201 4 hour 0.0180 0.0164 0.0142 0.0141 0.0183 6 hour 0.0167 0.0161 0.0266 0.0270 0.0214 8 hour -12 hour 24 hour 48 hour 0.0206 0.0191 0.0848 0.0339 0.0192 0.0177 0.0340 0.0321 0.0145 0.0145 0.0292 0.0297 0.0218 0.0148 0.0282 0.0359 0.0260 0.0164 0.0342 0.0280 34-ppm-1 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 NORMAN SERUM CURVE 1 ism ............................. :.Op .. .. 0.07175 0.1 2.0 0.05614 0.1 2.0 0.06462 0.1 2.0 0.08668 0.1 2.0 0.06728 0.1 2.0 0.05939 0.1 2.0 0.06385 0.1 2.0 0.07291 0.1 2.0 0.0753 0.1 2.0 0.07839 0.1 2.0 0.0902 0.1 2.0 0.1026 0.1 2.0 0.1126 0.1 2.0 0.1371 0.1 2.0 0.1451 0.1 2.0 0.1617 0.1 2.0 0.3217 0.1 2.0 0.2447 0.1 2.0 0.3078 0.1 2.0 0.4438 0.1 2.0 0.4584 0.1 2.0 0.4888 0.1 2.0 ................. 0.004 34 0.004 34 0.004 34 0.004 40 0.004 40 0.004 40 0.004 40 0.004 62 0.004 62 0.004 62 0.004 100 0.004 100 0.004 100 0.004 124 0.004 124 0.004 124 0.004 250 .0.004 250 0.004 250 0.004 500 0.004 500 0.004 500 176% 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 ............... 0.0817 0.0817 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 0.6004 1.2008 1.2008 1.2008 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 0.6156 0.8876 0.9168 0.9776 STDEV: %RSD: STDEV: %RSD: STDEV: %RSD: STDEV: %RSD: STDEV: %RSD: STDEV: %RSO: STDEV: %RSD: 0.015629 12 0.024057 17 0.005495 3.6 0,022443 11 0.025096 .8.5 0.082072 14 0.045915 5.0 SERUM CURVE 1 NORMAN (07/25195) 0.9-- 0.8-. 0.70.6LaU: wlx (A 0.40.3-02 0 0.0000 0.2000 y - 0.74U + O.OU Rz 0.9722 0.4000 0.6000 0.8000 MASS SPIKED (ug) 1.0000 1.2000 1.4000 SERUM CURVE I NORMAN (07/25/95) 0.9.- 0.8 0.7 0.6-0 LU lx w 0.5 co 4c 0.4-- 0.3-- 0.2-- 0.1 0 0.0000 y 0.743x + 0.064 F12 -0.9722 0.2000 0.4000 0.6000 0.8000 MASS SPIKED (ug) 1 .0000 1.2DW STUDY# 6329-134 SERUM ................... ............................................ ................... ...............................%...............................A.1...1.........1.............. ............. . ........ ....t..d.a..d...i.'...n......:.:...................... ................................................P...p.. ..........k.*.*........r.e.c.o.. d' sam' BLANK-1 0.0830 0.1 2.0 1.66 0.166 BLANK-2 0.0314 0.1 2.0 0.627 0.0627 SPIKE 62-1 0.0635 0.1 2.0 0.004 62 85% 1.27 0.15 0.127 SPIKE 62-2 0.0644 0.1 2.0 0.004 62 86% 1.29 0.15 0.129 SPIKE250-1 0.157 0.1 2.0 0.004 250 52% 3.15 0.60 0.315 SPIKE250-2 0.239 0.1 2.0 0.004 250 80% 4.78 0.60 0.478 SPIKE250-3 0.240 0.1 F52548-2HR 0.0226 0.1 F525-49-2HR 0.0186 0.1 2.0 0.004 250 2.0 2.0 80% 4.80 0.60 0.480 0.453 0.0453 0.372 0.0372 F52559-2HR 0.0179 0.1 2.0 0.357 0.0357 F52566-2HR 0.0200 0.1 2.0 0.400 0.0400 F52567-2HR 0.0201 0.1 2.0 0.402 0.0402 62PPM-3 0.0560 0.1 2.0 0.004 62 75% 1.12 0.15 0.112 62PPM-4 25OPPM-3 250PPM-4 0.0834 0.1 0.215 0.1 0.240 0.1 2.0 0.004 62 112% 1.67 o.i@ 0.167 2.0 0.004 250 72% 4.29 0.60 0.429 2.0 0.004 250 80% 4.80 0.60 0.480 BLANK-1 0.0722 0.1 2.0 1.44 0.144 BLANK-2 0.0386 0.1 2.0 0.772 0.0772 BLANK-3 0.0317 0.1 2.0 0.633 0.0633 BLANK-4 0.0340 0.1 BLANK-5. 0.0247 0.1 SPIKE 62-1 0.0858 0.1 2.0 2.0 2.0 0.004 62 0.680 0.0680 0.493 0.0493 88% 1.32 0.15 0.132 SPIKE 62-2 0.0802 0.1 2.0 0.004 82 108% 1.60 0.15 0.160 SPIKE 250-1 0.200 0.1 2.0 0.004 250 67% 4.00 0.60 0.400 SPIKE 250-2 0.204 0.1 2.0 0.004 250 68% 4.07 0.60 0.407 SPIKE 250-3 0.253 0.1 2.0 0.004 250 84% 5.07 0.60 0.507 SPIKE 250-4 0.185 0.1 BLANK 0.0796 0.1 BLANK 0.0237 0.1 F52548-4HR 0.0180 0.1 F52549-4HR 0.0164 0.1 F52559-4HR 0.0142 0.1 F52566-4HR 0.0141 0.1 F52567-4HR 0.0183 0.1 F52548-6HR 0.0167 0.1 F52549-6HR 0.0161 0.1 F52559-6HR 0.0266 0.1 62-PPM-1 0.0542 0.1 62-PPM-2 0.0722 0.1 250-PPM-1 0.101 0.1 250-PPM-2 0.159 0.1 250-PPM-3 0.251 0.1 BLANK 0.786 0.1 F52566-6HR 0.0270 0.1 F52567-6HR 0.0214 0.1 F52548-8HR 0.0206 0.1 F52549-8HR 0.0192 0.1 F52559-8HR 0.0145 0.1 2.0 0.004' 250 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 0.004 62 2.0 0.004 62 2.0 0.004 250 2.0 0.004 250 2.0 0.004 250 2.0 2.0 2.0 2.0 2.0 2.0 61% 3.69 0.60 0.369 1.59 0.159 0.475 0.0475 0.359 0.0359 0.329 0.0329 0.283 0.0283 0.282 0.0282 0.367 0.0367 0.334 0.0334 0.322 0.0322 0.532 0.0532 73% 1.08 0.15 0.108 97% 1.44 0.15 0.144 33% 2.01 0.60 0.201 53% 3.17 0.60 0.317 84% 5.03 0.60 0.503 15.'7 1.57 0.539 0.0539 0.427 0.0427 0.411 0.0411 0.384 0.0384 0.291 0.0291 STUDY# 6329-134 SERUM .98 ........s.......................................... Sarnpld:'::::,:@-@:;.re.a..d.i.i.i... ....... ........I............................................................%..:..:..:...."..'...:...:..:...:.:....:.A....................... k..i.W.......................... ppjm@:F,.*.......... .................r..e.dd ...... d* F52566-8HR 0.0218 0.1 F52567-8HR 0.0260 0.1 F52548-12HR 0.0191 0.1 F52549-12HR 0.0177 0.1 F52559-12HR 0.0145 0.1 F52566-12HR 0.0148 0.1 F52567-12HR 0.0164 0.1 62-PPM-1 0.0744 0.1 62-PPM-2 0.0915 0.1 250-PPM-1 0.284 0.1 250-PPM-2 0.339 .0.1 BLANK 0.0485 0.1 BLANK 0.0471 0.1 BLANK 0.0295 0.1 BLANK 0.0304 0.1 BLANK 0.0301 0.1 62-PPM-1 0.0583 0.1 62-PPM-2 0.0803 0.1 250-PPM-1 0.186 0.1 250-PPM-2 0.175 0.1 250-PPM-3 0.201 0.1 250-PPM-4 0.247 0.1 250-PPM-5 0.228 0.1 BLANK 0.105 0.1 BLANK 0.0205 0.1 F52548-24HR 0.0848 0.1 F52549-24HR 0.0340 0.1 F52559-24HR 0.0292 0.1 F52566-24HR 0.0282 0.1 F52567-24HR 0.0342 0.1 F52548-48HR 0.0339 0.1 F52549-48HR 0.0321 0.1 F52559-48HR 0.0297 0.1 F52566-48HR 0.0359 0.1 F52567-48HR 0.0280 0.1 BLANK 0.0613 0.1 BLANK 0.0396 0.1 BLANK 0.0383 0.1 BLANK 0.0412 0.1 BLANK 0.0319 0.1 62-PPM-1 0.0786 0.1 62-PPM-2 0.0934 0.1 250-PPM-1 0.304 0.1 250-PPM-2 0.269 0.1 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 0.004 62 2.0 0.004 62 2.0 0.004 250 2.0 0.004 250 2.0 2.0 2.0 2.0 2.0 2.0 0.004 62 2.0 0.004 62 2.0 0.004 250 2.0 0.004 250 2.0 0.004 250 2.0 0.004 250 2.0 0.004 250 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 0.004 62 2.0 0.004 62 2.0 0.004 250 2.0 0.004 250 0.436 0.0436 0.519 0.0519 0.383 0.0383 0.353 0.0353 0.290 0.0290 0.296 0.0296 0.327 0.0327 100% 1..49 0.15 0.149 123% 1.83 0.15 0.183 95% 5.68 0.60 0.568 113% 6.79 0.60 0.679 0.970 0.0970 0.942 0.0942 0.590 0.0590 0.607 0.0607 0.602 0.0602 78% 1.17 0.15 0.117 108% 1.61 0.15 0.161 62% 3.72 0.60 0.372 58% 3.50 0.60 0.350 67% 4.02 0.60 0.402 82% 4.94 0.60 0.494 76% 4.56 0.60 0.456 2.10 0.210 0.410 0.0410 1.70 0.170 0.680 0.0680 0.584 0.0584 0.564 0.0564 0.684 0.0684 0.678 0.0678 0.642 0.0642 0.594 0.0594 0.718 0.0718 0.560 0.0560 1.23 0.123 0.792 0.0792 0.766 0.0766 0.824 0.0824 0.638 0.0638 106% 1.57 0.15 0.157 125% 1.87 0.15 0.187 101% 6.08 0.60 0.608 90% 5.38 0.60 0.538 9.11.5Summary and raw data;ppm F-inserum as determinedby thermalextractiofnollowedby analysis usingSkalarsegmented flow analyzerwith ion selectiveelectrode. T hisdata,althoughsupportivei,nthe opinionof the Study Directorisnot requiredtoreachtheconclusion statedinFinalReportSection6.0,and thereforeisnot discussedindetail. RE: 6329-134 SERUM SAMPLES AMDT 111694.1 Date ofAnalysis:8/8/95 Analyst:DDW The samples are burned inthe Dohrman at950 C using0.10 mL ofthe serum. The gas iscollectedin2.0 enLof 1:1TISAB/Milli-Qwater. The samples are then analyzed on a SkalarSegmented Flow Analyzer usingthe IonSpecificElectrode (ISE)Method. TISAB bufferisadded toeach sample as itproceeds throughthe system. The sample then goes througha heated mixing coilbeforethe potentialbetween the ion-selectiveelectrodeand thereferenceelectrodeismeasured. The signalis amplifiedand relatedtothefluorideconcentration. The instrumentwas calibratedinthe ranges of0.015 -0.15 ppm and 0.15 - 1.50 ppm fluoride.The standardcurve forthe high range was plottedusing the inverselogarithmoption.The standardcurve forthe low range islinear.All standardsand samples were then calculatedby the Skalarsoftwareusing these curves.Allresultsbelow 0.0001 ppm appear on the raw data as A qualitycontrolstandardwas analyzed every 10 samples tocheck foraccuracy and drift. Raw data istaken from the appropriatecalibraterdange ofthe Skalarprintout and summarized on an Excel spreadsheet. The finalresultsare adjustedforthe collectiovnolume and any subsequent dilutions. ;2 GROUP 1 Dose Level:0 SUMMARY SERUM AMDT OF 6329-134 SAMPLES 111694.1 Sample IID Fluoridein Fluoridein Fluoridein Fluoridein Fluoridein Fluoridein Fluoridien Sample Sample Sample Sample Sample Sample Sample (ppm) 2 hr (ppm) 4 hr (ppm) 6 hr (ppm) 8 hr (ppm) 12 hr (ppm) 24 hr (ppm) 48 hr F52548 0.79 0.46 0.43 0.77 0.44 2.26 0.88 GROUP2 Dose Level: 2 mgtkg F52549 0.52 0.55 0.40 0.57 0.39 0.91 0.83 GROUP3 Dose Level: 20 mgtkg F52559 0.52 0.39 0.75 0.35 ND 0.87 0.76 GROUP4 Dose Level: 200 mgtkg F52566 0.72 0.34 0.86 0.56 0.31 0.77 0.94 GROUPS F52567 0.62 0.51 0.65 0.66 0.42 0.89 0.72 Dose Level: 1000 mgtkg 134S-RPT.XLS Page I SUMMARY SERUM AMDT OF 6329-134 SAMPLES 111694.1 MENEM= F52548-2 0.04 2.0 0.10 0.79 F52549-2 0.03 2.0 0.10 0.52 F52559-2 0.03 2.0 0.10 0.52 F52566-2 0.04 2.0 0.10 0.72 F52567-2 0.03 2.0 0.10 0.62 F52548-4 0.02 2.0 0.10 0.46 F52549-4 0.03 2.0 0.10 0.55 F52559-4 0.02 2.0 0.10 0.39 F525664 .0.02 2.0 0.10 0.34 F525574 0.03 .2.0 0.10 0.51 F52548-6 0.02 2.0 0.10 0.43 F52549-6 0.02 2.0 0.10 0.40 F52559-6 0.04 2.0 0.10 0.75 F52566-6 0.04 2.0 0.10 0.86 F52567-6 0.03 2.0 0.10 0.65 F52548-8 0.04 2.0 F52549-8 -0.03 2.0 F52559-8 0.02 2.0 F52566-8 0.03 2.0 F52567-8 0.03 2.0 0.10 0.77 0.10 0.57 0.10 0.35 0.10 0.56 0.10 0.66 F52548-12 0.02 2.0 0.10 0.44 F52549-12 0.02 2.0 0.10 0.39 F52559-12 ND 2.0 0.10 ND F52566-12 0.02 2.0 0.10 0.31 F52567-12 0.02 2.0 0.10 0.42 F52548-24 0.11 2.0 0.10 2.26 F52549-24 0.05 2.0 0.10 0.91 F52559-24 0.04 2.0 0.10 0.87 F52566-24 0.04 2.0 0.10 0.77 F52567-24 0.04 2.0 0.10 0.89 F5254848 0.04 2.0 0.10 0.88 F5254948 0.04 2.0 0.10 0.83 F5255948 0.04 2.0 0.10 0.76 F5256648 0.05 2.0 0.10 0.94 F5256748 0.04 2.0 0.10 - 0.72 134S-SUM.XLS Page 1 SERUM CURVE 1 7-31-95 NORMAN Sample ID Spk 34-1 Spk 34-2 Spk 34-3 Spk 40-1 Spk 40-2 Spk40-3 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 mL FC 95 Conc Mass Average % Result fmalvol SolutionFC 95 Soln Spiked Mass Recovery (ppm) (mL) Spiked (ppm) (ugF-) Recovered (ugF-) 0.09 2.0 0.004 34.00 0.07 2.0 0,004 34.00 0.08 0.15 188% 0.08 2.0 0.004 34.00 0.08 2.0 0.004 40.00 0.07 2.0 0.004 40.00 0.10 0.15 155%. 0.07 2.0 0.004 40.00 0.09 2.0 0.004 62.00 0.09 2.0 0.004 62.00 0.15 0.18 121% 0.09 2.0 0.004 62.00 0.11 2.0 0.004 100.0 0.12 2.0 0.004 100.0 0.24 0.24 99% 0.13 2.0 0.004 100.0 0.16 2.0 0.004 124.0 0.17 2.0 0.004 124.0 0.30 0.34 115% 0.19 2.0 0.004 124.0 0.33 2.0 0.004 250.0 0.26 2.0 0.004 250.0 0.60 0.61 102% 0.32 2.0 0.004 250.0 OA7 2.0 0.004 500.0 0.49 2.0 0.004 500.0 1.20 0.99 82% 0.52 2.0 0.004 500.0 SERUM CURVE I (NORMAN) 7-31-95 y-0.7743+x0.086 0.9881 1.20 i.oo-- 0.8-0- 0.60- 0.40-- 0.20 0.00 0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 MASS SPUCED (ug) SERCRV1 N.AVE Page 1 SERUM CURVE 1 7-31-95 NORMAN Sample ID Spk 34-1 Spk 34-2 Spk 34-3 Spk 40-1 Spk 40-2 8pk 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 Result (ppm) 0.09 0.07 0.09 0.08 0.07 0.07 0.09 0.09 0.09 0.11 0.12 0.13 0.16 0.17 0.18 0.33 0.26 0.32 0.47 0.49 0.52 DI:TISAB mL FC 95 Conc finalvol Solution FC 95 Soin (mL) Spiked (ppm) Mass Spiked (ugF-) Mass Recovered (ugF-) % Recovery 2.0 0.004 34.00 0.08 0.17 211% STANDARD DEVIATION: 2.0 0.004 34.00 0.08 0.13 163% % RSD: 2.0 0.004 34.00 0.08 0.16 191% 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% 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 O.OG4 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% 2.0 0.004 250.0 0.60 0.67 111% STANDARD DEVIATION: 2.0 0.004 250.0 0.60 0.52 87% % RSD: 2.0 0.004 250.0 0.60 0.65 108% 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 1 (t4ORMAN) 7/31195 y 0.7743x + 0.086 R 2. 0.9762 1-00 0.80 w 0.50 0.40 0.20 0-00.000 0-20 0.40 0.00 0.80 1.00 1.20 1.40 MASS SPIXED (ug) SERCRV1 N.SUM Page 1 134S-A.XLS 1995-08-08 10:58 output of: 95080SAI Operator :DDW Date of theAnalysis 1995-08-0807:01 IDATA\SERUM\950808Al AnalysisFileName C:\SKALAR\I)ATA\HW I Traccr 1.50 1.44 960/a 2 Drift 1.50 1.47 98% 3 Wash ND 4 Standard 1 0.015 0.015 970/9 5 Standard2 0.03 0.03 990/0 6 Standard 3 0.06 0.06 103% 7 Standard4 0.09 0.09 1000/0 a standard5 0.12 0.12 99% 9 Standard 6 0.15 0.15 1000/0 10 Standard7 0.30 0.29 93% 11 Standard8 0.60 0.62 103% 12 Standard 9 1.20 1.24 103% 13 Standard10 1.50 1.46 97% 14 Drift 1.50 1.53 1020/a 15 Wash ND 16 SERUM BLK 1 0.12 2.0 0.10 2.46 2.0 0.10 1.04 17 SERUM BLK 2 0.05 19 SPK 62-1 0.09 .2.0 0.10 1.86 0.004 62.00 0.15 0.1 2.0 0.10 1.90 0.004 62.00 0.15 0.1 19 SPK 62-2 20 SPK 250-1 0.10 0.28' 2.0 0.10 5.58 0.004 250.0 0.60 0.5 2.0 0.10 3.98 0.004 250.0 0.60 0.4 21 SPK 250-2 0.20 22 SPK 250-3 0.25 2.0 0.10 5.08 0.004 250.0 0.60 0.5 2.0 0.10 0.79 23 F52549-2 0.04 24 F52549-2 0.03 2.0 0.10 0.52 2.0 0.10 0.52 25 F52559-2 0.03 26 Drift 1.50 1.53 102% 27 Wash ND 28 F52566-2 o.04 2.0 0.10 0.72 2.0 0.10 o.62 29 F52567-2 0.03 30 SPK 62-3 0.08 2.0 0.10 1.61 0.004 62.00 0.15 0.1 2.0 0.10 2.28 0.004 62.00 0.15 0.11 31 SPK 624 0.11 32 SPK 250-1 0.24 2.0 0.10 4.86 0.004 250.0 0.60 0.9 2.0 0.10 5.56 0.004 250.0 0.60 0. 33 SPK 250-2 0.28 34 BLK 1 0.10 2.0 0.10 2.03 Page 1 134S-A.XLS 35 BLK 2 0.06 2.0 0.10 1.11 36 BLK 3 0.04 2.0 0.10 0,88 37 BLK 4 0.05 2.0 0.10 0.95 38 Drift 1.50 1.55 103% 39 Wash ND 40 BLK 5 0.04 2.0 0.10 0.84 41 SPK 62-1 42 SPK 62-2 43 SPK 250-1 44 SPK 250-2 45 SPK 250-3 46 SPK 250-4 47 BLK 0.09 0.11 0.25 0.25 0.30 0.23 0.12 2.0 0.10 1.86 0.004 62.00 0.15 0.19 2.0 0.10 2.29 0.004 62.00 0.15 0.23 2.0 0.10 4.94 0.004 250.0 0.60 0.49 2.0 0.10 4.98 0.004 250,0 0.60 0.50 2.0 0.10 5.94 0.004 250.0 0.60. 0.59 2.0 0.10 0.004 250.0 0.60 0.46 2.0 0.10 2.34 48 BLK 0.04 2.0 0.10 0.80 49 F52548-4 0.02 2.0 0.10 0.46 so Drift i.so 1.56 104% 51 Wash ND 52 F52549-4 0.03 2.0 0.10 0.55 53 F525594 0.02 2.0 0.10 0.39 54 F525664 0.02 2.0 0.10 0.34 55 F525574 0.03 2.0 0.10 0.51 56 F52548-6 0.02 2.0 0.10 0.43 57 F52549-6 0.02 2.0 0.10 0.40 58 F52559-6 0.04 2.0 0.10 0.75 59 SPK 62-1 0.09 2.0 0.10 I.S7 0.004 62.00 0.15 0.16 60 SPK 62-2 0.10 2.0 0.10 2.06 0.004 62.00 0.15 0.21 61 SPK 250-1 0.11 2.0 0.10 2.21 0.004 250.0 0.60 0.22 62 Djift 1.50 1.57 105% 63 Wash ND 64 SPK 250-2 65 SPK 250-3 66 BLK 0.20 0.31 0.12 2.0 0.10 3.96 0.004 250.0 0.60 0.40 2.0 0.10 6.16 0.004 250.0 0.60 0.62 2.0 0.10 2.38 67 F52566-6 0.04 68 F52567-6 0.03 2.0 0.10 0.86 2.0 0.10 0.65 69 Drift 1.50 1.57 105% 70 Wash ND Page 2 134S-B.XLS 1995-08-0814:27 OutPut of: 950808BI Operator :DDW Date of theAnalysis 1995-08-0810:58 AnalysisFileName C:\SKALAR\DATA\HWEDATA\SERUM\950808BI ........... I Tracer 1.50 1.47 98% 2 Drift 1.50 1.48 99% 3 Wash ND 4 Standard 1 0.015 0.015 97% 5 Standard 2 0.03 0.03 99% 6 Standard3 0.06 0.06 104% 7 Standard4 0.09 0.09 100% 8 Standard5 0.12 0.12 98% 9 Standard6 0.15 0.15 101% 10 Standard7 0.30 0.28 94% 11 Standard 8 0.60 0.62 103% 12 Standard 9 1.20 1.23 103% 13 Standard 10 1.50 1.47 98% 14 Drift 1.50 1.49 99% 15 Wash ND 16 F52549-8 0.03 17 F52559-8 0.02 18 F52566-8 0.03 19 F52567-8 0.03 20 F52548-12 0.02 21 F52549-12 0.02 22 F52559-12 ND 23 F52566-12 0.02 24 F52567-12 0.02 25 SPK 62-1 0.10 26 Drift 1.50 1.48 99% 27 Wasli ND 28 SPK 62-2 0.12 29 SPK 250-1 0.33 30 SPK 250-2 0.39 31 F52548-8 0.04 32 BLK 0.07 33 BI,K 0.07 34 BLK 0.04 2.0 0.10 0.57 2.0 0.10 0.35 2.0 0.10 0.56 2.0 0.10 0.66 2.0 0.10 0.44 2.0 0.10 0.39 2.0 0.10 ND 2.0 0.10 0.31 2.0 0.10 0.42 2.0 0.10 1.95 0.004 62.00 0.15 0.2 2.0 0.10 2.49 0.004 62.00 0.15 0.2 2.0 0.10 6.68 0.004 250.0 0.60 0.6 2.0 0.10 7.84 0.004 250.0 0.60 0.7 2.0 0.10 0.77 2.0 0.10 1.43 2.0 0.10 1.36 2.0 0.10 0.84 Page 1 ...../... 134S-B.XLS ,X/d.irW"$ 35 BLK 0.04 2.0 o@io 0.80 36 BI,K 0.04 2.0 0.10 0.82 37 SPK 62-1 0.08 2.0 0.10 1.54 0.004 62.00 0.15 0.15 38 Drift 1.50 1.49 99% 39 Wash ND 40 SPK 62-2 0.11 2.0 0.10 2.16 0.004 62.00 0.15 0.22 41 SPK 250-1 0.21 2.0 0.10 4.28 0.004 250.0 0.60 0.43 42 SPK 250-2 0.21 2.0 0.10 4.12 0.004 250.0 0.60 0.41 43 SPK 250-3 0.23 2.0 0.10 4.68 0.004 250.0 0.60 0.47 44 SPK 2504 0.28 2.0 0.10 5.66 0.004 250.0 0.60 0.57 45 SPK 250-5 0.25 2.0 0.10 5.06 0.004 250.0 0.60 0.51 46 BLK 0.15 2.0 0.10 2.90 47 BLK ND 2.0 0.10 ND 48 F52548-24 0.11 2.0 0.10 2.26 49 F52549-24 0.05 2.0 0.10 0.91 50 Drift 1.50 1.49 99% 51 Wash ND 52 F52559-24 0.04 2.0 0.10 0.87 53 F52566-24 0.04 2.0 0.10 0.77 54 F52567-24 0.04 2.0 0.10 0.89 55 F5254848 0.04 2.0 0.10 0.88 56 F5254948 0.04 2.0 0.10 0.83 57 F5255948 0.04 2.0 0.10 0.76 58 F5256648 0.05 2.0 0.10 0.94 59 F5256748 0.04 2.0 0.10 0.72 60 BLK 0.08 2.0 0.10 1.58 61 BLK 0.06 2.0 0.10 1.13 62 Drift 1.50 1.49 99% 63 Wash ND 64 BLK 0.06 2.0 0.10 1.21 65 BLK 0.06 2.0 0.10 1.23 66 BLK 0.05 2.0 0.10 1.06 67 SPK 62-1 0.10 2.0 0.10 1.93 0.004 62.00 0.15 0.19 68 Drift 1.50 1.48 99% 69 Wash ND Page 2 1995-08-08 10:58 OutPut of : 950808AI Software Operator version 6.1 cl990,93 : DDW Date of the Analysis : 1995-08-08 07:01 Analysis File Name : C:\SKALAR\DATA\HWIDATA\SERUM\950808Al CLQ,.-) 96 AIA'BT iii(,c-i4- WL4T. Fluoride 1.5 Calibration order = Inverse Logarithm Slope s x - cl Result = 101 s 1 xc, = corrected value of the sample = corrected value of the concentration 1 s = Slope of the electrode a2 = al = aO = -0.00000 0.00074 -1.18614 Fluoride L Calibration order = 2 Co.r.ra.Zat.ionr = 0.99946 Result = a2 X2 + al * x + aO a2 = al = aO = -0.00000 0.00030 0.00019 Sampler Type Number Sample Time Wash Time Air Time Take up special needle Height : SA1000 :1 : 50 sec. : 120 sec. : 1 sec. : Single : None : 70 mm. Diluter needle Height : 80 mm dilution Factor : 10 dilution Volume : 2.5 ml. Resample :1 Dilution runs : 1 User file TXT Reproces No /0 1995-08-08 10:58 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 : Ignore s2 standard : Ignore s3 standard : Ignore s4 standard : Ignore s5 standard : Ignore s6 standard : 0.150 7 standard : 0.300 :8 standard : 0.600 9 standard : 1.200 :10 standard : 1.500 Order : Inverse Logarithm Dimension : PPM start Value : 500 DU trigger Limit : 1800 Sec Peak shape : Pointed start ignore : 60 Sec eNd ignore : 120 Sec Measure window : 75 % Filter : No Regeneration : No formula output 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 950808Al 1995-08-08 10:58 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 : 500 DU trigger Limit : 1800 Sec Peak shape : Pointed start ignore : 60 Sec eNd ignore : 120 Sec Measure window : 75 % Filter : No Regeneration : No formula c4:=c3 output 950808Al 1995-08-08 10:58 OutPut of : 950808Al Fluoride 1.5 Fluoride L PPM PPM Pos Typ Ident Ch Result F Time wt iw Initial Wash 3 0-.065 1t Tracer 3 1.436 2d Drift 3 1.468 3w Wash 3 0.065 4 sl Standard 1 3 0.071 6 s2 Standard 2 3 0.077 6 s3 Standard 3 3 0.093 7 s4 Standard 4 3 0.109 8 s5 Standard 5 3 0.129 9 s6 Standard 6 3 0.156 10 s7 Standard 7 3 0.279 11 s8 Standard 8 3 0.617 12 s9 Standard 9 3 1.239 13 slO Standard 10 3 1.460 14 d Drift 3 1.532 15 w Wash 3 0.065 16 u SERUM BLK 1 3 0.133 17 u SERUM BLK 2 3 0.088 18 u SPK 62-1 3 0.112 19 u 20 u SPK 62-2 SPK 250-1 3 0.113 3 0.279 21 u SPK 250-2 3 0.199 22 u SPK 250-3 3 0 254 23 u F52548-2 3 24 u F52549-2 3 0.076 25 u F52559-2 3 0.076 26 d Drift 3 1..533 27 w Wash 3 0 065 28 u F52566-2 3 0.080 29 u F52567-2 3 0.078 30 u SPK 62-3- 3 0.104 31 u SPK 62-4 3 0.126 32 u SPK 250-1 3 0.243 33 u SPK 250-2 3 0.278 34 u BLK 1 3 0.117 35 u BLK 2 3 0.090 36 u BLK 3 3 0.084 37 u BLK 4 3 0.085 38 d Drift 3 1.548 39 w Wash 3 0.065 40 u BLK 5 3 0.083 41 u SPK 62-1 3 0.112 42 u SPK 62-2 3 0.126 43 u SPK 250-1 3 0.247 44 u SPK 250-2 3 0.249 45 u SPK 250-3 3 0.297 46 u SPK 250-4 3 0.228 47 u BLK 3 0.128 48 u BLK 3 0.082 49 u F52548-4 3 0.074 50 d Drift 3 1.557 51 w Wash 3 0.065 52 u 53 u F52549-4 F52559-4 3 0.076 3 0.073 65 209 383 620 734 909 1081 1259 1433 1609 1783 1959 2133 2307 2483 2725 2834 3008 3182 3360 3536 3710 3886 4060 4234 4408 4584 4825 4926 5109 5284 5463 5636 5811 5985 6161 6337 6511 6687 6917 7035 7212 7388 7562 7738 7912 8087 8263 8437 8613 8787 9019 9136 9305 Ch Result F Time 4 0.0002 0 4 0.4779 0 4 0.4793 0 4 0.0002 0 4 0.0146 0 4 0.0297 0 4 0.0618 0 4 0.0896 0 4 0.1185 0 4 0.1507 0 4 0.2466 0 4 0.3715 0 4 0.4644 0 4 0.4790 0 4 0.4816 0 4 0.0002 0 4 0.1231 0 4 0.0521 0 4 0.0931 0 4 0.0950 0 4 0.2470 0 4 0.1908 0 4 0.2317 0 4 0.0397 0 4 0.0259 0 4 0.0259 0 4 0.4816 0 4 0.0002 0 4 0.0360 0 4 0.0311 0 4 0.0805 0 4 0.1141 0 4 0.2239 0 4 0.2460 0 4 0.1013* 0 4 0.0554 0 4 0.0442 0 4 0.0473 0 4 0.4819 0 4 0.0002 0 4 0.0419 0 4 0.0931 0 4 0.1146 0 4 0.2272 o@ 4 0.2282 0 4 0.2569 0 4 0.2136 0 4 0.1170 0 4 0.0402 0 4 0.0228 0 4 0.4821 0 4 0.0002 0 4 0.0277 0 4 0.0193 0 Page 1 of 2 1995-08-08 10:58 Output of : 95080SAl Fluoride 1.5 Fluoride L ppm ppm Pos Typ Ident Ch Result F Time 54 u 55 u 56 u 57 u 58 u 59 u 60 u 61 u 62 d 63 w 64 u 65 u 66 u 67 u 68 u 69 d 70 w wt rw F52566-4 3 0.072 F52557-4 3 0.075 F52548-6 3 0.074 F52549-6 3 0.073 F52559-6 3 0.081 SPK 62-1 3 0.102 SPK 62-2 3 0.118 SPK 250-1 3 0.124 Drift 3 1.571 Wash SPK 250-2 3 0.065 3 0.198 SPK 250-3 3 0.308 BLK 3 0.130 F52566-6 3 0.083 F52567-6 3 0.078 Drift 3 1.572 Wash 3 0.065 RunOut Wash 3 0.065 9488 9664 9834 10013 10190 10364 10540 10715 10889 11100 11239 11415 11591 11764 11940 12115 12351 12590 Ch Result F Time 4 0.0169 0 4 0.0254 0 4 0.0216 0 4 0.0201 0 4 0.0377 0 4 0.0783 0 4 0.1032 0 4 0.1107 0 4 0.4823 0 4 0.0002 0 4 0.1899 0 4 0.2627 0 4 0.1190 0 4 0.0431 0 4 0.0323 0 4 0.4823 0 4 0.0002 0 4 0.0002 0 Page 2 of 2 Calibration curve of 950808Al : Fluoride L mjqr-Fr2-7mmmiN;M-!r-MT-TR'R 6 Raiii-z:4i&l-kidZT4TmM 0.0019642 6-4- 0.2301773 c 0 c 0.00018640 Order 2 Measured 900 r 0.99946 1.71!50844 Calibration curve of 950808Al : Fluoride I.S N -lrr_IT17-- rp-twr-r,mma Y6 S8180 Mrs?-3rPg-64-M 4-1 0.06sisol 0 Order 1 Measlured I Inverse Logarithm 1 s 4095 5894 4OS5 E;Mi,7m7--i@=WrM Raw data of 950808AI : Fluoride 1.5 CS 400 -M 64 M- IV 0 0 Esc=Exit Time FI=Help 1 Crtl-P=Edit peaks 5000 4035 M;F'tiTIT-V, RM@ Raw data of 950808AI : Fluoride 1.5 STIr--mlqssosum[EM 2S94 64 0 4S60 Time Esc=Exit 1 FI=Help 1 Crtl-P=Edit peaks 9s6o Raw data of 950808AI : Fluoride I.S 893S 160 HIM 64 4035 P4 0 8935 Time Esc=Exit 1 FI=Help 1 Crtl-P=Editpeaks 1 13935 1995-08-08 14:27 OutPut of : 950808Bl Software version 6.1 cl990,93 Operator Date of the Analysis Analysis File Name DDW 1995-08-08 10:58 C:\SKALAR\DATA\HWIDATA\SERUM\950808Bl. ct4 Ww-!. (.-3Z9-141 Fluoride 1.5 Calibration order = Inverse Logarithm Slope s = I.##### x - cl Result = 101 s 1 x = corrected value of the sample cl = corrected value of the concentration 1 s = Slope of the electrode a2 = al = aO = -0.00000 0.00069 -1.21965 Fluoride L Calibration order = 2 Cox-.relation r = 0.99948 Result a2 x2 + al * x + aO a2 = al = aO = 0.00000 0.00023 0.00544 Sampler Type Number Sample Time Wash Air Time Time Take up special needle Height : SA1000 :1 : 50 sec. : 120 sec. : 1 sec. : Single : None : 70 mm. Diluter needle Height : 80 mm dilution Factor : 10 dilution Volume : 2.5 ml. Resample :I Dilution runs 1 User file : TXT Reproces : No 1995-08-08 14:27 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 : sa 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 soc 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 950808Bl 1995-08-08 14:27 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 7 standard : Ignore :8 standard : Ignore S9 standard : Ignore slO standard : Ignore Order : 2 Dimension : PPM start Value : 500 DU trigger Limit : 1800 Sec Peak shape : Pointed start ignore : 60 Sec eNd ignore : 120 Sec Heasure window : 75 Filter : No Regeneration : No formula c4:=c3 output 950808Bl 1995-08-08 14:27 OutPut of : 950808Bl Fluoride 1.5 Fluoride L ppm ppm Pos Typ Ident Ch Result F Time wt iw Initial Wash 3 0.060 1t Tracer 3 1.468 2d Drift 3 1.480 3w Wash 3 0.060 4 si Standard 1 3 0.064 5 s2 Standard 2 3 0.071 6 s3 Standard 3 3 0.089 7 s4 Standard 4 3 0.107 8 s5 Standard 5 3 0.126 9 s6 10 s7 Standard 6 3 0.155 Standard 7 3 0.281 11 s8 Standard 8 3 0.616 12 s9 Standard 9 3 1.230 13 slO Standard 10 3 1.468 14 @d Drift 3 1.487 15 w Wash 3 0.060 16 u F52549-8 3 0.071 17 u P52559-8 3 0.066 18 u F52566-8 3 0.071 19 u F52567-8 3 0.073 20 u F52548-12 3 0.068 21 u F52549-12 3 0.067 22 u F52559-12 3 0.064 23 u F52566-12 3 0.065 24 u F52567-12 3 0.067 25 u SPK 62-1 3 0.112 26 d Drift 3 1.480 27 w Wash 3 0.060 28 u SPK 62-2 3 0.132 29 u SPK 250-1 3 0.334 30 u SPK 250-2 3 0.392 31 u F52548-8 3 0.076 32 u BLK 3 0.095 33 u BLK 3 0.092 34 u BLK 3 0.078 35 u BLK 3 0.076 36 u BLK 3 0.077 37 u SPK 62-1 3 0.098 38 d Drift 3 1.489 39 w Wash 3 0.060 40 u SPK 62-2 3 0.119 41 u SPK 250-1 3 0.214 42 u SPK 250-2 3 0.206 43 u SPK 250-3 3 0.234 44 u SPK 250-4 3 0.283 45 u SPK 250-5 3 0.253 46 u BLK 3 0.150 47 u BLK 3 0.061 48 u F52548-24 3 0.123 49 u F52549-24 3 0.080 50 d 51 w Drift 3 1.488 Wash 3 0.060 52 u F52559-24 3 0.079 53 u F52566-24 3 0.076 65 210 386 628 737 911 1085 1261 1437 1613 1787 1961 2137 2311 2487 2729 2840 3010 3188 3362 3534 3712 3878 4063 4235 4413 4589 4824 4939 51-13 5289 5459 5642 5814 5990 6164 6340 6514 6690 6929 7040 7216 7390 7565 7741 7915 8089 8241 8439 8610 8788 9015 9138 9310 Ch Result F Time 4 0.0054 0 4 0.7420 0 4 0.7466 0 4 0.0054 0 4 0.0145 0 4 0.0297 0 4 0.0621 0 4 0.0904 0 4 0.1172 0 4 0.1512 0 4 0.2577 0 4 0.4307 0 4 0.6533 0 4 0.7417 0 4 0.7496 0 4 0.0054 0 4 0.0283 0 4 0.0174 0 4 0.0281 0 4 0.0328 0 4 0.0220 0 4 0.0195 0 4 0.0134 0 4 0.0156 0 4 0.0208 0 4 0.0977 0 4 0.7468 0 4 0.0054 0 4 0.1247 0 4 0.2919 0 4 0.3253 0 4 0.0387 0 4 0.0716 0 4 0.0682 0 4 0.0419 0 4 0.0399 0 4 0.0412 0 4 0.0770 0 4 0.7503 0 4 0.0054 0 4 0.1081 0 4 0.2072 0 4 0.1997 0 4 0.2231 0 4 0.2589 0 4 0.2377 0 4 0.1451 0 4 0.0066 0 4 0.1129 0 4 0.0457 0 4 0.7498 0 4 0.0054 0 4 0.0437 0 4 0.0387 .0 Page 1 of 2 1995-08-08 14:27 output of : 95080SB1 Fluoride 1.5 Fluoride L PPM PPM Pos Typ Ident Ch Result F Time Ch Result F Time 54 u F52567-24 3 0.079 9487 4 0.0446 0 55 u F52548-48 3 0.079 9662 4 0.0439 0 56 u F52549-48 3 0.077 9837 4 0.0417 0 57 u F52559-48 3 0.075 10013 4 0.0378 0 58 u F52566-48 3 0.080 10187 4 0.0471 0 59 u F52567-48 3 0.074 10361 4 0.0358 0 60 u BLK 3 0.099 10539 4 0.0788 0 61 u BLK 3 0.085 10706 4 0.0564 0 62 d Drift 3 1.487 10886 4 0.7496 0 63 w Wash 3 0.060 11127 4 0.0054 0 64 u BLK 3 0.088 11236 4 0.0607 0 65 u 66 u BLK 3 0.088 11412 4 0.0614 0 BLK 3 0.084 11586 4 0.0532 0 67 u SPK 62-1 3 0.111 11760 4 0.0963 0 68 d Drift 3 1.482 11935 4 0.7475 0 69 w Wash 3 0.060 12166 4 0.0054 0 wt rw Runout Wash 3, 0.060 12410 4 0.0054 0 Page 2 of 2 1.78876741 Calibration curve of 950808Bl : Fluoride 1.5 6 ff!EERMRE@@ 0. 0608836 0.06031149 0 Order Measured Inverse Logarithm 409S s 5911 Calibration curve of 950808BI : Fluoride L gNir-rIT17-:3MlBirMreZ-p"--p-T 0.2096763- 997-irk"-sTIM - ITTm'g 0.0068003 rPE 94 0.0054427- 0 Order 2 Measured 0.99948 9"0 Raw data of 9SO80881 : Fluoride 1.5 E:n4s7ir-@@WMM 409S 0 MUPVU?-M 272 FIT-3TM64 P4 O@ C4 1 97 '7\(i -%\'\ () 4 0 Esc=Exit Time Fi=Help 1 Crtl-P=Edit peaks 1 do sooo E;Fliii7 409S Raw data of 9SO808BI : Fluoride 1.5 4990 I 3 P4 0 4560 Esc=Exit FI=Help T ime Crtl-P=Edit peaks 9s6o 4095 Raw data of SS080881 Fluoride I.S 4v LOS I P4 0 843S Time Esc=Exit ; FI=Help 1 Crtl-P=Edit peaks 139351