Document b5rjnGpamJE8KyLxMeOErZ4ko

3M EnvironmentaLlaboratory FinalReport-AnalyticaSltudy Single-DosDeermalAbsorption/ToxiciStyudyofT-6052 inRabbits In-VivoStudyReferenceNumber: HWI#6329-135 StudyNumber: AMDT-020795.1 TestSubstance:FC-120(T-6052) Name and AddressofSponsor: 3M SCD Division 367 Grove Street St.Pau'1,MN 55106 Name and Address of TestingFacility: 3M Environmental Technology & Services 935 Bush Avenue St.Paul,MN 55106 Method Numbers AMDT-M-1-0, AMDT-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 Dohrrnann DX2000 OrganicHalideAnalyzer-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: es D.irJo r n @t#ud-y @Dsi@r r Completion Date 1.0 SLTMMARY Samplesofliveart28 dayspostdermaldosesofFC-120(T-6052w)ereanalyzebdy combustionfortotalorganicfluorineE.ven thehighestdose of 1000 mg/kg (50ug/kg)resulteidnno organicfluorinaetpracticaqluantitatiolnevels.There isa traceof some fluorinedetectabliefone usesjustrelativmeeter readings. Electrospraymass spectrometrywas ableto detectni/z--59i9onwhich isthe perfluorodecanesulfonaatneion. The doses were too low toassessdermal absorptionwiththistestmethod. 2.0 INTRODUCTION The pharmacokineticstudyforFC- 120 was not successful.The highestdose was 50 ug/kg. Thus,inthisstudy,iftherewas a substantiaalmount of organicfluorine presentat28 days itwould indicattehata significanatbsorptionofFC-120 had occurred.However, intheeventof very littloerganicfluorinaet28 days,itwould notbe possibleto make an assessmentof dermal absorption.Liversamples and serum sampleswere availablfeoranalysisoftotalorganicfluorineand perfluorodecanesulfonaatneion.The samples were analyzed.Because thedosesare quitelow (highdose 50 ug/kg),itwas notexpectedthatfluorinweould be detected. 3.0 TEST MATERIALS 3.1Test,Control,and ReferenceSubstancesand Matrices 3.1.1AnalyticalReference Substance: FC-95, lot161 or 171. They are equivalent. 3.1.2AnalyticalReference Matrix: Bovine liverb,ovine serum, rabbitserum 3.1.3AnalyticalControl Substance: None 3.1.4AnalyticalControl Matrix: Bovine liverb,ovine serum and rabbit serum 3.2 Source of Materials:3M ICP/PCP DivisionforFC-95, bovine liverfrom grocerystoreb,ovineserum from Sigma Chemical Company, rabbitserum from AMDT-1 10394.1(HWI#6329-123) controlgroup animals. 3.3.Purityand StrengthofReference Substance:Responsibilitoyf Sponsor. 3.4Stabilitoyf ReferenceSubstance: To be determinedby Sponsor. -2- 3.5StoragCeonditionfsorTestMaterialRso:om temperatufroerFC-95.For biologicalsamples the storageis-20@+-0l0 C. 3.6 DispositionSpecimens: Biologicaltissuesand fluidswillbe retainedper GLP Regulationforthetimeperiodrequiredforstudieslongerthan28 days. 4.0 EXPERIEMENTAL -Overview The tissuesfrom animalsdosed as described(HWI#6329-135), were availablefor analysisforfluorinceompounds. At thediscretioonfthestudydirectora,seriesof analyticatlestscouldbe performed.The screeningforfluoridienlivervia combustion was themost likelyanalysistopresentdefinitivdeataforabsorptionif thepharmacokinetitcest(IV administratiowna)s positiveforfluorinientheliver. Otheravailablteestswere electrospramyass spectroscopy.However, ifthe definitivreesultcsouldbe obtainedwith combustionanalysisalone,only theliver samples would be analyzedand any othertestswould be forconfirmation. 5.0 EX]PERIMENTAL -METHODS 5.1AMDT-M-1-0, ThermalExtractioonfFluoridbey Means ofa Modified Dohrinann DX2000 OrganicHalideAnalyzer-Liver 5.2AMDT-M-2-0, FluorideMeasurement by Means ofan Orion EA940 Expandable Ion Analyzer 5.3 AMDT-M4-0, ExtractionofFluorochemicalsfrom RabbitLiver 5.4 AMI)T-M-5-0, AnalysisofRabbitLiverExtractforFluorochemicalsUsing ElectrosprayMass Spectrometry 5.5AMDT-M-8-0, Analysisof FluorideUsing the SkalarSegmented Flow Analyzer with Ion SelectiveElectrode 5.6 AMDT-M-14-0, Thermal Extractionof Fluorideby Means of a Modified Dohrmann DX2000 OrganicHalideAnalyzer-Serum 6.0 DATA ANALYSIS The datafromcombustioannalysiasreattachedT.heredoesnotappeartobe any totalorganicfluorineabove thepracticaqluantitatiolnimitforany of the liver samples at48 hourspostintravenousdose forany of the dosing regimens. Even the 50 ug/kg dose isnot above thepracticaqluantitatiolnimit.However, ifone usesjust the meter readingsand compares thosereadingtothe valuesforthe controlst,hereis possiblya traceof fluorinebeing detected. Electrospraymass spectrometryanalysisisattached.The electrosprawyas ableto detectperfluorodecanesulfonataenion inthe 50 ug/kg dose livers.The amount presentwas not quantitated. In view of the failuroef thepharmacokineticstudy(HWI#6329-134) to show a good marker for FC-120 ata dose of 50 ug/kg,itisnot reasonableto make an assessment of the extentof dermal absorptionfrom thisstudyatthe same dose level. Other datawas collectedusingSkalarsegmented flow analyzerwith ion selective electrode(seeappendices).This data,althoughsupportivei,ntheopinionof the Study Directorisnot requiredto reachtheconclusionstatedhere and thereforeisnot discussedin detail. 6.1 Circumstances that May Have Affected the Quality of the Data: The problem with thisanalysisisthatthepharmacokineticstudydidnot providea good marker fordermal absorptionbecause thedose levelwas too low. The dermal study isnot ata higherdose. 7.0 CONCLUSION This study does not providea usefulassessmentof dermal absorptionof FC-120. There isnot a usefulmarker. 8.0 MAINTENANCE OF RAW DATA AND RECORDS-- 8.1 Raw Data and Data: Raw data,approved protocol,approved finalreport, appropriatespecimens,and electronidcatawillbe maintainedintheAMDT archives. 9.0APPENDICES 9.1Protocoalnd Amendments 9.1.1ProtocoalndFinaRleportH:WI#6329-135":Single-DoDseenmi Absorption/ToxiciSttyudyof T-6052 inRabbits"(Protocotlype T?3016.AB fordosingof animals,tissuecollectione,tc.) 9.1.2 AnalyticaplrotocolAMDT-020795.1 9.1.3Amendment to AnalyticaPlrotocolAMDT-020795.1 9.2 Signed Reports from IndividualScientistsN:one 9.3 QualityAssurance Unit Statement:See attached 9.4 Key PersonnelInvolvedinthe Study: See attached 9.5 Materialsand Equipment: See methods 9.6 Solutions,Reagents,and Standards:See methods 9.7 Sample Preparation:See methods 9.8 QualityControlPractices:See methods 9.9 TestMethods: See ProtocolAMDT-020795.1 9.10 Instrument Settings:See methods 9.11 Data: See attached. 9.11.1Summary and raw data;ug F-inwhole liverasdeterminedby thermal extractiofnollowedby analysisusingOrion ion analyzer. 9.11.2Sununary and raw data;analysisof liverextractussingelectrospray mass spectrometry. 9.113 Summary and raw data;ppm F-inserum asdeterminedby thermal extractiofnollowedby analysisusingOrion ionanalyzer. 9.11.4Sununary and raw data;ppm F-inserum as determinedby thermal extractiofnollowedby analysisusingSkalarsegmented flow analyzerwith ion selectivelectrode. 9.1.1 Protocoland FinalReport:HWI#6329-135: "Single-DoseDermal Absorption/ToxicitSytudy of T-6052 inRabbits"(ProtocoltypeTP3016.AB for dosingof animals,tissuecollectione,tc.) HAZLC-MCON W IS C 0 N S IN POST OFFICE BOX 7545 IViADISOli. kNi 53707-.7545 Sponsor: 3M Toxicology Service Medical Department St. Paul, Minnesota a CORNING Coinnany FINAL REPORT Study Title: Single-DoseDermal Absorption/Toxicity Study of T-6052 in Rabbits Author: Steven M. Glaza Study Completion Date: June 27, 1995 Performing Laboratory: Hazleton Wisconsin, Inc. 3301 Kinsman Boulevard Madison, Wisconsin 53704 LaboratoryPro.iectIdentification: HWI 6329-135 P ho n e 608 24 1 44 71 E XF,r,[SS MAII DF I ivr ry Page I of 41 ii@,1,AA IJ l@I \1r) F a x 60 M A f)IS C)IJ. v@ . Page 2 of 41 HWI 6329-135 QUALITY ASSURANCE STATEMENT This report has been reviewed by the Quality AssuranceUnit of Hazleton Wisconsin, Inc., in accordancewith the Food and Drug Administration (FDA) Good LaboratoryPracticeRegulations,21 CFR 58-35 (b) (6) (7). The followinginspectionswere conductedand findingsrepqrtedto the Study Director and management. Written status reports of inspections and findings are issued to Hazleton management monthly according to standard operating procedures. Inspection Dates From To Phase 12/21/94 12/21/94 01/30/95 01/30/95 02/02/95 02/02/95 03/29/95. 03/29/95 03/29/95 03/29/95 06/27/95 06/27/95 Protocol Review Protocol Amendment Body Weight Data/Report Review Data Review Report Rereview Date Reported to Date to Study Director Management 12/22/94 01/30/95 02/02/95 03/29/95 03/29/95 06/27/95 01/10/95 02/10/95 03/10/95 04/10/95 04/10/95 07/10/95 Te-presenfative,Quality Assurance Unit 2 7Date Page 3 of 41 STUDY IDENTIFICATION Single-Dose Dermal Absorption/Toxicity Study of T-6052 in Rabbits HWI 6329-135 Test Material Sponsor Sponsor's Representative Study Director Study Location Study Timetable Study InitiationDate Experimental(In-life)Start Date In-life End Date Experimental Termination Date Study Completion Date T-6052 3M Toxicology Service Medical Department 3M Center, Bldg. 220-2E-02 P.O. Box 33220 St. Paul, MN 55133-3220 John L. Butenhoff, PhD 3M Toxicology Service Medical Department 3M Center, Bldg. 220-2E-02 P.O. Box 33220 St. Paul, MN 55133-3220 (612) 733-1962 Steven M. Glaza Hazleton Wisconsin, Inc. P.O. Box 7545 Madison, WI 53707-7545 (608) 241-7292 Hazleton Wisconsin, Inc. Building No. 3 3802 Packers Avenue Madison, WI 53704 December 30, 1994 January 5, 1995 February 2, 1995 June 27, 1995 June 27, 1995 Page 4 of 41 KEY PERSONNEL HWI 6329-135 Acute Toxicology Laboratory Animal 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-lifeSupervisor Thomas E. Palmer, PhD Anatomical Pathologist Rose M. Bridge Report Supervisor Ouality Assurance Sherry R. W. Petsel Manager Jack Serfort/ Deborah L. Pirkel Supervisors Necropsy Anne Mosher Supervisor Pathology Data Page 5 of 41 CONTENTS Quality Assurance Statement Study Identification Key Personnel Summary Objective Regulatory Compliance Test and Control Materials Test System Procedures Results Discussion Signature Reference Pathology Report Table I Individualand Mean Body Weights (g) 2 IndividualClinicalSigns 3 IndividualDermal IrritationScores 4 Individual Pathology Comments 5 IndividualAnimal Tissue Weights and Bile Volumes Appendix A Protocol TP3016.AB Protocol Amendment No. 1 HWI 6329-135 Paqe 2 3 4 6 8 8 8 9 10 13 13 14 14 15 16 17 19 23 25 27 40 Page 6 of 41 SUMMARY HWI 6329-135 .Thisstudy was done to assess the systemicabsorption/toxicityand relative skin irritancyof T-6052when appliedto the skin of rabbits. The study was conducted using three male and three female acclimated rabbits of the Hra:(NZW)SPFstrain for each treatmentgroup. GrouR Test Material 1 (Control)Distilledwater 2 T-6052 3 T-6052 4 Dose Level (mg/kq) oa 2 200 1,000 Number of Animals Males Females 3 3 3 3 3 3 3' 3 a Administered at a dose volume of 2.0 mL/kg. The back of each rabbit was clipped free of hair and a single dose of the respectivematerial at the indicateddose level was administeredto the skin of the rabbits. The treatmentsites remainedintact. The area of application was covered with a gauze bandage secured with paper tape around all edges and overwrapped with Saran Wrap'Oand ElastoplastOtape to provide an occlusive dressing for a 24-hour exposure period. Clinical observationswere conducted predose and at approximately1, 2.5, and 4 hours after test or controlmaterialadministration.Additionalclinical observationsand twice a day mortalitychecks were conducteddaily thereafter for 28 days. Body weights were determined on Day -8 for randomization purp' oses,before test or controlmaterialadministration(Day 1), and at in-lifetermination(Day29). The initialdermal irritationreadingwas made before test or controlmaterialadministration(recordedas the Day I reading). Subsequentreadingsof dermal irritationwere made approximately 30 minutes after bandage removal (Day 2) and on Days 4 and 8. Blood samples were collectedfrom a marginalear vein of the animals before in-life initiation(Day 1), approximately24-hourspostdose(Day 2), on Days 4, 8, 15, and 22. In addition,at the time of necropsyon Day 29, approximately20 mL of blood was obtained from each animal. All sampleswere centrifugedand separatedinto serum and cellularfractions. All animalswere euthanizedat terminationof the in-lifephase and necropsied. The whole liver,bile, an approximate1-cm x 1-cm section of the dermal applicationsite from all animals, and both kidneys from one male and one female in each group were collectedat necropsyand weighed (volumeonly determinedfor bile). The blood samples(serumand cellularfractions),livers,bile, dermal application sites, and kidneyswere sent frozento the Sponsorafter terminationof the in-lifephase. Page 7 of 41 HWI 6329-135 Application of T-6052 did not result in any test material-relatedchanges in body weight gain or macroscopic findings at necropsy. All animals appeared clinically normal throughout the study. No dermal irritationwas observed at the dermal scoring intervalsas a result of the applicationof distilled water or T-6052 at any of the dose levels. Page 8 of 41 HWI 6329-135 OBJECTIVE The objectiveof this study was to assess the systemictoxicity/absorptionand relativeskin irritancyof a test materialwhen appliedto the skin of rabbits. REGULATORY COMPLIANCE This study was conducted in accordance with the U.S. Food and Drug Administration'sGood LaboratoryPracticeRegulationsfor Nonclinical LaboratoryStudies,21 CFR 58, with the exceptionthat analysisof the test materialmixture preparedfor the Group 2 animals for concentration, homogeneity/solubilitya,nd stabilitywas not conducted. All proceduresused in this study are in compliancewith the Animal WelfareAct Regulations. In the opinionof the Sponsorand study director,the study did not unnecessarily duplicate any previous work. TEST AND CONTROL MATERIALS Identification The test materialwas identifiedas T-6052 and describedas a clear, colorless ,liquid. The controlmaterialwas distilledwater and was describedas a clear, colorlessliquid. Purity and Stability The Sponsor assumesresponsibilityfor test materialpurity and stability determinations(includingundertest conditions).Analysisof the test material mixture preparedfor the Group 2 animals for concentration, homogeneity/solubilitya,nd stabilitywas not conductedor requestedby the Sponsor. The purity and stabilityof the controlmaterialwere consideredto be adequatefor the purposesof this study. Storage and Retention The test and controlmaterialswere stored at room temperature. A reserve sample of each test and controlmaterialwas taken and will be retained in a freezer set to maintain a temperatureof -20*C 10'-for 10 years in accordance with Hazleton Wisconsin (HWI) Standard Operating Procedure (SOP). Any unused test materialwas returnedto the Sponsor after completionof the in-life phase according to HWI SOP. Any remaining controlmaterial is retainedfor other testingand will not be discardedafter issuanceof the final report. Page 9 of 41 Safety Precautions HWI 6329-135 The test and control material handling procedureswere according to HWI Sops and policies. Test Animal TEST SYSTEM Adult albino rabbits of the Hra:(NZW)SPFstrainwere procuredfrom HRP, Inc., Denver, Pennsylvania on December 28, 1994 and maintained at the Hazleton Wisconsin facility at 3802,PackersAvenue, Madison, Wisconsin. Housing After receipt,the animalswere acclimatedfor a period of at least 7 days. During acclimationand throughoutthe study,the animalswere individually housed in screen-bottomstainless steel cages in temperature-and humiditycontrolledquarters. Environmentalcontrolsfor the animal room were set to maintain a temperatureof 19* to 23*C, a relativehumidityof 50% 20%, and a 12-hour light/12-hourdark lightingcycle. In cases where variationsfrom these conditions existed, they were documented and considered to have had no adverse effect on the study outcome. Animal Diet The animals were provided access to water ad libitum and a measured amount of LaboratoryRabbit Diet HF #5326, PMI Feeds, Inc. The feed is routinely analyzed by the manufacturerfor nutritionalcomponents and environmental contaminants. Samples of the water are periodicallyanalyzedby HWI. There were no known contaminantsin the feed or water at levels that would have interferedwith or affectedthe resultsof the study. Selection of Test Animals The animals were identifiedby-animalnumber and correspondingear tag and were placed into study groups using a stratified body weight randomization program. The randomizationbody weights were determined on Day -8. The weight variation of the animalsfor each group of E@a,cshex selected for the study did not exceed 2 standarddeviationsof the mean weight, and the mean body weights for each group of each sex were not statisticallydifferentat the 5% probabilitylevel. One female animal (No. F53409)was replaced in the study prior to treatmentdue to poor health. This animal was replaced with another female (No. F52982) which was treated in the same manner. Page 10 of 41 Study Design HWI 6329-135 Animals weighing from 2,052 to 2,471 g at initiation of treatment were placed into the following study groups: Group Dose Level Test Material (mg/kq) I (Control) Distilledwater 2 T-6052 3 T-6052 4 T-6052 oa 2 200 1,000 Number of Animals Males Females 3 3 3 3 3 3 3 3 a Administered at a dose volume of 2.0 mL/kg. Justificationfor Species Selection Historically, the New Zealand White albino rabbit has been the animal of choice because of the large amount of background information on this species. PROCEDURES Preparation of Exposure Area On the day before test material application,the back and, if necessary (to obtain unblemishedskin), the flanks of each rabbit was clipped free of hair. The clipped area made up approximately 20% of the total body surface area. The intact skin of the test sites was inspectedfor interferinglesions, irritation,or defects that would preclude the use of any of the animals. The animals were clipped on Days 8 and 29 to aid in visualizing the application sites. Dose Administration All animals received a single administrationof the respectivetest or control material. The day of treatment was designated as Day 1. Group 1. An individual dose (2.0 mL/kg) was calculated and measured based on each animal's body weight on the day of treatment. The control material (distilledwater) was applied evenly to the test7-siteat a rate of approximately 0.04 mL/cm - Page 11 of 41 HWI 6329-135 Groups 2. 3. and 4. For the Group 2 animals (2 mg/kg), the test material (T-6052) was mixed with distilled water to a concentration of 200 mg/mL and applied at a dose volume of 0.01 mL/kg. The mixture was stored at room temperature until administered. The test material was administered undiluted to the test sites of the Groups 3 and 4 animals (200 or 1,000 mg/kg, respectively)using the average bulk density of 0.98 g/mL to determine the dose volume for each dose level (0.20 and 1.02 mL/kg, respectively). An individualdose of the respectivetest material or test material mixture was calculated for each animal based on its body weight on the day of treatment. The area of exposure for the 2, 200, and 1,000 mg/kg dose levels was 4, 25, and loo CM2 , respectively. The approximate rate of application ranged from 0.006 to 0.024-mL/CM2. Each area of application was covered with a 10-cm x 10-cm gauze bandage secured with paper tape around all edges and overwrapped with Saran WrapO and ElastoplastQDtape to provide an occlusive dressing. Collars were used to restrain the animals during the 24-hour exposure period. Approximately 24 hours after test or control material application,the restraining collars and bandages were removed and any residual test material was removed with tap water and disposable paper towels. Reason for Route of Administration The dermal route is a potential route of exposure in humans. Observations of Anig@h Clinical observations were conducted predose and at approximately 1, 2.5, and 4 hours after test or controlmaterial administration. Additionalclinical observations and twice a day mortality checks (morning and afternoon) were conducted daily thereafter for 28 days. Body weights were determined for randomization purposes on Day -8, before test material administration(Day 1), and at in-life termination(Day 29). The initial demal irritationreading was made before test or control material administration according to the Draizel technique (recorded as the Day 1 reading). Subsequent readings of dermal irritation were made approximately 30 minutes after bandage removal (Day 2) and on Days 4_and 8. Page 12 of 41 Sample Collections HWI 6329-135 Blood samples (approximately4 mL) were collectedfrom a marginalear vein of all animals beforeexperimentalinitiation(Day 1). Subsequentcollectionof blood was conducted approximately24-hours postdose (Day 2), and on Days 4, 8, 15, and 22. In addition,at the time of necropsyon Day 29, approximately 20 mL of blood was obtainedfrom the posteriorvena cava of each-animal. All samples were centrifugedand separated into serum and cellularfractions. These samples were then stored in a freezer set to maintaina temperatureof -20'C 10'C until shippedto the Sponsor. Pathology At terminationof the experimentalphase (Day 29), animalswere anesthetized with sodium pentobarbital,bled via the posteriorvena cava, exsanguinated, and necropsiedin random order. The sites of test and controlmaterial application were washed with lukewarm tap water before the necropsy procedure. All animals were subjectedto an abbreviatedgross necropsy examinationand any abnormalitieswere recorded. The whole liver, bile, an approximate 1-cm x 1-cm sectionof the dermal applicationsite from all animals,and both kidneys from the first male and female in each group were collected. The tissue samples were weighed (volumeonly determinedfor bile) and immediately placed on dry ice, then placed in a freezerset to maintaina temperatureof -200C 100C. After necropsy,the animalswere discarded. Shipment of Blood, Bile, and Tissues After experimentaltermination,the blood samples (serumand cellular fractions),livers,bile,dermal applicationsites, and kidneyswere sent frozen (on dry ice) to the Sponsor (JamesD. Johnson,3M E.E. & P.C., Bldg. 2-3E-09, 935 Bush Avenue, St. Paul, MN,.55106),along with their corresponding weights or volumes. The Sponsor is responsiblefor the retentionand dispositionof the samples. HWI does not accept any responsibilityfor the analysisof the tissue samplescollectedin this study nor are these results presentedin this report. StatisticalAnalyses No statisticalanalyseswere requiredby the protocol. Location of Raw Data, Records, and Final Report The raw data, records,and an originalsigned copy of the final report will be retained in the archives of HWI in accordancewith HWI SOP. Page 13 of 41 RESULTS HWI 6329-135 Body Weights Individual and mean body weights are in Table 1. All animals exhibited body weight gains from Day 1 to Day 29. Clinical Observations Individualclinical signs are in Table 2. All animals appeared normal throughout the study. Dermal Irritation Individualdermal irritationscores are in Table 3. The control material produced no dermal irritation. No dermal irritationwas observed in the animals treated with T-6052 at any of the dose levels. Pathology Individual animal pathology comments are presented in Table 4. Individual animal tissue weights and bile volumes are in Table 5. There were no lesions observed in any of the animals. Page 15 contains a pathology report by the study pathologist. DISCUSSION The acute systemic absorption/toxicityand relative skin irritancy of T-6052 were evaluated in male and female albino rabbits when administered as a single dermal application. Applicationof this material did not result in any dermal irritationor test material-relatedin-lifeclinicaleffects. There were no effects on body weight gain or macroscopic findings at necropsy. Page 14 of 41 SIGNATURE HWI 6329-135 Steven M. Glaza Date Study Director Acute Toxicology REFERENCE 1. Draize, J. H., "Acute Dermal Toxicity (Single Exposure)," In: Appraisal of the Safety of Chemicals in Foods, Drugs and Cosmetics - Dermal Toxicity, Association of Food and Drug Officials of the U.S., pp. 54-56 (1959). Page 15 of 41 HWI 6329-135 PATHOLOGY REPORT There were six rabbits (threemales and three females)each from four dose levels euthanizedand necropsiedat the terminationof the study. The test .material,dose level, day of death, and gross observationsrecordedfor each -animalare in the IndividualPathologyCommentsthat follow this-report. At necropsy,there were no visiblelesionsin any of the animals. The liver, bile, an approximate1-cm x 1-cm sectionof the dermal applicationsite from all animals, and both kidneys from one male and one female in each group were collected. The tissue sampleswere weighed (volumeonly determinedfor bile), frozen, and sent to the Sponsor. After necropsy,the animalswere discarded. Thomis-E.-Palmer,-PhD' Pathologist (6329-135.slh) 031695 7- @PS Date Animal Number F52979 F52972 F52973 Mean Page 16 of 41 HWI 6329-135 Table 1 Individualand Mean Body Weights (g) Male Randomization Day -8 Day 1 29 Animal Number Female Random- ization Day Day -8 1 29 Group 1 (Control)- DistilledWater (0 mg/kq) 1,903 2,090 1,921 2,072 2,362 2,211 2,559 3,041 2,818 F52976 F52983 F52975 2,151 2,116 2,043 2,259 2,296 2,265 2,681 2,863 2,671 1,971 2,215 2,806 2,103 2,273 2,738 F52990 F52997 F52986 Mean 2,095 2,031 2,034 2,053 Group 2 - T-6052 (2 mg/kq) 2,351 2,205 2,332 2,863 2,928 2,816 F52982a F52994 F53410 1,885 2,022 2,220 2,296 2,869 2,042 2,145 2,261 2,471 2,292 2,681 2,914 2,875 2,823 F52996 F52992 F52984 Mean 2,190 1,889 1,950 2,010 Group 3 - T-6052 (200 mg/kq) 2,302 2,052 2,257 2,897 2,729 2,993 F52989 F52993 F52977 2,097 1,993 2,049 2,204 2,873 2,046 2,316 2,234 2,323 2,291 2,874 2,651 2,687 2,737 F52980 F52978 F52991 Mean 1,936 2,181 2,108 2,075 Group 4 - T-6052 (1,000mg/kq) 2,184 2,384 2,351 2,637 3,063 3,142 F52995 F52987 F52988 2,131 2,140 2,188 2,306 2,947 2,153 2,249 2,274 2,423 2,315 2,644 2,817 3,015 2,825 a Animal No. F53409 was originallyselected by the randomizationprogram for use in the study but was replacedprior to treatmentwith No. F52982 due to poor health. Sex Male Female Page 17 of 41 Table 2 IndividualClinicalSigns HWI 6329-135 Animal Number Observation 1-4 Hours Day (Day 1) . 2-throucih2@ Group I (Control)- DistilledWater (0 mg/kq) F52979 Appeared normal F52972 Appeared normal F52973 Appeared normal F52976 Appeared normal F52983 Appeared normal vi F52975 Appeared normal Male Female F52990 F52997 F52986 F52982 F52994 F53410 Group 2 - T-6052 (2 mg/kq) Appeared normal Appeared normal Appeared normal Appeared normal Appeared normal Appeared normal Condition existed.. -Sex Male Female Animal Number F52996 F52992 F52984 F52989 F52993 F52977 Page 18 of 41 Table 2 (Continued) IndividualClinical Signs HWI 6329-135 Observation 1-4 Hours - (Day 1) Group 3 - T-6052 (200 mg/kq) Day 2 through 29 Appeared normal Appeared normal Appeared normal Appeared normal Appeared normal Appeared normal Male Female F52980 F52978 F52991 F52995 F52987 F52988 Group 4 - T-6052 (1,000 mg/kq) Appeared normal Appeared normal Appeared normal Appeared normal Appeared normal Appeared normal Condition existed. Page 19 of 41 Table 3 IndividualDermal IrritationScores HWI 6329-135 Group 1 (Control)- DistilledWater (0 mg/kg) Dermal Reaction Males Study Day 1 2 4 8 Animal No. F52979 Females Study Day 1 2 4 8 Animal No. F52976 Erythema Edema Atonia Desquamation Coriaceousness Fissuring 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Erythema Edema Atonia Desquamation Coriaceousness Fissuring Animal No. F52972 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Animal No. F52983 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Erythema Edema Atonia Desquamation Coriaceousness Fissuring Animal No. F52973 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Animal No. F52975 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Page 20 of 41 Table 3 (Continued) IndividualDermal IrritationScares HWI 6329-135 Group 2 - T-6052 (2 mg/kg) Dermal Reaction Erythema Edema Atonia Desquamation Coriaceousness Fissuring Males Study Day 1 2 4 8 Animal No. F52990 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Females Study Day 1 2 4 8 Animil No. F52982 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Erythema Edema Atonia Desquamation Coriaceousness Fissuring Animal No. F52997 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Animal No. F52994 0 0 0 0 0 0 .0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Erythema Edema Atonia Desquamation Coriaceousness Fissuring Animal No. F52986 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Animal No. F53410 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Page21 of 41 HWI 6329-135, Table3 (Continued) IndividuaDlermalIrritatioSncores Group3 - T-6052(200mg/kg) DermalReaction Males StudyDay 12 4 8 Animal No. F52996 Females StudyDay 1 2 4 8 Animal No. F52989 Erythema Edema Atonia Desquamation Coriaceousness Fissuring 0 0 0 0 0000 0000 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 .0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Erythema Edema Atonia Desquamation .Coriaceousness Fissuring Animal No. F52992 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Animal No. F52993 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Erythema Edema Atonia Desquamation Coriaceousness Fissuring Animal No. F52984 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Animal No. F52977 0 0 0 0 0 0 0 0 0 .0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Page 22 of 41 HWI 6329-135 Table 3 (Continued) IndividualDermal IrritationScores Group 4 - T-6052 (1,000 mg/kg) Dermal Reaction Males Study Day 1 2 4 8 Females Study Day 1 2 8 Animal No. F52980 Animal No. F52995 Erythema Edema Atonia Desquamation Coriaceousness Fissuring 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Erythema Edema Atonia Desquamation Coriaceousness Fissuring Animal No. F52978 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0. 0 0 0 0 0 0 Animal No. F52987 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Erythema Edema Atonia Desquamation Coriaceousness Fissuring Animal No. F52991 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Animal No. F52988 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Animal Number F52979 F52972 F52973 F52976 F52983 F52975 F52990 F52997 F52986 F52982 F52994 F53410 F52996 F52992 F52984 F52989 F52993 F52977 Page 23 of 41 Table 4 Individual Pathology Comments HWI 6329-135 Test Day Sex Died Sacrificed Necropsy Observation GrouR I (Control)- DistilledWater (0 mg/kq) m - 29 No visiblelesions. m - m - F - F - 29 No visiblelesions. 29 No visible lesions. 29 No visiblelesions. 29 No visiblelesions. F - 29 No visiblelesions. Group 2 T-6052 (2 mg/kq) m - 29 No visiblelesions. m - m - 29 No visiblelesions. 29 No visiblelesions. F 29 No visiblelesions. F 29 No visiblelesions. F 29 No visiblelesions. Group 3 - T-6052 (200 mg/kq) m m - m - F - F - F - 29 No visible lesions. 29 No visiblelesions. 29 No visiblelesions. 29 No visibte.lesions. 29 No visiblelesions. 29 No visiblelesions. - Not applicable. Page 24 of 41 HWI 6329-135 Table 4 (Continued) Individual Pathology Comments Animal Test Day Number Sex Died Sacrificed NecroDsy Observation F52980 m F52978 m F52991 m F52995 F F52987 F Group 4 - T-6052 (1,000 mg/kq) - 29 No visible lesions. - 29 No visible lesions. - 29 No visible lesions. - 29 No visible lesions. - 29 No visible lesions. F52988 F 29 No visible lesions. Not applicable. SexMale Female Male Female Male Female Page 25 of 41 HWI 6329-135 Table 5 Individual Animal Tissue Weights and Bile Volumes Animal Number Liver Weight (g) Dermal Appli- Kidneys cation Site Bile Volume (mL) Group 1 (Control)- DistilledWater (D mg/kq) F52979 91.387 - 0.593 F52972 83.176 16.196 0.358 F52973 76.442 - 0.800 0.9 1.15 1.2 F52976 61.443 0.445 0.8 F52983 98.109 - 0.393 0.9 F52975 87.984 14.617 0.608 0.4 Group 2 T-6052 (2 mqlkq) F52990 81.033 0.281 0.4 F52997 92.537 - 0.421 0.5 F52986 16.924 0.632 1.1 F52982 84.395 - 0.568 1.1 F52994 83.650 15.618 0.432 1.4 F53410 91.541 - 0.386 1.1 GrouR 3,- T-6052 (200 mg/kql F52996 78.540 F52992 80.845 - 0.673 0.511 0.46 1.31 F52984 F52989 F52993 F52977 89.277 98.425 88.925 72.840 15.044 16.883 - 0,421 0.837 0.848 0.526 0.75 0.7 0.55 0.9 Not applicable. Sex Male Female Page 26 of 41 HWI 6329-135 Table 5 (Continued) IndividualAnimal Tissue Weights and Bile Volumes Animal Number F52980 F52978 F52991 F52995 F52987 F52988 Liver. Weight (g) Dermal Appli- Kidneys cation Site Grou 4 T-6052 (1,000 mcizkci) 83.049 15.630 0.896 84.235 - 88.738 - 0.547 0.439 63.956 82.019 14.923 - 0.508 0.287 83.911 0.875 Bile Volume (mL) 1.0 1.6 0.9 0.25 1.35 1.2 Not applicable. Page 27 of 41 HWI 6329-135 APPENDIX A Protocol TP3016.AB Protocol Amendment No. 1 - W IS C 0 N S IN POST OFFICE BOX 754f) MADISON. Wl 53707 7!)4b Page 28 of 41 a COFTNNYG Cornpany Soonsor: 3M Toxicology Service Medical Department St. Paul, Minnesota PROTOCOL TP3016.AB Study Title: Single-Dose'DermalAbsorption/ToxicityStudy of T-6052 in Rabbits Date: December 30, 1994 PerformingLaboratory: HazletonWisconsin, Inc. 3301 Kinsman Boulevard Madison, Wisconsin 53704 LaboratoryProjectIdentification: HWI 6329-135 P lic)ri 41 4 1 U 0 Fi ,,I Page 29 of 41 STUDY IDENTIFICATION TP3016.AB Page 2 Single-Dose Dermal Absorption/ToxicityStudy 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 6329-135 T-6052 3M Toxicology Service Medical Department 3M Center, Bldg. 220-2E-02 P.O. Box 33220 St. Paul, MN 55133-3220 John L. Butenhoff, PhD 3M Toxicology Service Medical Department 3M Center, Bldg. 220-2E-02 P.O. Box 33220 St. Paul, MN 55133-3220 (612) 733-1962 Steven M. Glaza Hazleton Wisconsin,,Inc. P.O. Box 7545 Madison, Wl 53707-7545 (608) 241-7292 Hazleton Wisconsin, Inc. Building No. 3 3802 Packers Avenue Madison, WI 53704 January 5, 1995 February 2, 1995 March 16, 1995 Page 30 of 41 TP3016.AB Page 3 1. Study Single-Dose Dermal Absorption/Toxicity Study in Rabbits 2.. PurRose To assess the systemicabsorption and toxicity and relative skin irriiancyof a test materialwhen applied to the skin of rabbits 3. Regulatory-COMDliance This study will be conducted in accordance with the followingGood Laboratory Practice Regulations/Standards/Guidelines: C ] 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) Notification No. 313 (Japanese MOHW) All procedures in this protocol are in compliance with the Animal Welfare Act Regulations. In the opinion.of the Sponsor and study director, the study does not unnecessarilyduplicate any previous work. 4. Ouality Assurance The protocol,study conduct, and the final reportwill be audited by the Quality Assurance Unit in accordance with Hazleton Wisconsin (HWI) Standard Operating Procedures (SOPS) and policies. 5. Test Material A. Identification T-6052 B. Physical Description (To be documented In the raw data) C. Purity and Stability The Sponsor assumes responsibilityfor purity and stability determinations(includingunder test conditions). D. Storage Room temperature Page 31 of 41 TP3016.AB Page 4 E. Reserve Samples Reservesample(s)of each batch/lotof test and control materialswill be taken for this study. The test and controlmaterialreservesampleswill be storedat HWI in a freezerset to maintain a temperatureof -20'C tlo*C for 10 years per HWI SOP. The Sponsorwill be contactedafter 10 years for dispositionin accordancewith the appropriate regulatoryGood LaboratoryPractices. F. Retention Any unusedtest materialwill be returnedto the Sponsorafter completionof the in-lifephaseof the study. G. Saifet-Precautions As required by HWI SOPs and policies 6. Control-Material A. Identification .Distillewdater B. PhvsicalDesgrigtion Clear,colorlessliquid C. Purityand-Stability The purityand stabilityof thismanufacturedmaterialis consideredto be adequatefor the purposesof this study. D. Stora e Conditions Room temperature E. Reserve SamRIes See Section 5. E. Reserve Samples F. Rete-ntion Any remainingcontrolmaterialmay be used for other testing and will not be discardedafter issuanceof the finalreport. G. Safei:fPrecautions As requiredby SOPs and policies 7. ExRerimentalDesi A. Animals (1) Species Rabbit (2) Ltrai_nLS_oLrce Hra:(NZW)SPF/HRP, Inc. Page 32 of 41 TP3016-AB Page 5 (3)Age at Initiation Adult (4)Weightat Initiation 2.0 to 3.0 kg (5) Number and Sex 12 males and 12 females (6) Identification Individualnumberedear tag (7) HklLa-n-d-rY (a) Housing Individually,in screen-bottomstainlesssteelcages (heavygauge) (b) Food A measured amount of Laboratory Rabbit Diet HF #5326 (PMIFeeds,Inc.). The food is routinelyanalyzedby the manufacturerfor nutritionalcomponentsand environmentalcontaminants. (c)Water Ad libitum from an automatic system. Samples of the water are analyzedby HWI for total dissolvedsolids, hardness,and specifiedmicrobiologicalcontentand for selectedelements,heavy metals, organophosphates,and chlorinated hydrocarbons. (d)Contaminants There are no known contaminantsin the food or water thatwould interferewith this study. (e) Environment Environmentalcontrolsfor the animal room will be set to maintaina temperatureof 19*C to 23*C, a relativehumidity of 50% 20%, and a 12-hour light/12-hourdark cycle. (f)Acc Iimati-m At least 7 days (8) Selectionof Test Animals Based on health and body weight according to HWI SOPS. An adequatenumber of extra animals will be purchasedso that no animal in obviouslypoor health is placedon test. The animalswill be placed into study groups using a stratifiedbody weight randomizationprogramwithin nine daysof studyinitiation. Page 33 of 41 TP3016.AB Page 6 (9)Justificationfor SpeciesSelection Historically,the New Zealand White albino rabbit has been the animal of choice because of the large amount of backgroundinformationon this species. B. Dose Administration (1) Test Groups Group_ Test Material 1 (Control)Distilledwater 2 T-6052 3 T-6052 4 T-6052 Dose Level (mg/kq) 0* 2** 200 1000 Number of Animals Males Females 3 3 3 3 3 3 3 3 To be administeredat a dose volume of 2.0 mL/kg To.be administeredat a dose volume of .01mL/kg (2) Preparationof Exposure Area On the day beforetest material application,the back and, if necessary(to obtainunblemishedskin),the flanksof each rabbitwill be clippedfree of hair. The shavedarea will constituteapproximately20% of the total body surfacearea. The treatmentsites (intactskin)will be inspectedfor interferinglesions,irritation,or defects that would precludethe use of any of the animals. The animalswill be clipped as needed throughoutthe study. (3)Dose Administration All animalswill receivea singleadministratioonf the respectivetest or controlmaterial. The day of treatment will be designatedas Day 1. The dose for each animal in Group 2 will be dilutedwith distilledwater and applied at a dose volumeof .01 mL/kg. The respectivedose for each animalin Groups 3 and 4 will be appliedundiluted. All doses in Groups1-4 will be based on the animal'sbody weight just before administrationand will be spread onto the area of exposurein a thin and uniforma layer. The area of application(Groups1-4)will be coveredwith a lo-cm x 10-cm gauze bandage secured with gaper tape around all edges and overwrappedwith Saran Wrap and Elastoplast tape to providean occlusivedressinq'.The rabbitswill be collaredduringthe 24-hour applicationperiod. (4)Reason for Route of,Administration The dermal route is a potentialrouteof exposure in humans. Page 34 of 41 TP3016.AB Page 7 (5) Removal of Test Material Approximately24 hours after test or control material applicationthe bandages and collars will be removed and the residualtest material will be removed using water or an appropriatesolvent, if necessary. C. Observation of Animals (1) Clinical Observations For clinicalsigns before test or control material administrationand for clinicalsigns and mortalityat approximately1, 2.5, and 4 hours after test material administration(Day 1) and daily thereafterfor clinical signs,and twice daily (a.m. and p.m.) for mortalityfor at least 28 days. Observations may be extended when directed by the study director. (2) Reading of Dermal Irritation Before test or control material administrationthe initial dermal irritationreadingwill be made and recordedas the Day I reading (Attachment1). Additionaldermal irritationreadings will be made approximately30 minutes after bandageremoval (Day 2) and on Study Days 4 and 8. individualdermal irritationrecords will be maintained for each animal. (3) Body Weights For randomization,before test or control material application (Day 1), on Day 29, and at unscheduleddeath (when survivalexceeds I day) (4) Sample C21lections (a) Freguency Before initiation(Day 1), approximately24 hours post-dose (Day 2), Days 4, 8, 15, 22, and at experimentaltermination (Day 29) (b) Number of Animals All Page 35 of 41 TP3016-AB Page 8 (c)Method of Collection Blood samples (approximately4 mL) will be collected from the marginal ear vein of eitherear on Days 1, 2, 4, 8, 15, and 22. Approximately20 mL of blood (actualvolume to be documentedin the raw data)will be obtainedfrom the posteriorvena cava of each animalsacrificedin a moribundconditionorsacrificedat the time of necropsy(Day29). The sampleswill be stored at room temperatureand then centrifuged,and the separateserum and cellular fractionsstoredin a freezerset to maintain -20'CilO*C. The separatedserum and cellular fractionswill be sent frozenon dry ice to the Sponsorafter experimentaltermination. Sampleswill be shippedto: James D. Johnson 3M E.E. & P.C. Bldg. 2-3E-09 935 Bush Avenue St. Paul, MN 55106 James D. Johnson or alternatewill be notifiedby telephoneat (612)778-5294prior to the shipmentof the samples. D. Pathology (1)UnscheduledSacrifjces and Deaths Any animaldying during the study or sacrificedin a moribundconditionwill be subjectedto an abbreviated gross necropsyexaminationand all abnormalitieswill be recorded. Animals in a moribundconditionwill be anesthetizedwith sodiumpentobarbital(viainjectionin the marginalear vein), bled via the vena cava,and exsanguinated.Tissues, as describedin section D. Pathology,(3) SampleCollection,will be collected. After necropsy,the animalswill be discarded. (2) Scheduled Sacrifice At terminationof the experimentalphase (Day29), survivinganimalswill be anesthetizedwith sodium pentobarbita(lvia injectionin theigarginaelar vein), bled via the vena cava, exsanguinated-a,nd subjectedto an abbreviatedgross necropsyexamination. The animalswill be necropsiedin randomorder and all abnormalitieswill be recorded. Page 36 of 41 TP3016.AB Page 9 (3) Sami)leCollection The sites of test and controlmaterialapplicationwill be washed with lukewarm tap water prior to the necropsy procedure. The whole liver, bile, an approximate1-cm x 1-cm sectionof the dermal applicationsite from all animals,and both kidneys from the firstmale and female necopsiedin each group will be collectedand immediately placed in a freezerset to maintain a temperatureof -20*C 100C. After necropsy,the animalswill be discarded. The tissues(liver,bile,dermalapplicationsite, kidneys)will be sent frozenon dry ice to the Sponsor after experimentaltermination. The sampleswill be shippedto the personlistedin Section7.C (4).(c). The Sponsoris responsiblefor the retentionan@ disposition of the samples. E. StatisticalAnalyses No statisticalanalysesare required. 8. Report A final reportincludingthose items listedbelowwill be submitted. Descriptionof the test and controlmaterials Descriptionof the test system Procedures Dates of experimentalinitiationand termination Tabulationof mortalitydata by sex and dose level Descriptionof any toxiceffects/dermalirritation Tabulationof mean body weights by sex and dose level Gross pathologyfindings/grosspathologyreport 9. Locationof Raw Data, Records, and Final Report Originaldata, or copiesthereof,will be availableat HWI to facilitateauditingthe studyduring its progressand before acceptanceof the finalreport. When the finalreportis completed, all originalpaperdata, includingthose item listedbelowwill be retained in the archivesof HWI accordingto HWI SOP. Protocol and protocol amendments Dose preparationrecords In-liferecords Body weights Dose administration Observations Anatomical pathologyrecords Sample collectionrecords Shipping records Study correspondence Final report(originalsignedcopy) Page 37 of 41 TP3016.AB Page 10 The followingsupportingrecordswill be retainedat HWI but will not be archivedwith the study data. Animal receipt/acclimatiornecords Water analysis records Animal room temperature *andhumidity records Refrigeratorand freezertemperaturerecords Instrumentcalibrationand maintenancerecords Page 38 of 41 PROTOCOL APPROVAL TP3016.AB Page 11 John L. Butenhoff, PhD Date Sponsor's Representative 3M Toxicology Service Medical Department @t-evinM. Glaza Study Director Acute Toxicology Hazleton Wisconsin, Inc. /, i' LA presentative Qu lity Assurance Unit Hazleton Wisconsin, Inc. (6329-135.protdsk2) Date 1.24n Date Page 39 of 41 Attachment I Scoring Scale for Acute Dermal Reactions TP3016.AB Page 12 Erythema 0 - None I - Slight 2 - Moderate 3 - Severe Edema 0 - None I - Slight (barelyperceptibleto well definedby definiteraising) 2 - Moderate (raised approximatelyI mm) 3 - Severe (raisedmore than I mm) Atonia 0 - None I - Slight(slightimpairmentof elasticity) 2 - Moderate (slowreturn to normal) 3 - Marked (no elasticity) Oesguamation 0 - None I - Slight(slightscaling) 2 - Moderate (scalesand flakes) 3 - Marked (pronouncedflaking with denuded areas) Coriaceousness 0 - None 1 - Slight (decreasein pliability) 2 - Moderate (leatherytexture) 3 - Marked (toughand brittle) Fissuring 0 - None I - Slight (definitecracks in epidermis) 2 - Moderate (cracksin dermis) 3 - Marked (crackswith bleeding) Page 40 of 41 'HAZLCMCN W WI S C ON S IN POST OFFICE BOX 7545 MADISON.WI 53707 7545 a CORNING Company PROTOCOL'TP3016.AB Single-DoseDermal Absorption/ToxicityStudy of T-6052 in Rabbits Sponsor 3M Toxicology Service Medical Department 3M Center, Bldg. 220-2E-02 P.O. Box 33220 St. Paul, MN 55133-3220 HWI 6329-135 Contractor Hazleton Wisconsin,Inc. 3301 Kinsman Boulevard Madison, WI 53704 Sponsor's Representative John L. Butenhoff,PhD Study Director Steven M. Glaza Amendment No. I This amendmentmodifiesthe followingportionsof the protocol: EffectiveJanuary 24, 1995 At the request of the Sponsor,the weights of tissuescollectedand the volume of bile collectedwill be documentedin the raw data. Theseweightsand volumeswill be includedwith the sample shipment. Modify the following sectionsof the protocolto includethese-additions. 1. Page 9. 7. ExperimentalDesign;D-.Pathology-,(3) SamRle Collection. Modify the secondsentencein the first and secondparagraphsof this sectionwith the followingunderlinedadditions: The whole liver,bile, an approximate1-cm x 1-cm sectionof the dermal applicationsite from all animals,and both kidneysfrom the first male and femalenecropsiedin each groupwill be collected,weighed(volumeonly determinedfor bile),and immediatelyplaced in a freezerset to maintaina temperatureof -20*C 10*C. The samples and their carreS-Dondingweights or volumeswill be shippedto the person listedin Section7.C-.14).(c). 2. Page 9, 8. Report. Add the followingto this section: Individualanimaltissue weights and bile volumes P h o n e f;08 2,1 1 d4 / I F a x 6 Oll .14 1 1:1 7 7 Page 41 of 41 Amendment No. I PROTOCOL AMENDMENT APPROVAL John L. Butenhoff, PhD Date Sponsor's Representative 3M Toxicology Service Medical Department Steven M. Glaza Date Study Director Acute Toxicology Hazleton Wisconsin, Inc. Represenfativ Date Quality Assur@nce Unit Hazleton Wisconsin, Inc. (6329-135.Aml.dsk2) @HWI 6329-135 Page 2 9.1.2 AnalyticalprotocolAMDT-02.0795.1 3M Environmental Laboratory Protocol- AnalyticaSltudy Single-DoseDermal Absorption/ToxicityStudy ofT-6052 in Rabbits In-Vivo Study Reference Number: HWI#6329-135 Study Number: AMDT-020795.1 Test Substance: FC-120 (T-6052) Name and Address of Sponsor: 3M SCD Division 367 Grove Street St.Paul,NIN 55106 Name and Address of TestingFacility: 3M EnvironmentalTechnology and Services 935 Bush Avenue St.Paul,NM 55106 Proposed InitiatioDnate: July25, 1995 Proposed Completion Date: August 25,1995 Method Numbers AMDT-M-1-0, AMDT-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 Dohrrnann DX2000 OrganicHalideAnalyzer-Liver FluorideMeasurement by Means of an Orion EA940 Expandable Ion Analyzer Extractionof Fluorochemicalsfrom Rabbit Liver Analysis'ofRabbitLiverExtractforFluorochemicalsUsing ElectrosprayMass Spectrometry Analysisof FluorideUsing the SkalarSegmented Flow Analyzer with Ion SelectiveElectrode Thermal Extractionof Fluorideby Means of a Modified Dohnnann DX2000 Organic HalideAnalyzer-Serum Author: James D. Johnson Approved By: 2. "rs,- @a tem@eDsy@D4 . son tmudys D*4ctLor Date John Butenhoff,PhD Date Sponsor Representative. 1.0 PURPOSE ThisstudyisdesignetdoprovidienformatiaosntowhetheFrC-120(T-6052i)s dermallyabsorbed.The analyticaalspectofthisstudyistodeterminefluorinecontainingcompounds intheliverand serum of rabbitsB.y comparisonof thedata obtainedafterdermal absorptionwiththatobtainedafterintravenousinjection, assessmentof theextentof dermal absorptioncan be performed. 2.0 TEST MATERIALS 2.1 Test,Control,and Reference Substances and Matrices 2.1.1AnalyticalReference Substance: FC-95, lot161 or 171. They are equivalent. 2.1.2AnalyticalReference Matrix: Bovine liverb,ovine serum and rabbit serum 2.1.3AnalyticalControl Substance: None 2.1.4AnalyticalControl Matrix: Bovine liver,bovine serum and rabbit - serum 2.2 Source of Materials:3M ICP/PCP Division(2.1.1),grocery store(2.1.2,2.1.4 liver)S,igma Chemical Company (2.1.22,.1.4bovineserum),AMDT 110394.1 (Hwi#6329-123)controlgroup animals(2.1.22,.1.4rabbitserum) 2.3Number ofTest and ControlSamples: Tissuesand fluidfsrom 18 testanimals and 6 controlanimals.Tissuesand fluidsincludeliverk,idney,serum,cellular fractiond,ermal applicatiosniteand bile.Analysisofthesetissueswillbe atthe discretioonftheStudy Director. 2.4IdentiflcatioonfTest and Control Samples: The samplesareidentifieudsing theHWI animalidentificatinounmber which consistosf a lettearnd fivedigit number, plusthetissueidentitaynd day identit(yserum). 2.5Purity and StrengthofReference Substance: To be determinedby Sponsor. 2.6Stabilitoyf ReferenceSubstance:To be determinedby Sponsor. 2.7Storage ConditionsforTest Materials:Room temperature(2.1.1), -20 100C (2.1.22,.1.4).Testand Controlsampleswillbe receivedaccordingto ANMT-S- 10-0. 2.8DispositionofSpecimens: Biologicatlissueasnd fluidswillbe retainepder GLP Regulationforthetimeperiodrequiredforstudieslongerthan28 days. 2.9SafetyPrecautions:RefertoappropriatMeSDS. Wear appropriatleaboratory attireU.se cautionwhen handlingknivesforcuttingthesamples. 3.0 EXPERIMENTAL -Overview The tissuesfrom animalsdosed asdescribed(HWI#6329-135), areavailablfeor analysisforfluorinecompounds. At thediscretioonf theStudy Directora, seriesof analyticatlestscan be performed.The screeningforfluoridienliverviacombustion (SeeMethods-next Section)istheappropriataenalysistopresentdefmitivedatafor fluorineintheliverT.o confirmtheidentitoyf fluorine-containcionmgpoundspresentinliver(ifany at28 days)and serum atvariousintervalse,lectrospramyass spectrometrymay be selectedas one of theanalyticatlechniquesemployed. Not all ofthetissuesand fluidsampleswillbe analyzed.When sufficiendtatahas been collectedtomeet theobjectiveosfthestudyintheopinionofthe Study Director, analysiswillcease. 4.0 EXPERIMENTAL -Methods 4.1Liver and Serum screeningmethods: (attached) 4.1.1AMDT-M-1-0, Thermal ExtractionofFluorideby Means ofa Modified Dohrmann DX2000 OrganicHalideAnalyzer-Liver 4.1.2AMDT-M-2-0, FluorideMeasurement by Means of an Orion EA940 Expandable Ion Analyzer 4.1.3AMDT-M-4-0, ExtractionofFluorochemicalsfrom RabbitLiver 4.1.4AMDT-M-5-0. Analysisof RabbitLiverExtractforFluorochemicals Using ElectrospraMyass Spectrometry 4.1.5AMDT-M-8-0, AnalysisofFluorideUsing theSkalarSegmented Flow Analyzer withIon SelectivEelectrode 4.1.6AMDT-M-14-0, Thermal Extractioonf Fluorideby Means ofa ModifledDohrmann DX2000 OrganicHalideAnalyzer-Serum 5.0DATA ANALYSIS 5.1DataReportingD:atawilblereporteadsa concentrat(iwoenight/weigohft) fluoridepertissueor fluido,r asFC-95 (electrospramyass spectrometryp)erunitof tissueor fluid.Statistiucssed,atthediscretioonf theStudy Directorm,ay include regressionanalysisof serum concentrationwsithtimeand averagesand standard deviationsofconcentrationfsordifferendtose groups.Ifnecessary,simple statistictaelstssuchas Student'sttestmay be appliedto determinestatistical difference. 6.0 MAINTENANCE OF RAW DATA AND RECORDS 6.1 Raw Data and Records: Raw data,approved protocola,ppropriatsepecimens, approved finalreporta,nd electronidcatawillbe maintainedintheAMDT archives. 7.0 REFERENCES 7.1AMDT-S-10-0, Sample TrackingSystem 8.0 ATTACHMENTS 8.1ANMT-M-1-0, ThermalExtractioonfFluoridbey Means ofa Modified Dohnnann DX2000 OrganicHalideAnalyzer-Liver 8.2AMDT-M-2-0, FluorideMeasurement by Means of an Orion EA940 Expandable Ion Analyzer 8.3 AM:DT-M4-0, Extractionof Fluorochemi-calfsrom RabbitLiver 8.4 AMDT-M-5-0, AnalysisofRabbitLiverExtractforFluorochemicalsUsing ElectrosprayMass Spectrometry 8.5AMDT-M-8-0, AnalysisofFluorideUsing theSkalarSegmented Flow Analyzer with Ion SelectivEelectrode 8.6AMDT-M-14-0, Thermal Extractioonf Fluorideby Means of a Modified Dohrmann DX2000 OrganicHalideAnalyzer-Serum 3M E1nvi0ronmental Laboratory Method Thermal Extractionof Fluorideby Means of a Modified Dohrmann DX2000 Organic HalideAnalyzer - Liver Method IdentificatiNounmber: AMDT-M- I RevisionNumber: 0 AdoptionDate: RevisionDate: None Author RichYoungblom Approved by: ?oup7roLuepeLea 77 /0 /3 Dafe QualityAssurance Date Software:MS Word 5.la AffectedDocuments: AMDT-M-2 FluorideMeasurementby Means ofan OrionEA940 ExpandableIonAnalyzer AMDT-EP-3 RoutineMaintenanceofa ModifiedDohrmann DX2000 OrganicHalideAnalyzer 1.0 SCOPE. APPLICABLE COMPOUNDS, AND MATRICES - 1.1Scope:Thismethod isfortheoperatioonfa Dohrmann DX2000 when itisusedtoextract fluoridferom variousmatrices.The fluoridiestypicallcyollecteidnTISAB solutiofnoranalysis withan ionselectiveelectrode. 1.2'ApplicablCeompounds: Fluorochemicalosrotherfluorinatecdompounds. 1.3Matrices:Biologicatlissuepsa,rticularlliyver. 2.0 KEYWORDS 2.1Fluoridef,luorine,xtractiopny,rolysisi,onizatioino,nselectiveelectrodeD,ohrmann, halide, DX2000, fluorochenicals. 3.0 PRECAUTIONS 3.1Glasswareand exhaustgasescan be extremelyhot. 3.2Glasswareisfragileb,rokenglassr@aycauseinjuries. 3.3Pressurizedgases,propercompressedgashandlingpracticersequired. 3.4Solventbasedsamplesmay flashm,ay need toallowthem todrydown beforestartinrgun. 3.5Potentiabliohazarddsue tothebiologicamlatrices.Use appropriatpeersonalprotective equipment. 4.0 S12PLIES AND MATERIALS 4.1Compressed Oxygen, Hydrocarbonfreer,egulatedto30 PSI. 4.2Compressed Helium,High PurityGrade,regulatetdo45 PSI. 4.3QuartzglasssampleboatwithTeflonTmtubing,Dohrmann 890-097 orequivalent. 4.4Quartzglasscombustiontube,RelianceGlass G-9405-012 orequivalent. 4.5Orion940999 TotalIonicStrengthAdjustmentBuffer(TISAB 11)orequivalent. 4.6Sample collectiovnialsH,DPE. 4.7Mlli-QTM water 4.8Polystyrenepipettes. 4.9ActivatedCharcoal,E. Merck 2005 orequivalent. 4.10Hamilton Syringeor equivalent. 4.11Miscellaneouslaboratorgylassware 5.0 EQUIPMENT 5.1Rosemount Dohrmann DX2000 OrganicHalideAnalyzerm,odifiedforfluorideextraction. 5.2IBM compatible386 or486 computer. 5.3DX2000 softwarev,ersion1.00,modifiedforfluoridextraction. 5.4Excel Spreadsheetv,ersion5.0or greater 6.0 INTERFERENCES- 6.1 Sample sizeislimitedtoapproximately150mg, dependingon samplemoisturecontent.This may vary from matrixtomatrix. 2 7.0 SAMPLE 14ANDLING 7.1Samples arenottobe handledwithbarehands. Fluoridemay leachfrom theskintothe sample. Use forcepsorprobe totransfetrissues. 7.2Samplesofliverarecutfrom frozenliverand placedinataredand labeledweigh boat.use a cleanscalpealnd cuttingboard.The cuttinbgoardand scalpeslhouldbe cleanedwithwater, methanol,or methanol-watesrolutioanftereachliveriscut. 8.0 CALIBRATION AND STANDARDIZATION 8.1PreparatioonfCalibratiSotnandards 8.1.T1hestandarrdesquirfeodreachprojecwtilnleedtobeapproprifaotrethaitndividuparloject. Refertoprotocolforthatproject. 8.1.2Typically50-500ppm FC-95 inmethanolstandardsareused. 8.1.3For rabbiltiverstudiesu,sebeeflivearsthematrix.Cut apieceoffrozenbeefliver(100150mg) and weigh itina labeledandtaredweigh boat. 8.2 Calibration-Overview The normal cahbratioinsthefluoridceurve(AMDT-M-2). However, ifan optionalspikedliver curveisrequiredtheprocedurelistebdelow isused. 8.2.1A calibraticounrvefortheDX2000 isgeneratedby spikingsampleswithknown standards and combustingthem usingthesame methods and matrixtypeasthesamplestobe tested. 8.2.2Typicallyt,hreereplicateosfeachstandardand 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)= (Amount spiked'mmL) x (Conc.ofstandaridnppm) x (0.6004)* *FC-95 is60.04% F therefor0e.6004isthefactorusedtoconvertFC-95 toF 8.2.5StandardMass RecoveredF (ug)= (TISAB volumeinmL) x (Orionreadinginppm) 8.3Calibratio-nProcedure 8.3.1StartUp 8.3.1.1Run 2 ormore CleanCycleswhen startinignstrumenetachday. More cleancyclesmay be usediftheprevioussamplescontainedhighconcentratioonfsfluoride. 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 eachsampleasdescribedinsection9.0and analyzesample accordingtomethod AMDT-M-2 fortheionselectiveelectrodaenalysis. 3 8.3.2.F4orrabbisttudietsh,emeterreadinfgorablanksampleshoulbde0.03ppm orlower beforeproceedingwiththecalibrationB.um samplesuntilthislimitisreached,or untilin the judgement of theoperatorthereadingisstablewithrespecttohistoricraeladings(previous48 hours). 8.3.2.5For non-rabbitstudiest,heblankreadingsshouldreacha predeterminedionconcentration beforeproceedingwith thecalibration. 8.3.2.6Itmay be necessarytomix approximately50 mg of charcoalwiththesample to aid combustion. 8.3.3 Standard Curve 8.3.3.1Weigh outatleast15 matrixsamples(5standardswith3 replicateesach)intaredand labeledweigh boats.For rabbitstudiesw,eigh 100-150mg beefliversamples.Record weightsin studydata.Storethematrixsampleson dry iceor icepackstokeep them frozenuntilused. 8.3.3.2 Placeweighed beefliversample inDohrmann sample boat. 8.3.3.3StartwiththeloweststandardconcentrationU.sing a Hamiltonsyringe,ejecta fixed quantityof thestandardon or inthematrix.For rabbitstudiesu,se4 uL of standardand ejectiton or inthebeefliver. 8.3.3.4At least3 replicatesshouldbe usedforthelowest standardconcentrationm;ore replicates may be used atthediscretioonftheanalyst. 8.3.3.5Combust thesample asdescribedinsection9.3and analyzeaccordingtoAMDT-M-2. 8.3.3.6Run all15 standards.Ifone replicatiessignificantdliyfferenftrom theothertwo replicatesr,un anothersample forthatstandard.Indicateindatathatthenew replicatreeplacesthe oldreplicataend thatthenew replicatweillbe used to calculatteheregressiocnurve. 8.3.3.7When allstandardshave been run,calculattehe r2.r2 must be atleast0.95.Ifitisnotat least0.95,consultwith supervisor. 8.3.3.8A new standardcurveshouldbe runwhen thecombustiontubeor sample matrixis changed. New standardcurvemay alsobe runatthediscretioonftheanalyst. 8.4 Storage Conditions forStandards 8.4.1Storagerequirementsforstandardsaredependenton theindividuasltandardsused. Typicallys,tandardsarestoredatroom temperatureinplastisccrew topbottles. 8.4.2New FC-95 standardsshouldbe preparedatleastonce a month. 9.0 PROCEDURES 9.1TypicalOperatingConditions: 9.1.1Combustion tubetemperature= 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,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 found intheCELL CHECK menu. 9.2.6 See AMDT-EP-3 fordetailosftheDohrmann software. BOAT program 9.3Sample ExtractionProcedure: 9.3.1Open theSAMPLE HATCH and placethesample intheBOAT. Itmay be necessaryto mix approximately50 mg of charcoalwiththesample toaidcombustion.Ifthisisdone,charcoal shouldalsobe mixed inwhileestablishintghebaselineand when generatingthestandardcurve. 9.3.2Close SAMPLE HATCH. 93.3 Add appropriatveolume ofTISAB solutioonr 1:1TISAB:Milli-QTmwatermixturetoa labeledsample collectiovnial.Typically0.6mL to 15 n-iLareused. For rabbitstudiesu,se 1.0or 2.0mL of 1:1 TISAB:Milli-QTM watermixture. 9.3.4Placethevialso thatthetipoftheCOMBUSTION TUBE isintheTISAB atleast0.25 inches.Gases releasedduringpyrolysimsust bubble throughtheTISAB. 9.3.5Run theEOX-SOLIDS program found intheRUN menu. 9.3.6When theEOX program isfinishedr,emove thecollectiovnialfrom thecombustiontube. 9.3.7IfundilutedTISAB was used tocollecthesample,add an equalvolume ofNfilli-wQaTtmer totheTISAB tomake 1:1 TISAB:Milli-QTm. 9.3.8Rinsetheend of thecombustiontubewithMilli-QTmwaterand wipe witha KIMWIPE to remove any TISAB remainingon thetube. 9.3.9Open thesample hatchand remove any remainingash from theboat. Ash can be removed witha cottontippedapplicatoorrvacuumed out. Itmay be necessarytoscrapparticleosffthe bottom witha spatulaor othersimilardevice.A drop of Milli-QTMwater may be added tothe boattoaidintheClean Cycle. 9.3-10 Close thehatch. 9.3.11Run theCLEAN BOAT program. 9.3.12Sample isreadyforanalysibsy ionselectiveelectrod(eAMDT-M-2). 9.4 Sample Calculations 9.4.1Use thestandardcurvetocalculattehesample value. 9.4.2Sample Mass RecoveredF (ug)= (TISAB volinmL) x (Orionreadi ntercgpt) (slope) 10.0 VALIDATION 10.1QualitCyontrol 10.1.D1ailyStartUp Check Samples:Once thestandarcdurveisestablisheda,chdayof analysisisstartedby analyzingQC samples.The QC samplesaretobe thesame asthelowest concentratiosnpikedsamplesused togeneratethestandardcurve.Each concentratiomnust be done intriplicautnelessthefinttwo replicateasrewithin20% of thestandardcurve,thena third replicatiesnot necessary. 10.2 Precisionand Accuracy: See method developmentanalysisand sample analysisin FluorideNotebooks 2,3,and 5. Precisionand accuracyvarieswhm analyzingsamplesof different matricesand differenrteferencecompounds. 10.3 Other ValidationParameters:NA 5 11.0 DATA ANALYSIS 11.1Calculations 11.1.F1orthestandacrudrvues,eregressainoanlysiinEsxcelv,ersi5o.n0orgreater. 11.1.2To calculattehefluoridceontractioinnthesample,seemethod AMDT-M-2. 11.2 Analyzing theData 11.2.1r2must be atleast0.95orgreater".Outliersm"ay be excludediftwo ofthethreereplicates arewithin20% ofeachotherand theoutlieirsgreaterthan200% oftheaverageofthosetwo or lessthan50% oftheaverageofthosetwo. Any suchoutliersshouldbe pointedoutinthedataand notedintheFinalReportalongwiththereasonitwas considereadn outlier. 12.0 ATTACHMENIS None 13.0 REFERENCES 13.1Rosemount Dohrmann DX2000 OrganicHalideAnalyzerOperatoz'Msanual (Manual915349,revisionB, December 1993) 13.2 AMDT-M-2 FluorideMeasurementby Means of an OrionEA940 ExpandableIon Analyzer 13.3AMDT-EP-3 RoutineMaintenanceofa ModifiedDohrmann DX2000 OrganicHalide Analyzer 14.0 REVISIONS Revision Numbe Reason forChange Revision ]2ate 6. 3M Environmental Laboratory Method FluorideMeasurement by Means ofan Orion EA940 ExpandableIon Analyzer Method Identiricatoin Number: ANMT-M-2 RevisionNumber: 0 AdoptionDate: ID-,f-fs- RevisionDate: None Author Rich Youngblom Approved By: G@, Leader 161-3 Date wla QualitAyssurance Lo- 4,-1 Date Software: MS Word 5.la AffectedDocuments: AMDT-M-1 Thermal Extractionof Fluorideby Means of a Modif'ied Dohrmann DX2000 Organic HalideAnalyzer 1.0 SCOPE, APPLICABLE !COMPOUNDS3 AND MATRICES 1.1SCOPE: This method isforthecalibratioand operationof an Orion EA940 Expandable Ion Analyzer. 1.2APPLICABLE COMPOUNDS: Fluoride. 1.3APPLICABLE pH of 6.0. MATRICES: Liquidsamplesinan appropriatbeuffersolutionP.referred 2.0 KEYWORDS 2.1Fluoridef,luorinei,onselectiveelectrode 3.0 PRECAUTIQNS 3.1No hazardsidentifiewdiththismethod. 4.0 SUPPLTES AND MA ERIALS 4.1Orion 940999 TotalIonicStrengthAdjustmentBufferH (TISABII)orequivalent. 4.2 Orion Model 900001 electrodfeillinsgolutio(nAgCl)orequivalent. 4.3 Orion 940907 100 ppm fluoridsetandardor equivalent. 4.4Milh-QTm wateror equivalent. 4.5Magnetic stirbars. 4.6Lab tissues. 4.7Sample collectiovnials. 4.8 Plastic100 mL voluinetrifclasks. 4.9 Polystyrenepipettes. 4.10 Miscellaneouslaboratorgylassware. 5.0 EQUTPMENT S.I Orion Model EA940 ExpandableIon Analyzeror equivalent. 5.2 Orion Model 960900 SolidStateCombinationFluorideelectrodoer equivalent. 53 Magnetic StirPlate. 5.4 IBM compatible386 or486 computer(onlyneeded ifusingOrion 3E software). 5.5Orion RS232 interfacceable(onlyneeded ifusingOrion3E software). 5.6MicrosoftExcel5.0(onlyneeded ifusingOrion3E software). 6.0 INTERFERENCES 6.1Itisrecommended thatthepH be ator near6.0.A 1:1 mixtureofTISAB and sample/MilliQTI,Lwaterwillgenerallybringsample topH of 6.0. 6.2Sample temperaturemay effectfluoridmeeasurement. Itis-recommendedthatthesample be atroom temperatureas thestandardswere when themeter was calibrated. 6.3The ratethesamplesarestirreadtshouldbe consistenwtith theratethestandardswere stirred. 2 6.4Airbubblestrappedunder electrodcean giveerroneousreadings.Make sureno airistrapped under electrode. 7.0 SAMPLE HANDLING 7.1 No specialhandlingnecessary. 8.0 CALIBRATION AND STANDARDIZATION 8.1 PreparatioonfCalibratiSotnandards 8.1.M1easure50mL ofTISAB H into5 100mL plastvioclumetrfilcasks. 8-.1L.a2beltheflaskass0.050,.10,.5,1.0a,nd1.5ppm F-,alongwiththedateandyourinitials. 8.1.P3ipett0e.050,.1,0.5,1.0a,nd1.5mL of100ppm fluorisdteandaridntotheappropriately labeledflasks. 8.1.4Add approximately30 mL of Nfilh-QT?w4atertoeach flask. 8.1.5Shake theflaskstomix thesolutions. 8.1.6Eliminateairbubblesfrom theflasksby tippingtheflaskson theirsidesand rollingtheairin theflasksover theairbubbles. 8.1.7Bringthevolume intheflasksup tothe 100 mL mark withNfini-QTMwater. 8.1.8Invertand shake theflasksforthefinalmixing. 8.1.9Record standardsinStandardsLog Book. 8.2 Calibration 8.2.1Ifnecessar-yr,emove tapefrom electrodfeillinhgole. 8.2.2Invertprobe to wet topseal. 8.2-3Ejecta few dropsof fillinsgolutionfrom bottomof electrodteowet lowerseal. 8.2.4Filltheelectrodewithfillinsgolution. 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 theslopeintheappropriatleogbook. 8.2.7Clean theelectrodeby rinsingwithMilli-QTMwaterand wiping thesidesdown 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 stirbarinthesample and placethesample on thestirplate. 9.1.3Allow thesample ton-dxfora few secondsbeforeinsertintgheelectrode.When the electrodeisinsertedm,ake surethereareno airbubblestmpped under theelectrode. 9.1.4The sample shouldbe thesame temperatureas thecalibratior-standaarnddsstirreadtthe same mte as thecalibratiosntandards. 9.1.5When thereadingshave stabilizerde,cordthereadingintheappropriatleogbook. 3 9.2CalibrationAnd Nleasurement,Using Orion 3E Software: 9.2.1Calibration: 9.2.1.1Follow steps8.2.1to8.2.4. 9.2.1.2PressFunctionKey #8 (F8). 9.2-1.3The computer screenwillaskyou toconfirm thenumber of standardstobe used, concentratioonf thestandardsa,nd whetheror nota blankistobe includedinthecalibration. Make any necessarychangestotheinformationpresentedand clickon CONTINUE. 9.2.1.4Placetheelectrodienthefirssttandardon thestiprlateand clickon CONTINUE. 9.2.1.5Observe thereadingson thegraphicdisplayon thecomputer. When thereadingshave stabilizepdr,essACCEPT READING. 9.2.1.R6epeat step9.2.1.4and 9.2.1.f5ortheremainingstandards. 9.2.1.7Afterthefinalstandardt,hecomputer willdisplaytheslopeof thecurve,as wellas the intercepatnd correlationR.ecord theslope,intercepta,nd correlatioinntheappropriatleogbook and clickon CONTINUE. The calibratiodnatai.sautomaticallcyopiedtoC:\Orion\Data\Calib.txt 9.2.2Data Spreadsheet: 9.2.2.1SelecteitherNEW or OPEN from theFILE menu toopen a new orexistingspreadsheet tostoredatain. 9.2.2.2Record thename of thespreadsheetused intheappropriatleogbook. 9.2.3FluorideMeasurement: 9.2.3.1Follow steps9.2.1through9.2.4 9.2.3.2Enterthename of thesample intheappropriateplaceon thescreen. 9.2.3.3Clickon theNEW SAdvfPLE button 9.2.3.4When thereadingshave stabilizecdl,ickon theRECORD buttonand writetheresultinthe appropriatleog book. 10.0 VALIDATION 10.1.QualityControl: 10.2 Precisionand Accuracy 10.3 Other ValidationParameters AccordingtoReference13.2,therangeofdetectioins0.02 ppm fluorideup toa saturatesdolutioonf fluoride. 11.0 DATA ANALYSIS 11.1 CalculationsNonenecessary. 11.2 Analyzing theData Nonenecessary. 12.0 ATTACHMENTS None 13.0 REFERENCES 4 13.1Orion Model EA940 Expandable[onAnalyzer InstructioMnanual,Orion Research Incorporated1,99 1. 13.2Orion Model 960900 SolidStateCombination FluorideElectrodeInstructioMnanual,Orion Research Incorporated1,991. 14.0 REVTSTONS Revision Numbe Reason forChange Revision Date 5 3M Environmental Laboratory Method Extractionof Fluorochemicalsfrom Rabbit Livers 90P IdentiricatioNnumber: AMDT-M-4 RevisionNumber: 0 Adoption Date: la - 7 rRevisionDate: None Author-Dave Christenson/CyndiWieaber Approved By: up Leeaadd@er /0 Date -3 Qualit@Assurance Date Software:MS Word, 6.0 AffectedDocuments: M-5, Analysisof RabbitExtractforFluorochemicalUssing Electrospray Mass Spectroscopy. 1.0 SCOPE 1. 1 1.2 1.3 Scope: Thismethod isfortheextractioonf fluorocheniicaflrsom rabbitlivers. Ethyl acetateisused toextractfluorochemicalsfrom theliversforanalysisby electrospraymass spectroscopy. Applicable Compounds: Fluorochemicalsor otherfluorinatecdompounds. Matrices: RabbitLivers. 2.0 KEYWORDS 2.1 Fluorocherr@calsr,abbitliverse,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 Syringe,capableofmeasuring100pL 4.1.2 Eppendorf typeor disposablepipets 4.1.3 Gloves 4.1.4 Plasticgrindingtubes 4.1.5 Plasticentifuge tubes,15 mL 4.1.6 Labels .4.1.7 Nitrogen 4.1.8 Timer 4.1.9 FilterTsi,tannylonsyringef:dter0s.,2pm. 4.1.10 Analyticaplipetsg:lassvolumetripcipets. 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 acetaieby adding 250 mg ammonium acetatetoa 100 mL volunietdcflaskand dilutetovolume withMMI-Q water.Dilutethissolution1:10 fora 250 ppm solution. 4.2.2 Sodium carbonate/SodiumBicarbonateBuffer(I.T.Baker), (Na2CO)NaHC03) 0.25M: Weigh 26.5 g of sodium carbonate(,Na2CO3) and 21.0g of sodium bicarbonate(NaHC03) intoa I L volumetricflaskand bringtovolume withNfifli-wQater. 4.2.3 Diluteacetonitdlseolutiond,iluteacetonitril1e:1 with MMI-Q water. 4.2.4 EthylAcetate 4.2.5 Methanol 4.2.6 MiW-Q.water 4.2.7 IH,lH,2H,2H - perfluorooctanesulfoanciicd(Aldrich) 4.2.8 FC-95 (3M SpecialtCyhemical Division) 2 5.0 EQUIPMENT 5.1 Ultra-TurrTa2x5 Grinderforgrindinlgivesramples. 5.2 Vortexrmxer 5.3 Centrifuge 5.4 Shaker 5.5 AnalyticalEvaporator 6.0 INTERFERENCES 6.1 ,There areno known interferencaetsthistime. 7.0 SAMPLE HANDLING 7.1 ne rabbitliverasrereceivedfrozen,and must be keptfrozenuntiltheextractioins performed. 8.0 CALIBRATION AND STANDARDIZATION 8.1 Preparationof InternalStandards 8.1.1 Preparean internasltandardof approximately12 ppm 1H,lH,2H,2Hperfluomoctanesulphoniaccidtobe added toeach liversample. 8.1.2 Weigh atleast0.1 g of 1H,lK2H,2H-perfluorooctanesulphoniaccidintoa 100 mL volumetdc flaskR.ecord theactualweight. 8.1.3 Bring itup tovolume withmethanol,thisisthestockstandard. 8.1.4 To a 250 mL volumetricflask,add 3 mLs of thestockstandardand bringto volume with Milli-Qwater.Calculatetheactualconcentratioonf the standard. actualmg perfluoroctane- sulphonicacid X 0.1L 3 mL 250 mL actualconcentrationp,pm 8.2 Prepare FC-95 Anion Standards 8.2.1 PrepareFC-95 standardsforthestandardcurve. 8.2.2 Weigh appro),,imately100 mg of FC-95 intoa 100 n:LLvolumetdc flask. Record theactualweight. 8.2.3 Bringup tovolume withdilutaecetonitrile. 8.2.4 Dilutethesolutiownithdilutaecetonitri1l:e10fora solutioonf approximately100 ppm. Dilutethissolution1:10 withdiluteacetonitrifloer a solutionof approx.10 ppm. 8.2.5 Use the10 ppm solutiontomake working standardswithvaluescloseto 5.0ppm, 1.0ppm and 500 ppb. 8.3 Prepare Beef Liver Homogenate to Use for Standards 8.3.1 Weigh 40 g of Bovine liverintoa 250 mL Nalgenebottlceontaining 200 mLs Nhlli-Qwater.Grind toa homogenous solution. 8.3.2 Add 1 niL of thesolutiontoa 15 mL cennifugetube.Preparea totaolf eight I mL aliquotosf thesolutioinn 15 mL centrifugteubes.Be suretore- suspendsolutionby shakingitbetween aliq@ots. 8.3.3 SpikesevenoftheImL aliquotwsith thefollowingamounts ofworldng standardsin step9.12of theprocedure.One I mL aliquotservesas the blank. Worldng Standard (ApproximateConc.) 500 ppb 500 ppb 500 ppb 500 ppb PPM ppm 5 ppm UL w 200 300 400 502ff300 Approximatefinal concentratioonf FC-95 inliver Blank 0.292 ppm 0.584 ppm 0.877 ppm 1.168ppm 2.924 ppm 5T4-8ppm 8.772 ppm 8.4 Calculatetheactualvalueofthestandards: uL of standardx concentratio(ninppm) = finacloncentratio(nppm) 171 ;ngliver"I/rnlhomogenate ofFC -95inliver *Average weightof bovineliverin solutioans determinedby weighing I mL homogenatesof40 mg liverin200 m-L ofMM-Q water.ne amount of FC-95 isreportedas equivalentosf FC-95 potassiumsalt. 8.5 Calibration 8.5.1 Extractthespikedbeefliverhomogenate following9.13to9.23of this method.Use thesestandardstoestablisyhour curveon themass spectrometer. 8.5.2 Alternativelay,standardcurve may be generatedusingratiosof responses of theperfluorooctansulfonaatneion and theinternasltandardanionversus concentratioonftheperfluorooctanesulfonaatneion. 8.6 Storage Conditions for Standards S.6. 1 New standardsarepreparedwith each analysisS.tandardsarestoredin coveredplasticentrifugteubesuntiltheanalysison themass spectrometer isperformed. 8.7 Storage Conditionsfor Standards 8.7. 1 Beef liverhomogenatesmay be frozenafterpreparation. 9.0 PROCEDURES 9.1 Obtainfrozenliversamples.In spenttissuen,ote thattheliverhasnotbeen packaged with othertissues. 9.2 Use a dissectinsgcalpeland cutoffapproximately-l-ogf liver. 9.3 Weigh thesampledirectliyntoa tar-epdlastigcrindingtube. 9.4 Record theliverweightinthestudynotebook. 9.5 Put a labelon thevialwiththestudynumber, weight,rabbitID,dateand analyst initials. 9.6 9.7 9.8 9.9 9.10 9.11 9.12 9.13 9.14 9.15 9.16 9.17 9.18 9.19 9.20 9.21 9.22 9.23 9.24 9.25 Add 2.5mLs water. Grind thesample.Put thegrinderprobe inthesample and grindforabout 2 minutes,untilthesample isa homogeneous solutionwithno largechunks. Rinse theprobe offintothesample with2.5MJ-swaterusinga pipet. Take thegrinderapartand cleanitwith methanol aftereach sample.Follow AMDT-EP-22. Cap thesample and vortexfor15 seconds. PipetI rnL intoa 15 mL centrifugteube.Label thecentrifugteubewiththeidentical informationasthegrindingtube.(SeeAMDT-M-4 Worksheetfordocumentingthe remainingsteps.) Spike thebeefliverhomogenates withtheappropriataemount of FC-95 standard as describedin 8.3. Spike thesamplesand beef liverhomogenates with 100 uL of internasltandard. Add 1 mL of thesodium carbonate/sodiumbicarbonatbeufferand I mL ariumonium acetate. Using an analyticaplipet,add 5 naL ethylacetate. Cap thesample and vortex20 to30 seconds. Put them in theshakerfor20 min. Centrifugefor20 to25 minutes,untilthelayersarewellseparatedS.et thepower on thecentrifugteo25. Remove 4 mLs of thetoporganiclayerto a fresh15 mL centrifugteubewitha 5 na]Lgraduatedglasspipet.Transferthelabeltothefreshtube. Blow thesample down on theanalyticaelvaporatortoneardrynesswith nitrogen, approximately30 to40 minutes. Bring theremainingsampleup in 1 mL diluteacetonitffwiiethan analyticaplipet. Vortex 15 seconds. Transferthesample toa 3 mL syringeA.ttacha 0.2@= nylon mesh filtera,nd filter thesampleintoa freshcentrifugteubeor a autovialL.abel thetubeorvialwith the studynumber and animalnumber. Cap and hold foranalysisby electrospramyass spectroscopy. Complete AMDT-M-4 worksheetand attachtopage of studynotebook. 10.0 -VALIDATION 10.1 QualityControl-notapplicable 10.2 Precisionand Accuracy-not applicable 10.3 Other ValidationParameters-notapplicable 11.0 DATA ANALYSIS 11.1 None 12.0 ATTACHMENTS 12.1 WorksheetAMDT-M-4 13.0 REFERENCES 13. 1 AMDT-EP-22 RoutineMaintenanceofUltra-Tun-aTx-25 14.0 REVISIONS Revision Number Reason for-C-h@Ln= Revision Date Study Sample Number set Rl;inkT.iver Worksheet AMDT-M-4 FC-95 approx0.5ppm actual ppm #W - IOiltir, ?nn ,T. Inn ilf. 4nO ul, FC-95 approx I ppm actual ppm #W 500 i]T. - FC-95 approx.5 ppm actual PPM #W Date and InitialfsorStd. 7nn 100 i]T. ghitlyniirnher3xhL-.trhpt-nripinalworlmheet iglomti-eqind nlnre a mnv_ T.ivprPxtm nn Pmr&qq. PinetI mT, nf f,iveSrolution PinetlOOiiT,nf 12nnrnTntt-rnalqt-Anci2rd "qtd- Vnrtry IS gee- Pinet I mT, of 75tinnm Ammnniilm Aretvite qtd 0 Pinet1 mT.of 0-215Na.CO.ln 75M N:iT4('O.Riiffer Pir.-ti mT. of 1P@thyAlri-.tate- Vnrtex7t)-10 ,;h:;k7t0-min- rentriflive')0m-i7n5 Remnye ;14 MT, alintinotf oryanirI,;iver Binw down tnnegrtiryneg(g<O 25 mL) withN. Add I m- nf 1-1Aretnnitrile/14.0 TN# VnrtpxI 'ge Filteirtgin;aiIcc B-D Rvrinptw-itha O-2Ltm';RTfilteirntoa 1-5m I,nitto-q;lmvnil2el. T)ntek Initini@q 3M Environmental Laboratory Method Analysisof Rabbit Liver Extract for Fluorochemicalsusing Electrospray Mass Spectroscopy SOP IdentiricatioNnumber: AMDT-M-5 RevisionNumber: 0 Adoption Date: RevisionDate: None Author Dave Christenson/CynthWieaber Approved By: ?u2pdLrlo@ue@paL-edaeeerr Date QualityAssurance Date Software:MS Word, 6.0 AffectedDocuments: M-4, ExtractioonfFluorochemicalfsrom RabbitLivers 1.0 SCOPE--- I Scope: This method isfortheanalysisof extractosf rabbitliveror othertissuesor fluidsforfluorochemicalussingthe electrospramyass spectrometerT.he analysis isperformedby singleionmonitoringof FC-95 anion,M/Z= 499,theinternal standardM/Z = 427,and otherappropriatemasses. 1.2 Applicable Compounds: Fluorochemicalsor otherfluorinatedcompounds. 1.3 Matrices: RabbitLivers(samples),Beef Liver (standards)o,thertissuesand fluids. 2.0 KEYW!12RDS 2.1 Fluorochemicalsf,luorinatecdompounds, electrospramyass spectroscopym,ass spectrometerr,abbitlivers. 3.0 PRECAUTIONS 3.1 Use cautionwiththevoltagecablefortheprobe.When thevoltagecableisplugged intotheprobeDO NOT TOUCH THE PROBE, thereisriskof electricsahlock. 3.2 Do notrun thepump above it'csapacityof 4000 psi.Ifpressuregoesover 4000 psi stopand releasepressureT.he peak tubingmay be plugged.Troubleshootback to findtheplug and replacethepluggedtubing.See ANOT-EP- 15 3.3 Do notrun thepump todryness. 4.0 SIIPPLIES AND MATERIALS 4.1 Supplies 4. 1.1 Nitrogengasregulatedto 140 psi. 4.1.2 Fluofixcolumn orequivalenl 4.1.3 100uL or250 uL flatipsyringeforsampleinjection. 4.2 Reagents 4.2. 1 Diluteacetonitrimloebilephase,diluteacetonitri1l:e1 withMilli-Qwater. 4.2.2 Milli-Qwater,allwaterusedinthismethod shouldbe Milli-Qwater. 5.0 EQUIPMENT 5.1 VG Trio2000 ElectrospraMyass Spectrometeror equivalent. 5.2 ISCO SyringePump 5.3 SpectraphysicAsS300 Autosampler 5.4 100 uL Assembly 5.5 Autovialsorcapped centrifugteubes. 6.0 INTERFERENCES 6.1 There areno known interferenceastthistime. 7.0 SAMPLE HANDLING 7.1 Keep theextractesdamplesincapped 15 mL centrifugteubesor incapped autovials untilreidy foranalysis. -2 8.0 CALIBRATION AND STANDARDIZATION 8.1 Preparationof CalibrationStandards 8. 1.1 Seven beefliverstandardsand one blankbeefliverarepreparedduringthe extractiopnrocedure(.SeeAMDT-M-4, section8.0) 8.2 Calibration 8.2.1 Run theseven beefliverstandardstwice,startinwgiththeloweststandard to obtainthe standardcurve. S.2. 2 Typicallyone standardisrun aftereach 5 to7 samples.Choose a standard inthesame rangeof concentratioans thesamples. 8.3 Storage Conditionsfor Standards 8.3.1 Freshstandardsarepreparedwith each analysisS.tandardsarestoredin coveredplasticentrifugteubesuntiltheanalysison themass spectometeris performed.Samples and standardsareNOT refrigerated. 8.4 Storage Conditionsfor Beef Liver Homogenates 8.4. 1 Beef liverhomogenatesmay be frozenafteprreparation. 9.0 PROCEDURE 9.1 InitialSet-up 9.1.1 Setsoftwareto"Operateon",Ion Mode ES-. 9.1.2 Record backingpressureintheinstrumentlog. 9.1.3 Fillthesolventcylinderwithmobilephase. 9.1,4 Setthepump to"Run". Settheflowto 1000 uI-IrninO.bservesdroplets coming outof thetipof theprobe.The pressureshouldbe at 1700 to 1800 psi. 9.1.5 Check thefusedsilicaattheend of theprobe.Use an eye piecetocheck for chips.The tipshouldbe flatwithno jaggededges.Ifany chipsarefound cutoffthetipofthesilicwaitha column cutterand pullthesilictahroughto the appropriatleength. 9.1.6 Check your nitrogensupply.Turn on thenitrogenT.here shouldbe no nitrogenleakingAroundthetipof theprobe.A finemistshouldbe coming outof thetip. 9.1.7 Carefuhyguidetheprobeintotheopening.Insertituntilitwon't go any furtherC.onnect thevoltagecableto theprobe. 9. 1.8 Go tothe"Editor"page,and setIonizatioMnode toES-,and the appropriatemasses to427 and 499. 9.1.9 Ifitisnotinsingleion mode go to"Option"and setSIR. 9.1. IOStartAcquisitionA.ssigna filename, MO-DAY-YR + letterR.ecord itin thelogbook. 9. 1.11 Run thebeefliversamplesfirstr,unningeach standardtwiceatthe beginningof therun..Run a QC check by runningone standardafterevery 5 to7 samples. 9.2 Manual Injection 9.2. 1 Draw 150 uL of sample intoa syringeI.njeci-thseample intotherheodyne injectiopnort.Injectslowly.Record thesampleID inthelogbook. 9.2. 2 Turn thevalveto"On". 9.2.3 Wait two minutes,and injecthenextsample. 9.2.4 Record thescannumber foreach sample inthelogbook. -3 9.3 Using the Autosampler 9.3.1 Setup sampletrayA, B, or C. 9.3.2 Record thesamplesand theirpositionisntheinstrumentlog book. Up to 17 vialsmay be ineach run. 9.3.3 Set-upthesampler: 9.3.3.1 Push thesample'button 9.3.3.2 Setsample loop size= 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 9.3.3.7 Identiftyhetrayused Add thesamplestoQueue by prrssing"Enter-" 9.3.3.8 Press"Run" tostart 10-0 VALIDATION 10.1 QualityControl 10. 1.1Run a standardevery5 to7 samples.Ifa significacnhtange( 50%) in peak heightoccursstoptherun.Only thesamplesbeforethelastacceptable standardwillbe used.The remainingsampleswillbe reanalyzed. 10.2 Precisionand Accuracy 10.2. 1 See Method ValidationReportnumber AMDT-M-5-O.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 of thetwo valuesobtainedforeach standard. 11. 2.1Read peak heightsor areasforthesamplesfrom theprintoutU.se linear regressio,tnodeterminethesample concentrations. 11. 2.2 Calculatethemg of FC-95 anion,or otherfluorocheniicianlthetotalrabbit liver mg FC-95 anioninthetotalrabbitliver me FC-95 anionfrom std.curve gms of liverusedforanalysis x Totalmass ofliverg,ms 11.3 Make a -resultasbleand enteritin thestudybook. 11.4 Printa chromatogram foreach sample,withthepeaks labeledwiththesample or standardID.Write thestudynumber on theprintouti,nitiadla,te,and put itin the studyfolderS.tapleallchromatograrnstogetherand number pages. . 12.0 ATTACHMENTS None 13.0 REFERENCES 13.1 AMDT-EP- 17 1"4.0 REVISIONS Revision Num Reason forchangg Revision DAte 3M Environmental Laboratory Method Analysisof FluorideUsing the Skalar Segmented Flow Analyzer With Ion SelectiveElectrode Method IdentiricatioNnumber: AMDT-M-8 Adoption Date: RevisionNumber: 0 RevisionDate: None Author:Deb Wright CynthiaWeber Approved By: up Leader Date QualityAssurance Date Software:IBM MS Word, 6.0 AffectedDocuments: AMDT-EP-26, Operationand Maintenanceof theSkalarSegmented Flow Analyzer 1.0 SCOPE 1. 1 1.2 This method isfortheanalysisforfluoridet,hermallyextractedfrom samples using theDohrmann DX2000 (AMDT-M-1), and collectedinTISAB foranalysiswith an Ion SelectiveElectrode(ISE).The analysisisperformed usingtheSkalar Segmented Flow Analyzer with ISE. Samples can be tissuess,erum, biologicalmaterial,or othermaterialsextractedon theDohrmann. 2.0 KEYWORDS 2.1 Skalar,segmented flow,fluoride. 3.0 PRECAUTIONS 3.1 Follow standardlaboratorsyafetypractices. 4.0 SUPPLIES AND MATERIALS 4.1 Supplies 4.1.1 Sample cups,4 mL plasticups with caps 4.1.2 Autopipets,oxford orequivalentwith plastictips 4.1.3 Polypropylenevolumetricflasks,100 mL 4.1.4 Cartridgecomponents, refertotheSkalarMethods forcomponents and part numbers. 4.1.5 Sample prefilterEsv,ergreen 4.2 Reagents 4.2.1 Brij35, 30% S.F.A.S.Detergent 4.2.2 TISAB IIbuffersolutionP:urchaseTISAB R from Orion.To I liteorf TISAB IIadd 2.5mL or 100 ppm fluoridesolutionand 1 mL Brij. 4.2.3 Sampler rinsingsolutionD:iluteTISAB 111:1 with NElli-Qwater. 4.2.4 Ninic acidsolutiofnordecontamination,I N (labgrade):Slowly add 64 mLs concentratenditriaccid(HN03) to250 mLs of Milli-Qwater.Bring thevolume up to I L with Milli-Qwater. 4.3 Standards 4.3.1 Stock solution1,00 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 Worldng standard:Make up thefollowingworking standardsby adding the volumes ofintermediateor stockstandardindicatedon the table,using oxford orpumpmate pipets,to50 mLs of TISAB and dilutingto 100 n-J-s ithMilli-Qwater. Working Standard 0.015 ppm 0.03 ppm mLs of Stock Standard - - mLs of IntermediateStandard 0.15 0.3 0.06 ppm - 0.6 0.09 ppm - 0.9 0.12 ppm - 1.2 0.15 ppm - 1.5 0.3 ppm 0.3 - 0.6 ppm 0.6 1.2ppm 1.2 1.5ppm 1.5 5.0 EQUTPMENT 5.1 SkalarSegmented Flow Auto AnalyzerSansp'"System equippedwithISE 6.0 INTERFERENCES 6.1 High concentrationosf alkalinitcyh,loridep,hosphate,sulfateor ironcan cause interferences. 7.0 SAMPLE HANDLING 7.1 Samples shouldbe storedinpolyethylenebottlesS.amples shouldbe analyzed within30 days. 8.0 CALTBRATION AND STANDARDIZATION 8.1 Preparationof CalibrationStandards 8. 1.1 Preparecalibratiosntandardsas in section4.3. 8.2 Calibration 8.2. 1 The standardsareanalyzedatthebeginningof therun. 8.3 Storage Conditionsfor Standards 8. 3. 1 Standardsarestoredincapped polypropylenevolumetricflasksN.ew standardsarepreparedata minimum ofevery 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 thefluoridelectrodeisntheelectrodechamber. 9.1.3 Turn on thepower of thesampler,pumps, offsetpotentiometearnd headng bath. 9.1.4 Put thereagent-lineisn theappropriatebottles. 9.1.5 Turn on theinterfacec,omputer,displayand printerM.ake turn on the interfacebeforethe computer. 9.1.6 Letthesystemstabilifzoerapproximatel3y0 minutes. sure you 9.2 Startinga Run 9.2.1 Createa sample tableby selectinFgELES, TABLE, and CREATE, typein thename of thefilea,nd pressENTER. 9.2.2 Printthesample table,insertedin thesystem tableby pushingESC, PRINT, GROUP 1.This willprinttheentirerun. 9.2.3 Dialthesamplersettingtso theappropriatenumber of samples,number of secondsforsample wash, and number of !@econdsforthesample. 9.2.4 Fillthesample traywiththestandardss,amples,washes and driftsI.W and FW/RUNOUT cups on thesamplerdo notneed tobe filled. 9.2.5 Set thebaseline. 9.2.5. 1SelectGRAPHICS, REAL TIME. Ifyou cannotgetreal-timey,ou may be intheData HandlingPanel.Switch totheAnalysisPanelby selectinCgONTROL PANEL and pushingF7. 9.2.5.2 Use thesmallscrewdriverfortheoffsetpotentiometertosetthe baseline.Adjustthebaselineuntilitisapproximately3/4 inch from thebottom of thescreen. 9.2.5.3Check thehigheststandardand adjustthegain,ifnecessaryw,ith theinterfacsecrew #3. 9.2.6 Go toCONTROL PANEL, and toanalysipsanel.Deselecttheanalysisthat willnotbe run.(Selector deselectanalysisby pressingENTER.) PressTab toreturntotheAnalysisPanel. 9.2.7 Pressthespacebartobringup thelocalmenu. 9.2.8 SelectSTART tostartheanalysis. 9.2.9 Type yourID (initialtsh)e,sampletablewhich you createdunder9.2.1(or pressENTER forchoices)c,hooserunningwithor withoutthesystemtable and selecStTART ANALYSIS. 9.2. 10 Afterstartintghesoftware,starthesampler.Make surethatthesampleris settotherightnumber of samplesand thatthesample/wash/airtimesare 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. SelectAUTOCALCULATION and enterthefilename(or highlightthefileto be printedand pressENTER). 9.3.2 To view thecalibratiocnurve,go toGRAPHICS, CALIBRATION CURVE. 9.3.3 To printthehighlevelcurve,push PRINT SCREEN. 9.3.4 To printthelow levelscreen,push ESC togetoutof graphicsS.elect SETTINGS. Change themax y valuetoapproximately900.Go to CAL CURVE and pressESC, and Enter.PressPRINT SCREEN. 9.3.5 ReturntoSETTINGS and change themax valueback to4095,go toEDIT, pressENTER and PRINT SCREEN toprintsample peaks. 9.3.6 To printtheresultsgo toCONTROL PANEL, SPACEBAR, OUTPUT, OUTPUT. SelectPRINTER fortheEpson or PRN fortheLaser. 9.4 Shutdown 9.4.1 Put allthereagent-liniensMilli-Qwater. 9.4.2 Let thesystemrinseforapproximately30 minutes. 9.4.3 Afterthesystem hasrinsedcompletely,turnoffthesampler,pump and offsetpotentiometerT.urn offtheheatingbathon weekends.Leave liquidin the lines. 9.4.4 Take theelectrodoeut and soak in 100 ppm F overnight. 9.4.5 Releasethepump-decks, airbarsand samplerpump-tubing. 9.4.6 SelectFILES, pressALT F and selectQUIT toexittheproamm. 9.4.7 On Friday,turnoffthecomputer,displayand interfacfeortheweekend. 10.0 VALIDATION 10.1 Quality Control 10. 1.IRun a standard(mid tohigh concentratione)very 10 samples.Ifa significancthange inpeak heightoccurs,only thesamplesbeforethelast acceptablestandardwillbe used.The remainingsampleswillbe reanalyzed. 10.2 Precisioannd Accuracy 10.2.1SeeMethodValidatiRoenporntumberAMDT-M-8.0.VI 10.3 Other ValidationParameters 10.4 RefertoMethod ValidatioRneportNumber AMDT-M-8.0.Vl 11.0 DATA ANALYSIS 11.1 11.2 11.3 11.4 Calculations 11. 1.1 The standardcurve isplottedby theS kalarsoftware. 11. 1.2 Allcalculationasredone by theSkalarsoftware.r2 shouldbe 0.995 or better. Preparespreadsheetstosummarize data.Includesample volume, weightsused etc. Write thestudynumber on theprintoutsi,nitiadla,tetheprintouta,nd bind together withallpackage documents and placeinthestudyfolderM.ake a copy of the summary sheetand tapeintothestudynotebook.Back up alldataand spreadsheets onto studydiskand backup disks. ElectroniDcata 11.4. 1GLP studiesE:lectronicdataiscopiedonto theStudy floppydiskforeach study,and alsodataiscopiedontoa floppydiskthatisstoredinthelab. 11. 4.2 Other studiesA:lldataiscopiedontoa floppydiskthatisstoredin thelab. 12.0 ATTACHMENTS None 13.0 REFERENCES 13. 1 13.2 13.3 AMDT-M- 1,Thermal ExtractioonfFluorideby Means ofa ModifiedDohrmann DX2000 OrganicHalideAnalyzer-Liver SkalarMethods,#335,SkalarMethods Manual AMDT-EP-26, Operationand Maintenanceof theSkal,aSregmented Flow Analyzer 14.0 REVISTONS Revision NumbC Reason forchan Revision Date 3M EnvironmentalLaboratory Method TherznalExtractionofFluorideby Means ofa ModifiedDohrmann DX2000 Organic HalideAnalyzer- Serum Method IdentificatNiuomnber: AMDT-M-14 RevisioNnumber: 0 AdoptionDate: /0 -3 -1 r RevisionDate: None Author RichYoungblom Approved by: 60"4.-1 c:@F up Leader Date QualityAssurance z -;;?Date Software:MS Word 5.la AffectedDocuments: AMDT-M-2 FluoridMeeasurementby Means ofan OrionEA940 ExpandableIon Analyzer AMDT-EP-3 RoutineMaintenanceofa ModifiedDohrinannDX2000 OrganicHalideAnalyzer 1.0 SCOPE .APPLICABLE COMPOUNDS, AND MA RICES 1.1Scope:Thismethod isfortheoperatioonfa DohrrnannDX2000 when itisusedtoextract fluoridferom variousmatrices.The fluoridiestypicallcyollecteidnTISAB solutiofnoranalysis withan ionselectiveelectrode. 1.2ApplicableCompounds: Fluorocheniicaolrsotherfluorinatecdompounds. 1.3Matrices:Biologicaflllddsp,articularsleynun. 2.0 KEYWORDS 2.1Fluoridef,luorinee,xtractiopny,rolysisi,onizatioino,nselectiveelectrodeD,ohrmann, halide, DX2000, fluorocherr@dcals. 3.0 PRECAUTIONS 3.1 Glasswareand exhaustgasescan be extremelyhot. 3.2Glasswareisfragileb,rokenglassmay causeinjuries. 3.3Pressurizedgases,propercompressedgas handlingpracticersequired. 3.4Solventbasedsamplesmay flashm,ay need toallowthem todrydown beforestartinngm. 3.5Potentiabliohazardsduetothebiologicamlatrices.Use appropriatpeersonalprotective equipment. 4.0 SU-PPLIES AND MATERIALS 4.1Compressed Oxygen, Hydrocarbonfree,regulatetdo30 PSI. 4.2Compressed Helium,High PurityGrade,regulatetdo45 PSI. 4.3Quartzglasssample boatwithTeflonTmtubing,Dohrmann 890-097orequivalent. 4.4Quartzglasscombustiontube,RelianceGlass G-9405-012 orequivalent. 4.5Orion940999TotalIonicStrengtAhdjustmentBuffer(TISAB II)orequivalent. 4.6Sample collectiovnialsH,DPE. 4.7Nfilli-QTwmater 4.8Polystyrenpeipettes. 4.9ActivatedCharcoalE,.Merck 2005 orequivalent. 4.10 Hamilton Syringeor equivalent. 4.11 Miscellaneouslaboratorgylassware 5.0 EQLZTPMENT 5.1Rosemount Dohnnann DX2000 OrganicHalideAnalyzer,modifiedforfluorideextraction. 5.2MM compatible386 or486 computer. 5.3DX2000 softwarev,ersion1.00,modifiedforfluorideextraction. 5.4Excel Spreadsheetv,ersion5.0or greater 6.0 INTERFERENCES 6.1 Sample sizeislimitedtoapproximatel1y00 @Ll.Thismay varyfrom matrixtomatrix. 2 7.0 SAMPLE HANDLING 7.1Samples aretobe handledwithplastipcipettes.A new pipetteistobe used foreach sample. 8.0 CALIBRATION AND STANDARDTZATION S.iPreparatioonfCalibratioSntandards 8.1.1The standardsrequiredforeachprojectwillneed tobe appropriatfeorthatindividuaplroject. Refertoprotocolforthatproject. 8.1.2Typically50-500 ppm FC-95 inmethanol standardsareused. 8.1.3For rabbitserum studiesu,se beef serum as.therhatrix. 8.2 Calibration-Overview The normal calibratioinsthefluoridceurve(AMDT-M-2). However, ifan optionalspikedserum curveisrequiredtheprocedurelistebdelow isused. 8.2.1A calibratiocnurvefortheDX2000 isgeneratedby spilingsampleswithknown standards and combusting them usingthesame methods and matrixtype asthesamplesto be tested. 8.2.2Typically,threereplicateosf eachstandardand fiveconcentrationosf standardswillbe spiked. 8.2.3Standardcurvewillbe plottedas Mass SpikedF (ug)on thex-axisand StandardMass Recovered F (ug)on they-axis.Generatea regressioncurveand calculatteheequationfortheline and ther2 value. 8.2.4Mass SpikedF (ug)= (Amount spikedinmL) x (Conc.ofstandardinppm) x (0.6004)* *FC-95 is60.04% F therefor0e.6004 isthefactorusedto convertFC-95 toF 8.2.5StandardMass RecoveredF (ug)= (TISAB volume inmL) x (Orionreadinginppm) 8.3Calibration- Procedure 8.3.1StartUp 8.3.1.1Run 2 ormore CleanCycleswhen startinignstrumenetachday. More cleancyclesmay be usediftheprevioussamplescontainedhigh concentrationosf fluoride. 8.3.2Blanks 8.3.2.1Preparesample usingthesame methods and type of matrixas thetestsample. 83.2.2 For rabbitstudiesu,se beefserum as thematrix. 8.3.2.3Put serum blank-inDohrinannboat. Combust sample as describedin section9.0and analyzesample accordingtomethod AMDT-M-2 fortheionselectiveelectrodaenalysis. 8.3.2.4For rabbitstudiest,hemeter readingfora blank sample showd be 0.03ppm or lower beforeproceedingwiththecalibrationB.um samplesuntiltMs lin@tisreached,or untilinthe judgement of theoperatorthereadingisstablewithrespecttohistoricarleadings(previous48 hours). 8.3.2.5For non-rabbitstudiest,heblankreadingsshouldreacha predetem-@nedionconcentration beforeproceedingwith thecalibration. 8.3.2.6Itmay be necessarytomix approximately50 mg of charcoalwiththesample to aid combustion. 3 8.3.3Standard Curve 8.3.3.1Ifbeefserum isfrozen,thaw atleastenough tocompletethestandardcurve analysisforthe day (-30mL). 8.3.3.2 Pipette100@LLofbeefserum intoDohrmann sample boat. 8.3.3.3StartwiththeloweststandardconcentrationU.sing a Hamilton syringe,ejecta fixed quantityof thestandardon orin thematrix.For rabbitstudiesu,se 4 uL of standardand ejectiton or inthebeefserum. 8.3.3.4At least3 replicatesshouldbe used forthelowest standardconcentrationm;ore replicates may be used atthediscretioonf theanalyst. 8.3.3.5Combust thesampleas 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 tocalculatteheregressioncurve. 8.3.3.7When allstandardshave been run,calculattehe r2.r2 must be atleast0.95.Ifitisnot at least0.95,consultwith supervisor. 8.3.3.8A new standardcurveshouldbe run when thecombustion tubeor sample matrixis changed. New standardcurvemay alsobe run atthediscretioonf theanalyst. 8.4 StorageConditionsforStandards 8.4.1Storagerequirementsforstandardsaredependenton theindividuasltandardsused. Typically,standardsarestoredatroom temperatureinplastisccrew top bottles. 8.4.2New FC-95 standardshouldbe 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/D.ryingtime 240 seconds. 9.1.4Bake time= 300 seconds. 9.2 StartUp Procedure: 9.2.1Iftheprogram isnotstarteds,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. 9.2.5)When theoven has reachedtheREADY 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 pipetteIOOGL ofsampleintotheBOAT. Itmay be necessarytomix approximately50 mg of charcoalwiththesampleto aidcombustion.Ifthisis done,charcoalshouldalsobe mixed inwhileestablishintghebaselineand when generatingthe standardcurve. 9.3.2Close SAMPLE HATCH. .4 9.3.3Add appropriatveolume ofTISAB solutioonr 1:1TISAB:Milli-QTM watermixturetoa labeledsample collectiovnia].Typically0.6mL to 15 mL areused. For rabbitstudiesu,se 1.0or 2.0mL of 1:1TISAB:Milli-QTM watern-dxture. 9.3.4Placethevia]so thatthetipof theCOMBUSTION TUBE isintheTISAB atleast0.25 inches.Gases releaseduringpyrolysismust bubblethroughtheTISAB. 9.3.5Run theEOX-WATER program foundintheRUN menu. 9.3.6When theEOX program isfinishedr,emove thecollectiovnialfrom thecombustiontube. 9.3.7IfundilutedTISAB was used tocollecthesample,add an equalvolume of Milli-QTmwater totheTISAB tomake 1:1 TISAB:Milli-QTm. 9.3.8Rinse theend of thecombustiontubewithMilli-QTmwaterand wipe witha.KIMWIPE to remove any TISAB remainingon thetube. 9.3.9Open thesample hatchand remove any remainingashfrom theboat. Ash can be removed witha cottontippedapplicatoarnd/orvacuumed out.Itmay be necessarytoscrapparticleosffthe bottom witha spatulaor othersimilardevice.A drop of Milli-QTmwater may be added to the boattoaidintheClean Cycle. 9.3.10Close thehatch. 9.3.11Run theCLEAN BOAT program. 9.3.12Sample isreadyforanalysibsy ionselectiveelectrod(eANDT-M-2). 9.4 Sample Calculations 9.4.1Use thestandardcurve tocalculattehesample value. 9.4.2SainpleMass Recovered F (ug)= (TISAB volinniL)x LOrionreadinginRp_m- intercot) (Slope) 10.0 VALIDATION 10.1 QualityControl 10.1.1DailyStartUp Check Samples:Once thestandarcdurveisestablisheeda,chday of analysisisstartedby analyzingQC samples.The QC samples aretobe thesame as thelowest concentratiosnpikedsamplesused togeneratethestandardcurve.Each concentratiomnust be done intriplicautnelessthefirstwo replicateasrewithin20% of thestandardcurve,thena third replicatiesnot necessary. 10.2 Precisionand Accuracy: See method developmentanalysisand 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,useregressioannalysiisnExcel,version5.0orgreater. 11.1.2To calculattehefluoridecontractioinnthesample,seemethod AMDT-M-2. 5 11.2AnalyzintgheData 11.2.r12mustbeatleas0t.95orgreate"rO.utliermsa"y beexcludeidftwoofthethrereeplicates arewithin20% of eachotherand theoutlieirsgreaterthan200% oftheaverageof thosetwo or lessthan50% of theaverageof thosetwo. Any such outliersshouldbe pointedoutinthedataand notedintheFinalReport alongwiththereasonitwas 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 ofan Orion EA940 ExpandableIon Analyzer 13.3AMDT-EP-3 RoutineMaintenanceofa ModifiedDohrmann DX2000 OrganicHalide Analyzer 14.0 REVISTONS Revision Numbe Reason forChange Revision Date 6 9.1.3 Amendment toAnalyticalProtocolAMDT-020795.1 I AttachmentI GLP Study ProtocolAmendment StudyNumber- AMDT-020795.1 Study Tide: Single-DoseDermal Absorption/ToxicityStudyofT-6052 inRabbits Study Director James D. Johnson Amendment Date: November 8,1995 Amendment Number: I This amendment modifiesthefollowingportionoftheprotocol: AMDT-M- 14-0specifieussingbovinesenun fortheblanksinthethermalextractionH.owever, rabbitserum from thecontrolanimals (AMDT-1 10394.1)was used becausethebovinesenan blankswere higherthanthesamples. Appmved by: dy Director liate 9.3 QualityAssurance Unit Statement I I - A=hmntD GLP Study QualityAssurance Statement 04 Study Tide: Single-doseDermal Absorption/ToxicitSytudy of T-6052 in Rabbits StudyNumber: AMDT-020795.1 Name ofAuditor KariRambo M studyhas been inspectedby theQualityAssuranceUnit asindicateidnthefollowingtable. The findingswere reportedtothestudydirectorand management. InspectioDnates Fj= IQ 10/13/95 10/19/95 Phase FinalReport Date InspectioRneportedto Manag=nt StudyDirector 10/19/95 10/19/95 QAU Auditor Date 9.4 Key PersonnelInvolvedinthe Study 3M Environmental Laboratory Key Personnel Thermal extractiofnollowedby analysiussingOrionIonanalyzer: Jim Johnson Deb Wright RichYoungblom Deann Plununer Analysisofliverextractussingelectrospramyass spectrometry: Jim Johnson Dave Christenson Thermal extractiofnollowedby analysiussingSkalarsegmentedflow analyzerwithIonselectiveelectrode: Jim Johnson ..DebWright Rich Youngblom Deann Plummer Documentationand Reporting: Jim Johnson Rich Youngbiom QualityAssuranceUnit: GaleVan Buskirk CynthiaWeber KariRambo . 9.11Data 9.11.1Summary and raw data; ug F- in whole liveras determinedby thermalextractiofnollowedby analysis using,Orionion analyzer. Summary ofCombustion Data - Liver AMDT-020795.1, HWI 6329-135 As ReferencedinFinalReport section6.0DATA ANALYSIS Totalug FluorideinWhole Liver -Mean perDose Group** ug Std.Dev. ControlGroup 16.7+ 5.9 2.0mg/kg dose(T6052) 12.6 +3.2 200 mglkg dose(T6052) 18.3* +4.2 1000 mg/kg dose(T6052) 24.1 +4.2 Calculatedasthemean oftriplicastaemplesfrom eachofthreemale and threefemalerabbits. One outlieorniittedV.alue ofre-analyzesdampleincludedinthisreporl RPTI 35L.XLS FC120 AB ID LiverBik-I LiverBlk-2 LiverSpk-I LiverSpk-2 LiverSpk-3 LiverSpk-4 LiverSpk-5 LiverSpk-8 Liverblank-3 Liverblank-4 F52972-1 F52972-2 F52972-3 F52973-1 F52973-2 F52973-3 F52979-1 F52979-2 F52979-3 LiverBlank-1 LiverBlank-2 LiverSpike-1 LiverSpike-2 LiverSpike-3 LiverSpike-4 LiverBlank-A LiverSpike-5 LiverSpike-6 LiverSpike-7 F52975-1 F52975-2 F52975-3 F52976-1 F52976-2 F52976-3 F52983-1 F52983-2 F52983-3 F52986-1 F52986-2 F52986-3 F52990-1 F52990-2 F52990-3 F52997-1 F52997-2 F52997-3 % rcvry 80% 90% 92% 94% 108% 94% 80% 89% 79% 77% 88% 90% 92% Actual ppm Finliver (W/W) 0.133 0.108 1.10 0.980 1.06 2.72 3.24 2.43 0.393 0.320 0.360 0.317 0.315 0.240 0.243 0.269 0.214 0.171 0.166 0.276 0.131 0.864 1.35 0.863 0.965 0.000 1.02 0.993 1.04 o.ie5 0.135 0.116 0.135 0.125 0.294 0.153 0.151 0.132 0.149 0.147 0.145 0.162 0.196 0.195 0.220 0.243 0.123 Average ppm Finliver (WNV) 0.331 0.251 0.183 0.138 0.185 0.145 0.147 0.184 0.195 Whole TotalF- in liver liver burned weight whole liver (grams) (grams) 0.112 (ug) 0.124 0.110 0.139 0.132 0.105 0.101 0.117 0.119 0.121 0.107 82.5 0.103 82.5 27.3 0.132 a2.5 0.148 75.8 0.150 75.8 19.0 0.131 75.8 0.119 90.6 0.148 90.6 18.6 0.133 90.6 0.114 0.135 0.141 0.100 0.138 0.121 0.132 0.130 0.138 0.134 0.136 86.4 0.135 86.4 12.0 0.139 86.4 0.150 60.8 0.141 60.8 11.2 0.127 60.8 0.134 96.5 0.116 96.5 14.0 0.128 96.5 0.125 82.8 0.108 82.8 12.2 0.135 82.8 0.145 80.4 0.120 80.4 14.8 0.107 80.4 0.114 91.7 0.116 91.7 17.9 0.150 91.7 Dosage (mg/kg) 0.0 0.0 0.0 0.0 0.0 0.0 2.0 2.0 2.0 Page I RPT135L.XLS FCI 20 AB ID F52982-1 F52982-2 F52982-3 F52994-1 F52994-2 F52994-3 F5341 0-1 F53410-2 F53410-3 Liver Blank-1 LiverBlank-2 LiverSpike-I LiverSpike-2 LiverSpike-3 LiverSpike-4 LiverSpike-5 LiverSpike-6 LiverSpike-7 F52984-1 F52984-2 F52984-3 F52992-1 F52992-2 F52992-3 F52996-1 F52996-2 F52996-3 F52977-1 F52977-2 F52977-3 F52989-1 F52989-2 F52989-3 F52993-1 F52993-2 F52993-3 liverblank-I liverspike-I liverspike-2 liverspike-3 liverspike-4 liverspike-5 liverspike-6 liverspike-7 liverspike-8 liverspike-9 liverspike-10 % rcvry 65% 65% 71% 85% 96% 90% 80% 72% 75% 67% 77% 77% 88% 152% 81% 88% 88% Actual ppm Fin liver (WNV) 0.136 0.153 0.113 0.121 .0.105 0.113 0.114 0.130 0.102 0.207 0.130 0.911 0.747 0.751 1.02 1.13 1.04 0.946 0.216 0.224 0.180 0.173 0.205 0.247 0.843 0.272 2.07 0.207 0.180 0.173 0.217 0.249 0.192 0.174 0.199 0.153 0.312 0.741 0.928 0.670 0.826 0.934 1.27 1.52 0.829 1.32 1.12 Average ppm Finliver (W/W) 0.134 0.113 0.115 0.200 0.209 1.06 0.187 0.219 0.175 liver burned (grams) 0.147 0.105 0.116 0.130 0.151 0.148 0.129 0.119 0.146 0.106 0.102 0.108 0.131 0.144 0.125 0.129 0.131 0.128 0.112 0.104 0.131 0.121 0.115 0.136 0.134 0.115 0.142 0.140 0.126 0.128 0.106 0.134 0.13S 0.141 0.129 0.147 0.148 0.148 0.123 0.151 0.141 0.124 0.105 0.151 0.148 0.101 0.118 Whole liver weight (grams) 82.4 82.4 82.4 82.9 82.9 82.9 91.0 91.0 91.0 88.5 88.5 88.5 80.6 80.6 80.6 77.8 77.8 77.8 72.0 72.0 72.0 97.9 97.9 97.9 88.3 88.3 88.3 TotalF- in whole liver (ug) 11.0 9.37 10.5 17.7 16.8 82.5* 13.5 21.5 15.5 Dosage (mg/kg) 2.0 2.0 2.0 200 200 *Sample 200 reanalyzed below 200 200 200 Page 2 RPT135L.XLS FC120 AS lo F52996-1 F52996-2 F52996-3 F52978-1 F52978-2 F52978-3 Blank liver1 Blank liver2 Blank liver3 Blank liver4 Blank liver5 Blank liver6 LiverSpike-I Liver Sp@ke-2 Uver Spike-3 LiverSpike-4 Uver Spike-5 F52980-1 F52980-2 F52980-3 F52991-1 F52991-2 F52991-3 F52987-1 F52987-2 F52987-3 F52988-1 F52988-2 F52988-3 F52995-1 F52995-2 F52995-3 liverblank-I liverspike-1 liverspike-2 liverspike-3 liverspike-4 liverspike-5 liverspike-6 liverspike-7 liverblank-2 % rcvry 130% 84% 87% 82% 77% 72% 81% 82% 77% 83% 84% 81% Actual ppm Finliver (WNV) 0.291 0.244 0.422 0.259 0.224 0.225 0.411 4.45 2.01 0.340 0.277 0.404 1.37 1.09 0.831 0.955 0.784 0.289 0.235 0.231 0.330 0.270 0.238 0.204 0.604 0.230 0.249 0.490 0.340 0.453 0.315 0.266 0.168 0.862 0.820 0.847 0.905 2.03 1.89 1.75 0.268 Average ppm Finliver (WNV) 0.319 0.236 0.252 0.279 0.346 0.360 0.345 river burned (grams) 0.136 0.141 0.103 0.148 0.141 0.143 0.129 0.146 0.149 0.137 0.145 0.114 0.143 0.117 0.158 0.130 0.150 0.133 0.148 0.158 0.134 0.147 0.120 0.122 0.150 0.150 0.118 0.138 0.109 0.147 0.136 0.113 0.146 0.126 0.150 0.146 0.129 0.123 0.135 0.141 0.131 Whole liver weight (grams) 77.8 77.8 77.8 83.2 83.2 83.2 82.0 82.0 82.0 88.9 86.9 86.9 81.2 81.2 81.2 83.2 83.2 83.2 63.1 63.1 63.1 TotalF- in whole liver (ug) 24.8** 19.6 20.7 24.3 28.0 29.9 21.8 Dosage (mg/kg) 200 1000 1000 1000 1000 1000 1000 **Repeat ofabove analysis Page 3 9.11.2Summary and raw data;analysisof liver extractsusingelectrospraymass spectrometry. e HM 6329-136 04- c- S": ProtocolNumber. TestMaterial: Matrix: R Squared Value: Rmponse FactorAmount: Armlyst: Date: Method: Instrument: LABOAS,E File: A-.ju\ Group DO" Sample Group 1: 0 mg/kg Group 2: 2 mg /kg Group 3: 200 mwkg Group 4: 1000 mg/kg F52975 F52976 F52983 F52972 F52973 F52979 F52986 F52990 F52997 F52982 F52994 F53410 F52984 F52992 F52996 F52977 F52989 F52993 F52978 F52980 F52991 F52987 F52988 F52995 Single-DoseDermalAbsorption TP3016.AB T-6052InRabbits(FC-120) Uver Screening N/A DLC 4/SM FisonsVG 2000 ElectrospraMyS 040695B 4AAP-e.-@AD &4"- M- S9i IonCount Extractewdt Area 9 Dilution ConcentrationTotalnum factor W9 ofliver 9 N.D. re-extract re-extract N.D. N.D. N.D. N.D. N.D. re-extract N.D. N.D. N.D. $ $ re-extract $ N.D. N.D. $ $ $ $ $ re-extract Totalamount of FC-95per liver rng % of FC-95 SIR MonitorinogfM199 and 598. 3 PositivreesponseforIonmonitored. Administeredata dose volumeof2.0mL/kg. Administereadta dose volume of0.01mukg. ****Theconcentratiownas calculatebdyusingthestandardcurveand multiplyintgheresultby4/5.The 4/5 factoIrstheresulotfa miscalculatiIonnapplyingformula8.4InMethod AMDT-M-4-0. 137 mg ofUverwas used In thiscalculatonratherthan 171 mg. The concentradonsIntheStandardcurvearetherefor5e/4Largetrhanthey shouldbe. By multiplyintghecalculatecdoncentratioinnthestandardcurveby 4/5,thecomectresulItsobtained. r4 6^Ar'$,^ ,,wo "Na- I.C-ltat File:040695B Sample:HWI 040695B 6329-135; 40 ZFS- 22 LAB-BASE COMPOUND 10 r4 LL 52 v- U) IL 71 - The MS Data Sgstem FC-120 (AB) [M- 5991 itL 8 ri 00 T N 0 LL 9 JL i tL kr ID %r tv m IP kr 13 8 L 1194 LosZFS' JL a 3 JL4@ 8 9 @7 7 8@8 31 9 8-18 JLa 1099 248 0, inin 5.9 10.6 15'.9 2@i. 0 FIO NOT DEFINED File:040695C Sample:HWI 040695C 6329-135; ri LAE-BASE - The MS Data Sgsten COMPOUND FC-120 (AB) EM- 5991 rn -r v- V.FS rr (r t%J In 21 to on tr u- 0- r-i ILL U- LL 9 T 7 7 8 69 7 54 in tL 14 IL 13 6 I@@- 11 3 16 4 ZFSImin 86 :3 !5 26.0 FIO NOT DEFINED LAB &Nm F-ILE 0140-3q5e' 3 2q - 135' /.T5 NI-etklc),D-LlCLIV Sainple DLCL'IV 0[-)erator Dl,(-Run d..ate 1)5-08-1,)95 11:39:38 Vei:zic.)ti: Printetl on 05-08-1995 A'r 11:39:58 StraitjiitLine Fit f(-)rcedthrougli Ori,-jjn. --------------------------------- jp,,+ . r 3 iL -,EVEL ----- 1 2 3 4 5 6 7 AMOUNT ----------- 0.4000 0.8000 1.2000 1.61)00 4.0000 8.()000 12.0000 Component EXTERNAL STANDARD AREA --------- 31156 77583 83946 160773 166388 449788 565003 1 CALIBRATION Y SLOPE x + INTERCEPT --------------------------------------------------- Are,:k Amount R s(quared 5.0159E+04 1.993-E.-05 = 0.9467 Amourt Area + o.0000E+00 + 0.0i)OUE+00 9.11.3Summary and raw data;ppm F-in serum as determinedby thermalextractionfollowedby analysisusing Orion ion analyzer. This data,althoughsupportive,intheopinionof the Study Directorisnot requiredto reachtheconclusionstatedin FinalReport Section6.0,and thereforeisnot discussedin detail. HWI 6329-135 AMDT 011095.1 Dohrmann Serum Analysis AnalysisDates:08/02/95-08/18/95 All serum sampleswere thermallyextractedby a modifiedDohrrnann DX2000 OrganicHalideAnalyzerand collectedina 1:1 milliQ waterand TISAB solution.'Me sampleswere measured on an Orion EA940 expandableion analyzer.The Dohrrnann was calibrateudsing34ppm, 40ppm, 62ppm, 100ppm, 124ppm, 250ppm, and 500ppm FC-95 standards forserum curve 1. The same FC-95 standardswere used tocalibrattehe Dohimann with theexceptionof250ppm and 500ppm forserum curve2. Ile Orion was calibratebdy directmeasurement with no blank correction using0.05ppm,O.lppm,0.5ppm, l.Oppm and 1.5ppm F-standards.Ile slope,intercepta,nd correlatiownere recordedinthe appropriatleogbook. A summary tableisincluded,showing theppm F- ineach sample (seepage 2).An initiaclalibratiocnurvewithstandarddeviation%,RSD, R 2 value and equationof thelineison pages 8 - 11. Pages 3 -7 show theexcelspreadsheetthatwas generatedwhen thesamples were being analyzed.The Dohrmann FC-95 initiaclalibratiocnurve was not used togeneratethedatainthe spreadsheet.Any Orion readingbelow 0.05 isbelow theOrion calibratioannd shouldbe consideredan estimate. The FC-95 initiaclalibratiocnurve 1 was spikedintobovineserum. Bovine serum was used asblanksand QC check samples on 08/02/95and 08/03/95. However, due toproblems withblankshavinghigherreadings thanthesamples,serum curve2 was analyzedusingrabbitserum from study# 6329-123 rabbit# F52346. On days 08/15/95- 08/18/95rabbit serum from study# 6329-123 rabbitsF52346, F52335, and F52332, were used forblanksand QC check samples We were unabletolocatedserum samplesforDay 2 throughDay 4. This studywas discontinuedafterday 8 becausenothingwas found in thefirst day of sampling. Page 1 of 11 STUDY # 6329-135 SERUM FC 120 AB HWI 6329-135 Fluoridceoncentratioinnrabbisterum (ppm F-) Group I Dosage: 0 mglkg Sample F52972 F52973 F52979 F52975 F52976 F52983 Day I 0.533 0.498 0.551 0.517 0.479 0.506 Day 8 0.311 0.178 0.490 0.436 0.323 0.286 Day 15 0.553 0.859 0.612 0.674 0.501 0.503 Day 22 0.582 0.712 0.561 0.524 0.472 0.677 Group 2 Dosage: 2 mglkg Sample F52986 F52990 F52997 F52982 F52994 F53410 Day 1 1.45 0.650 0.444 0.466 0.446 0.388 Day 8 0.338 0.277 0.305 0.283 1.27 0.697 Day 15 0.393 0.463 0.36 0.298 0.346 0.354 Day 22 0.610 0.466 0.529 0.438 0.620 0.677 Group 3 Dosage: 200 mglkg Sample F52984 F52992 F52996 F52977 F52989 F52993 Day I 0.594 0.550 0.568 0.602 0.642 0.642 Day 8 0.694 0.526 0.845 1.10 1.57 0.612 Day 15 0.271 0.281 0.257 0.274 0.641 0.777 Day 22 0.620 0.636 0.712 0.685 0.814 0.580 Group 4 Dosage: 1000 mgikg Sample F52978 F52980 F52991 F52987 F52988 Day I 0.762 1.01 1.07 0.886 0.549 0.491 Day 8 0.449 0.566 0.709 0.630 0.478 0.513 Day 15 0.777 0.807 0.752 0.700 0.668 0.57 Day 22 0.449 0.516 0.581 0.335 0.249 0.275 STUDY 6329-135 SERUM Sample ID BLANK BLANK BLANK BLANK BLANK 62-PPM-1 62-PPM-2 250-PPM-1 250-PPM-2 BLANK BLANK BLANK F52986-DAY1 F52990-DAYI F52997-DAY1 F52982-DAYI F52994-DAY1 F53410-DAYI 62-PPM-1 250-PPM-1 SERUM BLANK-1 SERUM BLANK-2 SERUM BLANK-3 SERUM BLANK-4 SERUM BLANK-5 SERUM BLANK-6 SERUM BLANK-7 SERUM BLANK-8 SERUM BLANK-9 SERUM BLANK-10 SERUM BLANK-11 SERUM BLANK-12 SERUM BLANK-13 SERUM BLANK-14 SERUM BLANK-15 SERUM BLANK-16 SERUM BLANK-17 SERUM BLANK-1 8 SERUM BLANK-19 SERUM BLANK-20 62PPM-1 62PPM-2 62PPM-3 250PPM-1 250PPM-2 F52984DAY1 Actual reading (ppm F-) 0.0613 0.0396 0.0383 0.0412 0.0319 0.0786 0.0934 0.304 0.269 0.0903 0.0527 0.0326 0.0723 0.0325 0.0222 0.0233 0.0223 0.0194 0.0760 0.246 0.207 0.176 0.107 0.0842 0.0745 0.0856 0.0755 0.0691 0.0615 0.0657 0.0592 0.0672 0.0703 0.0674 0.0655 0.0507 0.0434 0.0403 0.0398 0.0375 0.0969 0.0902 0.0869 0.262 0.270 0.0297 Sample TISAB mL FC95 Conc. FC95 Qty finavlol spiked solution (mL or g) (mL) (ppm) 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.004 62 0.004 62 0.004 250 0.004 250 0.004 62 0.004 250 0.004 62 0.004 62 0.004 62 0.004 250 0.004 250 % Actual recovery ppm F- (ug/ug) insample 1.23 0.792 0.766 0.824 0.638 106% 1.57 125% 1.87 101% 90% 6.08 5.38 1.81 1.05 0.652 1.45 0.650 0.444 0.466 0.446 0.388 102% 82% 1.52 4.92 4.13 3.52 2.15 1.68 1.49 1.71 1.51 1.38 1.23 1.31 1.18 1.34 1.41 1.35 1.31 1.01 0.868 0.806 0.796 0.751 -130% 1-21% 117% 87% 90% 1.94 1.80 1.74 5.24 5.40 0.594 Mass spiked (ug F-) 0.149 0.149 0.600 0.600 0.149 0.600 0.149 0.149 0.149 0.600 0.600 Mass recovered (ug F-) 0.123 0.0792 0.0766 0.0824 0.0638 0.157 0.187 0.608 0.538 0.181 0.105 0.0652 0.145 0.0650 0.0444 0.0466 0.0446 0.0388 0.152 0.492 0.413 0.352 0.215 0.168 0.149 0.171 0.151 0.138 0.123 0.131 0.118 0.134 0.141 0.135 0.131 0.101 0.0868 0.0806 0.0796 0.0751 0.194 0.180 0.174 0.524 0.540 0.0594 STUDY 6329-135 SERUM Sample ID F52992DAYI F52996DAYI F52977DAYI F52989DAYI F52993DAY1 F52978DAYI F529BODAY1 F52991 DAYI F52987DAYI 62PPM-1 25OPPM-1 .8/15/95 Blank Serum-1 Blank Serum-2 Blank Serum-3 F52988-dayl F52995-dayl F52972-dayl F52973-dayl F52979-dayl F52975-dayl F52976-dayl F52983-dayl 40ppm-1 looppm-1 serum blank serum blank serum blank spike 34-1 spike 34-2 spike iOO-l spike 100-2 spike 100-3 F52972 DAY 8 F52973 DAY 8 F52979 DAY 8 F52975 DAY 8 F52976 DAY 8 F52983 DAY 8 F52986 DAY 8 F52990 DAY 8 F52997 DAY 8 F52982 DAY 8 SPIKE 62-1 SPIKE 62-2 SPIKE124-1 Actual reading (ppm F-) 0.0275 0.0284 0.0301 0.0321 0.0321 0.0381 0.0507 0.0533 0.0443 0.089G 0.264 Sample TISAB ML FC95 Conc. FC95 Qty finavlol spiked solution (mL org) (MU (ppm) 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.004 62 0.004 250 0.0401 0.0365 0.0279 0.0275 0.0246 0.0266 0.0249 0.0276 0.0259 0.0240 0.0253 0.0388 0.0982 0.0201 0.0192 0.0147 0.0431 0.0435 0.0928 0.0905 0.0984 0.0155 0.00864 0.0245 0.0218 0.0161 0.0143 0.0169 0.0139 0.0153 0.0142 o.0343 0.0853 o.106 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 0.004 40 0.004 100 0.004 40 0.004 40 0.004 100 0.004 100 0.004 100 0.004 62 0.004 62 0.004 124 % Actual recovery ppm F- (ug/ug) insample 06550 0.568 0.602 0.642 0.642 0.762 1.01 1.07 0.886 120% 88% 1.78 5.28 81% 82% 90% 91% 77% 75% 82% 46% 115% 71% 0.803 0.731 0.558 0.549 0.491 0.533 0.498 0.551 0.517 0.479 0.506 0.776 1.96 0.403 0.383 0.294 0.863 0.870 1.86 1.81 1.97 0.311 0.173 0.490 0.436 0.323 0.286 0.338 0.277 0.305 0.283 0.685 1.71 2.11 Mass spiked (ug F-) 0.149 0.600 Mass recovered lug F-) 0.0550 0.0568 0.0602 0.0642 0.0642 0.0762 0.101 0.107 0.0886 0.178 0.528 0.096 0.240 0.096 0.096 0.240 0.240 0.240 0.149 0.149 0.298 0.0803 0.0731 0.0558 0.0549 0.0491 0.0533 0.0498 0.0551 0.0517 0.0479 0.0506 0.0776 0.196 0.0403 0.0383 0.0294 0.0863 0.087 0.186 0.181 0.197 0.0311 0.0173 0.0490 0.0436 0.0323 0.0286 0.0338 0.0277 0.0305 0.0283 0.0685 0.171 0.2-11 - STUDY # 6329-135 SERUM Sample ID SPIKE124-2 Blank F52994-day 8 F53410 day 8 F52984 day8 F52992 day 8 F52996 day 8 F52977 day 8 F52989 day 8 F52993 day 8 F52978 day 8 F52980 day 8 40ppm-1 124PPM-1 Blank F52991 day 8 F52987 day 8 F52988 day 8' F52995 day 8 F52972 day 15 F52973 day 15 F52979 day 15 F52975 day 15 F62976 day 15 F52983 day 15 40 ppm-1 124 ppm-1 serum blank serum blank spike 40-1 spike40-2 spike40-3 spike 124-1 spike 124-2 spike124-3 serum blank F52986 DAY15 F52990 DAY15 F52997 DAY15 F52982 DAY15 F52994 DAY15 F5341 0 DAY15 F52984 DAY1 5 F52992 DAY15 F52996 DAY15 F52977 DAY15 Actual reading (ppm F-) 0.134 0.0722 0.0634 0.0348 0.0347 0.0263 0.0422 0.0550 0.0407 0.0306 0.0272 0.0283 0.0530 0.124 0.0608 0.0354 0.0315 0.0239 0.0257 0.0277 0.0430 0.0306 0.0337 0.0250 0.0251 0.0452 0.123 0.0259 0.0199 0.0373 0.0390 0.0448 0.122 0.134 0.116 0.0302 0.0196 0.0231 0.0180 0.0149 0.0173 0.0177 0.0135 0.0141 0.0129 0.0137 Sample TISAB mL FC95 Conc. FC95 Qty finavlol spiked solution (mL or g) (mL) (ppm) o.i 2.0 0.004 124 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.004 40 0.004 124 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.004 40 0.004 124 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0,004 40 0.004 40 0.004 40 0.004 124 0.004 124 0.004 124 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 % Actual recovery ppm F- (ug/ug) insample 90% 2.67 1.44 1.27 0.697 0.694 0.526 0.845 110% 84% 94% 83% 78% 81% 93% 82% 90% 78% 1.10 0.814 0.612 0.544 0.566 1.06 2.49 1.22 0.709 0.630 0.478 0.513 0.553 0.859 0.612 0.674 0.501 0.503 0.903 2.47 0.518 0.398 0.745 0.781 0.896 2.45 2.68 2.33 0.605 0.393 0.463 0.360 0.298 0.346 0.354 0.271 0.281 0.257 0.274 Mass spiked (ug F-) 0.2918 0.096 0.298 0.096 0.298 0.096 0.096 0.096 0.298 0.298 0.298 Mass recovered lug F-) 0.267 0.144 0.127 0.0697 0.0694 0.0526 0.0845 0.110 0.0814 0.0612 0.0544 0.0566 0.106 0.249 0.122 0.0709 0.0630 0.0478 0.0513 0.0553 0.0859 0.0612 0.0674 0.0501 0.0503 0.0903 0.247 0.0518 0.0398 0.0745 0.0781 0.0896 0.245 0.268 0.233 0.0605 0.0393 0.0463 0.0360 0.0298 0.0346 0.0354 0.0271 0.0281 0,0257 0.0274 STUDY 6329-135 SERUM Sample ID SPIKE 40-1 SPIKE 124-1 serum blank F52989day15 F52993 day 15 F52978 day 15 F52980 day 15 F52991 day 15 F52987 day 15 F52988 day 15 F52995 day 15 F52972 day 22 F52973 day 22 40ppm spike-1 40ppm spike-2 124ppm spike-I F52979 day 22 F52975 day 22 F52976 day 22 F52983 day 22 F52986 day 22 F52990 day 22 F52997 day 22 F52982 day 22 F52994 day 22 F5341 0 day 22 40ppm spike-1 124 ppm spike-1 62ppm spike-1 serum blank F52984 day 22 F52992 day 22 F52996 day 22 F52977 day 22 F52989 day 22 F52993 day 22 40ppm spike-1 124 ppm spike-1 serum blank serum blank spike40-1 spike40-2 spike40-3 spike40-4 spike 124-1 spike 124-2 Actual reading (ppm F-) 0.0373 0.152 0.0465 0.0785 0.0389 0.0389 0.0404 0.0376 0.0350 0.0334 0.0285 0.0291 0.0356 0.0722 0.0460 0.110 0.0280 0.0262 0.0236 0.0339 0.0305 0.0233 0.0264 0.0219 0.0310 0.0339 0.0645 0.130 0.0898 0.0405 0.0310 0.0318 0.0356 0.0343 0.0321 0.0290 0.0616 0.116 0.0221 0.0172 0.0235 0.0333 0.0343 0.0371 0.0562 0.0910 Sample TISAB mL FC95 Conc. FC95 Qty finavlol spiked solution (mL or g) (mL) (ppm) 0.1 2.0 0.004 40 0.1 2.0 0.004 124 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.004 40 0.1 2.0 0.004 40 0.1 2.0 0.004 124 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.004 40 0.004 124 0.1 2.0 0.004 62 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.004 40 0.1 2.0 0.004 124 0.1 2.0 0.1 2.0 0.1 2.0 0.004 40 0.1 2.0 0.004 40 0.1 2.0 0.004 40 0.1 2.0 0.004 40 0.1 2.0 0.1 2.0 0.004 124 0.004 124 % Actual recovery ppm F(ug/ug) insample 78% 0.746 102% 3.04 0.931 1.57 0.777 0.777 0.807 0.752 0.700 0.668 0.570 0.582 0.712 150% 1.44 96% 0.919 74% 2.20 0.561 0.524 0.472 0.677 0.610 0.466 0.529 0.438 0.620 0.677 134% 1.29 88% 2.61 121% 1.80 0.810 0.620 0.636 0.712 0.685 0.641 0.580 128% 1.23 78% 2.32 0.442 0.344 -49% 0.471 99% 0.666 71% 0.686 77% 0.743 38% 1.12 61% 1.82 Mass spiked (ug F-) 0.096 0.298 0.096 0.096 0.298 0.096 0.298 0.149 0.096 0.298 0.096 0.096 0.096 0.096 0.298 0.298 Mass recovered (ug F-) 0.0746 0.304 0.0931 0.157 0.0777 0.0777 0.0807 0.0752 0.0700 0.0668 0.0570 0.0582 0.0712 0.144 0.0919 0.220 0.0561 0.0524 0.0472 0.0677 0.0610 0.0466 0.0529 0.0438 0.0620 0.0677 0.129 0.261 0.180 0.0810 0.0620 0.0636 0.0712 0.0685 0.0641 0.0580 0.123 0.232 0.0442 0.0344 0.0471 0.0666 0.0686 0.0743 0.112 0.18-2 Sample ID spike124-3 spike 124-4 SPIKE100-1 SPIKE100-2 SPIKEIOO-3 BLANK BLANK F52978 DAY22 F52980 DAY22 F52991 DAY22 F52987 DAY22 F52988 DAY22 F52996 DAY22 SPIKE 40-1 SPIKE 100-1 STUDY 6329-135 SERUM Actual reading (ppm F-) 0.205 0.0799 0.0659 0.0855 0.0851 0.0454 0.0222 0.0225 0.0258 0.0290 0.0168 0.0125 0.0138 0.0337 0.0850 Sample TISAB ML FC95 Conc. FC95 Qty finavlol spiked solution (mL or g) (mL) (ppm) 0.1 2.0 0.1 2.0 0.1 2.0 0.004 124 0.004 124 0.004 100 0.1 2.0 0.004 100 0.1 2.0 0.004 100 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 .0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.004 40 0.1 2.0 0.004 100 % Actual recovery ppm F(ug/ug) insample 138% 54% 55% 71% 4.10 1.60 1.32 1.71 71% 1.70 0.908 0.444 0.449 0.516 0.581 0.335 0.249 0.275 70% 0.673 71% 1.70 Mass spiked (ug F-) 0.298 0.298 0.240 0.240 0.240 0.096 0.240 Mass recovered (ug F-) 0.410 0.160 0.132 0.171 0.170 0.0908 0.0444 0.0449 0.0516 0.0581 0.0335 0.0249 0.0275 0.0673 0.170 NORMAN SERUM CURVE 1 Sample ID I-PPM-1 4-ppm-2 4-ppm-3 3-ppm-1 0-ppm-2 0-ppm-3 0-ppm-4 2-ppm-1 2-ppm-2 2-ppm-3 00-ppm-1 00-ppm-2 00-ppm-3 24-ppm-1 24-ppm-2 24-ppm-3 50-ppm-1 50-ppm-2 :50-ppm-3 00-ppm-1 00-ppm-2 .00-ppm-3 Actual Sample reading Oty (ppm F-) (mL org) TISAB finalvol (mL) mL FC95 spiked Conc. FC95 solution (ppm) % Actual recovery ppm F- (ug/ug) insample Mass Mass spiked recovered (ugF-) (ugF-) 0.07175 0.1 2.0 0.004 34 176% 1.4350 0.0817 0.1435 STDEV (34ppm): 0. 0.05614 0.1 2.0 0.004 34 138% 1.1228 0.0817 0.11228 %RSD: 0.06462 0.1 2.0 0.004 34 158% 1.2924 0.0817 0.12924 AVERAGE: 0.08668 0.1 2.0 0.004 40 .180% 1.7336 0.0961 0.17336 STDEV (4oppm): 0. 0.06728 0.1 2.0 0.004 40 140% 1.3456 0.0961 0.13456 %RSD: 0.05939 0.1 2.0 0.004 40 124% 1.1878 0.0961 0.11878 AVERAGE: 0.06385 0.1 2.0 0.004 40 133% 1.2770 0.0961 0.1277 0.07291 0.1 2.0 0.004 62 98% 1'.4582 0.1489 0.14582 STDEV(62ppm): 0.0 0.0753 0.1 2.0 0.004 62 101% 1.5060 0.1489 0.1506 %RSD: 0.07839 0.1 2.0 0.004 62 105% 1.5678 0.1489 0.15678 AVERAGE: 0.0902 0.1 2.0 0.004 100 0.1026 0.1 2.0 0.004 100 0.1126 0.1 2.0 0.004 100 75% 1.8040 0.2402 0.1804 STDEV (100ppm): 0. 85% 2.0520 0.2402 0.2052 %RSD: 94% @2.2520 0.2402 0.2252 AVERAGE: 0.1371 0.1 2.0 0.004 124 92% 2.7420 0.2978 0.2742 @TDEV (I24ppm): 0. 0.1451 0.1 2.0 0.004 124 97% 2.9020 0.2978 0.2902 %RSD: 0.1617 $o.i 2.0 0.004 124 109% 3.2340 0.2978 0.3234 AVERAGE: 0.3217 0.1 0.2447 0.1 0.3078 0.1 2.0 0.004 250 2.0 0.004 250 2.0 0.004 250 107% 82% 103% 6.4340 0.6004 0.6434 STDEV (250ppm): 0. 4.8940 0.6004 0.4894 %RSD: 6.1560 0.6004 0.6156 AVERAGE: 0.4438 0.1 2.0 0.004 500 0.4584 0.1 2.0 0.004 500 0.4888 0.1 2.0 0.004 500 74% 8.8760 1.2008 0.8876 STDEV (500ppm): 0. 76% 9.1680 1.2008 0.9168 %RSD: 81% 9.7760 1.2008 0.9776 AVERAGE: SERUM CURVE I NORMAN (07125195) STUDY# 0.9-- 0.8 - 0.7-tx ui re 0.60 0.5 fA LU 0.4 0.3 - 0.2 0.1 0 0.0000 0.2000 y 0.743x + 0.0639 R2 0.9847 0.4000 0.6000 0.8000 MASS SPIKED (ug) 1.0000 1.2000 Sample ID Actual Sample TISAB mL FC95 Conc. FC95 % Actual reading Qty finalvol spiked solution recovery ppm F- (ppm F-)(mL org) (mL) (ppm) (ug/ug) in Sample Mass Mass spiked recovered (ugF-) (ugF-) SPIKE 34-1 0.0330 0.1 2.0 0.004 34 SPIKE 34-2 0.0479 0.1 2.0 0.004 34 SPIKE 34-3 0.0430 0.1 2.0 0.004 34 81% 0.660 0.'0817 0.0660 STDEV (34ppm) 117% 0.957 0.0817 0.0957 %RSD: 105% 0.861 0.0817 0.0861 AVERAGE: SPIKE 40-1 0.0682 0.1 2.0 0.004 40 SPIKE 40-2 0.0459 0.1 2.0 0.004 40 SPIKE 40-3 0.0508 0.1 2.0 0.004 40 142% 95% iO6% 1.36 0.917 1.02 0.0961 0.0961 0.0961 0.136 0.0917 0.102 STDEV(40ppm) %RSD: AVERAGE: SPIKE 62PPM-1 0.0422 0.1 2.0 0.004 62 SPIKE 62PPM-2 0.0441 0.1 2.0 0.004 62 SPIKE 62PPM-3 0.0690 0.1 2.0 0.004 62 SPIKE 62PPM4 0.0922 0.1 2.0 0.004 62 57% 0.845 0.149 0.0845 STDEV(62ppm 59% 0.882 0.149 0.0882 %RSD: ' 93% 1.38 0.149 0.138 AVERAGE: 124% 1.84 0.149 0.184 SPIKE 10OPPM-1 0.0962 0.1 2.0 0.004 100 SPIKE IOOPPM-2 0.159 0.1 2.0 0.004 100 SPIKE 10OPPM-3 0.0774 0.1 2.0 0.004 100 SPIKE 10OPPM-4 0.113 0.1 2.0 0.004 100 SPIKE IOOPPM-5 0.0930 0.1 2.0' 0.004 100 SPIKE 10OPPM-6 0.100 0.1 2.0 0.004 100 80% 1.92 0.240 0.192 STDEv(ioopp 132% 3.17 0.240 0.317 %RSD: 64% 1.55 0.240 0.155 AVERAGE: 94% 2.25 0.240 0.225 77% 1.86 0.240 0.186 83% 2.00 0.240 0.200 SPIKE 124PPM-1 0.149 0.1 2.0 0.004 124 100% 2.97 0.298 0.297 SMEV(124pp SPIKE 124PPM-2 0.150 0.1 2.0 0.004 124 101% 3.00 0.298 0.300 %RSD: SPIKE 124PPM-3 0.140 0.1 2.0 0.004 124 94% 2.80 0.298 0.280 AVERAGE: 0.3 0.25 0.2 tu 0 0.15 to o.i 0.05 0i 0.0000 0.0500 SERUM CURVE 2 NORMAN (08115196) y 0.9145x + 0.0064 0.9679 0.1000 0.1500 MASS SPIKED (ug) 0.2000 STUDY 0.2500 9.11.4Summary and raw data;ppm F-inserum as determinedby thermalextractiofnollowedby analysisusing Skalarsegmented flow analyzerwith ion selectivelectrode. This data,althoughsupportivei,n theopinionof the Study Directorisnot requiredto reachtheconclusionstatedin FinalReport Section6.0,and thereforeisnot discussedin detail. RE:6329-135SERUM SAMPLES AMDT 20795.1 DateofAnalysis8:/16,8/21,and 8/22/95 Analyst:DDW The samplesareburnedintheDohrmanat.950C using0.10mL oftheserum. The gas iscollecteidn2.0mL of1:1TISAB/Milliw-aQter.The samplesarethen analyzedon a SkalarSegmentedFlowAnalyzeursingtheIonSpecifiEclectrode (ISE)Method. TISAB buffeirsaddedtoeachsampleas itproceedsthrougthhesystem.The samplethengoesthrougha heatedmixingcoibleforethepotentibaeltweenthe ionselectievleectrodaend thereferenceelectrodiesmeasured.The signails amplifieadnd relatetdothefluoricdoencentration. The instrumenwtas calibratiendtherangesof0.015-0.15 ppm and 0.15- 1.50 ppm fluoridTeh.e standarcdurveforthehighrangewas plotteudsingthe inversleogarithomptionT.he standarcdurveforthelowrange'ilsinearA.ll standardasnd sampleswerethencalculatebdy theSkalarsoftwaruesingthese curvesA.llresultbselow0.0001ppm appearon therawdataas #.####. A qualitcyontrosltandarwdas analyzedevery10 samplestocheckforaccuracy and drift. Raw dataistakenfromtheappropriactaelibratreadngeoftheSkalarprintout and summarizedon an ExcelspreadsheetT.he finarlesultasreadjustefdorthe collectivoonlumeand anysubsequentdilutions. SUMMARY SERUM AMDT OF 6329-135 SAMPLES 020795.1 ------------ LIUW 'IA(O-14 @i Atih-rLlclqs,t Hcd:L (,329 -@,35 GROUP 1 Dose Level:0 GROUP2 Dose Level:2 mgtkg GROUP3 Dose Level: 200 mgtkg GROUP4 Dose Level: 1000 mgtkg F52972 F52973 F52979 F52975 F52976 F52983 1.04 0.51 0.88 0.05 0.98 ND 1.36 0.05 1.02 0.78 1.00 0.04 0.88 0.69 1.09 0.03 0.84 0.52 0.82 0.03 0.96 0.46 0.91 0.05 F52986 F52990 F52997 F52982 F52994 F53410 1.78 0.54 0.25 0.04 0.75 0.46 0.33 0.03 0.47 0.61 0.23 0.04 0.49 0.50 0.12 0.03 0.56 1.85 0.33 0.04 0.46 1.06 0.27 0.05 F52984 F52992 F52996 F52977 F52989 F52993 0.81 1.05 0.14 0.05 0.71 0.96 0.17 0.05 0.62 1,29 0.18 0.05 0.73 1.65 0.22 0.05 0.71 1.26 2.07 0.04 0.70 0.98 1.12 0.04 F52978 F52980 F52991 F52987 F52988 F52995 0.81 0.82 1.17 0.03 1.25 0.90 1.12 0.04 1.31 1.22 1.06 0.03 1.03 1.09 0.94 0.02 1.02 0.84 0.95 0.02 0.92 0.86 0.77 0.03 135S-SUM.XLS p ,a- 135S-A.XLS 1995-08-16 14:08 OutPut of :950816AI Operator :DDW Date of the Analysis 1995-08-16 08:58 Analysis FileName C:\SKALAR\DATA\HWIDATA\SERUM\950816AI 1 Tracer 1.50 1.47 98% 2 Drift 1.50 1.49 99% 3 Wash ND 4 Standard 1 0.015 0.016 109% 5 Standard 2 0.03 0.03 94% 6 Standard 3 0.06 0.06 100% 7 Standard 4 0.09 0.09 99% 8 Standard 5 0.12 0.12 102% 9 Standard 6 0.15 0.15 99% 10 Standard 7 0.30 0.29 95% 11 Standard 8 0.60 0.61 102% 12 Standard 9 1.20 1.22 101% 13 Standard 10 1.50 1.48 99% 14 Drift 1.50 1.49 99% 15 Wash ND 16 1dPK ra2-1 17 SPK 250-1 18 SPK 250-2 19 BLANK 20 BLANK 21 BLANK 0.13 0.35 0.32 0.12 0.07 0.04 2.0 0.10 2.59 0.004 62.00 0.15 2.0 0.10 6.92 0.004 250.0 0.60 2.0 0.10 6.44 0.004 250.0 0.60 2.0 0.10 2.41 2.0 0.10 1.37 2.0 0.10 0.82 22 F52986-1 23 F52990-1 24 F52997-1 0.09 0.04 0.02 2.0 0.10 1.78 2.0 0.10 0.75 2.0 0.10 0.47 25 F52982-1 0.02 2.0 0.10 0.49 26 Drift 1.50 1.48 98% 27 Wash ND 28 F52994-1 0.03 2.0 0.10 0.56 29 F5341 0-1 0.02 2.0 0.10 0.46 Page 1 135S-A.XLS 30 SPK 62-1 31 SPK 250-1 32 BLANK SERUM 33 BLANK SERUM 34 BLANK SERUM 35 BLANK SERUM 36 SPK 62-1 37 SPK 62-2 38 Drift 39 Wash 40 SPK 62-3 41 SPK 250-1 42 SPK 250-2 43 F52984-1 44 F52992-1 45 F52996-1 46 F52997-1 47 F52984-1 48 F52993-1 49 F52978-1 50 Drift 51 Wash 52 k52980-1 53 F52991-1 54 F52967-1 55 SPK 62-1 56 SPK 250-1 57 Ddft 56 Wash 1.50 1.50 1.50 0.10 0.29 0.06 0.05 0.06 0.05 0.14 0.11 1.49 99% ND 0.12 0.25 0.27 0.04 0.04 0.03 0.04 0.04 0.03 0.04 1.49 99% ND 0.06 0.07 0.05 0.11 0.26 1.50 100% ND 2.0 0.10 1.96 0.004 62.00 0.15 2.0 0.10 5.88 0.004 250.0 0.60 2.0 0.10 1.25 2.0 0.10 1.04 2.0 0.10 1.13 2.0 0.10 0.97 2.0 0.10 2.80 0.004 62.00 0.15 2.0 0.10 2.29 0.004 62.00 0.15 2.0 0.10 2.39 0.004 62.00 0.15 2.0 0.10 5.08 0.004 250.0 0.60 2.0 0.10 5.42 0.004 250.0 0.60 2.0 0.10 0.81 2.0 0.10 0.71 2.0 0.10 0.62 2.0 0.10 0.73 2.0 0.10 0.71 '2.0 0.10 0.70 2.0 0.10 0.81 2.0 0.10 1.25 2.0 0.10 1.31 2.0 0.10 1.03 2.0 0.10 2.10 0.004 62.00 0.15 2.0 0.10 5.14 0.004 250.0 0.60 Page 2 135S-B.XLS 1995-08-2112:30 OutPut of:95O82lAl Operator DDW Date ofthe Analysis 1995-08-2107:55 AnalysisFileName C:\SKALAR\DATA\HWIDATA\SERUMX95082lAl -------- --------------------------- NMI Tracer 1.50 1.47 99% 2 Drift 1.50 1.48 99% 3 Wash ND 4 Standard 1 0.015 0.014 96% 5 Standard2 0.03 0.03 990/o 6 Standard 3 0.06 0.06 1040/a 7 Standard4 0.09 0.09 99% 8 Standard5 0.12 0.12 99% 9 Standard6 0.15 0.15 101% 10 Standard7 0.30 0.29 93% 11 Standard9 0.60 0.62 103% 12 Standard9 1.20 1.22 102% 13 Standard 10 1.50 1.47 98% 14 Drift 1.50 1.54 103% 15 Wash ND 16 S@KUM BLK 1 0.08 2.0 17 SERLTM BLK 2 0.07 2.0 18 SERLIM BLK 3 0.06 2.0 19 F52988-1 0.05 2.0 20 F52995-1 0.05 2.0 21 F52972-1 0.05 2.0 22 F52973-1 0.05 2.0 23 F52979-1 0.05 2.0 24 F52975-1 0.04 2.0 25 F52976-1 0.04 2.0 26 Drift 1.50 1.53 102%. 27 Wash ND 28 F52983-1 0.05 2.0 29 SPK 40-1 0.07 2.0 0.10 1.53 0.10 1.34 0.10 1.12 0.10 1.02 0.10 0.92 0.10 1.04 0.10 0.98 0.10 1.02 0.10 0.88 0.10 0.84 0.10 0.96 0.10 1.34 0.004 46.00 0.10 Page 1 135S-B.XLS . ................. MEE -M, 30 SPK 100-1 0.14 2.0 0.10 2.83 0.004 100.0 0.24 31 BLK-1 0.03 2.0 0.10 0.69 32 BLK-2 0.03 2.0 0.10 0.64 33 BLK-3 0.02 2.0 0.10 0.49 34 SPK 40-1 0.06 2.0 0.10 1.29 0.004 40.00 0.10 35 SPK 40-2 0.06 2.0 0.10 1.2.3 0.004 40.00 0.10 36 SPK 100-1 0.13 2.0 0.10 2.50 0.004 100.0 0.24 37 SPK 100-2 0.12 2.0 0.10 2.44 0.004 100.0 0.24 38 Drift 1.50 1.54 103% 39 Wash, ND 40 SPK 100-3 0.14 2.0 0.10 2.70 0.004 100.0 0.24 41 BLK 0.03 2.0 0.10 0.69 42 F52972-8 0.03 2.0 0.10 0.51 43 F52973-8 ND 2.0 0.10 ND 44 F52979-8 0.04 2.0 0.10 0.78 45 F52975-8 0.03 2.0 0.10 0.69 46 F52976-8 0.03 2.0 0.10 0.52 47 F52983-8 0.02 2.0 0.10 0.46 48 F52986-8 0.03 2.0 0.10 6.54 49 F52990-8 0.02 2.0 0.10 0.46 50 Drift 1.50 1.53 102% 51 Wash ND 52 152997-8 0.03 2.0 0.10 0.61 53 F52982-8 0.02 2.0 0.10 0.50 54 SPK 62-1 0.05 2.0 0.10 1.10 0.004 62.00 0.15 55 SPK 62-2 0.11 2.0 0.10 2.27 0.004 62.00 0.15 56 SPK 124-1 0.14 2.0 0.10 2.75 0.004 124.0 0.30 57 SPK 124-2 0.17 2.0 0.10 3.42 0.004 124.0 0.30 58 BLK 0.10 2.0 0.10 2.07 59 F52994-8 0.09 2.0 0.10 1.85 60 F53410-8 0.05 2.0 0.10 1.06 61 F52984-8 0.05 2.0 0.10 1.05 62 Drift 1.50 1.55 103% 63 Wash ND 64 F52992-8 0.05 2.0 0.10 0.96 65 F52996-8 0.06 2.0 0.10 1.29 Page 2 135S-B.XLS ----------------------------------------I--------------- 66 F52997-8 0.08 2.0 0.10 1.65 67 F52989-8 0.06 2.0 0.10 1.26 68 F52993-8 0.05 2.0 0.10 0.98 69 F52978-8 0.04 2.0 0.10 0.82 70 F52980-8 0.05 2.0 0.10 0.90 71 SPK 40-1 0.108 2.0 0.10 1.51 0.004 40.00 0.10 72 SPK 124-1 0.17 2.0 0.10 3.32 0.004 124.0 0.30 73 BLK 0.09 2.0 0.10 1.79 74 Drift 1.50 1.54 102% 75 Wash ND 76 F52991-8 0.06 2.0 0.10 1.22 77 F52987-8 0.05 2.0 0.10 1.09 78 F52988-8 79 F52995-8 80 F52972-15 0.04 2.0 0.10 0.84 0.04 2.0 0.10 0.86 0.04 2.0 0.10 0.89 81 F52973-15 82 F52979-15 0.07 2.0 0.10 1.36 0.05 2.0 0.10 1.00 83 F52975-15 0.05 2.0 0.10 1.09 84 F52976-15 0.04 2.0 0.10 0.82 85 F52983-15 0.05 2.0 0.10 0.91 86 Drift 1.50 1.57 105% 87 Wash ND 88 tPK 40-1 0.08 2.0 0.10 1.52 0.004 40.00 0.10 89 SPK 124-1 0.17 2.0 0.10 3.30 0.004 124.0 0.30 90 Drift 1.50 1.53 102% 91 Wash ND Page 3 1995-08-21 16:58 Operator : DDW OutPut of: 95082lBl Date ofthe Analysis 1995-08-2112:29 Anal3,sisFileName C:\SKALAR\DATA\RWEDATA\SERUMN95082lBl .......... 135S-C.XLS I Tracer 1.50 1.49 99% 2 Drift 1.50 1.48 99% 3 Wash ND 4 Standard 1 0.015 0.016 105% 5 Standard 2 0.03 0.03 95% 6 Standard 3 0.06 0.06 102% 7 Standard 4 0.09 0.09 101% 8 Standard 5 0.12 0.12 98% 9 Standard 6 0.15 0.15 100% 10 Standard 7 0.30 0.28 93% 11 Standard 8 0.60 0.62 103% 12 Standard 9 1.20 1.22 102% 13 Standard 10 1.50 1.47 98% 14 Drift 1.50 1.44 96% 15 Wash ND 16 SVITUM BLK 1 0.03 17 SERUM BLK 2 0.02 18 SPK 40-1 0.04 19 SPK 40-2 0.04 20 SPK 40-3 0.05 21 SPK 124-1 0,14 22 SPK 124-2 0.14 23 SPK 124-3 0.14 24 BLK 0.03 25 F52986-15 0.01 26 Drift 1.50 1.41 94% 27 Wash ND 28 F52990-15 0.02 29 F52997-15 0,01 2.0 0.10 0.63 2.0 0.10 0.37 2.0 0.10 0.81 0.004 40.00 0.10 2.0 0.10 0.86 0.004 40.00 0.10 2.0 0.10 1.03 0.004 40.00 0.10 2.0 0.10 2.76 0.004 124.0 0.30 2.0 0.10 2.88 0.004 124.0 0.30 2.0 0.10 2.77 0.004 124.0 0.30 2.0 0.10 0.65 2.0 0.10 0.25 2.0 0.10 0.33 2.0 0.10 0.23 Page 1 135S-C.XLS 30 F52982-15 0.01 2.0 0.10 0.12 31 F52994-15 0.02 2.0 0.10 0.33 32 F53410-15 0.01 2.0 0.10 0.27 33 F52984-15 0.01 2.0 0.10 0.14 34 F52992-15 0.01 2.0 0.10 0.17 35 F52996-15 0.01 2.0 0.10 0.18 36 F52977-15 0.01 2.0 0.10 0.22 37 SPK 40-1 0.04 2.0 0.10 0.86 0.004 40.00 0.10 38 Drift 1.50 1.43 96% 39 Wash ND 40 SPK 124-1 0.19 41 BLK 0.07 2.0 0.10 3.82 2.0 0.10 1.*43 42 F52989-15 0.10 2.0 0.10 2.07 43 F52993-15 0.06 2.0 0.10 1.12 44 F52978-15 0.06 2.0 0.10 1.17 45 F52980-15 0.06 2.0 0.10 1.12 46 F52991-15 0.05 2.0 0.10 1.06 47 F52987-15 0.05 2.0 0.10 0.94 48 F52988-15 0.05 2.0 0.10 0.95 49 F52995-15 0.04 2.0 0.10 0.77 50 Drift 1.50 1.45 97% 51 Wash ND 52 F!52972-22 0.05 2.0 0.10 0.91 53 F52973-22 0.05 2.0 0.10 1.02 54 SPK 40-1 0.10 2.0 0.10 2.03 0.004 40.00 0.10 55 SPK 40-2 0.07 2.0 0.10 1.31 0.004 40,00 0.10 0 56 SPK 124-1 0.14 2.0 0.10 2.78 0.004 124.0 0.30 0 57 BLK 0.06 2.0 0.10 1.19 58 F52979-22 0.04 2.0 0.10 0.82 59 F52975-22 0.03 2.0 0.10 0.68 60 F52976-22 0.03 2.0 0.10 0.65 61 F52983-22 0.05 2.0 0.10 0.91 62 Drift 1.50 1.42 95% 63 Wash ND 64 F52986-22 0.04 2.0 0.10 0.88 65 F52990-22 6.03 2.0 0.10 0.69 Page 2 135S-C.XLS .................. 66 F52997-22 0.04 2.0 0.10 0.73 67 F52982-22 0.03 2.0 0.10 0.57 68 F52994-22 0.04 2.0 0.10 0.89 69 F53410-22 0.05 2.0 0.10 0.92 70 SPK 40-1 0.09 2.0 0.10 1.79 0.004 40.00 0.10 71 SPK 124-1 0.17 2.0 0.10 3.36 0.004 124.0 0.30 72 SPK 62-1 0.12 2.0 0.10 2.42 0.004 62.00 0.15 73 BLK 0.06 2.0 0.10 1.24 74 Drift 1.50 1.47 98% 75 Wash ND 76 F52984-22 0.05 2.0 0.10 0.94 77 F52992-22 0.05 2.0 0.10 0.91 78 F52996-22 0.05 2.0 0.10 0.96 79 F52977-22 0.05 2.0 0.10 0.93 80 F52989-22 0.04 2.0 0.10 0.88 81 F52993-22 0.04 2.0 0.10 0.79 82 SPK 40-1 0.09 2.0 0.10 1.71 0.004 40.00 0.10 83 SPK 124-1 0.15 2.0 0.10 2.92 0,004 124.0 0.30 84 Drift 1.50 1.46 97% 85 Wash ND Page 3 1995-08-2209:05 OutPut of : 950822Al Operator DDW Date of theAnalysis 1995-08-2206:49 AnalysisFileName C:\SKALAR\DATA\HWEDATA\SERUM\950822Al ... ................................. 135S-D.XLS -0 ............ I Tracer 1.50 1.46 97% 2 Drift 1.50 1.48 98% 3 Wash ND 4 Standard 1 0.015 0.016 104% 5 Standard2 0.03 0.03 94% 6 Standard3 0.06 0.06 1040@. 7 Standard4 0.09 0.09 101% 8 Standard5 0.12 0.12 97% 9 Standard6 0.15 0.15 101% 10 Standard7 0.30 0.28 92% 11 Standard8 0.60 0.62 104% 12 Standard9 1.20 1.23 102% 13 Standard 10 1.50 1.47 98% 14 Drift 1.50 1.54 102% 15 Wash ND 16 SERUM BLK 1 0.05 2.0 17 SqltUM BLK 2 0.03 2.0 18 SPK 40-1 0.04 2.0 19 SPK 40-2 0.06 2.0 20 SPK 40-3 0.06 2.0 21 SPK 40-4 0.06 2.0 22 SPK 124-1 0.08 2.0 23 SPK 124-2 0.13 2.0 24 SPK 124-3 0.28 2.0 25 SPK 124-4 0.12 2.0 26 Drift 1.50 1.53 102% 27 Wash ND 28 SPK 100-1 0.11 2.0 29 SPK 100-2 0.13 2.0 30 SPK 100-3 0.13 2.0 0.10 0.94 0.10 0.67 0.10 0.86 0.004 40.00 0.10 0.10 1.14 0.004 40.00 0.10 0.10 1.15. 0.004 40.00 0.10 0.10 1.22 0.004 40.00 0.10 0.10 1.66 0.004 124.0 0.30 0.10 2.57 0.004 124.0 0.30 0.10 5.56 0.004 124.0 0.30 0.10 2.34 0.004 124.0 0.30 0.10 2.12 0.004 100.0 0.24 0.10 2.63 0.004 100.0 0.24 0.10 2.56 0.004 100.0 0.24 Page 1 . ............ 135S-D.XLS 31 BLK 0.08 2.0 0.10 1.51 ,2 BI.K 0.04 2.0 1 0.78 33 Fi2978-22 0.03 2.0 0.10 0.67 34 F52980-22 0.04 2.0 0.10 0.85 35 F52991-22 0.03 2.0 0.10 0.58 36 F52987-22 0.02 2.0 0.10 0.50 37 F52988-22 0.02 2.0 0.10 0.34 38 Drift 1.50 1.53 102% 39 Wash ND 40 F52995-22 0.03 2.0 0.10 0.54 41 BLANK TISAB ND 42 SPK 100-1 0.11 2.0 o.ib 2.12 0.004 100.0 0.24 43 Drift 1.50 1.55 103% 44 Wash ND Page 2 SERUM CURVE 1 7-31-95 NORMAN kjLUA.) '12Z hm,i@--Lro-,gS,J u.) Sample ID Spk 34@ 1 Spk 34-2 Spk 34-3 Spk 40-1 Spk 40-2 Spk 40-3 Spk 62-1 Spk 62-2 Spk 62-3 Spk 100-1 Spk 100-2 Spk 100-3 Spk 124-1 Spk 124-2 Spk 124-3 Spk 250-1 Spk 250-2 Spk 250-3 Spk 500-1 Spk 500-2 Spk 500-3 Sbdar Result (ppm) Dl:nSAB fmal vol (mL) mL FC 95 Conc Solution FC 95 Soin Spiked (ppm) Mass Spiked (ugF-) Average % Mass Recovery 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 0.12 0.13 2.0 0.004 100.0 2.0 0.004 100.0 0.24 0.24 99% 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 @50.0 0.26 2.0 0.004 250.0 0.60 0.61 102% 0.32 2.0 0.004 250.0 0.47 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 1.20. SERUM CURVE I (NORMAM y - 0.7743+x 0.086 R! 0.9981 741-95 1.0-0- 0.80 0.6D - 0.40-- oz 0.2-0 0.00 0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 MASS SPIKED (ug) SERCRVLN.AVE --..Z SERUM CURVE I 7-31-95 NORMAN Sample ID Spk 34-1 Spk 34-2 Spk 34-3 Spk 40-1 Spk 40-2 Spk 40-3 Spk 62-1 Spk 62-2 Spk 62-3 Spk 100-1 Spk 100-2 Spk 100-3 Spk 124-1 Spk 124-2 Spk 124-3 Spk 250-1 Spk 250-2 Spk 250-3 Spk 500-1 Spk 500-2 Spk 500-3 Skalar Result (ppm) 0.09 0.07 0.08 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 finalvol (mL) mL FC 95 Conc Solution FC 95 Soln Spiked (ppm) Mass Spiked (ugF-) Mass % Recovered Recovery (ugF-) 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 0.004 124.0 0.30 0.32 108% STANDARD DEVIATION: 2.0 0.004 124.0 0.30 0.34 114% % RSD: 2.0 0.004 124.0 0.30 0.37 124% 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 79% STANDARD DEVIATION: 2.0 0.004 500.0 1.20 0.99 82% % RSD: 2.0 0.004 500.0 1.20 1.04 87% 0.2450 12.9998 0.0826 5.3307 0.0263 2.1670 0.1138 11.4530 0.0778 6.7516 0.1318 12.9196 0.0442 5.3672 SERUM CURVE I (NCRMAN) 7/31195 y 0.7743x+ 0.086 R 2 0.9762 1.20 1.00 0.30 0.00 0.20 0.40 0.60 O.So -1.00 O.DO 0.20 MASS SPIKED (ug) 1.20 1.40 SERCRVLN.SUM SERUM CURVE 2 8-16-95 NORMAN SPK 34-1 0.05 2.0 SPK 34-2 0.07 2.0 SPK 34-3 0.06 2.0 0.004 34.00 0.004 34.00 0.09 0.004 34.00 SPK 40-1 0.10 2.0 SPK 40-2 0.07 2.0 SPK 40-3 0.08 2.0 0.004 40.00 0.004 40.00 0.10 0.004 40.00 SPK 62-1 0.05 2.0 0.004 , 62.00 SPK 62-2 0.07 2.0 0.004 62.00 0.15 SPK 62-3 0.09 '2.0 0.004 62.00 SPK 624 0.12 2.0 0.004 62.00 SPK 100-1 0.14 2.0 SPK 100-2 0.20 2.0 SPK 100-3 0.11 2.0 SPK 1004 0.16 2.0 SPK 100-5 0.14 2.0 SPK 100-6 0.14 2.0 0.004 100.0 0.004 100.0 0.004 100.0 0.24 0.004 100.0 0.004 100.0 0.004 100.0 SPK 124-1 0.19 2.0 SPK 124-2 0.23 2.0 SPK 124-3 0.19 2.0 0.004 124.0 0.004 124.0 0.30 0.004 124.0 0.12 150% 0.16 167% 0.17 112% 0.30 123% 0.40 134% SERUM CURVE 2 8-16-95 1.2112x + &0191 le 0,9562' 0.40 0.30 0.25 0.20 0.13 0.10 0*03 0.00 0.00 0.05 0.10 MASS 0.15 &20 VIKED (00 0.25 0." SERCRV2N.AVE Page I SERUM CURVE 2 8-16-95 NORMAN ------------------------------------- ----------------------------------- ----------- SPK 34-1 0.05 2.0 SPK 34-2 0.07 2.0 SPK 34-3 0.06 2.0 SPK 40-1 0.10 2.0 SPK 40-2 0.07 2.0 SPK 40-3 0.08 2.0 SPK 62-1 0.05 2.0 SPK 62-2 0.07 2.0 SPK 62-3 0.09 2.0 SPK 62-4 0.12 2.0 SPK 100-1 0.14 2.0 SPK 100-2 0.20 2.0 SPK 100-3 0.11 2.0 SPK 100-4 0.16 2.0 SPK 100-5 0.14 2.0 SPK 100-6 0.14 2.0 SPK 124-1 0.19 2.0 SPK 124-2 0.23 2.0 SPK 124-3 0.19 2.0 0.004 - 34.00 0.004 34.00 0.004 34.00 0.004 0.004 0.004 40.00 40.00 40.00 0.004 0.004 0.004 0.004 62.00 62.00 62.00 '62.00 0.004 0.004 0.004 0.004 0.004 0.004 100.0 100.0 100.0 100.0 100.0 100.0 0.004 0.004 0.004 124.0 124.0 124.0 0.09 0.09 0.08 0.10 0.10 0.10 0.15 0.15 0.15 0.15 0.24 0.24 0.24 0.24 0.24 0.24 0.30 0.30 0.30 0.11 0.14 0.12 0.20 0.13 0.15 0.10 0.14 0.18 0.25 0.27 OA 0.23 0-32 0.27 0.27 0.38 0.45 0.37 132% 1690/o 150010 STANDARD DEVIATION: % RSD: 204% 139% 159% STANDARD DEVIATION: % RSD: 66% 92% 122% 1670/o STANDARD DEVIATION: % RSD: 114% STANDARD DEVIATION: 168% % RSD: 95% 135% 114% 114% 126% STANDARD DEVIATION: 151% % RSD: 126% 0.1837 12.2150 0.3354 20.0349 0.4333 38.7472 0.2535 20.5563 0.1454 10.8282 0.45- SERUM CURVE 2 (NORMAN) Y-1.1992x+0.0179 R! 0.780 8-16-95 0.40- 0.35- 0.30 - 0.25 - 0 0.20- rA 0.15 -- oool- 0.10- 0.05 0.00 0.00 0.05 0.10 0.15 V20 0.25 0.30 MASS SPIXFD (ug) SERCRV2N.SUM P@ge 1 1995-08-16 14:10 OutPut of : 950816AI Software version 6.1. cl990,93 Operator DDW Date of the Analysis 1995-08-16 08:58 Analysis File Name C:\SKALAR\DATA\HWIDATA\SERUM\950816Al hu%b7 Za-L95-1 Fluoride 1.5 Calibration order = Inverse Logarithm Slope Result s lo[ x cl xl corrected value of the sample c corrected value of the concentration I s Slope of the electrode a2 -0.00000 al 0.00065 aO -1.15706 Fluoride L Calibration order = 2 Corz-elation Result = a2 z-= 0.99717 x2 + al x + aO a2 = al = aO = 0.00000 0.00018 0.00912 Sampler Type Number Sample Time Wash Time Air Time Take up special needle Height : SAIOOO :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 1995-08-16 14:10 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 s7 standard : 0.300 s8 standard : 0.600 S9 standard : 1.200 slO 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 950816Al 1995-08-16 14:10 OutPut of sl standard : 0.015 s2 standard : s3 standard : 0.030 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 eNd ignore : 60 Sec : 120 Sec Measure window : 75 % Filter : No Regeneration : No formula c4:=c3 output 950816Al 1995-08-16 14:10 OutPut of : 950816Al Fluoride 1.5 Fluoride L PPM Pos Typ Ident Ch Result F Time wt iw Initial Wash 3 0.070 1t Tracer 3 1.472 2d Drift 3 1.486 3w Wash 3 0.070 4 sl Standard 1 3 0.074 5 s2 Standard 2 3 0.081 6 r.3 Standard 3 3 0.100 7 s4 8 s5 9 s6 -10 s7 11 s8 Standard 4 3 0.117 Standard 5 3 0.137 Standard 6 3 0.154 Standard 7 3 0.285 Standard 8 3 0.614 12 s9 Standard 9 3 1.217 13 slO Standard 10 3 1.479 14 d Drift 3 1.490 15 w Wash 3 0.070 16 u SPK 62-1 3 0.142 17 u SPK 250-1 3 0.346 18 u SPK 250-2 3 0.322 19 u 20 u 21 u 22 u BLANK BLANK BLANK F52986-1 3 0.136 3 0.105 3 0.088 3 0.117 23 u 24 u F52990-1 F52997-1 3 0.086 3 0.078 25 u 26 d 27 w F52982-1 3 Drift 3 Wash 3 0.079 1.477 0.070 28 u F52994-1 3 0.081 29 u 30 u F53410-1 3 0.078 62-1 3 0.122 31 u SPK 250-1 3 0.294 32 u BLANK SERUM 3 0.101 33 u BLANK SERUM 3 0.095 34 u BLANK SERUM 3 0.098 35 u 36 u BLANK SERUM 3 0.093 SPK 62-1 3 0.148 37 u 38 d 39 w SPK 62-2 3 Drift 3 Wash 3 0.132 1.485 0.070 40 u SPK 62-3 3 0.136 41 u SPK 250-1 3 0.254 42 u SPK 250-2 3 0.271 43 u F52984-1 3 0.088 44 u F52992-1 3 0.085 45 u 46 u F52996-1 F52997-1 3 0.082 3 0.086 47 u 48 u F52984-1 F52993-1 3 0.085 3 0.085 49 u 50 d 51 w 52 u 53 u F52978-1 3 0.088 Drift 3 1.488 Wash 3 0.070 F52980-1 3 0.101 F52991-1 3 0.103 65 207 383 619 729 907 1081 1258 1434 1608 1784 1958 2133 2308 2484 2726 2830 3011 3187 3361 3536 3710 3886 4062 4236 4410 4586 4825 4934 5110 5288 5462 5635, 5810 5986 6161 6335 6510 6684 6911 7034 7210 7382 7557 7734 7910 8086 8258 8434 8608 8785 9004 9131 9311 PPM Ch Result F Time 4 0.0091 0 4 1.8335 0 4 1.8558 0 4 0.0091 0 4 0.0164 0 4 0.0281 0 4 0.0601 0 4 0.0894 0 4 0.1221 0 4 0.1488 0 4 0.3400 0 4 0.7483 0 4 1.4687 0 4 1.8442 0 4 1.8617 0 4 0.0091 0 4 0.1296 0 4 0.4211 0 4 0.3892 0 4 0.1206 0 4 0.0686 0 4 0.0410 0 4 0.0889 0 4 0.0377 0 4 0.0234 0 4 0.0247 0 4 1.8413 0 4 0.0091 0 4 0.0281 0 4 0.0228 0 4 0.0978 0 4 0.3525 0 -4 0.0625 0 4 0.0519 0 4 0.0566 0 4 0.0485 0 4 0.1400 0 4 0.1145 0 4 1.8539 0 4 0.0091 0 4 0.1194 0 4 0.2971 0 4 0.3208 0 4 0.0407 0 4 0.0356 0 4 0.0310 0 4 0.036_1 0 4 0.0356- 0 4 0.0348 0 4 0.0407 0 4 1.8587 0 4 0.0091 0 4 0.0625 0 4 0.0654 0 Page Of 2 1995-08-16 14:10 OutPut of : 950816Al Fluoride 1.5 Fluoride L PPM PPM Pos Typ Ident Ch Result F Time Ch Result F Time 54 u F52987-1 3 0.095 9486 4 0.0517 0 55 u SPK 62-1 3 0.127 9662 4 0.1051 0 56 u SPK 250-1 3 0.257 9838 4 0.3017 0 57 d Drift 3 1.503 10012 4 1.8831 0 58 w Wash 3 0.070 10247 4 0.0091 0 wt rw RunOut Wash 3 0.070 10487 4 0.0091 0 Page 2 of 2 Calibration curve of 950816AI Fluoride L m@ir-ii@- misiMMM7-r-H 6 0.2692961. 901 I- S4 0.0091180 0 Order Measured 900 r 0.99717 r t.8S4187S Calibration curve of 950816AI Fluoride I.S 9 IL[WriTu-7ilWM"-t 0.07O;.i-7S7@o 64 -W m L -W 0.06964SI 0 Order Measured Inverse Logarithm 4095 s 5671 4095 E;mil- Raw data of 950816AI : Fluoride 1.5 0 480 o 0 Time Esc=Exit 1 FI=Help 1 Crtl-P=Edit peaks Raw data of 950816AI : Fluoride 1.5 4095 mzmi,7,7mw@#Cr-M 4S60 v@@ 3050 .1FS-40 6 11 Iko Jd 0 4560 Time 9560 Esc=Exit 1 FI=Help 1 Crtl-P=Edit peaks 1 Raw data of 950816AI E%Ftit7t7a-MM@WTTM 1095 8935 IKT-UF-M Fluoride t.5 0 8935 Time 1393 Esc=Exit 1 FI=Help 1 Crtl-P=Editpeaks 1 1995-03-21 Software 12:30 version 6.1 OutPut of cl990,93 95082lAl Operator : DDW Date of the Analysis : 1995-08-21 07:55 Analysis File Name : C:\SKALAR\DATA\HWIDATA\SERUM\950821AI b T %019 Fluoride 1.5 Calibration order = Inverse Logarithm Slope s = 1.10100 x - cl Result = 101 s 1 a2 = al = aO = -1 0.00000 0.00071 -1.20061 x = corrected value of the sample cl = corrected value of the concentration 1 s = Slope of the electrode Fluoride L Calibration order = 2 Corz-elation r = 0.99953 Result = a2 x2 + al * x + aO a2 = al = aO = -0.00000 0.00025 0.00771 Sampler Diluter Type Number Sample Time Wash Time Air Time Take up special needle Height : SAIOOO :I : 50 sec. : 120 sec. : 1 sec. : Single : None : 70 mm. needle Height : 80 mm dilution Factor : 10 dilution Volume : 2.5 ml. Resample :1 Dilution runs : 1 User file : TXT Reproces : No 1995-08-21 12:30 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 : s7 standard : 0.150 0.300 ss standard : 0.600 S9 standard : 1.200 slO 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 95082lAl 1995-08-21 12:30 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 eNd ignore : 60 Sec : 120 Sec Measure window : 75 Filter : No Regeneration : No formula c4:=c3 output 95082lAl 1995-08-21 12:30 OutPut of : 95082lAl Fluoride 1.5 Fluoride L PPM PPM Pos Typ Ident Ch Result F Time Ch Result F Time wt iw Initial Wash 3 0.063 1t Tracer 3 1.468 2d Drift 3 1.482 3w Wash 3 0.063 4 sl Standard 1 3 0.066 5 s2 Standard 2 3 0.073 6 s3 Standard 3 3 0.090 7 s4 Standard 4 3 0.106 8 s5 9 s6 Standard 5 3 0.128 Standard 6 3 0.156 10 s7 Standard 7 3 0.279 11 S8 Standard 8 3 0.619 12 s9 Standard 9 3 1.224 13 slO Standard 10 3 1.471 14 d Drift 3 1.539 15 w Wash 3 0.063 16 u SERUM BLK 1 3 0.098 17 u SERUM BLK 2 3 0.092 18 u SERUM BLK 3 3 0.086 19 u F52988-1 3 0.083 20 u F52995-1 3 0.081 21 u F52972-1 3 0.084 22 u F52973-1 3 0.082 23 u F52979-1 3 0.083 24 u 25 u 26 d F52975-1 3 F52976-1 3 Drift 3 0.080 0.079 1.529 27 w Wash 3 0.063 28 u F52983-1 3 0.082 29 u SPK 40-1 3 0.092 30 u 31 u SPK 100-1 BLK-1 3 0.147 3 0.075 32 u BLK-2 3 0.074 33 u 34 u BLK-3 SPK 40-1 3 0.070 3 0.091 35 u SPK 40-2 3 0.089 36 u SPK 100-1 3 0.133 37 u 38 d SPK 100-2 3 0.131 Drift 3 1.540 39 w 40 u Wash 3 0.063 SPK 100-3 3 0.142 41 u 42 u BLK F52972-8 3 0.075 3 0.071 43 u 44 u F52973-8 P52979-8 3 0.064 3 0.077 45 u 46 u F52975-8 P52976-8 3 0.075 3 0.071 47 u F52983-8 3 0.070 48 u 49 u 50 d F52986-8 3 F52990-8 3 Drift 3 0.071 0.070 1.525 51 w 52 u 53 u Wash F52997-8 F52982-8 3 0.063 3 0.073 3 0.070 65 208 382 570 728 903 1083 1259 1435 1609 1783 1958 2133 2309 2483 2723 2832 3012 3184 3360 3534 3710 3885 4060 4234 4410 4583 4810 4933 5111 5285 5460 5632 5810 5988 6162 6336 6512 6684 6902 7037 7207 7384 7558 7736 7909 8085 8257 8437 8609 8785 9012 9136 9310 4 0.0077 4 0.5962 4 0.5992 4 0.0077 4 0.0144 4 0.0298 4 0.0624 4 0.0891 4 0.1184 4 0.1509 4 0.2480 4 0.3935 4 0.5438 4 0.5968 4 0.6121 4 0.0077 4 0.0765 4 0.0672 4 0.0559 4 0.0510 4 0.0462 4 0.0518 4 0.0488 4 0.0508 4 0.0440 4 0.0420 4 0.6099 4 0.0077 4 0.0481 4 0.0672 4 0.1414 4 0.0347 4 0.0322 4 0.0246 4 0.0643 4 0.0617 4 0.1251 4 0.1221 4 0.6123 4 0.0077 4 0.1352 4 0.0347 4 0.0256 4 0.0090 4 0.0388 4 0.0347 4 0.026.1 4 0.02314 0.0271 4 0.0231 4 0.6090 4 0.0077 4 0.0303 4 0.0248 0 0 0 0 0 0 0 0 0 0 0 0' 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 @o 0 0 0 0 0 0 0 0 0 0 0 Page of 2 1995-08-21 12:30 OutPut of : 950821AI Fluoride 1.5 Fluoride L PPM PPM Pos Typ Ident Ch Result F Time Ch Result F Time 54 u SPK 62-1 3 0.085 9488 4 0.0549 0 55 u SPK 62-2 3 0.124 9664 4 0.1135 0 56 u SPK 124-1 3 0.144 9838 4 0.1373 0 57 u SPK 124-2 3 0.171 10014 4 0.1659 0 58 u BLK 3 0.116 10189 4 0.1037 0 59 u F52994-8 3 0.109 10365 4 0.0927 0 60 u F53410-8 3 0.085 10539 4 0.0532 0 61 u F52984-8 3 0.084 10715 4 0.0527 0 62 d Drift 3 1.552 10888 4 0.6153 0 63 w Wash 3 0.063 11124 4 0.0077 0 64 u F52992-8 3 0.082 11239 4 0.0479 0 65 u F52996-8 3 0.091 11415 4 0.0646 0 66 u F52997-8 3 0.102 11589 4 0.0823 0 67 u F52989-8 3 0.090 11766 4 0.0629 0 68 u P52993-8 3 0.082 11938 4 0.0488 0 69 u F52978-8 3 0.078 12108 4 0.0410 0 70 u F52980-8 3 0.080 12290 4 0.0452 0 71 u SPK 40-1 3 0.098 12466 4 0.0756 0 72 u SPK 124-1 3 0.166 12642 4 0.1606 0 73 u BLK 3 0.106 12816 4 0.0894 0 74 d Drift 3 1.536 12990 4 0.6115 0 75 w Wash 3 0.063 13232 4 0.0077 0 76 u F52991-8 3 0.089 13341 4 0.0612 0 77 u P52987-8 3 0.085 13517 4 0.0544 0 78 u F52988-8 3 0.079 13683 4 0.0420 0 79 u F52995-8 3 0.079 13867 4 0.0432 0 80 u P52972-15 3 0.080 14042 4 0.0442 0 81 u F52973-15 3 0.093 14218 4 0.0679 0 82 u F52979-15 3 0.083 14392 4 0.0501 0 83 u F52975-15 3 0.085 14566 4 0.0547 0 84 u F52976-15 3 0.078 14740 4 0.0410 0 85 u P52983-15 3 0.080 14916 4 0.0454 0 86 d Drift 3 1.574 15092 4 0.6204 0 87 w Wash 3 0.063 15334 4 0.0077 0 88 u SPK 40-1 3 0.098 15441 4 0.0761 0 89 u SPK 124-1 3 0.165 15619 4 0.1601 0 90 d Drift 3- 1.534 15792 4 0.6110 0 91 w Wash 3 0.063 16032 4 0.0077 0 wt rw RunOut Wash 3 0.063 16267 4 0.0077 0 Page 2 of 2 Calibration curve of 950821A1 Fluoride L so lF7,7,aMImirwr--M7--T-6@rg 0.216!5250 030 I- 64 c c 0-0077061 0 Order 2 Measured Soo r 0.99953 1 1.7854700 Calibration curve of 950821AI Fluoride I.S 0 0.0630020,@-0 Order neasured Inverse Logarithm s A86 4095 4095 Raw data of 95082tAt Fluoride t.5 P4 o tu\ 0 Esc=Exit 1 FI=Help Time Crtl-P=Edit peaks scoo 4095 Raw data of 9508'41-lA:lFluoride 1.5 m;Fi4a7aT;@@P!IM 4560 lUngr-M 3270 Ft7-3t*M64 0 4560 Esc=Exit 1 FI=Help Time Crtl-P=Edit peaks 9560 4095 Raw data of 9508'@IlAt: Fluoride 1.5 893S 688 64 0 8435 T ime Esc=Exit FI=Help 1 Crtl-P=Edit peaks A4 4\ 13935 4095 Raw data of 350821AI : Fluoride 1.5 t3310SIM= 746 @@64 P4 tl 0 13310 Esc=Exit FI=Help T ime Crtl-P=Edit peaks 1 183101 1995-08-21 16:58 output of : 950821BI Software version 6.1 cl990,93 Operator : DDW Date of the Analysis : 1995-08-21 12:29 Analysis File Name : C:\SKALAR\DATA\HWIDATA\SERUM\95082lBl Fluoride 1.5 Calibration order Inverse Logarithm Slope s 0.00000 x - cl I R esult = 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 0.00068 -1.19997 Fluoride L Calibration order = 2 Co.rrelation z,= 0.99960 Result = a2 X2 + al x + aO a2 = al = aO = -0.00000 0.00025 0.00307 Sampler Type Number Sample Time Wash Time Air Time Take up special needle Height : SAIOOO :1 : 50 sec. : 120 sec. : I 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 1995-08-21 16:58 output of Fluoride 1.5 Path number Signal type Decolor system Number diluts 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 s7 standard : s8 standard : S9 standard : slO standard : 0.300 0.600 1.200 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 95082lBl 1995-08-21 16:58 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 89 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 95082lBl 1995-08-21 16:58 output of 95082lBl Fluoride 1.5 Fluoride L ppm PPM Pos Typ Ident Ch Result F Time Ch Result F Time wt iw Initial Wash 3 0.063 65 4 0.0031 0 1t 2d Tracer 3 1.488 208 4 0.6348 0 Drift 3 1.478 384 4 0.6322 0 3w Wash 3 0.063 625 4 0.0031 0 4 sl Standard 1 3 0.068 733 4 0.0157 0 5 s2 Standard 2 3 0.074 911 4 0.0284 0 6 s3 Standard 3 3 0.091 1085 4 0.06i2 0 7 s4 Standard 4 3 0.109 1261 4 0.0909 0 8 s5 Standard 5 3 0.129 1435 4 0.1181 0 9 s6 Standard 6 3 0.156 1611 4 0.1507 0 10 s7 Standa rd 7 3 0.279 1787 4 0.2517 0 11 s8 Standard 8 3 0.620 1960' 4 0.4063 0 12 s9 Standard 9 3 1.224 2135 4 0.-5703 0 13 slO Standard 10 3 1.472 2311 4 0.630'8 0 14 d Drift 3 1.435 2485 4 0.6213 0 15 w Wash 3 0.063 2662 4 0.0031 0 16 u SERUM BLK 1 3 0.075 2837 4 0.0314 0 17 u SERUM BLK 2 3 0.070 3013 4 0.0184 0 18 u SPK 40-1 3 0.080 3186 4 0.0406 0 19 u SPK 40-2 3 0.081 3362 4 0.0431 0 20 u SPK 40-3 3 0.086 3536 4 0.0515 0 21 u SPK 124-1 3 0.145 3714 4 0.1381 0 22 u SPK 124-2 3 0.150 3888 4 0.1441 0 23 u SPK 124-3 3 0.145 4062 4 0.1386 0 24 u BLK 3 0.076 4238 4 0.0326 0 25 u F52986-15 3 0.067 4411 4 0.0127 0 26 d Drift 3 1.408 4586 4 0.6146 0 27 w Wash 3 0.063 4732 4 0.0031 0 28 u F52990-15 3 0.069 4938 4 0.0164 0 29 u F52997-15 3 0.067 5109- 4 0.0115 0 30 u F52982-15 3 0.064 5285 4 0.0058 0 31 u F52994-15 3 0.069 5459 4 0.0167 0 32 u F53410-15 3 0.068 5635 4 0.0137 0 33 u F52984-15 3 0.065 5811 4 0.0070 0 34 u F52992-15 3 0.065 5987 4 0.0083 0 35 u F52996-15 3 0.066 6163 4 0.0090 0 36 u F52977-15 3 0.066 6334 4 0.0110 0 37 u SPK 40-1 3 0.081 6513 4 0.0431 0 38 d Drift 3 1.434 6686 4 0.6211 0 39 w Wash 3 0.063 6924 4 0.0031 0 40 u SPK 124-1 3 0.191 7036 4 0.1851 0 41 u BLK 3 0.097 7212 4 0.0715 0 42 u F52989-15 3 0.118 7386 4 0.1036 0 43 u F52993-15 3 0.088 7559 4 0.0561 0 44 u F52978-15 3 0.089 7736 4 0.0585 0 45 u F52980-15 3 0.088 7911 4 0.0559 0 46 u F52991-15 3 0.086 8083 4 0.053_0 0 47 u F52987-15 3 0.083 8263 4 0.0469- 0 48 u F52988-15 3 0.083 8437 4 0.0474 0 49 u F52995-15 3 0.079 8613 4 0.0385 0 50 d Drift 3 1.454 8787 4 0.6263 0 51 w Wash 3 0.063 9024 4 0.0031 0 52 u F52972-22 3 0.083 9137 4 0.0457 0 53 u F52973-22 3 0.085 9313 4 0.0511 0 Page I of 2 1995-08-21 16:58 OutPut of : 95082lBl Fluoride 1.5 Fluoride L ppm PPM Pos,Typ Ident Ch Result F Time Ch Result F Time 54 u SPK 40-1 3 0.117 9489 4 0.1017 0 55 u SPK 40-2 3 0.093 9662 4 0.0655 .0 56 u SPK 124-1 3 0.146 9838 4 0.1391 0 57 u BLK 3 0.090 10010 4 0.0593 0 58 u F52979-22 3 0.080 10186 4 0.0411 0 59 u P52975-22 3 0.077 10365 4 0.0341 0 @o u F52976-22 3 0.076 10537 4 0.0324 0 61 u F52983-22 3 0.082 10713 4 0.0453 0 62 d Drift 3 1.419 10886 4 0.6175 0 63 w Wash 3 0.063 11120 4 0.0031 0 64 u F52986-22 3 0.082 11237 4 0.0438 0 65 u F52990-22 3 0.077 11409 4 0.0343 0 66 u F52997-22 3 0.078 11583 4 0.0363 0 67 u F52982-22 3 0.074 11762 4 0.0284 0 68 u F52994-22 3 0.082 11936 4 0.0445 0 69 u F53410-22 3 0.083 12114 4 0.0460 0 70' u SPK 40-1 3 0.108 12288 4 0.0893 0 71 u SPK 124-1 3 0.168 12462 4 0.1627 0 72 u SPK 62-1 3 0.131 12638 4 0.1211 0 73 u BLK 3 0.091 12812 4 0.0621 0 74 d Drift 3 1.469 12985 4 0.6301 0 75 w Wash 3 0.063 13227 4 0.0031 0 76 u F52984-22 3 0.083 13337 4 0.0469- 0 77 u F52992-22 3 0.082 13511 4 0.0455 0 78 u F52996-22 3 0.084 13685 4 0.0479 0 79 u F52977-22 3 0.083 13858 4 0.0467 0 80 u F52989-22 3 0.082 14035 4 0.0440 0 81 u F52993-22 3 0.079 14211 4 0.0397 0 82 u SPK 40-1 3 0.106 14379 4 0.0857 83 u SPK 124-1 3 0.152 14559 4 0.1459 0 84 d Drift 3 1.461 14735 4 0.6280 0 85 w Wash 3 0.063 14976 4 0.0031 0 wt rw RunOut Wash 3 0.063 15210 4 0.0031 0 Page 2 of 2 0.2128165, Calibration curve of 95082181 6-- gir,@ Fluoride L 0.0045592=)MiPW-64-= c 0.0030712-,, 0 Order 2 Measured 900 r 0.99960 no 1.7656142 Calibration curve of 9SO821BI Fluoride I.S 0.063706OW-IMI 64M c 0 c 0.0631072 0 Order Measured Inverse Logarithm 409S S 6199 4095 Raw data of 95082181 : Fluoride 1.5 0 592 It-f-3V-M64 t.14 C7 0 0 Esc=Exit FI=Help Time Crtl-P=Edit peaks 5000 Raw data of 950821BI : Fluoride 1.5 mLgi4,7ir.@@VVM 4095 - 4560 3148 64 4p 0 4!5'E;O Time Esc=Exit FI=Help 1 Crtl-P=Edit peaks 1 17 9!560 Raw data of 9508*A'lBl Fluoride I.S 409S 4A 'Ci liv 0 8935 Esc=Exit : FI=Help Time Crtl-P=Edit peaks 41 1393S 4095 Raw data of 9SO82181 Fluoride l.S M:Fti.7.1;@MWMM 13310 NE@@ 79-2---Ft?-T(YWG-4-= A.' ij 0 13310 Time Esc=Exit FI=Help Crtl-P=Edit peaks Printout of Sample Table 6329135D Inserted in System Table of 0 iw 1t 2d 3w 4 sl 5 s2 s3 7 s4 8 s5 9 s6 10 s7 11 s8 4n -n Initial Wash Tracer Drift Wash Standard 1 Standard 2 Standard 3 Standard 4 Standard 5 Standard 6 Standard 7 Standard 8 1 1.0000 1 1.000 1 1.000 1 1.000 1 1.000 1 1.000 1 1.000 1 1.000 1.000 1.000 1.000 1.000 1831 o@ group 1 1995-08-22 09:05 OutPut of : 950822Al Software version 6.1 cl990,93 Operator DDW Date of the Analysis : 1995-08-22 06:49 Analysis File Name : C:\SKALAR\DATA\HWIDATA\SERUM\950822Al ow %Izs.@-t(s-b),@ 4mtT ?.o-ns Fluoride 1.5 Calibration order Inverse Logarithm Slope s 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 0.00075 -1.18734 Fluoride L Calibration order 2 Corz-elation r = 0.99917 Result = a2 x2 + al * x + aO a2 = al = aO = -0.00000 0.00027 0.00577 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 1995-08-22 09:05 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 s7 standard : 0.300 S8 standard : 0.600 S9 standard : 1.200 slO standard : 1.500 order : Inverse Logarithm Dimension : PPM start Value : trigger Limit : Peak shape : start ignore : eNd ignore : Measure window : 500 DU 1800 Sec Pointed 60 Sec 120 Sec 75 Filter : No Regeneration :.No formula output Fluoride L Path number Signal type Decolor system Number diluts Resample dil Threshold diG output Window event 0 : Debubbled : No :0 : No : No : 4095 :0 : Off 950822AI 1995-08-22 09:05 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 sa 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 950822AI 1995-08-22 09:05 OutPut of : 950822Al Fluoride 1.5 Fluoride L PPM PPN Pos Typ Ident Ch Result F Time Ch Result F Time wt iw Initial Wash 3 0.065 65 4 0.0058 0 1t Tracer 3 1.461 208 4 0.7660 0 2d Drift 3 1.475 383 4 0.7723 0 3w Wash 3 0.065 616 4 0.0058 0 4 sl Standard 1 3 0.069 729 4 0.0156 0 5 s2 Standard 2 3 0.075 907 4 0.0281 0 6 s3 Standard 3 3 0.093 1083 4 0.0623 0 7 s4 Standard 4 3 0.111 1259 4 0.0911 0 8 s5 Standard 5 3 0.129 1433 4 0.1167 0 9 s6 Standard 6 3 0.157 1609 4 0.1512 0 10 s7 Standard 7 3 0.277 1785 4 0.2587 0 11 ss Standard 8 3 0.621 1959 4 0.4443 0 12 s9 Standard 9 3 1.227 2134 4 0.6732 0 13 slO Standard 10 3 1.469 2309 4 0.7697 0 14 d Drift 3 1.536 2484 4 0.8009 0 15 w Wash 3 0.065 2722 4 0.0058 0 16 u SERUM BLX 1 3 0.085 2837 '4 0.0472 0 17 u SERUM BLK 2 3 0.078 3011 4 0.0334 0 18 u SPK 40-1 3 0.083 3187 4 0.0432 0 19 u SPK 40-2 3 0.090 3361 4 0.0570 0 20 u SPK 40-3 3 0.090 3533 4 0.0575 0 21 u SPK 40-4 3 0.092 3710 4 0.0609 0 22 u SPK 124-1 3 0.105 3886 4 0.0829 0 23 u SPK 124-2 3 0.138 4059 4 0.1283 0 24 u SPK 124-3 3 0.278 4236 4 0.2595 0 25 u SPK 124-4 3 0.129 4412 4 0.1172 0 26 d Drift 3 1.533 4584 4 0.7994 0 27 w Wash 3 0.065 4825 4 0.0058 0 28 u SPK 100-1 3 0.121 4936 4 0.1059 0 29 u SPK 100-2 3 0.141 5112 -4 0.1317 0 30 u SPK 100-3 3 0.137 5286 4 0.1278 0 31 u BLK 3 0.101 5460 4 0.0755 0 32 u BLK 3 0.080 5635 4 0.0390 0 33 u F52978-22 3 0.078 5811 4 0.0334 0 34 u P52980-22 3 0.082 5987 4 0.0424 0 35 u F52991-22 3 0.076 6151 4 0.0291 0 36 u F52987-22 3 0.073 6335 4 0.0249 0 37 u F52988-22 3 0.070 6511 4 0.0169 0 38 d Drift 3 1.532 6685 4 0.7989 0 39 w Wash 3 0.065 6927 4 0.0058 0 40 u F52995-22 3 0.074 7037 4 0.0268 0 41 u SPH 4 6 -CL n67- A 72 :1. 42 u SPK 100-1 3 0.121 7386 4 0.1059 0 43 d Drift 3 1.550 7561 4 0.8082 0 44 w Wash 3 0.065 7803 4 0.0058 0 wt rw RunOut Wash 3 0.065 8036 4 0.0058 0 Page 1 of Calibration curve of 850822Al Fluoride L uN irrort-m-ii ir--rr@47f-f-r-H 6 0.2430511 0. 0073651 64 c ,0pl C' Iu 0 0. 0057706 0 Order 2 tleasured Soo r 0.99917 1.6923981 Calibration curve of 950822AI 6 Fluoride 1.5 0.0656496 c 0 0.0649727 0 Order Measured Inverse Logarithm 409@ S 5996 Raw data of SS08',12AI: Fluoride 1.5 M;Fl;sill4@=IT:m 0 409S 336 F)T-4-tM 64 %IV 17 Esc=Exit Time FI=Help 1 Crtl-P=Edit peaks 1 sooo 4095 Raw data of 95082d'-'A:lFluoride 1.5 4SGO 2628 V)73!!M 64 P4 4560 Time Esc=Exit 1 FI=Help 1 Crtl-P=Edit peaks 1 9560