Document V3gBY8nbowRXXV9RVXDzXb75w

3M EnvironmentaLlaboratory FinalReport-AnalyticSatludy Single-DoDsermalAbsorption/ToxiSctiutdyyofT-6053inRabbits In-VivSotudyReferencNeumber:HWI#6329-137 StudyNumber: AMDT-022195.1 TestSubstanceF:C-99(T-6053) Name andAddressofSponsor: 3M SCD Division 367 Grove Street St.Paul,MN 55106 Name and Address ofTestingFacility: 3M EnvironmentalTechnology& Services 935 Bush Avenue St.Paul,MN 55106 Method Numbers AMDT-M-1-0, AMDT-M-2-0, AMDT-M-4-0, AMDT-M-5-0, AMDT-M-8-0, and Revisions: Thermal Extractionof Fluorideby Means of a Modified Dohrmann DX2000 OrganicHalideAnalyzer-Liver FluorideMeasurement by Means ofan Orion EA940 Expandable Ion Analyzer ExtractionofFluorochemicalsfrom RabbitLiver AnalysisofRabbitLiverExtractforFluorochemicalsUsing ElectrospraMyass Spectrometry Analysisof FluorideUsing the SkalarSegmented Flow Analyzer with Ion SelectivEelectrode InitiatioDnate: See attachedprotocol Author:James D. Johnson Approved By: c meys D-i.,ip*son tuS@tmduesdyD ir*or Completion Date tr 133 1.0 SUMMARY AftedrermalapplicatoifoFnC-99(T-6053t)i,ssuaendserumsamplewsere collecte(dHWI#6329-137). The liverat28 days was combusted and analyzedfor totalorganicfluorine.The resultosf thisanalysishowed no differencebsetween thecontrolsand treatedrabbitsincludingthehigh dose which were rabbitstreated with250 mg/kg (T-6053).Thisformulatioinsa diethanolaminsealtof perfluorooctanesulfonatTew.enty percentofthesesolidsaretheC8. -T-6053 isa solutionthatis0.04% ofFC-99 solids.The dose forthehigh group was 20 ug/kg with respectoC8 perfluorooctanesulfonatTeh.e dose was below detection limitsofthemethod even ifallofthesampleswere absorbed. 2.0 INTRODUCTION This studywas performedinordertoprovidedataforthe assessmentof dermal absorptionofFC-99. Data from otherforms ofperfluorooctanesulfona(tFeC-95, thepotassiumsaltw)ere availabl(eHWI#6329-130,HWI#6329-159). A pharmacokineticstudy(HWI#6329-136) had shown thatperfluorooc@tanesulfonate would be a convenientmarker forFC-99 ifthedosesadministeredwere high enough. 3.0 TEST MATERIALS 3.1Test,Control,and ReferenceSubstancesand Matrices 3.1.1AnalyticalReferenceSubstance:FC-95,lot161 or 171. They are equivalent. 3.1.2AnalyticalReference Matrix: Bovine liverand bovine serum 3.13 AnalyticalControl Substance:None 3.1.4AnalyticalControl Matrix: Bovine liverand bovine serum 3.2 Source of Materials:3M ICP/PCP DivisionforFC-95, bovine liverfrom grocerystoreb,ovine serum from Sigma Chemical Company. 3.3.Purity and Strength of Reference Substance:Responsibilitoyf Sponsor 3.4 Stabilitoyf Reference Substance: To be determinedby Sponsor 3.5 Storage Conditionsfor Test Materials:Room temperatureforFC-95. For biologicaslamplesthestorageis-20+100C. 2 3.6 Dispositionof Specimens: Biologicaltissuesand fluidswillbe retainedper GLP Regulationforthetime periodrequiredforstudieslongerthen 28 days. 4.0 EXPERIMENTAL -Overview Tissueand serum samples were availableforanalysisfrom HWI#6329-137. Liver samples collectedat28 days postdose were analyzedfortotalorganicfluorineby combustion and selectiveion electrode.The datawere used to assessthe extentof dermal absorption. 5.0 EXPER]IMENTAL -METHODS 5.1AMDT-M-1-0, Thermal ExtractioonfFluorideby Means of a Modified Dohrinann DX2000 Organic HalideAnalyzer-Liver 5.2AMDT-M-2-0, FluorideMeasurement by Means of an Orion EA940 Expandable Ion Analyzer 5.3AMDT-M-4-0, Extractionof Fluorochemicalsfrom RabbitLiver 5.4AMDT-M-5-0, Analysisof RabbitLiverExtractforFluorochemicalsUsing ElectrosprayMass Spectrometry 5.5 AMDT-M-8-0, Analysisof FluorideUsing the SkalarSegmented Flow Analyzer with Ion SelectiveElectrode 6.0 DATA ANALYSIS The datafrom combustion and subsequentcombustion analysisfortotalorganic fluorineof liversamples from rabbitsdosed derrnallwyith FC-99 isattached.The data from liversat28 days postdose show no differencebsetween dosing groups. Rabbitswere dosed at0, 10,125,250 mg/kg (0,and about 0.8,10,and 20 uglkg with respectto the C8 perfluorooctanesulfonatTeh)e. formulationof FC-99 isa diethanolaminesaltof perfluorooctanesulfonawtieth about25% solids.Twenty percentof thesesolidsaretheC8. T-6053 isa 0.04% solutionof FC,-99solids.The dose forthehigh group was 20 ug/kg with respecttotheperfluorooctanesulfonate. In previousstudies,FC-95 has been shown to be persistenitn rabbitsafteran intravenousdose (HWI#6329-159), notdermallyabsorbed atdoses of 0.30 mglkg (HWI#6329-130), and suitableas a marker to assessdermal absorption 3 (HWI#6329-159). In a pharmacokineticstudyofFC-99 (HWI#6329-136), itwas concluded thatperfluorooctanesulfonawtoeuld be a usefulmarker forassessmentof dermal absorptionof FC-99. Since FC-99 containsperfluorooctanesulfonathee same as FC-95 butjusta differenstaltform (FC-95 isthepotassiumsalt)i,tis consistenthatno dermal absorptionappearstooccur atthesedose levelsof T-6053. However, ifallof the20 ug/kg dose were absorbed,themethod would nothave been ableto detectit.SinceT-6053 isthe same preparationof FC-99 used in the phartnacokinetisctudy,a directcomparison isappropriate. Other datawas collectedusingelectrospramyass spectrometryand Skalar segmented flow analyzerwith ion selectivelectrode(seeappendices).This data, althoughsupportivei,n theopinionof the Study Directorisnot requiredto reachthe conclusionstatedhere and thereforeisnot discussedin detail. 6.1 Circumstances that May Affectthe Quality ofthe Data: The circumstances thatmay affectthisdataanalysisisthata longerterm phannacokineticsstudyon this saltform of perfluorooctanesulfonawtaes not performed. The dose was very low (below detectionlimits)H.owever, itseems unlikelythattherewould be a substantiadlifferenceinthe pharmacokineticsof two saltforms at28 days especially with respectto depositionin liver. 7.0 CONCLUSION There does not appeartobe dermal absorptionof FC-99 (T-6053)at dermal dose levelsat20 ug/kg. The dose was below thedetectionlimitof the method. 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. 9.0 APPENDICES 9.1 Protocol and Amendments 9.1.1Protocoland FinalReport:HWI#6329-137 "Single-Dose Dermal Absorption/ToxicitSytudy of T-6053 in Rabbits" (Protocoltype TP3016.AB fordosingof animals,tissuecollectione,tc.) 9.1.2 AnalyticalprotocolAMDT-022195.1 0 6-(1-1 4 9.2 SignedReportsfrom IndividuaSlcientistNso:ne 9.3 QualityAssuranceUnitStatementS:eeattached 9.4 Key PersonnelInvolvedintheStudy:Seeattached 9.5 Materialasnd Equipment:Seemethods 9.6 SolutionsR,eagentsa,nd StandardsS:eemethods 9.7 SamplePreparationS:eemethods 9.8 QualityControlPracticesS:eemethods 9.9 TestMethods:SeeProtocoAlMDT-022195.1 9.10InstrumentSettingsS:eemethods 9.11Data:Seeattached. 9.11.1Summary andraw data;ug F-inwholelivearsdeterminebdy thermal extractiofnollowedby analysisusingOrion ion analyzer. 9.11.2Raw data;analysisof liverextractussingelectrospramyass spectrometry. 9.11.3Summary and raw data; ug F-in whole liveras determined by thermal extractiofnollowedby analysisusing Skalarsegmented flow analyzerwith ion selectivelectrode. 00000:31 5 9.1.1Protocoland FinalReport:HWI#6329-137 "Single-DoseDermal Absorption/ToxicitSytudy of T-6053 inRabbits"(ProtocoltypeTP-3016.AB for dosing of animals,tissuecollectione,tc.) HAZLEOCON W IS C 0 N S IN POSF OFFICE 80\ 7545 MAD ISO @l.VV1 53 70 754 5 Sponsor: 3M ToxicologyService Medical Department St. Paul,Minnesota CORNING FINAL REPORT Study Title: Single-DoseDermal Absorption/Toxicity Study of T-6053 in Rabbits Author: Steven M. Glaza Study CompletionDate: July 11, 1995 PerformingLaboratory: HazletonWisconsin,Inc. 3301 KinsmanBoulevard Madison, Wisconsin 53704 LaboratoryProjectIdentification: HWI 6329-137 P h o ne E X P RE 6 0 B 2 4 1 -4 4 7 1 A IL D z-'-IVERY Page I of 45 3 r)I K IPI'@@A BLVD 0()O()()@i F a x 6 0 8 - 2 -I.1 - 7 2 2 7 M A D I S 0 @J. vv 1 53 704 Page 2 of 45 HWI 6329-137 QUALITY ASSURANCE STATEMENT This report has been reviewed by the Quality Assurance Unit of Hazleton Wisconsin, Inc., in accordancewith the Food and Drug Administration (FDA) Good LaboratoryPracticeRegulations,21 CFR 58.35 (b) (6) (7). The following inspectionswere conductedand findingsreported to the Study Directorand management. Written status reports of inspections and findings are issued to Hazleton management monthly according to standard operating procedures. Inspection Dates From To -- Phase Date Reported to Date to Study Director Management 12/21/94 01/25/95 01/30/95 02/10/95 04/10/95 04/20/95 04/20/95 07/10/95 12/21/94 01/26/95 01/30/95 02/10/95 04/10/95 04/20/95 04/20/95 07/10/95 Protocol Review Protocol Amendment Protocol Amendment Necropsy Protocol Amendment Data/Report Review Data Review Report Rereview 12/22/94 01/26/95 01/30/95 02/10/95 04/10/95 04/20/95 04/20/95 07/10/95 01/10/95 02/10/95 02/10/95 03/10/95 05/10/95 05/10/95 05/10/95 08/10/95 Represer(tativeQ,uality Assurance Unit Date Page 3 of 45 STUDY IDENTIFICATION Single-Dose Dermal Absorption/Toxicity Study of T-6053 in Rabbits HWI 6329-137 Test Material Sponsor Sponsor's Representative Study Director Study Locations T-6053 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 InitialTest: Hazleton Wisconsin, Inc. Building No. 3 3802 Packers Avenue Madison, WI 53704 Replacement Animal: Hazleton Wisconsin, Inc. 3301 Kinsman Boulevard Madison, Wl 53704 Study Timetable Study InitiationDate Experimental(In-life)Start Date In-lifeEnd Date Experimental Termination Date Study Completion Date December 30, 1994 January 13, 1995 February 17, 1995 July 11, 1995 July 11, 1995 Page 4 of 45 HWI 6329-137 KEY PERSONNEL Acute Toxicology Ouality Assurance Steven M. Glaza Study Director Manager Sherry R. W. Petsel Manager Francis (Bud) W. McDonald Study Coordinator Patricia Padgham In-lifeSupervisor Laboratory Animal Medicine Cindy J. Cary, DVM Diplomate, ACLAM Supervisor Rose M. Bridge Report Supervisor Anatomical Patholoq-y Toxicology Support Thomas E. Palmer, PhD Anatomical Pathologist Kathy Myers Manager Calvin L. Horton Supervisor Jack Serfort/ Deborah L. Pirkel Supervisors Necropsy Anne Mosher Supervisor Pathology Data ooo(ilLO Page 5 of 45 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 1 Individual and Mean Body Weights (g) 2 Individual Clinical Signs 3 Individual Dermal Irritation Scores 4 Individual Pathology Comments 5 Individual Animal Tissue Weights and Bile Volumes Appendix A Protocol Deviations Protocol TP3016.AB Protocol Amendment No. 1 Protocol Amendment No. 2 HWI 6329-137 Paqe 2 3 4 6 8 8 8 9 10 13 14 14 14 15 16 17 19 23 25 27 28 29 41 44 Page 6 of 45 HWI 6329-137 SUMMARY This study was done to assess the systemicabsorption/toxicityand relative skin irritancyof T-6053when appliedto the skin of rabbits. The study was conducted using three male and three female acclimatedrabbits of the Hra:(NZW)SPFstrain for each treatmentgroup. Groui) Test Material 1 (Control)Distilledwater 2 T-6053 3 T-6053 4 T-6053 Dose Level (mg/kq) oa 10 125 250 Number of Animals Males Females 3 3 3 3 3 3 3 3* a Administeredat a dose volume of 2.0 mL/kg. One animal sacrificedon Day 2 due to possible broken back and replaced with another female animal. The back of each rabbit was clippedfree 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 WrapO and ElastoplastOtape to provide an occlusive dressing for a 24-hour exposureperiod. Clinical observationswere conductedpredose 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 determinedon Day -9 for randomization purposes,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 collected from a marginal ear vein of the animals before experimental initiation(Day 1), approximately24-hourspostdose(Day 2), on Days 4, 8, 15, and 22. In addition,at the timeof necropsyon Day 29, approximatel2y0 mL of blood was obtained from each animal. All samples were centrifugedand separated into serum and cellularfractions. All animalswere euthanizedat terminationof the in-lifephase and necropsied. The whole liver,bile, a section of the dermal applicationsite from all animals,and both kidneys from one male and one female in each group were collected at necropsy and weighed (volumeonly determinedfor bile). The tissuesfrom the female dosed at 250 mg/kg and sacrificedon Day 2 were collectedbut were not weighed. The blood samples (serumand cellularfractions),livers,bile, dermal application sites, and kidneyswere sent frozen to the Sponsorafter terminationof the in-lifephase. Page 7 of 45 HWI 6329-137 Applicationof T-6053 did not result in any test material-relatedchanges in body weight gain. All animalsappearedclinicallynormal throughoutthe study with the exceptionof one Group 4 female animal that was sacrificedon Day 2 due to an injury (possiblebrokenback). This animalwas replacedin the study and the replacementanimal appeared normal throughout the study. No dermal irritationwas observedat the dermal scoring intervalsas a result of the applicationof distilledwater or T-6053 at any of the dose levels. There were no test material-relatedlesions observedat necropsy. Page 8 of 45 HWI 6329-137 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 accordancewith the U.S. Food and Drug Administration'sGood LaboratoryPracticeRegulationsfor Nonclinical LaboratoryStudies, 21 CFR 58, with the exceptionthat analysisof the test material mixture 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 opinion of the Sponsorand study director,the study did not unnecessarily duplicate any previous work. TEST AND CONTROL MATERIALS Identification The test materialwas identifiedas T-6053 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 prepared for 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 Sponsorafter completionof all in-life testing accordingto HWI SOP. Any remainingcontrol material is retained for other testingand will not be discardedafter issuanceof the final report. 000014 Page 9 of 45 HWI 6329-137 Safety Precautions The test and control material handling procedureswere according to HWI SOPs and policies. TEST SYSTEM Test Animal Adult albino rabbits of the Hra:(NZW)SPFstrain were procured from HRP, Inc., Denver, Pennsylvania on December 28, 1994 and maintained at the Hazleton Wisconsin facility at 3802 Packers Avenue, Madison, Wisconsin. The replacement animal was received from HRP, Inc., Kalamazoo, Michigan on January 11, 1995 and was maintainedat the HazletonWisconsin facility at 3301 Kinsman Boulevard, Madison, Wisconsin. Housing After receipt,the animalswere acclimatedfor a period of at least 7 days. During acclimationand throughoutthe study,the animals.wereindividually housed in screen-bottomstainlesssteel cages in temperature-and humiditycontrolledquarters. Environmentalcontrolsfor the animal room were set to maintain a temperatureof 19* to 23*C, a relativehumidity of 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 7ibitum and a measured amount of Laboratory Rabbit Diet HF #5326, PMI Feeds, Inc. The feed is routinely analyzed by the manufacturerfor nutritionalcomponents and environmental contaminants. Samples of the water are periodicallyanalyzed by HWI. There were no known contaminantsin the feed or water at levels that would have interferedwith or affectedthe resultsof the study. Selection of Test Animals The animals were identified by animal number and correspondingear tag and were placed into study groups using a stratifiedbody weight randomization program. The randomizationbody weights were determined on Day -9. The weight variationof the animalsfor each group of each sex selected for the study did not exceed 2 standard deviationsof the mean weight, and the mean body weights for each group of each sex were not statisticallydifferentat the 5% probabilitylevel. One female animal (No. F53454)was replaced after test material exposuredue to a possiblebroken back. This animalwas replacedwith another female animal (No. F53504). 000 0 IL Page 10 of 45 HWI 6329-137 Study Design Animals weighing from 2,311 to 2,609 g at initiation of treatment were placed into the following study groups: Group Dose Level Test Material (mg/kq) 1 (Control) Distilledwater oa 2 T-6053 10 3 T-6053 125 4 T-6053 250 Number of Animals Males Females 3 3 3 3 3 3 3 3* a Administered at a dose volume of 2.0 mL/kg. One animal sacrificed on Day 2 due to a possible broken back and replaced with another female animal. This animal weighed 2,352 g at initiation. 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 unblemished skin), 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 appliedevenly to the test site at a rate of approximately 0.05 mL/cm ()()0016 Page 11 of 45 HWI 6329-137 Groui)s2,-3, and 4. For the Group-2 animals (10 mg/kg), the test material (T-6053)was mixed with distilledwater to a concentrationof 100 mg/mL and applied at a dose volume of 0.1 mL/kg. This mixture was stored at room temperatureuntil administered. The test materialwas administered undilutedto the test sites of the Groups 3 and 4 animals (125 or 250 mg/kg, respectively)using the averagebulk densityof 0.975 g/mL to determine the dose volume for each dose level (0.13 and 0.26 mL/kg, respectively).An individualdose of the respectivetest materialor test material mixture was calculatedfor each animal based on its body weight on the day of treatment. The area of exposurefor the 10, 125, and 250 mg/kg dose levels was 4, 16, and 25 cm2 , respectively. The approximaterate of applicationranged from 0.02 to 0.06 mL/CM2. Each area of applicationwas covered with a 10-cm x 10-cm gauze bandage secured with paper tape around all edges and overwrapped with Saran WrapD and ElastoplastOtape to providean occlusivedressing. Collarswere used to restrain the animals during the 24-hour exposure period. Approximately24 hours after test or controlmaterialapplication,the restraining collars and bandageswere 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 potentialroute of exposure in humans. Observations of Animals Clinical observationswere conducted predose and at approximately1, 2.5, and 4 hours aftertest or controlmaterialadministration.Additionalclinical observations and twice a day mortality checks (morningand afternoon)were conducted daily thereafterfor 28 days. Body weights were determinedfor randomizationpurposes on Day -9, before test materialadministration(Day 1), and at in-lifetermination(Day 29). The initialdermal irritationreadingwas made before test or controlmaterial administrationaccordingto the Draizeltechnique(recordedas the Day I reading). Subsequentreadingsof dermal irritationwere made approximately30 minutes after bandage removal (Day 2) and on Days 4 and 8. Sample Collections Blood samples (approximately4 mL) were collectedfrom a marginal ear vein of all animalsbefore experimentalinitiation(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 0()0017 Page 12 of 45 HWI 6329-137 20 mL of blood was obtained from the posterior vena cava of each animal. All samples were centrifuged and separated into serum and cellular fractions. These samples were then stored in a freezer set to maintain a temperature of -200C IOOC until shipped to the Sponsor. Pathology At termination of the experimental phase (Day 29), animals were anesthetized with sodium pentobarbital, bled via the posterior vena cava, exsanguinated, and necropsied in random order. The sites of test and control material application were washed with lukewarm tap water before the necropsy procedure. All animals were subjected to an abbreviated gross necropsy examination and any abnormalitieswere recorded. The whole liver, bile, a section of the dermal application site from all animals, and both kidneys from the first male and female in each group were collected. The tissue samples for all animals, with the exception of the animal sacrificed and necropsied on Day 2, were weighed (volume only determined for bile). The samples were immediately placed on dry ice (initialtest animals only), then placed in a freezer set to maintain a temperature of -20*C 10*C. After necropsy, the animals were discarded. Shipment of Blood, Bile, and Tissues After in-life termination,the blood samples (serum and cellular fractions), livers, bile, dermal applicationsites, and kidneys were sent frozen (on dry ice) to the Sponsor (James D. Johnson, 3M E.E. & P.C., Bldg. 2-3E-09, 935 Bush Avenue, St. Paul, MN, 55106), along with their corresponding weights or volumes. The Sponsor is responsible for the retention and disposition of the samples. HWI does not accept any responsibilityfor the analysis of the tissue samples collected in this study nor are these results presented in this report. Statistical Analyses No statistical analyses were required by the protocol. Location of Raw Data, Records, and Final Report The raw data, records, and an original signed copy of the final report will be retained in the archives of HWI in accordance with HWI SOP. Page 13 of 45 HWI 6329-137 RESULTS Body Weights Individualand mean body weights are in Table 1. All animalsexhibitedbody weight gains from Day I to Day 29. Clinical Observations Individualclinical signs are in Table 2. All animals appearednormal throughoutthe study with the followingexception: One Group 4 female (No. F53454) treated with T-6053 at 250 mg/kg appeared to have injuredits back at the time of the 24-hourblood collection. This animal was sacrificed,necropsied,and replacedwith No. F53504. The replacement animal appeared normal throughout the study. Dermal Irritation Individualdermal irritationscoresare in Table 3. The controlmaterial producedno dermal irritation. No dermal irritation.wasobservedin the animalstreated with T-6053 at any of the dose levels. Pathology Individualanimal pathologycommentsare presented in Table 4. Individual animal tissue weights and bile volumesare in Table 5. The necropsy of the Group 4 female (No. F53454),treatedwith T-6053 at 250 mg/kg and sacrificed on Day 2, revealeda fractureof the first lumbar vertebra. There were no lesions observed in any of the remaininganimals. Page 15 contains a pathologyreport by the study pathologist. ooool!) Page 14 of 45 HWI 6329-137 DISCUSSION The acute systemicabsorption/toxicitaynd relativeskin irritancyof T-6053 were evaluated in male and female albino rabbitswhen administeredas a single dermal application. There were no test material-relatedchanges in body weight gain or in-lifeclinicalfindingsat any of the dose levels. No test material-relateddermal irritationwas observedduring the study. SIGNATURE Steven M. Glaza Study Director Acute Toxicology ----?t k-q @5 Date REFERENCE 1. Draize, J. H., "Acute Dermal Toxicity (Single Exposure)," In: Appraisa7 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 45 HWI 6329-137 PATHOLOGY REPORT There were six rabbits (threemales and three females)each from four dose levels of 0, 10, 125, and 250 mg/kg of body weight euthanized and necropsied at the terminationof the study. One female (AnimalNo. F53454)dosed at 250 mg/kg of body weight was sacrificedon Day 2 because of an apparent broken back. This animal was replacedwith anotherfemale (AnimalNo. F53504). The test material,dose level, day of death, and gross observationsrecorded for each animal are in the IndividualPathologyCommentsthat follow this report. At necropsy,there were no visiblelesionsin any of the animals sacrificedat study termination. The liver, bile, an approximate1-cm x 1-cm section of the dermal applicationsite from each of these animals,and both kidneys from one male and one female in each group were collected. The tissue samples were weighed (volumeonly determinedfor bile), frozen, and sent to the Sponsor. The tissues from the female (dosedat 250 mg/kg) sacrificedon Day 2 were collected but were not weighed. After necropsy, the animals were discarded. The animal sacrificedon Day 2 had a fractureof the first lumbar vertebra. This findingcorrelatedwith the clinicalobservationof an apparentbroken back. .of Tho t. eaimer.'P-h'D-@ Date Pathologist (6329-137.slh) 040695 Animal Number F53464 F53441 F53451 Mean F53447 F53463 F53452 Mean F53453 F53458 F53446 Mean F53459 F53445 F53439 Mean Page 16 of 45 Table 1 Individualand Mean Body Weights (g) HWI 6329-137 Male Randomization Day -9 Day 1 29 Animal Number Female Random- ization Day Day -9 1 29 Group 1 (Control)- DistilledWater (0 mg/kq) 2,210 2,253 2,285 2,378 2,413 2,536 2,923 3,030 3,058 F53461 F53455 F53442 2,428 2,347 2,437 2,597 2,484 2,456 2,891 2,929 3,097 2,249 2,442 3,004 2,404 2,512 2,972 Group 2 - T-6053 (10 mg/kq) 2,241 2,207 2,085 2,539 2,464 2,362 2,977 2,937 2,777 F53444 F53448 F53460 2,358 2,476 2,411 2,338 2,486 2,516 2,798 2,944 2,972 2,178 2,455 2,897 2,415 2,447 2,905 Group 3 - T-6053 (125 mg/kg) 2,275 2,265 2,156 2,497 2,425 2,323 2,944 2,863 2,743 F53443 F53467 F53450 2,434 2,476 2,427 2,544 2,581 2,537 2,844 2,965 3,003 2,232 2,415 2,850 2,446 2,554 2,937 Group 4 - T-6053 (250 mg/kq) 2,221 2,090 2,206 2,338 2,311 2,380 2,742 2,798 2,942 F53449 F53456 F53454 a F53504c 2,297 2,220 2,424 NA 2,390 2,336 2,609 2,352 2,660 2,782 2,627' 2,939 2,172 21343 2,827 21314 2,359 2,794 NA Not available. a Animal No. F53454 was originallyselected by the randomizationprogram for use in the study and was treated. This animalwas sacrificedafter completion of the exposure period due to a possible broken back and was replaced with No. F53504. The body weights for No. F53454 are not included in the Day I or Day 29 group means. b Day 2 body weight. c Replacement animal (not included in group assignmentrandomization). -Sex Male Female Page 17 of 45 HWI 6329-137 Table 2 IndividualClinicalSigns Animal Number Observation 1-4 Hours Day (Day 1) 2 3 through 29 Group I (Control)- Distilled Water (o Mq/kq) F53464 Appeared normal t F53441 Appeared normal t F53451 Appeared normal F53461 Appeared normal F53455 Appeared normal t F53442 Appeared normal V/ Male Female F53447 F53463 F53452 F53444 F53448 F53460 Group 2 - T-6053 Appeared normal Appeared normal Appeared normal Appeared normal Appeared normal Appeared normal (10 mqlkq) Condition existed. 00 Sex Male Female Page 18 of 45 Animal Number F53453 F53458 F53446 F53443 F53467 F53450 Table 2 (Continued) IndividualClinical Signs Observation 1-4 Hours (Day 1) Group 3 --T-6053 (125 mg/kq) Appeared normal Appeared normal Appeared normal Appeared normal Appeared normal Appeared normal HWI 6329-137 Day 2 3 through 29 Male Female Groug 4 - T-6053 (250 mg/kq) F53459 Appeared normal F53445 Appeared normal F53439 Appeared normal F53449 Appeared normal F53456 Appeared normal F53454 Appeared normal Possible broken back (at time of 24-hour bleeding interval) Moribund sacrifice F53504a Appeared normal Condition existed. Condition not evident. a Animal No. F53504 replaced Animal No. F53454. Page 19 of 45 HWI 6329-137 Table 3 IndividualDermal IrritationScores Group I (Control)- DistilledWater (0 mg/kg) Dermal Reaction Males Study Day 1 _L 4 8 Females Study Day 1 2 4 8 Animal No. F53464 Animal No. F53461 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. F53441 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. F53455 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. F53451 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. F53442 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 oooo;;5 Page 20 of 45 HWI 6329-137 Table 3 (Continued) IndividualDermal IrritationScores Dermal Reaction Erythema Edema Atonia Desquamation Coriaceousness Fissuring Erythema Edema Atonia Desquamation Coriaceousness Fissuring Erythema Edema Atonia Desquamation Coriaceousness Fissuring Group 2 - T-6053 (10 mg/kg) Males Females Study Day Study Day -1 -L -L -.9.- 1 2 4 -8 Animal No. F53447 Animal No. F53444 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 Animal No. F53463 Animal No. F53448 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 Animal No, F53452 Animal No. F53460 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 Page 21 of 45 HWI 6329-137 Table 3 (Continued) IndividualDermal IrritationScores Group 3 - T-6053 (125 mg/kg) . Dermal Reaction Males Study Day 1 2 4 8 Animal No. F53453 Females Study Day 1 2 4 8 Animal No. F53443 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 Animal No. F53458 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. F53467 Erythema Edema Atonia Desquamation Coriaceousness Fissuring 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 Animal No. F53446 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 Animal No. F53450 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 Page 22 of 45 HWI 6329-137 Table 3 (Continued) IndividualDermal IrritationScores Dermal Reaction Erythema Edema Atonia Desquamation Coriaceousness Fissuring Erythema Edema Atonia Desquamation Coriaceousness Fissuring Erythema Edema Atonia Desquamation Coriaceousness Fissuring Group 4 - T-6053 (250 mg/kg) Males Study Day -1 4 8 Females Study Day 1 2 4 -8 Animal No. F53459 Animal No. F53449 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 Animal No. F53445 Animal No. F53456 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 Animal No. F53439 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. F53454a 0 0 0 0 0 0 0 0 0 0 0 0 Animal No. - - - - - - - - - - - - F53504 b 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 a Animal replaced with No. F53504. b Replacement animal for No. F53454. - Not applicable. Animal Number F53464 F53441 F53451 F53461 F53455 F53442 F53447 F53463 F53452 F53444 F53448 F53460 Page 23 of 45 HWI 6329-137 Table 4 Individual Pathology Comments Test Day Sex Died Sacrificed Necrapsy Observation Group I (Control)- DistilledWater (0 mg/kq) m - 29 No visible lesions. m - 29 No visible lesions. m - 29 No visible lesions. F - 29 No visible lesions. F - 29 No visiblelesions. F - 29 No visiblelesions. Groug 2 - T-6053 (10 mqlkq) m - 29 No visiblelesions. m - 29 No visiblelesions. m - 29 No visiblelesions. F - 29 No visiblelesions. F - 29 No visiblelesions. F - 29 No visiblelesions. Not applicable. Page 24 of 45 HWI 6329-137 Table 4 (Continued) Individual Pathology Comments Animal Test Day Number Sex Died Sacrificed Necropsy Observation Group 3 - T-6053 (125 mg/kq) F53453 m - 29 No visiblelesions. F53458 m - 29 No visiblelesions. F53446 m - 29 No visiblelesions. F53443 F - 29 No visiblelesions. F53467 F - 29 No visiblelesions. F53450 F - 29 No visiblelesions. F53459 m F53445 m F53439 m F53449 F F53456 F F53454a F F53504 b F Group 4 - T-6053 (250 mg/kq) - 29 No visible lesions. - 29 No visible lesions. - 29 No visible lesions. - 29 No visible lesions. - 29 No visible lesions. - 2 The first lumbar vertebrais fractured. - 29 No visiblelesions. - Not applicable. a Animal replaced with No. F53504. b Replacement animal for No. F53454. Sex Male Female Page 25 of 45 HWI 6329-137 Table 5 Individual Animal Tissue Weights and Bile Volumes Animal Number Liver Weight (g) Dermal Appli- Kidneys cation Site Bile Volume (mL) GrouR 1 (Control)- DistilledWater (0 mg/kq) F53464 87.407 - 0.468 0.3 F53441 114.827 16.722 0.331 2.5 F53451 89.123 - 0.394 0.3 F53461 73.175 - 0.371 0.7 F53455 94.647 16.740 0.512 i.o F53442 91.590 - 0.391 1.5 Group 2 T-6053 (10 mg/kq) Male F53447 81.754 - 0.213 2 a F53463 83.148 15.119 0.357 0.6 F53452 82.687 - 0.535 0.3 Female F53444 70.587 F53448 74.702 - 0.719 0.7 0.361 0.7 F53460 92.881 15.925 0.368 0.7 - Not applicable. a Volume not measured to tenths of a mL. .ot)oo3lL Sex Male Female Page 26 of 45 HWI 6329-137 Table 5 (Continued) IndividualAnimal Tissue Weights and Bile Volumes Animal Number F53453 F53458 F53446 F53443 F53467 F53450 Liver Weight (g) Dermal Appli- Kidneys cation Site Group 3 - T-6053 (125 mg/kq) 96.249 77.755 - 0.612 0.390 83.574 79.767 17.429 - 0.321 0.602 78.684 79.782 15.937 - 0.467 0.580 Bile Volume (mL) 1.5 0.5 0.7 0.6 0.7 2a Group 4 - T-6053 (250 mg/kg) Male F53459 79.255 15.567 0.372 0.4 F53445 97.381 - 0.270 0.5 F53439 83.548 0.349 0.5 Female F53449 73.983 - 0.391 0.4 F53456 77.804 14.412 0.572 1.0 F53454' NA - NA NA F53504c 83.081 1.253 1.1 Not applicable. a Volume not measured to tenths of a mL. b Moribund sacrificeon Day 2 (01/14/95)due to possible broken back. Tissues were collected,however,their correspondingweights or volumes were not taken since this requirementbecame effective on 01/24/95. Animal replaced with No. F53504. c Replacement animal for No. F53454. Page 27 of 45 HWI 6329-137 APPENDIX A Protocol Deviations Protocol TP3016-AB Protocol Amendment No. 1 Protocol Amendment No. 2 ooo0a.@3 Page 28 of 45 Protocol Deviations HWI 6329-137 Protocol Page 8, 7. Experimental Design, C. Observation of Animals, (4) Sample Collections, (c) Method of Collection, Second Sentence. Approximately 20 mL of blood (actual volume to be documented in the raw data) will be obtained from the posterior vena cava of each animal sacrificed in a moribund condition or sacrificedat the time of necropsy (Day 29). Actual Procedure Blood was inadvertently not collectedat the time of necropsy from the female animal (No. F53454) treated at 250 mg/kg that was sacrificed on Day 2 due to an apparent broken back. Page 8, 7. Experimental Design, D. Pathology, (1) Unscheduled Sacrifices and Deaths, Second Sentence. Animals in a moribund condition will be anesthetized with sodium pentobarbital(via injectionin the marginal ear vein), bled via the vena cava, and exsanguinated. The female animal (No. F53454) treated at 250 mg/kg was sacrificed on Day 2 due to an apparent broken back using an overexposure to carbon dioxide. Page 9, 7. Experimental Design, D. Pathology, (3) Sample Collection, Second Sentence. The whole liver, bile, an approximate 1-cm x 1-cm section of the dermal applicationsite from all animals, and both kidneys from the first male and female necropsied in each group will be collected and immediately placed in a freezer set to maintain a temperature of -200C IOOC. The size of the dermal applicationsite collected at necropsy was inadvertently not documented for the female animal (No. F53454) treated at 250 mg/kg that was sacrificed on Day 2 due to an apparent broken back. Also, the tissues collected from the animalsin the initialtest were placed on dry ice (during transportation)prior to being stored in a freezer. These deviations are not considered to have had an adverse effect on the outcome of the study. oo00,3,1- HAZLE-CON W W IS C 0 N S IN POST OFFICF BOX 7545 MADISON. Wi 53707-7545 Page 29 of 45 1 CORNING Coinpifiy St)onsor: 3M Toxicology Service Medical Department St. Paul, Minnesota PROTOCOL TP3016.AB Study Title: Single-Dose Dermal Absorption/Toxicity Study of T-6053 in Rabbits Date: December 30, 1994 Performing Laboratory: Hazleton Wisconsin, Inc. 3301 Kinsman Boulevard Madison, Wisconsin 53704 LaboratoryProject Identification: HWI 6329-137 0()003:5 P h orie 6 0 8 2 4 1 4 4 7 1 E X P R E S S -M A IL D E L IV E R Y 3301 K IN S M A N 8 L V D F ax MADISON 6 08 @j-@ 2,1 1 1227 /,).l Page 30 of 45 STUDY IDENTIFICATION TP3016.AB Page 2 Single-Dose Dermal Absorption/ToxicityStudy of T-6053 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 6329-137 T-6053 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 Stev@-n M. Glaza Hazleton Wisconsin, Inc. P.O. Box 7545 Madison, Wl 53707-7545 (608) 241-7292 Hazleton Wisconsin, Inc. Building No. 3 3802 Packers Avenue Madison, Wl 53704 January 13, 1995 February 10, 1995 March 24, 1995 Page 31 of 45 TP3016. AB Page 3 1. Study Single-Dose Dermal Absorption/ToxicityStudy in Rabbits 2. Purpose To assess the systemic absorptionand toxicity and relative skin irritancyof a test materialwhen applied to the skin of rabbits 3. Regulatory Compliance This study will be conducted in accordance with the following Good Laboratory Practice Regulations/Standards/Guidelines: f ] 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 report will 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-6053 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. Stoomraoe Ro temperature 0()0037 Page 32 of 45 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 stored at HWI in a freezerset to maintain a temperatureof -200C tlo*C for 10 years per HWI SOP. The Sponsorwill be contactedafter 10 years for dispositionin accordancewith the appropriate regulatoryGood LaboratoryPractices. F. Retention Any unused test materialwill be returnedto the Sponsorafter completionof the in-lifephase of the study. G. Safety Precautions As required by HWI SOPs and policies 6. ControlMaterial A. Identification Distilledwater B. PhysicalDescription Clear,colorlessliquid C. Purityand Stability The purityand stabilityof this manufacturedmaterialis consideredto be adequatefor the purposes of this study. D. Storage Conditi-ons Room temperature E. Reserve Samples See Section 5. E. Reserve Samples F. Retention Any remainingcontrolmaterialmay be used for other testing and will not be discardedafter issuanceof the final report. G. Safety Precautions As required by HWI SOPs and policies 7. Experimental DesicLn A. Animals (1) Species Rabbit (2) Strain/Source Hra:(NZW)SPF/HRP,Inc. Page 33 of 45 TP3016.AB Page 5 (3) Age at Initiation Adult (4) Weight at Initiation 2.0 to 3.0 kg (5) Number and Sex 12 males and 12 females (6) Identification Individual numbered ear tag (7) Husbandry (a) Housing Individually,in screen-bottomstainless steel cages (heavy gauge) (b) Food A measured amount of Laboratory Rabbit Diet HF #5326 (PMI Feeds, Inc.). The food is routinelyanalyzedby the manufacturerfor nutritionalcomponents and environmental contaminants. (c) Water Ad 7ibftum from an automatic system. Samples of the water are analyzed by HWI for total dissolved solids, hardness, and specifiedmicrobiologicalcontent and for selected elements, heavy metals, organophosphates, and chlorinated hydrocarbons. (d) Contaminants There are no known contaminants in the.food or water that would interferewith this study. (e) Environment Environmentalcontrols for the animal room will be set to maintain a temperature of 19*C to 23*C, a relative humidity of 50% +20%, and a 12-hour light/12-hourdark cycle. (f) Acclimation At least 7 days (8) Selection of Test Animals Based on health and body weight according to HWI SOPS. An adequate number of extra animals will be purchased so that no animal in obviouslypoor health is placed on test. The animals will be placed into study groups using a stratified body weight randomizationprogram within nine days of study initiation. 0()003!) Page 34 of@45 TP3016.AB Page 6 (9) Justificationfor SRecies Selection Historically,the New Zealand White albino rabbit has been the animal of choice because of the large amount of background informationon this species. B. Dose Administration (1) Test Groups Group Test Material I (Control)Distilledwater 2 T-6053 3 T-6053 4 T-6053 Dose Level (mg/kq) 0* 10** 125 250 Number of Animals Males Females 3 3 3 3 3 3 3 3 To be administered at a dose volume of 2.0 mL/kg To be administered at a dose volume of .01 mL/kg (2) Preparation of Exposure Area On the day before test material application,the back and, if necessary(to obtain unblemishedskin), the flanks of each rabbit will be clipped free of hair. The shaved area will constituteapproximately20Y*of the total body surfacearea. The treatmentsites (intactskin) will be inspectedfor interferinglesions,irritation,or defects that would preclude the use of any of the animals. The animals will be clipped as,needed throughout the study. (3) Dose Administration All animalswill receive a single administrationof the -respectivetest or control material. The day of treatment will be designatedas Day 1. The dose for each animal in Group 2 will be diluted with distilledwater and applied at a dose volume of .01 mL/kg. The respective dose for each animal in Groups 3 and 4 will be applied undiluted. All doses in Groups 1-4 will be based on the animal's body weight just before administrationand will be spread onto ,.thearea of exposure in a thin and uniform a layer. The area of application(Groups 1-4) will be coveredwith a 10-cm x 10-cm gauze bandage secured with Raper tape around all edges and overwrapped with Saran Wrap and Elastoplasto tape to provide an occlusive dressing. The rabbits will be collaredduring the 24-hour applicationperiod. (4) Reason for Route of Administration The dermal route is a potentialroute of exposure in humans. 0()00,i0 Page 35 of 45- TP3016.AB Page 7 (5) Removal of Test Material Approximately24 hours after test or controlmaterial 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 clinical signs 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 controlmaterial administrationthe initial dermal irritationreading will be made and recordedas the Day I reading (Attachment1). Additionaldermal irritation readings will be made approximately30 minutes after bandage removal (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 unscheduled death (when survival exceeds 1 day) (4) SamRle Collections (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 oooo-Il Page 36 of 45 TP3016.AB Page 8 (c) 4ethod of Collection Blood samples (approximately4 mL) will be collected from the marginal ear vein of either ear on Days 1, 2, 4, 8, 15, and 22. Approximately20 mL of blood (actualvolume to be documented in the raw data) will be obtained from the posterior vena cava of each animal sacrificedin a moribund condition or sacrificedat the time of necropsy (Day 29). The samples will be stored at room temperature and then centrifuged,and the separate serum and cellular fractionsstored in a freezer set to maintain -20*C tlO*C. The separated serum and cellular fractionswill be sent frozenon dry ice to the Sponsor after experimentaltermination. Samples will be shipped to: James D. Johnson 3M E.E. & P.C. Bldg. 2-3E-09 935 Bush Avenue St. Paul, MN 55106 James D. Johnson or alternatewill be notified by telephoneat (612) 778-5294prior to the shipment of the samples. D. Pathology (1) Unscheduled Sacrifices and Deaths Any animal dying during the study or sacrificedin a moribund condition will be subjected to an abbreviated gross necropsy examination and all abnormalitieswill be recorded. Animals in a moribund conditionwill be anesthetizedwith sodium pentobarbital(via injectionin the marginal ear vein), bled via the vena cava, and exsanguinated. Tissues, as described in section D. Pathology,(3) Sample Collection,will be collected. After necropsy,the animals will be discarded. (2) Scheduled Sacrifice At terminatioonf the experimentaplhase(Day29), surviving animals will be anesthetizedwith sodium pentobarbital(via injectionin the marginalear vein), bled via the vena cava, exsanquinated,and subjected to an abbreviatedgross necropsy examination. The animals will be necropsied in random order and all abnormalitieswill be recorded. o o 0,.'. -Page 37 of 45- TP3016.AB Page 9 (3) Sample Collection The sites of test and control material applicationwill 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 first male and female necopsied in each group will be collected and immediately placed in a freezer set to maintain a temperatureof -20*C tlO*C. After necropsy, the animalswill be discarded. The tissues(liver,bile, dermal applicationsite, kidneys)will be sent frozen on dry ice to the Sponsor after experimentaltermination. The sampleswill be shippedto the person listed in Section7.C.(4).(c).The Sponsor is responsiblefor the retentionand disposition of the samples. E. Statistical-Analyses No statisticalanalyses are required. 8. Report A final report includingthose items listed below will be submitted. Descriptionof the test and control materials Descriptionof the test system Procedures Dates of experimentalinitiationand termination Tabulation of mortality data by sex and dose level Descriptionof any toxic effects/dermalirritation Tabulation of mean body weights by sex and dose level Gross pathology findings/grosspathology report 9. Location of Raw Data, Records, and Final Report Originaldata, or copies thereof,will be availableat HWI to facilitateauditing the study during its progress and before acceptanceof the final report. When the final report is completed, all originalpaper data, includingthose item listed below will be retained in the archives of HWI according to HWI SOP. Protocol and protocol amendments Dose preparation records In-liferecords Body weights Dose administration Observations Anatomical pathology records Sample collection records Shipping records Study correspondence Final report (originalsignedcopy) Page 38 of 45TP3016.AB Page 10 The following supportingrecords will be retained at HWI but will not be archived with the study data. Animal receipt/acclimationrecords Water analysis records Animal room temperature and humidity records Refrigerator and freezer temperature records Instrument calibration and maintenance records 0()001.11'1 Page 39 of 45-. PROTOCOL APPROVAL TP3016.AB Page 11 John L. Butenhoff,PhD Date Sponsor's Representative 3M Toxicology Service Medical Department Ttevin M. Glaza Study Director Acute Toxicology Hazleton Wisconsin, Inc. iz 5b-ei3. Date presentative Date Qu-alityAssurance Unit Hgzleton Wisconsin,Inc. (6329-137.protdsk2) 000045 Page 40 of 45- TP3016.AB Page 12 Attachment I Scoring Scale for Acute Dermal Reactions Erythema 0 - None I - Slight 2 - Moderate 3 - Severe Edema 0 - None I - Slight (barelyperceptibleto well definedby definiteraising) 2 - Moderate (raised approximately1 mm) 3 - Severe (raised more than I mm) Atonia 0 - None I - Slight(slightimpairmentof elasticity) 2 - Moderate (slow return to normal) 3 - Marked (no elasticity) Desouamation 0 - None 1 - Slight(slightscaling) 2 - Moderate (scalesand flakes) 3 - Marked (pronouncedflaking with denuded areas) Coriaceousness 0 - None I - Slight(decreasein pliability) 2 - Moderate (leatherytexture) 3 - Marked (toughand brittle) Fissuring 0 - None 1 - Slight (definitecracks in epidermis) 2 - Moderate (cracks in dermis) 3 - Marked (crackswith bleeding) Page 41 o@ 45 HwAZILS*CC-0mNC(SONI N POST OFFICE BOX 7545 MADISON, Wl @j3707 7545 a CCMNING Comp,)ny PROTOCOL TP3016.AB Single-DoseDermal Absorption/ToxicityStudy of T-6053 in Rabbits HWI 6329-137 Sponsor 3M Toxicology Service Medical Department 3M Center, Bldg. 220-2E-02 P.O. Box 33220 St. Paul, MN 55133-3220 Sponsor's Representative John L. Butenhoff,PhD Contractor Hazleton Wisconsin,Inc 3301 Kinsman Boulevard Madison, WI 53704 Study Director Steven M. Glaza Amendment No. 1 This amendmentmodifiesthe followingportionsof the protocol: EffectiveJanuary 13, 1995 1. Page 6. 7. ExperimentalDesign;B. Dose Administration;-J-IT)est Groups. The test materialmixture could not be preparedat the concentration needed to utilizea dose volumeof .01 mL/kg. Modify the double asteriskedfootnote(**) in this sectionwith the followingunderlined change: ** To be administeredat a dose volume of .10 mL/kg 2. Page 6. 7. ExperimentalDesign;B. Dose Administration;(3) Dose Administration.The test materialmixturecould not be preparedat the concentrationneededto utilizea dose volume of .01 mL/kg. Modify the third sentencein this sectionwith the followingunderlinedchange: The dose for each animalin Group 2 will be dilutedwith distilled water and applied at a dose volume of .10 mL/kg. P h o iie EXPRESS 6 0 8 2 4 1 -4 4 7 1 NIAIL D E L IV E R Y 330 1 KINSMAN B LV D Fa x tvIADISON. 000047 6 0 8 24 1 72 2 7 I/V 1 53 104 Page 42 of 45 Amendment No. I HWI 6329-137 Page 2 Effective January 17, 1995 3. Page 6, 7. ExperimentalDesign: B. Dose Administration:(3) Dose @dministration. Animal No. F53454 (Group 4 female) was sacrificed on Day 2 due to an injury (apparentbroken back). Add the following as the second paragraph to this section: Due to the sacrifice on Day 2 of one Group 4 female (AnimalNo. F53454)because of an injury (apparentbroken back), a replacement animal will be treated at the same dose level in the same manner as for the other animals in the study. The observations(clinicalobservations,reading of dermal irritation, body weights and sample collections) and the pathology of the animals (unscheduledsacrificesand deaths, scheduled sacrifice, and sample collection)will be conducted in the same manner as for the other animals in the study. Effective January 24, 1995 At the request of the Sponsor, the weights of tissues collected and the volume of bile collectedwill be documentedin the raw data. These weights and volumes will be includedwith the sample shipment. Modify the following sections of the protocolto includethese additions. 4. Page 9. 7. ExperimentalDesign; D. Pathology; (3) Sample Collection. Modify the second sentence in the first and second paragraphs of this section with the followingunderlinedadditions: The whole liver, bile, an approximate 1-cm x 1-cm section of the dermal applicationsite from all animals,and both kidneys from the first male and female necropsied in each group will be collected,weighed (volumeonly determinedfor bile) and immediatelyplaced in a freezer set to maintain a te"m-perature of -200C IOOC. The samples and their correspondingweights or volumes will be shippedto the personlisted in Section 7.C.(4).(c). 5. Page 9. 8. Report. Add the followingto this section: Individual animal tissue weights and bile volumes Amendment No. I Page 43 o@ 45PROTOCOL AMENDMENT APPROVAL John L. Butenhoff, PhD Date Sponsor's Representative 3M Toxicology Service Medical Department @@@ % - hsd-@ Steven M. Glaza Date Study Director Acute Toxicology Hazleton Wisconsin, Inc. Representativ Date Quality Assur nce Unit Hazleton Wisconsin, Inc. (6329-137.Aml.dsk2) HWI 6329-137 Page 3 7'@117 0()00,1!) Page 44 of 45 HAZLEECON @F4lV@ W I S C 0 N S I N P 0 s f 0 1 f I('f f x II MADIC @ntjwi .1CORNING PROTOCOLTP3016.AB Single-Dose Dermal Absorption/ToxicityStudy of T-6053 in Rabbits HWI 6329-137 Sponsor 3M Toxicology Service Medical Department 3M Center, Bldg. 220-2E-02 P.O. Box 33220 St. Paul, MN 55133-3220 Sponsor's Representative John L. Butenhoff, PhD Contractor Hazleton Wisconsin, Inc. 3301 Kinsman Boulevard Madison, Wl 53704 Study Director Steven M. Glaza Amendment No. 2 This amendment modifies the following portion of the protocol: Effective January 20, 1995 1. P-age2. Study Location., The replacement animal was treated and maintainedat the main facilityof HazletonWisconsin, Inc., 3301 Kinsman Boulevard, instead of at Hazleton Wisconsin's Acute Studies Laboratory, 3802 Packers Avenue, since the replacement animal was receivedand acclimatedin this facility. Replace this sectionwith the following: Study Location (Init-laTlest) Hazleton Wisconsin, Inc. Building No. 3 3802 Packers Avenue Madison, WI 53704 Study Location (Replacement Animal) Hazleton Wisconsin, Inc. 3301 Kinsman Boulevard Madison, WI 53704 p@i0 rie 6 0 8 2,1 E X P FiE 1;S .1 1 i iL) K iti @ll ri iiI oooo5o Fa M A D IS 0 @l @j 6 @',l1 2 2 I i/0 4 Amendment No. 2 Page 45 of 45 HWI 6329-137 Page 2 PROTOCOL AMENDMENT APPROVAL 2. John L. Butenhoff, PhD Date Sponsor's Representative 3M Toxicology Service Medical Department Steven M. Glaza Study Director Acute Toxicology Hazleton Wisconsin, Inc. x.-@ Representate Quality Asstrance Unit Hazleton Wisconsin, Inc. (6329-137.Am2.dsk3) Date @,1/:7-q5- Date 0 0 0%8-11- 9.1.2 AnalyticalprotocolAMDT-022195.1 o .0 () o 0 @l- *Aq 3M Environmentalaboratory Protoco-lAnalyticSatludy Single-DosDeermal Absorption/ToxicSituydyofT-6053inRabbits In-VivoStudyReferenceNumber: HWI#6329-137 StudyNumber: ANMT-022195.1 TestSubstance:FC-99 (T-6053) Name and AddressofSponsor: 3M SCD Division 367 Grove Street St.Paul,NIN 55106 Name and Address of TestingFacility: 3M EnvirorumentalTechnology and Services 935 Bush Avenue St.Paul,MN 55106 Proposed InitiatioDnate: July25, 1995 Proposed Completion Date: August 25, 1995 Method Numbers AMDT-M-1-0, AMDT-M-2-0, AMDT-M-4-0, AMDT-M-5-0, AMDT-M-8-0, and Revisions: Thermal Extractionof Fluorideby Means of a Modified Dohrmann DX2000 Organic HalideAnalyzer-Liver FluorideMeasurement by Means of an Orion EA940 Expandable Ion Analyzer Extractionof Fluorochemicalsfrom Rabbit Liver Analysisof RabbitLiverExtractforFluorochemicalsUsing ElectrosprayMass Spectrometry Analysisof FluorideUsing the SkalarSegmented Flow Analyzer with Ion SelectiveElectrode Author:James D. Johnson Approved By: @aSatmeesslII,DI ctoolson Date Stmuuddyy 3@iire@r 2 John Butenhoff,PhD Date Sponsor Representative oooo@-i,'3 1.0PURPOSE ThisstudyisdesignetdoprovidienformatiaosntowhetheFrC-99(T-6053i)s dermallyabsorbed.The analyticaalspectofthisstudyistodeterminefluorinecontainingcompounds (biotransforinatpiroonducts)inthetissueand serum of rabbitsatvarioustimespostdose dermalapplicatioonfFC-99. The dataaretobe used to make an assessmentof theextentof dermal absorptionof FC-99. 2.0 TEST MATERIALS 2.1Test,Control,and Reference Substances and Matrices 2.1.1AnalyticalReference Substance: FC-95, lot161 or 171. They are equivalent. 2.1.2AnalyticalReference Matrix: Bovine liverand bovine serum 2.1.3AnalyticalControl Substance:None 2.1.4AnalyticalControl Matrix: Bovine liverand bovine serum 2.2 Source of Materials:3M ICP/PCP Division(2.1.1),grocerystore(2.1.22,.1.4 liver)S,igma Chemical Company (2.1.22,.1.4serum) 2.3Number of Testand ControlSamples: Tissuesand fluidsfrom 18 testanimals and 6 controlanimals.One animalwas replacedon day 2 (animalF53454 inthe 250 mg/kg dose group was replacedwithanimalF53504).Tissuesand fluidsinclude liverk,idney,serum,cellulafrractiond,ermalapplicatiosniteand bile.Analysisof thesetissueswillbe atthediscretioonfthe StudyDirector. 2.4Identificatioonf Test and Control Samples: The samplesare identifieudsing theHWI animalidentificatinounmber which consistosf a lettearnd fivedigit number, plusthetissueidentitaynd day identit(yserum). 2.5Purityand Strengthof Reference Substance: To be determinedby Sponsor. 2.6StabilityofReference Substance:To be determinedby Sponsor. 2.7Storage ConditionsforTest Materials:Room temperature(2.1.1), -20 100C (2.1.22,.1.4).Test and Controlsampleswillbe receivedaccordingto AMDT-S-10-0. 0 ()0 2 2.8DispositionfSpecimens:Biologictailssueasndfluidwsillbe retainepder GLP Regulationforthetime periodrequiredforstudieslongerthan 28 days. 2.9SafetyPrecautions:Refer to appropriateMSDS. Wear appropriatelaboratory attire.Use cautionwhen handlingknivesforcuttingthesamples. 3.0 EXPERIMENTAL - Overview The tissuesfrom animalsdosed as described(HWI#6329-137), areavailablefor analysisforfluorinecompounds. At thediscretioonf the Study Director,a seriesof analyticatlestscan be performed. The screeningforfluoridein livervia combustion (See Methods--nextSection)istheappropriateanalysisto presentdefinitivdeatafor fluorineintheliver.When sufficiendtatahas been collectedto meet theobjectives of thestudyinthe opinionof the Study Director,analysiswillcease. 4.0 EXPERU%4ENTAL -Methods 4.1Liver and Serum screeningmethods: (attached) 4.1.1AMDT-M-1-0, Thermal Extractionof Fluorideby Means of a Modified Dohnnann DX2000 OrganicHalide Analyzer-Liver 4.1.2AMDT-M-2-0, FluorideMeasurement by Means of an Orion EA940 Expandable Ion Analyzer 4.1.3AMDT-M-4-0, Extractionof Fluorochemicalsfrom RabbitLiver 4.1.4AMDT-M-5-0, Analysisof RabbitLiverExtractforFluorochemicals Using ElectrosprayMass Spectrometry 4.1.5AMDT-M-8-0. Analysisof FluorideUsing the SkalarSegmented Flow Analyzer with Ion SelectiveElectrode 5.0DATA ANALYSIS 5.1 Data Reporting: Data willbe reportedas a concentration(weight/weight)of fluorideper tissueor fluid,or as FC-95 (electrospramyass spectrometry)per unitof tissueor fluid.Statisticused,atthediscretioonf theStudy Director,may include 0()()055 3 regressionanalysisof serum concentrationwsithtime and averagesand standard deviationosf concentrationfsordifferendtose groups.Ifnecessary,simple statistictaelstssuch as Student'sttestmay be appliedto determinestatistical difference. 6.0 MAINTENANCE -OF RAW DATA AND RECORDS 6.1 Raw Data and Records: Raw data,approved protocola,ppropriatespecimens, approvedfinalreporta,nd electronidcatawillbe maintainedintheAMDT archives. 7.0 REFERENCES 7.1AMDT-S-10-0, Sample TrackingSystem 8.0 ATTACHMENTS 8.1AMDT-M-1-0, Thermal ExtractioonfFluorideby Means of a Modified Dohnnann DX2000 OrganicHalideAnalyzer-Liver 8.2AMII)T-M-2-0,FluorideMeasurement by Means ofan OrionEA940 Expandable Ion Analyzer 8.3AMDT-M4-0, ExtractioonfFluorochemicalfsrom RabbitLiver 8.4AMDT-M-5-0, AnalysisofRabbitLiverExtractforFluorochemicalsUsing ElectrosprayMass Spectrometry 8.5ANMT-M-8-0, AnalysisofFluorideUsing theSkalarSegmented Flow Analyzer with Ion SelectivEelectrode 0()OOrJG 4 3M Environmental Laboratory --- Method Tbermal ExtractionofFluorideby Means of a ModifiedDohrmann DX2000 OrganicHalideAnalyzer- Liver Method IdentificatiNounmber: AMDT-M-1 RevisionNumber: 0 AdoptionDate: RevisionDate: None Author:RichYoungblom Approvedby: roup ea7"/ OUP Lt dor@ QualityAssurance /0 /3 Date Date Software:MS Word 5.la AffectedDocuments: AMDT-M-2 FluoridMeeasurementby Means ofan OrionEA940 ExpandableIonAnalyzer AMDT-EP-3 RoutineMaintenanceofa ModifiedDohrmann DX2000 OrganicHalideAnalyzer 1 0()0057 1.0 SCOPE, APPLICABLE COMPOUNDS, AND MATRICES 1.1Scope:Thismethod isfortheoperatioonfa Dohrmann DX2000 when itisusedtoextract fluoridferom variousmatrices.The fluoridiestypicallcyollecteidnTISAB solutiofnoranalysis withan ionselectiveelectrode. 1.2ApplicableCompounds: Fluorochen-dcalosrotherfluorinatecdompounds. 1.3Matrices:Biologicatlissuesp,articularlliyver. 2.0 KEYWORDS 2.1Fluoridef,luorinee,xtractiopny,rolysisi,onizatioino,nselectiveelectrodeD,ohrmann, halide, DX2000, fluorochemicals. 3.0 PRECAUTIONS 3.1Glasswareand exhaustgasescan be extremelyhot. 3.2Glasswareisfragileb,rokenglassmay causeinjuries. 3.3Pressurizegdases,propercompressedgashandlingpracticersequired. 3.4Solventbasedsamplesmay flashm,ay needtoallowthem todrydown beforestartinrgim. 3.5Potentiabliohazardsdue tothebiologicamlatrices.Use appropriatpeersonalprotective equipment. 4.0 SUPPLIES AND MATERIALS 4.1Compressed Oxygen, Hydrocarbonfree,regulatetdo30 PSI. 4.2Compressed Helium,High PurityGrade,regulatetdo45 PSI. 4.3QuartzglasssampleboatwithTeflonTMtubing,Dohnnann 890-097orequivalent. 4.4Quartzglasscombustiontube,RelianceGlass G-9405-012orequivalent. 4.5Orion940999 TotalIonicStrengtAhdjustmentBuffer(TISAB II)orequivalent. 4.6Sample collectiovnialsH,DPE. 4.7Milh-QTM water 4.8Polystyrenepipettes. 4.9ActivatedCharcoal,E.Merck 2005 orequivalent. 4.10HamiltonSyringeor equivalent. 4.11Miscellaneoulsaboratorgylassware 5.0 EQUIPMENT 5.1Rosemount Dohrmann DX2000 OrganicHalideAnalyzer,modifiedforfluoridextraction. 5.2IBM compatible386 or486 computer. 5.3DX2000 softwarev,ersion1.00,modifiedforfluoridextraction. 5.4Excel Spreadsheetv,ersion5.0or greater 6.0 INTERFERENCES 6.1 Sample sizeislimitetdoapproximatel1y50mg, dependingon samplemoisturecontent.This may varyfrom matrixtomatrix. o ()0 0 @-2 2 7.0 SAMPLE HANDLING 7.1Samples arenottobe handledwithbarehands.Fluoridemay ]eachfrom theskintothe sample. Use forcepsor probetotransfetrissues. 7.2Samplesofliverarecutfromfrozenliverand placedina taredand labeledweigh boat.Use a cleanscalpealnd cuttinbgoard.The cuttinbgoardand scalpeslhouldbe cleanedwithwater, methanol,or methanol-watesrolutioanftereachliveriscut. 8.0 CALIBRATION AND STANDARDIZATION 8.1PreparationfCaUbratioSntandards 8.1.T1hestandarrdesquirefdoreachprojecwtilnleedtobeapproprifaotrethaitndividuparloject. Refertoprotocolforthatproject. 8.1.2Typically50-500ppm FC-95 inmethanolstandardsareused. 8.1.3Forrabbiltiverstudiesu,sebeeflivearsthematrix.Cut apieceof frozenbeefliver(100150mg) and weigh itina labeledandtaredweigh boat. 8.2 Calibration- Overview The normal calibratiiosnthefluoridceurve(AMDT-M-2). However, ifan optionalspikedliver curveisrequiredtheprocedurehstedbelow isused. 8.2.1A calibraticounrvefortheDX2000 isgeneratebdy spikingsampleswithknown standards and combustingthem usingthesame methods and matrixtypeasthesamplestobe tested. 8.2.2Typicallyt,hreereplicateosfeachstandardand fiveconcentratioonfsstandardswillbe spiked. 8.2.3Standardcurvewillbe plotteadsMass SpikedF (ug)on thex-axisand StandardMass RecoveredF (ug)on they-axis.Generatearegressiocnurveand calculatteheequationfortheline and ther2 value. 8.2.4Mass SpikedF (ug)= (Amount spikedinmL) x (Conc.ofstandaridnppm) x (0.6004)* *FC-95 is60.04% F therefor0e.6004isthefactoursedtoconvertFC-95 toF 8.2.5StandardMass RecoveredF (ug)= (TISAB volumeinniL)x (Orionreadinginppm) 8.3Calibratio-nProcedure 8.3.1StartUp 8.3.1.1Run 2 ormore CleanCycleswhen startinignsn-=enteachday. More cleancyclesmay be usediftheprevioussamplescontainedhighconcentradonsoffluoride. 8.3.2Blanks 8.3.2.1Preparesample usingthesame methods andtypeofmatrixasthetestsample. 8.3.2.2Forrabbitstudiesu,sebeefliverasthematrix.Prepareatleast3 samplesofbeefliver (100- 150 mg) forblanks. 8.3.2.3PutsampleinDohrmann boat.Combust eachsampleasdescribedinsection9.0and analyzesampleaccordingtomethod AMDT-M-2 fortheionselectiveelectrodaenalysis. 0()oor-,4 3 8.3.2.4For rabbitstudiest,hemeter readingfora blank sample shouldbe 0.03ppm or lower beforeproceedingwiththecalibrationB.um samplesuntilthislimitisreached,or untilin the judgement of theoperatorthereadingisstablewith respectohistoricarleadings(previous48 hours). 8.3.2.5For non-rabbitstudiest,heblankreadingsshouldreacha predeterminedionconcentration beforeproceedingwiththecalibration. 8.3.2.6Itmay be necessarytomix approximately50 mg of charcoalwith thesample to aid combustion. 8.3.3Standard Curve 8.3.3.1Weigh outatleast15 matrixsamples(5standardswith3 replicateesach) intaredand labeledweigh boats.For rabbitstudiesw,eigh 100-150mg beefliversamples.Record weightsin studydata.Storethematrixsampleson dryiceoricepacks tokeep them frozenuntilused. 8.3.3.2Placeweighed beefliversample inDohnnann sample boat. 8.3.3.3StartwiththeloweststandardconcentrationU.sing a Hamilton syringe,ejecta fixed quantityof thestandardon or inthematrix.For rabbitstudiesu,se 4 uL of standardand ejectiton or inthebeefliver. 8.3.3.4At least3 replicatesshouldbe used forthelowest standardconcentrationm;ore replicates may be used atthediscretioonftheanalyst. 8.3.3.5Combust thesample as describedinsection9.3and analyzeaccordingto AMDT-M-2. 8.3.3.6Run all15 standards.Ifone replicatiessignificantdliyfferenftrom theothertwo replicatersu,n anothersample forthatstandard.Indicateindatathatthenew replicatreeplacesthe oldreplicataend thatthenew replicatweillbe used tocalculatteheregressioncurve. 8.3.3.7When allstandardhsave been run,calculattehe r2. r2must be atleast0.95. Ifitisnotat least0.95,consultwith supervisor. 8.3.3.8A new standardcurveshouldbe runwhen thecombustiontubeor sample matrixis changed. New standardcurvemay alsobe run atthediscretioonf theanalyst. 8.4 StorageConditionsforStandards 8.4.1Storagerequirementsforstandardsaredependenton theindividuasltandardsused. Typically,standardsarestoredatroom temperatureinplastisccrew topbottles. 8.4.2New FC-95 standardsshouldbe preparedatleastonce a month. 9.0 PROCEDURES 9.1TypicalOperating Conditions: 9.1.1Combustion tubetemperature= 950*C. 9.1.2Oxygen and Helium flow = 50 cc/minute. 9.1.3Vaporization/Dryintgime= 240 seconds. 9.1.4Bake fime = 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 theSYSTEM SETUP window. 0()00(3() 4 9.2.5When the oven has reachedthe READY temperature,run the CLEAN found in theCELL CHECK menu. 9.2.6 See AMDT-EP-3 fordetailosf theDohrmann software. BOAT program 9.3Sample ExtractionProcedure: 9.3.1Open theSAMPLE HATCH and placethesample intheBOAT. Itmay be necessaryto mix approximately50 mg of charcoalwiththe sample toaid combustion. Ifthisisdone,charcoal should alsobe mixed inwhile establishintghebaselineand when generatingthe standardcurve. 9.3.2 Close SAMPLE HATCH. 9.3.3 Add appropriatveolume ofTISAB solutionor 1:1 TISAB:Milli-QTIlwatermixture toa labeledsample collectiovnial.Typically0.6 mL to 15 mL areused. For rabbitstudiesu,se 1.0or 2.0mL of 1:1 TIS AB: Milli-QTIlwatermixture. 9.3.4Place thevialso thatthetipof theCOMBUSTION TUBE isintheTISAB atleast0.25 inches.Gases releasedduringpyrolysismust bubble throughthe TISAB. 9.3.5 Run theEOX-SOLIDS program found inthe RUN menu. 9.3.6When theEOX program isfinishedr,emove thecollectiovnialfrom the combustion tube. 9.3.7IfundilutedTISAB was used tocollecthe sample,add an equalvolume ofMilli-QTM water to theTISAB tomake 1:1 TISAB:Nfilli-QTM. 9.3.8Rinse theend of thecombustion tubewith Milli-(Ymwaterand wipe with a KIMWIPE to remove any TISAB remainingon the tube. 9.3.9 Open thesample hatch and remove any remaining ash from theboat. Ash can be removed with a cottontippedapplicatororvacuumed out. Itmay be necessarytoscrapparticleosffthe bottom with a spatulaor othersimilardevice.A drop of Milli-QTIIwIater may be added tothe boatto aidinthe Clean Cycle. 9.3.10 Close thehatch. 9.3.11 Run theCLEAN BOAT program. 9.3.12 Sample isready foranalysisby ionselectiveelectrode(AMDT-M-2). 9.4 Sample Calculations 9.4.1Use thestandardcurve tocalculatethesample value. 9.4.2 Sample Mass Recovered F (ug)= (TISAB vol inmL) x (Orion readinizin pl2m - intercept) (Slope) 10.0 VALIDATION 10.1QualityControl 10.1.D1ailyStartUp CheckSamples:Oncethestandarcdurveisestablisheda,chdayof analysisisstartedby analyzingQC samples. The QC samples are tobe the same as thelowest concentrationspiked samples used togeneratethestandardcurve. Each concentrationmust be done intriplicautnelessthefirstwo replicateasrewithin20% of thestandardcurve,thena third replicatiesnotnecessary. 10.2 Precisionand Accuracy: See method development analysisand sample analysisin FluorideNotebooks 2,3,and 5. Precisionand accuracy varieswhen analyzingsamples of different matricesand differentreferencecompounds. 10.3 Other ValidationParameters: NA 000oll. 5 11.0 DATA ANALYSIS 11.1 Calculations 11.1.1For thestandardcurve,useregressioannalysiisnExcel,version5.0orgreater. 11.1.2To calculattehefluoridceontractioinnthesample,seemethod AMDT-M-2. 11.2 Analyzing theData 11.2.1r2must beatleast0.95orgreater".Outliersm"ay be excludediftwo ofthethreereplicates arewithin20% ofeachotherand theoutlieirsgreaterthan200% oftheaverageofthosetwo or lessthan50% oftheaverageofthosetwo. Any suchoutliersshouldbe pointedoutinthedataand notedintheFinalReportalongwiththereasonitwas considereadn outlier. 12.0 ATTACHMENTS None 13.0 REFERENCES 13.1Rosemount Dohnnann DX2000 OrganicHalideAnalyzerOperator'Msanual (Manual 915349,revisioBn, December 1993) 13.2 ANMT-M-2 FluorideMeasurementby Means ofan OrionEA940 ExpandableIon Analyzer 13.3AMDT-EP-3 RoutineMaintenanceofa ModifiedDohrmann DX2000 OrganicHalide Analyzer 14.0 REVISIONS Revision Numbe Reason forChang& Revision Date 0()00("--" 6 3M Environmental Laboratory Method FluorideMeasurement by Means ofan Orion EA940 Expandable Ion Analyzer Method IdentificatioNnumber: AMDT-M-2 RevisionNumber: 0 Adoption Date: RevisionDate: None Author: Rich Youngblom Approved By: G@, Leader liate QualityAssurance Date Software:MS Word 5.la AffectedDocuments: AMDT-M-1 Thermal Extractionof Fluorideby Means of a Modified Dohrrnann DX2000 Organic HalideAnalyzer oooob..'j 1.0 SCOPE. APPLICABLE COMPOUNDS3 AND MATRICES 1.1SCOPE: Thismethod isforthecalibratiaond operatioonfan OrionEA940 Expandable Ion Analyzer. 1.2APPLICABLE COMPOUNDS: Fluoride. 1.3APPLICABLE MATRICES: Liquidsamplesinan appropriatbeuffersolutionP.referred pH of6.0. 2.0 KEYWORDS 2.1Fluoridef,luorinei,onselectiveelectrode 3.0 PRECAUTIONS 3.1No hazardsidentifiweidththismethod. 4.0 SUPPLIES AND MA- ERIALS 4.1Orion940999 TotalIonicStrengthAdjustmentBufferH (TISABII)or equivalent. 4.2OrionModel 900001 electrodfeillinsgolutio(nAgCl)orequivalent. 4.3Orion940907 100ppm fluoridsetandardorequivalent. 4.4Nfilli-QTwMateror equivalent. 4.5Magneticstirbars. 4.6Lab tissues. 4.7Sample collectiovnials. 4.8Plasti1c00 mL volumetricflasks. 4.9Polystyrenepipettes. 4.10Miscellaneoulsaboratorgylassware. 5.0 EQUIPMENT 5.1OrionModel EA940 ExpandableIon Analyzerorequivalent. 5.2Orion Model 960900 SolidStateCombinationFluorideelectrodoer equivalent. 5.3MagneticStirPlate. 5.4IBM compatible386 or486 computer(onlyneededifusingOrion3E software). 5.5OrionRS232 interfacceable(onlyneededifusingOrion3E software). 5.6MicrosoftExcel5.0(onlyneededifusingOrion3E software). 6.0 INTERFERENCES 6.1Itisrecommended thatthepH be atornear6.0.A 1:1 mixtureofTISAB and sample/MilliQTM waterwillgenerallybringsample topH of 6.0. 6.2Sample temperaturemay effectfluoridmeeasurement.Itisrecommended thatthesamplebe atroom temperatureasthestandardwsere when themeterwas calibrated. 6.3The ratethesamplesarestirreadtshouldbe consistenwtiththeratethestandardswere stirred. 2 0(.)0064 6.4Airbubblestrappedunder electrodcean giveerroneousreadings.Make sureno airistrapped under electrode. 7.0 SAMPLE- HANDLING 7.1 No specialhandlingnecessary. 8.0 CALIBRATION AND STANDARDIZATION 8.1PreparatioonfCaUbrationStandards 8.1.M1easure50mL ofTISAB IIinto5 100mL plastvioclumetrfilcasks. 8.1.L2abeltheflaskass0.050,.1,0.5,1.0a,nd1.5ppm F-,alongwiththedateandyourinitials. 8.1.P3ipett0e.050,.1,0.5,1.0a,fid1.5m.Lof100ppm fluorisdteandaridntotheappropriately labeledflasks. 8.1.4Add approximately30 niL of Milli-QTMwaterto each flask. 8.1.5Shake theflaskstomix thesolutions. 8.1.6Eliminateairbubblesfrom theflasksby tippingtheflaskosn theirsidesand rollingtheairin theflasksover theairbubbles. 8.1.7Bringthevolume in theflasksup tothe100 mL mark withMiUi-QTM water. 8.1.8Invertand shake theflasksforthefinalmixing. 8.1.9Record standardsin StandardsLog Book. 8.2 Calibration 8.2.1Ifnecessary,remove tapefrom electrodfeillinhgole. 8.2.2Invertprobe towet topseal. 8.2.3Ejecta few dropsof fillinsgolutionfrom bottomof electrodteowet lowerseal. 8.2.4Filltheelectrodweithfillinsgolution. 8.2.5The meter and theF- electrodaeretypicallcyalibratebdy directmeasurement withno blank correctionu,singstandardswithconcentrationosf 0.05,0.1,0.5,1.0,and 1.5ppm F-,following themanufacturer'sinstructions. 8.2.6Record theslopeintheappropriatleogbook. 8.2.7Clean theelectrodbey rinsingwithMilli-QTMwaterand wipingthesidesdown withlab tissues. 8.3 StorageConditionsforStandards 8.3.1Calibratiosntandardsarestoredatroom temperature. 9.0 PROCEDURES 9.1Calibrationand Measurement, Standard method: 9.1.1The sample to be 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 airbubblestrappedunder theelectrode. 9.1.4The sample shouldbe thesame temperatureas thecalibratiosntandardsand stirreadtthe same rateas thecalibratiosntandards. 9.1.5When thereadingshave stabilizerde,cordthereadingintheappropriatelogbook. 0()00(;.,l 3 9.2CalibratiAonnd MeasurementU,singOrion3E Software: 9.2.1Calibration: 9.2.1.F1ollowstep8s.2.t1o8.2.4. 9.2.1.P2resFsunctioKney #8(F8). 9.2.1T.h3ecomputesrcreewnilalskyoutoconfirtmhenumberofstandartdosbeused, concentratioonfthestandardsa,nd whetherornot a blankistobe includedinthecalibration. Make any necessarychangestotheinformationpresentedand clickon CONTINLTE. 9.2.1.4Placetheelectrodienthefirssttandardon thestirplateand clickon CONTRTUE. 9.2.1.5Observe thereadingson thegraphicdisplayon thecomputer.When thereadingshave stabilizepdr,essACCEPT READING. 9.2.1.R6epeat step9.2.1.a4nd 9.2.1.5fortheremair@ngstandards. 9.2.1.7Afterthefinalstandardt,hecomputerwilldisplaytheslopeof thecurve,aswellas the intercepatnd correlationR.ecord theslope,intercepta,nd correlatioin theappropriatleog book and clickon CONTINLTE. The calibratidoantaisautomaticallcyopiedtoC:\Orion\Data\Calib.txt. 9.2.2Data Spreadsheet: 9.2.2.1SelecteitherNEW orOPEN from theFILE menu toopen a new orexistinsgpreadsheet tostoredatain. 9.2.2.2Record thename of thespreadsheetused intheappropriatleogbook. 9.2.3Fluoride Measurement: 9.2.3.1Follow steps9.2.1through9.2.4 9.2.3.E2nter thename of thesample intheappropriatpelaceon thescreen. 9.2.3.3Clickon theNEW SANTPLE button 9.2.3.4When thereadingshave stabilizecdl,ickon theRECORD buttonand wtitetheresulitnthe appropriatleog book. 10.0 VALIDATION 10.1QualityControl: 10.2Precisioan d Accuracy 10.3OtherValidatioPnarametersAccordintgoReferenc1e3.2t,herangeofdetectiiosn0.02 ppm fluoriduep toa saturatesdolutioonf fluoride. 11.0 DATA ANALYSIS 11.1 CalculationsNonenecessary. 11.2 Analyzing theData Nonenecessary. 12.0 ATTACHMENTS None 13.0 REFERENCES 4 0(.)()0(;( 13.1Orion Model EA940 ExpandableIonAnalyzerInstructioMnanual,Orion Research Incorporated1,99 1. 13.2Orion Model 960900 SolidStateCombinationFluorideElectrodeInstructioMnanual, Orion Research Incorporated1,991. 14.0 REVISIONS Revision Number Reason forChanp-e Revision Date 0()0067 5 3M Environmental Laboratory Method Extractionof Fluorochemicalsfrom Rabbit Livers SOP IdentiricationNumber: AMDT-M-4 Revision Number: 0 Adoption Date: Revision Date: None Author: Dave Christenson/CynthiWaeber Approved By: roup Leader Date QualitfAs'surance iiate Software: MS Word, 6.0 AffectedDocuments: M-5, Analysisof RabbitExtractforFluorocheiiiicaUlssing Electrospray Mass Spectroscopy. t)()OOCL@ 1.0 SCOPE 1.1 Scope: This method isfortheextractioonf fluorochemicalfsrom rabbitlivers. Ethylacetateisused toextracftluorocheri-dcfarlosm theliversforanalysisby electrospraymass spectroscopy. 1.2 Applicable Compounds: Fluorochemicalsor otherfluorinatecdompounds. 1.3 Matrices: RabbitLivers. 2-0 KEYWORDS 2.1 Fluorochemicals,rabbitliverse,lectrospramyass spectrometer,fluorinated compounds, extraction. 3.0 PRECAUTIONS 3.1 Use gloveswhen handlingtherabbitliverst,heymay containpathogens. 4.0 SUPPLRES-AND MATERRALS-4.1 Supplies 4.1.1 Syringcea,pabloefmeasurin1g00uL 4.1.2 Eppendorf typeordisposablpeipets 4.1.3 Gloves 4.1.4 Plastigcrindingtubes 4.1.5 Plasticcentrifugteubes,15 niL 4.1.6 Labels 4.1.7 Nitrogen 4.1.8 Timer 4.1.9 FiltersT,itannylonsyringefilter0s.,2um. 4. 1.1OAnalyticaplipetsg:lassvolumetricpipets. 4. 1.11 Disposableplasti3ccc syringes. 4. 1.12 Crimp cap autovials. 4.2 Reagents 4.2.1 Aqueous Ammonium Acetate(Aldrich)a,pprox.250 ppm: Preparea 2500 ppm aqueoussolutioonfanimonium acetatbey adding250 mg ammonium acetatteo100mLs NElli-Qwater.Dilutethissolutio1n:10fora 250 ppm solution. 4.2.2 Sodium carbonate/SodiuBmicarbonatBeuffer(J.T.Baker), (Na2CO3/NaHC03)0.25 M: Weigh 26.5g ofsodium carbonate(Na2CO3) and2 1.0g ofsodiumbicarbonat(eNaHC03)intoa I L volumetrifclaskand bringtovolume withMilli-Qwater. 4.2.3 Diluteacetonitrsiolleutiond,ilutaecetonitri1l:1ewithMilli-Qwater. 4.2.4 EthylAcetate 4.2.5 Methanol 4.2.6 Milli-Qwater 5.0 EQUIPMENT 5.1 Ultra-TurTr2a5xGrindefrorgiindilnigvesramples. 5.2 Vortexmixer 5.3 Centrifuge 5.4 Shaker 0()OOC!) 2 5.5 AnalyticEavlaporator 6.0 INTERFERENCES 6.1 There areno known interferenceastthistime. 7.0 SAMPLE HANDLING 7.1 The rabbitliverasrereceivedfrozen,and must be keptfrozenuntiltheextractioins performed. 8.0 CALIBRATION AND STANDARDIZATION 8.1 Preparationof InternalStandards 8.1.1 Preparean internasltandardof approximately12 ppm 1H,IH,2H,2Hperfluoroctanesulphonaiccidtobe added toeach liversample. 8.1.2 Weigh atleast0.1 g of IH, IH,2H,2H-perfluoroctanesulphoniacid. Record theactualweight. 8.1.3 Bringitup tovolume with 100 mLs of methanol,thisisthestockstandard. 8.1.4 Dilute3 mLs ofthestockstandardto250 mLs finalvolume with Milli-Q water.Calculatetheactualvalueof thestandard. actualmg perfluoroctane- sull2honiaccid x 0.1L 3 mL 250 niL actualvalue,ppm 8.2 Prepare FC-95 Anion Standards 8.2.1 PrepareFC-95 standardsforthestandardcurve. 8.2.2 Weigh approximately100 mg of FC-95 and recordtheactualweight. 8.2.3 Bringup tovolume withdiluteacetonitriilnea 100 niL volumetricflask. 8.2.4 Dilutethesolutionwithdiluteacetonitri1l:e10 fora solutionof approx.100 ppm. Dilutethissolution1:10 withdilutaecetonitrifloera solutionof approx.10 ppm. 8.2.5 Use the10 ppm solutionto make 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 Boviiieliverintoa 250 mL Nalgenebottlecontaining200 mLs Milli-Qwater.Grind toa homogenous solution. 8.3.2 Add I mL of thesolutiontoa 15 mL centrifugteube.Preparea totaolf eight I mL aliquotosf thesolutionin 15 mL centrifugteubes.Be sureto resuspendsolutionby shakingitbetween aliquots. 8.3.3 Spikesevenof theI mL aliquotwsiththefollowingamounts of working standardsin step9.12of theprocedure.One I rnL aliquotservesas the blank. 3 Approximate Conc. Working Standard uL ug FC-95/ g liver (Approximate Conc.) Blank - - 400 ppb 500 ppb 100 800 ppb 500 ppb 200 1.2ppm 500 ppb 300 1.6ppm 500 ppb 400 4 ppm I ppm 500 8 ppm 5 ppm 200 12 ppm 5 ppm 300 8.4 Calculatetheactualvalueofthestandards: uL ofstandardx mg/1- x IL/1000000uL mg FC-95 miz FC-95 x A_(organilcayerrecoveryfactor) 137mg liver*/nliLsolution5 mgftnl FC-95 equivalentisn liver mg/mL x 1000 mg/L = ppm FC-95 inliver *Average weightof bovineliverinsolutionT.he amount of FC-95 is reportedasequivalentsofFC-95 potassiumsalt. 8.5 Calibration 8.5.1 Extractthespikedbeefliverhomogenate following9.13to9.23of this method. Use thesestandardstoestablisyhour curve on themass spectrometer. 8.6 Storage Conditions for Standards 8.6.1 New standardsarepreparedwith each analysisS.tandardsare storedin coveredplasticentrifugteubesuntiltheanalysison themass spectometeris performed. 8.7 Storage Conditions for Standards 8.7.1 Beef liverhomogCe2 natesmay be frozenafterpreparation. 9.0 PROCEDURES 9.1 Obtainfrozenliversamples.Inspenttissuen,ote thattheliverhas notbeen packaged with othertissues. 9.2 Use a dissectinsgcalpeland cutoffapproximatelyI g of liver. 9.3 Weigh thesampledirectliyntoa taredplastigcrindingtube. 9.4 Record theliverweightinthestudynotebook. 4 9.5 9.6 9.7 9.8 9.9 9.10 9.11 9.@12 9.13 9.14 9.15 9.16 9.17 9.1.8 9.19 9.20 9.21 9.22 9.23 9.24 Put a labelon thevialwith thestudyi-@umberw,eight,rabbitJ.D,dite and analyst initials. Add 2.5 mLs water. Grind thesample. Put the grinderprobe in thesample and grind for at-@f,2,)Llt minutes,untilthesample isa homogeneous solutionwith no largeChLinks. Rinse theprobe offintothe sample with 2.5 n-llswater using a pipet. Take thegrinderapartand cleanitwith methanol afteie-ach s@imple.1--ollow A MDT- EP-22. Cap the sample and vortexfor 15 seconds. PipetI mL intoa 15 niL centrifugetube.Label thecentrifugetubewitt@t@ieidentical infonnationas thegrindingtube.(SeeAMDT-M-4 Wc@)rksheeftordoci.@mentiritghe remaining steps.) Spike the beefliverhomogenates with theappropriateamount of FC-';)s5tandard as describedin 8.3. Spike thesamples and beef liverhoi-nogenatesA,ith1(X)uL of internal@;tmdar@d. Add 1 mL of thesodium carbonate/.sodiumbicarbonatebuffera-nd1.ml..a,mmonium acetate.9.15Using an analyticaplipet,add 5 mL ethylacetate. Cap thesample and vortex 20 to30 seconds. Put them in the shakerfor20 n-@n. Centrifugefor20 to25 minutes,untilthelayersare wellseparated.Sel thepower on thecentrifugeto25. Remove 4 mLs of the top organic layei-to a fresh 15 rrilc.e-n,trifuget@libf@.v@itha f@ mL gaduated glasspipet.Transferthe labeltothefreshtube. Blow the sarripldfo-w-n on theanalyticaelvaporatorto near drynes@@witt-niitrozgnen, approximately 30 to 40 minutes. Bring theremaining sample up in I ri-dd-iluteacetoriitiiwlieth an ana.1y@@icpail)e@. Vortex 15 seconds. Transferthesample toa 3 n-iLsyringe.Attach a 0.2 nm nylon mesli filtera,nd filter the sample intoa freshcentrifugetube or a autovialL.abel thetube or v@'@awlith the study number and animal number.. Cap and holdforanalysisby electrospi-amyass spectroscopy, Complete ANI.DT-M-4 worksheet and attachtopage of studyrioteboc@k, 10.0 VALIDATION 10.1 QualityControl 10.2 Precisionand Accuracy 10.3 Other ValidatioPnarameters 11.0 DATA ANALYSIS 11.1 None 12.0 ATTACHMENI'S 12. 1 WorksheetAMDT-M-4 13.0 REFERENCES 13. 1 AMDT-EP-22 14.0 REVISIONS Revision Number Reisori forChanjl_,c R(,@vis'i@:)n o@f 5 Study Sample Numbcr set0 Rlnnk I iver Worksheet AMDT-M-4 approx 0.5ppm actual PPM #W- FC-95 approx I ppm actual PPM #W approx. 5 ppm actual PPM Date and InitialfsorStd. 400 iiT, - 500 UT- - 00 IIT, 100 11T, Rtildyniimber 3gherethf-n.ricrinnlw rk-,;heeitglocatt-fgind nl:;ren rony, T.iverFxtrgction Proct-qq- PinetI mT, of T.ivergnlution Pinet100 uT,of 12 nnm TntemalStqnclqrti I;td# Vnrtex Ii gec, PinetI mT. of 750 nnm Ammonium Aret;;te I;td# PinetImT, of0 75 Nn,rO,10 25M NnT4CO. Buffer Pinet5 mT. of FthylAretgtt- Vnrti-x70-30 -;Pr, Shi;ke20 min Centrifiic?,0&-2'9min Remove a 4 mT. gliniinotf orty;anliacyi-r Blow dnwn to neardryne,-g(q<O-?imT,)with N. Atld I m- of I-IAcetonitrile/T4.0 TN# Vnrtex 15 ger Filterusing ;iIrr 11-Dgyrinpe%%,it;hi0')iimCRl filteirntoq I i mi ititogamnlevial T),ate Tnitinig 3M Environmental Laboratory Method Analysis of Rabbit Liver Extract for Fluorochemicals using Electrospray Mass Spectroscopy SOP IdentiricatioNunmber: AMDT-M-5 Revision Number: 0 Adoption Date: 6 .-1r Revision Date: None Author: Dave Christenson/CynthiWaeber Approved By: ?17aroduep Lrea@d@er Date QualityAssurance Date Software:MS Word, 6.0 AffectedDocuments:M-4, ExtractioonfFluorochemicalfsrom RabbitLivers 1.0 SCOPE 1. 1 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: Fluorochemicalsorotherfluorinatecdompounds. 1.3 Matrices:RabbitLivers(samples)B,eefLiver(standardso)t,hertissuesand fluids. 2.0 KEYWORDS 2.1 Fluorochemicalsf,luorinatecdompounds, electrospramyass spectroscopym,ass spectrometerr,abbitlivers. 3.0 PRECAUTIONS 3.1 Use cautiowniththevoltagceablefortheprobe.Yvlhetnhevoltagceableisplugged intotheprobe DO NOT TOUCH THE PROBE, thereisriskof electricsahlock. 3.2 Do notrun thepump above it'scapacityof4000 psi.Ifpressuregoes over4000 psi stopand releasepressureT.he peak tubingmay be plugged.Troubleshootback to findtheplugand replacethepluggedtubing.See AMDT-EP-15 3.3 Do notrun thepump todryness. 4.0 SUPPLIES AND MATERIALS 4.1 Supplies 4. 1.1 Nitrogengas regulatedto 140 psi. 4.1.2 Fluofixcolumn orequivalent. 4.1.3 100 uL or250 uL flatipsyringeforsampleinjection. 4.2 Reagents 4.2. 1 Diluteacetonitrimloebilephase,diluteacetonitri1l:1e withMilli-Qwater. 4.2. 2 Milli-Qwater,allwaterusedinthismethod shouldbe Milli-Qwater. 5.0 EOUIPMENT 5.1 VG Trio2000 ElectrospraMyass Spectrometerorequivalent. 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 centrifugteubesorincapped autovials untilready foranalysis. oo00,;'..3 2 8.0 CALIBRATION AND STANDARDIZATION 8.1 Preparation of CalibrationStandards 8. 1.1 Seven beefliverstandardsand one blankbeef liverarepreparedduring the extractiopnrocedure.(See AMDT-M-4, section8.0) 8.2 Calibration 8.2. 1 Run theseven beefliverstandardstwice,startinwgith the loweststandard toobtainthe standardcurve. 8.2.2 Typicallyone standardisrun aftereach 5 to7 samples.Choose a standard in thesame range of concentrationas thesamples. 8.3 Storage Conditions for Standards 8.3. 1 Fresh standardsare preparedwith each analysis.Standardsarestoredin covered plasticcentrifugetubesuntiltheanalysison themass spectometeris performed. Samples and standardsareNOT refrigerated. 8.4 Storage Conditions for Beef Liver Homogenates 8.4. 1 Beef liverhomogenates may be frozenafterpreparation. 9.0 PROCEDURE 9.1 InitialSet-up 9.1.1 Set softwarrt,o"Operate on",Ion Mode ES-. 9.1.2 Record backingpressureintheinstrumentlog. 9.1.3 Fillthesolventcylinderwithmobile phase. 9.1.4 Set thepump to"Run". Set theflow to 1000 uL/min. Observes droplets coming outof the tipof theprobe.The pressureshould be at1700 to 1800 psi. 9.1.5 Check thefusedsilicaattheend oftheprobe.Use an eye piecetocheck for chips.The tipshouldbe flatwith no jagged edges.Ifany chipsare found cut offthetipof thesilicawitha column cutterand pullthesilicathroughto the appropriatelength. 9.1.6 Check your nitrogensupply.Turn on thenitrogen.There should be no nitrogenleakingaround the tipof theprobe.A finen-iissthouldbe coming out of thetip. 9.1.7 Carefullyguidetheprobe intotheopening.Insertituntilitwon't go any further.Connect thevoltagecabletotheprobe. 9.1.8 Go tothe"Editor"page,and setIonizationMode toES",and the appropriatemasses to427 and 499. 9.1.9 Ifitisnot insingleion mode go to"Option" and setSIR. 9.1.lOStartAcquisitionA.ssigna filename, MO-DAY-YR + letterR.ecord itin the log book. 9. 1.11 Run thebeefliversamples firstr,unningeach standardtwice atthe be@nning of therun..Run a QC check by running one standardafterevery 5 to7 samples. 9.2 Manual Injection 9.2. 1 Draw 150 uL of sample intoa syringe.Injectthesample intotherheodyne injectiopnort.Injectslowly.Record thesample ID inthelog book. 9.2.2 Turn thevalveto"On". 9.2.3 Wait two minutes,and injecthe nextsample. 9.2.4 Record the scann Limberforeach sample inthe logbook. 0()()07(,; 3 9.3 Using the Autosampler 9.3.1 Set up sample trayA B, or C. 9.3.2 Record thesamplesand theirpositionisn theinstrumentlogbook. Up to 17 vialsmay be ineach run. 9.3.3 Set-upthesampler: 9.3.3.1 Push thesample button 9.3.3.2 Setsample loop size= 100 uL 9.3.3.3 9.3.3.4 Set inject/sampl=e 2 SetCycle time= 0 9.3.3.5 Name thefileL:ivers 9.3.3.6 9.3.3.7 9.3.3.8 Identifythetrayused Add thesamplestoQueue by pressing"Entee' Press"Run" tostart 10.0 VALIDATION-- 10. 1 Quality Control 10. 1.l Run a standardevery 5 to7 samples.Ifa significancthange(50%) in peak heightoccursstoptherun.Only thesamplesbeforethelastacceptable standardwillbe used.'Me remainingsampleswillbe reanalyzed. 10.2 Precisionand Accuracy 10.2. 1 See Method ValidadonReportnumber AMDT-M-5.0.V 1 10.3 Other ValidationParameters 10.4 RefertoMethod ValidatioRneportNumber AMDT-M-5.0.Vl 11.0 DATA ANALYSIS 11.1 11.2 Calculations Plotthestandardcurve,usingthemean of thetwo valuesobtainedforeach standard. 11.2. 1Read peak heightsor areasforthesamplesfrom theprintoutU.se linear regressiontodeterminethesample concentrations. 11.2.2Calculatethemg of FC-95 anion,orotherfluorochemicailnthetotarlabbit liver: mg FC-95 anioninthetotalrabbitliver mg FC-95 anionfrom std.curve gms of liverused foranalysis x Totalmass of liverg,ms 11.3 Make a resulttsableand enteritinthestudybook. 11.4 Printa chromatogram foreach sample,withthepeakslabeledwiththesampleor standardDD.Writethestudynumber on theprintouti,nitiadla,te,and putitinthe studyfolderS.tapleallchromatobrramstogetherand number pages. 4 12.0 ATTACHMENTS None 13.0 REFERENCES 13. 1 AMDT-EP- 17 14.0 REVISIONS IRevision Num Reason forchange- Revision Date 0()0071"@ 5 3M Environmental Laboratory Method Analysis of Fluoride Using the Skalar Segmented Flow Analyzer With Ion SelectiveElectrode Method IdentiricatioNnumber: AMDT-M-8 Adoption Date: Revision Number: 0 Revision Date: None Author: Deb Wright / CynthiaWeber Appmved By: up Leader Date QualityAssurance Date Software:IBM MS Word, 6.0 AffectedDocuments: AMDT-EP-26, Analyzer Operationand Maintenance of theSkalarSegmented Flow 1.0 SCOPE 1. 1 1.2 Thismethod isfortheanalysisforfluoridet,hermallyextractedfrom samplesusing theDohrmann DX2000 (AMDT-M- 1),and collecteidnTISAB foranalysiwsithan IonSelectiveElectrode(ISE).The analysisisperformedusingtheSkalar Segmented Flow AnalyzerwithISE. Samples can be tissuess,erum,biologicamlaterialo,r othermaterialsextractedon theDohrmann. 2.0 KEYWORDS 2.1 Skalar,segmented flow,fluoride. 3.0 PRECAUTIONS 3.1 Followstandarldaboratorsyafetpyractices. 4.0 SUPPLIES AND MATERIALS 4.1 Supplies 4.1.1 Sample cups,4 mL plasticcups withcaps 4.1.2 Autopipetso,xfordor equivalenwtithplastitcips 4.1.3 Polypropylenevolumetricflasks,100 niL 4.1.4 Caruidge components,refertotheSkalarMethods forcomponents and part numbers. 4.1.5 Sample prefilterEsv,ergreen 4.2 Reagents 4.2.1 Brij35, 30% S.F.A.S.Detergent 4.2.2 TISAB IIbuffersolutionP:urchaseTISAB IIfrom Orion.To I liteorf TISAB IIadd 2.5mL or 100 ppm fluoridseolutionand I mL Brij. 4.2.3 SamplerrinsingsolutionD:iluteTISAB 111:1 withMilli-Qwater. 4.2.4 NitricacidsolutiofnordecontaminationI,N (labgrade):Slowlyadd 64 mLs concentratenditriaccid(HN03) to250 mLs of Milli-Qwater.Bring thevolume up to 1L withMilli-Qwater. 4.3 Standards 4.3.1 Stock solution1,00 ppm F: purchasedfrom Orion., 4.3.2 Intermediatsetandard,10 ppm: Dilute10 mLs of stocksolutionto 100 mLs withMilli-Qwater.Ust.polypropylenevolumetricflasks. 4.3.3 Working standardM:ake up thefollowingworking standardsby addingthe volumes ofintermediatoer stockstandardindicatedon thetable,using oxfordor pumpmate pipetst,o50 mLs of TISAB and dilutingto 100 mLs withMilli-Qwater. Working Standard mLs of Stock Standard 0.015 ppm 0.03PPM 0.06 ppm 0.09 ppm 0.12 ppm 0.15 ppm - 0.3 ppm 0.3 mLs of IntermediatSetandard 0.15 0.3 0.6 0.9 1.2 1.5 - 0.6 ppm 0.6 2 1.2ppni 1.2 1.5ppm 1.5 5.0 E!QUTPMENT 5.1 SkalarSegmented Flow Auto Analyzer Sansp"-S'ystem equipped with ISE 6.0 INTERFERENCES 6.1 High concentrationosf alkalinitcyh,loridep,hosphate,sulfateor ironcan cause interferences. 7.0 SAMPLE HANDLING 7.1 Samples shouldbe storedinpolyethylenebottlesS.amples should be analyzed within30 days. 8.0 CALIBRATION AND STANDARDIZATION 8.1 Preparationof CalibrationStandards 8. 1.1 Preparecalibratiosntandardsasin section4.3. 8.2 Calibration 8.2.1 The standardsareanalyzedatthebeginningof therun. 8.3 Storage Conditionsfor Standards 8.3. 1 StandardsarestoredincappedpolypropylenevolumetricflasksN.ew standardsarepreparedata minimum of every sixmonths, or as necessary. 9.0 PROCEDURE 9.1 Start Up Procedure 9.1.1 Clamp down thepumpdecks, airbarsand sampler-pump tubing. 9.1.2 Put thefluoridelectrodeisntheelectrodechamber. 9.1.3 Turn on thepower ofthesampler,pumps, offsetpotentiometerand heating bath. 9.1.4 Put therea-ent-lineisntheappropriatbeottles. 9.1.5 Turn on theinterfacec,omputer,displayand printerM.ake sure you turn on the interfacebeforethe computer. 9.1.6 Let thesystemstabilizfeorapproximately30 minutes. 9.2 Startinga Run 9.2.1 Createa sample tableby selectinFgILES, TABLE, and CR.EATF-,typein thename of thefilea,nd pressENTER. 9.2.2 Printthesample table,inserteidn thesystem tableby pushingESC, PRINT, GROUP 1.This willprinttheentirerun. 9.2.3 Dialthesamplersettingtsotheappropriatneumber of samples,number of seconds forsample wash, and number of secondsforthesample. 9.2.4 Fillthesample traywiththestandirdss,amples,washes and driftsr.W and FW/RUNOUT cups on thesamplerdo not need tobe filled. 9.2.5 Set thebaseline. ()()OOSI 3 9.2.5. 1SelectGRAPHICS, REAL TIME. Ifyou cannot getreal-timey,ou may be intheData Handling Panel.Switch tothe AnalysisPanel by selectingCONTROL PANEL and pushin- F7. 9. 2.5.2 Use thesmallscrewdriverfortheoffsetpotentiometertosetthe baseline.Adjustthebaselineuntilitisapproximately3/4 inchfrom thebottom of thescreen. 9. 2.5.3Check thehigheststandardand adjustthe gain,ifnecessary,with theinterfacescrew #3. 9.2.6 Go toCONTROL PANEL, and toanalysispanel.Deselecttheanalysisthat willnot be run.(Selector deselectanalysisby pressingENTER.) PressTab to returntotheAnalysisPanel. 9.2.7 Press thespacebartobringup thelocalmenu. 9.2.8 SelectSTART tostartheanalysis. 9.2.9 Type your ID (initialst)h,esample tablewhich you createdunder 9.2.1(or pressENTER forchoices),choose runningwith or withoutthe system table and selectSTART ANALYSIS. 9.2. 10 Afterstartintghe software,starthesampler.Make surethatthesampler is settotherightnumber ofsamples and thatthesample/wash/airtimesare OK. 9.2.11 SelectGRAPHICS, REAL TIME toview theprogressof the analysis. 9.3 Loading and Printing the Data-File 9.3.1 Go to CONTROL PANEL, pressthespacebartobringup the localmenu and selecLtOAD. SelectAUTOCALCULATION and enterthefilename(or highlightthefiletobe printedand pressENTER). 9.3.2 To view thecalibratiocnurve,go toGRAPHICS, CALIBRATION CURVE. 9.3.3 To printthehigh levelcurve,push PRINT SCREEN. 9.3.4 To printthelow levelscreen,push ESC togetout of graphics.Select SETTINGS. Change themax y valuetoapproximately900. Go toCAL CURVE and pressESC, and Enter.PressPRINT SCREEN. 9.3.5 Return toSEMNGS and change themax valueback to4095, go toEDIT, pressENTER and PRINT SCREEN toprintsample peaks. 9.3.6 To printtheresultgso toCONTROL PANEL, SPACEBAR, OUTPUT, OUTPUT. SelectPRINTER fortheEpson or PRN fortheLaser. 9.4 Shutdown 9.4.1 Put allthereagent-lineisnMilli-Qwater. 9.4.2 Let the systemrinseforapproximately30 minutes. 9.4.3 After thesystem has rinsedcompletely,turnoffthesampler,pump and offsetpotentiometerT.urn offtheheatin-bathon weekends. Leave liquidin the lines. 9.4.4 Take theelectrodeout and soak in 100 ppm F overnight. 9.4.5 Release thepump-decks, airbirsand sampler pump-tubing. 9.4.6 SelectFILES, pressALT F and selectQUIT toexitthe program. 9.4.7 On Friday,turnoffthecomputer, displayand interfacfeor theweekend. 10.0 VALIDATION 10.1 Quality Control 10. 1.1Run a standard(mid tohi0-hconcentratione)very 10 samples.Ifa significancthange inpeak heightoccurs,only thesamples beforethe last acceptablestandardwillbe used.The remaininasamples willbe reanalyzed. oooo.-Soot@., 4 10.2 Precisionand Accuracy 10.2.1See MethodValidatiRoenportnumberAMDT-M-8.0.V 1 10.3 Other ValidationParameters 10.4 RefertoMethodValidatiRoenportNumber AMDT-M-8.0.Vl 11.0 DATA ANALYSIS 11.1 11.2 11.3 11.4 Calculations 11.1.1The standarcdurveisplottebdy theSkalasroftware. 11. 1.2 All calculationasredone by theSkalarsoftware.r2 should be 0.995 or better. Prepare spreadsheetsto summarize data.Includesample volume, weights used etc. Write the studynumber on theprintoutsi,nitiald,atetheprintout,and bind together with allpackage documents and placeinthestudyfolder.Make a copy of the summary sheetand tapeintothestudy notebook.Back up alldataand spreadsheets onto study diskand backup disks. ElectroniDcata 11. 4. 1 GLP studiesE:lectronicdataiscopiedonto theStudy floppydiskforeach study,and alsodam iscopied onto a floppydisk thatisstoredinthelab. 11. 4.2 Other studies:All dataiscopiedonto a floppydiskthatisstoredinthe lab. 12.0 ATTACHN4ENTS None 13.0 REFERENCES 13.1 13.2 13.3 AMDT-M-1, Thermal ExtractionofFluorideby Means of a ModifiedDohrmann DX2000 OrganicHalide Analyzer-Liver SkalarMethods, #335, SkalarMethods Manual AMDT-EP-26, Operationand Maintenanceof theSkalarSegmented Flow Analyzer 14.0 REVISIONS Revision Number Reason forchangg Revision Date 0()008.'l 5 --. ;@ I II - 9.3 QualityAssurance Unit Statement ()()00811 Amchmnt D GLP Study QualityAssurance Statement Study Title:Single-dose Dermal Absorption/ToxicityStudy of T-6053 in Rabbits StudyNumber: AMDT-022195.1 Name ofAuditor:KariRambo This studyhas been inspectedby theQualityAssuranceUnit asindicatedinthefollowingtable. The findingswere reportedtothestudydirectoarnd management. InspectionDates EM T-Q 10/14/95 11/03/95 Phase FinalReport Date InspectioRneportedto Managm=t StudyDirect 11/03/95 11/03/95 QAU Auditor Date 9.4 Key PersonnelInvolvedin the Study oo008(; 3M Environmental Laboratory Key Personnel Thermal extractionfollowedby analysisusingOrion ton analyzer: Jim Johnson Deb Wright Rich Youngblom Deann Plummer Analysisofliverextractsusingelectrospramyass spectrometry: Jim Johnson Dave Christenson Thermal extractionfollowedby analysisusingSkslar segmented flow analyzerwith Ionselectiveelectrode: Jim Johnson Deb Wright Rich Youngblom Deann Plununer Documentation and Reporting: Jim Johnson Rich Youngblom QualityAssuranceUnit: Gale Van Buskirk CynthiaWeber KariRambo 9.11 Data 0()oos,s 9.11.1Summary and raw data; ug F -in whole liveras determinedby thermalextractiofnollowedby analysis usingOrion ionanalyzer. 0()()O.S!) Summary ofCombustion Data -Liver AMI)T-022195.1, HWI 6329-137 As Referenced in FinalReport section6.0DATA ANALYSIS Totalug Fluoridein Whole Liver Mean per Dose Group* Control Group 99 Std.Dev. 20.2 + 7.9 10 mg/kg dose (T6053)** 18.5 + 8.7 125 mg/kg dose (T6053) 16.5 + 4.0 250 mg/kg dose (T6053) 17.2 + 6.6 *Calculatedas the mean of triplicatseamples from each of threemale and threefemale rabbits. **One outliernot includedin mean. RPTI 37L.XLS FC99 ID SPK 63-1 SPK 63-2 SPK 126-1 SPK 126-2 LIVER BLK 1 LIVER BLK 2 F53442-1 F53442-2 F53442-3 F53464-1 F53464-2 F53464-3 F53455-1 F53455-2 F53455-3 liverblank-1 liverblank-2 livesrpike-I livesrpike-2 livesrpike-3 F53441 -1 F53441-2 F53441-3 F53451 -1 F53451-2 F53451-3 F53461 -1 F53461-2 F53461-3 F53460-1 F53460-2 F53460-3 liverblank-1 liverblank-2 liverspike-1 liverspike-2 liverspike-3 liverspike-4 liverblank-3 F53447-1 F53447-2 F53447-3 F53452-1 F53452-2 F53452-3 LIVER BLK 1 LIVER BLK 2 LIVER SPK 1 LIVER SPK 2 % rcvry 95% 96% 81% 82% 93% 90% 90% 90% 63% 97% 95% 92% 100% Actual ppm Finliver (wtw) 0.940 0.912 1.755 2.180 0.373 0.265 0.365 0.231 0.189 0.194 0.170 0.162 0.210 0.145 0.177 0.436 0.200 1.231 0.960 0.950 0.399 0.271 0.232 0.211 0.235 0.181 0.193 0.165 0.168 0.182 0.202 0.181 0.604 0.277 1.013 0.875 1.450 1.397 0.265 0.233 0.152 0.170 0.143 0.152 0.146 0.202 0.131 1.128 1.074 Average ppm Finliver (wtw) 0.262 0.175 0.178 0.301 0.209 0.175 0.188 0.185 0.147 Whole TotalFLiver liver inwhole burned weight liver (grams)(grams) (@Lg) 0.1525 0.1593 0.1393 0.1136 0.1216 0.1145 0.1460 0.1132 90.68 23.75 0.1598 0.1240 0.1447 86.57 15.17 0.1223 0.1375 0.1558 93.31 16.57 0.1126 0.1099 0.1376 0.1140 0.1420 0.1440 0.1388 0.1435 114.12 34.35 0.1343 0.1398 0.1152 88.41 18.47 0.1413 0.1552 0.1277 72.71 12.73 0.1316 0.1230 0.1370 90.52 17.03 0.1265 0.1361 0.1326 0.1342 0.1087 0.1011 0.1028 0.1518 0.1007 0.1376 79.96 14.80 0.1161 0.1295 0.1159 80.42 11.85 0.1309 0.1580 0.1622 0.1240 0.1402 Dosage (mg/kg) SPIKE DATA FOR 63 AND 126 PPM FC95 COPIED FROM 6329136L ENDING QC DDW 4-5-95 0 0 0 0 0 0 10 10 10 Page 1 FC99 ID F53463-1 F53463-2* F53463-3 F53444-1 F53444-2 F53444-3 F53448-1 F53448-2 F53448-3 F53446-1 F53446-2 F53446-3 F53453-1 F53453-2 F53453-3 F53458-1 F53458-2 F53458-3 F53443-1 F53443-2 F53443-3 F53450-1 F53450-2 F53450-3 F53467-1 F53467-2 F53467-3 F53439-1 F53439-2 F53439-3 F54445-1 F54445-2 F54445-3 F54459-1 F54459-2 F54459-3 LiverBlank-2 LiverBlank-3 LiverSpike-I LiverSpike-2 LiverSpike-3 LiverSpike-4 Liverblank-4 F53449-1 F53449-2 F53449-3 % rcvry 83% 81% 90% 96% Actual ppm Finliver (w/w) 0.696 1.869 0.170 0.629 0.068 0.163 0.188 0.146 0.184 0.187 0.140 0.183 0.246 0.145 0.285 0.503 0.160 0.190 0.175 0.175 0.194 0.164 0.171 0.153 0.222 0.203 0.178 0.119 0.139 0.390 0.200 0.151 0.242 0.140 0.150 0.142 0.432 0.148 1.096 1.149 0.932 1.306 0.277 0.248 0.173 0.163 Average ppm Finliver (wtw) 0.433 0.287 0.172 0.170 0.225 0.284 0.181 0.163 0.201 0.216 0.198 0.144 0.194 Whole TotalF- Liver liver inwhole burned weight liver (grams) (grams) (n) 0.1285 0.0152 81.40 35.28 0.1283 0.1190 0.1399 69.27 19.85 0.1348 0.1252 0.1199 72.33 12.47 0.1155 0.1169 0.1332 82.78 14.07 0.1164 0.1196 0.1479 94.51 21.30 0.1513 0.1133 0.1332 76.79 21.84 0.1001 0.1461 0.1238 76.45 13.87 0.1082 0.1303 0.1270 76.89 12.53 0.1072 0.1365 0.1087 77.56 15.61 0.1004 0.1366 0.1419 81.71 17.63 0.1328 0.1320 0.1261 95.81 18.97 0.1324 0.1479 0.1273 78.24 11.26 0.1177 0.1255 0.1328 0.1143 0.1063 0.1466 0.1110 0.1079 0.1284 0.1257 70.80 13.76 0.1495 Dosage (mg/kg) 10 10 10 125 125 125 125 125 125 250 250 250 250 Outliernot includedinaverage. Page 2 RPT1 37L.XLS o oo09 RPTI 37L.XLS FC99 ID F53504-1 F53504-2 F53504-3 F53456-1 F53456-2 F53456-3 F53454-1 F53454-2 F53454-3 LiverBik-I LiverBlk-2 LiverSpike-1 LiverSpike-2 LiverSpike-3 LiverSpike-4 % rcvry 94% 108% 104% 94% Actual ppm Finliver (w/w) 0.138 0.141 0.166 0.188 0.730 0.220 0.126 0.111 0.215 0.146 0.140 Average Whole TotalF- ppm F- Liver liver inwhole inliver burned weight liver (wtw) (grams)(grams) (@Lg) 0.1274 0.148 0.1368 82.31 12.22 0.1116 0.1017 0.379 0.1398 77.10 29.25 0.1258 0.1304 0.151 0.1414 93.04 14.02 0.1245 0.1149 0.1029 0.1074 0.1378 0.1052 0.1200 Dosage (mg/kg) 250 Replacement forF54454 250 250 Replaced on Day 2 by F53504 Page 3 9.11.2Raw data;analysisof liverextractussingelectrospray mass spectrometry. Thisdata,althoughsupportivei,ntheopinionof theStudy Directorisnot requiredtoreachtheconclusionstatedin FinalReport Section6.0,and thereforeisnot discussedin detail. 0000,44 041095A Sm (Mn,2x3) 100- (.329 (AlEt,)IFC--c[cL 2744 524 384 176 837 614 1875 1713 1407 1263 2551 2364 2140 -e-,cw-41 4400 4232 3501 3JO6U0I4+ 3844 3289 4567 4895 0 1041095A Sm (Mn, 2x3) 10G- 1730 1580 3201 639 798 525 91328 9L951179 L 500 1000 1500 2588 2745 1948 2098 2284 3073 294a 2000 2500 3000 3533 342 7 4140 3897 4333 LJ374l1 j, 4809 4661 49 3500 4000 4500 50 041095A Sm (Mn,2x3) 100- 524 837 384 806 176 218 329 614 468 1576 1407 1541 1326 11761263 19$)921222140 17131792 1944 04109;,A,S',m'('M'n','2x3) 100- 1730 1580 LP ip LL 639 LO 798 -S 525 ul (A U- LO U- La 384 LL. 1328 148 2098 995 1179 260 400 .6.0.0......8.00. 1066 ......1'2'0-0'' 1466 ...... 1'6'0'0' 1'800 2000 22 041095A Sm (Mn,2x3) 100- 2744 - 2364 2551 0/0- LV@3289 5151 51 44330(7 4400 4567 4670 4895 5027 40066433@2432 32 33550@0l1 33660@044 36723@8348444@ 32839344@1l00 0 041095A Sm (Mn,2x3) IOG- in U. 2745 2588 3201 w tv to 3 73 212C943 Ln LL m LP LL 3533 tL 3427 L37r4 LO rtO 38f97 3741 Li cn $A (A. 4140 I In Lt. to 4333 LL 4494 o@* m LP U- kP 4809 4661 ip 14 0 iL t to 5151 ir 4998 2400 2600 2800 3000 3200 3400 3600 3800 4000 4200 4400 4600 4800 5000 520( 9.11.3Summary and raw data; ug F- inwhole liveras detenninedby thennalextractiofnollowedby analysisusing Skalarsegmented flow analyzerwith ion selectivelectrode. This data,althoughsupportivei,ntheopinionof the Study Directorisnot requiredto reachtheconclusionstatedin FinalReport Section6.0,and thereforeisnot discussedin detail. oo00!)@-i RE:6329-137 LIUER SRMPLES RMBT 22195.2 Date of Rnalysis:5-2-95 Rnalyst: DOW The samples are burned inthe Dohrman at 950 C using between IB.1and IB.2grams of the liver.The gas iscollectedin 1.0 mL of 1:1TISRB/Milli-Qwater then an additionalI mL of 1:1 TISRB/Milli-Qisadded to allow for sufficientvolume for Skalar analysis.The samples are then analyzed on a Skalar Segmented Flow Analyzer using the Ion SpecificElectrode(ISE)Method. TISRB buffer isadded to each sample as itproceeds through the system. The sample then goes through a heated mixing coil before the potentialbetween the ionselectiveelectrodeand the reference electrode ismeasured. The signalisamplified and relatedto the fluorideconcentration. The instrument was calibratedin the ranges of 0.015 - 0.15 ppm and 0.15 - 1.50 ppm fluoride.The standard curve for the high range was plotted using the inverse logarithm option.The standard curve for the low range islinear.RIIstandards and samples were then calculatedby the Skalar software using these curves. Rilresultsbelow 0.0001 ppm appear on the raw data as R qualitycontrolstandard was analyzed every 10 samples to check for accuracy and drift. Raw data is taken from the appropriatecalibratedrange of the Skalar printoutand summarized on an Excel spreadsheet. The finalresultsare adjusted for the collectionvolume and any subsequent dilutions. --- ---------------------------------------- ---------------------------------- SUMMARY of 6329-137 LIVER SAMPLES AMDT ---------------- 2219--5--.--2------------------------- GROUP 1 Dose Level 0 -------------- F53442-1 0.02 2.0 0.1460 0.33 F53442-2 ND 2.0 0.1132 ND ND F53442-3 ND 2.0 0.1596 ND F53464-1 ND 2.0 0.1240 ND F53464-2 ND 2.0 0.1447 ND ND F53464-3 ND 2.0 0.1223 ND F53455-1 ND 2.0 0.1375 ND F53455-2 ND 2.0 0.1558 ND ND F53455-3 ND 2.0 0.1126 ND F53441-1 0.03 2.0 0.1388 0.39 F53441-2 0.02 2.0 0.1435 0.23 0.21 F53441-3 ND 2.0 0,1343 ND F53451-1 ND 2.0 0.1398 ND F53451-2 ND 2.0 0.1152 ND ND F53451-3 ND 2.0 0.1413 ND F53461-1 ND 2.0 0.1552 ND F53461-2 ND 2.0 0.1277 ND ND F53461-3 ND 2.0 0.1316 ND ........-.-.-.-.-.-.--.-.------------------ 90.6763 29.6 90.6763 ND ND 90,6763 ND 86.5739 ND 86.5739 ND ND 86.5739 ND 93.3090 ND 93.3090 ND ND 93.3090 ND 114.1164 45.1 114.1164 26.1 23.7 114.1164 ND 88.4088 ND 88.4088 ND ND 88.4088 ND 72.7104 ND 72.7104 ND ND 72.7104 ND F53460-1 ND 2.0 0.1230 ND 90.5154 ND F53460-2 ND 2.0 0.1370 ND ND 90.5154 ND ND F53460-3 ND 2.0 0.1265 ND 90.5154 ND F53447-1 ND 2.0 0.1007 ND 79.9589 ND F53447-2 ND 2.0 0.1376 ND ND 79.9589 ND ND F53447-3 ND 2.0 0.1161 ND 79.9589 ND F53452-1 ND 2.0 0.1295 ND 80.4220 ND F53452-2 ND 2.0 0.1159 ND ND 80.4220 ND ND GROUP2 F53452-3 ND 2.0 0.1309 ND 80.4220 ND Dose Level: 10mgfkg F53463-1 0.06 2.0 0.1285 0.95 81.3971 77.7 F53463-2 ND 2.0 0.1516 ND 0.32 81.3971 ND 25.9 F53463-3 ND 2.0 0.1283 ND 81.3971 ND F53444-1 ND 2.0 0.1190 ND 69.2675 ND F53444-2 ND 2.0 0.1399 ND ND 69.2675 ND ND F53444-3 ND 2.0 0.1348 ND 69.2675 ND F53448-1 ND 2.0 0.1252 ND 72.3334 ND F53448-2 ND 2.0 0.1199 ND ND 72.3334 ND ND F53448-3 ND 2.0 0.1155 ND 72.3334 ND F53446-1 ND F53446-2 ND F53446-3 0.02 F53453-1 0.02 F53453-2 0.02 F53453-3 0.03 F53458-1 0.04 2.0 0.1169 ND 2.0 0.1332 ND 2.0 0.1164 0.30 2.0 0.1196 0.29 2.0 0.1479 2.0 0.1513 0.37 2.0 0.1133 0.75 HWI-1 37L.SUM 82.7817 ND ND 82.7817 ND ND 82.7817 24.5 94.5134 27.2 0.29 94.5134 20.2 27.4 94.5134 34.9 76.7873 57.9 o()()16TF I nt SUMMARY of 6329-137 LIVER SAMPLES AMDT 22195.2 -------------- GROUP3 F53458-2 0.02 2.0 0.1332 0.26 ------0--.-3-4------ 76.7873 19.8 25.9 Dose Level: 125 mgfkg F53458-3 ND 2.0 0.1001 ND 76.7873 ND F53443-1 ND 2.0 0.1461 ND 76.4455 ND F53443-2 ND 2.0 0.1238 ND ND 76.4455 ND ND F53443-3 ND 2.0 0.1082 ND 76.4455 ND F53450-1 ND 2.0 0.1303 ND 76.8935 ND F53450-2 ND 2.0 0.1270 ND ND 76.8935 ND ND F53450-3 ND 2.0 0.1072 ND 76.8935 ND F53467-1 0.02 2.0 0.1365 0.23 77.5604 17.6 F53467-2 ND 2.0 0.1087 ND ND 77.5604 ND ND F53467-3 ND 2.0 0.1004 ND 77.5604 ND F53439-1 ND 2.0 0.1366 ND 81.7065 ND F53439-2 ND 2.0 0.1419 ND ND 81.7065 ND ND F53439-3 ND 2.0 0.1328 ND 81.7065 ND F53445-1 ND 2.0 0.1320 ND 95.8121 ND F53445-2 ND 2.0 0.1261 ND ND 95.8121 ND ND F53445-3 ND 2.0 0.1479 ND 95.8121 ND F53459-1 ND 2.0 0.1479 ND 78.2415 ND F53459-2 ND 2.0 0.1273 ND ND 78.2415 ND ND F53459-3 0.02 2.0 0.1177 0.39 78.2415 30.3 GROUP4 F53449-1 ND 2.0 0.1284 ND 70.7962 ND Dose Level: 250 mgtkg F53449-2 ND 2.0 0.1257 ND ND 70.7962 ND ND F53449-3 ND 2.0 0.1495 ND 70.7962 ND F53504-1 ND 2.0 0.1274 ND 82.3142 ND F53504-2 ND 2.0 0.1368 ND ND 82.3142 ND ND F53504-3 ND 2.0 0.1116 ND 82.3142 ND F53456-1 ND 2.0 0.1017 ND 77.1014 ND F53456-2 0.06 2.0 0.1398 0.82 0.27 77.1014 62.9 21 F53456-3 ND 2.0 0.1258 ND 77.1014 ND F53454-1 ND 2.0 0.1304 ND 93.0439 ND F53454-2 ND 2.0 0.1414 ND ND 93,0439 ND ND F53454-3 ND 2.0 0.1245 ND 93.0439 ND HWI-1 37L.SUM Page 2 1995-06-1308:39 OutPut of :950502AI Operator DDW Date oftheAnalysis:199"5-02 08:48 AnalysisFileName :C:\SKALAR\DATA\HWIDATA\LIVERSW50502Al HWI-1 37L.XLS I 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 ,Ohs27 28 29 30 31 Tracer Drift Wash Standard 1 Standard2 Standard 3 Standard4 Standard5 Standard6 Standard7 Standard8 Standard9 Standard10 Drift Wash LIVER BLK 1 LIVER BLK 2 F53442-1 F53442-2 F53442-3 F53464-1 F53464-2 F53464-3 F53455-1 F53455-2 Drift Wash F53455-3 LIVER BLK 1 LIVER BLK 2 LIVER SPK 1 1.50 1.50 0.015 0.03 0.06 0.09 0.12 0.15 0.30 0.60 1.20 1.50 1.50 1.50 1.46 97% 1.48 99% 0.01 97% 0.03 108% 0.06 95% 0.09 101% 0.12 101% 0.15 103% 0.29 95% 0.61 102% 1.23 103% 1.47 98% 1.51 100% 0.00 0.00 0.00 0.02 0.00 0.01 0.00 0.01 0.00 0.01 0.01 1.48 99% 0.00 0.01 0.02 0.00 0.08 2.0 0.1216 2.0 0.1145 2.0 0.1460 0.33 90.6763 29.56 2.0 0.1132 0.06 90.6763 5.13 2.0 0.1596 0.12 90.6763 11.02 2.0 0.1240 0.06 86.5739 5.17 2.0 0.1447 0.13 86.5739 11.61 2.0 0.1223 0.04 86.5739 3.82 2.0 0.1375 0.10 93.3090 8.96 2.0 0.1558 0.07 93.3090 6.71 2.0 0.1126 0.19 93.3090 18.07 2.0 0.1099 0.41 2.0 0.1376 0.05 2.0 0.1140 1.40 0.004 63.00 Page I HWI-1 37L.XLS 32 LIVER SPK 2 33 F53441-1 34 F53441-2 35 F53441-3 36 F53451-1 37 F53451-2 38 Drift 1.50 39 Wash 40 F53451-3 41 F53461-1 42 F53461-2 43 F53461-3 44 F53460-1 45 F53460-2 46 F53460-3 47 F53447-1 48 F53447-2 49 F53447-3 50 Drift 1.50 51 Wash 52 F53452-1 53 F53452-2 54 F53452-3 55 BLK 1 56 BLK 2 57 SPK 1 58 SPK 2 59 F53463-1 60 F53463-2 61 F53463-3 62 Drift 1.50 63 Wash 64 F53444-1 65 F53444-2 66 F53444-3 67 F53448-1 0.08 0.03 0.02 0.01 0.01 0.01 1.50 0.00 0.01 0.01 0.01 0.01 0.00 0.01 0.00 0.00 0.00 0.01 1.52 0.00 0.01 0.00 0.00 0.01 0.00 0.08 0.09 0.06 0.01 0.01 1.52 0.00 0.04 0.00 0.01 0.00 100% 101% 101% 2.0 0.1420 1.09 0,004 63.00 2.0 0.1388 0.39 114.1164 45.05 2.0 0.1435 0.23 114.1164 26.08 2.0 0.1343 0.19 114.1164 22.09 2.0 0.1398 0.10 88.4088 8.85 2.0 0.1152 0.14 88.4088 11.97 2.0 2.0 2.0 0.1413 0.15 88.4088 13.26 2.0 0.1552 0.15 72.7104 10.68 2.0 0.1277 0.10 72.7104 6.95 2.0 0.1316 0.08 72.7104 5.64 2.0 0.1230 0.07 90.5154 6.48 2.0 0.1370 0.16 90.5154 14.67 2.0 0.1265 0.05 90.5154 4.87 2.0 0.1007 0.07 79.9589 5.88 2.0 0.1376 0.07 79.9589 5.35 2.0 0.1161 0.10 79.9589 7.99 2.0 0.1295 0.12 80.4220 9.69 2.0 0.1159 0.05 80.4220 4.16 2.0 0.1309 0.06 80.4220 4.79 2.0 0.1580 0.10 2.0 0.1622 0.03 2.0 0.1240 1.35 0.004 63.00 2.0 0.1402 1.27 0.004 63.00 2.0 0.1285 0.95 81.3971 77.66 2.0 0.1516 0.15 81.3971 12.46 2.0 0.1283 0.09 81.3971 7.36 2.0 0.1190 0.61 69.2675 42.26 2,0 0.1399 0.00 69.2675 0.00 2.0 0.1348 0.11 69.2675 7.71 2.0 0.1252 0.05 72.3334 3.70 Page 2 HWI-137L.XLS 68 F53448-2 69 F53448-3 70 Drift 1.50 71 Wash 72 RunOut Wash 0.00 0.00 1.53 102% 0.00 0.00 2.0 0.1199 0.00 72.3334 0.00 2.0 0.1155 0.07 72.3334 4.98 Page 3 1995-06-13 09:41 Output of : 950502BI Operator DDW Date ofthe Analysis :1995-05-02 12:41 AnalysisFileName :C:ISKALAR\DATA\HWIDATA\LIVERS\950502BI HWI137L2.XLS 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 ,aft 27 28 29 30 Tracer Drift Wash Standard 1 Standard 2 Standard 3 Standard 4 Standard 5 Standard 6 Standard 7 Standard 8 Standard 9 Standard 10 Drift Wash F53446-1 F53446-2 F53446-3 F53453-1 F53453-2 F53453-3 F53458-1 F53458-2 F53458-3 F53443-1 Drift Wash F53443-2 F53443-3 F53450-1 1.50 1.50 0.015 0.03 0.06 0.09 0.12 0.15 0.30 0.60 1.20 1.50 1.50 1.50 1.49 99% 1.49 99% 0.00 0.01 95% 0.03 108% 0.06 96% 0.09 100% 0.12 102% 0.15 103% 0.29 95% 0.61 102% 1.24 103% 1.47 98% 1.51 101% 0.00 0.01 0.01 0.02 0.02 0.02 0.03 0.04 0.02 0.01 0.01 1.48 98% 0.00 0.02 0.01 0.02 2.0 0.1169 0.22 82.7817 18.41 2.0 0.1332 0.15 82.7817 12.43 2.0 0.1164 0.30 82.7817 24.46 2.0 0.1196 0.29 94.5134 27.18 2.0 0.1479 0.21 94.5134 20.19 2.0 0.1513 0.37 94.5134 34.86 2.0 0.1133 0.75 76.7873 57.88 2.0 0.1332 0.26 76.7873 19.83 2.0 0.1001 0.11 76.7873 8.59 2.0 0.1461 0.16 76.4455 12.24 2.0 0.1238 0.37 76.4455 28.16 2.0 0.1082 0.15 76.4455 11.16 2.0 0.1303 0.33 76.8935 25.14 Page 1 HWI137L2.XLS 31 F53450-2 0.01 2.0 0.1270 0.16 76.8935 12.11 32 F53450-3 0.00 2.0 0.1072 0.09 76.8935 6.74 33 F53467-1 0.02 2.0 0.1365 0.23 77.5604 17.61 34 F53467-2 0.00 2.0 0.1087 0.08 77.5604 6.14 35 F53467-3 0.00 2.0 0.1004 0.09 77.5604 6.95 36 F53439-1 0.00 2.0 0.1366 0.06 81.7065 5.14 37 F53439-2 0.01 2.0 0.1419 0.13 81.7065 10.59 38 Ddft 1.50 1.52 101% 39 Wash 0.00 40 F53439-3 41 F53445-1 42 F53445-2 43 F53445-3 44 F53459-1 45 F53459-2 46 F53459-3 0.03 0.01 0.00 0.01 0.01 0.00 0.02 2.0 0.1328 0.49 81.7065 40.11 2.0 0.1320 0.21 95.8121 19.74 2.0 0.1261 0.07 95.8121 6.23 2.0 0.1479 0.13 95.8121 12.44 2.0 0.1479 0.15 78.2415 11.53 2.0 0.1273 0.06 78.2415 4.55 2.0 0.1177 0.39 78.2415 30.31 47 BLK 2 0.02 2.0 0.1255 0.34 48 BLK 3 49 SPK 1 0.00 2.0 0.1328 0.00 0.06 2.0 0.1143 1.12 0.004 63.00 50 Drift 1.50 1.48 99% 51 Wash 0.00 52 SPK 2 0.06 2.0 0.1063 1.13 0.004 63.00 53 SPK 3 54 SPK 4 55 LIVER BLK 0.07 2.0 0.1466 0.94 0.08 2.0 0.1110 1.42 0.00 2.0 0.1079 0.03 0.004 0.004 63.00 63.00 56 F53449-1 57 F53449-2 58 F53449-3 0.01 0.00 0.00 2.0 0.1284 0.12 70.7962 8.27 2.0 0.1257 0.00 70.7962 0.00 2.0 0.1495 0.06 70.7962 4.45 59 F53504-1 60 F53504-2 0.00 2.0 0.1274 0.00 82.3142 0.00 0.00 2.0 0.1368 0.00 82.3142 0.00 61 F53504-3 0.00 2.0 0.1116 0.00 82.3142 0.00 62 Ddft 1.50 1.46 97% 63 Wash 64 F53456-1 0.00 0.00 2.0 0.1017 0.01 77.1014 1.06 65 F53456-2 0.06 2.0 0.1398 0.82 77.1014 62.87 66 F53456-3 0.01 2.0 0.1258 0.12 77.1014 9.44 Page 2 HWI137L2.XLS 67 F53454-1 0.00 68 F53454-2 0.00 69 F53454-3 0.01 70 BLK 1 0.00 71 BLK 2 0.00 72 SPK 1 0.08 73 SPK 2 0.10 74 Drift 1.50 1.49 100% 75 Wash 0.00 76 SPK 3 0.19 77 SPK 4 0.16 78 Drift 1.50 1.52 101% 79 Wash 0.00 wt RunOut Wash 0.00 2.0 0.1304 0.00 93.0439 0.00 2.0 0.1414 0.00 93.0439 0.00 2.0 0.1245 0.08 93.0439 7.77 2.0 0.1149 0.02 2.0 0.1029 0.00 2.0 0.1074 1.56 0.004 63.00 2.0 0.1378 1.38 0.004 63.0( 2.0 0.1052 3.52 2.0 0.1200 2.73 0.004 0.004 126.0 126.0 Page 3 1995--05-03 08:38 OutPut of 950502Al Software version 6.1 cl990,93 Operator Date of the Analysis DDW 1995-05-02 08:48 Analysis File Name C:\SKALAR\DATA\HWIDATA\LIVERS\950502Al leito AMB-T Omt-195'- Fluoride 1.5 Calibration order = Inverse Logarithm Slope Result s = #.##### x - cl s x = corrected value of the sample cl = corrected value of the concentration 1 s = Slope of the electrode a2 = al = aO = -0.00000 0.00066 -1.20681 Fluoride L Calibration order = 2 Correlation Result = a2 r = 0.99926 X2 + al * x + aO a2 = al = aO = 0.00000 0.00024 -0.00641 Sampler Type Number Sample Time Wash Time Air Time Take up special needle Height SA1000 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 :I User file : TXT Reproces : No 1995-05-03 08:38 OutPut of Fluoride 1.5 Path number Signal type Decolor system Number dilute Resample dil Threshold diG output Window event 3 Debubbled Yes 0 No No 4095 0 Off sl standard s2 standard s3 standard s4 standard s5 standard s6 standard s7 standard s8 standard S9 standard slO standard Order : Inverse Dimension : PPM start Value trigger Limit Peak shape start ignore eNd ignore Measure window Filter Regeneration formula output Ignore Ignore Ignore Ignore Ignore 0.150 0.300 0.600 1.200 1.500 Logarithm 500 DU 1800 Sec Pointed 60 Sec 120 Sec 75 % No No Fluoride L Path number Signal type Decolor system Number dilute Resample dil Threshold diG output Window event 0 Debubbled No 0 No No 4095 0 Off 950502Al 1995-05-03 08:38 OutPut of sl standard s2 standard s3 standard s4 standard s5 standard s6 standard s7 standard S8 standard S9 standard slO standard 0.015 0.030 0.060 0.090 0.120 0.150 Ignore Ignore Ignore Ignore Order : 2 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 c4:=c3 output 950502AI 1995-05-03 08:38 OutPut of 950502Al Fluoride 1.5 Fluoride L PPM Pos Typ Ident Ch Result F Time wt iw Initial Wash 3 0.062 65 1t Tracer 3 1.458 211 2d Drift 3 1.481 386 3w Wash 3 0.062 626 4 sl Standard 1 3 0..071 738 5 s2 Standard 2 3 0.079 910 6 s3 Standard 3 3 0.091 1084 7 s4 Standard 4 3 0.112 1261 8 s5 Standard 5 3 0.133 1437 9 s6 Standard 6 3 0.154 1611 10 s7 Standard 7 3 0.285 1787 11 S8 Standard 8 3 0.611 1961 12 s9 Standard 9 3 1.234 2135 13 slO Standard 10 3 1.465 2311 14 d Drift 3 1.505 2485 15 w Wash 3 0.062 2721 16 u LIVER ELK 1 3 0.061 A 2835 17 u LIVER ELK 2 3 0.060 3018 18 u F53442-1 3 0.075 3187 19 u 20 u F53442-2 F53442-3 3 0.066 3 0.069 3360 3536 21 u F53464-1 3 0.066 3708 22 u F53464-2 3 0.069 3879 23 u F53464-3 3 0.066 4063 24 u F53455-1 3 0.067 4237 25 u F53455-2 3 0.067 4411 26 d Drift 3 1.479 4587 27 w Wash 3 0.062 4812 28 u F53455-3 3 0.069 4925 29 u LIVER ELK 1 3 0.074 5109 30 u LIVER ELK 2 3 0.066 5291 31 u LIVER SPK 1 3 0.105 5463 32 u LIVER SPK 2 3 0.103 5641 33 u F53441-1 3 0.077 5815 34 u F53441-2 3 0.072 5987 35 u F53441-3 3 0.070 6167 36 u 37 u F53451-1 F53451-2 3 0.068 3 0.068 6335 6515 38 d Drift 3 1.502 6689 39 w Wash 3 0.062 6927 40 u F53451-3 3 0.069 7029 41 u F53461-1 3 0.069 7214 42 u F53461-2 3 0.067 7386 43 u F53461-3 3 0.067 7563 44 u F53460-1 3 0.066 7740 45 u F53460-2 3 0.069 7908 46 u F53460-3 3 0.066 8088 47 u F53447-1 3 0.066 8262 48 u F53447-2 3 0.067 8440 49 u F53447-3 3 0.067 8616 50 d Drift 3 1.517 8790 51 w Wash 3 0.062 9029 52 u F53452-1 3 0.068 9135 53 u F53452-2 3 0.066 9314 PPM Ch Result F Time 4 #.#### 0 4 0.7832 0 4 0.7926 0 4 #.#### 0 4 0.0145 0 4 0.0325 0 4 0.0567 0 4 0.0908 0 4 0.1213 0 4 0.1492 0 4 0.2708 0 4 0.4552 0 4 0.6976 0 4 0.7863 0 4 0.8025 0 4 0 4 0 4 0 4 0.0238 0 4 0.0032 0 4 0.0097 0 4 0.0037 0 4 0.0097 0 4 0.0027 0 4 0.0066 0 4 0.0056 0 4 0.7919 0 4 #.#### 0 4 0.0109 0 4 0.0224 0 4 0.0034 0 4 0.0798 0 4 0.0776 0 4 0.0274 0 4 0.0164 0 4 0.0130 0 4 0.0070 0 4 0.0078 0 4 0.8013 0 4 #.#### 0 4 0.0106 0 4 0.0114 0 4 0.0061 0 4 0.0051 0 4 0.0044 0 4 0.0111 0 4 0.0034 0 4 0.0037 0 4 0.0046 0 4 0.0058 0 4 0.8078 0 4 #.#### 0 4 0.0078 0 4 0.0030 0 Page 1 of 2 1995-05-03 08:38 OutPut of 950502Al Fluoride 1.5 Fluoride L Pos Typ Ident PPM Ch Result F Time PPM Ch Result F Time 54 u F53452-3 3 0.066 9485 4 0.0039 0 55 u BLK 1 3 0.068 9661 4 0.0078 0 56 u BLK 2 3 0.066 9839 4 0.0025 0 57 u SPK 1 3 0.107 10016 4 0.0834 0 58 u SPK 2 3 0.110 10191 4 0.0887 0 59 u F53463-1 3 0.094 10367 4 0.0613 0 60 u F53463-2 3 0.070 10537 4 0.0116 0 61 u F53463-3 3 0.067 10715 4 0.0058 0 62 d Drift 3 1.519 10889 4 0.80'85 0 63 w Wash 3 0.062 11126 4 #.#### 0 64 u F53444-1 3 0.081 11239 4 0.0363 0 65 u F53444-2 3 0.062 11425 4 #.#### 0 66 u F53444-3 3 0.068 11593 4 0.0075 0 67 u F53448-1 3 0.066 11765 4 0.0032 0 68 u F53448-2 3 0.064 11941 4 #.#### 0 69 u F53448-3 3 0.066 12118 4 0.0039 0 70 d Drift 3 1.531 12291 4 0.8135 0 71 w Wash 3 0.062 12529 4 0 wt rw RunOut Wash 3 0.062 12766 4 0 Page 2 of 2 Calibration curve ot 950SO@@@'Al Fluoride L C -0.006409 0 Order 2 Measured 900 0.9992S Calibration curve of 9SOSO'-)AI Fluoride I.S c, 0 U.UbZll-') 0 Measured Or-14.--r Inve,-c-Leogari.+Ihm 4035 oool.l,l 41j9S Raw data of 95050'@'Al Fluoride I.S t39 Inirg 64 1'5 14 Cl Esc=Exit FI=Help Time Crtl-P=Editpeakc. f 5 oo011!-3 4095 Raw data of 950501@Al Fluoride 1-S M:Fll.I.R,.TWMlrr-9 l4-q7SE@N[M 722 1 t-7-I3TH C-4 fI I t3710@ Esf-=@-xit Fl=Help Time Crtl-P=Edit peaks -1f 3998 4095 Raw data of 9SO502AI 4OC-,3 Fluor-ide t.S 29 gA '30 0 3998 Esc=Exit : FI=Help T imrCrtl-P=Edi@t-peaks 1 C. -S7 oo01.IL7 40!3 Raw data of 95050'-@@At Fluoride I.S c4 't2- 4(.,4? Sr 4-@ 6 C'@'3 Time -q 4 1 FI=Help oool.ll-. 409S Raw data of 9Sa-50@'Al Fluoride 1-@7-Y 91-)48 !-'sc=LC-ixt :.FI=Help Ct-tl-P=Edit peaks T ime ooolLIL!) 409S Raw data of 91@O'@OA':At Fluoride I..' 11941W-@ 21-0 964 0 11873 tse@txlt 1 tl--HelP1 Urtl-r--tditpeaks Time 14498i o ()o I'A-0., 1995-05-03 09:05 OutPut of 950502Bl Software version 6.1 cl990,93 Operator Date of the Analysis DDW 1995-05-02 12:41 Analysis File Name C:\SKALAR\DATA\HWIDATA\LIVERS\950502Bl Fluoride 1.5 Calibration order = Inverse Logarithm Slope : s = #.##### t = lo[ x - cl Resul s 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 -1.24604 Fluoride L Calibration order = 2 Coz,relation r = 0.99932 Result = a2 X2 + al * x + aO a2 = al = aO = 0.00000 0.00021 -0.00305 Sampler Type Number Sample Time Wash Time Air Time Take up special needle Height : SA1000 :I : 50 sec. : 120 sec. : 1 sec. : Single : None : 70 mm. Diluter needle Height dilution Factor dilution Volume Resample Dilution runs 80 mm 10 2.5 ml. 1 1 User file TXT Reproces No O.Z.2 j 1'0-I1L 1995-05-03 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 s2 standard s3 standard s4 standard s5 standard s6 standard s7 standard s8 standard S9 standard slO standard Order : Inverse Dimension : PPM start Value trigger Limit Peak shape start ignore eNd ignore Measure window Filter Regeneration formula output Ignore Ignore Ignore Ignore Ignore 0.150 0.300 0.600 1.200 1.500 Logarithm 500 DU 1800 Sec Pointed 60 Sec 120 Sec 75 % No No Fluoride L Path number Signal type Decolor system Number dilute Resample dil Threshold diG output Window event 0 Debubbled No 0 No No 4095 0 Off 950502Bl 1995-05-03 09:05 OutPut of sl standard 0.015 s2 standard s3 standard 0.030 0.060 s4 standard s5 standard S6 standard 0.090 0.120 0.150 s7 standard Ignore S8 standard Ignore S9 standard Ignore slO standard Order : 2 Ignore Dimension : PPM start Value trigger Limit 500 DU 1800 Sec Peak shape Pointed start ignore eNd ignore 60 Sec 120 Sec Measure window 75 % Filter No Regeneration No formula c4:=c3 output 950502Bl 1995-05-03 09:05 output of 950502Bl Fluoride 1.5 Fluoride L PPM PPM Pos Typ Ident Ch Result F Time Ch Result F Time wt iw Initial Wash 3 0.057 65 4 0 1t 2d Tracer 3 1.486 211 4 0.8506 0 Drift 3 1.491 387 4 0.8536 0 3w Wash 3 0.057 628 4 #.#### 0 4 sl Standard 1 3 0.064 737 4 0.0143 0 5 s2 Standard 2 3 0.073 913 4 0.0324 0 6 s3 Standard 3 3 0.087 1089 4 0.0576 0 7 s4 Standard 4 3 0.107 1264 4 0.0896 0 8 s5 Standard 5 3 0.131 1440 4 0.1221 0 9 s6 Standard 6 3 0.154 1613 4 0.1490 0 10 s7 Standard 7 3 0.286 1788 4 0.2673 0 11 s8 Standard 8 3 0.609 1964 4 0.4541 0 12 s9 Standard 9 3 1.235 2138 4 0.7300 0 13 slO Standard 10 3 1.467 2312 4 0.8407 0 14 d Drift 3 1.514 2488 4 0.8662 0 15 w Wash 3 0.057 2723 4 #.#### 0 16 u F53446-1 3 0.064 2829 4 0.0130 0 17 u F53446-2 3 0.062 3004 4 0.0100 0 18 u F53446-3 3 0.066 3191 4 0.0172 0 19 u F53453-1 3 0.066 3360 4 0.0172 0 20 u F53453-2 3 0.065 3539 4 0.0158 0 21 u F53453-3 3 0.071 3713 4 0.0279 0 22 u F53458-1 3 0.079 3891 4 0.0427 0 23 u F53458-2 3 0.066 4065 4 0.0172 0 24 u F53458-3 3 0.060 4237 4 0.0056 0 25 u F53443-1 3 0.063 4405 4 0.0117 0 26 d Drift 3 1.476 4587 4 0.8457 0 27 w Wash 3 0.057 4826 4 #.#### 0 28 u F53443-2 3 0.068 4941 4 0.0228 0 29 u F53443-3 3 0.062 5113 4 0.0079 0 30 u F53450-1 3 0.068 5289 4 0.0213 0 31 u F53450-2 3 0.062 5463, 4 0.0100 0 32 u F53450-3 3 0.060 5636 4 0.0047 0 33 u F53467-1 3 0.065 5817 4 0.0155 0 34 u F53467-2 3 0.060 5987 4 0.0043 0 35 u F53467-3 3 0.060 6162 4 0.0045 0 36 u F53439-1 3 0.060 6337 4 0.0043 0 37 u F53439-2 3 0.062 6516 4 0.0092 0 38 d Drift 3 1.515 6688 4 0.8668 0 39 w Wash 3 0.057 6927 4 #.#### 0 40 u F53439-3 3 0.073 7038 4 0.0326 0 41 u F53445-1 3 0.064 7214 4 0.0136 0 42 u F53445-2 3 0.060 7384 4 0.0041 0 43 u F53445-3 3 0.062 7564 4 0.0096 0 44 u F53459-1 3 0.063 7738 4 0.0109 0 45 u F53459-2 3 0.060 7918 4 0.0037 0 46 u F53459-3 3 0.068 8090 4 0.0228 0 47 u BLK 2 3 0.068 8266 4 0.0215 0 48 u BLK 3 3 0.056 8436 4 #.#### 0 49 u SPK 1 3 0.091 8616 4 0.0640 0 50 d Drift 3 1.483 8790 4 0.8492 0 51 w Wash 3 0.057 8930 4 0 52 u SPK 2 3 0.089 9141 4 0.0603 0 53 u SPK 3 3 0.094 9317 4 0.0688 0 Page I of 2 1995-05-03 09:05 OutPut of 950502Bl Fluoride 1.5 Fluoride L PPM ppm Pos Typ Ident Ch Result F Time 54 u 55 u 56 u 57 u 58 u 59 u 60 u 61 u 62 d 63 w 64 u 65 u 66 u 67 u 68 u 69 u 70 u 71 u 72 u 73 u 74 d 75 w 76 u 77 u 78 d 79 w wt rw SPK 4 3 LIVER BLK 3 F53449-1 3 F53449-2 3 F53449-3 3 F53504-1 3 F53504-2 3 F53504-3 3 Drift 3 Wash 3 F53456-1 3 F53456-2 3 F53456-3 3 F53454-1 3 F53454-2 3 F53454-3 3 BLK 1 3 BLK 2 3 SPK 1 3 SPK 2 3 Drift 3 Wash 3 SPK 3 3 SPK 4 3 Drift 3 Wash 3 RunOut Wash 3 0.100 0.059 0.061 0.056 0.060 0.056 0.054 0.057 1.459 0.057 0.058 0.087 0.061 0.057 0.057 0.060 0.058 0.057 0.103 0.111 1.494 0.057 0.185 0.164 1.518 0.057 0.057 9493 9665 9841 10013 10189 10361 10539 10711 10889 11044 11237 11413 11593 11763 11933 12108 12288 12463 12641 12817 12991 13222 13345 13517 13691 13890 14166 Ch Result F Time 4 0.0786 0 4 0.0018 0 4 0.0075 0 4 #.#### 0 4 0.0047 0 4 0 4 0 4 0 4 0.8366 0 4 #.#### 0 4 0.0007 0 4 0.0570 0 4 0.0077 0 4 0 4 0 4 0.0052 0 4 0.0009 0 4 #.#### 0 4 0.0839 0 4 0.0954 0 4 0.8550 0 4 #.#### 0 ta. 4 0. 1815 u5*@,a.L-L 4 0.1599 0 4 0.8686 0 4 0 4 0 Page 2 of 2 QQFZ44.@Rr Calibration curve of 9SO50.'st Fluoride L Pd 0.1960632i 0 0 -4 ly -o.oo-0050 0 0--der Measured Soo r 0 .9990-,,Ll 27, Calibration curve of 9SOS04-:81 Fluoride 1.5 t" 0.0567586 Order Measured Inverse Lc-,arithm 409S c-'070 j 409S Raw data of 95050@'81 ss Flijor-idIe.S i0 tzc=Exi@ Fl=Hei@o IC) 4p Time 4095 Raw data o'il9:SO501@81 -@i 4 Fluoride I.E-; P.4 lien D4 Ci FI=He.@Lp Time Ct-tl-P=Editpeaks 1 a -LC> 01 40,35 Raw data of 950SO'-Bl Fluoride I.S piirg 64 -998 Time Esc=Exit ! F!=Help Crti-P=Editpeaks T7 662q- 0()01:; 4095 Raw data of 950502Bl : Fluoride 1.5 50 qo 0 Time FI=4elp Crtl-P=Edit peaks g?48 3z Raw Liata of 3SOSGIAl Fluoride I 4095 T T, I gq-17 8"48 i-: c x i T ime Crtl-P=E-'difpea@ks ooojL"J,A@ 4095 Ft;3wtl;.@itraf Fluot-iilIe 7,24 JA 0 11873 Ec-c=Exit FI=Help F T ime Crti-P=Edit Peaks to 144!-