Document zd54djn6D2Z60KVbmoKn76O5B

3M EnvironmentaLlaboratory FinalReport-AnalyticaSltudy Single-dosIentravenouPsharmacokinetiSctudy ofT-6049inRabbits In-VivoStudyReferenceNumber: HWI#6329-129 StudyNumber: AMDT-112294.1 TestSubstance:FC-95 (T-6049) Name and AddressofSponsor: 3M SCD Division 367 Grove Street St.Paul,NIN 55106 Name and Address of Testing Facility: 3M Environmental Technology & Services 935 Bush Avenue St.Paul,MN 55106 Method Numbers AMDT-M-1-0, AMDT-M-2-0, AMDT-M4-0, AMDT-M-5-0, AMDT-M-14-0, 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 RabbitLiver Analysisof RabbitLiverExtractforFluorochemicalsUsing ElectrosprayMass Spectrometry Thermal Extractionof Fluorideby Means of a Modified Dohrmann DX2000 Organic HalideAnalyzer-Serum InitiationDate: See attachedprotocol Author: James D. Johnson Approved By: meess DDi-rJo*on j ttuumddyyD eddr Compl@tion Date ()()0001 1.0 SUMMARY Rabbitserum and liverwere analyzedforfluorinceontentatvarioustimesafter rabbitswere dosed intravenouslwyitha singledoseof FC-95 (T-6049).The fluorine inliverwas detectedaftera singledoseof 100 mg/kg (60ug/kgFC-95) or after 500 mg/kg (300ug/kg FC-95). The liverfluoridieon concentratioinntheGroup 5 animal (300 ug/kg),at48 hourspostdose was 1.0ppm and fortherabbitdosed with 60 ug/kg the concentratiownas about0.4ppm. The presenceof perfluorooctanesulfonawtaes confirmedwith electrospramyass spectrometry. The method would probablybe ableto detecta dose somewhere between 10 and 60 ug/kg. Analysisoffluorineat28 days inliverand serum willlikelyprovidea marker fordermal absorptionfordermallyappliedfluorochemicaltshatare biotransformedtoperfluorooctanesulfonaatneion. The dataagreewith a second pharmacokineticstudyofFC-95 which was carriedoutto28 days with more animals (HWI#6329-159). 2.0 INTRODUCTION This studywas designedtoprovideinformationastowhether theperfluorooctanesulfonataeniondoes go totheliverand othertissueswhen thematerialis administeredin an intravenousdose,and to ascertaitnhe change inconcentration with time afterdose inserum and liver. Itisknown from studiesdone previouslywithratsthatthehalf-lifoef perfluorooctanesulfonataenionisquitelong(>1 month). Itwas expectedthatthehalf-lifien rabbitswould alsobe long. Perfluorooctanesulfonatneion isa likelybiotransformationproductof severalfluorochemicaltshataretobe testedfordermal absorptioninotherstudies.The pharmacokineticosftheperfluorooctanesulfonate anion isvery relevanttothesedermalabsorptionstudies. 3.0 TEST MATERIALS 3.1Test,Control,and ReferenceSubstancesand Matrices 3.1.1AnalyticalReferenceSubstance:FC-95,lot161 or 171.They are equivalent. 3.1.2AnalyticalReferenceMatrix: Bovine liverand bovineserum 3.1.3AnalyticalControl Substance: None 3.1.4AnalyticalControlMatrix: Bovine liverand bovineserum 0 ()0 ()0 O'Ll-,Wo 2 3.2SourceofMaterial3sM: ICP/PCPDivisiofnorFC-95,bovinelivefrrom grocerystore,bovine serum from Sigma Chemical Company 3.3.Purity and Strength of Reference Substance: Responsibilitoyf Sponsor. 3.4 StabilityofReference Substance: To be determinedby Sponsor. 3.5 Storage Conditions for Test Materials:Room temperatureforFC-95. For biologicalsamples thestorageis-20@+100C. 3.6 Dispositionof Specimens: Biologicaltissuesand fluidswillbe retainedper GLP Regulationforthetime periodrequiredforstudieslongerthan28 days. 4.0 EXPERIMENTAL-Overview Serum and tissuefsrom animalsdosed as described(HWI#6329-129), were available foranalysisforfluorinceompounds. Sinceperfluorooctanesulfonatneion isnot biotransformedt,heanalysiswas accomplishedwithcombustion and subsequent analysisforfluorine.The fluorinedataarerelatedirectltyoperfluorooctanesulfonateconcentrationT.he fluorineanalysisof serum collecteadtdifferenttime intervalasfterdosingprovidesdatawhich can be interpretepdhannacokinetically. 5.0 EXPEREKIENTAL - Methods 5.1AMDT-M-1-0, Thermal Extractionof Fluorideby Means ofa Modified Dohrmann DX2000 OrganicHalideAnalyzer-Liver 5.2AAI[DT-M-2-0, FluorideMeasurement by Means of an Orion EA940 ExpandableIon Analyzer 5.3 AMDT-M-4-0, Extractionof Fluorochemicalsfrom RabbitLiver 5.4AMDT-M-5-0, Analysisof RabbitLiverExtractforFluorochemicalsUsing ElectrosprayMass Spectrometry 5.5AMDT-M-14-0, Thermal Extractioonf Fluorideby Means of a Modified Dohrmann DX2000 OrganicHalideAnalyzer-Serum 0()O()O"j 3 6.0DATA ANALYSIS ThemateriTa-l6049wasa solutiofnFC-95dilutetdo0.06%inwater.Thedoses of5,10,100,and500mg/kgarethus3,6,60,and300ug/kg.Theseareverylow doses. The liverfluorideionconcentratioinntheGroup 5 animal(300ug/kg),at48 hours postdose was 1.0ppm and fortherabbitdosed with 60 ug/kg theconcentratiownas about0.4ppm. The controlrabbitt,he5 mglkg rabbita,nd the 10 mg/kg rabbit concentrationwsere notabove thepracticallimitof quantitation. For liversof thetwo highestdosesusinganalysisby electrospramyass spectrometry, the'perfluorooctanesulfonwaatsedetectablaend theestimatedamounts forthe 100 and 500 mg/kg rabbitswere 0.3and 0.6ppm, respectively. These datashow thatperfluorooctanesulfonawtiellbe a very sensitivmearker for dermal absorptiontestsofFC-95 and forany fluorochemicaltshatare biotransforinetdoperfluorooctanesulfonatTeh.e factthatthematerialcan be observedinliveraftera 60 ug/kg dose issuggestivethata very largeportionofthe dose willbe inliverifthematerialispresentsystemically. Otherdatawas collecteodn serum samplesusingthermalextractioonffluorideby means of a modifiedDohnnann organichalideanalyzerand fluoridemeasurement by means ofa Orion expandableionanalyzer(seeappendices)T.his data,although supportivei,nthe opinionof the StudyDirectorisnot requiredto reachthe conclusionstatedhereand thereforeisnotdiscussedindetail. 6.1 Circumstances That May Affectthe Quality of the Data: These valuesfor concentratioanrefrom combustionanalysesofthebiologicamlaterial.The recoverieasrebased on spikingthebiologicamlaterialwith known amounts of FC-95. The fluorineismeasured by selectivieonelectrodeand theOrion meter is calibrateodver a 5 pointrange.However, ifthereisa differenrtesponseatdifferent concentrationosf FC-95 otherthanthepointused as a calibratiocnheck,therecould be a biasatthoseconcentrationesven though theamount of fluorineisbeing measured accurately.This can come from othervariablesin themethod such as the efficiencoyf combustion.Thus, thevaluesreportedforconcentratioanrenot absolutevalues.The valuesarecertainlsyufficienttomake thepointthatthe perfluorooctanesulfonaatneionispresentat48 hoursinliver. ooo004 4 7.0CONCLUSION The methodwouldprobablbye abletodetecatdosesomewherbeetween10and 60 uglkg. Analysisoffluorinaet28 days inliverand serum willlikelyprovidea marker fordermal absorptionfordermallyappliedfluorochemicaltshatare biotransformedtoperfluorooctanesulfonaatneion.The dataagreewith a second phannacokineticstudyofFC-95 which was carriedoutto28 days with more animals (HWI#6329-159). 8.0 MAINTENANCE OF RAW DATA AND RECORDS 8.1 Raw Data and Data: Raw data,approvedprotocola,pprovedfinalreport, appropriatespecimens,and electronidcatawillbe maintainedintheAMDT Archives. 9.0 APPENDICES 9.1 Protocol and Amendments 9.1.1Protocoland FinalReport:HWI#6329-129 "SingleDose Intravenous PharmacokineticStudy ofT-6049 inRabbits"(ProtocoltypeT?8084.PK for dosingof animals,tissuecollectione,tc.) 9.1.2 AnalyticalprotocolAN4DT- 112294.1includingmethods. 9.2 Signed Reports from IndividualScientistsN:one 9.3 Quality Assurance Unit Statement:See attached 9.4 Key Personnel Involvedinthe Study: See attached 9.5 Materials and Equipment: See methods 9.6 Solutions,Reagents,and Standards: See methods 9.7 Sample Preparation:See methods 9.8 Quality Control Practices:See methods 9.9 Test Methods: See ProtocolAMDT-1 12294.1 9.10 Instrument Settings:See methods 00000%-j 5 9.11Data:Seeattached: 9.11.1Summary and raw data;ug F-inwhole liverasdeterminedby thermal extractiofnollowedby analysisusingOrion ionanalyzer. 9.11.2Summary and raw data;analysisof liverextractussingelectrospray mass spectrometry. 9.11.3Summary and raw data;ppm F-inserum as determinedby thermal extractiofnollowedby analysisusingOrion ionanalyzer. 0()000(; 6 9.1.1Protocoland FinalReport:HWI#6329-129 "SingleDose IntravenousPharmacokineticStudy of T-6049 in Rabbits"(ProtocoltypeTP8084.PK for dosingof animals,tissuecollectione,tc.) HAZLaOCON W W IS C 0 N S IN Pos r OFFICE BOX 75-15 MADISON, Wl 53 10 7 7545 Sponsor: 3M St. Paul, Minnesota a CORNING Crxnpiny FINAL REPORT Study Title: Single-Dose Intravenous Phamacokinetic Study of T-6049 in Rabbits Author: Steven M. Glaza Study COMDletion Date: February 1, 1995 Performing Laboratory: Hazleton Wisconsin, Inc. 3301 Kinsman Boulevard Madison, Wisconsin 53704 LaboratoryProject Identification: HWI 6329-129 Page I of 24 P h o n e 6 0 8 2 4 1 -,14 7 1 E X P R E S S -k4l.1AL D E L Iv E R Y 330 1 K I @jS M A ri BLVD Fax MADISON. 6 0 8 -2 4 1 7 2 2 7 Wl 53704 Page 2 of 24 QUALITY ASSURANCE STATEMENT HWI 6329-129 This report has been reviewed by the Quality Assurance Unit of Hazleton Wisconsin, Inc., in accordancewith the Food and Drug Administration (FDA)Good LaboratoryPracticeRegulations,21 CFR 58.35 (b) (6) (7). The following inspectionswere conducted and findings reported to the Study Director and management. Written-statusreports of inspections and findings are issued to Hazleton management monthly according to standard operating procedures. Inspection Dates From To Phase Date Reported to Date to Study Director Management 11/06/94 11/06/94 Protocol Review 11/16/94 11/16/94 Animal Observation 01/10/95 01/10/95 Data/ReportReview 01/30/95 01/30/95 Report Rereview 11/08/94 11/16/94 01/10/95 01/30/95 12/10/94 12/10/94 02/10/95 02/10/95 *liaM. Danner Date Representative,Quality Assurance Unit 0()000!) Page 3 of 24 STUDY IDENTIFICATION Single-Dose Intravenous Pharmacokinetic Study of T-6049 in Rabbits HWI 6329-129 Test Material Sponsor Sponsor's Representative Study Director Study Location Study Timetable Experimental Start Date Experimental Termination Date T-6049 3M Toxicology Services 220-2E-02 3M Center St. Paul, MN 55144 John L. Butenhoff, PhD 3M Toxicology Services 220-2E-02 3M Center St. Paul, MN 55144 (612) 733-1962 Steven M. Glaza Hazleton Wisconsin, Inc. P.O. Box 7545 Madison, WI 53707-7545 (608) 241-7292 Hazleton Wisconsin, Inc. Building No. 3 3802 Packers Avenue Madison, WI 53704 November 15, 1994 November 17, 1994 Page 4 of 24 HWI 6329-129 KEY PERSONNEL Acute Toxicology Laboratory Animal Medicine Steven M. Glaza Study Director Manager Cindy J. Cary, DVM Diplomate, ACLAM Supervisor Francis (Bud) W. McDonald Study Coordinator Anatomical Pathology Patricia Padgham In-lifeSupervisor Rose M. Bridge Report Supervisor Oua iiy Assurance Jack Serfort/ Deborah L. Pirkel Supervisors Necropsy Anne Mosher Supervisor Pathology Data Sherry R. W. Petsel Manager 000()IIL Page 5 of 24 CONTENTS Quality Assurance Statement Study Identification Key Personnel Summary Objective Regulatory Compliance Test and Control Materials Test System Procedures Results Discussion Signature Reference Table 1 IndividualBody Weights (g) 2 IndividualClinicalSigns Appendix A Protocol TP8084.PK HWI 6329-129 Page 2 3 4 6 7 7 7 8 9 11 11 11 11 12 13 14 15 ooool'A. Page 6 of 24 HWI 6329-129 SUMMARY This study was done to assess the level of systemic exposure of T-6049 when administeredby intravenousinjectionto rabbits. Female Hra:(NZW)SPF rabbits were assigned at random to five groups (one/group). On Day 0, the animalsreceiveda single intravenousinjectionof the vehicle (sterilewater for injection)or 5, 10, 100, or 500 mg of T-6049/kg of body weight (Groups1 through 5, respectively). The dose volume was 0.5 mL/kg for all groups. Clinical observationswere conducted at approximately0.5, 2, 4, 24, and 48 hours after intravenousinjection. Body weights were determinedjust before test material administration(Day 0). A blood sample (approximately 4 mL) was collectedfrom an auriculararteryor marginalear vein of the animalsat 2-, 4-,'6-,8-, 12-, and 24-hourspost-injection.In addition,at the time of experimentaltermination(48-hourspost-injection),approximately 20 mL of blood was obtained from each animal. All samples were centrifuged, separated into serum and cellular fractions,and sent to the Sponsor. Approximately48 hours post-injection,the animalswere anesthetizedwith sodium pentobarbital,bled via the posteriorvena cava, and exsanguinated. An abbreviated gross necropsy examinationwas not done, however, tissues were collected. The whole liver, bile, and both kidneys from each animal were collectedand sent frozen to the Sponsorafter terminationof the in-life phase. The animals treatedwith sterilewater for injection,5 mg T-6049/kg,and 10 mg T-6049/kg appeared normal throughout the study. At the 0.5, 2, and 4 hour post-dose observation,the animal treated with 100 mg T-6049/kg appeared hypoactive and the animal treated with 500 mg T-6049/kg exhibited a staggeredgait. In addition,the animal treated with 500 mg/kg showed a decrease in food consumptionat the 48-hour post-dose observation. Page 7 of 24 HWI 6329-129 OBJECTIVE The objective of this study was to assess the level of systemicexposure to the test material,T-6049, when administeredas a single intravenousinjection to rabbits. REGULATORY COMPLIANCE This study was conducted in accordance with the U.S. Food and Drug Administration'sGood LaboratoryPractice Regulationsfor Nonclinical LaboratoryStudies,21 CFR 58, with the exceptionthat analysisof the test mixtures for concentration,homogeneity/solubilitya,nd stabilitywas not conducted. All proceduresused in this study were in compliancewith the Animal Welfare Act Regulations. In the opinion of the Sponsor and study director, the study did not unnecessarilyduplicate any previouswork. TEST AND CONTROL MATERIALS Identification The test materialwas identifiedas T-6049 and described as a clear, colorless liquid. The controlmaterialwas SterileWater for Injection,USP (Abbott Laboratories,Lot No. 86-748-DM-02;Exp. March 1, 1996), and was described as a clear, colorlessliquid. Purity and Stability The Sponsor assumesresponsibilityfor test materialpurity and stability determinations(includingunder test conditions). A sample of the test material/vehiclemixturesfor concentration,solubility,homogeneity,and stability analyseswas not taken before administrationas this was not requestedby the Sponsor. The purity and stabilityof the USP grade control material were consideredto be adequate for the purposes of this study. Storage and Retention The test material was stored at room temperature. The control material was stored refrigerated. Any unused test material will be returnedto the Sponsor after completionof all testing accordingto HazletonWisconsin (HWI) Standard Operating Procedure (SOP). Any remainingvehicle may be used for other testing and will not be discardedafter issuanceof the finalreport. ()()00111 Page 8 of 24 HWI 6329-129 Safety Precautions The test and control material handling procedures were according to HWI SOPs and policies. TEST SYSTEM Test Animal Adult albino rabbitsof the Hra:(NZW)SPFstrain were received from HRP, Inc., Kalamazoo, Michigan on October 19, 1994 and maintained at the Hazleton Wisconsin facility at 3802 Packers Avenue, Madison, Wisconsin. Housing After receipt,the animalswere acclimatedfor a period of at least 7 days. During acclimationand throughoutthe study, the animalswere individually housed in screen-bottomstainlesssteel cages in temperature- and humiditycontrolledquarters. Environmentalcontrolsfor the animal room were set to maintain.a temperatureof 19* to 23*C, a relative humidity of 50% t20%, and a 12-hour light/12-hourdark lightingcycle. In cases where variationsfrom the required temperature and humidity conditions existed, they were documented and considered to have had no adverse effect on the study outcome. Animal husbandry and housing at HWI complied with standards outlined in the "Guide for the Care and Use of LaboratoryAnimais".1 Animal Diet The animals were provided access to water ad 7ibitum and a measured amount of Laboratory Rabbit Diet HF #5326, PMI Feeds, Inc. The feed is routinely analyzed by the manufacturerfor nutritionalcomponents and environmental contaminants. Samplesof 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 identifiedby animal number and corresponding ear tag and were selected at random based on health and body weight requirements. 0()OOILIJ Page 9 of 24 HWI 6329-129 Study Design Female animals weighing from 2,587 to 2,857 g at initiationof treatmentwere placed into the followingstudy groups: Group Dose Level Dose Volume Number Treatment (mg T-6049/kg) (mL/kq)- of Animals I (Control) 0 2 T-6049 5 3 T-6049 10 4 T-6049 100 5 T-6049 500 0.5 1 0.5 1 0.5 1 0.5 1 0.5 1 Sterile Water for Injection,USP. Justificationfor S2eciesSelection Historically,the New ZealandWhite albino rabbit has been the animal of choice because of the large amount of background informationon this species. PROCEDURES Dose Pre2aration and Administration The test materialwas dilutedwith SterileWater for Injectionto achieve a specific concentrationfor each dose level in Groups 2 through4. The test material was administeredundilutedat the 500 mg/kg dose level, using the bulk density of 0.98 g/mL to determinethe dose volume. An individualdose of each respective test solutionor controlwas calculatedfor each animal -based on its body weight on the day of treatment. The respectivetest solutionwas administeredby intravenousinjectioninto a marginalear vein. The dose was given as a slow push (approximately30 to 60 seconds in duration). The prepared test solutionswere stored at room temperatureuntil administered. After administration,any remainingtest solutionswere discarded. Reason for Route of Administration Intravenous injectionis an acceptableroute to assess systemicexposure. Observations of Animals Clinical observationswere conductedat approximately0.5, 2, 4, 24, and 48 hours after intravenousinjection. Body weights were determinedjust before test material administration(Day 0). 0()()01(@ Page 10 of 24 HWI 6329-129 Sample Collection A blood sample (approximately4 mL) was collected from either ear via the catheterizationof the auricular artery or from the marginal ear vein of all animals at 2, 4, 6, 8, 12, and 24 hours post-injection. At the time of necropsy (approximately 48-hours post-injection), approximately 20 mL of blood was obtained from the posterior vena cava of each animal. All samples were stored at room temperature until centrifuged and separated into serum and cellular fractions. The blood samples were then stored in a freezer set to maintain a temperature of -20*C tlO*C until shipped to the Sponsor. Pathology At termination of the experimental phase (approximately 48-hours post-injection), animals were anesthetizedwith sodium pentobarbital,bled via the posterior vena cava, and exsanguinated. An abbreviated gross necropsy examination was not conducted, however, tissues were collected. The whole liver, bile, and both kidneys from each animal were collected and immediately placed on dry ice, then frozen by placing in a freezer set to maintain a temperature of -200C 100C. After tissue/bile collection, the animals were discarded. Shigment of Tissues After completion of the in-life phase the blood samples, livers, bile, and kidneys were sent frozen (on dry ice) to the Sponsor (James D. Johnson, 3M E.E. & P.C., Bldg. 2-3E-09, 935 Bush Avenue, St. Paul, MN, 55106). The Sponsor is responsible for the retention and disposition of the samples. HWI does not accept any responsibility for the analysis of the samples collected in this study nor are these results presented in this report. Statistical Analyses No statistical analyses were required by the protocol. Location of Raw Data, Records, and Final Report The raw data, records, and an original signed copy of the final report will be retained in the archives of HWI in accordance with HWI SOP. Page 11 of 24 RESULTS Body Weights Individualbody weightsat initiationare in Table 1. HWI 6329-129 Clinical Observations Individualclinicalsigns are in Table 2. The animalstreatedwith sterile water for injection, 5 mg T-6049/kg, and 10 mg T-6049/kg appeared normal throughoutthe study. At the 0.5, 2, and 4 hour post'-doseobservation,the animal treated with 100 mg T-6049/kg appeared hypoactive and the animal treatedwith 500 mg T-6049/kgexhibiteda staggeredgait. In addition,the animal treated with 500 mg/kg showed a decrease in food consumption at the 48-hour post-dose observation. Pathology All animals survived to terminationof the experimentalphase and were not examined grossly when sacrificed. DISCUSSION The level of systemicexposure of T-6049 was evaluated in female albino rabbitswhen administeredas a single intravenousinjectionat levels of 0, 5, 10, 100, and 500 mg/kg. Administrationof this material resultedin hypoactivityat the 100 mg/kg dose level, and staggeredgait and a decrease in food consumption at the 500 mg/kg dose level. SIGNATURE Steven M. Glaza Date Study Director Acute Toxicology REFERENCE 1. NIH PublicationNo. 86-23 (revised1985). Page 12 of 24 GrouD 1 2 3 4 5 Table 1 IndividualBody Weights (g) Dose Level (mg/kci) Sex Animal Number 0 Female F52652 5 Female F52653 10 Female F52654 100 Female F52665 500 Female F52666 Day 0 2,634 2,587 2,620 2,857 2,794 HWI 6329-129 Dose Level Group (mg/kq) Sex 1 0 Female Page 13 of 24 Table 2 IndividualClinicalSigns HWI 6329-129 Animal Number F52652 -Observation Appeared normal Hour 0.5 2 4 24 48 2 5 Female F52653 Appeared normal 3 10 Female F52654 Appeared normal 4 100 Female F52665 Appeared normal Hypoactivity 5 500 Female F52666 Appeared normal Staggered gait Decreased food consumption Indicatesconditionexists. Not applicable. 0 0 O'A.-I0ol Page 14 of 24 APPENDIX A Protocol TP8084.PK HWI 6329-129 0()0();41 HAZLR;NCCN W IS C 0 N S IN POST OFFICE BOX /&).15 MAR)ISON. Wi r)3707 Ir,,Il, Page 15 of 24 CORNING (:ottil);itiv Sponsor: 3M St. Paul, Minnesota PROTOCOL TP8084.PK Study Title: Single-Dose IntravenousPharmacokineticStudy of T-6049 in Rabbits Date: November 9, 1994 PerformingLaboratory: Hazleton Wisconsin,Inc. 3301 Kinsman Boulevard Madison, Wisconsin 53704 LaboratoryProjectIdentification: HWI 6329-129 p ri o ii e 6 0 8 I X p R E@ 5 S Ni F 2 t) M A I) Page 16 of 24 STUDY IDENTIFICATION TP8084.PK Page 2 Single-Dose Intravenous Phamacokinetic Study of T-6049 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 6329-129 T-6049 3M Toxicology Services 220-2E-02 3M Center St. Paul, MN 55144 John L. Butenhoff, PhD 3M Toxicology Services 220-2E-02 3M Center St. Paul, MN 55144 (612) 733-1962 Steven M. Glaza Hazleton Wisconsin, Inc. P.O. Box 7545 Madison, Wl 53707-7545 (608) 241-7292 Hazleton Wisconsin, Inc. Building No. 3 3802 Packers Avenue Madison, Wl 53704 Week of November 14, 1994 Week of November 14, 1994 Week of December 19, 1994 Page 17 of 24 TP8084.PK Page 3 I. Study Single-DoseIntravenousPharmacokineticStudy in Rabbits 2. Purpose To assess the level of systemicexposure when the test material is administeredas a singleintravenousinjectionto rabbits 3. Regulatorv omvliance This study will be conductedin accordancewith the followingGood LaboratoryPracticeRegulations/Standards/Guidelinweisth the exceptionthat analysisof the testmaterialmixturesfor concentration,solubility,homogeneity,and stabilitywill not be conducted: ( ] Conduct as a NonregulatedStudy [XI 21 CFR 58 (FDA) 40 CFR 160 (EPA-FIFRA) 40 CFR 792 (EPA-TSCA) C(81)30(Final)(OECD) 59 Nohsan No. 3850 (Japanese MAFF) NotificationNo. 313 (JapaneseMOHW) All proceduresin this protocolare in compliancewith the Animal Welfare Act Regulations. In the opinion of the Sponsorand study director,the studydoes not unnecessarilyduplicateany previous work. 4. AgalitLAssuranc, The protocol,study conduct,and the final reportwill be auditedby the QualityAssuranceUnit in accordancewith HazletonWisconsin (HWI) StandardOperatingProcedures(SOPS) and policies. S. Test Material A. Identification T-6049 B. PhysicalDescription (To be documentedin the raw data) C. Purityand StabilitY The Sponsorassumesresponsibilityfor purity and stability determinations(includingunder test conditions). Samplesof testmaterial/vehiclmeixture(s)for concentrations,olubility, homogeneity,and stabilityanalyseswill be takenbefore administrationif requestedby the Sponsor. These samples(if taken)will be sent to the Sponsorafter experimental terminationfor possibleanalysis. Page 18 of 24 0. Storage Room temperature TP8084.PK Page 4 E. Reserve Samples Reservesampleswill not be requiredfor this study. F. Retention Any unusedtest materialwill be discardedafter issuanceof the finalreport,unlessdirectedotherwiseby the Sponsor. G. Safety Precautions* As required by HWI SOPs and policies 6. ControlMaterial A. Identification Sterilewaterfor injection B. PhysicalDescription Clear,colorlessliquid C. Purityand Stability The purityand stabilityof this USP grade materialis consideredto be adequatefor the purposes of this study. D. Storage Refrigerated E. Reserve Sam2les See Section,5. E. Reserve Samples F. Retention Any remainingcontrolmaterial may be used for other testing and will not be discardedafter issuanceof the final report. G. Safety Precautions As required by HWI SOPs and policies 7. ExperimentalDesign A. Animals (1)Species Rabbit (2) Strain/Source Hra:(NZW)SPF/HRP,Inc. (3)Age at Initiation Adult oooo;@.j Page 19 of 24 (4)Weightat Initiation 2.5 to 3.5 kg TP8084.PK Page 5 (5)Number and Sex 5 females (6) Identification Individualnumbered ear tag (7) Husbandry (a) Housing Individually,in screen-bottomstainlesssteel cages (heavygauge) (b) Food A measured amount of Laboratory Rabbit Diet HF #5326 (PMIFeeds, Inc.). The food is routinelyanalyzedby the manufacturerfor nutritionalcomponentsand environmentalcontaminants. (c)Water Ad libitum from an automatic system. Samples of the water are analyzedby HWI for total dissolvedsolids, hardness,and specifiedmicrobiologicalcontent and for selectedelements,heavy metals, organophosphates,and chlorinatedhydrocarbons. (d) Contaminants There are no known contaminantsin the food or water that would interferewith this study. (e) Environment Environmentalcontrolsfor the animal room will be set to maintain a temperatureof 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) Selectionof Test Animals Based on health and body weight according to HWI SOPS. An adequatenumber of extra animalswill be purchasedso that no animalin obviouslypoor health is placed on test. (9) Justificationfor SpeciesSelection Historically,the New ZealandWhite albinorabbithas been the animalof choice becauseof the large amount of backgroundinformationon this species. 0()00;@G Page 20 of 24 B. Dose Administration TP8084.PK Page 6 (1) Test Groups GrouD Dose Level (mg/kg)a Number of Females 1 0 (Control) 2 5 3 10 4 100 5 500 a The dose volumewill be 0.5 ml/kg for Groups 1-4 and approximately0.5 mL/kg of body weight (dependingon the bulk densityof the test material)for Group S. C. Dosing Procedures (1)Dosing Route Intravenousinjectioninto a marginalear vein over approximately30 to 60 seconds. (2)Reason for Dosina Route Intravenousinjectionis an acceptableroute to assess systemic exposure. (3)Dosing Duration Single dose (4)Dose Preparation The test materialwill be dilutedwith sterilewater for injectionto achievea specificconcentrationfor each dose level in Groups 1-4. The testmaterialwill be administeredundilutedat the 500 mg/kg dose level,using the bulk density to determinethe dose volume. Individual doses will be calculatedbased on the animal'sbody weight taken just beforetest materialadministration.The preparedtest mixtureswill be storedat room temperature until administration. D. Observationof Animals (1) ClinicalObservations The animalswill be observedfor clinicalsignsof toxicityat approximately0.5, 2.0, 4.0, 24, and 48 hours after treatment. 0()001;47 Page 21 of 24 (2) Body Weights Just beforetestmaterialadministration. TP8084.PK Page 7 (3) Sample Collections (a) Freauency 2, 4, 6, 8, 12, 24, and 48 hours post-injection (b) Number of Animals All (c)Method of Collection Blood samples(approximately4 mL) will be collected from eitherear via the catheterizatioonf the auriculararteryor from the marginalear vein at 2, 4, 6, 8, 12, and 24 hours post-injection. Approximately20 mL of blood (actualvolume to be documentedin the raw data) will be obtainedfrom the posteriorvena cava of each animal at the time of necropsy(48 hours post-injection).Approximately 20 mL of blood will be collectedfrom moribund animalsduringthe study,also, if possible. The sampleswill be stored at room temperatureand then centrifuged,and the separateserum and cellular fractionsstored in a freezer set to maintaina temperatureof -20'C tlO*C. The separatedserum and cellularfractionswill be sent frozento the Sponsor after experimentaltermination. Sampleswill be shippedto: James D. Johnson 3M E.E. & P.C. Bldg. 2-3E-09 935 Bush Avenue St. Paul, MN 55106 James D. Johnsonwill be notified by telephoneat (612)778-5294prior to the shipmentof the samples. E. Termination (1) Unscheduled Sacrifices and Deaths Any animaldying during the studyor sacrificedin a moribundcondition,will be subjectedto an abbreviated gross necropsyexaminationand all abnormalitieswill be recorded. Animalsin a moribundconditionwill be anesthetizedwith sodiumpentobarbital,bled via the vena cava, and exsanquinated. 0()00%,Ll Page 22 of 24 TP8084.PK Page 8 (2) ScheduledSacrifice At approximately48 hours Post-injection,animals survivingto terminationwill be anesthetizedwith sodium pentobarbital,bled via the vena cava, and exsanguinated. An abbreviatedgross necropsyexaminationwill not be done,however,tissueswill be collected. (a) Sample Collection The whole liver and bile from each animaldying duringthe study,sacrificedin a moribundcondition, or survivingto terminationwill be collected. Both kidneysfrom each animalwill also be collected. The tissueswill be placedon dry ice immediatelyafter collectionand then placedin a freezerset to maintain a temperatureof -20*C 10*C. The tissues(liver,bile,kidneys)will be sent frozenon dry ice to the Sponsor after experimental termination.The sampleswill be shippedto the personlistedin Section7.D.(3).(c).The Sponsoris responsiblefor the retentionand dispositionof the samples. F. StatisticalAnalyses No statisticalanalysesare required. 8. Report A final reportincludingthose items listedbelow will be submitted. Descriptionof the test and controlmaterials Descriptionof the test system Procedures Dates of experimentalinitiationand termination Descriptionof any toxiceffects Gross pathologyfindings/grospsathologyreport,(ifapplicable) Page 23 of 24 TP8084.PK Page 9 9. Location of Raw Data, Records.-and Final Report Original data, or copies thereof, will be availableat HWI to facilitate auditingthe 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-life records Body weights Dose administration Observations Sample collection records Pathology records Study correspondence Final report (originalsigned copy) The following supportingrecords will be retained at HWI but will not be archivedwith 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 Page 24 of 24 TP8084.PK Page 10 PROTOCOL APPROVAL 7John L: Butenhoff, PhD Sponsor's Representative 3M Steven M. Glaza Study Director Acute Toxicology Hazleton Wisconsin, Inc. -R@presentative Quality Assurance Unit Hazleton Wisconsin, Inc. (6329-129.protdskl) Date I\-g@-Otli Date L,7 Date 0()001*il 9.1.2 AnalyticaplrotocolAMDT-1 12294.1 0()0()3;4 3M EnvironmentalLaboratory Protoco-lAnalyticSatludy Single-dosIentravenouPsharmacokinetiSctudyofT-6049inRabbits In-VivoStudyReferenceNumber: HWI#6329-129 StudyNumber: AMDT-112294.1 TestSubstance:FC-95 (T-6049) Name and AddressofSponsor: 3M SCD Division 367 Grove Street St.Paul,MN 55106 Name and Address of TestingFacility: 3M EnvironmentalTechnology and Services 935 Bush Avenue St.Paul,NfN 55106 Proposed InitiationDate: July25, 1995 Proposed Completion Date: August 25,1995 Method Numbers AMDT-M-1-0, AMDT-M-2-0, AMDT-M4-0, AMDT-M-5-0. AMDT-M-14-0, and Revisions: Thermal Extractionof Fluorideby Means of a Modified Dohrrnann DX2000 OrganicHalide Analyzer-Liver FluorideMeasurement by Means of an Orion EA940 Expandable Ion Analyzer Extractionof Fluorochemicalsfrom RabbitLiver Analysisof RabbitLiverExtractforFluorochemicalsUsing ElectrosprayMass Spectrometry Thermal Extractionof Fluorideby Means of a Modified Dohrmann DX2000 Organic Halide Analyzer-Serum Author:James D. Johnson Approved By: ? JSatomu2ed@sye-:.isro@eshtononsron Stmud 'r0ector Date John Butenhoff,PhD Date Sponsor Representative 0()0()33 1.0 PURPOSE Thisstudyisdesignetdoprovideinformatiaosntowhetherthe perfluorooctanesulfonataenion does go to therabbitliverand othertissueswhen the materialisadministeredin an intravenousdose,and toascertainthe change in concentrationwith time afterdose in serum and liver.These datawillbe used to interpredtermal absorptionstudieswhere perfluorooctanesulfonaitsedirectly appliedor ispresentas a biotransformatiopnroduct.The persistenceof perfluorooctanesulfonataes a marker in liverand serum or possiblyothertissuesisa criticaplartforthe interpretationf thesedermal studies. 2.0 TEST MATERL4LLS 2.1Test,Control.,and Reference Substances and Matrices 2.1.1AnalyticalReference Substance: FC-95, lot161 or 171. They are equivalent. 2.1.2Analytical Reference Matrix: Bovine liverand bovine serum 2.1.3Analytical Control Substance: None 2.1.4Analytical Control Matrix: Bovine liverand bovine serum 2.2 Source of Materials: 3M ICP/PCP Division (2.1.1),grocery store(2.1.2,2.1.4 liver)S,igma Chemical Company (2.1.22,.1.4serum) 2.3 Number of Test and Control Samples: Liverand serum from 4 testanimals and I controlanimal. Otherbiologicatlissues(kidney,bile,cellulafrractionw)ill be availableforanalysisifdeemed appropriateby theStudy Director. 2.4 Identiflcationof Test and Control Samples: The samples are identifieudsing theHWI animal identificatinounmber which consistsof a lettearnd fivedigit number, plusthetissueidentitaynd day identit(yserum). 2.5 Purity and Strength of Reference Substance: To be determinedby Sponsor. 2.6 Stabilityof Reference Substance: To be determinedby Sponsor. 2.7 Storage Conditions forTest Materials: Room temperature(2.1.1), -20 IOOC (2.1.22,.1.4).Test and Controlsamples willbe receivedaccordingto AN4]DT-S- 10-0. 0()0()"'1 2 2.8DispositioofnSpecimens:Biologictailssueasndfluidwsilble retainpeedr GLP Regulationforthetime periodrequiredforstudieslongerthan28 days. This studyisinparallewlith a 28 day dermalabsorptionstudyso alltissueswillbe retained. 2.9SafetyPrecautions:RefertoappropriatMeSDS. Wear appropriatleaboratory attireU.se cautionwhen handlingknivesforcuttingthesamples. 3.0 EX]PERRMENTAL - Overview The tissuesfrom animalsdosed as described(HWI#6329-129), areavailablfeor analysisforfluorinecompounds. At thediscretioonftheStudy Directora, seriesof analyticatlestscan be performed.The screeningforfluorideinliverviacombustion (SeeMethods@next Section)istheappropriataenalysistopresentdefmitivedatafor fluorineintheliver.For theseanalysesb,ovine liverappearstobe a good approximationtorabbitliverformatrixspikesand recoverydeterminations.Since perfluorooctanesulfonatneion isnotbiotransformedf,luorinecontentwillbe an accurateestimateof theconcentratioonfthe compound. 4.0 EXPEPUvtENTAL -Methods 4.1Liverand Serum screeningmethods: (attached) 4.1.1AMDT-M-1-0, Thermal ExtractionofFluorideby Means of a Modifled Dohrmann DX2000 OrganicHalideAnalyzer-Liver 4.1.2AMDT-M-2-0, FluorideMeasurement by Means ofan OrionEA940 Expandable Ion Analyzer 4.1.3AMDT-M4-0. Extractioonf Fluorochemicalfsrom RabbitLiver 4.1.4AMDT-M-5-0, AnalysisofRabbitLiverExtractforFluorochemicals Using ElectrosprayMass Spectrometry 4.1.5AMDT-M-14-0, Thermal Extractioonf Fluorideby Means of a ModifiedDohnnann DX2000 OrganicHalideAnalyzer-Serum 3 5.0DATA ANALYSIS 5.1DataReportingD:atawillbereporteadsa concentrat(iwoenight/weigohft) fluorideper tissueor fluido,r as FC-95 pertissueor fluidH.alf-lifoef disappearancefrom serum willbe determinedfrom the linearegressionof the averagesof theconcentrationastdifferenttimepointsforthepost distributiopnart ofthe serum concentratiovnersustime curve. 6.0 MAIINTENANCE OF RA-W DATA AND RECORDS 6.1 Raw Data and Records: Raw data,approvedprotocol,appropriatespecimens, approved finalreport,and electronidcatawillbe maintainedintheAMDT Archives. 7.0 REFERENCES 7.1AMDT-S-10-0, Sample TrackingSystem 8.0 ATTACEMENTS 8.1AMDT-M-1-0, ThermalExtractioonfFluoridbey Means ofa Modified Dohrmann DX2000 OrganicHalideAnalyzer-Liver8.2AMDT-M-2-0, FluorideMeasurement by Means ofan OrionEA940 Expandable Ion Analyzer 8.3AMDT-M-4-0, Extractionof Fluorochemicalsfrom RabbitLiver 8.4AMDT-M-5-0, Analysisof RabbitLiverExtractforFluorochemicalsUsing ElectrosprayMass Spectrometry 8.5 AMDT-M-14-0, Thermal ExtractionofFluorideby Means of a Modified Dohrmann DX2000 OrganicHalideAnalyzer-Serum 000(),'1(; 4 3M Environmental Laboratory Method Thermal Extractionof Fluorideby Means of a ModifiedDohrmann DX2000 OrganicHalideAnalyzer- Liver Method IdentificatiNounmber: AMDT-M-1 RevisionNumber: 0 AdoptionDate: RevisionDate: None Author:Rich Youngblom Approved by: roupLea Dafe roUP L( QualityAssurance Date Software:MSWord5.la ArfectedDocuments: AMDT-M-2 FluoridMeeasurementby Means ofanOrionEA940 ExpandableIonAnalyzer AMDT-EP-3 RoutineMaintenanceofa ModifiedDohrmann DX2000 OrganicHalideAnalyzer 1 0000"17 1.0 SCOPE, APPLICABLE COTMPOUNDS. AND MATRICES 1.1Scope:Thismethod isfortheoperatioonfa DohrrnannDX2000 when itisusedtoextract fluoridferom variousmatrices.The fluoridiestypicallcyollecteidnTISAB solutiofnoranalysis withan ionselectiveelectrode. 1.2ApplicableCompounds: Fluorochemicalosrotherfluorinatecdompounds. 1.3NlatricesB:iologicatlissuesp,articularlliyver. 2.0 KEYWORDS 2.1Fluoridef,luorine,xtractiopny,rolysisi,onizatioino,nselectiveelectrodeD,ohrmann, halide, DX2000, fluorochen@cals. 3.0 PRECAUTIONS 3.1Glassware and exhaustgasescan be extremelyhot. 3.2Glasswareisfragileb,rokenglassmay causeinjuries. 3.3Pressurizedgases,propercompressedgas handlingpracticersequired. 3.4Solventbasedsamplesmay flashm,ay need toallowthem todrydown beforestartinrgun. 3.5Potentiabliohazardsdue tothebiologicamlatrices.Use appropriatpeersonalprotective equipment. 4.0 SUPPLIES AIN@D MATERIALS 4.1Compressed Oxygen, Hydrocarbonfree,regulatedto30 PSI. 4.2Compressed Helium,High PurityGrade,regulatetdo45 PSI. 4.3Quar-tgzlasssampleboatwithTeflonTMtubingD,ohrmann 890-097orequivalent. 4.4Quartzglasscombustiontube,RelianceGlass G-9405-012orequivalent. 4.5Orion940999 TotalIonicStrengtAhdjustmentBuffer(TISAB 11)or equivalent. 4.6Sample collectiovnialsH,DPE. 4.7NEIH-QTIIIwater 4.8Polystyrenepipettes. 4.9ActivatedCharcoalE,. Merck 2005 orequivalent. 4.10Han-LiltoSnyringeor equivalent. 4.11Miscellaneouslaboratorgylassware 5.0 EQUIPMENT 5.1Rosemount Dohrmann DX2000 OrganicHalideAnalyzer,modifiedforfluorideextraction. 5.2IBi\4compatible386 or486 computer. 5.3DX2000 softwarev,ersion1.00,modifiedforfluorideextraction. 5.4ExcelSpreadshee@version5.0orgreater 6.0 INTERFEREIN'CES 6.1 Sample sizeislin-Liteodapproximately150mg, dependingon sample moisturecontent.This may varyfrom matrixtomatrix. 2 7.0 SAMPLE HANDLING 7.1Samplesarenottobe handledwithbarehands.Fluoridmeay leachfrom theskintothe sample. Use forcepsor probetotransfetrissues. 7.2Samplesofliverarecutfrom frozenlivearnd placedina taredand labeledweigh boat.Use a cleanscalpeland cuttingboard.The cuttingboardand scalpelshouldbe cleanedwithwater, methanol,or methanol-watersolutioanftereach liveriscut. 8.0 CALIBRATION AND STANDARDIZATION 8.1PreparatioonfCalibratiSotnandards 8.1.T1hestandarrdesquirfeodreachprojecwtilnleedtobeapproprifaotrethaitndividuparloject. Refertoprotocolforthatproject. 8.1.2Typically50-500 ppm FC-95 inmethanolstandardasreused. 8.1.3Forrabbitlivesrtudiesu,sebeeflivearst-hematrix.Cut a pieceoffrozenbeefliver(100150mg) and weigh itina labeledand taredweigh boat. 8.2 Calibratio-nOverview The normalcalibratiiosnthefluoridceurve(AMDT-M-2). However,ifan optionaslpikedliver curveisrequiredtheprocedurelistebdelow isused. 8.2.1A calibraticounrvefortheDX2000 isgeneratedby spikingsampleswithknown standards and combustingthem usingthesame methods and matrixtypeasthesamplestobe tested. 8.2.2Typicallyt,hreereplicateosfeachstandardand fiveconcentrationosfstandardswillbe spiked. 8.2.3Standardcurvewillbe plotteadsMass SpikedF (ug)on thex-axisand StandardMass RecoveredF (ug)on they-axis.Generatea regressiocnurveand calculatteheequationfortheline and ther2 value. 8.2.4Mass SpikedF (ug)= (Amount spikedinmL) x (Conc.of standaridnppm) x (0.6004)* *FC-95 is60.04% F therefor0e.6004isthefactoursedtoconvertFC-95 toF 8.2.5StandardMass RecoveredF (ug)= (TISAB volumeinmL) x (Orionreadinginppm) 8.3Calibratio-nProcedure 8.3.1StartUp 8.3.1.1Run 2 ormore Clean Cycleswhen startinignstrumenetachday. More cleancyclesmay be usediftheprevioussamplescontainedhighconcentratioonfs fluoride. 8.3.2Blanks 8.3.2.1Preparesample usingthesame methods and typeofmatrixasthetestsample. 8.3.2.2Forrabbitstudiesu,sebeeflivearsthematrix.Prepareatleast3 samplesofbeefliver (100 - 150 mg) forblanks. 8.3.2.3Putsample inDohrrnannboat.Combust eachsampleasdescribedinsection9.0and analyzesample accordingtomethod AMDT-i'vf-2fortheionselectiveelectrodaenalysis. 3 0()0()"1!) 8.3.2.4For rabbitstudiest,hemeterreadingfora blanksample shouldbe 0.03ppm or lower beforeproceedingwiththecalibrationB.um samplesuntilthislimitisreached,or untilin the judgement of theoperatorthereadingisstablewithrespecttohistoricarleadings(previous48 hours). 8.3.2.5For non-rabbitstudiest,heblank readingsshouldreacha predeterminedion concentration beforeproceedingwiththecalibration. 8.3.2.6Itmay be necessarytomix approximately50 mg of charcoalwiththesample toaid combustion. 8.3.3 Standard Curve 8.3.3.1Weigh outatleast15 matrixsamples(5 standardswith3 replicateesach)in taredand labeledweigh boats.For rabbitstudiesw,eigh 100-150mg beefliversamples.Record weightsin studydata.Storethematrixsampleson dryiceor icepacks tokeep them frozenuntilused. 8.3.3.2 Placeweighed beefliversample inDohrmann sampleboat. 8.3.3.3StartwiththeloweststandardconcentrationU.sing a Hamilton syringe4,@ecta fixed quantityof thestandardon or inthematrix.For rabbitstudiesu,se 4 uL of standardand ejectiton or inthebeefliver. 8.3.3.4At least3 replicatesshouldbe used forthelowest standardconcentrationm;ore replicates may be used atthediscretioonf theanalyst. 8.3.3.5Combust thesample asdescribedinsection9.3and analyzeaccordingtoAMDT-M-2. 8.3.3.6Run all15 standardsI.fone replicatiessignificantdliyfferenftrom theothertwo replicatesr,un anothersample forthatstandard.Indicateindatathatthenew replicatreeplacesthe oldreplicataend thatthenew replicatweillbe used tocalculatteheregressiocnurve. 8.3.3.7When allstandardhsave been run,calculattehe r2. r2 must be atleast0.95.Ifitisnot at least0.95,consultwith supervisor. 8.3.3.8A new standardcurveshouldbe run when thecombustiontubeor sample matrixis changed. New standardcurvemay alsobe run atthediscretioonf theanalyst. 8.4 StorageConditionsforStandards 8.4.1Storagerequirementsforstandardsaredependenton theindividuasltandardsused. Typically,standardsarestoredatroom temperatureinplastisccrew topbottles. 8.4.2New FC-95 standardsshouldbe preparedatleastonce a month. 9.0 PROCEDURES 9.1 TypicalOperating 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 time = 300 seconds. 9.2StartUp' Procedure: 9.2.1Iftheprogram isnotstarteds,tartheEOX proaram on thePC. 9.2.2Open theSYSTEM SETUP window. 9.2.3Put thefurnacemodule and thecellintheREADY mode. 9.2.4Close theSYSTEM SETUP window. 4 00 9.2.5When the oven has reachedtheREADY temperature,run theCLEAN 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 ingof charcoalwith thesample toaidcombustion. Ifthisisdone, charcoal should alsobe mixed inwhile establishintghebaselineand when generatingthe standardcurve. 9.3.2 Close SAMPLE HATCH. 9.3.3 Add appropriatevolume of TISAB solutionor 1:1 TISAB:Milli-QTI"water mixtureto a labeledsample collectiovnial.Typically0.6n-lLto 15 mL areused. For rabbitstudiesu,se 1.0or 2.0mL of 1:1 TISAB:Milli-QTItwater mixture. 9.3.4 Place thevialso thatthetipof theCOMBUSTION TUBE isintheTISAB atleast0.25 inches.Gases releasedduringpyrolysismust bubble throughthe TISAB. 9.3.5 Run theEOX-SOLIDS program found in theRUN menu. 9.3.6 When theEOX program isfinishedr,emove thecollectiovnialfrom thecombustion tube. 9.3.7IfundilutedTISAB was used tocollecthe sample,add an equal volume of Milli-QTMwater tothe TISAB to make 1:1 TISAB: Nfilli-QTM. 9.3.8Rinse theend of the combustion tubewith Milli-QTMwaterand wipe with a KIMWIPE to remove any TISAB remaining on the tube. 9.3.9 Open the sample hatchand remove any remaining ash from the boat. Ash can be removed with a cottontippedapplicatoror vacuumed out. Itmay be necessaryto scrapparticleosffthe bottom with a spatulaor othersimilardevice.A drop of Milli-QTMwater may be added to the boattoaid inthe Clean Cycle. 9.3.10 Close thehatch. 9.3.11 Run the CLEAN BOAT program. 9.3.12 Sample isready foranalysisby ion selectiveelectrode(ANMT-M-2). 9.4 Sample Calculations 9.4.1 Use the standardcurve tocalculatethesample value. 9.4.2Sample Mass Recovered F (ug)= (TISAB volinmL) x (Orionreadiniian ppm - intercepo (Slope) 10.0 VALIDATION 10.1 QualityControl 10.1.1Daily Start Up Check Samples: Once the standardcurve isestablishede,ach day of analysisisstartedby analyzingQC samples. The QC samples are tobe the same as the lowest concentrationspiked samples used togeneratethe standardcurve. Each concentrationmust be done intriplicatuenlessthe firsttwo replicateasre within20% of thestandardcurve,thena third replicateisnot necessary. 10.2 Precisionand Accuracy: See method development analysisand sample analysisin FluorideNotebooks 2,3,and 5. Precisionand accuracy varieswhen analyzingsamples of different matricesand differentreferencecompounds. 10.3 Other ValidationParameters: NA 5 0 ()ILII 11.0 DATA ANALYSIS 11.1 Calculations 11.1.1For thestandardcurve,useregressioannalysisinExcel,version5.0or greater. 11.1.2To calculattehefluoridceontractioinnthesample,seemethod AMDT-M-2. 11.2 Analyzing theData 11.2.1r2must be atleast0.95orgreater".Outliersm"ay be excludediftwo of thethreereplicates arewithin20% ofeachotherand theoutlieirsgreaterthan200% oftheaverageofthosetwo or lessthan50% of theaverageofthosetwo. Any suchoutliersshouldbe pointedoutinthedataand notedintheFinalReportalongwiththereasonitwas consideredanoutlier. 12.0 ATTACHMENTS None 13.0 REFERENCES 13.1Rosemount DohrrnannDX2000 OrganicHalideAnalyzerOperator'Msanual (Manual 915349,revisionB, December 1993) 13.2 AMDT-M-2 FluorideMeasurementby Means of an OrionEA940 ExpandableIon Analyzer 13.3AMDT-EP-3 RoutineMaintenanceofa ModifiedDohrmann DX2000 OrganicHalide Analyzer 14.0 REVISIONS Revision Numbe Reason forChange Revision Date 6 00004Avw 3M Environmental Laboratory Method FluorideMeasurement by Means ofan Orion EA940 Expandable Ion Analyzer Method IdentificatiNounmber: AMDT-M-2 RevisionNumber: 0 AdoptionDate:ID-,f-fsRevisionDate: None Author-Rich Youngblom Approved By: Qlaw-., G@, Leader /,0-3 Date QualityAssurance Date Software:MS Word 5.1a AffectedDocuments: AMDT-M-1 ThermalExtractionfFluoridbey Means ofa Modified Dohrmann DX2000 OrganicHalideAnalyzer 0 0 11@'i 1.0 SCOPE, APPLICABLE COMPOUNDS. AND MATRICES I.ISCOPE: Thismethod isforthecalibratiaond operatioonfan OrionEA940 Expandable [onAnalyzer. 1.2APPLICABLE COMPOUNDS: Fluoride. 1.3APPLICABLE MATRICES: LiquidsamplesinanappropriabtueffesrolutionP.referred pH of6.0. 2.0 KEYWORDS 2.1Fluoridef,luorinei,on selectiveelectrode 3.0 PRECAUTFONS 3.1No hazardsidentifiewdiththismethod. 4.0 SUPPLIES AIND MATERIALS 4.1Orion940999 TotalIonicStrengthAdjustmentBufferII(TISABII)orequivalent. 4.2OrionModel 900001electrodfeillinsgolutio(nAgCl)orequivalent. 4.3Orion 940907 100 ppm fluoridsetandardorequivalent. 4.4Milli-QTmwateror equivalent. 4.5Magneticstirbars. 4.6Lab tissues. 4.7Sample couectionvials. 4.8Plastic100mL volumetricflasks. 4.9Polystyrenpeipettes. 4.10Miscellaneouslaboratorgylassware. 5.0 EQUIPMENT 5.1OrionModel EA940 ExpandableIonAnalyzerorequivalent. 5.2OrionModel 960900 SolidStateCombinationFluoridelectrodoerequivalent. 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 1:1n-@xturoefTISAB and sample/MilliQ T,'Ilwatweirllgenerallybringsample topH of6.0. 6.2Sample temperaturmeay effecftluoridmeeasurement. Itisrecommended thatthesamplebe atroom temperatureas thestandardswere when themeterwas calibrated. 6.3The ratethesamplesarestirreadtshouldbe consistenwtiththeratethestandardswere stirred. 2 000044 6.4Air bubblestrappedunder electrodcean giveerroneousreadings.Make sureno airistrapped under electrode. 7.0 SAMPLE HANDLING 7.1 No specialhandlingnecessary. 8.0 CALIBRATION AND STANDARDIZATION 8.1 PreparationofCalibratioSntandards 8.1.1Measure 50 mL ofTISAB 11into5 100niLplastivcolumetrifclasks. 8.1.2Labelthef.laskass0.05,0.1,0.5,1.0a,nd 1.5ppm F-,alongwiththedateand yourinitials. 8.1.3Pipett0e.05,0.1,0.5,1.0a,nd 1.5mL of 100ppm fluoridsetandaridntotheappropriately labeledflasks. 8.1.4Add approximately30 mL of Milli-QTMwaterto each flask. 8.1.5Shake theflaskstomix thesolutions. 8.1.6Eliniinataeirbubblesfrom theflasksby dppingtheflaskson theirsidesand rollingtheairin the flasksover theairbubbles. 8.1.7Bring thevolume intheflasksup tothe100 niL mark withMi]li-QTMwater. 8.1.8Invertand shaketheflasksforthefinalmixing. 8.1.9Record standardsinStandardsLog Book. 8.2 Calibration 8.2.1Ifnecessary,remove tapefrom electrodefillinhgole. 8.2.2Invertprobe towet topseal. 8.2.3Ejecta few dropsof fillinsgolutionfrom bottom of electrodteo wet lowerseal. 8.2.4Filltheelectrodweithfillinsgolution. 8.2.5The meter and theF- electrodaeretypicallcyalibratebdy directmeasurement withno blank correctionu,singstandardswithconcentrationosf 0.05,0.1,0.5,1.0,and 1.5ppm F-,following themanufacturer'sinstructions. 8.2.6Record theslopein theappropriatleogbook. 8.2.7Clean theelectrodbey rinsingwith Milli-QTMwaterand wipingthesidesdown withlab tissues. 8.3 StorageConditionsforStandards 8.3.1Calibrationstandardsarestoredatroom temperature. 9.0 PROCEDURES 9.1Calibrationand i'vleasuremenStt,andard method: 9.1.1The sample tobe measured needsto be mixed withTISAB usingtheproportions recommended by theTISAB manufacturer. 9.1.2Place a stirbarinthesample and placethesample on thestirplate. 9.1.3Allow thesample torr@xfora few secondsbeforeinsertintgheelectrode.When the electrodeisinsertedm,ake surethereareno airbubblestrappedunder theelectrode. 9.1.4The sample shouldbe thesame temperatureasthecalibratiosntandardsand stirreadtthe same rateas thecalibratiosntandards. 9.1.5When thereadincshave stabilizerde,cordthereadingintheappropriatelogbook. 3 0()0()1-15 9.2 Calibration And LNleasurement,Using Orion 3E Software: 9.2.1 Calibration: 9.2.1.1 Follow steps8.2.1to 8.2.4. 9.2.1.2Press FunctionKey #8 (F8). 9.2.1.3The computer screenwillask you toconfirm thenumber of standardstobe used, concentrationof thestandards,and whether or not a blank istobe includedinthe calibration. Make any necessarychanges tothe informationpresentedand clickon CONTINUE. 9.2.1.4Place the electrodeinthe firssttandardon the stirplateand clickon CONTINUE. 9.2.1.5Observe thereadingson thegraphicdisplayon thecomputer. When the readingshave stabilizedp,ressACCEPT READING. 9.2.1.6Repeat step9.2.1.4and 9.2.1.5forthe remaining standards. 9.2.1.7Afterthe finalstandard,the computer willdisplaythe slopeof the curve,as well as the interceptand correlation.Record theslope,intercepta,nd correlatioinnthe appropriatelog book and clickon CONTINUE. The calibratiodnataisautomaticallcyopiedto C:\Orion\Data\Calib.txt. 9.2.2Data Spreadsheet: 9.2.2.1SelecteitherNEW or OPEN from theFILE menu to open a new or existingspreadsheet tostoredata in. 9.2.2.2Record the name of thespreadsheetused in theappropriatelog book. 9.2.3Fluoride Nleasurement: 9.2.3.1Follow steps9.2.1through9.2.4 9.2.3.2Enter the name of the sample inthe appropriateplaceon the screen. 9.2.3.3Click on theNEW SAMPLE button 9.23.4 When the readingshave stabilizedc,lickon theRECORD buttonand writetheresultinthe appropriatelog book. 10.0 VALIDATION 10.1 QualityControl: 10.2 Precisionand Accuracy 10.3 Other ValidationParameters According to Reference13.2,therange of detectionis0.02 ppm fluorideup to a saturatedsolutionof fluoride. 11.0 DATA ANALYSIS 11.1 Calculations None necessary. 11.2 Analyzingt-,Pthe Data None necessary. 12.0 ATTACH.NIENTS None 13.0 REFERE@NCES 4 0()004(; 13.1Orion Model EA940 Expandable IonAnalyzerInstructioMnanual,Orion Research Incorporated1,991. 13.2Orion Model 960900 SolidStateCombinationFluorideElectrodeInstructioMnanual, Orion Research Incorporated,1991. 14.0 REVISIONS Revision Numbe Reason forChange Revision Date 5 000047 3M Environmental Laboratory Method Extractionof Fluorochemicalsfrom Rabbit Livers SOP IdentiricatioNnumber: AMDT.M.4 RevisionNumber: 0 Adoption Date: RevisionDate: None Author* Dave Christenson/CynthiWaeber Approved BY: roup Leader Date i5u-alitAys-surance -3 /-1.5'Date Software:MS Word, 6.0 AffectedDocuments: M-5, AnalysisofRabbitExtractforFluorochemicalUssing Electrospray Mass Spectroscopy. 1.0 SCOPE 1.1 Scope: Thismethod isfortheextractioonf fluorochemicalfsrom rabbitlivers. Ethylacetateisusedtoextractfluorocheniicaflrsom theliversforanalysisby electrospramyass spectroscopy. 1.2 ApplicableCompounds: Fluorochemicalsor otherfluorinatecdompounds. 1.3 Matrices:RabbitLivers. 2.0 KEYWORDS 2.1 Fluorochemicalsr,abbitliverse,lectrospramyass spectrometerf,luorinated compounds, extraction. 3.0 PRECAUTIONS 3.1 Use gloveswhen handlingtherabbiltivertsh,eymay containpathogens. 4.0 SUPPLIES AND MATERIALS 4.1 Supplies 4.1.1 Syringe,capableof measuring 100 pL 4.1.2 Eppendorf typeor disposablepipets 4.1.3 Gloves 4.1.4 Plasticgrindingtubes 4.1.5 Plasticentrifugteubes,15 mL 4.1.6 Labels 4.1.7 Nitrogen 4.1.8 Timer 4.1.9 FiltersT,itannylon syringefilter0s.,2 gm. 4. 1.10 Analyticaplipetsg:lassvolumetricpipets. 4. 1. 11 Disposableplasti3c cc syringes. 4. 1.12 Crimp cap autovials. 4.2 Reagents 4.2.1 Aqueous Ammonium Acetate(Aldrich)a,pprox.250 ppm: Preparea 2500 ppm aqueoussolutioonf ammonium acetateby adding250 mg ammonium acetatetoa 100mL volumetdcflaskand dilutteovolume withNM-Q water.Dilutethissolution1:10fora 250 ppm solution. 4.2.2 Sodium carbonate/SodiumBicarbonateBuffer(J.T.Baker), (Na@C03/NaHC03)0.25 M: Weigh 26.5g of sodium carbonate(Na2CO3) and 21.0g of sodium bicarbonat(eNAHCO@ intoa I L volumetdc flaskand bringtovolume withNEHI-Q water. 4.2.3 Diluteacetonitrisloelutiond,iluteacetonitri1l:e1withWli-Q water. 4.2.4 EthylAcetate 4.2.5 Methanol 4.2.6 Milli-Qwater 4.2.7 IH,IH,2H,2H -perfluorooctanesulfoanciicd(Aldrich) 4.2.8 FC-95 (3M SpecialtCyhemicalDivision) 2 5.0 EQUIPMENT 5.1 Ultra-TurraxT25 Grinderforgrindingliversamples. 5.2 Vortexmixer 5.3 Centrifuge 5.4 Shaker 5.5 AnalyticaElvaporator 6.0 INTERFERENCES 6.1 nere areno known interferenactetshistime. 7.0 SAMPLE HANDLING 7.1 Ile rabbitliverasrereceivedfrozen,and must be keptfrozenuntiltheextractioins performed. 8.0 CALIBRATION AND STANDARDIZATION 8.1 Preparationof InternalStandards 8.1.1 Preparean internasltandardofapproximately12 ppm 1H,lH,2H,2Hperfluorooctanesulphonaiccidtobe added toeach liversample. 8.1.2 Weigh atleast0.1 g of IH,IH,2H,2H-perfluorooctanesulphoniacidintoa 100 mL volumetdc flaskR.ecord theactualweighl 8.1.3 Bringitup tovolume withmethanol,thisisthestockstandarcl 8.1.4 To a 250 mL volumetricflaska,dd 3 mLs ofthestockstandardand bringto volume with Milli-Qwater.Calculatetheactualconcentratioonf the standard. actualmg perfluoroctane- sulphonicacid X 3 mL 0.1 L 250 mL actualconcentrationp,pm 8.2 Prepare FC-95 Anion Standards 8.2.1 PrepareFC-95 standardsforthestandardcurve. 8.2.2 Weigh approximately100 mg of FC-95 intoa 100 mL volumetricflask. Record theactualweight. 8.2.3 Bringup tovolume withdiluteacetonitrile. 8.2.4 Dilutethesolutionwithdiluteacetonitri1l:e10 fora solutionof approximately100 ppm. Dilutethissolution1:10 with diluteacetonitfffloer a 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 ofBovine liverintoa 250 mL Nalgenebottlecontaining 200 mLs Milli-Qwater.Grind toa homogenous solution. 8.3.2 Add ImL of thesolutiontoa 15 mL centrifugteube.Preparea totalof eight I mL aliquotosf thesolutionin 15 mL centrifugteubes.Be sureto resuspendsolutionby shakingitbetween aliquots. 3 8.3.3 SpikesevenoftheImL aliquowtisththefollowianmgountsofworldng standardsinstep9.12of theprocedure.One I mL aliquotservesas the blank. Working Standard UL Approximate final (ApproximateConc.) concentratioonf FC-95 inliver - Blank 500 ppb 100 0.292 ppm 500 ppb 2 0.584 ppm 500 ppb 300 0.877 ppm 500 ppb 400 1.168 ppm I PPM 500 2.924 ppm- 5m 200 i 5.848 ppm- 5 pFmpmmm I 300 8.772 ppm 8.4 Calculatetheactualvalueof thestandards: uL ofstandardx concentratio(ninRRm) 171 mg liveF/1 ml homogenate finacloncentratio(nppm) ofFC -95inliver *Average weightof bovineliverinsolutionas determinedby weighing 1 mL homogenatesof40 mg liverin200 mL ofMilli-Qwater.The amount of FC-95 isreportedas equivalentsof FC-95 potassiumsalt. 8.5 Calibration 8.5.1 Extractthespikedbeefliverhomogenate following9.13to9.23of this method. Use thesestandardstoestablisyhour curve on themass spectrometer. 8.5.2 Alternativelay,standardcurvemay be generatedusingratiosof responses of theperfluorooctansulfonaatneionand theinternasltandardanion versus concentratioonf theperfluorooctanesulfonatneion. 8.6 Storage Conditions for Standards 8.6. 1 New standardsarepreparedwitheach analysisS.tandardsarestoredin coveredplasticentrifugteubesuntiltheanalysiosn themass spectrometer isperformed. 8.7 Storage Conditionsfor Standards 8. 7. 1 Beef liverhomogenates may be frozenafterpreparation. 9.0 PROCFDXTRFS 9.1 Obtain frozenliversamples.Inspenttissuen,otethattheliverhas notbeen packaged withothertissues. 9.2 Use a dissectinsgcalpeland cutoffapproximatelyIg of liver. 9.3 Weigh thesample directliyntoa taredplastigcrindingtube. 9.4 Record theliverweightinthestudynotebook. 9.5 Fluta labelon thevialwiththestudynumber, weight,rabbitD:),dateand analyst initials. 0()Oor.-I 4 9.6 9.7 9.8 9.9 9.10 9.11 9.12 9.13 9.14 9.15 9.16 9.17 9.18 9.19 9.20 9.21 9.22 9.23 9.24 9.25 Add 2.5 mLs water. Grind thesample.Put thegrinderprobe inthesample and grindforabout 2 minutes,untilthesample isa homogeneous solutionwithno largechunks. Rinsetheprobe offintothesamplewith2.5 mLs waterusinga pipet. Take thegrinderapartand cleanitwithmethanolaftereach sample.Follow AMDT-EP-22. Cap thesample and vortexfor 15 seconds. PipetI niL intoa 15 mL centrifugteube.Labelthecentrifugteubewith theidentical informationas thegrindingtube.(SeeAMDT-M-4 Worksheet fordocumentingthe remainingsteps.) Spike thebeefliverhomogenates withtheappropriataemount of FC-95 standard as describedin 8.3. Spike thesamplesand beefliverhomogenates with100 uL of intemalstandard. Add 1 mL of thesodium carbonate/sodiubmicarbonatbeufferand I mL ammonium acetate. Using an analyticaplipet,add 5 mL ethylacetate. Cap thesample and vortex20 to30 seconds. Put them in theshakerfor20 iriin. Centrifugefor20 to25 minutes,untilthelayersarewellseparatedS.et thepower on thecentrifugteo25. Remove 4 mLs of thetoporganiclayertoa fresh15 mL centrifugetubewitha 5 mL graduatedglasspipet.Transferthelabeltothefreshtube. Blow thesample down on theanalyticaelvaporatortoneardrynesswithnitrogen, approximately30 to40 minutes. Bzing theremainingsampleup in I niL diluteacetonitriwliethan analyticaplipet. Vortex 15 seconds. Transferthesample toa 3 mL syringeA.ttacha 0.2pm nylonmesh filtera,nd filter thesample intoa freshcentrifugteubeor a autovialL.abel thetubeor vialwiththe studynumber and animalnumber. Cap and holdforanalysisby electrospramyass spectroscopy. Complete AMDT-M-4 worksheetand attachtopage of studynotebook. 10.0 VALIDATION 10.1 QualityControl- notapplicable 10.2 Precisionand Accuracy- not applicable 10.3 Other ValidationParameters-notapplicable 11.0 DATA ANALYSIS 11.1 None 12.0 ATTACHMENTS 12.1 WorksheetAMDT-M-4 13.0 RMRENCES 13.1 ANMT-EP-22 RoutineMaintenanceofUltra-TurraTx-25 14.0 REVISION'S Revision Number Reason -foCrhange Revision Date 5 Study Sample Number set Blank T-ivt-r Worksheet AMDT-M-4 FC-95 approx 0.5 ppm actual PPM #W 100 ilf, 700 tiT@ 100 "T. 41)0iil, - FC-95 approx I ppm actual ppm #W if)OiiT, FC-95 approx.5 ppm actual PPM #W Date and InitialfsorStd. 2(10iiT, 100 iiT. -qudv nilm r 3ght-rtehe oripinglwo Liver1Pxtrn on Prn(-Pqq- beet iglorAtt--.-tnil-aaenda cony Pirk-It mt.-nfLiverSoliitinn Pinet100 iiLof 12 nnm Tntern;Sitiandard Vnrtex 11gre- stti -PinetI mT.nf 2';0nnm A mmnniiim Arft:lti- PinetI mT. of0 2,1Nn.('O,/0-25MN:IHCO. Ruffer Pineti mT. nf Rthyl Art-intLVnrtt-x70-10--Rer Sh:ike-'20min, -Centrifiiore-7(m)i-n25 Remnyp ;14 mL gliniinntf rognirI.nver Blnw down-tn negrcirvnrq(!<;0-25ml.)withN. AtltlI m- nf-1-1Acetnnitrile/74.0 TN# Vnrtex Ii qt-r tertiging;iI,ce R-T)gynnee with2 0 ')iimSRI filtii-mrn a I i ml. autog:imnivti-:;l T)Rtt- Tnitialq 00 6 3M Environmental Laboratory Method Analysisof Rabbit Liver Extract for Fluorochemicalsusing Electrospray Mass Spectroscopy SOP IdentiricatNiuomnber: AMDT-M-5 RevisionNumber: 0 Adoption Date: RevisionDate: None Author:Dave Christenson/CynthWieaber Appmved By: mup Leader Date ssurance Date Software:MS Word, 6.0 AffectedDocuments:M-4,ExtractioonfFluorocherriicfarlosm RabbitLivers 0 0 ILI 1.0 SCOPE 1. 1 1.2 1.3 Scope: Thismethod isfortheanalysisof extractosf rabbitliveror othertissuesor fluidsforfluorochemicalussingthe electrospramyass spectrometern.e analysis isperformed by singleionmonitoringofFC-95 anion,M/Z= 499, theinternal standardM/Z = 427, and otherappropriatemasses. Applicable Compounds: Fluorochemicalsor otherfluorinatecdompounds. Matrices: RabbitLivers(samples),Beef Liver(standards)o,thertissuesand fluids. 2.0 KEYWORDS 2.1 Fluorochemicals,fluorinatecdompounds, electrospraymass spectroscopy,mass spectrometer,rabbitlivers. 3.0 PRECAUTIONS 3.1 Use cautionwiththevoltagecablefortheprobe.When thevoltagecableisplugged intotheprobe DO NOT TOUCH THE PROBE, thereisriskof electricaslhock. 3.2 Do not run thepump above it'scapacityof 4000 psi.Ifpressuregoes over 4000 psi stop and releasepressure.The peak tubingmay be plugged.Troubleshootback to findtheplug and replacetheplugged tubing.See AMDT-EP-15 3.3 Do not run thepump to dryness. 4.0 SUPPLIES AND MATERIALS 4.1 Supplies 4. 1.1 Nitrogengas regulatedto 140 psi. 4. 1.2 Fluofixcolumn or equivalent. 4.1.3 100 uL or 250 uL flattipsyringeforsample injection. 4.2 Reagents 4. 2. 1 Diluteacetonitrimloebile phase,diluteacetonitri1l:e1 with Milli-Qwater. 4. 2.2 Milli-Qwater,allwaterused in thismethod shouldbe Milli-Qwater. 5.0 EQUIPMENT 5.1 VG Trio 2000 ElectrosprayMass Spectrometeror equivalent. 5.2 ISCO SyringePump 5.3 SpectraphysicsAS300 Autosampler 5.4 100 uL Assembly 5.5 Autovialsor capped centrifugetubes. 6.0 INTERFERENCES 6.1 There areno known interferenceastthistime. 7.0 SAMPLE HANDLING 7.1 Keep the extractedsamples incapped 15 mL centrifugetubesorincapped autovials untilready foranalysis. 0()OO-J,j 2 8.0 CALTBRATION AND STANDARDIZATION 8.1 Preparation of CalibrationStandards 8. 1. 1 Seven beefliverstandardsand one blank beef liverarepreparedduring the extractiopnrocedure.(See AMDT-M-4, section8.0) 8.2 Calibration 8.2. 1 Run theseven beef liverstandardstwice,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 standardsareprepared witheach analysisS.tandards are storedin covered plasticcentrifugetubesuntiltheanalysison the mass 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 softwareto"Operate on", Ion Mode ES-. 9.1.2 Record backing pressurein theinstrumentlog. 9.1.3 Fillthesolventcylinderwith mobile phase. 9.1.4 Set thepump to"Run". Set theflow to 1000 ul.Anin.Observes droplets coming out of thetipof the probe.The pressureshould be at 1700 to 1800 psi. 9.1.5 Check thefused silicattheend of theprobe.Use an eye piecetocheck for chips.The tipshould be flatwith no jagged ed-es.Ifany chipsare found cut offthetipof thesilicwaith a column cutterand pullthesilictahroughto the appropriatelength. 9.1.6 Check your nitrogensupply.Turn on the nitrogen.There should be no nitrogenleakingaround the tipof theprobe.A finen-@stshould be coming out of the tip. 9.1.7 Carefullyguide theprobe intotheopening.Insertituntilitwon't go any further.Connect the voltagecableto theprobe. 9.1.8 Go tothe"Editot"page,and setIonizatioMnode toES-,and the appropriatemasses to427 and 499. 9.1.9 Ifitisnotin singleion mode go to"Option" and setSIR. 9. 1. lOStartAcquisitionA.ssign a filename, MO-DAY-YR + letterR.ecord itin the log book. 9. 1.11 Run thebeefliversamples firstr,unningeach standardtwiceatthe beginning of the run..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 intothe rheodyne injectionport.Injectslowly.Record thesample ID in the log book. 9. 2.2 Turn thevalveto"On". 9.2.3 Wait two minutes,tndinjecthenextsample. 9.2.4 Record the scan number foreach sample inthelogbook. 3 0 ()0 0 9.3 Using the Autosampler 9.3.1 Set up sample trayA, B, or C. 9.3.2 Record thesamplesand theirpositionisn theinstrumentlog book.Up to 17 vialsmay be ineach run. 9.3.3 Set-upthesampler: 9.3.3.1 Push thesample button 9.3.3.2 Setsample loop size= 100 uL 9.3.3.3 Setinject/sampl=e2 9.3.3.4 SetCycle time= 0 9.3.3.5 Name thefileL:ivers 9.3.3.6 Identiftyhetrayused 9.3.3.7 Add thesamplestoQueue by pressing"Entee' 9.3.3.8 Press"Run" tostart 10.0 VALIDATION 10.1 QualityControl 10. 1. 1Run a standardevery5 to7 samples.Ifa significancthange( 50%) in peak heightoccursstoptherun.Only thesamplesbeforethelastacceptable standardwillbe used.ne remainingsampleswillbe reanalyzed. 10.2 Precisionand Accuracy 10. 2. 1See Method ValidatioRneportnumber AMDT-M-5.0.Vl 10.3 Other ValidationParameters 10.4 RefertoMethod ValidatioRneportNumber ANOT-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. 2.2 Calculatethemg ofFC-95 anion,or otherfluorochemicailnthetotarlabbit liver- mg FC-95 anioninthetotalrabbitliver mg FC-95 anionfrom std.curve gms of liverused foranalysis x Totalmass ofliverg,ms 11. 3 11.4 Make a resulttsableand enteritinthestudybook. Printa chromatogram foreach sample,Mth thepeaks labeledwiththesampleor standardD:).Writethestudynumber on theprintouti,nitiadla,te,and putitin the studyfolder.Stapleallchromatograms togetherand number pages. 4 12.0 ATTACHMENTS None 13.0 REFERENCES 13. 1 AMDT-EP- 17 14.0 REVISIONS Revision Num Reason forchangg Revision Date 0 0 0 0 -S'5 r'A,'. 3M Environmental Laboratory Method Thermal Extractionof Fluorideby Means of a ModifiedDohrmann DX2000 Organic HalideAnalyzer-Serum Method IdentificatiNounmber: AMDT-M-14 RevisionNumber: 0 AdoptionDate: RevisionDate: None Author:Rich Youngblom Approvedby: roupLeader Date dr@ p@L @ 4QualityAssurance z 7yDate Software:MS Word 5.1a AffectedDocuments: AMDT-M-2 FluoridMeeasurementby Means ofan OrionEA940 ExpandableIonAnalyzer AMDT-EP-3 RoutineMaintenanceofa ModiriedDohrmann DX2000 OrganicHalideAnalyzer 0 0 0 0."d@ 1.0 SCOPE, APPLICABLE COMPOUNDS, AND NIATRICES 1.1Scope:Thismethod isfortheoperationofa Dohrmann DX2000 when itisusedtoextract fluoridferom variousmatrices.The fluoridiestypicallcyollecteidnTISAB solutiofnoranalysis withan ionselectiveelectrode. .1.2ApplicableCompounds: Fluorochemicalsorotherfluorinatecdompounds. 1.3Matrices:Biologicaflluidsp,articularsleynim. 2.0 KEYWORDS 2.1Fluoridef,luorine,xtractiopny,rolysisi,onizatioino,nselectiveelectrodeD,ohrmann, halide, DX2000, fluorochemicals. 3.0 PRECAUTIONS 3.1Glassware and exhaustgasescan be extremelyhot. 3.2Glasswareisfragileb,rokenglassmay causeinjuries. 3.3Pressurizedgases,propercompressed gas handlingpracticersequired. 3.4Solventbasedsamplesmay flashm,ay need toallowthem todrydown beforestartinngm. 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-9405-012 orequivalent. 4.5Orion940999 TotalIonicStrengthAdjustmentBuffer(TISAB II)or equivalent. 4.6Sample collectiovnialsH,DPE. 4.7 NEIH-QTM water 4.8Polystyrenepipettes. 4.9ActivatedCharcoal,E.Merck 2005 orequivalent. 4.10Hamilton Syringeor equivalent. 4.11 Miscellaneouslaboratorgylassware 5.0 EQUTPNIENT 5.1Rosemount Dohrmann DX2000 OrganicHalideAnalyzer,modifiedforfluorideextraction. 5.2IBM compatible386 or486 computer. 5.3DX2000 softwarev,ersion1.00,modifiedforfluorideextraction. 5.4Excel Spreadsheetv,ersion5.0orgreater 6.0 INTERFERENCES 6.1 Sample sizeislimitedtoapproximately100 41. Thismay varyfrom matrixtomatrix. 0000(*() 2 7.0 SAMPLE HANDLING 7.1Samples aretobe handledwithplastipcipettesA. new pipetteistobe usedforeachsample. 8.0 CALIBRATION AND STANDARDIZATION 8.1PreparatioonfCalibratiSotnandards 8.1.1The standardrsequiredforeachprojecwtillneed tobe appropriatfeorthatindividuaplroject. Refertoprotocolforthatproject. 8.1.2Typically50-500ppm FC-95 inmethanolstandardasreused. 8.1.3Forrabbitse= studiesu,sebeefserum asthematrix. 8.2 Calibration-Overview The normal calibratioinsthefluoridceurve(AMDT-M-2). However, ifan optionalspikedserum curveisrequiredtheprocedurelistebdelow isused. 8.2.1A calibratiocnurvefortheDX2000 isgeneratedby spikingsampleswithknown standards and combustingthem usingthesame methods and matrixtypeasthesamplestobe tested. 8.2.2Typicallyt,hreereplicateosfeachstandardand fiveconcentratioonfsstandardwsillbe spiked. 8.2.3Standardcurvewillbe plotteadsMass SpikedF (ug)on thex-axisand StandardMass RecoveredF (ug)on they-axis.Generatea regressiocnurveand calculatteheequationfortheline and ther2 value. 8.2.4Mass SpikedF (ug)= (Amount spikedinmL) x (Conc.ofstandaridnppm) x (0.6004)* *FC-95 is60.04% F therefor0e.6004isthefactoursedtoconvertFC-95 toF 8.2.5StandardMass RecoveredF (ug)= (TISAB volumeinmL) x (Orionreadinginppm) 8.3Calibratio-nProcedure 8.3.1StartUp 8.3.1.1Run 2 or more CleanCycleswhen startinignstrumenetachday.More cleancyclesmay be usediftheprevioussamplescontainedhighconcentrationosffluoride. 8.3.2Blanks 8.3.2.1Preparesample usingthesame methods and typeofmatrixasthetestsample. 8.3.2.2Forrabbitstudiesu,sebeefserum asthematrix. 8.3.2.3Putserum blanki-nDohn-nannboat.Combust sample asdescribeidnsection9.0and analyzesampleaccordingtomethod AMDT-M-2 fortheionselectiveelectrodaenalysis. 8.3.2.4For rabbitstudiest,hemeterreadingfora blanksample shouldbe 0.03ppm orlower beforeproceedingwiththecalibratioBnu.m samplesuntitlhislimitisreached,oruntilinthe judgement of theoperatorthereadingisstablweith respect ohistoricraeladings(previou4s8 hours). 8.3.2.5Fornon-rabbisttudiest,heblankreadingsshouldreacha predetermineidonconcentration beforeproceedin_waith thecalibration. 8.3.2.6Itmay be necessarytomix approximately50 mg ofcharcoalwiththesampletoaid combustion. OOOOC-1 3 8.3.3Standard Curve 8.3.3.1Ifbeefserum isfrozen,thaw atleastenough tocompletethestandardcurve analysisforthe day (=30 mL). 8.3.3.2 Pipette100@LLof beefserum intoDohnnann sample boat. 8.3.3.3Startwith theloweststandardconcentrationU.sing a Hamilton syringe,ejecta fixed quantityof thestandardon or inthematrix.For rabbitstudiesu,se4 uL of standardand ejectiton or inthebeefserum. 8.3.3.4At least3 replicatesshouldbe used forthelowest standardconcentrationm;ore replicates may be used atthediscretioonf theanalyst. 8.3.3.5Combust thesample as describedin section9.3and analyzeaccordingtoAMDT-M-2. 8.3.3.6Run all15 standards.Ifone replicatiessigrdficantdliyfferenftrom theothertwo replicatesn,m anothersample forthatstandard.Indicateindatathatthenew replicatreeplacesthe oldreplicataend thatthenew replicatweillbe used tocalculatteheregressiocnurve. 8.3.3.7When allstandardshave been ran,calculattehe r2. r2must be atleast0.95.Ifitisnot at least0.95,consultwith supervisor. 8.3.3.8A new standardcurveshouldbe run when thecombustion tubeor sample matrixis changed. New standardcurvemay alsobe run atthediscretioonf theanalyst. 8.4 StorageConditionsforStandards 8.4.1Storagereqw rements forstandardsaredependenton theindividuasltandardsused. Typically,standardsarestoredatroom temperatureinplasticscrew top bottles. 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/n-dnute. 9.1.3Vaporization/Dryintgime= 240 seconds. 9.1.4Bake time = 300 seconds. 9.2StartUp Procedure: 9.2.1Iftheprogram isnot starteds,tartheEOX program on thePC. 9.2.2Open theSYSTEM SETUP window. 9.2.3Put thefumace module and thecellintheREADY mode. 9.2.4Closethe SYSTEM SETUP window. 9.2.5When the oven has reachedtheREADY temperaturer,un theCLEAN BOAT program found intheCELL CHECK menu. 9.2.6 See AMDT-EP-3 fordetailosftheDohrinann software. 9.3Sample ExtractionProcedure: 9.3.1Open the SANIPLE HATCH and pipette100@iLof sample intotheBOAT. Itmay be necessarytomix approximately50 mg of charcoal%iththesampleto aidcombustion. Ifthisis done,charcoalshouldalsobe mixed inwhileestablishintghebaselineand when generatingthe standardcurve. 9.3.2 Close SA',IVIPLHEATCH. 00 4 9.3.3 Add appropriatevolume ofTISAB solutionor 1:1 TISAB:Milli-QTM water mixture toa labeledsample collectiovnial.Typically0.6mL to 15 mL areused. For rabbitstudies,use 1.0or 2.0mL of 1:1 TISAB:Milli-QTM water mixture. 9.3.4 Place thevia]so thatthetipof theCOMBUSTION TUBE isintheTISAB atleast0.25 inches.Gases releasedduring pyrolysismust bubble through theTISAB. 9.3.5 Run theEOX-WATER program found intheRUN menu. 9.3.6 When theEOX program isfinishedr,emove thecollectiovnialfrom thecombustion tube. 9.3.7 IfundilutedTISAB was used tocollecthesample,add an equalvolume of Milli-QTMwater to theTISAB tomake 1:1 TISAB:Milli-QTM. 9.3.8Rinse theend of thecombustion tube with Milli-QTmwaterand wipe with a KIMWIPE to remove any TISAB remaining on thetube. 9.3.9 Open the sample hatchand remove any remainingash from the boat. Ash can be removed with a cottontippedapplicatorand/orvacuumed out. Itmay be necessaryto scrapparticleosff the bottom with a spatulaor othersimilardevice.A drop of Milli-QTmwater may be added to the boatto aidintheClean Cycle. 9.3.10 Close thehatch. 9.3.11 Run the CLEAN BOAT program. 9.3.12 Sample isready foranalysisby ionselectivelectrode(ANOT-M-2). 9.4 Sample Calculations 9.4.1 Use thestandardcurve to calculattehe sample value. 9.4.2 Sample Mass Recovered F (ug)= (TISAB volinmL) x (Orionreadiniizn 1?12m- intercept) (Slope) 10.0 VALIDATT N 10.1 QualityControl 10.1.1Daily Start Up Check Samples: Once thestandardcurveisestablishede,ach day of analysisisstartedby analyzingQC samples. The QC samples aretobe the same as thelowest concentrationspikedsamples used togeneratethestandardcurve. Each concentrationmust be done in triplicatuenlessthe fusttwo replicateasrewithin20% of thestandardcurve,thena third replicateisnot necessary. 10.2 Precision and Accuracy: See method development analysisand sample analysisin FluorideNotebooks 2,3,and 5. Precisionand accuracyvarieswhen analyzingsamples of different matricesand differenrteferencecompounds. 10.3 Other ValidationParameters: NA 11.0 DATA ANALYSIS 11.1 Calculations 11.1.1 For thestandardcurve,use regressionanalysisinExcel,version5.0 or greater. 11.1.2To calculatethe fluohdecontractioninthe sample,seemethod AMDT-M-2. 0()006"3 5 11.2 AnalyzingtheData 11.2.1r2mustbeatleas0t.95orgreater".Outliermsa"y beexcludediftwo ofthethreereplicates are within20% of each otherand the outlierisgreaterthan200% of theaverage of those two or lessthan 50% of theaverage of thosetwo. Any such outliershould be pointedout in thedata and noted intheFinalReport along with the reasonitwas consideredan outlier. 12.0 ATTACHMENTS None 13.0 REFERFNCES 13.1 Rosemount Dohnnann DX2000 Organic Halide Analyzer Operator'sManual (Manual 915349,revisionB, December 1993) 13.2 AMDT-M-2 FluorideMeasurement by Means of an Orion EA940 Expandable Ion Analyzer 13.3AMDT-EP-3 Routine Maintenance of a Modified Dohrmann DX2000 Organic Halide Analyzer 14.0 REVISIONS Revision Numbe Reason for Change Revision Date 0()0064 6 9.3 QualityAssurance Unit Statement 0()0065 At=h=nt D GLP Study QualityAssurance Statement Study Title: Single-dose Intravenous Pbarmacokinetic Study of T-6049 in Rabbits StudyNumber AMDT- 112294.1 Name ofAuditor:KariRambo Thisstudyhas been inspectedby theQualityAssuranceUnitas indicateidnthefollowingtable. The findingswere reportedtothestudydirectoarnd management. InspectionDates E= TO 10-11-95 11-03-95 Phase FinalReport Date InspectioRneported to Manag=ot StudyDirector 11-03-95 11-03-95 QAU Auditor Date 9.4 Key Personnel Involved in the Study oooo(;7 3M Environmental Laboratory Key Personnel Thermal extractiofnollowedby analysiussingOrion ionanalyzer: Jim Johnson Deb Wright Rich Youngblom Deann Plummer Analysisofliverextractussingelectrospramyass spectrometry: Jim Johnson Dave Christenson Documentation and Reporting: Jim Johnson Rich Youngblom QualityAssuranceUnit: Gale Van Buskirk CynthiaWeber KariRambo 9.11.1Summary and raw data;ug F-inwhole liveras determinedby then-naelxtractiofnollowedby analysis usingOrion ion analyzer. 0(')0()()*!) Summary ofCombustion Data - Liver AMDT-112294.1, HWI 6329-129 As Referenced in FinalReport section6.0DATA ANALYSIS Total ug Fluoridein Whole Liver Mean per Dose Group ED Control group 5.0mg/kg dose (T6049) 10.0mg/kg dose (T6049) 100 mgtkg dose (T6049) 500 mg/kg dose (T6049) ug 23.37 23.28 16.01 35.87 92.03 RPTI 29L.XLS FC95PK ID BLNK LIVER 1 BLNK LIVER 2 BLNK LIVER 3 BLNK LIVER 4 BLNK LIVER 5 LIVER SPK 63-1 LIVER SPK 63-2 LIVER SPK 63-3 LIVER SPK 126-1 LIVER SPK 126-2 LIVER SPK 126-3 LIVER SPK 253-1 LIVER SPK 253-2 LIVER SPK 253-3 LIVER SPK 506-1 LIVER SPK 506-2 LIVER SPK 506-3 LIVER SPK 1012-1 LIVER SPK 1012-2 LIVER SPK 1012-3 BLANK LIVER-1 BLANK LIVER-2 BLANK LIVER-3 F52652-1 F52652-2 F52652-3 F52653-1 F52653-2 F52653-3 F52654-1 F52654-2 F52654-3 F52665-1 F52665-2 F52666-1 F52666-1 F52666-2 F52666-3 LIVER SPK-A LIVER SPK-B LIVER SPK-C LIVER SPK-D LIVER SPK-E LIVER SPK-F LIVER BLNK-1 % rcvry 77% 86% 83% 34% 83% 86% 82% 80% 86% 69% 68% 69% 63% 75% 64% 66% 67% 73% 74% 76% 74% Actual Average ppm F- ppm Fin liver in liver (W/W) (W/W) 1.052 0.423 0.758 0.473 0.308 0.895 1.024 1.045 2.192 1.616 2.353 3.580 3.966 4.195 6.493 6.338 7.100 12.884 14.244 13.635 0.432 0.303 0.219 0.332 0.242 0.273 0.245 0.319 0.217 0.254 0.226 0.174 0.206 0.204 0.231 0.480 0.384 0.434 0.437 0.973 1.043 0.988 0.949 0.791 0.895 liver burned (grams) 0.1216 0.1551 0.1135 0.1502 0.1473 0.1300 0.1274 0.1205 0.1159 0.1554 6.iiio 0.1399 0.1228 0.1242 0.1292 0.1297 0.1175 0.1186 0.1277 0.1139 0.1144 0.1223 0.1402 0.1329 0.1202 0.1013 0.1385 0.1118 0.1155 0.1464 0.1173 0.1168 0.1411 0.1502 0.1325 0.1415 0.1146 0.1161 0.1269 0.1126 0.1290 0.1397 0.1457 0.1351 0.1232 Whole liver weight (grams) 85.62 91.68 78.65 82.72 93.11 TotalF- in whole liver (ug) 23.37 23.28 16.01 35.87 92.03 Dosage (mglkg) 0.0 5.0 10.0 100.0 500.0 Page 1 9.11.2Summary and raw data;analysisof liver extractussingelectrospramyass spectrometry. .P 0()()()I-e"$- HWI# 6329-129 A- I 0-aftIea" p Study: ProtocolNumber: Test Material: Matrix: R Squared Value: Re"nso FactorAmount: Analyst: Date: Method: Instrument: LABBASE File: Single-Dose IntravenousPharmacokinetic TP8084.PK T-6049 inRabbits(FC-95) Liver 0.9717 1.07E-05 DLC 3/28195 FisonsVG 2000 ElectrosprayMS 032895C %I-to - 195 Group Dose Group 1: 0 mg/kg Group 2: 5 mg /kg Group 3: 10mg/kg Group 4: 100 mgtkg Group 5: 500 mg/kg Sample F52652 IonCount Extractedwt Area 9 N.D. 0.9979 Dilution factor Concentration Totalmass @49 of liver 9 1 N.D. 85.619 Totalamount of FC-95 per liver mg N.D. F52653 N.D. 1.061 1 N.D. 91.683 N.D. F52654 6056 1.1129 1 0.0466 78.656 0.004 F52665 39065 1.0209 1 0.3274 82.718 0.027 F52666 71977 1.1125 1 0.5536 93.111 0.052 The concentrationwas calculatedby using the standardcurve and multiplyingthe resultby 4/5.The 415 factorIsthe resultof a miscalculationInapplyingformula8.4InMethod AMDT-M-4-0. 137 mg of liverwas used In thiscalculatiornatherthanl7l mg. The concentrationsInthe standardcurvearetherefore5/4largerthan they should be.By multiplyingthecalculatedconcentrationInthe standardcurve by 4/5,the co.. tresultIsobtained. It PC i,@@ File:03209SC LAB-BASE - The MS Bata System Sample:pat liver frost abaci-ptiowlZitudit;s 11/10/415 occ_ 032895C I@@- 12 1103 402 6 7 1 2383 23 2657 3 ZFS- 28/03/1 3 4628 48 928525 134 676067 1943 1826 zFS- 4998 403\3 221289 104778 18102 \1341 Scn 5 its 67950 1 2775 2do@--'-'2-5'030080 58175 3TI * 35'80 14452097852258 4Z7:1"i17@5 t 4000 4500 5''0'0-0 009T 00.OT 008 2 W6 44 TaH IVO,E T t,G9TTT T SIT T T 05099 9T E TT OLL,OT T6 OrsTE9 60Tee8g i 009 L9 VTLEE 6co9c gjjoil, 066V -P-25, TTOCE E9- T T ET 51 ET e TT 9IP6 T8 L9 ss:BT SGGT/Co/OZ waisfisviva sm aql asua-gul OS68 File:032895C LAB-BASE - The MS Sample: liver frtm absorp+ilpiistudtor 032095C 1010- JL3 JL 1 17 a 16 1 Is 6 Data System 632q IZI 1943 2 4 28/03/ %1/10/q5 OLC_ 23 2184 ZFS54 Is@- -900?JLJL 920525 26 4 21 4 676067 11376JL,,L6117969'340637 531717 Is 6 ZFS- 207922 224289 1341 149607 1 1601 1 7 176@965 41 2 SLO !n'.'.3184000 JL580 JL6'00.-1.'7.0.8....1.8.0..0....1..9.0..0....2.0.9.....2JLGQ 22'00 2 3 File:032895C LAB-BASE The MS Data System Sample:"* 4pt0 ab jr-."-Io-l.ltoudies 6329 - i2li 03ZB95C JLQO- JL7jLo j2@393 253 7 24.06 26@57t@LL3306089t 31 2 2*?66 34 9 3350 36 V.FS- 28/03/ I1/to/95 !i @JIlN I'll 01- 5 39 7 ------------ - -r-@ -T- ie@ .a 1 ........ 4 ZFS 91613 0322L3 1 1.33552233 22 5 67g5 62008665,65 62779755a 2890 5155 22.668A 58175 3 131 0; 1 ......... .....-.----- .Scn2290 i4-iii i68O 2 8 Oii 'iiii6 3200 3;iiii File:032895C Sample: 03289sc-- I@@3892 37 LAB-BASE 4 2 4142 The NS Data Sgsten ZI 20/03 It/(0/115 -*43 3 42 2 sp IL (-J 46 8 4 2 to V.FS- V.FS- 5346-L 4 55 754't 421R9 8722 43 14290 4507 12669 4644 .Son 3898 @41(i"i'i4880 ;iiii"i42'60 43'iiii;i,400 4@Iiii1 4@,00 1 4 File:032895C Sample:@-4 1 Is@- 4830 47 5 4970 ZFS- L I@@- 52256 47 5 Y.FS - LAB-BASE - The MS +Itd!ies (\I LL ri li) ,65170 tA 5295 5350 Data Sgstem to32Q - 12--t 28/03 '(/10/.7,5 5 3 5596 5688 58 a (L r4 U) tp U. LL 71977 413698 5608 5701 6563 5548 5 658 Sen 5089 5'2'9@S' 5 56 09 58,0, 9.11.3Summary and raw data;ppm F-inserum as determinedby thermalextractiofnollowedby analysisusing Orion ionanalyzer. Thisdata,althoughsupportivei,ntheopinionof theStudy Directorisnot requiredtoreachtheconclusionstatedin ,FinalReport Section6.0,and thereforeisnot discussedin detail. Summary of Combustion Data - Liver AMDT-112294.1, HWI 6329-129 Concentrationof Fluoridein Serum Mean per Dose Group per Sampling Interval* 500 mgtkg dosed rabbit Average ppm Fin sernm 48 EIR 24 HR 12 HR 8 HR (W/W) 0.633 0.707 0.789 0.620 6 HR 0.595 4 HR 0.614 2 HR 0.705 100 mgtkg dosed rabbit 48 HR 24 HR 12 HR 8 HR 6 HR 4 HR 2 HR 0.341 0.289 0.395 0.293 0.246 0.265 0.255 *2-24hour calculatedas the mean ofduplicatesamples,48 hour analyzedintriplicate. RPT129.XLS FC95 PK ID Serum blk 1 Serum bik 2 Serum blk 3 Serum blk4 Serum SPK 1 Serum SPK 2 Serum SPK 3 Serum SPK 4 Serum SPK 5 Serum SPK 6 SERUM BLANK-1 SERUM BLANK-2 F52666-F5-48 HR - 1 F52666-F5-48 HR - 2 F52666-F5-48 HR - 3 F52666-F5-24 HR - 1 F52666-F5-24 HR - 2 F52666-F5-12 HR - 1 F52666-F5-12 HR - 2 F52666-F5-8 HR - 1 F52666-F5-8HR - 2 F52666-F5-6HR - 1 F52666-F5-6HR - 2 F52666-F5-4HR - 1 F52666-F5-4HR - 2 F52666-F5-2HR - 1 F52666-F5-2HR - 2 SERUM BLANK 1 SERUM BLANK 2 F52665-F4-48 HR-1 F52665-F4-48 HR-2 F52665-F4-48 HR-3 F52665-F4-24HR-1 F52665-F4-24HR-2 F52665-F4-12HR-1 F52665-F4-12HR-2 F52665-F4-8HR-1 F52665-F4-8HR-2 F52665-F4-6HR-1 F52665-F4-6HR-2 F52665-F4-4HR-1 F52665-F4-4HR-2 F52665-F4-2HR-1 F52665-F4-2HR-2 % rcvry 69% 184% 79% 78% 103% 98% Actual ppm Fin serum (W/W) 0.931 0.590 0.391 0.321 2.10 5.58 2.40 2.36 3.13 2.99 0.511 0.375 0.689 0.582 0.628 0.735 0.680 0.835 0.743 0.636 0.605 0.617 0.573 0.580 0.649 0.755 0.655 0.575 0.314 0.333 0.350 0.339 0.303 0.276 0.464 0.326 0.288 0.299 0.242 0.249 0.272 0.257 0.254 0.256 Average ppm Finserum (W/W) 0.633 0.707 0.789 0.620 0.595 0.614 0.705 0.341 0.289 0.395 0.293 0.246 0.265 0.255 serum burned (mL) 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 Dosage (mg/kg) 500 500 500 500 500 500 500 100 100 100 100 100 100 100 Page 1