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3M EnvironmentaLl boratory FinalReport-AnalyticaSltudy Single-DosDeermal Absorption/ToxiciStyudyofT-6051and T-6054 in Rabbits In-VivoStudyReferenceNumber: HWI#6329-133 StudyNumber: AMDT-013195.1 TestSubstance:FC-129 (T-6051andT-6054) Name and AddressofSponsor: 3M SCD Division 367 Grove Street St.Paul,MN 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-M-4-0, AMDT-M-5-0, AMDT-M-8-0, and Revisions: Thermal Extractionof Fluorideby Means of a Modified Dohrmann DX2000 Organic Halide Analyzer-Liver FluorideMeasurement by Means of an Orion EA940 Expandable Ion Analyzer Extractionof Fluorochemicalsfrom RabbitLiver Analysisof RabbitLiverExtractforFluorochemicalsUsing ElectrospmyMass Spectrometry Analysisof FluorideUsing the SkalarSegmented Flow Analyzer with Ion SelectiveElectrode InitiatioDnate: See attachedprotocol Author:James D. Johnson Approved Bvl ,@/amSetsudy D. Jgti@don Dirdpeor Completion Date 1.0 SUMMARY SamplesoflivefrromrabbiatdsministerFeCd-129(T-6054o)rFC-129treated fabric(T-6051)were analyzedat28 days postdermal administratiofnortotal organicfluorinaend perfluorooctanesulfonatTeh.e resultshow thatforthehighest liquidformulationdose group (12.8mg/kg) thereison theaverageabout0.2% of thedose inwhole liverat28 days. Thus, dermal administratioonfFC-129 athigherlevelsresultsinsome dermal absorption. 2.0 INTRODUCTION Two studieswere performed on FC-129. A pharmacoldneticstudy(HWI#6329138)and thisdermalabsorptionstudy(HWI #6329-133).The pharmacokinetic studyshowed thatperfluorooctanesulfonaitsea usefulmarker to assessthe dermal absorptionof FC- 129. Liver,serum,and othertissueswere availableforanalysisby combustionfortotalorganicfluorineand electrospramyass spectrometryfor analysisof specifimcoleculessuch asperfluorooctanesulfonatBey. obtainingand thenanalyzingdatafrom rabbitsat28 days postdermal dose,informationforthe assessmentoftheextentofdermal absorptionofFC- 129 isprovidedinthisstudy. 3.0 TEST MATERL4,LS 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 3.1.3AnalyticalControl Substance: None 3.1.4AnalyticalControl Matrix: Bovine liverand bovine serum 3.2 Source of Materials:3M ICP/PCP DivisionforFC-95, bovine liverfrom grocerystore,bovine serum from Sigma Chemical Company. 3.3.Purity and Strength of Reference Substance: Responsibilitoyf Sponsor. 3.4 Stabilityof Reference Substance: To be determinedby Sponsor. 2 3.5StorageConditionfsorTestMaterialRso:om temperatufroerFC-95.For biologicalsamples the storageis-20-+0l0 C. 3.6Dispositionof Specimens: Biologicatlissueasnd fluidswillbe retainedper GLP Regulationforthetime periodrequiredforstudieslongerthan28 days. 4.0 EXPERIMENTAL -Overview The tissuesfrom animalsdosed asdescribed(HWI#6329-133), were availablfeor analysisforfluorinceompounds. At thediscretioonf theStudy Directora,*seriesof analyticatlestscouldbe performed.The screeningforfluoridienlivervia combustion was the most likelyanalysistopresentdefmitivedataforabsorption. Other availabletestswere electrospramyass spectroscopyand gas chromatography/massspectrometryformetabolitesL.iversampleswere analyzedby both combustion and electrosprayD.ata were thenanalyzedto assesstheextentof dermal absorption. 5.0 EXPERIMENTAL -METHODS 5.1 AMDT-M-1-0, Thermal ExtractioonfFluorideby Means ofa Modified Dohrmann DX2000 OrganicHalideAnalyzer-Liver 5.2ANMT-M-2-0, FluorideMeasurement by Means of an Orion EA940 Expandable Ion Analyzer 5.3AMDT-M4-0, Extractioonf Fluorochemicalfsrom RabbitLiver 5.4AMDT-M-5-0, Analysisof RabbitLiverExtractforFluorochemicalUssing ElectrosprayMass Spectrometry 5.5AMDT-M-8-0, AnalysisofFluorideUsing theSkalarSegmented Flow Analyzer with Ion SelectiveElectrode 6.0 DATA ANALYSIS The dataareattached.The leveloftotalorganicfluorinienwhole liverforthe control,0.128,1.28,and fabricdosesarebelow the practicaqluantitatiolnimitfor thismethod. Justusingmeterreadingsand extrapolatinfgrom the standardcurve, thevaluesareon theorderof 16 ug/whole liver.The 12.8mg/kg dermal dose however, resultsindetectablaemounts oforganicfluorideinwhole liverat28 days 3 postdose. The valuesrange from below thepracticaqluantitatiolnimitforone of the 6 rabbits(F52895) to 75 ug/whole liverforrabbitF52889. The averageis 45 ug/whole liverwith a standarddeviationof 21 ug. Electrospraymass spectrometrydataare in agreement with thecombustion data; thereisvery littlpeerfluorooctanesulfonatienthegroups otherthan the 12.8mg/kg group. Small detectableamounts areobservedin the 1.28mglkg and fabricgroups; however, theseareestimatedto be on the orderof 17 ug/whole liveror less.For the 12.8mg/kg dose group,perfluorooctanesulfonaitsedetectedin allrabbitliver samples at28 days postdermal dose. The amounts are estimatedto range from 25 to 85 ug/whole liver(mean of 48 ug/whole liver).The rabbitthatshowed 75 ug/whole liverof totalorganicfluorineforcombustionanalysis(F52889),had 56 ug/whole liverperfluorooctanesulfonate. From thepharmacokineticstudyon FC-129 (HWI#6329-138), itisknown thata good portionof the intravenousdose willbe biotransformedto perfluorooctanesulfonatIet.isknown thatthehalf-lifoef perfluorooctanesulfonate in rabbitsis>1 month. Thus, ifFC-129 isdermallyabsorbeda portionof itwill appear in liverat28 days as perfluorooctanesulfonate. Fiftyug perfluorooctanesulfonate/whollieveris0.2% of thedose forthe 12.8mg/kg rabbitsassuming a body weight of 2 kg and expressingthe dose inpotassium perfluorooctanesulfonaetqeuivalents(FC-95). Afteran intravenousdose of 12.8 mg/kg a rabbithad 1.05% of the dose in liverat48 hours. For comparison,1.05% with a biologicalhalf-lifoef 30 days would be approximately0.5% of thedose at day 28. Thus, estimatedlevelsafteran intravenousdose of 12.8mg/kg would be 0.5% of thedose in whole liverand estimatedlevelsafterdermal administratioonf 12.8mg/kg would be 0.2% of the dose in whole liverifthelevelsare compared at 28 days. Other datawas collectedusing Skalarsegmented flow analyzerwith ionselective electrode(seeappendices).This data,althoughsupportivei,n theopinionof the Study Directorisnot requiredto reachtheconclusionstatedhereand thereforeisnot discussedin detail. 6.1 Circumstances that May Have Affected the Quality of the Data: The problem with thisanalysisisthatthe extentof biotransformatioonf the restof the fluorinatecdompounds inthe liverat48 hours inthe pharmacokineticstudy (HWI#6329-138) to perfluorooctanesulfonaitsenot known. There couldbe considerablebiotransformatioonf theseveralpercentof dose thatfluorinated molecules otherthan perfluorooctanesulfonartepresent.However, the 1.05% of the dose observed at48 hours stililndicatesthatperfluorooctanesulfonaitsea sensitive marker to assessbiotransformatioonf thiscompound. At 28 days,thevaluecould 4 be somewhat higherthan theabove estimateof 0.5% of thedose due to delayed metabolism.Ifthiswere true,thevaluefordermal absorptionhas by definition (sinceitismeasured at28 days)a builtincompensationforthisdelayand thevalue of 0.2% ofthedose afterdermal administratioinsbeingcompared with a percentage of dose from the intravenousdose thatistoo low. 7.0 CONCLUSION There isevidenceof dermal absorptioninrabbitsof FC-129 afterdermal administratioonfa 12.8mglkg dose. 8.0 MAE'ITENANCE OF RAW DATA AND RECORDS 8.1 Raw Data and Data: Raw data,approved protocol,approved finalrepom appropriatsepecimens,and electronidcatawillbe maintainedintheAMDT archives. 9.0 APPENDICES 9.1 Protocol and Amendments 9.1.1Protocoland FinalReport:HWI#6329-133: "Single-DoseDermal Absorption/ToxicitSytudy ofT-6054 and T-6051 inRabbits"(Protocoltype TP3016.AB fordosingofanimals,tissuecollectione,tc.) 9.1.2 AnalyticaplrotocolAMDT-013195.1 9.2 Signed Reports from IndividualScientistsN:one 9.3 QualityAssurance Unit Statement:See attached 9.4 Key PersonnelInvolvedinthe Study: See attached 9.5 Materialsand Equipment: See methods 9.6 Solutions,Reagents,and Standards:See methods 9.7 Sample Preparation:See methods 9.8 QualityControl PracticesS:ee methods 5 9.9TestMethods:SeeProtocoAlMDT-0 13195.1 9.10 Instrument Settings:See methods 9.11 Data: See attached. 9.11.1Summary and raw data;ug F-inwhole liverasdeterminedby thermal extractiofnollowedby analysisusingOrion ionanalyzer. 9.11.2Sununary and raw data;analysisof liverextractussingelectrospray mass spectrometry. 9.11.3Sununary and raw data;ug F inwhole liverasdeterminedby thermal extractiofnollowedby analysisusingSkalarsegmentedflow analyzerwith ion selectivelectrode. 6-- 9.1.1Protocoland FinalReport:HWI#6329-133: "Single-DoseDermal Absorption/ToxicitSytudy of T-6054 and T-6051 in Rabbits"(Protocoltype TP3016.AB fordosingof animals,tissuecollection,, etc.) HAZLrzoCCN W IS C 0 N S IN POST OFFICE BOX 7545 MADISON,WL 53707-7545 Sponsor,: 3M St. Paul, Minnesota a CORNING Coni;),iny 11 2 FINAL REPORT Study Title: Single-Dose Dermal Absorption/Toxicity Study of T-6054 and T-6051 in Rabbits Author: Steven M. Glaza Study Completion Date: June 16, 1995 Performing Laboratory: Hazleton Wisconsin, Inc. 3301 Kinsman Boulevard Madison, Wisconsin 53704 Laboratory-ProjectIdentification: HWI 6329-133 P h o n e 6 0 8 - 2@4 I---4-4.-7-1 E X P R E S S - M A I L DELIVERY Page I of 46 3301 Ki@JS@ilA@l BLVD Fax MADISOrl. 6-.08- - 2 4 1 - 7 2 217 wi 537n4 Page 2 of 46 HWI 6329-133 QUALITY ASSURANCE STATEMENT This report has been reviewed by the Quality Assurance Unit of Hazleton Wisconsin, Inc., in accordance with the Food and Drug Administration (FDA) Good LaboratoryPractice Regulations, 21 CFR 58.35 (b) (6) (7). The following inspectionswere conducted and findings reported to the Study Director and management. Written status reports of inspections and findings are issued to Hazleton management monthly according to standard operating procedures. Inspection Dates - From To Phase 12/08/94 12/28/94 01/09/95 01/30/95 03/17/95 03/17/95 06/14/95 06/16/95 12/09/94 12/28/94 01/09/95 01/30/95 03/21/95 03/21/95 06/15/95 06/16/95 Protocol Review Dose Administration Protocol Amendment Protocol Amendment Data/Report Review Data Review Report Rereview Report Rereview Date Reported to Study Director Date to Management 12/09/94 12/28/94 01/09/95 01/30/95 03/21/95 03/21/95 06/15/95 06/16/95 01/10/95 01/10/95 02/10/95 02/10/95 04/10/95 04/10/95 07/10/95 07/10/95 ( 4xez--,,- @'e-Mc.i Dl ai na or Representative, Quality Assurance Unit 5ate Page 3 of 46 STUDY IDENTIFICATION Single-Dose Dermal Absorption/Toxicity Study of T-6054 and T-6051 in Rabbits HWI 6329-133 Test Materials 1. T-6054 2. T-6051 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 3M Toxicology Service Medical Department 3M Center, Bldg. 220-2E-02 P.O. Box 33220 St. Paul, MN 55133-3220 (612) 733-1962 Study Director Steven M. Glaza Hazleton Wisconsin, Inc. P.O. Box 7545 Madison, WI 53707-7545 (608) 241-7292 Study Location Hazleton Wisconsin, Inc. Building No. 3 3802 Packers Avenue Madison, WI 53704 Study Timetable Study InitiationDate Experimental (In-life)Start Date In-life End Date Experimental Termination Date Study Completion Date December 13, 1994 December 28, 1994 January 25, 1995 June 16, 1995 June 16, 1995 Page 4 of 46 HWI 6329-133 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-life Supervisor Thomas E. Palmer, PhD Anatomical Pathologist Rose M. Bridge Report Supervisor Ouality Assurance Sherry R. W. Petsel Manager Jack Serfort/ Deborah L. Pirkel Supervisors Necropsy Anne Mosher Supervisor Pathology Data Page 5 of 46 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 Deviation Protocol TP3016.AB Protocol Amendment No. 1 Protocol Amendment No. 2 HWI 6329-133 Page 2 3 4 6 8 8 8 9 10 13 13 14 14 15 16 18 20 25 27 29 30 31 42 45 Page 6 of 46 SUMMARY HWI 6329-133 This study was done to assess the systemic absorption/toxicityand relative skin irritancy of T-6054 and T-6051 when applied to the skin of rabbits. The study was conducted using three male and three female acclimated rabbits of the Hra:(NZW)SPF strain for each treatment group. - Groui) Test Material 1 (Control) Sterilewater 2 T-6054 3 T-6054 4 T-6054 5 T-6051 Dose Level (mg/kq)- oa 0.128 1.28 12.8 b Number of Animals Males Females 3 3, 3 3 3 3 3 3 3 3 a Administered at a dose volume of 2.0 mL/kg. b Administered as a 10-cm x 10-cm section of test material (fabric). The back of each rabbit was clipped free of hair and a single dose of the respective material at the indicateddose level was administeredto the skin of the rabbits. The treatmentsites remained intact. The area of application was covered with a gauze bandage secured with paper tape around all edges and overwrapped with Saran WrapO and ElastoplastO tape to provide an occlusive dressing for a 24-hour exposure period. Clinical observations were conducted predose and at approximately 1, 2.5, and 4 hours after test or control material administration. Additionalclinical observations and twice a day mortality checks were conducted daily thereafter for 28 days. Body weights were determined on Day -9 for randomization purposes, before test or control material administration(Day 1), and at in-life termination (Day 29). The initial dermal irritationreadingwas made before test or control material administration(recorded as the Day 1 reading). Subsequent readings of dermal irritation were made approximately 30 minutes after bandage removal (Day 2) and on Days 4 and 8. Blood samples were collected from a marginal ear vein of the animals before in-life initiation (Day 1), approximately24-hours postdose (Day 2), on Days 4, 8, 15, and 22. In addition, at the time of necropsy on Day 29, approximately 20 mL of blood was obtained from each animal. All samples were centrifuged and separated into serum and cellular fractions. All animals were euthanized at termination of the in-life phase and necropsied. The whole liver, bile, an approximate 1-cm x 1-cm section of the dermal application site from all animals, and both kidneys from one male and one female in each group were collected at necropsy and weighed (volume only determined for bile). The blood samples (serum and cellular fractions),livers, bile, dermal application sites, and kidneys were sent frozen to the Sponsor after termination of the in-life phase. Page 7 of 46 HWI 6329-133 Application of T-6054 and T-6051 did not result in any test material-related changes in body weight gain or macroscopic findings at necropsy. All animals appeared clinically normal throughout the study with the exception of one female animal treated with T-6054 at 1.28 mg/kg that exhibited weakened hind limbs the last 22 days of study. This animal was also noted as having small feces on Day 8. These findings are considered to be due to an injury incurred during the sample collection procedures and are not considered to be test material-related. The control material and test material T-6051 did not produce any dermal irritation. No dermal irritationwas observed as a result of T-6054 at a dose level of 0.128 or 1.28 mg/kg. T-6054 produced very slight dermal irritation in five animals at the 12.8 mg/kg dose level. Page 8 of 46 OBJECTIVE HWI 6329-133 The objectiveof this study was to assess the systemictoxicity/absorptionand relativeskin irritancyof test materialswhen appliedto the skin of 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 analysis of the test material mixtures prepared for the Groups 2, 3, and 4 animals for concentration,homogeneity/solubilitya,nd stabilitywas not conductedand the originaltest materialusage log can not be located althougha copy is retainedin the study file. All proceduresused in this study are in compliancewith the Animal Welfare Act Regulations. In the opinion of the Sponsor and study director,the study did not unnecessarilyduplicate any previous work. Identification TEST AND CONTROL MATERIALS The test materialswere identifiedand describedas follows: Identification Physical Descrigtion T-6054 T-6051 Amber liquid White plastic sheets 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 assumes responsibilityfor test materialpurity and stability determinations(includingundertest conditions).Analysisof the test material mixtures prepared for the Groups 2, 3, and 4 animals for concentration,homogeneity/solubilitya,nd stabilitywas not conductedor requestedby the Sponsor. The purity and stabilityof the controlmaterial were considered to be adequatefor the purposesof this study. Storage and Retention The test materialswere stored at room temperature. The controlmaterial was stored refrigerated. A reserve sample of each test and controlmaterialwas Page 9 of 46 HWI 6329-133 taken and will be retained in a freezer set to maintain a temperature of +100 for 10 years in accordance with Hazleton Wisconsin (HWI) Standard Operating Procedure (SOP). Any unused test material was returned to the Sponsor after completion of all in-life phase according to HWI SOP. Any remaining control material is retained for other testing and will not be discarded after issuance of the final report. -200C Safety Precautions The test and control material handling procedures were according to HWI Sops and policies. TEST SYSTEM Test Animal Adult albino rabbits of the Hra:(NZW)SPF strain were procured from HRP, Inc., Kalamazoo, MI, on December 14, 1994 and maintained at the Hazleton Wisconsin facility at 3802 Packers Avenue, Madison, Wisconsin. Housing After receipt, the animals were acclimatedfor a period of at least 7 days. During acclimation and throughout the study, the animals were individually housed in screen-bottom stainless steel cages in temperature- and humiditycontrolled quarters. Environmental controls for the animal room were set to maintain a temperature of 19* to 230C, a relative humidity of 50% 20%, and a 12-hour light/12-hourdark lighting cycle. In cases where variations from these conditions existed, they Were documented and considered to have had no adverse effect on the study outcome. Animal Diet The animals were provided access to water ad libitum and a measured amount of Laboratory Rabbit Diet HF #5326, PMI Feeds, Inc. The feed is routinely analyzed by the manufacturer for nutritional components and environmental contaminants. Samples of the water are periodically analyzed by HWI. There were no known contaminants in the feed or water at levels that would have interferedwith or affected the results of the study. Selection of Test Animals The animals were identified by animal number and corresponding ear tag and were placed into study groups using a stratified body weight randomization program. The randomization body weights were determined on Day -9. The Page 10 of 46 HWI 6329-133 weight variationof the animalsfor each group of each sex selectedfor the study did not exceed 2 standard deviations of the mean weight, and the mean body weights for each group of each sex were not statisticallydifferentat the 5% probabilitylevel. One female animal (No. F52890)was replacedin the study prior to treatment due to poor health. This animal was replacedwith another female (No. F52877). Study Design Animals weighing from 2,157 to 2,508 g at initiationof treatmentwere placed into the followingstudy groups: Group Dose Level Test Material (mg/kq) 1 (Control) Sterilewater 2 T-6054 3 T-6054 4 T-6054 5 T-6051 oa 0.128 1.28 12.8 b Number of Animals Males Females 3 3 3 3 3 3 3 3 3 3 a Administered at a dose volume of 2.0 mL/kg. b Administered as a 10-cm x 10-cm section of test material(fabric). Justificationfor SRecies Selection Historically,the New ZealandWhite albino rabbit has been the animal of choice because of the large amount of backgroundinformationon this species. PROCEDURES PreRaration of Exposure Area On the day before test materialapplication,the back and, if necessary(to obtain unblemishedskin),the flanksof each rabbit was clippedfree of hair. The clipped area made up approximately20% of the total body surface area. The test sites (intactskin)were inspectedfor interferinglesions, irritation,or defects that would precludethe use of any of the animals. The animals were clipped on Days 8 and 29 to aid in visualizingthe application sites. Dose Administration All animals receiveda single administrationof the respectivetest or control material. The day of treatment was designated as Day 1. Page 11 of 46 HWI 6329-133 Groug 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 (sterilewater for injection)was appliedevenly to the test site at a rate of approximately0.05 mL/CM2. Groups 2. 3. and 4. For the Groups 2, 3, and 4 animals (0.128, 1.28, 12.8 mg/kg, respectively),the test material (T-6054)was mixed with. sterile water for injection to a concentrationof 99, 990, and 9,920 mg/mL, respectively, and applied at a dose volume of 0.01 mL/kg. The mixtures were stored at room temperature until administered. An individualdose of the respective test material mixture was calculated for each animal based on its body weight on the day of treatment. For all three groups, the area of exposure was 4 CM2 and the approximate rate of application was 0.006 mL/cm 2 . Group 5. The test material (T-6051) was applied to each animal's skin as a 10-cm x 10-cm section of material that was moistened with distilled water. Each area of application was covered with a 10-cm x 10-cm gauze bandage secured with paper tape around all edges and overwrapped with Saran Wrapo and Elastoplast'Otape to provide an occlusive dressing. Collars were used to restrain the animals during the 24-hour exposure period. Approximately 24 hours after test or control material application,the restraining collars and bandages were removed and any residual test material was removed with tap water and disposable paper towels. Reason for Route of Administration The dermal route is a potential route of exposure in humans. Observations of Animals Clinical observations were conducted predose and at approximately 1, 2.5, and 4 hours after test or control material administration. Additionalclinical observations and twice a day mortality checks (morning and afternoon) were conducted daily thereafter for 28 days. Body weights were determined for randomization purposes on Day -9, before test material administration(Day 1), and at in-lifetermination(Day 29). The initial dermal irritationreading was made before test or control material administration according to the Draizel technique (recorded as the Day 1 reading). Subsequent readings of dermal irritation were made approximately 30 minutes after bandage removal (Day 2) and on Days 4 and 8. The only exception to this was the Day 8 erythema score for one female animal (No. F52889) in Group 4 was inadvertently not recorded. Page 12 of 46 HWI 6329-133 Sample Collections Blood samples (approximately4 mL) were collected from a marginal ear vein of all animals before experimentalinitiation(Day 1). Subsequent collection of blood was conducted approximately 24-hours postdose (Day 2), and on Days 4, 8, 15, and 22. In addition, at the time of necropsy on Day 29, approximately 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 100C until shipped to the Sponsor. Pathology At termination of the experimentalphase (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 abnormalities were recorded. The whole liver, bile, an approximate 1-cm x 1-cm 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 were weighed (volume only determined for bile) and immediately placed on dry ice, 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 experimental termination,the blood samples (serum and cellular fractions),livers, bile, dermal applicationsites, and kidneyswere 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 46 HWI 6329-133 RESULTS Body Weights Individual and mean body weights are in Table 1. All animals exhibited body weight gains from Day I to Day 29. Clinical Observations Individual clinical signs are in Table 2. All animals appeared normal throughout the study with the exception of one female animal (No. F52900) treated with T-6054 at 1.28 mg/kg that exhibitedweakened hind limbs during the last 22 days of study. This animal also had small feces on Day 8. These findings are considered to be due to an injury incurred during the sample collection procedures and are not considered to be test material-related. Dermal Irritation Individualdermal irritationscores are in Table 3. The control material and test material T-6051 produced no dermal irritation. No dermal irritationwas observed in the animals treated with T-6054 at a dose level of 0.128 or 1.28 mg/kg. T-6054 produced slight to moderate erythema reactions at Days 2 and 4 only in five animals at the 12.8 mg/kg dose level. Pathology Individual animal pathology comments are presented in Table 4. There were no lesions observed in any of the animals. Page 15 contains a pathology report by the study pathologist. DISCUSSION The acute systemic absorption/toxicityand relative skin irritancy of T-6054 and T-6051 were evaluated in male and female albino rabbits when administered as a single dermal application. Applicationof the these materials did not result in any test material-relatedeffects on in-life clinical findings, body weight gain or macroscopic findings at necropsy. The control material and test material T-6051 did not produce any dermal irritation. No dermal irritation was observed with T-6054 applied at a dose level of 0.128 or 1.28 mg/kg. T-6054 produced very slight dermal irritation in five animals at the 12.8 mg/kg dose level. Page 14 of 46 SIGNATURE HWI 6329-133 Steven M. Glaza Date Study Director Acute Toxicology REFERENCE 1. Draize, J. H., "Acute Demal 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 46 HWI 6329-133 PATHOLOGY REPORT There were six rabbits (three males and three females)each from five dose levels euthanizedand necropsiedat the terminationof the study. The test material,dose level, day of death, and gross observationsrecordedfor each animal are in the IndividualPathologyCommentsthat follow this report. . At necropsy,there were no visible lesionsin any of the animals. The liver, bile, an approximate1-cm x 1-cm sectionof the dermal applicationsite from all animals, and both kidneys from the first male and female in each group were collected. The tissue samples were weighed (volumeonly determinedfor bile), frozen,and sent to the Sponsor. After necropsy,the animalswere discarded. L 4A z 4@@ mas E. Palmer PhDt/- Date thologist P (6329-133.slh) 031095 Animal Number F52885 F52873 F52898 Mean Page 16 of 46 HWI 6329-133 Table 1 Individualand Mean Body Weights (g) Male Randomization Day -9 Day 1 29 Animal Number Female Random- ization Day Day -9 1 ..29 Group I (Control)- SterileWater for Injection(0 mg/kq) 2,292 2,204 2,169 2,483 2,321 2,366 2,853 2,745 2,745 F52967 F52878 F52883 2,204 2,331 2,274 2,368 2,475 2,499 2,863 2,978 2,985 2,222 2,390 2,781 2,270 2,447 2,942 F52887 F52893 F52897 Mean 2,272 2,204 2,338 2,271 Group 2 - T-6054 (0.128 mg/kg) 2,235 2,260 2,423 2,621 2,603 2,913 F52901 F52876 F52882 2,281 2,270 2,127 2,306 2,712 2,226 2,340 2,358 2,323 2,340 2,839 2,842 2,814 2,832 F52965 F52891 F52892 Mean 2,248 2,077 2,261 2,195 Group 3 - T-6054 (1.28 mg/kq) 2,369 2,383 2,333 2,966 2,757 2,614 F52884 F52900 F52968 2,351 2,210 2,166 2,362 2,779 2,242 2,508 2,460 2,289 2,419 2,928 2,588 2,822 2,779 F52886 F52880 F52899 Mean 2,286 2,161 2,353 2,267 Group 4 - T-6054 (12.8 mg/kq) 2,417 2,219 2,393 2,793 2,525 2,741 F52889 F52894 F52895 2,292 2,160 2,219 2,343 2,686 2,224 2,357 2,489 2,462 2,436 2,853 2,837 2,791 2,827 Animal Number F52879 F52963 F52874 Mean Page 17 of 46 HWI 6329-133 Table I (Continued) Individualand Mean Body Weights (g) Male Randomization Day -9 Day 1 29- Animal Number Female Random- ization Day Day -9 1 29 Group 5 - T-6051 (10-cm x 10-cm Section) 2,278 2,173 2,048 2,398 2,258 2,265 2,923 2,622 2,495 F52877a F52888 F52966 2,021 2,191 2,264 2,157 2,273 2,322 2,782 2,752 2,792 2,166 2,307 2,680 2,159 2,251 2,775 a Animal No. F52890 was originallyselected by the randomizationprogram for use in the study but was replacedwith No. F52877 due to poor health. Page 18 of 46 Table 2 IndividualClinical Signs HWI 6329-133 Animal Sex Number Observation 1-4 Hours Day (DayI)- 2 - 7 8 9 29 Group 1 (Control)- SterileWater for Injection(0 mg/kq) Male F52885 Appeared normal t F52873 Appeared normal F52898 Appeared normal Female F52967 Appeared normal 1( F52878 Appeared normal 1( F52883 Appeared normal it Male Female F52887 F52893 F52897 F52901 F52876 F52882 Grou2 2 - T-6054 (0.128 mg/kq) Appeared normal Appeared normal Appeared normal Appeared normal Appeared normal Appeared normal Male Female F52965 F52891 F52892 F52884 F52900 F52968 Grou2 3 - T-6054 (1.28mg/kq) Appeared normal 1( Appeared normal Appeared normal Appeared normal Appeared normal Weakened hind limbs Small feces Appeared normal Condition existed. Conditionnot evident. Sex Male Female Animal Number F52886 F52880 F52899 F52889 F52894 F52895 Page 19 of 46 Table 2 (Continued) IndividualClinicalSigns Observation 1-4 Hours (Day 1) 2 Group 4 - T-6054 (12.8 mg/kq) Appeared normal Appeared normal Appeared normal Appeared normal Appeared normal Appeared normal HWI 6329-133 Day 7 8 9 29 Male Female Group 5 - T-6051 (10-cm x 10-cm Section) F52879 F52963 F52874 Appeared normal Appeared normal Appeared normal F52877 F52888 F52966 Appeared normal Appeared normal Appeared normal 1( 1( Condition existed. Page 20 of 46 HWI 6329-133 Table 3 Individual Dermal Irritation Scores Group I (Control)- SterileWater for Injection(0 mg/kg) Dermal Reaction Males Study Day 1 2 4 8 Females Study Day 1 2 4 8 Animal No. F52885 --Animal No. F52967 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. F52873 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. F52878 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. F52898 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. F52883 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 46 HWI 6329-133 Table 3 (Continued) Individual Dermal Irritation Scores Group 2 - T-6054 (0.128 mg/kg) Dermal Reaction Males Study Day _L 4 8 Animal No. F52887 Females Study Day 1 2 4 Animal No. F52901 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. F52893 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. F52876 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. F52897 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. F52882 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 46 HWI 6329-133 Table 3 (Continued) IndividualDermal IrritationScores Group 3 - T-6054 (1.28 mg/kg) Dermal Reaction Males Study Day I _L_ 4 8 Females Study Day 1 2 4 8 Animal No. F52965 Animal No. F52884 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. F52891 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. F52900 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. F52892 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. F52968 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 23 of 46 HWI 6329-133 Table 3 (Continued) Individual Dermal IrritationScores Group 4 - T-6054 (12.8 mg/kg) Dermal Reaction Males Study Day 1 2 4 8 Females Study Day 1 2 4 8 Erythema Edema Atonia Desquamation Coriaceousness Fissuring Animal No. F52886 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. F52889 0 1 1 - 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. F52880 0 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Animal No. F52894 0 1 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. F52899 0 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Animal No. F52895 0 2 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Value not recorded. Page 24 of 46 HWI 6329-133 Table 3 (Continued) IndividualDermal IrritationScores Group 5 - T-6051 (10-cm x 10-cm Section) Dermal Reaction Males Study Day _1_ _L -.L- _L Females Study Day 1 -L -L 8 Animal No. F52879 Animal No. F52877 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. F52963 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. F52888 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. F52874 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. F52966 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Animal Number F52885 F52873 F52898 F52967 F52878 F52883 F52887 F52893 F52897 F52901 F52876 F52882 F52965 F52891 F52892 F52884 F52900 F52968 Page 25 of 46 HWI 6329-133 Table 4 Individual Pathology Comments Test Day sex Died Sacrificed Necropsy Observation Group I (Control)- SterileWater for Injection (o mg/kg) m - 29 No visible lesions. m - 29 No visible lesions. m - 29 No visible lesions. F - 29 No visible lesions. F - 29 No visible lesions. F - 29 No visible lesions. Group 2 - T-6054 (0.128 mg/kg) m - 29 No visible lesions. m - 29 No visible lesions. m - 29 No visible lesions. F - 29 No visible lesions. F - 29 No visible lesions. F - 29 No visible lesions. Group 3 - T-6054 (1.28 mg/kq) m - m - m - F - F - F - 29 No visible lesions. 29 No visible lesions. 29 No visible lesions. 29 No visible lesions. 29 No visible lesions. 29 No visible lesions. Not applicable. Animal Number sex F52886 m F52880 m F52899 m F52889 F F52894 F F52895 F F52879 m F52963 m F52874 m F52877 F F52888 F F52966 F Page 26 of 46 HWI 6329-133 Table 4 (Continued) Individual Pathology Comments Test Da Died Sacrificed Necropsy Observation Group 4 - T-6054 (12.8 mg/kq) - 29 No visible lesions. - 29 No visible lesions. - 29 No visible lesions. - 29 No visible lesions. - 29 No visible lesions. - 29 No visible lesions. Group 5 - T-6051 (10-cm x 10-cm Section) 29 No visible lesions. 29 No visible lesions. 29 No visible lesions. 29 No visible lesions. 29 No visible lesions. 29 No visible lesions. Not applicable. Page 27 of 46 Table 5 Individual Animal Tissue Weights and Bile Volumes HWI 6329-133 Sex Male Animal Number Liver Weight (g) Dermal Appli- Kidneys cation Site Bile Volume (mL) 'GroupI (Control)- Sterile Water for Injection(0 mg/kq) F52885 70.634 - 0.960 1.0 F52873 75-513 16.417 0.866 1.6 F52898 74.433 - 0.645 0.9 Female F52967 62.517 - 0.777 2.1 F52878 63.222 16.808 0.890 2.0 F52883 75.418 - 0.618 2.5 Group 2 - T-6054 (0.128 mg/kq) Male F52887 71.149 - 0.784 1.1 F52893 64.537 13.509 0.836 0.6 F52897 73.622 - 0.604 1.5 Female F52901 71.908 14.610 0.640 1.7 F52876 66.359 - 1.179 1.2 F52882 77.959 1.337 1.6 GrouR 3 T-6054 (1.28 mg/kq) Male F52965 69.880 18.191 0.452 1.8 F52891 65.695 - 0.525 1.1 F52892 69.216 - 0.949 1.1 Female F52884 65.907 - 0.590 1.2 F52900 61.937 - 0.710 1.0 F52968 67.347 12.513 0.866 1.3 Not applicable. Sex Male Female Male Female Page 28 of 46 HWI 6329-133 Table 5 (Continued) Individual Animal Tissue Weights and Bile Volumes Animal Number Liver Weight (g) Dermal Appli- Kidneys cation Site Bile Volume (mLl Group 4 - T-6054 (12.8 mg/kq) F52886 77.219 - 0.857 0.6 F52880 59.985 12.254 0.634 0.8 F52899 89.883 - 1.391 0.4 F52889 69.288 0.953 0.5 F52894 75.086 - 1.000 1.4 F52895 59.174 15.603 0.635 0.9 Group 5 - T-6051 (10-cm x 10-cm Section) F52879 89.874 0.884 1.0 F52963 71.814 - 1.535 0.6 F52874 71.657 15.784 0.990 0.6 F52877 71.284 - 1.154 0.6 F52888 65.633 - 1.231 0.5 F52966 68.719 15.855 0.898 1.5 - Not applicable. Page 29 of 46 HWI 6329-133 APPENDIX A Protocol Deviation Protocol TP3016.AB Protocol Amendment No. I Protocol Amendment No. 2 I Page 30 of 46 Protocol Deviation HWI 6329-133 Protocol Page 7, 7. Experimental Design, C. Observation of Animals, (2) Reading of Dermal Irritation, Second Sentence. Additional dermal irritationreadingswill be made approximately 30 minutes after bandage removal (Day 2) and on Study Days 4 and 8. Actual Procedure The Day 8 erythema score was inadvertentlynot recorded for one Group 4 female (No. F52889). This deviation is not considered to have had an adverse effect on the outcome of the study. W HWAI s LOUN'TS(OINN POST OFFICE BOX 7545 @ilADISON. Wl 53707-75,15 Page 31 of 46 CORNING cc);1,4x)ny Sponsor: 3M St. Paul, Minnesota PROTOCOL TP3016.AB Study Title: Single-Dose Dermal Absorption/ToxicityStudy of T-6054 and T-6051 in Rabbits Date: December 13, 1994 Performing Laboratory: Hazleton Wisconsin, Inc. 3301 Kinsman Boulevard Madison, Wisconsin 53704 LaboratoryProject Identification: HWI 6329-133 F'kiclIte F4()S 14 1 4 4 1 D E L I\/E R N I v I) 2 VV 1 53/0.1 Page 32 of 46 STUDY IDENTIFICATION TP3016.AB Page 2 Single-Dose Dermal Absorption/ToxicityStudy of T-6054 and T-6051 in Rabbits HWI No. Test Materials Sponsor Sponsor's Representative Study Director Study Location Proposed Study Timetable Experimental Start Date Experimental Termination Date Draft Report Date 6329-133 1. T-6054 2. T-6051 3M Toxicology Service Medical Department 3M Center, Bldg. 220-2E-02 P.O. Box 33220 St. Paul, MN 55133-3220 John L. Butenhoff, PhD 3M Toxicology Service Medical Department 3M Center, Bldg. 220-2E-02 P.O. Box 33220 St. Paul, MN 55133-3220 (612) 733-1962 Steven M. Glaza Hazleton Wisconsin, Inc. P.O. Box 7545 Madison, Wl 53707-7545 (608) 241-7292 Hazleton Wisconsin, Inc. Building No. 3 3802 Packers Avenue Madison, Wl 53704 December 28, 1994 January 25, 1995 March 8, 1995 Page 33 of 46 TP3016.AB Page 3 1. Study Single-DoseDermal Absorption/ToxicityStudy in Rabbits 2. Purpose To assessthe systemicabsorptionand toxicityand relativeskin irritancyof test materialswhen appliedto the skinof rabbits 3. Regulatory Com2liance This study will be conductedin accordancewith the followingGood LaboratoryPractice Regulations/Standards/Guidelines: [ ] Conduct as a Nonregulated Study CXI 21 CFR 58 (FDA) 40 CFR 160 (EPA-FIFRA) 40 CFR 792 (EPA-TSCA) C(81)30(Final)(OECD) 59 Nohsan No. 3850 (Japanese NAFF) NotificationNo. 313 (JapaneseMOHW) All proceduresin this protocolare in compliancewith the Animal WelfareAct Regulations. In the opinionof the Sponsorand study director,the study does not unnecessarilyduplicateany previous work. 4. Ouality Assurance The protocol,study conduct,and the final reportwill be auditedby the QualityAssurance Unit in accordancewith HazletonWisconsin (HWI) StandardOperating Procedures(SOPS) and policies. 5. Test Materials A. Identification I. T-6054 2. T-6051 B. PhysicalDescription 1. (To be documentedin the raw data) 2. (To be documentedin the raw data) C. Purityand Stability The Sponsor assumes responsibilityfor purityand stability determinations(includingunder testconditions). D. Storage Room temperature Page 34 of 46 TP3016.AB Page 4 E. Reserve Samples Reserve sample(s)of each batch/lotof test and control materialswill be taken for this study. The test and control material reserve samples will be stored at HWI in a freezer set to maintain a temperature of -20*C IOOC for 10 years per HWI SOP. The Sponsor will be contacted after. 10 years for disposition in accordancewith the appropriate regulatory Good Laboratory Practices. F. Retention Any unused test materials will be returned to the Sponsor after completionof the in-life phase of the study. G. Safety Precautions As required by HWI SOPs and policies 6. Control Material A. Identification Distilledwater B. Physical Description Clear, colorlessliquid C. Purity and Stabili-t-Y The purity and stabilityof this manufacturedmaterialis considered to be adequate for the purposes of this study. D. Storage Conditions Room temperature E. Reserve Samples See Section 5. E. Reserve Samples F. Retention Any remaining control material may be used for other testing and will not be discardedafter issuanceof the finalreport. G. Safety Precautions As required by HWI SOPs and policies 7. Experimental Design A. Animals (1) Species Rabbit (2) Strain/Source Hra:(NZW)SPF/HRP, Inc. Page 35 of 46 TP3016.AB Page 5 (3)A-cLe-aItnitiation Adult (4)Weightat Initiation 2.0 to 3.0 kg (5)Number and Sex 15 males and 15 females (6) Identification Individualnumberedear tag (7) Husbandry (a) Housing Individually,in screen-bottomstainlesssteel cages (heavy gauge) (b) Food A measured amount of Laboratory Rabbit Diet HF #5326 (PMI Feeds,Inc.). The food is routinelyanalyzedby the manufacturerfor nutritionalcomponentsand environmentalcontaminants. (c)Water Ad 7fbitum 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 Environmentalcontrols for the animal room will be set to maintain a temperatureof 19*C to 23*C, a relativehumidity 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 healthis placedon test. The animalswill be placed into study groups using a stratifiedbody weight randomizationprogramwithin ni,ne days of study initiation. Page 36 of 46 TP3016.AB Page 6 (9) 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. B. Dose Administration (1) Test Groups GrouD Dose Level Test Material --(mg/kq)_ I (Control)Distilledwater 2 T-6054 3 T-6054 4 T-6054 5 T-6051 0* 0.128 1.28 12.8 Number of Animals Males Females 3 3 3 3 3 3 3 3 3 3 To be administered at a dose volume of 2.0 mL/kg To be administered as a 10.0-cm x 10.0-cm piece of test material(fabric) (2) Preparation of Exposure Area On the day before test material application, the back and, if necessary (to obtain unblemished skin), the flanks of each rabbitwill be clippedfree of hair with an electric clipper. The shaved area will constituteapproximately 20% of the total body surface area. The treatmentsites (intactskin)will be inspectedfor interferinglesions, irritation,or defects that would precludethe 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 respectivedoses for the animals in Groups 1, 2, 3, and 4 will be based on the animal's body weight just before administrationand spread onto the area of exposure in a thin and uniform layer. The Group 1, 2, 3, and 4 materialswill be applied undiluted. The Group 5 material will be applied as a 10.0-cm x 10.0-cm piece of the test material moistened with distilledwater. The area of application (Groups 1-5) will be covered with a 10-cm x 10-cm gauze bandage secured with paper 0tape around all etdges and overwrapped with Saran Wrap and Elastoplast tape to provide an occlusivedressing. The rabbitswill be collared during the 24-hour applicationperiod. Page 37 of 46 TP3016-AB Page 7 (4) Reason for Route of Administration The dermal route is a potential route of exposure in humans. (5) Removal of Test Material Approximately24 hours after test or control material application the bandages and collars will be removed and. the residual test material will be removed using water or an appropriatesolvent, if necessary. C. Observation of Animals (1) Clinical Observations For clinicalsigns before test or control material administrationand for clinicalsigns and mortalityat approximately1, 2.5, and 4 hours after test material administration(Day 1) and daily thereafterfor clinical signs, and twice daily (a.m.and p.m.) for mortalityfor at least 28 days. Observations may be extended when directed by the study director. (2) Reading of Dermal Irritation Before test or controlmaterialadministrationthe initial dermal irritationreading will be made and recorded as the Day 1 reading (Attachment1). Additional dermal 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 unscheduleddeath (when survival exceeds 1 day) (4) Sample Collections (a) Freauency 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 (c) Method 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 sacrificed in a moribund conditionor Page 38 of 46 TP3016.AB Page 8 sacrificedat the time of necropsy (Day 29). The sampleswill be stored at room temperature and then centrifuged,and the separate serum and cellular fractionsstored in a freezer set to maintain -20*C tlOOC. The separated serum and cellular fractionswill be sent frozenon dry ice to the Sponsor after experimental termination. 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) 778-5294 prior to the shipmentof the samples. D. Pathology (1) Unscheduled Sacrifices and Deaths Any animal dying during the study or sacrificedin a moribund conditionwill be subjected to an abbreviated gross necropsyexamination and all abnormalitieswill be recorded. Animals in a moribund conditionwill be anesthetizedwith sodium pentobarbital(via injectionin the marginalear 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 terminationof the experimentalphase (Day 29), surviving animals will be anesthetizedwith sodium pentobarbital(via injectionin the marginalear vein), bled via the vena cava, exsanguinated,and subjected to an abbreviatedgross necropsy examination. The animals will be necropsiedin random order and all abnormalitieswill be recorded. (3) Sample Collection The sites of test and control materialapplicationwill 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 freezerset to maintain a temperatureof -20*C tlO*C. After necropsy,the animalswill be discarded. Page 39 of 46 TP3016.AB Page 9 The tissues (liver,bile,dermal applicationsite, kidneys)will be sent frozenon dry ice to the Sponsor after experimentaltermination. The samples will be shippedto the person listed in Section 7.C.(4).(c). The Sponsoris responsiblefor the retention and dispositionof the samples. E. StatisticalAnalyses No statisticalanalyses are required. 8. Report X final report includingthose items listed below will be submitted. Description of the test and control materials Description of 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 5riginal data, or copies thereof,will be availableat HWI to facilitate auditingthe study during its progressand before acceptance of the final report. When the final report is completed, all original paper data, includingthose item listedbelow 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 Anatomical pathology records Sample collection records Shipping records Study correspondence Final report (originalsigned copy) 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 temperaturerecords Instrument calibration and maintenance records Page 40 of 46 PROTOCOL APPROVAL TP3016.AB Page 10 @ohn L. Butenhoff, PhD Date Sponsor's Representative 3M Toxicology Service Medical Department Steven M. Glaza Date Study Director Acute Toxicology HazletonWisconsin, Inc. Representa ve Date Quality Asvurance Unit HazletonWisconsin, Inc. (6329-133.protdsk2) Page 41 of 46 TP3016-AB Page 11 Attachment I Scoring Scale for Acute Demal Reactions Erythema 0 - None I - Slight 2 - Moderate 3 - Severe Edema 0 - None 1 - Slight(barelyperceptibleto well definedby defin*ltreaising) 2 - Moderate (raisedapproximately1 mm) 3 - Severe (raisedmore than I mm) Atonia 0 - None I - Slight(slightimpairmenotf elasticity) 2 - Moderate(slow returnto normal) 3 - Marked(no elasticity) Desguamation 0 - None 1 - Slight(slightscaling) 2 - Moderate(scalesand flakes) 3 - Marked (pronouncedflakingwith denuded areas) Coriaceousness 0 - None I - Slight(decreasein pliability) 2 - Moderate(leatherytexture) 3 - Marked (toughand brittle) FissuLLng 0 - None I - Slight(definitecracksin epidermis) 2 - Moderate(cracksin dermis) 3 - Marked (crackswith bleeding) Page 42 of 46 HAZLEECC:N W IS C 0 N S IN POST OFFICE BOX /545 MADISON,WL 53707 75,15 CORNING Ctxnpanv PROTOCOL TP3016.AB Single-Dose Dermal Absorption/ToxicityStudy of T-6054 and T-6051 in Rabbits HWI 6329-133 Sponsor 3M Toxicology Service Medical Department 3M Center, Bldg. 220-2E-02 P.O. Box 33220 St. Paul, MN 55133-3220 Sponsor's ReRresentative John L. Butenhoff, PhD Contractor Hazleton Wisconsin, Inc 3301 Kinsman Boulevard Madison, Wl 53704 Study Director Steven M. Glaza Amendment No. I This amendment modifies the following portions of the protocol: Effective December 23, 1994 In order to obtain a measurable amount of test material (T-6054)for applicationin Groups 2, 3, and 4, the test materialwill be dilutedwith sterile water for injection and applied at a common dose volume of .01 mL/kg. Modify the following two sections of the protocol (protocol amendment items #1 and 02) to indicate these changes. I. Page 6. 7. Experimental Design; B. Dose Administration;(1) Test Groups. Add the following shaded additionsto this section: Grout) Test Material Dose Level Number of Animals (mg/kq) Males Females 1 (Control) Distilledwater 0* 3 3 2 T-6054 0. 12a@@.,', 3 3 3 T-6054 1. 28"' 3 3 4 T-6054 12.8.*... 3 3 5 T-6051 3 3 To be administered at a dose volume of 2.0 mL/kg To be administered as a 10.0-cm x 10.0-cm piece of test material(fabric) P tin rie EXPF,FSS 608 2 4 1 4-171 M A IL F L Iv E 8 y 1) 1 K IN S tviA rl ft1 1) ce Ni 1)1 1-(11 tiU ti wi 1 2'2 @310 4 Page 43 of 46 Amendment No. 1 HWI 6329-133 Page 2 2. Page 6. 7. Experimental Design: B. Qoso AdMi@nistration-,-(3I)ose Administration. Delete the fourth sentencein this sectionand then add the followingas the third and fourth sentencesto this section. The control material (Group1) will be appliedundiluted. The dose for each animal in Groups 2, 3, and 4 will be diluted with sterile water for injectionand applied at a dose volume of .01 mL/kg. Effective December 28, 1994 Sterilewater for injectionwill replacedistilledwater as the control material based on the fact that sterilewater for injectionwill also be the vehicle in the test mixtures for Groups 2, 3, and 4 (see protocol amendment items #1 and #2). Modify the followingthree sectionsof the protocolto indicate this change. 3. Page 4. 6. Control Material-,A. Identification. Replace distilled water with the following: SterileWater for Injection 4. Page 4. 6. Control Material% D. Storage Conditions. Replace Room temperature with the following: Refrigerated 5. Page 6. 7.-LX-DerimentalDesign; B. Dose Administration; (1) Teat Groups. Modify the table in this sectionwith the following shaded change: Group Test Material 1 (Control) 2 3 4 5 W,,e w ater 54 T-6054 T-6054 T-6051 Dose Level Number of Animals (mg/kq)- Males Females 0* 3 3 0.128*** 3 3 1.28*** 3 3 12.8*** 3 3 3 3 To be administered at a dose volume of 2.0 mL/kg To be administered as a 10.0-cm x 10.0-cm piece of test material(fabric) To be administered at a dose volume of .01 mL/kg page 44 of 46 Amendment No. 1 PROTOCOL APPROVAL 2. John L. Butenhoff, PhD Date Sponsor's Representative 3M Toxicology Service Medical Department Steven M. Glaza Date Study Director Acute Toxicology Hazleton Wisconsin, Inc. @eprei'e'ntatifte Date Quality AssfiranceUnit Hazleton Wisconsin, Inc. (6329-133.Aml.dsk2) HWI 6329-133 Page 3 Page 45 of 46 HAZLE-CC:N W IS C 0 N S IN POST OFFICF flOX 7545 MADISON.WI 1,@3707 /545 j CORNING Company PROTOCOL TP3016.AB Single-Dose Dermal Absorption/Toxicity Study of T-6054 and T-6051 in Rabbits HWI 6329-133 Sponsor Contractor 3M Toxicology Service Medical Department 3M Center, Bldg. 220-2E-02 P.O. Box 33220 St. Paul, MN 55133-3220 Hazleton Wisconsin, Inc. 3301 Kinsman Boulevard Madison, Wl 53704 Sponsor's Representative Study Director John L. Butenhoff, PhD Steven M. Glaza Amendment No. 2 This amendment modifies the followingportions of the protocol: Effective January 24, 1995 At the request of the Sponsor, the weights of tissues collectedand the volume of bile collectedwill be documentedin the raw data. These weights and volumes will be included with the sample shipment. Modify the following sections of the protocol to includethese additions. 1. Page 8, 7. Experimental Design; D. Patholooy; (3) Sample Collection. Modify the second sentence in the first paragraphof this section with the following underlined addition: 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 bilel and immediatelyplaced in a freezer set to maintain a temperatureof -20*C 10*C. 2. Page 9. 7. Experimental Design; D. Pathology; (3) Sample Collection. Modify the second sentence in the second paragraph of this section with the following underlinedaddition: The samples and their correspondingweights or volumes will be shippedto the person listedin Section7.C.(4).(c). P h o n e 606 EX P R E S F Fi'f 33i*lI F a t i(,,i ).1 1 1227 k! tl.-I I MADISON V%O 1 F)1/04 Page 46 of 46 Amendment No. 2 HWI 6329-133 Page 2 3. Page 9, 8. Report. Add the followingto this section: Individualanimal tissueweights and bile volumes PROTOCOL AMENDMENT APPROVAL John L. Butenhoff,PhD Date Sponsor's Representative 3M Toxicology Service Medical Department Steven M. Glaza Date Study Director Acute Toxicology Hazleton Wisconsin, Inc. Representat e Date Quality Assirance Unit HazletonWisconsin, Inc. (6329.-133.Am2.dsk2) 9.1.2 AnalyticaplrotocolAMDT-013195.1 3M EnvironmentaLlaboratory Protoco-lAnalyticaSltudy Single-DosDeermal Absorption/ToxicSituydyofT-6051and T-6054in Rabbits In-VivoStudyReferenceNumber: HWI#6329-133 StudyNumber: AMDT-013195.1 TestSubstance:FC-129(T-6051andT-6054) Name and AddressofSponsor: 3M SCD Division 367 Grove Street St.Paul,MN 55106 Name and Address of TestingFacility: 3M EnvironmentalTechnology and Services 935 Bush Avenue St.Paul,MN 55106 Proposed 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: J mes D7rgnstoonr Date tu@dy Dij John Butenhoff,PhD Date Sponsor Representative 1.0 PURPOSE ThisstudyisdesignetdoprovideinformatiaosntowhetherFC-129(T-6051and T-6054) isdermallyabsorbed. The analyticaalspectof thisstudyisto determine fluorine-containicnogmpounds (biotransformatipornoducts)inthetissueand serum of rabbitsatvarioustimespost dose dermal applicatioonf FC-129. 2.0 TEST MATERIALS 2.1Test,Control,and ReferenceSubstancesand Matrices 2.1.1AnalyticalReferenceSubstance: FC-95, lot161 or 171. They are equivalent. 2.1.2AnalyticalReference Matrix: Bovine liverand bovine serum 2.1.3Analytical Control Substance: None 2.1.4Analytical Control Matrix: Bovine liverand bovine serum 2.2 Source of Materials: 3M ICP/PCP Division(2.1.1),grocerystore(2.1.2,2.1.4liver)S,igma Chemical Company (2.1.22,.1.4-serum) 2.3 Number of Test and Control Samples: Tissuesand fluidfrom 24 testanimals and 6 controlanimals. Tissuesand fluidsincludeliver,serum,cellulafrraction, dermal applicationsiteand bile.Analysisof thesetissueswillbe atthediscretioonf the Study Director. 2.4Identificatioonf Test and Control Samples: The samplesare 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 100C (2.1.22,.1.4).Testand Controlsamples willbe receivedaccordingto AMDT-S-10-0. 2.8 Dispositionof Specimens: Biologicatlissuesand fluidswillbe retainedper GLP Regulationforthetime periodrequiredforstudieslongerthan 28 days. 2.9 Safety Precautions: Refer to appropriateMSDS. Wear appropriatelaboratory attire.Use cautionwhen handlingknivesforcuttingthe samples. 2 3.0 EXPERIMENTAL -Overview The tissuesfrom animalsdosed as described(HWI#6329-133), areavailablefor analysisforfluorinecompounds. At thediscretioonf theStudy Director,a seriesof analyticatlestscan be performed. The screeningforfluoridein livervia combustion (seeMethods--nextsection)istheappropriateanalysisto presentdefinitivdeatafor fluorineinthe liver.To confirm theidentitoyf fluorine-containicnogmpounds presentin liver(ifany at28 days)and serum atvariousintervalse,lectrospraymass spectrometrymay be selectedas one of the analyticatlechniquesemployed. Not all of thetissuesand fluidsamples willbe analyzed.When sufficiendtatahas been collectedto meet theobjectivesof thestudy intheopinionof theStudy Director, analysiswillcease. 4.0 EXPERIMENTAL - Methods 4.1Liver and Serum screeningmethods: (attached) 4.1.1AMDT-M-1-0, Thermal Extractionof Fluorideby Means of a Modified Dohrmann DX2000 OrganicHalideAnalyzer-Liver 4.1.2AMDT-M-2-0, FluorideMeasurement by Means of an Orion EA940 Expandable Ion Analyzer 4.1.3AMDT-M-4-0, 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 5.1Data Reporting: Data willbe reportedasa concentratio(nweight/weighto)f fluorideper tissueor fluid,or as FC-95 (electrospramyass spectrometry)perunitof tissueor fluid.Statisticused,atthediscretioonf theStudy Director,may include regressionanalysisof serum concentrationswith time and averagesand standard deviationsof concentrationfsordifferendtose groups. Ifnecessary,simple statistictaelstssuch as Student'sttestmay be appliedtodeterminestatistical difference. 3 6.0MAINTENANCE OF RAW DATA AND RECORDS 6.1Raw Data and Records:Raw dataa,pprovedprotocola,ppropriastpeecimens, approved finalreport,and electronidcatawillbe maintainedinthe AMDT archives. 7.0 REFERENCES 7.1 AMDT-S-10-0, Sample TrackingSystem 8.0 ATTACHMENTS 8.1ANMT-M-1-0, Thermal Extractionof Fluorideby Means of a Modified Dohrmann DX2000 OrganicHalideAnalyzer-Liver 8.2ANMT-M-2-0, FluorideMeasurement by Means of an Orion EA940 Expandable Ion Analyzer 8.3AMDT-M4-0, Extractionof Fluorochemicalsfrom Rabbit Liver 8.4AATDT-M-5-0, Analysisof RabbitLiver ExtractforFluorochemicalsUsing ElectrosprayMass Spectrometry 8.5 AMDT-M-8-0, Analysisof FluorideUsing the SkalarSegmented Flow Analyzer with Ion SelectiveElectrode 4_ 3M Environmental Laboratory Method Thermal Extractionof Fluorideby Means of a Modified Dohrmann DX2000 Organic HalideAnalyzer -Liver Method IdentiflcatNiuomnber: AMDT-M-1 RevisionNumber: 0 AdoptionDate: /6-f-CCRevisionDate: None Author:Rich Youngblom Approved by: 13 roup Lear Dafe roUP L( QualityAssurance Date Software:MSWord5.la AffectedDocuments: ANIDT-M-2 FluoridMeeasurementby MeansofanOrionEA940 Expandable[onAnalyzer AMDT-EP-3 RoutineMaintenanceofa ModifiedDohrmann DX2000 OrganicHalideAnalyzer 1.0 SCOPE. APPLICABLE COMPOUNDS, AND MATRICES 1.1Scope: Thismethod isfortheoperatioonfa Dohrmann DX2000 when itisusedtoextract fluoridferom variousmatricesT.he fluoridiestypicallcyollecteidnTISAB solutiofnoranalysis withan ionselectiveelectrode. 1.2ApplicableCompounds: Fluorochemicalosrotherfluorinatecdompounds. 1.3rvlatriceBsi:ologicatlissuesp,articularlliyver. 2.0 KEYWORDS 2.1Fluoridef,luorine,xtractiopny,rolysisi,onizatioino,nselectiveelectrodeD,ohnnann, halide, DX2000, fluorochemicals. 3.0 PRECAUTIONS 3.1 Glasswareand exhaustgasescan be extremelyhot. 3.2Glasswareisfragileb,rokenglassmay causeinjuries. 3.3Pressurizedgases,propercompressedgas handlingpracticersequired. 3.4Solventbased samplesmay flashm,ay need toallowthem todrydown beforestartinngm. 3.5Potentiabliohazardsdue tothebiologicamlatrices.Use appropriatpeersonalprotective equipment. 4.0 SUPPLIES AIND MATERIALS 4.1Compressed Oxygen, Hydrocarbon free,regulatetdo30 PSI. 4.2Compressed Helium,High PurityGrade,regulatetdo45 PSI. 4.3QuartzglasssampleboatwithTeflonTmtubing,Dohrmann 890-097or equivalent. 4.4Quartzglasscombustiontube,RelianceGlass G-9405-012 orequivalent. 4.5Orion940999TotalIonicStrengtAhdjustmentBuffer(TISAB II)orequivalent. 4.6Sample collectiovnialsH,DPE. 4.7NElli-QTMwater 4.8Polystyrenepipettes. 4.9ActivatedCharcoalE,. Merck 2005 orequivalent. 4.10HamiltonSyringeor equivalent. 4.11Miscellaneouslaboratorgylassware 5.0 EQUTPNIENT 5.1Rosemount Dohrmann DX2000 OrganicHalideAnalyzer,modifiedforfluorideextraction. 5.2IBM compatible386 or486 computer. 5.3DX2000 softwarev,ersion1.00,modifiedforfluorideextraction. 5.4Excel Spreadshee@version5.0or greater 6.0 INTERFERENCES 6.1 Sample sizeislirriiteodapproximately150mg, dependingon samplemoisturecontent.This may varyfrom matrixtomatrix. 2 7.0 SAMPLE HANDLING 7.1Samples arenottobe handledwithbarehands. Fluoridemay leachfrom theskintothe sample. Use forcepsor probe totransfetrissues. 7.2Samples ofliverarecutfrom frozenliverand placedina taredand labeledweigh boat.Use a cleanscalpeland cuttinbgoard.The cuttinbgoardand scalpeslhouldbe cleanedwithwater, methanol,or methanol-watersolutioanftereachliveriscut. 8.0 CALIBRATION AND STANDARDTZATION 8.1PreparatioonfCalibratiSotnandards 8.1.T1hestandarrdesquirefdoreachprojewcitlnleedtobeapproprifaotrethaitndividuparloject. Refertoprotocolforthatproject. 8.1.2Typically50-500 ppm FC-95 inmethanolstandardsareused. 8.1.3For rabbiltiverstudiesu,sebeefliverasthematrix.Cut a pieceof frozenbeeflive(r100150mg) and weigh itina labeledand taredweigh boat. 8.2 Calibration-Overview The normal calibratioinsthefluoridceurve(AMDT-M-2). However, ifan optionalspikedliver curveisrequiredtheprocedurelistebdelow isused. 8.2.1A calibraticounrvefortheDX2000 isgeneratedby spflcinsgampleswithknown 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.Generatea regressiocnurveand calculatteheequationfortheline and ther2 value. 8.2.4Mass SpikedF (u,,=c,()Amount spikedinmL) x (Conc.ofstandardinppm) x (0.6004)* *FC-95 is60.04% F therefor0e.6004isthefactorusedtoconvertFC-95 toF 8.2.5StandardMass RecoveredF (ug)= (TISAB volumeinML) x (Orionreadinginppm) 8.3Calibratio-nProcedure 8.3.1StartUp 8.3.1.1Run 2 ormore CleanCycleswhen startinignstrumenetachday.More cleancyclesmay be usediftheprevioussamplescontainedhighconcentrationosffluoride. 8.3.2Blanks 8.3.2.1Preparesampleusingthesame methods and typeofmatrixasthetestsample. 8.3.2.2For rabbitstudiesu,sebeefliverasthematrix.Prepareatleast3 samplesofbeefliver (100- 150 mg) forblanks. 8.3.2.3PutsampleinDohrmann boat.Combust each sampleasdescribedinsection9.0and analyzesampleaccordingtomethod AMDT-M-2 fortheionselectiveelectrodaenalysis. 3 8.3.2.4For rabbitstudiest,hemeter readingfora blanksampleshouldbe 0.03 ppm or lower beforeproceedingwiththecalibrationB.um samplesuntitlhislimitisreached,or untilinthe judgement of theoperatorthereadingisstablewithrespecttohistoricarleadings(previous48 hours). 8.3.2.5For non-rabbitstudiest,heblankreadingsshouldreacha predeterminedion concentration beforeproceedingwith thecalibration. 8.3.2.6Itmay be necessarytomix approximately50 mg of charcoalwiththesample toaid combustion. 8.3.3 Standard Curve 8.3.3.1Weigh outatleast15 matrixsamples(5standardswith3 replicateesach)intaredand labeledweigh boats.For rabbitstudiesw,eigh 100-150mg beefliversamples.Record weightsin studydata.Storethematrixsampleson dryiceor icepacks tokeep them frozenuntilused. 8.3.3.2Placeweighed beefliversample inDohrmann sample boat. 8.3.3.3Startwith theloweststandardconcentrationU.sing a Hamfiton syringe,ejecta fixed quantityof thestandardon orin thematrix.For rabbitstudiesu,se 4 uL ofstandardand ejectiton or inthebeefliver. 8.3.3.4At least3 replicatesshouldbe usedforthelowest standardconcentrationm;ore replicates may be used atthediscretioonf theanalyst. 8.3.3.5Combust thesample asdescribedinsection9.3and 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 allstandardshave been run,calculattehe r2.r2 must be atleast0.95.Ifitisnot at least0.95,consultwith supervisor. 8.3.3.8A new standardcurveshouldbe run when thecombustiontubeorsample matrixis changed. New standardcurvemay alsobe run atthediscretioonf theanalyst. 8.4 StorageConditionsforStandards 8.4.1Storagerequirementsforstandardsaredependenton theindividualstandardsused. 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 time= 300 seconds. 9.2StartUp Procedure: 9.2.1Iftheprogram isnotstarteds,tartheEOX program on thePC. 9.2.2Open theSYSTEM SETUP window. 9.2.3Put thefurnacemodule and thecellintheREADY mode. 9.2.4Close theSYSTEM SETUP window. 4 9.2.5When theoven has reachedtheREADY temperaturer,un theCLEAN found intheCELL CHECK menu. 9.2.6 See AMDT-EP-3 fordetailosftheDohrmann software. BOAT program 9.3Sample ExtractionProcedure: 9.3.1Open theSAM.PLE HATCH and placethesample intheBOAT. Itmay be necessaryto mix approximately50 mg of charcoalwiththesample toaidcombustion.Ifthisisdone,charcoal should alsobe mixed inwhileestablishintghebaselineand when generatingthestandardcurve. 9.3.2 Close SAMPLE HATCH. 9.3.3Add appropriatveolume ofTISAB solutioonr 1:1TISAB:Milli-QTI"watermixturetoa labeledsample collectiovnial.Typically0.6mL to 15mL areused. For rabbitstudiesu,se 1.0or 2.0niL of 1:1 TISAB:Milli-QT@,wlatermixture. 9.3.4Placethevialsothatthetipof theCOMBUSTION TUBE isintheTISAB atleast0.25 inches.Gases releaseduringpyrolysimsust bubblethroughtheTISAB. 9.3.5Run theEOX-SOLIDS program found intheRUN menu. 9.3.6When theEOX program isfinishedr,emove thecollectiovnialfrom thecombustiontube. 9-3.7IfundilutedTISAB was used tocollecthesample,add an equalvolume ofNfilli-QTwMater totheTISAB tomake 1:1 TISAB:Milli-QTm. 9.3.8Rinse theend of thecombustiontubewithMilli-QTMwaterand wipe with a KIMWIPE to remove any TISAB remainingon thetube. 9.3.9Open the sample hatchand remove any remainingash from theboat. Ash can be removed witha cottontippedapplicatoorr vacuumed out.Itmay be necessaryto scrapparticleosffthe bottom witha spatulaor othersimilardevice.A dropof Milli-QTbwlater may be added tothe boattoaidintheClean Cycle. 9.3.10 Closethehatch. 9.3.11 Run the CLEAN BOAT program. 9.3.12 Sample isready foranalysisby ionselectivelectrod(eAMDT-M-2). 9.4 Sample Calculations 9.4.1 Use thestandardcurvetocalculattehesample value. 9.4.2Sample Mass RecoveredF (ug)= (TISAB volinmL) x LQrionreadinginRpm -intercepo (Slope) 10.0 VALIDATION 10.1QualityControl 10.1.D1ailyStartUp CheckSamples:Oncethestandarcdurveisestablisheeadc,hdayof analysisisstartedby analyzingQC samples.The QC samples areto be thesame as thelowest concentratiosnpikedsamplesused togeneratethestandardcurve.Each concentratiomnust be done in triplicautnelessthefirsttwo replicateasrewithin20% of thestandardcurve,thena third replicatiesnot necessary. 10.2 Precisionand Accuracy: See method developmentanalysisand sample analysisin FluorideNotebooks 2,3,and 5. Precisionand accuracyvarieswhen analyzingsamplesof different matricesand differenrteferencecompounds. 10.3 Other ValidationParameters:NA 11.0 DATA ANALYSIS 11.1Calculations 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 arewit@dn20% of eachotherand theoutlieirsgreaterthan200% oftheaverageofthosetwo or lessthan50% of theaverageofthosetwo. 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 AMDT-M-2 FluorideMeasurementby Means ofan OrionEA940 ExpandableIon Analyzer 13.3AMDT-EP-3 RoutineMaintenanceof a ModifiedDohrmann DX2000 OrganicHalide Analyzer 14.0 REVISIONS Revision Numbe Reason forChancre Revision Date 3M EnvironmentalLaboratory Method FluorideMeasurement by Means ofan Orion EA940 Expandable Ion Analyzer Method IdentificatioNnumber: AMDT-M-2 RevisionNumber: 0 Adoption Date: RevisionDate: None Author Rich Youngblom ApprovedBy: G@, Leader /-3/ Date QualityAssurance Date Software: MS Word 5.1a AffectedDocuments: AMDT-M-1 Thermal Extractionof Fluorideby Means of a Modified Dohnnann DX2000 Organic HalideAnalyzer 1.0 SCOPE, APPLICABLE COMPOUNDS, AND MATRICES 1.1SCOPE: This method isforthecalibratioannd operationof an Orion EA940 Expandable Ion Analyzer. 1.2APPLICABLE COMPOUNDS: Fluoride. 1.3APPLICABLE pH of 6.0. MATRICES: Liquidsamplesin an appropriatbeuffersolutionP.referred 2.0 KEYWORDS 2.1 Fluoride,fluorinei,on selectiveelectrode 3.0 PRECAUTIONS -- 3.1 No hazardsidentifiewdiththismethod. 4.0 SUPPLIES AND MATERIALS 4.1Orion 940999 TotalIonicStrengthAdjustment BufferII(TISABII) or equivalent. 4.2Orion Model 900001 electrodefillinsgolution(AgCl) orequivalent. 4.3 Orion 940907 100 ppm fluoridestandardor equivalent. 4.4 NElli-QTIlwateror equivalent. 4.5Magnetic stirbars. 4.6 Lab tissues. 4.7 Sample collectiovnials. 4.8Plastic100 mL volumetricflasks. 4.9 Polystyrenepipettes. 4.10 Miscellaneouslaboratoryglassware. 5.0 EQUIPNIENT 5.1 Orion Model EA940 Expandable Ion Analyzer or equivalent. 5.2 Orion Model 960900 SolidStateCombination Fluorideelectrodeor equivalent. 5.3 Magnetic StirPlate. 5.4IBM compatible386 or 486 computer (onlyneeded ifusingOrion 3E software). 5.5Orion RS232 interfacceable(onlyneeded ifusingOrion 3E software). 5.6MicrosoftExcel 5.0(onlyneeded ifusingOrion 3E software). 6.0 INTERFERENCES 6.1 Itisreconunended thatthepH be ator near6.0. A 1:1 mixtureof TISAB and sample/Milli- Q Tlll water will generally bring sample to pH of 6.0. 6.2 Sample temperaturemay effectfluoridemeasurement. Itisrecorrunendedthatthe sample be atroom temperatureas the standardswere when the meter was calibrated. 6.3The ratethesamples are stirredatshould be consistentwith the ratethestandardswere stirred. 2 I 6.4Air bubblestrappedunder electrodcean giveerroneousreadings.Make sureno airistrapped under electrode. 7.0 SAMPLE HANDLING 7.1 No specialhandlingnecessary. 8.0 CALIBRATION AND STANDARDIZATION 8.1PreparatioonfCalibratioSntandards 8.1.1Measure50mL ofTISAB IIinto5 100mL plastivcolumetrfilcasks. 8.1.L2abeltheflaskass0.050,.1,0.5,1.0a,nd1.5ppm F-,alongwiththedateandyourinitials. 8.1.P3ipett0e.050,.1,0.5,1.0,and1.5mL of100ppm fluorisdteandaridntotheappropriately labeledflasks. 8.1.4Add approximately30 niL of Milli-QTmwatertoeach flask. 8.1.5Shake theflaskstomix thesolutions. 8.1.6Eliminateairbubblesfrom theflasksby tippingtheflaskson theirsidesand rollingtheairin theflasksover theairbubbles. 8.1.7Bringthevolume intheflasksup tothe100 mL mark withMUI-QTM water. 8.1.8Invertand shake theflasksforthefinalmixing. 8.1.9Record standardsinStandardsLog Book. 8.2 Calibration 8.2.1Ifnecessary,remove tapefrom electrodfeillinhgole. 8.2.2Invertprobe towet topseal. 8.2.3Ejecta few dropsof fillinsgolutionfrom bottomof electrodetowet lower seal. 8.2.4Filltheelectrodweithfillinsgolution. 8.2.5The meter and theF- electrodaeretypicallcyalibratebdy directmeasurement withno blank correctionu,sing standardswithconcentrationosf 0.05,0.1,0.5,1.0,and 1.5ppm F-,following themanufacturer'sinstructions. 8.2.6Record theslopein theappropriatleogbook. 8.2.7Clean theelectrodeby rinsingwithMilli-QTMwaterand wipingthesidesdown withlab tissues. 8.3 StorageConditionsforStandards 8.3.1Calibratiosntandardsarestoredatroom temperature. 9.0 PROCEDURES 9.1Calibrationand infeasurementS,tandard method: 9.1.1The sample to be measured needs tobe n-LixewdithTISAB usingtheproportions recommended by theTISAB manufacturer. 9.1.2Placea stirbar inthesample and placethesample on thestirplate. 9.1.3Allow thesample tomix fora few secondsbeforeinsertintgheelectrodeW.hen the electrodeisinsertedm,ake surethereareno airbubblestrappedundertheelectrode. 9.1.4The sample shouldbe thesame temperatureasthecalibratiosntandardsand stirreadtthe same rateas thecalibratiosntandards. 9.1.5When thereadingshave stabilizerde,cordthereadingintheappropriatleogbook. 3 9.2 CalibrationAnd Nleasurement,Using Orion 3E Software: 9.2.1 Calibration: 9.2.1.1Follow steps8.2.1to 8.2.4. 9.2.1.2PressFunctionKey #8 (F8). 9.2.1.3The computer screenwillask you toconfirm thenumber of standardsto be used, concentratioonf thestandards,and whether ornot a blankistobe includedinthecalibration. Make any necessary changes totheinformationpresentedand clickon CONTINUE. 9.2.1.4Place the electrodeinthe firssttandardon thestirplateand clickon CONTINUE. 9.2.1.5Observe the readingson thegraphicdisplayon thecomputer. When the readingshave stabilizedp,ressACCEPT READING. 9.2.1.6Repeat step9.2.1.4and 9.2.1.5fortheremaining standards. 9.2.1.7Afterthe finalstandard,thecomputer willdisplaytheslopeof thecurve,aswell as the interceptand correlationR.ecord theslope,intercep@and correlatioinnthe appropriatelog book and clickon CONTINUE. The calibratiodnataisautomaticallcyopiedto C:\Orion\Data\Calib.tKt. 9.2.2Data Spreadsheet: 9.2.2.1SelecteitherNEW or OPEN from theFILE menu toopen a new or existingspreadsheet to storedata in. 9.2.2.2Record the name of the spreadsheetused inthe appropriatelog book. 9.2.3FluorideMeasurement: 9.2.3.1Follow steps9.2.1through 9.2.4 9.2.3.2Enter the name of thesample intheappropriateplaceon thescreen. 9.2.3.3Clickon theNEW SA-NTLE button 9.2.3.4When thereadingshave stabilizedc,lickon theRECORD buttonand writetheresultinthe appropriatelog book. 10.0 VALIDATION 10.1 QualityControl: 10.2 Precisionand Accuracy 10.3 Other ValidationParameters AccordingtoReference 13.2,therange of detectionis0.02 ppm fluorideup toa saturatedsolutionof fluoride. 11.0 DATA ANALYSIS 11.1 Calculations None necessary. 11.2 Analyzintg,the Data None necessary. 12.0 ATTACHNIENTS None 13.0 REFERENCES 4 13.1Orion Model EA940 Expandable IonAnalyzerInstructioMnanual, Orion Research Incorporated1,991. 13.2Orion Model 960900 SolidStateCombinationFluorideElectrodeInstructioMnanual, Orion Research Incorporated1,991. 14.0 REVTSTONS Revision Number Reagn forChanee Revision Date 5 3M EiivironmentalLaboratory Method Extractionof Fluorochemicalsfrom Rabbit Livers SOP IdentiricatioNnumber: AMDT-M-4 RevisionNumber: 0 Adoption Date: RevisionDate: None Audior.Dave Christenson/CyndiWieaber Approved By: roupLeader lo - 3rl-tS Date QualityAssurance Date Software:MS Word, 6.0 AffectedDocuments: M-5, Analysisof RabbitExtractforFluarochemicalUssing Electrospray Mass Spectroscopy. 1.0 SCOPP, 1. 1 Scope: This method isfortheextractioonf fluorochemicalsfrom rabbitlivers. Ethyl acetateisused toextractfluorochemicalsfrom theliversforanalysisby electrospraymass spectroscopy. 1.2 Applicable Compounds: Fluorochemicalsor otherfluorinatecdompounds. 1.3 Matrices: RabbitLivers. 2.0 KEYWORDS 2.1 Fluorochemicals,rabbitliverse,lectrospraymass spectrometer,fluorinated compounds, extraction. 3.0 PRECAUTIONS 3.1 Use gloves when handlingtherabbitliverst,heymay containpathogens. 4.0 SUPPLIES AND MATERIALS 4.1 Supplies 4.1.1 Syringe,capableof measuring 100;iL 4.1.2 Eppendorf typeor disposablepipets 4.1.3 Gloves 4.1.4 Plasticgrindingtubes 4.1.5 Plasticentrifugetubes,15 mL 4.1.6 Labels 4.1.7 Nitrogen 4.1.8 Timer 4.1.9 FiltersT,itannylon syringefilter0s.,2 4. 1.10 Analyticalpipets:glassvolumetricpipets. 4. 1.11 Disposableplastic3 cc syringes. 4. 1.12 Crimp cap autovials. 4.2 Reagents 4.2.1 Aqueous Ammonium Acetate (Aldrich)a,pprox.250 ppm: Preparea 2500 ppm aqueous solutionof ammonium acetateby adding 250 mg ammonium acetatetoa 100 mL volumetricflaskand dilutetovolume withMM-Q water.Dilutethissolution1:10 fora 250 ppm solution. 4.2.2 Sodium carbonate/SodiumBicarbonateBuffer(J.T.Baker), (Na2CO3/NaliCO3) 0.25 M: Weigh 26.5 g of sodium carbonate(Na2CO3) a.nd21.0g of sodium bicarbonate(NaHC03) intoa I L volumetricflaskand bringtovolume with NERI-Q water. 4.2.3 Diluteacetonitrisloelutiond,iluteacetonitril1e:1 with Milli-Qwater. 4.2.4 EthyL Acetate 4.2.5 Methanol 4.2.6 Milli-Qwater 4.2.7 1H,IH,2H,2H - perfluorooctanesulfonaiccid(Aldrich) 4.2.8 FC-95 (3M SpecialtyChemical Division) 2 5.0 EOUIPMENT S-1 Ultra-TurraTx25 Grinderforgrindinglivesramples. S.2 Vonex mixer 5.3 Centrifuge 5.4 Shaker 5.5 AnalyticaElvaporator 6-0 INTERFERENCES 6.1 There areno known interferenceastthistime. 7.0 SAMPLE HANDLING 7.1 ne rabbitliversarereceivedfrozen,and must be kept frozenuntilthe extractioins performed. 8.0 CALIBRATION AND STANDARDIZATION 8.1 Preparation of InternalStandards 8.1.1 Prepare an internasltandardof approximately 12 ppm 1H,IH,2H,2H- perfluorooctanesulphoniacidtobe added toeach liversample. 8.1.2 Weigh atleast0.1 g of 1H,1H,2H,2H-perfluorooctanesulphonicacidintoa 100 mL volumetricflask.Record the actualweight. 8.1.3 Bring itup tovolume with methanol,thisisthestockstandard. 8.1.4 To a 250 mL volumetdc flaska,dd 3 mLs of thestockstandardand bringto volume with N4illi-wQater.Calculatetheactualconcentrationof the standard. actualmg perfluoroctane- sull2honiaccid X 0.1 L 3 mL 250 mL actualconcentrationp,pm 8.2 Prepare FC-95 Anion Standards 8.2.1 Prepare FC-95 standardsforthe standardcurve. 8.2.2 Weigh approximately100 mg of FC-95 intoa 100 niL volumetdc flask. Record theactualweight. 8.2.3 Bring up tovolume with diluteacetonitdle. 8.2.4 Dilutethesolutionwith diluteacetonitril1e:10 fora solutionof approximately100 ppm. Dilutethissolution1:10 with diluteacetonitdlfeor a solutionof approx. 10 ppm. 8.2.5 Use the 10 ppm solutiontomake working standardswithvaluescloseto 5.0 ppm, 1.0ppm and 500 ppb. 8.3 Prepare Beef Liver Homogenate to Use for Standards 8.3.1 Weigh 40 g ofBovineliveirntoa 250 niLNalgenebottlceontaining 200 mLs Mlli-Q water.Grind to a homogenous solution. 8.3.2 Add I mL of thesolutiontoa 15 mL centrifugetube.Preparea totalof eight I mL aliquotsof the solutionin 15 mL centrifugetubes.Be suretoresuspend solutionby shaking itbetween aliquots. 3 8.3.3 SpikesevenoftheI mL aliauotwsiththefollowingamounts ofworking standar-disnstep9.12of thiprocedure.One I mL aliquotservesas the blank. Worldng Yt-a-n-dard -uL (ApproximateConc.) - Soo 100 500 200 500 ppb 300 500 pRb 400 I ppm 500 5 ppm 200 5 ppm 300 Approximate final concentratioonf FC-95 inliver Blank 0.292 ppm 0.584 ppm 0.877 ppm 1.168 ppm 2.924 ppm 5.848 ppm 8.772 ppm 8.4 Calculatetheactualvalueof thestandards: uL ofstandardx -. i (inppm) 171 mg liver"1/ ml h-omogenate finacloncentratio(nppm) ofFC -95inliver *Average weightofbovineliverinsolutioansdeterminedby weighing I mL homogenates of40 mg liverin200 mL of NIMI-Q water.The amount of FC-95 isrepor-teads equivalentsof FC-95 potassiumsalt. 8.5 Calibration 8.5.1 Extractthespikedbeefliverhomogenate following9.13to9.23of this method. Use thesestandardstoestablisyhour curveon themass spectrometer. 8.5.2 Alternativelya,standardcurvemay be generatedusingratiosof responses of theperfluorooctansulfonantieonand theinternasltandardaiiionversus concentratioonf theperfluorooctanesulfonatneion. 8.6 Storage Conditions for Standards 8.6. 1 New standardsarepreparedwitheach analysisS.tandardsarestoredin coveredplasticentrifugteubesuntiltheanalysison themass spectrometer isperformed. 8.7 Storage Conditions for Standards 8.7. 1 Beef liverhomogenatesmay be frozenafterpreparation. 9.0 PROCEDURES 9.1 Obtainfrozenliversamples.In spenttissuen,otethattheliverhas notbeen packaged withothertissues. 9.2 Use a dissectinsgcalpeland cutoffapproximatelyIg of liver. 9.3 Weigh thesample directliyntoa taredplastigcrindingtube. 9.4 Record theliverweightinthestudynotebook. 9.5 Put a labelon thevialwiththestudynumber,weight,rabbitID, dateand analyst initials. 4 9.6 9.7 9.8 9.9 9.10 9.11 9.12 9.13 9.14 9.15 9.16 9.17 9.18 9.19 9.20 9.21 9.22 9.23 9.24 9.25 Add 2.5 mLs water. Grind thesample.Put thegrinderprobe in thesample and grindforabout 2 minutes,untilthesample isa homogeneous solutionwithno largechunks. Rinse theprobe offintothesample with2.5mLs waterusing a pipet. Take thegrinderapartand cleanitwithmethanolaftereach sample.Follow AMDT-EP-22. Cap thesample and vortexfor15 seconds. PipetI mL intoa 15 mL centrifugteube.Labelthecentrifugteubewiththeidentical informadonas thegrindingtube.(SeeAMDT-M-4 Worksheet fordocumentingthe remainingsteps.) Spike thebeefliverhomogenates withtheappropriataemount of FC-95 standard as describedin 8.3. Spike thesamplesand beefliverhomogenates with 100 uL of internasltandard. Add I mL of thesodium carbonate/sodiubmicarbonatbeufferand I mL ammonium acetate. Using an analyticaplipet,add 5 mL ethylacetate. Cap the sample and vortex20 to30 seconds. Put them intheshakerfor20 min. Centrifugefor20 to25 minutes,untilthelayersarewellseparated.Setthepower on thecentrifugeto25. Remove 4 mLs of thetoporganiclayertoa fresh15 mL centifugetubewitha 5 mL graduatedglasspipet.Transferthelabeltothefreshtube. Blow thesample down on theanalyticaelvaporatortoneardrynesswithnitrogen, approximately30 to40 minutes. Bringtheremainingsampleup in I mL dilutaecetonitdlweithan analyticaplipet. Vortex 15 seconds. Transferthesample toa 3 mL syringeA.ttacha 0.2gm nylonmesh filtearn,d filter thesample intoa freshcentrifugteubeor a autovialL.abel thetubeor vialwiththe studynumber and animalnumber. Cap and holdforanalysisby electrospmymass spectroscopy. Complete AMDT-M-4 worksheetand attachtopage of studynotebook. 10.0 VALIDATION 10.1 QualityControl-notapplicable 10.2 Precisionand Accuracy-not applicable .10.3 Other ValidationParameters-notapplicable 11.0 DATA ANALYSIS 11.1 None 12.0 ATTACHMENTS 12.1 WorksheetAMDT-M-4 13.0 REFERENCES 13.1 ANMT-EP-22 RoutineMaintenanceofUltra-Tun-axT-25 14.0 RE ISIONS Revision Number Reason forChange Revision Date 5 Study Sample Number set Riank T-iver Worksheet AMDT-M-4 FC-95 approx0.5ppm actual PPM #W IfltI)II, 7t)(1)1T. 00 uL 4()0i]T, FC-95 approx I ppm actual PPM #W 500 tiT. - FC-95 approx.5 ppm actual PPM #W Date and InitialfsorStd. - - 200 uL 100 iiT. qudy numher 3gheretheorivinawlork-gheeit%locatedond nl;iraeconv. T.ivePrxtrnctinnPmr&-,q- Pimt 1mT. nfT,iveIr;nliitinn Pinet100 uL nf 12 nnm Tntern;Ii;ltand;irfi stri VnrtpxIi Rt-f-@ PirieIt rnT.nf 750 nnm Ammnnitim Aretntf.. 'It;ti PinetI mT.nf 0 25 NarO,10 25M NaHrO. Riiffer Pimt i mT.nf FthylAretnti- Vnrtex?0-30 q&r ,gh;tk7t0- min- Centrifii7a0e-75 min Remnye :14 rnT.;ifiniinnftorg;inilraver Rlnw down tnneiieriryneg(s<O')imL) withN. Add I m- nf 1-1Ar-etnnitrile,-/14.0 TN# VnrtexIi ser Filteur-;inao lee B-D gyringewith2 0-2timgRl filteirntoa I i mT. ;Itito-;:;vminalle T)ntek. Tniti;il-q 6 3M- Environmental Laboratory. Method Analysis of Rabbit Liver Extract for Fluorochemicalsusing Electrospray Mass Spectroscopy SOP IdentiricatioNnumber: AMDT-M-5 Revision Number: 0 Adoption Date: RevisionDate: None Author. Dave Christenson/CynthiWaeber Approved By: tup.LroeuapdLeeard"er Date QualityAssurance Dam Software:MS Word, 6.0 AffectedDocuments: M-4, ExtractionofFluorocherrAcalfsrom RabbitLivers 1.0 SCOPE 1. 1 Scope: This method isfor theanalysisof extractsof rabbitliveror othertissuesor fluidsforfluorochemicalsusingthe electrospramyass spectrometerT.he analysis isperformed by singleion monitoringof FC-95 anion,M/Z= 499, theinternal standardM/Z = 427, and otherappropriatemasses. 1.2 Applicable Compounds: Fluorochemicalsor otherfluorinatecdompounds. 1.3 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 intothe probe DO NOT TOUCH THE PROBE, thereisriskof electricaslhock. 3.2 Do not run thepump above it'scapacityof4000 psi.Ifpressuregoes over 4000 psi stopand releasepressure.The peak tubingmay be plugged.Troubleshootback to findtheplug and replacetheplugged tubing.See AMDT-EP-15 3.3 Do not run thepump todryness. 4.0 SUPPLIES AND MATERIALS 4.1 Supplies 4. 1.1 Nitrogen gas regulatedto 140 psi. 4.1.2 Fluofixcolumn orequivalent. 4.1.3 100 uL or250 uL flatfipsyringeforsample injection. 4.2 Reagents 4.2. 1 Diluteacetonitrimloebilephase,diluteacetonitril1e:1 with Milli-Qwater. 4. 2.2 Milli-Qwater,allwaterused inthismethod 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 are no known interferenceastthistime. 7.0 SAMPLE HANDLING 7.1 Keep the extractedsamples incapped 15 mL centrifugetubesor incapped autovials untilready for analysis. 2 8.0 CALIBRATION AND STANDARDIZATION 8.1 Preparationof CalibrationStandards 8.1.1 Seven beefliverstandardsand one blankbeefliverarepreparedduringthe extractiopnrocedure.(SeeAMDT-M-4, section8.0) 8.2 Calibration 8.2. 1 Run thesevenbeefliverstandardstwice,startinwgiththeloweststandard toobtainthestandardcurve. 8.2.2 Typicallyone standardisrunaftereach5 to7 samples.Choose a standard in thesame rangeof concentratioans thesamples. 8.3 Storage Conditionsfor Standards 8.3.1 Freshstandardsarepreparedwitheach analysisS.tandardsarestoredin coveredplasticentrifugteubesuntiltheanalysison 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 Setsoftwareto"Operateon",Ion Mode ES-. 9.1.2 Record backingpressurein theinstrumentlog. 9.1.3 Fillthesolventcylinderwithmobilephase. .9.-1.4Set thepump to"Run". Settheflow to 1000 uLAnin.Observes droplets coming outof thetipof theprobe.The pressureshouldbe at1700 to 1800 psi. 9.1.5 Check thefusedsilicattheend of theprobe.Use an eye piecetocheck for chips.The tipshouldbe flatwithno jaggededges.Ifany chipsarefound cutoffthetipof thesilicwaitha column cuttearnd pullthesilictahroughto theappropriatleength. 9.1.6 Check your nitrogensupply.Tum on thenitrogenT.here shouldbe no nitrogenleakingaroundthetipof theprobe.A finemistshouldbe coming out of thetip. 9.1.7 Carefullyguidetheprobe intotheopening.Insertitundl itwon't go any fur-theCro.nnect thevoltagecabletotheprobe. 9.1.8 Go tothe"Editot"page,and setIonizatioMnode toES*,and the appropriatemasses to427 and 499. 9.1.9 Ifitisnotinsingleion mode go to"Option"and setSIR. 9. 1.lOStartAcquisiEionA.ssigna filename, MO-DAY-YR + letterR.ecord itin the logbook. 9. 1.11 Run thebeefliversamplesfirstr,unningeachstandardtwiceatthe beginningof therun..Run a QC check by runningone standardafterevery 5 to 7 samples. 9.2 Manual Injection 9. 2. 1 Draw 150 uL of sampleintoa syringe.Injecthesampleintotherheodyne injectiopnort.Injecstlowly.Record thesampleID inthelog book. 9.2.2 Turn thevalveto"On". 9.2.3 Wait two minutes,and injecthenextsample. 9.2.4 Record thescannumber foreich sampleinthelogbook. 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 Setinject/sampl=e2 9.3-3.4 SetCycle time= 0 9.3-3.5 Name thefileL:ivers 9.3.3.6 Identiftyhetrayused 9.3-3.7 Add thesamplestoQueue by pressing"Enter" 9.3.3.8 Press"Rurf'tostart 10.0 VALIDATION -- ILO1. Quality Control 10. 1.1Run a standardevery5 to7 samples.Ifa significancthange(50%) in peak heightoccursstoptherun.Only thesamplesbeforethelastacceptable standardwillbe used.The remainingsampleswillbe reanalyzed. 10.2 Precisionand Accuracy 10. 2. 1See Method ValidatioRneportnumber AINMT-M-5.0.V 1 10.3 Other ValidationParameters 10.4 RefertoMethod ValidatioRneportNumber AMDT-M-5.0.Vl 11.0 DATA ANALYSTS 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 of FC-95 anion,or otherfluorocherriiicnatlhetotarlabbit liver mg FC-95 anionin thetotarlabbitliver mg FC-95-anionfrom std.curve gms of liverusedforanalysis x Totalmass ofliverg,ms 11. 3 11. 4 Make a resultstableand enteritinthestudybook. Printa chromatogram foreach sample,withthepeakslabeledwiththesampleor standardD:).Write thestudynumber 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 REVTSIONS Revision Numbe Reason forchangc Revision Date - 5 __ __ 3M Environmental Laboratorv Method Analysis of Fluoride Using the Skalar Segmented Flow Analyzer With Ion SelectiveElectrode Method IdentiricatiNounmber: AMDT-M-8 AdoptionDate: /o-s--t5- RevisionNumber: 0 RevisionDate: None Author.Deb Wright/ CynthiaWeber Approved By: up Leader Date QualityAssurance Date Software:IBM MS Word, 6.0 AffectedDocuments: AMDT-EP-26, Operationand MaintenanceoftheSkalarSegmented Flow Analyzer 1.0 SCOPE 1. 1 This method isfortheanalysisforfluoridet,hermallyextractedfrom samples using theDohrmann DX2000 (AMDT-M-1), and collectedinTISAB foranalysiswithan Ion SelectiveElectrode(ISE).The analysisisperformed usingtheSkalar Segmented Flow Analyzer with ISE. 1.2 Samples can be tissuess,erum, biologicalmaterialo,rothermaterialsextractedon the Dohrmann. 2.0 KEYWORDS 2.1 Skalar,segmented flow,fluoride. 3.0 PRECAUTIONS 3.1 Follow standardlaboratorysafetypractices. 4.0 SUPPLIES AND MATERIALS 4.1 Supplies 4.1.1 Sample cups,4 mL plasticups withcaps 4.1.2 Autopipets,oxford orequivalentwithplastictips 4.1.3 Polypropylenevolumetricflasks,100 mL 4.1.4 Cartridgecomponents,refertotheSkalarMethods forcomponents and part numbers. 4.1.5 Sample prefilterEsv,ercrreen 4.2 Reagents 4.2.1 Brij35,30% S.F.A.S.Detergent 4.2.2 TISAB IIbuffersolutionP:urchaseTISAB E from Orion.To 1 literof TISAB 11add 2.5mL or 100 ppm fluoridesolutionand 1 mL Brij. 4.2.3 Sampler rinsingsolutionD:iluteTISAB 111:1with Milli-Qwater. 4.2.4 Nitricacidsolutiofnordecontamination,1 N (labgrade):Slowly add 64 mLs concentratenditriaccid(HN03) to250 mLs of Milli-Qwater.Bring thevolume up to 1 L withMilli-Qwater. 4.3 Standards 4.3.1 Stock solution,100 ppm F: purchased from Orion. 4.3.2 Intermediatestandard,10 ppm: Dilute10 mLs of stocksolutionto 100 mLs with Milli-Qwater.Use polypropylenevolumetricflasks. 4.3.3 Working standard:Make up thefollowingworking standardsby adding the volumes of intermediateor stockstandardindicatedon thetable,using oxford or pumpmate pipets,to50 mLs of TISAB and dilutingto 100 mLs with Milli-Qwater. Working Standard 0.015 ppm PPM 0.06 ppm 0.09 ppm 0.12 ppm 0.15 ppm 0.3 ppm 0.6 ppm --i=S of Stock Standard o.3 0.6 mLs of IntermediateStandard 0.15 0.3 0.6 0.9 1.2 1.5 - 1.2ppm 1.2 1.5ppm 1.5 5.0 E!QUTPMENT 5.1 SkalarSegmented Flow Auto Analyzer Sansp"'System equipped with ISE 6.0 TNTERFERENCES 6.1 High concentrationsof alkalinityc,hloride,phosphate,sulfateor ironcan cause interferences. 7.0 SAMPLE HANDLING 7.1 Samples should be storedinpolyethylenebottlesS.amples should be analyzed within30 days. 8.0 CALIBRATION AND STANDARDIZATION 8.1 Preparation of CalibrationStandards 8.1.1 Preparecalibratiosntandardsas in section4.3. 8.2 Calibration 8.2. 1 The standardsare analyzedatthebeginningof therun. 8.3 Storage Conditions for Standards 8.3. 1 Standards arestoredincapped polypropylenevolumetricflasks.New standardsareprepared ata n-dnimum of every sixmonths, oras necessary. 9.0 PROCEDURE 9.1 Start Up Procedure 9.1.1 Clamp down thepumpdecks, airbarsand sampler-pump tubing. 9.1.2 Put thefluorideelectrodesintheelectrodechamber. 9.1.3 Turn on thepower of the sampler,pumps, offsetpotentiometerand heating bath. 9.1.4 Put thereagent-lineisntheappropriatebottles. 9.1.5 Turn on the interfacec,omputer, displayand printer.Make sure you turn on the interfacebefore the computer. 9.1.6 Letthesystemstabilifzoerapproximatel3y0 minutes. 9.2 Startinga Run 9.2.1 Createa sample tableby selectingFILES, TABLE, and CREATE, typein the name of the filea,nd pressENTER. 9.2.2 Printthesample table,insertedinthe system tableby pushing ESC, PRINT, GROUP 1.This willprinttheentirerun. 9.2.3 Dialthesampler settingstotheappropriatenumber of samples,number of seconds forsample wash, and number of seconds forthe sample. 9.2.4 Fillthesample traywith thestandards,samples,washes and driftsr.W and FW/RUNOUT cups on the samplerdo not need tobe filled. 9.2.5 Set thebaseline. 3 9.2.5. 1SelectGRAPHICS, REAL TIME. Ifyou cannot getreal-timey,ou may be inthe Data Handling Panel.Switch totheAnalysisPanelby selectingCONTROL PANEL and pushing F7. 9.2.5. 2 Use thesmallscrewdriverfortheoffsetpotentiometertosetthe base line.Adjustthebaselineuntilitisapproximately3/4 inchfrom the bottom of thescreen. 9.2.5.3Check the higheststandardand adjustthegain,ifnecessary,with theinterfacescrew #3. 9.2.6 Go toCONTROL PANEL, and toanalysispanel.Deselectthe analysisthat willnot be run.(Selector deselectanalysisby pressingENTER.) PressTab toreturnto theAnalysisPanel. 9.2.7 Press thespacebar tobrin-up the localmenu. 9.2.8 SelectSTART tostartheanalysis. 9.2.9 Type your ID (initialst)h,esample tablewhich you createdunder 9.2.1(or pressENTER forchoices),choose runningwith or without thesystem table and selectSTART ANALYSIS. 9.2. 10 Afterstartintghe software,starthe sampler.Make surethatthe sampleris settotherightnumber of samples and thatthesample/wash/airtimesare OK. 9. 2.11 SelectGRAPHICS, REAL TIME toview theprogressof theanalysis. 9.3 Loading and Printing the Data-File 9.3.1 Go toCONTROL PANEL, pressthe spacebartobrincyup the localmenu and selecLtOAD. SelectAUTOCALCULATION and enterthefilename(or highlicrhtthefiletobe 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 SET'RINGS. Change themax y value toapproximately900. Go toCAL CURVE and pressESC, and Enter.PressPRINT SCREEN. 9.3.5 Return toSETMNGS and change the max valueback to4095,go toEDIT, pressENTER and PRINT SCREEN toprintsample peaks. 9.3.6 To printtheresults-o toCONTROL PANEL, SPACEBAR, OUTPUT, OUTPUT. SelectPRINTER forthe Epson orPRN for theLaser. 9.4 Shutdown 9.4.1 Put allthereagent-lineisnMilli-Qwater. 9.4.2 Let the system rinseforapproximately30 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, airbarsand sampler pump-tubinc, 9.4.6 SelectFILES, pressALT F and selectQUIT toexitthe program. 9.4.7 On Friday,turnoffthecomputer, displayand interfaceforthe weekend. 10.0 VALIDATION 10.1 Quality Control 10. 1.f Run a standard(mid tohi0ah concentratione)very 10 samples.Ifa sianificancthange inpeak heichtoccurs,only the samples beforethe last acceptablestandardwillbe used.The remainin- samples willbe reanalyzed. 4 10.2 Precisionand Accuracy 10. 2. 1See Method ValidationReport number ANMT-M-8.0.V 1 10.3 Other ValidationParameters 10.4 Referto Method ValidatioRneportNumber AMDT-M-8.0.Vl 11.0 DATA 11.1 11.2 11.3 11.4 ANALYSIS Calculations 11. 1.1 The standardcurve isplottedby the Skalarsoftware. 11. 1.2 All calculationasre done by the Skalarsoftware.r2 should be 0.995 or better. Prepare spreadsheetstosummarize data.Includesample volume, weights used etc. Write the study number on theprintoutsi,nitiald,atetheprintout,and bind together withallpackage documents and placeinthe studyfolder.Make a copy of the summary sheetand tapeintothestudynotebook.Back up alldataand spreadsheets onto study disk and backup disks. ElectroniDcata 11. 4.1 GLP studies:Electronicdam iscopied onto the Study floppydiskfor each study,and alsodataiscopied onto a floppydisk thatisstoredin thelab. 11. 4. 2 Other studiesA:ll dataiscopied onto a floppydiskthatisstoredinthe lab. 12.0 ATTACHMENTS None 13.0 REFERENCES 13.1 13.2 13.3 AMDT-M-1, Thermal Extractioonf Fluorideby Means of a ModifiedDohrmann DX2000 OrganicHalideAnalyzer-Liver SkalarMethods, #335, SkalarMethods Manual AMDT-EP-26, Operationand Maintenance of theSkalarSegmented Flow Analyzer 14.0 REVISTONS Revision Numbe Reason forchange Revision Date 5 9.3 QualityAssurance Unit Statement A=hrmntD GLP Study QualityAssuranceStatement Study Title:Single-doseDermal Absorption/ToxicityStudy of T-6051 and T-6054 in Rabbits Study Number. AMDT-013195.1 Name ofAuditor:Kari Rambo This study has been inspectedby theQualityAssurance Unit asindicatedinthefollowingtable. The findingswere reportedtothe studydirectoarnd management. InspectionDates EM 12 10/12/95 10/19/95 Phase FinalReport Date InspectionReported to Man D=t StudyDim 10/19/95 10/19/95 VA Auditor le-19-@5 Date 9.4 Key Personnel Involvedin the Study 3M Environmental Laboratory Key Personnel I Thermal extractiofnollowedby analysiussingOrion Ionanalyzer: Jim Johnson Deb Wright RichYoungblom Deann Plununer Analysisofliverextractussingelectrospramyass spectrometry: Jim Johnson Dave Christenson Thermal extractiofnollowedby analysiussingSkalarsegmentednow analyzerwithionselectiveelectrode: Jim Johnson Deb Wright RichYoungblom Deann Plununer Documentationand Reporting: Jim Johnson Rich Youngblom QualityAssuranceUnit: GaleVan Buskirk CynthiaWeber KariRambo 9.11 Data 9.11.1Summary and raw data;ug F- in whole liveras determinedby thermalextractiofnollowedby analysis using Orion ionanalyzer. Summary ofCombustion Data -Liver AMEDT-013195.1, HWI 6329-133 As Referenced in FinalReport section6.0DATA ANALYSIS Total ug FluorideinWhole Liver Mean per Dose Group* 99 Std.Dev. ControlGroup 16.2 + 5.5 0.128 mg/kg dose (T6054) 11.1 + 2.0 1.28mg/kg dose(T6054) 17.1 + 2.6 12.8mg/kg dose(T6054) 45.2 + 20.5 FabricExposure (T6051) 14.7 + 2.7 *Calculatedasthe mean oftriplicatseamples from each ofthreemale and threefemalerabbits. RPT1 33L.XLS FC129 AB ID liverblank-3 livesrpike-4 livesrpike-5 livesrpike-6 livesrpike-7 livesrpike-8 livesrpike-9 livebrlank-4 F52873-1 F52873-2 F52873-3 F52885-1 F52885-2 F52885-3 F52898-1 F52898-2 F52898-3 liverblank-1 liverblank-2 livesrpike-I livesrpike-2 F52878-1 F52878-2 F52878-3 F52883-1 F52883-2 F52883-3 F52967-1 F52967-2 F52967-3 F52887-1 F52887-2 liverblank-I liverspike-1 liverspike-2 liverspike-3 liverspike-4 liverspike-5 liverspike-6 F52887-3 F52893-1 F52893-2 F52893-3 % rcvry 83% 83% 102% 94% 84% 83% 91% 95% 69% 85% 83% 109% 101% 101% Actual ppm Finliver (WNV) 0.230 0.950 1.06 1.28 1.00 2.34 2.49 0.328 0.245 0.334 0.329 0.449 0.222 0.308 0.139 0.131 0.103 0.228 0.298 1.36 0.995 0.286 0.230 0.176 0.211 0.242 0.179 0.158 0.194 0.200 0.252 0.189 0.281 0.968 0.973 0.875 1.29 1.11 1.26 0.173 0.188 0.147 0.175 Average ppm Finliver (WNW) 0.303 0.326 0.124 0.231 0.211 0.184 0.205 0.163 Whole liver liver burned weight (grams) (grams) 0.140 0.132 0.118 0.121 0.142 0.108 0.101 0.116 0.121 74.2 0.107 74.2 0.104 74.2 0.150 69.2 0.114 69.2 0.108 69.2 0.150 73.3 0.148 73.3 0.146 73.3 0.152 0.134 0.101 0.145 0.105 70.2 0.150 70.2 0.151 70.2 0.115 73.8 0.107 73.8 0.135 73.8 0.144 61.5 0.118 61.5 0.108 61.5 0.108 70.6 0.128 70.6 0.129 0.108 0.132 0.143 0.128 0.138 0.121 0.148 70.8 0.133 63.5 0.135 63.5 0.135 63.5 TotalF- in whole liver (ug) 22.4 22.5 9.11 16.2 15.6 11.3 14.5 10.4 Dosage (mg/kg) 0.0 0.0 0.0 0.0 0.0 0.0 0.128 0.128 Page I RPT1 33L.XLS FC129 AB ID F52897-1 F52897-2 F52897-3 F52876-1 F52876-2 F52876-3 F52882-1 F52882-2 F52882-3 F52901-1 F52901-2 F52901-3 Liverbik 1 Liverbik2 Liverbik 1 Liverbik2 Liverbik 3 Liverspk-1 Liverspk-2 Liverspk-3 F52965-1 F52965-2 F52965-3 F52891-1 F52891-2 F52891-3 F52892-1 F52892-2 F52892-3 F52884-1 F52884-2 F52884-3 F52968-1 F52968-2 F52968-3 F52900-1 F52900-2 F52900-3 F52880-1 F5288G-2 F5288G-3 F52886-1 F52886-2 F52886-3 F52899-1 F52899-2 F52899-3 % rcvry 92% 89% 83% Actual ppm Fin liver (WM) 0.156 0.174 0.179 0.117 0.155 0.146 0.112 0.154 0.169 0.126 0.127 0.142 0.331 0.218 1.22 0.322 0.280 1.10 0.869 0.864 0.235 0.205 0.216 0.239 0.197 0.239 0.333 0.270 0.273 0.206 0.255 0.230 0.299 0.261 0.242 0.335 0.390 0.259 0.487 0.548 0.579 0.611 0.728 0.490 0.428 0.366 0.340 Average ppm Fin liver (WNW) 0.169 0.139 0.145 0.132 0.219 0.225 0.292 0.231 0.267 0.328 0.538 0.610 0.378 Whole liver liver burned weight (grams) (grams) 0.134 72.4 0.113 72.4 0.123 72.4 0.149 65.2 0.119 65.2 0.138 65.2 0.146 76.1 0.130 76.1 0.112 76.1 0.140 70.5 0.143 70.5 0.112 70.5 0.112 0.137 0.146 0.159 0.139 0.127 0.156 0.145 0.125 68.7 0.163 68.7 0.151 0.114 68.7 64.5 0.144 64.5 0.128 64.5 0.132 68.7 0.134 68.7 0.102 68.7 0.146 65.0 0.101 65.0 0.135 65.0 0.145 66.6 0.116 66.6 0.121 66.6 0.147 61.4 0.115 61.4 0.115 61.4 0.117 59.6 0.142 59.6 0.138 59.6 0.137 75.3 0.114 75.3 0.113 75.3 0.103 89.1 0.115 89.1 0.126 89.1 Total F- in whole liver (ug) 12.3 9.09 11.0 9.29 15.0 14.5 20.1 15.0 17.8 20.1 32.1 46.0 33.7 Dosage (mg/kg) 0.128 0.128 0.128 0.128 1.28 1.28 1.28 1.28 1.28 1.28 12.8 12.8 12.8 Page 2 RPTI 33L.XLS FC129 AB ID % rcvry livebrlank 1 livebrlank2 liverblank 3 liverspike-1 87% liverspike-2 0.7786 liverspike-3 0.7852 liverspike-4 0.9656 liverspike-5 0.8486 liverspike-6 82% F52889-1 F52889-2 F52889-3 F52894-1 F52894-2 F52894-3 F52895-1 F52895-2 F52895-3 F52874-1 F52874-2 F52874-3 F52879-1 F52879-2 F52879-3 F52963-1 F52963-2 F52963-3 F52877-1 F52877-2 F52877-3 F52888-1 F52888-2 F52888-3 F52966-1 F52966-2 F52966-3 Liverspike-7 60% Liverspike-8 71% Liverspike-9 75% Liverspike-10 98% Liverspike-11 103% Liverspike-12 101% Liverspike-I3 78% Liverspike-14 107% Liverspike-I5 102% Actual ppm Fin liver (W/W) 0.265 0.327 0.220 0.921 1.05 0.823 1.06 0.859 0.978 0.812 0.896 1.59 0.835 0.794 0.917 0.307 0.523 0.276 0.331 0.249 0.173 0.168 0.194 0.185 0.216 0.175 0.154 0.222 0.190 0.160 0.158 0.211 0.142 0.321 0.277 0.167 0.881 1.05 0.775 0.990 1.15 1.16 2.16 2.46 2.39 Average ppm Finliver (W/W) 1.10 0.849 0.368 0.251 0.182 0.182 0.191 0.170 0.255 liver burned (grams) 0.125 0.111 0.123 0.143 0.112 0.144 0.138 0.149 0.127 0.114 0.113 0.141 0.127 0.119 0.118 0.151 0.109 0.154 0.144 0.122 0.145 0.134 0.105 0.147 0.130 0.123 0.135 0.119 0.118 0.141 0.144 0.104 0.139 0.123 0.131 0.132 0.102 0.103 0.146 0.150 0.136 0.133 0.110 0.131 0.130 Whole liver weight (grams) 68.7 68.7 68.7 74.0 74.0 74.0 58.1 58.1 58.1 69.7 69.7 69.7 89.1 89.1 89.1 70.2 70.2 70.2 69.9 69.9 69.9 64.2 64.2 64.2 67.6 67.6 67.6 TotalF- in whole liver (ug) 75.4 62.8 21.4 17.5 16.3 12.8 13.3 10.9 17.3 Dosage (mg/kg) 12.8 12.8 12.8 Fabric Fabric Fabric Fabric Fabric Fabric Page 3 9.11.2Summary and raw data;analysisof liver extractsusing electrospramyass spectrometry. HWI 0 6329-133 S": ProtocolNumber: Test Matedal: matrix: R Squared Value: Response Factor Amount: Analyst: Date: Method: Instrument: LABBASE FILE SingleDoes Dermal Absorpbon TP3016.AB T-6054 & T-6051 inRabbits(FC-129) Uver 0.9889 1.15E-05 DLC 4aW AMDT-M-4 Fams VG 2000 ElectrosprayMS 0403950 A-1 10- 31 - Group Dome Group 1: Stedle Water 2.0mlkg Group 2: 0.128 mg /kg Group 3: 1.28 mgfkg Group 4: 12.8mgtkg Group 5: Fabric Sample F52883 F52M F52885 F52967 F52873 F52878 F52893 F52887 F52901 F52876 F52892 F52891 F52965 F52BB4 F52895 F52899 F52894 F52880 F52BB6 F52889 F52879 F52877 F52874 F52963 F52888 IonCount Extractedwt Area 9 Dilution Concentradon factor @W9 Totalmass of liver Totalamount of FC-95 per liver mg NID N/D NID N/D NID N/D N/D N/D N/D N/D N/D N/D N/D N/D N/D N/D N/D NIO N/D N/D N/D N/D N/D N/D N/D N/D N/D N/D N/D N/D 11413 1.0139 1 14922 1.0615 1 8191 1.1217 1 12572 1.0881 1 0.1033 0.1290 0.0670 0.1060 68.707 64.487 68.667 64.989 0.007 0.008 0.005 0.007 51584 1.117 1 58481 1.1894 1 127876 1.0236 1 58961 1.0931 1 84958 1.0915 1 93984 1.0615 1 0.4237 0.4511 1.1462 0.4949 0.7142 0.8124 58.094 89.078 73.983 59.624 75.341 68.670 0.025 0.040 0.085 0.030 0.054 0.056 16734 1.2566 1 26249 1.0106 1 N/D 9735 1.0772 1 N/D 0.1222 0.2383 N/D 0.0829 N/D 89.125 69.913 70.161 0.011 0.017 N/D 0.006 N/D The concentrationwas calculatedby usingthe standard curve and multiplyingthe resultby 4/5.The 415 factortothe resultofa miscalculationInapplyingformula 8.4InMethod AMDT-M-". 137 mg of liverwas used In thiscalculationratherthanI7l mg. Th* concentradons Inthe standard curve are therefore5/4largerthan they should be. By multiplyingthe calculatedconcentrationIn the standard curve by 415,the correctresultIsobtained. HWI # 6329-133 Study: ProtocolNumber: Test Material: Matrix: R Squwod Value: Response FactorAmount: Analyst: Date: Method: Instrument: LABBASE FILE SingleDose Dermal Absorption TP3016.AB T-6054 & T-6051 inRabbits(FC-129) Uver 0.9946 9.1OE-06 DLC 4/"5 AMDT-M-4 Fisons VG 2000 ElectrospraMyS 040495B Group Dose Group 2: 0.128 mg /kg Group 3: 1.28 mgtkg Group 5: Fabric Sample F52897 F52882 Ion Count Area 18736 N/D Extractedwt 9 1.1033 1.1025 Dilution factor Concentration Totalmass of pgtg liver 9 1 0.1236 72.433 1 N/D 76.060 Totalamount of FC-95 per liver mg 0.009 N/D F52900 16432 1.3161 1 F52968 27181 1.2011 1 0.0909 0.1648 61.356 66.642 0.006 0.011 F52966 8521 1.1457 1 0.0542 67.02 0.004 Administered as a lo.ocm x 10.0cm pieceof testfabric The concentrationwas calculatedby using the standardcurve and multiplyingthe resultby 4/5. The 415 factorIsthe resultof a miscalculationInapplyingformula8.4InMethod AMDT-M4-0. 137 mg of livewras used In thiscalculationratherthanl7l mg. The concentrationsInthe standard curve are therefore514 largerthan they should be. By multiplyingthe calculatedconcentrationInthe standard curve by 4/5,the correctresultIsobtained. DL(-'LIV Sample DL(:LIV Operator DLC Run (-late 05-()@)-1995 06:49:44 Ver:3ioti: 12 Printed on 05-09-1995 AT 06:49:56 Straight Line Fit forced throuqii ()ri(7in. --------------------------------- Ld 47 R,EVEL 1 3 AMOUNT ----------- 0.4000 -1.8000 1.2000 Component 1 EXTERNAL STANDARD CALIBRATICDN AREA --------- 47743 85693 132059 Y SL(DPE x + INTERCEPT --------------------------------------------------- Area Amount = 1.098SE-*05 9.1012E-06 Amount Area + O.OUOOE+00 + O.OOOOE-00 R s,luare@J = 0.9946 FILF-- o,4o-6q,so F- iZct 5 1 F-- D I.C- L .1k) iJpierat(3r LLC % f-.'iljina-te 05-()9-1995 O."@-:4'4:4V5erziori: L;'p Printed an 05-09-1995 AT 07:24:56 Straight Line Fit forced throu-.IhOr*iiiii-i. - - - - - - - - - - - - - - ---- - - - - - - - - - - - - - - - -- A-4 x C) m 74069 1.2 EVEL 1 2 3 My' AMOUNT ----------- 0.4000 0.8000 1.2000 Component EXTERNAL STANDARD AREA --------- 33695 74069 102142 l@ u CALIBRATION SLOPE x + INTERCEF'T --------------------------------------------------- Area m 8.7189E+04 @ln-ioijrit 1.1469E-05 R s-qijartz--=,J0.9889 Amount Area + O.OOOOE+00 + O.OOOOE+00 6,32q- 1'33 Fc-i2g COPY,.F C tFlifid,.-.'640395DTJ LAB-BASE - The MS Data System Sample:6329-133 RABBIT LIVER EXTRACTS; FC-129 (AB) 040395D ZFS- 777 9 5 085 616 39 25 22 3778 1 37 03 4 44 4 830644' JIL 1245 ZFSlsen 223935112570056788A655I03 925 616 5 -.1880 127816 5848 2 %OF 34489 1 "5L 27 4 22A7,0 -2-o's, i@Gi -0- 3 Cie@ 3500 26 84958-7515 43 k4O9L@'4j594L 4000 45'oo *5 File:040395D LAB-BASE The NS Data Sgsten c Sample:6329-133 RABBIT LIVER EXTRACTS; FC-129 (AB) 040395D Los. 319 3 9 j@ 4 J86 146_L66 ZFSisen 38577 49 498 72382 66 7489 596 660 LLi x2.i 95 1218235 P? 14 a 612 95 235768 see lose 12 b0 1 File:040395D LAB-BASE The NS Data System 03 Sample:6329-133 RABBIT LIVER EXTRACTS; FC-129 (AB) 040395D 1 57 loo jL g IS 6. w- 15 co %r 1 2145 2335 2 + 29 it 100- 98 12 A6 2 51584 15 7 99233 I 11959?522?2 Son -1610-0- lose 58481 19 5 age 16734 2305 22'61i 24iiii@- 2@Cili 0- File:840395D Sample:6329-133 040395D LAB-BASE The MS Data System RABBIT LIVER EXTRACTS; FC-129 (AB) iss- 31 31 9 34 8 3a 32 37 43 5 4 JL V.FS- t4 Uk t4 03 4 94 JLOG- 34489 32 8 x4.9 ZFS- 5325 15 i@,io 386i 0 "tit .4iicili 84958 43 6 14@Cii 75JL5 44 44F6i 0 File:040395D Sample:63Z9-133 040395D .too- 925 ZFS- 46 7 5 4656 LAD-BASE - The MS Data System RABBIT LIVER EXTRACTS; FC-129 (AB) 4E 9 4 7 5J.48 L 53 5 54 7 3 r4 261016 46 LNp, L 93984 53 4 x6.9 03 5 65 0 + ip ip ZFS- lson 11413 4676 4600 14922 4839 4800 5006 8191 51 6 @2@i6 9735 52 12572 5650 A 375 :166 @;ii6 tkVZ 432ck e3 63Zct 1143 E C o py of lnigal F'32'1 (II f:52 SSZ F'52li66 File:0410495B Sample:6329-133 840495B Is@- LAB-BASE - The MS Data System o@ EFC-1291 & 6329-143 IFC-1367F] RABBIT LIVER E) 19616776 22 6 7297995 14 9 0932777 26 6 1 ZFS1850346 37@lgg 611", 7 427 Is 394 410 v.FS- 36 41 62 7 49 7 19 1253 70J6LSt 203 3 2k I 2v'Wee 1862 2 4 1534 17 7 21 22 6 322 28-41 3 2E 2 3 isen 500 1000 1500 2800 2500 3000 0 File:048495B Sample:6329-133 040495B 161883 2841 LAB-BASE [FC-1291 & 63Z9-143 213251 30 5 xJL. 9 322568 3163 zF-S- 222848445 39270 3121 \ The MS Data [FC-1367F] (4. Ul@! 26429 33-61JL System 84 RABBIT LIVER EX 454 37 lei 3!546 42976 38 36 100- ............. 28-42- . v.FS- 3086 3164 37 32 35 7 Son 2800 3000 3200 3400- 3600 9.11.3Summary and raw data;ug F- inwhole liveras determinedby thermalextractiofnollowedby analysis using Skalarsegmented flow analyzerwith ion selectivelectrode. This data,althoughsupportivei,ntheopinionof the Study Directorisnot requiredto reachthe conclusion statedin section6.0 and thereforeisnot discussedin detail. RE: 6329-133 LIUER SRMPLES RMDT 13195.1 Date of Rnalysis:3/28, 3/29 and 3/30/95 Rnalyst: ODW 'li@rlcis The samples are burned in the Bohrman at 950 C using between 0.1 and 8.2 grams of the liver.The gas iscollectedin 1.0 mL of .1:1TISHB/Milli-Owater then an additional2 mL of 1:1 TISRB/Milli-Oisadded to allow for sufficientvolume for Skalar analysis.The samples are then analyzed on a Skalar Segmented Flow Rnalyzer 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 ion selectiveelectrodeand the reference electrode ismeasured. The signalisamplifiedand relatedto the fluorideconcentration. The instrument was calibratedin the ranges of 0.015 - 0.15 ppm and 0.15 - 1.50 ppm fluoride.The standard curve for the high range was plotted using the inverse logarithm option.The standard curve for the low range is linear.Rilstandards and samples were then calculatedby the Skalar software using these curves. Hilresultsbelow 0.0081 ppm appear on the raw data as #.####. R qualitycontrolstandard was analyzed every 10 samples to check for accuracy and drift. Raw data istaken from the appropriate calibratedrange of the Skalar printoutand summarized on an Excel spreadsheet. The finalresultsare adjusted for the collectionvolume and any subsequent dilutions. SUMMARY of 6329-133 LIVER SAMPLES AM DT 013195.1 -------------------------------------------------------------------- Thisisonlya partiasltudy.Allsampleswere notsavedfrom theDohrmann analysis.DDW F52884-1 ND F52884-2 ND F52884-3 ND 3.0 0.1463 ND 3.0 0.1011 ND 3.0 0.1345 ND F52891-1 ND F52891-2 ND F52891-3 ND 3.0 0.1140 ND 3.0 0.1443 ND 3.0 0.1284 ND GROUP3 Dose Level: 1.28mgtkg F52892-1 0.02 F52892-2 ND 3.0 0.1322 0.43 3.0 0.1342 ND F52900-1 ND F52900-2 ND F52900-3 ND 3.0 0.1474 ND 3.0 0.1153 ND 3.0 0.1150 ND F52965-1 0.02 F52965-3 0.02 3.0 0.1246 0.40 3.0 0.1510 0.31 F52968-1 ND F52968-2 ND F52968-3 ND 3.0 0.1448 ND 3.0 0.1159 ND 3.0 0.1211 ND 64.9989 ND 64.9889 64.9889 64.4869 ND 64.4869 64.4869 0.20 68.7067 68.7067 61.3555 ND 61.3555 61.3555 0.36 68.6665 68.6665 66.6415 ND 66.6415 66.6415 133-L.SUM SUMMARY of 6329-133 LIVER SAMPLES AM DT 013195.1 GROUP4 Dose Level: 12.8mp,/kg F52889-1 0.04 F52989-2 0.05 F52889-3 0.10 F52894-1 0.05 F52894-2 0.06 F52894-3 0.05 F52895-1 0.03 F52895-2 0.03 F52895-3 0.02 F52874-1 0.02 F52874-2 0.02 F52874-3 0.02 GROUPS F52879-1 0.02 Dose Level 10.0cm x 10.0cm fabric F52879-2 ND F52888-1 ND F52963-2 ND F52963-3 ND 3.0 0.1139 1.17 3.0 0.1125 1.34 3.0 0.1410 2.18 3.0 0.1272 1.20 3.0 0.1183 1.43 3.0 0.1185 1.17 3.0 0.1513 0.62 3.0 0.1091 0.87 3.0 0.1537 0.44 68.6699 1.56 68.6699 68.6699 73.9834 1.27 73.9834 73.9834 58.0944 0.65 58,0944 58.0944 3.0 0.1438 0.46 3.0 0.1221 0.50 3.0 0.1454 0.35 3.0 0.1338 0.34 3.0 0.1050 ND 3.0 0.1438 ND 3.0 0.1228 ND 3.0 0.1345 ND 69.6893 0.44 69.6893 69.6893 0.17 89.1246 89.1246 ND 64.2262 ND 70.1610 70.1610 133-L.SUM -------------------- I Tracer 1.50 1.20 800/0 2 Drift 1.50 1.23 82% 3 Wash ND 4 Std 1 0.015 0.016 104% 5 Std 2 0.03 0.03 101% 6 Std 3 0.06 0.06 97% 7 Std 4 0.09 0.09 99% 8 Std 5 0.12 0.13 104% 9 Std 6 0.15 0.15 98% 10 Std 7 0.30 0.29 98% 11 Std 8 0.60 0.60 101% 12 Std 9 1.20 1.23 102% 13 Sid 10 1.50 1.47 98% 14 Drift 1.50 1.25 83% is Wash ND 16 Blk-I ND 3 17 Blk-2 ND 3 18 Spk-I ND 3 19 Spk-2 ND 3 20 Spk-3 0.06 3 21 F52900-1 ND 3 22 F52900-2 ND 3 23 F52900-3 ND 3 24 F52968-1 ND 3 25 F52968-2 ND 3 26 Drift 1.50 1.26 84% 27 Wash ND 28 F52968-3 ND 3 29 F52884-1 ND 3 30 F52884-2 ND 3 31 F52884-3 ND 3 32 F52892-2 ND 3 33 F52548-2 ND 3 34 F52888-1 ND 3 35 F52963-2 ND 3 36 F52963-3 ND 3 Li33-I.XLS 0.1474 ND 0.1153 ND 0.1150 ND 0.1448 ND 0.1159 ND 61.3555 ND 61.3555 ND 61.3555 ND 66.6415 ND 66.6415 ND ND 0.1211 ND 0.1463 ND 0.1011 ND 0.1345 ND 0.1342 ND ND 0.1438 ND 0.1228 ND 0.1345 ND ND 66.6415 ND 64.9889 ND 64.9889 ND 64.9889 ND 68.7067 ND ND 64.2262 ND 70,1610 ND 70.1610 ND Page 1 0.004 63 0.004 63 0.004 63 ------------- 37 38 39 Bik Drift Wash 1.50 ND 1.26 84% ND k;@- 3 Ll33-I.XLS Page 2 A L133-2.XLS --- ------------------------- ------------------------------ I Tracer 1.50 1.21 81% 2 Drift 1.50 1.23 82% 3 Wash ND 4 Std 1 0.015 0.016 108% 5 Std 2 0.03 0.03 98% 6 Std 3 0.06 0..0578 7 Std 4 0.09 0.09 100% 8 Std 5 0.12 0.12 104% 9 Std 6 0.15 0.15 98% 10 Std 7 0.30 0.29 98% 11 Std 8 0.60 0.60 101% 12 Std 9 1.20 1.23 102% 13 Std 10 1.50 1.47 98% 14 Drift 1.50 1.25 83% 15 Wash ND 16 Bik-I 0.07 3.0 17 Blk-2 0.02 3.0 18 Blk-3 0.02 3.0 19 Spk-I 0.05 3.0 0.004 63.00 20 Spk-2 0.05 3.0 0.004 63.00 21 Spk-3 0.05 3.0 0.004 63.00 22 F52891-1 ND 3.0 0.1140 ND 64.4869 ND 23 F52891-2 ND 3.0 0.1443 ND 64.4869 ND 24 F52891-3 ND 3.0 0.1284 ND 64.4869 ND 25 F52892-1 0.02 3.0 0.1322 0.43 68.7067 29.31 26 Drift 1.50 1.25 83% 27 Wash ND 28 F52965-1 0.02 3.0 0.1246 0.40 68.6665 27.78 29 F52965-3 0.02 3.0 0.1510 0.31 68.6665 21.15 30 Drift 1.26 31 Wash ND Page I Ll 33-3.XLS fffff#Fffafffm=ins m---- I Tracer 1.50 1.25 83% 2 Drift 1.50 1.28 85% 3 Wash 0.02 4 Sid 1 0.015 0.018 123% 5 Sid 2 0.03 0.03 88% 6 Sid 3 0.06 0.06 100% 7 Sid 4 0.09 0.09 98% 8 Sid 5 0.12 0.12 104% 9 Sid 6 0.15 0.15 98% 10 Sid 7 0.30 0.29 96% 11 Sid 8 0.60 0.61 101% 12 Sid 9 1.20 1.24 104% 13 Sid 10 1.50 1.46 97% 14 Drift 1.50 1.31 87% 15 Wash 0.02 16 Bik-I 0.02 17 Blk-2 0.02 18 Blk-3 ND 19 Spk-I 0.06 20 Spk-2 0.05 21 Spk-3 0.05 22 Spk4 0.06 23 Spk-5 0.06 24 Spk-6 0.06 25 F52874-1 0.02 26 Drift 1.50 1.27 84% 27 Wash 0.02 28 F52874-2 0.02 29 F52874-3 0.02 30 F52879-1 0.02 31 F52879-2 ND 32 F52889-1 0.04 33 F52889-2 0.05 34 F52889-3 0.10 35 F52894-1 0.05 36 F52894-2 0.06 3.0 3.0 3.0 3.0 0.004 63.00 3.0 0.004 63.00 3.0 0.004 63.00 3.0 0.004 63.00 3.0 0.004 63.00 3.0 0.004 63.00 3.0 0.1438 0.46 69.6893 32.13 3.0 0.1221 0.50 69.6893 34.93 3.0 0.1454 0.35 69.6893 24.59 3.0 0.1338 0.34 89.1246 30.17 3.0 0.1050 ND 89.1246 ND 3.0 0.1139 1.17 68.6699 80.31 3.0 0.1125 1.34 68.6699 91.74 3.0 0.1410 2.18 68.6699 149.47 3.0 0.1272 1.20 73.9834 88.64 3.0 0.1183 1.43 73.9834 105.44 Page 1 PA ----------------- 37 F52894-3 0.05 38 Drift 1.50 1.28 85% 39 Wash 0.02 40 F52895-1 0.03 41 F52895-2 0.03 42 F52895-3 0.02 43 Drift 1.50 1.31 87% 44 Wash Ll 33-3.XLS --------------------------------------------- 3.0 0.1185 1.17 73.9834 96.91 3.0 0.1513 0.62 58.0944 36.17 3.0 0.1091 0.87 58.0944 50.64 3.0 0.1537 0.44 58.0944 25.74 Page 2 1995-03-28 17:30 OutPut of : 95032SPI Software version 6.1 cl990,93 Operator Date of the Analysis : DDW : 1995-03-28 15:28 Analysis File Name : C:\SKALAR\DATA\950328Fl i3tcks.l Fluoride 1.5 Calibration order = Inverse Logarithm Slope : a = #.##### Result 6 x - cl -@ 0 Aaid&i a = 105 s i x = corrected value of the sample cl = corrected value of the concentration 1 s = Slope of the electrode a2 al aO - -0.00000 0.00092 -1.24810 Fluoride L Calibration order = 2 Correlation r = 0.99716 Result a2 x* + al * x + aO a2 = al = aO = 0.00000 0.00022 0.00604 Sampler Type Number Sample Time Wash Time Air Time Take up special needle Height SA1000 :1 : 50 sec. : 120 sec. : 1 sec. : Single : None : 70 mm. Diluter needle Height : 80 mm dilution Factor : 10 dilution Volume : 2.5 ml. Resample :1 Dilution runs :1 User file : TXT Reproces : No 1995-03-28 17:30 OutPut of Fluoride 1.5 Path number Signal type Decolor system Number dilute Resample dil Threshold diG output Window event 3 Debubbled Yes 0 No No 4095 0 Off sl standard s2 standard s3 standard s4 standard ss 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 t 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 950328FI 1995-03-28 17:30 OutPut of sl standard : 0.015 s2 standard : 0.030 s3 standard : 0.060 s4 standard : 0.090 s5 standard : 0.120 s6 standard : 0.150 s7 standard : Ignore S8 standard : Ignore S9 standard : Ignore slO standard : Ignore Order : 2 Dimension : PPM start value 500 DU trigger Limit 1800 Sec Peak shape Pointed start ignore 60 Sec eNd ignore 120 Sec Measure window 75 Filter No Regeneration No formula output C4:=c3 950328PI 199S-03-28 17:30 OutPut of : 9SO328Fl Fluoride l.S Fluoride L Page 1 of 3 PPM PPM Poo Typ Ident Dil Weight Ch Result F Cor. Valu Time wt iw Initial Wash 1 1.000 3 O.OS6 4 0.0060 0 219 65 0 0 0 1t Tracer 1 1.000 3 1.199 4 0.9756 2065 2282 203 2065 0 0 2d Drift 1 1.000 3 1.230 4 0.9981 2096 2312 380 2096 0 0 3w Wash 1 1.000 3 0.056 4 0.0060 0 214 557 0 0 0 -rV,- 7- -rv- i.-Lpt-- 4 sl 5 s2 Standard 1 Standard 2 1 1.000 3 0.062 4 0.0156 1 1.000 3 0.070 4 0.0303 43 258 732 43 0 0 106 322 907 106 0 0 6 s3 Standard 3 1 1.000 3 0.088 4 0.0579 214 432 1081 214 0 0 7 s4 8 s5 Standard 4 Standard 5 1 1.000 3 0.109 4 0.0893 1 1.000 3 0.135 4 0.1250 325 544 1257 325 0 0 440 661 1432 440 0 0 9 s6 10 s7 11 s8 Standard 6 Standard 7 Standard 8 1 1.000 3 0.152 4 0.1469 1 1.000 3 0.293 4 0.2979 1 1.000 3 0.604 4 0.5548 506 728 1606 506 0 0 895 1121 1781 895 0 0 1410 1641 1956 1410 0 0 12 s9 Standard 9 13 slO Standard 10 14 d Drift 15 w Wash 1 1.000 3 1.229 4 0.9974 1 1.000 3 1.473 4 1.1797 1 1.000 3 1.248 4 1.0106 1 1.000 3 0.056 4 0.0060 2095 2332 2132 2095 0 0 2335 2576 2305 2335 0 0 2113 2338 2482 2113 0 0 0 226 2661 0 0 0 -T4,L 1995-03-28 17:30 Output of : 95032SP1 Fluoride 1.5 Fluoride L Page 2 3 PPM PPM Pon Typ Ident Dil Weight Ch Result P Cor. Valu Time 16 u BLK 1 1 1.000 3 too e < 14 240 2779 4 0.0091 14 0 0 Wb 17 u BLK 2 1 1.000 3 Absen A -32 194 3007 Pb 4 #.#### -32 0 0 18 u SPK 1 1 1.000 3 Absen A -23 202 3182 4 0.0011 -23 0 0 Pb 19 u SPK 2 1 1.000 3 Absen A -33 192 3357 4 #.#### -33 0 0 20 u SPK 3 1 1.000 3 0.087 4 0.0566 209 436 3533 209 0 0 21 u F52900-1 1 1.000 3 too 1 > 17 242 3765 4 0.0098 17 0 0 22 u F52900-2 1 1.000 3 too e < -1 224 3820 4 0.0058 -1 0 0 23 u F52900-3 1 1.000 3 Absen A -33 192 4057 4 #.#### -33 0 0 24 u F52968-1 1 1.000 3 0.058 4 0.0087 12 236 4184 12 0 0 25 u F52968-2 1 1.000 3 Absen A -32 192 4407 vb 4 #.#### -32 0 0 26 d Drift 1 1.000 3 1.259 4 1.0187 2124 2348 4581 2124 0 0 27 w Wash 1 1.000 3 0.056 4 0.0060 0 224 4751 0 0 0 28 u F52968-3 1 1.000 3 too e < 32 256 4871 4 0.0131 32 0 0 29 u F52884-1 1 1.000 3 0.061 4 0.0133 33 256 5089 33 0 0 30 u 31 u F52884-2 F52884-3 1 1.000 3 0.061 4 0.0135 1 1.000 3 0.061 4 0.0135 34 256 5282 34 0 0 34 256 5435 34 0 0 01 c A)D 1995-03-28 17:30 output of : 95032SF1 Fluoride 1.5 Fluoride L PPM PPM Poo Typ Ident Dil weight Ch Result F Cor. Valu Time 32 u 33 u F52892-2 F52548-2 1 1.000 3 0.061 4 0.0144 1 1.000 3 0.061 4 0.0147 38 260 5635 38 0 0 39 260 5805 39 0 0 34 u 35 u F52888-1 F52963-2 1 1.000 3 0.061 4 0.0140 1 1.000 3 0.061 4 0.0140 36 256 5983 36 0 0 36 256 6159 36 0 0 36 u 37 u F52963-3 1 1 SA,r@ 1.000 1.000 3 0.061 4 0.0140 3 Absen A 4 0.0011 36 256 6333 36 0 0 -23 196 6506 -23 0 0 38 d 39 w Drift Wash 1 1.000 3 1.257 4 1.0172 1 1.000 3 0.056 4 0.0060 2122 2340 6684 2122 0 0 0 218 6858 0 0 0 wt rw RunOut Wash 1 1.000 3 0.056 4 0.0060 0 0 7159 0 0 0 Page 3 of 3 .A-t 'AID .0 %4"L- ,vp OJ-L- .0 1c-)L Raw ctats of SS032WI : FILsorida I.S 10 0 Esc-F-Xit I Fl-Help I Crtl@P-Edit peaks T i.. I 437S 4000 Raw data of SSCK328FI FILsorlds 1.5 L,v 0 2423 F-sc-Exit I Fl-Help @L%3 I Crtl-P-Edit peaks Ti.. I 6798 - 1.8804347- Calibraticounrveof 9503213FFIluorid1e.5 4 L. c O.OS64972 0 Order Inverse Logerithn MaasLred 400i) s 3381 2.E=1643. Calibration curve of SSCG28FI Fluoride L 5 1 C3 0.0060376 Order 2 Measured 4000 r 0.98716 1995-03-29 12:46 OutPut of : 950329Al Software version 6.1 cl990,93 Operator : DDW Date of the Analysis : 1995-03-29 10:24 Analysis File Name : C:\SKALAR\DATA\950329Al Fluoride 1.5 Calibration order = Inverse Logarithm .@lope : B = #.##### Result 6 x - cl c@ 0 didddd 0 = 10& s i x = corrected value of the sample cl = corrected value of the concentration i s = Slope of the electrode a2 m al m aO = -0.00000 0.00087 -1.21638 Fluoride L Calibration order =2 Correlation r = 0.99645 Result a2 = al aO - = a2 x* + al * x + aO 0.00000 0.00020 0.00843 Sampler Type Number Sample Time Wash Time Air Time Take up special needle Height : SA1000 :1 : 50 sec. : 120 sec. :1 sec. : Single : None : 70 mm. Diluter needle Height : 80 mm dilution Factor : 10 dilution Volume : 2.5 ml. Resample :1 Dilution runs :1 User file : TXT Reproces : No 1995-03-29 12:46 OutPut of Fluoride 1.5 Path number Signal type Decolor system Number dilute Resample dil Threshold diG output Window event 3 : Debubbled : Yes :0 : No : No : 4095 :0 : Off sl standard : Ignore s2 standard : Ignore s3 standard : Ignore s4 standard : Ignore S5 standard : Ignore s6 standard : 0.150 s7 standard : 0.300 S8 standard : 0.600 S9 standard : 1.200 slO standard : 1.500 Order : Inverse Logarithm Dimension : PPM start Value : 500 DU trigger Limit : 1800 Sec Peak shape : Pointed start ignore : 60 Sec eNd ignore : 120 Sec Measure window : 75 Filter : No Regeneration : No formula output Fluoride L Path number Signal type Decolor system Number dilute Resample dil Threshold diG output Window event 0 : Debubbled : No :0 : No : No : 4095 :0 : Off 950329AI 1995-03-29 12:46 output of sl standard : 0.015 s2 standard : 0.030 s3 standard : 0.060 s4 standard : 0.090 ss standard : 0.120 s6 standard : 0.150 s7 standard : Ignore s8 standard : Ignore S9 standard : Ignore sio standard : Ignore order : 2 Dimension : PPM start Value 500 DU trigger Limit 1800 Sec Peak shape Pointed start ignore 60 Sec eNd ignore 120 Sec Measure window 75 Filter No Regeneration No formula c4:=c3 output 950329Al 7-1 1995-03-29 12:46 output of : 950329AI Fluoride 1.5 Fluoride L PPM PPM Pos Typ Ident Dil Weight Ch Result F Cor. Valu Time wt iw Initial Wash 1 1.000 3 0.061 4 0.0084 0 216 65 0 0 0 1t 2d 3w Tracer Drift Wash 1 1.000 3 1.209 4 1.1307 1 1.000 3 1.234 4 1.1510 1 1.000 3 0.061 4 0.0084 2068 2285 208 2068 0 0 2091 2308 382 2091 0 0 0 218 555 0 0 0 4 sl Standard 1 1 1.000 3 0.065 4 0.0162 37 258 732 37 0 0 5 s2 6 s3 Standard 2 Standard 3 1 1.000 3 0.073 4 0.0294 1 1.000 3 0.091 4 0.0578 96 320 901 96 0 0 208 436 1086 208 0 0 7 s4 Standard 4 1 1.000 3 0.112 4 0.0902 320 552 1260 320 0 0 8 s5 9 s6 Standard 5 Standard 6 1 1.000 3 0.136 4 0.1244 1 1.000 3 0.152 4 0.1470 425 660 1436 425 0 0 489 728 1608 489 0 0 10 s7 11 s8 Standard 7 Standard 8 1 1.000 3 0.293 4 0.3207 1 1.000 3 0.603 4 0.6246 893 1136 1784 893 0 0 1415 1664 1957 1415 0 0 12 s9 Standard 9 1 1.000 3 1.228 4 1.1457 2085 2342 2131 2085 0 0 13 siO Standard 10 14 d Drift 1 1.000 3 1.474 4 1.3519 1 1.000 3 1.247 4 1.1617 2308 2570 2309 2308 0 0 2103 2356 2483 2103 0 0 15 w Wash 1 1.000 3 0.061 4 0.0084 0 256 2663 0 0 0 Page -ro -TV 1 of 3 1.995-03-29 12:46 output of : 950329Al Fluoride 1.5 Fluoride L Page 2 of 3 PPM PPM Poe Typ ident 16 u Dil Weight Ch Result F Cor. Valu Time. W060-0-:@aLf BLK 1 1 1.000 3 0.100 4 0.0709 255 512 2829 ZJ. ixrl-% 255 0 0 17 u BLK 2 1 1.000 3 0.070 4 0.0230 68 321 3010 68 0 o .3s% 18 u BLK 3 1 1.000 3 0.067 4 0.0186 48 300 3185 48 0 0 19 u SPK 1 1 1.000 3 0.086 4 0.0503 180 432 3357 180 0 0 20 u SPK 2 1 1.000 3 0.087 4 0.0519 186 436 3533 186 0 0 21 u SPK 3 1 1.000 3 0.085 4 0.0485 173 422 3711 173 0 0 22 u F52891-1 1 1.000 3 Absen A 13 260 3883 4 0.0111 13 0 0 o 3 3 -3 23 u F52891-2 1 1.000 3 Absen A 13 258 4058 4 0.0111 13 0 0 24 u F52891-3 1 1.000 3 0.064 4 0.0142 28 272 4231 28 0 0 25 u 26 d F52892-1 Drift 1 1.000 3 0.067 4 0.0188 1 1.000 3 1.252 4 1.1653 49 292 4410 49 0 0 2107 2348 4584 'TV 2107 o 0 27 w 28 u Wash F52965-1 1 1.000 3 0.061 4 0.0084 1 1.000 3 0.066 4 0.0168 0 240 4747 0 0 0 40 280 4938 40 0 0 29 u 30 d 31 w F52965-3 Drift Wash 1 1.000 3 0.065 4 0.0155 1 1.000 3 1.263 4 1.1752 1 1.000 3 0.061 4 0.0084 34 274 5110 34 0 0 &o@46 S 2118 2358 5284 -rV-.i.-L 2118 o 0 0 240 5455 0 0 0 1995-03-29 12:46 OutPut of : 950329AI Fluoride 1.5 Fluoride L PPM PPM Poo Typ Ident Dil Weight Ch Result F Cor. Valu Time wt rw RunOut Wash 1 1.000 3 0.061 4 0.0084 0 256 S759 0 0 0 Page 3 of 3 R40C)o FLIDW cliato of 95032SAt Fluoride 1.5 0 nK1m2zE3mmL-- 14 CIO '7 0 Ese-Exit Fl-Help I Crtl-P=Edit Peaks T 1.. I 4375 Z5 4000 Raw date of SS032SAI FILmoricia 1.5 l@64 C) 0 423 Tine 6798 Ese-Fxit 3 Fl-Help I Ortl-P-Edit Peaks off 2.4168969- Calibration curve of SS032SAI @miiriviTNTr@ s Fluoride t.S 4 6 0.0607697 Order M..urd Inverse Logarithn 4006 s 3()34 2,-7 3.4480882-- Calibration curwe of SSM28AI Fluoride L c ci 0.0084278 a Order 2 Measured 4000 r 0.98645 1995-03-30 10:59 output of : 95033OAl Software version 6.1 C1990,93 Operator : DDW Date of the Analysis Analysis File Name : 1995-03-30 08:43 : C:\SKALAR\DATA\95033OAl cai -T@ ,akct -,33 Fluoride 1.5 Calibration order = Inverse Logarithm Slope Result : s = #.##### 6 x - cl l@ a dclag&A a = 101 s i x = corrected value of the sample cl = corrected value of the concentration 1 s = Slope of the electrode a2 = al m aO = -0.00000 0.00088 -1.12348 Fluoride L Calibration order n 2 Correlation r = 0.99781 Result = a2 x* + al * x + aO a2 = al = aO = 0.00000 0.00033 0.01842 Sampler Type Number Sample Time Wash Air Time Time Take up special needle Height SA1000 1 : 50 sec. : 120 sec. : 1 sec. : Single : None : 70 mm. Diluter needle Height dilution Factor dilution Volume Resample Dilution runs 80 mm 10 2.5 ml. 1 1 User file : TXT Reproces : No -ZC( 1995-03-30 10:59 OutPut of Fluoride 1.5 Path number Signal type Decolor system Number dilute Resample dil Threshold diG output Window event 3 : Debubbled : Yes :0 : No : No : 4095 :0 : Off sl standard : Ignore s2 standard : Ignore s3 standard : Ignore s4 standard : Ignore ss standard : Ignore s6 standard : 0.150 s7 standard : 0.300 s8 standard : 0.600 S9 standard : 1.200 slO standard : 1.500 Order : Inverse Logarithm Dimension : PPM start Value : 500 DU trigger Limit : 1800 Sec Peak shape : Pointed start ignore : 60 Sec eNd ignore : 120 Sec Measure window : 75 Filter : No Regeneration : No formula output Fluoride L Path number Signal type Decolor system Number dilute Resample dil Threshold diG output Window event 0 : Debubbled : No :0 : No : No : 4095 :0 : Off 95033OAl 1995-03-30 10:59 OutPut of sl STandard 0.015 s2 standard 0.030 s3 standard 0.060 s4 standard 0.090 S5 standard 0.120 s6 standard s7 standard 0.150 Ignore s8 standard Ignore S9 standard slO standard Ignore Ignore order : 2 Dimension : PPM start Value : 500 DU trigger Limit : 1800 Sec Peak shape : Pointed start ignore : 60 Sec eNd ignore : 120 Sec Measure window : 75 Filter : No Regeneration : No formula C4:=c3 output 95033OAl 1995-03-30 10:59 OutPut of : 95033OAl Fluoride 1.5 Fluoride L PPM PPM Poo Typ Ident Dil Weight Ch Result F Cor. Valu Time wt iw Initial Wash 1 1.000 3 0.075 4 0.0184 0 155 65 0 0 0 1t Tracer 1 1.000 3 1.248 2101 2274 211 4 b.8362 2101 0 0 2d Drift 1 1.000 3 1.279 2139 2329 386 4 0.8533 2139 0 0 3w Wash 1 1.000 3 0.075 4 0.0184 0 208 569 0 0 0 4 sl Standard 1 1 1.000 3 0.075 4 0.0184 0 209 755 0 0 0 5 s2 Standard 2 1 1.000 3 0.079 4 0.0264 24 234 906 24 0 0 6 s3 Standard 3 1 1.000 3 0.096 4 0.0599 124 336 1084 124 0 0 7 s4 Standard 4 1 1.000 3 0.113 4 0.0882 207 420 1262 207 0 0 8 S5 Standard 5 1 1.000 3 0.137 4 0.1248 313 528 1435 313 0 0 9 s6 Standard 6 1 1.000 3 0.154 4 0.1472 377 594 1612 377 0 0 10 s7 Standard 7 1 1.000 3 0.287 4 0.2835 753 976 1785 753 0 0 11 S8 Standard 8 1 1.000 3 0.606 1308 1540 1962 4 0.4990 1308 0 0 12 s9 Standard 9 1 1.000 3 1.242 2093 2338 2137 4 0.8327 2093 0 0 13 siO Standard 10 1 I.GOO 3 1.461 2383 2636 2312 4 0.9645 2383 0 0 14 d Drift 1 1.000 3 1.306 2173 2392 2487 4 0.8686 2173 0 0 15 w Wash 1 1.000 3 0.075 4 0.0184 0 220 2729 0 0 0 Page 1 of 3 1995-03-30 10:59 OutPut of : 95033OAl Fluoride 1.5 Fluoride L PPM PPM Poo Typ Ident Dil Weight Ch Result P Cor. Valu Time 16 u blk 1 1 1.000 3 0.076 4 0.0204 6 224 2822 6 0 0 17 u blk 2 1 1.000 3 0.074 4 0.0161 -7 208 3013 -7 0 0 18 u blk 3 1 1.000 3 0.072 4 0.0115 -21 192 3183 -21 0 0 19 u spk 1 1 1.000 3 0.095 4 0.0572 116 328 3363 116 0 0 20 u spk 2 1 1.000 3 0.091 4 0.0505 96 304 3536 96 0 0 21 u spk 3 1 1.000 3 0.093 4 0.0539 106 312 3706 106 0 0 22 u spk 4 1 1.000 3 0.097 4 0.0606 126 330 3888 126 0 0 23 u spk 5 1 1.000 3 0.098 4 0.0623 131 332 4063 131 0 0 24 u spk 6 1 1.000 3 0.096 4 0.0589 121 320 4235 121 0 0 25 u F52874-1 1 1.000 3 0.077 4 0.0221 11 208 4410 11 0 0 26 d Drift 1 1.000 3 1.265 4 0.8456 2122 2316 4587 2122 0 0 27 w Wash 1 1.000 3 0.075 4 0.0184 0 192 4766 0 0 0 28 u F52874-2 1 1.000 3 0.076 4 0.0204 6 196 4935 6 0 0 29 u F52874-3 1 1.000 3 0.075 4 0.0171 -4 184 5116 -4 0 0 30 u F52879-1 1 1.000 3 0.074 4 0.0151 -10 176 5274 -10 0 0 31 u F52879-2 1 1.000 3 0.073 4 0.0131 -16 169 5464 -16 0 0 Page 2 of 3 1995-03-30 10:59 OutPut of : 95033OAl Fluoride 1.5 Fluoride L PPM PPM Poo Typ Ident Dil Weight Ch Result F Cor. Valu Time 32 u F52889-1 1 1.000 3 0.088 4 0.0444 78 261 5640 78 0 0 33 u F52889-2 1 1.000 3 0.091 4 0.0501 95 276 5812 95 0 0 34 u F52889-3 1 1.000 3 0.122 4 0.1023 248 426 5990 248 0 0 35 u F52894-1 1 1.000 3 0.091 4 0.0508 97 274 6164 97 0 0 36 u F52894-2 1 1.000 3 0.094 4 0.0562 113 288 6340 113 0 0 37 u F52894-3 1 1.000 3 0.089 4 0.0464 84 258 6514 84 0 0 38 d Drift 1 1.000 3 1.276 4 0.8519 2136 2308 6688 2136 0 0 39 w Wash 1 1.000 3 0.075 4 0.0184 0 170 6872 0 0 0 40 u F52895-1 1 1.000 3 0.081 4 0.0314 39 208 7034 39 0 0 41 u F52895-2 1 1.000 3 0.082 4 0.0317 40 208 7212 40 0 0 42 u F52895-3 1 1.000 3 0.077 4 0.0227 13 180 7388 13 0 0 43 d Drift 1 1.000 3 1.310 4 0.8708 2178 2344 7565 2178 0 0 44 w Wash 1 1.000 3 0.075 4 0.0184 0 165 7796 0 0 0 wt rw RunOut Wash 1 1.000 3 0.075 4 0.0184 0 138 8040 0 0 0 Page 3 of 3 4000 mzma@ Raw data of 95033OAt 0 Fluoricto I.S Ezc=Exit Fl-Holp Crtl-P-Edit peaks Ti" 1 437S 4 coo Raw data of 950330AI : FILoorlds 1.5 4 D \ell np 0 2423 'Eac-Exit I Fl-Help I Crtl-P-Edit Peaks Ti I 6798 4000 Raw data of 950330AI Fluoride 1.5 0 8798 Ese-Exit I FI=Help I Crti-P-Edit Peaks Time "A'Lol12 1.2520844, Calibration curve of SS033GAI FILsoricis 1.5 0.0752354i 0 Orcgmr Inverse Heamuremi Logarithn a 4921 1.7848539- Calibration curve of 950=Al Fluoride L c a 0.0184248; a Order 2 fleasured r 0.99781 4000