Document NGYXyV7j0qXw88gNqMOgRb7My

3M Environmental L boratory FinalReport- AnalyticalStudy Single-doseDermal Absorption/ToxicitSytudy ofT-6049 inRabbits In-Vivo Study ReferenceNumber: HWI#6329-130 Study Number: AMDT-020895.1 Test Substance: FC-95 (T-6049) Name and Address of Sponsor: 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, ANMT-M-4-0, AMDT-M-5-0, and Revisions: Thermal Extractionof Fluorideby Means of a Modi:fied Dohrmann DX2000 Organic Halide Analyzer-Liver FluorideMeasurement by Means of an Orion EA940 Expandable Ion Analyzer Extractionof Fluorochemicalsfrom Rabbit Liver Analysisof RabbitLiver ExtractforFluorochemicalsUsing ElectrosprayMass Spectrometry InitiationDate: See attachedprotocol Author: James D. Johnson Approved By: Y Satmuedesys ID..:'Yonson Stmudy DiWctor Completion Date 0()oool- 1.0 SUMMARY Samples of rabbitliverat28 days postdermal administratioonf 0, 0.003,0.06,and 0.30 mg/kg FC-95 (T-6049)were analyzedby combustion fortotalorganicfluorine. There was no detectablelevelof totalorganicinany of thetreatedgroups above that observedin controlrabbitliver.Sincein a pharmacokineticstudy(HWI#6329-159), itwas establishetdhatperfluorooctanesulfonaitsepersistenitn rabbitliverwith a biologicalhalf-lifien excessof one month and thatperfluorooctanesulfonawtiell serveas a good marker forabsorptionof FC-95, itisconcluded thatdetectable dermal absorptiondoes not occuratthesedose levels.These dose levelsare very low and possiblytoo low to make an assessmentof dermal absorption. 2.0 INTRODUCTION FC-95 isthe potassium saltof perfluorooctanesulfoniacid.In a previous pharinacokinetisctudy(HWI#6329-159), itwas shown thatthebiologicahlalf-life is>1 month. Itispersistenitnserum and liverafteran intravenousdose. From previousstudiesinrats,themolecule isnot known tobiotransformor conjugate. Thus, analysisby combustion with subsequentmeasurement of fluoridewillestimate the amount of perfluorooctanesulfonaptreesent.At latertimes,theconcentrationin liverishigherthan theamount in serum on theaverage. For use as a marker to assessdermal absorption,thelevelof totalorganicfluorinein liveristhereforea more sensitivetestat28 days postdose. 3.0 TEST MATERIALS 3.1Test,Control,and ReferenceSubstancesand Matrices 3.1.1AnalyticalReferenceSubstance: FC-95, lot161 or 171.They are equivalent. 3.1.2AnalyticalReference Matrix: Bovine liverand bovine serum 3.1.3Analytical Control Substance: None 3.1.4Analytical Control Matrix: Bovine liverand bovine serum 3.2 Source of Nlaterials3:M 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 3.5StorageConditionfsorTestMaterialsR:oom temperaturfeorFC-95. For biologicalsamples,thestorageis-201 00C. 3.6 Dispositionof Specimens: Biologicaltissuesand fluidswillbe retainedper GLP Regulationforthetime periodrequiredforstudieslongerthan 28 days. 4.0 EXPERIMENTAL - Overview Samples of tissueand serum were availablefrom HWI#6329-130 foranalysisfor totalorganicfluorine.The samples were combusted and analyzedfortotalorganic fluorine.The datawere assessedforextentof dermal absorptionof FC-95. 5.0 EXPERIMENTAL - Methods 5.1 AMDT-M-1-0, Thermal Extractionof Fluorideby Means of a Modified Dohrmann DX2000 Organic HalideAnalyzer-Liver 5.2 AMDT-M-2-0,, FluorideMeasurement by Means of an Orion EA940 Expandable Ion Analyzer 5.3 AMDT-M-4-0, Extractionof Fluorochemicalsfrom Rabbit Liver 5.4 AMDT-M-5-0, Analysisof Rabbit LiverExtractforFluorochemicalsUsing ElectrosprayMass Spectrometry 6.0 DATA ANALYSIS The data from combustion analysisat28 days postdermal administratioonf FC-95 (T-6049) are attached.Tle dosingmaterialT-6049 isa 0.06% solutionof FC-95. Doses inthisanalysisare expressedas the FC-95 equivalents.The liverwas combusted and subsequentlyanalyzedfortotalorganicfluoride.There isno evidence of dermal absorptionatany dose levelforthe groups of rabbitsinthe control,0.003 mg/kg, 0.06mg/kg, and 0.30mg/kg groups. In allcasestheamount of organicfluorinewas below thepracticaqluantitatiolnevelsand apparentlybelow the detectionlimits. Electrospraymass spectrometrydid detectperfluorooctanesulfonaatneion,but again theresultsare nebulousand below thepracticaqluantitatiolnimits. There does not appear tobe den-nalabsorptionof FC-95 atthesedose levels. 0 0 0,'l 3 6.1CircumstanceTshatMay Have AffectetdheQualitoyftheData:No problems with thisanalysisarerecognizedthatcouldaffecttheconclusion. However, thedosesadministrateadreso low thateven ifallthedose had been absorbeditwould be difficulttoquantitateI.nthehigherdose group,2 kg rabbits would receiveonly 600 ug total.For dermal dosingwith analysisat28 days and a halflifeon theorderof a month, therewould be lessthan 300 ug presentinliver even ifallthe absorbedperfluorooctanesulfonahtaed gone totheliver. 7.0 CONCLUSION FC-95 isnotdennallyabsorbedat0.30 mg/kg dose. This isa very low dose and possiblytoo low to make an assessmentof dermal absorption. 8.0 MAINTENANCE OF RAW DATA AND RECORDS 8.1 Raw Data and Data: Raw data,approvedprotocola,pprovedfinalreport, appropriatespecimens,and electronidcatawillbe maintainedintheAMDT archives. 9.0 APPENDICES 9.1 Protocoland Amendments 9.1.1Protocoland FinalReport:HWI#6329-130 "SingleDose Dermal Absorption/ToxicitSytudyof T-6049 inRabbits(ProtocoltypeT?-3016.AB fordosingof animals,tissuecollectione,tc.) 9.1.2AnalyticalprotocolAMDT-020895.1 includingmethods. 9.2 Signed Reports from IndividualScientistNso:ne 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 0()000'1 4 9.8QualitCyontroPlracticeSse:emethods 9.9 TestMethods: See ProtocolAMDT-020895.1 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. 9.11.2Summary and raw data;analysisof liverextractsusingelectrospray mass spectrometry. 5 9.1.1 Protocoland FinalReport:HWI#6329-130 "SingleDose Dermal Absorption/ToxicitSytudy of T-6049 inRabbits(ProtocoltypeTP3016.AB for dosingof animals,tissuecollectione,tc.) 0()0()06 HAZLEOCON W IS C 0 N S IN POS r OFFIR.E BOX 7545 MADISON. Wl 53707-7545 St)onsor: 3M Toxicology Service Medical Department St. Paul, Minnesota a CORNING Conip@inv FINAL REPORT 0 Study Title: Single-Dose Dermal Absorption/Toxicity Study of T-6049 in Rabbits Author: Steven M. Glaza Study Completion Date: June 27, 1995 Performing Laboratory: Hazleton Wisconsin, Inc. 3301 Kinsman Boulevard Madison, Wisconsin 53704 LaboratoryProject Identification: HWI 6329-130 P h o n e 6 0 8 - -11 --14 7 1 E X P R E S S - M A I L D E L IV E R Y Page 1 of 42 330 1 K Iti:'@AA ti 6 L V D F a x 6 0 8 -2 .11 - 7 2 2 7 rvlA D IS ()@l, w 1 5 ',)7 0 4 Page 2 of 42 HWI 6329-130 QUALITY ASSURANCE STATEMENT This report has been reviewed by the Quality Assurance Unit of Hazleton Wisconsin, Inc., in accordancewith the Food and Drug Administration (FDA)Good LaboratoryPracticeRegulations,21 CFR 58.35 (b) (6) (7). The following inspectionswere conductedand 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 Date Reported to Date to Study Director Management 12/21/94 01/10/95 01/24/95 04/03/95 04/03/95 06/27/95 12/21/94 01/10/95 01/24/95 04/13/95 04/13/95 06/27/95 Protocol Review Animal Observation Protocol Amendment Data/Report Review Data Review Report Rereview 12/22/94 01/10/95 01/24/95 04/14/95 04/14/95 06/27/95 01/10/95 02/10/95 02/10/95 05/10/95 05/10/95 07/10/95 @epresen(atlv-e,Quality Assurance Unit Date Page 3 of 42 STUDY IDENTIFICATION Single-Dose Dermal Absorption/Toxicity Study of T-6049 in Rabbits HWI 6329-130 Test Material Sponsor Spontor's Representative Study Director Study Location Study Timetable Study InitiationDate Experimental(In-life)Start Date In-life End Date Experimental Termination Date Study Completion Date T-6049 3M Toxicology Service Medical Department 3M Center, Bldg. 220-2E-02 P.O. Box 33220 St. Paul, MN 55133-3220 John L. Butenhoff, PhD 3M Toxicology Service Medical Department 3M Center, Bldg. 220-2E-02 P.O. Box 33220 St. Paul, MN 55133-3220 (612) 733-1962 Steven M. Glaza Hazleton Wisconsin, Inc. P.O. Box 7545 Madison, Wl 53707-7545 (608) 241-7292 Hazleton Wisconsin, Inc. Building No. 3 3802 Packers Avenue Madison, WI 53704 December 30, 1994 January 7, 1995 February 10, 1995 June 27, 1995 June 27, 1995 Page 4 of 42 HWI 6329-130 KEY PERSONNEL Acute Toxicology Laboratory Animal Medicine Steven M. Glaza Study Director Manager Cindy J. Cary, DVM Diplomate, ACLAM Supervisor Francis (Bud) W. McDonald Study Coordinator Anatomical Pathology Patricia Padgham In-lifeSupervisor 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 000()I() Page 5 of 42 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 IndividualClinicalSigns 3 IndividualDermal IrritationScores 4 Individual Pathology Comments 5 IndividualAnimal Tissue Weights and Bile Volumes Appendix A Protocol Deviations Protocol TP3016.AB Protocol Amendment No. 1 HWI 6329-130 Paqe 2 3 4 6 8 8 8 9 10 13 14 14 14 15 16 17 19 23 25 27 28 29 41 Page 6 of 42 HWI 6329-130 SUMMARY This study was done to assess the systemic absorption/toxicityand relative skin irritancyof T-6049 when appliedto the skin of rabbits. The study was conducted using three male and three female acclimated rabbits of the Hra:(NZW)SPFstrain for each treatmentgroup. Group Test Material 1 (Control)Distilledwater 2 T-6049 3 T-6049 4 T-6049 Dose Level (mg/kq) oa 5 100 500 Number of Animals Males Females 3 3 3 3* 3 3* 3 3 a Administered at a dose volume of 2.0 mL/kg. One animal sacrificed on Day 2 due to possible broken back and replaced with another female animal. The back of each rabbit was clipped free of hair and a single dose of the respectivematerial at the indicateddose levelwas administeredto the skin of the rabbits. The treatmentsites remainedintact. The area of application was covered with a gauze bandage secured with paper tape around all edges and overwrapped with Saran WrapO and ElastoplastOtape to provide an occlusive dressing for a 24-hour exposure period. Clinical observationswere conductedpredose and at approximately1, 2.5, and 4 hours after test or controlmaterialadministration.Additionalclinical observationsand twice a day mortalitychecks were conducteddaily thereafter for 28 days. The only exceptionsto this were on Days 4 and 6 when the afternoon mortalitycheck was inadvertentlynot conducted for the two replacement animals. Body weights were determined on Day -4 for randomization purposes,before test or control,materialadministration(Day 1), and at in-lifetermination(Day 29). The initialdermalirritationreadingwas made before test or controlmaterialadministration(recordedas the Day I reading). Subsequent readings of dermal irritationwere made approximately 30 minutes after bandage removal (Day 2) and on Days 4 and 8. Blood samples were collectedfrom a marginalear vein of the animalsbefore in-life initiation(Day 1), approximately24-hourspostdose (Day 2), on Days 4, 9, 16, and 23. Replacement animals were bled on Days 1, 2, 4, 8, 15, and 22. In addition, at the time of necropsy on Day 29, approximately20 mL of blood was obtained from each animal. All sampleswere centrifugedand separated into serum and cellularfractions. All animalswere euthanizedat terminationof the in-life phase and necropsied. The whole liver, bile, an approximate1-cm x 1-cm section of the dermal applicationsite from all animals,and both kidneys from one male and one female in each group were collected at necropsy Page 7 of 42 HWI 6329-130 and weighed (volumeonly determinedfor bile). The blood samples (serum and cellularfractions),livers,bile, dermalapplicationsites,and kidneyswere sent frozen to the Sponsor after terminationof the in-lifephase. Applicationof T-6049 did not result in any test material-relatedchanges in body weight gain. All animals appearedclinicallynormal throughoutthe study with the exception of one Group 2 and one Group 3 female animals that were sacrificedon Day 2 due to injury (possiblebroken backs). These animals were replaced in the study and the replacement animals appeared normal throughout the study. No dermal irritationwas observedat the dermal scoringintervals as a result of the applicationof distilledwater or T-6049 at any of the dose levels. There were no test material-relatedlesions observedat necropsy. 0 ()IL"i Page 8 of 42 HWI 6329-130 OBJECTIVE The objectiveof this study was to assess the systemictoxicity/absorptionand relativeskin irritancyof a test materialwhen appliedto the skin of rabbits. REGULATORY COMPLIANCE This study was conducted in accordancewith the U.S. Food and Drug Administration'sGood LaboratoryPractice Regulationsfor Nonclinical LaboratoryStudies, 21 CFR 58,-withthe exceptionthat analysisof the test materialmixture prepared for the Group 2 animals for concentration, homogeneity/solubilitya,nd stabilitywas not conducted. 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 unnecessarily duplicate any previous work. TEST AND CONTROL MATERIALS Identification The test materialwas identifiedas T-6049 and describedas a clear, colorless liquid. The controlmaterialwas distilledwater and was describedas a clear,colorlessliquid. Purity and Stability The Sponsor assumes responsibilityfor test materialpurity and stability determinations(includingunder test conditions). Analysisof the test material mixture prepared for the Group 2 animals for concentration, homogeneity/solubilitya,nd stabilitywas not conductedor requestedby the Sponsor. The purity and stabilityof the controlmaterialwere consideredto be adequate for the purposes of this study. Storage and Retention The test and control materialswere stored at room temperature. A reserve sample of each test and control materialwas taken and will be retained in a freezer set to maintain a temperatureof -20*C 10' for 10 years in accordance with Hazleton Wisconsin (HWI) StandardOperating Procedure (SOP). Any unused test material was returnedto the Sponsorafter completionof the in-life phase accordingto HWI SOP. Any remainingcontrol material is retained for other testing and will not be discardedafter issuanceof the final report. Page 9 of 42 HWI 6329-130 Safety Precautions The test and control material handling procedureswere according to HWI SOPs and policies. TEST SYSTEM Test Animal Adult albino rabbits of the Hra:(NZW)SPFstrain were procured from HRP, Inc., Denver, Pennsylvania on December 28, 1994 and maintained at the Hazleton Wisconsin facility at 3802 Packers Avenue, Madison, Wisconsin. Housing After receipt, the animalswere acclimatedfor a period of at least 7 days. During acclimationand throughoutthe study, the animalswere individually housed in screen-bottomstainlesssteel cages in temperature- and humiditycontrolledquarters. Environmentalcontrolsfor the animal room were set to maintain a temperatureof 19' to 23*C, a relativehumidity of 50% 20%, and a 12-hour light/12-hourdark lightingcycle. In cases where variationsfrom these conditionsexisted,they were documentedand consideredto have had no adverse effect on the study outcome. Animal Diet The animals were provided access to water ad 7ibitum and a measured amount of Laboratory Rabbit Diet HF #5326, PMI Feeds, Inc. The feed is routinely analyzed by the manufacturerfor nutritionalcomponents and environmental contaminants. Samples of the water are periodicallyanalyzed by HWI. There were no known contaminantsin the feed or water at levels that would have interferedwith or affectedthe resultsof the study. Selection of Test-Animals The animals were identifiedby animal number and corresponding ear tag and were placed into study groups using a stratifiedbody weight randomization program. The randomizationbody weights were determined on Day -4. The weight variation of the animalsfor each group of each sex selectedfor the study did not exceed 2 standard deviationsof the mean weight, and the mean body weights for each group of each sex were not statisticallydifferentat the 5% probabilitylevel. Two female animals (Nos. F53398 and F53355) were replaced after they were treateddue to possible broken backs. These animals were replaced with two other female animals (Nos. F52999 and F53351) which were treated in the same manner. Page 10 of 42 HWI 6329-130 Study Design Animals weighing from 2,018 to 2,625 g at initiationof treatment were placed into the following study groups: Group Test Material 1 (Control) Distilled water 2 T-6049 3 T-6049 4 T-6049 Dose Level (mg/kq) oa 5 100 500 Number of Animals Males Females 3 3 3 3* 3 3* 3 3 a Administered at a dose volume of 2.0 mL/kg. One animal sacrificed on Day 2 due to possible broken back and replaced with another female animal. This animal weighed 2,625 g at initiation. Justificationfor Species Selection Historically, the New Zealand White albino rabbit has been the animal of choice because of the large amount of background information on this species. PROCEDURES Preparation of Exposure Area On the day before test material application,the back and, if necessary (to obtain unblemished skin), the flanks of each rabbit was clipped free of hair. The clipped area made up approximately 20% of the total body surface area. The intact skin of the test sites was inspectedfor interferinglesions, irritation,or defects that would preclude the use of any of the animals. The animals were clipped as needed throughout the study to aid in visualizing the application sites. Dose Administration All animals received a single administrationof the respective test or control material. The day of treatment was designated as Day 1. Group 1. An individual dose (2.0 mL/kg) was calculated and measured based on ea animal's body weight on the day of treatment. The control material (distilledwater) was ap?lied evenly to the test site at a rate of approximately 0.05 mL/cm 0()O()IG Page 11 of 42 HWI 6329-130 Groups 2, 3, and 4. For the Group-2 animals (5 mg/kg), the test material (T-6049) was mixed with distilled water to a concentration of 500 mg/mL and applied at a dose volume of 0.01 mL/kg. The mixture was stored at room temperature until administered. The test material was administered undiluted to the test sites of the Groups 3 and 4 animals (100 or 500 mg/kg, respectively)using the average bulk density of 0.99 g/mL to determine the dose volume for each dose level (0.10 and 0.51 mL/kg, respectively). The area of exposure for the 5, 100, and 500 mg/kg dose levels was 4, 25 (16 for replacement animal), and 100 cm2 , respectively. The approximate rate of application ranged from 0.006 to 0.014 mL/cm'. An individual dose of the respectivetest material or test material mixture was calculated for each animal based on its body weight on the day of treatment. 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 WrapD and ElastoplastODtape 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 controlmaterial administration. Additionalclinical observations and twice a day mortality checks (morning and afternoon) were conducted daily thereafter for 28 days. The only exceptions to this were on Days 4 and 6 when the afternoon mortality check was inadvertently not conducted for the two replacement animals. Body weights were determined for randomization purposes on Day -4, before test material administration(Day 1), and at in-life termination(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. Page 12 of 42 .HWI 6329-130 Samgle Collections Blood samples (approximately4 mL) were collectedfrom a marginal ear vein of all animalsbefore experimentalinitiation(Day 1). Subsequentcollectionof blood was conducted approximately24-hours postdose (Day 2), and on Days 4, 9, 16, and 23. Replacementanimalswere bled on Days 1, 2, 4, 8, 15, and 22. In addition, at the time of necropsyon Day 29, approximately20 mL of blood was obtained from the posteriorvena cava of each animal. All sampleswere' centrifugedand separated into serum and cellular fractions. These samples were then stored in a freezer set to maintain a temperatureof -200C IOOC until shipped to the Sponsor. Pathology At terminationof the experimentalphase (Day 29), animalswere anesthetized with sodium pentobarbital,bled via the posteriorvena cava, exsanguinated, and necropsiedin random order. The sites of test and controlmaterial applicationwere washed with lukewarm tap water before the necropsy procedure. All animals were subjected to an abbreviated gross necropsy examination and any abnormalitieswere recorded. The whole liver, bile, an approximate1-cm x 1-cm section of the dermal applicationsite from all animals,and both kidneys from the first male and female in each group were collected. The tissue samples for all animals,with the exceptionof the animal sacrificedand necropsiedon Day 2, were weighed (volume only determinedfor bile) and immediatelyplaced on dry ice, then placed in a freezer set to maintain a temperature of -200C IOOC. After necropsy, the animals were discarded. Shipment of Blood, Bile, and Tissues After experimentaltermination,the blood samples (serumand cellular fractions),livers,bile,dermal applicationsites,and kidneyswere sent frozen (on dry ice) to the Sponsor (JamesD. Johnson,3M E.E. & P.C., Bldg. 2-3E-09, 935 Bush Avenue, St. Paul, MN, 55106), along with the collected correspondingweights or volumes. The Sponsor is responsiblefor the retentionand dispositionof the samples. HWI does not accept any responsibilityfor the analysisof the tissue samplescollectedin this study nor are these resultspresentedin this report. StatisticalAnalyses No statisticalanalyseswere requiredby the protocol. Location of Raw Data, Records, and Final Report The raw data, records,and an originalsignedcopy of the final report will be retained in the archives of HWI in accordancewith HWI SOP. Page 13 of 42 Body Weights RESULTS HWI 6329-130 Individualand mean body weights are in Table 1. All animals exhibitedbody weight gains from Day 1 to Day 29. Clinical Observations Individualclinicalsigns are in Table 2. All animals appearednormal throughout the study with the followingexceptions: One Group 2 female (No. F53398) treatedwith T-6049 at 5 mg/kg appeared to have injuredits back at the time of the 24-hourblood collection. This animal was sacrificed,necropsied,and replacedwith No. F52999. The replacement animal appeared normal throughout the study. One Group 3 female (No. F53355) treated with T-6049 at 100 mg/kg was found prior to completionof the exposureperiodwith its leg caught in the cage floor, appearedto have injuredits back, and was sacrificed. Since this animal did not receive its full test materialexposure,the animalwas discarded without a gross necropsy examination. The animal was replaced with No. F53351. The replacement animal appeared normal throughout the study. Dermal Irritation Individualdermal irritationscores are in Table 3. The controlmaterial produced no dermal irritation. No dermal irritationwas observed in the animals treated with T-6049 at any of the dose levels. Pathology Individual animal pathology comments are presented in Table 4. Individual animal tissue weights and bile volumes are in Table 5. The treated skin in two animals dosed at 0 mg/kg, two animalsdosed at 5 mg/kg, and one animal dosed at 500 mg/kg was diffuselyred or had multiple red areas of variable size. These findingsare not consideredto be test material-related. There were no lesions observed in any of the remaininganimals. Page 15 contains a pathologyreport by the study pathologist. 000()JL!) Page 14 of 42 HWI 6329-130 DISCUSSION The acute systemicabsorption/toxicityand relativeskin irritancyof T-6049 were evaluated in male and female albino rabbits when administeredas a single dermal application. There were no test material-relatedchanges in body weight gain or in-lifeclinicalfindingsat any of the dose levels. No test material-relateddermal irritationwas observedduring the study. There were no test material-relatedlesionsobserved at necropsy. SIGNATURE l@,@ Steven M. Glaza D Date Study Director Acute Toxicology REFERENCE 1. Draize, J. H., "Acute Dermal Toxicity (Single Exposure)," In: Appraisa7 of the Safety of Chemica7s in Foods, Drugs and Cosmetics - Derma7 Toxicity, Association of Food and Drug Officials of the U.S., pp. 54-56 (1959). Page 15 of 42 PATHOLOGY REPORT HWI 6329-130 There were six rabbits (three males and three females) each from four dose levels of 0, 5, 100, and 500 mg/kg of body weight euthanizedand necropsied at the terminationof the study. One female (AnimalNo. F53398)dosed at 5 mg/kg of body weight was sacrificed on Day 2 because of an apparent broken back. This animal was replacedwith another female (AnimalNo. F52999). The test material,dose level, day of death, and gross observationsrecorded for each animal are in the IndividualPathologyCommentsthat follow this report. At necropsy, the treated skin in some animalswas diffusely red or had multiplered areas of variablesize. This includedtwo animalsdosed at 0 mg/kg, two animals dosed at 5 mg/kg, and one animal dosed at 500 mg/kg. There were no visiblelesionsin any of the remaininganimals. The liver, bile, an approximate1-cm x 1-cm sectionof the dermal applicationsite from each of these animals, and both kidneys from one male and one female in each group were collected. The tissue samples were weighed (volume only determined for bile), frozen, and sent to the Sponsor. The tissuesfrom the female dosed at 5 mg/kg and sacrificedon Day 2 were collectedbut were not weighed. After necropsy, the animals were discarded. The animal sacrificedon Day 2 had multipledark red areas of variable size within the skeletalmuscle of the lumbar-sacralregion of the spinal column. These findingscorrelatedwith the clinicalobservationof an apparentbroken back. Pathologist (6329-130.slh) 03/22/95 7,.2 Date Animal Number F53358 F53359 F53353 Mean F53402 F53364 F53352 Mean F53360 F53400 F53346 Mean F53365 F53354 F53347 Mean Page 16 of 42 HWI 6329-130 Table I Individualand Mean Body Weights (g) Male Randomization Day -4 Day 1 29 Animal Number Female Random- ization Day Day -4 1 29 Group I (Control)- DistilledWater (0 mg/kq) 2,066 2,030 2,136 2,231 2,093 2,312 2,816 2,670 3,012 F53404 F53405 F53356 2,200 2,426 2,110 2,286 2,474 2,224 2,997 2,911 2,685 2,077 2,212 2,833 2,245 Group 2 - T-6049 (5 mg/kq) 2,328 2,864 2,288 2,244 2,102 2,382 2,364 2,018 2,938 2,923 2,622 F53399 F53349 F53398 a F52999 2,095 2,376 2,331 2,485 2,122 2,492 2,419 2,625 2,580 2,919 - 3,272 2,211- 2,255 2,828 2,319 2,413 2,924 Group 3 - T-6049 (100 mg/kq) 2,157 2,076 2,115 2,194 2,122 2,211 2,943 2,696 2,694 F53350 F53355' F53397 F53351 2,320 2,280 2,145 2,059 2,409 2,297 2,245 2,253 2,996 - 2,706 2,606 2,116 2,176 2,778 2,175 2,302 2,769 Group 4 - T-6049 (500 mg/kq) 2,126 2,325 2,103 2,276 2,478 2,152 2,951 3,008 2,646 F53403 F53000 F53345 2,170 2,241 2,306 2,270 2,268 2,341 2,860 2,914 2,951 2,185 2,302 2,868 2,239 2,293 2,908 a Animal No. F53398 was originallyselected by the randomizationprogram for use in the study and was treated. This animal was sacrificedafter completionof the exposure period due to a possible broken back and was replaced with No. F52999. The body weights for No. F53398 are not included in the group means. b Animal No. F53355 was originallyselected by the randomizationprogram for use in the study and was treated. This animal was sacrificedbefore completionof the exposure period due to a possible broken back and was replaced with No. F53351. The body weights for No. F53355 are not included in the group means. - Not applicable. Sex Male Female Page 17 of 42 Table 2 IndividualClinicalSigns HWI 6329-130 Animal Number Observation 1-4 Hours Day (Day IL -L 3 through29 Group 1 (Control)- DistilledWater (0 mg/kg) F53358 Appeared normal F53359 Appeared normal F53353 Appeared normal F53404 Appeared normal F53405 Appeared normal F53356 Appeared normal .1 Male Female Group 2 - T-6049 (5 mg/kq) F53402 Appeared normal F53364 Appeared normal F53352 Appeared normal F53399 Appeared normal F53349 Appeared normal F53398 Appeared normal Possible broken back (at time of 24-hour bleedinginterval) Moribund sacrifice F52999a Appeared normal Condition existed. Condition not evident. a Animal No. F52999 replaced Animal No. F53398. Sex Male Female Page 18 of 42 Table 2 (Continued) IndividualClinicalSigns Animal Number Observation 1-4 Hours (Day 1) 2 Group 3 - T-6049 (100 mg/kq) F53360 Appeared normal F53400 Appeared normal F53346 Appeared normal F53350 Appeared normal F53355 Appeared normal Leg caught in cage floor (possible broken back) Moribund sacrifice F53397 Appeared normal F5335,a Appeared normal HWI 6329-130 Day 3 through 29 Male Female F53365 F53354 F53347 F53403 F53000 F53345 Group 4 - T-6049 (500 mg/kq) Appeared normal Appeared normal Appeared normal Appeared normal Appeared normal Appeared normal Condition existed. Conditionnot evident. Animal No. F53351 replaced Animal No. F53355. Ooo( .),.o Page 19 of 42 Table 3 IndividualDermal IrritationScores HWI 6329-130 Group 1 (Control)- DistilledWater (0 mg/kg) Dermal Reaction Erythema Edema Atonia Desquamation Coriaceousness Fissuring Males Study Day 1 2 4 8 Animal No. F53358 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Females Study Day 1 2 4 -8 Animal No. F53404 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. F53359 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. F53405 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. F53353 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. F53356 0 0 0 of 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 ()()0025 Page 20 of 42 Table 3 (Continued) IndividualDermal IrritationScores HWI 6329-130 Group 2 - T-6049 (5 mg/kg) Dermal Reaction Males Study Day 1 2 4 8 Animal No. F53402 Females Study Day 1 2 4 -8 Animal No. F53399 Erythema Edema Atonia Desquamation Coriaceousness Fissuring 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Animal No. F53364 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. F53349 Erythema Edema Atonia Desquamation Coriaceousness Fissuring Erythema Edema Atonia Desquamation Coriaceousness Fissuring 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Animal No. F53352 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 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. F53398' 0 0 - - 0 0 - - 0 0 - - 0 0 - - 0 0 - - 0 0 - - Animal No. F52 gggb Erythema Edema Atonia Desquamation toriaceousness Fissuring 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 a Animal replaced with No. F52999. b Replacement animal for No. F53398. - Not applicable. 000 0 Page 21 of 42 Table 3 (Continued) IndividualDermal IrritationScores HWI 6329-130 Group 3 - T-6049 (100 mg/kg) Dermal Reaction Erythema Edema Atonia Desquamation Coriaceousness Fissuring Erythema Edema Atonia Desquamation Coriaceousness Fissuring Males Study Day 1 2 4 8 Animal No. F53360 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. F53400 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Females Study Day 1 2 4 -8 Animal No. F53350 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. F53355a 0 - - - 0 - - - 0 - - - 0 - - - 0 - - - 0 - - - Animal No. F53346 Animal No. F53397 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 Animal No. F5335 lb Erythema Edema Atonia Desquamation Coriaceousness Fissuring 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 a Animal replacedwith No. F53351. b Replacement animal for No. F53355. Not applicable. 0 0 0 0'^."d7 Page 22 of 42 HWI 6329-130 Table 3 (Continued) IndividualDermal IrritationScores Group 4 - T-6049 (500 mg/kg) Dermal Reaction Males Study Day 1 2 4 8 Females Study Day 1 2 4 8 Animal No. F53365 Animal No. F53403 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 Animal No. F53354 Animal No. F53000 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 Animal No. F53347 Animal No. F53345 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 0()00%113 Page 23 of 42 HWI 6329-130 Table 4 Individual Pathology Comments Animal Test Day Number Sex Fl ed .Sacrificed Necropsv Observation F53358 m Group I (Control)- DistilledWater (0 mg/kq) - 29 No visible lesions. F53359 m - F53353 m - F53404 F - F53405 F - F53356 F - 29 No visible lesions. 29 The skin has multiple red areas within the dermal application site, up to 3 cm x 2 cm. Collected with routine section. 29 No visible lesions. 29 No visible lesions. 29 The treated skin area appears diffusely dark red. Collectedwith routine section. F53402 m - F53364 m - F53352 m - Group 2 T-6049 (5 mg/kq) 29 No visible lesions. 29 No visible lesions. 29 No visible lesions. F53399 F - F53349 F - F53398 a F - 29 The skin has diffusely dark red areas along the entire dorsal lumbar and thoracic region. Collectedwith routine section. 29 The skin has multiple red areas on the applicationsite up to I cm in diameter. Collected with routine section. 2 There are multiple dark red areas within the skeletal muscle of the lumbar sacral region surrounding the spinal column, up to 1.5 x 2.0 cm. F52999' F 29 No visible lesions. - Not applicable. a Animal replaced with No. F52999. b Replacement animal for No. F53398. 0()OO'A'O@ Page 24 of 42 Table 4 (Continued) Individual Pathology Comments Animal Test Day Number Sex Died Sacrificed Necropsy Observation F53360 m - F53400 m - F53346 m - F53350 F - F53397 F - F53351 F - Group 3 - T-6049 (100 mg/kq) 29 No visible lesions. 29 No visible lesions. 29 No visiblelesions. 29 No visiblelesions. 29 No visiblelesions. 29 No visiblelesions. HWI 6329-130 F53365 m - F53354 m - F53347 m - F53403 F - F53000 F - F53345 F - Group 4 29 29 29 29 29 29 T-6049 (500 mg/kq) No visiblelesions.. No visible lesions. No visible lesions. No visible lesions. The skin has multiple red areas on the applicationsite, up to I cm x 3 cm. Collectedwith routine section. No visible lesions. - Not applicable. Sex Male Female Male Female Page 25 of 42 Table 5 Individual Animal Tissue Weights and Bile Volumes HWI 6329-130 Animal Number Liver Weight (g) Dermal Appli- Kidneys cation Site Bile Volume (mL) Group I (Control)- DistilledWater (0 mg/kq) F53358 81.381 F53359 73.857 - 0.783 1.4 0.761 0.8 F53353 77.411 15.761 0.644 0.6 F53404 70.277 12.585 0.620 1.8 F53405 78.270 - 0.775 1.1 F53356 74.687 0.915 0.5 Group 2 T-6049 (5 mg/kg) F53402 78.731 14.611 0.468 2.3 F53364 84.099 - 0.641 1.8 F53352 67.613 0.706 1.8 F53399 65.818 - 0.546 0.8 F53349 77.596 14.211 0.489 0.9 F53398a - - - - F52999' 87.510 0.660 0.5 - Not applicable. a Moribund sacrificeon Day 2 (01/08/95)due to possible broken back. Tissues were collected, however, the correspondingweight or volume was not taken since this requirementbecame effective on 01/24/95. Animal replaced with No. F52999. b Replacement animal for No. F53398. 0()001031 Sex Male Female Page 26 of 42 Table 5 (Continued) IndividualAnimal Tissue Weights and Bile Volumes HWI 6329-130 Animal Number Liver Weight (g) Dermal Appli- Kidneys cation Site Group 3 - T-6049 (100 mg/kq) Bile Volume (mL) F53360 76.347 15.873 0.802 0.9 F53400 74.958 - 0.395 1.6 F53346 75.414 0.570 1.3 F53350 74.050 15.1@O 0.784 1.4 F53397 74.882 0.801 0.9 F53351 80.686 0.279 0.5 Group 4 T-6049 (500 mg/kq) Male F53365 82.598 - 0.441 1.8 F53354 70.968 15.234 0.540 1.3 F53347 83.696 - 0.716 2.0 Female F53403 70.624 15.427 0.910 1.4 F53000 79.617 - 0.714 0.8 F53345 81.341 0.633 1.8 Not applicable. 0()OO'JA'W Page 27 of 42 HWI 6329-130 APPENDIX A Protocol Deviations Protocol TP3016.AB Protocol Amendment No. 1 0()0033 Page 28 of 42 Protocol Deviations HWI 6329-130 Protocol Page 7, 7. Experimental Design, C. Observationof Animals, (1) ClinicalObservations,First Sentence. For clinicalsigns before test or controlmaterial administrationand for clinical signs and mortalityat approximately1, 2.5, and 4 hours after test material administration(Day 1) and Daily thereafterfor clinical signs, and twice daily (a.m. and p.m.) for mortalityfor at least 28 days. - Actual Procedure On Day 4 and 6, the afternoon mortality check was inadvertentlynot conducted for the two replacement animals (Group 2, No. F52999, and Group 3, No. F53351). Page 7, 7. Experimental Design, C. Observationof Animals, (3) Body Weights. For randomization,before test or controlmaterial application (Day 1), on Day 29, and at unscheduled death (whe.n survivalexceeds I day). The body weight of the Group 2 animal that was sacrificedon Day 2 was inadvertentlynot documented. These deviations are not consideredto have had an adverse effect on the outcome of the study. 00()034 HAZLrm-C(ON W IS C 0 N S IN POST OFFICE BOX 7),15 MADISON.WI 53707 75,15 Page 29 of 42 CORIYING Cotilr."Ifly Sponsor: 3M Toxicology Service Medical Department St. Paul, Minnesota PROTOCOL TP3016.AB Study Title: Single-Dose Dermal Absorption/ToxicityStudy of T-6049 in Rabbits Date: December 30, 1994 Performing Laboratory: Hazleton Wisconsin, Inc. 3301 Kinsman Boulevard Madison, Wisconsin 53704 LaboratoryProject Identification: HWI 6329-130 P h o n e 608 241-4411 E X P R E S S -M A IL DELIVF-RY 3301 K IN S ivi\ @j B!vn Fax MADISON OOOOZJ5 604 Y@11-7227 @N 1 53 704 Page 30 of 42 STUDY IDENTIFICATION TP3016.AB Page 2 Single-Dose Dermal Absorption/ToxicityStudy of T-6049 in Rabbits HWI No. Test Material Sponsor Sponsor's Representative Study Director Study Location Proposed Study Timetable Experimental Start Date Experimental Termination Date Draft Report Date 6329-130 T-6049 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 January 7, 1995 February 4, 1995 March 18, 1995 0()003(3' Page 31 of 42 I. Study Single-Dose Dermal Absorption/ToxicityStudy in Rabbits TP3016.AB Page 3 2. Purpose To assess the systemic absorptionand toxicity and relative skin irritancyof a test materialwhen appliedto the skin of rabbits 3. Regulatory COm2liance This study will be conducted in accordance with the following Good Laboratory Practice Regulations/Standards/Guidelines: ( ] Conduct as a Nonregulated Study [X] 21 CFR 58 (FDA) 40 CFR 160 (EPA-FIFRA) 40 CFR 792 (EPA-TSCA) C(81)30(Final)(OECD) 59 Nohsan No. 3850 (Japanese MAFF) Notification No. 313 (Japanese MOHW) All proceduresin this protocol are in compliancewith the Animal Welfare Act Regulations. In the opinion of the Sponsor and study director, the study does not unnecessarilyduplicate any previous work. 4. Ouality Assurance The protocol,study conduct, and the final report will be audited by the Quality Assurance Unit in accordance with Hazleton Wisconsin (HWI) Standard Operating Procedures (SOPS) and policies. 5. Test Material A. Identification T-6049 B. Physical Description (To be documented in the raw data) C. Purityand Stability The Sponsor assumes responsibilityfor,purity and stability determinations(includingunder test conditions). D. Storage Room temperature 0()0037 Page 32 of 42 TP3016.AB Page 4 E. Reserve Samples Reserve sample(s) of each batch/lot of 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 10*C for 10 years per HWI SOP. The Sponsor will be contacted after 10 years for disposition in accordance with the appropriate regulatory Good Laboratory Practices. F. Retention Any unused test material 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. ControlMaterial A. Identification Distilledwater B. Physical Description Clear, colorlessliquid C. Purity and Stability The purity and stabilityof this manufacturedmaterial is 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 final report. 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. 00()03.S Page 33 of 42 (3) Age at Initiation Adult TP3016.AB Page 5 (4) Weight at nitiation 2.0 to 3.0 kg (5) Number and Sex 12 males and 12 females (6) Identification Individual numbered ear tag (7) Husbandry (a) Housing Individually,in screen-bottomstainlesssteel cages (heavy gauge) (b) Food A measured amount of Laboratory Rabbit Diet HF #5326 (PMI Feeds, Inc.). The food is routinelyanalyzedby the manufacturerfor nutritional components and environmental contaminants. (c) Water Ad libitum from an automatic system. Samples of the water are analyzed by HWI for total dissolved solids, hardness, and specified microbiologicalcontent and for selected elements, heavy metals, organophosphates, and chlorinated hydrocarbons. (d) Contaminants There are no known contaminants in the food or water that would interferewith this study. (e) Environment Environmentalcontrols for the animal room will be set to maintain a temperature of 19'C to 23*C, a relative humidity of 507.+20%, and a 12-hour light/12-hourdark cycle. (f) Acclimation At least 7 days (8) Selection of Test Animals Based on health and body weight according to HWI SOPS. An adequate number of extra animals will be purchased so that no animal in obviouslypoor health is placed on test. The animals will be placed into study groups using a stratified body weight randomization program within nine days of study initiation. 0()003!) Page 34 of 42 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 informationon this species. B. Dose Administration (1) Test Groups Group Test Material 1 (Control) Distilledwater 2 T-6049 3 T-6049 4 T-6049 Dose Level Number of Animals (mg/kq-)- Males Females 0* 3 3 5** 3 3 100 3 3 500 3 3 To be administered at a dose volume of 2.0 mL/kg To be administered at a dose volume of .01 -mL/kg (2) Preparation of ExRosure Area On the day before test material application,the back and, if necessary(to obtain unblemishedskin), the flanks of each rabbit will be clipped free of hair. The shaved area will constituteapproximately20% of the total body surfacearea. The treatmentsites (intactskin)will be inspectedfor interferinglesions,irritation,or defects that would preclude the use of any of the animals. The animals will be clipped as needed throughout the study. (3) Dose Administration All animalswill receivea singleadministrationof the respectivetest or control material. The day of treatment will be designated as Day 1. The dose for each animal in Group 2 will be diluted with distilledwater and applied at a dose volume of .01 mL/kg. The respective dose for each animal in Groups 3 and 4 will be applied undiluted. All doses in Groups 1-4 will be based on the animal's body weight just before administrationand will be spread onto the area of exposure in a thin and uniform a layer. The area of application(Groups1-4) will be covered with a lo-cm x 10-cm gauze bandage secured with gaper tape around all edges and overwrapped with Saran Wrap and Elastoplast'o tape to provide an occlusivedressing. The rabbits will be collaredduring the 24-hour application period. (4) Reason for Route of Administration The dermal route is a potential route of exposure in humans. 0()OO-i Page 35 of 42 TP3016.AB Page 7 (5) Removal of Test Material Approximately24 hours after test or control material applicationthe bandages and collars will be removed and the 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 controlmaterial administrationand for clinical signs and mortalityat approximately1, 2.5, and 4 hours after test material administration(Day 1) and daily thereafterfor clinical signs, and twice daily (a.m.-and p.m.) for mortality for at least 28 days. Observations may be extended when directed by the study director. (2) Reading of Dermal Irritation Before test or controlmaterial administrationthe initial dermal irritationreading will be made and recorded as the Day I reading (Attachment1). Additionaldermal irritationreadingswill 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) Frequency Before initiation(Day 1), approximately24 hours post-dose(Day 2), Days 4, 9, 16, 23, and at experimental termination (Day 29) (b) Number of Animals All 0()OOILII Page 36 of 42 TP3016.AB Page 8 (c) Method of Collection Blood samples (approximately4 mL) will be collected from the marginal ear vein of either ear on Days 1, 2, 4, 9, 16, and 23. Approximately 20 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 condition or sacrificedat the time of necropsy (Day 29). The samples will be stored at room temperature and then centrifuged, and the separate serum and cellular fractionsstored in a freezer set to maintain -200C tIOeC. The separated serum and cellular fractionswill be sent frozen on 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 telephone at (612) 778-5294 prior to the shipment of 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 necropsy examinationand all abnormalitieswill be recorded. Animals in a moribund condition will be anesthetizedwith sodium pentobarbital(via injectionin the marginal ear vein), bled via the vena cava, and exsanguinated. Tissues, as described in section D. Pathology,(3) SampleCollection,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 abbreviated gross necropsy examination. The animals will be necropsied in random order and all abnormalitieswill be recorded. 00004'00 Page 37 of 42 TP3016.AB Page 9 (3) Sami)leCollection The sites of test and control material applicationwill be washed with lukewarm tap water prior to the necropsy procedure. 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 necopsied in each group will be collected and immediately placed in a freezer set to maintain a temperatureof -20*C 10*C. After necropsy, the animals will be discarded. The tissues (liver,bile, dermal applicationsite, kidneys)will be sent frozen on dry ice to the Sponsor after experimentaltermination. The samples will be shippedto the person listed in Section7.C (4).(c). The Sponsor is responsiblefor the retention an@ disposition of the samples. E. StatisticalAnalyses No statisticalanalyses are required. 8. Report A final report includingthose items listed below will be submitted. Descriptionof the test and control materials Description of the test system Procedures Dates of experimentalinitiationand termination Tabulation of mortalitydata 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 Original data, or copies thereof,will be availableat HWI to facilitateauditingthe study during its progressand before acceptanceof the final report.:When the final report is completed, all originalpaper data, includingthose item listedbelow will be retained in the archives of HWI according to HWI SOP. Protocol and protocol amendments Dose preparation records In-liferecords Body weights Dose administration Observations Anatomical pathology records Sample collection records Shipping records Study correspondence Final report (originalsignedcopy) 00004,,'l Page 38 of 42 TP3016.AB Page 10 The following supportingrecordswill be retained at HWI but will not be archivedwith the study data. Animal receipt/acclimationrecords Water analysis records Animal room temperature and humidity records Refrigerator and freezer temperature records Instrument calibration and maintenance records 0()004,1 Page 39 of 42 PROTOCOL APPROVAL TP3016.AB Page 11 z. John L. Butenhoff, PhD Date Sponsor's Representative 3M Toxicology Service Medical Department Steven' M. Glaza Study Director Acute Toxicology Hazleton Wisconsin, Inc. @gesentative Quality Assurance Un-tt:j Hazleton Wisconsin, Inc. (6329-130.protdsk2) Date 117,1 Date 00004@-i Page 40 of 42 Attachment I Scoring Scale for Acute Dermal Reactions TP3016.AB Page 12 Erythema 0 - None I - Slight 2 - Moderate 3 - Severe Edema 0 - None 1 - Slight (barelyperceptibleto well definedby definiteraising) 2 -.Moderate (raised approximately1 mm) 3 - Severe (raised more than 1 mm) Atonia 0 - None I - Slight (slightimpairmentof elasticity) 2 - Moderate (slow return to normal) 3 - Marked (no elasticity) Desnuamation 0 - None 1 - Slight(slightscaling) 2 - Moderate (scales and flakes) 3 - Marked (pronounced flaking with denuded areas) Coriaceousness 0 - None 1 - Slight (decreasein pliability) 2 - Moderate (leatherytexture) 3 - Marked (tough and brittle) Fissuring 0 - None I - Slight (definitecracksin epidermis) 2 - Moderate (cracks in dermis) 3 - Marked (cracks with bleeding) 00004()' Page 41 of 42 HAZLC-CC:N W IS C 0 N S IN POST OFFICE BOX 7545 MADISON. Wi 53707-7545 CORNUYG Company PROTOCOL TP3016.AB Single-Dose Dermal Absorption/Toxicity Study of T-6049 in Rabbits Sponsor HWI .6329-130 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 Soonsor's Representative John L. Butenhoff, PhD Study Director Steven M. Glaza' Amendment No. I This amendment modifies the following portions of the protocol: Effective January 10, 1995 1. Page 6. 7. Experimental Design; B. Dose Administration,(3) Dose Admi-nistration.Animal Nos. F53355 (Group 3 female) and F53398 (Group 2 female) were sacrificedon Day 2 due to injury (apparent broken backs). These animals will be replaced in this study. Replacement animals will be dosed, bled, and necropsied (includes tissue collection) at the same time as the animals in another 3M sponsored study (6329-137). Add the following as the second paragraph to this section: Due to the sacrifice on Day 2 of one Group 2 female (Animal No. F53398) and one Group 3 female (Animal No. F53355), replacementanimals will be treated at the respective dose levels tn the same manner as for the other animals in the study. The observations(clinicalobservations,reading of dermal irritation,and body weights) and the pathologyof the animals (unscheduledsacrifices and deaths, scheduled sacrifice, and sample collection)will be conducted in the same manner as for the other animals in the study. The method of blood sample collection will be in the same manner as for the other animals in the study. The frequency of blood collectionwill be as follows: Before initiation(Day 1), approximately24 hours post-dose (Day 2), Days 4, 8, 15, 22, and at experimentaltermination (Day 29). P h o n e 608 2 4 1 4 -17 1 E X P R E S S -M A IL D E L iv E R Y 3,'Ol K IN S M A N BLVD -@00004"t F a x ,6 0 8 _24 1:.2 MADIC.ON wi 53704 Page 42 of 42 Amendment No. I HWI 6329-130 Page 2 Effective January 24, 1995 At the request of the Sponsor, the weights of tissues collected and the volume of bile collected will be documentedin the raw data. These weights and volumes will be included with the sample shipment. Modify the following sections of the protocolto includethese additions. 2. Page 9. 7. ExperimentalDesign: D. Pathology: (3) Sample Collection. Modify the second sentence in the first and second paragraphs of this section with the followingunderlinedadditions: The whole liver, bile, an approximate 1-cm x 1-cm section of the dermal applicationsite from all animals, and both kidneys from the first male and female necropsied in each group will be collected,weighed (volume only determinedfor bile). and immediatelyplaced in a freezer set to maintain a temperature of -200C tlO*C. The samples and their correspondingweights or volumes will be shippedto the personlisted in Section7.C.(4).(c). 3. Page 9. 8. Report. Add the followingto this section: Individual animal tissue weights and bile volumes PROTOCOL AMENDMENT APPROVAL -7. /* Joh'nL. Butenhoff, PhD Sponsor's Representative 3M Toxicology Service Medical Department '6at-e Steven M. Glaza L3 Date Study Director Acute Toxicology Hazleton Wisconsin, Inc. Representativfi Quality Assurance Unit Hazleton Wisconsin, Inc. Date (6329-130.Aml.dsk2) 00004,S 9.1.2 AnalyticalprotocolAMDT-020895.1 000(-).I!) 3M Environmental Laboratory Protocol- AnalyticalStudy Single-dosDeermal Absorption/ToxiciSttyudy ofT-6049inRabbits In-VivoStudy ReferenceNumber: HWI#6329-130 StudyNumber: AMDT-020895.1 TestSubstance:FC-95 (T-6049) Name and Address ofSponsor: 3M SCD Division 367 Grove Street St.Paul,NlN 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, and Revisions: Thermal ExtractionofFluorideby Means ofa Modified Dohnnann DX2000 OrganicHalideAnalyzer-Liver FluorideMeasurement by Means ofan OrionEA940 Expandable Ion Analyzer ExtractionofFluorochemicalsfrom RabbitLiver Analysisof RabbitLiverExtractforFluorochenmicalsUsing ElectrosprayMass Spectrometry Author:James D. Johnson Approved By: # ttuuddeesysy D:. DDirrJeeo n ro Date John Butenhoff,PhD Date Sponsor Representative 0 0 0.,-0J- 1.0 PURPOSE The purposeofthisstudyistoassestshedermalabsorptioonfFC-95(T-6049)in rabbitsaftera singledermal dose of FC-95. 2.0 TEST MATERIALS 2.1Test,Control, and Reference Substances and Matrices 2.1.1AnalyticalReference Substance: FC-95, lot161 or 171.They are equivalent. 2.1.2AnalyticalReference Matrix: Bovine liverand bovine serum - 2.1.3 Analytical Control Substance: None 2.1.4 Analytical Control Matrix: Bovine liver and bovine serum 2.2 Source of Materials: 3M ICP/PCP Division (2.1.1), grocery liver)S,igma Chemical Company (2.1.22,.1.4serum) store (2.1.2, 2.1.4 2.3Number ofTest and Control Samples: Tissuesand fluidsfrom 18 test animals and 6 controlanimals.One animal was replacedon day 2 (animalF53398 inthe 5 mg/kg dose group replacedby animal F52999). Tissuesand fluidsinclude liverk,i,dney,serum, cellulafrractiond,ermal applicatiosniteand bile.Analysisof thesetissueswillbe atthediscretioonf the Study Director. 2.4Identificationof Test and Control Samples: The samples areidentifieudsing theHWI animal identificatinounmber which consistsof a lettearnd fivedigit number, plusthetissueidentityand day identity(serum). 2.5Purity and Strenegpth of Reference Substance: To be determinedby Sponsor. 2.6 Stability of Reference Substance: To be determined by Sponsor. 2.7 Storage Conditions for Test Materials: Room temperature (2.1.1), -20 100C (2.1.22,.1.4).Testand Controlsamples willbe receivedaccordingto AMDT-S-10-0. 2.8DispositionofSpecimens: Biologicaltissueasnd fluidswillbe retainedper GLP Regulationforthetime periodrequiredstudieslongerthan 28 days. 2.9 Safety Precautions: Refer to appropriateMSDS. Wear appropriatelaboratory attire.Use cautionwhen handlin(kynivesforcuttinct,hesamples. 2 3.0 EXPERIMENTAL -Overview Rabbitswere dosed with T-6049 in a controlgroup,5 mg/kg, 100 mg/kg, and 500 mg/ka dennally. (See HWI#6329-130 fordosing,animal weitc:rp htst,issuecollection, etc.)0Tissuesand serum areavailablferom theseanimalsforanalysisof total organicfluorinecontent.At thediscretioonf theStudy Directorsamples willbe analyzed to the extentnecessarytoprovideinformationto assessthe extentof dermal absorption. 4.0 EXPERIMENTAL - Methods 4.1 Liver and Serum screening methods: (attached) 4.1.1AMDT-M-1-0, Thermal Extractionof Fluorideby Means of a Modified Dohrmann DX2000 OrganicHalide Analyzer-Liver 4.1.2AMDT-M-2-0, FluorideMeasurement by Means of an Orion EA940 Expandable Ion Analyzer 4.1.3AMDT-M-4-0, Extractionof Fluorochemicalsfrom Rabbit Liver 4.1.4AMDT-M-5-0, Analysisof RabbitLiverExtractforFluorochenmicals Using ElectrosprayMass Spectrometry 5.0DATA ANALYSIS 5.1Data Reporting: Data willbe reportedas a concentratio(nweight/weighto)f fluorideper tissueor fluid,or as FC-95 per tissueor fluid.Statisticussed,atthe discretionof the Study Director,may includeserum averagesand standard deviationsof concentrationfsordifferendtose groups. Ifnecessary,simple statistictaelstssuch as Student'sttestmay be appliedto determinestatistical difference. 6.0 MAINTENANCE OF RAW DATA AND RECORDS 6.1 Raw Data and Records: Raw data,approved protocol,appropriatespecimens, approved finalreport,and electronidcatawillbe maintainedin the AMDT Archives. 0()0() 3 7.0 REFERENCES 7.1 AMDT-S-10-0, Sample Tracking System 8.0 ATTACHMENTS 8.1 AMDT-M-1-0, Thermal Extractionof Fluorideby Means of a Modified Dohrmann DX2000 Organic HalideAnalyzer-Liver 8.2 AMDT-M-2-0, FluorideMeasurement by Means of an Orion EA940 Expandable Ion Analyzer 8.3 AMDT-M4-0, ExtractionofFluorochemicalsfrom RabbitLiver 8.4 AMDT-M-5-0, Analysisof RabbitLiverExtractforFluorochenmicalsUsing ElectrosprayMass Spectrometry 0000ri,-j 4 3M Environmental Laboratory Method Thermal ExtractionofFluorideby Means of a ModifiedDohrmann DX2000 OrganicHalideAnalyzer- Liver Method IdentificatiNounmber: AMDT-M-1 RevisionNumber: 0 AdoptionDate: RevisionDate:None Author:Rich Youngblom Approved by: roup Lea 7@/ ro1u4 p@,L( 1,9/.3 Dafe QualityAssurance Date Software:MS Word 5.1a AffectedDocuments: AMDT-M-2 FluoridMeeasurementby Means ofanOrionEA940 ExpandableIonAnalyzer AMDT-EP-3 RoutineMaintenanceofa ModifiedDohrrnannDX2000 OrganicHalideAnalyzer 1 @.,l 00oo..j 1.0 SCOPE. APPLICABLE COMPOUNDS, AND MATRICES 1.1Scope:Thismethod isfortheoperatioonfa Dohrmann DX2000 when itisused toextract fluoridferom variousmatrices.The fluoridiestypicallcyollecteidnTISAB solutiofnoranalysis withan ionselectiveelectrode. 1.2ApplicableCompounds: Fluorochemicalosr otherfluorinatecdompounds. 1.3NtatricesB:iologicatlissuesp,articularlliyver. 2.0 KEYWORDS 2.1Fluoridef,luorine,xtractiopny,rolysisi,onizatioino,nselectiveelectrodeD,ohmiann, halide, DX2000, fluorochemicals. 3.0 PRECAUTIONS 3.1 Glasswareand exhaustgasescan be extremelyhot. 3.2Glasswareisfragileb,rokenglassmay causeinjuries. 3.3Pressurizedgases,propercompressedgashandlingpracticersequired. 3.4Solventbasedsamplesmay flashm,ay need toallowthem todrydown beforestartinrgun. 3.5Potentiabliohazardsdue tothebiologicamlatrices.Use appropriatpeersonalprotective equipment. 4.0 SUPPLIES AIND MATERIALS 4.1Compressed Oxygen, Hydrocarbon freer,egulatedto30 PSI. 4.2Compressed Helium,High PurityGrade,regulatetdo45 PSI. 4.3QuartzglasssampleboatwithTeflonTMtubingD,ohrmann 890-097orequivalent. 4.4Quartzglasscombustiontube,RelianceGlass G-9405-012orequivalent. 4.5Orion940999TotalIonicStrengthAdjustmentBuffer(TISAB 11)orequivalent. 4.6Sample collectiovnialsH,DPE. 4.7Milli-QT@,wiater 4.8Polystyrenpeipettes. 4.9ActivatedCharcoalE,. Merck 2005 orequivalent. 4.10HamiltonSyringeor equivalent. 4.11Miscellaneouslaboratorgylassware 5.0 EQUIPMENT 5.1Rosemount Dohrmann DX2000 OrganicHalideAnalyzer,modifiedforfluorideextraction. 5.2IBM compatible386 or486 computer. 5.3DX2000 softwarev,ersion1.00,modifiedforfluorideextraction. 5.4Excel Spreadsheetv,ersion5.0orgreater 6.0 INTERFERENCES 6.1 Sample sizeislimitedtoapproximatel1y50 mg, dependingon samplemoisturecontent.This may vary from matrixtomatrix. 2 0()Oo.j@j 7.0 SAMPLE HANDLING 7.1Samples arenottobe handledwithbarehands.Fluoridemay leachfrom theskintothe sample. Use forcepsor probetotransfetrissues. 7.2Samplesof liverarecutfrom frozenliverand placedina taredand labeledweigh boat.Use a cleanscalpeland cuttinaboard.The cuttingboardandscalpelshouldbe cleanedwithwater, methanol,or methanol-watersolutioanftereach liveirscut. 8.0 CALIBRATIOIN AND STANDARDIZATION 8.1PreparatioonfCalibratioSntandards 8.1.T1hestandarrdesquirefdoreachprojecwtilnleedtobeappropriaftoerthaitndividuparloject. Refertoprotocolforthatproject. 8.1.2Typically50-500ppm FC-95 inmethanolstandardasreused. 8.1.3For rabbitliverstudiesu,sebeefliverasthematrix.Cut a pieceoffrozenbeefliver(100150 mg) and weigh itina labeledand taredweighboat. 8.2 Calibratio-nOverview The normalcalibratiiosnthefluoridceurve(AMDT-M-2). However,ifan optionaslpikedliver curveisrequiredtheprocedurelistebdelow isused. 8.2.1A calibratiocnurvefortheDX2000 isgeneratedby spikingsampleswithknown standards and combustingthem usingthesame methods and matrixtypeasthesamplestobe tested. 8.2.2Typicallyt,hreereplicateosfeachstandardand fiveconcentratioonfsstandardswillbe spiked. 8.2.3Standardcurvewillbe plotteadsMass SpikedF (ug)on thex-axisand StandardMass RecoveredF (ug)on they-axis.Generatea regressiocnurveand calculatteheequationfortheline and ther2 value. 8.2.4Mass SpikedF (uL,=) (Amount spikedinmL) x (Conc.ofstandardinppm) x (0.6004)* *FC-95 is60.04% F therefor0e.6004isthefactoursedtoconvertFC-95 toF 8.2.5StandardMass RecoveredF (ug)= (TISAB volumeinnlL)x (Orionreadinginppm) 8.3Calibratio-nProcedure 8.3.1StartUp 8.3.1.1Run 2 ormore CleanCycleswhen startinignstrumenetachday. More cleancyclesmay be usediftheprevioussamplescontainedhighconcentrationosffluoride. 8.3.2Blanks 8.3.2.1Preparesample usingthesame methods and typeofmatrixasthetestsample. 8.3.2.2For rabbitstudiesu,sebeefliverasthematrix.Prepareatleast3 samplesofbeefliver (100- 150 mg) forblanks. 8.3.2.3Put sample inDohrmann boat.Combust eachsampleasdescribedinsection9.0and analyzesample accordinatomethod AMDT-NI-2 fortheionselectiveelectrodaenalysis. 3 8.3.2.F4orrabbisttudiest,hemeterreadinfgora blanksampleshouldbe0.03ppm orlower beforeproceedingwith thecalibrationB.um samples untilthislimitisreached,or untilinthe judgement of theoperatorthereadingisstablewith respecttohistoricarleadings(previous48 hours). 8.3.2.5For non-rabbitstudies,theblank readingsshould reach a predetem-dnedionconcentration beforeproceedingwith thecalibration. 8.3.2.6 Itmay be necessaryton-Liaxpproximately50 mg of charcoalwith the sample toaid combustion. 8.3.3 Standard Curve 8.3.3.1Weigh out atleast15 matrixsamples (5 standardswith 3 replicateesach) intaredand labeledweigh boats.For rabbitstudies,weigh 100-150 mg beef liversamples. Record weights in studydata. Storethematrix samples on dry iceor icepacks tokeep them frozenuntilused. 8.3.3.2 Place weighed beef liversample inDohrmann sample boat. 8.3.3.3Startwith thelowest standardconcentration.Using a Hamilton syringe,ejecta fixed quantityof the standardon or inthematrix.For rabbitstudiesu,se 4 uL of standardand ejectiton orinthe beefliver. 8.3.3.4At least3 replicatesshould be used forthe lowest standardconcentrationm;ore replicates may be used atthediscretionofthe analyst. 8.3.3.5Combust the sample asdescribedin section9.3 and analyzeaccordingto AMDT-M-2. 8.3.3.6Run all15 standards.Ifone replicatiessignificantdliyfferenftrom theothertwo replicatesr,un anothersample forthatstandard.Indicateindatathatthenew replicatreeplacesthe old replicateand thatthenew replicatweillbe used to calculatetheregressioncurve. 8.3.3.7When allstandardshave been run,calculatethe r2. r2 must be atleast0.95.Ifitisnot at least0.95,consultwith supervisor. 8.3.3.8A new standardcurveshould be run when the combustion tubeor sample matrixis changed. New standardcurve may alsobe rwi atthe discretionof theanalyst. 8.4 Storage Conditions for Standards 8.4.1Storage requirementsforstandardsare dependent on the individualstandardsused. Typically,standardsare storedatroom temperatureinplasticscrew topbottles. 8.4.2 New FC-95 standardsshould be preparedatleastonce a month. 9.0 PROCEDURES 9.1TypicalOperating Conditions: 9.1.1Combustion tubetemperature= 9500C. 9.1.2Oxygen and Helium flow = 50 cc/minute. 9.1.3Vaporization/Dryingtime= 240 seconds. 9.1.4Bake time = 300 seconds. 9.2StartUp Procedure: 9.2.1Ifthe program isnot starteds,tarthe EOX program on the PC. 9.2.2Open the SYSTEM SETUP window. 9.2.3Put the furnacemodule and thecellinthe READY mode. 9.2.4Close the SYSTEM SETUP window. 4 0()0()%.1 9.2.5When theoven has reachedtheREADY temperature,run theCLEAN found in theCELL CHECK menu. 9.2.6 See ANIDT-EP-3 fordetailosf theDohrmann software. BOAT program 9.3Sample ExtractionProcedure: 9.3.1Open theSAMPLE HATCH and placethesample inthe BOAT. Itmay be necessaryto n-dxapproximately50 mg ofcharcoalwiththesample to aidcombustion. Ifthisisdone, charcoal shouldalsobe mixed in while establishintghebaselineand when generatingthestandardcurve. 9.3.2 Close SAMPLE HATCH. 9.3.3 Add appropriatevolume ofTISAB solutionor 1:1 TISAB:Milli-QTN'water mixture toa labeledsample collectiovnial.Typically0.6niL to 15 mL areused. For rabbitstudiesu,se 1.0or 2.0mL of 1:1 TISAB:Milli-QTIIIwatermixture. 9.3.4Place the vialso thatthetipof theCOMBUSTION TUBE isin theTISAB atleast0.25 inches.Gases releasedduringpyrolysismust bubble throughthe TISAB. 9.3.5 Run theEOX-SOLIDS program found in theRUN menu. 9.3.6 When theEOX program isfinishedr,emove thecollectiovnialfrom thecombustion tube. 9.3.7 IfundilutedTISA.B was used to collecthesample,add an equalvolume of Milli-QTm water to theTISAB tomake 1:1 TISAB:Milli-QTM. 9.3.8Rinse the end of the combustiontube with &Elli-QTMwater and wipe with a KIMWIPE to remove any TISAB remaining on thetube. 9.3.9 Open the sample hatchand remove any remaining ash from theboat. Ash can be removed with a cottontippedapplicatororvacuumed out. Itmay be necessarytoscrapparticleosffthe bottom with a spatulaor othersimilardevice.A drop of Milli-QTMwater may be added to the boatto aidinthe Clean Cycle. 9.3.10 Close the hatch. 9.3.11 Run the CLEAN BOAT program. 9.3.12 Sample isready foranalysibsy ionselectiveelectrode(AMDT-M-2). 9.4 Sample Calculations 9.4.1 Use the standardcurve tocalculatethe sample value. 9.4.2 Sample Mass Recovered F (ug)= (TISAB volin mL) x (Orion readingin ppm - intercept (Slope) 10.0 VALIDATION 10.1QualityControl 10.1.1Daily Start Up Check Samples: Once the standardcurve isestablishede,ach day of analysisisstartedby analyzingQC samples. The QC samples are tobe the same as the lowest concentrationspikedsamples used togeneratethestandardcurve. Each concentrationmust be done in triplicatuenlessthe firsttwo replicateasrewithin20% of the standardcurve,then a third replicateisnot necessary. 10.2 Precisionand Accuracy: See method development analysisand sample analysisin FluorideNotebooks 2,3,and 5. Precisionand accuracy varieswhen analyzingsamples of different matricesand differentreferencecompounds. 10.3 Other ValidationParameters: NA 5 0()()Oli 11.0 DATA ANALYSIS 11.1 Calculations 11.1.1Forthestandarcdurve,useregressioannalysiisnExcel,versio5n.0or greater. 11.1.2To calculattehefluoridceontractiionthesample,seemethodAMDT-M-2. 11.2 Analyzing theData 11.2.1r2must be atleas0t.95orgreater".Outliersm"ay be excludediftwo of thethreereplicates arewithin20% ofeachotherand theoutlieirsgreaterthan200% oftheaverageofthosetwo or lessthan50% oftheaverageofthosetwo. Any suchoutliersshouldbe pointedoutinthedataand noted intheFinalReportalongwiththereasonitwas consideredan outlier. 12.0 ATTACHMENTS None 13.0 REFERENCES 13.1Rosemount Dohnnann DX2000 OrganicHalideAnalyzerOperator'Msanual (Manual 915349,revisioBn, December 1993) 13.2 ANMT-M-2 FluorideMeasurementby Means ofan OrionEA940 ExpandableIon Analyzer 13.3AMDT-EP-3 RoutineMaintenanceofa ModifiedDohrrrianDnX2000 OrganicHalide Analyzer 14.0 REVISIONS Revision Numbe Reason forChange Revision Date 6 0000!3!) 3M Environmental Laboratory Method FluorideMeasurement by Means ofan Orion EA940 ExpandableIon Analyzer Method IdentificatioNnumber: AMDT-M-2 RevisionNumber: 0 Adoption Date: Revision Date: None Author: Pich Youngblom Approved By: Qlalv@ G@, Leader Date Quality Assurance Lo- 4, Date Software: MS Word 5.1a Affected Documents: AMDT-M-1 Thermal Extractionof Fluorideby Means of a Modified Dohrmann DX2000 Organic HalideAnalyzer 0()OOC() 1.0 SCOPE. APPLICABLE COMPOUNDS, AND MATRICES 1.1SCOPE: Thismethod isforthecalibratiaond operatioonfan OrionEA940 Expandable Ion Analyzer. 1.2APPLICABLE COINTPOUNDS: Fluoride. 1.3APPLICABLE MATRICES: Liquidsamplesinan appropriatbeuffersolutionP.referred pH of 6.0. 2.0 KEYWORDS 2.1Fluoridef,luorinei,on selectiveelectrode 3.0 PRECAUTIONS 3.1No hazardsidentifiewdiththismethod. 4.0 SUPPLIES AND MATERIALS 4.1Orion940999 TotalIonicStrengtAhdjustmentBuffern (TISABII)orequivalent. 4.2OrionModel 900001 electrodfeillinsgolutio(nAgCl)orequivalent. 4.3Orion940907 100ppm fluoridsetandardorequivalent. 4.4Milli-QTIwIaIteror equivalent. 4.5Magneticstirbars. 4.6Lab tissues. 4.7Sample collectiovnials. 4.8Plastic100 mL volumetricflasks. 4.9Polystyrenpeipettes. 4.10Miscellaneouslaboratorgylassware. 5.0 EQUIPMENT 5.1OrionModel EA940 ExpandableIonAnalyzerorequivalent. 5.2OrionModel 960900 SolidStateCombinationFluorideelectrodoerequivalent. 5.3MagneticStirPlate. 5.4IBM compatible386 or486 computer(onlyneededifusingOrion3E software). 5.5Orion RS232 interfacceable(onlyneededifusingOrion3E software). 5.6MicrosoftExcel5.0(onlyneededifusingOrion3E software), 6.0 INTERFERENCES 6.1Itisrecommended thatthepH be atornear6.0.A 1:1 mixtureofTISAB and sampleIN4illiQ TlllwatewrillgenerallbyringsampletopH of6.0. 6.2Sample temperaturemay effecftluoridmeeasurement.Itisrecommended thatthesamplebe atroom temperatureasthestandardswere when themeterwas calibrated. 6.3The ratethesamplesarestirreadtshouldbe consistenwtiththeratethestandardswere stirred. 2 0()OOC-l 6.4Air bubblestrappedunder electrodcean giveerroneousreadings.Make sureno airistrapped under electrode. 7.0 SAINIPLE HANDLING 7.1 No specialhandlingnecessary. 8.0 CALIBRATION AND STANDARDIZATION 8.1 Preparation of CaUbration Standards 8.1.1Measure 50 m]L of TISAB IIinto5 100 mL plastivcolumetricflasks. 8.1.2Label theflasksas 0.05,0.1,0.5,1.0,and 1.5ppm F-,along withthedateand your initials. 8.1.3Pipette0.05,0.1,0.5,1.0,and 1.5niL of 100 ppm fluoridsetandardintotheappropriately labeledflasks. 8.1.4Add approximately30 mL of Milli-QTMwatertoeach flask. 8.1.5Shake theflaskstomix thesolutions. 8.1.6Eliminateairbubblesfrom theflasksby tippingtheflaskson theirsidesand rollingtheairin theflasksover theairbubbles. 8.1.7Bring thevolume intheflasksup tothe 100 mL mark witliMiUi-QTM water. 8.1.8Invertand shake theflasksforthefinalmixing, 8.1.9Record standardsin StandardsLog Book. 8.2 Calibration 8.2.1Ifnecessary,remove tapefrom electrodfeillinhgole. 8.2.2Invertprobe towet topseal. 8.2.3Ejecta few dropsof fillinsgolutionfrom bottom of electrodteo wet lowerseal. 8.2.4Filltheelectrodweith fillinsgolution. 8.2.5The meter and theF- electrodaeretypicallcyalibratebdy directmeasurement withno blank correctionu,singstandardswithconcentrationosf 0.05,0.1,0.5,1.0,and 1.5ppm F-,following themanufacturer'sinstructions. 8.2.6Record theslopein theappropriatleogbook. 8.2.7Clean theelectrodeby rinsingwithMilli-QTIwIaIterand wipingthesidesdown with lab tissues. 8.3 StorageConditionsforStandards 8.3.1Calibrationstandardsarestoredatroom temperature, 9.0 PROCEDURES 9.1Calibrationand inleasurementS,tandard method: 9.1.1The sample tobe measured needstobe mixed withTISAB usingtheproportions recommended by theTISAB manufacturer. 9.1.2Placea stirbarinthesample and placethesample on thestirplate. 9.1.3Allow thesample to mix fora few secondsbeforeinsertintgheelectrode.When the electrodeisinsertedm,ake surethereareno airbubblestrappedunder theelectrode. 9.1.4The sample shouldbe thesame temperatureas thecalibratiosntandardsand stirreadtthe same rateas thecalibratiosntandards. 9.1.5@Vhen thereadingshave stabilizerde,cordthereadingintheappropriatelogbook. 3 9.2CalibratioAnnd NleasurementU,singOrion 3E Software: 9.2.1Calibration: 9.2.1.1 Follow steps8.2.1to8.2.4. 9.2.1.2Press FunctionKey #8 (F8). 9.2.1.3The computer screenwillask you to confirm the number of standardstobe used, concentrationof thestandards,and whether or not a blank istobe includedinthe calibration. Make any necessarychanges tothe informationpresentedand clickon CONTINUE. 9.2.1.4Placethe electrodeinthe firssttandardon the stirplateand clickon CONTINUE. 9.2.1.5Observe thereadingson thegraphicdisplayon thecomputer. When thereadingshave stabilizedp,ress ACCEPT READING. 9.2.1.6Repeat step9.2.1.4and 9.2.1.5forthe remaining standards. 9.2.1.7Afterthe finalstandard,the computer willdisplaythe slopeof thecurve,aswell as the interceptand correlationR.ecord theslope,intercepta,nd correlatioinntheappropriatelog book and clickon CONTINUE. The calibratiodnataisautomaticallcyopiedtoC:\Orion\Data\Calib.txt. 9.2.2Data Spreadsheet: 9.2.2.1SelecteitherNEW or OPEN from theFILE menu toopen a new or existingspreadsheet tostoredata in. 9.2.2.2Record thename of thespreadsheetused in theappropriatelog book. 9.2.3Fluoride Measurement: 9.2.3.1Follow steps9.2.1through9.2.4 9.2.3.2Enter thename of thesample inthe appropriateplaceon thescreen. 9.2.3.3Click on theNEW SAMPLE button 9.2.3.4When the readingshave stabilizedc,lickon the RECORD buttonand writetheresultinthe appropriatelog book. 10.0 VALIDATION 10.1 QualityControl: 10.2 Precisionand Accuracy 10.3 Other ValidationParameters According toReference 13.2,therangeof detectionis0.02 ppm fluorideup to a saturatedsolutionof fluoride. 11.0 DATA ANALYSIS 11.1 Calculations None necessary. 11.2 Analyzina theData None necessary. 12.0 ATTACH,,NIENTS None 13.0 REFERENCES 4 0()00(;@"I 13.1Orion Model EA940 Expandable IonAnalyzer InstructioMnanual,Orion Research Incorporated,199 1. 13.2Orion Model 960900 SolidStateCombination FluorideElectrodeInstructioMnanual, Orion Research Incorporated1,991. 14.0 REVISTONS Revision Numbe Reason forChanize Revision Date 5 00006-1 3M Environmental Laboratory Method Extractionof Fluorochemicalsfrom Rabbit Livers SOP IdentiricatioNnumber: AMDT-M-4 RevisionNumber: 0 Adoption Date: RevisionDate: None Author: Dave Christenson/CynthiWaeber Approved By: roLu7peLaedad,er /0 Date QualityAssurance Date Software:MS Word, 6.0 AffectedDocuments: M-5, AnalysisofRabbitExtractforFluorochemicalsUsing Electrosp.-ay Mass Spectroscopy. 0000(.)Aj 1.0 SCOPE 1.1 Scope: This method isfortheextractioonf fluorochemicalfsrom rabbitlivers. Ethyl acetateisused toextractfluorocherrficaflrsom theliversforanalysisby electrospraymass spectroscopy. 1.2 Applicable Compounds: Fluorochemicalsor otherfluorinatecdompounds. 1.3 Matrices: Rabbit Livers. 2.0 KEYWORDS 2.1 Fluorochemicals,rabbitliverse,lectrospramyass spectrometerf,luorinated compounds, extraction. 3.0 PRECAUTIONS 3.1 Use gloveswhen handlingtherabbitliverst,heymay containpathogens. 4.0 SUPPLIES AND MATERIALS 4.1 Supplies 4.1.1 Syringe,capable ofmeasuring 100 gL 4.1.2 Eppendorf typeor disposablepipets 4.1.3 Gloves 4. IL.4 Plasticgrindingtubes 4.1.5 Plasticcentrifugetubes,15 niL 4.1.6 Labels 4.1.7 Nitrogen 4.1.8 Timer 4.1.9 FiltersT,itannylon syringefilter0s.,2 gm. 4. 1.10 Analydcal pipets:glassvolumetdc pipets. 4. 1.11 Disposableplastic3 cc syringes. 4. 1.12 Crimp cap autovials. 4.2 Reagents 4.2.1 Aqueous Ammonium Acetate(Aldrich)a,pprox.250 ppm: Preparea 2500 ppm aqueous solutionof ammonium acetateby adding 250 mg ammonium acetatetoa 100 mL volumetricflaskand dilutetovolume with Milli-Q water.Dilutethissolution1:10 fora 250 ppm solution. 4.2.2 Sodium carbonate/Sodium BicarbonateBuffer(J.T.Baker), (Na2CO3/NaHC03) 0.25 M: Weigh 26.5 g of sodium carbonate(Na2CO3) and 21.0g of sodium bicarbonate(Na]HC03) intoa 1 L volumetricflaskand bringtovolume withMilli-Qwater. 4.2.3 Diluteacetoniffisloelutiond,iluteacetonitri1l:e1 with Milli-Qwater. 4.2.4 EthylAcetate 4.2.5 Methanol 4.2.6 MIW-Q water 4.2.7 IH,IH,2H,2H - perfluorooctanesulfonaiccid(Aldrich) 4.2.8 FC-95 (3M SpecialtyChemical Division) 2 0()006(; 5.0 E!QUIPMENT 5.1 Ultra-TurraTx25 Grinderforgrindingliversamples. 5.2 Vortexmixer 5.3 Centrifuge 5.4 Shaker 5.5 AnalyticaElvaporator 6.0 INTERFERENCES 6.1 nere areno known interferencaetsthistime. 7.0 SAMPLE HANDLING 7.1 Ile rabbitfiverasrereceivedfrozen,and must be keptfrozenuntiltheextractioins performed. 8.0 CALIBRATION AND STANDARDIZATION 8.1 Preparationof InternalStandards 8.1.1 Preparean internasltandardofapproximately12ppm 1H,IH,2H,2Hperfluorooctanesulphonaiccidto be added toeach liversample. 8.1.2 Weigh atleast0.1 g of 1H,1H,2H,2H-perfluorooctanesulphoniaccidintoa 100 mL volumetricflaskR.ecord theactualweight. 8.1.3 Bringitup tovolume withmethanol,thisisthestockstandarcl 8.1.4 To a 250 mL volumetricflaska,dd 3 mLs of thestockstandardand bringto volume with Milli-Qwater.Calculatetheactualconcentratioonf the standard. actualmg perfluoroctane- sulphonicacid X 0.1 L 3 mL 250 mL actualconcentrationp,pm 8.2 Prepare FC-95 Anion Standards 8.2.1 PrepareFC-95 standardsforthestandardcurve. 8.2.2 Weigh approximately100 mg of FC-95 intoa 100 mL volumetricflask. Record theactualweight. 8.2.3 Bringup tovolume withdiluteacetonitrile. 8.2.4 Dilutethesolutionwithdiluteacetonitdl1e:10 fora solutionof approximately100 ppm. Dilutethissolution1:10 withdiluteacetoniagefor a solutionof approx.10 ppm. 8.2.5 Use the10 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 mL Nalgenebottlceontaining 200 mLs Milli-Qwater.Grind toa homogenous solution. 8.3.2 Add 1mL of thesolutiontoa 15 mL centrifugteube.Preparea totalof eight I n-lLaliquotosf thesolutionin 15 mL centrifugteubes.Be suretoresuspend solutionby shakingitbetween aliquots. oooog-) 8.3.3 SpikesevenoftheI mL aliquotwsiththefollowingamountsofworldng standardsin step9.12of theprocedure.One I mL aliquotservesas the blank. Working Standard UL (ApproximateConc.) - 500 ppb 100 500 ppb 2W- 500 ppb 3W- 500 ppb 400 1 ppm 500 55 ppp1m1mPEMMMEEI 200 300 Approximatefinal concentratioonf FC-95 inliver Blank 0.292 ppm 0.5 4 ppm 0.877 ppm 1.168 ppm 2.924 ppm I 5.848 ppm 8.772 ppm 8.4 Calculatetheactualvalueof thestandards: uL of standardx concentratio(nin1212m)= finalconcentratio(nppm) 171 mg liveF/1 ml homogenate ofFC -95inliver *Average weightof bovineliverin solutionasdeterminedby weighing I mL homogenatesof40 mg liverin200 mL ofNOI-Q water.The amount of FC-95 isreportedas equivalentosf FC-95 potassiumsalt. 8.5 Calibration 8.5.1 Extractthespikedbeefliverhomogenate following9.13to9.23of this method. Use thesestandardsto establisyhour curve on themass spectrometer. 8.5.2 Alternativelay,standardcurve may be generatedusingratiosof responses of theperfluorooctansulfonatneion and theinternasltandardanion versus concentratioonf theperfluorooctanesulfonatneion. 8.6 Storage Conditionsfor Standards 8. 6.1 New standardsarepreparedwith eachanalysisS.tandardsarestoredin coveredplasticentrifugteubesuntiltheanalysison themass spectrometer isperformed. 8.7 Storage Conditions for Standards 8.7.1 Beef liverhomogenates may be frozenafterpreparation. 9.0 PROCEDURLFS 9.1 Obtain frozenliversamples.In spenttissuen,otethattheliverhas notbeen packaged withothertissues. 9.2 Use a dissectinsgcalpeland cutoffapproximatelyI g of liver. 9.3 Weigh thesampledirectliyntoa taredplastigcrindingtube. 9.4 Record theliverweightin thestudynotebook. 9.5 Put a labelon thevialwiththestudynumber, weight,rabbitDD,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 inthesample and grindforabout 2 minutes,untilthesample isa homogeneous solutionwithno largechunks. Rinsetheprobeoffintothesample with2.5mLs waterusinga pipet. Take thegrinderapartand cleanitwithmethanolaftereach sample.Follow AMDT-EP-22. Cap thesample and vortexfor15 seconds. Pipet1 mL intoa 15 mL centrifugteube.Label thecentrifugteubewiththeidentical infor-matioansthegrindingtube.(SeeAMDT-M-4 Worksheet fordocumenting the r-emainingsteps.) Spike thebeefliverhomogenates withtheappropriataemount of FC-95 standard as describedin 8.3. Spike thesamplesand beefHver homogenates with 100 uL of internasltandard. Add I mL of thesodium carbonate/sodiubmicarbonatbeufferand ImL 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 cenftifugetubewith a 5 mL graduatedglasspipet.Transferthelabelto thefreshtube. Blow thesample down on theanalyticaelvaporatortoneardrynesswithnitrogen, approximately30 to40 minutes. Bring theremainingsampleup in 1 mL diluteacetoniauewithan analyticaplipet. Vortex 15 seconds. Transferthesampletoa 3 mL syringe.Attacha 0.2gm nylon mesh filtera,nd filter thesample intoa freshcentrifugteubeor a autovialL.abel thetubeor vialwiththe studynumber and animalnumber. Cap and holdforanalysibsy electrospramyass spectroscopy. Complete AMDT-M-4 worksheetand attachtopage of studynotebook. 10.0 VALIDATION 10.1 QualityControl- notapplicable 10.2 Precisionand Accuracy-not applicable 10.3 Other ValidationParameters-notapplicable 11.0 DATA ANALYSIS 11.1 None 12.0 ATTACHMENTS 12.1 WorksheetAMDT-M-4 13.0 REFERENCES 13. 1 AMDT-EP-22 RoutineMaintenanceofUltra-Tun-aTx-25 14.0 REVISIONS Revision Number Reason forChange Revision Date 0()O()U!j 5 Study Sample Number set# Rl:;nkT-iver Worksheet AMDT-M-4 FC-95 approx0.5ppm actual ppm Inn Ill. 700 UT 100 u@ 400 uT. - FC-95 approx Ippm actual ppm #w iflti)iT. FC-95 approx.5 ppm actual PPM #W Date and InitialfsorStd. - 200 iiT. 300 iiT. ------------ qtudynumber 3ghertt-henrityinwaolrk-gheeitgIneati-oini(inigren mny, I I I T.iver'F.xtrAc-bPomnre@gg- Pimt I mT.nfT.iveIr;nlutinn- Pinet100 uT,of I? nnm Internnlgt2ndard Std-# VortL-x15 -q Pinet I mf. of 2r%Onnm Ammnniiim ArL-tate. Std # Pit)eItmT.nf 0 219N&CO,10 25M Na]4('O.Ruffer Pineti mT. of FthylAreta Vnrtt-x*)0-10gec I,;h,a-kL20 min Centrifiip7e0-25 min Re.mnye:;4 mT. aliniinft orp;tni1c;;ver Blow fiownto near(iryne-(-<RO@;25 ml-)withN. AtlilI m- nf 1-1Acetnnitrilt-114.0 TNO$ Vnrte-x11;-ger. Filteurging2 3c!cB-T),;Yrincweitha 0')itmSRI filteirntc;)tI i mT,atitnq;imnvlie;;t T)attk- Tnitiniq 6 3M Environmental Laboratory Method Analysisof 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: @u ro/upp:Leaddeerr Date QualityAssurance Date Software:MS Word, 6.0 AffectedDocuments: M-4, Extractionof Fluorochemicalsfrom RabbitLivers 1.0 SCOPE 1. 1 Scope: This method isfortheanalysisof extractsof rabbitliveror othertissuesor fluidsforfluorochemicalsusingthe electrospraymass spectrometer.The analysis isperformed by singleionmonitoringofFC-95 anion,M/Z= 499, theinternal standardM/Z = 427, and otherappropriatemasses. 1.2 Applicable Compounds: Fluorochemicalsor otherfluorinatecdompounds. 1.3 Matrices:RabbitLivers(samples)B,eef 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 the voltagecable isplugged intothe probe DO NOT TOUCH THE PROBE, thereisriskof electricaslhock. 3.2 Do not run thepump above it'scapacityof 4000 psi.Ifpressuregoes over 4000 psi stopand releasepressure.The peak tubingmay be plugged.Troubleshoot back to findtheplug and replacetheplugged tubing.See AMDT-EP-15 3.3 Do not run thepump todryness. 4.0 SUPPLIES AND--MATERIALS 4.1 Supplies 4. 1.1 Nitrogengas regulatedto 140 psi. 4. 1.2 Fluofixcolumn or equivalent. 4.1.3 100 uLor250 uL flatdp syringeforsample injection. 4.2 Reagents 4.2. 1 Diluteacetonitrimloebile phase,diluteacetonitril1e:1 withMilli-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 theextractedsamples incapped 15 mL centrifugetubesor incapped autovials untilready foranalysis. 0000-i;@ 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 to obtainthe standardcurve. 8.2.2 Typicallyone standardisrun aftereach 5 to7 samples.Choose a standard inthesame range ofconcentratioans thesamples. 8.3 Storage Conditions for Standards 8.3. 1 Fresh standardsarepreparedwitheach 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 Set softwareto"Operateon",IonMode ES-. 9.1.2 Record backingpressureintheinstrumentlog. 9.1.3 Fillthesolventcylinderwithmobilephase. 9.1.4 Setthepump to"Run".Set theflowto 1000 uL/niin.Observes droplets coming outof thetipof theprobe.The pressureshould be at1700 to 1800 psi. 9.1.5 Check thefusedsilicaattheend of theprobe.Use an eye piecetocheck for chips.The tipshouldbe flatwithno jaggededges.Ifany chipsarefound cutoffthetipof thesilicwaitha column cutterand pullthesilictahroughto the appropriatleength. 9.1.6 Check your nitrogensupply.Turn on thenitrogenT.here shouldbe no nitrogenleakingaroundthetipof theprobe.A finemistshouldbe coming out of thetip. 9.1.7 Carefullyguidetheprobeintotheopening.Inserittuntilitwon't go any furtherC.onnect thevoltagecabletotheprobe. 9.1.8 Go tothe"Editor"page,and setIonizatioMnode toES-,and the appropriatemasses to427 and 499. 9.1.9 Ifitisnotinsingleionmode go to"Option"and setSIR. 9. 1.10 StartAcquisitionA.ssigna filename, MO-DAY-YR + letterR.ecorditin thelogbook. 9. 1.11 Run thebeefliversamplesfirstr,unningeach standardtwiceatthe beginningof therun..Run a QC check by runningone standardafterevery 5 to7 samples. 9.2 Manual Injection 9.2. 1 Draw 150 uL of sampleintoa syringeI.njecthesample intotherheodyne injectiopnort.Injecstlowly.Record thesample ID inthelogbook. 9.2.2 Turn thevtlveto"On". 9. 2.3 Wait two nlinutes,,indinjecthenextsample. 9. 2.4 Record thescannumber foreachsimple inthelogbook. 3 0 ()0 0 I-ilj 9.3 Using the Autosampler 9.3.1 Set up sample trayA, B, or C. 9.3.2 Record thesamplesand theirpositionisntheinstrumentlogbook. Up to 17 vialsmay be ineach run. 9.3.3 Set-upthesampler: 9.3.3.1 Push thesample button 9.3.3.2 Setsample loopsize= 100 uL 9.3.3.3 Setinject/sampl=e2 9.3.3.4 SetCycle time= 0 9.3.3.5 Name thefileL:ivers 9.3.3.6 Identifythetrayused 9.3.3.7 Add thesamplestoQueue by pressing"Entee' 9.3.3.8 Press"Run" tostart 10.0 VALIDATION 10.1 Quality Control 10. 1.1Run a standardevery5 to7 samples.Ifa significancthange(50%) in peak heightoccursstoptherun.Only thesamplesbeforethelastacceptable standardwillbe used.The remainingsamples willbe reanalyzed. 10.2 Precisionand Accuracy 10. 2.1See Method ValidatioRneportnumber AMDT-M-5.0.vi 10.3 Other ValidationParameters 10.4 RefertoMethod VadidadonReportNumber ANOT-M-5.0.Vl 11.0 DATA ANALYSIS 11.1 11.2 Calculations Plotthestandardcurve,usingthemean ofthetwo valuesobtainedforeach standard. 11. 2. 1Read peak heightsor areasforthesamples from theprintoutU.se linear regressiontodeterminethesample concentrations. 11. 2.2 Calculatethemg of FC-95 anion,or otherfluorocherr@cailnthetotalrabbit liver. mg FC-95 anioninthetotarlabbitliver mg FC-95 anionfrom std.curve gms of liverusedforanalysis x Totalmass ofliverg,ms 11.3 Make a resulttsableand enteritinthestudybook. 11.4 Printa chromatogram foreach sample,withthepeakslabeledwiththesample or standardD:).Writethestudynumber on theprintouti,nitiadla,te,and putitin the studyfolder.Stapleallchromatograms togetherand number pages. 4 12.0 ATTACHMENTS None 13.0 REFERENCES 13.1 AMDT-EP- 17 14.0 REVISIONS Revision Number Reason forchana Revision Date 000()?,- 4.J 5 9.3 QualityAssurance Unit Statement 0()Ool-if.kl Amchmnt D GLP Study QualityAssuranceStatement gw .... ......... ........................... Study Title:Single-doseDermal Absorption/ToxicityStudy of T-6049 in Rabbits Study Number. AMDT-020895.1 Name of Auditor KariRambo Ibis studyhas been inspectedby the QualityAssurance Unit asindicatedinthefollowingtable. The findingswere reportedto thestudydirectorand management. InspectionDates 10/13/95 10/19/95 Phase FinalReport Date InspectionReported to Manag=nt StudyDirect 10/19/95 10/19/95 QAU Auditor Date 9.4 Key Personnel Involvedin the Study 000()"f",Ll 3M Environmental Laboratory Key Personnel Thermal extractionfollowedby analysisusingOrion Ion analyzer: Jim Johnson Deb Wright Rich Youngblom Deann Plununer Analysisofliverextractsusingelectrospraymass spectrometry: Jim Johnson Dave Christenson Documentation and Reporting: Jim Johnson Rich Youngblom QualityAssurance Unit: Gale Van Buskirk Cynthia Weber Kari Rambo 00 9.11 Data 0()0011%i() 9.11.1Summary and raw data;ug F-inwhole liveras determinedby thermalextractiofnollowedby analysis usingOrion ionanalyzer. 00001,SIIL Summary ofCombustion Data - Liver AMDT-020895.1, HWI 6329-130 As ReferencedinFinalReport section6.0DATA ANALYSIS Totalgg FluorideinWhole Liver Mean perDose Group* ControlGroup 99 Std.Dev. 22.4 +5.7 5.0mglkg dose(T6049) 17.6+4.9 (0.003mg/kg)** 100 mg/kg dose(T6049) 15.8 + 2.6 (0.06mg/kg)** 500 mg/kg dose(T6049) 22.3+2.2 (0.30mg/kg)** *Calculatedasthe mean oftriplicatseamplesfrom each ofthreemale and threefemalerabbits. **Testmaterialisa 0.06% solutionofT6049, actualdose inparenthesis. FC95 AB % ID rcvry Livebrlnk-1 Liverblnk-2 Liverspk-1 106% Liverspk-2 99% F53405-1 F53405-2 F53405-3 F53356-1 F53356-2 F53356-3 F53404-1 F53404-2 F53404-3 F53353-1 F53353-2 F53353-3 Liverbik1 Liverblk2 Liverspk 1 91% Liverspk 2 102% F53358-1 F53358-2 F53358-3 F53359-1 F53359-2 F53359-3 F53402-1 F53402-2 F53402-3 F53364-1 F53364-2 F53364-3 F53352-1 F53352-2 F53352-3 F53349-1 F53349-2 F53349-3 F53399-1 F53399-2 F53399-3 F53346-1 F53346-2 F53346-3 F53350-1 F53350-2 F53350-3 F53397-1 F53397-2 F53397-3 Actual ppm Finliver (WNv) 0.835 0.287 1.140 1.200 0.474 0.281 0.305 0.212 0.249 0.198 0.684 0.244 0.254 0.232 0.205 0.18i 0.272 0.129 1.111 1.096 0.402 0.320 0.283 0.336 0.209 0.197 0.149 0.136 0.157 0.275 0.285 0.335 0.257 0.241 0.267 0.180 0.242 0.280 0.238 0.209 0.155 0.167 0.172 0.328 0.284 0.266 0.174 0.238 0.291 0.230 Average ppm Finliver (WNV) 0.353 0.220 0.394 0.206 0.335 0.247 0.147 0.298 0.255 0.234 0.200 0.222 0.241 0.253 liver burned (grams) 0.1138 0.1347 0.1403 0.1248 0.1141 0.1270 0.1322 0.1431 0.1458 0.1367 0.1279 0.1186 0.1230 0.1441 0.1096 0.1355 0.1165 0.1550 0.1237 0.1408 0.1346 0.1164 0.1370 0.1143 0.1174 0.1142 0.1518 0.1645 0.1434 0.1097 0.1140 0.1085 0.1685 0.1418 0.1401 0.1390 0.1010 0.1437 0.1014 0.1021 0.1290 0.1340 0.1396 0.1283 0.1296 0.1477 0.1375 0.1273 0.1367 0.1128 Whole TotalF- in liver whole weight liver (grams) (ug) 78.27 74.69 70.28 77.41 27.65 16.40 27.68 15.93 81.38 27.27 78.86 19.52 78.73 11.61 84.10 25.09 67.61 17.26 77.60 18.15 65.82 13.20 75.41 16.75 74.05 17.87 74.88 18.93 RPT1 30L.XLS Dosage (mglkg) 0 0 0 0 0 0 5 5 5 5 5 100 100 100 Page 1 000()N"j FC95 AB ID F53400-1 F53400-2 F53400-3 F53360-1 F53360-2 F53360-3 Liverbik 1 Liverbik2 Liverspk 1 Liverspk 2 F53347-1 F53347-2 F53347-3 F53365-1 F53365-2 F53365-3 F53345-1 F53345-2 F53345-3 F53354-1 F53354-2 F53354-3 F53403-1 F53403-2 F53403-3 F53000-1 F53000-2 F53000-3 F52999-1 F52999-2 F52999-3 F53351 -1 F53351-2 F53351-3 liverspk-3 liverspk-4 liverspk-5 liverspk-6 liverspk-7 % rcvry 95% 95% 94% 89% 71% 119% 100% Actual ppm Finliver (WM) 0.157 0.191 0.165 0.144 0.209 0.151 0.432 0.366 1.389 1.266 0.344 0.268 0.195 0.206 0.289 0.154 0.261 0.251 0.335 0.420 0.364 0.216 0.323 0.344 0.340 0.307 0.272 0.301 0.243 0.236 0.211 0.193 0.188 0.203 1.079 0.997 3.467 5.930 4.330 Average ppm Finliver (wfw) 0.171 0.168 0.269 0.216 0.282 0.333 0.336 0.293 0.230 0.195 liver burned (grams) 0.1418 0.1233 0.1356 0.1420 0.1057 0.1309 0.1137 0.1019 0.1036 0.1132 0.1221 0.1073 0.1415 0.1301 0.1212 0.1486 0.1438 0.1499 0.1352 0.1120 0.1091 0.1272 0.1126 0.1375 0.1378 0.1407 0.1384 0.1257 0.1369 0.1040 0.1218 0.1419 0.1449 0.1210 0.1312 0.1355 0.1253 0.1215 0.1401 Whole liver weight (grams) 74.96 76.35 83.70 82.60 81.34 70.97 70.62 79.62 87.51 80.69 TotalF- in whole liver (ug) 12.80 12.86 22.50 17.88 22.96 23.66 23.71 23.36 20.13 15.71 RPT1 30L.XLS Dosage (mg/kg) 100 100 500 500 Soo 500 500 500 5 100 Page 2 000(),Slll. 9.11.2Summary and raw data;analysisof liverextracts usingelectrospramyass spectrometry. r- HWI# 6329-130 Study: ProtocolNumb*r: Test Material: Matrix: R Squared Value: Response FactorAmount: Analyst: Date: Method: lnstruffwnt: LABBASE File: Single-DoseDermal Absorpton TP3016.AB T-6049 inRabbits(FC 95) Uver 0.9965 1.27E-05 DLC 4/3/95 AMDT-M-4 FisonsVG 2000 ElectrospraMyS 04039SAIS to-'31-q5 Group Dose Group 1: 0 mg/kg DistilleWdater Group 2: 5 mg /kg Group 3: 100 mg/kg Group 4: 500 mg/kg Sample F63404 F53366 F63359 F53368 FSUOS F53353 F53402 'F53352 F53399 F53349 F53364 F53400 F63360 F53350 F63346 F53397 F53403 F53347 F63000 F53345 F53365 F53354 IonCount Area N/D N/D N/D 6571 N/D N/D F-xtractwetd 9 Dilution ConcentrationTotalmass factor pgig ofliver 9 1.0422 1 1.1273 1 1.2009 1 1.0779 1 1.2166 1 1.0734 1 N/D N/D N/D 0.0618 N/D N/D 69.156 73.403 72.193 69.308 77.164 75.915 Togi amount of FC-95 per liver mg N/D N/D N/D 0.004 N/D N/D 6646 1.1472 1 58373 1.1648 1 N/D 1.1776 1 N/D 1.0143 1 22296 1.0259 1 0.0587 0.5080 N/D N/D 0.2203 76.184 81.324 63.494 76.006 80.666 0.004 0.041 N/D N/D 0.018 12849 1.3817 1 11111 1.054 1 18253 1.3119 1 N/D 1.3537 1 N/D 1.2903 1 0.0943 0.1069 0.1410 N/D N/D 73.828 74.574 72.534 73.666 73.063 0.007 0.008 0.010 N/D N/D 59686 1.0511 1 N/D 1.0447 1 34396 1.1242 1 4623 1.2143 1 N/D 1.0281 1 N/D 1.194 1 0.5756 N/D 0.3101 0.0386 N/D N/D 69.012 83.100 77.255 77.250 66.252 80.146 0.040 N/D 0.024 0.003 N/D N/D RabbitsF52999 (G2)and F53351 (G3)were notanalyzed The concentrationwas calculatedby usingthestandardcurveand multiplyintghe resultby 415.The 4/5 factoristheresultof a miscalculatioinnapplyingformula8.4inMethod AMDT-M-4-0. 137 mg of liverwas used in thiscalculatiornatherthani71 mg. The concentrationisnthe standardcurveare therefore5/4largerthanthey shouldbe.By multiplyintghecalculatedconcentratioInnthestandardcurveby 4/5,thecorrectresultisobtained. E ( 3 2el- 130 FC -1!@- SampLe DLCLIV Operator DLC Run -iate 05-09-1,),35 06:46:52 Versi,:)n: 11 Printet-I on 1)5-09-1995 AT 06:47:07 Str-aiqht Line Fit forcecl throuah Orilin. --------------------------------- A Atl..r OL 1 7 ,EVEL 4 AMOUNT ----------- 0.401D(D 1.21)00 1.61)00 Component E@^'TERNAL STANDARD AREA --------- 29977 6'@142C! 1)8594 124122 1 CALIBRATION Y SLOPE x INTERI--EPT --------------------------------------------------- Area Anioi.Lnt = 7.8924E@04 1.2670E-05 Amount Area O.OC)i:iOE+Of) 1).OOOOE+00 R s,4uared = 0.9955 PC rn IOIL,% 1.15 (Sc,,.* L116ck6, 5 A a&bAx.1 a.-A V-.t raWAAG'En- to File:040395A Sample!GA ROPOB 040395A 100- RABBIT LAB-BASE The NS Data System LIVEP, -EXT--R-Ar-T--Rtoll-tilsov- 23 2142 ZFS- 16 8 1964JIL 3 3 6 553 36 26 6 3415 \ 3764 4 03/0 513 787439 15 2 1386 v.FS,Son 217242 1 06 908 251031 @,513 logo 5966858696 5JL.L916659 6Z64_35g@ 3 _9 2090 129723 JL2299909 3000 34395 :3765 4000 8 9423 462 44L9@ 495 4? JL/ 5000 File:040395A Sample:GARBOPOL 040395A LAD-BASE - The MS Data Sgsten RABBIT LIVER ENTf*k9@g- to/i%lgsOLc 03/0 ZFS- 36 53 ?9 98 9? 86 V.FS- 456? 228 ,Scn 30 25031 5-3 4,it9-6 61 58696 ?9 Ogg 1106665 98 4961 1042 900 logo File:040395A Samp Ie:,GARBOPOi.1-@RAB, 040395A les- 9 fpv., 13 6 v.FS- LAB-BASE The MS Data System S to/q /q5 IZ K@V1541 03/0 FS340 16 8 looZFS- . ............ 641643 1386 -iO-74@- 9 ... 154 2 soni-30-0,--I--3,59 140i iW@o JL5'80-''-JL5'501600 59686 1"58 1650 7 ID File:040395A Sample *,GARB^PGL RABB14 040395A .too P531403 16 8 ZFS- 18 9 LAB-BASE - The MS Data Sgstem LIVE-.R.EXT-RAGT-s ioftwl-tr- D(-C- JL98 5 2JL42 2214 t Cr-2- 03/0 Gq 'PS334:1 24 59686 loo-I 5 8 L It V.FSisen' 12849 isea 1985 6645 2590 r4- File:040395A Sample:eARBepeL-RABBIT 040395A 2 cy-, %4 29 9 F 5 W 57Gr?- 36 5 LAB-BASE - The 17!U it-EXTRAC= 3174 MS Data Sgsten ML IC/li(tis F5 3344 G-Z 3-4 5 ;753-Mo 36 5 ZFS- 03/0 Fq3d 8-. JL2@7'23 29 v.FS - 58373 35 lscn2bilei 3808 i2,o6 34 18253 3 36 6 @6'06 DO File:040395A LAB-BASE The NS Data Sgstem Samp Ie: GAR *PGL-iWdflltT-L-TVZR-EXTRAM c@LC- 040395A s= 6 Ga- 4=9 T:Sss,99 ZFS- 4109 Fg33" 42 3 44 0 4 1 F*-3:@6'9 FS-33&46 cr( 47 4 r--4 4943 Fs34,o5 Cr I 51 7 FS33LIL rs's 5291 03/0 4pp,,/tsrci 57 6 irs 56 89423 44 9 x2.3 57 v.FS 22296 47 5833 6571 41,?gl 4623 4956 \ 0 Son4.o.o.,o.'-2.00 4400 4600 4890 5800 52010 5400 56 File:04039SA LAB-BASE Sample:CARBGPOL--RABBIT-L4VER-EXIlVeM 04039SA Y.FS - 'PS339-4- 59 2 The MS Data System ic>/icz 4m fv6 S-rc& 6 -t 03/0 vl5z(o*3ic, 623 8 V.FS - -------------------------------------------- 41944 61 39399 6236 6249 7180 6265 ISON 5900 5950 6900 6850 6100 6 JL50 6 2'00 6 25 0 630 File:040395B Sample:--------- AMA,InvbA%Vb.ftv 040395B ZFS- LAB-BASE The MS Data Sgstem lolisfy- DLC- 7'2 03/0 U) rL 10 fn LeeZFS,min 5373 L 180849 69 119112 72 65836 46 31889 2@ 1 9 40E;3 16 21L 6644889944@1.66t @4@4596 651 5 3 206865 5692 4956 tjLy' JL1.66jL 8 5 94f. L 8.0 iLe.0 iL a FIO NOT DEFINED