Document 6b9a1KBOE0dXX4MZdLOOrjYB1

3M MEDICAL DEPARTMENT, CORPORATE TOXICOLOGY ProtocolforStudyNo. T-7132.2;ST-39 PHARMACOKINETIC STUDY OF PERFLUOROOCTANE SULFONAMI]DE IN RATS Study Objective: The objectivoefthisstudyistoassessthepotentiaflororalabsorptionu,rinaryand fecal clearanceand biologicalpersistenceof PerfluorooctaneSulfonamide (PFOSA) inmale Sprague Dawley ratsaftera singleoraldose. Analysisof the serun-4liver,urineand feces forpotentialmetabolitesof PFOSA willbe performed by LCMS and perhaps other methods. Previous studiesof theN-ethyl derivativeof PFOSA have concluded that PFOSA isthe ultimatemetaboliteinrats(1,2);however, the analyticatlechniqueused in thesestudieswas unable to detectallpotentialmetabolites,including perfluorooctanesulfonat(ePFOS). No pharmacokineticstudieshave yet been done on PFOSA to resolvethisissueand searchfor other possiblemetabolitesinthe urineand feces.Recently,validatedmethods have been developed forthe quantitatioonf PFOSA and itspotentialmetaboliteP,FOS, inserum and liverdown to the low partper billion level(3).One goalof thisstudyisto determinethepotentialfor,ifoccurring,theextentof conversionof PFOSA to PFOS. Thisinformationwillhelpto expwn the pharmacokineticsof NETFOSE and itsmetabolitesand providedata forproper risk characterization. Research Client. 3M SpecialtyChemicals Division 3M Center,Building236 SaintPauL MN 55144 ponsor.,'36@-L"ediycai;on: 3M SpecialtyChemicals Division 3M Center,Building236 SaintPaul,NIN 55144 3i"vSiti-aieg'lc'"J'"oxicLualbogryaiciy 3M Center,Building270-3S-06 room SB314 SaintPaul,NN 55144 Stua@,Director.- Andrew M. Seacat,Ph.D. Toxicology Specialist 3M Medical Dept. /Corporate Toxicology 3M Center,Building220-2E-02 SaintPaul,N4N 55144 Ph.:651-575-3161 FAX: 651-733-1773 Study Toxicologist: Deanna Luebker, MS Advanced Research Toxicologist 3M Medical Dept. / Corporate Toxicology 3M Center,Building220-2E-02 SaintPaul,MN 55144 T-7132.2:ST-39 PFOSA PK Ph: 651-737-l')74FAX: 651-733-1773 Proposed,Stu4T,imeline In-LifSetartDate:October4th,1999 In-LifeEnd Date: November 2nd, 1999 AnalyticaClompletionDate:TBA FinalReport CompletionDate:TBA RegulatoryCompliance: Thisstudywillbeperformedinthe3M StrategiTcoxicologyLaboratoryundera defined protocoland classifiaesda "ClassB Study" asexplainedinTOX SOP 0950,Strategic ToxicologyLab GLP Program Procedure. TestMaterial: Dan Hakes. Product Resi)onsibilitL%i,aison3M Chemicals Division.willfumish hip-h-Duritv PFOSA. Identification: Name: Perfluorooctanesulfonan-@de Molecular Formula: CsFl7SO2NH2 Lot Number: L-10009(Preparedby George Moore, 3M SMD Lab,Bldg 236,April1996) Purity:Analysisby GCMS determinedthatthestarinmgaterialwas over 991/opure (4).Qualitativaend quantitativceompositionarlesulttshatwere derivedfrom thesingletrial'I-1/19F-NMcRross-integratiaonnalysis revealedthatthecompositionwas 65.8% CF3(CF2)x-SO2-NH2 (Normal chain)1,8.7%CF3(CF2)x-CF(CF3)-(CF2)y-SO2-NH2(Internal monomethylbranch)1,1.2% (CF3)2CF-(CF2)x-SO2-NH2 (Isopropyl branch),3.5% CxF2x+ I-CF(CF3)-SO2-NH2 (Alphabranch)and 0.28% (CF3)3C-(CF2)@,-SO2-NH2(t-Bburtaynlch)(5)H.PLC/NIS characterization ofthePFOSA samplerevealed9,600ppm ofPFOS, 1,100 ppm of C7F ISS02NH2, 5 10 ppm ofC9Fl9SO2NH2, 6,600ppm of C8F 16HS02NIH2, 24,000ppm of Cl&F36HS02NH2, 1,200ppm of CgFl5H2SO2NH2and lower concentrationosf severalotherarru*deBsa.sed on the sum of theimpurities, thepurityofthePFOSA samplewould be approximatel9y6 % (6 & appendix1). Stability: Documentationwillbe kept on filewiththeSponsor. StorageConditions: Upon receiptt,estmateriawlillbe storedtightlsyealedatroom tcmperature. Characteristics: Informationon synthesismethods,compositionor othercharacteristitchsat definethetestmateriawlillbe kept on filweiththeSponsor. 2 T-7132.2,ST-39 PFOSA PK Animals: Species: Rat Strain: Sprague Dawley Source: Harlan Age at initiationof treatment: Weight at initiationof treatment: Number and sex: 30 males 6-8 weeks approximately 150-250g Table I - Dose Groups Group Dose N 0 mg/kg 15 2 5 mg/kg I Euthanasia 5 each on days 1, 4 and 29 post dose 5 each on days 1,4 Iand 29 post dose Identification: AUA Number: eartag with animal number or unique tailmark. 2246 Husbandry: Housing: All ratsfrom groups I and 2, which are to be sacrificeodn day 29 post dose,willbe housed individualliynmetabolism cages for portionsof the stud,,(yseeTable 2). When not inmetabolism cages,theseratswillbe group housed instandardcages. All otherratswillbe group housed in standardcages throughout the study. Diet/Water: Harlan Teklad LM-485 Mouse/Rat SterilizablDeiet,suppliedby Harlan Teklad,Madison, WI, and tap water willbe provided to allratsad libitum throughout the stt!dy. Environment: Environmental controlsforthe animal room willbe setto maintaina temperature of 72 3'F, humidity of 30-701/o,a n-@m*mum of 10 exchanges of room airper hour and a 12 hour light/darckycle. Dose and Dosing P@-ocedures: Method of administration/Dose preparation: A single5mg/kg dose of PFOSA willbe admirn'sterevdia oralgavage to ratsingroup 2 on day zero of the study.The PFOSA willbe prepared from a stocksolut;-,o-f InOm2,/mi PFOSA in acetone A fin:z0l.1% (Imcylml) uniform suspension(or emulsion) of PFOSA in2% Tween 80 and 1% acetone willbe prepared using a 15 n-ddounce tissuegrinder.A volume of 5 ml suspension/ kg body weight willbe administeredto each rat.Resuspensionof solidswillbe performed with 5 strokesof the tissuegrinder pestelbefore each sample isdrawn-up inthe syringefor dosing.A single5 3 T-7132.2-S,T-39 PFOSA PK n-@/kg body weight dose of vehicle,2% Tween 80 and I% acetone,willbe administeredvia oralgavage to ratsingroup I on day zero of the study. Obsen,ation ofanimais: ClinicalObservations: Each animal willbe observed day (excludingweekends and holidays)for mortalityand morbidityand notablefindingswillbe recorded. Additional findingsN@,ilble recorded as they are observed. Body Weights: Each aru'malwillbe weighed immediatelypriorto treatment,weekly thereafterand immediately priorto euthanasia. Specimen Collection: Frequency (See table2): 13rineand feces collectionmsillbe made on dqvs I - 4 po,;tdoee Necropsies willbe performed on days 1,4 and 29 post dose. Table 2 - Schedule Sun Mon Oct 3 Oct 4 day 0 DOSING Oct 10 day 6 PD Oct 17 day 13 PD Oct 11 day 7 PD Oct 18 day 14 PD Oct 24 day 20 PD Oct 31 day 27 PD Oct 25 day 21 PD Nov I day 28 PD Tues Oct 5 day I PD Collection, 1>@,I PD sac Wed Oct 6 day 2 PD Collection Oct 12 day 8 PD Oct 19 day 15 PD Oct 13 day 9 PD Oct 20 day 16 PD Oct 26 day 22 PD Nov 2 day 29 PD I)N,29 PD sac. Oct 27 day 23 PD Thurs Oct 7 day 3 PD Collection Oct 14 day 10 PD Oct 21 day 17 PD Oct 28 day 24 PD Fri Oct 8 dav 4 PD C@llection Switchto reg cages. Dy 4 PD sac. Oct 15 day 11 PD Oct 22 day 18 PD Oct 29 day 25 PD Sat Oct 9 day 5 PD Oct 16 day 12 PD Oct 23 day 19 PD I Oct 30 day 26 PD Method of Specimen Collection: Urine and feceswillbe collectedfrom each metabolism cage atthe designatedtimes. The initiavlolume of urinewillbe recorded,the sidesof the urine collectionapparatuswillbe washed with approximately 5-1On-d delor@izewdate!-and the @nal volume of ufinc -1-1@4-1,1bcrcught to 15 rnlwith additionaldeionizedwater. Dailyfecesweight willbe recorded foreach arnmal. At the designatedtimes,animalswillbe euthani ed by C02 and gross necropsy performed. During necropsy,blood (@:6L-mi) willbe collectedviathe abdominal aortaand transfer-retdo blood collectiontubes without anticoagulant.Blood samples willbe aflowed to clotfora period 4 T-7132.2:ST-39 PFOSA PK of 15 to 30 minutes atroom temperatureand the clotwillbe spun down in a centrifugeat I100 x g for 5 minutes. The serum wfllbe transferredto labeled1.5H n-@crofugetubes and centrifugedagain at 2000 x g to remove any remainingred blood cells.Each serasample willthen be transferretdo a separatelabeledpolypropylenemicrofuge tube and frozen in dry ice.Liverswillbe removed, weighed, placedindividuadyinto labeledsterilesample bags and flashfrozen in liquidnitrogenthen maintainedon dry ice. Specimen Handling: Specimens willtemporarilybe storedina freezersetto maintain-60 to 80'C. For metaboliteanalysist,hesespecimens willbe packed indry ice and shipped to: Kris Hansen, Ph.D. 3M Environmental Technology and SafetyServices 935 Bush Avenue St.Paul,NfN 55133-3331 Ph: 612-778-6081, FAX: 612-778-6176. Allresults.Nilb]e provided forinclusioninthe finalreport. The number, type and date of collectionof specimens to be generated for analysisare as follows: Table 3 - Specimens Specime day I post dose Serum 10 (5/group/day) Liver 10 (5/group/day) Urine 10 (5/group/day) Feces 10 1 (5/group/day) day 2 post dose 10 10 Day 3 post dose 10 10 day 4 post dose 10 10 10 10 day 29 post dose 10 Total 30 10 30 40 40 Data Analysis: Data collecteodn parentrompotindand ;df-.ntifimgebtlaebolitewsillbe analyzedfnr toxicokinetipcarametersand for statisticalsliygnificandtifferencesbetween groups using ANOVA and /or StudentsT-test. 5 T-7132.2.ST-39 PFOSA PK Responsibilities: Deanna Luebker and Andrew Seacat Ail]be responsiblefor dosing the animals, collectingin-fifsepecimens,performing the necropsiesand collectingand sending tissuespecimens for analysis. Kris Hansen, 3M EnvironmentaL willbe responsiblefor analyticaelvaluationof the Andrew Seacatwilldrafta finalreportand ensurethe reportreceivesappropriate3M review before a finalreportisissued. 6 ,'Vignatures: e@@V,@4aO /;@. AndrewM. Seacat Ph.D. Toxicology Specialist Study Director Deanna Luebker, MS Advanced Research Toxicologist Study Toxicologist Sponsor Representative T-7132.2, ST-39 PFOSA PK it,za@ L Date Date D@te 7 T-7132.2;ST-39 PFOSA PK References: I Grossman MR. and Bowen J.M. (1990) Tissueanalysisof fluorinatedsulfonamide pesticide-a.n evaluationof distributioenl,iminationa,nd potentialfor bioaccumulationinorallyexposed rats.M.S. Thesis,Univ. of Georgia, Athens, GA. (alsopossiblypublishedas:Grossman Mark R. and Bowen J.M. (1990) Tissue distributioannd elirm'nationf a fluorinatedsulfonamidepesticideinrats. F@utidam.AppL Toxicol.,but notfoutid). 2. Grossman MR., Nbspagel,M.E. and Bowen J.M. (1992) Distributioannd tissuein ratsduring and afterprolonged dietaryexposure to a highlyfluorinated sulfonamidepesticideJ. Agric.Food Chem. 40, 2505 - 2509. 3. K.J.Hansen, L.A. Clemen, M.E. Ellefson,H.O. Johnson.(1999).Compound St)ecifiCcharacterizatioonf OrizanicFluorochemicalsinGeneral Population Human Sera Samples. 3M Environmental Lab, St.Paul,MN 55133. 4. PayferR.M. GC/MS analysesof PFOSA (L-10009). SA&C AnalyticalRequest No. 59426. Report 9/24/99.3M SA&C Lab Building236-2B-1 1. 5. Tom KestnerChemical Characterizatioonf PFOSA, L-10009, by IH and 19FNMR SpectroscopyReguests 59426. 3M SpecialtyAdhesives & Chemicals AnalyticalLaboratory / SNPvID-236-2B-1 1.September 25, 1999. 6. DeRoos F.L. Characterizatioonf PFOSA Samples, T-7132-1 (L-10009) and TN- A- 1584. Request # A- 151254. Report 10/7/99.Corporate AnalyticalTechnology Center,Building201-1-29,CATC - Chromatography Group. 8 Appendix 1: Teli736-0665 201-1W-29 T-7132.2-ST-39 PFOS'A PK CorporateAnaMicalTechnologyCenter To: LarryA. Wendling/US-Corporate/3M/US cc: Andrew Seacat/US-Corporate/3M/US subject-.CharacterizatioonfPFOSA Sample forToxicology Larry, Ihave completed the HPLC/MS characterizationfthePFOSA sample (T-7132-,L-10009 preparedby G. Moore 4/96)thatisproposed tobe used forthe animialfeedingstudy. I found 9,600 ppm of PFOS, 1,100 PPm Of C7FISS02NH2, 510 PPm Of C9Fl9SO2NH2, 6,600 ppm of CSF16HS02NH2, 24,000 ppm of ClSF36HS02NH2,1,200 ppm of C8Fl5H2SO2NH2 and lower concentrations of severalother amides. Based on the sum ofthe impurities,the purityof the PFOSA sample would be approximately 96 %. I'vethoughta littmloere about apparentpresence ofC18F36HS02NH2 inthe sample. The identificatioofnthiscompound was based primariloyn the observed molecularweightand the relativeelutionorderinthe chromatogram. RatherthanC18F36HS02NH2, itseems more probablethatthiscompound isactually(C8FI 7SO2)2NH. Confirmationofthis identificatiwoinllrequireadditionaalnalyses. Ialsoneed tothinkmore about whether the otherpartiallhydrogenatedperfluoroamidesmay actuallybe ofthis The PFOS was quantifieudsinga standardcurve preparedby analyzingPFOS standardsso itsconcentratiosnhouldbe accurate.The amide impuritiesh,owever, includingthepartially hydrogenatedamides,were allquantifieudsinga PFOSA standardcurve assuming thatthey had thesame mass spectralresponse factoras didPFOSA. While thisassumption will introducesome errortothe quantitativdeata,itisthebestwe can do sincewe do nothave standardsforeach ofthese amides. Also,thiscalculatedpurityassumes thatallofthe impuritieasreamenable to HPLC and are detectedby theanalysis.While not alwaystrue, thissample was analyzedby Rick PayferusingGC/MS and by CATC usingourGC method. Neitherofthe analysesfound volatiloersemivolatiliempuritiese,.g.,N-methyl FOSE, NethylFOSE, etc.at concentrationsgreaterthan 50 ppm. Ialsocarriedouta semiquantitativaessessment ofthepurityofthePFOSA by comparing thePFOSA responsetoa PFOSA standardcurve. Usingthistechnique,the purityofthe PFOSA sample was found to be 118 %. These analyses were carriedoutinduplicatew,ith triplicaitnejectionosfeach solutions,o they shouldbe relativelayccurateas analytical variatiownould be averaged. Ingeneral,however, itisnothighlyaccuratetouse a chromatographicmethod toquantifya relativelpyure materialdue tothe extremelylarge dilutiofnactori,nthiscase > 100,000,thatmust be applied.Itisnotexpected that instrumentavlariatiownould biasthe Durityhigh. Itispossiblethatthe puritvofthe sample isactuallhyigherthan thepurityofthe PFOSA thatwe are usingas a standard! Ifthiswere true,the puritwyould be determinedto be > 100%. Andrew Seacat has reviewedthe concentrationsofthe impuritietshatwere determined and calculatetdhattheconcentratioonf PFOS willnotadverselyaffectthe study. At 9,600 ppm, he calculatetshatthe ratsreceiving3 mg/kg/day would ingestapp.0.20 mg PFOS totalin 9 T-7132.2.ST-39 PFOSA PK Appendix I Continued: 28 days assuming a 0.25 kg rat. Ifwe furtherassume that30 % ofthat isdeposited inthe liver(based on previousfeedingstudies)and liverisapproximately12 grams, then the predictedconcentrationinthe liverfrom the residualPFOS would be 200 ug PFOS x 0.3 / 12 g liverweight= 5 ugigram,or 5 ppm, which initselsfhouldnotimpose any toxicological consequences. Any PFOS measured significantolvyerthatcouldbe attributetdo metabolism of PFOSA to PFOS. He believesthatthe 1-H and 2-H perfiuoroamides are interestinags they may be subjectto metabolicattack,however these amides should be analyticaldliystincftrom the Perfluorinatesdpecieson down the line,and would not amount to a foreseeabletoxic concentrationand thereforewould be acceptableifno othersubstitutcean be prepared. Andrew isstiltlhinkingaboutwhether the (C8FI7SO2)2NH willhave a negativeimpacton the study. Pleasegive me a callat6-0665 ityou have any questionsconceming ourdiialysesoritican provideadditionailnformation. Fred L.DeRoos CATC 10