Document nqYOjaK5KXOrB9nwE6w5Jzk1

3M MEDICAL DEPARTMENT, CORPORATE TOXICOLOGY ProtocoflorStudyNo. T-7132.2S;T-39 PRAR,MACOKINE]NC STUDY OF PERFLUOROOCTAN-E SLLFONAMIDE IENRATS Study Objective: The objectiveof thisstudy isto assessthe potentialfororalabsorption,urinaryand fecal clearanceand biologicalpersistenceofPerfluorooctaneSulfonan@de(PFOSA) inmale Sprague Dawley ratsaftera singleoraldose.Analysisofthe serurfll,iveru,rineand feces for potentiamletabolitesofPFOSA wU be performed by LCMS and perhaps other methods. Previousstudiesofthe N-ethylderivativeofPFOSA have concluded that PFOSA isthe ultimatemetaboliteinrats(1,2);however, theanalyticatlechniqueused in thesestudieswas unable to detectallpotentialmetabolitesi,ncluding perfluorooctanesulfonat(ePFOS). No pharmacokineficstudieshave yetbeen done on PFOSA to resolvethisissueand searchforotherpossiblemetabolitesintheurineand feces.Recently,validatedmethods have been developed forthe quantitatioonfPFOSA and itspotentialmetaboliteP,FOS, inserum and liverdown tothe low partperbilbon level(3).One goal of thisstudyisto determinethepotentiaflor,ifoccurringt,heextentof conversionof PFOSA to PFOS. This informationwillhelpto explainthe pharmacokineticsofNETFOSE and itsmetabolitesand providedataforproper risk characterization. Research Client. 3M SpecialtyChemicals Division 3M Center,Bading 236 SaintPauL MN 55144 Sponsor.Study Location: 3M SpecialtyChemicals Division 3M Center,Building236 SaintPaul,NlN 55144 3M StrategicToxicology Laboratory 3M Center,Building270-3S-06 room SB314 SaintPauL MN 55144 Study Director: Andrew M. Seacat,Ph.D. Toxicology Specialist 3M MedicalDept. / CorporateToxirology 3M Center,Building220-2E-02 SaintPaul,MN 55144 Ph,-6.51-575-3161 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 Ph: 651-737-1374 FAX: 651-733-1773 T-7132.2;ST-39 PFOSA PK Proposed Study 7-imetine In-Life StartDate: October 4th, 1999 In-LifeEnd Date: November 2nd, 1999 AnalyticalCompletion Date: TBA Final Report Completion Date: TBA Regulatory ConWliance: This studywillbe performed inthe3M StrategicToxicologyLaboratoryunder a defined protocol and classifieads a "ClassB Study" as explainedin TOX SOP 0950, Strategic Toxicology Lab GLP Program Procedure. TestMate7ial: Dan Hakes,ProductResponsibiliLtiyaison3M ChemicalsDivision,,%Nfiilmliishigh-purity PFOSA. Identification: Name: Per-fluorooctanesulfonarnide Molecular Formula: CsFl7SO2NH2 Lot Number: L-10009 (Preparedby George Moore, 3M SNM Lab, Bldg 236, April 1996) Purity: Analysisby GCMS determined thatthe staringmaterialwas over 990/opure (4).Qualitativeand quantitativecompositionalresultsthatwere derivedfrom the singletrial'W'9F-NMR cross-integratioannalysis revealedthatthe compositionwas 65.8% CF3(CF2)x-SO2-NH2 (Normal chain),8].7% CF3(CF2)x-CF(CF3)-(CF2)y-SO2-.NH2 (Internal monomethyl branch),11.2% (CF3)2CF-(CF2)x-SO2-NH2 (Isopropyl branch),3.5% CxF2x+ 1-CF(CF3)-S02-NH2 (Alpha branch)and 0.28% (CF3)3C-(CF2),-SO2-NH2(t-Butylbranch)(5)B.PLC/MS charactefintion of thePFOSA sample revealed 9,6W ppm of PFOS, 1,100 ppm of C7Fl5SO2NH2, 5 10 ppm of C9Fl9SO2NH2, 6,600 ppm of C&Fl6HSo2NH2, 24,WO ppm of C,sF-;6HS02NH2,1,200 ppm of CgFl5H2SO2NH2 and lower concentrationsof severalother an-@des.Based on the sum of the impurities, the purityof thePFOSA sample would be approximately96 % (6 & appendix 1). Stability: Documentation vallbe kept on filewith the Sponsor. Storage Conditions: Ul>on receipt,testmaterialwillbe storedtightlysealedatroom temperature. Characteristics: Informationon synthesismethods, composition or other characteristictshat definethe testmaterialwillbe kept on filewith the Sponsor. 2 Animah: Species: Rat Strain: Sprague Dawley Source: Fhulan Age at initiationof treatment: Weight at initiationof treatment: Number and sex: 30 males 6-8 weeks approximately 150-250g T-7132.2; ST-39 PFOSA PK Table 1 - Dose Groups Group Dose IN 1 0 mg/kg 15 1 2 5 mg/kg 15 Euthanasia I 5 each on days 1,4 and 29 post dose 5 each on days 1, 4 and 29 post dose I dentif-i cation: AUA Number: ear tag with animal number 2246 or ur@que tail mark. Husbandry: Housing: All ratsfrom groups 1 and 2,which areto be sacn'ficeodn day 29 post dose,-,kiblel housed individualliynmetabolism cagesforportionsofthe study(seeTable2). When not inmetabolismcages,theseratswillbe group housed instandardcages. AU otherratswillbe group housed in standardcages throughoutthe study. Diet/Water: Harlan Teklad LM-485 Mouse/Rat SterilizabDlieet,suppliedby Harlan Teklad,Madison, WL and tapwater willbe provided to allratsad libitum throughout thestudy. Environment: Environmental controlsfortheanimal room willbe setto maintaina temperatureof 72 3'F,humidityof 30-700/ca, minimum of 10 exchanges of room airper hour and a 12 hour light/darckycle. Dose and Dosing Procedures: Method of administration/Dosepreparation: A single5mg/kg dose of PFOSAAifl be administeredviaoralgavage to ratsingroup 2 on day zero of the study.The PFOSA willbe preparedfrom a stocksolutionof I00mg/ml PFO SA inacetone.A final0.1% (1mg/H) uniform suspension(oremulsion)of PFOSA in2% Tween 80 and 1% acetone willbe preparedusinga 15 n@ dounce tissuegrinder.A volume of 5 rrdsuspension/ kg body weight NVIIbIe administeredto each rat.Resuspensionof solidswillbe performed with 5 strokesofthe tissuegrinder pestelbeforeeach sample isdrawn-up inthe syringefordosing.A single5 3 T-7132@2; ST-39 PFOSA PK n@ / kg body weight dose of vehicle,2% Tween 80 and 1% acetone,willbe administeredviaoralgavage to ratsin group I on day zero of the study. Obsen,ation of Animals: ClinicalObservations: Each animal willbe observed daily(excludingweekends and holidays)for mortalityand morbidityand notablefindingswillbe recorded. Additional findingswillbe recorded as they are observed. Body Weights: Each animal willbe weighed immediately priorto treatment,weekly thereafterand immediately priorto euthanasia. Specimen Collection: Frequency (See table2): Urine and feeescollectionswillbe made on days I - 4 post dose. Necropsies willbe performed on days 1,4 and 29 post dose. Table 2 - Schedule Sun Oct 3 Mon Oct 4 day 0 D,OSFNG 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 Wed Tlurs Fri Sat Oct 5 Oct 6 Oct 7 Oct 8 Oct 9 day I PD day 2 PD day 3 PD dav 4 PD day 5 PD Collection, Collection Collection Collection Dy IPD sac Switchto reg cages. D@, 4 PD Oct 12 day 8 PD Oct 19 day 15 PD Oct 13 dav 9 PD Oct 20 day 16 PD Oct 14 day 10 PD Oct 21 day 17 PD sac. Oct 15 day 11 PD Oct 22 day 18 PD Oct 16 dav 12 PD Oct 23 day 19 PD Oct 26 Nov 2 day 29 PD Dy 29 PD sac. Oct 27 day 23 PD Oct 28 dav 24 PD Oct 29 day 25 PD Oct 30 day 26 PD Method of Specimen Collection: Urine and feceswillbe collectedfi-omeach metabolism r-ageatthe designatedtimes. The initiavlolume of urinewillbe recorded,the sidesof theurine collectionapparatuswillbe washed Aithapproximately5-1Oml deioru*zedwater and the finalvolume of urine %xilble brought to 15 H with additionaldeionizedwater. Daily fecesweight willbe recorded for each animal. At the designatedtimes,animalswillbe euthanizedby C02 and gross necropsy performed. During necropsy,blood (@t6 mi) willbe collectedviathe abdominal aortaand transferredto blood collectiotnubes without anticoagulant.Blood samples willbe allowed to clotfora period 4 T-7132.2; ST-39 PFOSA PK of 15 to 30 minutes at room temperature and the clotwillbe spun down in a centrifugeat I 100 x g for 5 minutes. The serum wU] be transferredto labeled 1.5ml microfuge tubes and centrifugedagain at 2000 x g to remove any remaining red blood cells.Each sera sample N"'Ilthen be transferredto a separate labeled polypropylene n-@crofugetube and frozen in dry ice. Livers willbe removed, weighed, placed individuady into labeled stefflesample bags and flashfrozen in fiquidnitrogen then maintained on dry ire. Specimen Handting: Specimens wflltemporar@y be stored in a freezersetto maintain -60 to 80'C. For metabofite analysis,these specimens wi-Bbe packed in dry ice and shipped to: Kris Hansen, Ph.D. 3M Environmental Technology and Safety Services 935 Bush Avenue St.Paa MN 55133-3331 Ph. 612-778-6081, FAX: 612-778-6176. All resultswillbe provided for inclusionin the finalreport. The number, type and date of coflectionof 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 __C5/group/day) Feces 10 1 (5/group/d@y)_ 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 collectedon parent compound and identifiablmeetaboliteswiflbe analyzed for toxicokineticparameters and for statisticafsliygnificantdifferencesbetween groups using ANOVA and /or Students T-test. 5 T-7132.2; ST-39 PFOSA PK Responsibiades: Dleama Luebker and Andrew Seacat wiE be responsiblefor dosing the animals, r-ofleefinign-fifespecimens, performing the necropsies and coflectingand sending tissuespecimens for analysis. Kris Hanser@ 3M EnvironrnentaL wiflbe responsiblefor analyticalevaluation of the Andrew Seacat wifldrafta finalreportand ensure the reportreceivesappropriate 3M review before a finalreport isissued. 6 Signatures: Andrew M. Seacat Ph.D. Toxir-ologySpecialist Study Director Dearma Luebker, MS Advanced Research Toxir-ologist Study To)dcologist at@ Sponsor Representative T-7132.2,ST-39 PF)OSA PK Date -7 Date D@te 7 T-7132.2;ST-39 PFOSA PK References: I. Grossman MR. and Bowen J.M. (1990) Tissueanalysiosf fluorinatesdulfonamide pesticidea:n evaluationof distribu6oreil,iminationa,nd potentiaflor bioaccumulationinorauy exposed rats.@4S. Thesis,Univ. of Georgia, Athens, GA. (alsopossiblypubhshed as:Grossman Mark R. and Bowen J.M. (1990) Tissuedistributioannd efiminatioonf a fluorinatedsulfonarr@dpeesficideinrats. Fundwn. AppL Toxicol.,but notfound). 2. Grossman M.R-, NfispageLM.E. and Bowen J.M. (1992) Distributioannd tissuein ratsduringand afterprolonged dietaryexposure to a l@ghlyfluorinated sulfonan-@dpeesticideJ..Agric.Food Chem. 40, 2505 - 2509. 3. K.J.Hanser@ L.A- Clemen, M.E. Ellefson,FLO. Johnson-(1999).Compound SpecificCharacterizatioonfOrganic Fluorochen@calsinGeneral Population Human Sera Samples.3M EnvironmentalLab, St.Paa MN 55133. 4. Payf@rR-M. GC/MS analysesof PFOSA (L-10009). SA&C AnalyticaRlequest No. 59426. Report 9/24/99.3M SA&C Lab Budding 236-2B-1 1. 5. Tom Kestner Chemical Characteri7AtioonfPFOSA, L-10009, by IH and 19FNMR SpectroscopyRNuests 59426.3M SpecialtAydhesives& Chemicals AnalyticalLaboratory / SNPvff)-236-2B-11,September25, 1999. 6. DeRoos F.L. CharactefimtionofPFOSA Swnples,T-7132-1 (L-10009) and TNA-1 584.Request iiA-1 51254. Report 10/7/99.Corporate AnalyticalTechnology Center,Bu@ding 201-1-29,CATC - Chromatography Group- Appendix 1: Tel:736-0665 201-lW-29 T-7132.2; ST-39 PFOSA PK CorporateAnaykal Technobgy Center To: LarryA. Wendling/US-Corporate/3M/US cc: Andrew SeacatfUS-Corporate/3MAJS Subject: Characterizationof PFOSA Sample forToxicology LarTy, I have completed the HPLC/MS characterizationofthe PFOSA sample (T-7132-,L-10009 prepared by G. Moore 4/96) thatisproposed to be used forthe animialfeedirigstudy. I found 9,600 ppm of PFOS, 1,100 PPm Of C7FISS02NH2, 510 PPm Of C9Fl9S02NH2, 6,600 ppm of C8Fl6HS02NH2,24,000 ppm of Cl8F36HS02NH2, 1,200 ppm of C8F 1SH2S02NH2 and lower concentrations of several other amides. Based on the sum of the impurities,the purityof the PFOSA sample would be approximately 96 %. I'vethought a littlmeore about apparent presence of C18F36HS02NH2 inthe sample. The identificationf thiscompound was based primarilyon the observed molecular weight and the relativeelutionorder inthe chromatogram. Rather than C18F36HS02NH2, itseems more probable thatthiscompound isactually(C8FI 7SO2)2NH. Confirmationof this identificativoanllrequireadditionalanalyses. Ialso need to thinkmore about whether the otherpartiallhyydrogenated perfluoroamides may actuallybe of this The PFOS was quantifiedusing a standard curve prepared by analyzing PFOS standards so itsconcentrationshould be accurate. The amide impuritiesh,owever, includingthe partially hydrogenated amides, were allquantifiedusing a PFOSA standard curve assuming thatthey had the same mass spectralresponse factoras did PFOSA. While thisassumption will introducesome errorto the quantitativedata, itisthe best we ran do sincewe do not have standards foreach of these amides. Also, thiscalculatedpurityassumes thatallof the impuritiesare amenable to HPLC and are detected by the analysis.While not always true, thissample was analyzed by Rick Payfer using GC/MS and by CATC using our GC method. Neither of the analyses found volatiloer semivolatileimpuritiese,.g.,N-methyl FOSE, NethylFOSE, etc.at concentrationsgreaterthan 50 ppm. I alsocarriedout a semiquantitativeassessment of the purityofthe PFOSA by comparing the PFOSA response to a PFOSA standard curve. Using thistechnique,the purityof the PFOSA sample was found to be 118 %. These analyses were carded out induplicate,with triplicatienjectionsof each solution,so they should be relativelayccurate as analytical variationwould be averaged. Ingeneral,however, itisnot highlyaccurate to use a chromatographic method to quantifya relativelypure materialdue tothe extremely large dilutiofnactor,inthiscase > 100,000,that must be applied. Itisnot expected that instrumentalvariationwould biasthe purityhigh. Itispossiblethatthe purityof the sample isactuallyhigherthan the purityofthe PFOSA thatwe are usingas a standard! Ifthiswere true,the puritywould be determined to be > 100%. Andrew Seacat has reviewed the concentrationsof the impuritiesthatwere determined and calculatedthatthe concentrationof PFOS willnot adverselyaffectthe study. At 9,600 ppm, he calculatesthatthe 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 % ofthatisdeposited inthe liver(based on previousfeeding studies)and liverisapproximately12 grams, then the predictedconcentrationin the liverfrom the residualPFOS would be 200 ug PFOS x 0.3 112 g liverweight = 5 ug/gram, or 5 ppfn,which in itselsfhould not impose any toxicological consequences. Any PFOS measured significantloyver thatcould be attributetdo metabolism of PFOSA to PFOS. He believesthatthe 1-H and 2-H pertluoroamides are interestinags they may be subjectto metabolic attack,however these amides should be analyticalldyistincftrom the Perfluodnatedspecies on down the line,and would not amount to a foreseeable toxic concentrationand thereforewould be acceptable ifno othersubstitutecan be prepared. Andrew isstiltlhinkingabout whether the (CBFI 7SO2)2NH willhave a negative impact on the study. Please give me a callat 6-0665 ifyou have any questions conceming our analyses or ifIcan provide additionalinformation. Fred L.DeRoos CATC 10