Agenda/l3MEPAScienceSeminar,Apri4,2012•Introductions/SigninAll(5min)Introductions/Sign‐‐•IntroductoryRemarks‐LZ/JS(5min)•CommentsOnGrandjean EtAl.StudyOfImmuneResponseMarkersinFaroeseChildren(LRZ,10min)•IsClearanceACommonExplanationAcrossTheEpidemiologicalAssociationsReportedAt“Low”PFOAAndConcentrationsAssociationsReportedPFOSConcentrations(GWO,120min)•DietaryFactorsAsPotentialEffectModifiersInEpidemiologicalStudies(SC20,min)•GeneralDiscussion•AdjournCommentsGrandjeanetonGrandjeanal.SerumVaccineAntibodyConcentrationsinChildrenExposedtoPerfluorinatedCompoundsJAMA,Jan25,2012,307(4):391‐397BriefSummary•656consecutivesingletonbirthsinFaroeIslands,1999–2001•Vaccinationsat3,5and12monthsagainstdip,tetpertpolioandHfluBboosterage5,,;DTbooster;DPTboosterafter2003;titersmeasuredage5prebooster,4weekspostboosterandage7•PFOS,PFOA,PFHxS,PFNA andPFDAmeasuredinmaternalseraweek32andchildrenage5prebooster•GMPFOSwas27.3ng/mL inmaternalseraand16.7ng/mL inchildrenatage5BriefSummary•Actualdata(tirv.fluorochemicalte soc e caconcentrations)notpresented•Resultsofmodelshowingimpactoftwofoldchangeineachfluorochemicalontitersisshown•Resultsofa“structuralequationmodel”usingPFOS,PFOAandPFHxStogetheralsoshownintabularform.Oddtiff llib ltil lf•sraoorangeowprotecveevesoantibodiesagainstdiphtheriaandtetanusatages5and7ispresentedtextpresentedintheBriefSummary•Intheirmodel,adoublingofPFOSseraconcentrationeitherinmaternalorchildseratendedtonegativelyimpacttiters(14negand2posslopes),buttheimpactincludedthenullin13of16comparisons•ForPFOS:theoddsratioforfallingbelowtheprotectivelevelofdiphtheriaantibodyatage5werepositiveandexcludedpositiveexcludedthenullbasedoneithermaternalorchildserameasurement;theoddsratioincludedthenullatage5fortetanustitersfallingbelowtheprotectivelevelandalsoincludedthenullprotective,includedforthiseffectatage7forbothtetanusanddiphtheriatitersComments•NocaseofdiphtheriareportedinDenmarksince1985(WHO)•TheauthorsinexplicablyfailtociteFeietal,2010,whofoundmaternalPFOSconcentrationsnoimpactofmaternalconcentrationsonhospitalizationforinfectiousdiseaseuptoage3inasimilarsizeDanishcohort•Theepisodesincreased3Mepisodesofcarestudyshowednoincreasedriskforanyinfectiousdiseaseepisodeofcareinchemicalvfilmplantemployeesbetween1993and1998•SOTposter– lackofimpactoninfectiousdisease•Nojustificationforrouingfluorochemicalsg pgComments•TheimmunotoxicologyliteratureforPFOSisnotaccuratelyrepresented;theystatethatimmunotoxiceffectshavebeenfoundinmiceat“...serumconcentrationssimilarreportedpresentconcentrationssimilartothosereportedinthepresentstudy...”(27and16ng/mL)•Peden‐Adamsisreportedaneffectonhumeralimmunityat92ng/mL;Qazicouldnotreplicatethisresultandreportedthiseffectatover11,000ng/mL•NoseraconcentrationsareotherstudyinwhichPFOSconcentrationsmeasuredreportedaneffectonanimmuneendpointatlessthan600ng/mLIs Clearance a Common ExplanationAcross the EpidemiologicalAssociations Reported at “Low”PFOA and PFOS Concentrations?Geary Olsen*, Sue Chang, John Butenhoff,Carol Ley, Larry ZobelApril 4, 2012*PresenterBiomonitoring30.434.9253035404550

Concentration (ng/mL)PFHxSPFOSPFOAGeometric Mean Perfluoroalkyl Biomonitoring Trends inNHANESand the American Red Cross Studies, 1999 – 2010Olsen et al. Environ Sci Technol 2012 (submitted)2.12.251.931.671.521.961.3420.717.114.513.28.35.24.73.953.923.444.132.440510152025NHANES1999-2000Am RedCross 2000-2001NHANES2003-2004NHANES2005-2006Am RedCross 2006NHANES2007-2008Am RedCross 2010

Concentration (ng/mL)30.434.93035404550

Concentration (ng/mL)PFHxSPFOSPFOAGeometric Mean Perfluoroalkyl Biomonitoring Trends inNHANESand the American Red Cross Studies, 1999 – 2010Olsen et al. Environ Sci Technol 2012 (submitted)Since 20002.12.251.931.671.521.961.3420.717.114.513.28.35.24.73.953.923.444.132.440510152025NHANES1999-2000Am Red Cross2000-2001NHANES2003-2004NHANES2005-2006Am Red Cross2006NHANES2007-2008Am Red Cross2010

Concentration (ng/mL)76% ↓in PFOS40% ↓inPFHxS48% ↓in PFOA30.434.920.7253035404550PFHxSPFOSPFOAGeometric Mean Biomonitoring Trends in NHANESand the American Red Cross Studies, 1999 – 2010NEW NHANES DATASupports American Red Cross 2010 AnalysesNewNHANESData2.1 2.25 1.93 1.67 1.52 1.96 1.52 1.3420.717.114.513.28.78.35.24.73.953.923.444.132.842.4405101520NHANES1999-2000Am RedCross 2000-2001NHANES2003-2004NHANES2005-2006Am RedCross 2006NHANES2007-2008NHANES2009-2010Am RedCross 201020253035404550

Concentration (ng/mL)PFOSPFOAGeometric Mean Perfluoroalkyl Biomonitoring Trends by Location,American Red Cross Studies, 1999 – 2010Olsen et al. Environ Sci Technol 2012 (submitted)05101520 Boston 2000-2001 Boston 2006 Boston 2010 Charlotte 2000

- 2001 Charlotte 2006 Charlotte 2010 Hagerstown 2000

- 2001 Hagerstown 2006 Hagerstown 2010 Los Angeles 2000

- 2001 Los Angeles 2006 Los Angeles 2010

Mpls

- St. Paul 2000

- 2001 Mpls St. Paul 2006 Mpls-St. Paul 2010 Portland 2000

- 2001 Portland 2006 Portland 2010

Concentration (ng/mL)CONCLUSIONS(Olsen et al. Environ Sci Technol 2012 , submitted)•Population halving time for PFOS is approximately 4.3 to 5.0 years.•This is similar to the individual geometric mean serum elimination half-life of ≈ 4.8 years.•This similarity suggests the dominant PFOS-relatedexposures to humans in the United Stateswere greatly mitigated during the phase-out period.•The PFOA decline observed between 2006 -2010 may represent the initial reflection of successof the U.S. EPA’s 2010/2015 PFOA Stewardship Program.Epidemiology Associations Reportedwith PFOS and PFOAPharmacokinetic Variability and the Miracle of Modern Analytical ChemistryMatthew Longnecker (NIEHS) Epidemiology 2006;17:350-351“The impressive capabilities of modern analytical chemistry are being applied with increasingfrequency in epidemiologic studies.With lower exposures being evaluated, andlowerconcentrations being measured, it seems opportune to bear in mind that a great proportionof variation in measured levels among subjects maybe accounted for by differences inmetabolism and excretion.The measurements we obtain may afford only a glimpse of abyproduct of the underlying pharmacokinetics, systems, biology, and pathogenesis. Whilethese are not entirely new concepts, their importance may be clearer today than everbefore.”Pharmacokinetic Variability and the Miracle of Modern Analytical ChemistryMatthew Longnecker (NIEHS) Epidemiology 2006;17:350-351“The impressive capabilities of modern analytical chemistry are being applied with increasingfrequency in epidemiologic studies.Withlowerexposures being evaluated, andlowerconcentrations being measured, it seems opportune to bear in mind that a great proportionof variation in measured levels among subjects maybe accounted for by differences inmetabolism and excretion.The measurements we obtain may afford only a glimpse of abyproduct of the underlying pharmacokinetics, systems, biology, and pathogenesis. Whilethese are not entirely new concepts, their importance may be clearer today than everbefore.”Pharmacokinetic Variability and the Miracle of Modern Analytical ChemistryMatthew Longnecker (NIEHS) Epidemiology 2006;17:350-351“The impressive capabilities of modern analytical chemistry are being applied with increasingfrequency in epidemiologic studies.Withlowerexposures being evaluated, andlowerconcentrations being measured, it seems opportune to bear in mind that a great proportionof variation in measured levels among subjects maybe accounted for by differences inmetabolism and excretion.The measurements we obtain may afford only a glimpse of abyproduct of the underlying pharmacokinetics, systems, biology, and pathogenesis. Whilethese are not entirely new concepts, their importance may be clearer today than everbefore.”Pharmacokinetic Variability and the Miracle of Modern Analytical ChemistryMatthew Longnecker (NIEHS) Epidemiology 2006;17:350-351“The impressive capabilities of modern analytical chemistry are being applied with increasingfrequency in epidemiologic studies.Withlowerexposures being evaluated, andlowerconcentrations being measured, it seems opportune to bear in mind that a great proportionof variation in measured levels among subjects maybe accounted for by differences inmetabolism andexcretion.The measurements we obtain may afford only a glimpse of abyproduct of the underlying pharmacokinetics, systems, biology, and pathogenesis. Whilethese are not entirely new concepts, their importance may be clearer today than everbefore.”Pharmacokinetic Variability and the Miracle of Modern Analytical ChemistryMatthew Longnecker (NIEHS) Epidemiology 2006;17:350-351“The impressive capabilities of modern analytical chemistry are being applied with increasingfrequency in epidemiologic studies.Withlowerexposures being evaluated, andlowerconcentrations being measured, it seems opportune to bear in mind that a great proportionof variation in measured levels among subjects maybe accounted for by differences inmetabolism andexcretion(clearance).The measurements we obtain may afford only aglimpse of a byproduct of the underlying pharmacokinetics, systems, biology, andpathogenesis. While these are not entirely new concepts, their importance may be clearertoday than ever before.”Pharmacokinetic Variability and the Miracle of Modern Analytical ChemistryMatthew Longnecker (NIEHS) Epidemiology 2006;17:350-351“The impressive capabilities of modern analytical chemistry are being applied with increasingfrequency in epidemiologic studies.Withlowerexposures being evaluated, andlowerconcentrations being measured, it seems opportune to bear in mind that a great proportionof variation in measured levels among subjects maybe accounted for by differences inmetabolism andexcretion(clearance).The measurements we obtain may afford only aglimpse of a byproduct of the underlying pharmacokinetics, systems, biology, andpathogenesis.While these are not entirely new concepts, their importance may be clearertoday than ever before.”Pharmacokinetic Variability and the Miracle of Modern Analytical ChemistryMatthew Longnecker (NIEHS) Epidemiology 2006;17:350-351“The impressive capabilities of modern analytical chemistry are being applied with increasingfrequency in epidemiologic studies.Withlowerexposures being evaluated, andlowerconcentrations being measured, it seems opportune to bear in mind that a great proportionof variation in measured levels among subjects maybe accounted for by differences inmetabolism andexcretion(clearance).The measurements we obtain may afford only aglimpse of a byproduct of the underlying pharmacokinetics, systems, biology, andpathogenesis.While these are not entirely new concepts, their importance may be clearertoday than ever before.”Epidemiologic associationsare not equivalent tocausation.Pharmacokinetic Variability and the Miracle of Modern Analytical ChemistryMatthew Longnecker (NIEHS) Epidemiology 2006;17:350-351“The impressive capabilities of modern analytical chemistry are being applied with increasingfrequency in epidemiologic studies.Withlowerexposures being evaluated, andlowerconcentrations being measured, it seems opportune to bear in mind that a great proportionof variation in measured levels among subjects maybe accounted for by differences inmetabolism andexcretion(clearance).The measurements we obtain may afford only aglimpse of a byproduct of the underlying pharmacokinetics, systems, biology, andpathogenesis.While these are not entirely new concepts, their importance may be clearertoday than ever before.”Epidemiologic associationsare not equivalent tocausation.May expect to observephysiologically-basedassociations.Pharmacokinetic Variability and the Miracle of Modern Analytical ChemistryMatthew Longnecker (NIEHS) Epidemiology 2006;17:350-351“The impressive capabilities of modern analytical chemistry are being applied with increasingfrequency in epidemiologic studies.Withlowerexposures being evaluated, andlowerconcentrations being measured, it seems opportune to bear in mind that a great proportionof variation in measured levels among subjects maybe accounted for by differences inmetabolism andexcretion(clearance).The measurements we obtain may afford only aglimpse of a byproduct of the underlying pharmacokinetics, systems, biology, andpathogenesis.While these are not entirely new concepts, their importance may be clearertoday than ever before.”Epidemiologic associationsare not equivalent tocausation..May expect to observephysiologically-basedassociations.“Reverse causation”Pharmacokinetic Variability and the Miracle of Modern Analytical ChemistryMatthew Longnecker (NIEHS) Epidemiology 2006;17:350-351“The impressive capabilities of modern analytical chemistry are being applied with increasingfrequency in epidemiologic studies.Withlowerexposures being evaluated, andlowerconcentrations being measured, it seems opportune to bear in mind that a great proportionof variation in measured levels among subjects maybe accounted for by differences inmetabolism andexcretion(clearance).The measurements we obtain may afford only aglimpse of a byproduct of the underlying pharmacokinetics, systems, biology, andpathogenesis.While these are not entirely new concepts, their importance may be clearertoday than ever before.”Epidemiologic associationsare not equivalent tocausation.May expect to observephysiologically-basedassociations.“Reverse causation”Pharmacokinetic Variability and the Miracle of Modern Analytical ChemistryMatthew Longnecker (NIEHS) Epidemiology 2006;17:350-351“The impressive capabilities of modern analytical chemistry are being applied with increasingfrequency in epidemiologic studies.Withlowerexposures being evaluated, andlowerconcentrations being measured, it seems opportune to bear in mind that a great proportionof variation in measured levels among subjects maybe accounted for by differences inmetabolism andexcretion(clearance).The measurements we obtain may afford only aglimpse of a byproduct of the underlying pharmacokinetics, systems, biology, andpathogenesis.While these are not entirely new concepts, their importance may beless cleartoday than ever before.”Epidemiologic associationsare not equivalent tocausation.May expect to observephysiologically-basedassociations.“Reverse causation”Is Clearance a Common Explanation Across the Epidemiological AssociationsReported at “Low” PFOA and PFOS Concentrations?Specifically, Is Clearancea Common Explanation Across These Reported Epidemiological Associations?Definition of Clearance1.the act of clearing.2.a quantitative measure of therate at which a substance isremoved from the blood, as by thekidneys, the liver, gut.3. expressed as the volume ofplasma cleared per unit time.Cl = Kel x VdStudy Populations and MethodsStudy Populations•General populationNHANESDenmarkOther•CommunityParkersburg, W.V. (mid-Ohio River)C8 Health ProjectEpidemiologic Study Designs•Cross-sectionalGeneral populationCommunityOccupationalMedical surveillance•Case-controlGeneral populationLongitudinal (cohort)C8 Health ProjectC8 Science Panel•Occupational3M CompanyDecatur, ALCottage Grove, MNAntwerp, BelgiumDuPontWashington Works (W.V.)Miteni (Italy)•Longitudinal (cohort)CommunityOccupational•Case-cohort•Cohort mortalityCommunityOccupationalStudy Populations and MethodsStudy Populations•General populationNHANESDenmarkOther•CommunityParkersburg, W.V. (mid-Ohio River)C8 Health ProjectEpidemiologic Study Designs•Cross-sectionalGeneral populationCommunityOccupationalMedical surveillance•Case-controlGeneral populationLongitudinal (cohort)C8 Health ProjectC8 Science Panel•Occupational3M CompanyDecatur, ALCottage Grove, MNAntwerp, BelgiumDuPontWashington Works (W.V.)Miteni (Italy)•Longitudinal (cohort)CommunityOccupational•Case-cohort•Cohort mortalityCommunityOccupationalApproximate Average PFOAConcentrations (ng/mL)50005600300040005000600070008000900010000

Concentration (ng/mL)5152867224500650150010000100020003000 General Popula t ion Washingt o n Coun t y, MN

O t her W. Va W ater D i st ri cts

Lu beck wa t er d istrict

Lit t l e H oc ki ng W at er A ssoc i ati on

D uPon t Washington Wor k s 3M A nt werp 3 M D ecat ur

3M Cott a ge G r o ve (ECF c ells)

3M Cot t age G r o ve ( s pray d ry er) Miteni (Italy)

Concentration (ng/mL)Approximate Average PFOSConcentrations (ng/mL)300900 9003004005006007008009001000

Concentration (ng/mL)203520 20 20 20500100200300

G ene r al Po p ul a t i on Washington Cou n ty, M N Other W. Va L ube c k

Li t t le H oc k in g W a t er A ss ocia t i on

D u P o nt Washingto n Works

3 M C o t ta g e G rov e

3M Cottage G r ove (work-up) 3M Antwerp 3 M D e c at u r

Concentration (ng/mL)Comparison of Human PFOA Biomonitoring Data toBenchmark Lower Bound Internal Concentrations from ToxicologicalStudy Endpoints3000060000125000400006000080000100000120000140000

Concentration (ng/mL)ToxicologyHuman Biomonitoring5 15 2867224500650150010005000560019000230003000002000040000

Ge ne ral Population

Wash i n gto n C o u nt y , MN

O ther W. V a W at er D i s t ri ct s

L ub ec k w a t er di stri ct

L itt le H o cki n g Water A ss ociat io n

D uP o nt W a sh i n gton Works 3M A n t w erp 3M D ec at u r

3M Cott a ge G rov e (ECF c el l s )

3M Cot t ag e Gr o ve (s pr a y dr y er ) M it en i (Ital y ) Post-natal survival at weaning (mouse)

Li ve r wei g ht- t o - b r a i n - weig h t ratio ( mon k eys)

Pos t -weani n g m o rtality (rat )

B o dy w e i ght change ( mo nkey s)

Tu o mr s (r a ts, Leydig cell)

Concentration (ng/mL)Comparison of Human PFOS Biomonitoring Data toBenchmark Lower Bound Internal Concentrations from ToxicologyStudy Endpoints16,00031,00044,00049,00056,00062,000200003000040000500006000070000

Concentration (ng/mL)HumanBiomonitoringToxicology20 35 20 20 20 20 50300900 90016,00001000020000

G e ner a l Pop ulat i o n

Wash i ngton County, MN Oth e r W. Va Lubec k

Litt le Hoc k i ng Wa t e r Ass o cia t i o n DuPont Wa s hington Works 3M Co t tage Grov e

3 M Cottag e G rove ( w ork-up )

3 M Antw e rp 3M D e catur Postnatal survival to day 8 (rats)

Pup w e i g ht ga in t hr ou gh w ea n i n g ( ra t s )

Live r w eight (ma l e rats)

L i v er w e i g ht (f e ma l e monk e y s )

Postn a tal surv i v a l to da y 6 (mouse )

Live r t umo rs (ma l e ra t s )

Concentration (ng/mL)Is Clearancea Common Explanation Across the Reported Epidemiological Associations?Delayed PubertyLopez-Espinosa et al. Environ Sci Technol 2011;45:8160-8166C8 Health ProjectCross Sectional Study3076 boys2931 girls8 – 18 years of ageGeometric Mean (GSD)QuartileBoysGirlsLopez-Espinosa et al. Conclusion:“Results suggest later age at having reached puberty of 3 to 6 months across the range ofconcentrations found in this population for both boys and girls.”Considerations re Delayed Puberty AssociationFor those who had reached puberty, the measure of serum perfluoroalkyls was takenafter puberty had been reached.Physiological changes may explain the associationsreportedMenstruationExpanding muscle/tissue massTime to Pregnancy(subfecundity)Definition: >12 months to become pregnantOdds Ratio for Subfecundity (TTP > 12 months).Fei et al. Human Repro 2009;24:1200-1205Study Pop: DenmarkStudy Design: CohortN = 1240PFOS < 26.0 ng/mLPFOA < 3.91 ng/mLPFOS 26. 1 – 33.3ng/mLPFOA 3.91 – 5.20ng/mLPFOS 33.4 –43.2 ng/mLPFOA 5.21 –6.96 ng/mLPFOS ≥ 43.3 ng/mLPFOA ≥ 6.97 ng/mLSuggested causal diagram of maternal perfluorochemical concentrations and time to pregnancy (TTP).Olsen GW, Butenhoff JL, Zobel LR. Repro Toxicol 2009.Acknowledgement: Dr. Matt Longnecker (NIEHS). Presented at EPA PFAA Days III (June 2010). See Whitworth et al. Epidemiology 2012:23:257-263.Odds Ratio for Subfecundity (TTP > 12 months).Whitworth et al. Epidemiology 2012;23:257-263Study Pop:NorwayStudy Design: Case-ControlN = 421 cases, 504 controlsPFOS < 10.34 ng/mLPFOA < 1.66 ng/mLPFOS 10.34 –13.09 ng/mLPFOA 1.66 – 2.24ng/mLPFOS 13.10 –16.60 ng/mLPFOA 2.25 –3.02 ng/mLPFOS ≥ 16.61 ng/mLPFOA ≥ 3.03 ng/mLOdds RatioOSubfecundity (TTP > 12 months) Stratified by Parity.Whitworth et al. Epidemiology 2012;23:257-263Study Pop: NorwayStudy Design: Case-controlN: 421 cases, 504 controlsOlsen, Butenhoff, Zobel Repro Toxicol 2009Fei et al. Epidemiology 2012“A subtlety not captured by the directed acyclic graph is that PFOA/PFOS were not measured at the beginningof the attempt at conception(which would have beenideal), but at the end, after a pregnancy had beenachieved.Thus, in the available data, the measurement of PFOA/PFOS can potentially be influenced by TTP for parous womenthrough reaccumulation of the chemicals. Such influence produces a cycle in the graph through the arrow from TTPto the measured PFOA/PFOS. However, for nulliparous women, that arrow does not exist in a model thatadjusts for age.”Olsen, Butenhoff, Zobel Repro Toxicol 2009Fei et al. Epidemiology 2012parouswomen“A subtlety not captured by the directed acyclic graph is that PFOA/PFOS were not measured at the beginningof the attempt at conception(which would have beenideal), but at the end, after a pregnancy had beenachieved.Thus, in the available data, the measurement of PFOA/PFOS can potentially be influenced by TTP for parous womenthrough reaccumulation of the chemicals. Such influence produces a cycle in the graph through the arrow from TTPto the measured PFOA/PFOS. However, for nulliparous women, that arrow does not exist in a model thatadjusts for age.”parouswomenOdds Ratio for Subfecundity (TTP > 12 months) Stratified by Parity.Fei et al. Epidemiology 2012:23:264-266Study Pop: DenmarkStudy Design: CohortN = 1240PFOS QuartileConclusionsTime to PregnancyWhitworth et al. “Among parous women, increased body burden may be due to a longinterpregnancy interval rather than the cause of a long time to pregnancy. Therefore, datafrom nulliparous women may be more informative regarding toxic effects ofperfluorinatedcompounds. Our results amongnulliparouswomen did not support anassociation with subfecundity.”Fei et al. “We found limited evidence for reverse causation as an explanation of our results.”Lower BirthweightAuthor Type Journal Year LocationOlsen et al. Occupational JOEM 2004 3M Decatur, ALGrice et al. Occupational JOEM 2007 3M Decatur, ALApelberg et al.(2 papers)GeneralpopulationES&T and EHP 2006, 2007 Baltimore, MDFei et al. (4papers)GeneralpopulationEHP (2), Am J Epi(1), Human Repro(1)2007, 2008,2008, 2009DenmarkInoue et al. GeneralpopulationEHP 2004 Hoikado, JapanMonroy et al. GeneralpopulationEnvironmentalResearch2008 Ontario, CanadaNolan et al. (2papers)Community ReproductiveToxicology2008, 2009 WashingtonCounty, Ohio (C8Health Project)Stein et al. Community Am J Epidemiology 2009 “6 waterdistricts”C8 Health ProjectHamm et al. GeneralpopulationJ Exp Sci Env Epi 2009 Edmonton, AlberttaCovariates included in statistical models of birthweight andPFOS or PFOA, by study investigatorAdjustedFor:Olsen Grice Inoue Apelberg Fei Monroy Washino Nolan Stein HammMaternal ageYes Yes Yes Yes Yes Yes YesYesMaternal BMIYes Yes Yes YesYesMaternal net wtgainYesMaternal heightYesYesMaternal raceYesYesYesMaternalsmokingYes Yes Yes Yes Yes YesYesEducationYes YesSESYes YesGestational ageYes Yes Yes Yes YesGetational ageat blood drawYes YesParityYes Yes Yes Yes Yes YesYesInfant sexYes Yes Yes Yes YesDiabetesYesHypertensionYesEndpointPFC Olsen Grice Inoue Apelberg Fei(4 papers)Monroy Washino Nolan(2 papers)SteinHammGestational agePFOSN.S. N.S. N.S. N.S. N.S.PFOAN.S. N.S. N.S. N.S. N.S. N.S.Birth Weight (g)PFOSN.S. N.S. N.S.* N.S. N.S.S.S.femalesS.S.N.S.PFOAN.S N.S.*S.S.N.S. N.S. N.S. N.S. N.S.Birth Length (cm)PFOSN.S. N.S. N.S.PFOAN.S.S.S.N.S.Head Circumference(cm)PFOSS.S.N.S. N.S.PFOAS.S.N.S. N.S.Abd/chestCircumference (cm)PFOSN.S. N.S.PFOAS.S.N.S.Apgar ScorePFOSN.S.PFOAN.S.Ponderal IndexPFOSS.S.N.S.PFOAS.S.N.S.Placental Weight (g)PFOSN.S.PFOAN.S.MiscarriagePFOSN.S.N.S.N.S.PFOAN.S.N.S.N.S.Birth defects (nonspecific)PFOSN.S.N.S.PFOAN.S.N.S. N.S.PreeclampsiaPFOSS.S.PFOAN.S.DevelopmentalmilestonesPFOSN.S.PFOAN.S.C8 Science Panel Very Recent PublicationsSavitz et al. Epidemiology 2012 doi:L10.1097/EDE.0b013e31824cb93bSavitz et al. Environ Health Perspect 2012 doi:org/10.1289/ehp.1104752C8 Health Project enrollees (Savitz et al. Epidemiology 2012)Self-reported pregnancy histories form the C8 Health Project participantsReconstructed historical exposures for C8 Health Project participants for each yearbeginning in 1951.Use modeled estimates of PFOA exposure at the timeof pregnancy to examinePFOA and pregnancy outcomes within this C8 Health Project population.1990 – 2006 time frame; 11,737 pregnancies1) Adjusted OR (95% CI) for IQR (lnPFOA)increase where = 2.192) Quintiles (2 lowest quintiles used as reference)3) Adjusted OR (95% CI) for 100 ng/mL increase in PFOAOR 95% CIMiscarriage 0.98 0.93 – 1.03Stillbirth 0.91 0.76 – 1.10Preeclampsia 1.08 1.01 – 1.15Preterm Birth 0.97 0.93 – 1.02Term Low Birthweight 0.96 0.79 – 1.16Birth Defect 0.97 0.90 – 1.06Conclusion: “No associations between estimated serum PFOA levels and adversepregnancy other than possibly preeclampsia.Savitz et al. Environ Health Perspect 2012TWO StudiesSTUDY 1.Examine birth certificate (OH and WV) and fetal death records (WV) from fivecounties (3 Ohio, 2 WV)Restricted to pregnancies from 1990 – 200410% random sampling of term births ≥2500 grams (n =3616) selected forgeocodingfor PFOA exposurePFOA exposure history considered at time of pregnancy and assumed motherlived at residence for 6 years prior to pregnancy1) Adjusted OR (95% CI) for IQR (lnPFOA) increase where = 2.192) Quintiles (2 lowest quintiles used as reference)3) Adjusted OR (95% CI) for 100 ng/mL increase in PFOASTUDY 1(change in 100 ng/mL)OR 95% CIStillbirth 1.20 0.86 – 1.68Pregnancy-inducedhypertension 1.06 0.86 – 1.31Preterm births<37 weeks gestation 1.02 0.94 – 1.10<32 weeks gestation 0.90 0.74 – 1.10Term low birthweight 1.00 0.86 – 1.15Term SGA0.960.67–1.11Term birthweight(Change in Birthweight, g) -14.8 -43.28, 13.68(By Quintiles of PFOA Exposure) OR 95% CIQ1-Q2 (<6.1 ng/mL) referenceQ3 (6.1 - < 10.2 ng/mL) 22.8 -32.9, 78.5Q4 (10.2 – 21.0 ng/mL) 2.3 -50.3, 54.8Q5 (21.0 – 717.6 ng/mL) -9.5 -58.4, 39.4STUDY 2Linked birth records from 1990 – 2004 for 13 counties (5 Ohio, 8 WV) to self-reported birthsfrom the C8 Health Project (5 of these counties)Linked (matched) 4547 birth certificates linked tothe C8 Health ProjectUsed lifetime residential history to calculate annual exposure PFOA estimatesSTUDY 2(change in 100 ng/ml)OR 95% CIPregnancy-inducedhypertension 1.01 0.91 – 1.12Preterm births<37 weeks gestation 1.09 1.00 – 1.18<32 weeks gestation 1.10 0.86 – 1.40Term low birthweight 1.00 0.82 – 1.21Term SGA 1.07 0.98 – 1.17Term birthweight(Change in Birthweight, g) -9.14g -20.30, 2.02(By Quintiles of PFOA Exposure) OR 95% CIQ1-Q2 (<8.9 ng/mL) referenceQ3 (8.9 - < 21.8 ng/mL) -3.8 -40.4, 32.8Q4 (21.8 – 83.3ng/mL) -25.4 -63.7, 12.9Q5 (83.3 – 921.3 ng/mL) -33.3 -73.1, 6.5ConclusionSavitz et al. (Environ Health Perspect)Little support for PFOA having adverse effects on pregnancy-inducedhypertension or preterm birthInconsistent evidence of reduced fetal growthImprecise indications of an association with earlypreterm birth.The potential association of PFOA with small decrements in birthweight is ofunknown significance, but is perhaps the most readily amenable to morerefined studies.Conclusion(continued)Savitz et al. Environ Health Perspect, 2012“If an impact on birth weight is causal, which is yet to be established,it reflects a biological process that is worth elucidating even if notof direct health concern.”Considerations(Olsen et al. Reprod Toxicol 2009;27:212-230)•PFOA and PFOS distributed primarily in extracellular fluid•Protein bound•Increased maternal plasma volume expansion and placental volumeare strongly correlated with birth weight.Other referencePlasma Volume Expansion in Pregnancy: Implicationsfor Biomarkers in Population StudiesFaupel-Badger et al. Cancer Epidemiology Biomarkers & Prevention 2007:16:17201725.Duration of Breastfeeding(Fei et al. Scand J Work Environ Health 2010;36:413-421)DenmarkCohort study1400 pregnant womenHigher maternal PFOS and PFOA concentrations were associated with shorter durationof breastfeeding. For example, median PFOS concentration was 37.0 ng/mL for womenwho breast fed < 3 months compared to 32.3 ng/mL forwomen who breast fed ≥ 6 months.For PFOA, these values were 5.90 ng/mL and 4.96 ng/mL, respectively.The duration of breastfeeding decreased with increasing concentrations of pregnancyPFOS and PFOA among multiparous women but not amongprimiparous women.Fei et al. conclusion. “These findings suggest thatPFOA and PFOS may reduce theability to lactate, but could equally reflect reverse causation since no associationwas seen in primiparous women.”MenopauseKnox et al. Implications of early menopause inwomen exposed to perfluorocarbons. J ClinEndocrinol Metab 2011;96:1747-1753C8 Health ProjectCross Sectional Study25,957 women aged 18 - 65Menopause is the permanent end ofmenstruation and fertility, defined asoccurring 12 months after the lastmenstrual period.Menopause in the Knox et al analysis wasdefined by the participant’s answer to thequestion of whether she had experiencedthe “change of life” (menopause).They were not asked the age of menopause.TABLE 2.Odds ratios for menopause by quintile of PFOA/PFOSWomen 18≤42 yrWomen > 42≤51 yrWomen > 51≤65 yrPoint Confidence Point Confidence Point Confidenceestimate limitsΧ2estimate limitsΧ2estimate limitsΧ2PFOAaPFOS quintiles.Quintiles in ng/mL. Quintile 1: 0.25 - 11.8,Quintile 2: 11.9 - 17.0, Quintile 3: 17.1 -22.4,Quintile 4: 22.5 - 30.7, Quintile 5: 30.8 - 564.3.PFOA quintiles.Quintiles in ng/mL. Quintile 1: 0.25 - 11.2,Quintile 2: 11.3 - 19.8, Quintile 3: 19.9 - 36.7,Quintile 4: 36.8 - 84.9, Quintile 5: 85.0 - 22412.0.PFOAa2 vs, 1 0.9 0.5 – 1.6 0.009 1.4 1.1 – 1.80.839 1.5 1.1 – 2.1 0.0413 vs. 1 0.9 0.5 – 1.5 1.2 0.9 – 1.6 1.6 1.2 –2.24 vs. 1 0.9 0.5 – 1.7 1.4 1.1 – 1.9 1.4 1.1 –1.95 vs. 1 1.2 0.7 – 2.1 1.4 1.1 – 1.8 1.7 1.3 –2.3PFOSa2 vs, 1 1.1 0.7 – 1.9 0.804 1.2 0.9 – 1.50.028 1.5 1.1 – 2.1 <0.00013 vs. 1 0.8 0.4 – 1.4 1.4 1.1 – 1.8 1.8 1.3 –2.54 vs. 1 1.0 0.6 – 1.8 1.4 1.1 – 1.8 2.0 1.5 –2.65 vs. 1 1.1 0.6 – 2.1 1.4 1.1 – 1.8 2.1 1.6 –2.8Results are adjusted for age, BMI, alcohol consumption, smoking, and exercise. Women with hysterectomy were excluded from analyses.a.Quintiles.“Mean PFC concentrations in women with and without hysterectomy werecompared in women 40-55 yrs of age to see whether hysterectomy made adifference. Both PFOS and PFOA were significantly higher (P < 0.0001) in womenwith a hysterectomy.”Knox et al. J Clin Endocrinol Metab 2011“Thus, the argument could be made that some of thesignificance associated with anincreased risk of menopause associated with increased PFC might be due to reversecausation, i.e., the disappearance of menses causing increasing PFC concentrationsrather than an increase in PFC causing early menopause.Knox et al. J Clin Endocrinol Metab 2011This argument is given additional support by the fact that women who had experiencedhysterectomy in our sample had higher mean levels of PFC than women who hadnot experienced hysterectomy.”“However even if the results reported here could besolely explained by reversecausation, they would still be cause for alarm because it would imply that increasedPFC exposure is the natural result of menopause orhysterectomy.”Knox et al. J Clin Endocrinol Metab 2011Is Clearancea Common Explanation Across the Reported Epidemiological Associations?Chronic Kidney Disease (CKD)Estimate of Net Renal Tubular Secrtion or Reabsorption ofPFOAin Different Species(Han et al. Chem Res Toxicol 2012;25:35-46)Species Gender GFR (L/d/kg) CLR(mL/d/kg)Net secretion(mL/d/kg)Netreabosprtion(mL/d/kg)PercentageofreabsoprtionRabbit Female 4 670 590 N.A.Male 4 640 560 N.A.Rat Female 14.4 666 378 N.A.Dog Female 5.3 50.8 55 52%Male 5.3 43 63 59%J.macaque Female 8.5 32 138 81.2%Male 8.5 15 155 91.2%Rat Male 14.4 18.2 270 93.7%Mouse Female 16.7 16 318 95.2%Male 16.7 10 324 97.0%Human 2.57 0.03 51 99.94%glomerular filtration rate(GFR) an expression of the quantity of glomerular filtrateformed each minute in the nephrons of both kidneys,usually measured by the rateof clearance of creatinine . Ideal measures of GFR includes use (infusion) of inulinas it is freely filtered but not secreted or reabsorbed in tubules.Creatinine-based approximations of GFR (eGFR)Filtered freely by the glomerulus, but also actively secreted by theperitubular capillaries in very small amounts such that creatinineclearance overestimates GFR by 10 to 20 percent.Creatinine levels affected by muscle mass, ingetion of meats1) 24-hour urine collection to measure creatinine2) Several indirect formulasCockgroft-GaultCKD-EpiMDRDModification of Diet in Renal Disease (MDRD) formulaestimated Glomerular Filtration Rate (eGFR) =175 x (serum creatinine in mg/dL)-1.154x (age in years)-0.203x (0.742 if female) x (1.21 if black)Chronic Kidney Disease (CKD) defined as eGFR < 60mL/minute/1.73m2Study Pop: NHANES 1999-2000, 2003-2004, 2005-2006Study Design: Cross-sectionalAssociation Between Plasma Perfluoroalkyl Level andeGFRShankar et al. Am J Epidemiol 2011;174:893-900Shankar et al. conclusions:•Results suggest that elevated PFC levels are associated with CKD•Cross-sectional study does not address temporality.•In CKD, when eGFR is reduced, the total filtration of PFOA and PFOS will also bereduced. This could lead to higher concentrationsof PFOA and PFOS and thereforeresult in a positive association between theseperfluoroalkylsand CKD.This is referred to as reverse causation.•An argument against ‘reverse causality’ is that theinverse association was strongeramong persons without CKD.•A ‘reverse causality’ association could be of clinical and/or public health importancebecause if subjects with CKD retain these compounds, the toxicities of PFOA andPFOS on the kidneys or other organs could be accentuated among CKD subjects.8010012014080100120140Study PopScatterplot of eGFR by PFOA and PFOS Serum ConcentrationsOlsen and Zobel Int Arch Occup Environ Med 2007;81:231-246Study Pop: OccupationalStudy Design: Cross-sectional0204060801 10 100 1000 10000 100000eGFRPFOA (ng/mL)0204060801 10 100 1000 10000PFOS (ng/mL)Study Pop: OccupationalStudy Design: Cross-sectionalPFOA and PFOS Serum Concentrations by DecilesOlsen and Zobel Int Arch Occup Environ Med 2007;81:231-246Adjusted Mean eGFR by PFOA and PFOS DecilesOlsen and Zobel Int Arch Occup Environ Med 2007;81:231-246Study Pop: OccupationalStudy Design: Cross-sectionalAdjusted Odds Ratio for Chronic Kidney Disease by PFOA and PFOS DecilesOlsen and Zobel Int Arch Occup Environ Med 2007;81:231-246Study Pop: OccupationalStudy Design: Cross-sectionalChronic Kidney Disease (eGFR) Results from C8 Health Project?Chronic Kidney Disease (eGFR) Results from C8 Health Project?Never ReportedC8HealthProjectAssociationBetweenC8DecilesandCreatinine,Serum(mg‐dL)S ifi db A &G dC8HealthProjectStratifiedbyAge&GenderOriginalslidecontainingfigureinpresentationwasremovedduetocopyright;however,imagecanbeviewedatthefollowinglink:http://www.hsc.wvu.edu/som/cmed/c8/results/otherLaboratoryValues/pdfs/Creatinine.pdfC8HealthProjectAssociationBetweenC8DecilesandAlbumin,Serum(g‐dL)S ifi db A &G dC8HealthProjectStratifiedbyAge&GenderOriginalslidecontainingfigureinpresentationwasremovedduetocopyright;however,imagecanbeviewedatthefollowinglink:http://www.hsc.wvu.edu/som/cmed/c8/results/otherLaboratoryValues/pdfs/Albumin_Deciles.pdfNHANES2009-2010NHANES 2009 – 2010eGFR by ln PFOA eGFR by ln PFOSCONCLUSIONS regarding Chronic Kidney Disease (CKD)No evidence of CKD via medical surveillance where workers’ serumconcentrations of PFOS and PFOA much higher than NHANES population.No evidence toxicologically.NHANES results by Shankar et al likely an association of renal clearance(eGFRand PFOA/PFOS) and NOT CKDNHANES 2009-2010 results continue to show eGFR association at evenlower levels of PFOA/PFOS than reported by Shankaret al.eGFR and PFOA/PFOS are positively associated which is biologicallyplausible.Inference:Health outcomes associated with eGFRcould therefore also be found to be associated withlowconcentrations of PFOA/PFOS.HyperuricemiaSteenland et al. Environ Health Perspect 2010;118:229-233Shankar et al. Clinical Epidemiol 2011;3:251-258NHANES 1999-2000 and 2003-2006 (Shankar et al.)C8 Health Project (Steenland et al.)Shankar et al. (NHANES, Cross-sectional)Steenland et al. (C8 Health Project, Cross-sectional)Is uric acid associated with eGFR?NHANES 2009 - 2010eGFR by Uric Acid eGFR by ln Uric AcideGFR and Systolic BPSystolic BP and ln PFOA Systolic BP and ln PFOSNHANES 2009 - 2010Steenland et al.“Only 119 people (0.2%) had creatinine>2.5 mg/dL, which suggests kidney diseasethat can affect uric acid; excluding theseparticipants from the analyses did not changethe results. A broader exclusion cut point of1.5 mg/dL excluded 739 and again had noeffect on results.”“We also analyzed a subset of participants whohad a PFOA level < 20 ng/mL, dividing them intoquartiles of PFOA exposure, so that the referentgroup had levels similar to those of the U.S.general population. Those with 5-9.9 ng/mL(n = 2,012), 10-14.9 ng/mL (n= 6,841), and15-20 ng/mL (n=7,387) had predicted increasesin uric acid of 0.14, 0.21, and 0.24 mg/dL,above those with PFOA levels < 5 ng/mL.”C-reactive ProteinC8HealthProjectAssociationBetweenC8DecilesandC‐ReactiveProtein,Quant(L)C8HealthProject(mg‐L)StratifiedbyAge&GenderOriginalslidecontainingfigureinpresentationwasremovedduetocopyright;however,imagecanbeviewedatthefollowinglink:http://www.hsc.wvu.edu/som/cmed/c8/results/otherLaboratoryValues/pdfs/CRP.pdfC8HealthProjectAssociationBetweenC8DecilesandCreatinine,Serum(mg‐dL)S ifi db A &G dC8HealthProjectStratifiedbyAge&GenderOriginalslidecontainingfigureinpresentationwasremovedduetocopyright;however,imagecanbeviewedatthefollowinglink:http://www.hsc.wvu.edu/som/cmed/c8/results/otherLaboratoryValues/pdfs/Creatinine.pdfDiabetes1.01.52.0OddsRaOdds Ratio for Medically Verified Type II Diabetes,C8 Health ProjectMacNeil et al. Environ Res 2009;109:997-10030.00.5Decile 1(<7.9ng/mL)Decile 2(8.0 - 11.6ng/mL)Decile 3(11.7 -15.6ng/mL)Decile 4(15.7 -20.8ng/mL)Decile 5(20.9 -28.0ng/mL)Decile 6(28.1 -39.4ng/mL)Decile 7(29.5 -57.5ng/mL)Decile 8(57.6 -89.7ng/mL)Decile 9(89.8 -191.2ng/mL)Decile 10(>191.2ng/mL)atioPFOA DecileC8HealthProjectAssociationBetweenC8DecilesandGlucose,Serum(mg‐dL)S ifi db A &G dC8HealthProjectStratifiedbyAge&GenderOriginalslidecontainingfigureinpresentationwasremovedduetocopyright;however,imagecanbeviewedatthefollowinglink:http://www.hsc.wvu.edu/som/cmed/c8/results/otherLaboratoryValues/pdfs/Glucose.pdfC8HealthProjectAssociationBetweenC8DecilesandCreatinine,Serum(mg‐dL)S ifi db A &G dC8HealthProjectStratifiedbyAge&GenderOriginalslidecontainingfigureinpresentationwasremovedduetocopyright;however,imagecanbeviewedatthefollowinglink:http://www.hsc.wvu.edu/som/cmed/c8/results/otherLaboratoryValues/pdfs/Creatinine.pdfC8HealthProjectAssociationBetweenC8DecilesandAlbumin,Serum(g‐dL)S ifi db A &G dC8HealthProjectStratifiedbyAge&GenderOriginalslidecontainingfigureinpresentationwasremovedduetocopyright;however,imagecanbeviewedatthefollowinglink:http://www.hsc.wvu.edu/som/cmed/c8/results/otherLaboratoryValues/pdfs/Albumin_Deciles.pdfMacNeil et al. Conclusion“Our findings do not demonstrate abn association between PFOA and either type IIOr fasting glucose level. Our data are limited bytehir cross-sectional nature, and doNot preculde the possibility of a causal relationship.”What does an increased risk for diabetes (type II)in the lowest PFOA decileof the C8 Health Project database meanwhen other health conditions are cross-sectionallyanalyzedwith respect to concentrations of PFOA and PFOS?Occupational Cohort Mortality StudiesDiabetesStudy Categorization Obs Exp SMR 95%CIAlexander et al. Decatur Facility(AL comparison)0 1.5 0.0 -Lundin et al. Cottage Grove Facility(MN comparison)23 20.0 1.2 0.7 – 1.7“Not exposed PFOA job”59.60.50.2-1.2“Not exposed PFOA job”Ever “Probable exposure” 18 8.9 2.0 1.2 - 3.2Ever “Definite exposure” 0 1.5 0.0 0.0 - 2.4Leonard et al. Washington Works Facility(W.V. comparison)22 31.5 0.7 0.4 - 1.1Washington Works Facility(DuPont comparison)22 11.2 2.0 1.2 - 3.0Is Clearancea Common Explanation Across Many of these Epidemiological Associations?High CholesterolPFOA and the Linear Associations (Slope) Between Cholesterol and PFOA in 10 Epidemiologic StudiesSteenland et al. Environ Health Perspect 2009;118:1100-1108 and Olsen et al. J Occup Environ Med 2012 (inpress)Nonlinear Association Between Cholesterol and PFOAand PFOSSteenland et al. Am J Epidemiology 2009;170:1268-78Study pop: C8 Health ProjectStudy Design: Cross-sectionalNote: Scale ofX axis for PFOSNonlinear Association Between Cholesterol and PFOAand PFOSSteenland et al. Am J Epidemiology 2009;170:1268-78Study pop: C8 Health ProjectStudy Design: Cross-sectionalNote: Scale ofX axis for PFOSAdjusted Mean Lipid Values by PFOA DecilesOlsen and Zobel Int Arch Occup Environ Health 2007;81:231-246150200250

Lipid valueStudy pop: 3M occupationalStudy Design: Cross-sectionalN = 506 male employees nottaking cholesterol loweringmedications050100Decile 1median 60range 7-130Decile 2median 190range 131-290Decile 3median 360range300-440Decile 4median 540range 450-710Decile 5median 910range 720-1110Decile 6median 1250range 1120-1400Decile 7median 1630range 1420-1850Decile 8median 2180range 1860-2500Decile 9median 2960range 2510-3690Decile 10median 4940range 3710-92,030Decile Number ( PFOA median and range in ppb for each decile)

Lipid valueCholesterolLDLHDLTriglyceridesOlsen et al. Longitudinalassessment of lipid and hepatic clinical parameters in workersInvolved with the demolition of PFOA and PFOS manufacturing facilities. JOEM (in press, 2012)-120-100-80-60-40-20020406080100120-3 -2 -1 0 1 2 3 4 5 6 7

Change in Non-HDL mg/dLChange in ln PFOA ng/mL-120-100-80-60-40-20020406080100120-3 -2 -1 0 1 2 3 4 5

Change in Non-HDL mg/dLChange in ln PFOS ng/mL-60-40-200204060-3 -2 -1 0 1 2 3 4 5 6 7

Change in HDL mg/dLChange in ln PFOA ng/mL-60-40-200204060-3 -2 -1 0 1 2 3 4 5

Change in HDL mg/dLChange in ln PFOS ng/mLSteenland et al. Am JEpidemiol 2009; 170:1268-78Study pop: C8 Health ProjectStudy Design: Cross-sectionalNonlinear Association Between PFOA, Total Cholesterol and Uric AcidSteenland et al. Environ HealthPerspect 2010;118:229-233Study pop: C8 Health ProjectStudy Design: Cross-sectionalSteenland et al. Am JEpidemiol 2009; 170:1268-78Nonlinear Association Between PFOS, Total Cholesterol and Uric AcidSteenland et al. Environ HealthPerspect 2010;118:229-233Is nonHDL cholesterol associated with the glomerularfiltration rate?Morita et al. Atherosclerosis 2010.Decrease in glomerular filtration rate by plasmalow-density lipoprotein cholesterol in subjects with normal kidney function assessedby urinalysis and plasma creatinine . Atherosclerosis 2010;210:602-606.Subjects were examinees at a wellness and promotionprogram in Japan during 2007.Patient levels < 1 mg/dLfor men and 0.8 mg/dLfor females w/oalbuminuriaPatient levels < 1 mg/for men and 0.8 mg/for females w/oalbuminuria4602 males; 2920 females.Cross-sectional analysisMorita et al. Atherosclerosis 2010.Decrease in glomerular filtration rate byplasma low-density lipoprotein cholesterol in subjects with normal kidneyfunction assessed by urinalysis and plasma creatinine . Athersclerosis2010;210:602-606.Morita et al. Atherosclerosis 2010Morita et al. Atherosclerosis 2010;210:602-606Conclusion:“We conclude that the high plasma level of LDL-C was the major riskfactor among coronary risk factors to reduce GFR probably due to impairing thefunction of renal arterioles and capillaries of glomeruli in subjects with normal kidneyfunction assessed by urinalysis and plasmacreatinine.”Lo et al. GFR, body mass index, and low high-density lipoprotein concentration inadults with and without CKD. Am J Kidney Dis 2007:50:552-558.Cross sectional analysis of 380,207 individuals from Kaiser Permanente who had anoutpatient measured of serum creatinine from 2003 – 2004.Conclusion: Decreased eGFR is independently associated with greater odds ofhaving a low HDL level (men < 40 mg/dl; females < 50 ng/mL).“Thyroid Disorders”Knox et al. J Toxicol Sci 2011Melzer et al Environ Health Perspect 2010Lopez-Espinosa et al. Environ Health Perspect 2012C8 Science Panel. Status Report 2011.Olsen andZobe. lIntArchOccupEnviron Health 2007Olsen andEnviron Health 2007NHANESC8 Health Project(adults and children)OccupationalAll cross-sectional studiesMelzer et al. Environ Health Perspect 2010;NHANES Data 1999-2000, 2003-2004 and 2005-2006Cross-sectionalKnox et al. J Toxicol Sci 2011;36:403-410C8 Health ProjectCross-sectionalN = 52,296No association with TSHLopez-Espinosa et al. Environ Health Perspect 2012C8 Health ProjectCross-sectional10,725 children (aged 1 – 17 years)Lopez-Espinosa et al. Environ Health Perspect 20121.01.52.02.53.0OddsRatiWomenMenC8 Science Panel Status Report 2011. Odds Ratios for Thyroid Disorders and PFOA DecileC8 Health Project0.00.5 Decile 1 (< 42 ng/mL) Decile 2 (42

- 93 ng/mL)

Decile 3 (94

- 127 ng/mL) Decile 4 (128

- 167 ng/mL) Decile 5 (168

- 238 ng/mL) Decile 6 (238

- 400 ng/mL) Decile 7 (401

- 821 ng/mL) Decile 8 (822

- 2,226 ng/mL) Decile 9 (2,227

- 6,028 ng/mL) Decile 10 (≥ 6,029 ng/mL)ioCumulative PFOA DecileAdjusted Mean Thyroid Values by PFOA DecilesOlsen and Zobel (Int Arch Occup Environ Health 2007)5.06.07.08.09.010.0

Value of Thyroid TestTSHT4Free T4OccupationalCross-sectional0.01.02.03.04.05.0Decile 1median 60range 7-130Decile 2median 190range131-290Decile 3median 360range300-440Decile 4median 540range450-710Decile 5median 910range 720-1110Decile 6median 1250range 1120-1400Decile 7median 1630range 1420-1850Decile 8median 2180range 1860-2500Decile 9median 2960range 2510-3690Decile 10median 4940range 3710-92,030Decile Number (median and range PFOA in ppb for each decile)

Value of Thyroid TestT3 (x 100)Are thyroid diseases related to eGFR?Åsvold et al. Association of thyroid function withestimatedglomerular filtration rate in a population-based study: the HUNT study.Eur J Endocrinol 2011;164:101-105Cross-sectional, Norwegian population based study of 29,480 individuals above 40 yearsof age without previously known thyroid disease.Calculated a geometric mean eGFR and odds ratio of chronic kidney disease (CKD: eGFR <60 ml/min per 1.73 m2) according to categories of thyroid function, using people withTSH in the lower third of the reference range (0.50– 1.4mU/L) as the comparison group.Åsvold et al., Eur J Endocrinol 2011.Åsvold et al., Eur J Endocrinol 2011Lo et al. Increased prevalence of subclinical and clinical hypothyroidism in persons withchronic kidney disease. Kidney International 2005;67:1047-1052NHANES 1988 – 1994N 14,623Cross-sectionalPrevalence of hypothyroidism by level of estimated glomerular filtration rate.P<0.0001 for trend.Lo et al. . Increased prevalence of subclinical andclinical hypothyroidism in persons withchronic kidney disease. Kidney International 2005NHANES 1988 – 1994N 14,623Cross-sectionalLee-O!eGFR Cardiac Output Resistance Blood FlowVascularSUMMARYHypothyroidism, Hyperthyroidism, and eGFRHypothyroidismHyperthyroidismeGFR Cardiac Output Resistance Blood Flow [PFAA]Arteriole VascularSUMMARYHypothyroidism, Hyperthyroidism, and eGFRHypothyroidismHyperthyroidism“To the best of our knowledge, this is the first large-scale report of an association betweenPFAAs in serum and thyroid function impairment in children aged 1 – 17 years. In a groupof 10,725 children from the Mid-Ohio Valley, measured serum PFOA concentrations in2005 were positively associated with thyroid disease (mostly hypothyroidism).Serum PFOS and PFNA concentrations were associatedwith slightly higher levels of TT4in children 1 – 17 years, but were not positively associated with subclinicalhyperthyroidism.”Lopez-Espinosa et al. Environ Health Perspect 2012Hepatic Enzymes (ALT)Lin et al. Am J Gastroenterology 2009Gallo et al. Environ Health Perspect 2012Olsen andZobel2007NHANESC8 Health ProjectOccupationalCross-sectional2025303540PFOA QuartilesALTGGT2025303540PFOS QuartilesALTGGTLin et al. Unadjusted Geometric Mean of ALT and GGT(NHANES, Cross-sectional)051015Q1 (<2.91ng/mL)Q2 (2.91 -4.2 ng/mL)Q3 4.21 -5.95 ng/mL)Q4 (>5.95ng/mL)051015Q1 (<15.51ng/mL)Q2 (15.51-23.50ng/mL)Q3 (23.51-33.80ng/mL)Q4 (>33.80ng/mL)Gallo et al. (C8 Health Project, Cross-sectional)Fitted values of ALT from fully adjusted regressionmodels by deciles of PFOS and PFOS giventhe mean values of the other covariatesAdjusted Mean Liver Clinical Chemistry Values by PFOA DecilesOlsen and Zobel (Int Arch Occup Environ Health 2007)5060708090

Value of Liver TestAlk PhosASTALTGGTOccupational, Cross-sectional010203040Decile 1median 60range 7-130Decile 2median 190range131-290Decile 3median 360range300-440Decile 4median 540range450-710Decile 5median 910range 720-1110Decile 6median 1250range 1120-1400Decile 7median 1630range 1420-1850Decile 8median 2180range 1860-2500Decile 9median 2960range 2510-3690Decile 10median 4940range 3710-92,030Decile Number (median and range PFOA in ppb for each decile)

Value of Liver TestLin et al. (NHANES data): “In conclusion, using the NHANES data from the US adultpopulation, we found that a higher serum concentration of PFOA was associated withElevated liver enzymes. These findings provide clues to the adverse effects of low-dose PFOAIn humans. Although the potential biological significance between PFOA and liver enzymes issmall and subclinical in the general US population,our data suggest that it would be prudentto monitor the liver enzymes of people with low level exposure to PFOA, particular in subjectswho are obese.”Gallo et al. (C8 Health Project) “A small but clear linear association between PFOA and PFOSSerum concentrations and ALT, a marker ofhepatocelluarinjury, was observed in this largeAuthors’ Interpretation of Datapopulation-based sample of individuals with exposure to perfluoroalkyl acids. These resultsare consistent with previous findings and warrant further investigation, in particular on thepotential health consequence of long-term exposureand potential accumulation of damage.”Olsen and Zobel (Occupational) “Measured hepatic clinical chemistry enzymes were notconsistently associated with employees’ serum PFOAconcentrations adjusted for age,alcohol, and either BMI or serum triglycerides. Aweakly positive association betweenalkaline phosphatase, ALT and GGT with PFOA was observed among the Decatur maleparticipants. Several epidemiologic research studies of the Decatur workforce have notreported associations with liver disease (malignantor non-malignant conditions) using avariety of data sources Including death certificates, episodes of care, and self-reports.Is ALT associated with the glomerular filtration rate?Alanine CatabolismAlanine is also important in intertissue nitrogen transport as partof the glucose-alanine cycle (see above). Alanine's catabolicpathway involves a simple aminotransferase reactionthat directlyproduces pyruvate. Generally pyruvate produced by this pathwaywill result in the formation of oxaloacetate, although when theenergy charge of a cell is low the pyruvate will beoxidized to CO2and H2O via theTCA cycle. This makes alanine a glucogenic aminoacid.Is Clearancea Common Explanation Across Many of these Epidemiological Associations?Pharmacokinetic Variability and the Miracle of Modern Analytical ChemistryMatthew Longnecker (NIEHS) Epidemiology 2006;17:350-351“The impressive capabilities of modern analytical chemistry are being applied with increasingfrequency in epidemiologic studies.With lower exposures being evaluated, andlowerconcentrations being measured, it seems opportune to bear in mind that a great proportionof variation in measured levels among subjects maybe accounted for by differences inmetabolism and excretion.The measurements we obtain may afford only a glimpse of abyproduct of the underlying pharmacokinetics, systems, biology, and pathogenesis. Whilethese are not entirely new concepts, their importance may be clearer today than everbefore.”Pharmacokinetic Variability and the Miracle of Modern Analytical ChemistryMatthew Longnecker (NIEHS) Epidemiology 2006;17:350-351“The impressive capabilities of modern analytical chemistry are being applied with increasingfrequency in epidemiologic studies.Withlowerexposures being evaluated, andlowerconcentrations being measured, it seems opportune to bear in mind that a great proportionof variation in measured levels among subjects maybe accounted for by differences inmetabolism andexcretion(clearance).The measurements we obtain may afford only aglimpse of a byproduct of the underlying pharmacokinetics, systems, biology, andpathogenesis.While these are not entirely new concepts, their importance may beless cleartoday than ever before.”Epidemiologic associationsare not equivalent tocausation.May expect to observephysiologically-basedassociations.“Reverse causation”Ingredients to Detect Associations Based on ClearanceLarge Study Populations“Low” exposures and concentrations (PFOA/PFOS)The miracle of modern analytical chemistryIngredients to Detect Associations Based on ClearanceLarge Study Populations“Low” exposures and concentrations (PFOA/PFOS)The miracle of modern analytical chemistryPhysiologyEffectofDietaryFactorsonPlasmaPFOAandNon‐HDLCholesterolinApoE*3.Leiden.CETPMiceJohnButenhoff,Ph.D.,DABTGearyOlsen,D.V.M,Ph.D.SueChang,Ph.D.BACKGROUNDCross‐sectionalstudieshaveobservedpositiveassociationsofserum[PFOA]and[PFOS]withnon‐HDLcholesterol(non‐HDL‐C).AssociationsweakensubstantiallyatserumPFOAandAssociationssubstantiallyPFOSconcentrations>50ng/mL.ToxicologicalandmechanisticstudiessuggestloweringofcholesterolaspotentialconsequenceofexposuretoPFOAdanPFOS.LongitudinaloccupationaldidnotassociationLongitudinaloccupationalstudyfindassociationofincreasingserum[PFOA]and[PFOS]withnon‐HDL‐C.HYPOTHESISSerumlipidproteinsmayserveasastoragecompartmentforPFOSandPFOA.Differencesindietarycompositionmayleadtosimilarnon‐causalassociationsbetweennon‐HDL‐CandPFOA/PFOSfromlowlevelsofexposureindiet.STUDYDESIGNFourdietarydosegroups(n=10♂ApoE*3.Leiden.CETPmice/group).StudyWeek‐4‐3‐2‐10 123 4NoPFOA/PFOS addedtodiets ~30ppbPFOA/~30ppbPFOSaddedtodiets[PFOA]indietswere81± 4%oftargetedconcentration.[PFOS]indietswere57± 27%oftargetedconcentration.DietaryTargetDietary[PFOA]dMeanM dMeanM dyGroup[PFOA]and[PFOS],ng/gMeasured[PFOA],ng/gMeasured[PFOS],ng/g1 36 28.4 17.823427 412 1..3 32 25.3 15.54 30 26.2 28.8ApoE*3‐Leiden.CETPMICEHuman‐likelipoproteinprofilesandlipidlevels.↓clearanceofapoB‐containinglipoproteins.Atherosclerosischaracteristicsofhumanlesions.Responsetotreatmentwithdrugs(i.e.,statins,fibratesandniacin)atsimilardosagestohumans.DIETCOMPOSITION(kCal/gdiet)6534 l/g Diet2

Kca011234DietaryGroupCALORICCONSUMPTION181416

e1012

K cal/d/mous468

Average K0241234DietaryGroupEXPERIMENTStudyWeek‐4‐3‐2‐10 123 4NoPFOA/PFOS addedtodiets ~30ppbPFOA/~30ppbPFOSaddedtodiets

e mentRandomizationXBodyWeight X X X XFoodIntakeXXXXXXX

M easur eFoodIntakeXXXXXXXPlasma Lipids X X X XHDL X X X X

MPlasma[PFOA] X X X X XStudyWeek‐4‐3‐2‐10 123 4NoPFOA/PFOS addedtodiets ~30ppbPFOA/~30ppbPFOSaddedtodiets2 02.53.0

o l/L)Groupbaselinelipoproteinprofile14161820

l /L)TotalCholesterolNon‐HDL‐CHDL‐C1.01.52.0

Cholesterol (mm oDietaryGroup1DietaryGroup2DietaryGroup3DietaryGroup4681012

olesterol (mmo l0.00.50 5 10 15 20 25FractionVLDLLDLHDL0246DietaryGroup1DietaryGroup2DietaryGroup3DietaryGroup4

ChFractionTherewerenodifferencesinbaselinecholesterolconcentrationsDietaryGroup1DietaryGroup2DietaryGroup3DietaryGroup4andprofilespriortoplacementonmodifiedcocoabutter/sucrosediets.StudyWeek‐4‐3‐2‐10 123 4NoPFOA/PFOS addedtodiets ~30ppbPFOA/~30ppbPFOSaddedtodiets14161820

/ L)TotalCholesterolNon‐HDL‐CHDL‐C2 02.53.0

/L)DietaryGroup1VLDL200250SerumPFOAConcentration(t=Week0)8101214

o lesterol (mmol /1.01.52.0

Cholesterol (mmolDietaryGroup1DietaryGroup2DietaryGroup3DietaryGroup4HDL050100150200DietaryGroup1 DietaryGroup2 DietaryGroup3 DietaryGroup4

PFOA Concentration (ng/mL)0246

Ch o0.00.50 5 10 15 20 25LDLHDLDietaryGroup1 DietaryGroup2 DietaryGroup3 DietaryGroup4FractionPriortoaddingPFOA/PFOSindiets,notabledifferencesbetweendietarygroupsincholesterolprofilesandconcentrationsbecameapparent.StudyWeek‐4‐3‐2‐10 123 420NoPFOA/PFOS addedtodiets ~30ppbPFOA/~30ppbPFOSaddedtodiets14161820

ol/L)TotalCholesterolNon‐HDL‐CHDL‐C2.02.53.0

m ol/L)DietrayGroup1+PFOA/PFOSDietaryGroup2+PFOA/PFOSVLDL150200250

ion (ng/mL)PlasmaPFOAConcentration(t=Week2)681012

C holesterol (mm1.01.5

Cholesterol (m mDietaryGroup2PFOA/PFOSDietaryGroup3+PFOA/PFOSDietaryGroup4+PFOA/PFOSLDLHDL050100DietaryGroup1 DietaryGroup2 DietaryGroup3 DietaryGroup4

PFOA Concentrat024DietaryGroup1 DietaryGroup2 DietaryGroup3 DietaryGroup4

C0.00.50 5 10 15 20 25FractionLDLMeanplasma[PFOA]rangedfrom65– 100ng/mL.PlasmacholesterollevelsandlipoproteinprofilesdidnotchangecholesterollipoproteinprofilessignificantlycomparedtoWeek0levels.Week0(noPFOA/PFOSindiet)Week2(PFOA/PFOSindiet)2 02.53.0

l /L)DietaryGroup1VLDL2.53.0

l /L)DietrayGroup1+PFOA/PFOSVLDL(noPFOA/PFOSindiet)(PFOA/PFOSindiet)0 51.01.52.0

Cholesterol (mmo lDietaryGroup1DietaryGroup2DietaryGroup3DietaryGroup4LDLHDL1.01.52.0

Cholesterol (mmo lypDietaryGroup2+PFOA/PFOSDietaryGroup3+PFOA/PFOSDietaryGroup4+PFOA/PFOSLDLHDL0.00.50 5 10 15 20 25Fraction0.00.50 5 10 15 20 25Fraction1214161820

o l/L)TotalCholesterolNon‐HDL‐CHDL‐C150200250

t ration (ng/mL)SerumPFOAConcentration(t=Week0)14161820

l/L)TotalCholesterolNon‐HDL‐CHDL‐C150200250

t ration (ng/mL)PlasmaPFOAConcentration(t=Week2)4681012

Cholesterol (mm o050100DietaryGroup1 DietaryGroup2 DietaryGroup3 DietaryGroup4

PFOA Concen t4681012

Cholesterol (mmo050100DietaryGroup1 DietaryGroup2 DietaryGroup3 DietaryGroup4

PFOA Concen t024DietaryGroup1 DietaryGroup2 DietaryGroup3 DietaryGroup4024DietaryGroup1 DietaryGroup2 DietaryGroup3 DietaryGroup4StudyWeek‐4‐3‐2‐10 123 4203 0NoPFOA/PFOS addedtodiets ~30ppbPFOA/~30ppbPFOSaddedtodiets12141618

m mol/)TotalCholesterolNon‐HDL‐CHDL‐C2.02.53.0

mmol/L)DietaryGroup1+PFOA/PFOSDi tG2+PFOA/PFOSVLDL150200250

e ntration (ng/mL)PlasmaPFOAConcentration(t=Week4)681012

Cholesterol ( m1.01.5

Cholesterol (DietaryGroup2+PFOA/PFOSDietaryGroup3+PFOA/PFOSDietaryGroup3+PFOA/PFOSLDLHDL050100DietaryGroup1 DietaryGroup2 DietaryGroup3 DietaryGroup4

PFOA Conc e024DietaryGroup1 DietaryGroup2 DietaryGroup3 DietaryGroup40.00.50 5 10 15 20 25FractionMeanplasmaPFOAcontinuedtorise(128– 195ng/mL).PlasmacholesterollevelsandlipoproteinprofilesdidnotchangecholesterollipoproteinprofilessignificantlycomparedtoWeek0andWeek2levels.Week0(noPFOA/PFOSindiet)Week4(PFOA/PFOSindiet)2 02.53.0

l /L)DietaryGroup1VLDL(noPFOA/PFOSindiet)(PFOA/PFOSindiet)2.53.0

l /L)VLDL0 51.01.52.0

Cholesterol (mmo lDietaryGroup1DietaryGroup2DietaryGroup3DietaryGroup4LDLHDL1.01.52.0

Cholesterol (mmo lDietaryGroup1+PFOA/PFOSDietaryGroup2+PFOA/PFOSDietaryGroup3+PFOA/PFOSDietaryGroup3+PFOA/PFOSLDLHDL0.00.50 5 10 15 20 25Fraction0.00.50 5 10 15 20 25FractionLDL1214161820

o l/L)TotalCholesterolNon‐HDL‐CHDL‐C150200250

r ation (ng/mL)SerumPFOAConcentration(t=Week0)1214161820

ol/)TotalCholesterolNon‐HDL‐CHDL‐C150200250

t ration (ng/mL)PlasmaPFOAConcentration(t=Week4)4681012

Cholesterol (mm o050100DietaryGroup1 DietaryGroup2 DietaryGroup3 DietaryGroup4

PFOA Concent r4681012

Cholesterol (mm050100DietaryGroup1 DietaryGroup2 DietaryGroup3 DietaryGroup4

PFOA Concen t024DietaryGroup1 DietaryGroup2 DietaryGroup3 DietaryGroup4024DietaryGroup1 DietaryGroup2 DietaryGroup3 DietaryGroup4161214

l , mmol/LWeek0Week2Week4810

C holestero l468

N on

‐ HDL

‐ C024

Plasma NDG1 DG2 DG3DG40012345Ratio,DietarykCalfromCocoaButter:DietarykCalfromSucrose↑kC lalfromCocoaButter↑kCalfromSucrose250200

p pb)Week1Week2Week4150

] , ng/mL ( p100

ma [PFOA ]50

PlasDG1DG2DG3DG40012345Ratio,DietarykCalfromCocoaButter:DietarykCalfromSucrose↑kC lfCBDG1DG2DG3DG4↑kCalfromCocoaButter↑kCalfromSucroseWeek2Week41416DG11012

, mmol/LDG268

N on

‐ HDL

‐ C ,DG3246

Plasma NDG4020 50 100 150 200PlasmaPFOAConcentration,ng/mLPOSSIBLEMODEOFACTIONFructoseandglucoseareknowntostimulatelipogenesis andtheproductionofVLDLcholesterol;therefore,increasingtheproportionfl ifhdl tifocaoresromsucrosemayaveresultetheup‐reguaonolipogenic pathways.Inaddition,withrespecttoplasma[PFOA],itisconceivablethatPFOA,whichresemblesfattyacids,maycompetewithdietaryfatforGIshownbasedbioavailabilitychartuptakeasonthebioavailability.Bothfactorsmayhavebeeninfluentialindeterminingthei tidthit dassocaonsfouninssuy.CONCLUSIONDifferencesinisocalorically‐balanceddietaryproportionsofsucroseandcocoabutterwereshowntoproducenon‐causal,cross‐sectional,positiveassociationsbetweenplasmaconcentrationsofnon‐HDL‐CandPFOAinApoE*3.Leiden.CETPmice.3.Leiden.CETPUncontrolleddifferencesindietarycompositionmaybeaneffectmodifierincrosssectionalepidemiologicalassociationshumansmodifier‐sectionalepidemiologicalassociationsinatlowserumconcentrationsofPFOAand,perhaps,otherperfluoroalkyls.THANKYOUSix‐MonthCapsule‐DosingStudywithlkPFOSinCynomogus MoneysSix‐MonthStudyofPFOSinlkCynomogus MoneyswithRecovery•Dailyintragastric capsuledosing–Control,0.03mg/kg,0.15mg/kg,0.75mg/kg•5‐foldincrementsindose•Malesandfemales6/sex/groupexcept0 03mg/kg(4/sex)females,6/sex/group.(4/sex)•Repeatedclinicalmeasuresover6‐monthsofdosing–5to6timepointsformostmeasurements•Hematology,serumclinicalchemistry,thyroidhormones,sexhormones•Baselinevaluesformostmeasurements–Allowedtrackingofindividual,withingroup,andbetweenhgroupcanges•Completehistologicalevaluations•Recoverysub‐groupswithliveratRecoverybiopsy7monthsEffectsonBodyWeightandLiverWeightinRelationtoDoseandSerum[PFOS]220 000240,000GroupMeanSerum[PFOS]inCynomolgusMonkeysafterSix‐MonthsofDosing30MeanPercentChangeinBaselineBodyWeightofMonkeysafterSixMonthsofDosingwithPFOS100,000120,000140,000160,000180,000200,000220,000

P FOS], ng/mL (ppb)0510152025

Percent020,00040,00060,00080,0000 0.03 0.15 0.75

Serum [ PDoseGroup,mg/kg/d‐20‐15‐10‐50 0.03 0.15 0.75DoseGroup,mg/kg/d**p,g/ g/33.5LiverWeighttoBodyWeightPercentinCynomolgusMonkeysafterSix‐MonthsofDosingwithPFOS120140LiverWeightinCynomolgusMonkeysafterSixMonthsofDosingwithPFOSp,g/ g/**11.522.5

Percent406080100

Liver Weight, g*00.50 0.03 0.15 0.75DoseGroup,mg/kg/d0200 0.03 0.15 0.75DoseGroup,mg/kg/dHistopathology•Histopathologygy–Tissuesexamined•Alltissuesfrom0.75mg/kggroupbylightmicroscopy•Liverthymusandspinalcordfrom03and0 15mg/kggroups,,0..bylightmicroscopy•Allliversbytransmissionelectronmicroscopy–Notreatment‐relatedfindingsat0 03and0 15mg/kgtreatmentrelatedfindings..–At0.75mg/kg•LM:Centrilobular hypertrophy,vacuolation andmildbilestasisdfdd l•EM:~50%incienceolipi‐ropetaccumulationandincreasedglycogen–Resolvedby7monthsinrecoveryLiverCellElectronMicroscopyinMaleCynomolgusMonkeysSix‐MonthCynomolgusMonkeysofStudyControl@6mos. 0.75mg/kgMale@6mos. 0.75mg/kgMale@6mos.RecoverySerum‐BasedToxicityLiverToxicityTests•Noincreasesinserumactivitiesof:increasesactivities–Alanine aminotransaminase (ALT)Aspartateaminotransaminase(AST)––γ‐Glutamyl transferase (GGT)Alk lih ht(ALP)–anepospaase–Sorbitol dehydrogenase (SDH)•NoindicationsofovertlivertoxicityMaleMonkeyLiverALTandAST‐27 37 62 91 153 182Group(mg/kg)StudyDay0 32 39 34 35 43 390.03 36 40 35 60 36 290.15 69 50 37 45 55 47ALT(IU/L)0.75 89 46 43 44 52 470 56 45 36 39 31 320 03443735353027( /)AST0.034437353530270.15 72 48 40 42 40 360.75 68 43 34 36 39 34AST(IU/L)Theonlystatisticallysignificantchangesobservedwerefordecreasedactivity,eitherwithingroupfrombaselineorbetweentreatedgroupsandtheirtime‐matchedcontrols.Thesedataargueagainstthepresenceofovertlivertoxicity.FemaleMonkeyLiverALTandAST27376291153182Group(mg/kg)StudyDay‐273762911531820 140 59 53 54 77 870.03 103 70 77 79 80 72(mg/kg)ALT/0.15 110 68 60 67 66 530.75 123 46 42 45 48 440 64 44 38 44 36 41(IUL)0.03 54 49 45 43 38 420.15 74 36 37 37 36 370.75 72 36 31 34 40 33AST(IU/L)Theonlystatisticallysignificantchangesobservedwerefordecreasedactivity,eitherwithingroupfrombaselineorbetweentreatedgroupsandtheirtime‐matchedcontrols.Thesedataargueagainstthepresenceofovertlivertoxicity.SerumLipids•Decreasedtotalcholesterol(CHOL)andcholesteroldecreasedhigh‐densitylipoproteincholesterol(HDL‐C)andfemalesat75mg/kginmalesfemales0.dose.•NobaselinevaluesforHDLbaseline.MaleMonkeySerumLipids‐27 37 62 91 153 1820 138 140 153 154 154 152StudyDayCommentsStatisticallysignificantdifferencesbetween0.03Group(mg/kg)0.03 110 118 114 126 120 1100.15 151 146 144 150 149 1470.75 138 130 125 112 65 480 69 63Nobaselinevalues.Differencesfromcontrolat0.03mg/kggroupandcontrolsbutnochangein0.03baseline.Reductionsin0.75mg/kggrouponDays91‐182aredifferentthanbaselineandcontrol.CHOL(mg/dL)0.03 46 420.15 55 480.75 19 130 2.2 2.4mg/kgareinroughproportiontodifferencestime‐matchedCHOL(seeratiobelow).Differencesfromcontrolat0.75mg/kgarelikelyreal.Valuesfor0.75mg/kggrouparesignificantlyHDL‐C(mg/dL)0.03 2.6 2.60.15 2.8 3.20.75 3.4 3.70 70 69 62 68 60 45differentfromcontrolvalues.Similaritiesofratiosin0,0.03,and0.15mg/kggroupssuggestslackofspecificHDLeffectin0.03and0.15mg/kggroups.ReductionsinTRIGfrombaselinevaluesincontrol,CHOL/HDL‐C0.03 61 47 60 54 60 330.15 56 58 57 50 65 360.75 67 91 76 73 60 300.15mg/kgand0.75mg/kggroupsaresimilar.Noobviouseffectoftreatment.TRIG(mg/dL)=Statisticallysignificantlydifferentthanwithin‐groupbaselinevalue(Dunnett'st‐test,p<0.05)=Statisticallysignificantlydifferentthantime‐matchedcontrolvalue(Dunnett'st‐test,p<0.05)=Statisticallysignificantlydifferentthanwithin‐groupbaselineANDtime‐matchedcontrolvalues(Dunnett'st‐test,p<0.05)FemaleMonkeySerumLipids‐27 37 62 91 153 1820 149 147 155 166 163 160CommentsDifferencefromcontrolvaluein0.03mg/kggroupStudyDayGroup(mg/kg)0.03 130 124 127 134 110 1220.15 144 133 137 140 130 1290.75 154 130 127 111 91 820 59 56onDay153isnotdifferentthatgroupbaseline.DecreasedCHOLin0.75mg/kggrouponDays153and182bothcomparedtocontrolandbaseline.Nobaselinevalues.DifferencesfromcontrolinareCHOL(mg/dL)0.03 47 420.15 41 360.75 23 210 2.8 2.9inroughproportiontodifferencesintime‐matchedCHOL(seeratiobelow).NosignificantdifferencesbetweentreatedgroupsHDL‐C(mg/dL)0.03 2.4 3.00.15 3.3 3.90.75 4.0 4.40 73 71 86 67 80 56Noevidencefortreatment‐relatedeffect.andcontrol.Similaritiesofratiosin0,0.03,0.15,and0.75mg/kggroupssuggestslackofspecificeffectonHDL.CHOL/HDL‐C0.03 62 64 76 61 82 490.15 58 57 64 56 69 490.75 61 66 61 60 64 53TRIG(mg/dL)=Statisticallysignificantlydifferentthanwithin‐groupbaselinevalue(Dunnett'st‐test,p<0.05)=Statisticallysignificantlydifferentthantime‐matchedcontrolvalue(Dunnett'st‐test,p<0.05)=Statisticallysignificantlydifferentthanwithin‐groupbaselineANDtime‐matchedcontrolvalues(Dunnett'st‐test,p<0.05)SerumThyroidHormones•AssayissuesleadingtoreanalysisbyMayoleadingreanalysisClinicendocrinologyunit.Ni llifiid•oclincaysigncantchangeinthyrohomeostasisbasedonreanalysisofsamplesbMiyayoClinc.•Nohistologicalevidenceofthyroidchangesconsistentwithalteredthyroidhomeostasis.MaleMonkeyThyroidHormonesStudyDayGroup‐27 37 62 91 182 1841840 0.00 0.34 0.57 0.00 0.43 0.370.550.03 0.00 0.35 0.42 0.00 0.34 0.560.560 150 050 330 980 030 740 701 38CommentsProblemswithassayusedatcontractlab.MayoClinicvaluesconsideredmorereliableandshownosignificantStudyDayTSH(U/ L)Group(mg/kg)0.150.050.330.980.030.740.701.380.75 0.00 0.21 0.22 0.00 0.93 0.931.430 1.011.400.03 1.211.530 150 941 47differencesfromcontrolatendofstudy.NegativebiasinFT4assayusedatcontractlabmayhavebeenpresent.Equilibriumdialysis‐RIAmethodatMayoClinic(μU/mL)FT4( /dL)0.150.941.470.75 1.121.500 3.49 4.06 2.27 3.89 4.38 3.253.240.03 4.46 5.11 3.91 5.30 4.72 3.853.680 154 634 003 084 473 992 713 00demonstratednodifferencesinmeans.Notreatment‐relatedeffectonTT4wasevident.TT4(μg/dL)(ng/dL)0.154.634.003.084.473.992.713.000.75 4.88 3.61 2.59 4.61 5.34 3.303.770 4.210.03 5.130 154 33AlthoughFT3groupmeanat0.75mg/kgislowerthancontrol,negativebiasmayhaveaffectedmethod.Resultat0.75mg/kgis(μg/dL)FT3(pg/mL)0.154.330.75 2.450 110 273 145 153 160 1151460.03 113 273 139 153 119 1101450 1511825514214712594129PFOSat0.75mg/kgreducedTT3basedoncomparisonswithbaseline,time‐matchedcontrols,andconfirmationatMayoClinic.equivocal.(pg/mL)TT3(ng/dL)=Statisticallysignificantlydifferentthanwithin‐groupbaselinevalue(Dunnett'st‐test,p<0.05)=Statisticallysignificantlydifferentthantime‐matchedcontrolvalue(Dunnett'st‐test,p<0.05)=Statisticallysignificantlydifferentthanwithin‐groupbaselineANDtime‐matchedcontrolvalues(Dunnett'st‐test,p<0.05)0.15118255142147125941290.75 133 239 121 118 66 4376Physiologicalconsequenceisequivocal.(ng/dL)FemaleMonkeyThyroidHormonesFemaleMonkeyThyroidHormonesGroupStudyDay‐27 37 62 91 182.00 184.00 184.000 0.21 0.73 1.19 0.00 0.73 0.531.020.03 0.00 0.24 0.46 0.00 0.68 0.432.010 150 340 610 820 021 270 471 33CommentsProblemswithcontractlabassay.NochangesinTSHbasedonMayoClinicdata.Group(mg/kg)TSH(U/ L)StudyDay0.150.340.610.820.021.270.471.330.75 0.01 0.59 0.74 0.00 0.84 1.031.860 1.061.530.03 1.011.700 150 901 35NochangesinFT4.(μU/mL)FT4( /dL)0.150.901.350.75 1.081.5706.46 5.82 3.53 4.775.66 4.143.780.037.06 5.06 4.07 6.004.33 3.482.800 157 133 682 773 773 912 883 23NochangesinTT4.(ng/dL)TT4(μg/dL)0.157.133.682.773.773.912.883.230.757.10 4.23 3.08 4.925.61 3.433.800 4.050.03 3.590 153 27AlthoughFT3groupmeanat0.75mg/kgislowerthancontrol,negativebiasmayhaveaffectedmethod.Resultat0.75mg/kgis(μg/dL)FT3(pg/mL)0.153.270.75 2.820 119 230 115 143 135 1061480.03 171 244 136 147 120 921390 151382141141109780116MayoClinicdatarevealnostatisticallysignificantdifferenceswithtime‐matchedcontrolsatterm.Baselinereductionatequivocal.TT3(ng/dL)(pg/mL)=Statisticallysignificantlydifferentthanwithin‐groupbaselinevalue(Dunnett'st‐test,p<0.05)=Statisticallysignificantlydifferentthantime‐matchedcontrolvalue(Dunnett'st‐test,p<0.05)=Statisticallysignificantlydifferentthanwithin‐groupbaselineANDtime‐matchedcontrolvalues(Dunnett'st‐test,p<0.05)0.1513821411411097801160.75 155 196 96 88 85 58990.75mg/kgisequivocal.(ng/dL)SteroidHormones•SteroidhormonescortisolestrogenestriolSteroidhormones(cortisol,estrogen,estriol,estrone,testosterone)–Onlyobserveddecreasedestradiolandonlyinobserveddecreasedestradiol0.75mg/kgmalesOtherMeasures•Notoxicologicallymeaningfulchangesin:toxicologicallymeaningfulchanges–Otherclinicalchemistrymeasures•SBACPKBILIBUNGLUCREATPROTGLOB,,,,,,,ALB,,CA,CL,K,NA,–Hematologicalparameters–Urinalyses–PCNAlabelinindex(livrpancreas,ttge,pa c eas,es es)–HepaticpalmitoylCoAoxidaseThankYouQuestionsandComments