Endocrine disruption : Fact or urban legend?

ToxicologyLetters223 (2013) 295–305

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Toxicology Letters

j o ur na l h o me p a g e : w w w . e l s e v i e r . c o m / l o c a t e / t o x l e t

Endocrine disruption: Fact or urban legend?

Gerhard J. Nohynek

, Christopher J. Borgert

, Daniel Dietrich

, Karl K. Rozman

aLesCaillons,58460Corvol,L’Orgueilleux,France

bCenterforEnvironmentalandHumanToxicology,DepartmentofPhysiologicalSciences,UniversityofFloridaCollegeofVeterinaryMedicine,Gainesville, 32605Fl,UnitedStates

cFacultyofBiology,UniversityofKonstanz,78457Konstanz,Germany

dUniversityofKansasMedicalCenter,DepartmentofPharmacology,ToxicologyandTherapeutics,KansasCity,KS66160,UnitedStates

h i g h l i g h t s

•Wereviewtheinternationaldefinitionsofendocrinedisruptors(EDs).

•WediscusstheassociationofEDswiththetesticulardysgenesissyndrome.

•WediscussthepivotalroleofpotencyinthesafetyassessmentofEDs.

•WediscussadditiveeffectsofhumansimultaneousexposuretoseveralEDs.

•WediscusswhetherhumanexposuretochemicalEDsposesahumanhealthrisk.

a r t i c l e i n f o

Articlehistory:

Available online 28 October 2013

Keywords:

Personalcareproducts Endocrinedisruptors Additiveeffects Potency

Testiculardysgenesissyndrome

a b s t r a c t

Endocrinedisruptors(EDs)aresubstancesthatcauseadversehealtheffectsviaendocrine-mediated mechanismsinanintactorganismoritsprogenyor(sub)populations.PurportedEDCsinpersonal careproductsinclude4-MBC(UVfilter)orparabensthatshowedoestrogenicactivityinscreeningtests, althoughregulatorytoxicitystudiesshowednoadverseeffectsonreproductiveendpoints.Hormonal potencyisthekeyissueofthesafetyofEDCs.Oestrogen-baseddrugs,e.g.thecontraceptivepillorthe syntheticoestrogenDES,possesspotenciesupto7ordersofmagnitudehigherthanthoseofPCPingredi- ents;yet,inuteroexposuretothesedrugsdidnotadverselyaffectfertilityorsexualorgandevelopment ofoffspringunlessexposedtoextremedoses.AdditiveeffectsofEDsareunlikelyduetothemultitudeof mechanismshowsubstancesmayproduceahormone-likeactivity;evenafteruptakeofdifferentsub- stanceswithasimilarmodeofaction,thepossibilityofadditiveeffectsisreducedbydifferentabsorption, metabolismandkinetics.ThisissupportedbyanumberofstudiesonmixturesofchemicalEDCs.Overall, despiteof20yearsofresearchahumanhealthriskfromexposuretolowconcentrationsofexogenous chemicalsubstanceswithweakhormone-likeactivitiesremainsanunprovenandunlikelyhypothesis.

© 2013 The Authors. Published by Elsevier Ireland Ltd.

Damnantquodnonintellegunt.

They seek condemnation of what they do not understand.

(Cicero)

Abbreviations: BfR,BundesamtfürRisikobewertung(Berlin,Germany);DES, Diethylstilbestrol;DEP,diethylphthalate;ECETOC,EuropeanCentreforEcotox- icologyandToxicologyofChemicals;ED,EndocrineDisruptor;EDC,Endocrine DisruptingChemical;EFSA,EuropeanFoodSafetyAgency;EPA,USEnvironmental ProtectionAgency;EU,EuropeanUnion;GLP,GoodLaboratoryPractice;IPCS,Inter- nationalProgramonChemicalSafety;4-MBC,4-methylbenzylidenecamphor;PCP, PersonalCareProducts/Cosmetics;TDS,TesticularDysgenesisSyndrome;WHO, WorldHealthOrganisation.

∗Correspondingauthorat:PreclinicalConsultant,LesCaillons,58460Corvol L’Orgueilleux,France.Tel.:+33386291078.

E-mailaddress:nepomuk@noos.fr(G.J.Nohynek).

Ithasbeenunderstoodforalongtimethatahighconsump- tionofhormonallyactiveplantconstituents,suchascoumestrol or daidzein containedin clover, can adversely affectreproduc- tionindomesticanimals,uptoinductionofpermanentinfertility (Lindner,1976).Therefore,itisnotsurprisingthathighdosesof industrialchemicals,suchasphthalates,chlorinatedcompounds aswellasnumerousothersubstanceswerealsofoundtoadversely affectreproductioninlaboratoryanimals.Fromtheearly1990sto thepresent,additionalconcernshavebeenraisedthatotherman- madechemicals,suchasphthalates,polychlorinatedbiphenylsor alkylphenolsmayalsoaffectthereproductioninhumans,wildlife oraquaticorganismbydisruptingtheirendocrinefunctions,e.g.

hormones secreted by human ovaries, testes, thyroid or other organs(Nilsson,2000;Safe,1997,2004).Thusanovelcategoryof potentiallyhazardoussubstances,endocrinedisruptorsorendocrine modulatorswasborn,althoughtheexactdefinitionofthemeaning ofthesetermsisstilldebatedandsomewhatunclear(Fosterand

0378-4274© 2013 The Authors. Published by Elsevier Ireland Ltd.

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Agzarian,2008).Indeed,theEuropeanUnion,theWHOandthe USEPAemployslightlydifferentdefinitionstherebyaddingtothe breadthofinterpretationalroomforanydatagenerated.Special concernshave beenraisedbysomescientistsand environmen- talactiviststhathumanexposuretohormonally-activeingredients usedinpersonalcareproducts/cosmeticswithpotentialhormonal activity,suchasparabens,phthalatesorcertainultravioletfilters, mayaffecthumanendocrine systemsandposea risktohuman health(WitorschandThomas,2010).Forexample,ithasbeensug- gestedthathumanexposure,inparticularpre-natalexposureofthe humanfoetus,tosuchsubstancesmayaffecthumansemenqual- ity,produceorcontributetomaleinfertility,birthdefectsinmale infants,breast andtesticularcancers,obesityand otheradverse healtheffects.

Given that chemicals with potential hormone-like activity, in particular personal care product ingredients, have a minute potency when compared with that of actual, mammalian hor- mones,suchasoestradiol,novelhypothesesweredevelopedthat human exposure to mixtures of a multitude of weakly active substancesmayproduceadditiveorevensynergisticeffectsand yetposearisk tohumanhealth(Witorsch,2002a;Myersetal., 2009).Consideringthesehypotheses,weattemptedtosummarise thepresentstate ofknowledge onEndocrinedisruptorsand to reviewwhetheringredientsofpersonalcareproducts/cosmetics mayproduceadversehumanhealtheffectssecondarytoendocrine disruption.

1. Whatare“Endocrinedisruptors”?

The Endocrine system is the term for multiple and diverse hormonal systems in the mammalian organism, such as thy- roidhormones,hormonesoriginatingfromthepancreas,ovaries, testes, adrenals or the brain. There have been a number of definitions of what is an “Endocrine disruptor” (ED).The most commonly agreed are the following: An ED is an exogenous substancethat causesadversehealth effects inthe intactorgan- ism oritsprogeny, secondary/consequent tochanges inendocrine function“(Weybridge, 1996).A similar definition hasbeen cho- senbytheWorldHealthOrganisation’sInternationalProgramon ChemicalSafety:Anendocrinedisrupterisanexogenoussubstance ormixturethataltersfunction(s)oftheendocrinesystemandcon- sequentlycausesadversehealtheffectsinanintactorganism,orits progeny,or(sub)populations(WHO/IPCS,2002).Ajointexpertgroup of the German Bundesamt fuer Risikobewertung (BfR)and UK healthauthorities(UK-BfR)regulatoryexpertgroupproposedan identicaldefinition:AnEDisanexogenoussubstanceormixturethat altersfunction(s)oftheendocrinesystemandconsequentlycauses adversehealtheffectsinanintactorganism,oritsprogenyor(sub) populations(BfR,2011).Themostrecentdefinitionissuedbythe EUisthatanEDisanexogenoussubstancethatcausesadversehealth effectsinanintactorganism,oritsprogeny,secondarytochangesin endocrinefunction(EU,2012);thisisalsoconsistentwiththerecent definitionbytheEuropeanFoodSafetyAgency(EFSA,2013).TheUS EPAdefinedEDCasfollows:Endocrinedisruptingchemicals(EDCs) havebeendefinedasexogenousagentsthatinterferewiththepro- duction,release,transport,metabolism,binding,action,orelimination ofthenaturalhormonesinthebodyresponsibleforthemaintenance ofhomeostasisandtheregulationofdevelopmentalprocesses(EPA, 2012).

Itisimportanttonotethatallmajorinternationaldefinitions stipulatethatanEDCmustcauseadverseeffectsviaanendocrine- mediatedmechanisminanintactorganism.Acommondefinition ofwhatconstitutesanadverse effecthasbeenproposedbythe BfR/UKexpert group,the WHO IPCS,ECETOC and independent authors(BfR,2011;WHO/IPCS,2002;ECETOC,2009;Lewisetal.,

2002). All current definitions agree that the definition of an

“adverseeffect”meanstoxicity,i.e.pathologyorfunctionalimpair- ment.Therefore,onlyasubstancethatproducestoxicityinanintact organismviaahormonalorhormone-likemechanismrepresents agenuineED.

Oftensubstanceshave beenreportedtobeEDbased onthe resultsofscreeningtests.Indeed,aconsiderablenumberofinvitro (sub-cellular or cellular) and in vivo (animal) screening tests for hormone-like activities of substances have been developed (Borgertetal.,2011).However,thesetestswereestablishedfor thepurposeofscreening,i.e. inorder toprioritisetoxicological testingofsubstancesthatmaypossesshormonalactivities.Given thatscreeningtestsdonotidentifytoxicity,theycannotdetermine whetherasubstanceisanEDornot.Screeningtestsdonoteven assurethatasubstancewillproduceahormonalactivityinhumans orotherorganisms;theymerelysuggestthatthetestsubstance mayhavesuchapotential.Therefore,whenasubstanceproduces changes in hormone-relatedparameters in screeningtests, this meansthatthetestsubstancehasabiologicalactivity,butitdoes notmeanthatitistoxicorisanED.Thisviewisalsosupported byarecentpositionoftheEuropeanFoodSafetyAgency(EFSA, 2013).Indeed,therearemorethanathousandnaturalorsynthetic substancesthathavebeenfoundtobepositiveinscreeningassays andtopossessweakhormone-likeactivitieswithoutcausingactual toxicityattheindividualorthepopulationlevel.Weakhormonal activitycanbeadvantageous,detrimentalorneutralfortheorgan- ism.Toillustratethis:achangeinroomtemperature,amealor daylightmayinducechangesincirculatinglevelsofhormones,such asthyroidhormones,insulinormelatonin,respectively(Fosterand Agzarian,2008).Thisdoesnotmeanthattheseinnocuousfactors shouldbeconsideredtobeEDs.

Similarly,ourfoodisfullofhormonallyactivesubstances:for example,soycontainssubstances(isoflavones)thatpossesspow- erfuloestrogenic activityin screening assays,which have been showntoproduceadversereproductiveeffectsinanimaltoxicity studies(Delclosetal.,2001;McClainetal.,2007).Thus,bydefini- tion,soyisoflavones,suchasgenistein,aregenuineEDs.However, isoflavonesdonotproduceoestrogeniceffectsinhumansornon- humanprimatesat dietarylevels(Clineetal.,2001).Moreover, Asianpopulationswithahighdietaryintakeofsoyorsoy-based foodtendtohavelower cancerratesofreproductiveorgans or breast,whencomparedwithEuropeanorUSpopulations(Peeters etal.,2003),butdonotpresentanincreasedincidenceoflower spermcountorTDS(testiculardysgenesissyndrome).Ithasbeen suggestedthatphytoestrogensmaybeusefulforthepreventionof breastandothercancers(Humphrey,1998;Menseetal.,2008).

Naturaloestrogensor similarhormone-like substancesare con- tainedinclover,hops,Brusselssprouts,beer,wine,walnuts,linseed andmany otherplantfoods(Kurzerand Xu,1997).It hasbeen knownforcenturiesorlongerthatgrazingsheeporcattleonclover mayresultininfertility,theso-calledcloverdisease.Today,weknow thatthisisduetocoumestrolanddaidzein,naturalconstituentsof cloverandnaturalcontraceptives(Lindner,1976;Oellermannetal., 1987).Giventhatcloverorsoyphyto-oestrogenshaveoestrogenic activityinvitroandbeenshowntoadverselyaffectreproduction in animals,theyshouldbeconsideredgenuine EDs –although, atnormalconcentrationspresentinanimalfeedorhumanfood, there is noevidence that theypose a risk to health. A human healthriskfromsoyisoflavonesisalsoreduced bythefactthat humanshaveagreaterabilitytothanlaboratoryrodentstodetoxify ingestedisoflavonesviaglucuronidationinthegutwallortheliver (Pritchettetal.,2008;Setchelletal.,2008;Rozmanetal.,2006).

However,these examplesdemonstrate that mammalianorgan- ismsdonotdifferentiatebetweennaturalandsyntheticsubstances withrespecttopotentialadverseeffects.Overall,hormonalactiv- ityofasubstancerepresentsabiologicalactivityoramechanism

that can begood, bad or neutral for the intactorganism. Hor- monalactivityonitsownhaslittletodowithhazard(toxicity) orevenlesssuggestsahealthriskfortheintactorganismunless itleadsadverseoutcomes,forexamplecarcinogenic,reproductive, ordevelopmentaleffectsthatareroutinelyconsideredinreaching regulatorydecisions(DekantandColnot,2013;Testaietal.,2013;

EPA,2012).

2. Isthereanassociationofendocrinedisruptorsand breastcancer?

Certainpersistentorganochlorinesubstances,suchaspesticides (DDT)or polychlorinated biphenyls(PCB), have beenshown to haveaweakpotentialforhormonalactivity.Thesesubstanceswere neverused inPersonalCareProductsand havebeenbannedin theUS,EuropeorJapanformorethan30years,althoughdeclin- ing residuescontinue to bepresent in theenvironment. It has beensuggestedthat residuesofthesesubstancesin thehuman organismcombinedwiththeirhormonalactivitymayplayarole intheaetiologyofbreastcancer.Indeed,in1992,USresearchers reportedelevatedlevelsofPCBsandDDTinarelativelysmallgroup ofwomenwithbreastcancer;subsequentUSstudiessuggesteda slightassociationbetweenbreastcancerandelevatedserumDDT, butnotserumPCB,levels(Kriegeretal.,1994;ACSH,1999).How- ever,follow-up studieson a largenumber of US breast cancer patientsfoundnocorrelationbetweenthehumanbodyburdenof organochlorinesubstancesandbreastcancer.Subsequentofficial USreviewsoftheassociationofbreastcancer,endometrialcan- cerandendometriosiswithorganochlorineresiduesconcludedthat nosuchrelationshipcouldbesupportedbytheexistingevidence (Ahlborgetal.,1995;Safe,1997).

In2004,astudyreportingparabenresiduesinhumanbreast tumour tissues re-opened the mediaattention and subsequent debateabouta possiblelinkbetweenendocrine disruptionand breastcancer.Giventhatsomelong-chainparabenspossessavery weakpotential for hormonal activity,the authors of thestudy suggestedthatparabenscontainedinunderarmdeodorantsmay migrateintothebreastandthereforemayhaveacausalrolein theaetiologyofbreastcancer(Darbreetal.,2004).However,that studyhadseriousweaknesses:thedetectedparabenresidueswere minute(intherangeofnanogramspergramoftissue),thestudy didnotinvestigateparabenlevelsinhealthytissuesandthereis noknowndirecttransportmechanismofexternallyappliedunder- armsubstancesintothebreast.Finally,thestudydetectedthesame parabensintumoursandblanksolventsthatwereusedfortissue extraction.Moreover,theauthorsfailedtonoticethatdeodorants donotcontainparabenssincetheyrequirenopreservativesdue totheirhighcontentofaluminium.Thestudyanditsclaimswere rejectedasflawedbyanumberofexpertsandinternationalhealth authoritieswhoconcludedthatthereisnoscientificevidencelink- ingunderarmdeodorantsorparabenswithbreastcancer(Witorsch and Thomas, 2010). Thisview is supportedby theresults of a largeepidemiologicalinvestigationthatshowednoevidencefor anincreasedriskofbreastcancerandunderarmantiperspirantuse (Miricketal.,2002).Nevertheless,anurbanlegendwasborn.

Indeed, long-chain parabens (butylparaben, propylparaben) possessextremelyweakestrogenicpotencies,i.e.about10,000- to1,000,000lowerthanthatofoestradiolorsynthetichormones, suchasethinylestradiol,an activeagent ofcontraceptive drugs (Routledgeetal.,1998;Goldenet al.,2005;CIR,2008).Yet,the humancontraceptivepillisuseddaily,chronically,andcontains largedosesofhighlypotentoestrogensandotherhormones.How- ever,useofthecontraceptivepillhasnotbeenassociatedwitha significantincreaseintheriskofbreastcancer.Itisthereforesci- entificallyimplausiblehowtheminuteactivityofparabenscould

posearisktohumanhealth,particularlywhentheyarecontained inlowconcentrationsinproductsthatareappliedtohumanskin.

Inthiscontextitshouldalsobementionedthattheincidenceof breastcancerintheUSandotherindustrialisedcountrieshasbeen stablesincethe1980s,whichisalsoinconsistentwiththealleged humanexposuretoEDCs(SEER,2010;Ahlborgetal.,1995;Safe, 1997).

3. Thehypothesisofthe“Testiculardysgenesissyndrome”

(TDS)

Acurrenthypothesishassuggestedthat,inmodernindustrial societies,humanmalefertilityisdeclining,whereastheincidence of diseases or birthdefects ofthe male reproductivesystemis increasing. The hypothesis that humansperm count is declin- ing whereas the incidence of testicular cancer, cryptorchidism (undescendedtestis)andhypospadias(abnormallyplacedurethral openingattheundersideofthepenis)isincreasingandthatthese changesareassociatedwithhumanexposuretohormonallyactive chemicals.Thehypothesiswaspopularisedin1992,whenagroup of Danish researchersreported a 50% decrease in sperm count fortheperiodof1938–1992(Carlsenetal.,1992),althoughthat studyhadsignificantweaknesses(Handelsman,2001;Fish,2000 and2008).Inthemeantime,thishypotheticalsyndromehasbeen namedthe“Testiculardysgenesissyndrome”(TDS).Itpostulates that,inWesternindustrialisedcountriesa)humanmalefertility, inparticularspermcount,hasdeclinedandcontinuestodecline, b)theincidenceofhumantesticularcancerisincreasing,c)the incidenceofcryptorchidismandd)hypospadiasinnewbornmale infantshasincreased(Thorupetal.,2010).Anadditionalhypothesis isthattheincreasedincidenceintheTDSmaybeduetochemical EDCinthehumanenvironment.

First,itshouldberealisedthatspermcountisnotequaltomale fertility.Itisacommonfallacythatmalefertilitycanbemeasured bycountingsperm.Spermcount/outputisactuallyasurrogatevari- ableformalefertility,butnotnecessarilyagoodone(Handelsman, 2001).Forexample,studiesinSweden(Akreetal.,1999;Scheike etal.,2008)ortheUK(Joffe,2000)suggestedthathumanfertil- ityhasincreasedinthepastdecades.Second,thereisnoevidence forageneraldeclineinspermcount(Saidietal.,1999;Fish,2000, 2008;Thorupetal.,2010;Itohetal.,2001;Handelsman,2001;Safe, 2004,2005;Greim,2005).Althoughsomestudiesfoundadecline (Carlsenetal.,1992;Rollandetal.,2012),othersfoundanincrease inspermcount(Saidietal.,1999;Fish,2008).Arecentstudy,one ofthelargest,longestandbest-controlledinvestigationseverper- formedonthisissue,foundnodecrease,butaslight,butsignificant increase in themean spermcount ofnearly 5000Danish mili- taryrecruitsovertheperiodof1996–2010(somestudydatafirst reportedbyBondeetal.,2011).Althoughtheresultsofthatstudy havebeenavailableforseveralyears,thecompletestudywasonly publishedin2012(Jorgensenet al.,2012),possibly inresponse topressurefromthescientificcommunity(Anon.,2011;Wilcox, 2011;Bondeetal.,2011).Nevertheless,anumberoffarsmaller studiesthatclaimeddecliningspermcountswerepublishedbythe sameresearchers–andwidelyreportedbythemedia.

Clearly,sucha“publicationbias”distortsthepublicriskpercep- tionandmayresultinconjuringimaginaryhealthrisksanddisease associationswithexposuretochemicals.Giventhatcurrentdata suggestthatspermcountinDenmarkhad remainedunchanged orevenslightlyimprovedoverthepast15years,this findingis nowconsistentwithsimilardatafromJapan,SwedenandtheUS, whichshowedthatspermcounthasremainedstableforthepast 20years(Itohetal.,2001;Axelssonetal.,2011;Fish,2008;Saidi etal.,1999).Hereitalsoshouldbeconsideredthatspermcounts reportedpriortothe1990smustberegardedwithgreatcautiondue

tomethodologicalshortcomingsand non-standardisedmethods (Handelsman,2001;Fish,2008).Eveninindustrialisedcountries alackofcompliancewithstandardisedspermmorphology/count methodswasreportedasrecentlyas2012or2005(Mallidisetal., 2012;Riddelletal.,2005).Finally,thenotionthatprenataloestro- genexposurehasadverseeffectsonmalefertilityhasbeenrefuted by studies on boys born to women exposed to high oral DES dosesduringpregnancy.Neitherfertilitynorspermoutputwere adverselyaffecteddespitemassiveinuterooestrogenexposure, althoughminorurogenitalmalformationsdidoccurinthispopu- lation(Learyetal.,1984;Handelsman,2001).Itisnoteworthythat therewasanapproximate14-folddifferencebetweenthehighest andthelowestclinicaldoseofDES;reproductivemalformations wereobservedonlyamongtheoffspringofwomenwhoreceived high-doseregimens(Borgertetal.,2012).

Moreover,thereisnoscientificevidenceforageneralincreasein theincidenceofcryptorchidismorhypospadiasinmaleinfants;in addition,ithasbeenarguedthatthesetwopathologiesarecaused bydifferentmechanisms,whichcastevenmoredoubtonacom- monoriginoracommoncausalagent(Thorupetal.,2010).An increasedriskofhpospadiasorcryptorchidismappearstobeasso- ciatedwithincreasingmaternalageand bodymass (Fish etal., 2001;McGlynnetal.,2006),whereasahighincidenceinhypospa- diashasalsobeenlinkedwithamaternallowproteinorvegetarian dietsduringpregnancy(Akreetal.,2008;NorthandGolding,2000).

Ontheotherhand,thereappearstobeanincreaseintheincidence intesticularcancerinsomeWesterncountries,althoughtheinci- denceinotherindustrialisedcountriesandregions,suchasAsia, appearstobestable.Althoughmaternalbodyweightmayplaya roleintheaetiologyoftesticularcancer(Aschimetal.,2005)the reasonforthisdiscrepancyremainsunknown;however,thereis noevidence fora causalrelationtochemicalEDCs,tothecon- trary:forexample,alargefollow-upstudyinmorethan1,300,000 Danishman foundnocorrelationbetweentesticularcancerand peri-nataloestrogenexposure(Ramlau-Hansenetal.,2009).Sim- ilarly,a review of81 epidemiology publicationsconcluded that thereisnostrongepidemiologicalevidencetoindicatethatprenatal exposuretooestrogenislinkedtodisturbeddevelopmentofthemale reproductiveorgans(Storgaardetal.,2006).Thisnotionisconsistent withobservationsinapopulationofmalesthatwereexposedin uterotomaternaldosesofDES,whichobservednomajorincrease intheincidenceoftesticularcancer(Learyetal.,1984).Overall,itis uncertainwhethertheTDSactuallyexistsandevenmoreuncertain thatsyntheticchemicalEDCsareassociatedwithit(Thorupetal., 2010;Safe,2005).

4. Personalcareproductingredients:purportedendocrine disruptors?

A series of substances in personal care products have been brandedasputativeEDC.Theyincludesolvents,suchasdiethyph- thalate,preservatives(long-chainparabens,triclosan),fragrances (polycyclicmusks),UVfilters(4-methylbenzylidenecamphor)or long-chainphthalateesters(WitorschandThomas,2010).Itistrue thatcertainlongside-chainphthalates,suchasdiethylhexyl-or butylphthalates,haveweakhormonalactivitiesandmayaffect male fertility in rats – when given at high oral doses. In con- trast,diethylphthalate,which hasbeenwidelyusedasavehicle infragrances,isdevoidofhormonalactivityorothersignificant toxicities.Diethylphthalatewasshowntobenon-toxicinalarge numberofsafetyevaluationsandwasratedtobesafeforusein cosmeticsbyinternationalexpertgroups,suchastheEUSCCSor theUSCosmeticIngredientReview(WitorschandThomas,2010).

However,thechemicalnamephthalatemadethissubstancetarget ofattacksbyNGOsduetoanallegedendocrinedisruptingactivity, inotherwords:healthriskassessmentbasedonthenamephthalate.

In2001,theUVfilter4-methylbenzylidenecamphor(4-MBC) wasreportedtobeactiveinvitroaswellasinvivo(uterotrophictest inimmaturefemalerats)screeningstudiesforoestrogenicactivity (Schlumpfetal.,2001).However,whenthesubstancewastested underconditionsofGoodLaboratoryPractice(GLP)inacomplete 1-generationreproductivetoxicitystudy,noadverseeffectswere found(Broschardetal.,2004).Therefore,perdefinition,4-MBCis notanED.Inaddition,anin-depthevaluationofthemetabolism of4-MBCinhumansandratsafterdermalapplicationaswellas atoxicokinetic-basedsafetyassessmentconcludedthatthesub- stanceposesnohealthriskforhumans(Schaueretal.,2006).

Longside-chainparabens,suchasbutylparaben,butnotmethyl- orethylparaben,havealsobeenreportedtopossessweakoestro- genicactivityinratswhengivensubcutaneously(butnotwhen givenorallyordermally)athighdoses(Routledgeetal.,1998).

Otherstudiesreportedthatrepeatedoraldosesofbutyl-andpropy- lparabenadverselyaffectedmalefertilityparametersinjuvenile rats(Oishi,2001).However,whenthelatterstudieswererepeated inlargerinvestigationsandunderconditionsofGoodLaboratory Practice,noeffectwasfoundatoraldosesofupto1000mg/kg/day suggestingthatpropyl-orbutylparabenshavenoEDactivity(Gazin etal.,2012;Hobermanetal.,2008).

Inaddition,ithasbeenshownthatwhole-bodyapplicationfor two weeks of a cream containing2.0% butylparaben(10 times themaximumconcentrationpermittedintheEUandappliedat 2mg/cm²)didnotaffectreproductiveandthyroidhormonelev- els in humans (Janjua et al., 2007).A study in 332 consumers (post-menopausalwomen)foundnopresenceofbutyl-orbenzyl parabensintheirplasma(Sandangeretal.,2011).Subsequently,the USCosmeticIngredientReviewreviewed thesafetyofparabens in-depth and concluded that all parabens are safe as used in personalcareproducts(CIR,2008).Incontrast,intheEU,themax- imal contentofpropyl-and butylparabensin PCPwerelimited toconcentrationsof0.2%.Inaddition,thereisgrowingevidence that parabens, when used in productsapplied to theskin, are hydrolysed(de-toxified) inhumanand animal skinresultingin para-hydroxybenzoicacid(Jewelletal.,2007;Aubertetal.,2012;

CIR,2008),anaturalsubstancethatisubiquitousinplants,veg- etablesandhumanfood.Para-hydroxybenzoicacidalsooccursin humanbreastmilkatconcentrationsofapproximately600␮g/kg, whereasinfantformulascontainupto3590␮g/kg(Lietal.,2009).

Takingintoaccountthatitisunlikelythatthehumanorganism issystemicallyexposedtoparabensafterapplicationofparaben- containingPCPproductstotheskin,thepotentialhumanhealthrisk fromparabensinPCPsshouldberatednegligibleorabsent.Inthis context,itisinterestingtonotethatmethylparabenisanatural, sex-attractantpheromoneinfemaledogs(Goodwinetal.,1979).

Thusitmaybearguedthat,atleastincanines,certainparabens furtherreproductionratherthanimpedeit.

5. Hormonalpotency:akeytopicofsafetyassessment

Thekeyquestiononthesafetyofsubstanceswithahormone- likeactivityistheirindividualhormonalpotency.Theimportance of potency of potential EDC for their humanand environmen- talsafetyassessmenthasbeenreviewedbyseveralauthorsand expertgroups(Calabreseetal.,1997;Borgertetal.,2013;Fegert, 2013;Testaietal.,2013;Dietrichetal.,2013).Toillustratepotency aspects:bothcyanideandtablesaltcanbetoxic,butcyanideisfar morepotentthantablesalt.Consequently,usingcommonsenseor riskmanagement,peopletendtobemorecautiouswhentheyhan- dlecyanidethanwhentheyusetablesalt.Inanalogy,oestradiol,the mammalianfemalesexhormoneorsyntheticoestrogenscontained inoralcontraceptivesareextremelypotentandactiveinmicro- gramdosesorwhenpresentinhumanbloodatng/mLlevels,i.e.in

Table1

Comparativeoestrogenicpotencyofnaturalorsyntheticsubstancesintherodentuterotrophicassayafteroraldoses(adaptedfromGoldenetal.,2005;Nilsson,2000;

WitorschandThomas,2010).

Substance Use/Origin Effectivedose(mg/kg/day) Relativepotency

Diethylstilbestrol(DES) Drug 0.0001 3,000,000

Ethinylestradiol Contraceptivepill 0.0003 1,000,000

Estrone Humanoestrogen 0.0012 250,000

Coumestrol Legumes(clover) 0.03 10,000

Genistein Soybeans 8 37

Daidzein Soybeans 12 25

4-MBC UVfilter 300 1.0

Butylparaben Preservative 600^a 0.5

Benzylparaben Preservative 2500 0.12

aSubcutaneousdoses,rats.

vivo.Indeed,naturalorsynthetichormones(e.g.ethinylestradiol) are10,000to1,000,000-foldmorepotentthanman-madechem- icalswithanoestrogenicactivity,suchaslong-chainparabensor ultravioletfilters(Goldenetal.,2005;Witorsch,2002a;Witorsch andThomas,2010;Table1).Thesedifferencesinpotencyareby severalordersofmagnitudehigherthanthosebetweenthetoxic- ityoftablesaltandcyanide.Whenimaginingthatoneoestrogenic potencyunitcorrespondstoonehorsepower,ethinyloestradiol (ingredientofthecontraceptivepill)wouldhavethepowerofa supertanker,coumestrol(clover)thatofabomber,genistein(soy beans)thatofasmallcar,whereasbutyl-orbenzylparabenwould beinthepowerrangeofthatofakitchenmixerorachildren’s bicycle,respectively(Fig.1).

Here it should be noted that butylparaben, although falsely brandedtobean“EndocrineDisrupter”,onlyshowedextremely weakpotentialforoestrogenicactivity(ratuterotrophictest)when administeredsubcutaneously(injectedundertheskin)atdosesof 800mg/kgandhigher(Routledgeetal.,1998).Thiswouldcorre- spondtoahumansubcutaneousdoseof48gramsina60kghuman beinginordertoproducea potentialactivity(correspondingto 50kgofacream,containing0.2%ofbutylparaben),nottomention thathumansarelesssensitivetosomehormonaleffectsthanrats (Borgertetal.,2012;Witorsch,2002b)).Whenbutylparabenwas givenorallytoratsorappliedtotheskinofratsithadnooestrogenic

Fig.1. Assumingthatoneoestrogenpotencyunit(seeTable1)correspondstoone horsepower,thepower/potencyofsomeofthesubstancesinTable1mayberanked asfollows.

activity(Routledgeetal.,1998).Inareproductivestudyinyoung malerats,butylparabenproducednoadversechanges inrepro- ductiveorgansorreproductivehormonelevelsatdosesexceeding 1000mg/kg/day(Hobermanetal.,2008).Consideringthis,itisbio- logicallyimplausiblehowbutylparaben,whenincludedat0.2%in acream,couldhaveanyeffectatallorevenposeahumanhealth risk.Overall,whentakingintoaccounttheirlimitedskinpenetra- tion,theirmetabolismintheskinand theirminute oestrogenic potencyofsubstancesusedascosmeticingredients,arisktohuman healthmaybeexcludedaltogether(Goldenetal.,2005;Witorsch andThomas,2010).Thisviewwasalsosupportedbytheresultsof biomonitoringstudiesonactualbloodlevelsofparabensinhuman consumers.Giventhatdetectedlevelswereabsentornegligible,a hormonaleffect,nottomentionahumanreproductiverisk,maybe clearlyexcluded(Sandangeretal.,2011).Ithasbeenarguedthat thepotencyofthemostactiveenvironmentaloestrogenswould needtobeatleast1000-timeshigherinordertopresenthuman reproductiverisks(Borgertetal.,2012).

Oestrogen-containingdrugs(e.g.thecontraceptivepill)orthe syntheticoestrogenDESpossesspotenciesthatareby6or7orders ofmagnitudehigherthanthatoflong-chainparabensorUVfil- terswith“estrogenicactivity” (Table 1; Fig.1).Yet,prenatalin uteroexposureofmentooestrogendrugsorDESdidnotadversely affecttheirfertility(Hemminkietal.,1998;Wilcoxetal.,1995;

Learyetal.,1984)orspermparameters(Schumacheretal.,1981).

ItisalsonoteworthythatDES,althoughitisseveraltimesmore potentthanoestradiol,appearstohaveano-effectlevel:human inuteroexposuretomaternaldosesofa totalof1.4gDESover 101days(approximately0.25mg/kg/day),ahugedoseinterms ofoestrogenicpotency,didnotproduceurogenitalabnormalities orabnormalspermparametersinmaleoffspring(Fish,2000).A no-effectlevelwasalsoobservedforethinyloestradiol(anactive ingredientofthecontraceptivepill):wheninjectedinadultmen athighdoses(60␮g/day)itaffectedspermmotilityanddensity;in contrast,20␮g/day,stillahugedoseintermsofhormonalpotency, hadnoeffectonspermmotilityanddensity(Lübbertetal.,1992).

Takingthesedataintoaccount,thehypothesisthatthenegligible exposureofhumanstochemicalsofnegligiblehormonalpotency couldhaveaneffectonhumanfertilityisabsurddefyingascientific basisaswellascommonsense.

6. Healthrisksofsimultaneousexposuretoseveral substanceswithweakhormone-likeactivities(“Cocktail Effects”)

Ithasbeenhypothesisedthat,althoughindividualman-made substancesgenerallyhaveaveryweakhormonalpotency,thecom- binationofseveralweaklyactingsubstancesmaybeadditiveor evensynergisticandtherebyyetproduceadverseeffectsonthe organism.Thishypothesishasbeenraisedasearlyas1996when Arnoldetal.(1996)claimedthatmixturesofseveralchemicalshad

synergisticpotencyonhumanoestrogenreceptors.Thesedatapro- ducedamajordebatebythescientificcommunityandthemedia (Anon., 1997).However,theresultscouldnot bereproducedin otherlaboratoriesbecausethedatawerefabricated,andthepre- viousarticlewaswithdrawnbytheauthors(McLachlan, 1997).

Recently,thehypothesisofapossibleadditionorsynergyoflow dosesofEDshasbeenraisedagain(Vandenbergetal.,2012;Zoeller etal.,2012).These,albeithypothetical,effectswerenamedLow- Dose Effects, Dose Addition of substances that produce Common AdverseOutcomes(DA-CAOS)orCocktailEffects.

However,theseeffectsarehighlyimprobable,ifnotimpossi- ble,onthebasisoftheoreticalaswellaspracticalconsiderations (Borgertetal.,2005,2012;RhombergandGoodman,2012).First of all, there is clear evidence that reproductive toxins includ- ingthosewithahormone-mediatedmechanismhaveathreshold of adversity (Piersma et al., 2011). To be additive,they would havetopossessexactlythesamemodeofaction(Borgertetal., 2005).Moreover,thereisamultitudeofdirectorindirectmech- anismsbywhichsubstances mayaffecthormonesor producea hormone-likeactivity.For example,substancesthat producean oestrogen-likeeffectmayhaveanaffinityfor cellularoestrogen receptorsoroestrogensub-receptors;theymayhaveagonisticor antagonisticproperties, theymay have oestrogenic, androgenic oranti-androgenicactivityortheymayproduceindirecteffects, suchasdirecttoxicityto,orstimulation/inhibitionofoestrogen- generatingtissues.Evenwhenthesametargetorganorhormonal systemisaffected,themodeofactionofadversitymaybequitedif- ferent:forexample,inrats,thereareamultitudeofmechanisms wherebysubstancesmayaffectthethyroidorthyroidhormonelev- els,althoughmanyofthesemechanismsarenotrelevantforman.

Inaddition,ithasbeenrecognisedthattheratisaninadequate modeltopredictadverseeffectsonthyroidhormonesofdrugsin man(WuandFarelly,2006).

Intheorganismtherearemanydifferenthormonesthatmayact ondifferentreceptorsorsub-receptors.Toillustratethis,noteven thenaturaloestrogenoestradiolandthepowerfulsyntheticoestro- genDESshareanidenticalmodeofaction(Safe,1998).Evenafter simultaneousuptakeofdifferentsubstancesthathavethesameora similarmodeofaction,thepossibilityofadditiveorcocktaileffects isreducedbydifferentabsorption,metabolismandkineticpath- ways,whichareneveridenticalfordifferentsubstances.Moreover, therearemajorquantitativeandqualitativedifferencesintheaffin- ityoractivitybetweenweakandstrongligands(substanceswith affinitytocellreceptors)tooroncellreceptors.Basedonphar- macologicalprinciples,weakligandswillonlyoccupyandtrigger cellreceptorswhenpresentathigh,near-toxicconcentrations,but theyhavenoeffectwhenpresentatsmallconcentrations.Over- all,thepresenceofmyriadsofweakhormonereceptoragonistsor antagonistsintheenvironmentandfoodisnotexpectedtoachieve physiologicalsignificance,sincemanywouldbecompetitiveago- nists/antagoniststheirlowpotencieswouldprecludeactivityvia thereceptor(Borgertetal.,2005,2012).

Thecomplexityoftheseinteractionshasbeendemonstratedin anumber ofstudies. Forexample, athighconcentrations(EC25

toEC₅₀)a mixtureof different-,anti-androgenicphthalates and bisphenol Ahad additive androgeniceffects onMDA-kb2 cells, whereas low concentration mixtures (<EC25) had antagonistic activity(Christenetal.,2012).Despitethefactthatdifferentmix- tureratiosneartheobservableresponserangecanbeinterpreted as concentrationadditive, there is no evidence of additivity at dosesintherangehumansmightbeexposed,andnotheoretical justificationforextrapolatingsuchdatatohumanexposurelev- els(Borgertetal.,2012).Asimilarphenomenonwasconfirmed inanotherstudyonmixtures ofweaklyestrogenicUVfilters in fish:mixtures containinghighconcentrations (EC₁₀ to EC₃₀)of individualUVfiltersshowedadditiveactivity,whereasmixtures

containinglowconcentrations(range:NOECtoEC₀₅)hadalower activitythanpredictedbythedoseadditionmodel, whichsug- gestsantagonism(KunzandFent,2009).Noadditiveeffectswere observedinratsafteroraladministrationofcombinationsofhigh dosesofethinylestradiol(apowerfuloestrogencontainedincon- traceptivepills),andgenistein,anoestrogenicisoflavonecontained insoy(Takagietal.,2004).Anadditiveeffectwasobservedwhen highdosesofgenisteinwerecombinedwiththerapeuticdosesof ethinylestradiol,whereaslowdosesshowednoadditivity(Charles etal.,2007).Thisobservationsuggeststhatadditivitymaybecon- strainedtosubstanceswithmoderatetohighpotencywhengiven neartheirindividualresponselevel,andwouldbeunlikelytooccur withsubstancespossessinglowpotencyorwhengivenatlowdoses (Borgertetal.,2012).Sincehumanexposuresaretypicallyorders ofmagnitudebelowtheobservableresponserange,mostmixture studiesarenotrelevanttohumanhealthrisks.

No evidence of synergy wasfound when weakly estrogenic hydroxylatedpolychlorinatedbiphenylsandpesticidesweretested incombination (Arcaroetal.,1998).Anotherlargestudyinves- tigatedbinarymixtures of six“endocrinedisrupting”chemicals (organochlorine compounds, phytoestrogens and actual hor- mones)that were administeredat five differentconcentrations to bobwhite quail; some combinations were additive, others wereantagonistic.Althoughgroupsizeswerelowandtherefore, conclusionsuncertain,noindicationsfor synergyweredetected (McMurryandDickerson,2001).

Anotherstudyinvestigatedoestrogenicresponsestomixtures ofsyntheticchemicalscombinedwithphytoestrogensatvarious doselevels.Aswouldbepredictedbytheoreticalpharmacologi- calconsiderations,low concentrationsofthesyntheticchemical mixturesfailedtoincreaseinvitroorinvivoestrogenicresponses relativetophytoestrogensalone.Significantlyincreasedresponses occurredonlywheneachsyntheticchemicalinthemixturewas nearoraboveitsindividualresponsethreshold.Invitro,highdoses ofchemicalsandphytoestrogensproducedgreaterthanadditive responses,whereasmixturesofsyntheticchemicalsproducedless thanadditiveresponsesintheabsenceofphytoestrogens.Invivo, thecombinedeffects wereconsistentwithadditivity.Thestudy concludedthat mixtureeffects are likely tobeof concernonly whenmixturecomponentsarepresentatorneartheirindividual responsethresholds,andthatextrapolationofmixtureeffectsfrom invitrotoinvivoshouldbeapproachedwithcaution(Charlesetal., 2007).

Similarly,in anotherstudy,rat uterotrophictestsona num- berofmixturesofsyntheticchemicals(nonylphenol,octylphenol,

␤-hexachlorocyclohexane,methoxychlor,bisphenolA,dibutylph- thalate) in combination with estradiol showed no additive or synergisticeffects,whereassomecombinationsofphytoestrogens (coumestrol,genistein,narigenin,catechin,epicatechin,quercetin) withestradiolactedadditively.Interestingly,allsyntheticchemi- cals,whentestedalonewereinactiveintheratuterotrophictest evenathighdoselevelsrelativetohumanexposure(VanMeeuven etal.,2007).

Theresultsof90differentstudiesonendocrineeffectsofchem- ical mixtures were recentlyreviewed by an expert group who concludedthatsynergyisextremelyrareandthattheamountof synergyatlowdoses,whenoccurring,didnotexceedthelevels predictedbyadditivemodelsbymorethanafactoroffour(Boobis etal.,2011).

Overall,thereisnogenuineinvivoevidenceforadditiveorcock- taileffectsofsmalldosesofman-made,chemicalsubstanceswith hormonalactivity.Takingintoaccountthefactthatanimalsand humansareexposedtothousandsof naturalhormone-likesub- stancesintheirfoodandenvironmentthat arecapableofovert hormone-relatedtoxicityathighlevelsofexposureinvitro,itisdif- ficulttoexplainhowthesespecieshavesurvivedifsuchsubstances

significantlyaddtoorsubtractfromendogenoushormonalactivity, whichisvitalforlife(Borgertetal.,2012).

7. Discussion:factsversusfears

Labelslike“Endocrinedisruptor”or“hormone-likesubstances”

arestigmaticterms;theysounddangerous,raisemediaattention andprovokehumanfears.Yet,intheabsenceofrelevanthuman exposureandpotencydata,thesetermsaremeaninglessinterms ofhumanhealthrisks.Overall,theentirediscussionwhetherman- madechemicals withhormone-like activitymaypose a riskto humanhealthhasa paradoxicalaspect:ifsuchactivities,how- ever small, could actually pose a potentialhealth risk, then it wouldmakesensetoworryaboutallsubstancesthatpossesssuch activities,particularlywhenpotentoestrogens,suchasthecontra- ceptivepill,aretakenorallyorwhentheyarepresentinhuman food,suchasphytoestrogens.Tothecontrary,anumberofepi- demiologystudiessuggest that thepotentcontraceptive pillor naturallyoccurringsoyisoflavonesorotherphytoestrogens,pose noornegligiblerisktohumanhealthorthatofhumanprogeny.

Thus,itisdifficulttoconceivehowsyntheticsubstancesthatare noteatenandpossessonlyatinyfractionoftheactivityofphar- maceuticalorsomenaturalsubstances couldbedangerous(see Fig.1).Hereitshouldbeconsideredthataveragehumansconsume about100␮gofoestrogenequivalentsadayfromnaturalsources (e.g.soyflavonoids),whereaschemicals,suchasbutylphthalate, in humanfoodamountto about0.02␮g oestrogenequivalents (Nilsson,2000).Yet,activistsandopportunisticandmedia-cited scientistsfocusonthattinynumber.Toputthesefiguresintoper- spective:asinglecontraceptivepillcontainsthestaggeringamount ofabout17,000␮gofoestrogenequivalents,reflectingthestriking potencyofgenuinehormones.

Scienceisaboutestablishingcauseand effect,itisnotabout guessing. Scientists develop a hypothesis – substance x causes observationy-andthenshouldrigorouslytestthehypothesisto determinewhetheritisvalidornot.Ifthehypothesisistestedrig- orouslyandcannotberefuted,itmustbetentativelyacceptedthat thehypothesismayberight(Taubes,2012).Ontheotherhand,if repeatedtestingfailstogenerateunequivocalsupport,thehypoth- esisshouldbeviewedwithscepticism.Letusputtheman-made environmentaldisruptor hypothesis to thetest:the hypothesis hasnowbeenevaluatedexperimentallyandepidemiologicallyfor nearly20yearsandnoconvincingevidencehasbeenfoundofan actualdeclineinhumanfertility,andevenlessofacausalrelation withsynthetichormonallyactivesubstances.

Thisraisesanotherimportantissue:epidemiologyattemptsto determinethecause(s)ofanestablisheddisease(Susser,1991).

Bacteria,virusesorexposuretotoxicsubstancesmaycausehuman diseases.Toillustratethis,inthe1950s,acausalrelationwasestab- lishedforlungcancerandtobaccosmoking.Indeed,lungcanceris agenuinediseasewithmeasurablefrequency.Itsincidencedra- maticallyincreasedinthe50s,whereascigarettesmokingbecame increasinglypopularintheprecedingdecades.Exposurewascer- tain,giventhattobaccosmokeisdirectlyinhaledintothelungs.

Thusthehypothesisforacausalrelationmadebiologicalsenseand causalitywasconfirmedbyanumberofsubsequentinvestigations thatinvolvedmillionsofsubjectsunequivocallyexposedtodirect inhalationoftobaccosmoke.Buthowcanonedetermineacause ofadiseasewhentheexistenceofthediseaseitselfisuncertain?

Forexample,theTesticulardysgenesissyndrome(TDS)ismerelya hypotheticaldisease,inotherwords:nobodyknowswhetherthis diseaseexistsornot-someexpertsinthefielddoubtwhetherTDS existsatall(Thorupetal.,2010).Scientificallyandphilosophically, thesearchforahypotheticalcauseofahypotheticaldiseasemakes nosense–woulditnotmakemoresensetofirstmakesurethat

thediseaseactuallyexists,beforespendingmillionsonthequest ofitscause?Withgoodreason,thequestforenvironmental,man- madeEDhasrightlybeentitledbytheEuropeanMolecularBiology OrganisationasACausewithoutaDisease(Breithaupt,2004).Never- theless,wearenowwitnessingtheadventofamassiveregulatory programmeinsearchofajustifiablepublichealthpurpose(Gori, 2007).Finally,evenwhena substanceisactiveinaninvitroor invivoEDassay,itisgenerallyverydifficulttoprovethattheeffect wasactuallycausedbyanendocrinemechanism,sinceaconcomi- tanteffectonendocrinefunctiononitsownisnotproofofcausality perse.Asanexample,manysubstancesmayaffecttheratthyroid andratthyroidhormonesbyanumberofdiversemodesofaction manyofwhicharenotendocrine-relevant(WuandFarelly,2006).

Thisposesanadditional,yetunresolved,problemofhowtoprove thatasubstanceisanEDinactualpractice.

Thehypothesisofandsubsequentsearchforman-made(syn- thetic),chemicalEDs intheenvironment,foodorpersonalcare productsbeganintheearly1990s.Uptodate,this researchhas spenthundredsofmillionsofEurosorDollarsoftaxpayer’smoney.

IntheEUalone,morethan150millionEuroshavebeenspenton researchintopotentialhealthrisksofEDCs(JensenandvanVliet, 2012).Giventhislargeamountofresearchfundingtheremayalso beavested interestofscientists intheEDfieldtokeep theED hypothesisontheagendainordertostayinbusiness.

InJanuary2013,asearchinTOXLINEfortheterm“endocrinedis- ruptors”yielded4278differentarticles(TOXLINE,2013)reflecting theenormousamountofscientificresearchdevotedtothistopic.

Asearchin MEDLINEfor theterm“endocrinedisruption”listed 33annualpublicationsin1992withanincreaseto290in2011 (MEDLINE,2013).Takingintoaccountthelargeresourcesspent onthistopic,oneshouldexpectthat,inthemeantime,someEDs thatcauseactualhumaninjuryordiseaseshouldhavebeeniden- tified.However,thisisnotthecase.Todate,withtheexceptionof naturalorsynthetichormones,notasingle,man-madechemical EDhasbeenidentifiedthatposesanidentifiable,measurablerisk tohumanhealth(theadverseeffectsofiatrogenicDESwerelong knownbeforethetermendocrinedisruptorwascoined).Certainly, therehasbeenmuchmediahypeaboutimaginaryhealthrisksfrom bisphenolA,parabensorphthalates.However,noactualevidence ofadversehumanhealtheffectsfromthesesubstanceshasever beenestablished.Tothecontrary,thereisincreasingevidencethat theirhealthrisksareabsentornegligible–orimaginary.Itisinter- estingtonotethatevensubstances,suchaslong-chainphthalates, whichwereconfirmedtobeEDs(anti-androgenic)intherat,were largelyinactiveinthemouseorinvivoexsituhumanfoetaltestis (Johnsonetal.,2012).Giventhatthehumanfoetusappearstobeat leastanorderofmagnitudelesssensitivetoDES-inducedreproduc- tivetractmalformationsthantheratfoetus(Borgertetal.,2012), suchspeciesdifferencesraisedoubtastotherelevanceofcurrent ratinvivotestdataforhumanswho,inaddition,areexposedenvi- ronmentallytofarlowerlevelsofsuchsubstancesthanlaboratory rats.

The largest human experiment on endocrine disruption includedmorethanabillionwomenandhascontinuedforalmost halfacentury:thecontraceptivepill.However,thecontraceptive pilldoes nosignificantharmto humanhealth, although it has a powerfuloestrogenic activity,is taken dailyand is usedover longperiodsoftime.It hasbeenestimatedthatabout500Mil- lionwomenintheEUandtheUSannuallyuseoralcontraceptives with approximately5% or 25 million women per year becom- ing pregnantand unknowinglyexpose theirfoetuses to potent oestrogenicandprogestindrugs(Takeda,2001).Yet,noadverse effectswereobservedinmenorwomenafterpreviousinutero exposuretooestrogen-containingdrugs(Hemminkietal.,1998;

Storgaardetal.,2006).ThecontraceptivepillisagenuineEndocrine Disruptor:itaffectshumanreproductivephysiologyandcontains

oestradiols(naturalorsynthetic)that aretoxic toreproduction whengivenathighdoses.Humanurinaryexcretionofresiduesof thecontraceptivepillaswellasnaturalhumanandanimaloestro- gensinwatermayalsoaffecttheenvironmentandarethought toberesponsibleforthefeminisationofmalefishthathasbeen observedinlargeriversdownstreamtheeffluentwaterofmajor citiesormassproductionoffarmanimals(Vajdaetal.,2011;Jobling etal.,2006).Interestingly,thecontraceptivepillhasrarely,ifever, beenatargetoftheEDC-lobby.Itisoutsidethescopeofthispaper tospeculateaboutthereasons.Possibly,focusingonchemicals,and notpharmaceuticalsprovidestheemotionalargumentthatthese areeverywhereandeverybodyisexposedorattackingthecompar- ativelytinyoestrogenicactivityofsomechemicalsthatproduceno ornegligiblehumanexposuremaybemorepoliticallyacceptable thanblamingthepill.Chemicalsubstancesare,afterall,perceived toposeahumanandenvironmentalhealthrisk,whetherthisis supportedbyscientificfactsornot.Blamingthecontraceptivepill foradverseenvironmentaleffectsiscertainlylessopportune.

Itisnotsurprisingthat,whenitwasrealisedthatitisimplausible thathormonallyactivechemicalsposeahumanhealthriskgiven thesmall humanexposure and theirnegligible potencies, new hypotheseshadtobeinventedinsupportofthenotionthatthey poseariskanyway,suchastheDoseAdditionofsubstancesthatpro- duceCommonAdverseOutcomes(DA-CAOS)(KortenkampandFaust, 2010)orCocktailEffects(Vandenbergetal.,2012).Despiteofheroic effortstofindinvivoobservationalvalidationforthesehypotheses, theinvivoevidencecontinuestoaccumulatethattheseeffectsare absentatlowdoses/concentrations,whichisconsistentwithphar- macologicaltheory(Borgertetal.,2012;RhombergandGoodman, 2012).Anotherinterestinghypothesisisthenon-monotonicdose- relationship,whichpostulatesthat,forEDs,high-doseeffectsmay benon-predictiveforeffectsobservedforlowdoses(Vandenberg etal.,2012;Myersetal.,2009).Althoughthishypothesisiscon- sistentwiththeideasofhomoeopathy,itcontradictscenturiesof toxicologicalandpharmacologicalexperiencedemonstratingthat activesubstancesproduceaspecificdose-responseintheaffected organism.Therearesomeexceptionstothisrule,althoughthey aregenerallyduetodifferentmechanismsoftherespectivesub- stance,suchashypervitaminosisAversusvitamin Adeficiency, bothofwhichcanbeteratogenic.Althoughnon-monotonicdose response curves for hormonaleffects had been postulated in a limitednumberof studies,thedatacouldnotbereproducedin largerin vivo studiesthat includedadequate qualityassurance (Dekant andColnot, 2013;Dietrich etal., 2013).Here aninter- estingpointmayberaised:ifnon-monotonicdose-relationships wererelevanttothesafetyassessmentofman-made,chemicalEDs, surelythesameconceptshouldalsoapplytothecontraceptivepill orphytoestrogens:accordingly,couldasingleChinesemealora cupofcoffeewreakhavocwithourendocrinesystems?Doesthis assumptionappearrealistic?

Inourview,theEDCsagamaybeapolitical,ratherthanasci- entificproblem.Paradoxically,todaysoyoestrogens(isoflavones) are marketed in health food stores with the claim that they relievemenopausalsymptoms.Yet,womeninmenopausehavean increasedriskofbreastcancer,whereasisoflavoneshaveapotential topromotemammaryandendometrialcarcinogenesis(Kakehashi etal.,2012).Soy-phytoestrogenshavealsobeenreportedtoinhibit theactivityofanti-cancerdrugs,whichmaybeofparticularcon- cernforwomen withoestrogen-dependent breastcancer(Gallo etal.,2007).Inrats,inuteroexposuretohighmaternaldosesofthe soyphytoestrogensgenisteinhasbeenshowntoadverselyaffect reproductiveparametersintheirmaleandfemaleoffspring;thus soyisoflavonesaregenuineEDs(McClainetal.,2007;Delclosetal., 2001).Atdietarylevels,however,phytoestrogensdonotappearto induceoestrogeniceffectsontheuterusofhumansornon-human primates(Clineet al.,2001).Anotherinterestingexample ofan

endocrine-activesubstanceiscaffeine:caffeinewasembryo-and foeto-toxicinratreproductionanddevelopmentaltoxicitystud- ies,itaffectedspermqualityinmice,increasedtheincidenceof tumoursinendocrineorgans(pituitaryadenomasandmammary tumoursinmiceandrats),andwaspositiveinaninvitrosteroido- genesisassay.Inaddition,themarginofexposureforhumancoffee consumers(meandailyintake:240mgcaffeine)relativetoadverse effectsobservedinanimalsisaslowas15-to35-fold(Barsetal., 2012).Veryrecently,anotherseriesofsubstances(vitaminsC,B9, B6,B3,sucrose,caffeine,gingerol,xanthanegum,paracetamoland ibuprofen)weretestedinaseriesofinvitroassaysforendocrine activity. Paracetamol, gingerol, caffeine and vitamin C affected stereoidogenesisinvitrofrom250,25,500and750␮M,respec- tively.Caffeine,whentestedinvivo(ratpubertalassay)atdose levelsrelevantforhumanconsumptionaffectedvaginalopening, oestruscycleandovarianweightinfemalesaswellasplasmapro- gesteronelevels,prostateandseminalweightsinmales(Tinwell etal.,2013).Surely,bytoday’sstandards,caffeinewouldperfectly qualifyasagenuineEDatdoselevelsrelevanttohumanexposure.

Today,ahugenumberofcellularandnuclearreceptorsandsub- receptorsareknown,manyof whichrespondtohormones.For example,theInternationalUniononBasicandClinicalPharmacol- ogy(IUPHAR)hasestablishedadatabasethatincludes3500ligand classes(IUPHAR,2013).Takingintoaccounttheubiquitouspres- enceofhormonesintheorganismandtheirroleinitsregulation onacellularlevel(OECDDRP,2012;GodmanandGilman,2011), onecouldaskaprovocativequestion:wouldnotmostsubstances, whenadministeredatdosessufficientlyhightoaffectatargetorgan ultimatelydisplayanendocrinedisruptingpotentialsomewherein theorganism?Forexample,itisknownthattablesaltintakeand subsequentchangesinsodiumplasmaconcentrationsmayaffect renalhormoneswithsubsequenteffectonbloodpressure(Isozaki etal.,1995).Tablesaltmayalsointeractwiththeactivityoforal contraceptives(Pechère-Bertschietal.,2003).Waterintakemay affecthumanadrenal,renalandhepatichormones,suchasaldos- terone,reninorangiotensinlevels(Testaietal.,2013).Wouldthis maketablesaltorwaterpotentialEDs?Inotherwords,themajority ofknownsubstancescouldpossiblybeclassifiedaspotentialEDs whentestedindepth.Yet,wouldsuchaclassificationcontributeto ahumanhealthbenefit?

Paradoxically,oursocietyanditsregulatorsappeartobequite toleranttowardspotentsubstancesthat areconfirmedEDs and aretakenorallyby alargenumber ofhumans.These examples showhowthefocusonpurportedman-made,endocrinedisrupting chemicals(EDCs)distractsfrompotentialhealthrisksofhuman exposuretoothersubstanceswithfargreaterpotentialforhor- monalactivity.Perhapstheentireissueofpurportedhealthrisksof chemicalEDsisjustanotherversionofthetrivialdichotomynatural isgoodversusman-madeisbad.

Finally,whenreflectingonthepresenceofalltheseman-made, albeithypothetical,EDCsin theenvironment,whyshouldEDCs primarilyaffectthemalesex,suchasapotentialdeclineinmalefer- tility,ahypotheticalincreaseintheincidenceofhypospadiasand cryptorchidisminmalebabies,increaseintheincidenceofmaletes- ticularcancers,areducedpenislengthinmalealligators(Guilette etal.,1996),orcausefeminisationofmalefish?Whyshouldfemales belessaffected?Arefemaleslesssusceptibleand,ifso,why?Onthe otherhand,couldtherebeamalesex-biasconcerningtheseimag- inaryrisks?Recently,ithasbeenclaimedthatboysfrommothers withhigherphthalatelevelsintheirurineshowedareducedten- dencyformasculineplaybehaviour,suchapreferenceoftoysover masculineplay. Interestingly,girlsseemedunaffectedbymater- nalphthalatelevels(Swanetal.,2009).Trueornot,mothersmay ratherberelievedaboutreducedmasculineplaybehaviouroftheir sons.However,somefatherswouldprobablybemoreconcerned aboutsuchdata:myboyshowingun-masculine,i.e.feminisedplay