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Toxicology 314 (2013) 51– 59

Contents lists available at ScienceDirect

Toxicology

jo u r n al homep age: www.elsev ier .com/ locate / tox ico l

plea for risk assessment of endocrine disrupting chemicals

manuela Testaia, Corrado L. Galli b,∗, Wolfgang Dekantc, Marina Marinovichb,ldert H. Piersmad, Richard M. Sharpee

Istituto Superiore di Sanità, Department of Environment and Primary Prevention, Mechanisms of Toxicity Unit, Viale Regina Elena, 299, Rome, ItalyUniversity of Milan, Department of Pharmacological and Biomolecular Sciences, Faculty of Pharmaceutical Sciences, Via Balzaretti 9, Milan, ItalyUniversität Würzburg, Versbacherstr 9, 97078 Würzburg, GermanyNational Institute for Public Health and the Environment RIVM, Laboratory for Health Protection Research, Antonie van Leeuwenhoeklaan 9, Bilthoven,he NetherlandsMRC Centre for Reproductive Health, The Queen’s Medical Research Institute, University of Edinburgh, 47 Little France Crescent, Edinburgh EH16 4TJ,cotland, UK

r t i c l e i n f o

rticle history:eceived 29 July 2013vailable online 11 August 2013

eywords:ndocrine active substances (EASs)ndocrine disrupters (EDs)onotonic dose–response

a b s t r a c t

Some recent EU Regulations have focused on the potential risks posed by the presence of endocrine dis-rupters (ED) into the environment. However there are conflicting opinions on how to assess the risk fromexposure to these molecules that can reversibly modulate hormonal activity, endocrine active substances(EAS) rather than causing irreversible damage (ED).

The present paper attempts to discuss that perturbation of normal endocrine homeostasis in itselfmay not be an adverse effect, since the endocrine system is naturally dynamic and responsive to variousstimuli as part of its normal function and it is modulated according to the characteristic trend of the

isk assessmentxposureegulatory decisions

dose–response curve.EDs should be evaluated using a weight-of-evidence (WoE) approach. If a chemical meets the criteria

to be defined as an ED in experimental animals, the relevance of observed effects to the human thenneeds to be addressed.

Hazard-based risk management is therefore not justified since does not meet the criteria for a soundscientifically based assessment.

. Introduction

In recent years, following evidence that exposure to certainnvironmental chemicals has led to interference with endocrineunction in a number of wildlife species, such as molluscs, crus-acean, fish, and birds in various parts of the world, concernas been expressed by the scientific community that comparableffects might also affect human health, as endocrine systems areargely conserved.

The endocrine system (as described in WHO/UNEP, 2012;HO/IPCS, 2002) is a complex network of glands, hormones

nd receptors, playing a crucial role in maintaining the physio-

ogical equilibrium of the human body as well as in regulatingody growth, metabolism and sexual development and function.he endocrine system acts through chemical messengers, the

∗ Corresponding author. Tel.: +39 025031 8315.E-mail addresses: emanuela.testai@iss.it (E. Testai), corrado.galli@unimi.it,

orradolodovico.galli@gmail.com (C.L. Galli), dekant@toxi.uni-wuerzburg.deW. Dekant), marina.marinovich@unimi.it (M. Marinovich), aldert.piersma@rivm.nlA.H. Piersma), r.sharpe@ed.ac.uk (R.M. Sharpe).

300-483X/$ – see front matter © 2013 Elsevier Ireland Ltd. All rights reserved.ttp://dx.doi.org/10.1016/j.tox.2013.07.018

© 2013 Elsevier Ireland Ltd. All rights reserved.

hormones, which are secreted by endocrine glands into the blood(or other extracellular fluids) reaching all parts of the body, andmodulating cellular or organ functions by binding to receptors intarget cells. Once the receptors are activated, they may interactwith DNA or other intracellular signalling processes, to controland coordinate physiological processes within the body. Theendocrine systems operate largely on a ‘push–pull’ system thatis characterized by compensatory feedback mechanisms, thusproviding homeostatic capacity, which is adaptive (Kortenkampet al., 2011; Goodman and Gilmans, 2013).

Chemicals can interfere with the functioning of the complexendocrine network. Some interactions result in homeostaticresponses only and are therefore physiological and non-adverse,whilst others are crossing what has been named the “threshold ofadversity” (Piersma et al., 2011), overwhelming the homeostaticcontrol, leading to adverse health effects. The term ‘endocrinedisrupter (ED)’ was first used at the Wingspread Conference in1991, and referred to those endocrine active substances which

may lead to an adverse health effect (Colburn and Clement, 1992).Distinct from ED, the Scientific Committee of EFSA in 2010 defined‘endocrine active substance (EAS)’ as “any chemical that caninteract directly or indirectly with the endocrine system, and

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ubsequently result in an effect on the endocrine system, targetrgans and tissues” (EFSA, 2010a,b). In other words, an EAS in oner more ways interferes with the endocrine system, producingn effect which, thanks to the above mentioned compensatoryeedback mechanisms, may prove to be temporary, if appropriateorrection occurs due to the homeostatic capacity of the endocrineystem (endocrine modulation or adaptive effect). However,oncern about EAS/ED has focused mostly on ‘critical periods’ inife when homeostatic function is not yet operational or is beingstablished, for example during foetal development, as effects athis time, may be uncorrected or have long-lasting consequenceslthough maternal homeostatic control is an important gatekeeperf hazardous foetal exposures. One example would be any com-ound that reduced androgen production/action in a male foetusuring the masculinization period, as any resulting deficits inasculinization cannot be corrected at a later time point.EFSA’s Scientific Committee has been requested by the Com-

ission to prepare an opinion related to potential hazards due toD, which has been adopted in March 2013 (EFSA, 2013). The WG,ppointed to prepare the draft, included also members from otherU agencies (i.e. the European Medicines Agency, the Europeanhemicals Agency, the European Environment Agency, the Euro-ean Commission’s Scientific Committees and the Joint Researchentre), which therefore have been asked to contribute as well.

n addition, in the last few years, the European Commission hasublished in 2012 the report on “State of the Art Assessmentf Endocrine Disrupters” (Kortenkamp et al., 2011), and works ongoing at the Organization for Economic Co-operation andevelopment as well as at US EPA (having established a specificrogramme named EDTA) with the organization of a number ofeetings and workshops on the issue. Very recently, a WHO report

WHO/UNEP, 2012) analyzed in detail the issue of endocrine dis-uption in humans, animal experimental models and wildlife, inhich the IPCS definition for ED (see below) has been still consid-

red as the most appropriate.Chemicals defined as EAS are not limited to man-made chemi-

als and by-products released into the environment, such as someesticides (e.g. DDT and other chlorinated compounds) or industrialhemicals (e.g. polychlorinated biphenyls, dioxins). Some synthet-cally produced pharmaceuticals (e.g. the contraceptive pill andreatments for hormone-responsive cancers) have been designedo be highly hormonally active (similarly or even higher than phys-ological hormones, which can be considered as the archetypicalAS) so as to exert their therapeutic action; they can in turn beeleased into the environment via sewage effluent, having effectsn environmental species. In addition, a number of natural chem-cals produced by plants and fungi are EAS, called phytoestrogens,uch as genistein, coumestrol or isoflavones in soya, and expo-ure to these occurs with comparably high doses (Safe, 1995) viaood or supplements. They may behave similarly to human hor-

ones or influence hormone levels in the body, triggering theomeostatic mechanisms of the body and thus possibly havingffects on human health and organisms in the environment, par-icularly at critical stages of development. As an example, it isnown that in rats genistein (contained in soy milk and a num-er of Asian foods, among others) can result in endocrine effectst human exposure levels and has been advocated for use in post-enopausal hormone therapy, although with very mixed results

egarding efficacy (Delclos et al., 2001, 2009; Latendresse et al.,009).

The EAS, which may belong to a variety of chemical classes,an exert their effects through a number of different mechanisms.

hey may mimic the biological activity of a hormone by bindingo the specific cellular receptor (agonistic effect), thereby settingff similar chemical reactions in the body, or they can bind tohe receptor without activating it (antagonistic effect), thereby

y 314 (2013) 51– 59

preventing or diminishing the action of the corresponding endoge-nous hormone. Alternatively, they can interfere with the kineticsof the natural hormones, thus altering their concentration by: (i)binding to specific transport proteins in the blood or located inthe membrane of target cells, thus altering levels of the naturalhormone in the circulation or inside the cells, or (ii) interferingwith the metabolic processes, through inhibition/induction of theenzymes normally involved in the synthesis or breakdown of thenatural hormone. Although attention has been mainly focusedon receptor mediated EAS, examples such as some phthalatesclearly indicate that environmental chemicals with the potentialto alter endogenous hormone production or metabolism maypose a greater risk than weak agonists or antagonists of hormonalreceptors (Sharpe and Irvine, 2004). For both types of mechanismthe importance of exposure level, ADME parameters and specificsusceptibility should never be forgotten. For example, some PCBsare potent inhibitors of sulphotransferases (Liu et al., 2009; Ekuaseet al., 2011), which conjugates estradiol before it is excreted. Suchan inhibition can prolong the actions of endogenous oestrogen, achange potentially relevant for breast cancer.

2. ‘Endocrine disrupter’: effect or adverse effect?

Since EAS exposure may or may not result in endocrine disrup-tion, it seems crucial in a regulatory context to define exactly whatis meant by the term endocrine disrupter (ED).

A number of definitions for EDs have been proposed during thepast two decades:

• “An ED is an exogenous agent that interferes with the produc-tion, release, transport, metabolism, binding, action or eliminationof natural hormones in the body responsible for the maintenanceof homeostasis and the regulation of developmental processes.”(Kavlock et al., 1996) endorsed by US EPA.

• “An ED is an exogenous substance that causes adverse health effectsin an intact organism, or its progeny, secondary to changes inendocrine function. A potential ED is a substance that possesses prop-erties that might be expected to lead to endocrine disruption in anintact organism.” (EC-Weybridge, 1996)

• “EDs are synthetic chemicals that when absorbed into the bodyeither mimic or block hormones and disrupt the body’s normalfunctions through altering hormone levels, halting or stimulatingthe production of hormones, or changing the way hormones travelthrough the body.” (NRDC, 1998)

• EDSTAC describes an endocrine disrupter as an exogenous chem-ical substance or mixture that alters the structure or function(s)of the endocrine system and causes adverse effects at the level ofthe organism, its progeny, populations, or subpopulations of orga-nisms, based on scientific principles, data, weight-of-evidence, andthe precautionary principle. (EDSTAC, 1998)

• “An ED is an exogenous substance or mixture that alters function(s) ofthe endocrine system and consequently causes adverse effects in anintact organism, or its progeny, or (sub)populations.”(WHO/IPCS,2002)

• “An ED is an exogenous chemical, or mixture of chemicals, that inter-feres with any aspect of hormone action” (Zoeller et al., 2012),endorsed by the Endocrine Society

The US EPA has further detailed its position on adversity and EDstating to “. . .not consider endocrine disruption to be an adverse effect

per se, but rather to be a mode or mechanism of action potentiallyleading to other outcomes, for example carcinogenic, reproductive,or developmental effects, routinely considered in reaching regulatorydecisions”.

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For regulatory purposes, some definitions are ambiguous sincehey fail to discriminate between alterations of the endocrineystem which fall within the range of physiological variation ofesponses (endocrine modulation or adaptive responses) and alter-tions that either overwhelm the homeostatic equilibrium or whichccur in the absence of a homeostatic process, thus leading ton adverse effect. Adaptive responses can be considered as part ofhe normal function of the organism, and the endocrine system isot an exception. A perturbation of normal endocrine homeostasis

n itself may not be an adverse effect, since the endocrine systems naturally dynamic and responsive to various stimuli as part of itsormal function and it is modulated according to the characteristicrend of the dose–response curve.

In this context, endocrine perturbation, as highlighted by EPAnd taken on board by WHO/IPCS, can be considered as a mode ofction (MoA), potentially leading to other outcomes, rather than aoxicological endpoint in itself (BfR, 2011; Kortenkamp et al., 2011;

arx-Stoelting et al., 2011). Nevertheless, chronic perturbation of mature endocrine system (due to chronic exposure to a com-ound(s)) so as to trigger chronic adaptation, can potentially leado long-term adverse effects; an example would be the triggering ofeydig cell tumours in rats via compounds that chronically loweredestosterone production by these cells.

The Authors endorse the WHO/IPCS (2002) scientific definitionf an ED, as well as the EPA consideration of this phenomenon as aoA.The WHO/IPCS definition, besides considering both human and

cosystem health, by adding the concept of effects on (sub) popu-ations, points out two aspects relevant for regulatory purposes:nduction of adverse effects in an intact organism, consequent toxposure.

Again, with reference to WHO/IPCS, its general definition ofdverse effect (that is not specific for EDs but usable for them too)s the following: “a change in the morphology, physiology, growth,evelopment, reproduction, or life span of an organism, system, orsub)population that results in an impairment of functional capacity,n impairment of the capacity to compensate for additional stress, orn increase in susceptibility to other influences” (WHO/IPCS, 2004).

The difference between endocrine activity and endocrine dis-uption is determined by the plasticity of the endocrine systemnd its limits of compensatory properties neutralizing potentiallyazardous exposures and maintaining homeostasis.

Many adaptive, compensatory, and physiological processesecessary for the correct functioning of an organism result ineasurable endocrine changes: this is the case for reactions to

ormal eating (e.g. insulin response to eating a sugar-rich food),o emergency situations (stress, infection), or the basis for sexualehaviour. It is only when these natural mechanisms are affectedy endogenous and/or exogenous factors to such a degree that therganism cannot compensate, that adverse effects are induced dueo an unwarranted response at the wrong time or to an excessiver chronic extent, and as endocrinology textbooks show, resultingn a disturbance of hormonal homeostasis.

In this respect, it is noteworthy to consider that, in order toffect the mature endocrine system, exogenous chemicals must actgainst the background of circulating levels of endogenous hor-ones, which are usually considerably more potent than any ED.

otency is an important aspect which has to be taken into accounto evaluate the relevance of environmental exposure to ED forumans. In relation to the affinity for the oestrogen receptor andhe estrogenic induction of male reproductive disorders in rats, itas been evaluated that ‘based on estrogenic potency, human expo-

ure to the most potent environmental estrogens would need to bet least 1000-fold higher than this level, for adverse effects relevanto the human male to be induced, and such levels of exposure areemote (Sharpe, 2003). In other words, environmental estrogens

y 314 (2013) 51– 59 53

are ≥3 orders of magnitude weaker than the body’s endogenoushormones; for example, nonylphenol (an alkylphenol ethoxylatesurfactant found as a low level contaminant in some rivers inEurope), has an estrogenic activity only about 1/105 than that of17�-estradiol (E2; Jobling and Sumpter, 1993; Flouriot et al., 1995).

In addition, the external exposure to natural chemicals suchas some estrogenic isoflavones contained in human food (Courantet al., 2007, 2008) can be considerably higher than industrial chem-icals with endocrine activity, beside the fact that isoflavones are upto two orders of magnitude more potent estrogenically than knownenvironmental EDs and have a longer half-life (Safe, 2013).

Among the exposed population, individuals (or groups of them)may have different susceptibilities to ED for several reasons (e.g.developmental stage, age, patho/physiological or nutritional sta-tus), so adversity cannot be presumed or established from thehazard alone, but a complete risk assessment approach integratinginformation on the potency of the chemical, the exposure and thesusceptibility of the exposed population is necessary. This is statedalso in the National Research Council of the US National Academiesreport on toxicity testing in the 21st century (NRC, 2007), whichreported that ‘the consequences of a biologic perturbation depend onits magnitude, which is related to the dose, the timing and duration ofthe perturbation, and the susceptibility of the host’.

The reference of the WHO definition to the intact organism indi-cates not only that in vivo tests are, at least for the time being,necessary to identify an ED, but also the limited relevance of invivo ‘manipulated’ animal models such as castrated or ovariec-tomized animals (as used in the uterotrophic assay). Because theseanimals have lost integrity of their hypothalamic–pituitary–gonad(HPG) endocrine axis, the normal dynamic physiological capacityto compensate for the actions of external hormonal activity hasbeen compromised; whilst useful for identifying compounds withrelevant hormonal/anti-hormonal activity, such artificial systemshave limited utility in evaluating risk of the compound in ques-tion. To date, considering the tests available, evidence coming fromin vitro assays as stand alone testing cannot be considered sufficientfor the identification of ED, but, once better characterized for thequalitative and quantitative hormonal response, might be useful toidentify the ‘potential’ for interaction with the endocrine systems(EAS definition) and therefore to set priority lists for further testing.

Finally, a cause-effect relationship should be demonstratedbetween endocrine activity and an adverse health effect, as wit-nessed by the use of the word consequent in the WHO definition.

General criteria to establish causality between endocrine modu-lation and adverse effects are not available; our existing regulatorytoxicological tests are generally unable to show causal linksbetween disruption of an endocrine mechanism and an adverseeffect. Simple co-occurrence is not proof of causality, and opinionson this aspect vary widely. At present, the toxicological relevanceof such changes is left to expert judgement, which is applied, on acase-by-case basis.

Therefore, in conclusion there are clearly specific requirementsfor a substance to be defined as an ED: (1) the demonstration ofan adverse effect in an intact organism, or the high likelihood ofthis occurring; (2) an endocrine disruption mode-of-action (bio-logical plausibility); and (3) a causal link between the endocrineactivity and the adverse effect (strength of association).

This last consideration also points towards the specificity of theED action. ED-induced adverse effects should occur in the absenceof generalized ‘overt’ toxicity, as otherwise they could represent anonspecific response, secondary to the primary toxic effect inducedby the chemical, rather than a genuine, primary ED effect. Therefore,

ED induced adverse effects should be considered relevant for a reg-ulatory purpose and to be used for the derivation of reference valueswhen the specific requirements quoted above are satisfied. Exceptunder certain circumstances (see below), there is no special reason

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or there being any difference between the level of evidence (hazardharacterization) needed to demonstrate an endocrine mediateddverse health effect and that used to demonstrate any other kindf adverse effect.

Regarding the testing strategy available to identify ED, EPA haseveloped the Endocrine Disrupter Screening Programme (EDSP)o determine whether certain substances may have intrinsic EDctivity with the potential to affect humans; information on thisan be obtained from the EPA website.

Not limited to the US situation, according to the definitions reported above, the Organization for Economic Co-operationnd Development (OECD) has developed a conceptual frameworkor assessing EDs (OECD, 2002, 2010a, 2010b), consisting of fiveevels of information, of increasing complexity, on the basis of

hich it should be possible to draw conclusions on endocrineroperties. Level 1 information (not necessarily coming from exper-

mental sources, but including existing information and in silicopproaches) is useful for prioritization. Level 2 contains studiesgenerally in vitro) from which initial information on the abilityf a chemical to interact with the endocrine system are obtained.n vitro models to check possible interaction with hormone recep-ors are available, indicating potential interference with the activityf endogenous hormones. However, as stated above, results fromhese studies can be used only as indicators for potential endocrine-

ediated effects and cannot be used in isolation, since theseystems lack toxicokinetics information, which will determine theose of the compound at the receptor site. Level 3 includes short-erm in vivo screens for specific single level effects on the endocrineystem, indicative of the potential for a response in an intact organ-sm and not necessarily of an adverse effect (Karbe et al., 2002). The

echanistic studies in level 2 and 3 are relevant for hazard identifi-ation: if results do not indicate any potential for endocrine activity,o further testing is needed. Positive results can be used for priori-ization for further testing with in vivo studies on apical endpointsncluded in levels 4 and 5, providing the required information forazard characterization (i.e. adverse effects induced, the relativeotency and the consequent functional and pathological disrup-ion that it causes) to be used in risk assessment. OECD prepared auidance Document, in which it is clearly stated that data on EDshould be evaluated using a weight-of-evidence (WoE) approachOECD, 2012). According to the ED definition, both mechanisticnd apical information describing the type of interaction with thendocrine system, as well as the consequent adverse effects, areeeded; therefore, a combination of tests rather than a single assayhould be available. All the available studies (both positive and neg-tive) should be evaluated, but their quality and reliability has toe carefully assessed in order to give them the correct weight inhe WoE approach.

In following the OECD conceptual framework, if a chemicaleets the criteria to be defined as an ED in experimental ani-als, the relevance of observed effects to the human then needs

o be addressed (Boobis et al., 2008, 2009; Carmichael et al., 2011).his should follow established human relevant frameworks. Forxample, species-specific differences in toxicokinetics and bio-ransformation may be relevant (see the case of BPA, EFSA, 2010a;ang et al., 2013) and in addition the endocrine system of smallodents used for hazard identification differs in a number ofespects from that in humans (Goodman et al., 2006; Witorsch,002), mainly due to differences in endocrine signalling across ani-al species. The following are examples:

(i) Circulating oestrogen concentrations during pregnancy are

∼100 times lower in mice than in women and for that rea-son pregnant mice may be far more susceptible than pregnantwomen to the adverse effects of compounds that either ele-vate or decrease oestrogen activity. In addition, the source of

y 314 (2013) 51– 59

pregnancy oestrogen differs between humans, deriving fromthe placenta in humans (i.e. foetal in origin) and from the cor-pora lutea in rodents (i.e. maternal in origin), which can affectsusceptibility.

(ii) There may be differences in endocrine regulation that fun-damentally alter the susceptibility to certain ED of humanscompared with rodents. For example, testosterone productionby the foetal human testis appears to be unaffected by certainphthalates that cause pronounced suppression of testosteroneproduction by the foetal rat testis (Heger et al., 2012; Johnsonet al., 2012; Mitchell et al., 2012). Similarly, the human foetaltestis is unaffected by potent estrogens such as diethylstilboe-strol (DES), whereas exposure of foetal rats or mice causes >80%suppression of foetal suppression of foetal testosterone pro-duction (Mitchell et al., 2013); the latter difference appears tobe due to the absence of ESR1 from human foetal Leydig cells,which may mean that other estrogenic EDs will also exert noeffect.

iii) In rodents, perinatal oestrogen exposure within the brain isessential for inducing male-specific sexual behaviour, whereasno such role is known in humans, in whom this form ofbehaviour is programmed exclusively by androgens (Sharpe,2010a)

(iv) In humans, significant brain development occursin utero, and neuroendocrine development of thehypothalamic–pituitary–adrenal axis is largely completedduring gestation. In contrast, in rodents, which are born in avery immature state, most of the neuroendocrine develop-ment occurs postnatally. Thus humans and rodents may differin timing of their susceptibility to ED that might affect theseprocesses (Matthews, 2000). Similar arguments apply to thehypothalamic–pituitary–gonadal axis.

Within the European Commission various pieces of legislationcontain specific provisions on endocrine disruption in the area ofplant protection products (PPP, or pesticides), biocides, industrialchemicals (REACH), and also medicines and cosmetics.

Regulation (EC) No 1107/2009 on PPP makes specific referenceto ED stating that the approval of active substances, safeners or syn-ergists is not allowed “if it is considered to have endocrine disruptingproperties that may cause adverse effects in humans/on non-targetorganisms unless the exposure. . .is negligible”. A similar approachis taken for biocides (Regulation (EC) No 528/2012). In addition,EDs are addressed within the REACH Regulation (EC 1907/2006),according to which: “. . .substances - such as those having endocrinedisrupting properties [. . .] for which there is scientific evidence of prob-able serious effects to human health or the environment” may beincluded in the list of “substances of very high concern”.

As a consequence the regulation of PPP and Biocides in the Euro-pean Union would occur via a hazard-based approach, which isdifferent from the approach to be taken in the USA and Japan,where risk assessment of all potential EDs is to be conducted,i.e. integrating both hazard and exposure. Indeed, the risk assess-ment approach would address uncertainty related to the ability ofa chemical to interfere with hormone action (i.e. the hazard) andthe manifestation of an adverse consequence linked to the dose,duration and timing of exposure.

3. Risk assessment: dose–response relationships

The normal risk assessment paradigm consists of four steps,

namely hazard identification, hazard characterization, assessmentand risk characterization, of which the latter is an integration ofthe first three steps (European Commission, 2000). The proceduresfor assessing risks of chemicals independently of their use (e.g.

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ndustrial chemical, contaminants in food and feed, plant protec-ion products and biocides) applied by EU and international agen-ies takes into account both inherent hazards as well as exposure.

Regarding EDs, aspects of hazard evaluation and applicabilityf the established risk assessment process have been questionednd are the subject of a strong debate within the scientific com-unity (Ashby, 2003; Welshons et al., 2003; Phillips et al., 2008;

homberg and Goodman, 2012; Sharpe, 2010b; Vandenberg et al.,009, 2012; vom Saal et al., 2010; Zoeller et al., 2012). The majoroints are related to the hypothesis that ED could (or not) (i) actith a non-threshold mechanism, (ii) give rise to a non-monotonicose–response (NMDR) relationship, and (iii) at very low doses,articularly during critical windows of exposure.

The paradigm in toxicology and risk assessment is that the indi-idual response of an organism to a chemical increases/decreasesroportionally to the exposure (dose). This gives rise to a monotonicose–response relationship, in which the effect either increases orecreases over the full dose range tested. It is generally acceptedhat for most chemicals (with no genotoxic potential) there is ahreshold dose, below which no (adverse) effect is expected toccur, on the basis of which a NO(A) EL or a BMDL are obtained,s the point of departure for the derivation of health basedeference values (i.e. ADI, TDI, ARfD). To assess substances thatre present at low levels in the diet, on the basis of knowledge ofhe chemical structure of the substance concerned and informationn human exposure, the Threshold of Toxicological Concern (TTC)pproach has been evaluated by the EFSA Scientific Committee.he Committee concluded that many endocrine mediated adverseffects involving development would be adequately covered by theefined TTC values (EFSA, 2012b).

However, in toxicology the possibility exists that non-onotonic dose–response (NMDR) relationships can occur. A

ose–response curve is non-monotonic when the slope of the curvehanges sign somewhere within the range of doses examined, giv-ng rise to U-shaped or an inverted U-shaped profile. The case ofssential trace elements such as copper is well known: physiolog-cal homeostasis regulates the circulating levels of these elementsn a range of optimal exposures below and above which adversity

ay occur: e.g. the effects due to copper deficiency are much moreevere than those due to its excess. However, the dose–responseurve in the deficiency dose range, as well as in the excess one isssentially monotonic and with a clear distinction in the types ofymptoms that emerge due to hypo- and hyper-exposure to copper,espectively.

Non-linearities in toxicokinetics may be the cause of NMDRhen the mode of action is concentration dependent as in the

ollowing cases:

two receptors with different actions and different affinities orKd’s.Two enzymes with different affinity (Km) for a chemical involvedin its biotransformation, producing different metabolites withdifferent effects.Saturation, induction/inhibition of metabolizing enzymes of theunique metabolic pathway.

As an example, at low doses the effect induced by a chemicalay be strictly related to its transformation products, the forma-

ion of which increases with the dose, up to high doses at which theaturation of metabolite formation may occur. This could give riseo accumulation of the parent compound with induction of possiblenexpected effects (constant level of the already observed adverse

ffect and appearance of a new one) but also to effects counter-cting the one due to metabolites. This would result in a NMDRurve, as the sum of (at least) two curves with different mechanisticackground.

y 314 (2013) 51– 59 55

Non-monotonicity is not synonymous with low dose effects,although some authors use the terms with this meaning, becausethere are low dose effects that follow monotonic dose–responsecurves. In addition, the use of terms such as ‘low dose’ or ‘environ-mentally relevant dose’ in the literature has no consensus and candescribe doses of the same compound covering several orders ofmagnitude. In many cases ‘low doses’ are defined as those below thepresumed NO(A)EL or BMDL derived by animal testing. In the caseof an NMDR curve, the traditional NO(A)EL/BMDL point of depar-ture cannot be used to derive a health-based guidance value. Thisreflects the uncertainties regarding identification of an exposurelevel at which it can be concluded that the risk for the exposedpopulation is minimal or negligible.

In the case of EDs there is still a strong debate about the ‘lowdose hypothesis’, according to which “low dose effects”, whichare not present at higher doses may display a non-monotonicdose–response (NMDR). Therefore, for a given effect, a simplemonotonic extrapolation from high to low doses during risk assess-ment of those substances would no longer be valid. However, upto now no scientific consensus has been reached as to the validityof the studies supporting the low dose hypothesis. It is noteworthythat where data suggestive of NMDR have been generated in vivoin rodent studies, they have usually proved to be unrepeatablein other laboratories. Therefore, such apparent ‘low dose’ effectsand NMDRs may be incidental findings with no pathophysiologicalmeaning. Furthermore non-monotonic low dose effects are oftenfound in physiological parameters without any link to adversity,being simply part of homeostatic control. In general, all endocrinesystems exhibit different manifestations at abnormally low (‘lowdose’) hormone levels versus those that manifest at abnormallyhigh levels of the same hormone, similar to the example of cop-per above: examples are given by the effects associated to lowglucocorticoid levels (Addison’s disease) versus high glucocorticoidlevels (Cushing’s syndrome) and effects of hypothyroidism versushyperthyroidism. The symptoms that manifest differently at sub-normal versus supranormal hormone exposure is not evidence ofNMDR, but simply reflects the level of activity of the hormone inquestion.

A careful analysis of studies supposed to support the occurrenceNMDR is necessary, since there are phenomena that could con-tribute to generating an ‘apparent NMDR’. For example, an NMDRcan be observed in studies where high-doses alter the experimen-tal model (cell, organ or animal), thus decreasing the observedresponse. This could occur when very high doses, irrelevant toactual exposure are tested, which are cytotoxic doses in in vitrostudies or in vivo, when are excessively toxic to animals (dosesexceeding the maximum tolerable dose) and can reduce the onsetof an effect. The same could happen when the formation of aggre-gates, colloids or micelles at high concentrations can reduce thebioavailability of a chemical and therefore decrease the toxicitythat appeared at lower concentrations. Such examples are ratherdifferent from the form of NMDR that has been also claimed forsome ED, in which the same endpoint effect seen at high doses isalso claimed to be observable at doses that are below the thresholdof the normal dose–response effect curve.

Studies used to support the NMDR should meet the same qualitycriteria as for any other studies used in the risk assessment proce-dure, and therefore poorly described experiments in non-validatedmodels should not be considered. Of paramount importance isreproducibility of the phenomenon – if it is real, it should be repro-ducible from experiment to experiment and between differentlaboratories. So far, no evidence of this nature has been produced

for EDs using in vivo hormonally responsive, intact systems. Sta-tistical plausibility should be also assessed to demonstrate thenon-monotonic nature of each identified dose–response relation-ship, which is not always an easy task due to the limited raw data

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vailable in the studies published in the scientific literature. Thisan be a serious limitation, since NMDR can be originated by experi-ental parameter variability in the absence of decent statistics and

ound interpretation of biological relevance. In addition levels ofuman exposure should also be considered in order to determine

n which part of an NMDR exposure they fall.In vitro studies are often used to demonstrate NMDR, but as

escribed by the OECD conceptual framework, they can only besed for hazard assessment, priority testing and to study the MoA.

ndeed, for the time being they are not useful for dose–responseefinition and for risk assessment purposes, and therefore cannote the basis for demonstrating NMDR occurrence. The main reasonor this limitation at present is the difficulty of in vitro to in vivoxtrapolation, since it is a challenge to translate exposure in cellultures to internal dosing in the intact organism. However, recentevelopments in PBPK modelling have shown promise in evalu-ting the relevance of in vitro effective concentrations to in vivoxposure levels (Piersma et al., 2013). Therefore, in vitro biokineticata that provides the actual level of cell exposure producing an

n vitro observed effect can improve the in vitro-in vivo extrapola-ion through the use of PBPK modelling (Blaauboer, 2010; Coecket al., 2013; PredictIV web site).

However, other limitations exist, especially the adversity versusdaptation issue. Indeed, the application of advanced in vitro mod-ls and testing strategies is strictly dependent on the ability toranslate information from the cell level to organs, and subse-uently to individuals, and to distinguish between adaptationersus adversity, with the latter clearly identified by actual markersf adversity (Blaauboer et al., 2012). Low dose effects and NMDRan e.g. be observed by applying ‘omics’ techniques, which havehown effects on the level of gene expression at concentrationsrders of magnitude below those resulting in adverse effects onell differentiation and embryonic morphogenesis (van Dartel et al.,011; Hermsen et al., 2012). These findings point at the criticaleed to distinguish physiological from adverse effects also in theselternative models, before changes in molecular biomarkers can beeaningfully interpreted in view of hazard and risk assessment.The presence of a response in animal in vivo studies at one dose

evel only is not sufficient to demonstrate a causal relationship ando claim an effect at low doses. In addition, a wide dose range andeasonably closely spaced dose intervals (<10-fold within the sametudy) with sufficient replicates are necessary to demonstrate U-haped dose–responses; and to re-emphasize, reproducibility ofny observed U-shaped dose–response curve is mandatory.

It has been claimed that conventional in vivo testing is inad-quate for the identification of low-dose effects or NMDRCsisplayed by some EDs. In that view, to test ED-induced effectsostulating a NMDR would require a change in the testing pro-ocol, with more doses tested, in order to identify effects especiallyn the low dose area. However, to detect effects at low doses anncreased number of animals per group and increased number ofoses of treatment are needed, to increase the statistical power.uring the June 2012 EFSA Colloquium on low dose responses

n toxicology and risk assessment (EFSA, 2012a), an optimum ofeven doses was proposed. This goes in the opposite direction tohe increasing political pressure in the EU to reduce animal test-ng, unless the same total animal number is spread over moreose groups, thus keeping statistical power unaltered and improv-

ng dose–response estimation at the same time (Slob et al., 2005).everal OECD test-guidelines for in vivo toxicity testing alreadyention this possibility as an alternative study design, improv-

ng dose–response analysis using the benchmark dose approach

e.g. the OECD 443 extended one-generation reproductive toxicitytudy).

The recent WHO-UNEP expert report (2012), which reviewedll the available literature, entered the debate considering the

y 314 (2013) 51– 59

existence and relevance of low-dose effects and NMDR induced byED. However, further work is still necessary to consider whetherNMDR can occur in a normally functioning homeostatic matureendocrine system in vivo, because in such a system ‘low doses’ (i.e.subnormal hormone levels) will trigger compensatory changes torestore normal hormone levels, as is also the case if hormone levelsbecome too high. In view of the potential impact on testing andrisk assessment, further studies in this area to resolve the presentuncertainty are essential.

4. Other ED issues: critical windows of susceptibility andmixtures effects

The low dose-NMDR issue has to a large extent deflected atten-tion away from the two ED issues that do remain of concern, namelycritical windows of susceptibility and the issue of mixtures effects.A transient adaptive endocrine modulation due to ED exposurein the adult organism may have different effects in a develop-ing organism in which the endocrine homeostasis system has notcompletely developed or for which feedback mechanisms do notoperate (e.g. masculinization of foetal males by androgens). In thesesituations, ED effects could result in permanent adverse changes(WHO/UNEP, 2012). Critical time windows are generally coveredby the existing animal testing aimed to identify developmentaland reproductive toxicants; nevertheless, the current mammaliantests do not fully cover specific endpoints appearing later in life likecertain cancers. However, extrapolating the windows of exposureduring development in animal models to windows of exposure inhuman development could be problematic, due to species differ-ences. In view of the growing evidence for foetal origins of manycommon human disorders that emerge in adulthood or duringageing, susceptibility of the developing foetus to EDs remains animportant area of research. This is particularly pertinent consider-ing that most such disorders are becoming increasingly common inmodern societies, and although there may be other causes for thischange (e.g. diet and lifestyle), EDs are likely to remain a focus ofinterest. Therefore it would be important also to consider how cur-rent regulatory toxicity testing can be improved, e.g. by including inreproductive toxicity testing protocols additional novel parametersthat may be endocrine-related, such as mammary gland develop-ment and senescence, and some parameter for adrenal disruption.

At present, most chemicals are regulated on a case-by-case basis.In general, no account is taken of any potential additivity or syn-ergism between compounds with a similar mechanism or MoA orwith a common endpoint effect. For certain classes of ED, for exam-ple anti-androgenic compounds, there are well-conducted in vivoand in vitro studies that show unequivocally additivity for mixturesin which each individual chemical is present at its NOEL level, caus-ing adverse effects in vivo following exposure during pregnancy inrats. Such studies are reproducible between laboratories, and havebeen consistent in their findings; studies involving combinationsof up to 10 different anti-androgenic chemicals have been tested.Although such mixtures studies may still be using levels of EDsthat are higher than environmental/human exposure levels, theymake the point that exposure to the ‘real world’ mixture of 100sof chemicals that we experience each day, might combine theirindividually negligent effects into an adverse effect; in most of thedocumented cases, the combined effects are concentration additive(no synergism) and can therefore be predicted using appropriatemodelling approaches. Such mixtures effects would be of particularconcern in relation to a critical period such as foetal development.

The problems that ‘mixtures effects’ pose to regulators are consid-erable, as they challenge the credibility of the current case-by-caseapproach. A possible approach to toxicity and risk assessment ofmixtures is described in a recent SCHER opinion (SCHER, 2011).

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owever, it also raises significant issues in relation to using aazard-based approach, especially where this might rely on EAS/EDctivity.

. Hazard versus risk

A hazard based approach, as foreseen in the European Com-ission legislations that contain specific provisions on endocrine

isruption, is not suitable to address the issue of ED for the purposef risk management decisions. At face value this would similarlyabel compounds to which there is high human exposure (e.g.ertain phthalates) and those where it is minimal (e.g. many pesti-ides). In further considering the mixtures issue above, this wouldompound such disparities. In medicine, the first principle for doc-ors is ‘do no harm’. It seems that a simple hazard-based approacho regulation would do just this, leveraging equal ‘branding’ toompounds with disparate activities, potencies and exposures. Inssence it would be an approach that largely ignored the avail-ble evidence, which seems nonsensical. By using the proposedazard approach to identify and regulate EDs, we could haveo face ‘extreme consequences’. For instance, dihydrogen oxidei.e. water) exposure affects adrenal aldosterone, renal renin andepatic angiotensin levels, and in high doses causes death. There-

ore, based exclusively on the hazard approach, water should beonsidered (and labelled) as an endocrine disrupter.

The only sound alternative is to apply the full risk assessmentrocess, taking into account all the available information of suffi-iently good quality on mechanism, hazard, exposure and adverseealth effects observed in intact organisms (including data onhe low dose hypothesis, NMDR and the mixtures issue), using a

oE approach and expert judgement. This includes the processor characterizing the extent to which the available data support aypothesis that an agent causes a particular effect (US EPA, 2011;CENHIR, 2012), and uses this information in an integrated riskssessment, as the basis for well-informed risk management. Thispproach is not specific for ED, it is similar to that used for any otherhemical and is based on sound scientific principles and experi-nce in use; most importantly, it is an evidence-driven approach aspposed to the hazard-based approach which is not, as it ignoresuch of the evidence.The scientific literature on endocrine disruption is overwhelmed

ith ever increasing amounts of experimental studies of very vari-ble quality. Topical human health effects are being associatedith ED exposure, whilst causality is difficult to prove. Increasingly

ensitive exposure assessment methodologies allow the detectionf even very low human exposures to endocrine active com-ounds, the relevance of which in terms of health hazards andisks is unclear. Adverse health effects observed in experimentalnimal studies need inter-species extrapolation to the situationn man. A host of in vitro studies show endocrine activities ofhemicals, but their relation with adverse health effects is oftenbscure. Nevertheless, these studies generate genuine and warr-nted concern with the general public as with regulators. It ishe responsibility of the scientific community not to add to theonfusion, but to produce sound science and an unbiased assess-ent of actual human risk on the basis of the actual data. It is

mpossible to prove a compound safe, simply because the risk isetermined by the exposure. This holds for compounds both with,nd without, primary endocrine activity. Hazard-based risk man-gement is therefore not scientifically justified. Therefore, using aut-off procedure simply based on hazard identification, as pro-

osed in the PPP legislation (Reg. 1107/2009), according to whichll chemicals with endocrine activity will not enter into the autho-ization for marketing procedure, does not meet the criteria for

sound scientifically based assessment. Integrated weighing of

y 314 (2013) 51– 59 57

hazard, potency, and exposure is necessary for proper risk assess-ment and risk management of endocrine disrupters as for allchemicals.

Conflict of interest statements

Testai, Galli, Dekant, Marinovich, and Piersma: For the past 3years since the beginning of the work, all Authors declare as nothaving any financial, personal, or association with any of the indi-viduals or organizations, that could inappropriately influence thesubmitted work.

Sharpe: For the past 2 years, I have been a member of a scientificadvisory committee for BASF on the issue of endocrine-disruptingpesticides/chemicals, for which I have received financial remuner-ation. I performed a similar role for Makhteshim Agan of NorthAmerica in 2012 with financial remuneration. I was a paid consult-ant for Johnson & Johnson on the safety of certain phthalates inpaediatric medicines in the period up to 2011. I wrote a review forChemTrust on ‘Male reproductive health disorders and the poten-tial role of exposure to environmental chemicals’ in 2009, for whichI received financial remuneration.

Acknowledgements

The authors thank the Italian Society of Toxicology (SITOX) tohave promoted the feasibility of the position paper “A Report of theItalian Society of Toxicology Task Force on Endocrine DisruptingChemicals Expert Consensus Documents”.

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