Scholarly article on topic 'Assessment of three approaches for regulatory decision making on pesticides with endocrine disrupting properties'

Assessment of three approaches for regulatory decision making on pesticides with endocrine disrupting properties Academic research paper on "Clinical medicine"

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Abstract of research paper on Clinical medicine, author of scientific article — P. Marx-Stoelting, L. Niemann, V. Ritz, B. Ulbrich, A. Gall, et al.

Abstract Recent EU legislation has introduced endocrine disrupting properties as a hazard-based “cut-off” criterion for the approval of active substances as pesticides and biocides. Currently, no specific science-based approach for the assessment of substances with endocrine disrupting properties has been agreed upon, although this new legislation provides interim criteria based on classification and labelling. Different proposals for decision making on potential endocrine disrupting properties in human health risk assessment have been developed by the German Federal Institute for Risk Assessment (BfR) and other regulatory bodies. All these frameworks, although differing with regard to hazard characterisation, include a toxicological assessment of adversity of the effects, the evaluation of underlying modes/mechanisms of action in animals and considerations concerning the relevance of effects to humans. Three options for regulatory decision making were tested upon 39 pesticides for their applicability and to analyze their potential impact on the regulatory status of active substances that are currently approved for use in Europe: Option 1, based purely on hazard identification (adversity, mode of action, and the plausibility that both are related); Option 2, based on hazard identification and additional elements of hazard characterisation (severity and potency); Option 3, based on the interim criteria laid down in the recent EU pesticides legislation. Additionally, the data analysed in this study were used to address the questions, which parts of the endocrine system were affected, which studies were the most sensitive and whether no observed adverse effect levels were observed for substance with ED properties. The results of this exercise represent preliminary categorisations and must not be used as a basis for definitive regulatory decisions. They demonstrate that a combination of criteria for hazard identification with additional criteria of hazard characterisation allows prioritising and differentiating between substances with regard to their regulatory concern. It is proposed to integrate these elements into a decision matrix to be used within a weight of evidence approach for the toxicological categorisation of relevant endocrine disruptors and to consider all parts of the endocrine system for regulatory decision making on endocrine disruption.

Academic research paper on topic "Assessment of three approaches for regulatory decision making on pesticides with endocrine disrupting properties"

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Regulatory Toxicology and Pharmacology

journal homepage: www.elsevier.com/locate/yrtph

Assessment of three approaches for regulatory decision making on ■.

pesticides with endocrine disrupting properties ^

P. Marx-Stoelting *, L. Niemann, V. Ritz, B. Ulbrich, A. Gall, K.I. Hirsch-Ernst, R. Pfeil, R. Solecki

Bundesinstitut für Risikobewertung (Federal Institute for Risk Assessment), Max-Dohrn-Str 8-10, 10589 Berlin, Germany

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ARTICLE INFO

Article history:

Received 7 April 2014

Available online 17 September 2014

Keywords: Pesticides

Endocrine Disruption Decision criteria

ABSTRACT

Recent EU legislation has introduced endocrine disrupting properties as a hazard-based "cut-off" criterion for the approval of active substances as pesticides and biocides. Currently, no specific science-based approach for the assessment of substances with endocrine disrupting properties has been agreed upon, although this new legislation provides interim criteria based on classification and labelling.

Different proposals for decision making on potential endocrine disrupting properties in human health risk assessment have been developed by the German Federal Institute for Risk Assessment (BfR) and other regulatory bodies. All these frameworks, although differing with regard to hazard characterisation, include a toxicological assessment of adversity of the effects, the evaluation of underlying modes/mechanisms of action in animals and considerations concerning the relevance of effects to humans.

Three options for regulatory decision making were tested upon 39 pesticides for their applicability and to analyze their potential impact on the regulatory status of active substances that are currently approved for use in Europe: Option 1, based purely on hazard identification (adversity, mode of action, and the plausibility that both are related); Option 2, based on hazard identification and additional elements of hazard characterisation (severity and potency); Option 3, based on the interim criteria laid down in the recent EU pesticides legislation. Additionally, the data analysed in this study were used to address the questions, which parts of the endocrine system were affected, which studies were the most sensitive and whether no observed adverse effect levels were observed for substance with ED properties.

The results of this exercise represent preliminary categorisations and must not be used as a basis for definitive regulatory decisions. They demonstrate that a combination of criteria for hazard identification with additional criteria of hazard characterisation allows prioritising and differentiating between substances with regard to their regulatory concern. It is proposed to integrate these elements into a decision matrix to be used withina weight ofevidence approach for the toxicological categorisation of relevant endocrine disruptors and to consider all parts of the endocrine system for regulatory decision making on endocrine disruption.

© 2014 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND

license (http://creativecommons.org/licenses/by-nc-nd/3XI/).

1. Introduction

Recent EU legislation has introduced endocrine disrupting properties as one of a number of new hazard-based "cut-off" criteria for the approval of active substances in plant protection products (Reg. (EC) No 1107/2009; European Council, 2009).

Accordingly, substances with endocrine disrupting properties that may cause adverse effects in humans may only be approved if exposure is negligible.

However, no specific science-based criteria for the assessment of substances with endocrine disrupting properties have been agreed upon so far. For pesticides, the EU Commission is required

Abbreviations: ANSES, Agence Nationale de Securite Sanitaire de l' Alimentation de l'environment et du travail (French National Agency for Food Safety); BfR, Bundesinstitut für Risikobewertung (German Federal Institute for Risk Assessment); CLP, Classification, Labelling and Packaging (of Chemicals); CRD, Chemicals Regulations Directorate; EC, European Commission; ECETOC, European Centre for Toxicology and Ecotoxicology; ED, Endocrine Disruptor; EFSA, European Food Safety Authority; EPA, Environmental Protection Agency; ESIS, European Substance Information System; GHS, Globally Harmonized System (of Classification and Labelling of Chemicals); HPA, Hypothalamic Pituitary Adrenal Axis; HPG, Hypothalamic Pituitary Gonadal Axis; HPT, Hypothalamic Pituitary Thyroid Axis; IPCS, International Programme on Chemicals Safety; MRL, Maximum Residue Level; REACH, Registration, Authorisation and Evaluation of Chemicals; Reg, Regulation; WHO, World Health Organisation.

q Disclaimer: This paper presents results of a research project of the Pesticides Safety Department of the BfR and does not necessarily represent the regulatory position of the institute. * Corresponding author. E-mail address: philip.marx-stoelting@bfr.bund.de (P. Marx-Stoelting).

http://dx.doi.org/10.1016/j.yrtph.2014.09.001 0273-2300/® 2014 The Authors. Published by Elsevier Inc.

This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).

to present a draft of measures concerning specific scientific criteria for the determination of endocrine disrupting properties in the near future. Pending adoption of specific scientific criteria, the new plant protection products regulation provides interim criteria for identification of some substances with endocrine disrupting properties for non-approval, based on classification and labelling: ''.. .substances that are or have to be classified, in accordance with the provisions of Regulation (EC) No 1272/2008, as carcinogenic category 2 and toxic for reproduction category 2, shall be considered as having endocrine-disrupting properties. .. .substances such as those that are or have to be classified, in accordance with the provisions of Regulation (EC) No 1272/2008, as toxic for reproduction category 2 and which have toxic effects on the endocrine organs, may be considered as having endocrine-disrupting properties'' (Reg. (EC) No 1107/ 2009; European Council, 2009). Similar provisions for a regulatory "cut-off", based on endocrine disrupting properties and including interim criteria for the determination of endocrine disrupting properties are also provided in the new EU biocides regulation 528/2012 (European Parliament and Council, 2012).

Assessment and regulation of substances with potential endocrine disrupting properties is associated with a number of challenges. The endocrine system is extremely complex and is involved in virtually all functions of the vertebrate organism (WHO/IPCS, 2002; WHO/UNEP, 2013). Consequently, any effect observed in a toxicity study might theoretically be linked to an alteration of function of the endocrine system. Since administration of high doses of test chemicals in pre-approval animal studies is mandatory according to internationally harmonised test guidelines and data requirements under regulatory frameworks, the majority of active substances in plant protection or biocidal products have been found to show at least some significant toxicity. Currently, the mechanism or mode of action leading to a toxic effect is often not clear and judgement whether it is endocrine mediated or not is difficult.

A related challenge is that the process of endocrine disruption does not correspond to a single endpoint per se. Endocrine disruption includes a variety of different mechanisms of toxicity that may affect different individual endpoints. Moreover, even though in the past decade several assays have been developed and validated to detect substance-induced effects caused by interference with the hypothalamic-pituitary-gonadal (HPG) axis, there are many other components of the endocrine system, for which at present no or only insufficiently validated specific mechanistic assays are available. In fact, if endocrine disruption is discussed in the public, the focus is very often on substances potentially affecting fertility and reproduction via interaction with steroid hormone systems such as the estrogen or androgen systems. Sometimes additionally effects on the thyroid hormone system are discussed whereas other parts of the endocrine system like the adrenal gland or the pancreas are seldomly taken into consideration.

Although substances with endocrine disrupting properties have been addressed in different sections of EU chemicals regulation (e.g. plant protection products regulation, biocides regulation, REACH), current data requirements and options for management decisions differ significantly between regulations. While data requirements under the recent EU pesticides regulation cover an extensive set of toxicity studies in at least four animal species, the scope of the data package for chemicals under REACH (Regulation EC No. 1907/2006) depends on the production volume and may not be as comprehensive as for pesticides (Beronius et al., 2009). Furthermore, the downstream regulatory consequences for substances with endocrine disrupting properties currently appear to differ between regulations. Active substances, safeners or synergists with endocrine disrupting properties to be used in plant protection are not to be authorised even if risk assessment demonstrates that there is no risk associated because of very limited exposure. This "cut-off"

is to be applied "unless the exposure of humans to that active substance, safener or synergist in a plant protection product, under realistic proposed conditions of use, is negligible, that is, when the product is used in closed systems or in other conditions excluding contact with humans and where its residues in food and feed do not exceed the default value set in accordance with point (b) of Article 18(1) of Regulation (EC) No 396/2005" (European Council, 2009). By contrast, chemicals under REACH (Reg. (EC) No 1907/2006) that have endocrine disrupting properties for which there is scientific evidence of probable serious effects to human health may be nominated for a candidate list in accordance with Article 57f. That means that these substances should become subject to an authorisation procedure and may not be placed on the market unless they have been authorised (European Council, 2006).

Although conclusions on endocrine disruptors in a regulatory context are required under the European regulations on plant protection products and also on biocides (European Parliament and Council, 2012) the lack of agreed criteria makes implementation of these pieces of legislation difficult. Fortoxicological hazards likecar-cinogenicity, reproductive toxicity or specific target organ toxicity, criteria for categorisation have already been established and internationally agreed upon in the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) which was implemented into European legislation by Regulation (EC) No 1272/2008 (CLP regulation) (European Council, 2008). However, the CLP regulation does not define a specific hazard class of endocrine disruptors. In this context it has been argued that endocrine disruption is based on a variety of mechanisms of action that may lead to adverse health outcomes like cancer, reproductive disease or specific target organ tox-icity, but does not represent in itself a single endpoint that would require classification or labelling (ECETOC, 2009). Furthermore, any substance that causes cancer or effects on reproduction and development like reduced fertility or malformations by an endocrine mode of action would be expected to be classified accordingly. However, consideration of specific toxicity on endocrine-related target organs also seems to be important in assessment of endocrine disruptors. If the regulatory "cut-off" was based alone on current classification for carcinogenicity (C) and/or reproductive toxicity (R), a substance with specific toxic effects on the adrenals or the pancreas might not be banned as an endocrine disruptor, in contrast to a substance known or presumed to cause reproductive toxicity by an endocrine-related mechanism in humans.

Recently, different sets of criteria have been discussed, addressing the challenges mentioned above (Bars et al., 2011; Marx-Stoelting et al., 2011). Governmental agencies such as the Danish EPA (Danish Ministry of the Environment, 2011), French ANSES (ANSES, 2012), British CRD/German BfR (BfR and CRD, 2011) and non-governmental organisations like ECETOC (2009) or Chemtrust(2011) have made proposals available on their websites. A number of these proposals have been summarised in a EU report (Kortenkamp et al., 2012). Scientific issues on the identification and characterisation of endocrine disruptors have also been discussed by expert panels at the European level and results of these discussions have been summarised in recent reports (EFSA Scientific Committee, 2013; Endocrine Disrupters Expert Advisory Group, 2013). While most of these approaches have some principles in common regarding hazard identification, they also show differences especially when it comes to proposals for decision making concerning the regulatory "cut-off". Most proposals are based on the WHO/IPCS definition for endocrine disruptor as ''.. .an exogenous substance or mixture that alters function(s) of the endocrine system and consequently causes adverse health effects in an intact organism, or its progeny, or (sub)populations'' (WHO/IPCS, 2002), and as such concentrate on adverse effects caused by a substance, taking into account the mode or mechanism of action which has to be related to endocrine disruption. In addition, most

decision strategies propose to use a weight of evidence approach and to consider relevance to humans. Significant differences remain in the way by which additional criteria of hazard characterisation like specificity, severity of effects or doses at which effects occur could be integrated into the regulatory decision.

The primary aim of this study was to evaluate different options for hazard based regulatory decision making by applying them to a number of active substances currently approved in plant protection products in the EU. This approach has been suggested by the BfR in 2011 (Marx-Stoelting et al., 2011). To simulate the range of substances encountered in regulatory practice, known "positives" as well as substances with unknown endocrine disrupting properties and presumed controls were included in the study. In addition the data analysed in this study were also used to address the questions, which parts of the endocrine system were affected by ED most frequently, which studies were the most sensitive in detection of ED effects and whether or not no observed adverse effect levels (NOA-ELs) were observed for substance with ED properties. Since no gold standard for any regulatory decision on endocrine disruption exists, this exercise cannot formally validate any set of criteria. However, the results of this study will allow a comparison of discussed approaches for regulatory decision making in terms of numbers of substances sorted out, applicability of criteria and reproducibility of decisions based on them and also whether or not criteria allow prioritising substances for regulatory decision making.

2. Materials and methods

2.1. Substance selection

Three sets of substances, 39 substances in total, were selected. The first set consisted of substances potentially falling under the interim "cut-off" criteria for endocrine disruption set by Regulation EC No 1107/2009, (i.e. substances classified in accordance with Regulation EC 1272/2008 as carcinogenic category 2 and toxic for reproduction category 2 shall be considered to have endocrine disrupting properties and substances classified as toxic for reproduction category 2 and showing effects on endocrine organs may be considered to have endocrine disrupting properties (European Council, 2009)).

This first set of substances was selected by a classification-based search in the EU pesticides database (http://ec.europa.eu/ sanco_pesticides/public/index.cfm), accessed in January 2011 and re-accessed in July 2013. The search was limited to active substances having been approved for pesticidal use in the EU. Furthermore, it was limited to substances with a current legal classification of 'toxic for reproduction' category 2 and/or 'carcinogenic' category 2, not taking into account substances with proposed classifications. In addition to the EU pesticides database, Annex VI of Regulation EU 1272/2008 was searched for candidate pesticides where the status of classification and labelling was not clear and to confirm results obtained in the pesticides database by accessing the European Substance Information System (ESIS) on http:// esis.jrc.ec.europa.eu. Table 1 lists all substances retrieved by this strategy.

The second set (Table 2) consisted of substances selected from Annex 1 of Directive 91/414/EEC by use of a computer-based random generator. Substances in Annex 1 were numbered from 1 to 351 (the total number of approved active substances at the beginning of the study) and the random generator was used to generate 22 integral numbers between 1 and 351. The substances selected by this approach were chosen for analysis of potential endocrine disrupting properties. Randomisation was considered especially important since a selection based on classification for reproductive toxicity as applied to generate the first set of substances, clearly

shows a bias for substances potentially affecting the hypotha-lamic-pituitary-gonadal (HPG) axis of the endocrine system. For the intended analysis of questions (such as which percentage of all substances approved as active substances in plant protection products might be flagged for "cut-off" by applying the different criteria and if endocrine targets other than the HPG system might be affected by endocrine disruption) it was considered important to obtain an unbiased selection of substances.

A third set of three substances (listed in Table 3) approved for use in organic farming in the EU was selected randomly from annex II of Regulation (EC) No 834/2007 (European Council, 2007) (as amended by Regulation (EC) No 889/2008), accessed through the following web page: http://www.bmelv.de/ SharedDocs/Downloads/Landwirtschaft/OekologischerLandbau/ EGOekoVOAnhang2.html.

2.2. Data evaluation

All available data from regulatory studies were checked for indications of endocrine disrupting properties. As indications for ED properties any reported pathological or histopathological change in glands, any change in hormone levels, effects on reproduction or development or tumours in hormone dependent organs were taken into account. In addition, a literature search for published articles was performed through Pubmed (www.pub-med.com). The substance name was entered as the primary search term. If up to 20 results were found for the substance name, all abstracts were checked and papers were selected for a complete evaluation if they analysed mammalian toxicology and/or potential endocrine effects of the substance (e.g. publications on chemical synthesis, analytical methods or ecotoxicity were not taken into account). If more than 20 results were obtained '"substance name' AND 'toxicity'" or '''substance name' AND 'endocrine''' or '''substance name' AND 'hormone''' were entered into Pubmed. All abstracts found by this refined search strategy were then analysed and, again, papers were selected for a complete evaluation if their subject was mammalian toxicology and/or potential endocrine effects.

In addition, mechanistic information and information on relevance to humans was collected, if available. Since it would have been beyond the scope of this analysis to conduct a new evaluation of existing studies, the respective summaries and evaluations in EU monographs, EFSA conclusions or EC scientific conclusions were chosen as a main source for toxicological findings with the selected compounds. Data were summarised in tabular form by taking into account the animal species, type of study and route of exposure (e.g. rat, 90-d, oral), the reliability of the study the adverse effects observed and their respective dose levels.

In addition to testing the sensitivity, reproducibility and applicability of the suggested regulatory options, several other points were addressed in the evaluation. One important question was which part(s) of the endocrine system were affected by the substances analysed in this exercise. Because of the ongoing discussion whether to include all subsystems of the hormonal system, rather than to focus on components and targets of the HPG axis, all effects that could be associated with a hormonal subsystem were taken into account. Further questions were related to the types of effect which might lead to identification as an endocrine disruptor (e.g. weight changes of glands, histopathology of endocrine related organs, changed fertility parameters, etc) and to the most sensitive study designs for the detection of these effects (e.g. the two-generation reproductive toxicity study or a chronic or carcinogenicity study). Additionally, it was analysed whether a NOAEL was observed for the endocrine effects in the available toxicological database.

List of substances classified as 'toxic to reproduction' (Rep) category 2 and/or 'carcinogenic' (Carc) category 2, potentially falling under the preliminary cut-off criteria set by Regulation (EC) No 1107/2009 according to the EU Pesticides database accessed under http://ec.europa.eu/sanco_pesticides/public/index.cfm?event=activesubstance. Target organs and effects found in toxicity studies and/or leading to classification and labelling are presented in accordance with the EU List of Endpoints as published for the individual substances in the EFSA Journal or EFSA Scientific Reports or Review Reports of the European Commission. The 16 substances analysed by several risk assessors independently are listed first and are printed in bold and underlined. In addition to these 16 substances the table also contains some substances classified after January 2011 and some substances, labelled Carc category 2, showing potential indications for endocrine disruption, which would not fall under the preliminary cut-off criteria.

Substance name Classification (human health)

Target organs (effects considered for decision on ED)

Remarks

Arnitrol Rep 2 (H361d), STOT RE 2 (H373)

Bromoxvnil Rep 2 (H361d), Skin sens 1 (H317), Acute

Tox 2 & 3 (H301, 330) Chlorotoluron Carc 2 (H351), Rep 2 (H361d)

Dimoxvstrobin Rep 2 (H361d), Carc 2 (H351), Acute Tox 4 (H332)

Epoxiconazole Carc 2 (H351), Rep 2 (H361fd)

Fenpropimorph Rep 2 (H361d), Acute Tox 4 (H302)

Ioxvnil Rep 2 (H361d), Acute Tox. 3 & 4 (H301,

H331, H312), Eye Irrit 2 (H319), STOT RE 2 (H373) Isoxaflutole Rep 2 (H361d)

Linuron Rep 1B (H360Df), Carc. 2 (H351), Acute

Tox 4 (H302), STOT RE 2 (H373) Mancozeb Rep 2 (H361d), Skin Sens 1 (H317)

Maneb Rep 2 (H361d), Acute Tox. 4 (H332),

Eye Irrit 2 (H319), Skin Sens 1 (H317)

Metconazole Rep 2 (H361d), Acute Tox 4 (H302)

Molinat Rep2(H361f), Carc 2 (H351), Acute

Tox 4 (H332, H302), STOT RE 2 (H373), Skin Sens 1 (H317)

Oxadiargyl Rep 1A (H360Fd), STOT RE 2 (H373)

Tebuconazole Rep 2 (H361d), Acute Tox 4 (H302), Tepraloxvdim Carc 2 (H351), Rep 2 (H361d)

Thyroid (hormone levels, histopathology, tumours) Malformations at maternally toxic dose levels; liver (histopathology,

weight)

Liver (enzyme induction), kidney (tumours), spleen, developmental toxicity (resorptions, skeletal anomalies)

Duodenum (tumours), thyroid (tumours), developmental toxicity (slight microcytic hypochromic anaemia, reduced body weight gain, reduced thymus weight, discoloured liver, cardiomegaly) Liver (increased organ weight, clinical chemistry, chronic hepatitis), adrenals (histopathology), ovaries, reproductive toxicity (impaired fertility, prolonged gestation, dystocia), developmental tox. (malformations: cleft palates in rat; higher abortion and resorption rate)

Liver (weight, clinical chemistry), inhibition of cholinesterase, developmental toxicity (malformations)

Liver (histopathology, tumours), thyroid (histopathology, tumours), uterus (tumours), developmental toxicity (malformations at maternal toxic dose levels, variations)

Liver (histopathology, tumours); thyroid (histopathology, tumours), eye (ocular lesions), developmental toxicity (delayed ossification) Liver, red blood cells, testes (Leydig cell tumours) reproductive toxicity (infertility), developmental toxicity (malformations) Thyroid (inhibition of thyroid peroxidase, histopathology, tumours), developmental toxicity (malformations at high dose levels, abortions, delayed ossification)

Thyroid (inhibition of thyroid peroxidase, histopathology), liver (histopathology, tumours), developmental toxicity (malformations at high dose levels, resorptions, delayed ossification) Liver (histopathology, clinical chemistry), spleen (atrophy), adrenals (histopathology) developmental toxicity (embryonic death, postimplantation loss, malformations)

Testes (degenerative changes), liver (histopathology), adrenals (histopathology), ovaries (degenerative changes), kidneys (tumours), nervous system (degenerative changes), reproductive toxicity (decreased fertility)

Liver (histopathology); thyroid (histopathology), developmental toxicity (litter loss and growth retardation as a consequence of haem synthesis inhibition at maternal toxic dose levels) Adrenals (histopathology), liver (histopathology), developmental toxicity (resorptions, malformations) Liver (histopathology, tumours), bladder, thyroid, prostate epididimydes, reproductive toxicity (decreased pup weight, delayed eye opening), developmental toxicity (malformations)

Additional substances not considered in the exercise but meanwhile also labelled R2 or C2

Cyproconazole Rep 2 (H361d), Acute Tox 4 (H302)

Etridiazole Carc 2 (H351), Acute Tox 3 & 4 (H302,

H312, H331)

Diuron Carc 2 (H351), STOT RE 2 (H373), Acute

Tox 4 (H302)

Flusilazole Carc 2 (H351), Rep 1B (H360D), Acute Tox

4 (H302)

Myclobutanil Rep 2 (H361d), Acute Tox. 4 (H302), Eye Irrit 2 (H319)

Profoxydim Carc 2 (H351), Rep 2 (H361d)

Liver (histopathology, weight, tumours), slightly increased pre-, peri-and postnatal mortality, malformations (cleft palate, hydrocephaly) Testes (tumours), thyroid (tumours), liver (increased organ weight, histopatology), developmental toxicity (reduced body weight, retarded ossification)

Mammary (tumours), bladder (urothelial tumours), blood (haemolytic anaemia)

Liver (histopathology, tumours), blood (anaemia), bladder (tumours), testes (tumours), reproductive toxicity (Increased gestation length and dystocia, associated reduced pup viability and survival), developmental toxicity

Liver (weight, histopathology), testes (atrophy, oligospermia), developmental toxicity (altered viability index without maternal toxicity)

Liver (histopathology, tumours), testes (atrophy, tumours), developmental toxicity (reduced anogenital distance, hypospadias, undescended testes, testicular atrophy)

European Commission (2001) European Commission (2004a)

European Commission (2005a)

EFSA Scientific Report (2005) 46:1-82

EFSA (2008b)

EFSA (2008c)

European Commission (2004b)

European Commission (2003c)

European Commission (2002f)

European Commission (2009)

Review Report on the Active Substance Maneb

EFSA (2006)

European Commission (2003d)

Formerly classified Rep 2, reclassification after Jan 2011

EFSA (2008f)

European Commission (2004c)

Classified after Jan 2011; EFSA (2010c)

C2 with tumours in endocrine related organs EFSA (2010a)

C2 with tumours in endocrine related organs EFSA (2005a) European Commission (2007)

Classified after Jan 2011; EFSA (2010b)

Classified after Jan 2011

2.3. Decision making

Data evaluation and decision making for the 16 substances originally pre-selected according to classification and labelling (underlined in Table 1) was performed by two to three risk assessors independently. Evaluation and decision making for the random

selection and the organic pesticides was performed by one or two risk assessors. Decision strategies are presented in Fig. 1 and Table 4 and also in (Marx-Stoelting et al., 2011). Data were obtained as described in section 2.2 and basically, all available data were evaluated. With respect to decision strategies 1 and 2 (Options 1 and 2) adverse effects potentially related to endocrine

List of substances randomly selected from annex I of Dir 91/414/EEC. The table shows the classification as of July 2013 according to annex VI of Regulation (EU) No 1272/2008 as well as target organs and adverse effects reported in the respective EU monographs and/or EFSA conclusions.

Substance name Classification (human health)

Target organs (effects considered for decision on ED)

Remarks

Benzoic acid

Not classified

Chlorpyrifos Acute Tox 3 (H301)

Carbon dioxide Not classified

Clodinafop-p Acute Tox 4 (H302), STOT-RE 2

(H373), Skin sens 1 (H317) Cypermethrin Acute Tox 4 (H302), STOT-SE 3 (H335)

Deltamethrin Acute Tox 3 (H331, H301)

Dimethenamid-P Not classified

Ethephon

Fenpropidin

Famoxadone

Florasulam

Fluoxastrobin Not classified

Fluroxypyr Not classified for human health

Foramsulfuron Not classified

Glyphosat Eye dam 1 (H318)

Linuron Rep 1B (H360Df), Carc. 2 (H351),

Acute Tox 4 (H302), STOT RE 2 (H373)

Mancozeb Rep 2 (H361d), Skin Sens 1 (H317)

Metiram Not classified

Pethoxamid Acute Tox 4 (H302), Skin sens 1

(H317)

Propoxycarbazon Not classified for human health

Prosulfuron Acute Tox 4 (H302)

Triflusulfuron Not yet classified

Effects on liver kidney and brain at dose levels above 500 mg/kg bw/d in short term studies; no effects in long term or multigeneration studies

Nervous system/inhibition of acetyl-cholinesterase; adrenal gland (weight,

histopathology)

Liver peroxisome proliferation demonstrated in rats and mice. In vitro investigations with human hepatocytes demonstrated lack of relevance to human risk assessment Neurotoxicity, liver, thyroid effects at high dose levels in published literature

Acute Tox 4 (H332, H312) Skin corr

1B (H314)

Not yet classified

STOT-RE 2 (H373)

Not classified for human health

Nervous system (neurological effects)

Liver (biochemical and histopathological changes), decreased body weight gain

Inhibition of ChE activity in erythrocytes

Local irritation, body weight, liver (hypertrophy), spinal cord demyelination, corneal opacity

Liver (centrilobular hypertrophy, diffuse fatty changes, increased bile pigment and necrosis); blood (RBC, Heinz bodies, associated haemolysis) (spleen is a secondary target organ. Eye toxicity (lens effects) was observed in dogs)

Liver, kidney (collecting duct hypertrophy, papillary mineralisation, necrosis and inflammation), adrenals (vacuolation, dogs only, high dose effect)

Liver (main target organ in dogs, mice and rats, altered AP levels in dogs), Kidney/ urethra/bladder lesions (at high dose in rats; changes in calcium/phosphate metabolism in rat long term studies)

Kidney. No evidence for a carcinogenic or reprotoxic potential Unspecific effects: decreased body weight gain and food consumption

Liver (organ weight", clinicial chemistry, histology); salivary glands (organ weight", histology); stomach mucosa and bladder epithelium (histology); eye (cataracts)

Liver, red blood cells, testes (Leydig cell tumours), adrenal gland (histopathology) reproductive toxicity (infertility) developmental toxicity (malformations)

Thyroid (inhibition of thyroid peroxidase, histopathology, tumours), developmental toxicity (malformations at high dose levels, abortions, delayed ossification)

Thyroid (inhibition of thyroid peroxidase, hyperplasia/hypertrophy), liver (increased weight), atrophy of hindlimb muscles in rats. No evidence for a carcinogenic or reprotoxic potential

Rat: liver (induction of drug metabolising enzymes, hypertrophy) and thyroid (hypertrophy, hyperplasia, tumours*). Dog: gastrointestinal tract (stomach: vacuolation, atrophy of the muscle layer at the highest dose only). Mouse: liver (induction of drug metabolising enzymes, hypertrophy, tumours*), small intestine (swelling, rarefaction, hypertrophy of villous epithelial cells)

'tumours considered to be based on a rodent specific mechanism Decreased body weight gain, increased water intake; irritation of forestomach epithelium (rats); decreased food consumption and relative heart weights (dog)

Liver (hepatocyte hypertrophy), heart (myocardial degeneration), haematopoietic system (red blood cells decreased), uterus and mammary gland in rats at high dose levels

Body weight, food intake/decrease; liver: increased weight, elevated liver enzymes, hypertrophy; blood (erythrocytes): regenerative anaemia; Testis/increased incidence of Leydig cell hyperplasia and adenoma in male rat

European Commission (2003a) EFSA (2011a)

European

Commission

(2008)

EFSA (2005b)

European

Commission

(2005b)

European

Commission

(2002a)

European

Commission

(2003b)

EFSA (2008e)

EFSA (2007a)

(European

Commission

(2002b)

European

Commission

(2002c)

EFSA (2007b)

EFSA (2011b)

European

Commission

(2002d)

European

Commission

(2002e)

European

Commission

(2002f)

European

Commission

(2009)

European

Commission

(2005c)

European

Commission

(2006)

European

Commission

(2003e)

European

Commission

(2002g)

EFSA (2008d)

disruption were identified and mechanistic data were evaluated to see whether or not an endocrine mode of action could be plausible. If this was the case, it was analysed if human relevance should be assumed or could be excluded based on the present data as suggested by the WHO/IPCS mode of action framework (Boobis et al., 2008). If this was not the case, different regulatory conclusions were drawn. According to Option 1, the regulatory decision was based on mode of action and adversity (hazard identification)

only, taking into account human relevance but not dose levels at which effects occurred. Option 2 was based on Option 1 but additional elements of hazard characterisation were taken into account, including the dose levels at which the effects were observed and their severity to differentiate between endocrine dis-ruptors of different levels of regulatory concern (ED1 or ED2). Option 3 consisted of the classification-based interim decision criteria laid down in the plant protection products regulation. While

List of substances randomly selected from annex II of Regulation (EC) 834/2007 (i.e. substances approved for use in organic agriculture). The adverse effects and the studies these were observed in are presented as well as a potential mode of action, the LOAEL and decisions according to Option 1 and Option 2. Mode of action: ED = endocrine mode of action is plausible; ED? Endocrine mode of action cannot be excluded; ? = mode of action unclear but, considering the pathology of the adverse effects, most likely not endocrine; no ED = no endocrine mode of action. Effects are summarised in respective EFSA scientific reports for copper sulphate (EFSA, 2008a), ferric (III) phosphate (EFSA, 2004, 2005c) and rapeseed oil (RMS Spain, 2007).

Substance name Adverse effects (study)

Mode of action

Decision based on hazard identification (Option 1)

[mg/kg bw/d]

Decision based on hazard identification and characterisation (Option 2)

Copper sulphate Liver (histopathology); short- ? (EFSA, 2008a) term, oral, rat and mouse

Kidney (hyaline droplet No ED

nephropathy) short-term, oral,

Blood (changes in HGB, red ED?

blood cell and platelet counts) Developmental toxicity ED?

(malformations) non-GL dev. Tox study, ip and iv administration, mouse and hamster; effect not observed in an oral rat study

Testis (degeneration); peritoneal ED injection of copper

Ferric (III) Increased risk of diabetes ED

phosphate mellitus in patients with chronic

iron overload or long term ingestion of high doses of iron, epidemiology (EFSA, 2004) Liver (haemosiderosis) ?

Parathyroid (hyperplasia) ED?

(caused by phosphate) (EFSA, 2005c)

Rapeseed oil (RMS Body weight changes, long-term, ? Spain, 2007) oral, rat

Heart (histopathology, ?

degeneration of cardiac muscle fibre if oil contained high eruic acid levels), long-term, oral, rat

No cut-off >16

No cut-off >16

Consider cut-off >16

Consider cut-off?; Severe effect but >30 unrealistic exposure route (ip, iv) effect, not observed in an oral rat study

Consider cut-off? Severe effect but unrealistic exposure route, not observed in oral studies Cut-off

No cut-off Consider cut-off

160-1200 mg/d

No cut-off 20000 ppm

No cut-off 20000 ppm

No cut-off No cut-off

No cut-off

Severe effect but unrealistic exposure route (ip, iv), effect not observed in an oral rat study; high dose effect; no cut-off

Severe effect but unrealistic exposure route, not observed in oral studies, no cut-off Severe effect, however high dose effects; no cut-off

160 - 1200 mg/d No cut-off 160 - 1200 mg/d No cut-off

No cut-off No cut-off

the interim criteria provide clear "cut-off" for substances classified as carcinogenic category 2 (C2)/reprotoxic category 2 (R2), the decision on substances classified only as R2 and causing toxicity to endocrine organs (maybe "cut-off") would be made based on expert judgement. Box 1, Fig. 1 and Table 4 summarise the three options and compare the criteria they used. Adversity in this context has been used as defined by WHO/IPCS (2004). For severity in the present exercise the following working definition has proven helpful: ''A severe effect is an effect which is either graded as severe in nature or irreversible. Typical irreversible effects comprise (but are not limited to) tumours, irreversible forms of organ damage, malformations or infertility". In study reports an organ hyperplasia or structural change might be described as 'mild'/'slight', 'moderate' or 'severe' in accordance with respective guidelines for pathol-ogists. These descriptions were adopted when judging severity of effect.

To obtain information on the reproducibility of the respective evaluations and decisions on endocrine disruptors by the independent regulators from our department their final conclusions were compared for each of the three options. Regulators reported results for each of the substances they evaluated in keywords and in tabular form and a central evaluation table to summarise the results for each of the three options was compiled. Since only the first set of 16 substances was evaluated independently by two to three risk assessors, reproducibility has been checked only for this set of substances.

Additionally, risk assessors were asked to note difficulties occurring during the evaluation process with each of the different options. This was used to obtain information on applicability, which was examined for the first and the second set of substances accordingly. If experts disagreed in their conclusions, e. g. ''cut-off''

versus no ''cut-off'' and no majority for a certain position could be found, the stricter conclusion, i.e. ''cut-off", was chosen for decision on the substance.

2.4. Exposure calculation

According to annex II, point 3.6.5. of Reg. (EC) No 1107/2009, approval of substances with endocrine disrupting properties is not possible unless exposure under realistic conditions of use is assumed to be negligible (European Council, 2009). Hence it was important to estimate the expected dietary exposure, in order to find out for how many substances exposure was actually negligible. Calculation of overall chronic dietary exposure was performed with EFSA's pesticide residue intake model (PRIMo) for each of the 16 substances selected based on classification and labelling (Table 1) and thus potentially falling under the preliminary "cutoff" (EFSA, 2007c), but, due to limitations in time and resources not for the other substances. The maximum residue level as set by Reg. 396/2005 was compared to the default residue concentration of 0.01 mg/kg food, which is defined as negligible by Regulation (EC) 1107/2009 (European Council, 2009). In addition, with regard to operator exposure, the intended uses supported during the EU evaluation of the substances were checked to establish whether or not only applications in closed systems are foreseen and if consequently the second criterion for negligibility set by the new EU plant protection products regulation was met.

2.5. Questions addressed

The primary aim of this study was to test three different approaches of regulatory decision making on pesticides with

Fig. 1. Illustration of key elements in the decision process of each of the three tested options for regulatory decision making. ED: Endocrine disruptor; C2: Carcinogenic Category 2 according to Regulation (EC) No 1272/2008; R2: Toxic for Reproduction Category 2 according to Regulation (EC) No 1272/2008.

Table 4

Criteria used for decision making within a weight of evidence approach under the two regulatory options published by Marx-Stoelting et al., 2011 as compared to the interim criteria laid down in Reg. (EC) No 1107/2009. In this context weight of evidence should be understood as suggested by EChA 2010 as 'a process of considering the strengths and weaknesses of various pieces of information in reaching and supporting a conclusion concerning a property of the substance'.

Considered criterion BfR Option 1 BfR Option 2 (based on hazard Option 3 (interim criteria

(based on hazard identification and additional according to Reg. (EC)

identification) elements of hazard characterisation) No 1107/2009)

Option 1

Option 2

Hazard identification

Cut-off

Additional elements of hazard characterisation

Option 3

Classification-

based identification of substances

Cut-off Risk

assessment

Cut-off "Maybe" Cut off

Adverse effect Yes

Endocrine mode of action Yes

Relevance to humans Yes

Potency No

Severity No

Lead effect No

Current legal classification No

Consistency (i.e. No of species/tissues concerned) No

a Questionable.

endocrine disrupting properties for the potential impact these criteria might have on the approval of substances and also for the reproducibility and applicability of their criteria. In addition to the primary aim, the data analysed in this study were also used to address the questions, which parts of the endocrine system were affected by ED most frequently, which studies were the most sensitive in detection of ED effects and whether or not NOAELs were observed for substances with ED properties.

3. Results

3.1. Substance set pre-selected based on classification and labelling

3.1.1. Flagging of substances

In total 16 substances were pre-selected, based on their classification for carcinogenicity and/or reproductive toxicity, as by January 2011. Application of Option 1 (hazard identification only, based on adversity and mode of action) to these 16 substances resulted in the decision that 15/16 active substances have at least some endocrine disrupting properties, while 1/16 was considered not to be an endocrine disruptor. This is not surprising because the substances were selected based on the interim "cut-off" criteria which are biased towards substances affecting the HPG axis. The outcome for hazard identification did not change when the Option 2 was applied. With Option 2, it was, however, possible to further differentiate between the positive substances based on

Yes Yes

Yes No

Yes Yesa

Yes No

Yes Yes

No Yes

No No (but contributes to classification)

hazard characterisation, taking into account severity of effects and doses at which effects occur. According to the criteria for pri-oritisation of endocrine disruptors specified in the BfR proposal (Marx-Stoelting et al., 2011), 6 substances were regarded as endocrine disruptors of high priority ("ED1" category substances) and 9 as endocrine disruptors of lower priority (''ED2'' category substances). The third option (equalling the interim "cut-off" criteria) led to partially conflicting results, depending on interpretation of the interim criteria. See Table 6 for details.

3.1.2. Estimated exposure to substances

One out of the 16 substances met the definition of negligible exposure to consumers as set by Regulation EC No 1107/2009, i.e. its MRLs were at or below 0.01 mg/kg food. Consumer dietary exposure for the 16 substances was calculated according to EFSA PRIMo (rev.2), based on diets of European consumer groups and the uses granted in EU member states as of September 2010. Three of the 16 substances had an exposure of below 10% of the respective ADI. For the present project, acute dietary exposure to the 16 substances was not estimated. These substances like all others were not restricted to application in closed systems only and consequently did not meet the criterion of negligible exposure to operators. Therefore, according to the legeal definition for negligible exposure, exposure to none of the 16 substances could be regarded as negligible.

Endocrine effects observed in vivo caused by 22 randomly selected pesticides and 3 organic pesticides. The most sensitive studies (i.e. in which effects occurred at the lowest dose) are listed as well as example effects observed in pathology and histopathology. Some substances affected more than one endocrine target organ, hence the number of cases listed exceeds the number of endocrine disruptors identified. Overall thyroid and adrenals were affected most often.

Affected endocrine-related endpoint Sensitive studies Example effects Cases detected [ntota] = 25]

Adrenal gland Chronic, short term Weight changes, tumours, hypertrophy, 4

vacuolisation

Endometrium Chronic Hyperplasia 1

Fertility Dev tox, 2-generation Reduced fertility 2

Malformations Dev tox, 2-generation Wolffian duct malformation cleft palate 2

Ovary Chronic Weight changes, tumours, cysts, hypertrophy 2

Pancreas Chronic Pale coloured, diabetes, p-cell degeneration 2

Pituitary Short term Weight changes 1

Prostate Chronic Weight changes 1

Parathyroid Chronic Hyperplasia 1

Testis Chronic, 2-generation Weight changes, altered sperm parameters, 3

tumours Leydig cell hyperplasia, atrophy

Thyroid Chronic Weight changes, tumours, hypertrophy, 5

hyperplasia

Uterus Chronic Weight changes, tumours 2

Table 6

Results of the evaluation according to a preliminary categorisation which must not be used as a basis for definitive regulatory decisions. If conclusions differed between regulators, all positions are printed. Results are underlined, if the majority of regulators came to this conclusion. If no majority was found the stricter option was applied for decision making on non-approval ("cut-off"). In option 1 ''ED'' means automatically ''cut-off'. In option 2 "ED1" equals ''cut-off' and in option 3 ''yes''.

Substance Option 1 Option 2 Option 3

Results for substance set selected based on classification (cf. Table 1)

Amitrole ED ED1 Yes

Bromoxynil ED ED2 Yes/No

Chlorotoluron ED ED2 Yes/?

Dimoxystrobin ED/no ED ED2/no ED No/Yes

Epoxiconazole ED ED1 Yes

Fenpropimorph ED ED2/ED1 Yes/No/?

Ioxynil ED ED1 Yes

Isoxaflutole ED ED2 Yes/?

Linuron ED ED1 ?/Yes

Mancozeb ED ED2/ED1 ?/Yes

Maneb ED ED2 ?/Yes/No

Metconazol ED ED1 Yes/?

Molinat ED ED1 Yes

Oxadiargyl ED/no ED ED2/no ED No/Yes

Tebuconazole ED ED2/ED1 Yes/?

Tepraloxydim ED ED2 Yes/?/No

Sum ED ("cut-off") 15/16 (15/16) 15/16 (6/16) 16/16 (16/16)

Similarity in decisions of independent evaluations for ED ("cut-off") 14/16 (14/1б) 14/16 (13/16) 4/16(5/16)

Results for randomly selected substance set (cf. Table 2)

Benzoic acid No ED No ED No

Chlorpyrifos ED ED2 No

CO2 No ED No ED No

Clodinafop-P No ED No ED No

Cypermethrin ED ED2 No

Deltamethrin No ED No ED No

Dimethenamid-P No ED No ED No

Ethephon No ED No ED No

Famoxadon No ED No ED No

Fenpropidin No ED? No ED? No

Florasulam No ED? No ED? No

Fluoxastrobin No ED No ED No

Fluroxypyr No ED No ED No

Foramsulfuron No ED No ED No

Glyphosat No ED? No ED? No

Linuron (also list 1) ED ED1 Yes/?

Mancozeb (also list 1) ED ED2 Yes/?

Metiram ED ED2 No

Pethoxamid ED ED2 No

Propoxycarbazon No ED? No ED? No

Prosulfuron No ED? No ED? No

Triflusulfuron ED ED2 No

Sum ED ("cut-off") 7/22 (7/22) 7/22 (1/22) 2/22 (1/22)

Results for substances approved for use in organic agriculture (cf. Table 3)

Copper sulphate ED? ED2? No

Ferric phosphate ED? ED2? No

Rapeseed oil No ED No ED No

3.2. Substances randomly selected

3.2.1. Endocrine disrupting properties of substances

Twenty-two randomly selected substances were subjected to

analysis and regulatory decision making on endocrine disrupting properties using the three different options. Random selection seemed the most appropriate way to sample substances for the testing of different sets of criteria in an unbiased way. When Option 1 (hazard identification only, based on mode of action and adversity) was applied, 7/22 substances (~32%) were considered to have endocrine disrupting properties. This was similar when the second option (hazard identification plus characterisation taking into account severity of effects and doses at which effects occur) was applied at the hazard identification stage. However, it was additionally possible via Option 2 to differentiate between one substance (''ED1'', ~5%) of higher regulatory priority and 6 substances (''ED2'', ~27%) of lesser priority. When the classification-based Option 3 was applied, 2 substances (~10%) were identified as candidates for endocrine disruption, with one substance classified C2/R1B (~5%, direct ''cut-off') and one substance classified R2 only (~5%, may be ''cut-off'). Detailed results are presented in Table 6.

3.2.2. Estimated numbers of pesticides falling under "cut-off"

When using Option 1 for a regulatory ''cut-off", approximately

32% of the substances were affected. Even if the relevance of this finding for the entirety of approved pesticide active substances is unclear because of the limited sample size [nsample = 22, ntotal = 351], it seems very likely that with this decision strategy based only on hazard identification a large percentage of active substances (20-40%) would be considered for banning.

When Option 2 was applied, one of the identified endocrine dis-ruptors was categorised as ''ED1'' and thus would be prioritised according to the BfR proposal. The remaining 6 substances identified as potential endocrine disruptors showed endocrine disrupting effects at generally higher dose levels in the toxicity studies. Rather than being considered for hazard-based banning, the BfR proposal suggests that they are subjected to a thorough risk assessment procedure to decide on approval or non-approval.

With Option 3, one substance directly fell under the ''cut-off'' due to its classification as carcinogenic category 2 and toxic for reproduction category 1B. Another substance, labelled toxic to reproduction category 2 would fit the criteria for ''may be cutoff", and thus might be considered for hazard-based prioritisation, depending also on expert judgement concerning toxicity towards endocrine organs. Overall, for the randomly selected set of 22 active substances, our estimation showed that the interim criteria set within Reg. (EC) 1107/2009 and the proposed Option 2 flagged similar numbers of pesticide active substances for consideration of hazard-based banning (~ 5% of substances), while Option 1 clearly flagged a higher percentage of active substances.

3.3. Substances approved for organic farming

Use of pesticides approved for organic agriculture is often perceived to carry a low potential to negatively affect human health or the environment. Three of these substances were included in the present study for purposes of comparison. Adverse effects of such compounds have been detected by toxicological studies submitted for regulatory purposes or published in the open literature. These data are summarised in Table 3, together with the modes or mechanisms of action suggested for these substances, the respective NOAELs and LOAELs and the regulatory conclusions resulting from application of either Option 1 or 2.

Based on Option 1, two of three substances might be regarded as having endocrine disrupting properties since they were shown

to cause some adverse effects in intact organisms that might be due to an endocrine mechanism of action. The level of evidence was, however, higher for one of the substances and less convincing for one of the others because in that case, an inappropriate (paren-teral) route of exposure was used in eliciting the adverse effects (see discussion and Tables 3 and 6 for details).

According to Option 2, the substances causing adverse effects on endocrine organs via a potential endocrine mode of action would also be considered as endocrine disruptors. However, due to the fact that all adverse effects were confined to higher dose levels none of the investigated organic pesticides would be likely to be regarded as being of high regulatory concern (''ED1''). Consequently, according to Option 2, all of these substances would be evaluated further by thorough risk assessment procedures.

Since the three example substances are not classified or labelled for reproductive toxicity category 2 or carcinogenicity category 2 according to Regulation (EC) No 1272/2008, none of them would be considered for ''cut-off" as endocrine disruptors under the classification-based Option 3. When interpreting these results, the very low sample size must be taken into consideration.

3.4. Reproducibility of decisions and ease of application of criteria

The criteria laid down in the three different regulatory options were applied by two to three regulators independently to the subset of 16 substances that had been pre-selected based on classification and labelling (Table 1). Decisions were compared among regulators to see whether they would come to similar scientific evaluations and regulatory conclusions. Additionally, regulators were asked to record the difficulties they had when applying each of the options to subset 1 and 2. Results on the reproducibility of the scientific evaluations and regulatory decisions and on the applicability (ease of decision making) are presented separately below.

With Option 1 and 2, the regulators came to similar conclusions on the identification of endocrine disrupting properties of the substances in 14/16 cases (87.5%). Concerning the regulatory consequence (''cut-off", no ''cut-off", no decision possible) all regulators arrived at similar conclusions in 14/16 cases (87.5%) with Option 1 and to a slightly lesser degree (13/16 cases; 81.3%) with Option 2. In cases for which regulators came to different results with Option 2 (3/16,18.7%), the regulatory conclusions differed only by one step ('ED1' vs. 'ED2').

Surprisingly, the classification-based Option 3 was the one where deviations between different regulators were most pronounced. This might be explained by the fact that the interim criteria toxic for reproduction category 2 and having toxic effects on the endocrine organs as a basis for consideration as having endocrine-disrupting properties (according to point 3.6.5 of annex 2 of Regulation (EC) 1107/2009) is prone to very different interpretations by different regulators.

With respect to applicability the main difficulties arose from uncertainties concerning the mode of action (50% of cases with adverse effects potentially related to ED), the judgement on human relevance (according to the WHO/IPCS human relevance framework used in this exercise, the default assumption always is relevance), and conflicting data. Problems in decision making were noted most often for Option 1, followed by Option 3. Fewer difficulties concerning the applicability in terms of final decision making were noted with Option 2, most likely because here, even in the presence of uncertainties, the inclusion of additional decision criteria (elements of hazard characterisation including potency) supported a higher level of confidence for regulatory decision making.

3.5. Illustration of decision process

In order to illustrate the application and comparative performance of different assessment and decision criteria, we present two practical examples from this study, one clearly positive substance and one substance used in organic farming. These examples also demonstrate the interaction of scientific evaluation and regulatory decision making.

3.5.1. Linuron

Linuron has been approved as an herbicide in the EU. It is known to have anti-androgenic properties and is therefore included as a positive control in the US EPA endocrine disruptor screening program (EDSP) or for validation of methods capable of detecting anti-androgenic properties such as the Hershberger assay (Moon et al., 2009, 2010). The decision process on linuron as conducted in the present study is illustrated in Fig. 2. The analysis was based on the EU monograph (RMS UK, 1996) and the respective EFSA conclusion (European Commission, 2002f). Additionally, a literature search was performed and several publications, especially on the mode of action, were taken into account for the decision process (Freyberger et al., 2010; Freyberger and Schladt, 2009; Lambright et al., 2000; McIntyre et al., 2002a,b; Tinwell et al., 2007; Turner et al., 2003; Vinggaard et al., 1999, 2000).

In the context of data evaluation, a number of adverse effects potentially related to endocrine disruption were detected, including tumours (Leydig cells, ovaries, uterus, endometrium, liver),

foetal malformations (Wolffian duct derivatives), reduced fertility or infertility, target organ effects (testis atrophy, adrenal gland hypertrophy).

Mechanistic data were evaluated, confirming an anti-andro-genic mode of action (Freyberger et al., 2010; Kang et al., 2004; Lambright et al., 2000; Tinwell et al., 2007; Wilson et al., 2009) and partially excluding other MoA like aromatase inhibition or estrogenicity (Vinggaard et al., 1999, 2000).

A further parameter addressed in the decision process was human relevance. Since hormone receptors are quite conserved among various species and one study showed a higher affinity of linuron to the human androgen receptor than to the rat androgen receptor (Lambright et al., 2000) it seems plausible to assume relevance to humans, which would have been the default assumption according to the WHO/IPCS mode of action framework (Boobis et al., 2008) in the absence of data anyway. To sum up the results of the evaluation with respect to hazard identification, linuron would have to be regarded as a substance causing adverse effects in intact organisms by an endocrine disrupting mode of action with relevance to humans.

In the last step of the analysis a regulatory decision has to be made according to one of the three options. According to Option 1, linuron would have to be regarded as an endocrine disruptor and would consequently fall under the "cut-off". Since linuron produced a range of severe effects at generally low exposure concentrations it also would be considered an endocrine disruptor of high regulatory concern ("ED1") according to Option 2, and would thus be proposed to be flagged for "cut-off" with Option 2. Similarly,

Rat reproductive toxicity Fertility Implant number litter size J., testicular atrophy, genital tract malformations in males, Leydlg cell hyperplasia, adrenal gland hypertrophy

Rat developmental toxicity Abortions t, skeletal defects t

2-yr rat Leydig cell adenoma, uterine adenocarcinoma and ovarian tumors, endometrial hyperplasia

18-mo mouse Liver adenomas

90-d rat RBC i, haemosiderin deposition

Published non-GL dev tox studies, rat Genital tract malformations In males, nipple retention, testis hypoplasia

Mechanistic data

Study Result

Hershberger bloassays Positive

In vitro receptor binding and transactivatlon assays AR binding and inhibition, higher affinity to hAR than to rAR

Published non-GL mech, rat Decrease In foetal testosterone production

90-d rat Methaemoglobin formation

Default: Human relevance

Option 1 : ED

Option 2: ED 1

Study ED LOAEL ED category

Rat reproductive toxicity >10, <100 mg/kg/day 1-2

Rat developmental toxicity >30, <300 mg/kg/day 2

2-yr rat < 5 mg/kg/day 1

18-mo mouse >50 mg/kg/day NA

Option 3: C2R1B

Fig. 2. Decision logic for the example substance linuron. After evaluation of all data an endpoint- based hazard analysis is conducted (grey boxes). This is followed by an analysis of the mechanisms, which might have caused toxicity (green boxes). This mechanistic evaluation includes criteria like adversity as well as the establishment of a mode/mechanism of action in animals. If this mechanism is related to ED, its relevance to humans will be analysed with the default assumption being relevance to humans (blue box). In the last step (red boxes) a regulatory decision is made. This could be based on hazard identification alone (Option 1). Alternatively, this decision might additionally take into account elements of hazard characterisation (Option 2). Option 3 represents the classification based decision as suggested by the interim criteria set by the EU regulation. GL: guideline, h: human, r: rat, AR: androgen receptor, yr: year, mo: month, d: day, RBC: red blood cells. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

when the third option was applied, linuron also fell under the ''cutoff' criterion for being labelled toxic to reproduction category 1B. Based on these findings, it can be concluded that all three sets of criteria were able to flag linuron as positive.

3.5.2. Ferric phosphate

Ferric phosphate has been approved as a molluscicide in the EU. It is additionally included in annex II of Regulation (EC) No 834/ 2007 for use in organic agriculture. Iron is an essential trace element. It is of physiological importance for oxygen transport and storage and acts as an essential co-factor in many enzymes, especially those involved in redox reactions. The WHO regards iron deficiency as a serious global health problem, leading to anaemia, adverse pregnancy outcomes and reduced immune function (WHO, 2008). However, free iron is highly reactive and needs to be closely controlled in living cells. Disorders associated with chronic iron overload in individuals homozygous for hereditary haemochromatosis include diabetes mellitus. In addition, some cases have been reported of patients who developed secondary haemochromatosis, among others with diabetic manifestation, due to long-term high-dose medical treatment with iron (e. g. 160-1200 mg iron/day for over a decade), although subjects with a disposition for hereditary haemochromatosis might have contributed to these cases (reviewed by EFSA, 2004). Further, a number of epidemiological studies have indicated an association between haem iron intake from red meat and type II diabetes mellitus (reviewed by EFSA, 2004). A more recent meta-analysis concluded that: ''Higher haem iron intake and increased body iron stores were significantly associated with a greater risk of type II diabetes mellitus.'' Dietary total iron, non-haem iron, or supplemental iron intake were not significantly associated with type II diabetes (Bao et al., 2012). Some experimental evidence is provided by studies involving animal models. For example, Awai et al. (1979) demonstrated induction of diabetes in animals by par-enteral administration of ferric nitrilotriacetate as a model of experimental haemochromatosis. Dietary iron restriction or iron chelation was shown to protect from diabetes and loss of beta-cell function in a mouse model for type II diabetes (obese ob/ob lep-/-mice; Cooksey et al., 2010).

Thus, although the information from epidemiological studies is partially conflicting, it appears plausible that excessive iron intake or iron overload may be associated with adverse effects on the endocrine system. Accordingly, both option 1 and option 2 might identify iron as an endocrine disruptor. Option 2, however, would tend to categorise iron as ED2, since diabetes has been associated with excessive iron intake or cases of iron overload.

With respect to the classification-based Option 3, neither iron nor phosphate nor ferric phosphate is classified for reproductive toxicity or carcinogenicity. Based on these findings, it can be concluded that ferric phosphate might be flagged for ''cut-off'' for endocrine disruption under Option 1, but not under the Options 2 and 3.

3.6. Effects considered suggestive of endocrine disrupting properties and sensitivity of studies

As suggested by the BfR criteria (Marx-Stoelting et al., 2011) all available data were included in the present study and all potentially endocrine-related effects were taken into account. Isolated effects on organs like liver or kidneys that are not only endocrine glands but have numerous other functions were not included automatically as endocrine effects, e.g. an increase in liver weight alone was not regarded as endocrine effect if no other findings were reported. However, if that increase in liver weight was accompanied for example by changes in blood hormone levels, a potential down-stream consequence of an altered metabolic capacity of

the liver, the effect was taken into account for the evaluation of endocrine disruptive properties. Table 5 summarises the effects leading to endocrine disruptor identification for the random selection [n = 22] and the organic pesticides [n = 3]. The first set of substances (classification-based selection) was excluded from this analysis because among substances already classified for reproductive toxicology a clear bias for substances affecting the HPG axis might be expected. The results show that even in this small group of substances, many tissues besides those with functional relation to the HPG axis are affected by endocrine mediated activity, especially the adrenals and the thyroid but also the pancreatic system.

Most interestingly, if adverse effects pointing to an endocrine mode of action occurred, they were usually seen in several studies or in several species or tissues and as part of a set of effects all supporting an endocrine-related mode of toxicity. As an example, linu-ron showed effects on various organs and functions, which is fully in line with the postulated anti-androgenic MoA. This observation could have two implications:

(A) As already practiced for carcinogenicity the number of affected species, sexes and tissues could contribute as an additional criterion ("consistency") for regulatory decision making on endocrine disruption (see Discussion).

(B) It may be possible to formulate a hypothesis on the mode of action and to test it for strength, coherence and plausibility by a thorough investigation of the toxicological data presented in the regulatory data package, in some cases even without the need for further mechanistic data. This could be applicable to some compounds for which extensive in vivo data are available, such as pesticides and biocides.

In the case of linuron and other substances it was found that the highest tier study from the OECD conceptual framework for the testing of endocrine disruptors (OECD, 2002) was not the most sensitive study. The OECD framework makes a distinction between different levels with the highest tier being multigeneration reproductive toxicity studies (in this specific case a two-generation reproductive toxicity study). Table 5 additionally summarises the study types (e.g. short term or chronic) that were found to be the most sensitive for detection of endocrine related effects in the data packages analysed within the present study. With the available data package some effects are easier to detect by a chronic toxicity study than by a two-generation reproductive toxicity study and vice versa.

3.7. No observed adverse effect levels for the evaluated substances

In this survey, reports summarising approximately 300 regulatory toxicity studies were evaluated. In only very few of these studies, no NOAEL was established. However, in virtually all such cases a follow-up study using lower dose levels was conducted to establish an NOAEL, e.g. the long-term NOAEL for linuron. In addition, several studies not showing a NOAEL were identified from the literature. However, virtually all of these latter studies were not conducted to determine a NOAEL but to analyse mechanisms of toxicity and most of them used only a single effective dose level. Such studies did not provide information on dose-response relationship. NOAELs were also found for the endocrine-related effects. NOAELs in one study were usually confirmed by other studies in the regulatory data package. The data requirements for pesticides include at least two to three studies on short-term toxicity in different species, studies on chronic toxicity and carcinogenicity (usually in rats and mice), studies on teratogenicity in two different species (usually rats and rabbits), a study on reproductive toxicity (generally a 2-generation study in the rat); all of them with at least three dose levels and a control group so that the complete package

covers a broad dose range, since dose levels often differ between the studies.

4. Discussion

4.1. Overall comparison of different sets of criteria

Since no gold standard for identification of substances with ED properties exists, a formal validation of any suggested set of criteria is not possible. However, application of the three options for criteria to a random selection of pesticides provides a rough estimate of an outcome regarding implementation in the context of a more extensive set of substances. Evaluation of a subset of substances selected based on classification and labelling by different regulators independently yielded an impression on reproducibility. In addition, the difficulties encountered with the application of the criteria for regulatory decision making could be compared for the three options applied to two subsets of substances. In conclusion, none of the sets of criteria could be considered perfect. Option 1 (hazard identification only) was associated with a high level of sensitivity (i.e. number of substances flagged for regulatory "cut-off" was high), and reproducibility of results was good. Problems generally associated with hazard identification included difficulties with mode of action or human relevance assessment. Option 2 (hazard identification plus characterisation) scored high on sensitivity for hazard identification, while it enabled prioritisa-tion of substances to be regarded as of lower or higher concern. In addition, reproducibility was comparable to Option 1. Applicability of Option 2 in terms of facilitating decision making was, however, regarded as better due to the integration of elements of hazard characterisation.

When applied within a random selection of substances, Option 3 (only consideration of substance classification) showed low sensitivity, presumably due to a focus on substances with reproductive toxicity. Application of Option 3 regarding the precisely defined "cut-off" criteria (classification as carcinogenic category 2 and toxic for reproduction category 2) can be regarded as easy since only the classification status or requirement for classification of the substance had to be checked to come to a conclusion. The other criteria within Option 3 (toxic for reproduction category 2 and toxic to endocrine organs), may be "cut-off", leave more room for interpretation and expert judgement. Accordingly, it was often difficult to come to a regulatory conclusion based on the may be "cut-off" criteria, and reproducibility was low.

4.2. Problems associated with different options for regulatory decision making

To examine the likelihood that substances should be flagged for endocrine disrupting properties and the reliability of such an identification, the present study tested different options for regulatory decision making. Several problems were identified as being associated with some of these different options in terms of sensitivity, reproducibility and applicability. Option 1 (taking into account elements of hazard identification only), while very sensitive, was prone to flag a high number of substances for hazard-based banning as endocrine disruptors. Option 3 was shown to have a limited reproducibility if applied by different regulators concerning the interpretation of the "may be'' criteria and was also shown to be less sensitive for a random set of substances. In this context it should be noted that some substances showing tumours in endocrine organs are classified as carcinogenic category 2 but would be missed by Option 3. Some examples are listed in Table 1.

Reproducibility appeared similar for Option 1 and Option 2 if these options were applied to the set of 16 pre-selected substances

based on classification and labelling. This is not surprising, as this pre-selection is clearly biased for substances affecting the HPG axis, hence resulting in a high number of easily identifiable substances with endocrine disrupting properties. For a randomly-selected group of substances, less problems in decision making (as indicated by a better applicability) and a better reproducibility would be expected for Option 2, as more criteria are taken into account, supporting a higher confidence in decisions. Overall, Option 2 (taking into account hazard identification plus additional elements of hazard characterisation) enabled to differentiate between substances of various potencies and thus to prioritise for further regulatory measures.

4.3. Additional elements of hazard characterisation

Since a hazard-based approach for non-approval of substances with certain properties is taken by recent European regulations, it would be consistent to consider both, elements of hazard identification and hazard characterisation, including potency and severity of effects. Potency has been used in regulatory hazard assessment since the 1960s and has been implemented in internationally harmonised regulations for chemical hazard assessment (European Council, 1967). It is a critical criterion for determination of acute toxicity and (besides severity of effects) also for specific target organ toxicity after repeated dosing (European Council, 2008). Potency relates to the dose levels at which certain effects occur. Potency has long been discussed as a potential criterion for evaluation of EDs (e.g. Borgert et al., 2012; Sharpe, 2010). Application of a weight of evidence decision matrix has been suggested for the toxicological assessment of EDs by a EU report (Kortenkamp et al., 2012). Overall, it seems feasible to include potency when making regulatory decisions on endocrine disruptors in combination with other elements of hazard characterisation, such as severity, within such a weight of evidence matrix. Option 2 according to the BfR criteria suggests that besides the dose that is necessary to induce adverse effects also the severity of effects should be taken into account when a regulatory decision on substances with potential endocrine disrupting properties is required. This criterion has been demonstrated to be useful in the present exercise.

The consistency, e.g. number of species, sexes and animals affected as well as the number of studies or tissues showing positive results, might serve as an additional criterion to consolidate concern regarding a substance, i.e. a substance causing testis atrophy, Leydig cell hyperplasia and tumours in mice and rats at low dose levels might be associated with a higher level of concern than a substance causing only a minimal effect in one study. Thus, consistency could contribute as an additional element of evaluation within a weight of evidence approach.

4.4. Windows of susceptibility and data requirements

In keeping with the basic principles of toxicology, a recent evaluation of data on reproductive toxicants indicated that both the dose and the window of exposure determine the outcome (Piersma et al., 2011). Accordingly, if a substance causes critical adverse effects within a window of susceptibility, this still requires a dose above a certain level (Piersma et al., 2011). In the present evaluation, for the evaluated substances showing endocrine disruptive or developmental toxic properties, dose responses were reported for these adverse effects that yielded no observed adverse effect levels under the conditions of the available studies. The dose causing developmental effects might, however, be lower than the dose causing maternal toxicity. Hence the effects induced during critical windows of development must be analysed in addition to toxic effects observed in short- or long-term toxicity studies. For

both plant protection products and biocides this has been a mandatory requirement for more than two decades. Directive No 91/ 414/EC and Regulation (EC) No 1107/2009 stipulate that at least two studies on developmental toxicity in different species are conducted as well as at least one multigeneration reproductive toxicity study. In these studies animals are exposed in utero during critical windows of susceptibility.

The quality of data that is used for regulatory decision making is another field of discussion (Myers et al., 2009). Quality differences that impact the informative value of data for regulatory purposes are most obvious in the area of documentation and experimental design (use of relevant routes of exposure, group size). While it is not regarded as an important parameter, whether the experimental work was conducted in an industry laboratory or a university research environment, a comprehensive documentation and presentation of the important parts of an experimental work is indispensable if this should contribute to a regulatory evaluation. For example, a publication that does not allow to clearly distinguish whether the active substance or a plant protection product containing it was used in the experiments, has low value for the purpose of regulating the active substance. In this context, recent progress towards the development of guidelines for the reporting of non-standard tests has been made (Beronius et al., 2014; Kilkenny et al., 2010). Non-standard data may well be considered for regulatory risk assessment, especially for closing of potential data gaps on the mode or mechanism of action of a specific substance, which is regarded highly important in the context of endocrine disruption.

Furthermore, studies using appropriate routes of exposure have to be ascribed a higher regulatory value than studies with less relevant exposure routes. What will be considered appropriate depends on the protection goal and the realistic route of exposure. For the evaluation of the risk due to dietary exposure, oral studies are of highest relevance. However, operators spraying pesticides or biocides may not be appropriately protected when only oral studies are considered so that studies with dermal and inhalative routes of exposure might become preferable. Other routes of exposure (i.v., i.p.) may be considered for understanding a mechanism of toxicity but are otherwise of limited value for classification and labelling or derivation of reference values for pesticides. The necessity to apply the same restrictions to the evaluation of endocrine disrupting properties can be illustrated with copper sulphate. Copper compounds are approved as fungicides and are also used in organic agriculture. Studies are available in which copper was applied orally or dermally, i.e. via the relevant and realistic exposure routes under the proposed conditions of use showing no testicular toxicity. However, intraperitoneal injections of copper caused testis atrophy (Chattopadhyay et al., 2005). Testicular atrophy is a severe effect and indicative of a potentially endocrine-related mode of toxicity. Findings from studies using non-standard routes may provide useful information but should be considered with caution. A decision based on such results for any kind of endocrine disruptors should be taken case by case.

4.5. Considering modes of action

For nearly all substances analysed in this exercise, some adverse effects have been observed. While the detection of toxic effects is favoured by the design of regulatory studies, the identification of a mode/mechanism of action remains difficult, since mechanistic data are lacking for most substances. Therefore, it is unclear in many cases, whether an adverse effect is mediated by endocrine activity. Since mode/mechanism of action is central to decisions on potential endocrine disrupting properties, a pragmatic way to evaluate mode/mechanism of action based on limited data was used in the present study to come to conclusive decisions.

An important point is the level of evidence that should be regarded necessary to trigger the assumption of an endocrine mode of action. One option to address this issue would be to use endocrine disruption as a mechanistic default assumption as suggested in a previous publication (Marx-Stoelting et al., 2011). A second option would be to assign endocrine disrupting properties only if a respective mode/mechanism of action was proven. As an intermediate way between these extreme options we decided to assume endocrine disrupting properties for the purpose of this exercise if a respective mode of action seemed plausible as proposed previously (BfR and CRD, 2011), for example if glands were directly affected or a combination of observations suggested an endocrine mode of action (e.g. liver effects and changes in blood hormone levels).

Results of this study clearly show that substances may not only affect target organs related to the HPG axis but also other parts of the endocrine system. If regulating endocrine disruptors is taken seriously this needs to be addressed by increased efforts to develop and validate tests for other subsystems like the hypothalamic-pituitary-thyroid- or the hypothalamic-pituitary-adrenal-axis, the pancreatic system, or the hormonal regulation of metabolic homeo-stasis and cardiovascular parameters.

5. Conclusion

Overall, the exercise described in this paper demonstrates that the interim criteria do not seem sufficient to regulate ED but that a combination of criteria for hazard identification with additional criteria of hazard characterisation (such as severity and potency) allows prioritising and differentiating between pesticides for a hazard-based regulation. Hence, it is considered necessary to integrate such elements into a decision matrix to be used within a weight of evidence approach for the toxicological categorisation of relevant endocrine disruptors. Moreover, the necessity to consider all parts of the endocrine system for regulatory decision making on endocrine disruption is highlighted. However, to obtain a better understanding of the potential impact, a larger number of substances, preferably randomly selected, would have to be analysed as proposed in the 2014 roadmap for defining criteria for identifying endocrine disruptors by the European Commission (European Commission 2014).

List of regulations

Regulation (EC) No 1107/2009: European regulation concerning the placing of plant protection products on the market. Regulation (EC) No 1272/2008: European regulation on classification, labelling and packaging of substances and mixtures. Regulation (EC) No. 1907/2006: European regulation concerning the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH).

Regulation (EC) No 396/2005: European regulation on maximum residue levels of pesticides in or on food and feed of plant and animal origin.

Regulation (EC) No 528/2012: European regulation concerning the making available on the market and use of biocidal products.

References

ANSES, 2012. Opinion of the French Agency for Food, Environmental and

Occupational Health & Safety. ANSES Opinion Request No 2012-SA-0033. Awai, M., Narasaki, M., Yamanoi, Y., Seno, S., 1979. Induction of diabetes in animals by parenteral administration of ferric nitrilotriacetate. A model of experimental hemochromatosis. Am. J. Pathol. 95, 663-673.

Bao, W., Rong, Y., Rong, S., Liu, L., 2012. Dietary iron intake, body iron stores, and the risk of type 2 diabetes: a systematic review and meta-analysis. BMC. Med. 10, 119.

Bars, R., Broeckaert, F., Fegert, I., Gross, M., Hallmark, N., Kedwards, T., Lewis, D., O'Hagan, S., Panter, G.H., Weltje, L., Weyers, A., Wheeler, J.R., Galay-Burgos, M., 2011. Science based guidance for the assessment of endocrine disrupting properties of chemicals. Regul. Toxicol. Pharmacol. 59 (1), 37-46.

Beronius, A., Ruden, C., Hanberg, A., Hakansson, H., 2009. Health risk assessment procedures for endocrine disrupting compounds within different regulatory frameworks in the European Union. Regul. Toxicol. Pharmacol..

Beronius, A., Molander, L., Ruden, C., Hanberg, A., 2014. Facilitating the use of nonstandard in vivo studies in health risk assessment of chemicals: a proposal to improve evaluation criteria and reporting. J. Appl. Toxicol. 34, 607-617.

BfR and CRD, 2011. Regulatory Definition of an Endocrine Disrupter in Relation to Potential Threat to Human Health - Proposal Applicable in the Regulatory Context of Plant Protection Products, Biocidial Products, and Chemicals Targeted within Reach.

Boobis, A.R., Doe, J.E., Heinrich-Hirsch, B., Meek, M.E., Munn, S., Ruchirawat, M., et al., 2008. IPCS framework for analyzing the relevance of a noncancer mode of action for humans. Crit. Rev. Toxicol. 38 (2), 87-96.

Borgert, C., Sargent, E., Casella, G., Dietrich, D., McCarty, L., Golden, R., 2012. The human relevant potency threshold: reducing uncertainty by human calibration of cumulative risk assessments. Regul. Toxicol. Pharmacol. 62 (1), 313-328.

Chattopadhyay, A., Sarkar, M., Biswas, N.M., 2005. Dose-dependent effect of copper chloride on male reproductive function in immature rats. Kathmandu Univ. Med. J. (KUMJ) 3 (4), 392-400.

Chemtrust, 2011. CHEM Trust's Contribution to the Ongoing Debate on Criteria for EDCs.

Cooksey, R.C., Jones, D., Gabrielsen, S., Huang, J., Simcox, J.A., Luo, B., Soesanto, Y., Rienhoff, H., Abel, E.D., McClain, D.A., 2010. Dietary iron restriction or iron chelation protects from diabetes and loss of beta-cell function in the obese (ob/ ob lep-/-) mouse. Am. J. Physiol. Endocrinol. Metab. 298, E1236-E1243.

Danish Ministry of the Environment, 2011. Establishment of Criteria for Endocrine Disruptors and Options for Regulation.

ECETOC, 2009. Guidance on Identifying Endocrine Disrupting Effects. ECETOC. 106, 1-133. B. Technical Report.

EFSA, 2004. Opinion of the scientific panel on dietetic products, nutrition and allergies on a request from the commission related to the tolerable upper intake level of iron. EFSAJ. 125,1-34.

EFSA, 2005a. Conclusion on the peer review of diuron. EFSA Sci. Rep. 25, 1-58.

EFSA, 2005b. Conclusion regarding the peer review of the pesticide risk assessment of the active substance clodinafop. EFSA Sci. Rep. 34, 1-78.

EFSA, 2005c. Opinion of the scientific panel on dietetic products, nutrition and allergies on a request from the commission related to the tolerable upper intake level of phosphorous. EFSA J. 233, 1-19.

EFSA, 2006. Conclusion on the peer review of metconazole. EFSA Sci. Rep. 64,1-71.

EFSA, 2007a. Conclusion on the peer review of the pesticide risk assessment of the active substance fenpropidin. EFSA Sci. Rep. 124,1-84.

EFSA, 2007b. Conclusion regarding the peer review of the pesticide risk assessment of the active substance fluoxastrobin. EFSA Sci. Rep. 102,1-84.

EFSA, 2007c. Pesticide Residues Intake Model for assessment of acute and chronic consumer exposure to pesticide residues-rev.2.

EFSA, 2008a. Conclusion on the peer review of copper compounds. EFSA Sci. Rep. 187, 1-101.

EFSA, 2008b. Conclusion on the peer review of epoxiconazole. EFSA Sci. Rep. 138,180.

EFSA, 2008c. Conclusion regarding the peer review of the pesticide risk assessment of the active substance fenpropimorph. EFSA Sci. Rep. 144, 1-89.

EFSA, 2008d. Conclusion regarding the peer review of the pesticide risk assessment of the active substance triflusulfuron. EFSA Sci. Rep. 195,1-115.

EFSA, 2008e. Conclusion regarding the peer review of the pesticide risk assessment of the active substance ethephon. EFSA Sci. Rep. 174,1-65.

EFSA, 2008f. Conclusion regarding the peer review of the pesticide risk assessment of the active substance tebuconazole. EFSA Sci. Rep. 176,1-109.

EFSA, 2010a. Conclusion on the peer review of the pesticide risk assessment of the active substance etridiazole. EFSAJ. 8 (10), 1823.

EFSA, 2010b. Conclusion on the peer review of the pesticide risk assessment of the active substance myclobutanil. EFSAJ. 8 (10), 1682.

EFSA, 2010c. Conclusion on the peer review of the pesticide risk assessment of the active substance cyproconazole. EFSA J. 8 (11), 1897.

EFSA, 2011a. Conclusion on the peer review of the pesticide risk assessment of the active substance chlorpyrifos. EFSA J. 9 (1), 1-14.

EFSA, 2011b. Conclusion on the peer review of the pesticide risk assessment of the active substance fluroxypyr. EFSA J. 9 (3), 2091.

EFSA Scientific Committee, 2013. Scientific Opinion on the hazard assessment of endocrine disruptors: Scientific criteria for identification of endocrine disruptors and appropriateness of existing test methods for assessing effects mediated by these substances on human health and the environment. EFSAJ. 11 (3), 3132.

Endocrine Disrupters Expert Advisory Group, 2013. Key scientific issues relevant to the identification and characterisation of endocrine disrupting substances. JRC. JRC Scientific and Policy Reports EUR 25919 EN.

European Commission, 2001. Review report for the active substance amitrole. EC Review Reports 6839/VI/97 final.

European Commission, 2002a. Review report for the active substance deltamethrin. EC Review Reports 6504/VI/99 final, 1-78.

European Commission, 2002b. Review report for the active substance famoxadone. EC Review Reports 6505/VI/99 final, 1-32.

European Commission, 2002c. Review report for the active substance florasulam. EC Review Reports 1406/2001 final, 1-18.

European Commission, 2002d. Review report for the active substance foramsulfuron. EC Review Reports SANC=/10324/2002 final, 1-21.

European Commission, 2002e. Review report for the active substance glyphosate. EC Review Reports 6511/VI/99 final, 1-56.

European Commission, 2002f. Review report for the active substance linuron. EC Review Reports 7595/VI/97 final.

European Commission, 2002g. Review report for the active substance prosulfuron. EC Review Reports 3055/99 final.

European Commission, 2003a. Review report for the active substance benzoic acid. EC Review Reports 1396/2001 final, 1-22.

European Commission, 2003b. Review report for the active substance dimethenamid-p. EC Review Reports 1402/2001 final, 1-24.

European Commission, 2003c. Review report for the active substance isoxaflutole. EC Review Report SANCO/3136.

European Commission, 2003d. Review report for the active substance molinate. EC Review Reports SANCO/3047.

European Commission, 2003e. Review report for the active substance propoxycarbazone. EC Review Reports SANCO/4067/2001 final.

European Commission, 2004a. Review report for the active substance bromoxynil. EC Review Reports SANCO/4347/2000 final.

European Commission, 2004b. Review report for the active substance ioxynil. EC Review Reports SANCO/4349.

European Commission, 2004c. Review report on the active substance tepraloxydim. EC Review Report SANC0/10388(Rev 4), 1-56.

European Commission, 2005a. Review report for the active substance chlorotoluron. EC Review Reports SANCO/4329/2000 final.

European Commission, 2005b. Review report for the active substance cypermethrin. EC Review Reports SANC0/4333/2000 final, 1-41.

European Commission, 2005c. Review report for the active substance metiram. EC Review Reports SANCO/4059/2001 final.

European Commission, 2006. Review report for the active substance pethoxamid. EC Review Reports SANC0/10396/2002 final.

European Commission, 2007. Review report for the active substance flusilazole. EC Review Reports 6850/VI/97 final.

European Commission, 2008. Review report for the active substance carbon dioxide. EC Review Reports 2987/08 rev. 1,1-41.

European Commission, 2009. Review report for the active substance mancozeb. EC Review Reports SANC0/4058/2001 - final, 1-63.

European Commission, 2014. Defining criteria for identifying Endocrine Disruptors in the context of the implementation of the Plant Protection Product Regulation and Biocidal Products Regulation. Available at http://ec.europa.eu/smart-regulation/impact/planned_ia/docs/2014_env_009_endocrine_disruptors_en. pdf.

European Council, 1967. Council Directive 67/548/EEC.

European Council, 2006. Regulation (EC) No 1907/2006. 1907/2006.

European Council, 2007. Regulation (EC) No 834/2007. 834/2007.

European Council, 2008. Regulation (ec) No 1272/2008.1272/2008.

European Council, 2009. Regulation (ec) 1107/2009.1107/2009.

European Parliament and Council, 2012. Regulation (EC) No 528/2012. Regulation (EC) No 528/2012 concerning the making available on the market and use of biocidal products.

Freyberger, A., Schladt, L., 2009. Evaluation of the rodent Hershberger bioassay on intact juvenile males-testing of coded chemicals and supplementary biochemical investigations. Toxicology 262 (2), 114-120.

Freyberger, A., Witters, H., Weimer, M., Lofink, W., Berckmans, P., Ahr, H.J., 2010. Screening for (anti)androgenic properties using a standard operation protocol based on the human stably transfected androgen sensitive PALM cell line. First steps towards validation. Reprod. Toxicol. 30 (1), 9-17.

Kang, I.H., Kim, H.S., Shin, J.H., Kim, T.S., Moon, H.J., Kim, I.Y., Choi, K.S., Kil, K.S., Park, Y.I., Dong, M.S., Han, S.Y., 2004. Comparison of anti-androgenic activity of flutamide, vinclozolin, procymidone, linuron, and p, p'-DDE in rodent 10-day Hershberger assay. Toxicology 199 (2-3), 145-159.

Kilkenny, C., Browne, W.J., Cuthill, I.C., Emerson, M., Altman, D.G., 2010. Improving bioscience research reporting: the ARRIVE guidelines for reporting animal research. J. Pharmacol. Pharmacother. 1, 94-99.

Kortenkamp, A., Evans, R., Martin, 0. V., McKinlay, R., Orton, F., and Rosivatz, E., 2012. State of the Art Assessment of Endocrine Disrupters. EU Commission, DG Environment.

Lambright, C., 0stby, J., Bobseine, K., Wilson, V., Hotchkiss, A.K., Mann, P.C., Gray Jr., L.E., 2000. Cellular and molecular mechanisms of action of linuron: an antiandrogenic herbicide that produces reproductive malformations in male rats. Toxicol. Sci. 56 (2), 389-399.

Marx-Stoelting, P., Pfeil, R., Solecki, R., Ulbrich, B., Grote, K., Ritz, V., Banasiak, U., Heinrich-Hirsch, B., Moeller, T., Chahoud, I., Hirsch-Ernst, K.I., 2011. Assessment strategies and decision criteria for pesticides with endocrine disrupting properties relevant to humans. Reprod. Toxicol. 31 (4), 574-584.

McIntyre, B.S., Barlow, N.J., Foster, P.M., 2002a. Male rats exposed to linuron in utero exhibit permanent changes in anogenital distance, nipple retention, and

epididymal malformations that result in subsequent testicular atrophy. Toxicol. Sci. 65 (1), 62-70.

McIntyre, B.S., Barlow, N.J., Sar, M., Wallace, D.G., Foster, P.M., 2002b. Effects of in utero linuron exposure on rat Wolffian duct development. Reprod. Toxicol. 16 (2), 131-139.

Moon, H.J., Kang, T.S., Kim, T.S., Kang, I.H., Ki, H.Y., Kim, S.H., Han, S.Y., 2009. OECD validation of phase 3 Hershberger assay in Korea using surgically castrated male rats with coded chemicals. J. Appl. Toxicol. 29 (4), 350-355.

Moon, H.J., Kang, T.S., Kim, T.S., Kang, I.H., Kim, S.H., Han, S.Y., 2010. OECD validation of phase-3 Hershberger assay using the stimulated weanling male rat in Korea. J. Appl. Toxicol. 30 (4), 361-368.

Myers, J.P., Vom Saal, F.S., Akingbemi, B.T., Arizono, K., Belcher, S., Colborn, T., Chahoud, I., Crain, D.A., Farabollini, F., Guillette Jr., L.J., Hassold, T., Ho, S.M., Hunt, P.A., Iguchi, T., Jobling, S., Kanno, J., Laufer, H., Marcus, M., McLachlan, J.A., Nadal, A., Oehlmann, J., Olea, N., Palanza, P., Parmigiani, S., Rubin, B.S., Schoenfelder, G., Sonnenschein, C., Soto, A.M., Talsness, C.E., Taylor, J.A., Vandenberg, L.N., Vandenbergh, J.G., Vogel, S., Watson, C.S., Welshons, W.V., Zoeller, R.T., 2009. Why public health agencies cannot depend on good laboratory practices as a criterion for selecting data: the case of bisphenol A. Environ. Health Perspect. 117 (3), 309-315.

OECD, 2002. OECD Conceptual Framework.

Piersma, A.H., Hernandez, L.G., van, B.J., Muller, J.J., van Leeuwen, F.X., Vermeire, T.G., van Raaij, M.T., 2011. Reproductive toxicants have a threshold of adversity. Crit. Rev. Toxicol. 41 (6), 545-554.

RMS Spain, 2007. Monograph prepared in the context of the inclusion of the following active substance in Annex I of the Council Directive 91/414/EEC Rapeseed oil.

RMS UK, 1996. UK Rapporteur Monograph Directive 91/414/EC Linuron.

Sharpe, R., 2010. Is it time to end concerns over the estrogenic effects of bisphenol A? Toxicol. Sci. 114 (1), 1-4.

Tinwell, H., Friry-Santini, C., Rouquie, D., Belluco, S., Elies, L., Pallen, C., Bars, R., 2007. Evaluation of the antiandrogenic effects of flutamide, DDE, and linuron in the weanling rat assay using organ weight, histopathological, and proteomic approaches. Toxicol. Sci. 100 (1), 54-65.

Turner, K.J., McIntyre, B.S., Phillips, S.L., Barlow, N.J., Bowman, C.J., Foster, P.M., 2003. Altered gene expression during rat Wolffian duct development in response to in utero exposure to the antiandrogen linuron. Toxicol. Sci. 74 (1), 114-128.

Vinggaard, A.M., Breinholt, V., Larsen, J.C., 1999. Screening of selected pesticides for oestrogen receptor activation in vitro. Food Addit. Contam. 16 (12), 533-542.

Vinggaard, A.M., Hnida, C., Breinholt, V., Larsen, J.C., 2000. Screening of selected pesticides for inhibition of CYP19 aromatase activity in vitro. Toxicol. In Vitro 14 (3), 227-234.

WHO, 2008. Worldwide prevalence of anaemia. 1-51.

WHo/lPCS, 2002. The International Programme on Chemical Safety (IPCS): Global assessment of the state-of-the-science of endocrine disruptors. Damstra, T., Barlow, S., Bergmann, A., Kavlock, R., Van der Kraak, G. Geneva, World Health Organization.

WHO/IPCS, 2004. IPCS Risk Assessment Terminology.

WHO/UNEP, 2013. Endocrine disrupting chemicals 2012. Ake Bergman, Jerrold J. Heindel Susan Jobling Karen A. Kidd and R. Thomas Zoeller. Geneva, World Health Organization.

Wilson, V.S., Lambright, C.R., Furr, J.R., Howdeshell, K.L., Earl Jr., G.L., 2009. The herbicide linuron reduces testosterone production from the fetal rat testis during both in utero and in vitro exposures. Toxicol. Lett. 186 (2), 73-77.