Setting priorities for further development and validation of test methods and testing approaches for evaluating endocrine disruptors : final report.
Date: 2018-04-24
Institutions: Directorate-General for Environment
Subjects: endocrine disease, endocrine disruptor, health risk, research method, testing, toxic substance

Setting priorities for further 
development and validation of 
test methods and testing 
approaches for evaluating 
endocrine disruptors 

Final Report  

Written by Risk & Policy Analysts Ltd 
& wca Environment 
February 2018 

Environment 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Setting priorities for further development and validation 
of test methods and testing approaches for evaluating 
 endocrine disruptors 

EUROPEAN COMMISSION 

Directorate-General for Environment 
Directorate B — Circular Economy & Green Growth 
Unit B.2 — Sustainable Chemicals 

European Commission 
B-1049 Brussels 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
EUROPEAN COMMISSION 

Setting priorities for further 
development and validation of 
test methods and testing 
approaches for evaluating 
endocrine disruptors  

Final Report 

(study awarded under Framework Contract 
ENV.A.3/FRA/2014/0029 on implementation of the 
Community strategy on Endocrine Disruptors) 

Directorate-General for Environment 
Sustainable Chemicals, 2018 

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EUROPEAN COMMISSION 

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Luxembourg: Publications Office of the European Union, 2018 

PDF  ISBN 978-92-79-83076-1 

doi:10.2779/21828       KH-01-18-423-EN-N 

© European Union, 2018 

Key Contributors 

Max La Védrine 

Dr Pete Floyd 

Dr Owen Green 

Dr Becky Brown 

Dr Louise Youngs 

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EUROPEAN COMMISSION 

Executive summary 

Context 

Endocrine  disruptors  (EDs)  are  substances  that  can  cause  harmful  effects  to  the  endocrine 
(hormone)  system  of  both  humans  and  animals.  Within  human  and  animal  bodies,  hormones  will 
act  at  very  small  levels  and  at  critical  times  to  regulate  e.g.  development,  growth,  metabolism, 
immunity, behaviour and reproduction. Exposure to endocrine disrupting substances can have life-
long effects and the consequences may be passed onto the next generation. The EU has therefore 
introduced specific legislative obligations aimed at phasing out endocrine disrupting substances. 

An important factor in the legislative framework and the regulation of chemicals is the availability 
of  methods  to  identify  and  assess  the  hazardous  properties  of  substances.  The  OECD  helps  to 
ensure  that  test  methods  (TMs)  are  internationally  and  mutually  accepted  as  part  of  a  test 
guideline  (TG)  programme.  The  OECD's  Endocrine  Disruptor  Testing  and  Assessment  Advisory 
Group coordinates development of testing and assessment approaches for endocrine disruptors. 

Despite the progress achieved on the development and validation of TGs for evaluation of EDs and 
associated  guidance  documents  under  the  OECD  auspices  over  the  last  20  years,  there  are  still 
some gaps and weaknesses in the current test methods for evaluation of EDs.  

To address these gaps and weaknesses, the European Commission Services organised a European 
expert workshop in Brussels 30 May – 1 June 2017 with the objectives of: 

 

 

 

Identifying gaps in test methods;  

Identifying ways to address those gaps; and  

Prioritising further development and validation of TMs and testing approaches in this area. 

The  workshop  followed  an  earlier  JRC  expert  survey  on  identification  of  gaps  in  available  test 
methods for the evaluation of endocrine disruptors, the results of which were published in a 2017 
report. 

The Workshop 

Around 70 experts attended the workshop  from EU  institutions; Member State and US  authorities 
as well as a wide range of experts from universities, research centres, industry and NGOs from the 
EU, USA and Japan. All members of the Steering Group attended. 

The  workshop  started  with  presentations  and  moderated  discussion  to  set  the  scene,  including 
updates from other studies launched by the European Commission's DG ENV. This was followed by 
break-out  groups  to  discuss  key  questions  and  priorities.  The  break-out  groups  consisted  of  four 
groups  focusing  on  human  health  and  two  groups  focusing  on  environment.  In  Session  1 
participants  identified  the  adverse  outcomes  and  pathways  of  highest  concern.  This  was  followed 
by  break-out  group  Session  2  where  gaps  in  current  OECD  TGs  and  ideas  to  address  them  were 
brought  to  the  table  and  discussed.  The  third  and  final  break-out  group,  Session  3,  was  the 
prioritisation  itself,  which  aimed  to  prioritise  the  ideas  discussed  in  Session  2.  The  workshop  was 
moderated  and  facilitated  by  a  professional  moderator  with  support  from  the  project  team  and  a 
cartoonist who visually brought key issues into focus. 

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Adverse outcomes 

There  was  generally  a  consensus  among  the  human  health  groups  in  terms  of  the  main  adverse 
outcomes. The most important ones considered by the groups were: 

 

Thyroid-related Developmental Neurotoxicity 

  Metabolic Dysfunction 

 

Female Reproduction 

  Male Reproduction 

There  was  a  strong  consensus  among  the  two  environment  groups  in  terms  of  the  main  adverse 
outcomes, the most important ones were related to: 

  Reproductive Health 

  Growth and Development 

Overall,  the  highest  priorities  identified  at  the  workshop  largely  match  those  identified  by 
respondents to a respective survey published by the Joint Research Centre in 2017. 

Ideas to fill the gaps  

For each of the main adverse outcomes, the workshop experts considered various ideas on possible 
endpoints/tests  to  provide  useful  information  for  the  regulation  of  endocrine  disruptors.  It  was, 
however,  recognised  that,  in  some  areas,  more  basic  research  was  required  to  improve  the 
underlying science of endocrine disruption. 

Consequently,  while  some  of  the  ideas  could  be  developed  into  the  current  testing  strategy 
immediately, many others would require significant research and development. 

During the event over 70 ideas were discussed by the experts in varying degrees of detail. 

Prioritisation methodology 

In preparing for the workshop, a range of factors were considered and these were presented to the 
workshop in order to determine whether the most significant factors had been identified and what 
importance  should  be  attached  to  each.  Furthermore,  for  each  factor,  a  simple  rating  scale  was 
proposed – such as ‘low’, ‘moderate’ and ‘high’.  

At  the  workshop,  the  view  was  expressed  that  a  tiered  approach  was  needed  and  regulatory 
relevance  (mode  of  action  (MOA)  and  adversity)  and  the  societal/ecosystems  benefits  were 
considered to be the first tier (Tier 1) factors. Furthermore, there was strong support for a proposal 
to invoke a pass/fail screen on whether proposals would be of regulatory relevance by being rated 
‘high’ on providing useful information on either MOA and/or adversity. 

In relation to the second tier, there was agreement that the Technology Readiness Level (TRL) was 
important  –  since it was recognised  that the  TRL could  also be used as a filter for selecting long-
term research projects (low TRL values) from proposals which could be translated into TGs over a 
relatively  short  timescale  (high  TRL  values).  Similarly,  it  was  recognised  that  both  ‘costs’  and 
‘animal concerns’ were factors which needed to be considered as part of a ‘reality check’ and could 
also be considered Tier 2 factors. 

Prioritisation results 

Both the human and environmental health groups discussed ideas to fill gaps that they felt should 
be prioritised. There were a number of areas where there was a consensus amongst the separate 
human health groups, the separate environmental health groups and  in some cases between both 

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EUROPEAN COMMISSION 

human  and  environmental  health  groups.  There  were  suggestions  that  some  ideas  could  be 
progressed  more  quickly  than  other  ideas  and,  for  the  prioritised  ideas,  a  suggested  timeline  has 
been included in the report.  Brief summaries of  all  the main ideas discussed  at the workshop  are 
included in Annex 2. 

The  prioritised  ideas  could  be  summarised  in  terms  of  five  main  themes,  these  are;  the 
development  of  new  in  vitro  assays,  in  vivo  test  enhancements,  the  development  of  new  in  vivo 
tests, regulatory modifications and research ideas. 

For the development of new in vitro assays, the attendees suggested that a number of assays are 
already at an advanced stage of development. The largest consensus was for  the development of 
thyroid  assays,  retinoid  assays,  and  PPAR  assays  (relating  to  both  human  and  environmental 
health).  There  was  also  a  consensus  for  the  development  of  glucocorticoid  receptor  (GR)  and 
mineralocorticoid  receptor  (MR)  transactivation  assays  as  well  as  new  in  vitro  assays  relevant  to 
both female and male reproductive health. 

For  in  vivo  test  enhancements,  the  new  MEOGRT  test  guideline  (OECD  TG  240)  (relating  to 
environmental health) was highlighted as being one that would benefit from further refinement and 
performance  testing  as  more  of  these  studies  were  actually  carried  out.  From  the  human  health 
perspective,  EOGRTS  (OECD  TG  443)  was  seen  as  a  method  which  could  be  further  refined  and 
have  a  number  of  additional  measurements  included  (e.g.  performing  whole  mount  mammary 
gland  assessment,  assessing  brown  fat  and  measuring  various  hormones).  The  largest  consensus 
made  by  the  attendees  was  for  inclusion  of  thyroid  function  biomarkers  (e.g.  TSH,  T4  and  T3 
measurements), for an indication of effects via the thyroid system, in several TGs (relating to both 
human  and  environmental  health).  Many  of  the  environmental  health  suggestions  were 
enhancements  because  while  many  existing  guidelines  have  good  characterisation  of  apical 
endpoints,  supporting  information  on  the  mechanism  of  action  is  often  lacking.  Ideas  included 
enhancements  to  in  vivo  assays  to  inform  on  both  female  and  male  reproductive  health,  and 
growth and development (e.g. behavioural endpoints based on mating, ecdysis triggering hormone 
and ecdysterone levels, spawning activity). 

For the development of new in vivo tests for human health, a new OECD CF Level 3 study (based 
on OECD TG 407 – mammalian toxicity) was particularly highlighted. The purpose of this test would 
be to provide mechanistic information for all of the EATS modalities in one go. New test guidelines 
proposed  for  the  environment  included  fish  and  amphibian  thyroid  assays,  and  a  larval  fish 
behavioural assay possibly based on predator avoidance. 

For regulatory modifications, the main ideas were related to a reconsideration of the conditions for 
omitting/triggering of DNT and DIT cohorts in the TG 443, and considering dietary exposure testing 
of  substances  with  Log  Kow  >6,  for  the  environment.  Another  recommendation  for  the 
environment  was  to  routinely  keep  tissue  samples  from  long  term  studies,  in  the  same  way  that 
samples from mammalian toxicity studies are typically retained.  

For more research, the main ideas for human health were for research into new female models of 
reproductive  health, 
into 
epigenetics,  gene  markers  and  other  biomarkers.  For  the  environment,  the  main  ideas  were  for 
research into invertebrate endocrinology/physiology and research into bird  and reptile  (e.g.  mode 
of action compared with mammals and other taxa) endocrinology. 

functional  neurodevelopmental  toxicity  endpoints  and  research 

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CONTENTS 

EXECUTIVE SUMMARY .................................................................................................. III 

ABBREVIATIONS .........................................................................................................VII 

THE PROJECT STEERING GROUP .................................................................................... IX 

BACKGROUND .............................................................................................................. 1 

1. PRIORITISATION METHODOLOGY ................................................................................ 3 

2. DISEASES/DISORDERS AND PATHWAYS OF CONCERN ................................................... 6 

Introduction ................................................................................................. 6 
2.1 
2.2  Human Health .............................................................................................. 7 
Environmental Health .................................................................................. 11 
2.3 

3. IDENTIFICATION OF GAPS ........................................................................................ 13 

3.1 
Introduction ............................................................................................... 13 
3.2  Human Health ............................................................................................ 14 
Environmental Health .................................................................................. 15 
3.3 

4. IDEAS TO CLOSE THE GAPS – TG AND TM ENHANCEMENT AND DEVELOPMENT ............... 17 

4.1 
Introduction ............................................................................................... 17 
4.2  Human Health ............................................................................................ 18 
Environmental Health .................................................................................. 26 
4.3 

5. PRIORITISATION ..................................................................................................... 34 

5.1 
Introduction ............................................................................................... 34 
5.2  Human Health ............................................................................................ 34 
5.3  Human Health Summary ............................................................................. 37 
Environmental Health .................................................................................. 39 
5.4 
Environmental Health summary .................................................................... 42 
5.5 
5.6  Non-priorities ............................................................................................. 45 

6. DISCUSSION .......................................................................................................... 46 

6.1 
Introduction ............................................................................................... 46 
6.2  Context ..................................................................................................... 46 
Prioritisation Summary – Human and Environmental Health ............................. 46 
6.3 
Further Research ........................................................................................ 48 
6.4 

7. CONCLUSION .......................................................................................................... 50 

7.1  Overview ................................................................................................... 50 
Summary of adverse outcomes .................................................................... 50 
7.2 
Summary of priority ideas............................................................................ 50 
7.3 

8. REFERENCES .......................................................................................................... 52 

9. WORKSHOP PROGRAMME ......................................................................................... 61 

10. PARTICIPANTS LIST ............................................................................................... 64 

ANNEX 1 - WORKSHOP REPORT .................................................................................... 66 

ANNEX 2 - IDEAS TO FILL GAPS .................................................................................... 71 

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Abbreviations 

3Rs 

Replacement, reduction and refinement (for animal testing) 

ADHD   

Attention deficit hyperactivity disorder 

AOP 

CAR 

CF 

CYP 

DIT  

DNT 

Adverse outcome pathway 

Constitutive androstane receptor  

Conceptual framework 

Cytochrome P450 enzymes 

Developmental immunotoxicity  

Developmental neurotoxicity  

DOHaD  

Developmental origins of health and disease 

DRP 

EATS 

ED 

EDC 

Detailed review paper  

Estrogen, androgen, thyroid and steroidogenesis 

Endocrine disruptor  

Endocrine disrupting chemical 

EOGRTS 

Extended one-generation reproductive toxicity study  

ER 

FGF 

Estrogen receptor 

Fibroblast growth factor 

FOAD 

Foetal origins of adult disease  

F1, F2   

First generation, second generation, etc. 

HPA  

HPE 

HPG 

HPI 

HPT 

IATA 

IEAM 

Hypothalamic–pituitary–adrenal (axis) 

Holoprosencephaly  

Hypothalamic–pituitary–gonadal (axis) 

Hypothalamic-pituitary-interrenal (axis) 

Hypothalamic–pituitary–thyroid (axis) 

Integrated approaches to testing and assessment 

Integrated environmental assessment and management 

INSL3   

Insulin-like peptide 3 

ITS 

JRC 

KE 

Integrated testing strategy 

Joint Research Centre 

Key event 

MEOGRT  

Medaka extended one generation reproduction test  

MIE 

MOA  

Molecular initiating event 

Mode of action 

ncRNA   

Non-coding ribonucleic acid 

OCSPP   

(US) Office of Chemical Safety and Pollution Prevention  

PCOS 

PEC  

Polycystic ovaries syndrome  

Predicted environmental concentration 

PNEC     

Predicted no effect concentration 

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PPAR 

Peroxisome proliferator-activated receptor 

PSA 

PXR 

RAR 

RXR 

T3 

T4 

TBT 

TDS 

TG 

TGP 

TM 

TMR 

TR 

TRH 

TRL 

TSH 

TTR 

XETA 

WNT 

WoE 

Prostate-specific antigen 

Pregnane X receptor  

Retinoid acid receptor 

Retinoid X receptor 

Triiodothyronine  

Tetraiodothyronine (also called thyroxine) 

Tributyltin 

Testicular dysgenesis syndrome 

Test guideline 

Test guideline programme 

Test method 

Test Methods Regulation (Council Regulation (EC) No 440/2008) 

Thyroid hormone receptor 

Thyrotropin releasing hormone  

Technology readiness level  

Thyroid stimulating hormone  

Transthyretin 

Xenopus embryonic thyroid-signalling assay  

Working group of the national coordinators of the OECD test guidelines programme 

Weight of evidence 

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The Project Steering group 

The Steering Group for this project had responsibility for coordinating the implementation 
of this project. It was composed of: 

  Maiken Rasmussen, European Commission, DG ENV 

  Dr Peter Korytar, European Commission, DG ENV 

  Sharon Munn, European Commission, DG JRC 

  Dr Elise Grignard, European Commission, DG JRC 

  Dr Stephanie Bopp, European Commission, DG JRC 

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Background 

One  important  factor  in  the  regulation  of  chemicals  is  the  availability  of  methods  to  identify  and 
assess the hazardous properties of  chemical  substances. To ensure that  such  test  methods (TMs) 
are internationally and mutually accepted, a test guideline programme (TGP) has been established 
under  the  auspices  of  the  OECD.  EU  legislation  regulating  chemical  substances  often  includes 
testing  of  the  substances  according  to  the  EU  Test  Methods  Regulation  (TMR)1,  which 
predominantly contains test guidelines (TGs) developed under the OECD. Therefore, the OECD TGP 
is of upmost importance for the EU regulatory processes on chemical substances. 

The  OECD's  Endocrine  Disruptor  Testing  and  Assessment  Advisory  Group  (EDTA  AG)  coordinates 
development of testing and assessment approaches for endocrine disruptors (EDs).  

The OECD’s Conceptual Framework2  (CF)  for the  Testing and Assessment  of Endocrine Disruptors 
was adopted in 2002 and updated in 2012. The OECD CF indicates the type of information the tests 
provide at the five different levels, moving from in silico to in vitro and in vivo. The five levels are 
described as follows:  

Level 1.  Existing data and non-test information 

Level 2.  In vitro assays providing data about selected endocrine mechanism(s)/pathway(s) 

Level 3.  In vivo assays providing data about selected endocrine mechanism(s)/pathway(s) 

Level 4.  In vivo assays providing data on adverse effects on endocrine relevant endpoints 

Level 5.  In  vivo  assays  providing  more  comprehensive  data  on  adverse  effects  on  endocrine 

relevant endpoints over extensive parts of the life cycle of the organisms 

The  information  from  the  TGs  in  the  OECD  CF  can  be  used  to  gain  more  information  about 
substances.  A  Guidance  Document  (GD),  OECD  GD  150,  was  developed  to  be  used  as  a  tool  to 
support  regulatory  authorities  by  helping  to  interpret  assay  results  and  by  suggesting  possible 
additional  studies  for  reducing  the  uncertainty  of  whether  a  substance  may  be  endocrine 
disrupting. 

The  primary  purpose  of  the  OECD  test  guidelines  is  mutual  recognition  of  data  across  regulatory 
programmes at regional and international level. 

Despite the progress achieved on the development and validation of TGs for evaluation of EDs and 
associated  guidance  documents  under  the  OECD  auspices  over  the  last  20  years,  there  are  still 
some gaps and weaknesses in the current test methods for evaluation of EDs. According to several 
scientific  reports  published  in  recent  years  (Kortenkamp  et  al.,  2011;  OECD,  2012;  EFSA,  2013; 
and WHO / UNEP, 2012) there is, in particular, a lack of: 

 

Tests both for human health and the environment covering additional hormonal pathways 
and modalities besides the estrogen, androgen, thyroid and steroidogenesis ('EATS') 
modalities; 

  Suitable animal models in relation to certain human endocrine disorders in which endocrine 
disruptors have been suggested to play a role, such as some mammary gland tumours and 
other hormonal cancers, endometriosis, metabolic syndrome or reproductive senescence; 

1   Council 

No 
lex.europa.eu/LexUriServ/LexUriServ.do?uri=OJ:L:2008:142:0001:0739:en:PDF  

Regulation 

440/2008. 

available 

(EC) 

TMR 

at: 

http://eur-

2   OECD  Conceptual  Framework  for  Testing  and  Assessment  of  Endocrine  Disrupters  (as  revised  in  2012). 
at: 

Available 
http://www.oecd.org/env/ehs/testing/OECD%20Conceptual%20Framework%20for%20Testing%20and%2
0Assessment%20of%20Endocrine%20Disrupters%20for%20the%20public%20website.pdf  

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  A single study involving observations through the complete life cycle of a mammal, from 
conception to old age, which would cover all specific windows of susceptibility in order to 
cover effects that might be induced by exposure during foetal or pubertal development that 
only emerge during later life stages e.g. cancer incidence in adult or aged animals; 

  Appropriate tests for environmental species (beyond fish and mammals) such as birds and 

invertebrates; and 

 

The thyroid modality is not yet well covered (a separate workshop was held on thyroid 
disruption (DG ENV/ANSES workshop on thyroid disruption (EC 2017)). 

The gaps in the current TGs for evaluation of EDs are a serious issue which needs to be adequately 
addressed as soon as possible. Considering the significant amount of work needed to address the 
gaps and the limited resources available, it is important to identify and set priorities for short- (less 
than  5  years),  medium-  (5-10  years)  and  long-  (>10  years)  term  development  and  validation  of 
test methods.  

To do so, the European Commission Services organised a European expert workshop in Brussels 30 
May – 1 June 2017 with the objectives of: 

 

 

 

Identifying gaps in test methods;  

Identifying ways to address those gaps; and  

Prioritising further development and validation of TMs and testing approaches in this area. 

The  workshop  was  structured  to  consider  these  objectives  in  chronological  order,  starting  with 
presentations  and  moderated  discussion  to  set  the  scene,  including  updates  from  other  studies 
launched by the European Commission, DG ENV. This was followed by break-out groups to discuss 
the core questions and priorities. The break-out groups consisted of four groups focusing on human 
health  and  two  groups  focusing  on  environment.  In  Session  1  participants  identified  the  adverse 
outcomes and pathways of highest concern. This was followed by break-out group Session 2 where 
gaps in current OECD TGs and ideas to address them were brought to the table and discussed. The 
third and final break-out group, Session 3, was the prioritisation itself, which aimed to prioritise the 
ideas  discussed  in  Session  2.  The  workshop  was  moderated  and  facilitated  by  a  professional 
moderator  (David  Rose)  with  support  from  the  project  team  and  a  cartoonist  (Floris  Oudshoorn) 
who visually brought key issues into focus. 

This report summarises the methodology used for prioritisation and the outcome of the  break-out 
group discussions including the application of the prioritisation methodology (sections 1 to  7). The 
workshop  programme  is  to  be  found  in  Section  9  and  the  participant  list  in  Section  10.  A  brief 
account of the workshop is presented in Annex 1, a description of the main ideas discussed at the 
workshop appears in Annex 2.  

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1. Prioritisation methodology 

Overview 

Proposals for new or enhanced test guidelines (TGs) or test methods (TMs) may be characterised 
by a range of factors. In order to prioritise such proposals, it is necessary to consider which factors 
are most likely to be of significance when considering the relative merits of different proposals. 

In preparing for the workshop, a range of factors were considered and these were presented to the 
workshop in order to determine whether the most significant factors had been identified and what 
importance  should  be  attached  to  each.  Furthermore,  for  each  factor,  a  simple  rating  scale  was 
proposed – such as ‘low’, ‘moderate’ and ‘high’. These factors are described below. 

Regulatory relevance 

Within the EU and elsewhere, the regulation of chemicals is, to some extent, dependent on a range 
of hazardous properties which are determined by the results of tests performed in accordance with 
internationally  recognised  test  guidelines.  Currently,  this  is  largely  achieved  through  the  use  of 
OECD  TGs.  As  such,  it  was  considered  that  proposals  for  future  enhancements  and/or  new  test 
guidelines/methods  for  evaluating  endocrine  disruptors  (EDs)  would  need  to  provide  information 
which  would  be  of  relevance  to  the  regulatory  framework.  More  specifically,  the  focus  of  the 
relevance would be on either the means by which an ED could produce an adverse effect (mode of 
action (MOA)) and/or the nature/extent of such an effect (adversity).  

Mode of Action (MOA) 

This  factor  informs  about  the  relevance  of  the  particular  option  to  elucidate  the  Mode  of  Action 
(mechanistic endpoints that may support identification of a plausible endocrine-related pathway to 
adversity). Suggested rating scale:  

 

 

 

‘Low’ (provides little information about the MOA) 

‘Moderate’ (informs to some extent about the MOA) 

‘High’ (provides good information about the MOA) 

Adversity  

This factor informs about the  relevance  of the  particular option to elucidate or define adversity in 
organisms (toxicology) or populations (ecotoxicology). Suggested rating scale:  

 

 

 

‘Low’ (provides little information about adversity) 

‘Moderate’ (informs to some extent about adversity)  

‘High’ (provides good information about adversity) 

Societal/ecosystem benefits of test enhancement (both real and perceived) 

This  factor  informs  about  the  potential  societal  benefits/ecosystem  benefits  of  particular  areas  of 
research.  The  benefits  represent  the  potential  avoidance  of  the  adverse  outcome  costs,  potential 
avoidance  of adverse effects and perceived  adversity.  By way of example, if a particular pathway 
was associated with a significant human disease burden, then a new/enhanced test method could 
result in ‘significant’ societal benefits. Suggested rating scale: 

 

 

 

‘Limited’ (few societal/ecosystem benefits possible) 

‘Some’ (few societal/ecosystem benefits likely OR some societal/ecosystem benefits 
possible) 

‘Significant’ (societal/ecosystem benefits likely OR significant societal/ecosystem benefits 
possible) 

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It  can  be  seen  that  these  rating  scales  account  for  both  the  (potential)  extent  of  the  benefits  as 
well as the degree of uncertainty that such benefits could be realised.  

Technology Readiness Level (TRL) 

For each idea of research or the development of a new/enhanced test method/guideline, it should 
be possible to classify the readiness with reference to the Technology Readiness Level (TRL). Based 
on  the  approach  used  across  the  Horizon  2020  research  programme,  TRLs  may  be  defined  as 
follows3: 

 

 

 

 

 

 

 

 

 

TRL 1 – basic principles observed (i.e. theoretically feasible in light of current knowledge) 

TRL 2 – technology concept formulated (i.e. possible test design identified) 

TRL  3  –  experimental  proof  of  concept  (i.e.  pilot  study  in  laboratory  demonstrating 
relevance of method) 

TRL  4  –  technology  validated  in  lab  (i.e.  test  system  validated  by  independent  research 
groups) 

TRL 5 – technology validated in relevant environment (i.e. test system validated in multiple 
labs) 

TRL  6  –  technology  demonstrated  in  relevant  environment  (i.e.  test  system  used  in 
regulatory/scientific  environment  (e.g.  OSCPP,  commercial  kit),  but  lacking  agreed 
reference substances for ED) 

TRL  7  –  system  prototype  demonstration  in  operational  environment  (i.e.  ready  for 
implementation in OECD Test Guideline) 

TRL  8  –  system  complete  and  qualified  (i.e.  already  implemented  in  some  OECD  Test 
Guideline (e.g. optional endpoints))  

TRL  9  –  actual  system  proven  in  operational  environment  (i.e.  regularly  and  successfully 
conducted in GLP compliant OECD guidelines) 

By inspection, it can be seen that existing and routinely used OECD TGs are TRL9 while theoretical 
research  into  modes  of  action  or  adversity  would  be  TRL1.  As  new/improved  test  methods  are 
researched and developed they will move through from low TRLs to lab validation (TRL4), through 
to working prototypes (TRL7), before being formally approved at OECD level. 

Consideration of the current TRL will provide an indication of its likely readiness. In essence, a high 
TRL (within the context of prioritising improvements in testing) is more likely to lead to  a tangible 
improvement.  

It  is  worth  noting  that  high  TRL  levels,  TRL6  and  above,  are  more  likely  to  be  implemented  in  a 
shorter  timescale  (e.g.  0-5  years),  TRL  levels  4  to  5  are  more  likely  to  be  implemented  in  the 
medium term (e.g. 5-10 years) and the lowest TRL level 1 to 3 are more likely to be implemented 
in the long term (e.g. >10 years). Clearly, further factors may influence the actual time scales for 
implementation. 

Costs of performing the tests 

This  factor  indicates  whether  the  associated  costs  (with  consideration  being  given  to  the  costs  of 
development and of performing the test) are likely to be classified as ‘low’, ‘moderate’ or ‘high’. By 
way of example, costs were considered ‘high’ if the option would require additional satellite groups, 
significant development and validation, large animal numbers (or new species) and/or take a long 
time.  

3   Based  on  those  used 

framework  programme,  available  here: 
https://ec.europa.eu/research/participants/data/ref/h2020/wp/2014_2015/annexes/h2020-wp1415-annex-
g-trl_en.pdf 

in  the  Horizon  2020  research 

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Animal concerns 

This factor informs about the level of concerns associated with the use of animals in testing. This 
accounts  for  the  3Rs  concept  (replacement,  reduction  and  refinement  in  relation  to  the  use  of 
animals in research4). As such, it would be expected that consideration would be given to whether 
animals are required for experimentation or tissue collection. In addition, consideration may need 
to  be  given  to  whether  additional  animals  might  be  required  for  the  additional  endocrine-related 
endpoints to be reliable (e.g. for hormone measurements). The likely level of animal concerns may 
be rated as ‘low’, ‘moderate’ or ‘high’.  

As  an  example,  the  animal  welfare  considerations  of  the  in  vitro  tests  are  low,  whereas  a  new 
reptilian reproductive toxicity screening test would be rated as ‘high’. 

Integration with other research  

Although it was not applied by all groups, in some cases (if known by the experts), the proposed 
research  into  new/enhanced  test  methods  may  form  part  of  an  integrated  research  programme 
allowing  for  synergies  with  results  from  other  research.  At  the  other  extreme,  the  proposed 
research  may  simply  duplicate  work  being  done  elsewhere  so  that  the  results  may  only  provide 
limited added value. With this in mind, a possible rating could be: 

 

 

 

‘Low’ (may duplicate other research) 

‘Moderate’ (potential synergies with other research)  

‘High’ (part of integrated research programme) 

Final Prioritisation Methodology 

At  the  workshop,  a  prioritisation  methodology  was  proposed  and  the  factors  were  explained.  The 
prioritisation  methodology  proposed  equal  weighting  to  be  applied  to  the  factors.  As  part  of  a 
session  where  groups  reviewed  ideas  to  fill  gaps  along  with  the  prioritisation  methodology  it  was 
felt  that improvements  could  be  made  to  the  methodology.  For  example,  equal  weighting  gave  a 
bias towards in vitro tests (generally CF Level 2). 

One  of  the  human  health  break-out  groups  explained  how  they  thought  a  tiered  approach  was 
needed  and  emphasised  the  importance  of  regulatory  relevance  with  respect  to  either  mode  of 
action (MOA) and/or adversity as well as the societal/ecosystem benefits. These were considered to 
be the first tier (Tier 1) factors – a view shared by other attendees. Furthermore, there was strong 
support  for  a  proposal  to  invoke  a  pass/fail  screen  on  whether  proposals  would  be  of  regulatory 
relevance by being rated ‘high’ on providing useful information on either MOA and/or adversity. 

In  relation  to  the  second  tier,  there  was  agreement  that  the  TRL  was  important  –  since  it  was 
recognised that the TRL could also be used as a filter for selecting long-term research projects (low 
TRL  values)  from  proposals  which  could  be  translated  into  TGs  over  a  relatively  short  timescale 
(high TRL values). 

Similarly, it was recognised that both ‘costs’ and ‘animal concerns’ were factors which needed to be 
considered as part of a ‘reality check’ and could also be considered Tier 2 factors. 

It  was  recognised  that  consideration  of  these  Tier  1  and  Tier  2  factors  may  still  not  provide  a 
comprehensive review of all relevant aspects. As such, further factors could be considered as Tier 3 
factors. These could include the factors relating to the ‘integration with other research’ and/or the 
proposed factor relating to the ‘redundancy screen’. 

This type of tiered approach was suggested as the approach that all groups  should follow in their 
subsequent discussions on prioritisation.  

4   As embodied in Directive 2010/63/EU of the European Parliament and of the Council of 22 September 2010 

on the protection of animals used for scientific purposes 

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2. Diseases/disorders and pathways of concern 

2.1 

Introduction 

As highlighted in the European Commission report on the State of the Art Assessment of Endocrine 
Disruptors  (Kortenkamp  et  al.,  2011),  there  are  various  adverse  outcomes  associated  with 
endocrine disruptors from both human health and environmental health perspectives.  

JRC had compiled a questionnaire to gather input from experts in the field on key issues to be used 
as a basis for the  further discussions at the workshop. An online survey with the  title ‘Identifying 
gaps  in  available  test  methods  for  evaluation  of  endocrine  disruptors’  was  performed  on  the  EU 
Survey platform and open for commenting from 19/05/2015 until 15/06/2015. A selected group of 
experts  from  EFSA,  ECHA  and  the  Working  Group  of  the  National  Coordinators  of  the  Test 
Guidelines  Programme  (WNT),  in  addition  to  experts  identified  in  Annex  3  of  the  ‘Roadmap  for 
setting priorities for further development and validation of test methods and testing approaches for 
evaluating endocrine disruptors’ were invited to participate  in the survey (JRC, 2017). 40 experts 
replied and were invited to the workshop.  

As  part  of  this  survey  experts  were  asked  to  indicate  their  priority  for  development  of  new  test 
methods  or  enhancement  of  existing  methods 
related 
diseases/disorders and any other disease/disorder considered to be of concern that were not listed. 
The survey responses are presented in JRC’s report and these can be seen in Table 2-1 along with 
additional comments made by the experts. The experts who completed the survey were invited to 
the workshop. 

relevant  endocrine 

listed 

for 

Table 2-1: List of diseases/disorders addressed in the JRC survey (according to 
WHO/UNEP, 2012) with additional comments as reported by JRC 
Disease/disorder 
Human female reproductive 
health 
Wildlife female reproductive 
health 
Human male reproductive 
health 

Examples 
Pubertal timing, fibroids, endometriosis, polycystic ovary 
syndrome, fecundity and fertility 
Uterine fibroids, lower reproductive success, egg shell 
thinning, imposex in snails 
Testicular cancer, semen quality, cryptorchidism, 
hypospadias, reduced anogenital distance, testicular 
dysgenesis syndrome 
Feminising disorders, cryptorchidism, low fertility and 
reproduction rates 
Cognitive and behavioural deficits, attention 
deficit/hyperactivity disorder (ADHD), autism 

Wildlife male reproductive 
health 
Thyroid-related disorders / 
neurodevelopmental disorders 
in children 
Hormone related cancers 
Adrenal disorders in humans 

Adrenal disorders in wildlife 

Metabolic disorders 
Immune related disorders in 
humans 
Immune related disorders in 
wildlife 
Wildlife toxicity (additional 
comments) 

Human/mammalian toxicity 
(additional comments) 

Breast, endometrial, ovarian, prostate, thyroid 
Adrenocortical hyperplasia, development of foetal adrenal 
cortex, effects on function of hypothalamic-pituitary-adrenal 
(HPA) axis 
Uterine occlusions/strictures, leiomyoma, adrenocortical 
hyperplasia, elevated baseline corticosterone levels 
Obesity, diabetes, metabolic syndrome 
Inflammatory disorders, immune cancers such as lymphoma 
and leukaemia, childhood respiratory disease 
Increased susceptibility to infectious diseases 

Screening methods for invertebrates;  
too little is known about the sensitivity of birds and the 
related MOAs; thyroid related disorders in wildlife including 
neurodevelopmental disorders 
Testicular and adrenal hormonal disorders, prostate 
adenomas and carcinomas, neurodevelopmental disorders, 
premature reproductive senescence, epigenetic MOAs and 
bone disorders 

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The attendees as part of the  first break-out group  session  were asked to review a list  of adverse 
human  health  effects  and  adverse  environmental  effects  associated  with  endocrine  disruption 
based  on  the  JRC  survey.  The  attendees  were  asked  to  consider  whether,  in  their  opinion,  any 
adverse  effects  were  missing  and  also  to  consider  which  were  the  most  important  adverse 
outcomes with respect to human health and environmental protection. 

2.2  Human Health 

The  adverse  outcomes  associated  with  human  health  as  identified  by  the  attendees  at  the 
workshop  are  presented  in  Table  2-2.  The  table  is  not  ranked  in  terms  of  prioritised  adverse 
outcomes, it is sorted in an order similar to that in Table 2-1. The workshop attendees attempted 
to rank the adverse outcomes and this is discussed below. 

Table 2-2: Overview of human diseases associated with endocrine disruption  
Adverse outcomes 
Female Reproductive 
Health 

Specific outcomes 
PCOS, uterine fibroids, endometriosis, precocious puberty, 
fecundity, adverse pregnancy outcomes and menopause-related 

Male and Female 
Reproductive Health 
Male Reproductive 
Health 
Neuro development 

Hormonal cancers 

Metabolic dysfunction  

Immune, Autoimmune 
and Inflammatory 
disorders 
Retinoid Target 
Malformations 
Other 

Impotence for both sexes 

Reduced semen quality and function, cryptorchidism, hypospadias, 
Benign Prostatic Hyperplasia (BPH) 
Hypothyroxinemia, autism, ADHD, neural defects, (potential) 
disruption of cortisol axis , mental health, neurodegenerative 
disease / peripheral neuropathy and altered stress response 

Female breast cancer, male breast cancer, prostate cancer, testis 
cancer, thyroid cancer, ovarian cancer, endometrial cancer, fallopian 
cancer, liver cancers (steroid induced) 
Obesity, diabetes and metabolic syndrome 

Hashimoto's disease, immune dysfunction, autoimmune disease, 
chronic inflammation, immune suppression, asthma, allergies and 
respiratory disease 
Craniofacial / cleft palate 

Osteoporosis, blood pressure, tachycardia and cardiovascular 
disease 

In  general,  the  list  in  Table  2-2  is  quite  similar  to  the  JRC  survey  (2017)  results  in  Table  2-1, 
although some allowance needs to be made for differences in the terminology used. For example, 
the inclusion of neurodevelopmental disorders in the JRC survey (2017) refers specifically to that in 
children,  whereas  neurological  adverse  outcomes  across  the  age  ranges  were  considered  by  the 
human  health  groups  and  additional  adverse  outcomes  such  as  mental  health  and 
neurodegenerative  disease,  or  (potential)  adverse  outcome  induced  via  disruption  of  the  cortisol 
axis, were included. Other adverse outcomes that are not mentioned in the JRC survey (2017) but 
were highlighted by workshop participants include fallopian cancer, liver cancers (steroid induced), 
Hashimoto’s  disease  (hypothyroidism),  immune  dysfunction,  autoimmune  diseases,  immune 
suppression, asthma, allergies, craniofacial/cleft palate, peripheral neuropathy, blood pressure and 
tachycardia. An attempt has been made to incorporate some of these adverse outcomes into more 
meaningful groups in Table 2-2. 

Details of the discussions about prioritising the adverse outcomes in the four human health break-
out groups are as follows: 

Human Health Group 1 

Attendees in this group indicated that it was challenging trying to rank the importance of different 
adverse outcomes.  The group  discussed  whether rankings should be based on the severity of the 
disease, cost burden or societal impacts. Further discussion centred around the need for estimation 
of the cost burden of the diseases and the consensus was that this element should not be included 

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as a major part of the decision-making process on prioritising the area(s) for development and that 
the severity of the disease was the most important factor. 

Human Health Group 2 

The  group  was  in  agreement  that  Adverse  Outcome  Pathways  (AOPs)  have  been  the  most 
significant advancement for the assessment of endocrine mode of action. 

When the group considered how adverse outcomes might be prioritised it was suggested that the 
decision is based on: 

 

 

Incidence – frequency at which specific adverse outcomes are observed 

Established endocrine MOA – this was considered to be an important feature 

  Reversibility – if regression of adverse effects from ED has been demonstrated 

Although  the  group  felt  that  it  was  hard  to  prioritise  the  adverse  outcomes  as  they  were  all 
important, the group suggested that the three most important might be those shown in Table 2-3. 

Table 2-3: Priority ranking of human diseases/disorders (Human Health Group 2) 
Adverse outcome 
Neuro-development 
Metabolic Dysfunction 
Female Reproduction 

Prioritisation 
High 
High 
High 

Concerning  the  neuro-developmental  adverse  outcome,  the  group  commented  that  it  was 
recognised as a difficult area in which to assess and diagnose the adverse outcomes, which could 
be  a  reflection  of  how  well,  or  otherwise,  the  effects  are  measured.  Concerning  female 
reproduction,  whilst  the  group  believed  that  evidence  of  adverse  effects  for  male  reproduction 
exists,  similar  reliable  evidence  for  cause/attribution  of  adverse  effects  is  currently  lacking  for 
female health and this is something that needs to be addressed. 

Human Health Group 3 

The  group  found  it  challenging  to  devise  a  way  in  which  adverse  outcomes  could  be  prioritised. 
There are clearly many ways that this could be achieved but it was a question of what would be the 
best way of doing this. The group felt that the impact on younger people, ‘the human potential’, is 
where  the  major  priority  should  be,  but  that  all  the  adverse  outcomes  are  important.  The  group 
tried to rank the ideas in terms of what they felt was of most importance. This is shown in Table 2-
4. 

Table 2-4: Priority ranking of human diseases/disorders (Human Health Group 3) 
Adverse outcome 
Thyroid axis / Neurodevelopment 
Metabolic dysfunction 
Female Reproductive Health 
Immune / Autoimmune / Inflammatory 
Hormone-related cancers 
Respiratory Disease 
Male Reproductive Health 
Neurodegenerative Disease 
Retinoid Target Malformations (e.g. craniofacial / cleft palate) 
Altered Stress Response (HPA targets) 
Cardiovascular Disease 
Bone disease / Osteoporosis 

Prioritisation 
High 
High 
High 
High 
Moderate/High 
Moderate/High 
Moderate 
Moderate 
Moderate 
Moderate 
Moderate 
Moderate 

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Human Health Group 4 

The group highlighted that there is now some evidence of endocrine related MOA for obesity based 
on  recent  work  that  has  indicated  the  significant  role  of  PPARɣ  (Peroxisome  proliferator-activated 
receptor gamma) in adipogenesis when forming a heterodimer with RXR. 

The  group  also  highlighted  how  work  by  Angel  Nadal’s  group  on  diabetes  (Type  II)  and  beta  cell 
function  and  Jean  Pierre  Bourguignon’s  group  on  precocious  puberty  in  females  (humans  and 
rodents), have both increased the endocrine MOA understanding and biological plausibility in these 
areas.  Additionally,  there  may  now  be  more  evidence  for  ED  involvement  in  ADHD/Autism  from 
studying  social  endpoints  in  rats.  When  the  group  considered  prioritisation,  they  did  this  in  part 
based  on  cost  burden  (cost  to  society,  e.g.  treatment)  and  incidence  of  diseases/conditions,  but 
also  considered  issues  that  are  easy  to  address  quickly  or  “low  hanging  fruit”.  For  example, 
investigations  into  semen  quality  were  seen  as  an  easy  option  since  human  samples  are  easily 
accessible and readily collected in clinics and, therefore, could be better used. 

Table 2-5: Priority ranking of human diseases/disorders (Human Health Group 4) 
Adverse outcome 
Male reproductive health 
Cancer - Breast cancer 
Cancer - Prostate cancer 
Female reproductive health - PCOS 
Metabolic - Obesity 
Neuro development - Neural defects, Autism and ADHD 
Female reproductive health - Uterine fibroids and endometriosis 
Cancer - Testicular cancer 
Cancer - Thyroid cancer 
Female reproductive health 
Adverse pregnancy outcomes 

Prioritisation 
High 
High 
High 
High 
High 
High 
Moderate 
Moderate 
Moderate 
Moderate 
Low 

The group provided reasons for some of their rankings: 

For male reproductive  health,  the group suggested  that cryptorchidism and hypospadias could  be 
detected in the Extended One-Generation Reproductive Toxicity Study (EOGRTS). However, it was 
considered  that  there  is  insufficient  investigation  into  the  reasons  for  reduced  semen  quality. 
Semen quality should be considered of high importance. 

For breast cancer, they believed that this disease should be a high priority since it is an important 
disease and understanding could easily be improved. They also highlighted how male breast cancer 
rates are also increasing. Attendees within the group believed that the method for mammary gland 
histology  in  animal  studies  is  often  conducted  incorrectly.  They  highlighted  that  when  using 
rodents,  it  is  necessary  to  ensure  that  the  appropriate  strain  of  rodent  is  used  for  breast  cancer 
assessment.  It  was  considered  that  the  most  sensitive  animal  model  was  not  always  being  used 
and this was a matter that could be improved. It has been shown that Sprague-Dawley and F344 
rats  have  differing  sensitivities  to  estrogen,  while  there  are  also  differences  between  inbred  and 
outbred strains (e.g. ACI and ACWi rats). 

In  relation  to  metabolic  dysfunction  –  obesity,  the  group  felt  that  there  are  good  models  for  the 
assessment of obesity (e.g. see OBELIX project). They believed an augmentation of current tests is 
possible to include more sensitive endpoints, which would increase the weight of evidence (WoE) of 
endocrine  related  effects.  There  are  also  a  number  of  biomarkers  for  obesity,  which  could  be 
incorporated  into  the  EOGRTS  study.  They  also  felt  that  there  is  a  need  to  consider  the 
interpretation  of  data.  For  example,  there  have  been  a  number  of  EOGRTS'  that  have  been 
conducted where effects on  body weight have  apparently been ignored. To identify an ED-related 
component  in  the  observed  weight  changes  the  development  of  circulating  biomarkers  would  be 
useful.  

For diabetes - type I, they felt that this was of high importance due to its increasing frequency and 
the  high  concerns  associated  with  the  disease.  They  also  felt  that  diabetes  was  perhaps  more 
relevant to immune dysfunction. Diabetes - type II was also felt to be of high importance, however, 

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there are no relevant methods for its detection in the standard data package. The group concluded 
that methods do exist for the testing of type II diabetes and these could be used. 

The group suggested that hypothyroidism should perhaps be rephrased as ‘hypothyroxinema’, the 
incidence  of  which  in  the  US  is  ~10%.  The  group  suggested  that  brown  fat  measurements 
(amount) could be a useful measure in F1 pups. 

For female reproductive health, the group indicated that Polycystic Ovarian Syndrome (PCOS) was 
of high importance. The group indicated that it can be considered a transgenerational effect, which 
is  not  necessarily  covered  by  the  current  test  guidelines  and  it  could  also  impact  on 
fertility/fecundity. They believed that an evaluation of this potential could be made by extrapolation 
from  Level  2/3  assays,  specifically  assays  that  assess  (or  could  be  enhanced  to  measure) 
androgenic  and  antiandrogenic  activity.  Also,  polycystic  ovaries  can  be  looked  at  histologically  in 
relevant  studies.  Animals  would  need  to  be  exposed  postnatally  and  viewed  for  up  to  90-days  to 
observe  such  effects.  However,  they  suggested  that  the  evidence  base  may  not  be  sufficient  to 
implement changes to the OECD TG and that the rat model is perhaps not a very sensitive model. 

A general consideration made by the group was how there are no good animal models for a lot of 
the  female  reproductive  endpoints.  Fecundity  may  also  not  be  covered  and  changing  the  mating 
procedures  may  be  useful.  The  estrous  cyclicity  was  highlighted  as  not  being  a  particularly 
sensitive marker. It was suggested that fish tests could be used to measure fecundity, which may 
be more practical or realistic. The group also felt that mammalian models are lacking. Overall the 
group felt that female reproductive health can be considered as moderately important when ranked 
against all the adverse outcomes discussed. 

For  testicular  cancer,  the  group  indicated  that  the  rat  is  not  susceptible  to  this  disease,  but  that 
this may be captured by other testicular dysgenesis syndrome (TDS) endpoints. The mechanism of 
Leydig cell tumour formation is completely different in rats compared to humans. 

For thyroid cancer, the group suggested that this is a very high concern; however, there are some 
sensitive assays available to detect these endpoints. The group suggested that humans do appear 
to get Thyroid Stimulating  Hormone  (TSH) induced thyroid cancer, as  observed in  rats. Thus, the 
group suggested that this was moderately important out of all the adverse outcomes discussed. 

The group also discussed adverse pregnancy outcomes. They felt that this is covered and thus can 
be considered to be low priority. They suggested that there can be a lot of contributing factors. 

Human Health Summary 

There  was  generally  a  consensus  among  the  human  health  groups  in  terms  of  the  main  adverse 
outcomes. The most important ones considered by the groups are: 

 

Thyroid-related Developmental Neurotoxicity 

  Metabolic Dysfunction 

 

Female Reproduction 

  Male Reproduction 

It  should  also  be  noted  that  some  groups  also  ranked  highly  hormone-related  cancers  and  one 
group highly ranked Immune / Autoimmune / Inflammatory disorders. 

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2.3 

Environmental Health 

Adverse outcomes associated with the environment  were presented to the workshop attendees as 
presented in Table 2-6. The adverse outcomes were categorised in groups along with taxa.  

Table  2-6:  Overview  of  environmental  adverse  outcomes  associated  with  endocrine 
disruption 
Reproductive Health 
Growth and Development 
Adrenal disorders 
Immune disorders 

Some information about biological plausibility of endocrine MOA is reported in the State of the Art 
Assessment  of  Endocrine  Disrupters  Annex  3:  comparative  analysis  of  endpoints  and  assays  by 
human  health  and  wildlife  endpoint.  Some  information  about  evidence  of  endocrine  attribution  is 
reported  in  the  State  of  the  Art  Assessment  of  Endocrine  Disrupters  Annex  1:  Summary  of  the 
State of the Science (Kortenkamp et al., 2011). 

The  adverse  outcomes  were  based  on  the  JRC  survey  (2017)  report  which  highlights  specific 
disorders in wildlife. An impact of endocrine disruption identified by attendees at the workshop that 
is not directly highlighted in the JRC survey (2017) is the impact on animal behaviour. An example 
might  be  effects  on  migration.  Glucocorticoids  are  essential  for  migrating  birds  (e.g.  white  crown 
sparrows) and developmental programming. 

As  part  of  the  first  break-out  group  sessions,  in  addition  to  identifying  any  currently  unidentified 
adverse  outcomes,  the  attendees  also  discussed  what,  in  their  opinion,  were  the  most  important 
adverse effects for the environment for which relevant TMs have to be developed or enhanced. 

Details of the discussions in the two environmental health break-out groups are summarised below. 

Environment Group 1 and Environment Group 2 

Both of the environment group discussions were very similar in what they thought  were the most 
important areas and these are shown in Table 2-7. 

Table 2-7: Priority ranking of environment outcomes (Environment Group 1 and 2) 
Adverse outcomes related to 
Reproductive Health 
Growth and Development 
Adrenal disorders 
Immune disorders 

Prioritisation 
High 
High 
Low 
Low 

A summary of the combined discussion from the environment groups is provided: 

Behaviour:  Environment  group  1  indicated  that  effects  on  behaviour  (including  effects  on 
migration) was also an important adverse outcome. However, in the plenary discussion the groups 
considered  that  although  such  behavioural  changes  may  impact  on  reproduction  there  were 
concerns that current test methods may not capture such indirect effects on reproductive success. 
For example, changes in behaviour or lipid metabolism in migratory birds might mean that they do 
not  make  it  to  their  destination  to  reproduce.  The  difficulty  in  assessing  behavioural  effects  in 
ecotoxicological studies was acknowledged. 

Adrenal disorders: There are a small number of examples (e.g. seal declines in the Baltic Sea) but 
the  adverse  outcome  is  also  linked  to  reproductive  effects  and  potentially  immune  disease.  In 
terms of laboratory test methods, effects on the adrenal system would be expected to be captured 
by reproductive studies. There are gaps in test systems for indicating this specific mode of action 
but  this  might  be  considered  a  lower  priority  because  there  are  few  examples.  Adrenal  disorders 
are not relevant for all groups (e.g. invertebrates).  

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Immune  disorders:  These  are  theoretically  very  important,  but  there  is  also  a  link  back  to  an 
integrated  reproductive and general  health  effect.  Reproductive  and  developmental  effects  should 
take priority because of regulation  at the population level (adverse effect). It can be very difficult 
to determine if immune effects observed in the field are due to chemical effects. There is a lack of 
specificity with the adverse outcome often being a secondary effect. For example, in  amphibians, 
there have been examples of chemicals affecting the immune system, which increases the number 
of  parasites  and  results  in  an  observed  adverse  effect  via  the  retinoid  system  (i.e.  growth  of  an 
additional limb). Fungal disease can also be exacerbated due to immune stress. Invertebrates have 
an immune system but it is much less well understood compared with vertebrates. 

The groups discussed which adverse outcomes were associated with wildlife population declines. It 
was  acknowledged  that  there  are  difficulties  in  field  evaluations  and  ecoepidemiology  in 
demonstrating  cause  and  effect.  Other  natural  environmental  factors  can  give  effects  consistent 
with  those  observed  for  endocrine  disruptors,  complicating  the  issue.  The  level  of  evidence  is 
therefore  not  always  sufficient  to  demonstrate  associations  to  a  causative  factor.  The  strongest 
links  for  wildlife  population  declines  are  for  adverse  outcomes  for  reproductive  health  but  the 
evidence for other outcomes is weaker or harder to prove. Laboratory evidence is therefore needed 
to imply potential population level effects. 

One  group  also  discussed  the  most  important  modalities  for  the  pathways  for  the  high  priorities; 
the group indicated that these were: 

  Reproductive health: HPG/HPA/HPE/HPT/RXR/PPAR/Somatotropin – i.e. all signalling 

pathways 

  Growth and development: HPT/HPG/Somatotropin/RXR/PPAR/HPA – again almost all of 

them  

  Behaviour: HPA/HPG/HPT 

The  following  aspects  were  highlighted  for  consideration  in  prioritising  adverse  outcomes  for  the 
environment; 

 

 

 

Plausibility – you need to connect adverse outcome to the pathway 

Pathway – some pathways are more relevant to the environment than others  

Ecosystem health 

  Biodiversity 

  Cost in terms of ecosystem services (e.g. pollinators/seafood)  

 

Food security and safety 

Environmental Health Summary 

There was a strong consensus among the two environment groups in terms of the  most important 
adverse outcomes, which were identified as being effects on: 

  Reproductive Health 

  Growth and Development 

This is because  the established  protection  goal for the environment  is  set  at  the population level. 
Therefore, the adverse outcomes of interest are reproduction and development as directly linked to 
population stability. Workshop attendees agreed that immune  and  adrenal effects were important 
but  that  the  adverse  outcomes  would  likely  be  captured  using  reproductive  and  developmental 
endpoints.  Changes  in  behaviour  might  ultimately  lead  to  reduced  reproductive  success  (e.g. 
changes in behaviour of  migratory  birds not  reaching  the relevant locations or changes  in mating 
behaviour of fish). Even if linked to reproductive effects in the end, current tests are not designed 
to  adequately  capture  such  effects.  However,  it  was  noted  that  this  is  a  general  issue  when 
measuring and interpreting behavioural outcomes. 

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3. Identification of gaps 

3.1 

Introduction 

For  the  JRC  survey  (JRC,  2017),  experts  were  asked  to  rank  endocrine  related  adversity,  in 
consideration of the predictability of existing test methods (TMs). They were further asked to rank 
endocrine related adversity by the need to develop new  TMs, thereby highlighting that the lowest 
predictability (i.e. biggest gaps) were not necessarily the highest priority. For example, endpoints 
indicative  of  thyroid  disruption  (i.e.  organ  weight,  histopathology  and  thyroxine  (T4)  levels)  are 
included  in  a  number  of  OECD  TGs,  however,  further  development  was  considered  to  be  the 
highest priority. Effects on senescence and latency of effects (e.g. Developmental Origins of Health 
and  Disease  (DOHaD)),  were  not  specifically  considered  in  the  survey,  however,  post-survey 
feedback on this issue indicated that many of these effects are likely to be covered by the identified 
disorders such as hormone-related cancers. The results of the survey are presented in Table 3-1. 

Table 3-1: Identification of gaps in available test methods for evaluation of ED 

Endocrine disease/disorder 
addressed in the survey 

State of the Science 
Evidence of ED 
attribution[1][2] 

Metabolic disorders 

Criteria partially met 

Immune related disorders in wildlife 
Adrenal disorders in wildlife 
Adrenal disorders in humans 

Thyroid-related disorders / 
neurodevelopmental disorders in 
children 
Immune related disorders in humans 
Hormone related cancers 

Human female reproductive health  
Wildlife male reproductive health 

Insufficient data 
Insufficient data 

Insufficient data 

Criteria partially met 

Insufficient data 

Criteria partially met 
Criteria partially met 
Criteria met 

JRC Expert Survey[3] 

Gaps in 
existing TM 

1 

2 
3 
4 

5 

6 
7 

8 
8 

Priority for 
developing 
TM 
2 

8 
11 
6 

1 

3 
7 

4 
9 

Wildlife female reproductive health 
Human male reproductive health 
[1] As stated in the State of the Art Assessment of Endocrine Disrupters Annex 1: Summary of the State of the 
Science. http://ec.europa.eu/environment/chemicals/endocrine/pdf/annex1_summary_state_of_science.pdf 
(Kortenkamp et al 2012).  

Criteria met 
Criteria met 

5 
10 

10 
11 

[2] When classifying the evidence of ED attribution, Kortenkamp et al. 2012, considered: (1) response in an 

intact whole organism; (2) response at multiple levels of biological organisation; (3) hormone action, release, 
metabolism or interactions; (4) dose-response observation; (5) comparison of phenotypes with 
pharmaceutical MOA; (6) life-stage sensitivity; (7) permanence; and (8) supporting data on endocrine activity 
from in vitro binding, transcriptional activation or cellular response studies.  

[3] JRC Expert survey on identification of gaps in available test methods for evaluation of endocrine disruptors.  

In this survey, it was evident that the top four areas in which gaps were defined in the evaluation 
of endocrine disruption involvement were: 

 

Thyroid-related disorders / neurodevelopmental disorders in children 

  Metabolic disorders 

 

Immune related disorders in humans 

  Human female reproductive health 

The  attendees, in  the  first  part  of  the  second  break-out  group  session,  were asked  to  review  the 
gaps  in  the  Test  Guidelines  for  the  identification  of  endocrine  disrupting  chemicals  in  those  key 
adverse outcome areas identified previously. 

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3.2  Human Health 

In  general,  all  human health  break-out  groups agreed  with  the JRC  survey  (2017)  ranking  of the 
perceived  gaps  in  the  regulatory  framework  for  the  assessment  of  potential  endocrine  disrupting 
chemicals. The comments made by the human  health break-out groups were quite similar and so 
these have been combined. 

From the human health break-out group discussions, some minor differences in the ranking of the 
gaps  in the testing strategy were  identified  but also, inevitably,  some  specific  gaps emerged  (see 
Table  3-2).  The  ultimate  objective  was  to  identify  the  top  20  to  30  proposals  for  testing 
improvements (details are presented in Section 4). However, from all groups the discussions led to 
the  grouping  of  many  of  the  proposals  into  more  general  aspects  of  importance  that  should  be 
further investigated, as well as some very specific areas where tests for particular parameters were 
requested.  According  to  the  discussions,  some  of  the  proposals  could  be  incorporated  into  the 
current testing strategy in the short term whereas many others would require significant research 
and  development.  The  principal  proposals  from  the  break-out  groups  considered  to  be  of  most 
importance  are  listed, in no particular order,  in Table 3-2  below.  As indicated  in Table 3-2,  these 
gaps  were  often  linked  with  a  potential  idea  about  how  these  gaps  could  be  addressed.  For 
example,  a  variety  of  additional  measurements  could  be  made,  including  markers  of  effects  on 
epigenetics, fat pads, bone, insulin and sperm. 

Approach 

Table 3-2: Most important developments from break-out groups 
Potential  enhancement  to  existing  TG  and  novel 
TM for development 
Progesterone receptor (PR) transactivation (in vitro) 
Sperm function markers 
Whole mount mammary gland histology 
Histological stains for brain tissue 
Harmonisation of behavioural assays5 
Serum bone biomarkers 
Bone breaking assay 
In vitro immune tests 
Immune cell markers in histopathology 
Inflammatory markers in serum 
Fasting glucose 
Insulin tolerance6 

New method 
Enhancement to TG 
Enhancement to TG 
Enhancement to TG 
Enhancement to TG 
Enhancement to TG 
Enhancement to TG 
New method 
Enhancement to TG 
Enhancement to TG 
Enhancement to TG 
Enhancement  to  TG 
/ new method 
Enhancement to TG 
Enhancement to TG 

Serum insulin levels 
Thyroid  and  neuro-developmental  markers  including: 
T3, T4, TSH measurements; pituitary TRH; brain size of 
F1  generation;  cognitive  function  in  offspring;  and 
thyroid TSH receptor expression 
peroxidase; 
Thyroid 
assays 
serum 
thyroxine-binding 
binding;  deiodinase  expression;  iodine  uptake;  T4 
binding protein displacement; TR reporter 
In vivo thyroid function screen7 

thyroid 
transthyretin 

including: 
globulin; 

New methods 

New method 

CF Level 

2 
4-5 
4-5 
4-5 
4-5 
4-5 
4-5 
2 
4-5 
4-5 
5 
4-5 

4-5 
4-5 

2 

3 

5   A wide range of behavioural and neurological observations are used in rodent toxicological tests and these 
are routinely investigated in 28-day or 90-day rodent toxicity tests (OECD TG 407 and OECD TG 408) and 
neurodevelopmental  studies.  There  is  a  call  for  more  widespread  use  of  these  non-invasive  assessments 
during longer term studies and a more rigid definition of appropriate tests. 

6  

It is recognised that Level 4 and 5 studies do not sufficiently investigate adversity related to the metabolic 
syndrome at present and this would be a significant development. 

7   For example, a study based on the existing OECD TG 407 which only investigates thyroid around 14 days 

long, including measurements of T4/T3/TSH and with detailed histopathological assessment. 

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Approach 

Table 3-2: Most important developments from break-out groups 
Potential  enhancement  to  existing  TG  and  novel 
TM for development 
PPARα,β/δ,ɣ transactivation assays 
CAR, RXR, RAR in vitro assays 
Epigenetic and molecular markers 
Serum chromogranin A 
Fat pad deposit weights/brown fat in pups 
Diagnostic  sub-acute  in  vivo  test  for  effects  on 
circulating hormones 
Combined  long-term  bioassay  and  reproductive  study 
with ED biomarker enhancements8 

New methods 
New methods 
Enhancement to TG 
Enhancement to TG 
Enhancement to TG 
New method 

New method 

CF Level 

2 
2 
All 
4-5 
4-5 
3 

5 

Another  point  raised  by  some  attendees  in  addition  to  the  lack  of  endocrine  specific  endpoints  in 
existing  test  methods  was  that  there  is  a  lack  of  exposure  during  sensitive  windows.  Although  a 
variety  of  in  vivo  regulatory  mammalian  toxicology  tests  do  together  cover  the  full  lifecycle, 
through the conduct of a number of different expensive studies. 

3.3 

Environmental Health 

The environment groups both agreed that there is real difficulty in proving that an adverse effect 
observed in the field is due to endocrine disruption. There is a need for basic biological research to 
understand  mechanisms  of  action  for  EDs  in  wildlife.  This  was  particularly  highlighted  as  an 
important  area  for  invertebrates  (specifically  molluscs).  The  disruption  of  the  retinoid  pathway  is 
the  most  likely  cause  of  tributyltin  (TBT)  effects  in  molluscs  but  is  still  not  well  understood  after 
decades  of  investigative  research.  In  general,  the  thyroid  system  needs  to  be  better  covered  in 
OECD tests. Where there is an opportunity to do so this is particularly important for fish. There are 
thyroid  assays  and  a  definitive  test  for  amphibians  (OECD  TG  231),  but  for  fish  which  are  more 
routinely used in testing, the available test methods are less specific. A need for thyroid endpoints 
for fish was also discussed at a recent DG ENV/ANSES workshop on thyroid disruption (EC 2017). 
The  behavioural  effects  of  EDs  were  also  identified  as  a  data  gap,  as  were  endpoints  for  the 
assessment of adrenal and immunotoxic effects. 

Some  of  the  commonly  used  OECD  guidelines  lack  relevant  endpoints  for  addressing  some 
endocrine modes of action. For example, many of the invertebrate species are parthenogenic and 
therefore  they  do  not  address  effects  on  the  male  (an  exception  being  the  lifecycle  test  with 
chironomids  (OECD  TG  223))  or  sexual  reproduction.  The  African  Clawed  frog  (Xenopus  laevis), 
which  is  the  model  species  used  for  amphibian  testing,  needs  to  be  induced  to  spawn  in  the 
laboratory so true reproductive testing is not possible. Further, the fish and amphibian models do 
not  include  species  that  only  reproduce  annually  for  logistical  reasons.  The  size  of  some  test 
species  can  be  a  limitation  when  it  comes  to  including  additional  endpoints.  For  example,  with 
zebrafish, there may be induction of the stress response during sample processing and only limited 
plasma  and  tissue  can  be  obtained  per  fish.  In  the  future  increasingly  sensitive  analytical 
techniques may help overcome some of these issues. 

A  proposed  test  method  for  birds  based  on  a  two-generation  test  with  the  Japanese  quail  is  not 
currently being pursued by the OECD because the test lacked reproducibility and it does not appear 
to  respond  to  known  endocrine  active  substances.  This  means  that  there  is  currently  no 
recommended  test  design  for  ED  assessment  with  birds  which  includes  supporting  mechanistic 
data.  There  are  also  currently  no  available  tests  for  reptiles.  In  general,  it  is  not  known  if  the 
current sentinel OECD species will sufficiently protect birds and reptiles from potential ED effects. 

8   The  identification  of  this  gap  was  mentioned  several  times  during  the  workshop.  The  perception  is  that, 
whilst  the  effects  of  a  substance  on  the  complete  mammalian  lifecycle  can  be  assimilated  over  the  full 
range  of  in  vivo  mammalian  toxicity  studies,  this  is  achieved  over  several  years  in  different  sources  of 
animals  and  at  considerable  expense  financially  and  in  terms  of  animal  use.  A  fully  integrated  study  is 
claimed to make considerable savings in both these areas (see Section 4). 

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There  was  discussion  regarding  improving  the  use  and  interpretation  of  existing  test  guidelines 
before  developing  new  ones.  An  example  was  the  recently  adopted  CF  Level  5  fish  Medaka 
Extended One Generation Reproduction Test (MEOGRT; OECD TG 240) which could be improved by 
understanding  differences  between  strains  and  getting  more  data  for  positive  control  substances. 
Indeed, there is currently a US/Japan collaborative effort to perform additional studies to improve 
interpretation.  

The  two  environment  groups  highlighted  similar  data  gaps.  Environment  group  1  identified  two 
major areas to focus on and an additional requirement for consideration: 

 

Thyroid effects (particularly in fish) 

  Research into mechanistic understanding for invertebrates, birds and reptiles 

  Some in the group thought that if the substance had a Kow >6 then dietary exposure to 

chemicals for endocrine testing should be considered 

Environment group 2 identified the following areas for which they considered data gaps existed:  

  Behavioural effects of EDs 

  Research into mechanistic understanding for invertebrates (molluscs) and birds  

 

Thyroid effects (particularly in fish) 

There were also some similarities for the research topics that the environment groups identified, 
for environment group 1 these were: 

 

 Invertebrates endocrinology/physiology (highest priority) 

o  Echinoderm research to look at development 

o  RXR research in invertebrates, specifically in molluscs as example of the most 

vulnerable species.  

o  No further endocrine mollusc guideline development until further research has 

described physiology and endocrinology (metabolic pathway).  

  Birds – A review of the effects of endocrine disruption in birds including analysis for which 
modes of action and associated specific endpoints might only be observed in birds (as 
opposed to mammals or other taxa) due to their unique characteristics. The review would 
include consideration of the outcomes of the US EPA validation work with the quail model 
including the impact of different diets. 

 

 

 

 More research on transgenic embryos/fish for screening  

 Reptiles (potentially connected to birds – for extrapolation) 

 Collect additional embryos for targeted analysis of DNA methylation marks, histone 
modifications and miRNAs - useful for assigning epigenetic effects in organisms 

The research needs identified by environment group 2 were: 

  Mollusc endocrinology 

 

Immunotoxicology in animals 

  Adrenals/interrenals 

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4. Ideas to close the gaps – TG and TM enhancement and development 

4.1 

Introduction 

The workshop participants discussed several ideas for addressing gaps. While most of these ideas 
were specific for human health assessment and environmental assessment, some screening assays 
were identified as useful from both perspectives. These ideas are listed in both Table 4-1 (human 
health) and Table 4-2 (environment). The in vitro assays are usually developed by using human or 
mammalian test systems. Many of these assays are already in development for use as part of the 
US ToxCast and Endocrine Disruptor Screening Program (EDSP). 

Mechanistic  information  to  support  an  endocrine  mode  of  action  is  a  challenge  for  both  human 
health  and  the  environment.  Sharon  Munn  (JRC)  presented  the  progress  to  date  with  adverse 
outcome  pathways  (AOPs)  within  the  framework  of  developing  integrated  approaches  to  testing 
and  assessment  (IATA).  An  AOP  is  a  conceptual  framework  that  portrays  existing  knowledge 
concerning the linkage between a molecular initiating event and an adverse outcome, at a level of 
biological  organisation  relevant  to  hazard  assessment.  AOPs  are  a  sequential  chain  of  causally 
linked  events,  starting  on  molecular  level  and  leading  through  different  levels  of  biological 
organisation (OECD AOP Development Programme 20129).  

AOPs  allow  for  the  mapping,  organisation  and  integration  of  various  types  of  information  derived 
from in vitro, in vivo and computational experimental systems.  

ED  identification  is  focussed  on  the  mechanism/mode  of  action  and  the  plausible  link  to  one  or 
more  adverse  outcomes.  AOPS  can  be  useful  in  helping  to  link  endocrine  mode  of  action  to  an 
adverse  outcome.  There  are  over  200  AOPs  in  the  OECD  knowledge  base10,  many  of  which  are 
endocrine relevant. AOPs describing an endocrine mode of action can be used to identify endocrine-
specific endpoints providing mechanistic and/or adverse effect data for inclusion in existing assays 
or as a basis for  the development of new  assays  for inclusion  at  any level  of the  OECD  CF.  AOPs 
may  also  provide  support  to  epidemiology  studies  by  using  mechanistic  data  to  strengthen 
associations between chemical exposure and adverse outcomes observed in populations.  

A preliminary screening list of proposals for testing improvements or enhancements was provided 
for review and discussion. These ideas to fill gaps had come from various sources including the JRC 
survey  (2017),  ideas  in  the  OECD  Work  plan  (2016a),  the  OECD  scoping  document  on  thyroid 
signalling  (OECD  2014),  The  State  of  the  Science  review  of  endocrine  disrupters  (WHO/UNEP 
2012),  and  a  recently  published  review  of  regulatory  test  methods  for  endocrine  adverse  health 
effects  (Manibusan  and  Touart  2017).  Other  potential  sources  of  ideas  include  the  Integrated 
Environmental  Assessment  and  Management  (IEAM)  Special  Series  on  Current  Limitations  and 
Recommendations  to  Improve  Testing  for  the  Environmental  Assessment  of  Endocrine  Active 
Substances by Coady et al. (2017) and OECD Detailed Review Paper (DRP) 178 (2012). Ultimately, 
the  objective  was  to  review  and  amend  this  list  based  on  the  expert  knowledge  of  the  attendees 
and  to  identify  those  options  which  were  likely  to  contribute  most  to  adverse  outcomes  of  the 
highest concern identified in the first break out session. The experts attending the workshop were 
also encouraged to introduce their own ideas. 

After the workshop, the attendees were also asked if they had any additional ideas that were not 
discussed  at  the  workshop;  the  attendees  were  also  asked  to  consider  the  prioritisation  factors 
when making suggestions. These additional ideas, provided by attendees, appear in Annex 2. 

9   For  further  information  see:  http://www.oecd.org/chemicalsafety/testing/adverse-outcome-pathways-

molecular-screening-and-toxicogenomics.htm  

10   OECD AOP knowledge base:  http://aopkb.org/  

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4.2  Human Health 

Details  of  the  discussions  in  the  four  human  health  break-out  groups  are  presented  below,  the 
ideas that were discussed by the attendees are summarised in Table 4-1. A more detailed outline 
of each idea also appears in Annex 2. 

Human Health Group 1 

The  group  discussed  how  in  order  to  bridge  the  gap  between  regulatory  needs  and  science,  it  is 
important  to  have/develop  a  testing  strategy.  Since  the  endocrine  disruptor  field  is  large,  it  was 
suggested  that  it  would  seem  sensible  to  start  with  the  EATS  axis  since  this  is  the  area  which  is 
most known about at present. It was suggested that it is important to have a strategy behind the 
test rather than simply trying to add tests to the OECD Conceptual Framework. 

It was also  discussed  that there was a clear  gap  of  data  on temporal aspects, thus  data  tracking 
the potential development of endocrine disruption effects during the lifespan of the test species are 
lacking.  It  was  suggested  by  some  of  the  attendees  to  combine  fertility  studies  with  that  of  the 
long-term bioassay such that the potential for developing cancers can also be studied from in utero 
exposure  onwards.  This  suggestion  was  based  on  the  plenary  presentation  about  an  integrated 
model  for  the  assessment  of  multiple  toxicological  endpoints  in  one  rat  bioassay  by  Fiorella 
Belpoggi. Such a study, it is claimed, would reduce the overall number of animals required by the 
current set of rodent OECD TGs used to cover different endpoints, but it would be a very expensive 
study. Not all of the attendees agreed that this would be a good approach as there is no regulatory 
need for such a study and may not actually reduce the number of animals as claimed. Under PPPR 
and BPR the OECD TG 451 (Carcinogenicity Studies) and OECD TG 452 (Chronic Toxicity Studies) 
are  already  combined  and  the  requirement  is  to  submit  an  OECD  TG  453  (Combined  Chronic 
Toxicity/Carcinogenicity  Studies).  It  was  suggested  that  the  dose  setting  rationale  for  a  cancer 
bioassay and a reproductive toxicity study is different, thus, there would most likely be a need to 
have separate dosing regimes within the study. 

Functional neurodevelopmental toxicity endpoints were identified as an area where further research 
is required. 

A member of the group highlighted the need for a Level 3 in vivo test which would help to bridge 
the  gap  to  the  studies  which  may  already  have  been  conducted,  i.e.  a  short  term  mechanistic  in 
vivo test. The group indicated that a similar idea was also raised in the thyroid workshop. This test 
should be based on the study design of the sub-acute repeated dose toxicity study (OECD TG 407). 
But, it may not have to be longer than 14 days to allow measurements of specific hormones and at 
the same time transcriptomic (micro arrays or next generation sequencing) could be conducted on 
a  few  organs,  thus  providing  mechanistic  information  for  all  EATS  modalities  in  one  go.  They 
suggested  that  in  a  relatively  short  timeframe  such  a  test  could  be  developed  as  most  of  the 
endpoints  needed  are  already  measured  in  other  assays  or  the  techniques  exist  to  make  the 
measurements.  The  study  protocol  would  only  need  modification  and  validation  in  terms  of 
hormone  measurements  and  positive  controls  for  the  gene  expression  (most  of  this  information 
already  exists  in  the  public  domain).  The  advantage  of  such  a  test  would  be  that  it  is  relatively 
cheap, quick and would provide in vivo mechanistic information on all endocrine modalities in one 
go (depending on the tissue). 

Human Health Group 2 

The  group  discussed  a  number  of  ideas  as  part  of  the  session.  The  group  indicated  that  whole 
mounted female and male mammary gland assessments  should be considered for inclusion  in the 
EOGRTS  study.  They  suggested  that  these  could  also  be  considered  for  inclusion  within  the 
repeated dose 90-day oral toxicity study in rodents (TG 408) and/or the repeated dose 28-day oral 
toxicity study in rodents (TG 407). 

It  was  discussed  how  the  thyroid  hormones  thyroxine  (T4)  and  triiodothyronine  (T3)  should  be 
measured as part of in vivo tests and a definition of degree of change which should be considered 
adverse  should  be  developed.  If  not  incorporated,  additional  parameters  indicative  of  adverse 
outcomes from thyroid hormones insufficiency may need to be incorporated into current TGs. 

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The  group  also  believed  that  a  number  of  enhancements  could  be  incorporated  into  the  existing 
TGs; this includes taking measurements of serum leptin, adiponectin, insulin and the lipid profile as 
part  of  in  vivo  tests.  Other  measurements  that  could  be  made  are  serum  oxytocin  and  the  Anti-
Mullerian  Hormone  (AMH)  as  a  marker  of  fertility.  Furthermore,  markers  could  be  measured  for 
potentially neuronal/behavioural effects. 

It was suggested that parameters informative of antiandrogenic effects on accessory glands should 
be  assessed  with  respect  to  potential  infertility.  Also,  measurements  of  insulin  release  by 
pancreatic  beta  cells,  beta  cells  division  and  beta  cell  death  could  be  made.  The  group,  as 
recognised  by  other  groups,  also  suggested  that  the  Developmental  Neurotoxicity  (DNT)  and 
Developmental Immunotoxicity (DIT) cohorts which are part of EOGRTS TG 443, perhaps should be 
mandatory, since currently under REACH they are triggered only if there are particular concerns. 

The group indicated that more time was needed to think about temporal aspects of ED testing and 
Foetal origins of adult disease/Developmental origins of health and disease (FOAD/DOHaD). 

Human Health Group 3 

The  approach  taken  by  this  group  was  to  agree  the  4/5  adverse  outcomes  considered  to  be  the 
most  important  from  break-out  session  1,  and  then  try  to  connect  the  assays  with  the  adverse 
outcomes.  The  adverse  outcomes  that  were  considered  by  the  group  were  neurodevelopment, 
female reproductive health, obesity (metabolic dysfunction) and hormonal cancers. 

For  neurodevelopment,  the  group  discussed  how  more  brain  endpoints  are  required.  In  vitro 
oligodendrocyte  and  neurite  development  assays  were  two  such  ideas  that  were  raised.  Other 
indicators  of  thyroid  toxicity  such  as  a  positive  result  in  the  amphibian  metamorphosis  assay 
(OECD  TG  231)  or  measures  of  thyroid  hormone  T4  increasing/decreasing  (±10%  up  or  down) 
were discussed as being a potential trigger for further investigation of neurodevelopmental effects. 
Gene markers were also discussed as perhaps being a useful indicator for further investigation. 

For  female  reproductive  health,  the  group  discussed  how  there  are  very  few  endpoints  that  are 
amenable  to  testing  at  this  time  (i.e.  PCOS,  endometriosis).  However,  there  are  methods  for 
precocious puberty. 

For  obesity,  there  were  discussions  about  how  endpoints  such  as  dyslipidemia  and  adipocyte 
differentiation could be incorporated into appropriate studies, however, it was not known whether 
suitable  models  were  either  available  or  in  development  for  a  lot  of  these  endpoints  (i.e.  likely 
deficit in basic research in these aspects). 

For hormonal cancers, it was suggested that efforts should be made to link  this adverse outcome 
with  other  established  diseases/adverse  outcomes  (e.g.  adverse  outcomes  based  on  male  and 
female  reproductive health  and metabolic dysfunction). If  such  a link  could be  established then it 
was  suggested  that  considerations  should  be  made  to  enhance  the  relevant  existing  assays  to 
include measurements for hormonal cancers. 

The group also considered  that there is a definite  need for more  research  into gene markers and 
other biomarkers since there was a level of agreement that there was a potential for these to aid 
interpretation of a number of studies. 

Human Health Group 4 

The group discussed a number of ideas; for reproductive health it was suggested that more should 
be  done  to  analyse  sperm  function.  There  was  a  discussion  about  whether  cryptorchidism  may 
already  be  covered,  but  it  was  suggested  that  perhaps  a  longer  observation  window  would  be 
useful. Histopathology of the ovaries was discussed. Because ovaries have an endocrine function it 
was suggested that an evaluation of a number of sections of ovaries from the test organisms used 
in in vivo studies would be advantageous as an endocrine effect may be detectable. However, the 
group questioned whether or not this would be feasible. 

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The  group  suggested that  research  into  in  vitro  inhibition  of  prostaglandin  synthesis  (PG2)  would 
support  in  vitro  assay  development11.  The  group  also  discussed  how  they  thought  that 
transactivation assays would provide more information than binding assays, which also frequently 
use  radioactivity.  They  were  of  the  opinion  that  a  higher  priority  may  therefore  be  given  to 
transactivation  assays  over  binding  assays.  However,  advantages  of  binding  assays  are  that  they 
are usually quick and they can be highly sensitive. 

For  the  adverse  outcome  hormonal  cancer,  there  was  agreement  that  the  two-hit  theory  of 
carcinogenesis  (initiation  and  promotion)  is  important.  It  was  suggested  that  whole  mount 
histopathology  of  mammary  tissues  should  be  included  in  studies  with  rats.  Perhaps  a  long  and 
expensive combined study right through to senescence is not the answer but rather use epigenetic 
markers, which will probably need to be developed.  It  was suggested  that  research to implement 
these omics technologies should be prioritised. 

Chromogranin  A  could  be  a  useful  serum  biomarker  to  introduce  into  in  vivo  studies  as  it  is 
considered  a  useful  indicator  of  carcinoid  syndrome  (carcinoid  tumours)  and  both  pancreas  and 
prostate cancers. 

The group suggested that the routine DNT assessment will miss a lot of hormonal mechanisms of 
action. 

The placenta could be used as a marker for female reproductive health. It was considered by the 
group to be a huge wasted resource. Placental perfusion studies  and  biomarkers/gene  expression 
studying  of  the  placenta  should  be  considered.  It  was  highlighted  that  a  consortium  is  trying  to 
address this as a COST action in the first place. However, there is a huge amount of variation and 
further research on the relevance and reliability of developing assays based on the human placenta 
within  the  context  of  endocrine  disruptor  assessment  is  required.  Such  examinations  of  the 
placenta  could  be  an  addition  to  the  prenatal  developmental  toxicity  study,  in  which  dams  are 
sacrificed  the  day  before  the  expected  delivery  date.  Histone  modification,  DNA  methylation,  and 
miRNA  epigenetic  markers  are  worthy  of  consideration;  however,  more  baseline  information  is 
needed  (in  vivo)  to  define  what  a  normal  range  is  and  what  would  be  considered  an  adverse 
outcome. In addition epigenetic markers could be studied (in vitro and in vivo). 

Immune disorders were discussed by the group and it was suggested that development of markers 
of  inflammation  are  required.  However,  it  was  mentioned  that  microarrays  of  cytokines  and 
biomarkers  of  inflammation  already  exist.  It  was  highlighted  that  even  if  rat  models  of  diabetes 
already  exist,  there  is  anyway  a  need  for  the  development of  primary  markers  for  non-genotoxic 
carcinogenesis pathways. 

The CAR, PXR and RAR/RXR transactivation assays, whilst not necessarily endocrine specific, have 
been  implicated  in  the  process  of  disruption  of  some  metabolic  processes  e.g.  lipid  metabolism, 
adipogenesis  and  steroid hormone  metabolism.  Therefore,  these  assays  were  suggested  as  being 
potentially useful in providing information on endocrine relevant mechanisms of action. 

When  considering  neurological  diseases,  harmonisation  of  assessments  of  cognitive  and 
behavioural  endpoints  in  in  vivo  studies  is  important.  Cortical  heterotopias  in  offspring  should  be 
considered,  a  change  in  the  structure  of  the  brain  sufficient  to  demonstrate  adversity.  The 
measurement  of  electrophysiological  endpoints  in  vivo  was  also  identified  as  being  relevant 
However,  while  the  measurement  of  electrophysiological  endpoints,  ex  vivo,  is  interesting,  it  is 
probably not relevant for regulatory test guidelines. Neuronal migration could also be included in in 
vivo studies. In the future, it may be interesting to look at sexually dimorphic regions in the brain, 
looking at specific regions. 

For assessment of metabolic dysfunction, it is considered that existing in vivo test guidelines could 
be enhanced by the addition of measurements and examination of brown fat. The fat pads should 
be excised at necropsy and examined histologically. 

11   Note that this idea is discussed in more detail in Table 4-2 (environmental heath) and Annex 2, but it could 

also be applicable to human reproductive health. 

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Regarding bone health, peripheral biomarkers of catabolism/metabolism of bone, Fibroblast Growth 
Factor  23  (FGF23),  and  additional  serum  markers  could  be  added  to  existing  TGs.  Although 
interesting,  peripheral  quantitative  computed  tomography  is  probably  not  practical  for  regulatory 
toxicology studies. Bone breaking might be a more useful endpoint. 

Human Health Group Ideas 

As indicated in the  break-out group summaries  above, ideas to close  the gaps were discussed by 
the  attendees  at  the  workshop.  A  summary  of  these  ideas  appears  in  Table  4-1  below.  Further 
information  on  the  various  ideas  is  presented  in  Annex  2.  The  cross  reference  shown  in  brackets 
correlates with the ideas  in Annex  2;  some ideas were also  highlighted by environmental groups. 
Ideas  highlighted  by  human  health  experts  may  also  be  applicable  to  environmental  health  and 
vice versa. 

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Table 4-1: Suggested ideas to close the gaps for human health 
Adverse 
outcome 

in vitro / 
in vivo 

Suggested  tests  to  fill  gaps  (with 
cross  references  to  Annex  2  and 
environmental ideas)  
Thyroid pathway screens: 
Deiodinase (1, see also environment idea 1) 

Test 

CF 
Level 

Further information 

Thyroid 

Thyroid pathway screens: 
TBG binding assay; TTR serum binding 
assay; T4 binding protein displacement 
assay; and TR reporter assay) (2, see also 
environment idea 2) 

Thyroid pathway screens: Thyroid 
peroxidase assay, Sodium Iodide Symporter 
(3, see also environment idea 3) 

Application of amphibian thyroid assays 
(XETA) since HPT axis is well conserved in 
vertebrates (4) 
Serum T4/T3/TSH assays in parents and 
juveniles (5) 

in  vitro  / 
ex vivo 

New 

in vitro 

New 

2 

2 

Both 

Both 

2 / 4 - 5 

in vivo 

New 

3 

in vivo 

Enhancement 

4 - 5 

Bone breaking assay (6) 

in vivo 

Enhancement 

4 - 5 

Thyroid-related 
developmental 
neurotoxicity 

Dendritic arborisation assay (7) 

in vitro 

New 

Neuronal progenitor cell proliferation/ 
differentiation assays; neurite extension (8) 

in vitro 

New 

2 

2 

Oligodendrocyte differentiation (9) 

Both 

New 

2 - 4 

Assays  already  exist,  as  described  in  OECD  207,  some 
more development and research may be required 

Screening  assays  would  be  useful.  Need  to  consider 
alongside the recommendations from the DG ENV / ANSES 
Thyroid Testing Workshop (2017) 
For each of these ideas suggested assays already exist, as 
described  in  OECD  207,  some  more  development  and 
research may be required 

Screening  assays  would  be  useful.  Need  to  consider 
alongside the recommendations from the DG ENV / ANSES 
Thyroid Testing Workshop (2017) 
Suggested  assays  already  exist,  as  described  in  OECD 
207,  some  more  development  and  research  may  be 
required,  this  would  be  a  new  in  vitro  TG  or  an 
enhancement if included in in vivo TGs 
A  draft  OECD  TG  exists  and  this  test  method  is  currently 
being validated by OECD 

Assays  for  rodent  T4/T3/TSH  are  in  place.  Decision  on 
which  TG  to  include  the  measurements  and  timing,  for 
example measuring levels in rodents at Post Natal Day 16  
Standardised  procedures  for  measuring  bone  density  are 
available  and  could  be  included  routinely  in  short  and 
long-term rodent studies 
After exposure  to  test  compounds  dendrite  arborisation 
can be assessed through immunocytochemical staining 

Cell  proliferation  can  be  measured  following  exposure  to 
T3  and  could  be  measured  by  total  metabolic  activity  of 
hNPCs cells 
Research  has  been  conducted  using  cell  cultures  and 
zebrafish,  they  are  suggested  as  being  well  suited  for 
investigating oligodendrocyte development 

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Table 4-1: Suggested ideas to close the gaps for human health 
Adverse 
outcome 

Suggested  tests  to  fill  gaps  (with 
cross  references  to  Annex  2  and 
environmental ideas)  
Cognitive function test (10) 

in vitro / 
in vivo 

in vivo 

Enhancement 

4 - 5 

Test 

CF 
Level 

Further information 

Thyroid-related 
developmental 
neurotoxicity 
(Cont) 

Cortical heterotopia in offspring (11) 

in vivo 

Enhancement 

4 - 5 

Other thyroid endpoints in F1 pup brains 
(12) 

in vivo 

Enhancement 

4 - 5 

Mandatory to include DNT and/or DIT 
cohorts in OECD TG 443 study unless a 
justification can be made to exclude(13) 
Electrophysiological endpoints (14) 

in vivo 

Enhancement 

5 

in vivo 

New 

4 - 5 

Adrenal / Steroid 

Glucocorticoid Receptor (GR), 
Mineralocorticoid receptor (MR) 
transactivation assays (15, see also 
environment idea 22) 
Adrenal steroid synthesis in vivo: data 
collection of a range of steroids is valuable 
for mechanistic information (not just 
adrenal) provided not too many extra 
animals are needed. (16, see also 
environment idea 24) 
More and better-defined assays are required 
(17) 

in vitro 

New 

2 

in vivo 

Enhancement 

3 - 5 

Both 

Both 

Placental model (18) 

Both 

Both 

Assays for PCOS/endometriosis using gene 
expression/transactivation (19) 
Progesterone receptor (PR) transactivation 
(20, see also environment idea 15) 

in vitro 

New 

in vitro 

New 

Female 
Reproductive 
Health 

All 

All 

2 

2 

Some tests known to be in regulatory use are considered 
by  some  to  lack  in  sensitivity.  Others  may  need 
development and decisions on which TG to include them 
Specific examination of brain tissue which may require 
special staining 
Similar to the previous suggestion, TGs (e.g. OECD TG 
421 and 443) could be enhanced by thyroid hormone 
measurements being performed in F1 pup brains. 
Oligodendrocyte analysis could also be performed 
This would be a change, to include the DNT and/or DIT 
cohorts by default, this might be more about modifying 
the regulatory requirements 
Research activity, electrophysiological techniques can be 
used to examine actions of compounds on e.g. 
hypothalamic neurons in vivo 
e.g. Marks et al. Tools for the Discovery and 
Characterization of GR Modulators: A Comparison of 
Binding, Co-regulator Interaction, and Transactivation 
Assays 
If you suspect alterations HPA axis take sample for steroid 
hormone analysis. 
It is increasingly possible for various hormone levels to be 
measured by LC-MS/MS methods using normal serum 
levels 

Research  activity,  these  could 
involve  new  TG  or 
enhancements, some assays have been highlighted by the 
OECD (2017) 
Research  activity.  Potential  assays  have  been  suggested 
by Wakx et al. (2014) and Rat et al. (2017) 
Research  activity,  some  work  has  been  published  in  this 
area (Salama et al. 2009 and Utsunomiya et al. 2008) 
Several PR transactivation assays have been developed. A 
PR  transactivation  assay  may  require  co-factors  to  be 
considered 
Include in OECD TG 408 and 443 studies 

Serum oxytocin and Anti-Mullerian Hormone 
(21) 
Histopathology of ovaries and follicle count 
(22) 
Whole mount (male and) female mammary 
gland assessment (23) 

in vivo 

Enhancement 

4 - 5 

in vivo 

Enhancement 

4  

Include in OECD TG 408 study 

in vivo 

Enhancement 

4 - 5 

Include in OECD TG 443, 407 and 408 studies 

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Table 4-1: Suggested ideas to close the gaps for human health 
Adverse 
outcome 

in vitro / 
in vivo 

Suggested  tests  to  fill  gaps  (with 
cross  references  to  Annex  2  and 
environmental ideas)  
Prostate-Specific Antigen (PSA) secretion, 
prostate model and antiandrogenic effects 
(24) 

Both 

New 

All 

Test 

CF 
Level 

Further information 

Male Reproductive 
Health 

Sperm function and morphology (25) 

in vivo 

Enhancement 

4 - 5 

Cryptorchidism endpoints (26) 
PPAR α, β/ɣ, δ transactivation assays, also, 
Retinoid X Receptor (RXR) and Retinoic Acid 
Receptor (RAR) transactivation assays (27, 
see also environment ideas 10 and 11) 
Evaluation of pancreatic beta cells (28) 

in vivo 
in vitro 

Enhancement 
New 

4 - 5 
2 

in vitro 

New 

2 

Adipocyte differentiation (29) 
Body fat estimation (30) 

in vitro 
in vivo 

New 
Enhancement 

2 
4 - 5 

Metabolic 
dysfunction 

Serum gut hormones and adipokines (31) 

in vivo 

Enhancement 

4 - 5 

Serum and liver retinoic acid levels (32) 

in vivo 

Enhancement 

4 - 5 

Evaluation of glucose, serum insulin and 
insulin tolerance (33) 
Lipoprotein profile (34) 

in vivo 

Both 

4 - 5 

in vivo 

Enhancement 

4 - 5 

Assessment of brown fat in F1 pups (35) 

in vivo 

Enhancement 

4 - 5 

Determination of endocrine MoA to aid test 
development (36) 

Both 

Both 

All 

Apoptosis in thymus (37) 

in vivo 

Enhancement 

3 - 5 

Serum chromogranin A (38) 

in vivo 

Enhancement 

4 - 5 

Epigenetics (39) 

Both 

Enhancement 

All 

Immune / 
Autoimmune / 
Inflammatory 

Hormonal cancer 

General 

in  vivo 

Research  activity.  Work  by  Lorenzetti  et  al.  (2010)  and 
Smeriglio  et  al.  (2014)  has  suggested  a  tiered  in  vitro 
approach  while 
rat  studies  have  shown 
antiandrogenic effects Mukherjee et al. (1992) and Gupta 
et al. (2017) highlight mutations in the PSA that correlate 
with male infertility 
Technology  already  exists  for  rodents  and  should  be 
incorporated  into  reproductive  toxicity  studies  in  rodents 
as  a  routine,  additional  miRNA  measurements  could  be 
made 
Endpoints in rodent offspring 
Transactivation  assays  for  the  different  PPARs  (also,  RXR 
and  RAR  assays)  exist  already  (see  OECD  DRP  178). 
OECD test guideline may need to be developed 

is 

of 

pads 

from 

animals 

research 

individual 

Further 
required,  some  studies  have 
highlighted  the  impact of BPA  on  the  insulin  synthesis  by 
pancreatic β-cells 
Distinguish obesity and diabetes 
Analysis 
fat 
(predominantly white adipose tissue) 
These  measurements  could  be  incorporated  into  rodent 
studies 
Assays  may  need  to  be  developed  for  use  in  rodent 
studies 
New  test  could  be  developed,  while  the  modification  of 
existing tests may need to be undertaken  
Include  in  OECD  TG  408  study  and  possibly  longer-term 
studies also 
For  example,  assessment  could  be  included  in  OECD  TG 
443 and perhaps other Level 4-5 studies 
Research  activity  e.g.  development  of  immune  in  vitro 
tests, 
and 
inflammatory markers in serum as part of in vivo assays 
Normal  H&E  histopathological  assessment  to  be  included 
in all in vivo rodent tests of the CF 
Suggested marker for carcinoid syndrome which could be 
included in medium to long-term rodent studies.  
Analysis  may  include  miRNA  of  sperm.  A  challenge  is  to 
link  epigenetic  changes 
to  adverse  outcomes  and 
identifying specific epigenetic endpoints 

biomarkers,  metabolomics 

cytokine 

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Table 4-1: Suggested ideas to close the gaps for human health 
Adverse 
outcome 

in vitro / 
in vivo 

Test 

CF 
Level 

Further information 

Suggested  tests  to  fill  gaps  (with 
cross  references  to  Annex  2  and 
environmental ideas)  
A new level 3 short term in vivo mechanistic 
test based on OECD TG 407 to measure 
EATS and transcriptomic analysis of organs 
(40) 

in vivo 

New 

3 

Gene markers (41) 

in vivo 

Enhancement 

3 - 5 

Combined long-term bioassay and 
reproductive study with ED biomarker 
enhancements (42) 
Delete the current OECD TG 416 2-
generation study from the CF (43) 

in vivo 

New method 

5 

in vivo 

Enhancement 

5 

The test would allow measurements of missing hormones 
at  the  same  time  transcriptomic  (micro  arrays  or  next 
generation  sequencing)  could  be  conducted  on  a  few 
organs thus providing mechanistic information for all EATS 
modalities in one go 
There  is  definitely  a  need  for  more  research  into  gene 
markers (and biomarkers) 
This  is  based  on  work  being  conducted  by  the  Ramazzini 
Institute, there are plans to use this method as part of an 
integrated project looking at hazardous substances 
It was suggested that OECD TG 416 could be replaced by 
OECD  TG  443  including  both  the  DNT  and  DIT  cohorts, 
however, it was also highlighted that OECD TG 416 is still 
used in certain regulatory programmes and that it should 
not be deleted 

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4.3 

Environmental Health 

The  ideas  suggested  by  the  break-out  groups  for  new  test  methods  and  enhancements  to  close 
gaps  identified  for  environmental  health  in  Section  3,  are  given  in  Table  4-2.  As  well  as  specific 
new  test  methods  and  enhancements  to  test  guidelines,  the  groups  made  recommendations  for 
testing approaches and had ideas for areas which should be a focus for research.  The discussions 
held in environment group 1 and environment group 2 were very similar. Therefore the discussions 
in both of the environmental break-out groups are summarised together below. 

The workshop participants from  the environment  groups identified several  in vitro  assays (i.e.  CF 
Level  2  assays)  which  might  provide  additional  screening  level  information  on  the  mechanism  of 
action of a substance. These include  in vitro assays also identified as useful by the human health 
groups  and  which  are  already  under  development,  using  human  or  mammalian  test  systems,  for 
use  as part of the US EPA ToxCast  Programme. It should be noted that while  in vitro  human cell 
line  assays  may  be  useful  for  a  screening  level  assessment  for  vertebrates  in  general,  specificity 
may  differ  amongst  different  taxa  and  species12.  Screening  level  in  vitro  assays  were  specifically 
suggested for the thyroid pathway, retinoid pathway and the HPA axis (i.e. corticosteroids) as well 
as  for  specific  endocrine  pathways  more  typical  for  invertebrates  (e.g.  moulting  hormones 
(ecdysteroids13), juvenile hormones). The regulatory use of in vitro screening assays was discussed 
during the plenary. No  in vitro  ED  tests are  currently  included  in the information requirements  of 
the EU regulations like REACH, PPPRs, and BPR (Dang et al., 2016). This type of information must 
be specifically requested by regulators and is not routinely generated by applicants. 

ToxCast  has  six  reporter  assays  for  trans-activation  of  retinoic  acid  receptors  which  could  be 
developed  and  validated  for  screening  (US  EPA  2017;  Coady  et  al.,  2017).  Both  Retinoic  X 
Receptors (RXR) and Retinoic Acid Receptors (RAR) are well conserved receptors across taxa, and 
heterodimerises  in  most  species.  Consequently,  the  development  of  receptor  assays  would  be 
relevant  for  multiple  taxa.  One  suggestion  was  about  research  into  effects  on  echinoderms  and 
their  development.  The  role  of  the  Retinoid  X  Receptor  (RXR)  in  invertebrates  should  also  be 
investigated, specifically in molluscs as an example of the most vulnerable taxa (i.e. effects of TBT 
contamination).  It  was  noted  that  the  RXR  receptor  may  be  cloned  for  more  than  one  species  of 
mollusc  (Bouton  et  al.  (2005),  Carter  et  al.  (2010)  and  Vogeler  et  al.  (2017)).  Participants  were 
keen to point out that some substances will affect enzyme systems and cause an ED effect which is 
not directly receptor mediated (e.g. dioxins can reduce retinoid levels but do not interact with the 
receptors).  CYP  26  can  be  measured  directly  as  an  indication  of  effects  via  the  retinoid  pathway 
and  CYP  1A  has  also  been  measured  as  a  less  reliable  surrogate.  Further  research  is  required  to 
improve  understanding.  There  is  considerable  cross  talk  with  other  pathways  as  discussed  in  the 
plenary presentation on retinoid pathways by Alice Baynes (Brunel University). 

There  are  also  specific  recommendations  with  respect  to  retinoid  pathways  for  developing  assays 
for  multiple  taxa.  This  is  because  ED  effects  via  the  RXR  are  relevant  across  a  range  of  taxa 
including invertebrates (specifically molluscs), but receptor activity will differ. 

In  vitro  assays  were  also  suggested  as  useful  for  providing  mechanistic  information  regarding 
invertebrate  endocrine  pathways  for  moulting  and  development.  Receptor  assays  for  juvenile 
hormone and ecdysteroids may be useful to link adverse outcomes to these pathways. The results 
from  these  assays  might  provide  additional  mechanistic  data  to  support  endpoints  for  male 
production in the Daphnia reproduction test (OECD TG 211; Annex 7) and in a proposed screening 
assay  for  juvenile  hormone  effects,  which  is  currently  being  developed  as  part  of  the  OECD  test 
guidelines  programme.  The  ecdysone  and  juvenile  hormone  receptors  have  been  cloned  for 
Daphnia magna. This has been a long process, starting with the genome project 15 years ago (see, 
for example, Kato et al., 2007). 

The  ideas  to  fill  gaps  associated  with  thyroid  signalling  were  mainly  based  on  the  outcomes  of  a 
recent  DG  ENV  /  ANSES  workshop  on  thyroid  signalling  and  the  OECD  thyroid  scoping  document 

12   Nevertheless,  in  vitro  assays  that  cover  EATS  mechanisms  of  action  are  likely  to  be  informative  (at  the 

screening level) for all vertebrate species.  

13   Although it is noted that some moulting hormones (ecdysteroids) also affect vertebrates via Erβ. 

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207 (OECD 2014). The outcomes from the workshop were presented during the plenary by Olwenn 
Martin  (Detecting  thyroid  disruption  –  recommendations  from  an  expert  workshop)  (DG  ENV  / 
ANSES  Thyroid  Testing  Workshop  2017).  The  gaps  that  were  most  relevant  for  the  environment 
were for in vitro assays (Level 2 of the CF) and for specific thyroid related endpoints in existing fish 
and amphibian tests (Level 3 to 5 of the CF). For the environment, thyroid assays are well covered 
for amphibians but not for fish. As most aquatic vertebrate regulatory testing is using fish it would 
be  useful  to  try  and  incorporate  thyroid  endpoints  into  existing  fish  tests  –  e.g.  thyroid 
histopathology.  Research  is  ongoing  in  this  area  and  for  example  a  specific  zebrafish  eleuthero 
embryo thyroid assay is under development (Thienpont et al., 2011; Raldua et al., 2012). 

Neuro-endocrine  effects  of  substances  acting  via  thyroid  signalling  was  an  area  considered  for 
further  research  with  a  focus  on  developing  behavioural  assays  predictive  of  adverse  effects. 
Specific  tests  for  behaviour  were  discussed  with  one  group  suggesting  a  new  “hypothetical”  high 
throughput  fish  larvae  behavioural  assay  which  ideally  could  be  linked  to  adversity  via  thyroid 
signalling pathways. The other group suggested a behavioural assay focussing on amphibians, but 
there were concerns about the costs of such an assay. 

Behavioural effects were not just proposed for neuroendocrine endpoints. Mating behaviour in fish 
was  also  suggested  as  an  option  being  more  indicative  of  estrogen  /  androgen  effects.  An 
evaluation of courtship behaviour could be an addition to existing OECD tests for fish reproduction; 
concerns  were  raised  that  the  endpoint  might  show  high  variability  and  be  difficult  to  practically 
implement. 

The  measurement  of  hormone  levels  within  existing  OECD  tests  was  a  suggestion  made  by  both 
environment groups and  which  was also discussed  in the plenary. The hormones to be measured 
should be guided by the expected mode of action and adverse outcome pathways can be used to 
guide selection of appropriate biomarkers. There was a specific suggestion to include measurement 
of  T4  levels  in  the  Larval  Amphibian  Growth  and  Development  Assay  (LAGDA)  study  (OECD  TG 
241).  Also,  thyroid  hormone  measurements  in  level  3-5  fish  assays  were  discussed  and  several 
research projects are ongoing in relation to thyroid endpoints in fish tests (see paragraph below). 
Coady  et  al.,  2017  pointed  out  that  in  order  to  improve  knowledge  on  lower  animal  toxicology, 
more  guidelines  on  best  practices  are  needed,  especially  involving  species  for  which  this  has  not 
been  a  routine  endpoint  in  the  past.  Corticosteroid  samples  in  fish  and  amphibians  could  be 
relevant  if  you  have  a  mode  of  action  via  the  HPA  axis  but  might  also  be  useful  in  general  to 
explain  stress  in  fish.  Invertebrate  specific  hormone  analysis  was  also  suggested  as  potentially 
useful  (e.g.  ecdysis  triggering  hormone  levels  or  Ecdysterone  levels  (20-EH)  in  arthropods).  As 
discussed  in  Section  3  there  can  be  limitations  to  how  much  tissue  or  plasma  is  available  for 
additional  hormone  measurement  depending on  the  test  species  and  life  stage  (e.g.  for  zebrafish 
embryos). There were also questions regarding background hormone levels and how such data will 
be interpreted  because  general stress like sampling influences  some hormone levels.  Further, the 
inability  to  generate  time  course  data  (sampling  is  typically  destructive)  greatly  limits 
interpretation.  There  were  some  concerns  that  if  hormone  analysis  were  added  to  TG  as  an 
“optional” endpoint that they would not generally be measured. A recommendation was that tissue 
samples from all endocrine long-term in vivo studies be retained and stored appropriately in case 
retrospective  analysis  were  required.  However,  this  may  have  its  own  limitations  in  regards  to 
storage stability. 

Existing tests with fish could be improved. It was discussed that there is a need to understand and 
interpret  the  existing  Level  5  Medaka  Extended  One  Generation  Reproduction  Test  (MEOGRT) 
(OECD TG 240) before adding additional endpoints. There is a need for high quality data and more 
proficiency in this test as currently there are performance issues and differences between strains of 
fish and hatching success. The US EPA is currently working on demonstration studies with Japan to 
improve  the  method.  It  was  suggested  that  the  existing  OECD  TG  229  (Fish  Short  Term 
Reproduction Assay) and the OECD TG 234 (Fish Sexual Development Test) could be combined to 
produce  a  partial  life  cycle  test.  This  test  would  potentially  alleviate  concern  regarding  fish 
fertilisation,  oocyte  and  sperm  evaluation.  The  purpose  would  be  to  have  a  mid-tier  (partial 
lifecycle)  test  that  covers  known  sensitive  windows  of  the  lifecycle  (reproduction  and  sexual 
development) in a single test. Under some circumstances it could possibly be used to replace OECD 
TG 240 (MEOGRT) unless multi-generational effects investigations are required. From a regulatory 
perspective,  it  would  be  useful  to  have  more  guidance  regarding  when  to  conduct  a  21-day  Fish 
Assay (OECD TG 230) rather than the Fish Short Term Reproduction Assay (OECD TG 229) or even 
if  a  screening  assay  is  actually  the  best  option  (i.e.  rather  than  conducting  the  fish  sexual 
development test (OECD TG 234)). 

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It  was  suggested  that  as  part  of  EOGRTS  (OECD  TG  443)  both  the  Developmental  Neurotoxicity 
(DNT) and Developmental Immunotoxicity (DIT) cohorts may presently be rarely triggered. It was 
suggested that this was partly due to the REACH regulations and its requirements for the EOGRTS 
(Commission  Regulation  (EU)  2015/282)  where  the  standard  information  requirements  are  the 
basic  configuration  of  EOGRTS  and  a  proposal  justifying  the  analysis  of  DIT  and/or  DNT  cohorts 
needs to be submitted to ECHA for approval. It was suggested that if the cohorts were allowed to 
be  triggered  more  often  this  would  allow  more  information  to  be  obtained  which  could  provide 
supporting  information  on  alternative  modes  of  action  for  both  human  health  and  environmental 
endocrine assessments. 

In one environment group,  there was considerable discussion regarding testing the relevant route 
of exposure for substances. A strong recommendation from the group was for more consideration 
to  be  given  to  the  relevance  of  exposure  route  for  substances  that  have  a  high  Log  Kow.  It  was 
suggested  that  substances  with  a  Log  Kow  >6  should  be  considered  for  endocrine  testing  via  a 
dietary exposure route. The reason for this is that bioaccumulation has been identified in oily fish 
and  top  of  the  food  chain  fish  such  as  tuna,  marlin,  herring  and  farmed  salmon.  Jacobs  et  al. 
(2002;  2004)  and  other  authors  have  shown  that  fish  are  susceptible  to  contamination  with 
lipophilic organic chemicals that are ubiquitous contaminants of the marine ecosystem. 

There  was  discussion  of  additional  endpoints  that  could  be  measured  in  existing  OECD  TGs  with 
molluscs  (OECD  TG  242  and  /  or  OECD  TG  243).  Histopathology  on  the  gonads  of  molluscs  was 
ruled out as it would be technically challenging.  Embryo tests with molluscs might be useful as a 
stand-alone or add-on to the partial life cycle tests but such a test may not provide any additional 
mechanistic  information.  A  specific  recommendation  from  one  break-out  group  was  that  there 
should  be  no  further  endocrine  mollusc  guideline  development  until  further  research  has  been 
conducted to understand endocrine pathways in this group. 

A key gap is the lack of understanding regarding invertebrate endocrinology. This was highlighted 
independently  by  both  environment  groups and  is also a conclusion in the JRC survey  (2017)  (as 
discussed  by  Stephanie  Bopp  (JRC)  in  her  plenary  presentation).  Molecular tools  have  assisted in 
improving the mechanistic understanding of invertebrate endocrine pathways and it is now known 
that invertebrates either lack receptors for human vertebrate sex steroids or the receptors are not 
active  in  these  species.  There  is  a  general  recommendation  for  further  work  in  this  area,  as 
indicated by Coady et al., 2017. 

As discussed in Section 3, it is difficult to predict whether the species that are currently used for ED 
testing  will  be  sufficient  to  protect  all  species  in  the  environment.  Specific  gaps  in  the  testing 
programme  exist  for  birds  and  reptiles.  Currently  birds  are  assumed  to  be  protected  through 
extrapolation  primarily  from  one-generation  avian  reproduction  studies  (OECD  TG  206)  and 
mammalian  data.  However,  cases  have  been  described  where  bird-specific  physiological  qualities 
have resulted in specific potential effects related to endocrine disruption. Therefore, it is essential 
that cross taxa comparisons are performed to inform the potential needs for bird specific endocrine 
testing  and  assessment  of  chemicals.  Given  the  similarities  between  birds  and  reptiles  it  was 
considered  that  reptiles  should  be  included  in  this  cross  taxa  comparison.  If  needed,  specific 
screens  should  be  developed  to  evaluate  the  impact  of  specific  endocrine  MOAs  on  targeted 
endpoints  so  that  links  from  mechanistic  to  apical  data  can  be  made  (Coady  et  al.,  2017).  The 
addition of mechanistic endpoints to OECD TG 206 might be of value as the currently available test 
focuses only on apical endpoints. Laboratory tests with reptiles (and land based amphibians) are a 
challenge, because they reproduce just once a year. 

Another area that was considered important for further research was immunotoxicity in wildlife and 
links  to  endocrine  disruption.  The  WHO/UNEP  (2012)  report  highlights  the  links  between  immune 
function  and  poor  reproductive  success  in  marine  mammals.  Hayes  et  al.  (2006)  discuss  the 
possible  links  between  endocrine  disruption  and  disease  in  amphibians,  and  more  recently 
Casanova-Nakayama et al. (2011) has reviewed the potential for endocrine disrupting compounds 
to target the immune  system  of  fish.  However,  at  the moment there is not enough  knowledge to 
link endocrine disruption and immunotoxicity in wildlife.  Diseases in the environment are different 
from  those  in  humans  and less well understood  and therefore further research in this area  would 
be valuable. The adrenals and interrenals also need to be better understood for wildlife. 

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Environment Group Ideas 

As indicated above, ideas to close the gaps were discussed by the attendees at the workshop and a 
summary  of  these  ideas  appears  in  Table  4-2  below.  Further  information  on  the  various  ideas  is 
presented in Annex 2. The cross reference shown in brackets correlates with the idea in Annex  2; 
some  ideas  were  also  highlighted  by  human  health  groups.  Ideas  highlighted  by  environmental 
experts may also be applicable to human health and vice versa. 

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Table 4-2: Suggested ideas to close the gaps for environmental health 

Data gap 

Suggested tests to fill gaps 
(cross reference to Annex 
2 and human ideas) 
Thyroid pathway screens:  
Deiodinase expression in vitro/ex 
vivo (1, see also human health 
idea 1) 

in vitro / 
in vivo 

in vitro / 
ex vivo 

Species 

Test 

CF 
Level 

Further information 

General 

New 

2 

Thyroid pathway screens:  
TBG binding assay; TTR serum 
binding assay; T4 binding protein 
displacement assay; and TR 
reporter assay) (2, see also 
human health idea 2) 

Thyroid pathway screens: Thyroid 
peroxidase assay, Sodium Iodide 
Symporter (3, see also human 
health idea 3) 
Sodium-dependent serotonin 
transporter activity (4) 

Growth and 
Development 

in vitro 

General 

New 

2 

Both 

General 

New / 
enhancement 

2 / 4 - 5 

Both 

General 

New 

2 - 3 

Ecdysis triggering hormone levels 
in vivo (5) 

in vivo 

Invertebrates  New 

Ecdysterone levels (20-EH) in 
arthropods (6) 

Using Transgenic 
embryos/fish/amphibians for 
screening: XETA / EASZY (7) 
Include T4 in the LAGDA study 
(perhaps also T3 and TSH) (8) 
Thyroid testing in fish (9) 

in vivo 

Invertebrates  New 

in vivo 

Fish / 
Amphibian 

New 

in vivo 

Amphibian 

Enhancement 

4 

in vivo 

Fish 

New or 
enhancement 

3 - 5 

3 

3 

3 

Assays already exist, as described in OECD 207, 
some more development and research may be 
required  

Screening assays would be useful. Need to consider 
alongside the recommendations from the DG ENV / 
ANSES Thyroid Testing Workshop (2017) 
For each of these ideas suggested assays already 
exist, as described in OECD 207, some more 
development and research may be required  

Screening assays would be useful. Need to consider 
alongside the recommendations from the DG ENV / 
ANSES Thyroid Testing Workshop (2017) 
Suggested assays already exist, as described in 
OECD 207, some more development and research 
may be required, this would be a new in vitro TG or 
an enhancement if included in in vivo TGs 
Neurotransmitters/neurohormones are important for 
the function of organisms, an assay analysing 
transporter protein (and other routine biomarkers) 
could be developed 
Substances which target the ecdysone receptor 
have the potential to interrupt the successful 
moulting of insects 
Ecdysterone is responsible for the moulting of 
arthropods, responses are accompanied by specific 
changes in gene expression in vivo 
Some test methods are already in development 

Hormone analysis in an existing test, with effects on 
hormone levels being indicative of adverse effects 
Some research is ongoing e.g. Zebrafish 
Eleutheroembryo Thyroid Assay (Thienpont et al. 
2011; Raldua et al. 2012). Also, endpoints like eye 
development in zebrafish (Baumann et al. 2016) 
swim bladder inflation (Stinckens et al. 2016) are 
being investigated 

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Table 4-2: Suggested ideas to close the gaps for environmental health 

in vitro / 
in vivo 

Species 

Test 

CF 
Level 

Further information 

Data gap 

Growth and 
Development 
(cont) 

Suggested tests to fill gaps 
(cross reference to Annex 
2 and human ideas) 
PPAR transactivation assays (for 
use with several taxa) (10, see 
also human health idea 27) 
Retinoid X Receptor (RXR) and 
Retinoic Acid Receptor (RAR) 
transactivation assays (for use 
with several taxa) (11, see also 
human health idea 27) 
In vitro receptor assays such as 
Constitutive Androstane Receptor 
(CAR), Pregnane X Receptor 
(PXR), Farnesoid X Receptor 
(FXR), Liver X Receptor (LXR), 
Vitamin D Receptor (VDR) (12) 
Daphnia in vitro juvenile hormone 
and ecdysone agonist (13) 

in vitro 

General 

New 

in vitro 

General 

New 

in vitro 

General 

New 

in vitro 

Invertebrates  New 

2 

2 

2 

2 

Reproductive 
Health / Growth 
and Development 

MEOGRT - Performance and 
possibly refinement (14) 

in vivo 

Fish 

Enhancement 

5 

Progesterone receptor (PR) 
transactivation (15, see also 
human health idea 20) 
Prostaglandin synthesis inhibitors 
in vitro (16) 

in vitro 

General 

New 

in vitro 

General 

New 

2 

2 

Reproductive 
Health 

Spawning assay in echinoderms in 
vivo (17) 

in vivo 

Marine 
invertebrate  

Enhancement 

3 

Combining OECD TG 229 and 
OECD TG 234 (18) 

in vivo 

Fish 

Enhancement 

3 - 4 

Transactivation assays for different PPARs exist 
already (see OECD DRP 178). OECD test guideline 
may need to be developed  
Transactivation assays for RXR and RAR assays 
exist already (see OECD DRP 178). OECD test 
guideline may need to be developed. The RXR 
receptor is cloned for various species 

In vitro receptor assays other than PPAR, RXR and 
RAR could be developed 

Ecdysone and juvenile hormone receptors may 
represent a useful molecular system for further 
examination of hormonal and chemical effects on D. 
magna 
Currently there are performance issues and 
differences between strains of fish and hatching 
success. The US EPA is working on demonstration 
studies with Japan to improve the method 
Several PR transactivation assays have been 
developed. A PR transactivation assay may require 
co-factors to be considered 
Assays are available for measuring levels of 
prostaglandin or prostaglandin metabolites by 
ELISA. These could be used as the basis for 
developing a new in vitro test 
Test guidelines already exist for a sea urchin 
fertilisation assay (EPA 1008.0 (2002)) or embryo-
larvae development assay (ASTM E1563 – 98 
(2012)), these could be investigated further for the 
inclusion of ED effects 
This may allow for the development of a mid-OECD 
CF level (partial lifecycle) test that covers both 
known sensitive windows of the lifecycle – namely 
reproduction and sexual development in 1 test 

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Data gap 

Reproductive 
Health (cont) 

Behaviour / 
Reproductive 
Health 

Adrenal / Steroid 

Table 4-2: Suggested ideas to close the gaps for environmental health 

Suggested tests to fill gaps 
(cross reference to Annex 
2 and human ideas) 
Behavioural assessments in vivo 
(frogs), e.g. via the assessment of 
the calling behavior of male frogs 
or via swimming behaviour (19) 

in vitro / 
in vivo 

Species 

Test 

CF 
Level 

Further information 

in vivo 

Amphibians  

New or 
enhancement 

3 - 5 

Fish behavioural assay (embryo) 
(20) 

in vivo 

Fish 

New 

3 - 5 

in vivo 

Fish 

Enhancement  

3 - 5 

in vitro 

General 

New 

in vitro 

General 

New 

2 

2 

in vivo 

General 

Enhancement 

3 - 5 

Fish behavioural assay (mating 
behaviour) (21) 

Glucocorticoid Receptor (GR), 
Mineralocorticoid receptor (MR) 
transactivation assays (22, see 
also human health idea 15) 
Adrenal steroidogenesis synthesis 
for wildlife species (23) 

Steroid hormone synthesis in 
vivo: data collection of a range of 
steroids is valuable for 
mechanistic information provided 
not too many extra animals are 
needed. (24, see also human 
health idea 16) 

fish 

throughput 

It may be possible to video-track swimming 
behaviour as part of the existing amphibian tests. 
Studies also exist which have investigated the 
effects of chemicals on calling in male frogs. 
Previous results in Xenopus laevis indicate that the 
frogs cannot be used again after being used in a 
calling behaviour assessment and this would add an 
additional expensive screening study. Research 
activity 
larvae 
A  hypothetical  high 
behavioural  assay.  Limitation 
that  behavioural 
effects  may  not  be  considered  as  a  measure  for 
adversity.  If  there  were  a  clear  link  to  the  thyroid 
then  an  argument  could  be  made  that  the  assay 
could  potentially  be  used  to  demonstrate  adversity 
(would  need  to  prove  the  behaviour  is  affected  by 
the plausible mechanism). This would be a research 
activity 
e.g. based on mating or courtship behaviour. To be 
incorporated  into  existing  fish  reproduction  TGs. 
This would be a research activity 
e.g. Marks et al. Tools for the Discovery and 
Characterization of GR Modulators: A Comparison of 
Binding, Co-regulator Interaction, and 
Transactivation Assays 
i.e. glucocorticoids, mineralocorticoids, progestins, 
aromatase, androgens, and estrogens (Manibusan 
and Touart 2017) 
If you suspect alterations HPA/HPI axis take sample 
for steroid hormone analysis (for example, possible 
fish TG include OECD TG 229 / OECD TG 230 / 
OECD TG 240, other tests could include this on 
amphibians and mammals). 
It is increasingly possible for various hormone levels 
to be measured by LC-MS/MS methods using 
normal serum levels 

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Table 4-2: Suggested ideas to close the gaps for environmental health 

Data gap 

Suggested tests to fill gaps 
(cross reference to Annex 
2 and human ideas) 
Biomarkers for immunity (25) 

in vitro / 
in vivo 

Species 

Test 

CF 
Level 

Further information 

in vivo 

General 

New 

3 - 5 

If Log Kow >6 then dietary 
exposure for endocrine testing 
should be considered (26) 

in vivo  

General 

Enhancement 

3 - 5 

General 

Keep tissues from all endocrine 
long-term in vivo studies (27) 

in vivo 

General 

Enhancement 

3 - 5 

Understand if current testing can 
be extrapolated to reptiles (28) 
Avian mechanistic screens / 
additional mechanistic endpoints 
could be added to OECD TG 206 
(29) 

in vivo 

Reptiles 

N/A 

in vivo 

Birds 

New or 
enhancement 

N/A 

3 - 5 

Research activity. Amphibians could be a priority for 
this because diseases associated with the immune 
system are seen in the environment for these 
species, however, research has also been conducted 
in fish and other species are at risk 
Recommendation based on the most scientifically 
valid approach however consideration should be 
given regarding how the information would be 
interpreted from a regulatory perspective 
This is a recommendation for keeping tissue 
samples from level 3-5 studies for the environment 
to enable additional analysis to be conducted at a 
later date, and is based on a practice that is already 
routine in some mammalian toxicity studies 
Research activity 

Review of bird testing including need for specific 
testing, relevant test species and availability of tests 
that include mechanistic data (or potential to 
include mechanistic endpoints in existing tests). 
Research activity 

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5. Prioritisation 

5.1 

Introduction 

During  the  morning  of  the  third  day  the  break-out  groups  considered  the  adverse  outcomes  and 
ideas that they had identified from the previous day. The groups also then made some suggestions 
for prioritising ideas to address gaps. The prioritisations made by each group are  indicated in the 
following sections. 

The  groups  were  all  encouraged  to  follow  the  tiered  prioritisation  approach  that  was  suggested. 
Tier one emphasises the importance of regulatory relevance with respect to either mode of action 
(MOA)  and/or  adversity  as  well  as  societal/ecosystem  benefits.  Tier  two  factors  considered  both 
‘costs’ and ‘animal concerns’, TRL was also considered as a factor to help define the timeline of the 
ideas. If experts were aware of further factors (e.g. integration with other research) these could be 
considered tier three factors. 

5.2  Human Health 

Human Health Group 1 

Human  Health  group  1  made  suggestions  for  ideas  that  could  help  address  gaps  for  adverse 
outcome pathways: 

Table 5-1: Human Health Group 1 priorities 
Adverse outcome 

Idea to fill the gap 

Thyroid-related 
developmental neurotoxicity 

Thyroid 

Oligodendrocyte differentiation 
Dendritic arborisation assay 
Cognitive function test 
Cortical heterotopia in offspring 
Serum levels T4/TSH 
Thyroid peroxidase/TTR/deiodinase/iodine 
uptake 

Female reproductive health  More/better defined endpoints and assays 
Metabolic dysfunction 

General 

Interference  with  adipocyte  differentiation  (in 
vitro) 
Body  composition/body  fat  estimation  (weight 
gain is monitored already) 
A new level 3 short term in vivo mechanistic test 
based  on  OECD  TG  407  to  measure  hormones 
and transcriptomic analysis of organs 

Additional 
information 
Long timeline 
Long timeline 
Medium timeline 
Medium timeline 
Short timeline 

Short timeline 

Long timeline 

Long timeline 

Short timeline 

Short timeline 

In addition to the ideas the group highlighted, they also made some general remarks: 

Another  important  consideration  is  (in  vivo)  gene  expression  (e.g.  ncRNA)  of  target  tissue  (e.g. 
brain, endometrium); it was suggested that these might give added value to the study but that this 
depends on the type of study and the relevant endpoints. ncRNA was highlighted as something that 
might be worth looking at more in the future, as presently we might not be there yet scientifically, 
but that it may be relevant for reproductive toxicity and development. 

The  screening  battery  tests  for  Nuclear  Receptors  (NR)  are  not  necessarily  linked  to  a  specific 
outcome.  The  group  also  suggested  that  transactivation  assays  provide  more  information  than 
binding  assays.  The  RAR/RXR  transactivation  assays  were  specifically  highlighted,  but  the  group 
recognised  there  were  several  other  suggestions.  The  group  also  suggested  that  if  several 
transactivation assays were to be developed, a requirement would not be that all of the screening 
assays have to be used, but that they would be available in case they needed to be used. 

The  group  highlighted that  immune  endpoints  were  important  but that  relevant  test  methods  are 
currently lacking and this should be area for further focus. 

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Human Health Group 2  

Human  Health  group  2  made  suggestions  for  ideas  that  could  help  address  gaps  for  adverse 
outcome pathways: 

Table 5-2: Human Health Group 2 priorities 

Adverse outcome 

Idea to fill the gap 

Thyroid-related 
developmental 
neurotoxicity 

Thyroid 

Female reproductive health 

Male reproductive health 

Metabolic dysfunction 

Thyroid endpoints in pup brain 

Include DNT mandatory (TG 443) 

In vitro for MOA (see thyroid scoping doc and 
Thyroid workshop report) TPO, NIS 
Measure Thyroid hormones 
In vitro assays PCOS / endometrium (gene 
expression/trans activation) 

  Goal OECD scoping document 

Placental model 

Measure: Serum Oxytocin and Anti-Mullerian 
Hormone and other hormones (in TG 408 and 
443) 

Additional 
information 
Long timeline 

Short timeline 

Short timeline 

Short timeline 

Long timeline 

Long timeline 
Medium timeline 
in TG 443 
Short timeline in 
TG 408 

Short timeline 

Histopathology of ovaries, follicle count (TG 408)  Short timeline 
Whole mount female (and male mammary 
gland) assessment and/or histopath. to be 
included in EOGRTS (possibly TG 408 and/or TG 
407) 
PSA secretion and prostate model (male 
accessory glands) 
In vitro pancreatic beta cells 
Adipocyte differentiation (distinguish obesity and 
diabetes) 
Glucose, serum insulin and insulin tolerance in 
vivo (high adversity) 
In vivo retinoic level (serum and liver) 

Long timeline 

Long timeline 

Long timeline 

Lipoprotein profile 

Medium timeline 

Medium timeline 
Short timeline TG 
408 but longer in 
other TGs 

General 

In vivo measurement of serum gut hormones 
and adipokines 
Delete The current OECD TG 416 (2 gen study) 
(from 2001) 

  Use OECD TG 443 (including as in OECD 
both DNT and DIT cohorts) (from 2012) 

Short timeline 

Short timeline 

Directorate-General for Environment 
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Setting priorities for EDC testing  
Page 35 

 
 
 
 
 
 
 
 
 
 
 
Human Health Group 3 

Human  Health  group  3  made  both  comments  and  suggestions  for  ideas  that  could  help  address 
gaps for adverse outcome pathways: 

Table 5-3: Human Health Group 3 priorities 

Adverse outcome 

Idea to fill the gap 

Thyroid-related 
developmental 
neurotoxicity 

Thyroid 

Metabolic dysfunction 

Immune / Autoimmune / 
Inflammatory 

Female reproductive 
health 

General 

In vitro and ex vivo oligodendrocyte 
proliferation/differentiation assays (long 
timeline); cortical heterotopia – would require 
additional animals to existing TGs, but very 
informative (medium timeline) 
Neuronal progenitor cell 
proliferation/differentiation assays; neurite 
extension 
Electrophysiological endpoints (very informative, 
but requires basic research which could be 
achieved in a short timeline) 
T4 assays in dams and juveniles  
TTR assay 
Application of Amphibian thyroid assays, notably 
XETA, since HPT axis conserved in vertebrates 
Adipocyte differentiation 
Fasting serum insulin 
PPAR  transactivation assays 

Weight gain (but more detailed, with analysis of 
individual fat pads from different areas- 
epididymal versus subcutaneous etc.) 
Dyslipidemia (lipid/protein profiles) 
Here there is a major research deficit- especially 
for endocrine related MOA 
Application of multiplexed cytokine assays 

Metabolomics 

Here there is a major research deficit, especially 
since many adverse outcomes (PCOS, fibroids, 
endometriosis) are not addressed by any existing 
endpoints/models, and many adverse outcomes 
are very human specific 
Appropriate use of gene markers 

Additional 
information 

Long timeline / 
Medium timeline 

Medium timeline 

Short timeline 

Short timeline 
Short timeline 

Short timeline 

Long timeline 
Medium timeline 

Short timeline 

Short timeline 

Short timeline 

Long timeline 

Long timeline 

Long timeline 

Long timeline 

Long timeline 

Biomarkers and Epigenetics: 

Appropriate  application  of  suitable  biomarkers,  epigenetics  methodology,  and  omics  approaches 
(especially transcriptomics) have large potential to provide information on many adverse outcomes 
relatively  simply  and  without  adding  to  cost  (can  use  existing  TG  and  TM  with  few,  if  any,  extra 
animals). 

Directorate-General for Environment 
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Setting priorities for EDC testing  
Page 36 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Human Health Group 4 

Table 5-4: Human Health Group 4 priorities 
Adverse outcome 

Idea to fill the gap 

Thyroid-related  developmental 
neurotoxicity 
Thyroid 
Male reproductive health 

Metabolism 

/ 

/  Autoimmune 

Immune 
Inflammatory 
Female reproductive health 
Cancer 
Adrenal / Steroid 

Brain  histopath,  cortical  heterotopia, 
oligodendrocyte (longer timeline) 
T4 at PND 16 
Cryptorchidism 
Sperm function & morphology 
Fat pad weight, animal weight 
PPAR, RXR transactivation assays 
Brown fat in pups 
Apoptosis in thymus 

PR transactivation assay 
Whole mount mammary 
GR,  MR  transactivation  assay  (in 
vitro)  

Additional 
information 
Medium timelines 

Short timeline 
Short timeline 
Short timeline 
Short timeline 
Short timeline 
Short timeline 
Long timeline 

Short timeline 
Short timeline 
Short timeline 

The group indicated that the societal value was high for all endpoints and that there was no redundancy for any 
of the ideas. 

The group suggested that longer term research areas should focus on: 

 

 

Placental models 

Early markers of cancer and other diseases (epigenetic, gene expression) 

  2 hit model of carcinogenesis including developmental exposures 

  Bone endpoints 

  Serum markers of immune function 

It  was  also  suggested  that  epigenetic  markers  of  cancer  were  ready  to  be  incorporated  into  the 
TGs. 

5.3  Human Health Summary 

There  was  a  consensus  among  the  human  health  groups  that  the  main  adverse  outcomes  for 
prioritisation  were  for  thyroid-related  developmental  neurotoxicity,  metabolic  dysfunction,  female 
reproduction  and  male  reproduction.  Some  also  suggested  that  hormone-related  cancers  and 
immune  /  autoimmune  /  inflammatory  disorders  should  also  be  prioritised.  Gaps  in  test  methods 
and guidelines were also identified for these adverse outcomes. The experts at the workshop were 
able to suggest a number of ideas for new test methods and test enhancements that they believed 
should be prioritised. 

There  is  some  overlap  associated  with  these  ideas,  for  example  whole  mount  mammary  gland 
histopathology  was  highlighted  by  one  group  as  being  a  priority  for  enhancement  for  cancer 
testing, while the idea was also highlighted by another group with respect to developmental tests. 
A number of groups also made suggestions to include measurements related to fat/lipids. 

All of the ideas highlighted by the experts in the four human health groups are briefly summarised 
below,  organised  by  the  adverse  outcome  and  sorted  by  those  ideas  highlighted  the  most  by  the 
groups. 

Directorate-General for Environment 
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Setting priorities for EDC testing  
Page 37 

 
 
 
 
 
 
 
 
Short Term Ideas 

Table 5-5: Short term human health priority ideas 

Ideas to fill gaps 

Idea type 

CF Level 

Serum T4 Measurements 
DNT/DIT mandatory testing  Additional testing 

Enhancement 

4 - 5 
4 - 5 

Number of 
groups 
highlighting 
the idea  
Four 
One 

Adverse 
outcome 

Thyroid-
related 
developmental 
neurotoxicity 

Thyroid 

Metabolic 
dysfunction 

Female 
reproductive 
health / 
Cancer 

Female 
reproductive 
health 

Male 
reproductive 
health 

Electrophysiological 
endpoints 

Thyroid in vitro screening 
assays  
XETA 
Fat measurements 
PPAR transactivation and 
related assays 
Lipoprotein profile 
Brown fat in pups 
In vivo measurement of 
serum gut hormones and 
adipokines 

Whole mount mammary 
gland 

Serum oxytocin, Anti-
Mullerian Hormone and 
hormone measurements 
(short to medium term) 
Ovary analysis 
Progesterone receptor (PR) 
transactivation 
Cryptorchidism 

Sperm function and 
morphology 

Adrenal / 
Steroid 

GR and MR transactivation 
assays 

General 

Delete TG 416 

A new level 3 short term in 
vivo mechanistic test based 
on OECD TG 407 to 
measure hormones and all 
EATS modalities in one test 

New Test Method 

4 - 5 

One 

New Test 
Methods 
New Test Method 
Enhancement 
New Test 
Methods 
Enhancement 
Enhancement 

Enhancement 

2 

3 
4 - 5 

2 

4 - 5 
4 - 5 

4 - 5 

Three 

One 
Three 

Two 

One 
One 

One 

Enhancement 

4 - 5 

Two 

Enhancement 

4 - 5 

Enhancement 

4 - 5 

New Test Method 

2 

Enhancement 

Enhancement 

New Test 
Methods 
Delete an 
existing test 
guideline 

New Test Method 

4 - 5 

4 - 5 

2 

5 

3 

One 

One 

One 

One 

One 

One 

One 

One 

Directorate-General for Environment 
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Setting priorities for EDC testing  
Page 38 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
Medium Term Ideas 

Table 5-6: Medium term human health priority ideas 

Adverse 
outcome 

Thyroid-
related 
developmental 
neurotoxicity 

Metabolic 
dysfunction 

Ideas to fill gaps 

Idea type 

CF Level 

Cortical heterotopia 
Cognitive function test 
Neuronal progenitor cell 
proliferation/differentiation 
assays; neurite extension. 
Serum insulin tolerance and 
insulin measurement 
Serum and liver retinoic acid 
levels 

Enhancement 
Enhancement 

4 - 5 
4 - 5 

New Test Method 

2 

Both 

Enhancement 

4 - 5 

4 - 5 

Long Term Ideas 

Table 5-7: Long term human health priority ideas 

Adverse outcome  Ideas to fill gaps 

Idea type 

CF Level 

Number of 
groups 
highlighting 
the idea  
Three 
One 

One 

Two 

One 

Number of 
groups 
highlighting 
the idea  

Thyroid-related 
developmental 
neurotoxicity 

Metabolic 
dysfunction 

Female 
reproductive health 

Male reproductive 
health 

Immune / 
Autoimmune / 
Inflammatory 

General 

Oligodendrocyte 
differentiation 
Thyroid endpoints in 
pup brain 
Dendritic arborisation 
assay 
Adipocyte 
differentiation 
Pancreatic beta cells 
More and better-
defined assays 
Placental model 
PSA secretion and 
prostate model 
Apoptosis in thymus 
More research 
Cytokine biomarkers 
Metabolomics 
Gene markers 

5.4 

Environmental Health 

Environment Group 1 

New Test Method 

2 - 4 

Three 

Enhancement 

4 - 5 

New Test Method 

New Test Method 

New Test Method 

New Test Methods 

Both 

New Test Method 

Enhancement 
New Test Method 
Enhancement 
Enhancement 
Enhancement 

2 

2 

2 

All 

All 

All 

3 - 5 
All 
All 
All 
4 - 5 

One 

One 

Three 

One 

Three 

One 

One 

One 
One 
One 
One 
One 

Environment  group  1  highlighted  ideas  they  thought  were  important,  made  an  indication  of  what 
they believe the TRL level is and what they felt the priority for the idea should be (C = high, B = 
higher, A = highest). 

Directorate-General for Environment 
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Setting priorities for EDC testing  
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Table 5-8: Environment Group 1 priorities 

Data gap 

Idea to fill the gap 

Environment Group 1 highest priority ideas (priority A) 

Additional 
information 
(TRL) 

CF 
Level 

Priority 

Growth and 
Development 

Thyroid pathway screens, including; 

  Deiodenase expression in vitro/ex 

vivo 
Iodine uptake assays 
Thyroid peroxidase assay 
TBG binding assay 
TTR serum binding assay 
T4 binding protein displacement 
assay 
TR reporter assay 

 
 
 
 
 

 

Sodium-dependent serotonin transporter 
activity 
Thyroid endpoints in fish – e.g. in TGs 
229/234/240, or by using zebra fish embryo 
to evaluate chemicals with thyroid activity 
(in development) 
Using Transgenic embryos/fish/amphibian 
for screening: XETA / EASZY + Spiggin 
Medaka 
Measure the TSH, T4 and T3 in the LAGDA 
study 
OECD validation of the in vitro assays for 
RXR and RAR 

OECD validation of PPARs in vitro assays 

Reproductive 
Health 

Prostaglandin synthesis inhibitors (in vitro) 

Progesterone Receptor (PR) transactivation 
assay 

Combine OECD TG 229 and 234 

Medium 
timeline (4-6) 

2 

A+ 

Long timeline 
(3) 

2 - 3 

A 

Medium 
timeline (5) 

3 - 5 

A 

Short timeline 
(9+7) 

Short timeline 
(9) 
Short timeline 
(7/8) 
Short timeline 
(7/8) 
Medium 
timeline (5) 
Short timeline 
(8) 
Short timeline 
(8) 

3 

4 

2 

2 

2 

2 

3 - 4 

Adrenal / 
steroid 

General 

Glucocorticoid Receptor (GR) and 
Mineralocorticoid receptor (MR) 
transactivation assays (in vitro) 
Keep tissues from all endocrine long-term in 
vivo studies 
If Kow > 6 consider dietary exposure for 
endocrine testing 

Short timeline 
(8) 

2 

Short timeline 
(9) 
Short timeline 
(9) 

3 - 5 

3 - 5 

Environment Group 1 high priority ideas (priority B) 

Growth and 
Development 

Ecdysis triggering hormone levels in vivo 

Ecdysterone levels (20-EH) in arthropods 

Juvenile hormone receptor activity and 
ecdysone agonist in vitro 
If you suspect alterations HPA/HPI axis take 
sample for steroid hormone analysis – e.g. 
OECD TG229/TG230 (CF Level 3) and OECD 
TG240 (CF Level 5) 

Long timeline 
(3) 
Long timeline 
(3) 
Medium 
timeline (4) 

3 

3 

2 

Short timeline 
(7) 

3 - 5 

B 

A 

A 

A 

A 

A 

A 

A 

A 

A 

A 

B 

B 

B 

Directorate-General for Environment 
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Setting priorities for EDC testing  
Page 40 

 
 
 
 
 
 
 
 
Table 5-8: Environment Group 1 priorities 

Data gap 

Idea to fill the gap 

Additional 
information 
(TRL) 

CF 
Level 

Priority 

Environment Group 1 high priority ideas (priority B) 

Reproductive 
Health 

Adrenal / 
Steroid 

Spawning assay in echinoderms in vivo 
(apical for now due to lack of 
endocrinological knowledge) 
Adrenal steroid synthesis (in vitro) for 
wildlife species 

Medium 
timeline (5) 

Medium 
timeline (6) 

3 

2 

B 

B 

Research Topics: 

 

Invertebrates endocrinology/physiology (Priority A+) 

o  Echinoderm research to look at development 

o  RXR  research  in  invertebrates,  specifically  in  molluscs  as  example  of  the  most 

vulnerable species 

o  No further endocrine mollusc guideline development until further research has been 

done in physiology and endocrinology (metabolic pathway) 

  Birds – A review of the effects of endocrine disruption in birds including  analysis for which 
modes  of  action  and  associated  specific  endpoints  might  only  be  observed  in  birds  (as 
opposed to mammals or other taxa) due to their unique characteristics. The review would 
include consideration of the  outcomes of  the US EPA  validation  work with the quail model 
including the impact of different diets. (Priority A) 

 

 

 

 More research on transgenic embryos/fish for screening (Priority A)  

 Reptiles (potentially connected to birds – for extrapolation) (Priority B) 

 Collect additional embryos for analysis of methylation marks - useful for assigning 
epigenetic effects in organisms (Priority B) 

Environment Group 2 

Out  of  all  the  ideas  that  environment  group  2  has  discussed,  they  decided  to  highlight  the  eight 
ideas that they thought were particularly important. They indicated what the gap was, ideas to fill 
the  gap  and  they  gave  an  indication  of  what  they  believed  the  timeline/TRL  level  was  and  the 
related  OECD  CF  level.  All  of  the  ideas  below  can  be  regarded  as  being  of  the  highest  priority 
(Priority A). 

Table 5-9: Environment Group 2 priorities 

Data gap 

Idea to fill the gap 

Additions to fish tests 
(e.g. thyroid) 
In vitro retinoid assays 
(excluding RXR and RAR, 
which are achievable in a 
short timeline) 

A pack of in vitro assays 
(receptors and enzyme)  

Daphnia in vitro juvenile 
hormone and ecdysone 
agonist  
Review of bird testing / 
mechanistic “add ons” 

Growth and 
Development 

Research 

Additional information 
(TRL) 

CF 
level 

Priority 

Long timeline (1) 

3 - 5 

A 

Medium/long timeline (4-2) 

2 

A 

Assays are at various stages 
of development 
Short/Medium/long timelines 
(7-3) 

Medium timeline (4) 

2 

2 

Long timeline (2) 

3 - 5 

A 

A 

A 

Directorate-General for Environment 
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Setting priorities for EDC testing  
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Table 5-9: Environment Group 2 priorities 

Data gap 

Idea to fill the gap 

Additional information 
(TRL) 

Behaviour / 
Reproductive 
Health 

Fish behavioural assay 
(mating behaviour) 

Long timeline (2) 

Fish behavioural (embryo)  Long timeline (1) 

CF 
level 

3 - 5 

3 - 5 

Priority 

A 

A 

MEOGRT - Performance 
and possibly refinement 

Reproductive 
Health / Growth 
and Development 
The group indicated that the societal value was high for all endpoints and that there was no redundancy for any 
of the ideas. 
There was a discussion as to if there was redundancy because amphibian tests already exist but it was agreed 
that there wasn’t redundancy because of the species differences and because it’s important to have these add 
ons to existing fish tests. 

Short timeline (9) 

A 

5 

Research Topics: 

  Mollusc endocrinology 

 

Immunotox (wildlife) 

  Adrenals/ interrenals 

Finally,  the  regulatory  acceptability  should  be  considered.  For  example,  there  is  a  proposal  to 
exempt moulting hormones from ED criteria. This decision may affect how assays for invertebrate 
specific  assays  relating  to  moulting  are  considered  with  respect  to  ED  from  a  regulatory 
perspective.  

5.5 

Environmental Health summary 

There  was a consensus among the two environmental groups that diseases and disorders are not 
really  relevant  for  environmental  health/animals,  however  it  is  possible  to  group  by  adverse 
outcomes for prioritisation. 

The  areas  that  the  two  environmental  groups  prioritised  were;  growth  and  development, 
reproductive  health,  adrenal  and  general.  Most  of  the  priority  ideas  were  based  around  the  most 
common adverse outcomes being related to growth and development, and reproductive health. 

Short Term Ideas 

Table 5-10: Short term environment priority ideas 

Adverse 
outcome 

Ideas to fill gaps 

Idea type 

CF Level 

OECD validation of in vitro assays 
for RXR / RAR (retinoid assays) 
OECD validation of in vitro assays 
for PPAR 
Using Transgenic 
embryos/fish/amphibians for 
screening: XETA / EASZY + 
Spiggin Medaka 
Measure the TSH, T4 andT3 in the 
LAGDA study 

New test 
method 
New test 
method 

Enhancement 

Enhancement 

MEOGRT - Performance and 
possibly refinement 

Enhancement 

2 

2 

3 

4 

5 

Growth and 
Development 

Reproductive 
Health / 
Growth and 
Development 

Number of 
groups 
highlighting 
the idea  

Two 

One 

One 

One 

One 

Directorate-General for Environment 
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Setting priorities for EDC testing  
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Table 5-10: Short term environment priority ideas 

Adverse 
outcome 

Reproductive 
Health 

Adrenal / 
Steroid 

General 

Ideas to fill gaps 

Idea type 

CF Level 

Progesterone Receptor (PR) 
transactivation 
Combine OECD TG 229 and 234 
Glucocorticoid Receptor (GR) and 
Mineralocorticoid receptor (MR) 
transactivation test (in vitro)  
Keep tissues from all endocrine 
long-term in vivo studies 
If Kow > 6 consider dietary 
exposure for endocrine testing 

New test 
method 
Enhancement 

New test 
method 

2 

3 - 4 

2 

Enhancement 

3 - 5 

Enhancement 

3 - 5 

Number of 
groups 
highlighting 
the idea  

One 

One 

One 

One 

One 

Medium Term Ideas 

Table 5-11: Medium term environment priority ideas 

Adverse 
outcome 

Ideas to fill gaps 

Idea type 

CF 
Level 

Number of 
groups 
highlighting 
the idea  

Thyroid pathway screens/pack, 
including (but not limited to); 

  Deiodinase expression in 

 

 
 
 
 
 

vitro/ex vivo 
Iodine uptake assays 
Thyroid peroxidase assay 
TBG binding assay 
TTR serum binding assay 
T4 binding protein 
displacement assay 
TR reporter assay 
If you suspect alterations HPG/HPI 
axis take sample for steroid 
hormone analysis. 
TG229/TG230/TG240 
Thyroid endpoints in fish – e.g. 
using zebra fish embryo to 
evaluate chemicals with thyroid 
activity (in development) OECD TG 
229 / TG 234 / TG 240 
Daphnia in vitro juvenile hormone 
and ecdysone agonist 
In vitro assays other than PPAR, 
RXR and RAR 
Prostaglandin synthesis (PG2) 
inhibitors (in vitro) 
Spawning assay in echinoderms in 
vivo (apical for now due to lack of 
endocrinological knowledge) 
Adrenal steroid synthesis (in vitro) 
for wildlife species 

New test 
methods 

2 

Two 

Enhancement 

3/5 

One 

Both 

3 - 5 

One 

New test 
method 
New test 
method 
New test 
method 

New test 
method 

New test 
method 

2 

2 

2 

3 

2 

Two 

One 

One 

One 

One 

Growth and 
Development 

Reproductive 
Health 

Adrenal / 
Steroid 

Directorate-General for Environment 
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Setting priorities for EDC testing  
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Long Term Ideas 

Table 5-12: Long term environment priority ideas 

Adverse 
outcome 

Ideas to fill gaps 

Idea type 

Growth and 
Development 

Sodium-dependent serotonin 
transporter activity 
Ecdysis triggering hormone levels 
in vivo 
Ecdysterone levels (20-EH) in 
arthropods 
Additions to fish tests (e.g. 
thyroid) 

Behaviour/ 
Reproductive 
Health 

Research 

Fish behavioural (embryo) 

Fish behavioural assay (mating 
behaviour) 
Review of bird testing / 
mechanistic “add ons” 

Research Ideas 

CF 
Level 

2 - 3 

3 

3 

New test 
method 
New test 
method 
New test 
method 

Enhancement 

3 - 5 

New test 
method 

3 - 5 

Enhancement 

3 - 5 

Enhancement 

3 - 5 

Number of 
groups 
highlighting 
the idea  

One 

One 

One 

One 

One 

One 

One 

The  environment  groups  also  highlighted  specific  ideas  for  environmental  research,  these  are 
shown below.  

Table 5-13: Priority ideas for environmental research 

Research area 

Ideas to fill gaps 

Idea type 
(Priority) 

Number of 
groups 
highlighting 
the idea  

Invertebrates endocrinology / physiology 

 

Echinoderm research to look at 
development 

  RXR research in invertebrates, 

specifically in molluscs as example 
of the most vulnerable species 

  No further endocrine mollusc 

guideline development until 
further research has been done in 
physiology and endocrinology 
(metabolic pathway) 
A review of the effects of endocrine 
disruption in birds including analysis for 
which modes of action and associated 
specific endpoints might only be observed 
in birds (as opposed to mammals or other 
taxa) due to their unique characteristics. 
The review would include consideration of 
the outcomes of the US EPA validation 
work with the quail model including the 
impact of different diets 
More research on transgenic embryos/fish 
for screening 
Immunotoxicology (wildlife) - research 
into immunotoxicity including potential 
assays 

Invertebrates 

Birds 

Transgenic 
embryos 

Immunotoxicology 

Research need 
(Priority A+) 

Two 

Research need 
(Priority A) 

One 

Research need 
(Priority A) 

Research need 
(Priority A) 

One 

One 

Directorate-General for Environment 
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Setting priorities for EDC testing  
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Table 5-13: Priority ideas for environmental research 

Research area 

Ideas to fill gaps 

Adrenal / steroid 

Reptiles 

Embryos for 
methylation 
marks 

Adrenals/interrenals - endocrine effects 
on the adrenal systems of non-target 
organisms 
Reptiles (potentially connected to birds – 
for extrapolation) 
Collect additional embryos for analysis of 
methylation marks - useful for assigning 
epigenetic effects in organisms 

Idea type 
(Priority) 

Research need 
(Priority A) 

Research need 
(Priority B) 

Research need 
(Priority B) 

Number of 
groups 
highlighting 
the idea  

One 

One 

One 

5.6  Non-priorities 

At  the  workshop,  there  was  also  some  discussion  about  non-priorities,  this  include  the  following 
comments: 

  Any test guidelines proposed using primates was going to be unfeasible 

 

 

For one guideline, the equipment (micro-computed tomography (micro CT)) and its 
associated costs was considered to be beyond what might be considered appropriate for a 
guideline 

It was suggested that binding assays should not be as highly prioritised as transactivation 
assays, as transactivation assays give more information 

  A  point  made  by  various  groups  was  how  the  current  TG  416  (2  gen  study)  (from  2001) 
should be removed, instead attendees suggested to use TG 443 (including as in OECD both 
DNT and DIT cohorts) (from 2012), however some attendees did not agree with the point 
about  removing  TG  416  and  they  highlighted  its  requirement  in  (e.g.)  the  PPP  and  BP 
Regulations 

  No  further  endocrine  mollusc  guidelines  should  be  developed  until  further  research  has 

been done into the physiology and endocrinology (metabolic pathway) of molluscs 

Directorate-General for Environment 
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Setting priorities for EDC testing  
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6. Discussion 

6.1 

Introduction 

The  priorities  highlighted  by  the  human  and  environmental  health  break-out  groups  were 
summarised in the previous section. Some of the underlying themes are highlighted below, taking 
account of the findings of the JRC Survey (2017). 

6.2 

Context 

There  was  recognition  amongst  various  attendees  (particularly  from  academia)  at  the  workshop 
that there is an ongoing trend for national and academic budgets to be increasingly squeezed and 
attendees did not anticipate that funding opportunities would become easier. As such, there would 
be  a  greater  reliance  on  cross  working  and  partnerships  while  the  European  Commission  and 
research budgets (e.g. Horizon 2020) will continue to be important. 

From the industry side, the view was expressed that  there was a desire to form a bridge between 
regulatory  bodies  and  industry/Contract  Research  Organisations  (CRO)  to  ensure  effective 
development of future ED testing. 

Against  this  background,  it  was  generally  acknowledged  that  it  would  be  necessary  to  prioritise 
ideas  for  improving  ED  testing.  More  generally,  it  was  also  clear  that  there  are  many  areas  of 
potential  endocrine  disruption  requiring  further  research  to  inform  the  underlying  scientific 
knowledge.  

6.3 

Prioritisation Summary – Human and Environmental Health 

Both the human and environmental health groups discussed ideas to fill gaps that they felt should 
be  prioritised.  There  were  a  number  of  areas  where  there  was  a  consensus  across  the  separate 
human  health  groups,  the  separate  environmental  health  groups  and  between  both  human  and 
environmental  health  groups.  The  groups  were  also  largely  in  agreement  with  respect  to  the 
anticipated  timelines  (in  the  short,  medium  or  long  term)  for  the  development  to  OECD  Test 
Guideline  status  of  the  proposed  new  test  methods/enhancements.  The  prioritised  ideas  in  this 
section  are  summarised  in  terms  of  in  vitro  ideas,  in  vivo  enhancements,  in  vivo  new  tests, 
regulatory modifications and research ideas. 

6.3.1 In vitro ideas 

All of the human and environmental health groups suggested that more should be done to prioritise 
the thyroid14. Most groups also suggested that there are  a number of  in vitro  thyroid assays that 
are currently under development and should be a priority for being made into new test guidelines. 
Thyroid  specific  in  vitro  assays  highlighted  by  both  human  health  and  environment  groups  were; 
thyroid  peroxidase  assay,  TTR  serum  binding  assay,  deiodinase  expression  in  vitro/ex  vivo,  TBG 
binding  assay,  T4  binding  protein  displacement  assay,  TR  reporter  assay  and  the  Sodium  Iodide 
Symporter (NIS) activation assay. As a number of these have been under development for a while 
it was felt that they had a high TRL and that these could be developed into new guidelines in the 
short to medium term. Human health groups suggested that this might be achievable in a shorter 
timeline  whereas  environmental  health  groups  suggested  a  medium  timeline.  Other  in  vitro 
transactivation  assays  that  were  indicated  as  being  priorities  by  both  human  and  environmental 
health groups were PR, GR and MR transactivation assays. 

Other  suggestions  for  new  assays  were  also  made  for  human  health  and  for  the  environment,  a 
high  priority  suggested  by  three  groups  was  for  the  OECD  validation  of  the  PPAR  (α,β/δ,γ) 
transactivation assays. It was felt that these could become TGs in the short term.  

14   The European Commission’s recent workshop on the thyroid, which many of the attendees at this workshop 
also attended, may have had an influencing factor on some of the attendee’s prioritisations. However, this 
is also built on the OECD thyroid scoping document. 

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Another  high  priority  suggested,  which  could  be  useful  for  human  and  environmental  health,  was 
the validation of RXR and RAR assays (some attendees indicated that in particular the validation of 
RXR  assays  was  their  top priority).  It  was  felt  that  both  of  these  could  become  test  guidelines  in 
the short term. 

The  creation  of  a  new  in vitro  test  method  involving  an  assay  looking  at  adipocyte differentiation 
was highlighted as a priority by three of the human health groups, although this was considered as 
a  long-term  idea.  Other  human  health  ideas  for  assays  that  should  also  be  developed  included: 
neuronal  progenitor  cell  proliferation/differentiation  assays  (medium  term),  cytokine  assays  (long 
term),  dendritic  arborisation  assay  (long  term),  pancreatic  beta  cell  test  (long  term),  assays  for 
PCOS and endometriosis (long term). 

Additional  environment  group  in  vitro  ideas  included  a  prostaglandin  synthesis  inhibition  test 
(medium term), and daphnia juvenile hormone  and ecdysone agonist receptor activity tests (both 
longer term). 

As indicated in the JRC survey (2017), an important aspect  was that in vitro tests should be used 
in a smart combination as part of a WoE approach (e.g. IATA) and that the tests should ideally be 
combined  in  a  mechanistically  based  framework  addressing  specific  key  events  in  relevant  AOPs. 
The  JRC  survey  (2017)  also  suggests  that  the  current  OECD  CF  tests  focus  on  estrogenic, 
androgenic and steroidogenic effects, and that this should be widened to other relevant pathways. 
A number of comments in the JRC survey (2017) indicated that more should be done to consider 
metabolism  when  using  in  vitro  tests.  Another  comment  referred  to  problems  with  false  positives 
and false  negatives,  which  would  need  to be dealt with as part  of  the performance  testing  of  the 
methods. 

6.3.2 In vivo enhancements 

All  four  of  the  human  health  groups  and  both  environmental  health  groups  specifically  suggested 
that thyroid hormones levels, in particular TSH, T4 and T3, should be more routinely measured in 
vivo (including T4 serum levels in dams and juvenile animals, and at specific times in studies – e.g. 
post  natal  day  16  in  rat  pups).  This  would  be  an  enhancement  to  existing  tests  and  could  be 
achieved quite quickly. Three groups suggested that fat measurements should be made in  in vivo 
test  organisms.  Two  groups  suggested  that  the  whole  mount  mammary  gland  histopathology 
should be assessed as an endpoint in higher CF level tests (e.g. TG 407, TG 408 and TG 443) and 
two  groups  suggested  that  brain  histopathology  and  specifically  examination  for  cortical 
heterotopias  in  offspring  should  be  carried  out.  Individual  groups  also  suggested  that  sperm, 
glucose, hormones (such as insulin) and the lipoprotein profile should be more routinely analysed 
as, generally, there are techniques widely available and the parameters are easy to analyse. It was 
also suggested by two groups that markers of gene expression should also be measured on a more 
regular  basis  as  this  could  improve  the  value  of  assays,  while  another  group  suggested  that  an 
enhancement of in vivo tests would be to take measurements of retinoid levels from serum and the 
liver of test organisms. 

It was also suggested by attendees that some performance improvement and refinements could be 
made to the MEOGRT (OECD CF Level 5) test guideline (OECD TG 240). The combination of TG229 
and  TG234  was  suggested  by  workshop  attendees  as  well  as  in  the  JRC  survey  (2017).  The 
potential  added  value  being  coverage  of  all  relevant  life  stages  with  endpoints  diagnostic  for 
endocrine disruptors in the environment. Other ideas for enhancements to in vivo assays included 
behavioural endpoints based on mating in long term fish studies, and measuring ecdysis triggering 
hormone and / or ecdysterone levels in Daphnia studies. A spawning assay in echinoderms based 
on adapting existing tests was also a suggestion. 

It  was  suggested  in  the  JRC  survey  (2017)  that  some  in  vivo  test  guidelines  are  limited  in  their 
capacity  to  detect  EDs  due  to  lack  of  (mandatory)  endocrine  specific  endpoints  and/or  to  lack  of 
exposure during sensitive windows of development. Although guidelines such as TG 407 have been 
updated, some endpoints are still only optional. A priority could be to make these (including weight 
of  uterus  and  ovaries,  oestrus  cyclicity,  histopathological  changes  in  mammary  glands  and 
pituitary, and circulating levels of T3, T4 and TSH) and other endpoints mandatory and to include 
some  of  these  measurements  in  other  existing  tests  (e.g.  TG  407,  TG  408,  TG  414,  TG  440,  TG 
441, TG 443). As suggested in the JRC survey (2017), the mechanistic information generated from 
the enhancement of in vivo tests could then be used to inform AOPs and to identify key events that 
could then be targeted by new or existing in vitro methods. 

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6.3.3 In vivo new tests 

Although  most  of  the  priority  ideas  for  new  tests  were  for  in  vitro  studies,  there  were  some 
suggestions for new in vivo studies that could be prioritised. For human health, new in vivo studies 
included developing a test with electrophysiological endpoints, a PSA secretion and prostate model 
and  the  development of  more and better-defined assays/tests for  investigation  of  disturbances  of 
female reproductive health. 

For environmental health, proposed new in vivo studies included a larval fish behavioural assay and 
an  amphibian  thyroid  assay,  notably  XETA.  XETA  could  also  be  applicable  to  human  health.  A 
specific thyroid test for fish was also suggested. 

It was indicated that a short term mechanistic in vivo test (CF Level 3) is needed. It was suggested 
that  the  test  should  be  based  on  the  study  design  of  OECD  TG  407  with  a  duration  of  not  more 
than 14 days to allow for measurements of specific hormones and to gather organ information for 
all  EATS  modalities  in  one  test.  However,  a  comment  made  by  an  attendee  during  a  break-out 
group  suggested  that  in  some  situations,  in  her  country,  there  was  a  tendency  for  skipping  the 
current  CF  Level  3  test  and  going  straight  to  a  CF  Level  4/5  test  as  this  saved  time,  money  and 
may  result  in  gathering  more  relevant  information  as  well  as  saving  animals.  Another  attendee 
within  the  group,  working  for  a  Member  State  Competent  Authority,  recognised  this  but  found  it 
was not always the case in his country. 

6.3.4 Regulatory modifications 

Other  suggestions  made  by  the  attendees  related  to  a  reconsideration  of  the  conditions  for 
omitting/triggering  of  DNT  and  DIT  cohorts  in  the  TG  443,  perhaps  these  should  be  mandatory, 
since currently under REACH they are triggered only if there are particular concerns. The triggering 
of  these  would  also  increase  the  evidence  base  for  detecting  developmental  immunotoxicants.  A 
further  point  raised  at  the  workshop  was  how  there  is  a  limited  understanding  and  very  little 
research publications considering the links between ED pathways and immunotoxicity. 

Another  suggestion  for  ENV  testing  was  for  the  consideration  of  dietary  exposure  for  endocrine 
testing for substances with a Log Kow >6 due to the accumulation of these substances in the food 
chain. 

A  suggestion  for  the  deletion  of  OECD  TG  416  was  made  on  a  number  of  occasions.  One  of  the 
main  reasons  for  this  suggestion  is  the  existence  of  OECD  TG  443  which  was  developed  as  an 
alternative to TG 416. However, this suggestion was seen by some as being not currently possible 
due  to  OECD  TG  416  being  a  standard  information  requirement  in  certain  regulatory  frameworks 
across the world. 

6.3.5 Other recommendations 

There  was  also  a  suggestion  that  tissues  from  all  endocrine  long-term  in  vivo  studies  should  be 
kept.  This was particularly relevant for environmental testing where this may not always be  done 
routinely. Typically tissue samples are stored for a finite time (e.g. in case re-analysis is required) 
before  being  destroyed.  There  would  be  additional  costs  associated  with  keeping  tissues  samples 
and  some  test  laboratories  may  also  lack  the  storage  capacity  to  do  this,  especially  for  studies 
involving a large number of test organisms. 

6.4 

Further Research 

An important area of further research that was identified by three of the human health groups was 
for  more  assays  and  models  for  female  reproductive  health.  Further  research  into  endocrine 
disruption and immune / autoimmune / inflammatory adverse outcomes was also highlighted by a 
human health and an environment group. 

For  the  environment,  the  highest  priority  identified  by  the  attendees  was  for  more  research  into 
invertebrate  endocrinology  and  physiology.  Other  highly  rated  priorities  were  bird  research 
(perhaps a meta-analysis) and research into transgenic embryos/fish for screening purposes. 

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Epigenetics  was  identified  as  being  an  area  of  research  that  might  benefit  both  human  and 
environmental health. The  use  of epigenetics might also be  a  relatively easy measure to  enhance 
the present TGs. 

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7. Conclusion 

7.1  Overview 

The  overall  conclusion  from  the  expert  workshop  is  that  prioritising  ideas  for  the  development  of 
new or enhancement of existing test methods and guidelines for evaluating endocrine disruptors is 
a complex task. 

This  is  primarily  because  adverse  outcomes  associated  with  endocrine  disruption  cover  an 
extremely wide range of human,  vertebrate and invertebrate  conditions and  behaviours.  As such, 
this  means  that  there  will  be  an  extensive  number  of  additional  parameters  required  to  address 
testing  gaps  associated  with  particular  outcomes.  Furthermore,  the  links  between  exposure  to 
certain chemicals and specific adverse outcomes associated with endocrine disruption are uncertain 
to  varying  degrees.  For  certain  adverse  outcomes,  further  research  may  be  needed  to  help 
substantiate  the  links  between  exposure  and  the  adverse  outcome.  The  research  would  increase 
knowledge  and  contribute  to  the  further  testing  of  substances  suspected  to  play  a  role  in  the 
etiology of the disease/conditions. 

Nevertheless, it is recognised that the regulation of chemicals relies on the availability of methods 
to  identify  and  assess  the  hazardous  properties  of  chemical  substances.  With  this  in  mind,  it  is 
possible to consider a diverse  range  of  ideas against criteria associated with regulatory relevance 
as  well  as  a  range  of  other  criteria.  Indeed,  it  was  recognised  that  ideas  for  new/enhanced  tests 
would have to be of regulatory relevance to be taken forward.  In this context, it was agreed that 
such  tests  would  have  the  capacity  to  provide  either  good  information  on  MoA  or  adversity  (or 
both) to be considered of regulatory relevance. 

While preference may be given to those ideas (of regulatory relevance) which can be developed in 
the  near  future  at  a  relatively  low  cost,  it  is  recognised  that  there  is  also  a  need  for  long-term 
research to explore some of the underlying science of endocrine disruption, and that both could be 
assigned a high priority but the nature of the funding and follow up activities would be different. It 
was noted by some groups that consideration of the Technology Readiness Level (TRL) provided an 
indicator of the likely associated timescale for the development of an idea into a new/enhanced ED-
relevant test guideline. 

7.2 

Summary of adverse outcomes 

At  the  workshop,  a  number  of  concerns  for  diseases/conditions  potentially  related  to  exposure  to 
endocrine  disrupting  substances  with  respect  to  both  human  and  environmental  health  were 
discussed.  Although  all  of  the  adverse  outcomes  were  recognised  as  being  important,  the  human 
and  environmental  health  groups  were  able  to  come  to  a  general  consensus  about  the 
diseases/conditions currently of highest priority.  

For human health, this was those related to thyroid-related developmental neurotoxicity, metabolic 
dysfunction, female reproduction and male reproduction. For environmental health, this was those 
related to reproductive health, growth and development. 

The main concerns closely matched those reported in the JRC survey. 

7.3 

Summary of priority ideas 

At the workshop, a range of ideas for addressing gaps in available test guidelines to better identify 
substances  that  may  contribute  to  the  etiology  of  diseases  or  conditions  related  to  endocrine 
disruption were also discussed. These ideas could be broadly categorised into five main themes: 

 

 

 

 

 

Theme 1: The development of more in vitro assays, 

Theme 2: Enhancing the current test guidelines by refining them and performing additional 
measurements/analyses, 

Theme 3: The development of new and appropriate in vivo methods, 

Theme 4: Modifications of regulatory data requirements, 

Theme 5: More research. 

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For Theme 1, the attendees suggested that a number of in vitro assays are already at an advanced 
stage  of  development.  The  largest  consensus  was  for  the  development  of  thyroid  assays 
(principally  TPO,  TTR,  TBG  and  NIS),  retinoid  assays  (principally  RXR  and  RAR),  and  the  PPAR 
assays (these all relate to human and environmental health). There was also a consensus for the 
development  of  the  GR  and  MR  transactivation  assays  as  well  as  new  in  vitro  assays  for  both 
female and male reproductive health. 

For Theme 2, the new MEOGRT test guideline (relating to environmental health) was highlighted as 
being  one  that  would  benefit  from  further  refinement  and  performance  testing  as  more  of  these 
studies  were  actually  carried  out.  From  the  human  health  perspective,  EOGRTS  (OECD  TG  443) 
was  seen  as  a  method  which  could  be  further  refined  and  have  a  number  of  additional 
measurements  performed  (e.g.  performing  whole  mount  mammary  gland  assessment,  assessing 
brown fat and measuring various hormones). The largest consensus made by the attendees was for 
inclusion of TSH, T4 and T3 measurements (relating to both human and environmental health). 

For  Theme  3,  the  development  of  a  new  OECD  CF  Level  3  study  (based  on  OECD  TG  407  – 
mammalian toxicity) was particularly highlighted. Other ideas included the development of new  in 
vivo  assays  for  both  female  and  male  reproductive  health,  spawning,  behaviour,  growth  and 
development  tests  for  the  environment  (e.g.  embryo  behavioural  assays,  ecdysis  triggering 
hormone and ecdysterone levels). 

For  Theme  4,  the  main  ideas  were  related  to  a  reconsideration  of  the  conditions  for 
omitting/triggering of DNT and DIT cohorts in the TG 443, and considering dietary exposure testing 
of substances with Log Kow >6. 

For  Theme  5,  the  main  ideas  for  human  health  were  for  research  into  new  female  models  of 
reproductive  health, 
into 
epigenetics,  gene  markers  and  other  biomarkers.  For  the  environment,  the  main  ideas  were  for 
research into invertebrate endocrinology/physiology and research into bird (e.g. mode of action in 
birds as opposed to mammals and other taxa), and reptile endocrinology. 

functional  neurodevelopmental  toxicity  endpoints  and  research 

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9. Workshop programme 

SETTING PRIORITIES FOR FURTHER DEVELOPMENT AND VALIDATION OF TEST 
METHODS AND TESTING APPROACHES FOR EVALUATING ENDOCRINE DISRUPTORS 

Marivaux Hotel, Boulevard Adolphe Max 98, Brussels 
30 May - 1 June 2017 

Day 1 – 30 May (afternoon only): 

11:30-13:30: Registration and lunch 

13:30:  Formal opening of expert conference 

  Overview  and  aim  of  the  workshop  –  Maiken  Rasmussen  (European  Commission, 

DG Environment) 

13:45:  Set the scene for the workshop 

 

‘What’s  the  concern?  An  overview  of  adverse  outcomes  associated  with 
endocrine  disrupting  chemicals’  (State  of  Science  review)  –  Andreas 
Kortenkamp (Brunel University) 

14:10:  OECD Test Guidelines and the Conceptual Framework  

 

 

‘An introduction to the Extended One-Generation Reproductive Toxicity Study 
(EOGRTS):  what  does  it  tell  us  and  what  can  we  learn  from  it?’  –  Manon 
Beekhuijzen (Charles River) 
‘To  cull  or  not  to  cull?  Considerations  for  Studies  of  Endocrine  Disrupting 
Chemicals’ –Laura N. Vandenberg (Tufts University) 

14:50:  Moderated  discussion  on  feedback  from  participants  on  experiences  of  using  the  OECD 

Conceptual Framework. The discussion will cover: 
1.  Your experience with using OECD guidelines for substance evaluation, regulation etc. 
2.  The process and progress of international validation and lessons learnt 
3.  Identifying  important  considerations  for  how  current  guidelines  could  be  improved  - 

whilst considering a balance between conclusive testing and screening 

15:25:  Substance evaluation and WoE assessment  

 

‘Adverse Outcome Pathways (AOP) and Integrated Approaches to Testing and 
Assessment (IATA)’ – Sharon Munn (JRC) 

15:45:  Coffee/tea 

16:05:  Updates from other studies launched by the European Commission, DG ENV 

 

 

 

 

‘Expert  survey  on  identification  of  gaps  in  available  test  methods  for 
evaluation of endocrine disruptors’ – Stephanie Bopp (JRC) 
‘Detecting  thyroid  disruption  –  recommendations  from  an  expert  workshop’  – 
Olwenn Martin (Brunel University) 
'Overview  of  the  Retinoid  System  draft-Detailed  Review  Paper  Project'  –  Alice 
Baynes (Brunel University) 
‘Temporal  aspects  in  the  testing  of  chemicals  for  endocrine  disrupting  effects 
in relation to human health and the environment’ – Anke Joas (BiPro) 

17:45:  Closing remarks 

19:30:  Informal dinner at Hotel Marivaux 

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Day 2 – 31 May (full day): 

08:40:  Introduction  to  break-out  group  session  1:  Prioritising  diseases/disorders  and 

pathways 

 

‘Introduction to break-out groups and expectations’  

There will be six groups – groups will separately focus on human health and environmental 
testing.  The  composition  of  the  groups  will  be  pre-determined  and  attendees  will  be 
expected to stay with the same group throughout the remainder of the workshop.  

09:00:  Break-out group session 1  

Discuss  the  core  questions  and  priorities,  collectively  reviewing  pre-prepared  lists  of 
diseases/disorders  and  the  most  relevant  endocrine  pathways  associated  with  endocrine 
disruption for both human and environmental health. The aim of the group discussions will 
be to:  

1. Review the list of diseases/disorders and pathways  
2. Check if something is missing 
3. Rate ‘importance’ of adverse outcomes (high/moderate/low) 

10:15:  Coffee/tea 

10:30:  Group  presentations  –  Human  health  (5  minutes  per  group)  followed  by  professionally 

moderated discussion to review key issues.  

11:00:  Group  presentations  –  Environment  (5  minutes  per  group)  followed  by  professionally 

moderated discussion to review key issues.  

11:20:  Development and validation of new/revised test methods for evaluation of ED 

 

 

 

 

‘What do OMIC data from sticklebacks exposed to EDCs tells us about AOPs’ – 
Ioanna Katsiadaki (Cefas) 
‘An  Integrated  Experimental  Design  for  the  Assessment  of  multiple 
Toxicological End Points in Rat Bioassays’ – Fiorella Belpoggi (Ramazzini Institute) 
‘Mammalian histopathology: can we get more out of what we already have?’ – 
John Foster (Regulatory Science Associates) 
‘Developments  in  thyroid  disrupter  testing’  –  Thomas  Zoeller  (University  of 
Massachusetts) 

12:45:  Lunch 

13:45:  ‘Method for Prioritisation’ – RPA 

Following the presentation, a discussion will cover: 
1.  Do you agree with the factors proposed for the prioritisation? 
2.  Do you agree with the ranking proposed? 

14:10:  Introduction to break-out group session 2: Gaps and ideas to fill them 

 

‘Brief  overview  of  approach  to  identifying  potential  enhancements  and 
associated ratings’  

14:25:  Break-out group session 2  

Discuss the identification and preliminary screening of potential ideas for improving testing 
and gaps and ideas identified by attendees’. As such, the aim of the group discussions will 
be to:  

1. Review the gaps 
2. Review the suggested enhancements and new tests to address the gaps, based on 
attendees’ experience  
3. Check if something is missing/add and discuss attendees’ ideas (gaps, enhancements 
etc.) 
4. Identify those options which are likely to offer the significant societal benefits 

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15:20:  Coffee/tea 

15:40:  Continuation of break-out group session 2  

As such, the aim of the group discussions will be to:  

1. Review the ratings  
2. Review the top 20/30 options (and attendees’ suggestions) for suggested enhancements  
3. Discuss whether the ranking looks about right 
4. Propose changes  

16:45:  Group  presentations  –  Human  health  (5  minutes  per  group)  followed  by  professionally 

moderated discussion to review key issues.  

17:20:  Group  presentations  –  Environment  (5  minutes  per  group)  followed  by  professionally 

moderated discussion to review key issues.  

17:45:  Close 

Day 3 – 1 June (morning only):  

Feedback forms regarding the content, quality and organisation of the workshop will be provided at 
the beginning of the day for anonymous submission.  

08:30:  Review of key themes to emerge from Day 2  

08:40:  Introduction to break-out group session 3: Prioritisation  

  Discuss the outcome of break-out group session 2 – RPA/wca 

The outcome of break-out group session 2 will be summarized in a short presentation, 
followed by moderated discussion regarding the prioritisation shortlist proposed.  

09:05:  Break-out group session 3 

Apply the prioritisation factors/methodology, and discuss the screening of shortlisted ideas 
for improving testing. 

As such, the aim of the group discussions will be to:  

1. Review the revised shortlist (taking account of Day 2 discussions) 
2. Review the ratings/scores for individual parameters 
3. Propose a ranking for the enhancement ideas A – C (A being the highest ranking) 

10:15:  Coffee/tea 

10:30:  Group  presentations  –  Human  health  (5  minutes  per  group)  followed  by  professionally 

moderated discussion to review key issues.  

11:20:  Group  presentations  –  Environment  (5  minutes  per  group)  followed  by  professionally 

moderated discussion to review key issues.  

12:00:  Closing comments from the chair 

12:30:  Lunch 

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10. Participants list 

Break out group moderators and rapporteurs are highlighted by * 

Name 

Institution 

Agnė Janonytė 

Lithuanian Environmental Protection Agency 

Alan Boobis 

Alice Baynes* 

Imperial College London, Faculty of Medicine 

Brunel University 

Andreas Kortenkamp* 

Brunel University  

Angel Nadal 

Endocrine Society 

Angeliki Lysimachou 

PAN Europe 

Anja Kehrer 

Anke Joas 

German Environment Agency (UBA) 

BiPro 

Anna Maria Andersson* 

Rigshospitalet 

Annegaaike Leopold* 

Calidris environment bv 

Anne-Laure Demierre 

Bundesamt für Gesundheit (BAG) 

Aude Kienzler 

DG JRC, European Commission 

Barbara Demeneix* 

National Museum of Natural History 

Becky Brown 

Corrado Galli 

wca 

University of Milan 

David Du Pasquier 

Watchfrog 

Elise Grignard 

Erik Rushton 

DG JRC, European Commission 

CEFIC - Endocrine Modulation Steering Group 

Esther Rosenthal 

Federal Institute for Risk Assessment (BfR) 

Fiorella Belpoggi 

Ramazzini Institute 

Francesca Pellizzato 

ECHA 

Gwynne Lyons 

ChemTrust 

Henrik Holbech* 

University of Southern Denmark 

Ilse Van Overmeire 

Scientific Institute of Public Health 

Ioanna Katsiadaki 

James Wheeler* 

Cefas 

Dow 

John Foster 

Judy Choi 

Laura Fabrizi 

Regulatory Science Associates 

BiPro 

DG SANTE, European Commission 

Laura Vandenberg* 

UMass School of Public Health 

Laure Geoffroy 

Leif Norrgren 

INERIS, France 

Swedish University of Agricultural Science (SLU) 

Linda-Jean Cockcroft 

Risk & Policy Analysts 

Louise Youngs 

wca 

Maiken Rasmussen 

DG ENV, European Commission 

Majorie van Duursen* 

Health and Environment Alliance 

Manon Beekhuijzen  

Charles River Laboratories 

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Name 

Margareta Halin 
Lejonklou 

Maria Uhl 

Institution 

Swedish Chemicals Agency 

Austrian Environment Agency 

Mariana F. Fernandez 

Spain Ministry of Health and University of Granada 

Mark Miller* 

Martina Rauch 

Martine Rohl 

National Institute of Environmental Health Sciences 

EFSA 

FPS Public Health 

Max La Védrine 

Risk & Policy Analysts 

Miriam Jacobs 

Public Health England 

Niklas Andersson 

ECHA 

Olwenn Martin 

Owen Green 

Patience Browne 

Pete Floyd 

Peter Korytar 

Brunel University  

wca 

OECD 

Risk & Policy Analysts 

DG ENV, European Commission 

Philip Marx-Stölting  

Federal Institute for Risk Assessment (BfR) 

Pia Juul Nielsen 

Danish EPA 

Poul Bjerregaard 

University of Southern Denmark 

Rebecca Halliday 

Risk & Policy Analysts 

Richard Ivell* 

University of Nottingham, School of Bioscience 

Seema Schappelle  

U.S. Environmental Protection Agency 

Sharon Munn 

DG JRC, European Commission 

Sofie Christiansen* 

National  Food  institute,  Technical  University  of  Denmark 
(DTU) 

Sofie Norager 

DG RTD, European Commission 

Stefania Barmaz 

EFSA 

Stefano Lorenzetti 

Istituto Superiore di Sanità (ISS) 

Stephanie Bopp 

DG JRC, European Commission 

Taisen Iguchi 

Ozaki National Research Institutes, Yokohama City University 

Tuomo Karjalainen 

DG RTD, European Commission 

Tom R Zoeller 

University of Massachusetts 

Ulla Hass  

National  Food  institute,  Technical  University  of  Denmark 
(DTU) 

Unni Cecilie Nygaard  

Norwegian Institute of Public Health 

Walter Lichtensteiger 

University of Zurich 

ZhiChao Dang 

RIVM, Netherlands 

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Annex 1 - Workshop report 

A1.1  Introduction 

This section describes the proceedings of the workshop on setting priorities for further development 
and validation of test methods and testing approaches for evaluating endocrine disruptors  held on 
30 May - 1 June 2017 at Marivaux Hotel, Brussels, Belgium. 

The  workshop  background,  its  objectives  and  its  agenda  can  be  found  in  the  background  section 
and section 9 of this report. 

The  workshop  was  conducted  on  the  basis  of  extensive  material  distributed  to  all  participants  in 
advance.  This  material  consisted  of  a  discussion  paper  and  a  breakout  group  guidance  document 
(not included in this report). 

A1.2  Workshop participants 

Invited workshop participants were from EU institutions, Member States and US EPA/NIEHS as well 
as  a  wide  range  of  experts  from  universities,  research  centres,  industry  and  NGOs  from  the  EU, 
USA and Japan. All members of the Project’s Steering Group attended. 

A list of participants can be found in section 10 of this report. 

A1.3  Workshop report: Summary of formal presentations 

In this section, a brief summary of formal workshop presentations is given.  

After  a  welcome  address  by  Maiken  Rasmussen  (DG  ENV)  that  highlighted  the  objectives  of  the 
workshop,  there  was  an  introductory  presentation  by  Andreas  Kortenkamp  (Brunel  University 
London,  UK)  that  provided  a  State  of  the  Science  overview  of  adverse  outcomes  associated  with 
endocrine  disrupting  chemicals.  The  presentation  highlighted  the  four-fold  increase  in  testicular 
germ  cell  cancer  incidence  witnessed  over  10  years,  and  how  hypospadias,  breast  cancer  and 
congenital hypothyroidism are also increasing; it was argued that this is not the result of improved 
diagnosis. He also  highlighted  that there is a total  lack of funding  for  research into the causes  of 
female  reproductive  health  disorders  and  how  women  are  more  prone  to  autoimmune  diseases. 
The  questions/comments  that  followed  the  presentation  included  how  cross-talk  should  be 
considered  and  that  there  is  already  sufficient  biological  plausibility  to  consider  this  potential.  It 
was  questioned  whether  the  increased  risk  of  thyroid  related  cancers  in  women  is  a  result  of 
varying expression of liver enzymes. 

Manon Beekhuijzen (Charles River) provided an overview of her experiences of the EOGRTS (OECD 
TG 443) and how it is superior to the two-generation study (OECD TG 416). In particular, it saves 
around  1200  animals  per  study  (when  second  generation  not  triggered),  more  information  is 
gained  from  less  animals,  it  uses  more  animals  per  litter,  more  detailed  examinations  are 
performed,  and  there  is  the  possibility  for  additional  examinations  (i.e.  by  creation  of  additional 
cohorts). Manon highlighted some issues of variability and stress as part of the study. Following the 
presentation  Thomas  Zoeller  (University  of  Massachusetts,  Amherst,  USA)  indicated  that  in  their 
studies  the  coefficient  of variance  is  very  low,  and  he  suggested  that  maybe they  could  help one 
another.  Miriam  Jacobs  (Public  Health  England,  UK)  reminded  attendees  that  anyone  can  contact 
their National Coordinators at the OECD Test Guidelines Programme (TGP) as well as the Business 
and Industry Advisory committee (BIAC), in order to give feedback on the practical implementation 
of the  OECD  TGs. It  was  suggested  that if  animals are  used for ED  studies, we  must harvest  the 
most  information  possible  from  these  animals,  and  therefore  should  the  neurotoxicity  and 
immunotoxicity cohort not be mandatory? Sharon Munn (JRC) indicated that it had been suggested 
at  the  recent  EC/ANSES  thyroid  workshop  that  the  sensitivity  of  DNT  may  be  too  low  to  detect 
certain  low  incidence  effects.  Sofie  Christiansen  (National  Food  institute,  Technical  University  of 
Denmark  (DTU),  Denmark)  indicated  that  the  National  Coordinator  of  the  OECD  Test  Guideline 
Programme for Denmark had suggested deleting the 2 Generation study (OECD  TG 416) in 2014. 
The OECD  conducted a survey  in 2016,  which  had  indicated support  for the deletion of  the OECD 
TG 415 (one-generation study), but some countries halted the deletion of OECD TG 416. 

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Laura  Vandenberg  (University  of  Massachusetts,  Amherst,  USA)  presented  a  summary  of  her 
endocrine disrupting studies using rats, and the impacts of culling and gender bias in litters. Laura 
indicated that culling may be appropriate for some endpoints, and less appropriate for others. 

There was a moderated discussion  which focused on the use  of the OECD  Conceptual Framework 
and the following points were raised: 

Experiences  with  using  the  OECD  TGs  for  regulatory  decision  making,  including  substance 
evaluation (under REACH): 

  An attendee suggested that what is currently missing for substance evaluation, is a testing 

strategy. 

  Some attendees indicated  that the process  is very  protracted  and  very frustrating.  It  was 
suggested that it is not the science, but it is the convenience and logistics that appears to 
be drive for a lot of the TG development. Also, adding more enhancements or endpoints to 
tests  is  not  always  allowed  by  national  authorities.  Another  attendee  highlighted  how  the 
OECD  Guidance  Document  150  is  a  document  that  provides  some  information  on  this 
matter. 

  An attendee highlighted a gap between ED testing and REACH, the attendee indicated that 
there are presently no OECD level 1, 2 or 3 tests included in the REACH regulation standard 
information requirements. The attendee suggested that it would be easy to add some OECD 
CF Level 1 and Level 2 information into the requirements. 

 

In  one  attendee’s  experience,  they  indicated  that  pubertal  assays  were  not  very 
reproducible.  They  suggested  that  an  increased  understanding  of  the  sensitivity  to 
moderate/mild effects would be beneficial. 

In terms of the process and progress of international validation and lessons learnt: 

  An  attendee  indicated  that  ECHA  frequently  asks  for  additional  endpoints  in  regulatory 
studies,  and  in  the  attendee’s  experience  some  companies  will  proactively  provide  this 
data,  which  helps  to  bridge  the  gap  between  science  and  the  regulatory  decision  making 
process.  They  also  suggested  that  there  may  be  better  analytical  techniques  and  better 
methods  than  those  that  are  presently  in  the  TGs,  for  example,  in  relation  to  making 
hormone measurements. 

 

Frustration was expressed with regards to the avian OECD TG. The development of this TG 
had been taken out of the hands of the avian task force and there was limited international 
discussion  on  TG  development.  The  two-generation  validation  was  completed  on  a 
substance  which  was  not  the  most  relevant  and  in  a  species  which  was  not  sufficiently 
relevant and the development of an OECD test guideline was not pursued. It was suggested 
by some attendees that we need to go back to the drawing board and consider whether or 
not  there  is  a  data  gap  or  whether  other  taxa  are  sufficient  to  cover  effects  in  birds  (i.e. 
sufficiently protective). 

  An  attendee  suggested  that  they  had  learnt  from  the  process  of  international  test 
validation,  but  they  suggested  that  it  really  does  take  a  long  time.  They  also  suggested 
that  this  may  be  exacerbated  when  trying  to  implement  validation  of  in  vitro  methods. 
They questioned how do we validate fit for purpose methods? As part of the discussion the 
CLARITY  project  was  highlighted  as  being  a  good  example  of  how  to  quickly  incorporate 
academic  studies,  however,  it  was  highlighted  that  this  was  a  very  expensive  study.  An 
attendee  indicated  that  it  would  be  beneficial  to  have  a  means  of  implementing  a 
performance based approach, to bring in these studies. 

 

It was suggested that there have been some issues in validation where the results have not 
always  been  reproducible.  However,  another  problem  highlighted  is  when  the  validation 
process is moving slowly due to a lack of funding, political reasons, it is being blocked by 
research  companies  or  by  a  lack  of  information.  It  was  highlighted  that  the  OECD  is  also 
considering “me-too" methods, where it will be possible to fast-track the validation process. 
An attendee indicated  that  The ToxCast programme  has cost  millions of  dollars,  however, 
we may not be able to directly use the data generated by the non-validated methods. 

  Some  attendees  felt  that  there  was  a  potential  for  lessons  to  be  learnt  from  the  clinical 
environment, where a number of biomarkers can be identified for use in both in vitro and in 
vivo assays, in order to inform on ED effects. They suggested that focus has been on test 

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methods  which  concentrate  on  adverse  outcomes,  however,  the  only  boundary  to  the  in 
vivo  test,  is  what  is  measurable  and  these  assays  can  be  enhanced  to  include  additional 
parameters occurring upstream from the adverse outcomes. They indicated that they have 
spent  a  lot  of  money  on  measuring  molecular  initiating  events,  but  little  on  the  way  this 
information translates to the whole animals. 

In  terms  of  identifying  important  considerations  of  how  current  guidelines  could  be  improved, 
whilst considering a balance between conclusive testing and screening: 

  An  attendee  indicated  that  the  US  EDSTAC  made  recommendations  on  testing  guidelines 
that  were  validated  over  a  10-year  period.  However,  none  of  the  assays  considered  by 
EDSTAC  were  less  than  50  years  old.  They  indicated  that  this  would  not  be  ideal  for  the 
development of new TG and the enhancement of existing TG. However, the attendee also 
indicated  that  the  CLARITY  project  conducted  by  NIEHS/FDA  showed  an  interesting 
approach  for  prioritising  according  to  statute  and  this  showed  that  we  can  cut  the  time 
period without affecting the validity and the efficacy of the results. 

Sharon  Munn  (JRC)  gave  an  informative  presentation  on  adverse  outcome  pathways  (AOP)  and 
integrated  approaches  to  testing  and  assessment  (IATA).  Sharon  indicated  that  the  integrated 
approach  weighs  all  relevant  existing  data  and  can  guide  the  targeted  generation  of  new  data 
where required to inform regulatory decision-making of a potential hazard and/or risk.  

Stephanie Bopp (JRC) provided an overview of the JRC survey on identification of gaps in available 
test methods for evaluation of endocrine disruptors and highlighted some of the main points raised 
by  the  respondents.  After  the  presentation,  follow  up  questions  were  asked  about  the  survey 
findings,  including  what  was  meant  by  practical  problems?  Stephanie  indicated  that  this  was 
intended to be  related  to the  practical problems of  implementing  new assays/endpoints. She  also 
indicated  that  the  JRC  was  grateful  to  the  survey  responders  who  had  invested  their  time  in 
answering the questions and providing numerous detailed comments. An attendee suggested that 
perhaps human toxicology should be separated from ecotoxicology when ranking the priorities.  

Olwenn  Martin  (Brunel  University,  UK)  presented  an  update  of  the  recommendations  emerging 
from  the  expert  thyroid  disruption  workshop.  Olwenn  highlighted  four  main  testing  gaps,  these 
were  in vitro  assays, thyroid related endpoints in OECD  CF Levels 3-5  assays, test  guidelines not 
considering  exposure  during  critical  windows  of  sensitivity,  and  downstream  effects  of  thyroid 
disruption. 

Alice  Baynes  (Brunel  University,  UK)  presented  an  overview  of  the  OECD  project  to  develop  a 
Detailed Review Paper on the retinoid system. This highlighted the absence of OECD test guidelines 
that cover retinoid system modulation but it was indicated that Swedish authorities are working on 
addressing this issue. 

Anke  Joas  (BiPro)  presented  an  overview  of  the  project  on  temporal  aspects  in  the  testing  of 
chemicals for endocrine disrupting effects (in relation to human health and the environment). This 
project  has  highlighted  that  there  remain  considerable  limitations  in  the  current  standardised 
methods,  which  hinder  the  identification  and  understanding  of  the  mechanisms  of  endocrine 
disrupting chemicals. As outcomes of the project they recommend that; foetal hormone levels are 
measured,  there  is  quantitative  analysis  on  mammary  gland  development  and  function,  there  is 
more  flexibility  in  test  durations,  and  there  is  further  research  on  epigenetics  and  the 
development/validation of suitable markers that can be incorporated in the OECD TG. 

Ioanna  Katsiadaki  (Cefas,  UK)  shared  her  experiences  of  using  transcriptomics,  metabolomics, 
morphometrics, behaviour and biomarkers as part of exposing sticklebacks to endocrine disrupting 
chemicals.  It  was  suggested  that  nonyl  phenol  and  ethoxylates  may  have  been  the  real  cause  of 
the intersex previously seen in fish in UK waters rather than the contraceptive pill. The reasoning 
for this is that the use of the contraceptive pill has increased while the regulation of nonyl phenols 
and ethoxylates has seen a decrease in their use and there has been a decrease in intersex. Ioanna 
also  highlighted  how  fish  have  different  reproductive  strategies  which  impacts  the  applicability  of 
AOPs developed from one class of fish to another. 

Fiorella  Belpoggi  (Ramazzini  Institute,  Italy)  shared her  experience  of  an  integrated experimental 
design for the assessment of multiple toxicological endpoints in rat bioassays. She highlighted how 
carcinogenicity,  toxicity  and  reproductive/developmental  toxicity  endpoints  are  not  currently 
investigated  in  a  single  protocol.  Fiorella  indicated  that  although  their  integrated  experimental 

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protocol required 1720 animals, the costs, both monetarily and ethically, may be lower than what 
would be required to conduct several separate studies. However, the protocol is not easy, it is also 
labour intensive, and validation is required. 

John  Foster  (Regulatory  Science  Associates)  gave  a  presentation  on  mammalian  histopathology 
and asked whether we can get more out of what we already have and gave a number of examples 
of additional measurements. 

Thomas Zoeller (University of Massachusetts, USA) gave a presentation on developments in thyroid 
disrupter testing. He suggested that we are not considering most of the possible sites of action of 
environmental contaminants on the HPT axis and presented a range of potential adverse outcomes 
that could be further investigated.  

A1.4  Workshop report: Deliberations of the discussion groups 

Workshop attendees were split into six groups (four  human  health and two  environment  groups), 
to discuss three topics, listed below, over three breakout group sessions: 

  Diseases, disorders and adverse outcomes related to endocrine disruption 

  Review and identification of ideas to fill gaps 

 

Prioritisation of the ideas to fill the gaps 

A1.4.1 Break-out group session 1: Diseases, disorders and adverse outcomes of 
endocrine disruptors 

During this breakout group session, the groups discussed a range of adverse outcomes of exposure 
to  endocrine  disruptors  for  both  human  health  and  the  environment.  Both  sets  of  groups  also 
attempted to rank the adverse outcomes according to what they felt should be the highest priority. 

In  the  plenary,  the  outcomes  of  these  discussions  were  presented  to  the  other  attendees;  these 
discussion outcomes are found in section 2 of this report. 

A1.4.2 Discussion about the prioritisation methodology 

There  was  a  discussion  about  the  prioritisation  methodology  suggested  by  the  project  team.  The 
human and environmental health groups were able to explore this further in the following breakout 
groups.  The  factors  proposed  (MOA,  adversity,  societal  benefits  (real  and  perceived),  technology 
readiness  level,  cost  of  performing  the  test,  animal  welfare  concerns  and  integration  with  other 
research) were generally recognised as being the main key factors. 

Amendments  to  the  prioritisation  methodology  were  applied  by  some  groups,  it  was  highlighted 
that the prioritisation methodology more favourably weighted in vitro studies due to the perceived 
lower financial costs and animal welfare concerns. A tiered approach was applied by one group, the 
most  important  factors  were  whether  it  gave  a  high  level  of  MOA/adversity  information  and  the 
potential societal benefits,  however, they indicated that their ranking still did not look quite right. 
This  tiered  approach  was  suggested  as  the  one  that  all  groups  should  follow.  There  are  some 
challenges  when  trying  to  develop  a  methodology  since  it  needs  to  cover  all  of  the  different  CF 
levels. Furthermore, considering benefits is challenging when the link between endocrine effect and 
severity  of  the  adverse  effect  is  not  always  clear  and  the  methodology  needs  to  consider  both 
human and environmental health. 

A1.4.3 Break-out group session 2: Review and identify ideas to fill gaps 

During this breakout group session, the groups highlighted their own ideas and reviewed a list of 
pre-identified ideas (from various sources) that would help to address gaps. 

In  the  plenary,  the  outcomes  of  these  discussions  were  presented  to  the  other  attendees;  these 
discussion outcomes are found in section 4 of this report. 

A1.4.4 Break-out group session 3: Prioritisation of the ideas to fill the gaps 

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During this breakout group session, the groups discussed ideas to fill the gaps that had previously 
been highlighted and attempted to prioritise the ideas. 

In the plenary, the outcomes of these discussions and the prioritised ideas were presented to the 
other attendees; the outcomes of these discussions and the groups’ priorities are found in section 5 
of this report. 

A1.5  Feedback on the workshop 

Attendees were asked to complete a short feedback questionnaire. 37 attendees kindly completed 
the questionnaire. The feedback was positive with 97% (strongly) agreeing with the statement: the 
workshop provided me with  the  opportunity  to share  my knowledge and experience  in relation to 
testing approaches for evaluating endocrine disruptors. 

In relation to addressing gaps, 81% (strongly) agreed with the statement: The workshop provided 
me  with  the  opportunity  to  tell  attendees,  the  project  team  and  the  Commission  what  I  felt  the 
priorities to address gaps should be and 78% (strongly) agreed with the statement: The workshop 
provided  me  with  the  opportunity  to  learn  about  ideas  to  address  gaps  in  testing  and  how  these 
might be prioritised. 

Several  attendees  commented  on  some  of  the  difficulties  in  trying  to  cover  testing  related  to  all 
areas  potentially  impacted  by  endocrine  disruption.  It  was  unfortunate  that  the  background 
materials  were  not  distributed  sufficiently  far  ahead  of  the  workshop  which  meant  that  many 
attendees  had  not  had  the  time  to  review  the  materials  before  the  workshop.  This  was 
compounded, to some extent, by a lack  of  clarity  in the presentations by the consultants on how 
the ideas to address gaps had been developed. 

Nevertheless,  76%  of  respondents  (strongly)  agreed  with  the  statement  that:  the  workshop 
process  was  fit  for  purpose  and  86%  (strongly)  agreed  with  the  statement:  the  workshop  was  a 
good use of my time. 

Finally, apart from some technical problems on the final day, the venue was conducive to positive 
discussions with 95% of respondents (strongly) agreeing with the statement that: the facilities and 
catering were suitable for the event. 

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Annex 2 - Ideas to fill gaps 

A2.1  Ideas discussed at the workshop 

Although this Annex has separated the ideas into human health and environmental health ideas, it 
should  be  noted  that  some  ideas  would  be  applicable  to  both  human  and  environmental  health. 
Also, it should be noted that there is read across from human health to the environment and vice 
versa.  In  some  cases  (particularly  for  human  health),  the  ideas  mentioned  at  the  workshop  have 
been  expanded  by  the  project  team  with  additional  information  obtained  after  the  workshop.  For 
ideas where such material has been provided by  one of the workshop moderators, rapporteurs  or 
attendees, these contributions are indicated in the ideas below by attendee appearing in brackets 
(Attendee).  For other ideas  where, based  on  the discussions by the experts at  the  workshop, the 
additional  research  and  material  has  been  identified  by  the  project  team,  these  are  indicated  by 
project team appearing in brackets (Project team). Also, an indication of the anticipated timeline to 
develop the idea to fill the gap also appears within brackets, the timelines were suggested by the 
attendees. 

In terms of adhering to the 3Rs principles, the extent to which this can be achieved will depend on 
the  idea  being  developed  and  its  implementation.  Ideas  that  are  for  the  development  of  in  vitro 
TGs  would  not,  by  definition,  require  additional  animals  or,  indeed,  any  animals  for  the  test  (an 
exception  being  those  studies  with  ex  vivo  aspects).  “Enhancements”  may  lead  to  additional 
information being obtained from the existing number of test organisms in the study design, or may 
require satellite group(s). This should make the test design more efficient and may in fact lead to a 
reduction  in  animal  testing  based  on  the  rationale  that  more  data  is  obtained  which  may  avoid 
additional testing that may have otherwise been required. Although new in vivo TGs may increase 
the  amount  of  animals  used  for  testing,  these  studies  may  also  provide  more  information  which 
may in some cases prevent the need for further testing. 

A2.2  Human Health ideas 

The human health ideas are preceded by the relevant adverse outcome as indicated in Table 4-1. 

1.  Thyroid - Thyroid pathway screens (Deiodinase expression) - in vitro/ex vivo 

Three out of the four human  health groups (and  both  of the environment groups) suggested that 
developing more thyroid screening assays should be a priority. Specific assays were highlighted at 
the workshop and most are described in the new scoping document on in vitro and ex vivo assays 
for the identification of modulators of thyroid hormone signalling, OECD no. 207:  

Deiodinase  expression 
types  of 
deiodinase/deiodination  assays;  radioactive  based  assays  monitoring  iodine  release  from  labelled 
T4,  HPLC  assays  based  on  competitive  inhibition  with  iodoacetate  measuring  T3  and  rT3, 
colorimetric assays measuring free iodine based on the Sandell-Kolthoff reaction. 

in  vitro/ex  vivo  assays.  There  are  currently 

three 

These assays all require further research. (Short-term ideas) (See also environment idea 1) 

2.  Thyroid - Thyroid pathway screens (TBG binding assay; TTR serum binding assay; T4 

binding protein displacement assay; and TR reporter assay) - in vitro 

TBG  binding  assays.  There  are  a  variety  of  assays  that  are  highlighted  in  OECD  207  and 
information on suggested assays has already been published. TBG binding assays are indicated as 
being assays that might be ready for validation in the near future. In addition to being relevant for 
the  environment,  TBG  has  the  highest  affinity  and  carries  the  majority  of  T4  in  blood  in  humans 
and it is the primary binding protein in humans. 

The  transthyretin  (TTR)  serum  binding  assays  were  highlighted  by  two  out  of  the  three  human 
health groups. Within OECD 207 two such TTR assays are described, these are the Combined TBG 
and Transthyretin TTR Binding Assays and the TransThyretin (TTR) Binding Assays, measuring the 
displacement of T4 from its binding site on the serum binding proteins. The TTR binding assays are 
indicated as almost being ready for validation in the short term. 

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T4  binding  protein  displacement  assay.  Several  of  these  methods  for  T4  displacement  from  TTR 
have been published; 1) radioligand binding assay: Somack et al. 1982, Brouwer and van den Berg 
1986, Lans et al. 1993, 1994, Hamers et al. 2006, Ucan-Marin et al. 2010. 2) plasmon resonance-
based biosensor: Marchesini et al. 2006, 2008. 3) Fluorescence displacement: Cao et al. 2010, Cao 
et  al.  2011  and  Ren,  Guo  2012,  Montano  et  al.  2012.  The  work  needed  for  validation  of  such 
assays include a survey of the most applicable and stable assay(s) and a validation of the selected 
assay(s). 

TR reporter assay. There are a variety of TR reporter assays, the Transactivation Reporter Assays 
with  TRα  and  TRβ  have  been  highlighted  as  being  highly  sensitive.  As  commercial  TR  reporter 
assays already exist the development and validation of an assay might be achievable in the short 
term. The assay would help with the  identification and quantification of thyroid  hormone receptor 
disrupting potency of compounds.  

The assays highlighted above are the ones discussed at the  workshop, however, within  OECD  no. 
207  additional  thyroid  assays  and  their  readiness  levels  are  highlighted.  (Short-term  ideas)  (See 
also environment idea 2) 

3.  Thyroid  pathway  screens:  Thyroid  peroxidase  assay,  Sodium  Iodide  Symporter  -  in 

vitro and in vivo 

The thyroid peroxidase assay is commonly referred to as the TPO assay. TPO assays are described 
in the new scoping document on in vitro and ex vivo assays for the identification of modulators of 
thyroid hormone signalling, OECD no. 207. There are commercially available TPO assays and some 
of these developed TPO assays may be ready for validation in the short term. However, in vivo TPO 
assays  may  also  be  developed.  Findings  by  Hornung  et  al.  (2015)  indicate  that  inhibition  of  TPO 
activity in vitro was a good indicator of a chemical's potential for thyroid hormone disruption in vivo 
and may be useful for prioritizing chemicals for further investigation. 

The Sodium Iodide Symporter Activity Assay is commonly known as the NIS activity assay. Three 
such assays are described in the scoping document OECD no. 207: 

Radioactive  Sodium/Iodide  Uptake  assay  using  transiently  transfected  FRTL-5  cells,  this  assay  is 
currently at the research stage and it could potentially be used to assess mechanisms of disruption 
that  involve  NIS  symporter,  but  it  should  be  specifically  designed  and  improved  for  endocrine 
disruptors. 

Radioactive  Sodium/Iodide  Uptake  Assay  using  hNIS  stably  transfected  in  FRTL5  cells  and  in 
HEK293 cells, there is a need to decide which stably transfected cell type should be taken forward 
for  validation;  either  FRTL5  cells  or  HEK293  and  then  whether  to  test  with  the  radio-isotope 
method or the colorimetric method. 

A  non-Radioactive  Sodium/Iodide  Uptake  Assay  based  on  Sandell-Kolthoff  reaction  and  using 
FRTL5 cells, is still at the research stage. 

The NIS assays are at different stages of development. (Short-term ideas) (Project team) (See also 
environment idea 3) 

4.  Thyroid  -  Application  of  the  amphibian  thyroid  assays  (XETA)  since  HPT  axis  is  well 

conserved in vertebrates - in vivo 

A  human  health  group  suggested  there  could  be  the  application  of  amphibian  thyroid  assays, 
notably  the  Xenopus  Embryonic  Thyroid-disruption  Assay  (XETA),  to  human  health  assessments 
since the HPT axis is conserved across vertebrates. A draft OECD TG exists and this test method is 
currently  being  validated  by  OECD  for  inclusion  as  a  Level  3  in  vivo  non-mammalian  toxicology 
study. The test is designed to be a screen for thyroid activity in  amphibians; it is not designed to 
provide  information  on  endocrine  activity  for  use  in  assessing  the  environmental  risks  of  an 
individual  chemical  based  on  a  PEC/PNEC  approach.  As  indicated  in  the  OECD  (2017)  guidance 
document  on  standardised  test  guidelines  for  evaluating  chemicals  for  endocrine  disruption,  the 
XETA  uses  transgenic  Xenopus  laevis  tadpoles  into  which  a  THbZIP  promoter  has  been  inserted, 
that  is  regulated  by  TH  and  other  TH  agonists  (Morvan-Dubois  et  al.,  2008).  Furthermore,  the 
promoter is linked to the gene coding for Green Fluorescent Protein (GFP), and thus the degree of 

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receptor  response  can  be  measured  by  fluorescence  in  the  transparent  tadpoles.  (Short-term) 
(Project team) 

5.  Thyroid - Serum T4/TSH assays in parents and juveniles - in vivo 

All four human health groups (and an environment group) suggested that more should be done to 
measure  hormones.  In  particular  thyroid-stimulating  hormone  (TSH),  T4  and  T3  levels  were 
suggested as being a priority. As suggested by one group, this could be an enhancement that could 
be  made  to  existing  test  guidelines  (e.g.  OECD  TG  443  (Extended  One  Generation  Reproductive 
Toxicity study) or OECD TG 416 (Two Generation Reproductive Toxicity study)) to measure T4 on 
appropriate post-natal occasions in such rodent studies. Also, it was suggested by a group that if a 
±10% T4 change is detected in the test species this could be an indication of an adverse signal or 
a trigger for further investigation and potentially  further testing to be performed. This will help to 
obtain  more  information  from  the  same  number  of  animals,  as  the  idea  is  to  perform  additional 
analysis as part of existing TGs. (Short-term) (Attendee) (Project team) 

6.  Thyroid - Bone breaking assay - in vivo 

A  human  health  group  highlighted  that  the  thyroid  hormone  is  known  to  affect  the  rate  of  bone 
replacement.  They  indicated  that  some  evidence  suggests  that  those  people  with  low  TSH  levels 
may lose bone at faster rates than those with normal TSH levels. In these situations, people may 
develop osteoporosis, a condition where bones become fragile and are more likely to break. A bone 
breaking  assay  based  on  known  endocrine  disrupting  chemicals  could  be  developed  with  bone 
breaking  as  the  endpoint.  An  interesting  technique  that  could  be  used  to  assess  bone  strength 
would  be  peripheral  quantitative  computed  tomography;  however,  this  may  not  be  practical  for 
regulatory toxicology studies. 

It  would  be  considered  an  enhancement  of  existing  TG  with  longer  test  durations  (e.g.  OECD  TG 
408  (Repeated  Dose  90-Day  Oral  Toxicity  Study  in  Rodent),  OECD  TG  452  (Chronic  Toxicity 
Studies) and OECD TG 453 (Combined Chronic Toxicity/Carcinogenicity Studies)) to conduct a bone 
breaking assessment, for  example,  a bone breaking assessment of femoral bone,  ash weight  and 
percentage,  x-ray  imaging  and  pathological  assessment.  The  current  TGs  (mentioned  previously) 
might  also  be  enhanced  by  measuring  peripheral  biomarkers  of  catabolism/metabolism  of  bone, 
such  as  FGF23  and  additional  serum  markers.  (Bone  breaking  assay  -  medium-  to  long-term) 
(Additional measurements as an enhancement - short-term) (Attendee) (Project team) 

7.  Thyroid-related developmental neurotoxicity – Dendritic arborisation assay - in vitro 

A  human  health  group  suggested  that  the  development  of  a  dendritic  arborisation  assay,  a  new 
test method, should be a priority. The idea would be for the development of a new OECD CF Level 
2 in vitro assay. The dendritic arborisation is described in the OECD (2012)  detailed review paper 
on  the  state  of  the  science  on  novel  in  vitro  and  in  vivo  screening  and  testing  methods  and 
endpoints for evaluating endocrine disruptors. It is indicated that several studies have investigated 
the  impact  of  thyroid  disrupting  compound  exposure  on  tyrosine  hydroxylase-dependent  dendrite 
arborisation  of  cerebellar  Purkinje  cells  (e.g.  Ibhazehiebo  et  al.  (2011)).  The  proposed  assay 
requires  isolation  of  primary  rat  or  mouse  Purkinje  cells,  as  described  by  Kimura-Kuroda  et  al. 
(2005),  and  subsequent  exposure  to  test  compounds  of  interest  for  17  days.  The  dendrite 
arborisation  is  then  assessed  through  immunocytochemical  staining,  and  the  extent  of  the 
arborisation  is  quantified  by  tracing  the  outline  of  the  cell  and  dendritic  branches  of  randomly 
selected Purkinje cells and computing the area using imaging software. (Long-term) (Project team) 

8.  Thyroid-related developmental neurotoxicity –Neuronal progenitor cell proliferation/ 

differentiation assays; neurite extension - in vitro 

A  human  health  group  suggested  that  a  test  should  be  developed  which  looks  at  neuronal 
progenitor cell proliferation.  

The  measurements  of  human  neural  progenitor  cells  (hNPCs)  are  discussed  as  part  of  Stoll  et  al. 
(2011),  which  indicates that  alterations/aged  hNPCs  have  decreased  regenerative  capacity,  fewer 
functional  mitochondria,  and  less  oxygen  consumption  compared  with  young  adult  hNPCs.  As 
indicated  by  Schreiber  et  al.  (2010),  it  is  also  possible  to  use  primary  foetal  hNPCs,  which  are 
cultured  as  neurospheres  to  mimic  basic  processes  of  brain  development  in  vitro.  The  assay 

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involves  the  growth  of  normal  hNPCs  cultured  as  free-floating  neurospheres  in  a  proliferation 
medium  and  plating  onto  a  poly-D-lysine/laminin  matrix.  In  this  assay,  cell  proliferation  is 
determined by measuring sphere size  and  identified  changes in cell number  by  measuring sphere 
diameter in contrast to the negative control without mitogens. 

Also, in terms of neurite extension estrogen replacement therapy, for example, has been shown to 
decrease  the  morbidity  from  several  chronic  neurological  diseases.  Nathan  et  al.  (2004),  using 
cultured adult mouse cortical neurons, conclude that estradiol facilitates neurite growth through an 
apolipoprotein E dependent mechanism. (Medium-term) (Project team) 

9.  Thyroid-related  developmental  neurotoxicity  -  Oligodendrocyte  differentiation  -  in 

vitro and in vivo 

The  main  function  of  oligodendrocytes  is  to  support  axons  in  the  central  nervous  system.  Axons 
transmit information in the form of electrical impulses over long distances and this is enhanced by 
myelin. Deficiencies in these processes are associated with developmental disorders.  

As suggested by Yoshida and Macklin (2005), green fluorescent protein (GFP) transgenic zebrafish 
technology has been used to directly visualise and analyse dynamic developmental processes. They 
established a stable transgenic zebrafish line, at the  F1  generation, that expresses  enhanced GFP 
(EGFP) that was visually detectable around 24-hr post fertilisation. 

Furthermore, Mathews and Appel (2016) suggested that an in vivo myelination model, such as the 
zebrafish, allows for live observation of cell dynamics and cell-to-cell interactions. They suggested 
that  this  is  well  suited  for  investigating  oligodendrocyte  development.  A  reason  for  this  is  that 
zebrafish  offer  several  advantages  to  investigating  myelination,  including  the  use  of  transgenic 
reporter  lines,  live  imaging,  forward  genetic  screens,  chemical  screens,  and  reverse  genetic 
approaches.  

Studies  have  indicated  oligodendrocyte  differentiation  as  part  of  in  vitro  analysis,  for  example, 
Pedraza  et  al.  (2013),  analysed  the  effects  of  modulating  the  Rho-A/Rho-associated  pathway,  by 
the  use  of  selective  inhibitors,  on  the  transformation  of  oligodendrocyte  precursor  cells  into 
mature,  myelinating  oligodendrocytes.  The  study  used  cellular  cultures  from  rodent  and  human 
origin and resulted in Rho-A/Rho-associated kinase inhibition of oligodendrocyte precursor cells and 
resulted  in  a  significant  generation  of  branches  and  cell  processes  in  early  differentiation  stages, 
followed by accelerated production of myelin protein as an indication of advanced maturation. 

Tests to identify oligodendrocyte differentiation could be based on new in vitro or in vivo models, 
these could be developed around zebrafish and/or rodent cultures. (Long-term) (Project team) 

10. Thyroid-related developmental neurotoxicity – Cognitive function test - in vivo 

A  human  health  group  suggested  that  more  should  be  done  to  investigate  whether  endpoints 
measuring cognitive function could be  developed and incorporated into an existing test guideline. 
The  reason  for  this  is  that  chemicals  have  been  shown  to  alter  cognitive  function  in  animals  and 
humans.  It  is  suggested  the  changes  in  cognitive  function  are  mediated  by  the  endocrine-like 
actions  of  these  chemicals  and  their  interaction  with  hormonally  mediated  events  in  the 
development and function of the central nervous system. It was mentioned that cognitive function 
tests are presently in regulatory use but considered by some to lack sensitivity. The idea is seen as 
enhancement  of  current  TGs.  However,  cognitive  function  endpoints  may  need  to  be  specifically 
developed and decisions on which TG to include them within would have to be made. Research has 
particularly focused on cognitive aging in rodents. (Medium-term) (Project team) 

11. Thyroid-related  developmental  neurotoxicity  –  Cortical  heterotopia  in  offspring  -  in 

vivo 

Three  human  health  groups  suggested  that  more  should  be  done  to  review  cortical  heterotopia. 
Work  published  in  this  field  includes  the  identification  of  cellular  malformations  in  the  corpus 
callosum,  by  Goodman  and  Gilbert  (2007),  which  could  provide  the  basis  of  a  model  of  cortical 
dysplasia/heterotopia.  In  this  paper,  they  report  on  serum  thyroid  hormone  measurements  and 
brain sectioning as well as immunohistochemistry.  

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A  human  health  group  suggested  that  this  analysis  should  be  done  in  the  offspring  of  test 
organisms,  in  particular  rodents;  one  group  went  further  suggesting  brain  histopathology  and  in 
particular oligodendroglioma analysis should be done as an enhancement to current TGs, the most 
relevant  TG  could  be  identified  as  part  of  further  work.  It  was  also  suggested  that  specific 
examination of brain tissue may require special staining. (Medium-term) (Project team) 

12. Thyroid-related  developmental  neurotoxicity  –  Other  thyroid  endpoints  in  vivo  in  F1 

pup brains - in vivo 

Similar  to  the  suggestion  above,  it  was  suggested  that  OECD  TG  443  (Extended  One-Generation 
Reproductive  Toxicity  Study)  (other  TG,  e.g.  OECD  TG  421,  may  also  be  relevant)  could  be 
enhanced  by  evaluation  of  thyroid  hormone  levels  in  rat  pup  brain  tissue  if  effects  on  thyroid 
hormones  (serum  concentrations  of  total  T4,  total  T3,  and  TSH)  are  not  considered  sufficient  to 
evaluate the potential for adverse effects on the developing brain. In addition to this analysis in F1 
pup brains, oligodendrocyte analysis could also be performed. (Long-term) (Project team) 

13. Thyroid-related  developmental  neurotoxicity  -  Mandatory  to  include  DNT  and  DIT 

cohorts in in vivo OECD TG 443 study - in vivo 

A  human  health  group  indicated  that  one  of  their  top  priorities  was  for  more  use  to  be  made  of 
OECD TG 443 in line with the TG text. Specifically, in TG 443 where it says: “Decisions on whether 
to  assess  the  second  generation  and  to  omit  the  developmental  neurotoxicity  cohort  and/or 
developmental immunotoxicity should reflect existing knowledge for the chemical being evaluated, 
as  well  as  the  needs  of  various  regulatory  authorities”.  The  assessment  of  these  DNT  and  DIT 
cohorts is needed because hormones are very important for proper development of the brain and 
immune system.  Presently, under REACH  they  are  triggered only  if  there are  particular concerns, 
whereas  they  could  be  included  by  default  unless  there  is  a  good  justification  to  exclude  them. 
(Short-term) (Attendee) 

14. Thyroid-related  developmental  neurotoxicity  -  Electrophysiological  endpoints  -  in 

vivo 

A  human  health  group  suggested  electrophysiological  endpoints  should  be  developed.  The  group 
mentioned that some basic research (that could be completed in the short-term) would need to be 
undertaken  but  that  the  information  could  be  very  informative.  For  example,  electrophysiological 
techniques can be used to examine actions of compounds on hypothalamic neurons  in vivo. Such 
an approach has been known for some time and it is seen as valuable for studies on magnocellular 
neurons  which  secrete  AVP  or  oxytocin  as  the  cells can  be  readily  identified on  the  basis  of  their 
firing  patterns  and  by  antidromic  stimulation  of  the  nerve  terminals  in  the  neurohypophysis 
(McKenzie et al., 1995). However, such approaches might be challenging and test organisms may 
require surgery prior to testing. (Short-term) (Project team) 

15. Adrenal  /  Steroid  -  Glucocorticoid  Receptor  (GR),  Mineralocorticoid  receptor  (MR) 

transactivation assays - in vitro 

A  human  health  group  (and  an  environment  group)  indicated  that  effects  on  glucocorticoids  and 
mineralocorticoids are not currently included in the OECD CF tests. In vitro glucocorticoid receptor 
bioassays could be developed and these has been suggested  (e.g. Marks et al). As highlighted by 
the  OECD  (2012),  receptors  for  glucocorticoids  and  mineralocorticoids  are  typically  cytoplasmic 
protein complexes that, when occupied, act as ligand-activated transcription factors that migrate to 
the nucleus, where they bind to glucocorticoid response elements and activate specific genes. New 
assays  might  include  a  Level  2  GR  and/or  MR  transactivation  reporter  assays.  (See  also 
environment idea 22) (Short-term) (Attendee) (Project team) 

16. Adrenal / Steroid - Adrenal steroid synthesis: data collection of a range of steroids is 
valuable for mechanistic information (not just adrenal) provided not too many extra 
animals are needed - in vivo 

Today LC-MS/MS methods for measuring simultaneously multiple steroids are becoming more and 
more  available.  By  using  such  methods,  it  is  possible  to  get  information  on  levels  of  multiple 
steroids including precursors of sex steroids and also on corticosteroids using no more serum than 
normally used for obtaining information on a single steroid; i.e. glucocorticoids, mineralocorticoids, 

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progestins,  androgens,  and  estrogens  (Manibusan  and  Touart  2017).  If  it  is  suspected  that 
alterations  to  the  HPG  axis  is  taking  place,  it  is  suggested  that  samples  for  steroid  hormone 
analysis are conducted. (See also environment idea 24) (Short-term) (Attendee) 

17. Female Reproductive Health  - More  and better-defined assays are  required - in vitro 

and in vivo 

Three  out  of  the  four  human  health  groups  suggested  that  there  is  a  lack  of  good  assays  and 
models to identify adverse outcomes for female reproductive health from endocrine disruptors. The 
three  groups  highlighted  that  developing  appropriate  assays  and/or  models  was  a  high  priority. 
Specifically,  one  group  suggested  that  there  was  a  need  for  more/better  defined  endpoints  and 
assays.  Another  group  suggested  that  this  is  a  major  research  deficit,  especially  since  many 
adverse  outcomes  (PCOS, 
fibroids,  endometriosis)  are  not  addressed  by  any  existing 
endpoints/models,  and  many  adverse  outcomes  are  very  human  specific.  Another  human  health 
group  had  similar  discussions  and  suggested  in  vitro  assays  are  needed  for  PCOS  and 
endometriosis,  the  group  suggested  that  gene  expression/transactivation  assays  should  be 
considered. This group also suggested  that a placental  model  should  be  developed  (see idea 18). 
They indicated that this would involve research and it would ideally result in a new test guideline. 

The development of more and better-defined assays may involve a mix of test enhancements and 
new TGs to be developed, both in vitro and in vivo. (Long-term) (Project team) 

18. Female Reproductive Health - Placental model - in vitro and in vivo 

For  the  placental  model,  more  than  one  approach  could  be  considered.  Research  could  be 
undertaken to see if simple in vitro models or more complex in vivo tests could be developed. One 
such  approach,  highlighted  by  Wakx  et  al.  (2014)  and  Rat  et  al.  (2017),  would  involve  human 
placental  cells  being  lined  for  a  set  duration  with  a  known  endocrine  disrupting  substance 
(reference),  a  blank  (control)  and  the  test  substance.  As  part  of  the  test  cell  viability  will  be 
assessed,  one  way  in  which  this  could  be  achieved  is  by  performing  cytofluorometric  neutral  red 
assay  and  P2X7  cell  receptor  activation  using  YO-PRO-1  probe.  The  release  of  the  placental 
hormones;  human  placental  lactogen  (hPl)  and  human  chorionic  gonadotropin  (hCG)  can  be 
determined  by  enzyme-linked  immunosorbent  assay  (ELISA).  The  activation  of  P2X7  and  altering 
of  hPl  and  hCG  levels  could  be  useful  for  screening  studies.  The  examples  suggested  are  in  vitro 
studies, however the placenta ideas developed may also include in vivo tests. (Long-term) (Project 
team) 

19. Female  Reproductive  Health  -  Assays 

for  PCOS/endometriosis  using  gene 

expression/transactivation - in vitro 

As  above,  for  idea  17;  it  was  highlighted  that  there  is  a  lack  of  good  assays  for  PCOS  and 
endometriosis. It was also highlighted that this was a research gap. It was suggested that specific 
in  vitro  assays  should  be  developed  and  that  gene  expression/transactivation  assays  should  be 
considered.  Work  published  in  this  field  has  focused  on  the  use  of  cell  lines  and  cell  cultures, 
plasmids construction, transient transfection, the use of luciferase assay, ChIP assay, the isolation 
of total RNA and real-time RT-PCR, siRNA and immunoblotting (Salama et al. 2009 and Utsunomiya 
et al. 2008). (Long-term) (Project team) 

20. Female Reproductive Health - Progesterone Receptor (PR) transactivation - in vitro 

For human health, this was suggested as being a priority assay for OECD validation, it was also a 
priority  suggested  by  an  environmental  health  group.  Attendees  suggested  that  a  PR 
transactivation assay may require co-factors to be considered, such factors are described by Li and 
O'Malley (2003). 

As highlighted by the OECD (2012), several PR transactivation assays have been developed; these 
assays are based either on yeast or human cell lines and are usually stably transfected with human 
PR-A or PR-B. Antigestagenic activities of polycyclic musks have been demonstrated by Schreurs et 
al.  (2005). Also,  a  recent study compared  a binding assay  and  two reporter gene assays,  with  in 
vivo effects of a variety of chemicals in the McPhail test (Sonneveld et al. 2011). The OECD (2012) 
indicated that findings from this study showed good correlation between PR binding, transactivation 
activity in both reporter gene assays, and the in vivo gestagenic response (Sonneveld et al. 2011).  

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The development of these assays into TG may therefore be achievable in the short term. (See also 
environment idea 15) (Short-term) (Attendees) (Project team) 

21. Female Reproductive Health - Serum oxytocin and Anti-Mullerian Hormone - in vivo 

Oxytocin is a mammalian hormone that also acts as a neurotransmitter in the brain. In addition to 
its well-known peripheral hormonal functions (i.e., induction of labour and milk ejection), oxytocin 
acts  as  an  important  neuronal  messenger  within  the  brain  regulating  social  and  emotional 
behaviours in a wide variety of animal species including humans (Lee  et al. 2009). Oxytocin plays 
an organisational role in the central nervous system and the oxytocin neural system is also thought 
to be involved in the underlying mechanisms that guide the development of social behaviours (e.g. 
maternal behaviour), reproduction and stress responses (Mogi et al. 2014).  

The anti-mullerian hormone has been shown  to be an endocrine  marker of ovarian  gonadotropin-
responsive  follicles  in  cattle  (Rico  et  al.  2008)  and  a  predictive  endocrine  marker  for  embryo 
production  in  goat  (Monniaux  et  al.  2011).  In  the  ovary,  the  anti-mullerian  hormone  has  been 
shown  to  cause  an  inhibitory  effect  on  primordial  follicle  recruitment  as  well  as  on  the 
responsiveness of growing follicles to follicle-stimulating hormone (Visser et al. 2006). A test could 
be based around the anti-mullerian hormone as a marker for ovarian dysfunction.  

Measurements  of oxytocin and anti-mullerian hormone  could be included in OECD  TG  408  (short-
term) and OECD TG 443 (medium-term) studies. (Short- to medium-term) (Attendee) 

However, another attendee indicated that oxytocin is a highly pulsatile hormone for which a single 
measure  can  never  be  obtained  with  reliability.  Although  the  attendee  also  indicated  that  in  a 
different context it may be possible to measure hypothalamic oxytocin mRNA. (Attendee) 

22. Female Reproductive Health - Histopathology of ovaries and follicle count - in vivo 

As TG 408 (90 days study) is on revision the inclusion of sensitive female histopathology would be 
relevant to include. The endpoints are already included in TG 443. (Short-term) (Attendee) 

23. Female  Reproductive  Health  -  Whole  mount  (male  and)  female  mammary  gland 

assessment - in vivo 

It  was  suggested  that  OECD  TG  443  should  include  mandatory  assessment  of  mammary  gland 
development  (whole  mount).  The  mammary  gland  is  a  target  of  endocrine  active  substances  and 
evaluation of this in  some protocols might provide information on developmental abnormalities or 
adverse outcomes not available from other endpoints. Assessment of mammary gland development 
(whole mount) may also provide relevant information concerning the adverse outcome cancer. The 
mammary gland whole mount has been proposed as a powerful method to evaluate organisational 
effects  during  development.  The  whole  mount  mammary  gland  could  be  assessed  in  both  males 
and females, in OECD TG 443, 407 and 408 studies. (Short-term) (Attendee) 

24. Male Reproductive Health - PSA secretion, prostate model and antiandrogenic effects 

- in vitro and in vivo 

After  the  workshop,  an  attendee  highlighted  the  publications  by  Lorenzetti  et  al.  (2010)  and 
Smeriglio  et  al.  (2014).  These  publications  outline  a  tiered  in  vitro  approach  for  the  screening  of 
chemicals  with  a  prostate-mediated  effect  and  intracellular  distribution  and  biological  effects  of 
plant  bioactives  in  a  sex  steroid-sensitive  model  of  human  prostate  adenocarcinoma.  It  is 
suggested  that  the  human  prostate  cell  line  LNCaP  may  be  utilised  for  the  identification  of 
molecules  targeting  prostate  function  by  the  integrated  assessment  of  cell  viability  (MTS  assay) 
and prostate-specific antigen (PSA) secretion as specific marker. Both cell viability and the level of 
PSA  can  be  identified  and  this  can  be  used  to  identify  chemicals  which  are  able  to  interfere  with 
prostate function.  

A  group  suggested  that  parameters  informative  of  antiandrogenic  effects  on  accessory  glands 
should also be assessed when investigating male fertility. Greaves (2007) indicates that drugs with 
anti-androgenic activity,  whether mediated directly or  through the  hypothalamus, produce  similar 
morphological changes in the male accessory sex glands to those produced by castration as part of 
their pharmacodynamic activity. Also, in some cases because of their use in prostate disease they 

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have  been  shown  to  produce  atrophy  of  the  prostatic  tissue  in  humans.  Mukherjee  (et  al.  1992) 
identified  that  flutamide,  a  pure  antiandrogen,  when  administered  to  rats  showed  antiandrogenic 
effects when weights of ventral prostate, seminal vesicles and epididymis were analysed. Research 
by  Gupta  et  al.  (2017)  analysed  the  complete  coding  region  of  the  kallikrein-3  (KLK3)  gene  in 
ethnically matched 875 infertile and 290 fertile men to find if genetic variations in  KLK3 correlate 
with  infertility,  their  research  indicated  that  mutations  in  the  KLK3/PSA  gene  correlate  with  male 
infertility. 

The ideas highlighted in the first paragraph are in vitro studies and the others are enhancements. 
(Long-term) (Attendee) (Project team) 

25. Male Reproductive Health - Sperm function and morphology - in vivo 

A human health group suggested that a priority should be to enhance the current test methods by 
analysing  sperm  function  and  morphology.  Male  fertility  is  not  only  sperm  count  and  morphology 
but also sperm function related to e.g. motility, acrosome reaction, the ability to penetrate the egg. 
Research into the identification of membrane progesterone receptor (mPR) alpha on human sperm 
and  its  association  with  sperm  motility  (Thomas  et  al.  2009)  and  methods  for  measuring  sperm 
quality  and  quantity  in  reproductive  toxicity  tests  (Hala  et  al.  2009)  has  also  been  conducted  by 
various  authors.  Methods  and  technology  for  monitoring  these  functions  exist,  furthermore  tests 
exist for rodents and these could be routinely incorporated into reproductive toxicity studies using 
rodents,  for  example  OECD  TG  488  (Transgenic  Rodent  Somatic  and  Germ  Cell  Gene  Mutation 
Assays) where sperm sampling at 49 days can take place, and a similar analysis can take place in 
fish tests, e.g. OECD TG 229 (Fish Short Term Reproduction Assay). (Short-term) (Project team) 

26. Male Reproductive Health - Cryptorchidism endpoints - in vivo 

A human health group suggested that an enhancement of current TG would be for the analysis of 
cryptorchidism and hypospadias in rodent offspring. It was suggested that those tests with longer 
observational windows would be useful. Potentially the most suitable TG for this analysis would be 
the Extended One-Generation Reproductive Toxicity Study (EOGRTS); however, this endpoint could 
be incorporated in other  in vivo  OECD  CF Level  4 and 5 studies.  These  studies could  also  include 
other  endpoints,  for  example  sperm  analysis  and  histopathological  studies  of  reproductive  tissue, 
as conducted by Dutta et al. (2013). (Short-term) (Project team) 

27. Metabolic dysfunction - PPAR α, β/ɣ, δ transactivation assays - in vitro 

The  peroxisome  proliferator-activated  receptors  (PPARs)  are  a  group  of  nuclear  receptor  proteins 
that function as transcription factors regulating the expression of genes and a range of EDCs have 
been  shown  to  interact  with  the  PPARs.  PPAR  activation  may  be  involved  in  metabolic  effects  of 
EDCs. Two human health groups  (and an  environment group)  suggested that  the development of 
test  methods  for  the  measurement  of  PPAR  transactivation  should  be  a  priority.  Both  groups 
suggested  that  the  PPAR  (α,β/δ,γ)  transactivation  assays  were  important  and  one  of  the  groups 
also  highlighted  the  RXR  transactivation  assay  as  being  an  important  assay.  Comments  made  by 
the  groups,  including  environment  groups,  included  how  these  assays  are  already  under 
development  and  are  not  far  from  becoming  test  guidelines.  Indeed,  there  are  already 
commercially available PPAR assays and these have been presented in peer reviewed literature. 

The OECD (2012) also highlighted that screening assays are available for the rapid assessment of 
PPAR  signal  disruptors,  as  are  apical  endpoints  that  could  be  incorporated  into  currently 
recommended OECD whole animal assays. Among the screening assays, the OECD suggested that 
prioritisation should be given to PPAR transactivation reporter assays. (See also environment ideas 
10 and 11) (Short-term) (Project team) 

28. Metabolic dysfunction - Evaluation of pancreatic beta cells - in vitro 

A  human  health  group  suggested  that  a  priority  should  be  to  develop  a  new  OECD  CF  Level  2  in 
vitro  test  method  that looks  at  pancreatic  beta  cells  (β  cell).  The  reason  for  this  was  that  type 1 
and type 2 diabetes are characterized by progressive β-cell failure. The development of an in vitro 
test may therefore be able to highlight areas of concern. Presently, research in humans has shown 
that  higher  BPA  exposure  in  adults  has  been  associated  with  a  higher  risk  of  developing  type  2 
diabetes (Lang et al. 2008). As part of in vivo rodent studies, it has been identified that the impact 

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of  BPA  on  the  insulin  synthesis  by  pancreatic  β-cells  is  equipotent  to  that  of  estradiol  (Alonso-
Magdalena et al. 2008 and 2011). (Long-term) (Project team) 

29. Metabolic dysfunction - Adipocyte differentiation - in vitro 

Three out of the four human health groups suggested that a new test method should be developed 
to  analyse  adipocyte  differentiation;  this  was  also  a  matter  raised  by  an  environment  group. 
Adipogenesis is the process of differentiation of different cell types into adipocytes, the primary fat 
storage cell type. Induction and inhibition of adipogenesis can be measured indirectly for example 
by quantifying triglyceride accumulation in cells and tissues. It was highlighted that commercial kits 
are available for such analysis (Kanayama et al. (2005)). A human health group suggested that a 
new test method could be developed to distinguish obesity and diabetes. Another group suggested 
that detecting interference  with  adipocyte differentiation as part of an  in vitro  (OECD  CF Level  2) 
test may give an indication of endocrine disruption, however they also noted that other tests may 
be more relevant. 

The OECD  (2012) has  highlighted a potential  adipocyte  differentiation  assay.  It  indicates  that the 
ability  of  chemicals  to  stimulate  adipocyte  differentiation  can  be  evaluated  in  cultured  cells. 
Specifically, preadipocyte cells, such as mouse 3T3-L1 or C3H10T1/2 preadipocyte cells, are ideally 
suited for this assay. In the assay, cultured cells can be treated with the putative RXR-ligand, and 
cells  are  monitored  for  several  indices  of  differentiation  into  adipocytes.  Markers  of  adipocyte 
differentiation,  such  as  induction  of  PPARγ  and  AP2  mRNA  levels  (Lehmann  et  al.  1995),  can  be 
measured  by  real  time  RT-PCR.  However,  the  adipocyte  differentiation  assay  may  not  be  able  to 
differentiate between RXR agonists and PPAR agonists. (Long-term) (Attendee) (Project team) 

30. Metabolic dysfunction - Body fat estimation - in vivo 

Body fat estimation is a procedure for calculating the total percentage body fat by measurement of 
skin-fold  thickness  in  various  parts  of  the  body.  Models  then  exist  for  various  species  to  convert 
these measurements into total percentage body fat; this will mainly be white adipose tissue which 
comprises  the  majority  of  the  total  body  fat.  They  can  be  used  as  a  marker  of  ED  targeting 
metabolism or fat tissue development. Three out of the four human health groups suggested that 
more should be done with the measurement of fat in test organisms. The three groups each made 
comments  about  how  enhancements  could  be  made.  One  group  suggested  that  the  body 
composition/body fat could  become an  estimation,  as presently  weight gain is monitored  already. 
Another group suggested that more information should be recorded about weight gain; specifically 
there should be analysis of individual fat  pads  from different areas, an example being epididymal 
compared to the subcutaneous. The third group suggested that fat pad weight and animal weight 
should be measured. (Short-term) (Attendee) (Project team) 

31. Metabolic dysfunction - Serum gut hormones and adipokines - in vivo 

A human health group suggested that a priority should be to make in vivo measurements of serum 
gut  hormones  and  adipokines  as  part  of  the  current  tests,  suggesting  that  this  could  be  an 
enhancement  to  the  current  tests.  Presently,  commercial  kits  for  the  analysis  of  metabolism  and 
endocrine  biomarkers  are  available,  these  kits  may  be  used  to  profile  gut  hormones  (glucagon, 
glucagon-like  peptide  1  (GLP-1),  leptin,  connecting-peptide  (C-peptide),  gastric  inhibitory 
polypeptide (GIP), insulin, peptide YY (PYY), ghrelin amylin) adipokines (adiponectin, interleukin-1β 
(IL-1β), interleukin 6 (IL-6), monocyte chemoattractant protein 1 (MCP-1), plasminogen activator 
inhibitor 1 (PAI-1), resistin, tumor necrosis factor-α (TNF-α)) and other biomarkers for metabolism, 
muscle  and  bone.  A  human  health  group  suggested  that  assays  could  be  developed  for  use  in 
rodent studies. As the idea is to perform additional analysis as part of  in vivo TG, this will help to 
obtain  more  information  from  the  same  number  of  animals  and  may  help  reduce  animal  testing 
numbers. (Short-term) (Project team) 

32. Metabolic dysfunction - Serum and liver retinoic acid levels - in vivo 

An  attendee  indicated  that  an  OECD  detailed  review  paper  is  currently  being  developed  and  it  is 
expected to provide a relevant  background  for scoping efforts in this area.  It  was suggested that 
assays could be developed for use in rodent studies. As the idea is to perform additional analysis as 
part of in vivo TG, this will help to obtain more information from the same number of animals and 
may help reduce animal testing numbers. (Medium-term) (Attendee) 

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33. Metabolic dysfunction - Evaluation of glucose, serum insulin and insulin tolerance - in 

vivo 

Two  human  health  groups  suggested  that  a  priority  should  be  to  take  serum  insulin  and  other 
related  measurements.  One  of  the  groups  suggested  levels  of  fasting  serum  insulin  should  be 
recorded, the other group suggested that glucose and insulin tolerance should be analysed as part 
of  in  vivo  studies,  this  could  be  an  enhancement  to  the  current  TGs  for  sub-chronic  to  chronic 
repeat-dose  toxicity  studies  which  could  provide  some  information  related  to  highly  ranked 
metabolic  disorders.  Foetal  and  neonatal  (lactational)  exposures  to  known  endocrine  disrupters 
have  been  shown  to  alter  body  weight,  body  composition,  and  glucose  homeostasis  in  rats. 
Although it was suggested that the idea could be an enhancement of the current TGs, a new in vivo 
TG could also be developed. Elevated growth hormone levels have been shown to result in insulin 
resistance, increased blood glucose, and increased lipid metabolism (Perrini et al. 2008). (Short- to 
medium-term) (Project team) 

34. Metabolic dysfunction - Lipoprotein profile - in vivo 

Dyslipidemia relates to an abnormal amount of lipids such as triglycerides or cholesterol (total, HDL 
and  LDL)  in  the  blood.  The  idea  to  enhance  in  vivo  studies,  by  including  measurements  of 
lipoproteins, is already an inclusion in the draft revised OECD TG 408 (Repeated Dose 90-Day Oral 
Toxicity  Study  in  Rodents).  Similar  assessments  could  also  be  included  into  other  longer-term 
studies. (Short-term) (Attendee) 

35. Metabolic dysfunction - Assessment of brown fat in F1 pups - in vivo 

Brown  fat  is  different  to  white  adipose  tissue  both  pathologically  and  anatomically,  it  has  a 
significant metabolic function, having higher concentrations of mitochondria and blood vessels. It is 
also mainly located between the shoulder blades in most mammals as well as being recognisable as 
brownish flecks in the white adipose tissue. 

Thyroid hormone action is involved in the development of brown fat and change in the volume of 
brown  fat  is  potentially  an  outcome  of  developmental  thyroid  hormone  disruption.  The  volume  of 
brown fat can be measured. This measurement could be included e.g. in the extended 1 generation 
test (OECD TG 443). (Short-term) (Attendee) (Project team) 

36. Immune / Autoimmune / Inflammatory - Determination of endocrine MoA to aid test 

development - in vitro and in vivo 

A  human  health  group  suggested  that  there  is  a  major  research  deficit  relating  to  knowledge  of 
endocrine disruption and immune / autoimmune / inflammatory effects as adverse outcomes. The 
group  especially  highlighted  that  the  endocrine  related  MOA  was  an  area  of  deficit  research; 
examples  highlighted  were  cytokine  biomarkers,  metabolomics,  development  of  immune  in  vitro 
tests  and  inflammatory  markers  in  serum.  Although  there  has  been  research  into  this  area 
(Boutzios  and  Kaltsas  2015,  and  Coutinho  and  Chapman  2011),  it  was  suggested  that  further 
research would aid the test development for assessment of immune / autoimmune / inflammatory 
adverse outcomes. (Long-term) (Project team) 

37. Immune / Autoimmune / Inflammatory - Apoptosis in thymus - in vivo 

The rationale behind including histopathology of the thymus is that the thymus is an organ vital to 
proper T cell development, and the regulation of cell survival and death contributes significantly to 
its  efficient  function  (resulting  in  a  strong  immune  response  towards  foreign  antigens  while 
avoiding auto immunity). The involution of the thymus during postnatal maturation is the result of 
changes in the rate of cell proliferation and apoptosis and deviations in the normal pattern/timing 
of involution of the thymus might be an adverse outcome. A human health group suggested that a 
priority  should  be  to  enhance  the  current  test  guidelines  by  investigating  apoptosis  within  the 
thymus.  Normal  H&E  histopathological  assessment  could  be  included  in  all  in  vivo  rodent tests  of 
the CF (CF Levels 3 – 5). (Long-term) (Attendee) 

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38. Hormonal cancer - Serum chromogranin A - in vivo 

A human health group suggested that chromogranin A (CgA) could be a useful serum biomarker to 
be introduced into OECD CF level 4 and 5  in vivo studies as a TG enhancement. CgA is an amino 
acid  protein  that  is  a  marker  of  neuroendocrine  tumours;  however,  because  CgA  has  a  wide 
spectrum,  together  with  other  regulatory  proteins,  there  are  several  causes  of  elevated 
chromogranin  unrelated  to  neuroendocrine  tumour  which  may  result  in  false  positives  (Gut  et  al. 
2016).  A  group  suggested  that  the  use  of  serum  CgA  as  a  biomarker  would  be  useful  as  an 
indicator of neuroendocrine neoplasms, including carcinoids, pheochromocytomas, neuroblastomas, 
medullary  thyroid  carcinomas,  some  pituitary  tumours,  functioning  and  non-functioning  islet  cell 
tumours, and other amine precursor uptake and decarboxylation tumours and carcinoid syndrome.  
(Short-term) (Project team) 

39. General - Epigenetics - in vitro and in vivo 

Epigenetics  involves  the  study  of  changes  to  organisms  by  the  modification  of  gene  expression. 
Research has been conducted and reported by various authors on molecular mediators (including: 
histone  post-translational  modifications,  histone  variants,  nucleosome  positioning,  chromatin 
looping,  DNA  modifications,  DNA  structural  variations  and  miRNA  molecules).  For  the  molecular 
mediators, there are recommended detection methods for their analysis. Strengths and limitations 
of  these  methods  have  been  recognised.  Cell  culture  systems  have  also  been  published  for  the 
testing of epigenetics effects of endocrine disruption. 

As part of the OECD CF Level 1, epigenetics can and have been identified in studies that reported 
both adverse effects and epigenetic changes in response to an environmental exposure. As part of 
in vivo TG epigenetic endocrine and reproductive end points could be incorporated into a number of 
tests, these include: 

Sperm analyses for miRNAs in OECD TG 483 (Mammalian Spermatogonial Chromosomal Aberration 
Test). Mature sperm would be collected and the analysis would include analysis of relevant histone 
modifications,  DNA  methylation  changes  or  expression  of  non-coding  RNAs  in  germ  cell 
suspensions  &  mature  sperm.  Additional  analysis  could  be  conducted  of  the  test  chemical  & 
metabolite exposure in the germ cell suspensions and mature sperm. 

Level 5 in vivo studies reproductive toxicity studies (OECD TG 443 and OECD TG 416). 

Level  4  in  vivo  studies  including  OECD  TG  407  (repeated  dose  28-day  oral  toxicity  study  in 
rodents),  OECD 
the 
reproduction/developmental  toxicity  screening  test),  OECD  TG  421  (reproduction/developmental 
toxicity  screening  test),  OECD  TG  422  (Combined  Repeated  Dose  Toxicity  Study  with  the 
Reproduction/Developmental  Toxicity  Screening  Test)  and  OECD  TG  426  (Developmental 
Neurotoxicity Study) 

study  with 

(combined 

repeated 

toxicity 

dose 

422 

TG 

Level  2  in  vitro  studies  to  which  epigenetic  endpoints  could  be  added  include  OECD  TG  493 
(performance-based  test  guideline  for  human  recombinant  estrogen  receptor  (hrER))  in  vitro 
assays  to  detect  chemicals  with  ER  binding  affinity,  OECD  TG  455  (performance-based  test 
guideline for stably transfected transactivation in vitro assays to detect estrogen receptor agonists 
and  antagonists),  OECD  TG  457  (BG1Luc  estrogen  receptor  transactivation  test  method  for 
identifying estrogen receptor agonists and antagonists) and OECD TG 456 (H295R steroidogenesis 
assay). Although not presently an OECD CF Level 2 test, OECD TG 236 (fish embryo acute toxicity 
(FET) test) could be adapted to use zebrafish embryos. 

However,  one  of  the  major  challenges  when  studying  epigenetic  changes  inferred  by  endocrine 
disrupting  chemicals  is  to  link  these  epigenetic  changes  to  adverse  outcomes.  (This  idea  has  a 
mixed  timeline;  the  studying  of  epigenetics  could  enhance  TG  and  this  could  be  achieved  in  the 
short term, but linking epigenetic changes to adverse outcomes and validating these as part of TG 
would have a longer timeline) (Attendee) (Project team) 

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40. General  -  A  new  level  3  short  term  in  vivo  mechanistic  test  based  on  OECD  407  to 
analyse  EATS  modalities  (hormone  measurements  and  transcriptomic  analysis  of 
organs) - in vivo 

A human health group suggested that a new OECD CF level 3 short term  in vivo mechanistic test 
should be developed. It was indicated that this test could be based on the study design of the sub-
acute repeated dose toxicity study (OECD  TG 407  -  Repeated Dose  28-day Oral Toxicity  Study in 
Rodents)  but  may  not  need  to  be  longer  than  14  days  to  allow  for  the  measurement  of  specific 
hormones. At the same time transcriptomic (micro arrays or next generation sequencing) could be 
conducted  on  a  few  organs  and  this  would  provide  mechanistic  information  for  all  of  the  EATS 
modalities in one go. 

The study protocol may only need modification and validation in terms of hormone measurements 
and  positive  controls  for  the  gene  expression.  Most  of  this  information  may  already  exist  in  the 
public domain. The advantage of such a test would be that it is relatively cheap, quick and would 
provide  in  vivo  mechanistic  information  on  all  endocrine  modalities  in  one  go  (depending  on  the 
tissue). (Short-term) (Attendee) 

41. General - Gene Markers - in vivo 

Several  attendees  suggested  that  there  is  definitely  a  need  for  more  research  into  gene  markers 
(and  biomarkers).  Various  studies  have  been  published  looking  at  gene  and  biomarkers,  these 
include  work  by  various  authors  looking  at  breast  cancer.  For  example,  Annaratone  et  al.  (2014) 
searching  for  neuro-endocrine  markers  in  breast  cancers  as  part  of  an  integrated  approach  using 
immunohistochemistry  and  gene  expression  profiling.  Work  looking  at  biomarker  genes  for 
detecting estrogenic activity of endocrine disruptors has been conducted by Jung et al. (2012) and 
research  by  Droździk  et  al.  (2015)  has  suggested  that  variants  in  the  TGFB3  gene  may  be 
associated with male infertility, however further research into this and other human health adverse 
outcomes is required. 

In  terms  of  the  environment,  work  by  Hutchinson  et  al.  (2006)  on  screening  and  testing  for 
endocrine  disruption  in  fish  —  biomarkers  as  “signposts,”  not  “traffic  lights,”  in  risk  assessment. 
Schiller  et  al.  (2013)  studied  the  effects  of  genistein  on  gene  expression  of  fish  embryos  as  an 
alternative  testing  approach  for  endocrine  disruption.  While  work  by  Muth-Köhne  et  al.  (2016) 
looked  at  linking  the  response  of  endocrine  regulated  genes  to  adverse  effects  on  sexual 
differentiation improves comprehension of aromatase inhibition in a fish sexual development test. 

Similar  to  epigenetics,  the  analysis  of  gene  and/or  biomarkers  as  part  of  a  current  TG  may  help 
enhance  the understanding of  specific genes for their  potential use.  However,  further studies  and 
research  would  be  needed  to  examine  the  accumulated,  synergistic,  and  additive  effects  of  EDs 
using biomarkers. (Long-term/research) (Project team) 

42. General  -  Combined  long-term  bioassay  and  reproductive  study  with  ED  biomarker 

enhancements - in vivo 

In  her  plenary  presentation,  Fiorella  Belpoggi  presented  the  Ramazzini  Institute's  work  and 
suggestion  for  an  integrated  experimental  design  for  the  assessment  of  multiple  toxicological 
endpoints  in  rat  bioassays.  The  suggested  approach  is  to  investigate  carcinogenicity,  toxicity  and 
reproductive/developmental  toxicity  endpoints  in  a  single  protocol.  The  animals  will  all  be  of  the 
same  generation  and  the  protocol  explores  windows  of  susceptibility  that  are  currently  not 
addressed 
includes  a  prolonged  period  of 
exposure/observation  of  experimental  animals,  starting  exposures  from  the  gestation  and 
continuing through lactation and weaning until at least 130 weeks or longer. 

the  other  guidelines.  The  protocol 

in 

toxicity/carcinogenicity  design 

is  based  on  OECD  TG  453 

The 
(combined  chronic 
toxicity/carcinogenicity  studies),  modified  only  for  the  earlier  start  and  longer  duration.  By 
beginning exposure in utero, especially for endocrine-disrupting chemicals that “act” preferentially 
in  early  life,  the  protocol aims  to  provide more  reliable  and  appropriate  indicators of  human  risk. 
Interim  kills  are  planned;  this  follows  OECD  TG  453  and  aims  to  provide  information  on 
progression/regression  of  non-neoplastic  events  and  neoplastic  changes  and  mechanistic 
information. 

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The  reproductive/developmental  toxicity  part  of  the  study  mimics  human  exposure  during  critical 
windows of susceptibility (WOS) for the developmental effects and other non-cancer effects. These 
adverse  effects  are  studied  in  the  rodent  prenatal,  neonatal,  prepubertal,  pubertal,  adult  parous 
and  adult  nulliparous  WOS.  These  effects  are  compared  between  each  WOS,  or  with  the  possible 
long-term  carcinogenicity  effect.  Also,  animals  are  bred  to  obtain  toxicity  information  throughout 
the reproductive cycle. 

It  was  suggested  that  the  design  has  the  ability  to  reduce  the  total  number  of  animals  required 
when compared to conducting each of the separate test protocols. 

However, it was suggested that the testing protocol is labour intensive, that there is no regulatory 
need  for  the  study  and  it  would  be  too  expensive  and  too  complicated  to  run.  Additionally,  new 
cancer studies under the pesticides and biocides regulations are already combined with the chronic 
toxicity study (OECD TG 451 and OECD TG 452 are combined and the requirement is to submit an 
OECD TG 453 (combined chronic toxicity/carcinogenicity studies)). It was also suggested that the 
dose setting rationale for a cancer and a reproductive toxicity study is different, thus, there will be 
a  need  to  have  separate  tiers  for  cancer  and  reproductive  toxicity  within  the  study.  (Long-term) 
(Attendee) (Project team) 

43. General - Delete the current OECD TG 416 2-generation study from the CF - in vivo 

It  was  suggested  that  OECD  TG  416  (Two-Generation  Reproduction  Toxicity)  should  be  deleted 
from  the  OECD  CF  as  the  OECD  TG  443  (Extended  One-Generation  Reproductive  Toxicity  Study) 
has been adopted. The background  for this is that  OECD  TG  443 compared with OECD  TG 416 is 
preferable  because  of  its  higher  sensitivity  due  to  the  inclusion  of  more  pups  for  analysis  and  its 
broader  scope  regarding  developmental  immuno-  (DIT)  and  neurotoxicity  (DNT)  and  endocrine 
disruption.  However,  despite  this  suggestion  being  raised  by  a  number  of  attendees,  it  was 
highlighted that OECD TG 416 is currently used as a standard information requirement in the PPPR, 
BPR  and  other  regulatory  frameworks  around  the  world  and  it  should  therefore  not  be  deleted. 
(Short-term) (Attendee) 

A2.3  Environmental Health ideas 

1.  Thyroid - Thyroid pathway screens (Deiodinase expression) - in vitro/ex vivo 

Both of the  environment  groups  (and three out of  the four  human  health groups) suggested that 
developing more thyroid screening assays should be a priority. Specific assays were highlighted at 
the workshop and most are described in the new scoping document on in vitro and ex vivo assays 
for the identification of modulators of thyroid hormone signalling, OECD no. 207:  

types  of 
Deiodinase  expression 
deiodinase/deiodination  assays;  radioactive  based  assays  monitoring  iodine  release  from  labelled 
T4,  HPLC  assays  based  on  competitive  inhibition  with  iodoacetate  measuring  T3  and  rT3, 
colorimetric assays measuring free iodine based on the Sandell-Kolthoff reaction. 

in  vitro/ex  vivo  assays.  There  are  currently 

three 

These assays all require further research. (Short-term ideas) (See also human health idea 1) 

2.  Growth and Development - Thyroid pathway screens (TBG binding assay, TTR serum 
binding assay, T4 binding protein displacement assay, TR reporter assay) -  in vitro 

TBG  binding  assays.  There  are  a  variety  of  assays  that  are  highlighted  in  OECD  207  and 
information on suggested assays has already been published. TBG binding assays are indicated as 
being assays that might be ready for validation in the near future. In addition to being relevant for 
the  environment,  TBG  has  the  highest  affinity  and  carries  the  majority  of  T4  in  blood  in  humans 
and it is the primary binding protein in humans. 

The  transthyretin  (TTR)  serum  binding  assays  were  highlighted  by  two  out  of  the  three  human 
health groups. Within OECD 207 two such TTR assays are described, these are the Combined TBG 
and  Transthyretin  TTR  Binding  Assays  and  the  TransThyretin  (TTR)  Binding  Assays.  The  TTR 
binding assays are indicated as almost being ready for validation in the short term. 

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T4  binding  protein  displacement  assay.  Several  of  these  methods  for  T4  displacement  from  TTR 
have been published; 1) radioligand binding assay: Somack et al. 1982, Brouwer and van den Berg 
1986, Lans et al. 1993, 1994, Hamers et al. 2006, Ucan-Marin et al. 2010. 2) plasmon resonance-
based biosensor: Marchesini et al. 2006, 2008. 3) Fluorescence displacement: Cao et al. 2010, Cao 
et  al.  2011  and  Ren,  Guo  2012,  Montano  et  al.  2012.  The  work  needed  for  validation  of  such 
assays include a survey of the most applicable and stable assay(s) and a validation of the selected 
assay(s). 

TR reporter assay. There are a variety of TR reporter assays, the Transactivation Reporter Assays 
with  TRα  and  TRβ  have  been  highlighted  as  being  highly  sensitive.  As  commercial  TR  reporter 
assays already exist the development and validation of  an assay might be achievable in the short 
term. The assay would help with the  identification and quantification of thyroid  hormone receptor 
disrupting potency of compounds.  

The assays highlighted above are the ones discussed at the workshop, however, within  OECD  no. 
207  additional  thyroid  assays  and  their  readiness  levels  are  highlighted.  (Short-term  ideas)  (See 
also human health 2) 

3.  Thyroid  pathway  screens:  Thyroid  peroxidase  assay,  Sodium  Iodide  Symporter  -  in 

vitro and in vivo 

The thyroid peroxidase assay, commonly referred to as TPO. TPO assays are described in the new 
scoping  document  on  in  vitro  and  ex  vivo  assays  for  the  identification  of  modulators  of  thyroid 
hormone  signalling,  OECD  no.  207.  There  are  commercially  available  TPO  assays  and  some  of 
these developed TPO assays may be ready for  validation in the short term. However, in vivo TPO 
assays  may  also  be  developed.  Findings  by  Hornung  et  al.  (2015)  indicate  that  inhibition  of  TPO 
activity in vitro was a good indicator of a chemical's potential for thyroid hormone disruption in vivo 
and may be useful for prioritizing chemicals for further investigation. 

The Sodium Iodide Symporter Activity Assay is commonly known as the NIS activity assay. Three 
such  assays  are  described  in  the  new  scoping  document  on  in  vitro  and  ex  vivo  assays  for  the 
identification of modulators of thyroid hormone signalling, OECD no. 207: 

Radioactive  Sodium/Iodide  Uptake  assay  using  transiently  transfected  FRTL-5  cells,  this  assay  is 
currently at the research stage and it could potentially be used to assess mechanisms of disruption 
that  involve  NIS  symporter,  but  it  should  be  specifically  designed  and  improved  for  endocrine 
disruptors. 

Radioactive  Sodium/Iodide  Uptake  Assay  using  hNIS  stably  transfected  in  FRTL5  cells  and  in 
HEK293 cells, there is a need to decide which stably transfected cell type should be taken forward 
for  validation;  either  FRTL5  cells  or  HEK293  and  then  whether  to  test  with  the  radio-isotope 
method or the colorimetric method. 

Non-Radioactive Sodium/Iodide Uptake Assay based on Sandell-Kolthoff reaction and using FRTL5 
cells, this idea is still at the research stage. 

The NIS assays are at different stages of development. (Short-term ideas) (Project team) (See also 
human health 3) 

4.  Growth and Development - Sodium-dependent serotonin transporter activity - in vitro 

and in vivo 

Generally,  neurotransmitters/neurohormones  as  serotonin  and  dopamin  are  very  important  in 
relation  to  the  endocrine  function  of  invertebrates,  for  example  in  molluscs.  The  receptors  are 
conserved  across  taxa  so  effects  on  these  systems  would  be  relevant  for  both  vertebrate  and 
invertebrate  taxa.  The  work  needed  includes  literature  survey  to  gather  existing  information, 
research on appropriate endpoints and validation of the selected endpoints. An OECD CF Level 3 in 
vivo  assay  involving  the  assay  for  the  transporter  protein  could  be  developed;  this  could  also 
incorporate  other  routine  biomarkers  such  as  cytoplasmic  electrolytes.  However,  it  may  also  be 
possible  that  an  in  vitro  epigenetic  assay  for  the  gene  that  encodes  the  serotonin  transporter  be 
developed. The idea is to develop a new in vitro and/or in vivo TG (however, elements of the work 
might also be used to enhance current TG). (Long-term) (Attendee) 

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5.  Growth and Development - Ecdysis triggering hormone levels - in vivo 

The  ecdysis  triggering  hormone  (ETH)  system  is  essential  for  successful  moulting  of  insects. 
Various  authors  have  studied  ecdysis  triggering  hormone  signalling  in  insects  (Roller  et  al.  2010, 
Lenaerts et al. 2017) and in D. magna (Sumiya et al. 2014, 2016). The interruption of this ETH or 
silencing of genes has the potential to disrupt and cause lethality at the expected time of ecdysis. 
Substances  which  are  known  to  target  ecdysone  receptor  have  the  potential  to  interrupt  this 
process. In some situations, such as targeted pest control, this property might be desirable. An in 
vivo test could be created to identify substance effects on the ETH system. As indicated by Song et 
al. 2017 based on a review, multiple targets of EDCs in the moulting processes were identified and 
the link between mode of action (MoA) and adverse effects characterized to inform future studies. 
An  adverse  outcome  pathway  (AOP)  describing  ecdysone  receptor  agonism  leading  to  incomplete 
ecdysis  associated  mortality  was  developed  according  to  the  OECD  guideline  and  subjected  to 
weight of evidence considerations by modified Bradford Hill Criteria. This review proposes the first 
invertebrate  ED  AOP  and  may  serve  as  a  knowledge  foundation  for  future  environmental  studies 
and  AOP  development.  The  idea  is  to  develop  a  new  in  vivo  TG  (however,  not  covered  by  the 
Directive 2010/63). (Long-term) (Attendee) 

6.  Growth and Development - Ecdysterone levels (20-EH) in arthropods - in vivo 

Similar  to  ecdysis  triggering  hormone  (ETH)  (see  environment  idea  5,  above),  ecdysterone  and 
juvenile hormone are  also responsible for  the  moulting of  arthropods.  OECD  test  guidelines could 
be developed for in vivo systems detecting ecdysone and juvenile hormone. The idea is to develop 
a new in vivo TG (however, not covered by the Directive 2010/63). (Long-term) (Attendee) 

7.  Growth and Development - Using transgenic embryos for screening: XETA / EASZY - 

in vivo 

Some assays are already in the OECD work programme i.e. the XETA (Xenopus Embryonic Thyroid-
signalling  Assay),  the  EASZY  (Endocrine  Active  Substances,  acting  through  estrogen  receptors 
using  transgenic  cyp  19a1b-GFP  Zebrafish  Embryos)  along  with  others.  The  finalization  of  these 
and  other  assays  will occur  within  the  next  1-2  years  if  the  validation  is performing  as  expected. 
Other  new  transgenic  assays  could  be  relevant  for  other  MoA  than  thyroid  and  estrogen,  such  as 
glucocorticoid  pathways.  The  time  line  for  such  developments  will  be  expected  to  be  4-8  years 
estimated from the time line for the XETA and EASZY assay developments. (See also human health 
idea 4) (Short-term) (Attendee) 

8.  Growth and Development - Include T4 in the LAGDA study (perhaps also T3 and TSH) 

- in vivo 

Methods for T4  quantification  in blood and plasma are  well known and for many years ELISA kits 
have  been  the  preferred  method  and  several  commercial  kits  are  available.  Within  recent  years, 
LC-MS/MS methods using ultrafiltration as a simple sample preparation have been developed, e.g. 
Wang  and  Stapleton,  2010;  Jarvis  &  McClure,  2014;  Saba  et  al.,  2014.  The  work  needed  for 
validation  of  such  assays  includes  a  survey  on  the  most  applicable  and  stable  assay(s)  and  a 
validation of the selected assay(s). Other thyroid hormones like T3 and TSH may also be relevant. 
Time frame 2-3 years. (Short-term) (Attendee) 

9.  Growth and Development - Thyroid testing in fish - in vivo 

In addition to thyroid screening tests an environment group felt that a priority would be to enhance 
the  current  fish  tests  by  adding  thyroid  endpoints,  for  example  OECD  TG  229  (Fish  Short  Term 
Reproduction Assay), TG 234 (Fish Sexual Development Test) and TG 240 (Medaka Extended One 
Generation  Reproduction  Test  (MEOGRT)).  The  Zebrafish  Eleutheroembryo  Thyroid  Assay 
(Thienpont  et  al.  2011;  Raldua  et  al.  2012)  is  also  being  developed  and  highlighted  by  OECD 
(2014).  This  assay  involves  the  measuring  of  T4  concentration  in  thyroid  follicles  of  5  days  post-
fertilization  zebrafish  following  3  days  of  exposure  to  a  water-borne  chemical.  Post  fertilization 
zebrafish  are  also  subjected  to  whole-mount  immunofluorescence  analysis.  The  assay  has  the 
potential  for  identifying  water  soluble  chemicals  that  directly  affect  the  thyroid  function,  and  the 
assay may be generally applicable to chemicals that have indirect function. 

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Other endpoints that may also be included in fish tests are eye development in zebrafish (Baumann 
et  al.  2016)  and  swim  bladder  inflation  (Stinckens  et  al.  2016)  or  histopathology.  The  research 
could  be  used  to  enhance  the  current  TG  or  to  develop  new  OECD  level  3  –  5  in  vivo  tests. 
(Medium-term) (Attendee) (Project team)  

10. Growth  and  Development  -  Peroxisome  proliferator-activated  receptors  (PPARs) 

assay (for use with several taxa) - in vitro 

An  environment  group  suggested  that  this  was  a  priority  for  OECD  validation,  this  was  also  a 
priority suggested by two human health groups. 

An attendee indicated that the activation of RXR–PPAR heterodimers has been shown to be effected 
by  endocrine  disruptors  in  the  environment  (Le  Maire  et  al.  2009).  Presently,  in  vitro 
transactivation assays for PPARs are already commercially available and several assays have been 
presented in  the  peer  reviewed  literature  (e.g.  Maloney  &  Waxman.,  1999;  Harada  et  al.,  2015). 
The  work  needed  for  these  new  OECD  CF  level  2  test  guidelines  would  be  validation  of  selected 
assays at OECD level. (See also human health idea 27) (Short-term) (Attendee) 

11. Growth  and  Development  -  Retinoid  X  Receptor  (RXR)  and  Retinoic  Acid  Receptor 

(RAR) transactivation assays (for use with several taxa) - in vitro 

For the environment, both the RXR and RAR assays were considered as being a particular priority, 
this was because of the link to a mode of action for the effects of TBT in molluscs. 

As indicated by OECD (2012): 

RXR  transactivation  reporter  assays  have  been  used  to  assess  both  agonistic  and  antagonistic 
activity  of  putative  RXR  ligands  (Li  et  al.  (2008);  Goldstein  et  al.  (2003);  Schoff  et  al.  (2004)). 
Commercial  kits  are  available  that  can  be  used  to  screen  chemicals  for  agonist  or  antagonist 
activity in humans and the environment. 

RAR  transactivation  reporter  assays  have  been  used  for  two  decades  to  evaluate  retinoid-like 
activity of chemicals (Bernard et al. 1992). More recently developed RAR assays use reporter genes 
that code for fluorescent proteins whereas the older ones used  chloramphenicol acetyltransferase. 
Presently there are RAR assays that are commercially available. 

As assays already exist, it was suggested that a priority was for these to undergo OECD validation. 
This was also a priority suggested by two human health groups. In vitro retinoid signalling system 
the  US  EPA  ToxCast  Programme 
assays  have  also  been  developed  as  part  of 
(https://cfpub.epa.gov/si/si_public_record_report.cfm?dirEntryId=336407).  Specific  assays 
for 
human health and wildlife species exist and the RXR receptor is cloned for more than one species 
of  mollusc.  Both  the  RAR  and  RXR  reporter  assays could  serve  as  a  screening  assay  to  discern  a 
potential  anchoring  molecular  event  that  would  trigger  assessment  along  the  relevant  adverse 
outcome pathway. (See also human health idea 27) (Short-term) (Project team) 

12. Growth and Development - Receptor assays other than PPAR, RXR and RAR - in vitro 

One  of  the  environment  groups  suggested  that  the  consideration  of  in  vitro  assays  other  than 
PPAR,  RXR  and  RAR  was  also  important.  It  was  suggested  that  there  may  be  other  assays  that 
would be important to develop. Such assays may include, but are not limited to: 

  Constitutive Androstane Receptor (CAR) 

 

 

 

Pregnane X Receptor (PXR) 

Farnesoid X Receptor (FXR) 

Liver X Receptor (LXR) 

  Vitamin D Receptor (VDR) 

As  highlighted  by  OECD  (2012),  more  is  known  about  some  of  the  potential  assays  than  others. 
Some PCBs are CAR activators and have been shown, in several different studies involving various 
vertebrate  species,  to  decrease  T4/T3  and  increase  thyroid  hypertrophy  and  TSH  (Hagmar  et  al. 
2001, Koopman-Esseboom et al. 1994, Dean et al. 2002, Gocmen et al. 1989 and Richardson et al. 

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2008). Stronger responses have been observed in females (Hagmar et al. 2001), which reportedly 
have greater CAR levels and activity in both humans and rodents (Lamba et al. 2003, Hernandez et 
al.  2009,  Ledda-Columbano  et  al.  2003  and  Petrick  et  al.  2007).  Bile  acid  receptors  such  as  FXR 
and  PXR  can  be  activated  by  lithocholic  acid,  but  VDR  is  activated  at  much  lower  concentrations. 
While  vitamin  D  is  ubiquitous  among  organisms,  VDR  is  only  found  in  vertebrates  (Owen  and 
Zelent  2000).  It  appears  that  there  may  be  limited  use  of  the  VDR  reporter  assay  to  evaluate 
responsiveness to potential EDCs. The high fidelity of the VDR may severely limit the promiscuity 
of the receptor. Further research into these other assays may lead to the development of new tests 
guidelines. (Medium-term) (Project team) 

13. Growth and Development - Daphnia juvenile hormone and ecdysone agonist - in vitro 

As  noted  in  the  main  report,  the  ecdysone  and  juvenile  hormone  receptors  have  been  cloned  for 
Daphnia magna. This has been a long process and it started with the genome project 12 years ago 
(Watanabe et al. (2005)). Iguchi et al. (2006) and other authors have written about the application 
of  ecotoxicogenomics  for  studying  endocrine  disruption  in  vertebrates  and  invertebrates. 
Tatarazako  et  al.  (2003)  and  others  have  shown  that  exposure  of  adult  daphnids  to  juvenile 
hormones and their analogues induces parthenogenetically reproducing D. magna to produce male 
neonates.  This  effect  of  juvenile  hormone  agonists  is  responsible  for  affecting  the  chemical 
signalling  responsible  for  inducing  the  production  of  male  offspring.  The  two-hybrid  system  and 
reporter  gene  assays,  such  as  those  being  developed  by  Miyakawa  et  al.  (2013)  using  juvenile 
hormone receptor and Kato et al. (2007) using ecdysone receptor, may represent a useful reporter 
system  for  further  examination  of  hormonal  and  chemical  effects  on  D.  magna  at  the  molecular 
level. (Medium-term) (Attendee) 

14. Reproductive  Health  /  Growth  and  Development  -  MEOGRT  -  Performance  and 

possibly refinement - in vivo 

A  group  suggested  that  what  was  needed  was  enhanced  usability  of  this  method  to  improve  the 
delivery  of  successful  studies.  The  group  suggested  that  demonstration  studies  (US-EPA)  would 
help  and  be  a  scaffold  for  targeted  mechanistic  investigations.  Increased  experience,  better 
validation and including as many endpoints as possible would be recommended as this is perhaps 
the main level 5 test for the environment assessment. (Short-term) (Attendee) (Project team) 

15. Reproductive Health - Progesterone receptor (PR) transactivation - in vitro 

The test method was seen  as a  priority for OECD  validation by  an environmental  health  group. It 
was a priority suggested by a human health group. Attendees suggested that a PR transactivation 
assay  may  require  co-factors  to  be  considered,  such  factors  are  described  by  Li  and  O'Malley 
(2003). 

As highlighted by the OECD (2012), several PR transactivation assays have been developed; these 
assays  are  based  either  on  yeast  or  human  cell  lines  which  are  usually  stably  transfected  with 
human  PR-A  or  PR-B.  Antigestagenic  activities  of  polycyclic  musks  have  been  demonstrated  by 
Schreurs  et  al.  (2005).  Also  a  recent  study  compared  a  binding  assay  and  two  reporter  gene 
assays,  with  in  vivo  effects  of  a  variety  of  chemicals  in  the  McPhail  test  (Sonneveld  et  al.  2011). 
The  OECD  (2012)  indicated  that  findings  from  this  study  showed  good  correlation  between  PR 
binding, transactivation activity in both reporter gene assays, and the  in vivo gestagenic response 
(Sonneveld et al. 2011).  

The development of these assays into TG may therefore be achievable in the short term. (See also 
human health idea 20) (Short-term) (Attendees) (Project team)  

16. Reproductive Health - Prostaglandin synthesis inhibitors - in vitro 

Kristensen  et  al.  (2011)  indicate  that  prostaglandins  play  key  roles  in  development  and 
maintenance of homeostasis of the adult body and known EDCs inhibit the prostaglandin pathway 
in a mouse Sertoli cell  line,  human primary  mast  cells  and reduced synthesis  in  ex  vivo  rat testis 
leading  to  reduced  testosterone  production.  Commercial  assays  are  available  for  measuring 
prostaglandin  E  metabolites  (PGEM)  using  ELISA  (Patel  et  al.  2016).  These  have  been  used  to 
measure PGEM levels in fish gills although the results showed high inter-individual variability (Patel 
et  al.  2016).  Similar  assays  are  available  for  prostaglandin  D2  (PGD2)  levels  which  have  been 

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measured  in  mouse  sertoli  cells  (Kugathas  et  al.  2016).  Further  work  is  required  to  evaluate  the 
most reliable endpoints and also to establish the extent of endocrine-disrupting effects that might 
arise  from  simultaneous  interference  with  PGD2  signalling  and  androgen  action.  The  knowledge 
from  these  and  other  in  vivo  studies  may  aid  the  development  of  in  vitro  tests.  (Medium-term) 
(Project team) 

17. Reproductive Health - Spawning assay in echinoderms - in vivo 

Test  guidelines  already  exist  for  sea  urchin  fertilisation  (EPA  1008.0  (2002)  or  embryo-larvae 
development  assays  (ASTM  E1563  -  98(2012))  which  could  be  investigated  for  the  potential  to 
include  ED  related  effects.  If  considered  ED  relevant,  an  adaption  of  the  test  to  cover  ED  effects 
with validation should be initiated. Time frame 2-6 years. The idea is to develop a new in vivo TG 
(however not covered by the Directive 2010/63). (Medium-term) (Attendee) (Project team) 

18. Reproductive Health - Combining OECD TG 229 and OECD TG 234 - in vivo 

OECD TG 229 is a 21-day fish assay (CF level 3) validated for fathead minnow, Japanese medaka 
and  zebrafish.  The  assay  covers  reproduction  by  measuring  fecundity  during  21  days  of  aquatic 
exposure. OECD TG 234 is a 60-day fish assay (CF level 4) validated for Japanese medaka, three 
spined  stickleback  and  zebrafish  (fathead  minnow  is  partly  validated  but  lacks  androgen  agonist 
validation).  The  assay  covers  the  period  of  development  from  fertilization  through  sexual 
development and differentiation including eventually maternal transfer. A combination of OECD  TG 
229 and OECD TG 234 would create a partial life cycle test of approximately 12 weeks that covers 
the  most  sensitive  life  stages  of  fish;  reproduction  and  development.  The  combined  test  could 
potentially reduce the total number of test animals and the costs of fish testing because currently, 
a  positive  OECD  TG  229  very  often  triggers  further  ED-related  fish  testing  (e.g.  OECD  TG  234, 
OECD  TG  240  or  OCSPP  850.1500  (Fish  Life  Cycle  Toxicity)).  The  work  needed  for  this  test 
enhancement  is  a  literature  survey  on  existing  data  from  OECD  TG  229  and  OECD  TG  234,  a 
comparison  of  endpoints  including  statistical  power  analysis,  development  of  an  OECD  SPSF 
(Standard  Project  Submission  Form)  and  finally  validation  of  the  combined  test  with  the  fish 
species of interest. Estimated time frame 3-4 years. (Short-term) (Attendee) 

19. Reproductive Health - Behavioural assessments (frogs) - in vivo 

Research activity with a time frame longer than 5 years. A possible intermediate activity could be 
to investigate if it would be possible to video-track behaviour (perhaps calling or swimming) in the 
existing amphibian tests (OECD TG 231 and OECD TG 241). Tests based on the effects of EDs on 
the  calling  behaviour  of  male  frogs  (X.  laevis)  are  discussed  by  Behrends  et  al.  2010,  and 
Hoffmann  and  Kloas  2010.  However,  although  a  good  idea,  there  is  a  suggestion  that  the  frogs 
cannot be used again and therefore this might mean that this endpoint could only be developed as 
an additional expensive standalone test  rather  than as an add on to an existing test.  Behavioural 
assessments  could  be  applied  to  any  in  vivo  exposure  involving  mammals,  fish,  or  amphibians. 
(Long-term) (Attendee) 

20. Behaviour / Reproductive Health - Fish behavioural assay (embryo) - in vivo 

At  the  workshop,  behavioural  assays  were  highlighted  as  being  an  important  gap.  The  gap  was 
highlighted as being significantly important to animal reproduction. A hypothetical high throughput 
fish  larvae  behavioural  assay  was  suggested  as  being  an  assay  that  could  be  developed.  If  there 
were a link to the thyroid then the assay could potentially be used to demonstrate adversity (would 
need to prove the behaviour is affected by the plausible mechanism). An example of an assay that 
could  potentially  be  developed  includes  a  predator  avoidance  test  with  larval  fathead  minnows 
(Painter et al. 2010). (Long-term) (Project team)  

21. Behaviour  /  Reproductive  Health  -  Fish  behavioural  assay  (mating  behaviour)  -  in 

vivo 

As indicated above, reproductive  behavioural  assays  were indicated  as being an  important  gap at 
the  workshop.  A  gap  exists  for  mating  behaviour  and  it  was  suggested  that  an  assay  could  be 
developed based on mating or courtship behaviour.  Studies in male sticklebacks have shown that 
both  estrogens  and  antiandrogens  can  interfere  with  reproductive  behavioural  patterns, 
differentially affecting aggressive behaviour towards male conspecifics and courtship behaviour, as 

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well as nest building (Sebire et al. 2008, and Wibe 2002). Garcia-Reyero et al. (2011) describe an 
assay  based  on  nest  guarding  behaviour  by  male  fathead  minnows.  It  was  suggested  that 
behavioural assessments could be incorporated into  existing fish  reproduction  TGs  and that  these 
may  also  be  applicable  to  other  in  vivo  studies  involving  mammals  and  amphibians.  (Long-term) 
(Project team) 

22. Adrenal  /  Steroid  /  Mechanistic  information  -  Glucocorticoid  Receptor  (GR), 

Mineralocorticoid receptor (MR) transactivation assays - in vitro 

An  environment  group  indicated  that  effects  on  glucocorticoids  and  mineralocorticoids  are  not 
currently  included  in  the  OECD  CF  tests.  In  vitro  glucocorticoid  receptor  bioassays  could  be 
developed  and  these  has been  suggested  (e.g.  Marks  et  al).  As  highlighted  by  the  OECD  (2012), 
receptors  for  glucocorticoids  and  mineralocorticoids  are  typically  cytoplasmic  protein  complexes 
that,  when  occupied,  act  as  ligand-activated  transcription  factors  that  migrate  to  the  nucleus, 
where they bind to glucocorticoid response elements and activate specific genes. New assays might 
include a Level 2 GR  and/or MR transactivation reporter assays. (See also human health idea 15) 
(Short-term) (Attendee) (Project team) 

23. Adrenal / steroid - Adrenal steroid synthesis - in vitro 

In vitro adrenal steroid synthesis screening assays relevant for wildlife species are required. Assays 
already exist and have  been reviewed by Manibusan  and  Touart,  2017.  For other species a novel 
assay could be developed or it could be based on existing assays based on human adenocarcinoma 
(H295R)  cells  for  rapid  in  vitro  determination  of  effects  on  steroidogenesis  (Hecker  et  al.  2006, 
OECD  TG  456:  H295R  Steroidogenesis  Assay)  but  modified  for  use  with  other  taxa,  such  as  fish. 
OECD  TG  456  suggests  that  there  are  species-dependent  differences  in  response  to  substances 
that  alter  steroidogenesis  in  vivo  for  humans  and  rats.  Fish  are  therefore  likely  to  have  a  further 
different response and therefore it is seen as useful to develop taxa specific assays. (Medium-term) 
(Attendee) 

24. Adrenal / steroid - Adrenal steroid synthesis: data collection of a range of steroids is 
valuable for mechanistic information (not just adrenal) provided not too many extra 
animals are needed - in vivo 

Hormone  level  measurements  in  OECD  CF  level  3-5  fish  and  amphibian  test  guidelines  are  not 
required.  Data  on  specific  steroid  hormone  levels  (including  adrenal/interrenal)  would  be 
informative in relation to the mechanism(s) of action of test chemicals and could be a valuable part 
of the WoE. If it is suspected that alterations to the HPA/HPI axis are taking place, it is suggested 
that  samples  could  be  taken  for  steroid  hormone  analysis  (for  example,  possible  fish  test  include 
OECD  TG  229,  OECD  TG  230  and  OECD  TG  240,  other  tests  could  include  amphibians  and 
mammals). A variety of methods for quantification of steroids in various tissues of mammals, fish 
and  amphibians  are  available  in  the  peer  reviewed  literature:  e.g.  in  fish  plasma  (Bussy  et  al. 
2017),  rat  tissue  (Maeda  et  al.,  2013),  brain  of  various  species  (Taves  et  al.  2011).  A  challenge 
could  be  the  available  amount  of  plasma  or  tissue  in  small  test  species  such  as  zebrafish  and 
Japanese  medaka,  but  new  highly  sensitive  chromatographic  methods  are  diminishing  this 
challenge.  Another  challenge  (for  both  the  environment  and  human  health)  would  be  stress 
response  (during  sampling  process)  that  may  interfere  with  the  plasma  hormone  levels  (e.g. 
cortisol).  Standardising  the  sampling  method  would  be  necessary  for  some  hormones.  The  work 
needed  for  this  general  test  enhancement  depends  on  the  specific  test  and  hormone,  but  would 
include  validation  of  methods  and  characterisation  of  control  hormone  level  variation.  Estimated 
time frame 2-4 years. (See also human health idea 16) (Short-term) (Attendee) 

25. General - Biomarkers for immunity - in vivo 

An  environment  group  indicated  that  an  area  of  interest  was  biomarkers  for  immunity.  It  was 
suggested  by  the  group  that  there  are  data  indicating  that  immunotoxicity  may  be  affected  by 
endocrine  disrupting  chemicals  in  wildlife  (fish,  amphibians  and  marine  mammals),  but  the 
relationship  is  unclear.  Amphibians  were  highlighted  by  attendees  as  being  a  potential  priority 
because  diseases  associated  with  the  immune  system  are  seen  in  the  environment  for  these 
species. However, it was highlighted that research has shown that fish are also susceptible, as well 
as other taxa. This is therefore an area for further research which could lead to the development of 
new in vivo OECD CF level 3 - 5 tests. (Long-term) (Attendee) (Project team) 

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26. General  -  If  Log  Kow  >6  then  dietary  exposure  for  endocrine  testing  should  be 

considered, this is done for the Bioconcentration Factor (BCF) - in vivo 

One environment group felt that  for many  substances the relevant exposure route was not being 
considered as part of the experimental design. There was a suggestion that for substances with a 
high log Kow (i.e.  >6),  dietary exposure for endocrine  testing should be considered. While this is 
scientifically  valid,  consideration  should  be  given  regarding  how  the  information  would  be 
interpreted from a regulatory perspective. (Short-term) (Attendee) (Project team)  

27. General - Keep tissues from all endocrine long-term studies - in vivo 

Depending on the animal this may include whole fish, liver samples or even plasma samples. This 
recommendation  is  directly  applicable  to  all  level  3-5  studies  because  the  samples  are  already 
present.  Practical challenges include storage capacity because such tissue samples take up space 
in  freezers.  The  work  needed  includes  discussion  in  OECD  expert  groups  and  WNT  if  such 
recommendations  should  be  included  in  the  text  of  the  TGs,  formulation  of  such  text  and 
implementation of the text in the TGs. Time frame 1-2 years. By keeping tissue samples for further 
analysis  this  may  help  reducing  animal  testing  by  obtaining  more  data  from  the  available  tissue 
and allowing for re-analysis of samples. This is mainly applicable for the environment because this 
is more common practice for mammalian testing (Short-term) (Attendee) 

28. Research - Understand if current testing can be extrapolated to reptiles - in vivo 

First activity would be a literature survey on differences and similarities of the endocrine system of 
reptiles  compared  to  mammals,  fish,  amphibians  and  birds.  Similarities  have  to  be  identified  and 
later experimental data need to  be obtained  to evaluate effects on reptiles of substances  causing 
ED  effects  in  the  other  vertebrate  taxa.  This  activity  would  most  efficiently  be  combined  with  an 
assessment of ED activity in birds. Time line 5-10 years. (Medium- to long-term) (Attendee) 

29. Research  -  Avian  mechanistic  screens  /  additional  mechanistic  endpoints  could  be 

added to OECD TG 206 - in vivo 

A review of the effects of endocrine disruption in birds including analysis for which modes of action 
and  associated  specific  endpoints  might  only  be  observed  in  birds  (as  opposed  to  mammals  or 
other  taxa)  due  to  their  unique  characteristics.  The  review  would  include  consideration  of  the 
outcomes  of  the  US  EPA  validation  work  with  the  quail  model  including  the  impact  of  different 
diets. (Short-term review, long term research) (Attendee) 

A2.4  Ideas identified by attendees after the workshop 

The prioritisation factors that were asked to be considered were  MoA, adversity, societal benefits, 
TRL, costs and animal concerns. All of the additional ideas highlighted are ideas for human health. 

1.  Male Reproductive Health - Serum insulin-like peptide 3 (INSL3) - in vivo 

Measurement of serum INSL3 as an enhancement in any of the relevant rodent studies, since it has 
been  shown  that  this  parameter  is  much  more  meaningful  about  testis  development  than  any 
measurements of steroid hormones such as testosterone. (Short- to medium-term) 

2.  Metabolic dysfunction - Histological examination of endocrine pancreas; alterations in 
structure  and  dimension  of  the  islet  of  Langerhans.  Measurement  of  total  beta  cell 
mass - in vivo 

Pancreas  samples are  removed and fixed in paraformaldehyde. Subsequently, pancreatic tissue is 
embedded  in  paraffin  and  sections  are  prepared.  Tissue  sections  are  stained  for  β-cells.  For 
morphometric analysis, 4 sections of each pancreas per animal, separated by 200 μm, are covered 
systematically  by  capturing  images  from  non-overlapping  fields  with  a  digital  camera.  The  islet 
cross-sectional area and total pancreatic area are measured using the analysis program Metamorph 
Software (or any other similar software). Pancreatic β-cell mass (mg per pancreas) is calculated by 
multiplying relative insulin-positive area (the ratio of insulin positive area over total pancreas area) 

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by the pancreas  weight. For quantification  of  the  number of islets per area, only islets with more 
than 5 positive-stained cells are scored. 

For  histological  examinations,  after  dehydration,  sections  will  be  stained  with  hematoxylin  and 
eosin or Masson’s trichrome. 

All these protocols could be included in the OECD TG 408 study. 

3.  Metabolic  dysfunction  -  Non-alcoholic  fatty 

liver  disease: 

liver  histological 

examination, presence of fat, necrosis, fibrosis and inflammation - in vivo 

For a first histological study a hematoxylin and eosin staining is ideal. 

To visualize liver triglycerides and lipids, an easy and highly used method is the Oil Red O Staining. 
This is performed using liver sections which are fixed and stained in red oil. 

The Sirius Red staining protocol is used to observe fibrosis in a lot of cases of inflammation caused 
by metabolic pathologies. 

All these protocols could be included in the OECD TG 408 study. 

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