Fourth annual forum on endocrine disruptors : exchanging knowledge, identifying challenges, building synergies : 21-22 September 2022 : forum report.
Date: 2023-07-14
Institutions: Directorate-General for Environment
Subjects: chemical product, chemicals legislation, conference proceedings, endocrine disease, endocrine disruptor, health legislation, health risk, labelling, packaging, scientific cooperation, toxic substance

FOURTH ANNUAL FORUM ON

Exchanging Knowledge, Identifying Challenges, Building Synergies

21-22 September 2022

FORUM REPORT

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KH-03-22-160-EN-N

Opening session

Session 1: 
Updates from the Commission and Agencies

Session 2: 
From bench to validated test guidelines: 
(pre)validation of test methods

Session 3: 
Focus on thyroid

Session 4: 
Developments on bisphenols 

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Conclusions and next steps

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Opening 
session 

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Virginijus Sinkevičius
European Commissioner for Environment, 
Oceans and Fisheries

Welcome  to  the  Fourth  Annual  Forum  on 
Endocrine Disruptors. If you are here, you 
know that these chemicals are a cause of 
concern.  I  myself  am  deeply  concerned  - 
not only as the Commissioner for the Envi-
ronment, but also as a citizen and a father. 

Endocrine  disruptors  affect  us  at  critical 
moments,  when  the  body  is  particularly 
vulnerable; during embryonic development 
or foetal development, during early child-
hood and puberty. 

The  problem  is  the  way  they  mimic  our 
body’s  hormones  with 
lasting  effects. 
Sometimes they are even passed on to the 
next  generation.  They  disrupt  critical  body 
functions, and they can lead to diseases like 
diabetes, obesity, and cancer. They weaken 
our immunity, and cause great suffering. 

Their economic effects are also considera-
ble, as they effect the workforce and pose 
challenges for healthcare systems. Studies 
put the costs to European society as some-
where  between  46  to  288  billion  euros, 
every single year.

In the EU, we have always been committed 
to  ensuring  a  high  level  of  protection  for 
citizens  and  their  environment.  We  have 
been acting on this problem for 25 years, 
since the days they were first discovered. 
That  long  commitment  has  led  to  many 
actions, but now it is time to move into a 
different gear.

Therefore,  we  are  delivering  a  break-
through regulatory change in the two most 
important  regulations  on  chemicals  man-
agement: REACH and the CLP regulation on 
the  classification,  labelling  and  packaging 
of substances. 

The draft Commission proposal for the CLP 
Delegated Act is being published this week. 
The idea is to introduce new hazard class-
es  for  endocrine  disruptors.  We  consulted 
the relevant stakeholders and the compe-
tent  authorities  of  the  Member  States  at 
the drafting stage. Right now, we are con-
sulting the public. 

Our plan is to have the Commission adopt 
the  proposal  by  the  end  of  November 
2022. After that, it goes to the Parliament 
and the Council for a scrutiny period of two 
months. If they have no objections, it will 
be published as adopted by all three insti-
tutions early next year. 

That  means  that  the  EU  will  finally  have 
horizontal criteria for endocrine disruptors. 
Some  Member  States  have  already  pub-
lished lists of endocrine disrupting chemicals. 
Those lists will help speed up our efforts at 
identification, classification and labelling. 

But we will not stop there. In the Chemicals 
Strategy for Sustainability, we also aim to 
ban  endocrine  disrupting  chemicals  from 
consumer  products.  The  only  exceptions 
will be if their use in a product is essential 
for  society.  This  will  be  made  possible  by 
the upcoming revision of REACH and also 
product-specific legislation.

And work continues on many more fronts. 
We  are  developing  and  validating  test 
methods,  funding  Horizon  research  pro-
jects  on  the  effects  of  endocrine  disrup-
tors,  consolidating  data,  and  improving 
data  transparency  with  “one  substance, 
one assessment”.

When  you  put  it  all  together,  the  EU 
clearly emerges as a driving global force 
on  hazard  and  risk  management  of  en-
docrine disruptors. 

This Forum is important because it is gen-
uinely international. It brings together our 
partners from around the globe, and their 
tremendous expertise. 

We are also here to learn from each other, 
and  to  pave  the  way  for  other  regions  to 
adopt  similar  measures.  We  are  here  be-
cause it is a battle we share; because we 
all want a toxic-free environment - for so-
ciety, for nature, and for the future health 
of our children.

The European Commission’s 
Communication of 7 November 
2018, “Towards a comprehensive 
European Union framework 
on endocrine disruptors”, 
committed the Commission to 
organising an Annual Forum on 
Endocrine Disruptors, to bring 
together all interested parties to 
help guide policy.

The Fourth Annual Forum 
was co-organised by DG 
Environment and ANSES, the 
French National Agency for 
Food, Environmental and Oc-
cupational Health and Safety. 
The Forum was held on 21-22 
September 2022 in the Char-
lemagne building in Brussels, 
and was also followed by a large 
online audience worldwide. It 
follows up a first segment of the 
event, which took place in Paris 
on 12 May 2022.

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“An endocrine 
disruptor is 
an exogenous 
substance or 
mixture that alters 
the function(s) 
of the endocrine 
system and 
consequently 
causes adverse 
effects in an intact 
organism, or its 
progeny, or (sub)
populations.”

Source: World Health 
Organisation / International 
Programme on Chemical Safety 
(WHO/IPCS) 2002.

4

Matthieu Schuler
Managing general Director, Science for expertise division, 
French National Agency for Food, Environmental and 
Occupational Health Safety (ANSES)

It  is  an  honour  for  France,  and  especially 
for  me  as  a  representative  of  ANSES,  the 
French “One health agency” to take part in 
the  organisation  and  introduction  of  this 
Fourth Endocrine Disruptors Forum. 

At  first  glance,  our  involvement  has  been 
due to the French Presidency of the EU. But 
I  feel  that  the  main  reason  is  that  ANSES 
has  played  a  key  role  in  showing  an  early 
path from scientific evaluation of endocrine 
disruption to regulatory acts with protective 
measures,  at  national  and  European  level, 
specifically for bisphenol A (BPA).

Endocrine  regulation  is  essential  for  life. 
Hormones are part of our biology, from our 
early  creation  steps,  through  childhood  to 
adulthood, and until the end of life. This reg-
ulation varies with the needs of our bodies, 
therefore leading to different susceptibility 
to endocrine active substances according to 
our age and biological status.

Our  “biological  clock”  has  evolved,  in  the 
long  term  with  human  evolution,  to  cope 
with  endocrine  active  substances,  partly 
generated internally and partly received as 
intakes  -  especially  via  food.  If  I  dare  use 
an  image,  the  “software”  of  our  organism 
is able to regulate an adequate level for a 
large number of endocrine active substanc-
es. But some substances overpass this reg-
ulation capacity, and lead to diseases. These 
are the so-called endocrine disruptors.

This overview explains why the WHO defini-
tion - which serves as the basis for the reg-
ulatory  definitions  of  endocrine  disrupting 
substances – can appear complex.

Moreover,  it  calls  for  a  precise  scientific 
evaluation  to  be  able  to  determine  the 
substances  that  are  endocrine  disruptors 
from  among  all  the  many  endocrine  ac-
tive  substances.  There  are  nearly  1  000 
substances,  for  instance,  in  the  long  list 
of “substances of interest” established by 
ANSES  in  2021,  requiring  an  evaluation 
with sufficient level of confidence.

One  more  aspect  of  endocrine  disrupting 
substances’  characteristics:  some  of  them 

show,  as  usual  toxic  substances,  their  ad-
verse  effects  at  high  levels  of  exposure  - 
precisely  when  the  intake  overwhelms  the 
regulating capacity, as is the case for resor-
cinol or cholecalciferol. But some may pro-
duce effects at chronic or low doses, such as 
BPA. This is why endocrine disruptors have 
to be considered as a distinct hazard class. 
In  particular,  it  does  not  mean  that  endo-
crine disrupting effects occur for every sub-
stance at every dose - the usual distinction 
between hazard and risk applies. 

Of  course,  these  characteristics  need  sci-
entific data at hand for the evaluators, like 
these now required for biocidal products or 
plant protection products along with their 
submission  dossiers.  The  question  is  now 
raised,  along  with  the  coming  addition  to 
the  CLP  Regulation,  what  will  be  the  re-
quirements  for  these  data  in  other  regu-
lated domains.

After  having  sharing  with  you  some  key 
characteristics of endocrine disruptors, I will 
conclude with keys for timely actions.

The  number  of  substances  for  which  as-
sessments  are  needed  represents  a  real 
challenge  for  health  and  safety  agencies 
across the EU, requiring them to join forces 
to  cope  with  the  evaluation  of  substances 
toward  endocrine  disruption  as  a  hazard 
class. It is important to speed up the “one 
substance,  one  assessment”  principle  and 
apply it for that purpose. Of course, this also 
requires  adjustment  in  the  regulatory  re-
quirements for data, and the development 
of the corresponding testing protocols.

Another  key  is  the  permanent  need  for 
precision,  transparency  and  information 
exchanges at all stages of the substance’s 
life cycle (from design to market) and, as a 
common backbone, a clear and widely un-
derstood regulatory framework.

I wish you all a fruitful Forum, an excep-
tional  occasion  for  all  stakeholders  to 
exchange  and  gain  mutual  understand-
ing  of  these  both  complex  and  concern 
raising questions.

 
 
 
Session 1: 
Updates from 
the Commission 
and Agencies

5

The Chemicals Strategy 
for Sustainability: 
state of play

oto: © European Commission

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Cristina de Avila gave an overview 
of progress on the implementation 
of the Chemicals Strategy for 
Sustainability (CSS). She highlighted 
the Delegated Act to introduce 
new hazard classes for endocrine 
disruptors in the CLP (classification, 
labelling and packaging) Regulation, 
published on 20 September 2022, 
the day before this Forum.

Cristina de Avila
Head of Unit, Safe and Sustainable Chemicals, 
DG Environment, European Commission

The Strategy is an offspring of the Euro-
pean  Green  Deal.  It  aims  to  improve  the 
legislative framework for chemicals in Eu-
rope to protect people, including the most 
vulnerable, and the environment. This will 
be achieved through a clear vision, objec-
tives,  and  a  concrete  action  plan  to  ad-
dress present and future challenges.

The vision is to ensure that by 2030 there 
is a toxic-free environment where chemi-
cals are produced and used in a way that 
maximises their benefits to society, while 
avoiding harm to people and the planet.

Key  objectives  are  to:  (i)  strengthen 
legislation  and  promote  innovation,  to 
ensure  all  chemicals  on  the  market  are 
used safely and sustainably; (ii) promote 
and  reward  substitution  of  chemicals 
causing  long-term  adverse  effects  on 
humans  and  the  environment;  and  (iii) 
phase out the most harmful chemicals in 
consumer products.

The Strategy has a specific place for en-
docrine  disruptors,  calling  for  a  ban  of 
them in consumer products, together with 
persistent and other harmful substances. 
There  is  also  a  place  for  specific  initia-
tives,  like  the  action  plan  for  PFAS  that 
will  only  allow  their  essential  uses.  In 
addition, the Strategy also addresses the 
combination effects of chemical mixtures.
To reduce the risk posed by chemicals of 
concern, the Commission is implementing 
amendments to REACH and CLP, the two 

main horizontal pieces of chemicals legis-
lation in Europe. This includes new hazard 
classes for endocrine disruptors.

REACH

In REACH, the Commission is updating the 
information requirements for the registra-
tion dossiers that companies have to pro-
vide for placing substances on the Europe-
an market. This will ensure these dossiers 
have  sufficient  information  to  allow  risk 
management of endocrine disruptors.

Specifically,  the  Commission  is  updating 
REACH Annexes I and VII to X to include 
new data requirements on endocrine dis-
ruption.  This  takes  into  consideration:  (i) 
the OECD Conceptual Framework for Test-
ing and Assessment of Endocrine Disrupt-
ing Chemicals; (ii) the update of biocides 
and  plant  protection  products  Annex-
es;  and  (iii)  the  tiered  obligations  under 
REACH requiring different levels of infor-
mation  depending  on  the  volumes  (ton-
nages) of the chemicals being registered. 

A  second  important  change  in  REACH  is 
that endocrine disruptors will have their 
own  place  as  Substances  of  Very  High 
Concern  (SVHC).  This  replaces  the  situ-
ation  where  endocrine  disruptors  are  of 
concern within other categories.

Substance evaluation in REACH is the tool 
to confirm or clear a potential concern for 
a substance. The REACH amendment will 

streamline  evaluation  procedures.  This 
includes  clarifying  how  to  establish  con-
cerns, what can be asked for, and stream-
lining the Community Rolling Action Plan 
(CoRAP);  and  also  supporting  chemical 
grouping,  to  move  away  from  the  more 
inefficient procedure of dealing with sub-
stances one-by-one.

The  REACH  revision  will  also  extend 
the  generic  risk  management  approach 
(followed  the  example  of  CMRs)  to  im-
plement  restrictions  on  endocrine  dis-
ruptors,  as  well  as  persistent,  bioaccu-
mulative  and  toxic  (PBT)  substances, 
very persistent and very bioaccumulative 
(vPvB)  substances, 
immunotoxicants, 
sensitis-
neurotoxicants, 
ers,  and  specific  target  organ  toxicants 
(STOT). In particular, the CSS calls for the 
avoidance of all these chemicals in con-
sumer products. 

respiratory 

There is a further call to extend this ge-
neric  risk  approach  to  professional  uses. 
All  restrictions  will  be  accompanied  by 
the possibility of derogations for essential 
uses for society, the details of which are 
still to be decided.

In terms of indicative timing of action, the 
Commission  has  launched  a  number  of 
supporting studies that are coming to an 
end. The draft proposal for the revision of 
REACH  is  expected  to  be  adopted  in  the 
first quarter of 2023.

6

CLP

The  Commission  has  committed  to  intro-
ducing  new  hazard  classes  for  endocrine 
disruptors under the CLP Regulation, as the 
repository of hazard classes in the EU. The 
call comes not only from the CSS, but also 
the  Commission’s  Communication  of  2018 
on endocrine disruptors that recognised the 
benefits  of  horizontal  identification  for  a 
coherent regulatory response.

The  Commission  shared  the  draft  hazard 
categories under the CLP Regulation on 20 
September 2022 for public consultation. It 
included  hazard  classes  for  endocrine  dis-
ruptors for human health and the environ-
ment within two categories (Cat 1 and Cat 
2). There will also be hazard classes for PBT 
and  vPvB  substances,  basically  bringing 
all  the  criteria  and  expertise  developed  in 
REACH  into  the  CLP  Regulation  for  a  more 
coherent approach. The endocrine disruptor 
hazard  classes:  (i)  are  based  on  the  WHO 
definition;  (ii)  build  on  existing  criteria  for 
pesticides  and  biocides;  (iii)  are  to  be  ap-
plied horizontally across all legislation; and 
(iv)  are  separated  for  human  health  and 
environment to aid their implementation in 
specific pieces of legislation.

Category 1 is for known or presumed endo-
crine disruption for human health. This will 
largely be based on evidence from human 
and/or  animal  data.  The  data  should  pro-
vide evidence that the substance meets the 
three  criteria:  (i)  endocrine  activity;  (ii)  an 
adverse effect in an intact organisms or its 
offspring  or  future  generations;  (iii)  and  a 
biologically  plausible  link  between  the  en-
docrine activity and the adverse effect.

Category 2 is for suspected endocrine disrup-
tors  for  human  health.  Again,  this  is  largely 
based  on  evidence  from  human  and/or  ani-
mal data. The difference between Categories 
1 and 2 is the level of evidence.

For  human  health,  a  new  hazard  statement 
will  be  printed  on  labels;  either  “may  cause 
endocrine  disruption  in  humans”  (Cat  1)  or 
“suspected of causing endocrine disruption in 
humans” (Cat 2).

The  deadline  for  commenting  on  the  draft 
hazard  categories  under  the  CLP  Regulation 
is 18 October 2022. Discussions will also be 
held with experts, with a view to adoption in 
November 2022.

Finally, concluded Ms de Avila, the EU is set-
ting an example globally, which is one of the 
building  blocks  of  the  CSS.  The  CPL  Regula-
tion  is  the  implementation  in  the  EU  of  the 
UN  Globally  Harmonised  System  of  Classifi-
cation and Labelling of Chemicals (GHS). The 
new hazard classes will be proposed for the 
GHS,  starting  in  the  discussions  of  the  GHS 
Sub-Committee on 7-9 December 2022.

Photo: © European Commission

Q&A

Katrina Sichel (moderator) 
directed questions to Cristina 
de Avila from the audience 
in Brussels and from online 
participants via SLIDO. 

Norbert Kaminski 
(Michigan State University): 
Could you give an update on the 
second part of the CLP revision via the 
Ordinary Legislative Procedure?

Cristina de Avila:  We are revising 
CLP with two proposals instead of just 
one. One was published yesterday (20 
September 2022), which is a Delegated 
Act to amend the Annexes in CLP to in-
troduce the new hazard classes. The other 
Act will amend the terms of the CLP itself 
through the Ordinary Legislative Proce-
dure, which cannot be done in any other 
way. The idea is to have both acts adopted 
at the same time in November 2022, 
and then it will continue to the 
Council and the Parliament. 

Norbert Kaminski 
(Michigan State University): 
You mentioned criteria that were being 
proposed, and one of them was “essen-
tial use”. Could you elaborate a bit on 
how that will be determined.

Cristina de Avila: We are talking about 
the concept of essential uses in existing 
processes that we have, in this case not 
only in REACH but also in other pieces 
of legislation, such as the Cosmetic Prod-
ucts Regulation and the Toys Regulation. 
In REACH we already have existing 
restriction and authorisation procedures, 
but what the Strategy is calling for is the 
introduction of essential uses in the case 
of restrictions based on generic approach-
es as the only way of derogating. So we 
need to find a way of operationalising 
this concept into our existing restrictions 
process. The final decision of our restric-
tion lies with the Commission, via an 
amendment to an Annex of REACH. 
If we are talking about an authorisation, 
it is also the decision of the Commission, 
but addressed to the company that has 
asked for the authorisation to use this 
SVHC. We are now looking at what are 
the elements of essential uses for their 
criteria, and how we will apply them in 
practice, and who will oversee 
the governance.

Malik Duhaut (Covestro): 
Is the Commission planning to apply 
generic approaches to endocrine 
disruptor category 1 (or both category 
1 and 2)?

Cristina de Avila: I think this is a deci-
sion that will have to be taken for each 
piece of legislation. What the Strategy 
calls upon is an extension of the current 
system for endocrine disruptors. 
In REACH we have generic approaches 
that apply to CMRs category 1. 
In other pieces of legislation, that may 
be different. Generic approaches rely 
on a harmonised classification. 

Caroline Bassoni (COSMED): 
The definition of “biologically plausi-
ble link” is quite vague and we feel it 
is open to different interpretations. Is 
there a plan to define more criteria to 
allow a fairer assessment of this link?

Cristina de Avila: There is always a 
possibility of developing a guidance of 
what it is going to be, if it is not clear 
in practice. That would be for industry, 
who need to self-classify, but also for ad-
ministrations, especially ECHA who are 
in the risk assessment committee taking 
steps for harmonised classification. 

Helene Loonen 
(European Environmental Bureau): 
When do you think that the new haz-
ard classes will be operational? We are 
also concerned about the slow progress 
made on the REACH revision, especial-
ly calls from the European Parliament 
to postpone it, so what will the Com-
mission do? Will it commit to deliver 
on the objectives of the Green Deal and 
Chemicals Strategy to rapidly phase 
out substances of concern including 
endocrine disruptors?

Cristina de Avila: The transitional 
period is maybe 18 months or longer. 
When you have hazard classes in place 
it is not only an option of the Commis-
sion, there is a scrutiny period with the 
Parliament and the Council. Once in 
law, in principle there is an obligation 
after the transition period to self-clas-
sify and we are free then as authorities 
to start harmonised classifications of 
endocrine disruptors. After that, it is up 
to Member States to introduce the new 
harmonised classifications. We are com-
mitted to deliver the REACH revision 
under this Commission. The calls from 
the Parliament are only from some sec-
tors. Overall, we have full support from 
the European Parliament in an opinion 
adopted in June 2022.

7

ED and non-ED active 
substances for plant 
protection and 
biocidal products

oto: © European Commission

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Karin Nienstedt provided an update on 
pesticides and biocides since 2018, as regards 
endocrine disruptors, in the context of the 
Biocidal Products Regulation (BPR) and the 
Plant Protection Products Regulation (PPPR). 

New criteria for endocrine disruptors were 
adopted  in  the  BPR  and  PPPR  in  2018. 
This  means  that  since  2018:  (i)  Member 
States, EFSA and ECHA have been apply-
ing the criteria for all active substances to 
be used for pesticides and biocides (dos-
siers submitted since 2018, and also dos-
siers that were pending at that time); (ii) a 
Joint EFSA/ECHA Guidance Document has 
been  available  to  help  apply  the  criteria; 
and (iii) training for risk assessors to help 
them  apply  the  new  criteria,  e.g.  Better 
Training for Safer Food (BTSF).

The new criteria for endocrine disruptors 
for  biocides  and  pesticides  are  based 
on  the  three  elements  of  the  WHO/IPCS 
definition:  endocrine  mode  of  action,  an 
adverse effect, and a plausible link. They 
identify  known  and  presumed  endocrine 
disruptors,  and  are  fully  equivalent  with 
the  two  categories  of  the  draft  criteria 
just published for the CLP Regulation. 

Beyond  the  criteria,  new  data  require-
ments  were  also  adopted  under  both 
pieces of legislation. In 2020, Annexes II 
and III of the BPR were amended, setting 
data requirements for determining on en-
docrine  disruptors.  Two  Communications 
led  to  revisions  in  data  requirements  for 
the  PPPR,  listing  the  relevant  guidance 
documents and test methods.

Biocides

In 2022, after four years of implementa-
tion for biocides, 42% of the Review Pro-
gramme is completed (for a combination 
of  active  substances  and  product  types). 
There  are  still  128  active  substances  in 
the  Review  Programme  under  assess-
ment by the evaluating Member States or 

ECHA for endocrine disrupting properties.
As regards ECHA opinions published since 
2018, four active substances were identi-
fied  as  endocrine  disruptors:  cholecalcif-
erol,  DBNPA,  cyanamide,  and  propicona-
zole.  For  cholecalciferol  (vitamin  D3),  a 
derogation was approved allowing its use 
as a rodenticide. 

Eight active substances were identified as 
clearly not being endocrine disruptors for 
human  health  or  the  environment.  There 
have been 19 ECHA opinions where eval-
uations were inconclusive, and these are 
being looked at case-by-case.

What is important for biocidal products is 
that there is a provision to trigger an ear-
ly review of approvals (BPR Article 15). For 
instance, an ongoing early review was trig-
gered for three substances to check for en-
docrine disruptor criteria in the light of re-
cent concerns: iodine, PVP-iodine and zineb.

Pesticides

The endocrine disruptor criteria has been 
applied  to  new  and  pending  dossiers  for 
pesticides  since  November  2018.  For 
pending  dossiers  (submitted  before  No-
vember 2018) a ‘stop-clock’ has been im-
plemented to enable time for conclusions 
to be reached. The criteria have also been 
applied to some MRLs (maximum residue 
level) processes, and updates of 90 sub-
stances  for  ongoing  MRL  processes  are 
published by EFSA.  

In a nutshell, processes have been initiated 
or finalised for 95 active substances used in 
pesticides:  40  finalised,  26  on  ‘stop-clock’, 
25  where  ‘stop-clock’  has  been  resumed, 
while a few dossiers were withdrawn.

8

Karin Nienstedt
Policy Officer, DG SANTE, 
European Commission

For  the  40  dossiers  where  EFSA  conclu-
sions  were  finalised:  28  active  substances 
were  clearly  identified  as  not  being  endo-
crine  disruptors,  6  were  identified  as  en-
docrine  disruptors  for  human  health  and 
3  for  non-target  organisms  (environment). 
There were a few others where no conclu-
sion could be reached and additional data 
is needed. In one case for human health, a 
substance was banned for other reasons.

Among the substances identified as endo-
crine  disruptors,  mancozeb  has  not  been 
approved  for  use,  a  decision  also  taking 
into  account  other  risks.  Currently,  three 
substances  are  still  under  discussion  in 
the PAFF Committee (The European Com-
mission’s  Standing  Committee  on  Plants, 
Animals, Food and Feed): asulam-sodium, 
benthiavalicarb, and clofentezine.

Q&A

Katrina Sichel (moderator):  
What is the timeline for the 
‘stop-clock’?

Karin Nienstedt:  The ‘stop-clock’ 
takes 3-30 months. This is a flexible 
timing that depends on the assessment 
process of EFSA and relevant Member 
States. They should look at what is in 
the dossier, what other data is needed, 
and should give time to applications 
to generate this data. We put an upper 
limit of 30 months so as not to delay 
the process for years.

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Highlights 
from the session 
on Endocrine 
Disruptors of 
the EFSA One 
conference 

Maria Arena
Scientific Officer, European Food 
Safety Authority (EFSA)

Maria Arena presented the highlights of the session on endocrine 
disruptors from the EFSA One conference held in June 2022.

associated  with  the  induction  of  periph-
eral  effects  is  important.  However,  the 
impact of the thyroid hormone T4 on the 
brain in the sensitive population (i.e. foe-
tus and new-borns) is still unknown, and 
models to define this impact represent a 
complex research task.

For  environmental  effects:  (i)  it  was 
agreed  that  identifying  endocrine  disrup-
tion in the environment is challenging be-
cause of a lack of methods and knowledge; 
(ii) more should be invested for advancing 
knowledge  and  methods  in  ecotoxicology; 
(iii)  mechanistic  understanding  should  be 
increased to enable extrapolation between 
taxa;  and  (iv)  the  breaking  of  walls  be-
tween mammalian toxicology and ecotox-
icology  should  be  encouraged,  with  data 
considered in a more holistic way. 

The  four-day  EFSA  One  (Health,  Envi-
ronment,  Society)  Conference  2022  was 
co-designed with its sister agencies: Euro-
pean Chemicals Agency (ECHA), European 
Centre for Disease Prevention and Control 
(ECDC),  European  Environment  Agency 
(EEA), European Medicines Agency (EMA), 
and the Joint Research Centre (JRC).

A  key  aim  was  to  foster  engagement 
among  experts,  Member  States  and 
stakeholders  from  diverse  backgrounds. 
The focus was on food safety in the con-
text  of  sustainable  food  systems,  devel-
opments in risk assessment and regulato-
ry science, and the European Green Deal. 
It also marked EFSA’s 20th anniversary.

Following the opening day’s plenary ses-
sions,  days  2  and  3  were  dedicated  to 
thematic  breakout  sessions  within  four 
parallel tracks (One Society, One Life, One 
Planet,  and  Many  Ways).  The  final  day 
comprised the closing plenary sessions.

The  endocrine  disruptors  session  was  in 
the  ‘Many  Ways’  track,  focusing  on  what 
works  and  what  needs  improving  in  en-
docrine  disruptor  assessment.  It  began 
with  a  keynote  presentation  by  Andreas 
Kortenkamp (Brunel University); then four 
posters  were  selected  for  special  pres-
entation;  followed  by  six  ‘flash  reports’ 

from  representatives  of  academia,  the 
private  sector,  EU  agencies,  European 
Commission,  NGOs  and  Member  States. 
Finally, there was a moderated panel dis-
cussion and a wrap up session with clos-
ing remarks.

The  main  outcomes  were:  (i)  agreement 
on the need for harmonised criteria across 
legislation for endocrine disruptor identi-
fication, though this is not always possi-
ble  or  warranted  (e.g.  low  tonnage  sub-
stances  under  REACH);  (ii)  hazard-based 
cut-off criteria are warranted in the vast 
majority  of  cases  and  across  legislation, 
though in some cases a risk assessment 
approach  might  be  an  option;  (iii)  ‘one 
substance,  one  assessment’  is  desirable, 
but requires mechanisms for sharing data 
across agencies so consistent conclusions 
can be reached.

Regarding  New  Approach  Methodologies 
(NAMs): (i) alternative methodologies (e.g. 
in vitro test batteries) increase mechanis-
tic  understanding  and  move  away  from 
animal  data;  (ii)  validation  of  NAMs  is  a 
key  step  for  their  regulatory  acceptance; 
and  (iii)  procedures  are  needed  to  speed 
up their regulatory implementation.

Concerning  human  health,  it  was  noted 
that the quantification of thyroid effects 

9

 
 
 
Sharing data on 
endocrine disruptors 
for research and 
regulatory purposes: 
EASIS

Siegfried Morath gave an overview 
of EASIS, the Endocrine Active 
Substances Information System. 
This freely-accessible web-based 
application was introduced a few 
years ago, but JRC have recently 
updated it with current technologies. 

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Siegfried Morath
Scientific Officer, Joint Research Centre 
(JRC), European Commission

generated  by  New  Approach  Methodolo-
gies (NAMs) is captured via the OECD Har-
monised Template 201 (OHT 201). 

The  insights  gained  can  facilitate  sci-
entific  development,  for  example  by:  (i) 
putting more standards into science (e.g. 
effect-based  reporting  data  formats);  (ii) 
associating  mechanistic  effects  with  ad-
verse  outcomes;  (iii)  supporting  the  pre-
diction  of  effects  from  chemical  struc-
tures; and (iv) enabling the use of Artificial 
Intelligence and Machine Learning tools.

Dr Morath invited participants in Brussels 
to try out an EASIS demonstration system 
on  both  days  of  the  Forum.  Anyone  can 
access the EASIS landing page online. 

Currently, 
the  EASIS  database  con-
tains  over  10  000  curated  entries  from 
peer-reviewed  scientific  publications  for 
more  than  600  substances.  The  data  in-
cludes  mechanistic  and  adverse  effects 
information,  relevant  to  disturbances  of 
the endocrine system in humans and the 
environment.

Key features of EASIS include (i) support 
for the “one substance, one assessment” 
approach  and  the  Chemical  Strategy  for 
Sustainability,  with  the  aim  of  making 
better use of academic data in regulato-
ry  assessment;  and  (ii)  IUCLID  software, 
which is internationally used for collecting 
and exchanging data on hazards of chem-
icals, and is a reporting tool of REACH.

Additionally, EASIS follows FAIR (Findable, 
Accessible,  Interoperable  and  Reusable) 
data principles, with the aim of improving 
the value of scientific data.

EASIS  integrates  different  types  of  data. 
On one hand, human health and environ-
mental  effects  data  are  obtained  main-
ly  using  conventional  approaches,  like  in 
vivo methods. On the other hand, mecha-
nistic information for endocrine disruptors 

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Discussion and Q&A
Katrina Sichel moderated the discussion at the end of the first 
session, with questions selected from the floor and via SLIDO.

Q&A

Q&A

Q&A

Daniela Fruth (knoell Germany): 
What exactly does peer-reviewed 
data mean - any authority review, or 
information from publicly available 
(active) substance dossiers included?

Siegfried Morath (JRC): It is primarily 
data from peer-reviewed scientific pub-
lications, after applying internal search 
terms and criteria for study selection 
and data extraction for our database.

Q&A

Yvonne Andersson 
(Swedish Chemicals Agency): 
Could you elaborate, or give exam-
ples, for which endocrine disrupting 
substances a risk assessment approach 
might be an option?

Maria Arena (EFSA): During the 
EFSA conference the risk assessment 
approach was mentioned for mixtures, 
and it could be used for other situa-
tions, but there were no discussions 
of how specifically this could be done. 
We don’t want to give the idea we are 
pushing for a risk assessment approach, 
because we have the hazard-based 
criteria, but there may be opportunities 
where risk assessment could be used.

Daniela Fruth (knoell Germany): 
Does it mean that currently identi-
fied endocrine disrupting substances 
under BPR and PPPR are considered 
as Cat 1 under the draft CLP update 
for Human Health and Non-Target 
Organisms?

Karin Nienstedt (DG SANTE):  From 
the scientific point-of-view, the criteria 
on biocides and pesticides are fully 
equivalent to Cat 1 under the draft 
proposal for CLP. However, from a 
regulatory point-of-view what has yet 
to be decided is that if a substance 
identified under biocides or pesticides 
legislation as an endocrine disruptor 
will then be fully taken over on CLP 
or if then a complete new dossier has 
to be submitted.

Q&A

‘Luca’: 
Regarding assessment of biocidal 
non-active substances (co-formu-
lants), are food flavourings/additives 
likely to not possess endocrine disrup-
tor properties?

Maria Arena (EFSA): At this stage, the 
criteria in place apply only to pesticides 
and biocides. Food ingredients and 
flavourings do not have such criteria for 
the identification of endocrine disrup-
tors. These substances are also used in 
low percentages. Though hazard-based 
criteria at the moment are just for 
pesticides and biocides, in CLP they 
could in practice be applied to other 
frameworks.

Sandra Jen (EDC-Free Europe): 
You mention 9 substances identified 
with ED properties and 3 under 
discussion, what was the timeline for 
the others? What is coming next for 
substance where no conclusions could 
be reached?

Karin Nienstedt (DG SANTE):   
To clarify, it is 6 substances for human 
health and 3 for environment, which 
are also included in human health, so 
in total it is 6 not 9. There are some 
conclusions of EFSA which are very 
recent, where we have not looked at the 
details for decision making. If no other 
risks are identified, these 4 will join 3 
other substances where case-by-case 
discussions are ongoing, such as for 
possible derogations. 

Q&A

Ninja Reineke (CHEM Trust): 
At the EFSA conference, there was a 
sense of urgency during the endocrine 
disruptor session, we need to get 
quicker and more efficient in their 
identification. What can EFSA do to 
achieve this?

Maria Arena (EFSA):  I think we can 
be faster based on regulations we have, 
and also using all available data in a 
holistic manner to conclude on them. 
When we cannot conclude, it is because 
there are no data to draw conclusion. 
There are many projects ongoing to 
improve the situation, such as those for 
AOPs and NAMs.

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Session 2: 
From bench to 
validated test 
guidelines: 
(pre)validation 
of test methods

12

The “integrated Fish 
Endocrine Disruptor 
Test” (iFEDT)

Lisa Baumann talked about 
ecotoxicological work with 
zebrafish, mainly resulting from 
the EU tender project iFEDT 
(“integrated fish endocrine 
disruptor test”). This work is 
leading to the development of a 
new fish test method, for use by 
industry to test for environmental 
effects of endocrine disruptors.

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Lisa Baumann
Heidelberg University, Germany

Endocrine  disruptor  testing  with  aquatic 
species  is  challenging  because:  (i)  many 
test  guidelines  for  fish  and  amphibians 
are  complex,  long,  expensive  and  use 
many  animals;  (ii)  only  a  few  tests  cur-
rently  cover  all  relevant  life  stages  and 
include  population-relevant  apical  end-
points; (iii) it can be difficult to distinguish 
endocrine  disruption  from  general  toxic-
ity;  and  (iv)  major  gaps  and  weaknesses 
exist regarding the different EATS (Estro-
gen,  Androgen,  Thyroid,  Steroidogenesis) 
modalities,  for  instance,  currently  EAS 
are mainly assessed in fish and T in am-
phibians, requiring the running of multiple 
tests to cover all modalities.

Therefore,  DG  Environment  issued  a  call 
in 2018 asking for improved Test Guide-
lines  for  endocrine  disruptors  to  address 
these  issues.  The  iFEDT  project  resulted 
from this call.

The most popular Test Guideline (TG) for 
fish, OECD TG 229 (fish short term repro-
duction assay), uses adult fish, often ze-
brafish, exposed for 21 days to potential 
endocrine  disruptors.  Different  endpoints 
are  checked  (e.g.  vitellogenin  biomarker, 

secondary sex characteristics, gonad his-
tology, and reproduction).

In OECD TG 234 (fish sexual development 
test), fish are exposed from early embryo 
up  to  juvenile  stage,  and  different  end-
points  are  assessed  that  are  potentially 
disrupted  by  endocrine  disruptors,  like 
growth,  hatch,  survival,  vitellogenin,  sex 
ratio, and gonad histology. 

The iFEDT project team suggested merg-
ing  the  TG  229  and  TG  234  protocols, 
starting with adult fish and going directly 
into the following generation. In total, for 
zebrafish  such  a  test  has  a  duration  of 
84 days (21+63 for the two parts of the 
merged  test).  This  is  considerably  short-
er  than  the  existing  medaka  extended 
one-generation  test  (MEOGRT,  TG  240) 
that runs over two generations, therefore 
reducing  time  taken  and  number  of  ani-
mals used.

The second key aspect of the merged test 
is  the  introduction  of  a  thyroid  modality 
endpoint,  which  currently  does  not  exist 
in fish tests. The reason the thyroid is ad-
dressed in amphibians is that they under-

go  a  clearly  visible  metamorphosis  from 
tadpoles  to  frogs,  but  fish  also  undergo 
a  metamorphosis  with  developmental 
changes regulated by thyroid hormones.

Research  at  the  Universities  of  Heidel-
berg,  Antwerp  and  Southern  Denmark 
(and  elsewhere)  has  shown  a  range  of 
thyroid endpoints in fish sensitive to en-
docrine  disruptor  exposure.  Eye  devel-
opment,  swimbladder  inflation,  pigmen-
tation,  thyroid  histopathology,  and  fin 
development are all regulated by thyroid 
hormones. The work in Heidelberg has fo-
cused on eye development, for which very 
specific endpoints have been defined.

The merged test - iFEDT - has new end-
points for adults (thyroid hormone levels 
and  thyroid  histopathology)  and  for  de-
veloping  embryos  in  the  second  phase 
(e.g.  thyroid  follicles,  eye  and  swimblad-
der development, and hormone levels). 

Therefore, iFEDT integrates all life stages 
and all relevant endpoints within one test. 

To validate the iFEDT test, it was run with 
the  model  thyroid  hormone  axis  disrup-

13

 
Q&A

Q&A

Andrew Turley (Chemical Watch):  
Where does this test fit into REACH 
and CLP? Where is the policy benefit?

Lisa Baumann: I am talking as a 
biologist, not a regulator, so I would 
hope with the iFEDT test we can, for 
example, reduce in a step-wise approach 
the number of tests and animals used. 
By testing early you also get early alerts 
of problems.

Mike Broderick 
(Health and Safety Authority, Ireland): 
The combination of the fish tests seems 
like a very good idea. However, are we 
sure that concentrating the ED tests 
on fish alone will protect amphibians?

Lisa Baumann (Heidelberg Universi-
ty): We are not suggesting skipping the 
amphibian tests entirely, for exactly that 
reason. Amphibians are very vulnerable 
to many chemicals, and they are highly 
endangered, especially in Europe. For 
that we need amphibian tests, but we 
think they can be substantially reduced. 
Running two tests unnecessarily for fish 
and amphibians is something we want 
to avoid for 3Rs reasons.

Q&A

Cécile Michel-Caillet (ANSES): 
Is the eye’s histopathology also 
usable for evaluating thyroid 
disruption in mammals?

Lisa Baumanm: For our AOP develop-
ment, we did not only collect evidence 
for fish, we also collected it for all other 
vertebrate classes, though for birds and 
reptiles it was limited. For mammals we 
know that thyroid hormones are very 
important for eye development. This is 
the concept we follow in the EURION 
project ERGO, to show that the effects 
we see in one species can be translated 
to others. We are developing adverse 
outcome pathways (AOPs) to show that 
what happens in a fish can also happen 
in a mammal or a human.

tor  propylthiouracil  (PTU),  which  inhibits 
thyroid hormone synthesis; and with the 
model estrogen disruptor ethinylestradi-
ol (EE2).

PTU  exposure  resulted  in  strong  effects 
on  endpoints,  down  into  the  sub-lethal 
range  (ruling  out  general  toxicity).  More-
over,  vitellogenin,  used  in  many  fish  and 
amphibian  guidelines  to  identify  estro-
gen-related effects, disrupted F1 females, 
and acted as expected in male fish.

Thyroid  histopathology  clearly  showed 
the  effects  of  high  PTU  exposure,  seen 
externally as a red outgrowth (goitre) due 
to proliferating thyroid follicles (trying to 
compensate for thyroid hormone synthe-
sis inhibition).

The eye development endpoint (thickness 
of  retinal  layers)  was  also  successfully 
validated.  This  endpoint  is  also  impor-
tant  for  population-level  assessments, 
because  fish  with  impaired  vision  will 
not survive in the environment. The iFEDT 
team  produced  a  new  adverse  outcome 
pathway  based  on  the  action  of  PTU  on 
eye development, proving the relationship 
between thyroid hormone synthesis inhi-
bition,  altered  eye  development,  and  be-
haviour that might lead to mortality.

Dr Baumann concluded by saying they are 
currently  finalising  the  iFEDT  test  proto-
cols.  There  is  one  remaining  challenge, 
which is to improve the method for meas-
uring thyroid hormone levels; this applies 
to all projects aiming to use measured TH 
levels  as  an  endpoint  in  Test  Guidelines 
for fish and amphibians. Two publications 
are currently been prepared that summa-
rise all the iFEDT results. The final stage 
will be validation to make it an OECD TG.

14

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Photo: © European Commission

Panel discussion

The panel discussion was moderated by 
Cécile Michel-Caillet, Head of the REACH-
CLP-Endocrine Disruptors Unit, French Agency 
for Food, Environmental and Occupational 
Health & Safety (ANSES), Paris, France. 

Panel members were: 

Philippe Hubert, Director, Pepper Platform, France; 
Anne Gourmelon, Principal Administrator, OECD Test Guidelines Programme; 
Sharon Munn, Senior Scientific Officer, Joint Research Centre (JRC), 
European Commission; and Lisa Baumann, Heidelberg University, Germany.

15

Joint Research 
Centre (JRC)

oto: © European Commission

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The  JRC  is  the  knowledge  service  of  the 
European Commission, supporting EU poli-
cies with independent evidence throughout 
the whole policy cycle. It has around 3 000 
staff,  almost  75%  of  them  scientists,  at 
research facilities across five EU Member 
States and at its headquarters in Brussels.

About forty staff work at JRC’s Chemical 
Safety  and  Alternative  Methods  unit  in 
Ispra, Italy. This unit contains the Europe-
an  Reference  Laboratory  for  Alternatives 
to  Animal  Testing  (EURL  ECVAM),  estab-
lished under the Directive on the protec-
tion  of  animals  for  scientific  purposes 
(2010/63), which focuses on the 3Rs: the 
Replacement,  Reduction  and  Refinement 
of animal testing.

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Sharon Munn 
(JRC)

In relation to endocrine disruptors: (i) the 
JRC  supports  the  updates  to  REACH  and 
the  CLP  Regulation;  (ii)  the  EURL  ECVAM 
Thyroid Validation Study in assessing 18 
methods;  (iii)  the  newly-released  EASIS 
(Endocrine  Active  Substances  Informa-
tion  System)  which  provides  structured 
data  on  endocrine  activity  and  adverse 

effects; (iv) the eight EURION Cluster pro-
jects funded under H2020 on methods for 
identifying endocrine disruptors; and (v) it 
also  supports  OECD  activities  on  endo-
crine disruptors.

Further  information  can  be  found  in  the 
EURL ECVAM Status Report.

OECD Test 
Guidelines 
Programme

Anne Gourmelon 
(OECD)

OECD works with its 38 member countries 
to provide harmonised methodologies and 
tools for chemical safety assessments.

crine  disruptor  testing  and  assessment, 
within  5  levels  of  increasing  biological 
complexity.

OECD’s  work  on  endocrine  disruptors 
started  in  1997,  with  the  creation  of  a 
dedicated  Task  Force.  Work  has  focused 
on  the  development  of  Test  Guidelines 
(TGs)  for  the  screening  of  endocrine  dis-
ruptors.  In  2002,  the  OECD  developed  a 
Conceptual  Framework  for  Testing  and 
Assessment of Endocrine Disrupters. This 
organised  knowledge  and  data  on  endo-

In  2012,  OECD  published  the  first  Guid-
ance Document 150 on Standardised Test 
Guidelines  for  Evaluating  Chemicals  for 
Endocrine  Disruption.  A Revised  GD  150 
was published in 2018. 

In  recent  years,  the  Adverse  Outcomes 
Pathway  (AOP)  concept  has  been  de-
veloped  to  better  understand  modes 

of action and therefore to identify new 
testing methods.

OECD’s  work  on  endocrine  disruptors 
involves  collaborations  with:  (i)  OECD 
member  and  partner  countries;  (ii)  ani-
mal welfare and environmental NGOs; (iii) 
business  and  industry  advisory  commit-
tees;  (iv)  private  and  public  laboratories 
(in member countries); (v) validation and 
evaluation bodies; (vi) private-public part-
nerships;  and  (vii)  several  Expert  Groups 
and Advisory Bodies.

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Pepper 
Platform, 
France

Philippe Hubert 
(Pepper Platform)

Pepper  (Public-private  platform  for  the 
pre-validation  of  endocrine  disruptor 
characterisation methods) is a non-profit 
public-private  association  established  in 
2020 to address the lack of pre-validat-
ed test methods for endocrine disruptors. 
In particular, it addresses the missing link 
between  experimental  design  and  vali-
dated methods; the aim is to fill this gap.

Pepper’s  tasks  are  to:  (i)  find  mature 
methods  that  are  pertinent  to  regulato-
ry  use;  (ii)  prepare  procedures  for  vali-

dation, quantify experimental plans, and 
select labs; (iii) prove the transferability 
of  the  methods  to  other  labs;  (iv)  test 
methods  in  blind  ring  trials  with  many 
substances  to  assess  reliability,  predic-
tive  capacity  and  applicability  domains; 
and  (v)  organise  submissions  to  OECD 
for their adoption as Test Guidelines.

Currently, Pepper  is  working  on  6  meth-
ods, involving 17 labs. Submissions have 
started  for  3  methods,  the  other  3  are 
in  a  transferability  assessment  phase. 

Work on a third series of methods starts 
later in 2022.

The  private  funding  organisations  in-
volved  in  this  private-public  platform 
include  BASF,  BAYER,  LVMH,  and  French 
councils  (chemical  industry,  beauty,  de-
tergent,  and  hygiene  products).  There  is 
a  need  to  extend  resources  not  only  to 
obtain more financial support, but also to 
address the discrepancy that arises from 
most of the money coming from France; 
Pepper needs to be equilibrated.

Cécile Michel-Caillet (ANSES): 
What does it mean in concrete terms 
to perform a validation of a test method?

Anne  Gourmelon  (OECD):  ‘The  process  by  which  the 
reliability and relevance of a particular approach, method, 
process or assessment is established for a defined purpose’. 
This is our definition of validation. Relevance relates to 
the biological and mechanistic meaning of the data. The 
reproducibility  and  reliability  relates  to  the  trust  in  the 
data, and the defined purpose relates to the regulatory rel-
evance of the data.

Then comes the Modular Approach, the process by which 
you  can  achieve  the  validation  of  a  method. This  advo-
cates  for  flexibility  in  undertaking  a  validation  exercise, 
and depends on the pre-existing information available for 
a method. 

There  are  different  ways  to  demonstrate  a  method  is 
valid:  (i)  prospective  validation  (lab  work);  (ii)  perfor-
mance-based  validation  (pre-existing  standard  and  simi-
lar methods); and (iii) retrospective validation (collecting 
existing data). However, in all cases it is necessary to have 

a good protocol description, transparent reporting of re-
sults, and methods that are accessible to future users.

Lisa Baumann (Heidelberg University): iFEDT repre-
sents the first steps of developing a test method. We used 
known methods and endpoints, but did a lot of lab work 
to extend existing guidelines. As researchers, it was a new 
world to us, and knowing that our research will one day 
be an OECD TG changes a bit the way we approach the 
work and how we want to publish it. 

Sharon  Munn  (JRC):  Validation  is  a  very  demanding 
process. It does not always go to plan, and it takes time to 
introduce a new method into a naïve lab. It should also 
be kept in mind that there might be proprietary elements, 
and this should be sorted out at an early stage if the test 
method is to be used commercially.

Cécile Michel-Caillet (ANSES): 
Is it easy to find methods mature enough to 
enter the validation process?

Philippe Hubert (Pepper Platform): The answer is no, to 
find mature methods there are a lot of things to consider. 

17

At Pepper we have designed the ReadEDTest self-assess-
ment questionnaire to help test developers know if their 
methods are mature enough. Factors asked about include 
a  clear  method  description,  the  operational  readiness  of 
a  laboratory,  reproducibility  and  transferability,  histori-
cal  data,  the  presence  of  standard  operating  procedures 
(SOPs), the relevance of methods to regulatory needs, and 
if labs have sufficient time to do prevalidations.

Sharon  Munn  (JRC):  During  the  thyroid  validation 
study we found that the scientific literature was not de-
tailed  enough  to  enable  implementation  in  the  lab,  for 
example, Standard Operating Procedures are often lack-
ing. We think that the application of technical readiness 
criteria will be very helpful, and we have asked scientists 
within the EURION projects to go through such criteria 
themselves, to identify gaps in their own methods.

Cécile Michel-Caillet (ANSES): 
What are the obstacles to the validation by 
the research labs themselves?

Lisa  Baumann  (Heidelberg  University):  Research  labs 
run experiments for publications, the main goal is not to 
produce  methods  that  can  be  run  in  lots  of  other  labs. 
University scientists are under pressure to publish and dis-
cover novel things, making it difficult to allocate funding 
and space to run experiments already performed in several 
other labs, as is required to validate Test Guidelines.

Cécile Michel-Caillet (ANSES): 
Can we take a bit more time to explain 
the differences between a validated 
method and a Test Guideline.

Sharon  Munn  (JRC):  For  a  validated  method  there  is 
a  rigorous  procedure  behind  it  that  shows  the  method 
is  reproducible  and  it  measures  what  it  is  supposed  to 
measure. A validation process also involves an independ-
ent peer review, which puts a quality stamp on it. A Test 
Guideline is the next stage in the process, bringing in reg-
ulatory acceptance and consensus building.

Anne Gourmelon (OECD): A Test Guideline answers a 
regulatory  question,  and  a  context  of  use  coming  from 
a regulatory framework. There is a bit of a ‘chicken and 
egg’ question here: what comes first the validated method 
or the regulatory need?  Regulatory frameworks have to 

be flexible to uptake innovations and new guidelines, to 
close the loop.

Cécile Michel-Caillet (ANSES): 
What is the added value of validated test 
methods per se?

Sharon Munn (JRC): The most important things are the 
quality  stamp;  the  reproducibility;  understanding  sensi-
tivity, specificity and the limitations of the method; and 
the applicability domain.

Lisa  Baumann  (Heidelberg  University):  At  last  week’s 
EURION meeting it was interesting to see the different 
perspectives. There is an idealistic value, to protect human 
health and the environment, and we want to have good 
methods to assess endocrine disruptors, but there are also 
regulatory and other pressures. I think there is added val-
ue in being very detailed in the lab regarding methods, it 
is sound science, and we should write everything we do 
down to ensure robust protocols.

Cécile Michel-Caillet (ANSES): 
What would happen if industry or an 
authority relied on unvalidated methods?

Sharon  Munn  (JRC):  Non-validated  methods,  such  as 
academic data and the scientific literature are used for the 
identification  of  endocrine  disruptors,  especially  when 
Test  Guidelines  are  not  there  to  base  decisions  on. The 
problem is that every time you have to assess if the meth-
od is good or if the results are as expected. With a validat-
ed method that has already been done for you, and you 
understand how to interpret the results.

Cécile Michel-Caillet (ANSES): 
At OECD, there is also the concept of the 
mutual acceptance of data, can you tell 
us a little more about that?

Anne  Gourmelon  (OECD):  The  system  of  mutual  ac-
ceptance of data relies very much on Test Guidelines and 
Good Laboratory Practice. The benefit is that “tested once, 
accepted everywhere” saves resources and avoids duplicate 
testing. So OECD member countries and partner countries 
are accepting data that has been generated in another coun-
try, if they have the same regulatory requirements. 

18

Philippe  Hubert  (Pepper  Platform):  Conversely, 
when    people  use  non-validated  methods,  it  can  be  
very costly.  Different methods (even when duly pub-
lished)  also  do  not  always  give  the  same  results;  you 
can start getting disagreements and controversies.

Anne  Gourmelon  (OECD):  There  are  ways  to  be-
come more efficient. More key players (e.g. contract 
research organisations) need to play a role in valida-
tion. They will make money afterwards selling meth-
ods as services.

Philippe  Hubert  (Pepper  Platform):  I  am  a  bit  less 
optimistic, because if you develop a method from zero 
and fund all the validation yourself you will never have 
payback. That  is  a  good  thing  per  se  in  that  it  is  an 
open system accessible to all; methods are not propri-
etary, but some proprietary elements are submitted to 
FRAND constraints. However, without external fund-
ing there will be nothing in the validation process.

Cécile Michel-Caillet (ANSES): 
How to optimise the existing process? 
Is it necessary to know who the method 
will be used by or what for to get a 
proper validation?

Sharon Munn (JRC): OECD ‘Guidance Document 
34 on the validation and international acceptance of 
new or updated test methods for hazard assessment’ 
talks about validating a method for a particular pur-
pose,  so  in  that  sense  yes;  though  sometimes  it  is 
not that easy. 

You  should  not  start  a  validation  if  you  don’t  know 
what  you  are  aiming  for. There  should  be  an  estab-
lished regulatory need before finding assays to validate, 
though there is a bit of interplay between the two. This 
issue is dealt with by relevance committees in EURL 
ECVAM and Pepper.

Cécile Michel-Caillet (ANSES): 
Do we have any words on how to optimise 
the process and mutualise the efforts?

Lisa  Baumann  (Heidelberg  University):  The  key  is 
the funding and also support while the methods are 
being developed. iFEDT and the EURION Cluster 
are good examples of that. In EURION projects to 
date,  over  40  new  endpoints  and  assays  have  been 
developed that will hopefully go into validation, in-
cluding 15 for the T modality where there are a lot 
of gaps.

Sharon  Munn  (JRC):  For  EURION,  to  bring  in 
prevalidation to get good method descriptions is re-
ally  helpful.  It  would  be  good  to  take  this  forward 
in future research activities, such as PARC, the Part-
nership for the Assessment of Risks from Chemicals 
(launched on 1 May 2022).

19

Discussion and Q&A
Katrina Sichel (moderator) directed audience questions to the panellists.

Q&A

Andrew Turley (Chemical Watch): 
The main beneficiary of new 
validated test methods would seem 
to be the chemical industry, because 
it is test data that enables market 
access under systems like REACH 
or BPR. Do you agree, and is the 
chemical industry pulling its weight 
regarding funding? Does Pepper have 
experiences to share of obtaining 
industry funding for validation?

Philippe Hubert (Pepper Platform): 
It is not that obvious that the chemical 
industry is the one with the most 
interest in this issue. Where you have 
a problem is also in industries selling 
things to people - toys, food, medical 
devices. These industries are faced with 
customers who do not trust products. 
What we are trying to do in Pepper 
is to convince all those industries that 
they have to take part in the fight 
against endocrine disruptors.

Sharon Munn (JRC): Companies are 
involved with validation activities, for 
example, in support of the Thyroid 
Validation Study. There is also a busi-
ness model called External Validation 
where a company can deliver a method 
and results to an independent body for 
peer review.

Anne Gourmelon (OECD): By 
being involved in validation, contract 
research organisations gain proficiency 
and competence in running the meth-
od, then have a competitive advance 
by participating in validation.

Q&A

Erik Prochazka (Cruelty Free Europe): 
With the new ED hazard criteria we are looking at a significant 
increase in animal testing. How can we speed up the validation 
process of new non-animal methods?

Ioana Bere (CHEM Trust): 
How much time does it take, on average, to validate a method 
and also to develop a method?

Judith Giernoth (Covestro): 
Reproducibility was mentioned as key for validation. 
How do you ensure reproducibility for academic studies, 
when they are included into assessments?

Maria Arena (EFSA): 
I agree that eye-related endpoints are population relevant, but don’t you 
think their reversibility can make the assessment challenging?

Lisa Baumann (Heidelberg University): I agree that the ability of animals to recov-
er from the eye damage induced by endocrine disruptors is a challenge in those test 
methods. The younger life stages are more sensitive, though effects are for all ages, so 
I would say it is population relevance. What we advertise in iFEDT and ERGO is 
that we use a combination of endpoints that are related to thyroid, for example, the 
swimbladder is also population relevant and very sensitive to endocrine disruptors. 
For these combinations, we have to assess which tests are best for which endpoints.

Anne Gourmelon (OECD): It takes a few years to validate a method, but we have to 
recognise that when there is a clear need and a clear interest in validating a method, it 
can go quickly if resources are mobilised, and can be done in a couple of years. Also a 
test taking 3 days is different to one taking 90 days, we have to weigh that in.

Sharon Munn (JRC): For academic studies that are not validated, if several papers 
say the same thing there is a Weight of Evidence but reproducibility is not being 
covered. That is what validation is about – to show reproducibility of a method.

Philippe Hubert (Pepper Platform): The reason why we say that it is important 
to have validation is to have the proof of reproducibility. In a published paper you 
usually do not have data to show reproducibility. There are almost no papers on the 
methods themselves; the focus is on substances. Reviewers of papers are not so keen 
on pinning the issues on the methods.

Sharon Munn (JRC): Validation of new non-animal methods (NAMs) is a demand-
ing process, but ways of speeding it up are being investigated and discussed. Usually 
we conclude that there are not many steps we can drop. There are some possible 
ways, such as the leaner Thyroid Validation Study. We could use retrospective data to 
help validation, to avoid big inter-laboratory reproducibility studies. We are constant-
ly looking at how to innovate, so that NAMs can be used as soon as possible.

Cécile Michel-Caillet (ANSES): It should also be quicker to validate in vitro studies 
than chronic in vivo studies.

Anne Gourmelon (OECD): It is a matter of knowing when there is enough data, 
and to maybe not separate too much validation from prevalidation. Maybe it is suffi-
cient to have prevalidation and some retrospective review of what’s out there. 
We need to be innovative in how to validate methods that are really needed.

20

Q&A

Tine Vandenbrouck 
(Apeiron-Team NV): 
How does combining two 
test methods like in iFEDT 
impact the validation? 
Is it a real added value?

Lisa Baumann (Heidelberg 
University): Regarding non-animal 
tests, there is high public pressure for 
good reasons to reduce animal use, 
so industry is facing pressure to use 
them. For combining existing valida-
tion Test Guidelines, the validation 
process should be shorter.

Anne Gourmelon (OECD): Hope-
fully we stop doing this dichotomy of 
prevalidation and then validation. We 
need to come back to the essentials of 
validation, the relevance and repro-
ducibility, and take a decision when 
data is enough, and have trust due to 
adequate protocols.

Q&A

Q&A

Norbert Kaminski 
(Michigan State University): 
I agree that reproducibility is a 
big part of validation, but another 
important part is to convince the 
scientific community that endpoints 
or methods are actually measuring 
what you think they are measuring. 
That is why we use specific model 
compounds that have already been 
well characterised to validate a 
methodology.

Philippe Hubert (Pepper Plat-
form): When we do validations, 
we are trying to find “positive” and 
“negative” substances. It is very im-
portant not to be limited to the old 
well-known ones, and to be open to 
other disciplines such as epidemiolo-
gy or clinical results.

Heather Patisaul 
(North Carolina State University): 
Regarding reproducibility, one 
of the potential advantages of 
moving from animal testing to in 
vitro testing is you can capture 
biological variability that you 
cannot capture in animal models. 
But as you aim for perfect 
reproducibility – are you down to 
one genome, one individual, one 
sex, one age, one moment in time? 
As we move to in vitro testing, there 
is an enormous opportunity to 
capture biological variability.

Anne Gourmelon (OECD): This is 
not part of the validation itself, but 
there are research groups looking at 
biological variability, and upscaling 
our models to what it means in the 
population.

Philippe Hubert (Pepper 
Platform): My last sentence would 
be that we have to stop having to play 
hot potato, validation is something 
expensive, which has to be done and 
financed, so we need to stop discuss-
ing who should do it (industry or 
administration or researchers), 
and involve everyone;  we are in 
this game at the moment.

Anne Gourmelon (OECD): I think 
the principles of validation, establish-
ing relevance, and reproducibility are 
clear, but let’s be more innovative in 
the process and approach so that we 
become more efficient.

Sharon Munn (JRC): Validation and 
regulatory acceptance are interrelated 
but are different processes, so keep 
that in mind.

Lisa Baumann (Heidelberg 
University): I would repeat that 
funding is the central issue, to 
support researchers who develop 
the methods.

21

Session 3: 
Focus on 
thyroid

22

European Cluster to Improve 
Identification of Endocrine 
Disruptors (EURION): 
Emerging results

oto: © European Commission 

h
P

Andreas Kortenkamp 
talked about the 
importance of the thyroid 
hormone system, its 
vulnerability to endocrine 
disruptors, and the work 
of EURION projects to 
develop tests for these 
harmful substances. 

Andreas Kortenkamp
Professor, Human Molecular Toxicology, 
Brunel University London, UK; EURION Cluster; 
European Society of Endocrinology

A shift in cognitive abilities at population 
level, due to impaired brain development 
caused by endocrine disrupting chemicals, 
would  massively  impact  the  number  of 
people with learning difficulties and intel-
lectual disabilities, and also have consid-
erable societal impacts. 

Proper  brain  development  depends  on 
thyroid hormones, which are essential for 
the  proliferation,  migration  and  position-
ing  of  brain  cells.  In  the  1990s,  work  by 
Gabriella  Morreale  de  Escobar  in  Madrid 
showed how important thyroid hormones 
were  to  early  brain  development;  previ-
ously, they were thought not to cross the 
placental barrier. Specialised transporters 
in the placenta are now known to enable 
these hormones to reach the foetus.

Thyroid hormones are thyroxine (T4) and 
triiodothyronine (T3), which differ only by 
one  iodine  atom.  T3  is  the  active  one  in 
circulation, but the most crucial one is T4. 
Three  essential  processes  for  brain  de-
velopment  are  dependent  on  thyroid 
hormones:  (i)  radial  cell  migration  (from 
base  to  brain  cortex);  (ii)  GABA  Switch 
(between  excitatory  and  inhibitory);  and 

(iii)  interneuron  differentiation  leading 
to  network  formation  (connecting  cells 
via synapses). The effects of thyroid hor-
mone  on  these  early  processes  extends 
into adulthood.

Follicular thyroid cells make little internal 
globules,  where  thyroid  hormone  is  syn-
thesised. The process begins when iodide 
is imported into cells. Synthesis is driven 
by  enzymes,  most  importantly  TPO  (thy-
roid  pyroxidase).  The  newly-synthesised 
thyroid  hormone  is  exported  out  of  the 
cell.  Iodide  is  recycled.  Interfering  with 
any of these stages can result in subopti-
mal thyroid hormone synthesis. 

Foetal  development  depends  on  thyroid 
hormones crossing the placenta. Mothers 
with too high or too low thyroid hormones 
in  pregnancy  give  birth  to  babies  with  a 
lower  IQ.  Another  factor  is  iodide  defi-
ciency, requiring supplements to be taken 
during  pregnancy.  Even  mild  iodine  defi-
ciency  can  affect  cognitive  development 
(e.g. language). 

In  the  ATHENA  (EURION  Cluster)  pro-
ject,  these  complex  processes  are  being 

bought  together  in  an  Adverse  Outcome 
Pathway  (AOP)  network.  Several  molec-
ular  initiating  events  lead  to  decreasing 
levels  of  T4  in  the  serum,  impacting  key 
events, eventually leading to adverse out-
comes such as learning and memory im-
pairment, and cognitive function declines. 
This  AOP  is  being  used  to  build  rational 
testing  strategies  for  thyroid  hormone 
disruption,  for  example,  which  assays  to 
deploy and in what sequence.

The  project  team  cover  several  molecu-
lar initiating events using high throughput 
assays  and  screen  tens  of  thousands  of 
chemicals to build up a knowledge base, 
which  feeds  into  work  on  quantitative 
structure-function  relationships.  A  topo-
logical analysis of the AOP revealed nodal 
points,  the  most  important  being  ‘hip-
pocampal gene expression altered’ where 
up  to  nine  pathways  converge,  followed 
by  decreases  in  ‘T3  in  neuronal  tissues’, 
‘T4 in neuronal tissue’, and ‘T4 in serum’.

However, it was found that there are cur-
rently no validated analytical methods for 
measuring  T4  or  T3  in  neuronal  tissues. 
It  is  important  to  get  information  on  T4 

23

and T3 in neuronal tissues and hippocam-
pal  gene  expression  to  interpret  current-
ly  available  assays,  because  there  is  no 
straightforward  linear  relationship  be-
tween T4 in serum and T4 in tissues.  

Key gaps in current OECD Test Guidelines 
and EU testing methods are: (i) no validat-
ed  in  vitro  methods  for  these  Molecular 
Initiating Events – ATHENA and other EU-
RION projects intend to make a substan-
tial contribution here; (ii) serum T4 chang-
es are measured in various guidelines, but 
not  in  pregnant  rats  (where  information 
is  needed);  (iii)  thyroid  histopathology  is 
measured (less usefully); (iv) no methods 
for Adverse Outcomes in the brain; and (v) 
no  consistent  timing  of  serum  T4  meas-
urements across guidelines. 

The  thyroid  gland  features  prominent-
ly  in  testing,  but  what  is  missing  is  the 
brain. One promising test approach could 
be based on ATHENA work at Denmark’s 
Technical  University  on  neuronal  migra-
tion  defects  in  the  brain,  and  the  clear-
ly  seen  histology  affects  as  a  result  of 
improper  thyroid  hormone  action  during 
development.

The EU endocrine disruptor criteria needs 
to show a mode of action (changes to se-
rum  T4),  an  adversity  (change  in  thyroid 
gland histopathology), and a link between 
mode of action and adversity. For thyroid 
hormone  disruption  and  brain  functions, 
advancement  of  the  methods  will  take 
years.  The  ATHENA  team  therefore  pro-
pose a change in the criteria with respect 
to  the  thyroid  hormone  system,  so  that 
serum  T4  and/or  thyroid  gland  histopa-
thology are considered as adverse. 

Clinical  endocrinologists  regard  changes 
in  T4  as  adverse,  and  in  and  of  itself  it 
triggers  clinical  interventions.  In  chem-
ical  regulation  that  is  not  the  case,  and 
we propose to bring the two in alignment 
until we know much more about the inter-
vening key events in these adverse path-
ways,  said  Prof  Kortenkamp.  In  addition, 
validation is proceeding too slowly, and it 
needs more funding at European level.

24

Q&A

Q&A

Martina Klaric (Huntsman): 
If serum T4 has no linear and clear 
relation to T4 in the brain (and 
hence adverse effect), why would 
serum T4 level be considered as 
adversity?

Andreas Kortenkamp: Because, first-
ly, that is all we can do at the moment 
and, secondly, if T4 is definitely low 
in serum in the clinic that is a warning 
sign and will trigger an intervention. 
It is not good news if your serum T4 
level is low, or too high.

Q&A

Sakina Mhaouty-Kodja (ANSES): 
Changes in thyroid hormone levels 
can also affect gene expression. Is it 
something planned in the analysis of 
neuronal tissues?

Andreas Kortenkamp: That’s again 
very tricky – we are measuring 
altered gene expression in neuronal 
tissues. These methods are far from 
validation, but they would be very 
important. You cannot properly regu-
late these substances just on the basis 
of knowledge of T4 levels in serum 
and histopathology. That is how it 
is currently set up, and it is totally 
inadequate.

‘CamilaQM’: 
What do you think about the use of 
ToxCast high throughput essays for 
the investigation of thyroid MIE for 
substances already under evaluation?

Andreas Kortenkamp: I think that 
is a good idea. It is the reason why in 
ATHENA we do not cover in vitro 
assays for molecular initiating events 
that are well advanced already, we are 
covering the gaps. I would support 
that, with one caveat. In work at 
the Danish Technical University in 
ATHENA on TPO enzyme, some 
of these chemicals were surprisingly 
found not to be active in an animal. 
This means that there is a step missing, 
not only in vitro assays but also input 
from toxicokinetics to tell us if these 
chemicals are likely to accumulate or 
reach tissues at the right concentration 
to produce an effect.

Q&A

Andrew Turley (Chemical Watch): 
You said that in a clinical setting 
serum T4 levels are indicative of 
adversity. Could you spell out what 
that means?

Andreas Kortenkamp: You will get 
medication, especially an expectant 
mother if a doctor finds T4 in blood 
serum is too low, with a drug that 
bumps up thyroid hormone levels.

Q&A

Cécile Michel-Caillet (ANSES): 
To clarify, if we need to measure things in neuronal tissues, it means we need in vivo 
studies, or would 3D models be sufficient to answer this request?

Andreas Kortenkamp: Models may help as well. Going back to basics, it may be 
a bit premature to advocate routine blanket measurement of these hormones in 
neuronal tissues. I would say, at the present time, we need as a minimum a couple 
of chemicals to know how changes in serum T4 translate into hormonal levels in 
neuronal tissues. We know this for some chemicals but not enough, it is not routine 
testing but the results will enable us to come up with better testing strategies.

oto: © Florian Caiment

h
P

Florian Caiment
Toxicogenomics department, Maastricht University

SCREENED 
project 
(EURION 
Cluster)

Florian Caiment presented 
an overview of SCREENED 
(Screening for Endocrine 
Disruptors), the smallest of the 
eight EURION Cluster projects. 

SCREENED has three main objectives: (i) 
develop  new  3D  in  vitro  assays/models 
that  mimic  the  complex  structure  and 
function  of  thyroid  glands;  (ii)  gain  un-
derstanding  of  adverse  effects  of  en-
docrine disruptors on thyroid, by testing 
compounds  in  assays;  and  (iii)  engage 
in  dialogue  with  regulatory  agencies,  to 
ensure that the assays developed are fit 
for risk assessment.

The  first  in  vitro  3D  model  developed  (in 
Sabine  Costagliola’s  lab  at  ULB)  was  an 
organoid,  using  murine  embryonic  stem 
cell-derived  thyroid  follicles;  then  hu-
man-derived  stem  calls  to  monitor  cell 
proliferation, differentiation and organisa-
tion; and lastly using functional thyroid fol-
licles from induced pluripotent stem cells.

The  second  3D  model  being  developed 
(by Roberto Toni’s team at the University 
of  Parma)  is  a  decellurised  thyroid.  The 
aim is to obtain sex-specific thyroid lobe 
matrices from young adult rats, destroy-
ing  the  cell  and  keeping  the  surrounding 
structure, so that human cell material can 
be added surrounded by functional tissue.

The  third  3D  model  being  developed  (by 
Lorenzo Moroni’s team in Maastricht) is a 
bioprinted thyroid. The aim is to optimise 
bioprinting processing parameters (e.g. fi-
bre size, type of hydrogel) and to develop 
a bioprinted perfusable vascular network. 
The  functional  bioprinted  thyroid  sits  in-
side a hydrogel, enabling it to be exposed 
to test substances.

There  has  also  been  significant  progress 

in  developing  other  highly  innovative 
technologies,  in  particular  a  microbiore-
actor that mimics the circulatory system 
in the body. Three sets of carrier module 
have  been  successfully  developed,  along 
with protocols for harvesting cells.

These new possibilities for a  microbiore-
actor were demonstrated (in Stefan Gisel-
brecht’s lab at Maastricht University), with 
different  cavity  shapes  (flat  carrier,  tray, 
μ-cavities)  and  different  membranous 
carriers for various cell models: (i) single 
flow  (below  or  above  cells);  (ii)  counter 
flow  (different  directions  above  and  be-
low cells), and (iii) cross-flow (same direc-
tions above and below cells).

All  the  models  were  challenged  with  se-
lected  endocrine  disruptors,  in  this  case 
4  phthalates  (used  in  the  plastics  indus-
try),  4  organophosphate  flame  retard-
ants (OPFRs), 4 polychlorinated biphenyls 
(PCBs),  and  4  polycyclic  aromatic  hydro-
carbons  (PAHs).  In  the  3D  models,  these 
16  compounds  were  screened  over  24 
hours.  Most  of  the  markers  used  were 
very highly expressed.

The project team looked at available omics 
data (e.g. genomics, proteomics) to identi-
fy gene and protein signatures for known 
classes of endocrine disruptors. Five com-
pounds were selected for cytotoxic studies 
(DEHP, TPP, PCB-118, PCB-153, BaP), using 
a freely-available culture of human local-
ised thyroid cells, in assays over 24 hours. 
Thyroglobulin was consistently detected in 
all samples with the selected substances, 
confirming it was a good thyroid model.

The  next  steps,  said  Dr  Caiment,  were 
setting up a pilot bioreactor inside an in-
cubator,  starting  with  different  doses  of 
substances  flowing  over  mouse  thyroid 
follicles  in  membranous  carriers.  Tran-
scriptomics  and  proteomics  are  being 
measured (compared to a static petri dish 
system).  The  team  are  also  working  to 
get omics accepted in regulatory risk as-
sessments. In addition, they will validate 
in  silico  models  with  experimental  data 
for  AOPs,  perform  in  vitro  testing  of  en-
docrine disruptors in the 3D models, and 
validate an integrated omics platform for 
endocrine disruptor screening.

Q&A

Katrina Sichel (moderator): 
SCREENED developing new assays 
for risk assessment using highly inno-
vative technologies, but also engaged 
with regulators to ensure that assays 
developed are fit for risk assessment. 
How do you see regulators respond-
ing to these models?

Florian Caiment: At first, we had 
ambitions to start validating the 
models against an in vivo rat model, 
to compare them. This is an impor-
tant step to have them accepted by 
regulators, but it had to be removed 
from the project due to the difficulty 
of engaging with animal models.

25

The pesticide database 
on thyroid hormone 
disrupting system

oto: © Andrea Terron 

h
P

Andrea Terron gave an 
overview of endocrine disruptor 
assessments conducted by 
EFSA up to September 2022. 
In total, 95 pesticide active 
substances were assessed 
for human health and/or non-
target organisms in line with 
the ECHA-EFSA Guidance 
Document (2018). 

Andrea Terron
Scientific Officer, European 
Food Safety Authority (EFSA)

For  human  health,  24  substances  were 
considered to have no endocrine disruptor 
activity;  for  27  a  waiver  was  considered 
(e.g.  inert  substances);  for  32  substanc-
es  additional  data  was  requested;  and 
10  substances  were  considered  to  have 
endocrine  disruption  properties.  Docu-
mentation on human health and non-tar-
get organisms are available on the EFSA 
website. 

Of the 10 substances identified as meet-
ing the criteria for endocrine disruptors, 7 
affected the T (thyroid) modality (benthi-
avalicarb, mancozeb, metiram, clofentez-
ine,  asulam,  metribuzin,  and  thiabenda-
zole),  with  one  each  affecting  the  E,  A, 
and S modalities.

The  T  modality  has  been  evaluated  for 
95  substances  so  far.  Additional  data 
was  requested  for  6  of  those  substanc-
es,  with  the  Comparative  Thyroid  Assay 
(CTA)  requested  in  5  cases  (focusing  on 
the  sensitive  population:  the  foetus  and 
new-borns). For one substance, the OECD 
TG 443, with inclusion of the assessment 
of the thyroid hormone system and of the 
second generation, was requested.

In line with EFSA/ECHA guidance, the com-
pleteness of the available dataset for the 
T  modality  is  generally  not  an  issue  for 
plant  protection  products;  for  both  sub-
stances under renewal as well as for new 
active substances. In the current dataset, 
the tests available are: Thyroid histologi-
cal  evaluation  (a  standard  requirement), 
thyroid weight (frequently assessed), and 
thyroid hormone (T3 and T4) and thyroid 
stimulating hormone (TSH) levels (gener-
ally included as additional information).

In the dataset of pesticides evaluated to 
date, there has been no case of a drop in 
T4 or increase in TSH without evidence of 
changes  in  the  thyroid  histology;  though 
there  have  been  perturbations  in  hor-
mone levels with concomitant changes in 
thyroid histology. 

There  are  several  factors  to  consider 
regarding  the  database;  including  lack 
of  robust  data  on  modes  of  action  (e.g. 
reliance  on  methods  to  measure  thyroid 
hormone  levels);  a  lack  of  data  in  the 
sensitive  population  (foetus,  new-borns); 
the  intrinsic  limitations  of  the  subjective 
thyroid histopathology evaluation and the 

possibility of false negatives for molecu-
lar  initiating  events  that  do  not  produce 
key  event  changes  in  TSH  and/or  thyroid 
histology.

Thyroid  histology  is  considered  a  valid 
endpoint  for  hazard  identification,  and 
when  positive  it  indicates  that  the  TSH 
pathway  is  affected.  The  comparative 
thyroid  assay  (CTA)  would  likely  repre-
sent  the  best  study  design  for  the  as-
sessment  of  thyroid  histopathology  and 
changes in thyroid hormones in the sen-
sitive population.

Indeed,  developmental  neurotoxicity 
(DNT)  is  the  real  adverse  outcome  of 
concern  for  thyroid  hormone  system 
disruption.  However,  the  sensitivity  of 
DNT  endpoints  for  changes  in  T4  is  still 
unknown,  and  endpoints  included  in  the 
DNT  studies  are  not  EATS  mediated  but 
EATS  sensitive.  Therefore,  T  modality 
conclusions  would  highly  benefit  from 
the inclusion of mechanistic data in the 
Weight of Evidence analysis.

In  conclusion,  looking  at  the  outlook  for 
the  T  modality,  Dr  Terron  noted:  (i)  it  is 

26

 
oto: © Andrea Terron 

h

P

Katrina Sichel (moderator) 
selected audience questions to ask.

Q&A

Q&A

Cécile Michel-Caillet (ANSES): 
You mentioned DNT endpoints 
coming from TG 443 or TG 426 
studies. Have you views on the quali-
ty of data you get from these studies? 
Can you comment further on the 
limits of histopathology?

Andrea Terron: Yes, moving from the 
OECD TG 426 to the OECD TG 
443, the quality of the study for the 
DNT is indeed an issue. We generally 
have many comments, for example, 
on how methods are described and 
how data is reported. We think that 
frequently more expertise or educa-
tional training are needed to conduct 
the DNT arm in the OECD TG 443. 
This issue should be resolved, because 
it concerns chemicals assessment and 
animal use.

On the intrinsic limitation of thyroid 
histopathology: The pathologist 
scores (e.g. minimal, mild, moderate, 
marked, or very marked) are subjec-
tive. This works well when the effect 
is evident, but the sensitivity of the 
eyes will never match the sensitivity 
of a morphometric analysis which is 
more objective and potentially able to 
capture effects at lower doses.

Svenia Jasper (Henkel): There is no 
Test Guideline listed under the PPP/
BP Guidance Document for T-relat-
ed mechanisms for human health. 
Which test would you recommend?

Andrea Terron: The best scenario 
would be to determine if the mo-
lecular initiating events have been 
affected; then thyroid hormones are 
very critical because the histology will 
work only for some specific pathway. 
It is practically difficult to move away 
from thyroid histological evaluation, 
but the match between histology and 
hormones in the sensitive population 
remains a critical step forward. I hope 
that projects like ATHENA will give 
the possibility of measuring the thy-
roid hormone in the brain or identify 
biomarkers to be used as an endpoint 
in the overall Weight of Evidence.

Q&A

Natacha Cingotti (Health and 
Environment Alliance - HEAL): 
Renewal of suspected thyroid 
disruptors is paused due to data 
gaps (e.g. ziram, cyprodynil, 
spinosad). So data completeness is 
also an issue for renewals?

Andrea Terron: As a matter of princi-
ple, I do not remember any dataset for 
which histology was not conducted. 
Sometimes we have less data on organ 
weight, but in the majority of pivotal 
studies thyroid gland histology is as-
sessed, and if positive this is sufficient, 
but we cannot exclude some false 
negatives. The dataset is frequently 
not adequate for non-target organisms 
(non-mammalian).

27

important  to  investigate  the  molecular 
initiating events to better tailor the test-
ing strategy, to minimise false negatives 
and help identify the mode of action; (ii) 
give  priority  to  the  CTA  as  a  critical  in 
vivo study and learn how to use both his-
tology and thyroid hormones as T-medi-
ated  adverse  endpoints  in  the  sensitive 
population; and (iii) increase the sensitiv-
ity of DNT endpoints indicative of disrup-
tion of the thyroid hormone system to be 
applied in the regulatory setting; though, 
it is appreciated that this is a very chal-
lenging research task.

Methods for the identification 
of thyroid hormone disrupting 
substances, Update on JRC 
coordinated activities

oto: © European Commission

h
P

Sharon Munn gave an overview of 
recent JRC activities on identifying 
endocrine disruptors affecting the 
thyroid hormone system. 

Sharon Munn 
Senior Scientific Officer, Joint Research 
Centre (JRC), European Commission

Thyroid  disruption  can  result  in  metabol-
ic  disorders,  problems  with  growth  and 
development,  learning  deficiencies,  hear-
ing  loss,  and  visual  defects.  The  thyroid 
hormone  (TH)  signalling  system  can  be 
perturbed  in  the  hypothalamus,  pituitary, 
thyroid  glands  (via  interference  with  hor-
mone  synthesis),  through  metabolism  of 
TH  in  the  liver,  transport  of  hormone  via 
serum binding proteins, transport into cells 
and through interaction with thyroid recep-
tor in the cells around the body. An OECD 
Scoping Document in 2014 describes the 
methods  available  to  measure  disruption 
to thyroid hormone activity, and their read-
iness for validation. 

The OECD Conceptual Framework lists all 
the  Test  Guidelines  (TGs)  for  measuring 
endocrine  disruption.  There  is  a  big  gap 
for Level 2 tests (in vitro mechanistic as-
says) for measuring effects on the thyroid 
pathway.  Therefore,  the  JRC’s  European 
Union  Reference  Laboratory  for  Alterna-
tives  to  Animal  Testing  (EURL  ECVAM), 
with  the  help  of  15  EU-NETVAL  labora-
tories and 14 method developers, carried 
out validation studies for 18 methods to 
address this gap.

Methods  were  selected  on  the  basis  of:  (i) 
level  of  readiness;  (ii)  suitability  for  imple-
mentation  in  a  wide  variety  of  laboratories 
(e.g. no complex equipment); (iii) confidence 
that test systems will be available in the fu-
ture (e.g. accessible cell lines); (iv) where pos-
sible, human-based cells and high through-
put  systems;  and  (v)  exclusion  of  human 
embryonic stem cells on ethical grounds. 

Other issues considered included the train-
ing  needs  of  EU-NETVAL  laboratories,  ap-
plication  of  OECD’s  Good  In  vitro  Method 
Practices (GIVIMP) Guidance Document, and 
using  these  methods  in  a  Good  Laborato-
ry  Practice  (GLP)-compliant  environment. 
In  the  move  to  non-animal  methods,  the 
substitution of animal-derived reagents for 

in vitro tests was also investigated and ap-
plied where practicable. 

The  18  methods  cover  all  8  blocks  of  the 
OECD  Scoping  Document:  higher  level  sig-
nalling from brain to thyroid gland (block 1); 
thyroid hormone synthesis (2); serum-bind-
ing proteins (3); metabolism and excretion 
by the liver (4); transport into cells (5); in-
side  cell  interaction  with  human  thyroid 
hormone  receptors  (6);  zebrafish  embryo 
assay for T4 (7); and integrative cellular as-
says looking at consequences of over/under 
stimulation of TH receptors (8).

The  validation  study  was  divided  into  two 
stages. Firstly, the definition of the in vitro 
method (and a set of SOPs) and five valid 
runs with a limited number of control and 
reference chemicals to examine robustness 
and  reliability.  If  successful,  the  method 
was  tested  with  a  wider  range  of  chemi-
cals – 30 blind-coded chemicals to achieve 
three valid runs per test chemical.

To select the 30 chemicals tested, experts 
were  consulted.  They  considered  existing 
evidence from in vitro and in vivo methods, 
clinical  and  epidemiology  data.  The  final 
selection  also  considered  distribution  in 
chemical space, solubility, and commercial 
availability.  At  least  two  chemicals  were 
selected  for  each  mode  of  action  tested. 
Progress  can  be  monitored  on  the  TSAR 
website  (Tracking  System  for  Alternative 
methods  towards  Regulatory  acceptance) 
– search for ‘thyroid’. 

The aim is to bring these methods forward 
into  regulatory  use,  as  Test  Guidelines.  To 
this end, the OECD Thyroid Methods Disrup-
tion Expert Group was established, in June 
2022,  to  coordinate  the  development  of 
Test Guidelines for thyroid-related methods. 

Ms Munn showed an image of an Adverse 
Outcome  Pathway  (AOP)  network  for  thy-
roid  hormone  system  disruption,  depicting 

all  the  points  a  chemical  may  interact  at 
the  molecular  level  to  impact  thyroid  hor-
mone  levels  in  serum  and  tissues,  leading 
to an array of adverse effects in mammals 
(including  humans),  fish  and  amphibians. 
The selected methods in the validation study 
were located at different points on this AOP 
network, with the aim of building an integrat-
ed  testing  strategy  to  identify  thyroid  hor-
mone  system  disruptors.  This  could  include 
the  use  of  toxicokinetic  models  to  predict 
what concentrations reach the brain and tis-
sues, compared to the effects shown in vitro.

In  summary,  Ms  Munn  said  that  to  accel-
erate assessments for endocrine disruptors, 
mechanistic (mode of action) knowledge is 
needed. New technologies can provide this 
mechanistic  information,  but  trust  in  their 
reliability and relevance is required. That is 
the reason for the EURL ECVAM validation 
study of methods to assess the disruption 
of  the  thyroid  hormone  signalling  system, 
which will be finalised in April 2023.

Q&A

Andreas Natsch (Givaudan): 
You have more than 10 tests and only 2 
chemicals per “Mode of Action”/assay: 
So these assays will only be validated 
for robustness but not for predictivity, 
how to deal with this?

Sharon Munn: We had that balance 
to make and the idea was to test these 
30 chemicals in all the assays. We were 
trying a ‘lean’ validation. Some aspects are 
missing, like transferability to other labs. 
BASF included extra chemicals, relevant 
to the particular mode of action. Data 
could also exist in the literature, so the 
OECD Expert Group is pulling this all 
together to identify remaining gaps and 
needs for further validation work.

28

oto: © European Commission

h

P

How to differentiate 
general toxicity-related 
endocrine effects from 
endocrine disruption: 
the case study of 
carbon disulfide

oto: © European Commission

h
P

Sakina Mhaouty-Kodja shared the 
experiences of the Endocrine Disrupting 
Chemicals Working Group at ANSES in 
identifying substances with endocrine 
disrupting activity.

Sakina Mhaouty-Kodja
Chairperson of the EDC Working 
Group, ANSES, France

The ANSES methodology asks: (Q1) what 
is a substance’s adverse effect? (Q2) does 
it  act  through  an  endocrine  mode  of  ac-
tion (MOA)? and (Q3) does the endocrine 
MOA induce the adverse effect identified? 

To answer these questions, data is need-
ed from regulatory and academic studies. 
However,  there  are  issues  with  appropri-
ate  analysis,  statistics,  information  on 
hormonal levels, weight of hormone-sen-
sitive organs, and (for females) oestrous 
stage  at  the  day  of  tissue  collection  or 
analysis. A particular issue with regulato-
ry studies is that huge doses of substanc-
es  are  often  given,  making  it  difficult 
to  discriminate  between  endocrine  and 
non-endocrine (general toxicity) modes of 
action on endocrine systems. 

This  can  be  illustrated  by  recent  ANSES 
work  on  carbon  disulfide  (CS2).  It  is  a 
highly volatile liquid, used in the industri-
al  manufacture  of  regenerated  cellulose 
and  viscose.  It  is  also  used  as  an  inter-
mediate in the manufacture of dithiocar-
bamate pesticides, and was considered a 
risk to workers, and to people and the en-
vironment near manufacturing facilities.

In  EU  regulations,  carbon  disulfide  is 
classified  as  a  suspected  substance  for 

reproductive  toxicity  (damaging  fertility 
and  unborn  child  development);  and  for 
severe health effects, in particular on the 
nervous and cardiovascular systems. That 
is  why  it  has  a  current  occupational  ex-
posure limit of only 15 mg/m3. It is listed 
as a potential endocrine disruptor in two 
lists: the ‘Database of endocrine disrupt-
ing  chemicals  and  their  toxicity  profiles’ 
(DEDuCT list), and ‘The Endocrine Disrup-
tion Exchange’ list (TEDX).

For the EDC Working Group evaluation of 
CS2,  all  available  information  was  gath-
ered  from  Test  Guidelines,  the  extend-
ed  one-generation  reproductive  toxicity 
study (2019), the scientific literature, and 
reports  of  other  agencies.  This  data  was 
summarised on the ECHA website and in 
a recent publication (2022).

Looking  at  the  adverse  effects  identi-
fied  for  CS2  from  available  data,  it  can 
induce  effects  on  male  and  female  re-
production  (e.g.  reduce  sperm  count  and 
embryonic  development).  Concerning 
the  nervous  system,  it  can  reduce  brain 
weight and change the thickness of some 
sub-regions  of  the  brain  associated  with 
retinal atrophy. Behavioural effects from 
academic  studies  on  developmental  ex-
posure,  for  lower  doses  than  used  in  TG, 

impaired 

showed  induced  delayed  sensorimotor 
development  and 
locomotor 
activity. More data is available for adults 
(using  rodents),  which  showed  impaired 
motor activity, visual response and spatial 
learning and memory; as well as reduced 
brain  weight,  retinal  atrophy,  and  axonal 
swelling of nerve fibres. Furthermore, hu-
man studies from workers exposed to CS2 
showed reduced nerve conduction veloci-
ty, impaired performance on psychomotor 
tests, altered autonomic nervous system, 
and retinopathy.

Data from a US EPA report did not show 
any effect of CS2 on EATS modalities, but 
CS2  is  metabolised  into  thiourea  which 
is  active  on  thyroid  peroxidase  (TPO),  an 
enzyme  important  in  thyroid  hormone 
synthesis,  which  may  explain  why  expo-
sure  to  CS2  lowers  T4  levels  in  rats  (in 
vivo mechanistic studies) and in humans 
(occupational  exposure).  From  this  data, 
we  can  suggest  that  CS2  fulfils  the  first 
two  criteria  for  an  endocrine  disruptor, 
and  has  a  possible  adverse  pathway  via 
thiourea  that  impacts  thyroid  hormone 
levels through TPO disruption. 

However, an analysis of all the data shows 
that CS2 has several other modes of ac-
tion. It can also: (i) interact with amino ac-

29

ids  to  form  dithiocarbamate,  these  have 
been  shown  to  trigger  axonal  swelling, 
and  to  reduce  dopamine  β-hydroxylase 
and noradrenaline levels associated with 
the  behavioural  impairments  observed 
in  experimental  studies;  (ii)  reduce  nitric 
oxide synthases activity, important in the 
production  of  the  brain  neurotransmit-
ter nitric acid; and (iii) increase oxidative 
stress in the brain. 

Therefore,  there  are  several  modes  of 
action, working in parallel. To understand 
the  most  plausible  mode  of  action  (e.g. 
endocrine  or  non-endocrine)  to  trigger 
the thyroid and nervous system effects, 
the  ANSES  group  used  the  European 
guidance  criteria  (temporality,  specifici-
ty, essentiality…).

In  summary:  (i)  neurotoxicity  induced 
behaviour  impairment  but  also  reduced 
brain weight and axonal swelling; (ii) thy-
roid  disruption  due  to  endocrine  activity 
was found at the same doses that trigger 
neurotoxicity; (iii) some of the neurotoxic 
effects preceded the reduced levels of T4; 
(iv) thyroid disruption did not appear as an 
initiating event; and (v) the high cytotoxic 
potential  of  the  substance  is  not  selec-
tive of endocrine cells. It was concluded, 
based on all the knowledge gathered, that 
CS2 does not fulfil the WHO definition to 
be classified as an endocrine disruptor (as 
implemented in EU regulation).

Dr  Mhaouty-Kodja  said  their  conclusion 
was  in  contradiction  with  DEDuCT  and 
TEDX  lists.  This  highlights  the  need  for 
thorough analysis using all available data 
and knowledge; and the relevance of Test 
Guidelines that can discriminate between 
endocrine versus non-endocrine modes of 
action, in particular TGs using doses that 
are lower than those inducing general or 
systemic toxicity. This is not just specific 
to the thyroid system, but can be applied 
to other endocrine systems.

30

Q&A

Q&A

Martina Klaric (Huntsman):
Is ECVAM planning to develop 
also the defined approaches out 
of the validated methods?

Sharon Munn: We have not 
made the decision to lead that 
activity at this point, we are just 
finishing off  the validation, but 
we would be interested in being 
involved in such activities. We 
look forward to ATHENA and 
ERGO delivering some proposals 
for what a testing strategy might 
look like.

Q&A

Barnali James 
(FMC Corporation): 
Is there any further information 
on why the NIS work stalled? 

Sharon Munn: The NIS is 
not stalled now, it has started 
again. It was mainly stalled 
due to COVID and should be 
completed in April next year.

Svenia Jasper (Henkel): 
In accordance with the horizontal 
approach, which T-related test is 
likely to be adopted into the PPP/BP 
Guidance Document? What is 
the proposed timeline?

Sharon Munn (JRC): T-related tests 
are used in in vitro tests. In that sense, 
REACH and the PPR and BPR regula-
tions are in the process of updating data 
requirements, and the concern is that 
timelines do not really match. A place-
holder was put into REACH informa-
tion requirements for the in vitro thyroid 
methods, and we probably won’t be in 
time for the next update, but we need 
to look at the possibilities for phasing in 
these tests when they become available.

Q&A

Andreas Kortenkamp 
(Brunel University): 
It seems to me that CS2 is a damn 
dangerous substance. You have 
produced data to show it may not be 
classed as an endocrine disruptor under 
EU law, the consequences may be that 
it is less restricted. Are we not dealing 
here with a crazy loophole in the whole 
regulatory arena? Is it not time to intro-
duce tougher criteria for developmental 
neurotoxicants?

Cécile Michel-Caillet (ANSES): 
The presentation focused on the analysis 
of all the data regarding the endocrine 
disruption endpoint. This case led us 
to question how to use ECHA/EFSA 
guidance, and to see the limits of data we 
have in hand to answer difficult questions 
such as secondary unspecific toxicity while 
working with highly toxic substances like 
CS2. On the side of regulation, we have 
produced a Risk Management Option 
Analysis (RMOA), so we do not foresee any 
relaxing of the regulation at all.

Sakina Mhaouty-Kodja: To complete 
the answer, with the TG 443 we did 
not have enough data on developmental 
neurotoxicity. I heard Lisa Baumann’s 
presentation about merging test guidelines 
and maybe we could consider this for oth-
er TGs, including neural toxicity which is 
lacking in data.

Session 4: 
Developments 
on bisphenols 

31

Keynote: Bisphenols 
Health Impacts 
on Children

oto: © Anne-Simone Parent

h
P

Anne-Simone Parent 
talked about the 
effects of bisphenols 
on children’s health. 

Anne-Simone Parent
European Society for Paediatric Endocrinology; 
European Society of Endocrinology; Endocrine 
Society; University of Liège, Belgium

We are all exposed to dozens of endocrine 
disrupting  chemicals,  she  said.  Some  of 
them  are  considered  non-persistent,  such 
as  bisphenols  that  are  rapidly  eliminated 
from the body but we are exposed to them 
every day. Others are persistent, such as the 
PCBs or flame retardants that accumulate 
in  the  adipose  tissue  and  can  stay  in  the 
body for several decades. 

Data  from  the  European  Human  Biomon-
itoring  Initiative  (HBM4EU)  show  that  all 
adult humans across Europe are exposed to 
at least low levels of bisphenol A (BPA). BPA 
is detected in more 90% of the population 
on  a  given  day.  Importantly,  replacement 
chemicals such as bisphenol S and bisphe-
nol F are also now detected in 50% of the 
samples analysed under HBM4EU.

There are two main windows of sensitivity 
to endocrine disruptors, the foetus and the 
child.  This  is  when  crucial  developmental 
processes are set up, such as the neuronal 
connections  that  will  control  reproduction, 
energy  balance  or  cognitive  function.  Hor-
mones play a crucial role in those process-
es,  and  therefore  the  foetus  and  the  child 
are extremely sensitive to very low doses of 
endocrine disrupting chemicals.

The  risk  is  high  because  endocrine  dis-
ruptors can cross the placenta; the foetus 
and children have high biological and met-
abolic  sensitivity  and  are  less  able  than 
adults  to  eliminate  endocrine  disruptors; 
and children use their mouths to discover 
the world, increasing the possibility of oral 
endocrine  disruptor  intake.  There  are  two 
important  concepts  when  discussing  the 
effects of endocrine disruptors on children. 
One  is  the  critical  window  of  sensitivity, 
meaning  that  developing  organisms  are 
very  sensitive  to  environmental  factors. 
The  other  is  the  development  origin  of 
health and disease, whereby our environ-
ment  during  these  sensitive  early  stages 
defines our health as adults.

Bisphenols are ubiquitous due to their use 
in  plastics.  They  mimic  estrogens,  but  can 
also  disrupt  thyroid  hormone  action.  They 
are  detected  in  amniotic  fluid  and  cord 
blood, so can pass from mother to unborn 
baby. They are present in more than 90% of 
the population in the EU. 

Studies have repeatedly shown that a few 
years after foetal exposure to BPA, children 
showed impaired working memory. Another 
study  showed  that  exposure  to  bisphenol 
F and other replacement chemicals during 
foetal  life  was  associated  with  impaired 
cognitive function in boys 7 years after ex-
posure. It has also been repeatedly shown 
in  laboratory  studies  that  developmental 
exposure to BPA is associated with abnor-
mal neuronal development.

BPA is also considered to be an obesogen. 
Development  exposure  is  associated  with 
increased  weight  gain  in  rodents,  and  lab 
studies  have  shown  that  exposure  in-
creased  food  intake  and  caused  abnormal 
fat  development.  In  epidemiological  stud-
ies, with children exposed to four different 
levels of BPA, obesity prevalence more than 
doubled in children with the highest meas-
ured levels of urinary BPA.

Another important concept is that different 
tissues and organs have different sensitiv-
ities  to  given  endocrine  disruptors.  This  is 
the case for bisphenols. A dose of 300 000 
μg/kg  bw/day  of  BPA  leads  to  an  increase 
to the weight of the ovary, but much lower 
levels of exposure (down to 0.03 μg/kg bw/
day), consistent with human exposure, are 
associated  with  disruption  of  genes  in  the 
hypothalamus  in  the  brain,  which  controls 
puberty  and  reproduction.  In  more  recent 
studies, exposure to such low doses for bi-
sphenol S are involved in the disruption of 
genes in the placenta and in foetal growth.

A final concept that I would like to illustrate, 
said Prof Parent, is the BPA legacy. DNA has 
an epigenetic 3D organisation that is trans-
mitted  from  one  generation  to  the  next. 
Several studies have shown that BPA mod-
ifies the genetic organisation of DNA. This 
can  be  transmitted  to  the  next  generation 
and, via exposure of the foetus’ reproduc-
tion cells is also observed in the third and 
fourth generations, even though they have 
never seen the compound. Transgeneration-
al effects of BPA have been shown for obe-
sity, behaviour, and reproduction.

In conclusion: (i) developing organisms are 
extremely sensitive to endocrine disruption; 
(ii) different organs/systems have different 
sensitivities  to  endocrine  disrupting  chem-
icals;  (iii)  and  in  particular  the  brain  is  ex-
tremely sensitive to BPA at low levels; and 
(iv)  replacement  chemicals  have  similar 
effects at environmentally relevant levels.

Q&A

Katrina Sichel (moderator): 
Are Test Guidelines sufficient to 
capture the effects that you describe?

Anne-Simone Parent: I think that the 
tests don’t cover all aspects, in particular 
they do not take into account the windows 
of sensitivity or transgenerational effects, 
or the different sensitivities of tissues and 
organs to a given compound. I would like 
to recommend the consensus statement 
that was published in Nature Review En-
docrinology by a group of endocrinology 
experts who established 10 key character-
istics of endocrine disruptors. If you look, 
only 3 of the 10 characteristics are covered 
by the current recommended tests; BPA 
corresponds to 9 of the key characteristics.

32

oto: © Anne-Simone Parent

h

P

New approaches for 
regulatory toxicology: 
the bisphenols experience 

This panel discussion on bisphenols was moderated by Cécile Michel-
Caillet (ANSES). The panel members first gave presentations of their work.

oto: © European Commission

h
P

Luísa  Camacho,  in  her  own  opinion, 
talked about work in the National Center 
for Toxicological Research (NCTR) on bi-
sphenol  A  (BPA).  The  NCTR  is  a  part  of 
the  U.S.  FDA  located  in  Arkansas  and, 
unlike  the  other  Washington  DC-based 
centers,  it  is  focused  on  research  rath-
er  than  regulation.  NCTR’s  mission  is  to 
conduct  scientific  research  to  support 
the decision-making of the FDA.

BPA  is  a  high-production  volume  indus-
trial  chemical  used  in  the  production  of 
many consumer products, including poly-
carbonate plastic food and drink contain-
ers,  and  epoxy  resins  used  to  line  cans. 
BPA  migration  to  foods  results  in  wide-
spread  low-level  human  exposure,  cur-
rently estimated to be lower than 0.5 μg/
kg body weight/day.

In  2018,  two  reports  from  independent 
expert  groups  (NTP/CERHR  and  FDA/CF-
SAN)  identified  data  gaps  for  BPA.  The 
FDA/NCTR Research Program on BPA was 

CLARITY BPA: 
FDA/NCTR 
conclusions

Luísa Camacho
Deputy Director, Division of Biochemical Toxicology, 
National Center for Toxicological Research (NCTR), 
Food and Drug Administration (FDA), USA

set  up  to  address  these  gaps,  including 
toxicokinetics  studies;  PBPK  (physiolog-
ically  based  pharmacokinetic)  models; 
and a 90-day toxicity study that included 
perinatal exposure and internal dosimetry 
measurements.  The  group  has  produced 
numerous peer-reviewed publications.

al  scientists  in  the  USA.  It  was  a  novel 
collaborative  research  model  to  bridge 
investigate  and  applied-regulatory  sci-
ence research. CLARITY-BPA had two key 
components:  (i)  NCTR  core  study  (2-year 
chronic  toxicity  study),  and  (ii)  grantee 
studies.

The  90-day  toxicity  rat  study  identified 
several  endpoints  affected  by  high  dos-
es of BPA (resulting in a serum free BPA 
maximum concentration of 300 000 pM) 
and the synthetic estrogen (control) ethi-
nylestradiol  (EE2).  The  BPA  effects  over-
lapped in most part with EE2, supporting 
the view that at high levels BPA acts as a 
weak estrogen. No effects were detected 
for low doses of BPA (2.5 up to 2 700 μg/
kg bw/day) or naïve (non-gavage) control.

CLARITY-BPA  (Consortium  Linking  Aca-
demic and Regulatory Insights on Bisphe-
nol A Toxicity) is a consortium of National 
Institute of Environmental Health Scienc-
es  (NIEHS)-funded  grantees  and  feder-

The  CLARITY-BPA  Grantee  Studies  eval-
uated  endpoints  (molecular,  structural 
and  functional)  not  typically  assessed  in 
guideline  studies  (brain  and  behaviour, 
cardiac, immune, mammary gland, ovary, 
penile  function,  prostate  gland  and  ure-
thra,  testis  and  epididymis,  metabolism 
and  thyroid  hormones,  and  uterus).  The 
published peer-reviewed papers, to Octo-
ber 2021, are compiled in National Tox-
icology Program (NTP) Research Report 
18, and all raw data is on NTP’s Chemical 
Effects  on  Biological  Systems  (CEBS) 
database to ensure transparency.

used 
Both  NCTR 
Sprague-Dawley  rats  from  the  same  lit-

grantees 

and 

33

ters,  housed  and  dosed  together,  to  op-
timise  animal  use  and  data  integration. 
Both  also  used  specially-selected  diets 
and housing to minimise background ex-
posure to phytoestrogens and BPA. Back-
ground exposure to BPA was monitored: in 
diets  it  was  0.03-0.2  μg/kg  bw/day,  with 
no detectable exposure from housing. 

25 000 μg/kg bw/day) and two doses of 
estrogen control (EE2 at 0.05 and 0.5 μg/
kg bw/day). Endpoints assessed included 
body  weight,  survival,  vaginal  opening 
and  cytology,  litter  parameters,  1-year 
sacrifice (e.g. organ weights, sperm anal-
ysis,  histopathology),  and  2-year  sacri-
fice (histopathology).

There  were  two  dosing  arms:  ‘continuous 
dosing’  (pregnant  mothers,  then  direct-
ly  from  birth  to  2  years);  and  ‘stop  dose’ 
(dosing stopped at weaning on day 21 and 
animals kept for 2 years) to identify peri-
natal exposure induced effects later in life.

There were several exposure groups: ve-
hicle  control,  5  doses  of  BPA  (2.5  up  to 

EE2 
induced  dose-dependent  adverse 
effects,  mainly  in  females  and  minimal 
effects in males. There were statistically 
significant  effects  at  all  BPA  doses,  but 
below  25  000  μg/kg  bw/day  they  were 
unlikely to be treatment-specific because 
of a lack of dose-response, inconsistency 
across sacrifice times/dosing arms, and a 
lack of pattern across biologically-related 

endpoints  and  organ  systems.  However, 
adverse  effects  on  female  reproductive 
tissues and male pituitary in the 25 000 
μg/kg bw/day group may be treatment re-
lated.

Overall, said Dr Camacho, our conclusion 
is  that  the  data  does  not  support  “low 
dose”  effects  or  non-monotonic  dose-re-
sponse  by  BPA.  The  main  goal  was  to 
have  these  data  integrated  with  all  the 
other  scientific  literature  on  BPA,  includ-
ing  grantee  studies  and  other  studies, 
across the world, to contribute to the on-
going safety assessment of BPA.

CLARITY BPA 
experience

oto: © European Commission

h
P

Norbert Kaminski
Director, Center for Research on Ingredient 
Safety, Michigan State University, USA

Norbert Kaminski, in his own opinion, gave 
a  grantee’s  perspective  of  the  CLARITY 
BPA  study.  He  identified  the  contribution 
of  academic  investigators  in  labs  across 
the US, provided an academic’s perspective 
on the major strengths of the CLARITY-BPA 
Program, and gave an overview of the im-
munotoxicological evaluation of BPA.

Academic  laboratories  were  included  in 
CLARITY-BPA to provide input beyond that 
typically  found  in  guideline  studies  that 
assess  chemical  safety.  Most  academic 
labs  focus  on  mechanism,  and  that  was 
a major contribution. In terms of the im-
munotoxicology  evaluation  at  Michigan 
State, for instance, it was a combination 
of  guideline/core  and  non-core  experi-
mental approaches.

Among the strengths of CLARITY-BPA: (i) it 
addressed one of the biggest challenges 
of working with BPA, its ubiquitous nature, 
with a protocol that went to great lengths 
to minimise and monitor background ex-
posure,  something  most  academic  labs 
are  unable  to  do;  (ii)  the  sharing  of  ani-
mals minimised their use (though a lot of 

animals  were  used)  and  enabled  tissues 
to be traced back to a particular animal; 
(iii)  tissues  received  by  grantees  were 
blinded,  so  investigators  did  not  know 
which tissues they were receiving (which 
is important in light of the AMGEN paper 
showing  that  when  studies  were  blinded 
they were more likely to be reproduced by 
another  laboratory);  and  (iv)  the  results 
and detailed methodologies are in a pub-
licly-accessible  centralised  database,  for 
anyone to further analyse.

A paper by Heindel et al. (2020) summa-
rised  work  on  data  integration,  analysis, 
and interpretation conducted by eight ac-
ademic (grantee) CLARITY-BPA studies.

On  the  immunotoxicological  evaluation 
of  BPA,  it  should  be  noted  that  there  is 
no  single  assay  as  the  immune  system 
comprises many different cell types with 
different functions. Our main interest was 
in  estrogen-related  receptors,  said  Prof 
Kaminski, due to several recent Japanese 
papers  suggesting  that  BPA  had  a  higher 
binding affinity to estrogen-related recep-
tors (ERR) than the estrogen receptor (ER). 

Starting  on  day  21,  the  Michigan  lab 
evaluated  leukocyte  composition  of  thy-
mus and spleen, for different cell types in 
terms  of  their  proportionality  in  the  two 
organs. They measured 21 different cellu-
lar phenotypes. On day 90, then 6 months 
and 1 year, they continued characterising 
leukocytes  cell  types  in  the  spleen,  but 
in  addition  quantified  various  leukocyte 
functions  in  these  spleen  subpopula-
tions.  116  different  functional  endpoints 
were measured per spleen, totalling 137 
endpoints  per  animal.  So  over  4  years, 
every time the lab received tissues from 
NCTR,  an  assembly  line  was  immediate-
ly formed, with people assaying different 
endpoints.  To  ensure  cell  viability,  the 
NCTR harvested spleens late in the after-
noon  for  shipping  overnight,  for  work  to 
begin by noon the next day.

For  the  immunological  characterisation, 
the  team  measured  lymphocyte  B  cells 
and T cells (and two subpopulations); NK 
(natural killer) T cells (and two subpopula-
tions); and assessed several endpoints for 
innate immune cells (myeloid and granu-
locytes).  In  terms  of  function,  measure-

34

ments were made for lymphocyte B cells 
(e.g. antibody production of IgM), T cells, 
myeloid activation, and NK activation.

For  the  immunological  characterisation, 
for  all  cell  types,  there  was  little  effect 
on innate immune cells. In terms of mye-
loid cells, the one interesting finding was 
for the classical dendritic cells, where the 
same effects occurred with estradiol, the 

positive  control  and  BPA.  There  were  no 
effects for antibody production by B cells. 
For activation of T cell responses, modest 
effects occurred with estradiol but not BPA.

In  conclusion,  Prof  Kaminski  said  that 
a  trend  was  seen  beginning  at  day  21, 
which  became  significant  at  90  days,  in 
splenic  classical  dendritic  cell  numbers 
was observed in female rats. However, no 

sustained effects were observed on either 
leukocyte  cell  subpopulations,  for  either 
total cell number or function, with either 
estradiol  or  BPA  treatment.  The  results 
were  published  in  companion  papers  in 
Toxicology in 2018: Part 1 and Part 2.

Q&A

Cécile Michel-Caillet (moderator) invited two further members of the 
CLARTY-BPA study, Heather Patisaul and Gale Prins, to contribute 
as part of this Q&A session, and also asked the first question: 

How many animals were used in the study?

Luísa Camacho (CLARITY-BPA): We have large animal 
rooms at NCTR and still we used all our animal space. 
That  is  why,  although  grantees  thought  it  important  to 
also run the estrogen control, initially not included in the 
study plan, we were only able to accommodate the EE2 
‘continuous dosing’ but not the ‘stop dose’ arm.

Heather Patisaul (CLARITY-BPA grantee, North Car-
olina  State  University),  a  neuroscientist,  talked  about 
other aspects of CLARITY-BPA. 

The study made very efficient use of animals and aligns 
with the 3Rs. It leveraged the unique strengths of guide-
line and academic studies. There is a need to expand tox-
icity  testing,  and  the  core  study  did  not  have  any  brain 
endpoints. This  is  something  as  a  grantee  I  and  Cheryl 
Rosenfeld  (University  of  Missouri)  could  bring  to  the 
table.  Between  us  we  published  seven  different  studies, 
looking  at  behaviour,  neuroanatomy,  gene  expression, 
brain development, and epigenetics. We did a lot of this 
work at NCTR. 

Another  unique  aspect  of  CLARITY-BPA  was  that  the 
tissues for diverse endpoint studies come from the same 
animals. This  permits  a  systems  level  analysis  of  all  the 
data, including data that can be difficult to interpret in 

isolation.  When  this  cohesive  systems-level  analysis  was 
done, what CLARITY-BPA showed was that there were 
very concordant effects on the organs that you would pre-
dict would be sensitive to BPA, namely the brain, ovary, 
prostate, and mammary gland. These results were consist-
ent with the literature and human data. So I think there is 
a lot of lessons learnt that can benefit others interested in 
pursuing this type of endeavour. 

Gale Prins (CLARITY-BPA grantee, University of Illi-
nois at Chicago) introduced her work as principal inves-
tigator of academic studies on the prostate gland. 

In  CLARITY-BPA,  we  were  able  to  repeat  our  previous 
studies that showed significant carcinogenic effects on the 
prostate gland with very low doses of BPA with additional 
estrogen exposure. A major strength of CLARITY-BPA is 
that it allowed evaluation of multiple organ endpoints, by 
sharing tissues from the same animals. This also enabled 
the integration of the statistically significant data from ac-
ademic scientists, and permitted the expansion of a study 
to  a  systems  biology  level.  A  group  of  us  published  our 
findings on this in the Heindel et al. (2020) paper.

The  findings  reveal  that  BPA  exposures  simultaneously 
affect numerous organs in individual rats, a strong indi-

35

we used dietary dosing, in which the exposure during the 
perinatal  period  was  through  the  dam/mother,  while  in 
the 90-day and CLARITY-BPA studies we directly dosed 
the pups rather than rely on milk transfer. The different 
routes of exposure resulted in very different internal do-
simetry to EE2 hence it is not surprising that the effects 
across studies were not 100% aligned. 

Robert Thomas Zoeller (University of Massachusetts): 
What kind of variability is imposed by 
the pattern of analysis, and how can you 
could measure it?

Norbert Kaminski: Surprisingly (or maybe not) the var-
iability in general was not much, mainly because most 
of the endpoints we measured were by flow cytology, so 
you are evaluating probably about 10 and 30 000 cells 
per endpoint. In the two publications from our lab, we 
show  graphically  those  endpoints  with  some  effects  or 
changes, giving an idea of the level of variability, which 
is very small.

Ninja Reineke (CHEM Trust): 
I understand one of the main aims of 
CLARITY was to compare academic and 
guideline studies. Why didn’t the FDA publish 
an integrated report to extract even more 
lessons from this innovative approach?

Luísa Camacho: We published our data not only in the 
NTP  Research  Report  9,  but  also  as  a  peer-reviewed 
publication.  In  that  manuscript  we  did  try  to  integrate 
our data with the grantees’ data published in the mean-
time.  Overall,  I  think  the  consortium  initially  intended 
to  publish  a  final  report  that  integrated  all  the  streams 
of data, but after 10 years of working together and since 
the regulators will reanalyse and reinterpret the data for 
their needs, I think there was a general consensus to do in-
stead a compendium of all the peer-reviewed publications 
(NTP Research Report 18). All the raw data is available 
for further analysis by others. So, the publication of the 
NTP compendium was the end of the consortium.

cation that the data are not spurious nor random events. 
Importantly, this approach confirmed that below 2.5 μg/
kg bw/day, dose effects of BPA were observed in multiple 
organs and tissues at once, thus shedding light on the in-
terconnectedness of these low dose effects across biologi-
cal systems. 

To summarise, the data indicates that BPA effects in one 
tissue  were  correlated  with  effects  in  another  tissue,  in-
cluding  immune  cells,  thus  providing  strong  evidence 
that the data are revealing true effects at a low BPA dose in 
this  guideline  Good  Laboratory  Practice  (GLP)-compli-
ant study. It also shows that these low dose effects lead to 
systemic health consequences. Importantly, the only way 
to obtain this information was by using a CLARITY-like 
design, and I strongly recommend that others use this ap-
proach in the future.

Cecile Michel-Caillet (ANSES): 
You have mentioned that it is really 
important that we now have data for multiple 
systems in one single animal. Are data 
published animal-by-animal?

Gail  Prins:  This  was  published  in  Reproductive Toxi-
cology (Heindel et al., 2020), where all the integration 
was done.

Katrina Sichel (moderator) addressed 
audience questions to the panel.

Laura Vandenberg (University of Massachusetts): 
Can you speak about reproducibility? 17a-EE 
was  included  in  CLARITY  and  a  prior  NTP 
study but numerous outcomes had opposite ef-
fects between these studies?

Luísa  Camacho  (CLARITY-BPA):  We  did  a  90-day 
study before CLARITY-BPA and in both studies we in-
cluded a reference estrogen control. The EE2 doses in the 
CLARITY study were ten-fold lower than in the previous 
study  to  really  understand  the  sensitivity  of  the  animal 
model  to  estrogenic  activity.  In  addition  to  these  stud-
ies,  we  also  conducted  NTP-funded  multi-generational 
studies using the same animal model and animal diet, but 
there was a major difference in study design between the 
previous  NTP  multi-generational  studies  with  EE2  and 
the  CLARITY-BPA  study.  In  the  previous  NTP  studies 

36

grated at the end. A weakness of the CLARITY study was 
that we did not have our FDA partners with us when we 
integrated the data.

Andreas Kortenkamp (Brunel University): 
To an outsider the contrast between the guide-
line studies and the grantee studies is consider-
able. Do you have any idea why you don’t see 
an effect and the grantees do?

Luísa Camacho: The endpoints are different, the inter-
pretations of data are different, so all together that comes 
to different findings for the same data. 

Malik Duhaut (Covestro): 
I see quite a discrepancy between your 
results and the results of EFSA. Very strong 
immunotoxin effects were detected by EFSA 
in their conclusion. How do you explain this 
discrepancy?

Norbert Kaminski: First of all, we did not evaluate the 
subpopulation of T cells called TH17 used in the EFSA 
study. The reason I think the EFSA conclusion is curious 
is that what they tried to do was link TH17 cells (which 
secrete a pro-inflammatory cytokine) with epidemiology 
studies on allergic respiratory airway disease. There is no 
scientific data cited in the EFSA report, or any in the liter-
ature, that show that TH17 calls have an effect on allergic 
airway  responses,  which  are  mediated  by TH2  cells  (so 
there is no plausible link). In addition, the epidemiology 
studies they try to link with the TH17 cells I suggest are 
very weak in terms of exposure, often having single data 
points,  either  for  urine  or  blood,  for  BPA  exposure.  Fi-
nally, there are as many epidemiological studies showing 
a link to allergic respiratory disease with exposure to BPA 
as there are those that do not. Therefore, I am puzzled by 
the findings and conclusions of the EFSA report.

Ana Soto (Tufts University School of Medicine): 
What statistical criteria do you use to assess 
non-monotonic dose responses (NMDR) and 
do these criteria ever detect NMDR?

Luísa  Camacho:  Test  Guideline  statistical  studies  usu-
ally  don’t  look  for  non-monotonic  dose  responses,  they 
assume a linear response. In our study, we did run addi-
tional  statistical  tests  despite  the  risk  of  maximising  the 
number of statistical findings that are not necessarily re-
lated to treatment. For instance, for the pathology data, in 
addition to the usual tests we also ran two additional tests, 
one being the relative treatment effect (RTE) method that 
does not assume a linear response and would probably be 
sensitive to a non-monotonic dose response.

Heather Patisaul: I just want to correct something that 
Luísa mentioned earlier, she said there was consensus on 
not producing an integrated study and that is inaccurate. 
Eight of the ten grantees took it upon themselves to do 
such a published study (i.e. Heindel et al., 2020). I think 
that  in  a  consortium  study  like  this,  there  needs  to  be 
accountability from start to finish to ensure data get inte-

37

An EU Member State 
experience on the 
grouping of bisphenols

oto: © European Commission

h
P

Christian Unkelbach gave a regulatory 
overview on bisphenol A (BPA) with a 
focus on REACH, and an update on the 
German restriction proposal on bisphenols. 
His organisation, BAuA, is the competent 
authority for REACH in Germany, working 
in close collaboration with the German 
Environment Agency. 

Christian Unkelbach
Federal Institute for Occupational Safety 
and Health (BAuA), Germany 

In  2012,  BPA  was  included  in  the  first 
Community  Rolling  Action  Plan  (CoRAP) 
for substance evaluation, which started its 
assessment in Germany for endocrine dis-
ruption and other concerns. This substance 
evaluation  was  concluded  in  late  2016, 
and published in 2017.

The  conclusion  of  this  substance  evalu-
ation  was  that  for  human  health  no  fur-
ther regulatory measures were necessary, 
though  there  were  some  risk  character-
isation  observations  for  specific  catego-
ries  in  worst-case  assumptions.  However, 
for  the  environment  the  information  was 
sufficient to conclude that BPA was an en-
docrine disruptor, and also a Substance of 
Very  High  Concern  (SVHC)  under  REACH; 
therefore requiring measures to minimise 
emissions to the environment. 

This  substance  evaluation  procedure  ini-
tiated a German Regulatory Management 
Option  Analysis  (RMOA),  an  informal  pro-
cess for directing regulatory action against 
BPA.  In  2017,  BAuA  started  a  two-month 
consultation  with  stakeholders  and  met 
with  commenting  parties,  leading  to  the 
publication  of  the  RMOA  conclusion  doc-
ument  on  ECHA’s  website  (PACT  section). 
This concluded the necessity to clarify the 
endocrine disruptor properties for the en-
vironment  of  BPA  at  the  EU  level  via  the 
SVHC  identification  process  under  REACH, 
as a first step, followed by a restriction to 
minimise exposure in the environment. 

French  colleagues  have  also  worked  on 
BPA  and  human  health,  and  they  identi-

fied BPA’s reprotoxic and endocrine disrup-
tor  properties  for  human  health  in  2016. 
Germany  added  the  label  of  endocrine 
disruptor for the environment to the can-
didate  list  entry  for  BPA  under  REACH  in 
late 2017.

recommendation 

A place on the candidate list requires ECHA 
to  recommend  substances,  at  least  bian-
nually, for inclusion in Annex XIV of REACH. 
These  are  the  substances  undergoing  au-
thorisation.  ECHA’s 
is 
based on a points system using three crite-
ria: hazard, tonnage, and uses (BPA scores 
quite  highly  on  this  prioritisation  scale). 
ECHA forwarded the list including BPA to the 
Commission in 2019. However, the German 
RMOA concluded that inclusion in Annex XIV 
was  the  second-best  regulatory  measure 
for BPA, after a tailored restriction to reduce 
emissions to the environment.

Restrictions  are  included  in  Annex  XVII  of 
REACH.  Restrictions  may  be  tailored  for 
the placing on the market of substances in 
mixtures, articles or on their own.

A  preliminary  wording  of  the  planned  re-
strictions on bisphenols (2021) centred on 
BPA, but can encompass other bisphenols 
of concern, if they fulfil structural require-
ments, and are identified as endocrine dis-
ruptors for the environment.

A core set of bisphenols will be addressed 
for  restriction:  (i)  BPA  and  bisphenol  B 
(already  identified  as  endocrine  disrup-
tors for the environment); (ii) bisphenol S 
(proposed  for  identification);  and  (iii)  bi-

38

sphenol F and bisphenol AF (and its salts) 
(special assessment under REACH by the 
Member  State  Committee).  Additional-
ly,  BAuA  adopts  a  semi-dynamic  mech-
anism,  to  enable  the  future  inclusion  of 
other substances in the bisphenol family 
with  endocrine  disruptor  properties  into 
the restriction. 

Therefore,  concluded  Mr  Unkelbach,  this 
is not a wholesale ban on bisphenols, or a 
ban  on  bisphenols  solely  based  on  struc-
tural  similarity  to  BPA.  To  qualify  for  re-
strictions, the bisphenol structure has to be 
linked with endocrine disrupting properties 
in the environment for which EU-wide con-
sensus has to be established.

The  ‘Planned  Restriction  on  Bisphenols’ 
will  proceed:  (i)  via  two  additional  infor-
mal ‘Calls for Evidence’ (launched Novem-
ber 2020 and October 2021); (ii) a public 
consultation  process  for  bisphenol  F  and 
bisphenol  AF  for  the  environment,  which 
will inform Member State opinion making; 
(iii)  the  Annex  XV  restriction  dossier  was 
to  be  submitted  by  7  October  2022;  and 
(iv)  a  formal  6-month  public  consultation 
will be launched after the dossier has been 
approved by ECHA‘s scientific committees 
(RAC risk assessment committee and SEAC 
Committee  for  Socio-economic  Analysis), 
which is expected to happen at the end of 
2022 or in early 2023.

oto: © European Commission

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European Chemicals 
Agency work on 
bisphenols 

oto: © European Commission

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Francesca Pellizzato 
presented work that ECHA has 
done on the bisphenol group 
with some Member States, in 
particularly Germany, Belgium, 
France and Sweden. 

Francesca Pellizzato
Scientific Officer, European Chemicals 
Agency (ECHA), Helsinki, Finland

ECHA’s  Integrated  Regulatory  Strategy  is 
a process under which authorities identify 
and address substances of concern. There 
are  different  interconnected  regulatory 
processes, but work on groups is central to 
the overall reiterative process. It is a trans-
parent  process,  as  ECHA  publishes  on  its 
website  the  Annual  Report,  the  Chemical 
Universe mapping of all REACH registered 
substances  to  the  respective  regulatory 
action pool, and the Assessments of Regu-
latory Needs (e.g. for bisphenols).

By  grouping  substances  we:  (i)  screen 
groups  of  substances  with  a  focus  on 
those with suspected priority hazards and 
highest  potential  for  exposure;  (ii)  bring 
consistency in how similar substances are 
treated; (iii) target the right substances at 
the right time; (iv) pool information to ena-
ble faster action; (v) increase predictability 
of authorities’ actions; and (vi) support in-
formed  substitutions,  and  avoid  regretta-
ble substitutions.

The  bisphenol  group  focuses  on  148  bi-
sphenols which share the same backbone, 
but  differ  in  the  bridge  between  the  two 
phenyl  rings,  or  have  different  substitu-
ents, or where phenolic hydroxyls could be 
derivatised. There is an upper cut-off value 
for Molecular Weight of 600. The bisphe-
nols list comprises BPA and its derivatives 
(half of the 148); BPS and its derivatives; 
BPF and its derivatives; BPAF and its salts; 
and two heterogeneous subgroups.

In terms of hazard, the focus is on endo-
crine disrupting properties for both human 

health and environment, but other hazards 
are  also  covered  (e.g.  reprotoxicity,  skin 
sensitisation).  The  methods  used  include 
finding patterns across groups, QSAR mod-
elling,  and  data  on  mammals  to  inform 
environmental considerations (holistic ap-
proach). The information sources included 
REACH  registration  dossiers,  other  REACH 
and CLP information, and previous assess-
ments under EU legislation. 

ing  a  proposal  to  restrict  use  of  BPA  and 
other  bisphenols  with  environmental  en-
docrine disruptor properties; (iii) once it is 
clearer which bisphenols the German pro-
posal covers, the Commission will consider 
any further needs for regulatory action; (iv) 
extending the restriction of BPA in thermal 
paper; and (vi) as BPAF and its salt are also 
defined as PFAS they will be considered in 
the planned PFAS restriction.

Ms  Pellizzato  concluded  that  assessing 
chemicals  in  groups  has  been  a  success-
ful approach. It makes it faster to identify 
which chemicals need regulatory action or 
more data, and those chemicals for which 
no  further  action  is  needed.  The  case  of 
bisphenols  has  proved  that  working  with 
groups  of  substances  improves  consist-
ency  and  coherence  of  regulatory  work, 
helps  to  avoid  regrettable  substitutions, 
and helps identifying early on substances 
registered  for  intermediate  uses  only,  or 
currently not registered but which could be 
potential substitutes for known substances 
of concern. 

Further  information  can  be  found  on 
ECHA’s website, including the assessment 
of  regulatory  needs  for  bisphenols  and 
the Integrated Regulatory Strategy.

Exposure  information  came  from  REACH 
registration  dossiers  (though  the  Chem-
ical  Safety  Reports  were  not  necessarily 
looked at), and information on uses is used 
as proxy for human exposure and releases 
to the environment. The potential for expo-
sure  was  considered  high  for  substances 
with widespread uses. 

Grouping for regulatory action divided the 
148 bisphenols into four groups: (i) based 
on ‘Need for EU regulatory action’ (34 bi-
sphenols); (ii) ‘Inconclusive - no hypothesis 
yet’ (22); (iii) ‘Currently no need for action’ 
(26);  and  (iv)  ‘Currently  no  action  –  not 
possible to conclude’ (66).

Of the 34 substances with a need for ac-
tion, hazards were confirmed for 3 (includ-
ing  BPA);  information  is  available  to  con-
firm  hazards  for  9  (actions  are  ongoing); 
and data for another 9 bisphenols may be 
required to confirm hazards.

The  proposed  actions  are:  (i)  a  group  re-
striction  has  been  identified  as  the  best 
way to manage the risks of 34 bisphenols; 
(ii) German authorities are already prepar-

39

Q&A

Q&A

Judith Giernoth (Covestro): 
You did not consider the REACH 
registration Chemical Safety 
Reports of the substances. 
Could you explain why?

Francesca Pellizzato: This is a screen-
ing exercise, so we don’t have time 
to go through the Chemicals Safety 
Report included in each substance 
dossier. We take uses as proxy for 
exposure information.

Cecile Michel-Caillet (ANSES): 
For the two groups of 26 and 66 
substances, which data did you con-
sider, those in the dossiers or those 
available in the literature?

Francesca Pellizzato: As it is a 
screening exercise, ECHA is not 
generally fishing for information 
outside of the registration dossiers. 
For bisphenols, Member States did 
alert us to relevant information out 
there useful for assessments, but as a 
general rule we stick to the informa-
tion in the registration dossier.

Q&A

Natacha Cingotti (HEAL): 
Why not already initiate a single 
restriction on all 34 bisphenols of 
concern to ECHA instead of duplicat-
ing efforts (with the limited German 
restriction for now)?

Francesca Pellizzato: As I said, it 
would be preferable to have a SVHC 
identification in REACH or a classifi-
cation in the CLP Regulation as a first 
step, before proceeding with a restric-
tion. I understand the urgency to act 
on these substances, but unfortunately 
we do not have the basis for acting 
on all of them immediately, although 
I think all of them would fulfil the 
definition of environmental endocrine 
disruptors, so they could go into the 
restriction from Germany.

Christian Unkelbach: This was con-
sidered as one of the many imaginable 
options, but data is lacking to really 
extend the restriction to all of the 
bisphenols identified at the screening 
level by ECHA.

Cecile Michel-Caillet (ANSES): 
For what was presented by Norbert 
Kaminski, I think there is one point we 
should probably highlight. It was said 
that based on the AMGEN paper, using 
blinded samples increases reproducibil-
ity of the data produced by others. We 
need to have validated Test Guidelines, 
but if we can have reproducible results 
it’s already a good point when consider-
ing the Weight of Evidence approach.

Q&A

Sandra Jen (EDC-Free Europe coalition): 
It is great we are having these scientific discussions on the effects of endocrine disrup-
tors on the foetus and children across generations, but maybe we are missing a bit 
the human rights perspective. There has been lots of developments on this front late-
ly, especially on children’s rights, and the right to a toxic-free environment. I think 
we could imagine that affected EU citizens start envisaging legal actions for breach 
of these human rights, like the right not to be exposed to hundreds of chemicals 
before even being born. Is this something that regulators are starting to consider?

Christian Unkelbach: We are proposing to restrict bisphenols with a risk for the 
environment, but there will obviously be an impact on humans via the environment, 
so there will be a reduction of bisphenol exposure to humans if the restriction proceeds 
as we intend it to. With regards to potential legal action that is not something we have 
deeply considered.

Cecile Michel-Caillet (ANSES): I hope you see that we are trying to speed up our 
work using the grouping approach, with the aim of protecting the environment now 
and for our children.

Katrina Sichel (moderator): 
Can you tell us a bit more about the 
semi-dynamic extension mechanism?

Christian Unkelbach: We did not 
want the restriction to just be buried 
in Annex XVII and stay there. We 
wanted to give the Commission an 
opportunity to add further bisphenols 
as they are identified as endocrine dis-
ruptors. There is a precedent for CMR 
(carcinogenic, mutagenic, or toxic for 
reproduction) substances in consumer 
products, where the Commission is 
able to add further substances.

Q&A

Andreas Natsch (Givaudan): 
Does the restriction in the environ-
ment consider the rapid biodegrada-
tion of BPA and hence low exposure?

Christian Unkelbach: The restriction 
does consider degradation data in BPA 
in the dossier. The second part alludes to 
a potential lack of risk, but for us even 
low exposures of environmental endo-
crine disruptors are a cause of concern, 
and would give rise to this restriction. 
Therefore, biodegradation of BPA is not 
a reason to exonerate this substance, 
which is ubiquitous in the environment.

Q&A

Andrew Turley (Chemical Watch): 
Regarding the restriction proposal 
and this dynamic mechanism, are 
you envisaging that the inclusion 
of new bisphenols would be an 
automatic thing, through a harmo-
nised classification, or would there 
be a procedural mechanism that the 
Commission would follow with some 
sort of consultation process?

Christian Unkelbach: In short, it is 
the second option you proposed, that 
is where the semi-dynamic comes 
in. The inclusion should not only be 
based on the hazard identification, 
which would occur for example under 
the CLH (harmonised classification 
and labelling) procedure. There would 
be some additional kind of consid-
eration with regard, for example, to 
transition periods for specific uses.

40

Q&A

Gale Prins (University of Illinois at Chicago): 
Samples were not only blinded when they came to us, but we had to upload all 
data into a CEBs database where it was locked down and no-one had access to 
it to change anything once quality control was done; it became a read only format. 
They did not release the results back to us until all the investigators locked down 
their data. Once everyone had finished, the data was decoded. It was tightly 
controlled in terms of making sure no-one was talking to each other behind the 
scenes about what they found in different groups. I see that as beneficial, 
it provides confidence and transparency. 

Erik Prochazka (Cruelty Free Europe): 
The regulatory action on bisphenols took a long time – did conflicting results 
from animal studies contribute to this, and could it be resolved using NAMs?

Adama Traore (Givaudan): 
EU vs USA strategies: might harmonisation be helpful for the industry, 
particularly for BPA.

Norbert Kaminski (Michigan State University): I think harmonisation is very impor-
tant, especially from the private sector perspective because it is very difficult to make 
specialised products that are just for certain countries, or certain parts of the world. I 
would also, regarding data not being consistent across studies, say that the results are 
still a little unsettled, and I think a very important aspect is exposure levels. One also 
needs to consider what levels people are exposed to, because every chemical at some 
dose is going to be toxic.

Luísa Camacho: To frame CLARITY-BPA data in terms of FDA vs grantees, I don’t 
think that is a correct dichotomy. I think there is space for discussion in how to 
interpret the data and that is part of the scientific process. I think our primary role 
as researchers is to generate data of sufficiently good quality for regulators to use for 
decision making, and I think we were very successful in CLARITY-BPA in that respect. 
FDA is also interested in NAMs and reducing animal use whenever possible, and efforts 
are ongoing in the US to improve reproducibility and other aspects of NAMs.

Francesca Pellizzato: I don’t think that conflicting results is the issue for why it takes 
so long to regulate substances. With regard to NAMs we have tried QSARs for the 
148 bisphenols to identify degradation products or metabolites. This exercise did not 
give very good results for the BPA derivatives. However, for the subgroup of TBBPA 
and its derivatives results were very encouraging and in the Assessment Report we 
based our conclusion a lot on QSARs, but we then need further data to confirm 
what NAMs are predicting.

Christian Unkelbach: With current legislation in place, NAMs can inform on a 
potential concern but it is then necessary to have other methods to come to a robust 
conclusion, and that is the framework we are working with right now.

Cecile Michel-Caillet (ANSES): NAMs are really useful for endocrine activity, but we 
have three parts in our definition, and it is more difficult for adverse effects. It did not 
prevent France from identifying bisphenol B, but animal data are still needed.

Luísa Camacho: Metabolism is important when defining toxicity and sometimes 
NAMs fail there; it is important to keep this in mind.

Q&A

Andreas Przybilla: 
If ECHA acknowledges the risk of bi-
sphenols as endocrine disruptors, but 
lacks data from registration dossiers, 
wouldn’t it be necessary to create the 
data themselves, for example, with 
direct requests to laboratories?

Francesca Pellizzato: ECHA is not 
currently asking laboratories to per-
form tests. I think there is some dis-
cussion at Commission level to funds 
regulators to test some substances, but 
at the moment there is nothing of this 
sort in REACH.

Cecile Michel-Caillet (ANSES): 
Maybe we could mention the PARC 
initiative that goes in that direction, to 
help us create data that are missing.

Q&A

Judith Giernoth (Covestro): 
You mentioned that in the 
CLARITY study you found effects 
that were maybe treatment related 
in the highest dose group, in 25 
000 μg/kg bw/day. Could you also 
say whether the effects could be 
considered as adverse?

Luísa Camacho: In the 90-day study 
the highest two doses were 100 000 
and 300 000 μg/kg bw/day of BPA, 
continuous doses. Those two doses, 
especially the highest one, induced 
several consistent adverse effects in 
related endpoints, so we are very 
confident those doses of BPA induced 
adverse effects. In the CLARITY-BPA 
chronic study, the highest BPA dose 
was 25 000 μg/kg bw/day. There 
were some signals in that dose group 
that may be interpreted as treatment 
effects, but they were subtle, for exam-
ple, in the male pituitary and uterus in 
continuous dose, but not as robust as 
in the 90-day study.

41

Conclusions 
and next steps

oto: © European Commission

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In the form of a conversation 
with moderator Katrina Sichel, 
Cristina de Avila summed 
up some of the Forum’s key 
outcomes and looked ahead to 
the next steps.

Cristina de Avila
Head of Unit, Safe and Sustainable Chemicals, 
DG Environment, European Commission

What were the key takeaways for you? 
What particularly resonated for 
you and for the Commission?

In  the  Forum,  we  focused  on  test  meth-
ods and research. There are some 40 test 
methods  for  endocrine  disruptors  in  the 
pipeline, but validation is crucial and this is 
a  process  that  needs  widespread  cooper-
ation  and  considerable  funding  going  for-
ward. The early integration of the regulato-
ry approach was also highlighted. 

We heard a very important and concerning 
message, which is the very specific effects 
of endocrine disruption on the thyroid and 
the  link  with  declining  IQ-levels.  This  is 
alarming news for our society, and it is ex-
tremely important that we make the right 
decisions now.

We  organised  the  Forum  to  listen  to  dif-
ferent perspectives, exchange information, 
and to seek synergies. We also learn from 
other regions, and I would like to thank in 
particular our US speakers for having joined 
us for this exchange.

What do you think are the main take-
aways to underline for the audience?

Endocrine  disruptors  are  a  very  specific 
form of hazard and they are probably the 
most complex. 

The  first  segment  of  this  year’s  Forum  in 
Paris was very informative on how to com-
municate on endocrine disruptors. The gen-
eral  public  has  been  made  aware,  and  is 
rightfully concerned about these hazards. It 
is our responsibility as a public authority to 
not only raise awareness, but also take the 
necessary regulatory actions. 

As an EU institution we feel it is beneficial 

to continue working closely with the Mem-
ber  States  that  are  willing  to  do  commu-
nication  and  awareness  raising,  to  better 
communicate with European citizens. 

The  European  Commission  is  very  com-
mitted  to  continue  working  intensively  to 
regulate endocrine disruptors. We will soon 
have  a  breakthrough  regulation  on  the 
identification of endocrine disruptors, with 
the  proposed  new  hazard  classes  to  be 
adopted by the Commission before the end 
of this year. We hope the European Parlia-
ment  and  Council  will  have  no  objections, 
so the Act can enter into force according to 
plan in early 2023. 

Where would you like to see more 
efforts focused in the coming year 
or two? 

We heard today that investing in and fund-
ing  validation  of  test  methods  is  crucially 
important  –  since  my  institution,  the  Eu-
ropean Commission, is one of the possible 
funders, we will be certain to pass on that 
message to our colleagues that distribute 
the funds.

Validated test methods are the basis of the 
process, to be able to identify endocrine dis-
rupting substances. We are focusing a lot on 
their classification into hazard classes.

Only  once  they  are  identified  and  classi-
fied,  can  we  regulate  them.  And  our  plan 
is to ban endocrine disruptors in consumer 
products  –  apart  from  where  their  use  is 
essential  for  society,  as  we  announced  in 
the Chemicals Strategy for Sustainability.

velopment of chemicals that are “safe and 
sustainable by design”, for which criteria will 
be coming out in the coming months.

Of what are you especially proud 
and optimistic?

Very broadly, I am proud of this Commis-
sion’s pioneering European Green Deal, be-
cause it generated the Chemicals Strategy 
for Sustainability which is really ambitious 
for the better protection of health and the 
environment.

For the implementation, we are delivering 
one action after the other but we are under 
extreme  time  pressure  to  make  regulato-
ry proposals in the first half of next year, 
notably  the  REACH  revision.  I  want  to  be 
optimistic that we will make it because we 
must make it.

I am also proud that the EU is a world-
wide frontrunner in regulating endocrine 
disruptors. 

This  Forum  has  made  it  clear  once  again 
that we all have much work ahead of us, 
but we are highly motivated and ambitious 
to carry on. 

I would also like to look ahead to the 5th 
Annual Forum on Endocrine Disruptors next 
year. This annual event will keep us on our 
toes, to keep presenting the latest develop-
ments and progress in a transparent way, 
possibly  together  with  the  Spanish  presi-
dency at that time. It would also be nice to 
continue an outreach to and an exchange 
with non-EU countries and regions.

Yesterday and today there was a lot of focus 
on  “the  criminals”,  but  with  the  Chemicals 
Strategy we also want to give a message of 
hope and stimulate innovation and the de-

For now, I would like to thank all our speak-
ers, the participants here in the room and 
the online audience for their active interest 
and contributions.

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