COMMISSION STAFF WORKING DOCUMENT Accompanying the document Communication from the Commission Roadmap towards phasing out animal testing for chemical safety assessments
Date: 2026-06-01
Institutions: Directorate-General for Internal Market, Industry, Entrepreneurship and SMEs, European Commission
Subjects: animal experimentation, animal welfare, chemicals legislation, dangerous substance, environmental impact, environmental protection, health risk, product safety, research method, risk management

EN 

  EN 

   EUROPEAN COMMISSION  Brussels, 1.6.2026  SWD(2026) 144 final  COMMISSION STAFF WORKING DOCUMENT Accompanying the document Communication from the Commission Roadmap towards phasing out animal testing for chemical safety assessments {C(2026) 3497 final}  
 
 
Contents 
Contents .......................................................................................................................................................... 1 

List of abbreviations ....................................................................................................................................... 3 

Introduction ..................................................................................................................................................... 6 

1.  Developing a roadmap – a collective effort ............................................................................................ 7 

1.1. Roadmap development through inter-service and inter-agency collaboration ......................... 7 

1.2. Building a community through dedicated conferences and workshops .................................. 7 

1.3. Listening to stakeholders – gathering evidence through consultations ................................... 7 

1.4. Understanding change – stakeholder perspectives .............................................................. 8 

2. 

Identifying opportunities towards phasing out animal use ..................................................................... 9 

2.1. Recommendation of actions for human health and environmental assessments ...................... 9 

2.2. Preparing for the transition to animal-free chemical safety assessment ............................... 16 

2.3. Overarching opportunities across Human Health and Environmental Safety ........................ 53 

3.  Recommendations and actions for environmental safety assessments .................................................61 

3.1. Acute fish toxicity ....................................................................................................... 61 

3.2. Aquatic and terrestrial bioaccumulation .......................................................................... 65 

3.3. Fish chronic toxicity ..................................................................................................... 78 

3.4. Endocrine disruption for environmental safety assessments ............................................... 90 

3.5. Birds and mammals ................................................................................................. 100 

3.6. Ecological thresholds of toxicological concern and species sensitivity distributions ........... 107 

4.  Recommendations and actions for human health assessments ...........................................................109 

4.1. Acute toxicity (human health assessment) ..................................................................... 109 

4.2. Genotoxicity ............................................................................................................. 116 

4.3. Carcinogenicity ......................................................................................................... 125 

4.4. Repeated dose toxicity ................................................................................................ 135 

4.5. Developmental neurotoxicity - the DNT in vitro battery ................................................. 163 

4.6. Developmental and reproductive toxicity (DART) assessments ....................................... 175 

4.7. Endocrine Disrupting Chemicals - Use of New Approach Methodologies for their assessment 
within European Union regulatory frameworks:  Recommendations from the 2024 EPAA Partners’ 
Forum............................................................................................................................. 186 

5. Working together to design and implement non-animal approaches in regulatory testing .....................196 

5.1. Role and composition of the roadmap steering team ....................................................... 196 

5.2. Roadmap steering team .............................................................................................. 196 

5.3. Engaging Member-State and stakeholder experts in agency collaborative structures ........... 198 

5.4. EU Agency Network (EUAN) – working group on animal-free approaches ...................... 199 

5.5. Translating innovative methods into regulatory applications – validation, standardisation, 
qualification .................................................................................................................... 201 

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5.6. Safe spaces and regulatory exploration spaces for animal-free methodologies ................... 207 

6. Indicators – managing change by measuring it .......................................................................................212 

Annexes ......................................................................................................................................................230 

Annex I – Chemical safety assessments under following legislations are in scope of the roadmap
 ...................................................................................................................................... 230 

Annex II - Ongoing agency activities (ECHA, EFSA and EMA) that support the phasing out of 
animal testing for chemical safety assessments .................................................................... 234 

Annex III - Engaging with the general public ...................................................................... 244 

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List of abbreviations 
3Rs – replacement, reduction and refinement 
3Rs WP – European Medicines Agency working party on the 3Rs 
ADME – absorption, distribution, metabolism and excretion 
ACR – acute-to-chronic ratio 
AFSA – animal-free safety assessment 
AI – artificial intelligence 
AOP – adverse outcome pathway 
APCRA – Accelerating the Pace of Chemical Risk Assessment 
ASPA – ASPIS-initiated alternative safety profiling approach 
ASPIS – animal-free safety assessment of chemicals: project cluster for implementation of novel 
strategies 
BCF – bioconcentration factor 
BfR – Bundesinstitut für Risikobewertung (German Federal Institute for Risk Assessment) 
BMF – biomagnification factor 
BPR - Biocidal Products Regulation 
CEN – European Committee for Standardization 
CF – conceptual framework 
CHMP – Committee for Medicinal Products for Human Use 
C&L – classification and labelling 
CLP – classification, labelling and packaging 
CRO – contract research organisation 
CSR – corporate social responsibility 
DA – defined approach 
DART – developmental and reproductive toxicity 
DNT – developmental neurotoxicity 
EBW – exposure-based waiving 
EC – European Commission 
ECHA – European Chemicals Agency 
ECI – European Citizens’ Initiative 
ecoTTC – ecological threshold of toxicological concern 
ED – endocrine disruption 
EFSA – European Food Safety Authority 
EMA – European Medicines Agency 
EPAA – European Partnership for Alternative Approaches to Animal Testing 
ERA – European Research Area 
ESA – environmental safety assessment 
ESEC – European specialised expert community 
EU – European Union 
EU NETVAL – EU network of validation laboratories 
EURL ECVAM – EU Reference Laboratory for Alternatives to Animal Testing 
FET – fish embryo test 
GD – guidance document 
GHS – globally harmonised system 

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GLP – good laboratory practice 
GMO – genetically modified organism 
HSI – Humane Society International 
IATA – integrated approaches to testing and assessment 
ICCS – International Collaboration on Cosmetics Safety 
ICCVAM – Interagency Coordinating Committee on the Validation of Alternative Methods 
ICH – International Council on Harmonisation 
IMI – Innovative Medicines Initiative 
IOC – ionisable organic compounds 
ISG – inter-service group of Commission services and the three EU-Agencies ECHA, EFSA and 
EMA  
ISO – International Organization for Standardization 
IVIVE – in vitro-in vivo extrapolation 
JRC – Joint Research Centre 
KE – key event 
KER – key event relationships 
LD50 – median lethal dose (lethal dose 50%) 
LERU – League of European Research Universities 
LoE – lines of evidence 
MAD – mutual acceptance of data 
MIE – molecular initiating event 
ML – machine learning 
MoA – mode of action 
MPS – micro-physiological systems 
NAM – new approach methodology 
NGO – non-governmental organisation 
NGRA – next-generation risk assessment 
NIVA – Norsk institutt for vannforskning (Norwegian Institute for Water Research) 
NOAEL – no observed adverse effect level 
NoG – notes of guidance 
OECD – Organisation for Economic Co-operation and Development 
OoC – organ-on-chip 
OSOA – one substance, one assessment 
PARC – Partnership for the Assessment of Risks from Chemicals 
PBT/PMT – persistent, bioaccumulative and toxic / persistent, mobile and toxic 
PBTK – physiologically-based toxicokinetic 
PBK – physiologically-based kinetic 
PEC – predicted environmental concentration 
PoD – point of departure 
PNEC – predicted no-effect concentration 
qAOP – quantitative adverse outcome pathway 
QIVIVE – quantitative in vitro-in vivo extrapolation 
OPPTS – Office of Prevention, Pesticides, and Toxic Substances 
QSAR – quantitative structure-activity relationship 

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REACH – registration, evaluation, authorisation and restriction of chemicals 
RES – regulatory exploration space 
SCCS – Scientific Committee on Consumer Safety 
SIR – standard information requirement 
SME – small and medium-sized enterprise 
SSbD – safe and sustainable by design 
SSD – species sensitivity distribution 
TD – toxicodynamics 
TGs – test guidelines 
TK – toxicokinetics 
TTC – threshold of toxicological concern 
UVCB – unknown or variable composition, complex reaction products, or biological materials 
UN – United Nations 
US – United States 
WHO – World Health Organization 
WoE – weight of evidence 

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Introduction 
In 2023, responding to the European Citizens’ Initiative (ECI) ‘Save Cruelty-Free Cosmetics – Commit 
to a Europe Without Animal Testing’, the Commission committed to developing a roadmap towards 
phasing out animal testing for chemical safety assessments. In response to both this commitment and 
repeated calls from the European Parliament for a strategic plan, the Commission is now introducing a 
roadmap  in  the  Communication  corresponding  to  this  staff  working  document  (SWD).  This  SWD 
contains more detailed information supporting each section of the Communication. 

The  roadmap  applies  to  all  pieces  of  EU  legislation  that  require  animal  testing  for  chemical  safety 
assessments. A total of 15 different legislative areas have been identified that fall under the roadmap’s 
scope. Annex I provides an overview of these legislative areas. 

The goal of this roadmap – to phase out animal testing for chemical safety assessments  – cannot be 
achieved solely through actions of the Commission. Therefore, numerous stakeholders have contributed 
to  the  roadmap’s  development  and  have  committed  to  undertake  many  of  the  individual  actions 
described in this SWD. 

This SWD outlines: 

- opportunities and concrete actions per toxicological endpoint across the main areas of human health 
and environmental safety assessment, identified by the working groups for the safety assessment of 
human health and the environment during roadmap development, as summarised under 2. Identifying 
opportunities towards phasing out animal use; 

- the proposed organisational structures to support the roadmap’s implementation, existing safe-space 
structures  and  processes,  and  agency  activities  that  support  the  phasing  out  of  animal  testing  for 
chemical safety assessments, as summarised under 5. Working together to design and implement non-
animal approaches in regulatory testing; 

- reflections on possible ways for engaging with the public to ensure broad acceptance of the proposed 
revised  approach  to  how  chemical  safety  assessments  are  conducted,  as  summarised  in  Annex  III: 
Engaging with the general public; 

- possible indicators for monitoring the progress of roadmap implementation, as summarised under 6. 
Indicators- Managing change by measuring it; 

-  ongoing  agency  activities  (the  European  Chemicals Agency  (ECHA),  the  European  Food  Safety 
Authority (EFSA) and the European Medicines Agency (EMA)) that support the phasing out of animal 
testing for chemical safety assessments (Annex II).  

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1. Developing a roadmap – a collective effort 

1.1. Roadmap development through inter-service and inter-agency 
collaboration 
The  Commission  set  up  an  inter-service  steering  group  consisting  of  relevant  services  1  as  well  as 
ECHA,  EFSA  and  the  EMA  to  guide  the  roadmap’s  development. Three  working  groups  were  also 
created to develop the specific recommendations described in the respective chapters of this SWD for 
safety assessments of human health (section 4) and environmental safety (section 5) and for change 
management.  The  individual  chapters  in  sections  4  and  5  on  selected  toxicology  endpoints  were 
developed  by  the  respective  working  groups,  where  stakeholders  contributed  to  drafting  specific 
chapters. 

1.2. Building a community through dedicated conferences and 
workshops 
To ensure that stakeholders were involved in the design of the roadmap, the Commission organised 
three workshops on this topic. 

The first workshop on 11-12 December 2023 sought to identify the major challenges in moving towards 
non-animal chemical safety assessments and the ways of achieving this goal. 

A second workshop organised on 25 October 2024 collected input from various stakeholders on how 
they addressed the transition from animal-based testing to innovative, reliable non-animal alternatives. 
Participants reported on progress in developing the roadmap and discussed actions and milestones for 
driving forward this transformation of chemical safety assessments. 

The third workshop on 16 -17 June 2025 presented a first draft of the roadmap’s content and collected 
feedback from stakeholders on the potential actions and milestones. 

A  three-day  conference  was  co-organised  by  the  Commission  and  the  European  Partnership  for 
Alternative Approaches  to Animal  Testing  (EPAA)  in  March  2025  to  develop  proposals  for  short-, 
medium- and long-term actions for potential inclusion in the roadmap. 

Several other events were also organised by stakeholders to provide information and exchange views 
on the development of the roadmap. 

All these events gathered a large number of experts from various stakeholder groups, bringing highly 
valuable insights to the roadmap’s development.2 

1.3. Listening to stakeholders – gathering evidence through 
consultations 
To ensure that a broad range of stakeholders were involved in the preparation of this roadmap, several 
consultation activities were carried out in addition to the events described above. 

First, a call for evidence was organised according to the Better Regulation guidelines3 via the ‘Have 
your Say’4 website, with a four-week consultation period in September – October 2024. As a result, 90 

1 DG GROW, ENV, JRC, RTD, SANTE and SG. 
2 https://single-market-economy.ec.europa.eu/sectors/chemicals/reach/roadmap-towards-phasing-out-animal-testing/events-
and-outreach-activities_en. 
3 https://commission.europa.eu/law/law-making-process/better-regulation/better-regulation-guidelines-and-toolbox_en. 
4 https://ec.europa.eu/info/law/better-regulation/have-your-say/initiatives/14281-Animal-testing-in-chemical-safety-
assessments-Commission-roadmap-to-phase-it-out_en. 

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responses were submitted and are available on the ‘Have your Say’ site. A summary of the received 
comments is available in a synopsis report 5. 

In addition, two targeted online stakeholder surveys and expert interviews were organised to collect 
feedback on more specific issues pertinent to the roadmap’s development. Feedback was received from 
a broad set of stakeholders and is summarised in several published reports 6. 

1.4. Understanding change – stakeholder perspectives 
Between October 2024 and March 2025, the Change Management Working Group held 20 bilateral 
meetings with diverse stakeholders (including major EU industry associations, small and medium-sized 
enterprises  (SMEs),  contract  research  organisations  (CROs),  consultants,  non-governmental 
organisations  (NGOs),  academia  and  regulators)  to  provide  input  into  the  roadmap.  These  bilateral 
meetings  supplemented  the  above-mentioned  stakeholder  consultations  and  the  consultations  with 
national  representatives  at  meetings  of  regulatory  committees  and  expert  groups  dealing  with  the 
legislative areas within the scope of the roadmap. The results of the bilateral meetings are described in 
Berggren & Worth (2026)7. 

5 https://webgate.ec.europa.eu/circabc-ewpp/d/d/workspace/SpacesStore/a96d57cb-85a5-48fb-bb03-
c751b8e39843/download. 
6 https://single-market-economy.ec.europa.eu/sectors/chemicals/reach/roadmap-towards-phasing-out-animal-testing_en. 
7 Berggren, E. and Worth, A .: Managing change while phasing out animal testing in chemical safety assessments – a 
collection of stakeholder experiences and expectations, NAM Journal, 2, 2026, p. 100079, 
https://data.europa.eu/doi/10.1016/j.namjnl.2026.100079, 

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2. Identifying opportunities towards phasing out 

animal use 

2.1. Recommendation of actions for human health and environmental 
assessments 
A core goal of the roadmap is to provide concrete recommendations for the transition to non-animal 
approaches  across  all  chemical  safety  assessment  areas.  The  recommendations  reflect  the  state  of 
knowledge  at  the  stage  of  drafting  the  Commission  Communication  and  its  accompanying  Staff 
Working Document. Recommendations will need to be adapted as scientific knowledge advances on 
how to phase out animal testing. They are operationally categorized based on different indicative time 
horizons for implementation: 

1.  Short-term  actions  –  available  approaches  for  which  the  process  of  implementation  into 
regulatory  practice,  including  by  omitting  redundant  tests,  can  be  launched  immediately  or 
within a short timeframe. 

2.  Medium-term  actions  –  approaches  for  which  further  steps  for  validation  or  regulatory 
adaptation are required before wider use. After finalising required steps, approaches will be 
implemented immediately when deemed acceptable for use in a given regulatory context. 
3.  Long-term  actions  –  redefining  safety  assessment  paradigms  and  performance  criteria  for 
future  non-animal  approaches.  Long-term  actions  will  include  the  need  to  develop  further 
approaches, which will be implemented in a given regulatory context when deemed acceptable. 
Work on a new scientific safety assessment framework will start immediately as a core activity 
for implementing the roadmap. 

During the development of the roadmap, the working groups of the Commission services with input 
from stakeholders, created different chapters of the SWD. These chapters contain recommendations for 
different areas, such as: 

1.  A long-term change towards a new scientific assessment framework that allows to switch 

to animal-free regulatory assessments (chapter 2.2.) 

2.  Overarching  opportunities  across  human  health  and  environmental  safety  assessments 

(chapter 2.3.) 

3.  Environmental safety assessments (chapter 3) 
4.  Human health safety assessments (chapter 4) 

The long-term goal requires development of a new scientific assessment framework, as outlined in the 
corresponding Commission Communication on the roadmap. Five sub-chapters of the SWD explain the 
development, conditions and parameters of a  new scientific assessment framework  further. The first 
sub-chapter provides background information, gives information on ongoing projects developing a Next 
Generation Risk Assessments (NGRA), and suggests steps necessary to reach the goal. The second sub-
chapter reflects upon reaching the protection goals with NGRAs and the level of confidence needed. 
The third sub-chapter outlines the path to a hazard classification for human systemic toxicity based 
solely on the use of alternative approaches to animal testing, while the fourth sub-chapter addresses the 
switch to a new regulatory system for environmental safety assessments. Finally, sub-chapter five offers 
reflections about the integration of human health and environmental safety assessments, with respective 
recommendations for implementation. 

The  working  groups  also  identified  a  number  of  areas  that  represent  overarching  opportunities 
applicable to both human health and environmental safety assessments, which are the following: 

1.  Replacing in vivo with in silico methods 
2.  Developing TK and ADME models for regulatory needs 

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3.  Determining  the  Point  of  Departure  (PoD)  and  signatures  of  toxicity  for  chemicals  through 

measured omics data 

4.  Fostering the use of alternatives to animal approaches for nanomaterials and nanoparticles Risk 

Assessment 

Short- to long-term opportunities for replacing, reducing or refining animal testing for environmental 
safety assessments is described in chapter 3 for the following different areas: 

1.  Fish acute toxicity 
2.  Aquatic and terrestrial bioaccumulation 
3.  Fish chronic toxicity 
4.  Endocrine disruption 
5.  Birds and mammals 

Finally, chapter 4 provides more detailed considerations on short- to long-term recommendations for 
moving towards phasing out animal testing for human health safety assessments in the following areas: 

1.  Acute toxicity 
2.  Genotoxicity 
3.  Carcinogenicity 
4.  Repeated dose toxicity 
5.  Developmental neurotoxicity 
6.  Developmental and reproductive toxicity 
7.  Endocrine disruption – human health 

The  following  tables  summarise  several  actions  proposed  for  (eco)toxicological  assessments.  The 
information included in the tables is abbreviated for the purposes of providing an overview and should 
be read in conjunction with the more detailed chapters of this SWD and the materials referenced therein 
to understand the recommendations in context. The actions should be taken as illustrative examples. 

10 

 
 
 
 
 
Table 1: Actions for implementation of alternatives and further development relevant to human health safety assessment 

High level goal  Means to achieve it   

Specific actions linked  

Sector 

Replacement 

Replacing in vivo study 
with computational 
methods   

Use of computational models for Acute oral toxicity, 
Pharmacokinetic modelling 

“ 

Use of PK modelling to predict residue depletion in target 
animal safety studies 

Replacing in vivo study 
with in vitro assays  

Use of in vitro assays to predict/measure developmental 
neurotoxicity, malformations and embryofoetal lethality, 
pyrogenicity, sensitisation  

Technical amendments 
to Annexes VII to X of 
Regulation (EC) No 
1907/2006 

Pharmaceuticals (V) 

 Cross-sectoral 

Short, mid or long 
term  

Short-term  

Short-term / mid-
term  

Short-term / mid-
term  

Reduction 

Waiving of in vivo tests 
based on historical 
experience  

Use existing information (e.g. on pesticide active substances) to 
support waiving or deletion of long-term systemic toxicity in 2nd 
species  

 Cross-sectoral 

Short-term / mid-
term 

Reduction 

Proposal to reduce and replace animal studies for genotoxicity or 
carcinogenicity 

Cross-sectoral 

Mid-term 

Reduction/waiving of 
studies based on target 
patient population 

Reduction of repeated-dose toxicology (RDT) studies for 
advanced cancers or severely debilitating/life threatening 
diseases  

Pharmaceuticals (H) 

Short-term / mid-
term  

Reduction based on 
additional data in vitro/in 
silico approaches 

Reduction through use of complex in vitro models to predict 
drug induced liver injury / pharmacokinetic parameters / 
cardiotoxicity / immunotoxicity. 

Cross-sectoral (H) 

Reduction based on in 
silico approaches 

Reduction of control animals included in RDT testing through 
use of virtual control groups 

Pharmaceuticals (H) 

Reduction through study 
design optimisation 

Several approaches (a priori statistical considerations, reductions 
of dose groups or recovery animals where possible) 

 Cross-sectoral 

Short-term / mid-
term  

Short-term / mid-
term  

Short-term / mid-
term  

11 

 
 
 
  
  
 
 
 
 
 
Reduction through 
incorporation of multiple 
readouts in one study 

Reduction of in vivo studies through inclusion of additional 
endpoints in repeated dose toxicity studies, based on 
retrospective data/experience 

 Cross-sectoral 

Short-term  

Refinement of in vivo 
studies where not yet 
replaceable   

Development of new 
assessment framework  

Refinement 

Establishing an 
overarching, non-
animal scientific 
assessment 
framework  

Waiving of long-term in vivo studies through shorter omics-
enhanced studies 

Use of evident toxicity rather than lethality as endpoint in acute 
toxicity studies  

 Cross-sectoral 

Short-term  

Characterising protection and confidence levels of traditional 
assessment and non-animal-based scientific assessments based 
on a new scientific assessment framework 

Cross-sectoral 

Long-term  

“ 

“ 

Developing a non-animal-based classification system  

Cross-sectoral 

Long-term  

(excluding 
pharmaceuticals) 

Describing toxicity through changes measured at molecular level 
rather than adverse effects in organisms  

Cross-sectoral 

Long-term 

Frameworks for specific 
endpoints 

To design (for selected endpoints below) a non-animal approach 
in vitro/in silico battery able to reliably distinguish between non-
toxic and potentially toxic substances. To obtain qualitative 
system with high sensitivity suitable for tiered hazard 
assessment and agree on characterising the endpoint, based on 
non-animal information feeding a WoE assessment. 

Cross-sectoral 

Mid-term  

- genotoxicity  
- carcinogenicity 
- reproductive toxicity 
- endocrine disruption 
- nanomaterial assessment 

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Table 2: Actions – further development and implementation of alternatives relevant to environmental safety assessment; the recommendations 
usually consist of different sub-actions, which can be found in more detail in the respective chapter on recommendations for the environmental safety 
assessment 

High level goal  

Means to achieve it   

Specific actions linked (described in subsequent SWD chapters) 

Short, mid or long term  

Acute aquatic toxicity 

Reduction / Replacement 

Adapt legislation and 
guidance  

Reduction or replacement based on available methods (in silico, in vitro, 
information from other taxa than fish or more sensitive endpoints) 

Short-term 

Reduction 

Approach development  Explore waiving options based on scientific considerations while maintaining 

Short-to mid-term 

an equivalent level of protection 

Reduction / Replacement 

Frameworks for specific 
endpoints   

Develop an approach for the assessment of acute aquatic toxicity based fully on 
non-animal approaches 

Mid-/ long-term 

Bioaccumulation 

Reduction / Replacement 

Adapt guidance and 
legislation 

Reduction or replacement based on available methods (in silico, in vitro, 
information from other taxa than fish) 

Short-term 

Reduction 

Approach development  Explore waiving options based on scientific considerations while maintaining 

Short-to mid-term 

an equivalent level of protection 

Replacement 

Chronic fish toxicity 

Reduction  

Frameworks for specific 
endpoints   

Develop an approach for the assessment of bioaccumulation based fully on non-
animal approaches 

Mid-to long-term 

Adapt test guidelines, 
guidance, legislation 

Clarify regulatory requirements for fish testing and waiving options (e.g. using 
information from other taxa than fish) based on scientific considerations while 
maintaining the level of protection 

Short-term  

Reduction / Replacement 

Approach development  Gain a better understanding of how to make maximal use of information from 

Mid-term 

existing in vivo and non-animal approaches  

•  Link different lines of evidence from non-animal approaches 

•  Develop  a  reference  dataset  of  in  vivo  chronic fish effects / endpoints 

inter alia for the generation of Acute to Chronic Ratios 

13 

 
 
•  Develop  a  reference  dataset  relating  chronic  invertebrate  effects  to  in 

vivo chronic fish effects 

Replacement 

Frameworks for specific 
endpoints  

Define a non-animal approach for assessing chronic fish toxicity based on in 
silico and in vitro methods 

Long-term 

Endocrine Disruption (ED) for environmental safety assessments (ESA) 

Reduction / Replacement 
/Refinement 

Adapt test guidelines, 
guidance, legislation 

Adapt test guidelines / guidance / legislation to maximise information from in 
vivo testing and use tests both for concluding on the ED properties and risk 
assessment  

Mid-term 

Reduction  

Validation, case studies  Validation of certain in vivo methods (EAMA, LATT, FET with thyroid 

Mid-term 

endpoints) as as a measure to potentially reduce animal testing 

Reduction / Replacement 

Guidance development 

Improve guidance to use the OECD conceptual framework rather as toolbox 
instead of a tiered approach and foster the application of non-animal approaches 

Mid-term 

Reduction  

Research, case studies, 
guidance development 

Link AOP pathways or networks to non-animal approaches; develop guidance 
on the use of AOP pathways or networks 

Mid-term 

Refinement 

Research, case studies  Map mechanisms and applicability domains of eleutheroembryo methods 

Mid-term 

(RADAR, REACTIV, XETA) 

Reduction / Replacement 
/Refinement 

Research, case studies  Gain a better understanding of how to make maximal use of information from 
existing non-animal approaches, including those for the assessment of human 
health, and by linking different information from non-animal approaches 
(different sub-actions, see chapter on ED for ESA)  

Mid-term / long-term 

Replacement 

Research, case studies  Build evidence on cross-species extrapolation by exploring key events/AOPs 

Mid-term / long-term 

for different species  

Replacement 

Frameworks for specific 
endpoints 

Define a framework for assessing endocrine disruption for environmental safety 
with a mechanism-based panel of in silico and in vitro assays taking into 
account knowledge of (quantitative) Adverse Outcome Pathways 

Long-term 

Long-term change for environmental safety assessment for all taxa (including mammals and birds) 

Replacement 

Development of new 
assessment framework 

Develop animal-free environmental Next Generation Risk Assessment (NGRA) 
and implement it for environmental safety assessments. 

Long-term 

14 

 
 
Harmonise human health 
and environmental safety 
assessment under One 
Health 

It is important to explore the links between environmental safety assessments 
and human health NGRA approaches under a one-health concept to ensure the 
best use of data. 

Mid- / Long-term 

15 

 
 
 
 
 
2.2. Preparing for the transition to animal-free chemical safety 
assessment 

2.2.1. The long-term shift towards “Next-Generation Risk Assessment” 
(NGRA) in EU chemicals legislation 
Terminology 
At multiple occasions during the development of the roadmap, e.g. at the workshops organised by the 
Commission, but also in the working groups for human health or environmental safety assessment, the 
terminology questions were raised. This includes definitions for test methods without the use of animals 
or for “animal” itself, the term “Next-Generation Risk Assessment” (NGRA)”. While definitions are 
important to clarify the subject of a discussion, these definitions often contain context-specific elements, 
and  a  universally  accepted  “one-size-fits-all”  definition  is  not  available.  Without  claiming  to  be 
exhaustive,  terminology  used  to  signify  a  test  system  that  is  not  using  animals  or  that  is  limited  to 
species not considered sentient includes 

•  Alternative test methods (to animal testing), alternative approaches or alternative test systems 
•  Non-animal methods, non-animal testing (system), non-animal approaches, or non-animal data 
•  Animal-free methods or animal-free approaches 
•  New Approach Methodologies (NAMs) 

EU legislation, while not defining the terms, is using the terms mentioned under the first two bullets 
above8, while scientific literature is commonly using the term NAM, albeit with different meanings.  

Throughout  the  roadmap  Commission  Communication  and  the  Staff  Working  Document, 
preference is given to: 

•  Non-animal approach, signifying methods or approaches that do not require the use of any live 
animal  including  in  their  early  life  stages.  They  comprise  in  vitro  and  in  silico  methods 
(including read-across and grouping), organs-on-a-chip etc. 

•  Alternative method or approach to animal testing, signifying  every method or approach that 
supports  replacing,  reducing  or  refining  animal  testing  (Three  Rs). As  such,  it  can  involve 
animal testing if it leads to a reduction or refinement of animal testing. 

Directive 2010/63/EU contains a Definition of the Three Rs: 

•  Replacement – a method or testing strategy not entailing the use of live animals. 
•  Reduction – reducing the number of animals used in a project for a certain purpose without 

compromising the project’s objectives 

•  Refinement – refinement of methods to eliminate or reduce pain, suffering, distress or harm to 
animals. The avoidance of testing of sentient species9, using instead species with a potentially 
lower capacity to experience pain, suffering and distress, or of early life stages of vertebrates 
or amphibians would therefore result in refinement.  

It is important to recognise that both reduction and refinement remain important stepping stones towards 
the goal of phasing out animal testing. By its very nature, however, it is also clear that the roadmap 
aims at ultimately relying on non-animal approaches only. 

8 E.g. the Regulation on Plant Protection Products (EC) No 1107/2009, Directive 2010/62/EU  on the protection of 
animals used for scientific purposes, or the REACH Regulation (Regulation (EC) 1907/2006) 
9 It is acknowledged that the question which species can be considered “non-sentient” e.g. fruit flies, water fleas, 
round worms and embryos of zebrafish and frogs as explored by the ASPIS cluster project PrecisionTox is the subject 
of an ongoing scientific debate beyond the scope of this text. The term is nevertheless used here to clarify that new in 
vivo testing in species that have been identified as “sentient” should not be performed under NGRA. 

16 

 
 
 
 
Last not least, EU legislation does not define the term “animal”. Directive 2010/63/EU applies to the 
animals and life-stages listed in its Article 1, which  excludes for instance animals in their early life 
stages  (eleuthero  embryos)  or  invertebrates.  Nevertheless,  animals  in  their  early  life  stages  or 
invertebrates  are  naturally  considered  “animals”.  Consequently,  the  term  “non-animal  approach” 
excludes testing with animals in their early life stages, invertebrates etc. 

The roadmap therefore assumes that in the long-term also testing with animals in their early life stages 
or invertebrates need to be replaced to reach the ultimate goal of phasing out animal testing for chemical 
safety assessments. However, in the short- to medium-term, testing with animals (vertebrates) in their 
early life stages or with invertebrates is considered an refinement and to be acceptable, if it reduces the 
reliance  on  vertebrate  testing  beyond  early  life  stages.  This  consideration  takes  into  account  that 
suffering of animals differs. 

Next-Generation Risk Assessment based on alternatives to animal testing  

A similarly recurring issue in the discussions around the innovation of chemical risk assessment10 is the 
request for clear definitions of the term “Next-Generation Risk Assessment” (NGRA). Various authors 
have  made  attempts  at  providing  direct  or  indirect  definitions  for  the  term,  e.g.  Dent  et  al.  (2018), 
Berggren and Worth (2023), Marx-Stoelting et al. (2023), Sewell et al. (2024) or Herzler et al. (2025), 
to name just a few. While the desire for exact definitions is understandable (and such definitions may 
eventually be formulated), they are not a prerequisite11 for constructing and implementing the roadmap.  

The following considerations have been discussed in the context of NGRA for the assessment of human 
health.  Similar  considerations  on  NGRA  for  the  environmental  safety  assessment  are  presented  in 
chapter. 2.2.4. Long-term change - A new paradigm for regulatory environmental safety assessments. 

As presented by members of the Commission working group on  Human Health at the  3rd Roadmap 
Conference in June 2025, the eventual full phasing out of animal testing for chemical safety assessments 
requires the transition to a “New Safety Paradigm” characterised by the following elements: 

- 

human-centric safety assessments12 using alternatives to animal testing; 

-  mechanistic understanding to distinguish relevant from irrelevant substance effects; 

- 

- 

- 

- 

- 

utilisation of up-to-date science; 

high throughput and efficiency; 

a high level of protection and confidence, demonstrated explicitly and transparently; 

consistency across regulatory sectors in line with the “One Substance, One Assessment” (OSOA) 
principle; 

integration with environmental safety in line with the “One Health” paradigm. 

10 Note that “chemical risk assessment” is used here as a collective term inclusive of partial aspects of hazard or exposure 
assessment and does not only refer to “full-scale” risk assessments. 
11 Of note, even pivotal NGRA-related reports by US EPA did not attempt to provide such a definition: (2014). Next 
generation risk assessment: incorporation of recent advances In molecular, computational, and systems biology (final 
report) (EPA/600/R-14/004). https://ordspub.epa.gov/ords/eims/eimscomm.getfile?p_download_id=520491 and Thomas, R. 
S., Bahadori, T., Buckley, T. J., Cowden, J., Deisenroth, C., Dionisio, K. L., Frithsen, J. B., Grulke, C. M., Gwinn, M. R., 
Harrill, J. A., Higuchi, M., Houck, K. A., Hughes, M. F., Hunter, E. S., III, Isaacs, K. K., Judson, R. S., Knudsen, T. B., 
Lambert, J. C., Linnenbrink, M.,Williams, A. J. (2019). The next generation blueprint of computational toxicology at the 
U.S. Environmental Protection Agency. Toxicological Sciences, 169(2), 317-332. https://doi.org/10.1093/toxsci/kfz058 . 
12 It should be noted that efficient testing strategies that provide information for both human health and 
environmental assessments might need to make compromises as regards the use of more human centric 
approaches. Elements for a NGRA for environmental assessments are discussed in Chapter 2.2.4. 

17 

 
 
 
 
For the purpose of this document, “NGRA” then refers to chemical risk assessment conceptual 
frameworks or practical workflows implementing one or more elements of this paradigm. In an 
interim phase, NGRA might also rely on animal testing that supports reducing or refining animal testing, 
if  that  testing  addresses  other  aspects  of  the  new  paradigm  (as  exemplified  by  the  augmentation  of 
“classical” in vivo rodent studies augmented with transcriptomics measurements to elucidate possible 
mechanisms/modes of action). However, the long-term goal remains to use non-animal approaches only. 

The novelty of this paradigm can best be understood in contrast to  present-generation chemical risk 
assessment frameworks/workflows which are largely characterised by 

- 

- 

- 

- 

- 

- 

- 

animal-centric testing and assessment strategies; 

empirical “black box” testing in whole organisms mostly without mechanistic understanding at the 
molecular, cellular, organ or system level; 

slow uptake of new scientific developments; 

slow and resource-intensive testing, often following an untargeted “shotgun” approach; 

an implicit and therefore often untransparent level of protection, with uncertainties not considered 
(or considered, but not reported) in a quantitative way; 

variability of concepts across regulatory sectors; 

compartmentalisation between human health and environment. 

Historical development and future perspectives 

United States, Canada and the UK 

More than a decade ago, the United States Environment Protection Agency (US EPA) published their 
pivotal  report  “Next  Generation  Risk Assessment:  Incorporation  of  Recent Advances  in  Molecular, 
Computational, and Systems Biology” (US EPA, 2014), and initiated the Tox21 programme, a joint 
effort  of  several  U.S.  federal  agencies.  This  programme  provided  the  backbone  for  introducing  a 
paradigm change in science-based chemical risk assessment in the U.S., with worldwide repercussions. 
A more detailed vision and strategy was provided in the landmark report “Toxicity Testing in the 21st 
Century. A Vision  and  a  Strategy”,  published  by  the  U.S.  National Academy  of  Sciences  (National 
Academies, 2007), and followed by the formal installation of an alliance of the involved U.S. agencies 
in 2008. This co-operation has provided an unprecedented dynamic for the science-based innovation of 
chemical risk assessments. 

In  2018,  ICCVAM,  the  United  States  Interagency  Coordinating  Committee  on  the  Validation  of 
Alternative Methods, published its “Strategic Roadmap for Establishing New Approaches to Evaluate 
the Safety of Chemicals and Medical Products in the United States” (ICCVAM, 2018). Most recently, 
in  January  2024,  the  US  National  Institutes  of  Health’s  “Complement-ARIE”  fund  (Sunderic  et  al., 
2025) was approved, which will provide a budget of $35-40 M per year over the next ten years, inter 
alia for technology development projects/centres, a data and resource coordinating centre, a validation 
network for regulatory implementation, community engagement and training and strategic engagement. 

In April 2025, the US Federal Drug Administration (US FDA) released its “plan to phase out animal 
testing  requirement  for  monoclonal  antibodies  and  other  drugs”.13  In  June  2025,  the  US  National 
Institutes of Health followed with the announcement to reduce use of animals in NIH-funded research 

13 https://www.fda.gov/news-events/press-announcements/fda-announces-plan-phase-out-animal-testing-requirement-
monoclonal-antibodies-and-other-drugs, last accessed 2025-07-12  

18 

 
 
 
 
by  focusing  its  funding  programs  on  “human-based  research  technologies”.14  In  July  2025,  Canada 
published  its  Strategy  to  replace,  reduce  or  refine  vertebrate  animal  testing  under  the  Canadian 
Environmental  Protection Act15  and  in  November  2025,  the  UK  published  its  Replacing  animals  in 
science strategy16. 

European research projects and partnerships 

In Europe, major EU projects, such as Predict-IV (Mueller et al., 2015), SEURAT-1 (Daston et al., 2015; 
Gocht et al., 2015) or EU-ToxRisk (Daneshian et al., 2016; Moné et al., 2020) have achieved great 
progress  on  the  scientific  side  and  have  successfully  started  addressing  (and  bridging)  the 
communication gap between academic researchers and regulatory practitioners. Several EU  projects 
active at the time of drafting this document, such as the ASPIS17 cluster featuring the human-health-
related  projects  RISK-HUNT3R18,  ONTOX19  and  PrecisionTox20,  the  ENKORE21  cluster  with  its 
projects  EDC-MASLD,  MERLON,  HYPIEND  and  ENDO-MIX,  or  the  EURION22  cluster  with  its 
projects ATHENA, EDCMET, ENDpoiNTS, ERGO, FREIA, GOLIATH, OBERON and SCREENED, 
focusing on the assessment of endocrine disruptors, are building on and complementing this work. 

i) 

ASPIS cluster 

A core initiative within the ASPIS cluster is the ASPIS Alternative Safety Profiling Approach (ASPA), 
a  structured  pragmatic  workflow  for  NGRA  developed  within  the  RISK-HUNT3R  project  which 
provides a tiered decision-making framework (see below for more details).  

The ONTOX project develops physiological maps, quantitative adverse outcome pathway networks and 
ontology frameworks with the aim of providing artificial intelligence (AI)-assisted probabilistic NGRA 
workflows.   

The PrecisionTox project, based on the hypothesis that fundamental mechanisms of toxicity have been 
conserved between species, inter alia applies omics technologies to non-traditional model organisms 
(e.g. invertebrates) to predict human-relevant toxicological effects. This cross-species approach seeks 
to  bridge  gaps  between  environmental  and  human  risk  assessment,  reinforcing  the  "One  Health" 
concept. 

ii) 

European Partnership for the Assessment of Risks from Chemicals (PARC)23  

The Partnership for the Assessment of Risks from Chemicals (PARC) contributes to the development 
of NGRA strategies on diverse levels, from Adverse Outcome Pathways (AOPs) to strategies for legal 
implementation  (Herzler  et  al.,  2025).  The  NGRAroute  activity,  a  strategic  initiative  aimed  at 
establishing  animal-free  NGRA  as  the  default  framework  under  EU  chemicals  legislation,  seeks  to 
integrate  NAMs  into  regulatory  workflows  while  addressing  scientific  and  policy  challenges.  Key 
priorities  cover  fundamental conceptual  questions around integrating  diverse  NAMs into  workflows 
that are still able to provide classical regulatory outputs such as classification and labelling, points of 

14 https://www.nih.gov/news-events/news-releases/nih-prioritize-human-based-research-technologies last accessed 2005-
07-12 
15 Strategy to replace, reduce or refine vertebrate animal testing under the Canadian Environmental Protection Act, 1999 
(CEPA) - Canada.ca 
16 Replacing animals in science strategy - GOV.UK 
17 https://aspis-cluster.eu 
18 https://www.risk-hunt3r.eu 
19 https://ontox-project.eu 
20 https://precisiontox.org 
21 https://enkore-cluster.eu/ 
22 https://eurion-cluster.eu/ 
23 https://eu-parc.eu 

19 

 
 
 
 
departure, reference/guidance values etc. In addition, the focus of the activity is on the legal uptake and 
implementation side, with its position at the border between science and policy. 

Based  on  the  scientific  progress  in  recent  years,  the  understanding  of  possible  ways  to  build  an 
integrated, overarching NGRA framework has greatly increased over the past decades. NGRAroute has 
tried to summarise these experiences and to bundle them into ten guiding principles for a future NGRA 
framework, which were first discussed during the 1st Roadmap workshop in December 2023. Following 
an  intensive  stakeholder  commenting  round,  a  revised  version  was  later  provided  as  an  Additional 
Deliverable24 from the PARC Partnership: 

1.  Protection: The framework allows to determine whether an adequate level of protection for human 

health and the environment is attained. 

2.  Confidence:  The  framework  allows  to  determine  whether  an  adequate25  level  of  confidence  is 

attained, especially when concluding on the absence of relevant hazard, exposure or risk. 

3.  Generation of information: The framework takes into account all relevant existing information. If 

additional information is required, it is generated without in vivo testing in sentient species. 

4.  Science:  The  framework  uses  adequate  and  reliable  modelling,  testing  and  assessment 
methodology, with high scientific relevance to the protection of human health and the environment, 
representing the current state of scientific knowledge. 

5.  Evidence integration: The framework can integrate all relevant lines of evidence with acceptable 

reproducibility. 

6.  Regulatory workflows: The framework is applicable to all relevant chemical hazard, exposure and 

risk assessment workflows. 

7.  Biology: The framework covers all relevant (eco)toxicological pathways and endpoints. 

8.  Exposure: The framework covers all relevant exposure levels, durations and routes. 

9.  Chemistry:  The  framework  is  applicable  to  substances  in  their  relevant  physical  forms,  their 

transformation products, groups of substances, mixtures26 and to substances in articles. 

10. Efficiency: The framework allows for assessments within an acceptable time- and cost-frame. It 
includes  integrated  testing  and  assessment  approaches  that  are  as  complex  as  scientifically 
necessary, but also as simple and straightforward as possible. 

The 10 guiding principles were proposed as a means to:  

- 

foster broad consensus on fundamental questions at an early developmental stage instead of getting 
into lengthy discussions on small details early on; 

-  define the political, scientific and regulatory boundaries of NGRA; thereby helping to  

-  structure the further roadmap work and provide a basis for focused topical discussions. 

It is noted that the principles describe an ideal chemical risk assessment framework, with some of them 
also not well addressed by present-day risk assessment schemes. In this regard, the principles may also 
be  used  to  highlight  possible  gains  to  be  made  by  switching  from  the  current  to  an  NGRA-based 
regulatory system. 

Following the publication of the roadmap, it is planned to further develop the NGRAroute activity into 
a platform for NGRA implementation in the EU. In this context, the project also intensified work on 
Next-Generation Environmental Risk Assessment. 

24 https://www.parcopedia.eu/wp-content/uploads/2024/10/PARC_AD2.1_submitted_approval_pending.pdf  
25 as defined by the respective regulatory context and problem formulation 
26 as defined by Art. 3 of the REACH Regulation 

20 

 
 
 
Broad consensus among all stakeholders will be key for acceptance of the shift towards NGRA. With 
the  intention  to  create  a  hub  bringing  together  the  various  factions  of  the  chemical  risk  assessment 
community for an open dialogue aiming at broad consensus solutions, PARC has created the online 
knowledge management and community platform PARCopedia.27  

iii) 

EPAA NAM Designathon 

The EPAA NAM Designathon is a collaborative initiative aimed at developing a novel framework for 
classifying for human systemic toxicity using NAMs (or alternatives to animal testing). The project 
seeks  to  transition  from  traditional  animal-based  classification  criteria  to  non-animal  systems  while 
maintaining or enhancing protection levels.   

The Designathon focuses on creating a systemic toxicity classification matrix based on alternatives to 
animal testing, designed to replace animal-derived criteria with mechanistic and kinetic data, targeting 
a  broad  basis  of  human  systemic  toxicity  endpoints  (e.g.  CMRs,  endocrine  disruption,  STOT),  but 
without  focussing  necessarily  on  the  current  classification.  By  calibrating  the  new  system  based  on 
already assessed chemicals, the initiative aims to ensure equivalent level of protection (i.e. the same 
risk  management  decisions  can  be  made)  and  to  inform  future  UN  GHS  and  broader  EU  chemical 
legislation revisions.   

The framework employs a classification matrix based on three levels of concern (low, medium, high 
concern)  using  only  toxicokinetic  (TK)  and toxicodynamic  (TD)  properties.  It prioritises flexibility, 
allowing integration of future regulatory needs and emerging alternative approaches. Though initially 
focused on human health, the framework is designed for extension to environmental safety. 

EU regulatory bodies 

In addition to the large-scale research projects named above (and others), regulatory bodies and agencies 
have started initiatives to actively investigate and promote  alternatives to animal  testing and NGRA 
based on alternative approaches.  

i) 

Scientific Committee on Consumer Safety (SCCS) 

In the EU, the Scientific Committee on Consumer Safety (SCCS), driven by the ban on animal use for 
the testing of cosmetic ingredients, has frequently adapted its Notes of Guidance with a view to non-
animal approaches and NGRA (most recently in SCCS (2023)). 

The EU Cosmetic Products Regulation (No 1223/2009) mandates that all cosmetic products must be 
safe, with safety evaluations based exclusively on data generated without using animal testing. This 
prohibition on animal testing extends to marketing bans, making the adoption of non-animal approaches 
unavoidable. The SCCS framework prioritises exposure-driven risk assessment, integrating hazard data, 
chemical characterisation, and exposure estimates. Key elements include:   

-  Validated non-animal methods: Priority is given to OECD-validated methods, though scientifically 
valid non-validated non-animal approaches (criteria under development) are also considered; 

-  Notes  of  Guidance:  The  SCCS’s  12th  revision  outlines  testing  requirements,  with  forthcoming 

updates addressing NGRA guidance; 

-  Threshold of toxicological concern (TTC): a risk assessment tool for low-exposure scenarios. 

The NGRA approach integrates various data sources, including in silico, in chemico, and in vitro studies, 
along with read-across and historical in vivo data. This strategy is designed to address gaps in systemic 
toxicity assessments, particularly in areas lacking (validated) tests for repeated-dose toxicity. A "safe 
harbour" approach allows for the parallel submission of NGRA alongside standard regulatory dossiers. 

27 https://parcopedia.eu  

21 

 
 
 
This dual-submission framework aims at fostering regulatory acceptance of novel methodologies while 
ensuring compliance with existing standards. 

A weight of evidence (WoE) approach strengthens confidence in non-animal approaches by integrating 
multiple  data  streams.  This  helps  mitigate  the  limitations  of  individual  assays.  WoE  is  considered 
particularly  robust  when  it  aligns  with  established  AOPs  and  key  biological  events,  providing  a 
structured, scientifically sound basis for regulatory decision-making. 

In terms of the development of non-animal-based NGRA, the SCCS advocates for: 

-  harmonised criteria for physiologically based pharmacokinetic (PBPK) modelling in NGRA. 

-  validation  or  scientifically  valid  status  of  non-animal  approaches  used  in  existing  NGRA  case 
studies,  many  of  which  derive  from  contract  research  organisations  (CROs)  but  lack  formal 
regulatory recognition. 

-  Differentiation between non-animal approaches and broader new approach definitions (in the sense 

of alternatives to animal testing) to avoid ambiguity. 

The SCCS framework balances regulatory rigour with scientific innovation, ensuring cosmetic safety 
through  quantitative  risk  assessment  rather  than  hazard  alone.  By  promoting  NGRA,  WoE,  and 
exposure-driven  strategies,  the  Committee  aims  to  advance  animal-free  methodologies  while 
maintaining consumer trust. Continued collaboration across sectors and refinement of validation criteria 
will be pivotal to overcoming current limitations and achieving harmonised implementation. 

Global developments 

i) 

APCRA 

As an important example of global collaboration among regulators from US, Canada, Europe, Asia and 
Australia, the initiative for “Accelerating the Pace of Chemical Risk Assessment28” (APCRA) aims to 
discuss “progress and barriers in applying new tools to prioritization, screening, and quantitative risk 
assessment of differing levels of complexity” and “opportunities to increase collaboration in order to 
accelerate  the  pace  of  chemical  risk  assessments”  by  means  of  case  studies  with  high  regulatory 
relevance (Kavlock et al., 2018; Paul Friedman et al., 2019; Paul Friedman et al., 2025). 

ii) 

OECD 

On a global scale, the Organisation for Economic Co-Operation and Development (OECD), supported 
by its member countries, with large contributions from EU Member States, the European Commission’s 
Directorate-General  Joint  Research  Centre  (JRC)  including  the  EU  Reference  Laboratory  for 
alternatives to animal testing (EURL ECVAM), and European agencies like the European Chemicals 
Agency  (ECHA)  and  the  European  Food  Safety Authority  (EFSA),  has  successfully  facilitated  the 
conceptual development of innovative chemical hazard and risk assessment methodologies, such as the 
Defined  Approaches  for  Skin  Sensitisation  (DASS)  Guideline  Document  497  published  in  2021 
(OECD, 2023a) or the Recommendations on Evaluation of Data from the Developmental Neurotoxicity 
(DNT)  In-Vitro  Testing  Battery  (OECD  2023c).  Via  the  “Integrated  Approaches  to  Testing  and 
Assessment”  (IATA)  case  studies  project,  the  OECD  continues  to  actively  explore  their  practical 
application in the regulatory context. The OECD also plays a key role in further developing concepts, 
e.g. the concept of Adverse Outcome Pathways (AOPs) and practical approaches to validate test as well 
as non-test methods regarding their fitness-for-purpose for regulatory chemical risk assessment and by 
developing relevant guidance in the field (e.g. (OECD, 2017a, 2017b, 2017c, 2023b). 

28 Home - APCRA 

22 

 
 
 
 
Towards an integrated, overarching NGRA framework 
The need for an overarching framework 

Current chemical risk assessments frameworks feature a number of traditional concepts, e.g. substance-
by-substance assessments or deterministic hazard/exposure/risk characterisation, as well as traditional 
test methods using experimental animals that have been challenged for, among others, ethical, scientific 
and performance-related reasons. 

To phase out animal testing from chemical risk assessments, as desired by the roadmap, there can in 
principle be two strategies: 

1.  The  chemical  risk  assessments  framework  as  such  is  maintained,  but  the  (animal-based)  test 
methodology is replaced one by one - with non-animal approaches, where possible. 

2. A new (“next-generation”) risk assessments framework is introduced. 

Regarding 1., ECHA has noted that 

“Until recently, NAMs development aimed to fully replace animal testing for each specific regulatory 
endpoint.  These  developments  have  been  successful  for  some  relatively  simple  endpoints  (like  skin 
sensitisation), where the adverse effect and the mechanism(s) leading to this effect are relatively well 
understood. Development of NAMs for more complex endpoints has so far been less successful. 
By now, the scientific community and regulators widely accept that it would be almost impossible to 
develop one-to-one replacements of animal tests by NAMs for more complex endpoints such as e.g., 
repeated dose toxicity or reproductive/ developmental toxicity.” (ECHA, 2025) 

As a consequence, and in line with 2. above, it has become increasingly clear that not only the methods, 
but also the risk assessment frameworks themselves need to be adapted to accommodate new paradigms 
and concepts and to respond to regulatory challenges not well addressed by the current frameworks, 
such as the efficient assessment and management of a huge number of chemicals on the market, possible 
combination effects, new types of chemicals and materials or chemical effects on biodiversity.   

In  addition,  the  development  of  a  new,  unified  NGRA  framework  also  offers  the  chance  to  better 
integrate  human  health  and  environmental  risk  assessment  workflows  across  the  diverse  chemical 
sectors,  resulting in  a more  holistic  approach  in  the spirit  of  the “One  Substance,  One Assessment” 
philosophy  formulated  inter  alia  in  the  EU  Chemicals  Strategy  for  Sustainability  (European 
Commission, 2020). Changing the current chemical risk assessment framework, however, comes at a 
price,  as  it  will  trigger  the  need  for  deeper  changes  in  the  current  chemicals’  legislation  and  the 
accompanying guideline and guidance documents, compared to a one-by-one test method replacement 
strategy.  

The  necessary  first  step  in  this  direction  consists  of  elaborating  the  conceptual  details  of  a  new, 
integrated, overarching NGRA framework, to better understand where (and which) legislative and other 
fundamental conceptual changes are needed. In addition, by structuring the framework and associated 
workflows, current knowledge gaps, where applicable, can be identified and targeted by future funding 
of corresponding research initiatives. 

Chemical risk assessment workflows and outputs 

A large variety of overarching as well as sector-specific EU chemicals legislation is in place and covers 
a wide range of different chemicals, uses and exposures (see Annex I). While chemical risk assessment 
under  some  of  the  sectorial  regulations  may  sometimes  address  issues  specific  for  that  sector  (e.g. 
certain unique exposure scenarios), in general only a limited set of basic risk assessment workflows will 
be used across all sectors in the vast majority of cases, such as: 

-  determination of the fate of a chemical in the human body (absorption, distribution, metabolism, 

excretion, bioaccumulation) or the environment (biodegradation partitioning, ADME, etc.); 

23 

 
 
 
-  hazard identification/classification and labelling; 

-  hazard  characterisation,  i.e.  identification  of  points  of  departure  (PoDs)  for  risk  assessment, 
derivation of safe exposure levels (health-based or environmental guidance values environmental 
quality standards, MRLs), including extrapolation across species, duration, and routes; 

-  exposure assessment (estimation of migration from products, single-source or aggregate internal 

human exposure in serum/plasma or in target tissues, in environmental compartments etc.); 

-  screening/prioritisation of substance of high concern; 

- 

full-scale risk characterisation / exposure-driven safety assessment. 

Under the current chemical risk assessment system, each of the above workflows is designed to provide 
specific, “tangible” outputs needed to address the risk assessment problem at hand. In addition, these 
workflows build on one another. Therefore, a future unified NGRA framework should in the end be able 
to integrate all relevant human health and environmental safety assessment workflows into a modular 
scheme in which some of the modules may or may not apply, depending on the specific chemical risk 
assessment problem formulation at hand. 

Understanding the outputs required by chemical risk assessment, e.g. an estimate of internal exposure, 
a  safe  dose  or  a  measure  of  risk  for  humans  and  the  environment,  and  how  these  outputs  can  be 
delivered, is an essential prerequisite for creating the future NGRA framework  based on non-animal 
approaches.  

Standardised NGRA workflows: the ASPIS Alternative Safety Profiling Approach (ASPA) 

One of the overall key success factors of the current chemical risk assessment frameworks lies in the 
highly  schematic  and  standardised  nature  of  the  regulatory  workflows  built  around  the  regulatory 
outputs described above. This standardisation as implemented by test guidelines and guidance at the 
sub-legal level, is crucial for: 

-  providing a common understanding between actors with multi-disciplinary backgrounds; 

- 

the global harmonisation of chemical risk assessment; 

-  schematic training including an efficient transfer – via guidance – of knowledge from specialised 
experts to risk assessors faced with the need to cover a broad range of interdisciplinary scientific 
questions; 

-  comparability of assessments and creating level playing fields for all market actors. 

The ASPIS cluster, and specifically the ASPIS cluster project RISK-HUNT3R, is currently developing 
the  so-called  “Alternative  Safety  Profiling  Approach”,  or  ASPA.  The  ASPA  attempts  to  lay  out  a 
modular NGRA framework as described in the previous section for addressing the outputs required by 
hazard/exposure/risk  assessment,  using  only  non-animal  approaches.  More  specifically,  it  aims  to 
standardise the processes for data collection, data integration and data reporting. Operationalisation of 
NGRA,  i.e.  the  use  of  non-animal  approaches  for  regulatory  purposes,  requires  that  outcome(s)  are 
provided with sufficient certainty,  that it is fully clear which  non-animal  approaches and tools were 
used  and  that  all  intermediate  and  final  decision  points  within  a  tiered  testing  strategy  process  are 
transparently  justified  and  documented.  In  other  words,  full  implementation  of  NGRA  –  and  thus 
phasing  out  animal testing  for  chemical  risk  assessment  –  would  be  greatly  accelerated  when  these 
aspects are addressed in a transparent and structured way.  

ASPA is a well-guided, adaptable workflow co-developed by a large group of scientists, and improved 
stepwise by stakeholder input and application in defined case studies. A first overview of the ASPA 
workflow, including its principles, structural outline, and its operability is given in Leist et al. (2025). 
Depending  on  the  specific  problem  formulation,  only  a  subset  of  the  ASPA  elements  may  apply. 
Accompanied by a dedicated software tool, ASPA-assist, one of the main aims of the ASPA is to guide 

24 

 
 
users through an NGRA workflow specific for their respective problem formulation and to support them 
in  deciding  whether,  and  if  so,  which  further  information  (derived  from  non-animal  approaches)  is 
needed to come to a conclusion. Guidance is given by different information layers within each step of 
ASPA. Also, the ASPA-assist provides a detailed report of the assessment done. 

Key components of NGRA in ASPA include: 

-  Hypothesis-driven  approach:  Data  is  collected  based  on  a  predefined  hypothesis,  ensuring 

relevance and efficiency; 

-  Exposure and kinetics: Understanding how chemicals interact with biological systems at different 

exposure levels (this includes ADME properties); 

-  Hazard identification and characterisation: The use of high-throughput methods, including in 
silico  (QSAR,  structure-based  modelling)  and  in  vitro  (mechanistic  assays,  cell  stress  reporters, 
transcriptomics), to determine potential toxic effects and the dose at which these may occur; 

-  Decision  logic  and  risk  assessment:  Integrating  deterministic  and/or  probabilistic  models  to 

determine the likelihood of hazard and exposure, thereby refining risk predictions. 

-  Case  studies: ASPIS  is  validating ASPA  through  diverse  case  studies  across  chemical  sectors, 

including CLP classification and risk assessments.  

-  Probabilistic  risk  assessment:  By  modelling  uncertainty  ranges  for  hazard  and  exposure  data, 
ASPA  provides  regulators  with  confidence  intervals  for  risk  outcomes,  facilitating  informed 
decision-making. 

-  Evolutionary toxicology: PrecisionTox contributes with omics data to identify molecular toxicity 
pathways shared across animal species , linking toxicogenomic responses in invertebrates to human 
AOPs, thereby bridging ecological and human health assessments. 

-  Stakeholder  collaboration:  Workshops  engage  regulators,  academia,  and  industry  to  refine 

workflows and address implementation barriers.   

While ASPA is still under construction, it is already clear that it can become of paramount relevance for 
the implementation of the roadmap, because it may inter alia serve: 

- 

- 

- 

to demonstrate how a future animal-free NGRA framework can produce the same or equivalent 
outputs to those provided by the current, largely animal-based framework; 

to provide a concrete blueprint for NGRA-based strategy to be incorporated into the legislations; 

to  help  identify,  by  means  of  dedicated  case  studies,  knowledge  or  methodological  gaps  in  the 
workflow, which can then be addressed in a targeted way by the Commission working groups, and 
to  support  these  groups  in  developing  concrete  decision  criteria  regarding  the  acceptability  of 
outputs generated by non-animal approaches and associated uncertainties. 

Formulating NGRA frameworks such as the ASPA is an indispensable prerequisite for the further work 
on the Commission roadmap: only if the details of such a framework have become sufficiently clear 
can  the  need  for  changing  the  legislation  and/or  respective  guideline  and  guidance  documents  be 
formulated. 

Stepwise, tiered strategies proposed in ASPA for assessment and generation of new information 
under NGRA 

A future NGRA framework as the basis for chemical risk assessments based on non-animal approaches 
will likely need to take into account data from different methods, including relevant (as defined by the 
problem formulation and regulatory context) existing data, and combine them in a stepwise approach. 
During the time of transitioning to chemical safety assessments without the use of animals, it may still 
be necessary to use in vivo testing where the problem formulation cannot be addressed adequately by 
use of existing data or new data generation with non-animal approaches. In the spirit of reducing or 

25 

 
 
refining animal testing, additional information might be generated with in vivo testing with less sentient 
species, e.g. conducting testing with early life stages.  

The following scheme29 30, without claiming to be exhaustive, describes potential elements of a stepwise 
strategy  for  chemical  risk  assessments  towards  phasing  out  animal  testing  based  on  a  synopsis  of 
approaches published so far. 

Step 1 

Step 2 

Step 3 

Step 433 

Assess  and  integrate  all  existing  and  relevant  hazard  and  exposure  data  of  sufficient 
quality, in order to identify any relevant knowledge gaps as well as any relevant regulatory 
concerns  triggering  the  need  for  further  data  generation.  In  cases  where  biological 
mechanisms or pathways are conserved between humans and other species, it could also 
be  justified  –  subject  to  a  case-by-case  analysis  –  to  use  existing  data  generated  for 
environmental  assessment  in  non-mammalian  species  to  fill  data  gaps  when  evaluating 
human  health  (and  similarly  using  mammalian,  including  human,  data  to  support 
environmental safety assessments).  

Already  in  the  near  future,  machine-learning  or  other  AI  methods  may  dramatically 
improve the consideration of existing toxicity or exposure data (provided that the required 
high-quality curated datasets are generated and become publicly available). 

Follow up on such gaps or triggers first by means of read-across from similar substances, 
where possible, see e.g. Patlewicz and Shah (2023)31. 

Depending  on  the  problem  formulation  and  regulatory  context,  use  filters  based  on 
physico-chemical, in silico models or ADME 32 properties to rule out relevant exposure, 
where  possible  (to  be  further  explored),  taking  into  account  all  potentially  relevant 
chemical species (parent, metabolites, abiotic degradation products). 

Next,  if  possible  or  required,  perform  a  bioactivity  screening  step  using  in  chemico,  in 
silico or in vitro models to identify possible relevant pathways of biological perturbation. 
This bioactivity screen is meant as a gatekeeper to focus further testing on any triggers that 
might have been detected during the previous steps and therefore, it 

-  needs  to  have  a  high  biological  coverage,  to  ensure  that  no  relevant  bioactivity  is 

missed, 

-  needs to be “protective”, i.e. it must have a high negative predictive value (likelihood 
that a negative outcome really is a negative), thereby providing high confidence when 
concluding that no relevant hazard/exposure/risk is present, and  

-  should preferably also have a high positive predictive value to keep potential positive 

follow-up testing (see next steps) within reasonable limits. 

Step 5 

Where possible and appropriate, follow up on any triggers from the screening approach or 
remaining  gaps  using  non-animal  approaches.  During  the  transition  period  towards  an 
animal-free chemical safety assessment, targeted in vitro testing or in vivo testing with a 
preference for testing with non-sentient animals might still be necessary. 

29 It is noted that the approach presented here is written from a human-health perspective. Also, for environmental risk 
assessments, new ways of tiered information generation e.g. based on monitoring data could be considered 
30 A similar stepwise approach forms the foundation of the ASPA 
31 The EU agencies published guidance on read-across, e.g. Guidance on the use of read‐across for chemical safety 
assessment in food and feed - - 2025 - EFSA Journal - Wiley Online Library 
32 Absorption, metabolism, distribution, excretion 
33 Within this step - in analogy to current risk assessment practice – a tiered approach could be applied, starting with less 
resource-intensive approaches the results of which have been demonstrated to be on the conservative side and then refining 
with more complex approaches only if necessary. 

26 

 
 
 
The methodology used should be “predictive”, i.e. it should have high predictive accuracy 
(i.e.  a  high  fraction  of  correctly  identified  positive  and  negatives)  regarding  adverse 
outcomes for human health (or other species or populations in case of environmental safety 
assessments).  It  should  allow  hazard  classification,  where  required,  cover  all  relevant 
mechanistical pathways, and consider the need for metabolic competence to account for 
relevant transformation products. 

It  should  also  provide  dose/concentration-response  information  to  derive  points  of 
departure  (PoDs)  for  further  risk  assessment,  where  this  is  required  by  the  problem 
formulation and regulatory context. 

(Only during the transition to NGRA:) Perform new in vivo testing in sentient animals after 
a case-by-case assessment concluding that the remaining knowledge gaps need to be closed 
because a) there is reason to assume that closing them will have a relevant impact on risk 
management, that b) alternative methodology is inadequate for addressing the knowledge 
gap, and c) there is a legal requirement to close the knowledge gap. If all of this is the case, 
the  in  vivo  testing  strategy  pursued  should  minimise  suffering  and  stress  of  the 
experimental animals to the maximum extent possible. It should also make best use of the 
animals, e.g. by adding relevant alternative testing components, such as omics (cf. concept 
of “TG+” studies, PARC (2023)). 

Step 6 

In the above concept, it is critical to define after each step whether the knowledge obtained is already 
sufficient to inform risk management (and therefore the assessment can stop). Non-animal approaches 
should  effectively  be  used  to  determine  the  presence  or  absence  of  hazards,  ultimately  resulting  in 
classification without testing on animals. For this, clear decision criteria are needed, in particular for 
reliably demonstrating “no relevant toxicity” (to be defined by the problem formulation and regulatory 
context). 

While the knowledge required for the hazard, exposure or risk assessment, as well as the potential areas 
of concern needs to be defined a priori, and a catalogue of established methods and models may be 
provided, the latter steps of the stepwise approach may also require case-by-case decisions which would 
need  to  be  standardised  as  much  as  possible  to  avoid  subjectivity  and  bias,  unnecessarily  lengthy 
assessments and delayed decisions due to the tiered approach. Inter alia PARC conducts projects that 
can provide insights on this matter. 

Incorporating a stepwise approach, such as the one described above, would likely require a revision of 
legislative texts34.  

During the transition period to NGRA, in vivo testing will still be required for some time. In the current 
risk assessment framework, however, a catalogue of standard in vivo test requirements is often in place, 
while  the  likelihood  that  such  tests  will  provide  added  value  to  risk  management  is  not  clear  in  an 
individual case. To better address this aspect already while in vivo testing cannot be completely replaced 
for certain areas of concern, criteria and methodology should be developed to determine the likelihood 
that additional in vivo testing in animals will provide added value for risk management.  

34 This SWD does not intend to pre-empt any decisions or discussions needed that could lead to a legislative 
proposal. 

27 

 
 
 
 
 
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30 

 
 
 
 
2.2.2. Characterising the protection level and level of confidence associated 
with next-generation risk assessments 
Introduction – the concept of adequate level of protection 
A  future  animal-free  Next-Generation  Risk  Assessment35  (NGRA)  framework  should  allow  to 
determine whether an “adequate level of protection” (as defined by the regulatory context and problem 
formulation)  for  human  health  and  the  environment  is  attained.  One  of  the  most  frequently  heard 
requirements for NGRA is that, compared to the current system, it needs to provide “the same or a 
higher level of protection for human health and the environment.”  

Risk can be defined as “a measure of the likelihood or probability that damage to life, health, property, 
and/or the environment will occur as a result of a particular hazard.”36 

It is also known from everyday experience that there is no such thing as zero risk in life. Chemical risk 
management is, therefore, tasked with reducing any risks from chemical exposure to an (albeit very 
low) residual level that is acceptable from a societal perspective. 

In addition, chemical risk management often faces risk-benefit considerations, since the removal of a 
hazardous chemical from the market might lead to undesirable consequences in other societal areas, 
e.g.  the  unavailability  of  certain  products  essential  for  the  functioning  of  society  as  a  whole.  Risk 
management then has to decide about the level of residual risk from uses of that chemical which might 
still be tolerable in view of the societal benefits. 

An “adequate level of protection” can then be defined in general terms as follows: 

An adequate level of protection represents a level of residual risk for which the combination of severity 
of effect and expected likelihood of occurrence is acceptable, or at least tolerable, from a societal risk-
benefit perspective. 

The  quantitative  benchmarks  for  “acceptable”  or  “tolerable”  need  to  be  defined  on  the  policy/risk 
management level. A clear and transparently communicated rationale in this respect will not only allow 
for more comparable and consistent risk assessments across chemicals and regulatory sectors, but it will 
also help enhance the acceptance of the resulting risk management measures by society as a whole. 

Once such benchmarks have been established, criteria for demonstrating that they have been attained 
by a given risk assessment need to be provided on the regulatory level. This will then provide the basis 
for  the  scientific  community  to  develop  and  propose  methods  and  workflows  within  an  NGRA 
framework which are able to demonstrate that these criteria can be met. 

However,  with  respect  to  the  current,  mostly  animal-based  human  health  chemical  risk  assessment 
framework, Chiu & Paoli have noted that: 

“Paradoxically, risk assessments for the majority of chemicals lack any quantitative characterisation 
as to the likelihood, incidence, or severity of the risks involved.” (Chiu and Paoli, 2021). 

As a consequence, any future NGRA framework is challenged with the “mission impossible” to prove 
that it lives up to a level of protection that currently is mostly not characterised explicitly for individual 
assessments, but rather defines safety in an indirect way, e.g. by absence of relevant effects in a set of 

35 In this section, the term “risk assessment” is used collectively to refer to full risk characterisation or its parts, i.e. it includes 
hazard  identification  and  characterisation  as  well  as  external  and  internal  exposure  assessment.  NGRA  then  refers  to  risk 
assessment strategies that do not yet represent the (present-generation) regulatory standard. In the context of chemical risk 
assessment  it  usually  refers  to  strategies  using  human-centred  test  methods  and  decision  logics  informed  by  mechanistic 
understanding. 
36 www.oxfordreference.com, last accessed 2025-07-08 

31 

 
 
 
standard tests or by ensuring, via monitoring programmes, that exposure of the human population or 
the environment is not exceeding “safe” levels. 

With  one  of  the  world’s  most  sophisticated  chemical  management  systems  in  place  in  the  EU,  the 
protection level ensured by the current chemical risk assessment framework is very high by comparison 
to the situation in many other regions of the world. However, this generic statement is of little help for 
assessing the level of protection present when trying to characterise the level of protection offered e.g. 
by a reference dose in relation to a specific scenario of exposure to a given hazardous chemical. 

Since the current risk assessment framework does not offer a protection level benchmark to which an 
NGRA result could be compared, there are two strategies for demonstrating that an adequate protection 
level has been reached:  

- 

- 

a relative approach, aiming to compare the protection level represented by NGRA outcomes 
with that obtained from traditional risk assessments and 
an  absolute  approach,  trying  to  characterise  the  protection  level  attained  by  a  given  risk 
assessment, whether NGRA or traditional, in absolute terms. 

The  long-term  roadmap  activity  described  in  this  section  is  proposing  to  develop  both  strategies  in 
parallel. 

Strategies for characterising the protection level provided by risk assessments based on non-
animal approaches 
“Relative” strategy 

Assuming that the protection level provided by the current chemical risk assessment framework is “in 
general very high,” a defensible scientific strategy can be to demonstrate that on average, over a large 
number of cases with sufficient coverage of chemical and biological space, the output from NGRA, e.g. 
in  the  form  of  a  hazard  classification result,  points  of  departure  (PoDs) for further  risk  assessment, 
Predicted  No-Effect  Concentration  (PNEC)  in  the  environment  or  Health-Based  Guidance  Value 
(HBGV), exposure estimates etc., coincides with that from traditional assessments. 

A corresponding relative strategy, therefore, compares outcomes from traditional animal-based tests 
with those from NGRA, based on alternatives to animal testing or ultimately on non-animal approaches. 
To this end, it uses reference datasets to evaluate the similarity in estimated toxicity and associated 
uncertainties. This approach can be summarised in the following steps: 

1.  Characterise the overall uncertainty of the outcomes from NAM-based human health and 

environmental risk assessments as well as that from traditional approaches. 
To allow comparison of non-animal approaches-based vs. traditionally derived Points of Departure 
(PoDs) for hazard characterisation, such as BMDLs, NOAELs/LOAELs, ECx values, first inherent 
uncertainties  of  these  approaches  need  to  be  characterised.  In  is  important  to  do  it  at  the  level 
suitable  to  address  given  hazard  concerns  as  defined  in  the  regulatory  system  (e.g.  allowing  a 
conclusive assessment at the GHS hazard class level). 

2.  Provide suitable, well-curated traditional reference datasets and make them broadly available 

to method developers. 

3.  Establish performance criteria with acceptance by regulators, but also a broad spectrum of 

stakeholders, which newly developed NGRA strategies would need to fulfil. 
For example, such criteria could consider uncertainty estimates from step 1. above, e.g. the overall 
uncertainty associated with traditional PoDs could be used as the basis for defining the required 
level of concordance between traditional and non-animal approaches-based PoDs). Both the criteria 
and reference data sets should be broadly disseminated to method developers. 

32 

 
 
 
 
 
4.  Demonstrate  the  acceptable  performance  of  newly  developed  NGRA  strategies  against the 

respective reference datasets.  
To address more complex endpoints and provide risk assessments and regulation with the required 
level of protection and confidence, non-animal approaches-based solutions will need to combine in 
vitro  assays,  in  silico  models,  knowledge-driven  frameworks  (e.g., AOPs)  and  data  integration 
logic. Moreover, since risk is defined as the combination of the severity of effect and the probability 
of  occurrence,  probabilistic  hazard,  exposure  and  risk  assessment  methodology  will  need  to  be 
developed and established in support of this approach to safeguard true comparability of traditional 
and non-animal approaches-based risk assessments. 
Reference  datasets  should  be  well-balanced 
the  chemical, 
mechanistic/biological and physicochemical property spaces, as well as biological effect space. At 
the same time, they must have realistic sample size – for endpoints which are toxicologically more 
diverse, it should be larger than for narrower ones. 

terms  of  coverage  of 

in 

To fully assess the protection level provided by a given risk assessment framework (or a regulatory 
measure  based  on  it),  further  elements  of  the  risk  assessment  process  need  to  be  considered,  when 
qualitatively or quantitatively extrapolating from measured or modelled (eco)toxicological PoDs to the 
real-life exposure situation in humans or the environment. In some of these areas, significant innovation 
has  taken  place  in  recent  years,  as  exemplified  by  the  probabilistic  characterisation  of  PoDs  and 
assessment factors, particularly those addressing population variability, the improvement of physiology-
based  kinetic  (PBK)  models  or  the  evolution  of  human  biomonitoring  as  a  tool  for  characterising 
internal exposure, all important building blocks of future NGRA frameworks. 

A great advantage of the relative approach is that it can be applied immediately, provided an adequate 
reference dataset is available, cf. (Paul Friedman et al., 2019) or (Lu et al., 2024). Nevertheless, there 
also inherent problems are associated with it:  

-  As in the current system, this strategy is not helpful when trying to assess the level of protection 
provided by a specific individual assessment, e.g. when trying to establish which percentage of an 
exposed population is protected from which severity of effect and likelihood of occurrence. 

-  Using the outcome of traditional risk assessments as benchmark propagates possible flaws of the 
previous  system,  e.g.  the  deterministic  combination  of  possibly  overconservative  worst-case 
assumptions or animal models with poor predictivity for the target species of the assessment. 

“Absolute” strategy 

1. 

Protection level 

The definition of risk given in the introduction to this section contains two complementary elements – 
potential damage and likelihood of occurrence. To fully characterise the risk from exposure to hazardous 
chemicals, the following aspects need to be considered: 

-  nature and extent or size of a (possible) effect; in chemical risk assessment, the term “endpoint”37 
is frequently used to qualitatively describe the nature of effect whereas “adversity”38 is used as the 
synonym  for  “damage”  in  the  above  definition  (i.e.  not  every  effect  caused  by  exposure  to  a 
chemical). 

37 Note that „endpoint“ in this sense may refer to different levels of biological organisation, i.e. molecular, cellular, organ, 
system, organism, population, ecosystem or even the planetary level. 
38 Cf. the definition of "adverse effect” in (WHO IPCS, 2004):“Change in the morphology, physiology, growth, 
development, reproduction, or life span of an organism, system, or (sub)population that results in an impairment of 
functional capacity, an impairment of the capacity to compensate for additional stress, or an increase in susceptibility to other 
influences”. 

33 

 
 
 
 
- 

likelihood  of  the  occurrence  of  an  adverse  effect  (over  time  depending  on  the  nature  of  the 
population or system exposed and the variability in exposure level, duration and frequency as well 
as susceptibility (sensitivity and vulnerability)39 of that population or system. 

As  the  term  “likelihood  of  occurrence”  implies,  the  protection  level  provided  by  an  individual  risk 
assessment  can  be  characterised  using  probabilistic  assessment  methodologies.  Maertens  and  co-
workers  have  recently  described  the  benefits  of  such  approaches  over  current  “deterministic”40  risk 
assessment outputs (Maertens et al., 2024; Maertens et al., 2022; Maertens et al., 2025). EU Horizon 
2020  projects  such  as  RISK-HUNT3R  (Moné  et  al.,  2020)  or  ONTOX  (Vinken  et  al.,  2021)  or  the 
European Partnership for the Assessment of Risks from Chemicals (PARC, Herzler et al. (2025))41 are 
currently  working  on  the  integration  of  probabilistic  methodology  into  NGRA  frameworks  and 
workflows. 

It may also be argued that probabilistic methods more adequately reflect the reality of potential chemical 
effects on populations. A conceptual framework has been provided by Chiu and Slob (2015) and further 
elaborated  on  by  WHO  IPCS  (2018),  who  also  developed  a  tool  for  practical  application  of 
“Approximate Probabilistic” risk characterisation, APROBA(-Plus)42. 

However, experience with the practical application of such methodology is currently limited (cf. e.g. 
EFSA (2019) or BfR (2023)) and required input data may be lacking or difficult to access. Alternatives 
to animal approaches, and in particular high-throughput methods, may be particularly suited to generate 
such data, since e.g. more concentrations or replicates could be tested at lower cost compared to adding 
further animals. In addition, alternatives to animal approaches can be utilised to assess e.g. intraspecies 
variability (Rusyn et al., 2022; Zeise et al., 2013) 

Consequently,  the  envisaged  absolute  strategy  aims  to  develop  an  overall  conceptual  approach  for 
probabilistic  characterisation  of  human  risk,  integrating  data  on  exposure,  effect  severity,  and 
population  sensitivity  and  vulnerability.  This  method  expresses  risk  as  a  probability  distribution, 
explicitly incorporating uncertainty (see section below) and variability. Probabilistic risk metrics –  e.g., 
95th  percentile  thresholds  –    can  be  used  to  standardise  protection  levels  across  different  regulatory 
applications.  This  approach  ultimately  allows  for  a  transparent  and  standardised  assessment  of  the 
protection  level.  Compared  to  the  relative  approach,  it  can  also  be  applied  –  once  established  –  in 
situations with sparse data or where PoDs are not directly available. 

2. 

Level of confidence 

Developing robust concepts and methodology for the probabilistic characterisation of exposure, hazard 
or risk is an important step, but not enough in itself. In addition, an adequate level of confidence (as 
defined  by  the  problem  formulation  and  regulatory  context)  needs  to  be  attained,  especially  when 
concluding on the absence of relevant hazard, exposure or risk. 

Approaches to achieve confidence in NGRA frameworks, non-animal approaches and results based on 
them in general terms, e.g. by validation, are addressed in other sections of this document. While  a 
prerequisite, this trust alone, however, does not help when trying to assess the confidence that one can 
place in a specific risk assessment outcome. 

39 In line with WHO IPCS (2018), more “sensitive” individuals in a population show the same effect size at a lower exposure 
than less sensitive ones; more “vulnerable” individuals are more strongly affected by the same effect size. 
40 In this context, deterministic refers to the unambiguous relationship between e.g. an HBGV, given as a single number, and 
the protection level of an exposed individual. In contrast, a probabilistic risk assessment describes this relationship in terms 
of a probability distribution, bearing witness to the fact that e.g. different individuals may possess different susceptibility to a 
chemical’s adverse effect.  
41 EU Horizon 2020 projects: RISK assessment of chemicals integrating HUman centric Next generation Testing 
strategies promoting the 3Rs | RISK-HUNT3R | Project | Fact Sheet | H2020 | CORDIS | European Commission, 
Ontology-driven and artificial intelligence-based repeated dose toxicity testing of chemicals for next generation 
risk assessment | ONTOX | Project | Fact Sheet | H2020 | CORDIS | European Commission, Partnership for the 
Assessment of Risks from Chemicals | PARC | Project | Fact Sheet | HORIZON | CORDIS | European 
Commission.  
42 https://www.rivm.nl/en/aproba-plus (accessed 2024-10-04) 

34 

 
 
 
All methodology used in the context of chemical risk assessment either represents models (in silico, in 
vitro, in vivo) or measurements (subject to experimental error) with limited sample size. Both are then 
used  to  make  inferences  about  larger  collectives  (of  chemicals,  populations  or  species).  Without 
exception, therefore, all chemical risk assessments are associated with uncertainty.  

Uncertainty has been defined by WHO IPCS as  

“imperfect knowledge concerning the present or future state of an organism, system, or (sub)population 
under consideration.” (WHO IPCS, 2004) 

Acc. to the WHO IPCS guidance document on characterising uncertainty in hazard characterisations, 

“it  can  be  further  defined  as  lack  of  knowledge  regarding  the  “true”  value  of  a  quantity,  lack  of 
knowledge  regarding  which  of  several  alternative  model  representations  best  describes  a  system  of 
interest, or lack of knowledge regarding which probability distribution function and its specification 
should represent a quantity of interest.” (WHO IPCS, 2018) 

In contrast, according to the same document, variability marks the 

“heterogeneity of values over time, space or different members of a  population, including stochastic 
variability  and  controllable  variability.  Variability  implies  real  differences  among  members  of  that 
population.  For  example,  different  individual  persons  have  different  intake  and  susceptibility.  In 
relation to human exposure assessment, differences over time for a given individual are referred to as 
intraindividual variability; differences over members of a population at a given time are referred to as 
interindividual variability.” (WHO IPCS, 2018) 

Biological variability is a reality that may be measured and  – e.g. in terms of experimental design – 
partly controlled but not changed. In contrast, uncertainties (e.g. about the variability of the response of 
a given population towards a chemical stressor) may in principle be remediated by generation of further 
knowledge, e.g. by applying a refined assessment strategy or by generating additional data. 

Under current chemical risk assessment frameworks, assessment outcomes are mostly only reported in 
a  deterministic43  way,  without  a  transparent,  quantitative  statement  regarding  the  associated 
uncertainties. This is often compensated by using what are supposed to be worst-case or justified generic 
assumptions, the combination of which, however, may result in highly conservative (and potentially 
overconservative) assessments.44 Notably, such (over–) conservatism may not be a problem or even be 
desired in certain cases but needs to be made at least transparent to avoid misinformed risk management 
decisions. An additional, undesirable consequence of this practice is that different risk assessments may 
differ vastly with respect to their inherent uncertainties, rendering their results incomparable. 

A  non-exhaustive  list  of  elements  in  current  chemical  risk  assessment  typically  associated  with 
uncertainties includes 

- 

the outcome of dose-response analyses, 

-  extrapolations across species, exposure routes or durations, 

-  assumed use patterns for chemicals/products, or 

- 

the variability with respect to susceptibility towards a certain chemical’s toxicity within the target 
population or between target species, 

43 For example, a health-based guidance value (HBGV, single number) is compared to an exposure estimate (single number), 
resulting in a single number (e.g. the risk characterisation ratio, RCR = exposure/HBGV) to characterise a possible risk. 
44 For example, the likelihood that, for a given substance, the discrepancy between the sensitivity of rats vs. humans towards 
that chemical’s toxicity is tenfold AND a person’s individual susceptibility to the effect is again tenfold that of the population 
median AND exposure of that person is at the maximum level experienced in the population is extremely low, yet, such a 
combined assumption is frequently made in CRA. 

35 

 
 
 
-  exposure assessments, as use tonnages, uses, associated use patterns, emissions, fate & leaching 

potential are often no/rarely published or known. 

In NGRA, some of these aspects, e.g. the extrapolation across species in human health assessments, 
will become less relevant. However, additional uncertainties will have to be considered, e.g. in relation 
to  extrapolating  the  results  from  in  vitro  experiments  to  the  in  vivo  situation  (in-vitro-to-in-vivo 
extrapolation, IVIVE). 

Qualitative  statements  to  the  end  that  uncertainties  are  present  are  of  limited  practical  help  in  risk 
management and communication. The same holds for semi-quantitative statements that uncertainty was 
“low”, “medium” or “high”, as long as no exact boundaries between these classes are provided (but 
then these boundaries could also be stated directly). In addition, acceptance of an assessment outcome 
implies that, whatever the uncertainty, it was considered acceptable in the end. 

The benefits of quantitative approaches to uncertainty assessment have again been described by WHO 
IPCS: 

“More transparently representing in quantitative form the confidence we can have in toxicological risk 
projections and estimates of the relationship between dose and health effect, thereby facilitating choices 
of preventive measures and/or further information gathering by risk managers. For instance, health-
based  guidance  values  […]  may  be  defined  based  on  a  pre-specified  and  harmonised  level  of 
conservatism, or estimated health risks for a given exposure situation can be expressed in terms of an 
uncertainty distribution or a confidence interval.” (WHO IPCS, 2018)  

Another noteworthy aspect of uncertainty in the context of chemical risk assessment is that it works in 
both directions, i.e. a statement on the risk posed by a certain exposure scenario is uncertain regarding 
a potential under- as well as overestimation of hazard, exposure and/or risk. 

The degree of uncertainty and confidence acceptable under a given problem formulation and regulatory 
context needs to be defined at the policy level and concrete benchmarks for acceptance need to be set 
at the regulatory level. The ability to provide a transparent measure of the uncertainties inherent in a 
risk  assessment  would  then  also  mark  a  superior  quality  of  NGRA  over  the  current  chemical  risk 
assessment framework, where such a measure is often not available or provided. Where uncertainties 
and confidence cannot be sufficiently quantified, (semi)qualitative methods may be applied, but this 
will  result  in  additional  uncertainty  about  the  outcome  of  the  assessment,  which  should  be  made 
transparent. 

3. 

Challenges and aspects to be considered under the “absolute” approach 

A  non-exhaustive  selection  of  specific  challenges  and  aspects  to  be  considered  when  developing 
strategies demonstrating the level of protection and increasing the level of confidence with NGRA based 
on non-animal approaches has been compiled by the PARC project.45 

Conclusion 
Since the absolute strategy relies on input from the relative approach, both methods are complementary 
and will be developed concurrently. The relative approach could be used immediately where appropriate 
reference datasets exist or can be curated within a reasonable period. In contrast, the absolute strategy 
requires initial consensus on the most effective conceptual framework before proceeding.  

Nevertheless,  introducing  such  concepts  requires  substantial  preparatory  work:  defining  reference 
datasets,  characterising  uncertainty  in  both  traditional  and  non-animal  method-based  estimates  and 
agreeing on performance benchmarks. Therefore, working on developing and introducing strategies for 
characterising the protection level provided by risk assessments with non-animal approaches will be a 
long-term  project,  which  will  need  to  be  supported  by  collaborations  like  PARC  and  EU  research 
projects and involving broad participation of regulators and stakeholders. Overall, the aim is to provide 
a  sound  scientific  basis  for  chemical  risk  assessment,  management  and  communication,  as  well  as 
practical methodological approaches for characterising uncertainty, variability, and protection level. In 

45 Source: PARC Deliverable AD2.1, submitted to the European Commission for approval, draft version available at 
https://www.parcopedia.eu/wp-content/uploads/2024/10/PARC_AD2.1_submitted_approval_pending.pdf.  

36 

 
 
 
this way, it will help to achieve regulatory and societal acceptance of non-animal approaches by making 
the concept of “protection” both scientifically tangible and transparently communicable. 

References 

BfR (2023): Bisphenol A: BfR proposes health based guidance value, current exposure data are needed 
for  a  full  risk  assessment  :  BfR  Opinion  no.  018/2023,  issued  19  April  2023.  DOI: 
10.17590/20230419-114234-0 

Chiu  W.A.  and  Paoli  G.M.  (2021):  Recent  advances  in  probabilistic  dose–response  assessment  to 
inform risk-based decision making. Risk Analysis 41 (4), 596 - 609. DOI: 10.1111/risa.13595 

Chiu W.A. and Slob W. (2015): A unified probabilistic framework for dose–response assessment of 
human  health  effects.  Environmental  Health  Perspectives  123  (12),  1241  -  1254.  DOI: 
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EFSA  (2019):  Establishment  of  cumulative  assessment  groups  of  pesticides  for  their  effects  on  the 

thyroid. EFSA Journal 17 (9), e05801. DOI: 10.2903/j.efsa.2019.5801 

Herzler M., Luijten M., Marx-Stoelting P., and Rivière G. (2025): The European Partnership PARC’s 
role in actively promoting the uptake of new approach methodologies and next-generation risk 
assessment into regulatory risk assessment practice. Current Opinion in Toxicology 42, 100517. 
DOI: 10.1016/j.cotox.2025.100517 

Lu  E.-H.,  Ford  L.C.,  Chen  Z.,  Burnett  S.D.,  Rusyn  I.,  and  Chiu  W.A.  (2024):  Evaluating  scientific 
confidence in the concordance of in vitro and in vivo protective points of departure. Regulatory 
Toxicology and Pharmacology 148, 105596. DOI: 10.1016/j.yrtph.2024.105596 

Maertens A., Antignac E., Benfenati E., Bloch D., Fritsche E., Hoffmann S., Jaworska J., Loizou G., 
McNally K., Piechota P., Roggen E.L., Teunis M., and Hartung T. (2024): The probable future 
of toxicology – probabilistic risk assessment. ALTEX - Alternatives to animal experimentation 
41 (2), 273-281. DOI: 10.14573/altex.2310301 

Maertens  A.,  Golden  E.,  Luechtefeld  T.H.,  Hoffmann  S.,  Tsaioun  K.,  and  Hartung  T.  (2022): 
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Maertens A., Kincaid B., Bridgeford E., Brochot C., de Carvalho e Silva A., Dorne J.-L.C.M., Geris L., 
Husøy T., Kleinstreuer N., Ladeira L.C.M., Middleton A., Reynolds J., Rodriguez B., Roggen 
E.L., Russo G., Thayer K., and Hartung T. (2025): From cellular perturbation to probabilistic 
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Paul Friedman K., Gagne M., Loo L.-H., Karamertzanis P., Netzeva T., Sobanski T., Franzosa J.A., 
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38 

 
 
 
 
 
2.2.3. Hazard classification for systemic toxicity based on non-animal data 
(EPAA Designathon) 
Rationale and background 
This chapter reflects on possibilities for hazard classification for human systemic toxicity solely based 
on the use of alternatives to animal testing (or new approach methodologies, NAMs). This involves the 
design  of  a  potential  future  classification  scheme  (Next  Generation  Classification  and  Labelling) 
ensuring equivalent protection, by calibrating the system with substances that are currently classified.  

Figure 1. Proposal for potential new classification scheme for chemicals based on three levels of 
concern (High, Medium and Low) according to Berggren E & Worth AP (2023). 

A proof-of-concept of this idea is the focus of the EPAA Designathon project, which started in 202346. 
Central to this project is a level of concern matrix (Figure 1). The idea is that results from eventual non-
animal approaches-based solutions should fall somewhere on the matrix, reflecting different levels of 
concern  (high,  medium,  low)  which  would  be  the  basis  for  different  risk  management  measures 
(Berggren and Worth, 2023; Worth and Berggren, 2025; Worth et al., 2025). The two dimensions of 
the matrix refer to intrinsic properties, one axis being information on toxicokinetics (TK) and ADME 
properties and the other on toxicodynamics (TD), both based on non-animal approaches. 
Moreover,  the  project  also  explores  the  possibility  for  a  systemic  toxicity  class  based  on  single 
alternative methods. This might be feasible since adverse outcomes are not mutually independent from 
a mechanistic point of view – a limited number of upstream key events are likely to underpin a spectrum 
of adverse outcomes due to divergent AOP networks (Figure 2). 

46 EPAA designathon challenge. https://single-market-economy.ec.europa.eu/calls-expression-
interest/epaa-designathon-human-systemic-toxicity_en 

39 

 
 
 
 
 
 
Figure 2. Hypothesis that adverse outcomes associated with systemic toxicity are underpinned by 
a limited number of upstream key events.  

Further considerations will be necessary as part of the roadmap implementation phase on how the new 
hazard class could be brought into operation. For instance, it could be beneficial to operate for an interim 
phase the current GHS system for classification and the future GHS hazard class in parallel (Figure 3). 
This  would  on  one  hand  ensure  continuity  with  the  current  GHS  system,  while  both  systems  could 
mutually inform each other. Whether to introduce such options after the development phase, requires 
careful reflection, including on operability and appropriateness. It will further require consultations of 
EU regulators and international partners. In addition, anticipating the need to protect for systemic health 
concerns not yet considered (e.g. immunotoxicity, endocrine disruption), the new system is designed to 
embrace any additional endpoint, while the current system tends to establish either new classes or define 
additional criteria that are included in current classes. 

Figure 3. Co-existence of a new NAM-based systemic toxicity class with existing hazard classes  

For other toxicological effects development of a similar classification strategy could be explored. 

References 
Berggren E & Worth AP (2023). Towards a future regulatory framework for chemicals in the European 

Union — Chemicals 2.0. Regulatory Toxicology & Pharmacology 142: 105431. 

40 

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    
 
 
 
 
 
Worth AP & Berggren E (2025). A twin transition in regulatory toxicology: moving toward Chemicals 

2.0 and phasing out animal testing. Toxicological Sciences 203: 160–165.  

Worth AP, Berggren E & Prieto P (2025). Chemicals 2.0 and Why We Need to Bypass the Gold Standard 

in Regulatory Toxicology. Alternatives to Laboratory Animals 53: 21-25.  

41 

 
 
 
 
2.2.4. Long-term change - A new paradigm for regulatory environmental safety 
assessments 
Introduction and recommendations  

Discussions with regulators and stakeholders during the development of the roadmap made it evident 
that  replacing  animal  testing  with  one-to-one  with  equivalent  non-animal  approaches  can  only  be 
achieved in specific and limited number of cases. Non-animal approaches provide information that is 
different from the animal tests that are currently used. Information from non-animal approaches is well 
suited to provide the information necessary to reach the policy goals of protecting human health and 
the environment. However, regulatory assessments, and in the end also chemical legislation, has to be 
re-designed and tailored to the information that non-animal approaches provide if a transition should 
succeed.  A  paradigm  shift  is  needed  for  regulatory  environmental  safety  assessments  (ESA)  that 
facilitates incorporation of non-animal approaches while guaranteeing a high level of environmental 
protection. 

To  achieve the paradigm shift, it is recommended to develop  and  implement the  use  of animal-free 
exposure-led  hypothesis-driven  environmental  Next  Generation  Risk  Assessment  (NGRA)  in  the 
regulatory  context.  The  new  system  should  include  considerations  of  how  to  define  a  system  for 
classification and labelling that relies on innovative non-animal approaches only but offers at least the 
same  level  of  protection.  The  new  system  based  on  NGRA  also  needs  to  provide  the  information 
required for hazard characterisation and the derivation of thresholds.  

Reaching such a system requires the following implementation and research activities: 

•  Development of a generic bioactivity battery covering the most relevant biological pathways to 

be used for the hazard identification of chemicals with unknown modes of action. 

•  Development of proposals for prioritising biological pathways, expected consequences, taxa 

and traits with expected higher risk. 

•  An  understanding  of  how  to  use  information  from  sub-organism  level  to  extrapolate  to  the 

population level or higher. 

•  Designing  and  implementing  cross-sector  case  studies  for  hazard  identification  and 
characterisation  considering  two  parallel  approaches,  one  using  exclusively  non-animal 
approaches, the second combining non-animal approaches and invertebrate testing. 

•  Developing tiered, hypothesis-driven strategies for the use of non-animal approaches for hazard 

• 

identification and characterisation. 
Integrating  information  on  distribution  and  fate  of  chemicals  to  upgrade  the  hazard 
characterisation case studies as exposure-led environmental NGRA assessments. 

•  Development and implementation of computational tools to facilitate gathering, organising and 

interpreting of anticipated datasets e.g., according to FAIR principles. 

•  Exploring  the  links  to  human  health  NGRA  tools/data  where  applicable  under  a  one-health 

concept. 

The above points require to extend the knowledge in multiple areas relevant for ESA, and inter alia the 
following: 

•  Extending  the  biological  and  ecological  knowledge  that  is  integrated  into  ESAs,  e.g., 
natural variability in impact on risk drivers due to life history traits of relevant organisms 
under different ecological scenarios and conditions; 

•  Extending the chemical and non-chemical stressor knowledge integrated into ESAs, e.g., 

spatial and temporal exposure patterns, multiple stressors on ecosystem services; 

•  Leveraging  computational  advances  to  build  tools  and  models  to  support  the  gathering, 
integration  and  interpretation  of  exposure  datasets,  as  well  as  biological  and  ecological 
knowledge, for the systems-based risk assessments, e.g., digital twins and AI advances.  

42 

 
 
 
 
 
The current regulatory system for environmental safety assessment 
The current ESA paradigm was developed around the 1980s. Since then, the knowledge on ecological 
stress  and  capacities  for  predicting  the  impacts  of  chemical  exposure  on  organisms,  populations  or 
ecosystems has been significantly improved. 

A main advantage of the current paradigm regarding regulatory use is the high level of predictability. 
The  combination  of  a)  preestablished  standard  study  protocols  conducted  on  selected  species  and 
endpoints, and b) agreed extrapolation factors, limits the needs for applying expert judgement to those 
cases with quality issues on the studies and the confirmation that the effect is associated to the exposure 
(e.g. unclear dose-response relationship). 

An advantage from the regulatory perspective is the possibility for conducting an ESA with a minimum 
set of data, e.g. under REACH for some substances, it is possible to derive PNECs for water, sediment 
and soil using just three aquatic short-term studies or acute toxicity QSAR estimations, if their reliability 
is demonstrated. This, however, does not apply to poorly water-soluble substances. The flip side of this 
coin is the limited relevance of the derived toxicity values, particularly for addressing delayed effects 
(i.e. effects seen after exposure has ended), at the ecosystem level, and long-term consequences at the 
population and/or community level.  

The methodological limitations can be grouped in the following aspects: 

•  Coverage of species, taxonomic groups and traits: e.g. a cladoceran crustacean represents all 

aquatic invertebrates.  

•  Coverage of ecologically relevant endpoints: e.g. immunotoxicity, sublethal morbidity, stress 

adaptation, etc. are not considered. 

•  Coverage  of  biological  mechanistic differences for  the  selected  endpoints:  e.g. reproduction 
effects linked to the reproductive approach of the selected species such as parthenogenicity for 
daphnids. 

•  Coverage of ecological conditions: laboratory assays conducted under optimised development 

conditions for the test species. 

•  Coverage of ecological interactions: single species laboratory test with no competition, etc. 

The conceptual limitations are mostly linked to the current regulatory approach, the first two in the list 
below  are  already  highlighted  in  the  EU  Chemicals  Strategy  for  Sustainability,  which  calls  for  the 
consideration of aggregate and combined exposures: 

•  Lack of integration of exposures to the same substance from different uses in different sectors 
(and in some cases also within the same sector, e.g. due to different applicants or to assessing 
each use individually in the case of pesticides and biocides). 

•  Lack of consideration of the combined effects linked to simultaneous or subsequent exposures 

to different substances. 

•  Focus  on  direct  toxic  effects,  do  not  consider  that  indirect  effects  not  identified  by  apical 
endpoints, such as increased susceptibility to  diseases or behavioural changes, may be more 
relevant for population and community dynamics. 

•  Overgeneralisation  and  insufficient  consideration  of  real  environmental  and  ecological 

variability within the EU. 

•  Approach,  based  on  setting  thresholds  for  each  individual  substance,  puts  the  focus  on  the 
regulatory decision, but does not provide information on the actual level of risk associated to 
the decision. 

•  There is no information related to the uncertainty associated to the ecotoxicological threshold. 

Due  to  the  above-mentioned  limitations,  the  current  approach  based  on  laboratory  studies  does  not 
inform on: 

•  Type,  likelihood  and  magnitude  of  the  potential  environmental  effects  linked  to  the  final 

• 

regulatory decision, 
Influence of the variability of  biotic (ecosystem structure and functions), and abiotic factors 
(including climate and landscape distribution) on the environmental risks characterisation, 

43 

 
 
•  Possibility  of  indirect  environmental  effects  that  may  affect  population  and  community 

dynamics and impact on biodiversity. 

The current regulatory environmental safety assessment includes several steps:  hazard identification, 
hazard characterisation that quantitatively assesses the magnitude of adverse effects (thresholds), and 
risk assessment, which combines hazard identification, hazard characterisation with information on fate 
and exposure of substances in the environment.  

Conceptually, the concept used in ESA for hazard characterisation and risk assessment is an adaptation 
of the system for human health assessments, based on the extrapolation of the no-effect level using 
generic extrapolation factors. The main modification is in the selection of the toxicity endpoints. The 
approach for environmental risk assessment (ERA) is to consider only those effects that are assumed to 
be relevant at population level, in particular survival, growth and reproduction. As a consequence, the 
assumed “no-effect level” from the most sensitive species used as point of departure may in fact produce 
significant  effects  at  individual  level  as  long  as  no  statistically  significant  effects  are  observed  on 
survival, growth and reproduction.  

The current extrapolation factors are a legacy from risk management decisions in the second half of the 
20th Century (Tarazona 1997), lacking a sound scientific basis (Chapman et al. 1998; Smith and Cairns 
1993). In addition, species sensitivity is usually distributed along several orders of magnitude (Newman 
et  al.  2000),  indicating  that  real  interspecies  variability  is  much  larger  than  that  covered  by  the 
interspecies extrapolation factors, thus the real level of protection depends on the availability or not of 
toxicity data for the most sensitive taxonomic groups. 

The current risk assessment paradigm includes parallel quantitative assessment for hazard and exposure, 
including environmental fate, which are then compared using their quotient to characterise the risk. The 
most  common  approach  for  hazard  characterisation  is  to  set  an  acceptability  threshold  for  each 
environmental  compartment  or  exposure  pathway,  i.e.  a  predicted-no-effect-concentration  (PNEC). 
PNECs  are  derived  by  applying  an  assessment  factor  (to  cover  intra/interspecies  and  lab-to  field 
variabilities) to the lowest level of effect or no effect, i.e. lowest relevant lethal (or effect) concentration 
that effects 50% or 10% of the animals (L(E)C50/L(E)C10) or no-observed-effect concentration (NOEC), 
obtained in experimental studies from pre-established standard species.  

Differences between legislative areas exist. In some areas, for instance plant protection products (PPPs), 
the  approach  is  modified.  The  acceptability  threshold  is  established  for  each  taxonomic  group  and 
exposure timeline (i.e. acute and chronic). Exceedance of this threshold requires further refinement or 
assumption of unacceptable risk. The problem formulation includes different exposure-to-target lines 
according to the use patterns.  

Under REACH, the main exposure-to-target lines are for the aquatic compartment (including sediment), 
soil, and predators exposed via the aquatic and terrestrial food chains. For PPPs, additional assessment 
lines are included for non-target terrestrial vertebrate and invertebrate groups. 

The interspecies assessment (or extrapolation) factors for PNEC derivation under REACH depend on 
the number of taxonomic groups with available information and, when it is assumed that steady-state 
can be achieved in acute tests, an additional acute to chronic extrapolation factor may be applied when 
only  acute  toxicity  information  is  available.  For  PPP,  the  uniform  principles  provide  acceptability 
thresholds, which conceptually are equivalent to the overall extrapolation factor, for each assessed taxa 
and  exposure  time.  Species  sensitivity  distributions  (SSDs)  and  higher-tier  studies  can  be  used  for 
refinement. 
There are no solid scientific bases for setting the interspecies extrapolation factors, and it should be 
noted  that  the  final  risk  acceptability  level  is  also  heavily  dependent  on  the  exposure  scenarios 
developed in the guidance documents. For example, the PECwater for industrial and domestic emissions 
is estimated after the mixing zone, using a dilution factor, thus the selection of the dilution factor is 
critical for understanding the overall level of protection. 

44 

 
 
 
Opportunities for a paradigm shift 
The  short-  and  medium-term  approaches  in  the  roadmap  towards  phasing  out  animal  testing  for 
chemical safety assessment focus on the replacement of animal testing (or reduction or refinement) with 
methods or a combination of methods that provide the same or similar information as the current animal 
testing.  

The long-term option addressed in this document explores the needs for a paradigm shift to develop 
non-animal ESA approaches, addressing, in addition, the limitations of the current system, through the 
use of recent scientific developments and novel approaches for supporting regulatory decisions. 

The  paradigm  shift  requires  to  explore  the  capacity  of  mechanistic  information  from  non-animal 
approaches  to  address  the  methodological  and  conceptual  limitations  listed  above.  The  proposed 
methodology  is  based  on  exposure-led,  hypothesis-driven,  environmental  next  generation  risk 
assessment methodologies using non-animal approaches.  

With  a  focus  on  non-animal  approaches,  environmental  NGRA  will  provide  more  informative  risk 
characterisations  of  the  assessed  chemical,  i.e.,  a  risk  profile  identifying  the  biological  pathways 
expected to be affected at the estimated exposure levels and the taxa, traits and functions with higher 
risk,  including  the  variability  associated  to  different  environmental  conditions  in  the  exposure 
assessment,  and  ecological  considerations  for  the  extrapolation  of  a  hazard  profiling  based  on  non-
animal approaches.  

The  basis  of  the  current  paradigm  is  the  extrapolation  of  effects  from  individual  organisms  to 
populations. The additional complexity for NGRA based on non-animal approaches is represented by 
the need to explore effects at the lower levels of biological organisation and link them, to assess impact 
at much higher levels, as depicted in Figure 4. The development of tools that enable to go beyond the 
impact on the single organism by extrapolating to population and community level should be a priority, 
as the aim of environmental NGRA should be increasing the environmental relevance, beside moving 
away from animal testing.  

Figure 4: The complexity line for NAM-based NGRA environmental assessments47  

47 Source of diagram: M. Hardy, College Biology I, modified. 

45 

 
 
 
 
 
The exposure-led and the hypothesis-driven NGRA is linked to more flexible problem formulations, 
replacing fixed information requirements by Integrated Approaches to Testing and Assessment (IATA). 
Following the IATA process, the problem formulation starts with the assessment of existing information 
and  the  identification  of  which  information  is  needed  for  regulatory  decision-making.  Exposure 
considerations should lead the assessment, and this represents a clear benefit in the EPAA context as 
emissions and most environmental fate processes can be addressed without the use of animal methods. 
In the implementation, it should be considered that the possibility for targeting the exposure assessment 
is  much  higher  in  some  sectors  than  in  others,  requiring  adaptations  to  the  specific  regulatory 
frameworks.  The  focus  of  the  exposure  assessment  should  be  to  identify  which  populations  and 
communities  could  be  exposed  and  under  which  circumstances,  targeting  the  identification  of  the 
information needs to cover the effect assessment. 

Several NAM-based approaches are under development to cover the effect assessment and, in general, 
these are linked to Adverse Outcome Pathway (AOP) frameworks (Brooks et al. 2023). The “offer” 
includes  advanced  molecular  methods  (Schumann  et  al.  2024b),  effects  at  genetic  level  and  the 
associated pathways (Rivetti et al. 2023), and high throughput in vitro bioactivity (Schaupp et al. 2023); 
frequently  supported  by  computational  methods  (Kosnik  et  al.  2024;  Schumann  et  al.  2024a).  The 
assessment  of  interspecies  similarities  and  differences  is  essential  in  all  cases,  and  evolutionary 
conservation represents a key element (Dufourcq Sekatcheff et al. 2024; Rivetti et al. 2023).   

These different options are complementary, and the new paradigm should allow the development and 
implementation  of  hypotheses  supported  by  any  of  the  options  in  a  weight  of  evidence  approach. 
Phylogenetic studies indicate that, in general, biological pathways are widely shared among organisms 
from different taxa, however, the same pathway may be associated to different physiological processes, 
consequently the impairment may result in very different adverse outcomes; e.g., the alteration of the 
thyroid  pathway  has  been  associated  with  neurodevelopmental  effects  in  mammals  (AOP  42), 
metamorphosis impairment in amphibians (AOP 176), and swimming alteration in fish (AOP 176)48. 
This situation facilitates the screening process for setting a battery of relevant bioactivities but requires 
sufficient knowledge on the biology and ecology of the different species in order to assess the possible 
outcomes and identify the risk drivers in terms of integration of exposure, biological role and ecological 
traits. 

In addition to cross-species extrapolation, improving ecological relevance requires a new brainstorming 
to  link  current  population  drivers  (survival,  reproduction,  growth  and  development)  with  species 
ecology  and  environmental  conditions,  as  well  as  to  consider  other  endpoints,  such  as  behavioural 
effects or disease susceptibility. The current paradigm is based on the consideration of the “population 
relevance” of the endpoints measured  in vivo studies, but population relevance is not unequivocally 
defined,  and  the  relative  relevance  of  each  endpoint  depends  on  the  species,  trait,  environmental 
conditions  and  ecological  status.  For  example,  behavioural  alterations  or  increased  susceptibility  to 
infectious diseases due to immunosuppression, may be much more relevant in some circumstances than 
effects  on  survival  or  reproduction. As  a  consequence,  a  well-designed  bioactivity  battery  linked  to 
sufficient knowledge on the biology and ecology of wildlife may be much more informative in terms 
of  potential  ecological  impacts  than  observational  laboratory  animal  studies  in  a  limited  number  of 
species.  

There are several activities already addressing this issue, e.g., Partnership for the Assessment of Risks 
from Chemicals | Parc (eu-parc.eu), and collaboration is essential. Some proposals keep exposure and 
effects  as  independent  assessments  (Rivetti  and  Campos  2023);  others  suggest  a  more  integrative 
approach of the exposure and effect assessments (Di Nicola et al. 2023). 

The proposed implementation is summarised in Figure 5. In addition to the integration of exposure and 
effects,  a  key  element  is  the  consideration  of  the  environmental  characteristics  and  the  ecological 
conditions, which obviously are interlinked.  

48 https://aopwiki.org/ accessed on 27 Feb 2025 

46 

 
 
 
 
Figure 5. Proposed implementation of environmental NGRA. 

In the regulatory context, in addition to exposure-led risk assessments, the identification of intrinsic 
hazards not linked to exposure assessments is also relevant, for example CLP aims to cover hazards of 
non-intended  emissions  as  a  result  of  accidents  or  spills.  Under  REACH,  the  hazard  identification 
triggers  exposure  assessment  and  risk  characterisation  when  certain  hazard  criteria  are  met.  The 
environmental  and  ecological  conditions  are also  relevant, for  example  for  assessing  the  hazards  of 
accidental non-intended emissions. The current technologies offer options for adapting the information 
relevant  for  different  spatial  scales  and  conditions.  Therefore,  the  environmental  NGRA  should  be 
complemented with innovative proposals for addressing the hazard identification requirements.  

The new paradigm should start from the regulatory need, supporting the regulatory decisions needed 
for individual chemicals and proposed uses, but exploring other options than those currently used. For 
example,  the  paradigm  should  support  hazard  identification  needs  and  the  associated  risk 
communication and “downstream” legislative measures but may suggest different hazard classes and 
categories than those currently used, e.g., informing on the relative vulnerability of taxonomic groups 
and environmental sub-compartments. 

47 

 
 
 
 
 
References 

Brooks BW, van den Berg S, Dreier DA, et al. (2023) Towards Precision Ecotoxicology: Leveraging 
Evolutionary Conservation of Pharmaceutical and Personal Care Product Targets to Understand 
Adverse Outcomes Across Species and Life Stages. Environmental Toxicology and Chemistry 
43(3):526-536 doi:10.1002/etc.5754 

Chapman PM, Fairbrother A, Brown D (1998) A critical evaluation of safety (uncertainty) factors for 
ecological  risk  assessment.  Environmental  Toxicology  and  Chemistry  17(1):99-108 
doi:10.1002/etc.5620170112 

Di Nicola MR, Cattaneo I, Nathanail AV, et al. (2023) The use of new approach methodologies for the 
environmental risk assessment of food and feed chemicals. Current Opinion in Environmental 
Science and Health 31 doi:10.1016/j.coesh.2022.100416 

Dufourcq  Sekatcheff  E,  Jeong  J,  Choi  J  (2024)  Bridging  the  gap  between  human  toxicology  and 
ecotoxicology  under  one  health  perspective  by  a  cross‐species  adverse  outcome  pathway 
network 
and  Chemistry 
doi:10.1002/etc.5940 

toxicity.  Environmental  Toxicology 

reproductive 

for 

Kosnik MB, Schuwirth N, Rico A (2024) Harnessing Computational Methods to Characterize Chemical 
Impacts  on  Biodiversity.  Environmental  Science  &  Technology  Letters  11(3):185-194 
doi:10.1021/acs.estlett.3c00865 

Newman  MC,  Ownby  DR,  Mézin  LCA,  et  al.  (2000)  Applying  species‐sensitivity  distributions  in 
ecological risk assessment: Assumptions of distribution type and sufficient numbers of species 
Environmental Toxicology and Chemistry 19(2):508-515 doi:10.1002/etc.5620190233 
Rivetti C, Campos B (2023) Establishing a NexGen, mechanism‐based environmental risk assessment 
paradigm  shift:  Are  we  ready  yet?  Integrated  Environmental  Assessment  and  Management 
19(3):571-573 doi:10.1002/ieam.4763 

Rivetti C, Houghton J, Basili D, Hodges G, Campos B (2023) Genes‐to‐Pathways Species Conservation 
Analysis:  Enabling  the  Exploration  of  Conservation  of  Biological  Pathways  and  Processes 
Across 
42(5):1152-1166 
doi:10.1002/etc.5600 

Species.  Environmental  Toxicology 

and  Chemistry 

Schaupp CM, Maloney EM, Mattingly KZ, Olker JH, Villeneuve DL (2023) Comparison of in silico, 
in vitro, and in vivo toxicity benchmarks suggests a role for ToxCast data in ecological hazard 
assessment. Toxicological Sciences 195(2):145-154  

Schumann  P,  Rivetti  C,  Houghton  J,  Campos  B,  Hodges  G,  LaLone  C  (2024a)  Combination  of 
computational  new  approach  methodologies  for  enhancing  evidence  of  biological  pathway 
conservation 
species.  Science  of  The  Total  Environment  912:168573 
doi:https://doi.org/10.1016/j.scitotenv.2023.168573 

across 

Schumann PG, Chang DT, Mayasich SA, Vliet SMF, Brown TN, LaLone CA (2024b) Cross-species 
molecular docking method to support predictions of species susceptibility to chemical effects. 
Computational Toxicology 30:100319 doi:https://doi.org/10.1016/j.comtox.2024.100319 

Smith EP, Cairns J (1993) Extrapolation methods for setting ecological standards for water quality: 

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Arch Toxicol Suppl 19:137-46 doi:10.1007/978-3-642-60682-3_12 

48 

 
 
 
2.2.5. Integration of Human Health and Environmental assessment 
Strong  scientific  evidence  supports  an  integrative  approach  (e.g.,  One  Health)  in  policymaking  and 
interventions49, including the increased use of information to evaluate chemical risks to humans and the 
environment. Health and ecological systems are deeply interconnected, with risks and impacts often 
overlapping between the two areas through the disruption of similar biological processes. Non-animal 
approaches  offer  opportunities  to  adopt  a  more  unified  approach  to  identifying,  predicting,  and 
managing  the  consequences  of  chemical  exposures  by  assessing  chemical  toxicity-based  chemical-
biology interaction and their multifaceted impacts on biological systems.  

The  shift  towards  integrated  assessment  methods  begins  with  replacing  traditional  animal  tests  by 
leveraging  significant  technological  advancements  in  bioscience,  including  molecular  toxicology, 
systems biology, and exposure science. These advancements have resulted in the development of data-
rich  tools  and  models  that  can  clarify  the  key  mechanistic  pathways  linking  chemical  exposure  to 
adverse health effects in both humans and ecological receptors. 

Transitioning to  integrated  assessment  strategies also  involves addressing the conceptual  and socio-
technological obstacles that stem from a conventional dependence on representative animal species, 
which  imperfectly  serve  as  substitutes  for  humans  and  natural  populations.  Greater  collaboration 
between human health and environmental scientists in this process should enhance the overall scientific 
robustness,  reproducibility,  and  translational  relevance  of  risk  assessments,  ultimately  supporting 
regulatory  frameworks  that  are  adaptable,  transparent,  and  protective  of  public  health  and  the 
environment. 

In addition to promoting ethical goals such as reducing animal testing, integrated assessments grounded 
in mechanistic evidence of chemical toxicity reduce duplication, encourage innovation, and support the 
EU’s strategic aims of leading on sustainability and competitiveness. 

Considering  the  many  opportunities  presented  by  scientific,  regulatory,  economic,  and  ethical 
imperatives,  promoting  further  integration  of  human  and  environmental  safety  should  be  a  key 
objective. This goal should, therefore, be fully incorporated into all relevant initiatives arising from this 
roadmap. 

Cross-cutting  recommendations,  while  essential  for  further  integrating  Human  and  Environmental 
safety assessments, are also relevant to other parts of the roadmap. 

Increase non-animal approaches familiarity through education and training.  

• 
•  Support the development of comprehensive professional and academic training programmes 

that integrate human health, environment, and regulation. 

•  Ensure that the Common Data Platform under one substance, one assessment (OSOA) 

contains high-quality human and environmental data, including complex datasets such as 
from non-animal approaches, OMICS, exposure, and others. 

•  Develop plans for the economic viability of non-animal approaches by creating opportunities 
for private investors and innovators to compete in providing non-animal approaches services 
and products that are regulatory fit for purpose and relevant to human and environmental safety. 

Background 

Rationale for Change – justifying a transition to non-animal approaches that are relevant for 
the protection of human health and the environment. 

49 European Commission: Group of Chief Scientific Advisors and Directorate-General for Research and 
Innovation, One Health governance in the European Union, Publications Office of the European Union, 2024, 
https://data.europa.eu/doi/10.2777/8697309 

49 

 
 
 
 
The European Commission's Group of Chief Scientific Advisors (GCSA), through the Scientific Advice 
Mechanism  (SAM),  issued  an  opinion  on  One  Health  governance  in  the  EU,  highlighting  the 
interconnectedness of human, animal, and environmental health. 

There is strong scientific evidence supporting the application of a One Health approach to policymaking 
and interventions, including the better use of relevant information to assess the risks of chemicals for 
protecting both humans and the environment. 

Ongoing regulatory changes present a unique opportunity to integrate human health and environmental 
risk assessments, with the OSOA approach serving as a facilitator for this more comprehensive and 
efficient framework. 

A shift to an integrated approach using non-animal approaches, and where assessments are based on 
mechanistic  understanding,  offers  the  best  chance  to  effectively  and  scientifically  extrapolate 
toxicological data from one species to another, including humans. 

An  integrated  assessment  using  non-animal  approaches  streamlines  and  improves  both  safety  and 
hazard assessments across all relevant regulations, resulting in a general reduction in animal testing 
across regulatory areas, while fulfilling the EU Commission's dual objectives: simplifying regulations 
and simultaneously maintaining or even improving protection levels. 

Integrating the One Health approach into existing EC health priorities, such as the EU Life Sciences 
Strategy  and the  EU  Biotech Acts,  will strengthen the  competitiveness  of  EU  SMEs  and  encourage 
sustainable innovation throughout the sector. To make One Health and OSOA operational, human and 
environmental  evidence  need  a  common  framing.  Integration  should  explicitly  link  to  ecology  and 
biodiversity research programmes (e.g., Biodiversa+, PARC, projects funded under the Horizon Europe 
programme)  and  develop  frameworks  for  coordinated  assessments  and  decisions  across  regulatory 
regulations. 

Critical core requirements for further integration of human and environmental safety assessment. 

Supporting greater integration of human and environmental risk assessment requires meeting several 
key requirements that are vital for the transition to non-animal approaches and their adoption across 
different regulatory sectors. These considerations should be addressed during the entire implementation 
of the roadmap, including scientific, regulatory, and educational efforts. Such considerations include, 
but are not limited to: 

•  Ensuring  that  opportunities  for  further  integration  of  human  and  environmental  safety  are 
woven into all actions related to scientific, educational, economic initiatives, and regulatory 
processes of the roadmap as core elements. 

• 

Improving  the  utilisation  of  exposure  information  by  enhancing  the  use  of  human  and 
environmental (bio)monitoring data. 

•  Supporting  a  greater  understanding  of  how  toxicologically  relevant  processes  essential  to 
human health and  environmental safety are evolutionarily conserved across distantly related 
species, and if and how those translate into possible adverse effects across organisms. 

•  Developing  integrated,  tiered  safety  assessment  frameworks  that  maximise  data  integration 

across human and environmental safety, including exposure information. 

•  Establish cross-sectorial organisational structures to foster closer collaboration among human 
health and environmental scientists to overcome conceptual, language and cultural barriers to 
unite these disciplines under the banner of 21st-century toxicology. 

50 

 
 
 
 
•  Enhancing confidence in alternatives to animal testing and their applications for human health 
and the environment by developing case studies and promoting regulatory alignment with the 
scientific evidence. 

Recommendations 
Towards  a  paradigm  shift:  To  fully  realise  the  goal  of  enabling  broader  integration  of  human  and 
environmental  safety,  a  progressive  regulatory  framework  is  essential  to  support  a  transition  to  an 
integrated  assessment  using  alternatives  to  animal  testing,  aligned  with  growing  confidence  and 
certainty in the results of cross-species extrapolation, which is based on a mechanistic understanding of 
physiological  and  ecological  similarities  and  differences  for  specific  regulatory  purposes.  For 
environmental assessments reality-checks that leverage the integration of existing data and knowledge 
from  environmental  monitoring  and  ecological  research,  is  an  opportunity  to  further  facilitate  a 
transition. Short-, medium-, and long-term objectives can be achieved through education, training, data 
and knowledge sharing, and scientific development, as outlined below. 

The  short-  and  medium-term  recommendations  are  based  on  existing  regulatory  frameworks  and 
initiatives;  however,  they  still  need  transdisciplinary  research  and  the  integration  of  these 
infrastructures. Longer-term recommendations will require changes to the current regulatory paradigm 
and policy. 

Short-term recommendations 

Develop and enable the necessary structures to facilitate further integration of human and 
environmental safety. 

• 

Identify  and  support  the  networks  that  can  build  consensus  (e.g., ASPIS,  EPAA,  EU  cross-
agency  One  Health  task  force,    and  related  structures).  Define  leadership  roles  and  employ 
change management practices to enable this integration. 

•  Foster closer collaboration between human toxicologists and environmental scientists to unify 

these disciplines while recognising and considering their specificities. 

•  Define the criteria and indicators most useful for monitoring and evaluating the effectiveness 

of an integrated approach to assessing chemical risks for regulation. 

•  Short-term  research  needs  which  can  be  delivered  by  relevant  partners,  including  research 

organisations, industry consortia and academia: 

I.  Define the “biological domains of applicability” for currently available assays. 
II. 

Identify data streams common to human and environmental species and focus on 
how to use them in an integration context. 

III.  Develop an ontology of data based on non-animal approaches that complies with 

regulatory requirements and are relevant to human health and the environment. 

IV.  Design and develop case studies that illustrate how the integration of human and 

environmental health would function in practice. 

Medium-term recommendations 

Support cross-disciplinary research and knowledge exchange. 

• 

Invest in research: 

•  Develop a reference list of mechanisms of toxicity and their roles in identifying adverse 
effects  on  different taxonomic  groups and  ecological  functions  and  establish testing 
strategies that consider their use for both humans and the environment. 

• 

• 

Identify current TD/TK approaches common to human and environmental species. 

Identify  mechanisms  of  toxicity  which  may  lead  to  adverse  effects  on  different 
taxonomic groups. 

51 

 
 
 
• 

Increase  knowledge  of  toxicity  mechanisms,  biological  roles  and  ecological 
interactions  for  understanding  the  potential  adverse  effects  at  different  levels  of 
biological organisation. 

•  Research in comparative toxicology and cross-cutting areas (e.g., broaden the number 
and diversity of representative non-animal-based test systems and PBK models). 

•  Expand research in comparative toxicology and cross-cutting areas (e.g., broaden the 
number  and  diversity  of  representative  non-animal-based  test  systems  and  PBK 
models). 

Long-term recommendations 

•  Phasing  out  animal  testing  across  regulatory  sectors  by  utilising  NAMs  data  that  meets 
regulatory  assessment  standards  for  comprehensive  protection  of  human  health  and  the 
environment.Facilitate a paradigm shift towards tiered safety assessments enabling complete 
integration of human and environmental data in regulatory risk assessment, e.g. an overarching 
next  generation  risk  assessment  (NGRA)  framework  which  describes  how  to  seamlessly 
integrate human health and environment workflows. 

•  Enable legislative change to adopt an integrative approach, including indicators and monitoring 
programmes  to  assess  the  actual  level  of  protection  achieved  and  implement  additional 
measures as necessary. 

52 

 
 
 
 
2.3. Overarching opportunities across Human Health and 
Environmental Safety 

2.3.1. Replacing in vivo with in silico methods 
Applying assessment elements from the QSAR Assessment Framework (QAF) to 
support increased regulatory use of QSARs 

Rationale and background  

Risk  assessors  may  face  situations  where  data  availability  ranges  from  scarce  to  abundant.  When 
toxicological data for substances are lacking, limited physico-chemical or toxicity information can be 
supplemented with QSAR models to support hazard identification and characterisation efforts. These 
predictive approaches help bridge data gaps by estimating toxicity or identifying analogous substances 
with  established  profiles.  Furthermore,  for  certain  toxicological  endpoints,  the  performance  and 
coverage  of  available  models  are  sufficiently  robust  to  warrant  their  consideration  as  the  default 
approach for meeting specific regulatory requirements. Historically, the use of QSARs as a primary 
source of information regarding hazardous properties was limited. This was mainly because developers 
provided information on model performance (e.g. sensitivity or specificity for qualitative properties and 
prediction  accuracy  for  quantitative  ones),  while  definition  of  applicability  domain  and  model 
limitations was often missing. Consequently, confidence in QSAR predictions was limited because e.g. 
the information that a model is performing well for 75% of chemicals from the validation set is not 
helping the user in concluding if a substance of concern will be included in this subset. As a result, this 
limitation  impeded  broader  acceptance,  as  both  end  users  and  regulatory  evaluators  lacked 
comprehensive methodology to assess the reliability of predictions for compounds of interest.  

The recently implemented OECD QSAR Assessment Framework (QAF) has not only updated criteria 
for model validity but also introduced new measures to evaluate the reliability of individual predictions. 
These newly introduced principles for predicting reliability and associated Assessment Elements (AEs) 
provide a comprehensive solution for determining whether a given prediction is appropriate and reliable 
for regulatory applications. Nonetheless, effectively applying these AEs currently demands significant 
computational expertise and in-depth understanding of the endpoint for which predictions are generated. 
Accordingly,  it  is  essential  to  establish  a  set  of  objective  criteria  which,  together  with  the  newly 
developed AEs for model validity and prediction reliability, will enhance the broader adoption of the 
QAF by both model developers and end-users. 

Proposed approach: A project team is planned to be set up to achieve that through the following 
actions: 

Identify which AEs would require such criteria. 

• 
•  For each identified AEs propose suitable criteria, check for every criterion if it can be 

applicable to all properties or needs to be endpoint-specific and whether that criterion needs 
to be software implementable. As an example: under prediction reliability, the Performance of 
the model for similar substances (AE 3.4 in the Prediction and Result Checklists of the OECD 
QAF) requires a clear definition of 'similar substances,' which can encompass structural, 
physicochemical, and mechanistic similarities. Moreover, this definition cannot be universal 
as different toxicological properties might be differently affected by various elements of 
similarity. Therefore, for this AE, there is a need for software implementable, endpoint-
specific similarity measures to assess the adequacy of the closest analogues used to assess 
performance for substances similar to the target substance.  

•  Build case studies to examine how newly proposed criteria work in practice. 
• 

In case of positive outcome, amend the OECD QAF and promote the solutions within the 
modelling community. 

Planned output(s)  

53 

 
 
 
 
•  QAF AEs assessed and amended by a set of suitable criteria, 
•  Set of case studies demonstrating usability of QAF and newly developed criteria QAF 

updated to increase its practical value. 

Intended outcome(s)  

It  is  anticipated  that  this  initiative  will  substantially  enhance  regulatory  adoption  of  QSARs  by 
increasing  awareness  of  the  importance  of  assessing  the  reliability  of  individual  predictions  and 
providing effective tools for this purpose. The capability to evaluate the reliability of each prediction is 
expected to bolster regulatory confidence in computational methods. Furthermore, promoting newly 
developed criteria among software developers will facilitate straightforward assessment in accordance 
with QAF, as all essential information required will be automatically generated by the software. 

References  

Benfenati E, Chaudhry Q, Gini G, Dorne JL. 2019. Integrating in silico models and read-across methods for 

predicting toxicity of chemicals: A step-wise strategy. Environ Int.131:105060. doi: 
10.1016/j.envint.2019.105060. 

(Q)SAR Assessment Framework: Guidance for the regulatory assessment of (Quantitative) Structure Activity 
Relationship models and predictions - Second edition, OECD Series on Testing and Assessment No. 
405, OECD Publishing, Paris. 

Ratier A, Lopes C, Multari G, Mazerolles V, Carpentier P, Charles S. 2022.New perspectives on the calculation 

of bioaccumulation metrics for active substances in living organisms. Integr Environ Assess Manag. 
18(1):10-18. doi: 10.1002/ieam.4439. 

54 

 
 
2.3.2. Developing TK and ADME models for regulatory needs 
Development of reliable ADME (absorption, distribution, metabolism and elimination) 
profiling, PBK (physiologically based kinetic) modelling and (Q)IVIVE tailored to 
regulatory needs 

Rationale and background  

Understanding what the body does to the chemical “toxicokinetics” (TK) and what the chemical does 
to  the  body  “toxicodynamics”  (TD)  are  the  fundamental  dimensions  of  toxicology.  TK  reflects  the 
processes by which a chemical substance enters, moves through, is degraded, and ultimately exits the 
body, either unchanged or as metabolic byproducts, and is fundamental to chemical hazard assessment. 
This  information  is  obtained  through  investigating  the  absorption,  distribution,  metabolism,  and 
excretion (ADME) of substances.  

Traditionally, animal models have been used in toxicology, where both TK and TD properties influence 
observable outcomes. However, these studies often do not distinguish the specific contributions of each 
property  or  their  quantitative  relationship.  This  presents  a  challenge  in  developing  non-animal 
approaches and comparing them to data from animal research. For example, low toxicity observed in 
vivo may result from a weak toxicodynamic profile of the parent compound, rapid detoxification, or 
limited  systemic  exposure.  In  contrast,  high  toxicity  may  result  from  bioactivation  of  the  parent 
compound  to  a  toxic  moiety  or  from  bioaccumulation.  Considering  both  TK  and  TD  properties  of 
chemicals is necessary when proposing non-animal approaches to reliably predict hazardous properties 
for chemicals. 

Therefore, understanding of these mechanisms is now integral to non-animal approaches that address 
complex toxicological endpoints, as reliable results require consideration and computational integration 
of all relevant elements to achieve regulatory relevance. Consequently, an ADME component forms 
part of all new non-animal approaches. Additionally, PBK models offer means to predict toxicity for 
hazard characterisation, and QIVIVE enables conversion of effect doses observed in vitro to external 
exposures (oral, dermal, or inhalation) for hazard and risk characterisation through reverse dosimetry. 

Despite the importance of ADME and TK data, many pieces of legislation (such as REACH) have not 
explicitly required such data, highlighting the need for standardised methodologies capable of reliably 
assessing  these  properties  across  diverse  physico-chemical  characteristics.  In  addition,  international 
standardisation  and  updated  OECD  guidance  are  necessary  to  expand  coverage  of  non-animal 
approaches suitable for determining ADME properties. 

Proposed approach 

This action will focus on four tasks: 

-  Standardising methods for reliable TK parameter determination across chemical classes and 

harmonising them internationally at the OECD level; 

-  Developing metabolically competent in vitro models and analytical techniques to accurately 

identify biotransformation products in vitro; 

-  Developing and integrating models in open access tools to assess bioaccumulation potential in 

humans and other representative environmental species, 

-  Developing PBK models and QIVIVE tools that can be parameterised using in vitro data and 

in silico results to apply them in NGRA. 

Standardisation of Methods: As ADME properties are increasingly recognised as critical components 
in risk assessment, the routine generation of physico-chemical properties and in vitro kinetic parameters 
is  crucial.  These  include  partition  coefficients  (octanol/water,  KoW),  intrinsic  clearance  (Clint), 
unbound fraction (Fu), and absorption rate. In a number of contexts, isoform-specific metabolism for 

55 

 
 
 
 
phase  I  and  phase  II  enzymes  and  transporter  data  are  also  significant,  particularly  given  that 
polymorphic enzymes can result in considerable inter-individual variability in human TK and TD. With 
these parameters, generic or chemical-specific physiologically based kinetic (PBK) models can be used 
to predict internal concentrations in body fluids (such as blood or urine) or target organs across species 
(forward dosimetry). Consequently, it will be necessary to identify appropriate models compatible with 
these properties, including thorough characterisation of the variability and uncertainty associated with 
model outcomes and applicability domains. These activities will subsequently inform the development 
of  relevant  test  guidelines  and  OECD  guidance  documents  and  update  of  the  relevant  OECD 
Harmonised  Templates  (OHTs)  to  facilitate  international  harmonisation  and  broad  regulatory 
acceptance. 

Identification  of  Biotransformation  Products:  Metabolic  detoxification  and  bioactivation  are  key 
processes  that  can  significantly  impact  toxicological  outcomes.  Primary  hepatocytes  are  commonly 
employed to assess hepatic clearance, but these are unsuitable for incubation periods exceeding four 
hours.  In  addition,  current  analytical  methods  predominantly  track  changes  in  parent  compound 
concentration and often lack robustness in detecting and quantifying relevant metabolites. This task will 
prioritise  the  identification  of  well-characterised,  metabolically  competent  in  vitro  models  with 
sustained  stability,  followed  by  the  selection  of  suitable  analytical  techniques  capable  of  detecting 
multiple biotransformation products using semi- or non-targeted approaches. 

Models for Assessing Bioaccumulation Potential: Accurately determining whether a substance may 
bioaccumulate  within  organisms  is  essential  not  only  for  PBT  assessment  but  also  for  predicting 
systemic  toxicity  using  non-animal  approaches.  The  toxicity  of  bioaccumulative  compounds  is 
frequently underestimated by most in vitro methods; therefore, reliable indicators of bioaccumulation 
potential  are  crucial  to  minimise  false  negatives. This  task  will  primarily  focus  on  optimising  PBK 
modelling approaches for assessing bioaccumulation while using data generated from in vitro models 
developed under other tasks within this action. 

Planned output(s) 

✓  In  the  short  term:  Development  of  OECD  Test  Guidelines  for  the  generation  of  metabolic 
clearance data in human hepatocytes and protein binding (fraction unbound) to predict human 
blood and plasma concentrations, along with a defined approach for estimating clearance (half-
life and other TK properties such as AUC and Css).  

✓  In the mid-term, develop additional defined approaches to cover dermal and inhalation routes 

of exposure and develop OECD TGs as well. 

✓  Stimulation of efforts aiming at generating TK and ADME data from in vitro systems. 
✓  Preparation of an in vitro TK package (annex update, validated test methods and guidance for 

registrants) to be used as standard information requirement in legislation.  

✓  Future TK packages should cover relevant species for risk assessment, including human but 
also consider environmentally relevant species, e.g, terrestrial and aquatic vertebrates, birds and 
mammals.  Initialisation  of  projects  aiming  at  identification  and  characterisation  of  models 
suitable for the identification of metabolites and bioaccumulation potential. 

✓  Development of an in vivo and in vitro ADME/TK database using OECD harmonised templates 
(e.g. OHT 58 and OHT 201 and any new OHTs for newly validated in vitro ADME methods) 
to support the use of ADME/TK data in NGRA across regulations. 

Intended outcome(s) 

This action aims to promote the widespread adoption and standardisation of in vitro ADME/TK methods 
across various regulatory frameworks. As this is a critical component for future non-animal approaches, 
the  routine  generation  of  TK/ADME  data  will  facilitate  the  development  of  New  Approach 

56 

 
 
 
 
 
 
Methodologies (NAMs) for assessing complex toxicological endpoints. Furthermore, the data produced 
through this initiative can support existing regulatory processes, thereby enhancing confidence in non-
animal  hazard  assessment  strategies.  Finally,  the  capability  to  identify  relevant  biotransformation 
products will allow for greater confidence in positive and negative findings where necessary. 

References 
Beaudoin  R,  Benfenati  E,  Billat  PA,  Buratti  FM,  Chiara  Dall’Asta,  KeyvinDarney,  Gianni  Galaverna,  Luca 
Dellafiora L, Pedroni L, Hoogenboom R, Lautz L, Louisse J, Roncaglioni A, Testai E, Tebby C, Thépaut 
E,  Vichi  S,  Zeman  F.  2023.  Case  studies:  Generic  kinetic  and  kinetic-dynamic  modelling  in  human 
subgroups of the population and animal species to support transparency in food and feed safety. EFSA 
supporting publication2024:EN-9010. 138 pp. doi:10.2903/sp.efsa.2024.EN-9010. 

Bossier,  H.,  J.  Chau,  C.  Ndour,  M.  Varewyck,  T.  Verbeke,  S.  Vergucht  A  Web-based  open  source  tool  for 
Toxicokinetic and Toxicodynamic modelling EFSA Supporting Publications, 17 (11) (2020), p. 1926E, 
10.2903/sp.efsa.2020.EN-1926  

Carnesecchi E, Langezaal I, Browne P, Batista-Leite S, Campia I, Coecke S, Dagallier B, Deceuninck P, Dorne 
JLC, Tarazona JV, Le Goff F, Leinala E, Morath S, Munn S, Richardson J, Paini A, Wittwehr C. 2023. 
OECD  harmonised  template  201:  Structuring  and  reporting  mechanistic  information  to  foster  the 
integration of new approach methodologies for hazard and risk assessment of chemicals. Regul Toxicol 
Pharmacol. 142:105426. doi: 10.1016/j.yrtph.2023.105426.   

Davidson-Fritz SE, Ring CL, Evans MV, Schacht CM, Chang X, Breen M, Honda GS, Kenyon E, Linakis MW, 
Meade  A,  Pearce  RG,  Sfeir  MA,  Sluka  JP,  Devito  MJ,  Wambaugh  JF.2025.  Enabling  transparent 
toxicokinetic  modeling  for  public  health  risk  assessment.  PLoS  One.  20(4):e0321321.  doi: 
10.1371/journal.pone.0321321. PMID: 40238721; PMCID: PMC12002443.   

Dorne JLCM, Cortiñas-Abrahantes J, Spyropoulos F, Darney K, Lautz L, Louisse J, Kass GEN, Carnesecchi E, 
Liem AKD, Tarazona JV, Billat PA, Beaudoin R, Zeman F, Bodin C, Smith A, Nathanail A, Di Nicola 
MR, Kleiner J, Terron A, Parra-Morte JM, Verloo D, Robinson T. TKPlate 1.0: An Open-access platform 
for toxicokinetic and toxicodynamic modelling of chemicals to implement new approach methodologies 
in chemical risk assessment. EFSA J. 2023 Nov 14;21(11):e211101. doi: 10.2903/j.efsa.2023.e211101.  
Bronzwaer S, Devos Y, Dorne JCM, Dujardin B, Dumont AF, Heppner C, Fernández EN, Paraskevopoulos K, 
das Neves CG. 2025. Advancing EFSA's regulatory science: Updated research and innovation needs. 
EFSA  J.  2025  Apr  16;23(4):e220401.  doi:  10.2903/j.efsa.2025.e220401.  PMID:  40242780;  PMCID: 
PMC12001418. 

Hines DE, Bell S, Chang X, Mansouri K, Allen D, Kleinstreuer N. 2022. Application of an accessible interface 
in  vivo  extrapolation.  Front  Pharmacol. 
in  vitro 

to 

for  pharmacokinetic  modeling  and 
https://doi.org/10.3389/fphar.2022.864742  

Ratier A, Lopes C, Multari G, Mazerolles V, Carpentier P, Charles S. 2022.New perspectives on the calculation 
of  bioaccumulation  metrics  for  active  substances  in  living  organisms.  Integr  Environ  Assess  Manag. 
18(1):10-18. doi: 10.1002/ieam.4439.   

57 

 
 
 
 
2.3.3. Determining the Point of Departure (PoD) and signatures of toxicity for 
chemicals through measured omics data 
Rationale and background   

The hazard assessment of complex systemic toxicity endpoints, such as developmental and reproductive 
toxicity, currently depends on apical adverse effects observed at the organ or organism level. In contrast, 
most  non-animal approaches  yield data  primarily  at the molecular level, often  lacking the  means to 
directly demonstrate toxic effects at the apical level. Hence, a robust understanding of the relationship 
between  molecular-level  changes  and  subsequent  adverse  outcomes  is  essential  for  the  regulatory 
adoption of alternatives to animal testing. 

A  number  of  challenges  remain  unresolved:  there  is  no  consensus  regarding  the  criteria  to  assess 
biological  relevance  of  models  used  to  generate  such  data,  sampling  and  sequencing  methods  are 
variable,  substantial  differences  exist  in  data  (pre)processing  workflows,  and  multiple  strategies  are 
employed  to  derive  points  of  departure  or  identify  toxicological  signatures. While  recent  efforts  by 
OECD WPHA and WNT have led to progress toward standardising sampling procedures, other aspects 
still  require  international  consensus  and  harmonisation. Addressing  these  challenges  may  facilitate 
broader adoption of animal-free approaches and has the potential to improve the efficiency of chemical 
risk  assessments.  Omics  data  can  be  generated  in  a  high-throughput  manner,  resulting  in  extensive 
datasets that may directly identify modes of toxicological action.  

Proposed approach 
This initiative aims to support international harmonisation efforts by concentrating on three primary 
areas:  

•  Establishing criteria to determine the biological relevance of models used for omics data 

generation.  

•  Standardising the omics data processing pipeline.  
•  Reaching consensus on methodologies for identifying toxicity signatures and deriving points 

of departure (PoD).  

Rather than initiating new activities, this action will identify and engage with relevant ongoing projects 
to ensure that the focus areas outlined above are adequately addressed. For example, the existing OECD 
working group responsible for developing guidance on omics-based read-across—an extension of the 
OECD Omics Reporting Framework (OORF)—is well positioned to address key areas 1 and 2. The 
main  questions  to  consider  regarding  biological  relevance  are:  how  to  demonstrate  that  key 
toxicological features of concern are addressed by the proposed biological model (in vitro assays), and 
to what extent this evidence should rely on mechanistic or scientific understanding versus empirical 
data.  

For the omics processing pipeline, the OECD Omics Reporting Framework (ORF) has identified core 
elements  of  data  processing  for  both  transcriptomics  and  metabolomics,  allowing  flexibility  in  the 
methods  used  to  address  these  elements.  In  this  action,  appropriate  data  processing  techniques  for 
different scenarios will be determined.  

For  key  area  3,  the  systemic  toxicity  working  group  under  the  OECD  WPHA,  comprising  leading 
international  experts  in  alternatives  to  animal  testing,  is  evaluating  various  approaches  to  address 
systemic toxicity and considering diverse strategies for PoD derivation and the identification of high-
concern  toxicity  indicators.  In  this  area,  the  most  important  elements  will  be  to  agree  the  assays 
composition  of  the  battery  to  address  wide  toxicological  coverage,  specific  hazard  identification, 
TK/IVIVe and ADME.  

Ongoing and future OECD projects are vital for achieving global harmonisation; however, this action 
is  not  confined  to  OECD  initiatives.  Other international  collaborations,  such as APCRA,  and  major 
scientific projects under Horizon Europe, PARC, CEFIC LRI, SCCS, and US funding schemes will also 
be systematically evaluated to identify opportunities for supporting these goals. 

58 

 
 
Planned output(s) 

•  To develop case studies with input from JRC, ECHA, and EFSA regarding the use of 

bioactivity dose-response data for relevant in vitro endpoints and submit them to the OECD 
IATA programme;  

•  To finalise the OECD Read Across Application Module under OORF;  
•  To support the development of guidance documents for applying Points of Departure (PoDs) 
in next generation risk assessment based on alternatives to animal testing, with the intention 
to reduce and ultimately phase out animal studies; 

•  To develop a practical workflow and toolbox to integrate in vitro and in silico non-animal 

approaches for deriving PoDs based on such approaches (e.g. by jointly work on regulatory 
specific adaptations of ASPA workflow). 

Intended outcome(s) 
This action is intended to support the regulatory application of omics and molecular data by first 
establishing standardised data (pre)processing workflows and agreeing on a set of techniques to derive 
PoDs and identify toxicological signatures. Case studies are to be used to illustrate the applicability of 
these techniques for various regulatory purposes. Guidance documents are also intended to promote 
consistent approaches for data processing among applicants, registrants, and regulators. 

References 
Abedini JA, Cook B, Bell SM, Chang XQ, Choksi NY, Daniel AB, Hines DE, Karmaus AL, Mansouri 

K, McAfee E, Phillips J, Rooney JP, Sprankle CS, Allen D, Casey WM, Kleinstreuer NC. 
2021. Application of new approach methodologies: ICE tools to support chemical evaluations. 
Comput Toxicol. 20:100184. https://doi.org/10.1016/j.comtox.2021.100184   

EFSA, Bronzwaer S, Devos Y, Dorne JCM, Dujardin B, Dumont AF, Heppner C, Fernández EN, 

Paraskevopoulos K, das Neves CG. 2025. Advancing EFSA's regulatory science: Updated 
research and innovation needs. EFSA J. 2025 Apr 16;23(4):e220401. doi: 
10.2903/j.efsa.2025.e220401. PMID: 40242780; PMCID: PMC12001418. 

Escher, S. E., Partosch, F., Konzok, S., Jennings, P., Luijten, M., Kienhuis, A., de Leeuw, V., Reuss, 

R., Lindemann, K., & Bennekou, S. H. Development of a Roadmap for Action on New 
Approach Methodologies in Risk Assessment. 2022. EFSA Supporting Publications. 
Friedman PK, Gagne M, Loo LH, Karamertzanis P, Netzeva T, Sobanski T, Franzosa JA, Richard AM, 

Lougee RR, Gissi A, Lee JJ, Angrish M, Dorne JL, Foster S, Raffaele K, Bahadori T, Gwinn 
MR, Lambert J, Whelan M, Rasenberg M, Barton-Maclaren T, Thomas RS. Utility of In Vitro 
Bioactivity as a Lower Bound Estimate of In vivo Adverse Effect Levels and in Risk-Based 
Prioritization. Toxicol Sci. 173(1):202-225. doi: 10.1093/toxsci/kfz201.  

59 

 
 
 
 
 
 
 
2.3.4. Fostering the use of alternatives to animal testing for nanomaterials and 
nanoparticles risk assessment 
Background  

EFSA’s Nano Guidance documents (EFSA Scientific Committee, 2021a,b) highlight the relevance in 
using alternatives to animal testing as part of Integrated Approaches to Testing and Assessment (IATA) 
for evaluating nano-specific toxicokinetic and toxicodynamic considerations. With the aim to develop 
further recommendations on the use of alternatives to animal testing for nanomaterials and small/nano 
particles risk assessment, EFSA has funded experimental projects established through partnership with 
EU MSs and extra EU organisations, and in collaboration with other EU agencies. Some examples are 
the  EFSA  NANOCELLUP  Project  (GP/EFSA/SCER/2020/04)  and  EFSA  NAMs4NANO  Project 
(GP/EFSA/MESE/2022/01). EFSA’s NANOCELLUP Project is an example of a recently finalised case 
study aimed to design an IATA based on alternatives to animal testing to fill data gaps identified during 
real  risk  assessment  practice.  EFSA’s  NAMs4NANO  Project is  still  ongoing  and  aims  to  develop  a 
“Qualification system for NAMs for EFSA” and, in parallel, a number of proof-of-concept case studies. 
While the ultimate goal would be to establish a system for broader use in the context of alternatives to 
animal testing for chemical risk assessment in general, nanomaterials risk assessment has been selected 
as starting point being an area in which the implementation of novel approaches is promising for several 
reasons,  including  the  general  unavailability  of  adapted  OECD  Test  Guidelines.  The  knowledge 
developed  from  EFSA  projects  on  the  use  of  alternatives  to  animal  testing  for  nanomaterials  risk 
assessment will be used in the context of the new self-task mandate from EFSA Scientific Committee 
to work on the update of 2021 EFSA’s Nano Guidances (M-2024-00062). 

Ongoing action 

EFSA started a multiannual project called NAMs4NANO (2023-2027, 5.3M). The project was granted 
to a large consortium involving several EU MSs and extra EU organisations and includes a partnership 
with  JRC. The main  goal of  this  project  is  to  promote  the  implementation  of  alternatives to  animal 
testing in the risk assessment of nanomaterials, and it is subdivided in three Lots (i.e. Lot 1: Review of 
NAM-based tools for nano-specific risk assessment & Develop a Qualification System for NAMs in 
EFSA using the experience from EMA & US FDA; Lot 2: Design and conduct a set of (5) case studies 
using NAM-based IATA to fill data gaps in nanomaterials risk assessment, covering regulatory areas 
within the EFSA's remit; Lot 3: Design and conduct a set of (5) case studies to improve NAM-based 
methodologies for nanospecific assessment). The first two Lots are led by BfR (Germany), and the third 
Lot  by  ISS (Italy). The  first  deliverable  of  this  project  is  a  ‘Review  of  NAM-based  tools  for  nano-
specific risk assessment’ and ‘Proposal for a qualification system for New Approach Methodologies 
(NAMs) in the food and feed sector: example of implementation for nanomaterial risk assessment’, 
which were published as External Report in the EFSA website. Finalisation of the project is expected 
by 2027. 

Cooperation with ECHA/EMA/SCCS is ongoing on this topic to ensure regulatory relevance of ongoing 
projects and align on methodologies, when appropriate. 

Additional recommendations for research 

Two new outsourced activities aimed at promoting the implementation of alternatives for animal testing 
for nanoparticles risk assessment were recently launched and are expected to start by the end of 2025. 
The  projects  are  related  to  ‘Developing  provisions  for  guidance  on  New Approach  Methodologies 
(NAMs)  data  integration  and  implementing  hypothesis-driven  Next  Generation  Risk  Assessment 
(NGRA), with specific focus on the area of food nanotechnology’ and ‘Integration of New Approach 
Methodologies  in  Chemical  Risk  Assessment:  Case  Studies  Addressing  Nanoplastics  in  Food 
(NAMs4NANOPlastics)’. 

Lastly, capacity building activities are recommended to support stakeholders in the implementation of 
methodologies for nanomaterials risk assessment.  

60 

 
 
 
3. Recommendations and actions for environmental 

safety assessments  

3.1. Acute fish toxicity 

Current data requirements relating to acute toxicity  
The OECD TG 203 fish, acute toxicity test (AFT) is the most commonly used standardised animal test 
method  to  assess  acute  fish  toxicity  (OECD,  2019).  Juvenile  fish  are  exposed  to  a  range  of 
concentrations of the test chemical in test solutions with regular observation of mortalities. The TG has 
been updated in 2019 adding as observations also the visible abnormalities (also known as sublethal 
clinical signs) related to equilibrium (e.g., loss of balance, floating at the surface or sinking), appearance 
(e.g., weak or dark pigmentation), ventilatory behaviour (e.g., hyper-, hypo- or irregular ventilation) 
and swimming behaviour (OECD TG 203, Annex 4). This was added in order to improve the ability to 
predict chemical toxicity and minimise suffering of animals. Acute fish toxicity is expressed in terms 
of an external concentration of the test substance in the water that is lethal to 50% of the test organisms 
(LC50) during the duration of the test; internal concentrations in the fish are uncertain. In addition, no 
mechanistic  information  can  be  obtained  from  the  endpoint  of  mortality.  OECD  TG  203  can  be 
conducted with a range of fish species.  

Recommendations – acute fish toxicity 
Short term: 

•  Waiving based on more sensitive taxa: Include waiving options for fish acute toxicity testing 
in  case  of  chemical  classes  for  which  testing  with  other  aquatic  organisms  from  different 
taxonomic levels are known to be more sensitive50.  
In silico (QSARs): Use as replacement when demonstrated that chemical is within applicability 
domain. 

• 

•  OECD TG 249: Use the fish cell line acute toxicity test (RTgill-W1 cell line assay) as default 
for  testing  for  fish  acute  toxicity  when  demonstrated  that  chemical  is  within  applicability 
domain. 

•  OECD  TG  236:  Use  the  fish  embryo  acute  toxicity  (FET)  test  as  refinement  when 

demonstrated that chemical is within applicability domain51. 

Medium term: 

• 

Increase the knowledge on sensitivities of species of different taxonomic groups and trophic 
levels with the aim to expand possibilities for waiving fish acute testing.  
In silico (QSARs): Expand applicability domains of existing QSARs   

• 
•  Explore and expand where possible chemical applicability domains of OECD TG 249 and 
OECD TG 236 through mapping and prediction model refinement (data review and discussion; 
data  generation,  where  required)  and  document  substance  (classes)  not  within  the  chemical 
applicability domain. 

•  Finalise development of the OECD guidance document on IATA for fish acute toxicity; 
Recommend  introducing  in  legislation  and  guidance  an  approach  that  replaces  fish  acute 
toxicity testing considering the OECD guidance document on an IATA for fish acute toxicity 

50 The action might also require to further clarify for which chemical classes or for which toxicities informaton 
from other taxa provide sufficient certainty that protection levels are fulfilled. In this case, the action should be 
considered a mid-term action.  
51 In the long-term, animal-free approaches not relying on embryos are preferred  

61 

 
 
 
 
and the domain of chemical applicability of alternatives. Information on the specify domain of 
chemical applicability space could be inlcuded in guidance. 
•  Weight of evidence (WoE) and defined approach (DA):52  

o  Acute fish toxicity D) for hazard classification: Evaluate with external datasets 
o  Perform case studies to gain experience with using different lines of evidence 
o 
o  Expand WoE approaches, also by increasing the understanding of MoAs  

Implement acute fish toxicity DA for hazard classification in regulatory frameworks  

Long term:  

In silico methods: Develop new in silico/ QSARs with wider applicability domain 

• 
•  OECD TG 249: Assay being completely animal-derived component free for GLP testing   
•  OECD TG 236: Develop a completely animal-free fish acute toxicity assessment to be able to 

eliminate this test  

•  Weight of evidence: Advance completely animal-free fish acute toxicity weight-of-evidence 

assessment 

General recommendations  

o 

Increase the understanding and consider inherent variability in acute fish toxicity testing to 
set  data driven  expectations  for validation  of alternatives to animal  testing  and  required 
precision 

o  Transparent criteria for applicability, and also documentation of excluded substance with 

development of guidance necessary re how to handle out of domain substances 

o  Validate  existing  mechanistic  alternatives  to  animal  testing  to  increase  mode  of  action 

(MoA) understanding (e.g. QSARs)  

o  Develop  additional  mechanistic  alternatives  to  animal  testing  to  increase  MoA 

understanding  

Description of method readiness, applicability domain and validation status  
In  silico  (QSAR  models)  are  already  available  to  predict  acute  toxicity  to  fish  for  some  chemical 
classes. In addition, some QSAR tools include mode of action profilers. The applicability domains of 
QSAR  generally  cover  organic  chemicals  with  log  Kow  values53  up  to  max.  approx.  6  (suggests 
substance is highly lipophilic). Inorganic chemicals, superhydrophobic, organometallic chemicals, ionic 
chemicals  (though  ionic  surfactants  are  covered  by  ECOSAR)  and  nanomaterials  are  generally  not 
within QSAR applicability domains. When QSARs are used, appropriate documentation of evaluation 
is  needed  in  terms  of  both,  the  validity  of  the  model  itself  (assessed  according  to  OECD  guidance 
document on the validation of QSAR models (OECD 2014)) and the reliability of the outcome (e.g. 
whether the substance is within the applicability domain of the QSAR). 

Within the Partnership for the Assessment of Risks from Chemicals (PARC) various models are being 
developed for different fish species (publication, in preparation).  

In vitro and assays using non-protected life stages: Assays for fish acute toxicity include the OECD 
TG 249 and the OECD TG 236. Whilst methods using (eleuthero-)embryos are in fact animal methods 
(such as the OECD TG 236), those are non-protected life stages as defined in the scope of Directive 
2010/63/EU before they start to be independently feeding larval forms. The FET and RTgill-W1 tests 

52 This also includes integrated approaches to testing and assessments (IATA) and defined approaches (DA) 
53 Octanol-water partition coefficient, measure between hydrophilicity and lipophilicity of a substance, with log Kow values 
typically between -3 very hydrophilic and +10 extremely lipophillic 

62 

 
 
 
 
 
 
have been validated and applied with different chemicals. Both, the FET and RTgill-W1 tests seem to 
have similar chemical applicability domains and limitations (e.g. neurotoxicity).  

Weight-of-evidence  approaches:  Tiered  testing  frameworks  and  integrated  testing  strategies  (ITS) 
exist for assessing acute fish toxicity, which aim to maximise use of existing tools and data, and integrate 
different data streams effectively (ECHA, 2023; OECD, 2012). An IATA is under development, and a 
score-based DA for acute fish toxicity has been published (Macmillan et al., 2025). A recent analysis of 
acute  fish  and  daphnid  data  from  the  EnviroTox  database  showed  that  daphnid  data  may  cover 
limitations of alternative methods (e.g., neurotoxicity), so that daphnid data could potentially safeguard 
the  use  of  FET  and  RTgill-W1 testing for environmental  protection, and fish  testing  would  only  be 
required in rare cases (Schür et al., 2025). 

Threshold  approach:  The  threshold  approach  for  acute  fish  toxicity  exists  as  a  reduction  method 
(OECD GD 126), whereby fish toxicity is initially evaluated at a single test concentration in an OECD 
TG 203 limit test, with the concentration set based on algae and daphnid toxicity, with additional fish 
testing only considered if mortality is observed.  

alternative 
approach 
Description 
incl. 
endpoint(s) 

OECD TG 126: Threshold approach for acute fish toxicity 

•  Threshold  approach  is  based  on  observation  that  fish  is  not  always  most 
sensitive group of aquatic organisms (among algae, invertebrates, and fish) 
(Rawlings et al., 2019) 

•  Fish toxicity is initially evaluated as a single concentration (limit) OECD TG 
203  test,  which  requires  fewer  fish  compared  to  a  full  acute  fish  toxicity 
study  

•  This  single  concentration  is  the  threshold  concentration  (TC)  identified 
based on lowest EC50 value from reliable algae (OECD TG 201) or acute 
invertebrate (e.g. OECD TG 202 daphnia) toxicity data  

References 
Bols, N. C., Barlian, A., Chirino-Trejo, M., Caldwell, S. J., Goegan, P., & Lee, L. E. J. (1994). Development of a 
cell line from primary cultures of rainbow trout, Oncorhynchus mykiss (Walbaum), gills. Journal of Fish 
Diseases, 17(6). https://doi.org/10.1111/j.1365-2761.1994.tb00258.x 

OECD. (2010). OECD GD 126: Short guidance on the threshold approach for acute fish toxicity. 
OECD. 

framework,  Series  on 

(2012).  Fish 
https://doi.org/https://doi.org/10.1787/9789264221437-en 

toxicity 

testing 

testing  and  assessment,  No  171. 

OECD. 

(2013).  Test  Guideline  No. 

(FET)  Test. 
https://www.oecd.org/en/publications/test-no-236-fish-embryo-acute-toxicity-fet-test_9789264203709-
en.html 

Fish  Embryo  Acute  Toxicity 

236: 

OECD. (2014). Guidance Document on the Validation of (Quantitative) Structure-Activity Relationship [(Q)SAR] 

Models. https://doi.org/10.1787/9789264085442-en 

OECD. (2019). Test No. 203: Fish, Acute Toxicity Test. http://www.oecd.org/termsandconditions/. 
OECD.  (2021a).  Test  Guideline  No.  249 :  Fish  Cell  Line  Acute  Toxicity:  The  RTgill-W1  cell  line  assay. 

http://www.oecd.org/termsandconditions/. 

ECHA. (2023a). Chapter R.7b: Endpoint specific guidance Guidance on Information Requirements and Chemical 
Safety  Assessment  Chapter  R.7b:  Endpoint  specific  guidance  (Version  5.0;  December  2023). 
https://doi.org/10.2823/161062 

Macmillan, D. S., Ambure, P., Aranda, V., Bayona, Y., Bonderovic, V., Dawick, J., Fabre, N., Fischer, S., Hodges, 
G.,  Llobet-Mut,  Á.,  Loisel-Joubert,  S.,  Rivetti,  C.,  Roberts,  J.,  Schirmer,  K.,  Serrano-Candelas,  E., 
Serrano  Ramón,  B.,  &  Stackhouse,  R. A.  (2025). Addressing  the  challenges  of  acute  toxicity  hazard 
classification using a non-animal defined approach. Environmental Toxicology and Chemistry, vgaf046. 
https://doi.org/10.1093/etojnl/vgaf046 

63 

 
 
 
 
 
Natsch, A., Laue, H., Haupt, T., von Niederhäusern, V., & Sanders, G. (2018). Accurate prediction of acute fish 
toxicity of fragrance chemicals with the RTgill-W1 cell assay. Environmental Toxicology and Chemistry, 
37(3). https://doi.org/10.1002/etc.4027 

Rawlings, J. M., Belanger, S. E., Connors, K. A., & Carr, G. J. (2019). Fish embryo tests and acute fish toxicity 
tests are  interchangeable in the  application of the threshold approach. Environmental Toxicology and 
Chemistry, 38(3). https://doi.org/10.1002/etc.4351 

Schug, H., Maner, J., Hülskamp, M., Begnaud, F., Debonneville, C., Berthaud, F., Gimeno, S., & Schirmer, K. 
(2020). Extending the concept of predicting fish acute toxicity in vitro to the intestinal cell line rtgutgc. 
Altex, 37(1). https://doi.org/10.14573/altex.1905032 

Schür,  C.,  Paparella,  M.,  Faßbender,  C.,  Stoddart,  G.,  Baity  Jesi,  M.,  &  Schirmer,  K.  (2025).  Daphnids  can 
safeguard  the  use  of  alternative  bioassays  to  the  acute  fish  toxicity  test:  A  focus  on  neurotoxicity. 
Environmental Toxicology and Chemistry, vgaf014. https://doi.org/10.1093/etojnl/vgaf014 

64 

 
 
 
 
3.2. Aquatic and terrestrial bioaccumulation 

Introduction 
Bioaccumulation  assessments  are  required  for  hazard  evaluation  (e.g.,  PBT/vPvB  assessment, 
classification, and labelling) as well as exposure modelling and risk assessment in wildlife and humans 
(ECHA 2023a; ECHA 2023b). These evaluations are mandatory under various EU regulations including 
REACH,  Biocidal  Product  Regulation  (BPR),  Classification,  Labelling  and  Packaging  (CLP), 
Veterinary Medicinal Product Regulation (VMPR), Food Additives (FA) and Plant Protection Products 
(PPP).  Bioaccumulation  data  also  provides  critical  insights  into  long-term  ecotoxicity,  as  the 
accumulation  of  substances  in  organisms  may  result  in  internal  concentrations  surpassing  toxicity 
thresholds and facilitate their transfer through the food web via biomagnification leading to adverse 
effects and secondary poisoning. 

The shift towards alternatives to animal testing highlights the need for reliable, robust, and biologically 
relevant alternative strategies for aquatic and terrestrial bioaccumulation assessments considering the 
substances’ physico-chemical  properties,  proposed  use  and exposure  routes. This  document  outlines 
current and future options for assessing bioaccumulation without vertebrate testing, aiming to eliminate 
in vivo testing. 

Provisions on the need to perform a bioaccumulation assessment differs slightly depending on the area 
of  legislation.  If required, information  on  aquatic  bioaccumulation is  usually  requested,  For  aquatic 
species, a bioconcentration factor (BCF) can be determined using OECD TG 305 or OECD TG 321 
HYBIT  bioconcentration  tests.  Also  other  species  are  used  (e.g.  mussels)  for  bioaccumulation 
assessment. 
Several  pieces  of  legislation  include  the  possibility  to  waive  the  bioaccumulation  assessment  if  the 
substance has certain properties. E.g., under REACH the assessment can be omitted if the substance has 
a low potential to cross biological membranes and/or an octanol-water partition coefficient (log KOW) 
≤ 3. In the case of human medicinal products (European Medicines Agency, 2024), actives with log KOW 
>4.5 require a full PBT/vPvB assessment. 

Some pieces of legislation like REACH or CLP apply a weight-of-evidence determination using expert 
judgement to conclude whether a substance may be bioaccumulative (B) or very bioaccumulative (vB) 
(ECHA  2023b;  ECHA  2024b).  Conclusions  can  also  be  drawn  from  information  such  as  terrestrial 
bioaccumulation  data,  biomagnification  factors  (BMFs),  field  bioaccumulation  assessment  factors 
(BAFs), trophic magnification factor (TMFs), QSAR and mass-balance model predictions, as well as 
in  vitro  biotransformation rates,  in  vitro–in  vivo  extrapolation  (IVIVE),  physicochemical  properties, 
toxicokinetic  studies,  and  biomonitoring  data.  Methods  for  collecting,  generating,  evaluating,  and 
integrating various lines of evidence (in vivo, in vitro, in silico bioaccumulation and TK data) to assess 
the bioaccumulation of organic chemicals in aquatic and air-breathing organisms have been developed 
and described (Arnot et al., 2022, ECHA 2023a, OECD 2024a)  

Legislation does not contain mandatory bioaccumulation data requirements for terrestrial organisms. 
However, in some legislative areas, such information might still be relevant e.g. for food chain exposure 
modelling and PBT assessments (ECHA 2023a, ECHA 2023b).  
Historically, aquatic bioaccumulation, which may be supported with terrestrial bioaccumulation data 
(e.g. earthworms), had been assumed to be protective of air breathing organisms. This assumption has 
come under scrutiny since some chemicals not considered bioaccumulative in the aquatic environment 
are still able to accumulate in air-breathing species, including birds and mammals (ECHA 2022a, Hoke 
et al 2015). The need to find improved methods to address this has been highlighted in ECHA’s Key 
Areas for Regulatory Challenge (KARC) report (ECHA 2023c).  

65 

 
 
 
 
 
 
 
Current vertebrate test methods used to meet standard information requirements 
for bioaccumulation testing 
The in vivo test OECD TG 305 (Bioaccumulation in Fish: Aqueous and Dietary Exposure) is resource-
intensive, animal reliant, time consuming and costly. The guideline allows for two exposure routes: 
aqueous or dietary. Dietary exposure studies derive a BMF instead of a BCF. Dietary exposure studies 
should  be  considered  for  substances  for  which  it  is  not  possible  to  maintain  and  measure  aqueous 
concentrations reliably and/or potential bioaccumulation may be predominantly expected from uptake 
via feed (e.g. low solubility and high affinity of adsorption to suspended solids or sediments leading to 
reduced  bioavailability  in  aquatic  media)  (ECHA  2023a  and  b).  For  example,  maintaining 
concentrations of highly hydrophobic chemicals (log KOW > 5 and a water solubility below ~ 0.01-0.1 
mg/L)  in  aquatic  media  can  become  technically  infeasible.  Nevertheless,  studies  using  aqueous 
exposure are preferred (ECHA 2023b) and regulatory classification criteria are based on aquatic BCFs. 
In  addition,  the  dietary  method  faces  uncertainties  in  calculating  uptake  rates  for  the  purpose  of 
calculating a BCF from the BMF and assumes first-order kinetics, which may not suit all substances, 
especially those with complex behaviours like ionisable organic compounds (IOCs) or those with rapid 
metabolism.  
Since the adoption of OECD TG 305 in 1996, numerous chemicals have been tested, but substantial 
variability  and  uncertainty  in  in  vivo  BCF  data  remain,  as  noted  by  Arnot  and  Gobas  (2006)  and 
Wassenaar  et  al.  (2020).  These  uncertainties,  particularly  with  empirical  BCF  measurements,  pose 
challenges when values approach B or vB thresholds (Camenzuli et al., 2019). 

There are no specific test guidelines for bioaccumulation in air breathing organisms.  Substances which 
screen based on physicochemical properties as potentially bioaccumulative in air-breathing organisms 
(see  above)  may  be  further  assessed  via  elimination/metabolism  half-lives  from  QSAR  or  in  vitro 
studies  (intermediate  assessment)  or  from  existing  in  vivo  toxicokinetic  (TK)  studies,  such  as  from 
OECD TG 417 (Hofer et al, 2021) (definitive assessment) (ECHA 2023b) 
However, in the EU, toxicokinetic and metabolism studies (OECD TG 417) are at present not required 
for REACH at any tonnage, whereas such studies are required for pesticides. In addition, the OECD TG 
417 is not designed for bioaccumulation assessment, and while the standard TK study in rats involves 
usually a single dose, repeated dose may be needed for assessing bioaccumulation in mammals (Hofer 
et al. 2021). 
Novel modelling and non-animal approaches have been also suggested recently and should be validated 
in near future (ECHA 2022a, Saunders et al. 2023). 

Alternative approaches for bioaccumulation  
Under Annex XI of REACH, registrants may adapt information requirements under Annex VII to X by 
using alternative information including weight-of-evidence, QSAR, in vitro, grouping/read-across and 
exposure-based waiving. 

Screening approaches/in-silico/read-across 

The use of log KOW is one of the main criteria used to screen for substances suspected to have a high 
bioaccumulation potential via partitioning to lipid storage.  Under REACH, the standard information 
requirement for bioaccumulation in aquatic species may be omitted if log KOW is below 3. Screening 
criteria exist for bioaccumulation in air-breathing mammals are based on log KOA and log KOW (log 
KOW>2  and  log  KOA  >5;  (ECHA  2023b).  However, these  cut-offs  only  apply  when the  potential for 
bioaccumulation of the substance is solely driven by lipophilicity. Therefore, the use of log KOW as a 
screening  criterion  is  not  considered  adequate  if  the  substance  is  surface  active  or  ionisable  at 
environmental pH (pH 5 – 9) as other mechanisms may drive their bioaccumulation potential. For these 
substances  information  on  the  pKa  is  needed  and  apparent  partitioning  information  for  the  charged 
form. In these instances, the use of membrane-water partitioning information log K/DMW, besides the 
log  KOW,  is  considered  a  more  biologically  relevant  alternative  and  is  gaining  increased  attention. 
However, there is no standard guideline available for its empirical determination and in silico methods 

66 

 
 
 
 
 
for  log  K/DMW  are  still  limited,  consisting  of  ppLFERS  (poly-parameter  Linear  Free  Energy 
Relationships),  which  have  been  shown  to  work  well  for  neutral  chemicals,  to other  more  complex 
computational approaches, which can be expensive and computationally slow. 

Whilst the use of physical-chemical partitioning properties provides a simple low-cost approach, none 
of these parameters take into account biotransformation which may result in an overestimation of the 
bioaccumulation potential of chemicals which are rapidly biotransformed. It should be noted that no 
harmonised regulatory cut-offs exist worldwide. Furthermore, specific cut-off values may lead to more 
or less false negative/positive outcomes (Nendza 2018, Gimeno et al 2024). No regulatory cut-offs have 
been agreed for log K/DMW. 

There are numerous in silico models commercially or freely available for predicting BCF with defined 
guidance  (OECD  2024b)  to  assess  their  robustness  and  reliability.  These  predictions,  along  with 
structurally based screening profilers i.e. OECD QSAR Toolbox, are accepted as part of a wider weight 
of evidence approach. In addition to QSAR models for fish BCFs, mass balance models for simulating 
BCFs, BAFs, BMFs, HLT and TMFs have been developed. An overview of the different models and 
how to apply them as Lines of Evidence (LoEs) is outlined in the OECD IATA on Bioaccumulation 
(OECD,  2024a).  However,  these  models  are  generally  not  considered  suitable  for  polymers,  highly 
hydrophobic chemicals, metals and complex UCVBs and many have a heavy reliance on use of log 
KOW and do not account for biotransformation. 

Several validated QSAR models for predicting whole-body biotransformation rate constants or half-
lives  have  been  developed  and  validated  following  OECD  guidance  for  QSAR  application  (OECD 
2014). These QSAR predictions can also be used and leveraged to refine BCF estimates and/or serve 
as a comparator to the OECD TG 319 data as outlined in the OECD IATA on Bioaccumulation (OECD, 
2024a). These models include BCFBAF v3.01 module in EPISuite which was developed based on a 
published database of fish biotransformation rate constants (Arnot and Mackay, 2008; Arnot et al. 2008) 
and the University of Insubria’s QSARINS model (Papa et al., 2014). This information can be combined 
and  compared  with  other  lines  of  evidence  within  the  Bioaccumulation  Assessment  Tool  (BAT) 
(Armitage et al. 2021). Several in silico models to predict biotransformation and half-lives are easily 
accessible  and  comparable  via  the  EAS-E  Suite  platform  (Eas-E-Suite  -  ARC  Arnot  Research  & 
Consulting). 

Under the Partnership for the Assessment of Risks from Chemicals (PARC), deep learning models for 
predicting bioconcentration factor (BCF) values are being developed. BCFpro, a model that considers 
test conditions and species used in addition to chemical structure, has been submitted for publication.  
The EFSA TK PLATE model (Dorne, 2023) is an open access platform developed by the European 
Food Safety Authority (EFSA) to enhance chemical risk assessment through toxicokinetic (TK) and 
toxicodynamic  (TD)  modelling.  This  platform  predicts  how  chemicals  are  absorbed,  distributed, 
metabolized,  and  excreted  in  various  species,  including  humans,  animals,  and  ecological  species  of 
relevance. Validation of the model is still on-going with one of the main limitations being the limited 
availability of TK/TD parameters for many species. Further work (long term) is required to develop the 
platform to include priorities identified by EFSA, ECHA and PARC including bioaccumulation models. 

Read across provides an accepted approach with defined guidance (ECHA 2017a, ECHA 2017b, ECHA 
2022b, OECD). 

In vitro assays/IVIVE (aquatic bioaccumulation) 

Biotransformation is one of the major uncertainties in mass-balance or QSAR models to predict BCFs 
based on log KOW. Thus, in vitro assays have been standardized and validated (Nichols et al. 2018) to 
determine biotransformation rates which can be incorporated into the models to refine BCF predictions. 
Currently, these in vitro assays (OECD TG 319 A/B; OECD 2018a & b) can be used as an alternative 
to the current fish in vivo study as a part of a weight of evidence approach (ECHA Guidance R7c, 2023). 

67 

 
 
 
 
 
 
 
 
 
The biotransformation rate (in vitro intrinsic clearance; CLIN VITRO, INT) of a test chemical is determined 
with either primary hepatocytes (RT-HEP) (OECD TG 319A) or liver S9 sub-cellular fractions (RT-S9) 
from Rainbow Trout (OECD TG 319B) using a substrate depletion approach. Both in vitro methods are 
highly reliable and reproducible (Nichols et al. 2018). No significant differences in performance have 
been observed between the two assays using hepatocytes or S9 sub-cellular fractions, thus there is no 
preference for one in vitro system or the other (Fay et al. 2017). Enzymatically inactive controls are 
included in the test protocol to detect potential abiotic losses due to adsorption or volatility ensuring 
that observed chemical loss in the active system is due to biotransformation. Whilst these assays have 
been  standardized  for  Rainbow  Trout,  they  can  also  be  applied  to  other  species  and  to  identify 
biotransformation products as outlined in the Guidance Document 280 (OECD 2018c).  

The measured in vitro intrinsic clearance can be extrapolated to a whole-body biotransformation rate 
constant (kB) using an appropriate  in vitro-in vivo extrapolation (IVIVE) model with log KOW as an 
input. The chemical concentration in fish is then simulated in a one-compartment model by Arnot and 
Gobas  (2003)  to  predict  the  BCF  (OECD  2018c).  IVIVE  models  have  been  developed  for  neutral 
organic chemicals with log KOW values ranging from 2 to 8 (Nichols et al. 2013). The Nichols et al. 
model  is  described  in  OECD  Guidance  Document  280  (OECD  2018c). An  updated  and  corrected 
version has been integrated in the Bioaccumulation Assessment Tool (BAT) (Arnot et al, 2023) and in 
EAS-E  Suite  (https://arnotresearch.com/eas-e-suite/).  Simple  one-compartment  models  are  most 
commonly used and are based on the assumption that the liver is the major organ for biotransformation 
(Nichols et al. 2013, Krause and Goss, 2018). However, complex multi-compartment models to generate 
organ-specific rate constants have also been developed (Krause and Goss, 2020). Generally, a simple 
one-compartment model is sufficient to predict a BCF unless specific information indicates that first-
pass  effects  or  direct  blood  flow  from  the  gastrointestinal  tract  (GIT)  to  the  liver  is  of  particular 
importance. In this instance, it may be more useful to use a multi-compartment model.  As a different 
approach, an empirical model was recently developed for neutral organic chemicals correlating in vitro 
biotransformation rates and in vivo BCFs (Laue et al 2023). 

The classical IVIVE models cannot directly be applied for BCF prediction of IOCs and surfactants, 
since  log  KOW  is  not  considered  a  reliable  parameter  for  these  substances.  IOCs  display  different 
behaviour  compared  to  neutral  organic  chemicals  such  as  preferential  sorption  to  membranes 
(phospholipids)  and  plasma  proteins,  the  influence  of  pH  on  uptake  and  elimination  rates,  and  the 
greater potential importance of active transport mechanisms (Armitage et al. 2017). A strong correlation 
between the log BCF and the membrane lipid−water distribution ratio (DMLW, R2 = 0.96) was found for 
10  anionic  surfactants  and  biotransformation  was  identified  as  the  major  mechanism  of  elimination 
(Ribbenstedt et al. 2021). In these instances, the BIONIC model, (Armitage et al, 2013 & 2017, CEFIC 
ECO37 project) provides a more appropriate method for calculating BCF, facilitating the use of log 
K/DMW  and  log  DOW  as  partitioning  descriptors  to  overcome  the  limitations  of  log  KOW.  Whilst  the 
BIONIC model provides a more suitable alternative, it is a complex model, and further work is required 
to  improve  the  transparency  of  and  demonstrate  robustness  and  applicability  to  a  wider  range  of 
chemicals. 

The incorporation of biotransformation rates for BCF predictions results in a better agreement to in vivo 
values compared to predictions solely based on log KOW (Cowan-Ellsberry et al. 2008, Dyer et al. 2008, 
Laue et al. 2014, Nichols et al. 2013, Nichols et al. 2018). Whilst these approaches are included in 
current guidance (ECHA 2023a), regulatory experience  with in vitro data and IVIVE is still limited 
therefore the resulting BCF estimate can only be used as part of a WoE approach rather than a direct 
replacement of a in vivo measurement. To date, > 250 unique chemicals have been tested in the TG 
319A/B assays including a broad range of chemical classes covering a range of hydrophobicity (log 
KOW ranging from 0.20 to 8.68). Approximately 1/3 of the tested chemicals are IOCs (Embry et al. MS 
in preparation), for which the 319 B assay has been shown to work well (Chen et al 2016, Droge et al. 
2021).  

While the OECD TG 319 assays offer an alternative to in vivo assays, there remain certain limitations 
which  include  uncertainties  regarding  their  applicability  to  chemicals  for  which  dietary  exposure  is 

68 

 
 
 
 
 
 
more  relevant,  such  as  highly  hydrophobic  substances,  the  lack  of  a  clearly  defined  domain  of 
applicability, and the need for easily accessible, up-to-date extrapolation models for BCF predictions. 
Furthermore, the applicability domain of alternative approaches (in silico, OECD TG 319 A/B, OECD 
TG 321) for vB classification needs to be compared and defined. Some  alternative approaches  (e.g. 
OECD 319 A/B) may not be suitable to provide sufficient confidence to determine whether the BCF is 
>5000 L/kg (e.g. for potentially very bioaccumulative chemicals with a relatively low log Kow (<ca. 
5)). Even if these chemicals are not biotransformed in vitro, log KOW dependent BCF predictions may 
result in BCFs <5000 L/kg. For these substances further testing in e.g. the HYBIT as a higher tier, may 
be needed. ECHA’s PBT Expert group has set up a working group to make recommendations on use of 
the OECD TG 319 A/B for bioaccumulation assessment under REACH. Target date for the final report 
is December 2025.  

Since the OECD TG 319 A/B as in vitro test is easier to use, needs less resources and is substantially 
faster compared to the TG 321, the TG 319 A/B is considered the more efficient alternative to animal 
testing to (de-)prioritize B testing. Therefore, it is recommended as  short-term goal to include these 
assays  in  chemical  legislation  to  enhance  the  regulatory  acceptance  of  in  vitro  data  to  assess 
bioaccumulation in aquatic species. 

There are also a number of additional in vitro approaches currently under development which warrant 
further research. These include: 

•  Use of S9 fractions from the gut to determine biotransformation for chemicals that undergo 
substantial  extra-hepatic  biotransformation,  for  which  current  'liver  only’  approaches  fail  to 
account for, leading to over prediction of bioaccumulation in particular for hydrophobic, poorly 
water-soluble chemicals for which dietary update is the relevant uptake route (Saunders et al 
2020). Organ specific biotransformation was observed in S9 subcellular fractions from liver, 
intestine, gills and brain for three chemicals in Brown Trout and Rainbow Trout (Franco et al. 
2024).  If there  is significant underestimation of the whole-body biotransformation rate (i.e. 
resulting in an overestimation of the BCF) extrahepatic biotransformation could optionally be 
determined to refine BCF predictions using multicompartment models in the long term.  This 
method is still under development and requires further standardisation and validation before 
use. 

•  Development  of  in  vitro  methods  to  measure  very  slowly  biotransformed  chemicals.  The 
relatively short lifespan of the hepatocytes and S9 fractions makes these methods unsuitable for 
substances  with  slow  biotransformation  rates.  Improvements  were  reported  for  S9  fractions 
expanding the  stability  by addition  of  protease  inhibitors  (Nichols et  al,  2021). However, it 
should be kept in mind that the IVIVE models have been developed for “well-metabolized” 
chemicals (Nichols et al. 2013) and slow biotransformation rates have only a minor impact on 
predicted  BCFs.  Nevertheless, 
to  measure 
biotransformation of very slowly biotransformed chemicals, e.g. using spheroids, is ongoing. 

investigate  methods 

further  work 

to 

•  Use  of  immortalised  Rainbow  trout  cell  lines  from  liver,  gut  and  gill  to  determine 
biotransformation, used in conjunction with PBTK models to predict bioconcentration in the 
whole  fish  (Stadnicka-Michalak  et  al.  2018).  This  assay  provides  a  relatively  simple  high 
throughput approach (dependant on analytical requirements) which can be adapted to allow for 
the measurement of depletion kinetics and analysis of biotransformation products. To date only 
a limited number of chemicals have been tested, and further work is required to investigate and 
compare  the  metabolic  activity  of  the  immortalised  cell  lines  with  those  of  primary  cells. 
Activities of organ specific biotransformation enzymes were demonstrated recently in different 
rainbow trout cell lines (Martin et al. 2025). Furthermore, standardization and validation are 
needed to facilitate use of this method in a longer term as part of a weight of evidence approach 
with the potential to complement and as a long-term goal to replace the current OECD 319 in-
vitro assays to not any longer rely on fish as source for hepatocytes or S9 fractions. 

In vitro assays/IVIVE (bioaccumulation in air-breathers) 

69 

 
 
 
 
 
As with aquatic bioaccumulation, the use of in vitro biotransformation methods using rat liver S9 sub-
cellular fractions and hepatocytes (equivalent to the existing OECD 319A/B for Rainbow Trout) has 
potential for use in a weight of evidence approach for air-breathing organisms. This method has been 
developed as part of CEFIC-LRI ECO 41 project for neutral hydrophobics (Lee et al. 2022) and has the 
advantage  of  being  a  simple,  high  throughput  assay  with  a  high  degree  of  reproducibility  as  an 
alternative approach to the OECD TG 417 rat in vivo study which currently has not been optimized for 
bioaccumulation assessment (Hofer et al. 2021, Arnot et al. 2022).   

The  pharmaceutical  industry  adopted  S9  assays  for  early-stage  drug  metabolism  studies,  as  a  cost-
effective, high-throughput alternative to hepatocyte and microsome assays, with the added benefit of 
covering both phase I and II metabolism. Liver cells ad S9 assays from different mammalian species 
have  been  further  optimized  for  automation,  reproducibility,  and  use  with  human-derived  material, 
improving their relevance for predicting human drug metabolism and inter-individual variability. This 
knowledge could be utilized for further method development. The clear advantage of these in vitro tests 
is that they could, in principle, cover a range of different cell lines from different species to facilitate 
the assessment of bioaccumulation potential in a more holistic manner without animal testing. Such 
methods could also be used to predict interspecies differences in metabolism and toxicity. 

The BMF is considered more appropriate for air breathing organisms therefore existing IVIVE models 
(which are predominately built for extrapolating in vitro data to a BCF) are also inappropriate. The use 
of IVIVE models developed for assessing dietary uptake of pharmaceuticals in rats is well established 
and with some modifications have the potential to provide a suitable approach. Inter species sensitivity 
also requires some consideration as in vitro biotransformation rates between terrestrial organisms has 
been demonstrated within pharmacological studies. The method could have merit as part of a weight of 
evidence screening approach (Lee et al 2017, 2022) for air breathing organisms for predicting BMF but 
still  requires  a  significant  amount  of  work  with  regards  to  validation,  understanding  interspecies 
sensitivity and use of IVIVE models. 

Research into non animal approaches for bioaccumulation assessments in birds is still very much in the 
early  stages  of  development  but  is  considered to  be a  key  requirement.  In August  2024  the  NC3Rs 
CRACK-IT Challenge "Wings of Change" initiative was launched with the specific focus of advancing 
alternatives  to  avian  animal  testing.  Although  primarily  focused  on  acute  and  chronic  endpoints, 
knowledge  of  biotransformation  will  be  required to  develop,  use  and  interpret  in  vitro  and  in  silico 
methods. Avian S9 assays are currently under development at the University of Saskatchewan as part 
of a HESI sponsored project. 

Use of other aquatic species  

The current information requirement in REACH is for bioaccumulation in aquatic species (preferably 
fish) therefore any aquatic organism may in theory be considered as relevant. However, established B 
and vB criteria for fish may be overconservative for invertebrate and may need further investigations.  
The use of Hyallela azteca (OECD TG 321) in bioaccumulation testing has recently (Oct 2024) been 
approved as a direct alternative to in vivo fish testing by the Member States Committee under REACH 
(ECHA 2024a). Whilst this provides a regulatory accepted alternative under REACH it has not yet been 
adopted into other regulations. However, for substances where it is not possible to maintain and measure 
aqueous  concentrations  reliably  and/or  where  potential  bioaccumulation  may  be  predominantly  via 
food, an OECD TG 305 via dietary exposure is still favoured. This is because the OECD TG 321 only 
allows to test chemicals via the aqueous route (ECHA 2024a).  

The test set-up of the HYBIT assay is very similar to the existing TG 305 bioconcentration test. The 
organisms (n= ca. 1200 - 1500) are exposed to the test chemical via water either in a semi-static or flow 
through  system  followed  by  a  depuration  phase  of  ca.  1  to  4  weeks  (or  longer)  depending  on  the 
hydrophobicity of the test chemical. According to the TG 321, only male amphipods should be tested 
involving manual sexing of the animals. Thus, the HYBIT is resource intensive and expensive with 
similar costs compared to the TG 305.  However, this approach has the advantage of avoiding vertebrate 

70 

 
 
 
 
 
 
 
testing.  Whilst the method has undergone validation, this was limited to only three stable substances 
with relatively low log KOW values chemicals (3.74 and 4.1) and one chemical (hexachlorobenzene (log 
Kow 5.86) which is clearly bioaccumulative (i.e. no biotransformation). Since the HYBIT is relatively 
new and there is limited data, the reliability and domain of applicability has to be further explored as a 
short-term goal. Approximately 30 chemicals have been tested in the HYBIT so far which are either 
non-bioaccumulative with a relatively low log KOW or (very) bioaccumulative. Since biotransformation 
is an important elimination mechanism and invertebrates may have limited biotransformation capability 
compared to fish, there is a need to focus on chemicals which are biotransformed in fish. Thus, further 
work is needed to widen the range of chemicals tested to build confidence in the method across a wider 
chemical space. Since the OECD TG 319 A/B as in vitro test is easier and substantially faster to use, 
and needs less resources compared to the TG 321, the TG 319 A/B is considered the more efficient 
alternative to animal testing to (de-)prioritize B testing in fish which should be considered in a tiered 
approach (see OECD Bioaccumulation IATA, OECD 2024a).  

Bioaccumulation tests using other aquatic invertebrates such as molluscs (ASTM E1022-94) may be 
relevant as part of a weight of evidence approach (ECHA 2023b) due to their ability to accumulate 
chemicals  primarily  through  direct  water  exposure  (bioconcentration)  and  ingestion  of  suspended 
particles.  However,  many  molluscs  lack  metabolic  pathways  to  biotransform  certain  contaminants, 
potentially  leading  to  overestimation  of  bioaccumulation.  This  highlights  the  issue  that  different 
invertebrate  species  have  varied  bioaccumulation  capabilities  due  to  differences  in  uptake  routes, 
metabolism, sensitivity and elimination rates. Thus, results from one species may not always be directly 
extrapolated  to  another,  necessitating  a  weight-of-evidence  approach  to  account  for  interspecies 
differences. 

Efforts are also underway relating to the potential use of Daphnia magna bioaccumulation testing as a 
screening test (UK Environment Agency, 2025a,b).  

Integrated Approaches for Testing and Assessment 

The recently published Integrated Approach for Testing and Assessment (IATA) for Bioaccumulation 
(OECD 2024a) utilizes a Weight of Evidence (WoE) framework to assess discrete organic chemicals in 
aquatic and terrestrial environments. This approach aligns with OECD guidelines and aims to provide 
clear  guidance  for  stakeholders  in  collecting  and  integrating  various  Levels  of  Evidence  (LoE)  for 
decision-making. 

The IATA combines existing OECD guidelines and Weight of Evidence (WoE) principles to facilitate 
clear and transparent decision-making in various contexts by both regulators and risk assessors. 
It  offers  examples  and  guidance  for  evaluators  on  how  to  collect  and  integrate  data  from  public 
databases and models for both data-poor and data-rich chemicals. The data evaluation criteria within 
the IATA are primarily developed from existing OECD TG for each LoE (e.g., OECD TG 319 A/B), 
providing a systematic approach to address uncertainty within each individual LoE. As each LoE is 
evaluated,  the  guidance  and  methods  in  this  IATA  provide  a  systematic,  pragmatic,  iterative,  and 
transparent  process  to  determine  if  there  is  sufficient  confidence  in  the  available  data  and/or  WoE 
approach  for  decision-making.  Additionally,  it  includes  three  case  studies  to  demonstrate  its 
applicability in assessing bioaccumulation. 

The Bioaccumulation Assessment Tool (BAT) is one way to operationalize this framework, which is 
meant to enable users to evaluate data from multiple sources, including in vitro, in silico, and field data, 
while addressing data gaps for chemicals with limited information (Arnot et al. 2022).  Additionally, 
the  Bioaccumulation  Estimation  Tool  (BET)  is  available  online,  allowing  users  to  obtain  model 
predictions based on chemical identifiers. The IATA emphasizes the importance of data reliability and 
uncertainty in the assessment process, following established OECD recommendations for a transparent 
and systematic evaluation. Guidance and training for regulators should be promoted soon.  

71 

 
 
 
 
 
  
 
 
The Bioaccumulation IATA is intended to guide the collection, generation, evaluation, and weighing of 
various types of bioaccumulation data including physical-chemical, in silico, in vitro and in vivo data. 
Central to this  IATA  are the  elements  and  principles of WoE  such  as problem formulation  and  data 
relevance and reliability.  

The  IATA  includes  different  LoEs,  including  a  variety  of  alternatives  to  animal  testing  (previously 
outlined in this document): 

•  Phys.-Chem. properties (e.g. log KOW as screening criteria for neutral organic chemicals) 
• 
•  OECD TG 319 A/B biotransformation assays in hepatocytes or liver S9 fractions from rainbow 

In silico prediction of biotransformation and/ or BCF using QSARs  

trout which can be combined with IVIVE or other models to predict BCFs in fish 

•  Laboratory bioaccumulation data generated in different species, e.g. invertebrates (e.g. OECD 

TG 321 Hyalella azteca bioconcentration test) and/or OECD TG 305 fish in vivo test 

•  Field data on bioaccumulation and trophic magnification 

Conclusions 

Bioaccumulation assessments can potentially be fulfilled WITHOUT the use of the OECD 305 in vivo 
assay,  primarily  based  on  the  availability  of  various  QSARs  to  predict  half-lives  and/or  BCFs,  the 
OECD  TG  319  A/B  assays  coupled  with  IVIVE  models,  and  the  recently  accepted  OECD  TG  321 
(OECD  2024a)  in  vivo invertebrate  test (HYBIT).  These  alternative  approaches can also  be  used  to 
support read across approaches and align with the OECD IATA for Bioaccumulation (OECD, 2024a) 
which promotes a weight-of-evidence approach for aquatic and terrestrial bioaccumulation assessment.  
The long-term focus should be on fully in vitro methods using that utilise immortalised cell lines from 
a  range  of  aquatic  and  terrestrial  supported  by  the  integration  of  in  silico  predictive  and  modelling 
approaches, thus eliminating the use  
To enable this transition, it is essential to support the training of stakeholders (regulators, industry) in 
the evaluation of in vitro data, applying toxicokinetic and IVIVE models, and understanding 
guidelines on applicability and limitations. The onus for this should lie with method developers, early 
adopters, trade organisations and other academic experts. Existing animal data on toxicokinetics, 
metabolism, or chronic studies for relevant substances or analogues should also be leveraged to 
minimise unnecessary animal testing. 

Recommendations  

Short term  

• 

introduce  into  legislation  and  guidance,  an  approach  that  replaces  the  in  vivo  fish 
bioaccumulation 
the  OECD  guidance  document  on  IATA  for 
bioaccumulation (2024a) and the domain of chemical applicability of alternatives.  Available 
alternative methods are inter alia the ones listed below. Domain of chemical applicability space 
should be explicit in guidance. 

testing  considering 

o  QSARs 
o  OECD 319 A/B with IVIVE 
o  OECD TG 321 

•  Evaluate and promote the applicability of OECD 319 A/B with IVIVE as an efficient and cost-
effective  approach  for  decision  making  on  the  need  for  further  higher  tier  bioaccumulation 
testing e.g. OECD 321.  

•  Amend Annexes VII to X of Regulation (EC) No 1907/2006 to allow the use of validated OECD 
methods (e.g., 319A/B, 321) which avoid in vivo vertebrate testing. Consider OECD TG 319 
A/B  as  lower  tier  compared  to  OECD  321  in  accordance  with  the  OECD  IATA  on 
bioaccumulation. 

72 

 
 
 
 
 
 
•  Evaluate and standardize IVIVE models to be used in conjunction with OECD 319 A/B for 
neutral  and  ionizable  organics  across  a  wider  chemical  space  to  improve  transparency  and 
regulatory  confidence.  Models  should  be  easily  accessible,  clearly  defined  for  regulatory 
applications and systematically account for potential sources of uncertainty in BCF predictions 
for a broader chemical space. 
Introduce log K/DMW baseline screening cut-off values for surfactants and IOCs, analogous to 
the log KOW-based criteria for Tier 1 screening of neutral organic chemicals. 

• 

•  Compare and evaluate applicability domain of alternatives to animal testing (in silico, OECD 
TG 319 A/B, OECD TG 321) for B and vB classification. Further investigation may be needed 
to define the domain of applicability and possible limitations of currently available alternatives 
to animal testing  for B and/ or vB classification. 

•  Superhydrophobic  substances  (Log  KOW>  8):  More  information  needs  to  be  gathered  on 
mechanisms,  matrices  and  parameters  enabling  assessment  of  bioaccumulation  of  super-
hydrophobic  substances.  This  will  allow  development  of  tools  and  methods  for  the 
bioaccumulation assessment of such substances (KARC, 2025).  

•  Utilise  the  results  and  outcome  of  an  ongoing  research  project  funded  by  the  German 
Environment Agency (Umweltbundesamt, FKZ 3723 64 405 0), running from April 2024 to 
March  2027.  This  project  specifically  focuses  on  "Bioaccumulation  of  superhydrophobic 
chemicals"   

Medium term  

•  Ensure  further confidence in  the  domain of applicability  of  the  OECD TG  321 by  reviewing 
current  data  on  tested  chemicals.  If  necessary,  conduct  additional  testing  across  a  broader 
chemical space, especially for chemicals that undergo biotransformation. 

•  Superhydrophobic  substances:  Improve  bioaccumulation  assessment  of  super-hydrophobic 
substances which feeds into the identification of substances of very high concern (SVHCs) and 
for  classification  of  substances  as  PBT/vPvB (KARC,  2025).  Phase  out  vertebrate  testing  by 
excluding current OECD TG 305 data requirements where there is sufficient confidence in using 
alternatives to animal testing as LoEs and their applicability to B/vB criteria. Existing in vivo 
fish bioaccumulation data may still be used for B/vB assessment and to train models. 
Increase confidence in logK/DMW, for screening of surfactants and IOCs, via standardisation and 
validation of experimental methods including the development of a new OECD Test Guideline. 
•  Define  superhydrophobic  chemicals  and  support  development  of  suitable  bioaccumulation 
assessment methods since they cannot be tested with standard in-vivo or in-vitro procedures due 
to technical challenges.  

• 

•  Evaluate  the role  of extrahepatic  biotransformation for  a  wider set  of  hydrophobic chemicals 
where dietary uptake is the relevant uptake route. If there is significant underestimation of the 
whole-body biotransformation rate (i.e. resulting in an overestimation of the bioaccumulation 
potential) in vitro intrinsic clearance determined in gut S9 fractions (or gill S9 fractions) can 
optionally be used to refine BCF predictions using multicompartment models. 

•  Evaluate the metabolic capabilities of invertebrates (H. azteca) compared to fish particularly for 
hydrophobic chemicals. If data demonstrate lower metabolic capabilities for invertebrates, the 
current ECHA B/vB criteria (developed for fish) may need to be adjusted, where BCFs have 
been determined in invertebrates.  

•  Standardize and validate in vitro methods for biotransformation in air-breathing organisms using 
rat hepatocytes and liver S9 fractions including the development of a new OECD Test Guideline. 
•  Support further development and implementation of IVIVE models) to predict bioaccumulation 

in air-breathing organisms for regulatory applications. 

•  Continue to research and develop alternatives to animal testing for avian species including S9 
and hepatocyte-based assays (NC3Rs CRACK-IT “Wings of Change”). Evaluate and if required, 

73 

 
 
 
 
build on the tiered approach for bioaccumulation assessment in air-breathers which is already 
integrated in the OECD IATA on bioaccumulation (e.g. building additional case studies focusing 
on air-breathers). 

Long term  

•  Further validate and develop relevant TK/TD models, e.g. EFSA TK PLATE model, addressing 

currently uncovered chemical and biological spaces.  

•  Continue  to  research  and  standardize  biotransformation  assays  in  immortalized  cell  lines  to 
move completely  away  from  the  need  of  animals to  isolate  S9 fractions  or  hepatocytes and 
further  develop  PBTK  models  for  bioaccumulation  prediction  based  on  cell  line  intrinsic 
clearance. 

•  Standardize  and  validate  in  vitro  methods  for  biotransformation  and  IVIVE  extrapolation 
models in mammals and birds including the development of a new OECD Test Guideline.  

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Predictivity of an Empirical Model Compared to In Vitro-In vivo Extrapolation Models', Environ 
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Lee, Y.-S., Lo, J. C., Otton, S. V., Moore, M. M., Kennedy, C. J., Gobas, F. A. P. C., (2017) In vitro to 
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Lee, Y.-S., Cole, T.R., Jhutty, M.S., Cantu, M.A., Chee, B., Stelmaschuk, S.C., Gobas, F.A.P.C. (2022). 
Bioaccumulation  Screening  of  Neutral  Hydrophobic  Organic  Chemicals  in  Air‐Breathing 
Organisms Using In Vitro Rat Liver S9 Biotransformation Assays. Environmental Toxicology and 
Chemistry. https://doi.org/10.1002/etc.5439 

Martin, B.P.M., Franco, M.E. & Schirmer, K. Comparative characterization of organ-specific phase I 
and II biotransformation enzyme kinetics in salmonid S9 sub-cellular fractions and cell lines. Cell 
Biol Toxicol 41, 37 (2025). https://doi.org/10.1007/s10565-025-09992-8 

Nendza, M., Kühne, R., Lombardo, A., Strempel, S., Schüürmann, G. PBT assessment under REACH: 
Screening for low aquatic bioaccumulation with QSAR classifications based on physicochemical 
properties to replace BCF in vivo testing on fish. Science of The Total Environment, Volumes 616–
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Nichols, J. W., Huggett, D. B., Arnot, J. A., Fitzsimmons, P. N., & Cowan‐Ellsberry, C. E. (2013). 
Toward  improved  models  for  predicting  bioconcentration  of  well‐metabolized  compounds  by 
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Nichols, J., Fay, K., Bernhard, M.J., Bischof, I., Davis, J., Halder, M., Hu, J., Johanning, K., Laue, H., 
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3.3. Fish chronic toxicity 
The recommendations provided describe general and effect-specific actions for advancing the weight-
of-evidence (WoE) for waiving of in vivo chronic fish testing based on different alternatives to animal 
testing as an integrated approach or group of methods. 

The  present  document  is  intended  to  support  chronic  fish  toxicity  information  requirements  under 
different  regulations.  The  term  WoE  is  therefore  considered  from  a  broad  perspective  without 
intentionally fully aligning with any specific definition or regulatory requirements. 

The  main  chronic  fish  toxicity  effects  covered  by  this  document  are:  growth,  developmental, 
reproductive, behavioural, and survival effects. Research needs and issues that require a paradigm shift 
are identified and suggested. 

Current animal test methods to meet information requirements for chronic fish 
toxicity  
Summaries of the current in vivo methods are set out below, with observed endpoints per study displayed 
in Table 1. 

Fish  chronic  tests  might  be  used  to  provide  information  on  different  endpoints.  Depending  on 
legislation, such tests might be triggered by assessments of chronic fish toxicity, endocrine disruption, 
or for example in the context of the assessment of persistent, bioaccumulative and toxic properties of 
substances. Opportunities for reducing the number of animal testing can be harvested if fish chronic 
tests  are  designed  to  provide  information  relevant  for  the  different  assessments  (e.g.  endocrine 
disruption,  growth,  survival  etc.),  which  might  require  modification  of  standard  test  protocols. The 
description of tests below is indication such opportunities. 

OECD TG 210: Fish, Early-life Stage Toxicity Tests (FELS)  

Test/exposure duration starts as soon as possible after fertilization (before cleavage of the blastodisc) 
and  ends  usually  28  –  60  days  post-hatch  (depending  on  species).  It  covers  newly  fertilized  egg, 
embryonic development, hatching and early stages of growth. 

Reporting of relevant effect concentrations and parameters may depend upon the regulatory framework. 

OECD TG 234: Fish Sexual Development Test (FSDT)  

The FSDT is essentially an extension of the FELS - exposure is continued until sexual maturation is 
completed (i.e. usually to around 60 days post fertilization depending on the species), with the aim to 
cover the sex-labile period in which fish are expected to be most sensitive to the effects of endocrine 
disrupting  chemicals that  interfere  with  sexual  development  (predominantly  estrogen,  androgen  and 
steroidogenesis modalities). 

Opportunities  exist  to  meet  FELS  data  requirement  while  investigating  EAS-mediated  endocrine 
activity via the OECD TG 234 (Estrogen-Androgen-Steroidogenesis (ECHA and EFSA, 2018)). For 
this purpose, the OECD TG 234 (point 30) recommends five test concentrations if the data are to be 
used for risk assessment purposes. 

OECD TG 215: Fish Juvenile Growth Test  

Some regulatory frameworks (e.g. REACH) may accept the OECD TG 215 as a long-term fish test if 
there is strong evidence that growth inhibition is the most relevant endpoint in fish (e.g. based on mode 
of  action)  and  that  early-stage  exposure  is  not  needed  (e.g.  if  embryonic  or  larval  exposure  is  not 
relevant or if these are not the most sensitive stages). 

OECD TG 229 (EPA compliant version OPPTS 890.1350) and OECD 230: Fish Short Term 
Reproductive Assays  

78 

 
 
These are 21-day group-spawning assays with adult fish. Growth endpoints are therefore not relevant, 
but mortality can occur due to physiological and behavioural stress arising from reproductive activity 
in the assay. The tests are usually used as screening assays. 

Fish full life cycle test, e.g. OPPTS 850.1500 

Test  exposure  starts  with  freshly  fertilised  eggs  (before  beginning  of  gastrulation)  and  covers  the 
hatching  of  larvae,  growth  of  juvenile  fish  and  reproduction  (egg  numbers,  hatching  success  and 
survival of the F1 generation). Depending on the fish species under investigation the study duration is 
100-190 days. 

It is noted that, the EFSA/ECHA guidance on identification of endocrine disruptors (ECHA and EFSA, 
2018) describes that it is possible to modify a fathead minnow Fish full life cycle test study by adapting 
the test design and including EATS parameters to cover the endpoints required, as recommended in a 
Medaka Extended One Generation study (OECD TG 240). Particularly the guidance recommends that 
the assessment of gonad histopathology (e.g. staging of gonads, severity of intersex) is systematically 
performed each time that the study is carried out. Furthermore, the Zebrafish Extended One Generation 
Reproduction Test (ZEOGRT) is undergoing validation (Teigeler et al., 2021).

79 

 
 
 
Table 1: Summary table in vivo and alternative tests  

Observed effects 

OECD 
210 
ELS 

OECD 
234 
Sexual 
develop
ment 

OEC
D 229 
Short 
term 

OECD 
230 
Short 
term 

OPPTS 
850.150
0 
FFLC 

OPPTS 
890.135
0 
Short 
term 

OECD 240 
MEOGRT 

OECD 
215 
Juvenile 
growth 

RaX 

TTC 

RTgil
l-W1 
cells 

OECD 
236 
FET 

QSARs* 
(e.g. 
ECOSAR, 
KATE, 
VEGA) 

ACR 

Lack of 
exposure/
waiving 

AOPs 

In vitro 
repro 
batteries 
(EAWAG) 

Glial cell 
line 

Brain 
fibroblast-
like cell line 

In vivo Methods 

Available Alternative Methods 

Other approaches 

Alternative Methods under development 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

Growth 

Development 

Dry weight 

Wet weight 

Standard length 

Total length 

Hatching 

Morphological 
abnormalities 
Deformed larvae 

Swim-up 

Fecundity 

Reproduction 

Fertility 

Sex ratio 

Behaviour 

Survival 

Chronic 
toxicity (not 
specific) 

EDs 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

*Some QSARs are still under development 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

80 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Recommendations 
Short term: 

Several short-term options have been identified that could help to reduce the need for fish chronic toxicity. 
The Commission intends to clarify the below points with support of EU agencies and discuss them with 
Member State authorities and stakeholders.  

•  General (chronic fish toxicity): Review possibilities to waive fish chronic toxicity testing based on 
mode of action or based on available data from aquatic organisms from different taxonomic groups 
than fish (e.g. Daphnia). 

•  Behavioural effects: Clarification on regulatory requirements to report behavioural effects  

•  General (chronic fish toxicity): Review possibilities to use exposure-based waiving of the chronic 
fish endpoint, permitted within some regulatory domains, via case studies and additional guidance. 

Milestones:  

1.  A report summarising the discussion with Member State authorities and stakeholders. 
2.  Update of guidance documents and legislation. 

Medium term: 

The implementation of the following three recommendations will be explored together with Member States 
and stakeholders:  

•  General (chronic fish toxicity): Develop case studies of how different alternative approaches can 

be strung together (as integrated approaches) to gain better understanding on the:  

(i) 

Strengths and limitations of existing methods (in silico, in vitro, and in vivo); 

(ii) 

Links to other endpoints (acute, non-fish species, endocrine, etc.); 

(iii) 

Information that these methods can provide regarding chronic mechanism / modes of action 
(e.g. by use of TK/TD, QIVIVE). 

•  General  (chronic  fish  toxicity):  Explore  possibilities  to  develop  a  reference  dataset  of  in  vivo 

chronic fish effects / endpoints to:  

(i) 

(ii) 

Identify which chronic effects and endpoints are more sensitive per different chemical classes;  

Improve the understanding of the inherent variability of in vivo chronic fish assays (which will 
support the validation of alternative approaches);  

(iii)  Generate Acute to  Chronic  Ratios (ACRs) for  different  fish  species and chemical  spaces to 

better understand where ACRs can be used as parts of WoE-approaches; 

(iv)  Develop integrated testing strategies covering different effects / endpoints. 

•  General  (chronic  fish  toxicity):  Explore  the  possibilities  to  develop  a  reference  dataset  relating 
chronic invertebrate (daphnid) effects / endpoints to in vivo chronic fish effects / endpoints and to 
identify  which  chronic  effects  and  endpoints  are  more  sensitive  per  different  chemical  classes 
across invertebrates and fish; 

The following medium-term recommendations are rather research oriented. The Commission will explore 
possibilities  for  implementing  the  recommendations,  together  with  partners  from  Member  States  and 
stakeholders, or e.g. via research funding:  

81 

 
 
 
•  Growth effects: Investigate further the predictive capacity of fish cell line proliferation for effects 
on  fish  growth,  by  combining  in  vitro  and  in  silico  approaches  (TK  and  growth  models). This 
includes testing further chemicals in the RTgill-W1 cell-line test, but also using other fish cell lines, 
cell culture models and effect endpoints. 

•  Growth effects: Support extrapolation of in vitro data to fish in vivo effects by comparing short and 
long-term cytotoxicity and cell proliferation in the RTgill-W1 cell line or on other relevant fish cell 
lines to chronic fish toxicity data, with focus on survival and fish growth effects. 

•  Developmental effects: Investigate further the predictive capacity of the OECD TG 236 (FET) for 
developmental effects by investigating the relationship between additional endpoints in the FET 
test and chronic fish toxicity, e.g. between teratogenicity endpoints and chronic fish developmental 
effects  

•  Mechanistic information – relevant for all chronic effects: Include additional endpoints in the FET 
test to obtain mechanistic information – e.g. transcriptomics endpoints for tPOD (transcriptomic 
Point of Departure) derivation, strengthening read-across and derive effect concentrations.  

•  Behavioural  effects:  Depending  on  regulatory  requirements,  explore  the  predictive  capacity  of 
alternative approaches for chronic fish behavioural effects (e.g. investigate if the OECD TG 236 
can be adjusted for incorporating endpoints of relevance for prediction of chronic fish behavioural 
effects). 

Long-term: 

The  following  long-term  recommendations  are  research  oriented.  The  Commission  will  explore 
possibilities  for  implementing  the  recommendations,  together  with  partners  from  Member  States  and 
stakeholders, or e.g. via research funding:  

•  Developmental effects: In vitro/ in silico: Develop and validate in vitro and in silico approaches to 

predict effects on fish development. 

•  Growth effects: Continue research to increase the functionality of RTgill-W1 assays by advancing 
experimental procedures as needed, e.g. on adding water flow and cell painting (Fenton et al., 2023; 
Nyffeler et al., 2025). 

82 

 
 
 
Description of method readiness, applicability domain and validation status 

Alternative approaches for chronic fish toxicity waiving and weight-of-evidence 

This section describes the currently accepted waiving approaches and  alternative approaches that can be 
used to support weight-of-evidence for chronic fish toxicity assessment as integrated approach/ group of 
methods. Although there is no one-to-one replacement, summary information is provided for the specific 
available alternative approaches per respective relevant effect/ endpoint. 

The information under this section might be regarding also from the perspective of recommendations for 
reconsidering the data requirements for chronic fish (i.e. by re-examination of the actual need for providing 
data on these different endpoints).  

Since  this  section  intends  to  reflect  on  chronic  fish  toxicity  information  requirements  under  different 
regulations, WoE is therefore considered from a broad perspective without intentionally fully aligning with 
any specific definition.  

Chronic fish data waivers and using other information 

Use of available data 

Use of available data is promoted or is being considered under different regulations by:  

•  Encouraging data sharing among applicants. Applicants are encouraged to share environmental risk 
assessment study data on the same active ingredients. Regarding pharmaceuticals this can be the 
case  between  originator  and  generic  companies.  Data  sharing  is  specifically  mentioned  in  the 
revised ERA guideline for human medicinal products (EMEA/CHMP/SWP/4447/00 Rev. 1). The 
REACH  regulation  contains  obligations  for  sharing  existing  data  involving  vertebrate  animals 
(REACH Art.  27). The  advantages  are  that  double  testing  can  be  avoided  and  therefore  animal 
testing can be reduced, and costs can be saved.  

•  Use of literature data: Regulatory assessments, including for fish chronic toxicity, can be based on 
literature data only, if relevant data are available. For PPPs, Regulation (EC) No 1107/2009 requires 
dossiers to include a systematic assessment of scientific peer-reviewed open literature on the active 
substance  and  its  relevant  metabolites  published  within  the  last  10  years  before  the  date  of 
submission of the dossier. The endpoints taken from publicly accessible publications must fulfil 
certain  criteria  and  must  be  assessed  for  relevance  and  reliability.  This  can  be  done  based  on 
standardised  assessment  methods  like  e.g.  CRED  (CRED:  Criteria  for  reporting  and  evaluating 
ecotoxicity  data  (Moermond  et  al.,  2015).  The  advantages  are  that  no  new  studies  need  to  be 
performed, and the amount of costs, labour and animals needed can be reduced. Challenges are to 
find literature studies which suits the regulatory purpose e.g. provides enough background and raw 
data and are of high quality. Contradictory results from multiple studies might create interpretation 
challenges. 

•  Adaptation of data requirements: REACH does not include an endpoint specific waiver for chronic 
fish  toxicity,  but  there  are  general  rules  for  adaptation  of  standard  information  requirements 
(REACH Annex  XI).  These  include  for  instance  the  use  of  existing  data,  weight  of  evidence 
approaches,  (Q)SAR,  in  vitro  methods,  grouping  and  read  across  approaches,  or  that  testing  is 
technically not possible. 

Grouping and Read-across  

83 

 
 
 
 
For  some  modes  of  action  available  data  indicating  that  toxicity  in  chronic  toxicity  relates  to  the  same 
mechanism  causing  acute  toxicity  (e.g.  for  Succinate  dehydrogenase  inhibitors),  read-across  supporting 
waiving of chronic based on (slightly prolonged) acute fish tests might be possible.  

Such an approach can be supported by validation of TK/TD models (e.g. GUTS) parameterised with acute 
toxicity data against chronic study data. Toxicity (survival) over long-term and time-varying exposure can 
then be modelled, avoiding the use of chronic studies for substances with the same mode of action. This 
approach may also be supported by modification of acute tests to better inform time-to-effect parameters 
for subsequent TK/TD modelling, at least for some known modes of action.  

Exposure based / environmental fate-based waiving  

Under Regulation (EC) 1107/2009, chronic fish toxicity is required for active substances where exposure 
in edge-of-field surface waters is likely, and the substance is stable in water (<90% loss by hydrolysis in 24 
h). 

Under  Regulation  (EU)  528/2012,  chronic  fish  toxicity  testing  is  required  if  the  results  of  the 
ecotoxicological studies, studies on fate and behaviour and/or the intended use(s) of the active substance 
indicate a risk for the aquatic environment, or if long-term exposure is expected.  

Under REACH, the general rules for adaptation in Annex XI allow for substance-tailored exposure-driven 
testing (Section 3 of Annex XI). Testing in accordance with Annex IX (the Annex at which chronic fish 
toxicity testing is required) may be omitted based on the exposure scenario(s) developed in the Chemical 
Safety Report, provided any of the criteria in Section 3.2 of Annex XI are fulfilled (i.e. there is an absence 
of or no significant exposure, a PNEC can be derived from the available test data, and exposures are always 
well below the derived PNEC; or strictly controlled conditions are applied throughout the life cycle) and 
adequate justification and documentation is provided, and the specific conditions of use are communicated 
through the supply chain. 

Waiving of chronic fish based on mode of action  

The  identification  of  the  most  sensitive  organism  /  taxonomic  group  based  on  mode  of  action,  and 
accordingly waiving of chronic fish testing when fish are not the most sensitive organism, is recommended. 
For example, the need and waiving of chronic fish testing may be relevant on a mode of action basis for 
certain pesticides when the risk is driven by other organisms due to mode of action e.g. herbicides and 
insecticides. Initiatives to deprioritise and waive chronic fish tests include: 

•  Waiving of chronic fish testing in case fish are not the most sensitive species: 

o  The  US  EPA  Office  of  Pesticides  Programs (OPP)  allows  waiving  chronic  fish toxicity 
testing of pesticides if there is no acute toxicity and little exposure, unless there is high 
bioaccumulation.  

o  As reported by Teixido et al. (2023), the FELS toxicity (OECD TG 210) endpoints were 
the most sensitive (10-fold) for 9.5% of analysed chemicals (n=223) when compared to 
chronic Daphnia and algae endpoints. Some of these compounds have a known or assumed 
specific  mode  of  action  such  as  endocrine  disrupting,  or  other  non-narcotic  MoA, 
indicating  that  the  higher  sensitivity  in  the  FELS  toxicity  test  is  related  to  a  specific 
mechanism of action. Thus, the use of existing data (i.e., chronic non-vertebrate data, and 
data  from  alternatives  to  animal  testing)  may  improve  (and  reduce  the  uncertainty 
associated  with)  assessments  for  a  more  integrated  approach  to  evaluate  chronic  fish 
toxicity.  

o  For neurotoxic chemicals, Schür, Paparella et al. (2025) analysed historic acute toxicity 
data  for  fish  and  daphnids  and  concluded  that  fish  rarely  represent  the  most  sensitive 
trophic level of the two, a conclusion which could be explored for considering also chronic 
fish toxicity.  

84 

 
 
o  For  human  pharmaceuticals  with  a  specific  toxicity  profile  (e.g.  endocrine  active 
substances,  antibacterials)  a  so-called  tailored  testing  strategy  is  applied,  which  differs 
from  the  standard  test  composition.  In  the  revised  ERA  guideline  for  human  medicinal 
products  (EMEA/CHMP/SWP/4447/00  Rev.  1)  fish  testing  (e.g.  OECD  210)  for 
antibacterials. 

•  Decision  tree  for  reducing  fish  testing  for  legacy  pharmaceuticals:  A  decision  tree  has  been 
proposed  by  Coors  et  al  (2023)  to  reduce  the  need  for  experimental  testing  on  fish  for  legacy 
pharmaceuticals.  If  substantial evidence  indicates that  a  pharmaceutical may  affect fish,  in vivo 
testing is warranted. In cases where such evidence is lacking, the decision tree employs a risk-based 
approach using two proxies to assess hazard, and both are compared to the predicted environmental 
concentration  (PEC)  as  a  conservative  estimate  of  environmental  exposure. The  first  proxy  for 
characterising hazard is the minimum no observed effect concentration (NOECmin) derived from 
non-vertebrate chronic toxicity tests (OECD TG 211 and OECD TG 201). The second proxy is 
derived by the fish plasma model (FPM) that suggests toxic effects in fish are unlikely below human 
therapeutic plasma concentrations. In the next step, both of these proxies for hazard (NOECmin 
and TWC, theoretical therapeutic water concentration) are related to the chemicals’ PEC in surface 
water. The  two  derived  risk  quotients  (PEC/NOECmin  and  PEC/TWC)  are  then  compared  to  a 
proposed  threshold  value  of  0.001.  If  at  least  one  of  the  two  risk  quotients  is  greater  than  the 
threshold value, the compound should be considered for in vivo testing in fish. If both quotients are 
below the threshold value, no in vivo fish testing has to be considered. The threshold value of 0.001 
which correspond to a safety factor of 1000 may be adjusted in the future based on new data.  

Extrapolation approaches 

Acute to Chronic ratios  

Acute-to-chronic ratios (ACRs) can be used to estimate chronic toxicity of chemicals in cases where only 
acute toxicity data are known, and chronic data are either limited or absent (Raimondo et al., 2007). They 
are empirically derived as ratios between the acute (represented by LC50 or EC50) and chronic (represented 
by NOEC or MATC) ecotoxicological thresholds (Brill et al., 2021; Raimondo et al., 2007). 

Extrapolations for fish chronic endpoints that are based on acute fish or Daphnia toxicity have been shown 
as a valid method by comprehensive data analysis provided by the EU Commission Joint Research Centre 
(Kienzler  et  al.,  2016).  In  general,  the  use  of  acute  to  chronic  ratios  has  been  demonstrated  as  a  valid 
replacement in the scientific literature (e.g. Brill et al., 2021; Raimondo et al., 2007, Scholz et al., 2018, 
Wheeler et al., 2014), particularly is the mode of action of chemicals has been accounted for. Whilst values 
can vary, if the MoA of a chemical is narcotic, its ACR is usually relatively constant (Wang et al., 2020). 

If reliably defined, ACRs can be used as screening tools or weight of evidence to waive need for chronic 
testing.  However,  one  of  the  key  challenges  is  the  fact  that  robust  and  reliable  data  is  often  lacking, 
particularly with respect to existing fish chronic toxicity data. Ideally, data should be matched by species 
to avoid issues around species sensitivity differences. No single ACR exists for all chemicals, leading to a 
lack of standardisation and potential challenges in knowing which factor to use.  

Currently, ACRs are not accepted by ECHA under REACH as a direct replacement for in vivo studies. Use 
of ACRs is most appropriate for chemicals with a narcotic mode of action under both acute and chronic 
exposures, thus their use also requires reliable determination of MoA.  

Given the inherent challenges of deriving robust ACR (data availability, a priori knowledge of MoA) it is 
unlikely that this approach will ever be considered reliable enough to be considered a direct replacement 
for an in vivo fish chronic study. However, the approach could have some merit as a screening tool or as 
part of a wider WoE argument. It is recommended to generate and publish more ACRs for different fish 

85 

 
 
 
species and chemical spaces to better understand where ACRs can be used as parts of WoE approaches 
including under REACH.  

Alternative  approaches  to  animal  testing  for  assessment  of  chronic  fish  toxicity 
including standardised stage and applicability domain  
Several alternative approaches to potentially predict chronic effects in fish are already available. However, 
they are in earlier stages of development than acute alternatives and require additional development and 
data collection efforts.  

This  section  summarises  the  information  on  available  alternative  approaches  according  to  respective 
relevant effect / endpoint, including also approaches that are still in an exploratory phase. It is expected that 
effect-specific WoE can be developed for some / each of the main effects / endpoints considering different 
alternative approaches (integrated approach / group of methods). 

Recommendation: As regards fish chronic endpoints, there is a strong need to develop case studies of how 
different methods can be strung together to gain better understanding on the strengths and limitations of the 
existing methods (in silico, in vitro, and in vivo), links to other endpoints (acute, non-fish species, endocrine, 
etc.) and information that these methods can provide regarding chronic mechanism / modes of action. A 
better understanding of different methods working together would also be required for the development of 
an IATA or Defined Approach. 

Growth  

Stadnicka-Michalak et al. (2015) presented an approach based on a fish cell line assay and PBTK modelling 
that  quantitatively  predicts  fish  growth  effects.  The  authors  exposed  RTgill-W1  cells  for  120  h  to  two 
fungicides, measured cell survival and proliferation, predicted long-term inhibition of cell proliferation, 
interpolated fish weight reduction to different chemical concentrations, and presented model predictions of 
reduced fish growth in agreement with in vivo data. The model is so far only applicable to rainbow trout 
and  fathead  minnow.  The  approach  would  need  to  be  further  developed  before  use  for  regulatory 
assessments is possible. In particular, data with further substances are required. 

Survival 

Hernández-Moreno  et  al.  exposed  RTgill-W1  cells  to  15  nanobiomaterials  (NBM)  and  measured 
cytotoxicity  (viability)  for  up  to  28  days.  The  cells  can  be  used  for  screening  of  long-term  toxicity  of 
nanobiomaterials. The approach evaluates the time needed to reduce by 20% or 50% the cell viability with 
respect to a control. To consider applying the approach in regulatory assessments, a comparison with the 
long-term survival of fish in vivo would be necessary. 

Development 

In addition to acute mortality, the OECD TG 236 allows for observation of hatching and lethal as well as 
sublethal developmental effects in zebrafish embryos up to 96 hours post fertilisation, or possibly longer. 
To use such data, a recommendation is to investigate the relationship between teratogenicity information 
from the OECD TG 236 and from chronic developmental fish toxicity.  

Another  recommendation  is  to  include  transcriptomics  endpoints  in  the  OECD  TG  236  to  also  obtain 
mechanistic information for tPOD derivation, strengthening read-across and derive effect concentrations. 
ECHA contracted the Fraunhofer Institute to explore including transcriptomics endpoints in TG 236 to also 
obtain  mechanistic  information  for  tPOD  derivation,  i.e.  alterations  of  gene  expression,  which  could 
strengthen  category  building  for  read-across.  The  aim  is  to  adapt  the  OECD  TG  236  to  use  it  as  an 

86 

 
 
 
alternative to chronic fish toxicity testing. According to ECHA, the approach could not only be used to 
derive effect doses from molecular data in fish, but potentially also inform about the mode of toxicological 
action. It is expected that it could potentially also be used to address endocrine disruption or other concerns 
which are based on known mechanisms of toxicity.  

Reproduction 

Reproductive  adversity  as  it  is  conceived  now  is  not  assessable  by  methodologies  other  than  in  vivo 
methods. This is also the case for generating adversity related to endocrine disruption. A better knowledge 
of adverse outcome pathways (AOP) could help to link information from in vitro methods to adverse effects.   

Behaviour 

It is recommended, in the short-term, to clarify the regulatory requirements to report behavioural effects. 
Based on the actual requirements, the predictive capacity of alternative approaches for chronic behavioural 
effects in fish can be explored in the medium-term by, e.g., investigating if endpoints can be included in the 
OECD TG  236  for  prediction  of  chronic  fish  behavioural  effects.  Furthermore,  available  literature  data 
could be assessed to explore the performance of fish behavioural studies to be used not only as a screening 
for neurotoxicity but also for endpoint derivation.  

The  following  considerations  are  based  on  that  the  roadmap  aims  to  phase  out  animal  tests  gradually, 
starting from protected fish life stages54 of vertebrates (i.e. from fish larvae with external feeding), then 
later progressing to non-protected life stages (embryos and early non-feeding larvae), and in the future also 
phasing out testing with invertebrates.  

In vitro and eleutheroembryos neurotoxicity assays available 

Recent publications have described the development and the application of brain cell lines of marine teleost 
fish to investigate metal neurotoxicity, namely glial cell line derived from the Red Sea Bream (Luo et al., 
2023) and brain fibroblast-like cell line derived from the European sea bass (Morcillo et al., 2017). 

AOPs 

The opportunities and challenges for defining AOPs that might be linked to neurotoxic and behavioural 
effects have been explored by review papers (Fitzgerald et al., 2021; Legardi et al., 2018) with a focus on 
the  use  of  behavioural  assays  with  fish  early  life  stages.  Identified  research  needs  to  include  the 
identification  of  molecular  targets,  validation  of  pathways,  and  determination  of  the  molecular  events 
causing the observed phenotype (Fitzgerald et al., 2021). Ultimately, the use of AOPs, also for cross-species 
extrapolations and ecological risk assessment, could be promoted if behavioural changes were incorporated 
in AOPs as whole organism responses (Legardi et al., 2018). 

In silico  

Quantitative structure-activity  relationship  models (QSAR)  are  available that  predict chronic toxicity  to 
fish, including the OECD QSAR toolbox, ECOSAR, KATE and VEGA. Some QSAR tools like iSafeRat 
include mode or mechanism of action profilers.  

The  applicability  domains of  QSARs  generally cover  organic  chemicals  up  to log  Kow  of  8.  Inorganic 
chemicals, organometallic chemicals, ionic chemicals (though ionic surfactants are covered by ECOSAR) 
and nanomaterials are generally not within QSAR applicability domains.  

54 According to Directive (EC) 2010/63 

87 

 
 
 
 
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Coors A, Brown AR, Maynard SK, Nimrod Perkins A, Owen S, Tyler CR. 2023. Minimizing Experimental Testing on 

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3.4. Endocrine disruption for environmental safety assessments 

Introduction 
Endocrine disruptors (EDs) are “exogenous substances or mixtures that alter the functions of the endocrine 
system and consequently cause adverse effects in intact organisms, or its progeny or population” (WHO, 
2002). According  to  the  ED  identification  criteria  as  defined  in  the  Guidance  to  Regulation  (EC)  No 
1272/2008 on classification, labelling and packaging (CLP) of substances and mixtures (ECHA, 2024), in 
the  Plant  Protection  Products  (PPP) regulation  605/2018  and  Biocical  Product Regulation  2100/2017, a 
substance shall be considered as having ED properties if it meets all the following criteria:  a) it shows an 
adverse  effect  in  an  intact  organism  or  its  progeny,  which  is  a  change  in  the  morphology,  physiology, 
growth,  development,  reproduction  or  life  span  of  an  organism,  system  or  population  that  results  in  an 
impairment of functional capacity, an impairment of the capacity to compensate for additional stress or an 
increase in susceptibility to other influences,  b) it has an endocrine activity, i.e. it alters the function(s) of 
the endocrine system, c) the adverse effect is a consequence of the endocrine activity. According to the 
above-mentioned regulations, a substance is considered to be an endocrine disruptor if the three criteria are 
met, unless the adverse effects identified are not relevant at the level of population. The CLP guidance 
(ECHA, 2024) reports that effects on growth (body weight and length), development and reproduction (such 
as fecundity, fertility, sex ratio, and hatching success) in single species in laboratory studies are generally 
regarded as adverse at the level of population. Therefore, establishing causal links between molecular-and 
individual-level  responses  is  crucial  to  conclude  on  ED  properties  as  well  as  to  further  elucidate 
extrapolation  from  individual  level  effect  to  population. It  is  important  to  recognise  that  the  domain  of 
applicability of the term ‘endocrine disruptor’ is contingent on the defined scope of the endocrine system. 
To date, most scientific and regulatory efforts related to EDs have been focused on interference with the 
estrogen (E), androgen (A), thyroid (T) and steroidogenesis (S) pathways, referred to as EATS modalities. 
However, the endocrine system comprises other hormone signalling systems beyond EATS modalities, that 
potentially could also be affected by exposure to xenobiotics – collectively these are considered ‘non-EATS 
modalities’.  Potential  non-EATS  modalities  include  progesterone  signalling,  and  the  Hypothalamic-
Pituitary-Adrenal  (HPA)  and  somatotropic  axes.  The  disruption  or  perturbation  of  those  pathways  can 
potentially  lead  to  a  high  variety  of  apical  effects  involving  growth,  development,  reproduction, 
metabolism,  behaviour,  etc. Moreover,  under  non-EATS-modalities,  endocrine  pathways  relevant  for 
invertebrates, e.g., ecdysone and retinoic acid should be also acknowledged. 

The  above-mentioned  identification  criterion  (a)  includes  the  notion  of  ‘intact  organisms’,  which  is 
understood to mean that the effect would occur in vivo, observable in a test animal system. However, it does 
not necessarily mean that the adverse effect has to be demonstrated in an intact test animal (ECHA, 2024) 
but may be shown in adequately validated alternative test systems predictive of adverse effects in humans 
and/or  wildlife  (Solecki  et  al.,  2017).  Operational  actions  towards  phasing  out  animal  testing  for  the 
environmental  safety  assessment  of  endocrine  disruptors  were  identified  and  outlined  in  the  following 
paragraphs in a stepwise progression. Each step (short-term, medium-term, and long-term) is building upon 
the  previous  one  to  ensure  a  cohesive  and  effective  approach  to  reach  the  overall  objective.  The 
recommendations provided apply across all chemical sectors. Nevertheless, the relevance and feasibility of 
these  recommendations  may  differ  between  sectors,  given  the  distinct  data  requirements  imposed  by 
different legislative frameworks governing each sector.  

Recommendations   
Reduction of animal testing 

90 

 
 
 
The  following  options  for reduction  or  refinement  of  animal  testing  in  the short- to medium term  were 
identified.  Reduction  of  animal  testing  would  be  an  interim  solution  until  a  full  transition  to  an 
environmental safety assessment that relies fully on the use on non-animal approach is possible. 

Adapt test guidelines or guidance so that information from in vivo testing is maximised and can be 
used both for concluding on the ED properties and risk assessment.  It is recognised that the testing 
strategy recommended for the investigation of the endocrine disrupting properties is not fully in line with 
the testing required for performing a risk assessment. However, the two strategies could be aligned since 
this will considerably reduce the use of animals. This would require adapting current in vivo testing inter 
alia  as  regards  number  of  dose  levels  applied,  since  the  derivation  of  reliable  no  observed  effect 
concentrations for use in risk assessment require at least four test concentrations or considered endpoints. 
Legislative requirements or guidance would need to be modified.  

A similar measure would be to complement the available study protocols with additional endpoints that 
can give indications on toxicity related to e.g. specific mode of action of a compound allowing regulators 
to  conclude  that  any  potential  ED  related  effects  would  be  secondary  to  other  toxicities  (i.e. 
acetylcholinesterase  activity  measurements  in  fish  in  regards  of  neurotoxicity).  Furthermore,  to  avoid 
unnecessary repetition of testing, test guidelines of in vivo studies, should contain all possible biological 
endpoints mentioned in current TGs. This would mean e.g. the inclusion of gonad histopathology in FSDT 
(OECD TG 234) and FSTRA (OECD TG 229). 

Better definition of test concentration range. In particular, a better definition of the Maximum Tolerated 
Concentration (as defined in the ECHA Guidance on the application of CLP criteria (ECHA, 2024)). This 
would avoid testing at unnecessarily high concentrations leading to excessive toxicity (e.g. mortality or 
severe suffering). This would also help to better discern ED mediated effects and effects related to other 
toxicities. In a similar way, low concentrations which are not sufficient to conclude on the lack of potential 
ED-related activities or adversity should be not tested to avoid the need of multiple repetitions of the test. 
The development of a document giving guidance on how to best define the concentration range to test ED 
properties is recommended. 

Foster the validation of modified protocols overcoming some of the main issues identified in available 
standard  tests,  e.g.,  Extended Amphibian  Metamorphosis Assay  (EAMA)  (Ortego  et  al.,  2021),  Larval 
Amphibian Toxicity Test (LATT), inclusion of thyroid endpoints in fish embryo acute toxicity test (OECD 
TG 236). Both EAMA and LATT assays generate data on apical endpoints which are sufficient to evaluate 
T‐mediated adverse effects that are potentially relevant at the population level. Their validation may result 
in the obsolescence of Larval Amphibian Growth and Development Assay (LAGDA, OECD TG 241).  

Currently under validation, the proposal of the OECD project “Inclusion of thyroid endpoints in OECD fish 
TGs” consists of the addition of assessment of posterior swim bladder inflation, thyroid hormone levels, 
eye  development,  thyroid  follicle  morphology.  It  is  still  unclear  how  the  inclusion  of  some  of  these 
endpoints may allow the collection of information useful to predict adversity due to endocrine (T-modality) 
and non-endocrine mechanisms in individuals starting from the embryonic life stage. Since those testing 
methods  employ  animals,  this  action  point  is  considered  to  represent  a  short-term  recommendation, 
although this does not reflect the potential foreseen date of validation according to OECD GD 34.  

Improvement  and  better  use  of  the  existing  OECD  conceptual  framework  (CF)  proposed  in 
ECHA/EFSA  guidance  (2018)  considering  it  as  toolbox  instead  of  a  tiered  approach  only.  This  would 
include consideration of whether it would be possible to use non-animal approaches to interrogate observed 
findings and further explore mechanisms following OECD CF level 3 in vivo studies. Uncertainty around 

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apparent activity indicated by an OECD CF level 3 test could be verified with an appropriate test at lower 
level, before resorting to higher level tests with concomitant animal use to address adversity. This could be 
facilitated  by  developing  Integrated  Approaches  to  Testing  and  Assessment  (IATA)  which  provide  a 
framework of available methods and recommendations on best methods depending on the specific case 
under assessment. 

Short-term recommendations  

Investigate  whether  the  current  human/mammalian  in  vitro  standard  methods  are  sufficiently 
predictive of EATS-related endocrine activity in non-mammalian species and identify sufficient in 
vitro datasets to support a conclusion on the absence of EAS-related endocrine activity. 
Currently, studies placed at the OECD CF level 2 for the investigation of endocrine activity, mainly through 
EAS-modalities, are specific for providing mechanistic information related to the assessment of endocrine 
disruption for human health, since they use either human or mammalian material. To date, no information 
is available on whether those can be considered predictive of possible endocrine activity in non-mammalian 
species. Therefore, a 2-step approach is proposed: 

A.  Investigate  extrapolation  of  findings  from  human/mammalian  cell-based  assays  to  non-
mammalian species through e.g. a meta-analysis comparing outcomes from in vitro test battery 
and OECD CF level 3 studies with fish, e.g. test done according to OECD TG 250, 251, 252, 229, 
230. 

B.  Review the sufficiency of the in vitro dataset for EAS-related endocrine activities as defined 
by ECHA/EFSA Guidance (2018) with the aim of supporting the conclusion on the absence of 
endocrine activity based on data derived from in vitro and non-animal methodologies, as already 
done in human health assessment by the use of the ToxCast ER model to conclude on the E modality 
without the need to perform a study in line with OECD TG 440. 

Better  understanding  of  how  alternatives  to  animal  testing  already  in  use  and  taken  up  in  the 
regulatory world are performing in the real world.   
A mapping exercise is advised to better understand identifiable mechanisms and applicability domains of 
recently adopted OECD TGs (e.g. RADAR and REACTIV assays). The performance of the XETA (OECD 
TG 248) with non-PPP compounds should also be evaluated.   

Development of guidance on the use of AOPs framework to map alternatives to animal testing.  
Recently,  there  has  been  a  growing  focus  on Adverse  Outcome  Pathways  (AOPs)  as  a  framework  for 
organizing mechanistic data, including data generated by alternatives to animal testing, to predict or explain 
adverse  outcomes  and  the  biological  plausible  link  between  endocrine  activity  and  adversity  and  more 
generally to facilitate decision-making regarding the safety of chemicals. However, a single AOP is often 
insufficient to fully explain an Adverse Outcome (AO), and a network approach may be required to capture 
the  complexity  of  the  underlying  biology.  Therefore,  while AOPs  are  valuable  for  the  development  of 
alternatives to animal testing, guidance on how to integrate individual non-animal methods within the AOP 
framework is needed to identify gaps in the methodology and inform its further development.  

Mid-term recommendations  

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Build solid evidence and gain further knowledge on cross-species extrapolation to reduce uncertainty 
linked to the lack of data of relevant species.  
Extrapolating  biological  data  across  species  is  a  crucial  aspect  of  biomedical  research,  pharmaceutical 
development and toxicology. This knowledge advancement in the field of human health has highlighted the 
potential to use similar methodologies to assess chemical risk across animal models used in ecotoxicology 
and  for  the  prediction  in  wildlife. Building  a  solid  case  for  the  application  of  in  silico  cross-species 
extrapolation,  starting  from  a  data  rich  chemical/species,  would  create  a  common  ground  for  both  the 
scientific  and  the  regulatory  relevance.  It  is  however  crucial  to  gain  further  knowledge  on  structural 
conservation of molecular key events (KEs) and KE relationships of an AOP of relevant species. This would 
allow to extend the biologically plausible taxonomic domain of an identified AOP. Simultaneously, the use 
of cross-species extrapolation based on conservation of (part of) pathways amongst species should be seen 
as part of a weight of evidence approach for safety decision making within the regulatory context, and as 
support  for  the  development  of  new  relevant  non-animal  approaches  with  high  potential  for  regulatory 
acceptance.  

Identify pathways other than EATS and develop  alternatives to animal testing and AOP for those 
non-EATS pathways, so that it is possible to distinguish endocrine from non-endocrine pathways.  
Identify  pathways  other  than  EATS  is  beneficial  to  different  aims,  1)  improve  the  overall  toxicological 
assessment  of  EDs,  taking  into  consideration  new  endocrine  pathways  and  excluding  non-endocrine 
pathways (e.g. distinguish systemic non-endocrine specific from endocrine-specific toxicity).   

Long-term recommendations  

Define mechanistic based panel of in silico and in vitro assays that allows for a hypothesis driven 
safety assessment  
A hypothesis-based safety assessment focuses on the potential bioactivity of chemicals and on key adverse 
outcome pathways (AOPs) relevant for environmental species. There are several benefits of this concept as 
recognized by pharmaceutical companies for human safety applications (Bowes et al., 2012). However, to 
be  suitable  for  environmental  safety,  substantial  work  would  be  needed  to  identify  critical  biological 
characteristics in keystone species that, if impaired, would affect population levels (a primary goal within 
environmental safety assessment). Additionally, robust in silico and in vitro assays able to quantify those 
critical biological characteristics need to be developed. Those assays could be employed in the first tier of 
safety assessment, emulating human safety processes  and potentially leveraging synergies with existing 
approaches in the human toxicology field.  

Develop quantitative Adverse Outcome Pathways (qAOPs)  
The AOP  concept  was  developed  to  organize  available  information  describing  how  the  interaction  of  a 
chemical with a molecular target (Molecular Initiating Event, MIE) can result in an adverse outcome (AO) 
in individuals and, by extension, in a population. In an AOP, the MIE is causally related to the AO through 
a series of KEs linked by key-event relationships (KERs). As they are based on a biologically plausible 
suite of KEs, AOPs can be used in weight-of-evidence approaches to identify endocrine disruptors. An AOP 
that is sufficiently populated with substance-specific data allows establishing plausible links between KEs 
leading to an individual-level AO. However, both potency and exposure are required for a KE to activate 
the next step in the chain, that is, to reach the “tipping point.” Many factors can interfere with KERs, so 
that an AO may not occur even if a MIE has been identified and effects on KEs are empirically observed. 
The integration of qAOP and TK-TD information could solve this issue (Perkins et al., 2019). Additionally, 

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development of qAOP networks would help in understanding extrapolation across species. Detailed qAOPs 
with established potency thresholds for each KE and AO prediction can however be technically challenging 
and resource intensive.  

General recommendations  

Building trust on in vitro and in silico testing.  

Following  scientific  progress,  non-animal  approaches  are  becoming  increasingly  used  by  industry  and 
consequently regulators are receiving more data from studies using non-animal approaches, which may not 
be validated methods because of lack of resources. The evaluation of these studies by a regulator can be a 
time-consuming  process,  based  on  the  level  of  details  and  transparency  of  reporting.  Moreover,  the 
complexity  of  different  approaches  may  have  an  impact  on  resources.    Addressing  and  increasing 
understanding in both scientific and regulatory defined questions such as ADME processes, applicability 
domain, actual exposure quantification and therefore decreasing the overall uncertainties would qualify the 
method for regulatory use.  

Beside this, maximising the effort to demonstrate the performance of new methods compared to in vivo 
methods (i.e. eleutheroembryos assays in parallel to Fish Short Term Reproduction Assay (OECD TG 229)) 
would strengthen confidence. 

Overview of the available and established methodologies using non-protected life 
stage animal for assessing endocrine disrupting potential of chemicals  
Considering the need to demonstrate activity, adversity and a plausible linkage, it appears unrealistic that a 
single  assay  can  provide  sufficient  evidence  to  identify  a  chemical  as  ED,  according  to  the  criteria. 
Furthermore, in some cases even when in vivo data are available, the following considerations may limit 
the  straightforward  interpretation  of  standard  (eco)toxicological  tests:  the  animal  model,  the  life  stages 
tested,  the  relevance  and  specificity  of  the  measured  endpoints  for  endocrine  activity/adversity,  or  the 
exposure design. Currently, in vitro methods are often used as supplementary or supportive information to 
the results of animal testing, highlighting the issue of comprehensiveness of animal testing, and the need 
for strategies based on integration of data from multiple test methods.   

In vivo assays utilizing organisms at non-protected life stages according to Directive 2010/63/EU  
Available alternatives for addressing the requirement to sufficiently investigate endocrine activity in non-
mammalian  species  are  assays  using  eleutheroembryos  at  stages  considered  in  the  legislation  not  to  be 
protected organisms. These mechanistic OECD CF level 3 assays do not investigate adverse effects (e.g. 
impaired growth and development, reduced number of offsprings). Currently there are five methods using 
either amphibian or fish eleutheroembryos : Xenopus Eleutheroembryonic Thyroid Assay (XETA, OECD 
TG  248),  measurement  of intrafollicular  thyroxine  (T4)  using  zebrafish  eleutheroembryos (ZETA,  non-
standardized),  Rapid  Androgen  Disruption  Activity  Reporter  assay  (RADAR  assay,  OECD  TG  251), 
Detection  of  Endocrine  Active  Substances,  acting  through  estrogen  receptors,  using  transgenic 
tg(cyp19a1b:GFP) Zebrafish embrYos (EASZY, OECD TG 250), Rapid Estrogen ACTivity In vivo assay 
(REACTIV assay, OECD TG 252). The first two assays are relevant for the thyroid modality while the last 
three assays can be used for EAS modalities (Annex 1, Table 2). These methods are considered alternatives 
to  vertebrate  testing  because  eleutheroembryos  used  in  the  different  assays  are  not  yet  feeding 
independently  (their  development  and  growth  still  depends  on  the  maternal  egg  yolk  present  in  their 

94 

 
 
 
 
digestive tract) and thus, they are not considered protected life stages according to EU Directive 2010/63 
in the EU. However, it is not always evident that such assays end before the  eleutheroembryos start free 
feeding. It has been observed that eleutheroembryos may start independent feeding earlier due to different 
testing conditions such as temperature.  

In vitro assays  
OECD CF Level 2 in vitro assays provide information on the mechanisms through which a substance could 
be considered endocrine active (e.g. by binding to and activating a receptor or interfering with hormone 
production). Some are listed in the ECHA/EFSA guidance (2018) (Annex 1, Figure 2) and others are under 
development and validation following the steps outlined in OECD GD 34 (Annex 1, Table 2). The in vitro 
assays are largely based on human cell test systems and as yet there is no validated test guideline for in vitro 
assays based on fish or amphibian cells for endocrine activity.   

Other methodologies or ongoing initiatives with potential regulatory relevance 
Other in vitro assays 
Some  fish  and  amphibian  in  vitro  assays  have  been  developed  for  use  in  the  Japanese  regulatory 
programmes  EXTEND  (e.g.,  Medaka  estrogen  receptor  α  reporter  gene  assay  and  Medaka  androgen 
receptor β reporter gene assay for estrogenic, anti-estrogenic and androgenic activities; Medaka androgen 
receptor β reporter gene assay for anti-androgenic activity; Xenopus tropicalis thyroid receptor β reporter 
gene assay for thyroid and anti-thyroid activity).    

Inclusion of endpoints in existing fish TGs and development of new assay with endpoints sensitive to 
thyroid disruptors 
The  ongoing  OECD  project  2.64  was  launched  in  2023  with  the  objective  of  validating  thyroid 
morphology/histopathology, swim bladder inflation, eye development/histopathology, and thyroid hormone 
levels  as  additional  endpoints  in  OECD  TGs  236  and  210.   However,  swim  bladder  inflation  and  eye 
development/histopathology are endpoints sensitive to, but not diagnostic of, the thyroid modality, meaning 
that they are not specific for thyroid disruptors (Jaka et al., 2023). The inclusion of (some of) these endpoints 
in the zebrafish eleutheroembryo test (FET; OECD TG 236) would be relevant for thyroid-mediated activity 
whereas it is still not clear how measurements of such endpoints in the fish early life-stage test (OECD TG 
210) could potentially provide information of adverse effects with population relevance.  

As the fish early life-stage test is a common data requirement for aquatic risk assessment, the inclusion of 
thyroid-related endpoints in the fish early life-stage test is expected to provide information on the thyroid 
modality,  thereby  potentially  reducing  the  need  to  conduct  additional  animal  studies  on  amphibians.  It 
might, however, increase the need of in vivo testing (for fish) under those areas of legislation that currently 
do not require a fish early life stage test or see it as optional. In addition, it needs to be underlined that using 
an in vivo test, including those based on eleutheroembryos, are considered as an intermediate solution and 
only if it reduces the overall need for animal testing until a full replacement is possible.  

Similar to the transgenic fish eleutheroembryonic assays developed to detect EAS-mediated activity (Annex 
1, Table 2), transgenic eleutheroembryonic assays were recently developed to investigated thyroid-mediated 
activity in zebrafish (Jaka et al., 2023) and Japanese medaka (Pesce et al., 2024; 2023). These assays are 
commercially available but have not yet undergone validation according to OECD GD34 and introduced as 
OECD TG.    

Current efforts for integrating these alternatives in the EU regulatory frameworks   

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For non-target organisms other than mammals, to consider the T-mediated endocrine activity sufficiently 
investigated, an Amphibian Metamorphosis Assay (AMA; (OECD TG 231) should be available according 
to  ECHA/EFSA  guidance (2018). Following the  publication  of the  OECD TG  248  (XETA) in 2019,  an 
annex (Annex A) was added to the ECHA/EFSA Guidance (2018) explaining in which circumstances the 
XETA can replace the AMA for having the thyroid-mediated activity sufficiently investigated in non-target 
vertebrates other than mammals. This shows that the XETA can be implemented in testing strategies to 
inform on certain thyroid mechanisms. Mechanisms not covered by the XETA, in particular early phases 
of thyroid hormones synthesis, could be covered by data from in vitro assays (Lagadic et al., 2024) (Annex 
1, Table 2). Based upon the estimates for pesticide active substances, the XETA was used for 61% of 56 
substances  evaluated  for  the  thyroid  modality.  An  AMA  was  conducted  as  a  follow-up  study  for 
approximatively 10% of the substances tested in the XETA.  Overall, this represents 31 AMAs avoided. 
Considering that a typical AMA requires approximatively 400 animals, the implementation of the XETA as 
defined in the ECHA/EFSA Annex A saved 12,400 animals for the evaluated pesticide active substances 
(Du  Pasquier  et  al.,  2025).  Up  to  now,  there  is  no  proposal  from  EFSA/ECHA  for  using  fish 
eleutheroembryonic assays as an alternative to the recommended OECD CF level 3 test with intact fish, 
e.g. the FSTRA, for EAS modalities. The applicability domains of the available fish eleutheroembryonic 
assays need to be further investigated especially with respect to the steps they are able to cover within the 
steroidogenesis pathway.  Some information on this aspect will be available in the EFSA technical report 
on  recurring  issue  in  the  context  of  ED  assessments  (in  preparation).  Nevertheless,  regulators  are 
recommending the use of the RADAR assay in case of specific concerns related to potential anti-androgenic 
properties of chemicals (Panter et al., 2023). 

Main obstacles and critical aspects  
Limited confidence in new methods   
In the case of eleutheroembryos assays, the current limited confidence is mainly related to the use of not 
fully  developed  animals  which  may  lead  to  (different)  uncertainties  compared  to  studies  with  more 
developed  or  adult  organisms,  in  e.g.  the AMA  and  the  FSTRA. There  is  indeed  evidence  of  not  fully 
functional endocrine systems (e.g., thyroid hormone synthesis in the eleutheroembryonic stages used in the 
XETA) or of unknowns or limited metabolic competencies. This is also the case for in vitro test systems 
where  metabolic  competencies  may  be  unclear  These  issues  can  be  addressed  by  combining  different 
methods.  For  example,  the  XETA  can  be  combined  with  in  vitro  thyroperoxidase  and  sodium-iodide 
symporter inhibition assays. Similarly, the RADAR and REACTIV assays can be associated with the  in 
vitro aromatase inhibition assay (OPPTS. 890.1200) and H295R steroidogenesis assay (OECD TG 456). 
More  confidence  can  also  be  gained  from  comparative  studies  between  eleutheroembryonic  assays  and 
chronic amphibian and fish assays (Du Pasquier et al., 2025; 2024). If we consider cell based/molecular 
assays, ADME aspects are generally lacking. There are different methods that aim to incorporate ADME in 
toxicity assays. If we consider fish embryos, the chorion could represent an effective barrier to chemical 
exposure, potentially leading to an unrealistic scenario.  

Importance of the Weight of Evidence  
Since the ED assessment is a complex assessment, it is important to stress that one-to-one replacement is 
unrealistic.  To  this  respect,  the  importance  of  the  weight  of  evidence,  where  all  evidence  is  used  and 
considered together with its uncertainties, is of pivotal importance. Work will be needed to better understand 
and  provide  guidance  on Weight  of  Evidence  application  particularly  in  situations  where  there  may  be 
batteries of assays incorporating many alternatives to animal testing (e.g. for thyroid).  

96 

 
 
  
 
Understanding of the applicability domains of available assays  
In the case of the fish eleutheroembryonic assays, these have been validated for the detection of E and A 
activity.  While  the  RADAR  and  REACTIV  assays  have  been  shown  to  be  able  to  detect  effects  of 
downstream steroidogenesis (inhibition of 5a-reductase and aromatase), it is unclear whether they are able 
to consistently do so for a wider range of chemicals. Simultaneously, the lack of validation and confirmation 
of  coverage  for  upstream  steroidogenic  effects  does  currently  not  allow  for  these  assays  to  cover 
steroidogenic  activity  in  fish.  Consequently,  substitution  of  fish  assays  such  as  the  21-day  Fish Assay 
(OECD TG 230) or the FSTRA (OECD TG 229) with eleutheroembryonic assays is currently not possible. 

Difficult to test substances/multi constituents  
The  applicability  domain  of  the  currently  available  in  vitro  assays  is  unlikely  to  cover  the  variety  of 
chemical structures and properties found in industrial chemicals. Experience based on other in vitro study 
types required under the current REACH information requirements (e.g., for skin sensitisation) has found 
that methods are not applicable to poorly soluble substances, metals, salts, and substances of Unknown or 
Variable  compositions,  Complex  reaction  products  and  biological  materials  (UVCBs).  Substances  with 
specific  physico-chemical  properties,  such  as  volatility  or  poor  solubility,  can  lead  to  false  positives  or 
render  in  vitro  studies  unfeasible. Volatile  chemicals  may  result  in  significant  chemical  losses  to  air  or 
contamination in neighbouring wells when using standard test systems like multi-well plates. Adaptations 
to these systems are necessary to ensure robust and reliable test results. However, many of those issues are 
not specific to in vitro methodologies and are applicable to in vivo assays (Burden et al., 2024). Additionally, 
a  significant  proportion  of  registered  substances  under  REACH  (up  to  30%)  are  multi-constituent 
substances and UVCBs (ECHA, 2017), which pose an extreme challenge for (eco)toxicity testing.  

Population relevance of endocrine-mediated adverse effects  
According to Commission Regulation 605/2018, a substance is considered to be and ED if the 3 criteria are 
met,  unless  there  is  evidence  demonstrating  that  the  adverse  effects  are  not  relevant  at  the  level  of 
population.  Generally,  effects  on  growth,  development  and  reproduction  in  single  species  in  laboratory 
studies  are  regarded  relevant  for  the  maintenance  of  the  wild  population.  Therefore,  when  effects  are 
observed  in  those  parameters  the  relevance  at  the  level  of  population  is  inferred  unless  the  contrary  is 
proven. According to the ECHA/EFSA guidance, field studies/monitoring and population modelling are 
possible approaches for further demonstrating whether an endocrine-mediated effect is relevant at the level 
of population level assessment of endocrine-mediated adverse effects. However, considering the limitations 
of  in  natura  approaches,  population  models  appear  as  the  most  suitable  tools  to  address  population 
relevance (Hazlerigg et al., 2025; Crane et al., 2019). The available population models use apical endpoints 
(e.g. fecundity, sex ratio, time-to-metamorphosis, sexual behaviour) measured in laboratory animal studies 
as entries to predict population dynamics in terms of abundance and biomass. If they cannot be obtained 
from animal studies, these endpoints need to be generated using in vitro-in vivo extrapolation methods that 
are  accurate  enough  to  fulfil  the  requirements  and  criteria  of  good  modelling  practices.   Recently  a 
workshop was organised under the umbrella of the Society of Environmental Toxicology and Chemistry, 
the MAPPED55, on the use of models to address the population relevance of endocrine-mediated effects in 
fish and amphibians. Publications should become available soon. 12 

55 https://www.setac.org/resource/update-from-the-inaugural-mapped-workshop.html 

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Guidelines for the Testing of Chemicals, Section 2. OECD Publishing, Paris, 2025. 

Organisation  for  Economic  Co-operation  and  Development,  Test  No.  229:  Fish  Short  Term  Reproduction Assay, 

OECD Guidelines for the Testing of Chemicals, Section 2. OECD Publishing, Paris, 2012. 

Organisation for Economic Co-operation and Development, Test No. 230: 21-day Fish Assay: A Short-Term Screening 
for  Oestrogenic  and Androgenic Activity,  and Aromatase  Inhibition,  OECD  Guidelines for  the Testing  of 
Chemicals, Section 2. OECD Publishing, Paris, 2009. 

Organisation for Economic Co-operation and Development, Test No. 231: Amphibian Metamorphosis Assay, OECD 

Guidelines for the Testing of Chemicals, Section 2. OECD Publishing, Paris, 2009. 

Organisation  for  Economic  Co-operation  and  Development, Test  No.  234:  Fish  Sexual  Development Test,  OECD 

Guidelines for the Testing of Chemicals, Section 2. OECD Publishing, Paris, 2011. 

Organisation for Economic Co-operation and Development, Test No. 236: Fish Embryo Acute Toxicity (FET) Test, 

OECD Guidelines for the Testing of Chemicals, Section 2. OECD Publishing, Paris, 2025. 

Organisation  for  Economic  Co-operation  and  Development,  Test  No.  240:  Medaka  Extended  One  Generation 
Reproduction  Test  (MEOGRT),  OECD  Guidelines  for  the  Testing  of  Chemicals,  Section  2.  OECD 
Publishing, Paris, 2023. 

Organisation  for  Economic  Co-operation  and  Development,  Test  No.  241:  The  Larval  Amphibian  Growth  and 
Development  Assay  (LAGDA),  OECD  Guidelines  for  the  Testing  of  Chemicals,  Section  2.  OECD 
Publishing, Paris, 2015. 

Organisation  for  Economic  Co-operation  and  Development,  Test  No.  248:  Xenopus  Eleutheroembryonic  Thyroid 

Assay (XETA), OECD Guidelines for the Testing of Chemicals, Section 2. 2019. 

Organisation for Economic Co-operation and Development, Test No. 250: EASZY assay  - Detection of Endocrine 
Active  Substances,  acting  through  estrogen  receptors,  using  transgenic  tg(cyp19a1b:GFP)  Zebrafish 
embrYos, OECD Guidelines for the Testing of Chemicals, Section 2. OECD Publishing, Paris, 2021. 
Organisation  for  Economic  Co-operation  and  Development,  Test  No.  251:  Rapid  Androgen  Disruption  Activity 
Reporter (RADAR)  assay, OECD Guidelines for the Testing of Chemicals, Section 2. OECD Publishing, 
Paris, 2022. 

Organisation  for  Economic  Co-operation  and  Development,  Test  No.  252:  Rapid  Estrogen  Activity  In  vivo 
(REACTIV) assay, OECD Guidelines for the Testing of Chemicals, Section 2. OECD Publishing, Paris, 2024. 
Organisation for Economic Co-operation and Development, Test No. 440: Uterotrophic Bioassay in Rodents: A short-
term screening test for oestrogenic properties, OECD Guidelines for the Testing of Chemicals, Section 4. 
OECD Publishing, Paris, 2007. 

Ortego,  L.S.,  et  al.,  The  extended  amphibian  metamorphosis  assay: A  thyroid‐specific  and  less  animal‐intensive 
alternative to the larval amphibian growth and development assay. Environmental Toxicology and Chemistry, 
2021. 40(8): p. 2135-2144. 

Panter, G.H., et al., Detection of anti-androgenic activity of chemicals in fish studies: a data review. Critical Reviews 

in Toxicology, 2023. 53(5): p. 326-338. 

Perkins, E.J., et al., Building and applying quantitative adverse outcome pathway models for chemical hazard and risk 

assessment. Environmental toxicology and chemistry, 2019. 38(9): p. 1850-1865. 

Pesce, E., et al., A novel transgenic model to study thyroid axis activity in early life stage medaka. Environmental 

Science & Technology, 2023. 58(1): p. 99-109. 

Pesce,  E.,  et  al.,  Testing  the  sensitivity  of  the  medaka  Transgenic  Eleuthero-embryonic  THYroid-Specific  assay 

(TETHYS) to different mechanisms of action. Aquatic Toxicology, 2024. 276: p. 107081. 

Solecki, R., et al., Scientific principles for the identification of endocrine-disrupting chemicals: a consensus statement. 

Archives of Toxicology, 2017. 91(2): p. 1001-1006. 

United  States  Environmental  Protection Agency,  Endocrine  Disruptor  Screening  Program Test  Guidelines  OPPTS 

890.1200: Aromatase (Human Recombinant). 2009. 

World  Health  Organization,  Global  assessment  of  the  state-of-the-science  of  endocrine  disruptors.  International 

Program on Chemical Safety, 2002. 

99 

 
 
3.5. Birds and mammals  

Introduction  
The  document  outlines  current  environmental  safety  assessment  (ESA)  approaches  using  birds  and 
mammals which are relevant to EU chemical safety regulations. It draws mostly from pesticide legislation, 
which has the highest demand for terrestrial vertebrate testing. To register a pesticide, applicants typically 
generate data using around 5,500 laboratory birds and re-use data from approximately 5,000 laboratory 
mammals in the human safety studies for the terrestrial vertebrate risk assessments (see details in Table 5).  

Field studies using wild animals can be requested on a case-by-case basis, typically employing low severity 
and non-lethal assessments, similar to conservation research (e.g., radio-tracking). Such studies are unlikely 
to be requested under some regulatory regimes, e.g. the REACH regulation. However, these studies are not 
mandatory and fall outside the scope of this document and thus roadmap. Existing data can also be used 
within Weight of Evidence. 

This document highlights current data requirements for laboratory in-vivo studies, as well as proposals for 
short-, mid- and longer-term action, to arrive at a new destination, where regulatory-relevant decisions can 
be made using data generated from best available non-animal-based scientific tools.  

Terrestrial wildlife is part of safety assessments in several chemicals risk assessment regulations, including 
plants, microorganisms  and  invertebrates.  However,  these  do  not trigger the  use  of  vertebrates  unless  a 
chemical product is intended for use in habitats they occupy—for example, birds and mammals in areas 
where pesticide or biocide products may be applied.  

The most stringent terrestrial wildlife chemical safety assessment in Europe is in the pesticide legislation, 
Regulation (EU ) No 1107/2009. The data requirements for terrestrial safety testing for pesticidal active 
substances are described in Regulation (EU) No 283/2013.  

In vivo testing requirements have been considered across all five vertebrate animal classes. For mammal 
safety, a wide range of studies are performed to investigate human safety across different life stages (in 
utero  to  breeding),  exposure  durations  (acute,  chronic,  lifespan)  and  exposure  routes  (oral,  dermal  or 
inhalation). These studies also consider interspecies concordance, typically involving rodents, rabbits, dogs, 
and occasionally other species. The data generated from these laboratory mammal studies are re-used as 
the foundation for assessing environmental safety of terrestrial vertebrates, including wild mammals. No 
additional laboratory mammal testing is required specifically for ecological effect assessments of pesticides.  

The only additional tests with terrestrial vertebrates required for ecological effect testing for pesticides are 
those with birds. Both acute and chronic toxicity data are required for at least one avian species as part of 
pesticide authorization/registration in Europe. For global registration of a pesticide, further acute or chronic 
testing may be triggered depending on regional requirements. In most cases, the number of laboratory birds 
used for ESA can be comparable to the number of laboratory mammals used for human safety assessments 
for a pesticide, though this equivalence is not widely acknowledged (Table 5).   

No additional laboratory birds or mammals are used for bioaccumulation testing, nor are they required for 
ecological endocrine screening. 

Extrapolation of effect information between animal classes or within species is currently feasible on a case-
by-case basis. However, our understanding of the physiological, anatomical, and biochemical differences 
among  terrestrial  vertebrates—particularly  in  terms  of  absorption,  metabolism,  toxicokinetics,  and 
toxicodynamics—remains  limited. Advancing  research  in  these  areas  could  support the  development  of 
more regionally tailored risk assessments, potentially accounting for local breeding patterns and migration 
behaviours.   

100 

 
 
Bird and mammal in vivo data are used to determine relevant endpoint values used in acute or chronic risk 
assessments. Under Regulation (EU) No 1107/2009, the risk assessment outcome for terrestrial vertebrates 
is based on a toxicity-exposure ratio (TER), which uses results from in vivo studies to establish the initial 
toxicity value—typically, the No Adverse Effect Level (NOAEL). One possibility to refine the risk could 
be to consider the ecological (population) relevance of laboratory observations or by applying mechanistic 
effects  knowledge,  such  as  those  derived  from  toxicokinetic-toxicodynamic  (TKTD)  models  or  other 
scientifically  justified  approaches,  although  currently  they  are  not  widely  used and/or  accepted. A TER 
above 10 for acute assessments is considered protective against visible mortality, while a TER above 5 for 
chronic  assessments  is  assumed  to  safeguard  against  long-term  impacts  on  population  abundance  and 
biodiversity. In chronic scenarios, the population is regarded as the ecological entity of concern.  

Beyond pesticide ERAs, data from laboratory or wild birds and mammals may occasionally be available 
for  ESAs  under  REACH.  However,  such  data  is  not  available  in  the  majority  of  cases.  The  REACH 
regulation requires ERA for predators, including birds and mammals, exposed via the terrestrial and aquatic 
food chains, referred to as ‘secondary poisoning’, in cases where a substance has potential to bioaccumulate 
and also a potential to cause toxic effects if accumulated in higher organisms. Usually, only mammalian 
toxicity information available from the human health risk assessment (not discussed further here) is used 
for such assessments. It is acknowledged in REACH guidance (Chapter R7c) that birds are fundamentally 
different from mammals in certain aspects of their physiology (e.g. the control of sexual differentiation, egg 
laying, etc.), and so mammalian toxicity data are of limited predictive value for birds. 

Bird and mammal testing specifically for ERA is not expected under EU regulations governing cosmetics, 
pharmaceuticals, or veterinary medicines. The use of animals for efficacy testing of regulated chemicals—
such as  human  or  veterinary medicines—is  considered  outside  the  scope  of  this  document.  In  addition, 
testing on target species is outside of the scope of the roadmap. 

A successful alternative test method should not require any new animal material—such as primary cells, 
plasma,  embryos,  or  unprotected  life  stages.  It  should,  either  alone  or  in  combination  with  other  tests, 
provide a level of protection that is equal to or greater than that provided by current approaches, and be 
suitable for regulatory decision-making.  

To  be  considered  successful,  the  method  must  undergo  suitable  validation  steps.  This  includes 
demonstrating that its biological and chemical applicability domains are well-defined, and that it performs 
reliably and reproducibly under testing conditions in a laboratory setting.  

While population-level effects are the ecological entity of concern, most in vivo studies primarily capture 
effects at the level of individual animals. To bridge this gap, computational models are already being used 
to support population-level effect assessments on a case-by-case basis. However, these models still face 
challenges in validation and currently have limited regulatory acceptance. Ongoing projects initiatives—
such as the PARC (Partnership for Assessment of Risk of Chemicals), SETAC Effect modelling interest 
group56 and the EFSA WG on effects models in ERA57—are actively working to advance the development, 
validation, and regulatory uptake of these tools. 

Progress  in  developing  animal-free  methods  to  predict  individual-level  effects  varies  across  the  five 
vertebrate classes. Significant advancements have been made in mammalian safety testing, largely driven 
by its relevance to human safety. In contrast, avian safety testing methods have seen limited innovation 
since the 1980s—though this is beginning to change. Within PARC various models are being developed for 
in total 18 domestic and wildlife birds (publication, in preparation), which might be used in screening and 
und in a weight of evidence, this document focuses specifically on these two terrestrial vertebrate classes. 

56 SETAC Working group: Model Acceptability and scenario Development (MAD) (https://www.setac.org/group/effect-
modeling/document.html?id=0332F6DE-F1F3-4621-A395E98FE6A9CE84). 
57 EFSA WG on effects models in ERA. Accessed via: https://open.efsa.europa.eu/working-group/300000037091043 

101 

 
 
 
Wild mammals  

For wild mammals (non-humans), individual-level effect assessments typically begin with the mammalian 
dataset originally generated for human safety evaluations. These datasets currently include in vivo studies 
addressing acute, chronic, developmental, and  reproductive outcomes. As in vivo tests are progressively 
replaced  by  non-animal  alternatives,  it  is  essential  to  carefully  assess  the  ecological  relevance  of  these 
replacement(s).  

Currently, no mammalian assays are in development specifically for ecological purposes that are not also 
intended for human safety assessment. However, assays developed for human health but later deemed to 
have limited human relevance may still hold value for ecological assessments. For example, the mouse 
embryonic  stem  cell  test  (Seiler  &  Spielmann,  2011)  may  be  considered  less  human-relevant  than  its 
human-derived counterpart (Gabdoulline et al., 2015), yet it could still be informative for ESA.  

To  support  progress  in  this  area,  continued  collaboration  between  stakeholders  focused  on  non-animal 
approaches for human safety and those working on wild vertebrate protection is strongly recommended.   

Wild birds 

Laboratory bird studies on wild birds typically investigate acute, chronic or reproductive outcomes. These 
studies are most commonly performed using the mallard duck (Anas platyrhynchos) and a quail (Coturnix 
japonica or Colinus virginianus), with other species such as canaries occasionally required outside Europe. 
As in vivo tests are gradually replaced by non-animal alternatives, it is essential to evaluate the ecological 
relevance of these replacement(s).  

The  core  in  vivo  test  methods  for  avian  toxicity  have  remained  largely  unchanged  since  their  initial 
publication  in  the  1980s. While  protocols  for  acute  testing  were  updated  in  OECD Test  Guideline  223 
(2016), they still rely on purpose-bred birds that do not survive the study. Currently, no non-animal studies 
are accepted by global regulatory authorities—excluding non-testing approaches such as read-across or data 
waiving,  if  agreed.  No  in  vitro  or  in  silico  assays  are  yet  sufficiently  advanced  to  serve  as  short-term 
replacements for existing in vivo models.  

However, promising developments are emerging:  

o  Commercial availability of avian cell line, such as chicken and quail for non-biomedical uses58 

o  Development of qPCR arrays using quail embryo hepatocytes (animal-based) (Crump et al., 

2023) 

o  Publication of the first avian physiologically based kinetics and bioenergetics models (Martin et 

al., 2024) 

o  The UK NC3Rs "Wings of Change" challenge (2024-2028), which funds the development of 

New Approach Methodologies (NAMs) to assess acute and chronic avian toxicity for chemical 
screening and environmental risk assessment59 

It is recommended to maintain awareness of these and other avian-specific developments, and to explore 
their potential for regulatory use. Additionally, leveraging cross-species research and concepts—such as 
virtual control groups, in vitro-in vivo extrapolation (IVIVE), and dynamic energy budget – toxicokinetic-

58 University of Edinburgh National Avian Research Facility https://vet.ed.ac.uk/roslin/engagement/facilities/national-avian-
research-facility/avian-resources 
59 UK National Centre for the replacement, refinement & reduction of animals in research (NC3Rs) CrackIT challenge 
https://nc3rs.org.uk/crackit/wings-change 

102 

 
 
 
 
toxicodynamic  models—can  accelerate  progress.  To  support  this,  effective  collaboration  between 
stakeholders  focussed  on  non-animal  approaches  for  all  wild  vertebrate  safety  should  be  actively 
encouraged and facilitated.  

Vision  
The  vision  centres  on  developing  and  implementing  non-animal  approaches  capable  of  predicting 
regulatory-relevant endpoints such as the acute LD50 and the chronic NOAEL. Achieving this requires a 
deep understanding of the biological effects observed in traditional (in vivo) studies and how they inform 
regulatory decisions, along with strategies to replicate or improve upon them using alternative approaches.  

Two complementary concepts have emerged to support this goal: the Adverse Outcome Pathway (AOP) 
framework, and an in vitro / in vivo testing strategy adapted from the human safety paradigm. These are 
briefly summarised below, with further details provided in the relevant Annex.  

The toxicology parallelogram adapted for ecotoxicology 

Figure 6:  a parallelogram view of how in vivo and in vitro data combine with ecological modelling 
to predict potential wildlife risk. 

For over a century, medical researchers and toxicologists have relied on laboratory animals as surrogates 
for humans, using in vivo animal studies to predict and extrapolate potential human health risks. Over time, 
the tools and methodologies available have evolved significantly.  

Today, emerging in vitro assays—both animal- and human-based—combined with advanced computational 
modelling, offer the potential to match or even surpass the predictive power of traditional animal tests for 
human risk assessment. This concept is visualised as a ‘parallelogram’ model, illustrating the relationship 
between different data sources and their predictive value.  

It is now proposed to adapt this parallelogram framework to illustrate how wildlife risk assessments could 
similarly benefit from the integration of diverse testing approaches. This adapted model would reflect the 
unique challenges and opportunities in predicting ecological effects using non-animal methods. 

The Adverse Outcome Pathway (AOP) framework  

103 

 
 
 
 
 
The Adverse Outcome Pathway (AOP) framework, first introduced by Ankley et al. (2010), was designed 
to  support  the  integration  of  emerging  mechanistic  data  into  ecological  risk  assessments.  It  provides  a 
structured approach to link molecular-level events to adverse outcomes that are meaningful for regulatory 
decision-making—specifically, effects on survival, development, and reproduction in individual organisms 
and, ultimately, impacts at the population level. 

Figure 7: The Adverse Outcome Pathway (AOP) concept illustrates how data from diverse test 
methods can be integrated to identify adverse effects relevant for risk assessment—specifically to 
protect wild bird populations from known chemical stressors. 

The  AOP  framework  anticipates  how  information  from  different  levels  of  biological  or  ecological 
complexity can be integrated to support regulatory decision-making. A central feature of this approach is 
the  identification  of  individual-level  endpoints  that  can  be  used  to  predict  population-level  responses 
through computational modelling.  

For a non-animal approach to be regulatory-relevant, it must generate the type of information needed by 
regulators. The AOP framework helps map how various types of biological data can be linked to a sub-
lethal adverse effect—such as those used to derive a NOAEL—or to lethality endpoints relevant for acute 
risk  assessments. To  optimise  this  approach,  current  scientific  knowledge  about toxicity  mechanisms  is 
being  compiled,  and  knowledge  gaps  are  being  identified.  This  results  in  a  plausible,  qualitative,  and 
chemically agnostic description of how different observations from in vivo studies are connected to adverse 
outcomes.  

However,  for  a  chemical-specific  risk  assessments,  qualitative  AOPs  are  not  sufficient.  Deriving  a 
numerical value equivalent to a NOAEL requires a quantitative AOP (qAOP), which incorporates chemical 
specific data for key events and parameters. Although several AOPs have emerged since their introduction 
to ESAs over 15 years ago, no validated qAOPs are currently available.  

To enable animal-free ESA, it is recommended that significant efforts be directed toward the development 
and  validation  of  relevant AOPs.  These  should  be  integrated  with  existing  and  emerging  ecologically 
relevant  computational  models  -  such  as  dynamic  energy-TKTD  modelling  -  to  support  their 
implementation in regulatory decision-making.   

104 

 
 
 
Optimising ecological realism in risk assessment 

Globally, there are approximately 11,000 bird species and 6,000 mammal species, with around 600 bird 
species  and  250  mammalian  species  found  in  Europe.  Despite  this  diversity,  current  global  testing 
requirements for chemical safety assessments typically involve only up to 4 mammalian species (rat, mouse, 
rabbit, dog) and one to three bird species (duck, quail, canary). Risk assessments rely on adjustment factors 
to extrapolate data from these few laboratory species to thousands of wild species. As ecological knowledge 
grows, the reliability of cross-species extrapolation continues to improve.  

In addition, even within a single species, individuals may inhabit diverse landscapes and be exposed to 
varying  stressors—chemical,  climatic,  or  ecological. To  enhance  the  relevance  of  environmental  safety 
assessments,  it  is  recommended  to  incorporate  species-specific  ecological  information,  including  life 
history  traits,  population  dynamics,  habitat,  and  landscape  context.  This  enables  more  accurate  and 
regionally tailored decisions at the country, zonal, or European level.  

While this recommendation may extend beyond the immediate scope of phasing out of animal testing for 
chemical safety assessments, it aligns with the overarching goal of the European Commission roadmap to 
protect wildlife using the best available science. Continued investment in ecological research and cross-
species extrapolation is essential to inform these assessments and to monitor their effectiveness as non-
animal test methods are adopted. 

Recommendations  
Currently, only a limited number of ready-to-implement animal-free approaches exist to fully replace  in 
vivo  testing  with  birds  and  mammals.  The  process  of  developing  the  roadmap  has  helped  identify  key 
methodological development gaps that, if addressed, could support the future phase-out of animal testing.  

The  main  recommendation  is  to  establish  a  structured,  collaborative  process—within  the  organisational 
framework implementing the roadmap and in partnership with stakeholders such as EPAA and the research 
community—to systematically work toward the goal of phasing out avian studies for regulatory purposes.  

Priority actions: 

•  Develop AOP models: Focus on regulatory-relevant effects by building AOPs that establish 

causal links between molecular or cellular data and adverse outcomes. Progress toward qAOPs to 
enable derivation of endpoint values for risk assessment.  

•  Leverage advances from human safety science: Apply and adapt concepts already in development 

for human toxicology, including: 

In vitro-in vivo extrapolation (IVIVE),  

o  Virtual control groups,  
o 
o  Dynamic energy budget-toxicokinetic-toxicodynamic modelling, 
o  qAOPs. 

These approaches should be applied across and between all vertebrate classes, wherever 
scientifically justified. 

•  Fund targeted research for ecological relevance: Support studies to identify key bird and mammal 

species for regulatory focus, including: 

o  Species-specific traits and population dynamics, 
o  Cross-species extrapolation methods, 
o 

Integration of habitat and landscape-level data to inform species prioritisation at national, 
zonal, or EU level 

105 

 
 
 
References 

Ankley GT, Bennett RS, Erickson RJ, Hoff DJ, Hornung MW, Johnson RD, Mount DR, Nichols JW, Russom CL, 
Schmieder PK, Serrrano JA, Tietge JE, Villeneuve DL. Adverse outcome pathways: a conceptual framework 
to support ecotoxicology research and risk assessment. Environ Toxicol Chem. 2010 Mar;29(3):730-41. doi: 
10.1002/etc.34. PMID: 20821501. 

Article  1  of  Directive  2010/63/EU  of  the  European  Parliament  and  of  the  Council  of  22  September  2010  on  the 
https://eur-lex.europa.eu/legal-

protection 
content/EN/TXT/PDF/?uri=CELEX:32010L0063 accessed February 2025. 

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Crump D, Hickey G, Boulanger E, Masse A, Head JA, Hogan N, Maguire S, Xia J, Hecker M, Basu N. Development 
and  Initial  Testing  of  EcoToxChip,  a  Novel  Toxicogenomics  Tool  for  Environmental  Management  and 
Chemical Risk Assessment. Environ Toxicol Chem. 2023 Aug;42(8):1763-1771. 

EFSA (European Food Safety Authority), Aagaard A, Berny P, Chaton PF, Antia AL, McVey E, Arena M, Fait G, 
Ippolito A, Linguadoca A, Sharp R, Theobald A and Brock T, 2023. Guidance on the risk assessment for 
Birds  and  Mammals.  EFSA  Journal  2023;  21(2):7790,  300  pp.  https://doi.org/10.2903/j.efsa.2023.7790. 
https://doi.org/10.2903/j.efsa.2023.7790 

EFSA.  2014.  Scientific  Opinion  on  good  modelling  practice  in  the  context  of  mechanistic  effect  models  for  risk 
assessment of plant protection products EFSA Panel on Plant Protection Products and their Residues (PPR). 
European Food Safety Authority (EFSA), Parma, Italy. EFSA Journal 2014;12(3):3589. 

Gabdoulline R, Kaisers W, Gaspar A, Meganathan K, Doss MX, Jagtap S, Hescheler J, Sachinidis A, Schwender H. 
Differences in the Early Development of Human and Mouse Embryonic Stem Cells. PLoS One. 2015 Oct 
16;10(10):e0140803. 

Kramer VJ,  Etterson  MA,  Hecker  M,  Murphy  CA,  Roesijadi  G,  Spade  DJ,  Spromberg  JA, Wang  M, Ankley  GT. 
Adverse  outcome  pathways  and  ecological  risk  assessment:  bridging  to  population-level  effects.  Environ 
Toxicol Chem. 2011 Jan;30(1):64-76. doi: 10.1002/etc.375. PMID: 20963853.  

Martin T, Bauer B, Baier V, Paini A, Schaller S, Hubbard P, Ebeling M, Heckmann D, Gergs A. Reproductive toxicity 
in birds predicted by physiologically-based kinetics and bioenergetics modelling. Sci Total Environ. 2024 
Feb 20;912:169096. 

Perkins,  E.J., Ashauer,  R.,  Burgoon,  L.,  Conolly,  R.,  Landesmann,  B.,  Mackay,  C.,  Murphy,  C.A.,  Pollesch,  N., 
Wheeler,  J.R.,  Zupanic,  A.  and  Scholz,  S.,  2019.  Building  and  applying  quantitative  adverse  outcome 
pathway models for chemical hazard and risk assessment. Environmental toxicology and chemistry, 38(9), 
pp.1850-1865. 

Schmolke A,  Thorbek  P,  DeAngelis  DL,  Grimm  V.  2010.  Ecological  models  supporting  environmental  decision 

making: A strategy for the future. Trends Ecol Evol 25:479–486. 

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interpretation of comparative in vitro metabolism studies | EFSA 

Seiler, A., Spielmann, H. The validated embryonic stem cell test to predict embryotoxicity in vitro. Nat Protoc 6, 961–

978 (2011).  

The validated embryonic stem cell test to predict embryotoxicity in vitro | Nature Protocols 

106 

 
 
 
3.6. Ecological thresholds of toxicological concern and species 
sensitivity distributions 

Background 
Ecological thresholds of toxicological concern (ecoTTC) and species sensitivity distributions (SSDs) are 
both  probabilistic  methods  that  use  mathematical  models  to  identify  chemical  concentration  thresholds 
below  which  a  minimal  probability  of  negative  effects  on  environmental  communities  is  expected. 
Deterministic  hazard  assessment  approaches  often  utilized  in  regulatory  schemes  determine  protective 
thresholds via the use of individual hazard data points (i.e., the most sensitive acute or chronic toxicity 
value)  and  uncertainty  factors  to  generate  predicted  no  effect  concentrations  (PNECs)  for  individual 
chemicals. Compared to that probabilistic methods such as ecoTTC and SSD leverage the full breadth of 
existing data and allow to easily integrate newly generated data.  

If sufficient data for deriving them are available, ecoTTCs and SSDs (as well as other probabilistic hazard 
assessment  approaches  such  as  chemical  toxicity  distributions)  are  robust  methods  that  can  be  used  to 
minimize the need for animal data when assessing chemical safety. The approaches can also be used to 
analyse the value of generating new data (e.g., fish toxicity data) for refining the assessment (i.e., identified 
protective thresholds). Thus, not only can new animal testing be avoided to safely support chemical use, 
but  the  value  in  additional  testing  can  be  quantified.  Integrating  these  approaches  in  chemical  safety 
assessments serve as a tool to reduce animal testing. 

Recommendations 
Medium-term actions 

•  Harmonisation of methods and applied criteria, improvement of methods 

o  For  SSD:  Harmonisation  is  needed  on  the  selection  and  use  of  assessment  factors  in 

addition to the x percentile of hazardous concentrations (HCx). 

o  For ecoTTC: Acceptable criteria for chemical grouping need to be defined in guidance.  
o  For ecoTTC: Comparisons against reference data are needed to characterize accuracy and 

to determine how conservative are thresholds determined with ecoTTC. 

•  Better integration of approaches in the chemical safety assessment framework 

o  Guidance is needed to clarify how to use the approaches in chemical safety assessment. 
Clarity and acceptance is needed regarding broader utilization of available data (e.g., acute 
toxicity data, non-standard species). 

o  Probabilistic hazard assessment  should be built into core tools  used for chemical safety 

assessments such as the OECD Toolbox. 

Ecological Thresholds of Toxicological Concern (EcoTTCs)  
The  ecoTTC  is  an  approach  for  identifying  a  single  environmental  concentration  below  which  adverse 
effects  on  environmental  communities  are  expected  to  be  minimal  for  a  group  of  toxicologically  or 
chemically similar substances. This method supports reduction in animal testing by leveraging existing data 
and minimizing the need for new ecotoxicity studies. Free software is available to support calculations of 
ecoTTCs. 

The  ecoTTC  is  a  threshold  concentration  using  a  selected  percentile  (often  the  5th  percentile)  of  the 
probability  distribution  of  Predicted  No  Effect  Concentrations  (PNECs)  derived  from  acute  or  chronic 
toxicity data for similar chemicals. This is a conservative approach that identifies a hazardous concentration 

107 

 
 
 
 
threshold  that  is  determined  based  on  a  distribution  of  concentrations  at  which  no  toxicity  has  been 
identified. Data leveraged are from chemicals that share relevant characteristics to the substance or chemical 
class  of  interest  (e.g.,  mode-of-action,  physicochemical  properties)  ensuring  that  the  derived  hazardous 
concentration is fit for purpose. Inclusion of data based on quality and appropriateness for grouping must 
be individually considered. 

Because  of  the  use  of  grouped  data,  the  ecoTTC  approach  is  valuable  in  providing  screening-level 
assessments for chemicals with minimal toxicity data and may be able to provide insight for more complex 
assessments (e.g., UVCBs). However, it does require the availability of robust, chemical-specific hazard 
data  with  sufficient  taxonomic  coverage  to  generate  high-quality  PNECs.  Additionally,  it  is  likely  a 
conservative approach given the inclusion of uncertainty factors in the derivation of PNECs. A similar, but 
alternative, approach that evaluates a distribution of effect concentration values without uncertainty factors 
– known as a chemical toxicity distribution (CTD) – might be more relevant when considering specific 
species or trophic levels, particularly if one trophic level is uniquely sensitive. 

The  ecoTTC  method  is  not  yet  leveraged  in  regulatory  frameworks  for  environmental  assessments. 
Opportunities  for  improving  robustness  include  the  need  for  broader  toxicity  data  on  non-standard  test 
organisms,  better-defined  criteria  for  chemical  grouping,  and  validation  through  comparisons  with 
reference data to characterize the method's accuracy. 

Species Sensitivity Distributions (SSDs) 
An SSD is an approach for representing differential sensitivity to a single toxicant across environmental 
species.  Similar  to  ecoTTC,  it  can  be  leveraged  to  identify  protective  chemical  environmental 
concentrations  beneath  which  a  minimal  probability  of  negative  effects  on  environmental  communities 
would be expected. However, in this case the focus is the hazard potential of an individual chemical. If 
sufficient data is available, analysis can be quickly done via freely available software and open-source tools. 

Protective thresholds identified using SSDs are also usually based on the 5th percentile, known as the 5th 
percentile  hazardous  concentration  (HC5),  though  the  probability  distribution  is  based  on  effect 
concentration values derived from acute or chronic environmental toxicity data across various species, not 
PNECs. SSDs are considered to be conservative and robust approach for assessing environmental hazards 
of individual chemicals.  

A drawback is that SSDs require a significant amount of data across several taxonomic groups to represent 
the  potential  diversity  of  sensitivity  to  a  toxicant  in  the  environment.  Hence,  the  method  will  only  be 
applicable  for  reduction  of  animal  testing  for  those  substances,  for  which  data  from  several  taxonomic 
groups  are  already  available.  Global  requirements  for  utilizing  SSDs  in  regulatory  toxicology  require 
vertebrate data (i.e., fish) to be included as part of the taxonomic coverage. Without consideration of data 
generated by non-standard species or guideline assays, an SSD can be difficult to construct. For example, 
an SSD cannot be generated based on data requirements for chemical registration under REACH alone.  

SSDs are used in some capacity for chemical safety assessment in various global regulatory frameworks, 
including the EU. However, the approaches are not harmonized. Additionally, the value of effort in these 
higher-tier approaches is not visible in current chemical safety requirements and guidance, thus creating 
challenges for implementation. Clarity on broader utilization of data (e.g., acute toxicity data, non-standard 
species) and use of application factors in addition to HCx (often removing the value of SSD assessment 
versus standardized deterministic approaches) represent some of the immediate needs for increased use. 

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4. Recommendations and actions for human health assessments 

4.1. Acute toxicity (human health assessment) 

Replacement of in vivo testing with in-silico methods: pilot project on acute oral 
toxicity 
Introduction 

This section describes a strategy and stepwise approach for replacing in vivo testing in animals to determine 
the acute oral toxicity (AOT) of chemicals with in silico methods60.  

As part of the work in the Human Health Roadmap Working Group (HH WG), an initiative has been started 
by stakeholders including the European Chemicals Agency (ECHA), the United States National Toxicology 
Program  Interagency  Center  for  the  Evaluation  of Alternative Toxicological  Methods  (NICEATM),  the 
German Federal Institute for Risk Assessment (BfR), the European Partnership for Alternatives to Animal 
Testing  (EPAA)  and  the  European  Food  Safety Authority  (EFSA)  to  evaluate  the  feasibility  of  such  a 
replacement. The  strategy includes legal  analysis,  performance  benchmarking,  evaluation  of  existing  in 
silico methods and a phased approach to regulatory integration. 

As a pilot, the activity is also meant to serve as a template for similar, possibly more complex cases in the 
future by 

- 

- 

- 

exploring the legal options for the use of in silico methods instead of in vivo testing for AOT assessment 
and to this end, the need for a corresponding adaptation of legal texts or guidance; 
implementing  a  first,  selected  in  silico  method  for  that  purpose;  further  methods  can  then  be 
implemented based on the experiences gained; 
exploring  and,  where  necessary,  proposing  adaptations  to  the  legal  text  and/or  guidance  to  enable 
options for making in silico methods the default approach for AOT assessment. 

Below, these steps are elaborated in more detail for two Regulations, i.e. Regulation (EC) 1272/2008 (CLP) 
and Regulation (EC) 1907/2006 (REACH). However, following initial discussions, the activity is meant to 
be extended to all other legislations in the scope of the Roadmap, which require61 – or use the results from 
- AOT testing.  

As the strategy will explore the use of already existing in silico tools, it addresses “basket” 1 (short-term 
actions) of the roadmap. 

Approach 

The stepwise approach followed in this activity can be summarised as follows: 

1.  Determine the generic regulatory needs and requirements with respect to AOT assessment. 

60 For this document, the term “in silico tool” refers to a computer model, or suite of models, available in the form of executable 
software and used to predict physico-chemical, toxicokinetic or toxicodynamic properties of chemicals. In contrast, the term “in 
silico method” refers to a conceptual approach for assessing such properties in the context of chemical risk assessment utilising 
one or more specific in silico tools. 
61 Of note, this activity is not applicable to the human pharmaceutical sector where conventional acute toxicity studies are no 
longer required (ref ICH M3(2)). For these products relevant information can be obtained from other toxicity studies carried out 
as part of the drug development program. Also for veterinary medicinal products it is recommended to derive toxicological 
reference values from other study types and waiving of acute oral toxicity studies is usually accepted. Especially data of LD50 
studies are of limited value and are normally not accepted as single basis for quantitative risk assessment 
(EMA/CHMP/CVMP/3Rs/164002/2016 Rev. 1). 

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2.  For each specific legislation: determine whether the legislation itself or associated guidance needs to 

be updated, or whether additional guidance needs to be developed to  

a) allow for the replacement of in vivo AOT testing with in silico methods in general and  

b) make in silico methods the default option for AOT characterisation under the specific legislation. 

3.  Develop generic performance/acceptability criteria and benchmarks for AOT in silico methods to 

meet in order to establish scientific and regulatory confidence in using these methods. 

4.  For  each  specific  in  silico  method  considered:  establish  scientific  confidence,  including  a 
characterisation of the method’s applicability domain and whether and how it meets the benchmarks 
under 3. 

5.  Formulate an overall implementation plan incl. action items, milestones and timelines, taking into 

account any open questions identified under steps 1 – 4. 

Regulatory needs and requirements 

In vivo AOT testing for chemical safety in the EU is currently performed acc. to OECD Test Guidelines 
(TGs) 420 (Acute Oral Toxicity - Fixed Dose Procedure), 423 (Acute Oral toxicity - Acute Toxic Class 
Method) or 425 (Acute Oral Toxicity: Up-and-Down Procedure). In addition, historical data generated by 
using  the  now  obsolete  OECD  TG  401  (Acute  Oral  Toxicity)  are  used  for  AOT  assessments,  where 
available.  These  methods  serve  to  determine  a  so-called  Acute  Toxicity  Estimate  (ATE),  i.e.  a 
(semi-)quantitative estimate of the single dose or a dose / dose range (in mg kg/bw), such as the LD50 
(lethal dose for 50% of the animals tested at that dose level) characterising the acute toxicity of a substance 
or mixture when administered orally to rodents. 

Need for updating existing legislation or guidance 

CLP Regulation 

Use of in silico tools for AOT assessment in general 

Under CLP, in silico tools may be used for AOT classification, on condition that 

- 
- 

they are scientifically valid (plausible, reliable, sufficiently predictive) and 
they  provide ATEs  suitable  for  deciding  on  placing  –  or not  –  chemicals into  one  of  the  CLP AOT 
categories.  

No  changes  of  the  CLP  legal  text  appear  necessary  to  allow  for  using  in  silico  methods  for  AOT 
classification and labelling, provided their adequacy and reliability is demonstrated. It should be considered, 
however, to remove explicit (albeit non-prescriptive) reference to in vivo methods from the text and to add 
the possibility to use in silico tools directly. 

In addition, once the adequacy and reliability of specific in silico methods has been agreed upon, it would 
seem helpful to perform (minor) updates of the CLP guidance document, explaining their applicability as 
well as potential caveats, if any. 

Make in silico tools the default option for AOT assessment 

To make the use of in silico methods the default approach for classification and labelling under CLP would 
require an update of Chapter 3.1 of the United Nations’ Globally Harmonised System and the corresponding 
Chapter 3.1 in Annex I of the CLP Regulation. 

REACH 

110 

 
 
With respect to REACH, no further requirements in addition to those already mentioned in the previous 
section on CLP have been identified. 

Use of in silico tools for AOT assessment in general 

No  changes of the  REACH  legal text appear  strictly  necessary to allow for  using  results  from  in  silico 
methods for AOT classification and labelling and/or risk assessment, provided their adequacy and reliability 
is demonstrated. It should be considered, however, to review and, where necessary, revise the respective 
REACH  guidance  document  on  Information  Requirements/Chemical  Safety  Assessment  (IR/CSA), 
Chapter  R.7a,  to  remove  explicit  (albeit  non-prescriptive)  reference  to  in  vivo  methods  and  to  add  the 
possibility to use in silico tools directly. 

In addition, once the adequacy and reliability of specific in silico methods has been agreed upon, it would 
seem helpful to perform a (minor) update of this part of the REACH guidance, explaining their applicability 
as well as potential caveats, if any. 

Make in silico tools the default option for AOT assessment 

To  make  in  silico  methods  the  default  approach  for AOT  characterisation  under  REACH,  an  update  of 
Annex VII of Regulation (EC) No 1907/2006 would be required. 

Other regulations in the scope of the COM roadmap  

Although the analysis of further regulations is pending, it is not expected that data from existing AOT in 
vivo tests are used in a significantly different way in other EU chemical legislations as compared to REACH 
and CLP. 

Scientific and regulatory confidence in in silico methods for AOT assessment 

To  evaluate  whether  in  silico  methods  can  be  trusted  scientifically,  their  adequacy  and  reliability  for 
providing  reliable  estimates  of  AOT  has  to  be  assessed.  The  QSAR  Assessment  Framework  (QAF), 
published by the Organisation for Economic Co-Operation and Development (OECD) in 2023, offers a 
number of Assessment Elements (AEs) for this purpose, as well as for assessing the reliability of predictions 
made using these models. 

However, the QAF itself does not provide performance benchmarks that have to be met by an  in silico 
method in order to be acceptable for a given regulatory purpose. 

Assuming a specific in silico method has been found scientifically acceptable in general, its suitability in 
regulatory terms62 is mainly determined by its applicability domain (AD) and predictive performance. In 
the context of replacing an existing in vivo test method, a comparative element is added, as expressed by 
the following questions: 

-  To what extent does the AD of the in silico method cover (or perhaps even exceed) that of the in vivo 
method to be replaced? Are there sectors of chemical space for which the use of the in silico method is 
not possible or advisable? 
Is the predictive accuracy of the in silico method, i.e. the degree to which the in silico method is capable 
of reproducing the results of the in vivo test, acceptable? 

- 

The assessment of predictive performance first requires the development of generic acceptance criteria. In 
other words, for AOT assessment, the question has to be answered what percentage of misclassifications 
vs. the previous method or what degree of deviation of the predicted ATE vs. the “true” ATE, e.g. a measured 

62 In cases where a 3R’s compliant methodology is considered to be used in regulatory testing in the pharmaceutical sector, the 
developer can apply for qualification advice or a qualification opinion through EMA’s Scientific Advice Working Party. EMA 
also offers early interactions to NAM developers via the Innovation Task Force. 

111 

 
 
 
LD50 value, is considered acceptable. Considerations regarding e.g. what degree of deviation was accepted 
when introducing OECD TGs 420, 423 and 425 to replace the previous, now obsolete OECD TG 401, as 
well as an analysis of variability in the outcomes from multiple AOT tests available for the same substance, 
can help in establishing such benchmarks. 

In principle, generic benchmarks developed in the context of CLP (for both, predicting ATE values/ranges 
or classifications) would also pertain to REACH, as far as classification and labelling are concerned. In the 
rare cases, where ATEs are used as PoDs for risk assessment, no additional benchmarks would seem to be 
required on first thought.  

Confidence in specific in silico methods 

As the most suitable in silico tool to be investigated by this pilot first, a specific in silico modelling suite, 
CATMoS, has been selected, because it 

a) represents a consensus suite of a variety of models developed in different regions of the world and  

b) has already undergone extensive prevalidation.  

It is expected that, once generic benchmarks have been defined and the performance of CATMoS has been 
assessed, other available models can be added with comparatively little extra effort. 

The Collaborative Acute Toxicity Modelling Suite (CATMoS) 

Description of the method 

Following  a  workshop  in  2018  (Kleinstreuer  et  al.,  2018),  the  U.S.  National  Toxicology  Program 
Interagency Center for the Evaluation of Alternative Toxicological Methods (NICEATM), together with 
many  international  collaborators,  has  built  a  suite  of  in  silico  tools  under  the  name  of  CATMoS 
(Collaborative Acute  Toxicity  Modelling  Suite).  CATMoS  is  part  of  the  in  silico  suite  OPERA  (OPEn 
structure–activity/property Relationship App) (Mansouri et al., 2018). OPERA is available as a standalone 
tool, but also as a Konstanz Information Miner (KNIME) workflow, a plugin for the OECD QSAR Toolbox 
and as part of NICEATM’s Integrated Chemical Environment (ICE) platform. 

Adequacy of the method for the intended purpose 

Where applicable, the CATMoS suite produces both, a predicted ATE value in the form of an LD50 estimate 
and a prediction of the appropriate GHS/CLP Acute Tox. sub-category. Therefore, it can be concluded that, 
in principle, CATMoS provides the acute toxicity outputs typically required by EU chemicals legislation. 

Reliability and accuracy of the method for the intended purpose 

The predictive performance of the CATMoS tool has been broadly investigated by Mansouri et al. (2021) 
based  on  a  dataset  of  >  11 000  chemicals. Another  evaluation  with  a  specific  focus  on  pesticides  was 
recently published by Bishop et al. (2024). 

Whether or not the predictive performance of the method is considered sufficient to replace the current in 
vivo  test  methods  under  EU  chemicals  legislation  remains  to  be  ascertained  based  on  an  independent 
assessment and the generic performance benchmarks yet to be developed.  

In addition, an analysis of the coverage of chemical space should be performed in order to describe the AD 
of the method and to identify sectors of chemical space, if any, where the predictivity of the method might 
not fulfil regulatory expectations. To some degree, such investigations have already been performed in the 
two publications cited above as well as in a recent publication by industry (Weyrich et al., 2025). 

Implementation 

112 

 
 
Based on the considerations above, it is proposed to implement this strategy in two phases. 

Preparatory phase 

A small informal working group including experts from ECHA, EFSA, NICEATM, BfR, industry and the 
European Commission has been set up in January 2024 to perform the following tasks: 

- 

Investigate the composition and quality training and test data sets which have been used to pre-
validate CATMoS 

-  Broaden the test data set with additional curated data from regulatory processes which were not 

- 

- 

known to the developers of CATMoS (e.g. from ECHA’s REACH database and EFSA’s openfoodtox 
database) (Dorne et al., 2021; Patlewicz et al., 2024). 
propose suitable predictivity benchmarks for the prediction of ATE values/ranges, LD50 values, and 
acute toxicity categories based on the performance of the existing in vivo methods and to 
analyse adequacy and reliability of the CATMoS model using the OECD QAF and assess its 
predictivity against the proposed benchmarks, incl. an estimate of the percentage of substances 
(within the chemical space relevant for REACH) for which the CATMoS model would provide 
reliable predictions (according to the benchmarks defined above), taking into account the pre-
validation work done already in Mansouri et al. (2018) and Bishop et al. (2024). 

Preliminary results  

ECHA has started an independent evaluation of AOT data similar to the work described by Karmaus et al. 
(2022), with the objective of establishing an AOT reference data set for LD50 values based on data from 
REACH registrations, quantification of the variability between these values and performing a preliminary 
analysis  of  performance  of  the  CATMoS  model  suite.  In  this  context,  also  general  technical  questions 
around building a reference data set, such as handling multiple data points per reference substance, are 
addressed with a view to extract learnings also relevant for further endpoints later in the roadmap work. 
Preliminary results indicate that acceptable (from a technical point of view, i.e. not necessarily correct) 
CATMoS predictions can be obtained for the bulk of REACH registration data, raising hope that, acceptable 
predictivity assumed, CATMoS may be suitable for replacing AOT in vivo testing for a significant part of 
REACH-relevant chemical space. 

Future work will then integrate the learnings from this exercise with that from other recent publications 
such as Bishop et al. (2024) or Weyrich et al. (2025). On these grounds, a first proposal for benchmark 
criteria will be developed which can then be carried over to the implementation phase. 

Implementation phase  

Once the preparatory work on performance benchmarks and the performance assessment of the CATMoS 
suite  will  be  completed,  which  is  expected  to  happen  in  the  course  of  2025,  the  process  of  regulatory 
implementation should be initiated. The table below provides a non-exhaustive list of ideas for actions that 
should be taken in this context, along with proposed main actors as well as third parties who should be 
consulted in the process.  

Action 
Technical expert group to 
continue work on performance 
benchmarks and performance 
assessment. 

Main actors 
ECHA, NICEATM, BfR, EPAA, 
EFSA, further parties have 
indicated their interest to join 

To consult with 
European Partnership for the Assessment of 
Risk s from Chemicals (PARC) 

It is anticipated that after a first proposal for 
benchmarks and a first assessment of 
CATMoS’ performance become available, 
these preliminary results will be discussed 

113 

 
 
 
 
Action 

Main actors 

To consult with 
with the OECD QSAR expert group (and, 
possibly, further OECD expert groups). 

Confirm the interpretation and 
analysis regarding the CLP and 
REACH legal texts and guidance 
as presented above with experts 
for classification & labelling. 
Perform analogous analyses for 
all other legislations in scope of 
the COM roadmap.  

European Commission and ECHA  Meeting of the Competent Authorities for 
REACH and CLP (CARACAL), ECHA’s 
Risk Assessment (RAC) and Member State 
Committees (MSC) 

COM and responsible European 
agencies (e.g. ECHA for biocidal 
products, EFSA for food-related 
regulations,63 etc.  

Relevant risk assessment and risk 
management bodies or committees, e.g. 
SCCS for cosmetics64, EFSA Scientific 
Committee for food-related legislation etc., 
ECHA’s Biocidal Products Committee etc., 
possibly also the OSOA65 working group. 
Various stakeholder representatives: 
academia, industry, NGOs and policymakers 

Consult with stakeholders. 

To be determined based on the 
organisational structure that will be 
established for  

Once this initial implementation phase has been concluded and the general acceptability of in silico methods 
as well as the availability of at least one suitable specific method has been ascertained, the above-mentioned 
minor updates of the REACH and CLP guidance documents, as well as to other legislation and guidance in 
the scope of the COM roadmap, where necessary, should be initiated to promote the use of in silico methods 
for AOT assessment as soon as possible. Possible ways to already promote use of in silico methods even 
while the official guidance has not yet been updated should be explored. 

In parallel, the necessary steps to initiate updates to the GHS, CLP and as well as other pieces of legislation 
in  the  scope  of  the  roadmap  in  order  to  implement  in  silico  methods  as  the  default  approach  could  be 
explored. Contact with regulators and stakeholders, including relevant working groups (e.g. at UN GHS 
level, the Informal Working Group on Non-Animal Test Methods, IWG NATM) will be made at an early 
time-point to receive feedback and buy-in. 

Outlook 

For the moment, the scope of this activity encompasses AOT assessment of substances as well as mixtures, 
following a component-based approach66. It remains to be explored to what extent also acute toxicity via 
the  dermal  and  inhalation  routes  can  be  covered  with  the  available  in  silico tools  and  whether  this  can 
already be included in the present activity or remains to be addressed separately. 

Further future challenges include handling substances with multiple or inconsistent LD50 values, extending 
methods  to  acute  dermal  and  inhalation  toxicity,  and  ensuring  coverage  of  out-of-domain  chemicals, 
including inorganics. Broader integration of additional models beyond CATMoS is also planned. 

64 Although the Cosmetic Products Regulation (EC) 1223/2009 (CPR) prohibits new in vivo testing in vertebrates, still further 
discussions about the acceptability of in silico methods in general, or of specific in silico tools in particular, might be required. 
65 One Substance, One Assessment 
66 Component-based approaches aim at determining mixture properties based on the properties of the mixture’s individual 
constituents, as opposed to a whole mixture approach, which can be followed with the in vivo test, but is not compatible with in 
silico methods. 

114 

 
 
 
 
 
References 

Bishop, P. L., Mansouri, K., Eckel, W. P., Lowit, M. B., Allen, D., Blankinship, A., Lowit, A. B., Harwood, D. E., 
Johnson, T., & Kleinstreuer, N. C. (2024). Evaluation of in silico model predictions for mammalian acute 
oral toxicity and regulatory application in pesticide hazard and risk assessment. Regulatory Toxicology and 
Pharmacology, 149, 105614. https://doi.org/10.1016/j.yrtph.2024.105614  

Dorne  JLCM,  Richardson  J,  Livaniou A,  Carnesecchi  E,  Ceriani  L,  Baldin  R,  Kovarich  S,  Pavan  M,  Saouter  E, 
Biganzoli F, Pasinato L, Zare Jeddi M, Robinson TP, Kass GEN, Liem AKD, Toropov AA, Toropova AP, 
Yang  C, Tarkhov A,  Georgiadis  N,  Di  Nicola  MR,  Mostrag A, Verhagen  H,  Roncaglioni A,  Benfenati  E, 
Bassan A. (2021). EFSA's OpenFoodTox: An open source toxicological database on chemicals in food and 
feed and its future developments. Environ Int. 146:106293. doi: 10.1016/j.envint.2020.106293.  

Karmaus, A. L., Mansouri, K., To, K. T., Blake, B., Fitzpatrick, J., Strickland, J., Patlewicz, G., Allen, D., Casey, W., 
&  Kleinstreuer,  N.  (2022).  Evaluation  of  Variability Across  Rat Acute  Oral  Systemic  Toxicity  Studies. 
Toxicological Sciences, 188(1), 34-47. https://doi.org/10.1093/toxsci/kfac042  

Mansouri,  K., Karmaus, A. L., Fitzpatrick, J., Patlewicz,  G., Pradeep, P., Alberga, D., Alepee, N., Allen, T. E. H., 
Allen,  D.,  Alves,  V.  M.,  Andrade,  C.  H.,  Auernhammer,  T.  R.,  Ballabio,  D.,  Bell,  S.,  Benfenati,  E., 
Bhattacharya, S., Bastos, J. V., Boyd, S., Brown, J. B.,  Kleinstreuer, N. C. (2021). CATMoS: Collaborative 
Acute Toxicity Modeling Suite. Environ Health Perspect, 129(4), 47013. https://doi.org/10.1289/ehp8495  

Patlewicz G, Karamertzanis P, Friedman KP, Sannicola M, Shah I.(2024) A systematic analysis of read-across within 

REACH registration dossiers. Comput Toxicol. 30:1-15. doi: 10.1016/j.comtox.2024.100304.  

Weyrich, A., Peter, N., Watzek, N., Michael, S., Lauer, B., Zwinkmann, J., & Teubner, W. (2025). Can acute oral in 
vivo toxicity testing for EU REACH be fully replaced by a QSAR method? Evaluation of the CATMoS model 
and  Pharmacology,  162,  105861. 
using 
https://doi.org/https://doi.org/10.1016/j.yrtph.2025.105861  

industry  data.  Regulatory  Toxicology 

chemical 

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4.2. Genotoxicity 

Background 
Genotoxicity  is  considered  an  important  toxicological  effect  in  the  safety  evaluation  of  chemicals  and 
pharmaceuticals. It encompasses three endpoints: gene mutation, clastogenicity (structural chromosomal 
aberrations), and aneugenicity (chromosome loss or gain). Please see the ‘Definitions’ section below for 
more detailed descriptions. Assessment of genotoxicity for regulatory purposes usually involves a tiered 
approach, starting with a standard battery of in vitro mutagenicity tests, followed by in vivo testing on the 
same mutagenicity endpoint in case of a positive in vitro test result (Cimino, 2006; Dearfield et al., 2011; 
Eastmond et al., 2009). Generally, the conclusion from this approach is limited to hazard identification: it 
relies on a yes/no binary decision. Characterisation of the genotoxic hazard using quantitative analyses has 
been  demonstrated  to  be  feasible  and  possibly  is  of  added  value  for  risk  assessment  of  chemicals  and 
pharmaceuticals (Beal et al., 2023; Chepelev et al., 2023; Luijten et al., 2020; Nicolette et al., 2021; White 
et al., 2020); however, this is not yet a standard requirement in the regulations currently in use, as several 
open questions and uncertainties remain to be addressed (Sachse et al., in preparation). 

The current standard battery of in vitro genotoxicity tests is rather consistent across different sectors and 
different  regulatory  jurisdictions.  Commonly  it  includes  a  bacterial  reverse  gene  mutation  test,  a 
mammalian  cell  gene  mutation  test,  and/or  a  mammalian  cell  chromosomal  aberration  test  (European 
Commission, 2008, 2009, 2013; ICH, 2008) or a bacterial reverse gene mutation test and a mammalian cell 
micronucleus test (EFSA Scientific Committee, 2011; SCCS, 2023). The performance of the battery for 
detecting  genotoxic  potential  has  been  evaluated  for  a  large  set  of  chemicals  that  are  known  to  be 
carcinogenic and/or to induce genotoxic effects in rodents (Kirkland et al., 2011). The results of this analysis 
indicated that  a  combination  of  the  bacterial  reverse  gene  mutation  test, i.e. the Ames test (OECD Test 
Guideline (TG) 471), and the in vitro mammalian cell micronucleus test (MNvit; OECD TG 487), would 
be sufficient to reliably predict genotoxic potential of chemicals. The MNvit was preferred over the in vitro 
mammalian chromosome aberration test (OECD TG 473), because the MNvit is capable of detecting both 
clastogenicity  and  aneugenicity (while  the  in  vitro mammalian  chromosome  aberration test  is  primarily 
suited for detecting clastogenicity). 

In the context of the European Commission's roadmap for phasing out animal testing for chemical safety 
assessments, a subgroup of the Human Health working group experts convened and discussed how in vivo 
testing for genetic toxicity could be minimised, whether classification and hazard or risk assessment would 
be possible based on in vitro outcomes only, and whether there is potential for harmonisation of testing 
requirements across different legislations in scope of the roadmap. The authors explicitly chose to keep the 
discussions at a relatively high level of abstraction. The reason for doing so is that genotoxicity is a very 
severe health effect. Many, if not all, of the proposed actions require detailed knowledge and extensive 
discussions, often complemented with in-depth analyses of large datasets, to ensure that modified or entirely 
new approaches for addressing genotoxicity still safeguard public health. Below, five different areas for 
potential changes are described with a brief summary of the current situation and the main opportunities 
for reducing animal testing, plus proposed actions for the short-, medium- and long-term. 

EPAA Team of experts 

The EPAA is currently establishing a new team of experts in genotoxicity that will extensively discuss the 
areas for potential changes and proposed actions, further define an implementation plan, set out clear goals 
to  deliver  non-animal-based  approach  across  sectors,  as  well  as  define  any  change  management 
requirements and responsibilities. The team could coordinate some activities, bring the required knowledge 
together  and  support  driving  forward  the  roadmap  goals  by  developing  a  consensus  approach  to 
genotoxicity assessment, taking into account related initiatives (see examples in Table  6), similar to the 
carcinogenicity team (see chapter on carcinogenicity). The team will include representatives of industries 

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across different sectors, EU agencies (EFSA, ECHA, EMA), the European Commission, Member States 
and academia. 

Table 6. EU and global activities and projects on genotoxicity relevant for the roadmap 

Ongoing revision of the GHS criteria for classification in the hazard class germ cell mutagenicity led 
by the European Union. 

OECD: Several projects focused on new test methods, which are at different stages of validation, 
such as ToxTracker (OECD Project 4.125), comet and micronucleus assays in reconstructed skin 
models (OECD Project 4.139), γH2AX/pH3 (OECD Project 4.168), in vitro alkaline comet assay 
(OECD Project 4.190), micronucleus test for nano, error-corrected next-generation sequencing 
(OECD Project 4.175) 

EFSA: current update of its guidance on genotoxicity assessment applicable to food and feed safety. 

PARC: IATA for genotoxicity; IATA for carcinogenicity 

HESI Genetic Toxicology Technical Committee: improving the scientific basis of the 
interpretation of results from genetic toxicology tests for regulatory purposes and promoting 
and monitoring the development of innovative tests and testing strategies for assessment of 
genotoxic potential 

Health Canada: GeneTox21 platform including high-throughput transcriptomic (TGx-DDI) 
and flow cytometry-based assays (flow cytometry-based micronucleus (MN) test, multiplexed 
flow cytometry-based reporter assay) 

Reducing in vivo testing through changing GHS classification criteria 
Under  the  United  Nations  Globally  Harmonized  System  for  classification  and  labelling  (GHS), 
classification  of  chemicals  for  germ  cell  mutagenicity  is  based  on  their  potential  to  induce  heritable 
mutations in germ cells, within two main categories: Category 1 including known (1A) or presumed (1B) 
human germ cell mutagens and Category 2 covering chemicals suspected of causing such effects (GHS, 
2025). Currently, classification in Category 1A is based on human evidence from epidemiological data, 
while Category 1B is based on information from animal testing or effects in human germ cells. In vitro 
alternatives for assessing germ cell mutagenicity do not currently exist. 

To  avoid  follow-up  in  vivo  germ  cell  testing  after  positive  results  from  in  vivo  genotoxicity  testing  in 
somatic cells, the Netherlands submitted a proposal to GHS in 2023 to classify also in vivo somatic cell 
mutagens  as  Category  1.  The  main  rationale  for  the  proposal  is  that  somatic  cell  mutagens  are  not 
appropriately risk managed. Classification of substances as Category 2 for carcinogenicity or reproductive 
toxicity  means  that  these  substances  are  suspected  to  cause  adverse  health  effects  due  to  insufficient 
evidence. Most of the substances suspected to cause germ cell mutagenicity and classified as Category 2 
are, however, proven to be somatic cell mutagens. They may be able to reach the gonads and potentially 
cause germ cell mutagenicity (even if not proven). Somatic cell mutagenicity can therefore be considered 
a critical health hazard in itself due to its high correlation with carcinogenicity (90% Annex VI to CLP 
substances67).  Therefore,  the  possibility  to  waive  in  vivo  studies,  such  as  the  two-year  rodent  cancer 
bioassay, based on evidence for somatic cell mutagenicity would lead to a considerable reduction of animal 

67 Annex VI to CLP, https://echa.europa.eu/information-on-chemicals/annex-vi-to-clp 

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testing.  The  discussion  on  the  extension  of  the  hazard  class  germ  cell  mutagenicity  to  mutagenicity 
(covering both germ cell and somatic cell mutagens) is proposed to be postponed until completion of the 
ongoing  revision  of  the  germ  cell  mutagenicity  chapter  in  the  GHS. The  ongoing  revision  of  the  GHS 
chapter  also  includes  exploring  possibilities  to  use  somatic  cell  mutagenicity  data  in  combination  with 
indirect evidence of interaction with germ cells to classify chemicals as Category 1B. In addition, non-
testing methods such as read-across and (Q)SARs are introduced in the chapter on germ cell mutagenicity 
in the GHS as suitable evidence for classification. 

Proposed Actions 

Short-term: 

•  Finalise  the  revision  of  the  GHS  chapter  on  germ  cell  mutagenicity:  reach  consensus  on 
incorporating information from in vitro and non-animal test methods wherever possible (Actors: 
GHS Informal Working Group). 

Medium -term: 

•  Restart the discussion at the GHS to either extend the current hazard class on germ cell mutagenicity 
to mutagenicity or to introduce an additional class for mutagenicity (Actors: GHS Subcommittee). 
•  Explore  options  at  EU  level  to  increase  risk  management  measures  for  Category  2  germ  cell 
mutagens, in case no consensus is achieved to change classification criteria or to introduce a hazard 
class for mutagenicity, i.e. trigger CMR Category 1 risk management measures for Category 2 germ 
cell  mutagenicity  in  EU  chemical  legislation.  This  would  lead  to  the  same  desired  level  of 
protection while omitting the need for in vivo germ cell genotoxicity testing (Actors: DG ENV, DG 
GROW, ECHA). 

Reducing or replacing in vivo testing through improved in vitro methods 
Various  in  vitro  test  methods  for  the  assessment  of  genotoxicity  are  available,  including  tests  that  are 
described  as  OECD  TGs  and  that  are  commonly  used  for  regulatory  purposes.  These  standard  in  vitro 
genotoxicity  tests  are  considered  sensitive,  but  their  specificity  is  relatively  low:  in  vitro  tests  for 
genotoxicity are optimised to identify the presence of a genotoxicity hazard. While this is a desired outcome 
from a regulatory perspective, it also leads to a relatively high false positive rate (although some recent 
dosing advice changes applied to the OECD TGs has substantially reduced the potential for false positives). 
A positive in vitro result may, depending on the regulatory jurisdiction, trigger in vivo follow-up testing, 
which is sometimes unnecessary in case this in vitro result is indeed a false positive (for example under 
REACH). Therefore, identifying the false positive in vitro results early on would avoid following up with 
in  vivo  studies  that  would  yield  negative  test  results.  As  such,  improvement  of  the  composition  of  the 
standard in vitro battery and/or providing in vitro alternatives to be used as follow-up to confirm (or deny) 
an  initial  positive  in  vitro mutagenicity  test  result  would  contribute  to  a reduction  in animal testing  for 
genotoxicity  assessment.  This  requires  the  development  and  validation/qualification  of  more  human-
relevant in vitro test methods (including but not limited to ongoing work at OECD, such as ToxTracker 
assay, MultiFlow assay, DNA sequencing, biomarker-based tests) addressing all genotoxic endpoints. Key 
features  of  more  human-relevant  in  vitro  test  methods  are  adequate  metabolic  capacity  as  well  as  high 
specificity (besides high sensitivity). Also, different test methods should cover different cell types. 

Improvement  of  the  standard  battery  for  genotoxicity  assessment,  and  thus  the  development  of  in  vitro 
follow-up tests, should go beyond simply enhancing test specificity. Currently, the standard battery consists 
of tests that offer limited mechanistic insight and are relatively low in throughput. To progress towards a 
paradigm where in vitro testing only is sufficient, new methods must deliver mechanistic data that reveal 
disruption in key biological events leading to genotoxic or mutagenic effects. In combination, these methods 
should encompass different modes of action (MoA) relevant for genotoxicity and more accurately replicate 

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the in vivo exposure environment, including factors such as metabolic activation, DNA repair capacity, and 
oxidative stress responses. 

Phasing  out  animal  tests  for  genotoxicity  assessment  requires  a  deeper  understanding  of  toxicokinetic 
(ADME) parameters and effective strategies for generating and applying this information to improve the 
predictive  power  of  in  vitro  methods.  While  progress  has  been  made,  current  advancements  remain 
insufficient to support the full implementation of an exclusively in vitro testing approach. More critically, 
transitioning  to  such  a  paradigm  demands  careful  consideration  of  the  intended  protection  goals.  This 
includes evaluating where and how quantitative analysis of genotoxic effects can add value. Ultimately, a 
robust  chemical  risk  assessment  framework  should  integrate  mechanistic  insights,  potency  data  and 
toxicokinetic profiles.  

Proposed Actions 

Short-term: 

•  Complete validation according to OECD GD 34 of alternative approaches for genotoxicity that are 

• 

already in the pipeline. 
(Further)  Develop  more  human-relevant  genotoxicity  in  vitro  test  methods  using  different  cell 
types.  Key  features  of  such  more  human-relevant  in  vitro  test  methods  are  adequate  metabolic 
capacity as well as high specificity (Possible actors: PARC, test method developers). 

•  Discuss with different stakeholders involved the desired level of protection and thus the adequate 
level of  prediction for innovative test methods for genotoxicity assessment (see also chapter on 
level of protection in this SWD). 

•  Develop case studies aimed at exploring how to best use toxicokinetic information to refine the 
prediction of in vitro test methods for genotoxicity assessment (Possible actors: PARC, EPAA, HESI 
GTTC). 
Introduce potency in the evaluation of in vitro tests for genotoxicity to allow distinguishing between 
weak/moderate and strong mutagens; case studies will be needed to demonstrate where and how 
this could be of added value. 

• 

•  Explore (sand box) the contexts of use in which a threshold for genotoxicity can be assigned and/or 

quantitative analyses can be used to derive a point of departure for risk assessment. 

Medium-term: 

•  Continue to develop advanced test systems with higher cellular complexity such as 3D models for 

liver, lung, gastrointestinal tract. 

•  Achieve  qualification  and/or  validation  of  promising  more  human-relevant,  metabolically 

competent genotoxicity in vitro test methods using different cell types.  

•  Discuss regulatory uptake of a threshold and/or point of departure for genotoxicity in chemical risk 

assessment. 

Reducing in vivo testing through application of a weight of evidence assessment in 
certain cases 
Negative results obtained from an adequate evaluation with the standard in vitro battery for genotoxicity 
can be considered sufficient to conclude that the substance is non-mutagenic in some but not all regulatory 
areas.  While there is  a  need  to modernise  the  standard  battery to  implement more  relevant  in  vitro  test 
systems with adequate metabolic capacity and improved specificity as outlined above, the high sensitivity 
of the current standard in vitro battery can be retained, while considering the possibility of false negatives 
(due to e.g. limited cellular uptake, in vitro-specific metabolism, low solubility, high volatility). If positive 
results  are  obtained  in  the  standard  in  vitro  battery,  in  vivo  follow-up  testing  may  be  required.  Before 

119 

 
 
 
 
proceeding directly to in vivo testing, a weight of evidence (WoE) assessment may be performed in certain 
scenarios, considering both existing and newly generated non-animal information to evaluate the possibility 
of  avoiding  in  vivo  confirmation.  When  routinely  integrated  into  the  follow-up  testing  and  assessment 
strategy, the WoE approach may, in some cases, allow the conclusion that a substance is (not) mutagenic 
without the need to conduct in vivo genotoxicity tests. 

The WoE approach should address whether the positive or negative in vitro result is predictive of mutagenic 
effects  in  humans.  Relevant  information  to  be  considered  in  the  WoE  approach  may  be  obtained  from 
existing  data  (e.g.  testing  data  on  physico-chemical,  toxicokinetic,  and  toxicodynamic  properties;  non-
testing data such as (Q)SAR, kinetic simulations, and read-across) or by generating new data using a Non-
Animal  Method-based  strategy.  The  possibility  of  false/misleading  positives  should  be  addressed 
considering factors known to influence the specificity of in vitro assays (e.g. in vitro specific metabolism, 
cytotoxicity, altered testing conditions like pH, osmolality, precipitates, etc.). Information on the expected 
absorption, distribution, metabolism, and excretion (ADME) of the substance in humans is required. Such 
toxicokinetic information is needed, for example, to evaluate systemic availability and identify potential 
target  tissues  (initial  site  of  contact,  such  as  skin,  respiratory  tract,  upper  gastrointestinal  tract)  versus 
systemic targets. Information on the metabolism of the substance should be gathered to decide whether the 
in  vitro  system  adequately  covers  the  relevant  metabolic  pathways.  Toxicokinetic  information  may  be 
obtained from available in vivo ADME data or may be generated in vitro or through kinetic modelling. 
Existing information on toxicological endpoints other than genotoxicity may be used to better understand 
the toxicokinetic (e.g. systemic availability) and toxicodynamic (e.g. target tissue/organ) properties. Non-
testing  data  obtained  through  e.g.  read-across  or  (Q)SAR  applications  may  provide  information  on  the 
mutagenic mechanism. 

To follow-up and confirm the mutagenic effects (or lack thereof) observed with the standard in vitro battery, 
higher-tier 3D tissue models can be used, which are generally considered to be a more biologically relevant 
in vivo-like test system due to their higher cellular complexity. Advanced 3D in vitro models are already 
recommended in the cosmetics sector, and the development of OECD TGs for 3D Skin models is underway 
(OECD Project 4.139: 3D reconstructed skin micronucleus test and comet assay). Other 3D models (e.g. 
liver, lung) are expected to become available in the near future. The mutagenic mode of action (MoA) is 
another important line of evidence for assessing relevance to humans. While several alternatives to animal 
approaches (e.g. omics methods, error-corrected sequencing (ECS, OECD Project 4.175) or reporter assays 
(e.g.  Toxtracker,  OECD  Project  4.125)  have  been  developed  to  elucidate  the  MoA,  ongoing  efforts are 
underway to develop Adverse Outcome Pathways (AOPs) and AOP networks to provide a framework for 
organising mechanistic information. Methods employing ECS in cell lines of different origin (rodent vs. 
human cell lines) may be used to inform cross-species extrapolation. The Notes of Guidance published by 
the SCCS (2023) already provides valuable advice. 

If  the  WoE  assessment  does  not  confirm  the  initial  positive  or  negative  result,  further  investigation, 
including in vivo testing, may be needed. Once validated, non-animal testing methods that reliably predict 
in vivo effects with high sensitivity and specificity; may be sufficient to overrule positive results from the 
initial  in  vitro  test  battery.  Such  methods  could  support  a  conclusion  that  the  substance  lacks  relevant 
mutagenic properties in humans. 

Proposed Actions 

Short-term: 

•  Perform evaluation of available non-animal strategies, e.g. Cosmetics (Possible actor: EPAA). 
•  Conduct case studies for different contexts of use to identify scenarios in which an  in vitro-only 
testing strategy may provide sufficient WoE to conclude on genotoxic potential (Possible actors: 
PARC, EPAA). 

120 

 
 
 
•  Continue to develop and improve toxicokinetic models, e.g. to better predict systemic availability 

and tissue/organ distribution (Possible actors: academia, industry). 

•  Continue to develop AOPs and AOP networks for genotoxicity (Possible actors: PARC, 

academia, industry). 

•  Discuss with different stakeholders involved the desired level of protection and thus the adequate 
level of prediction for innovative test methods for genotoxicity assessment (see also chapter on 
level of protection in this SWD). 

Medium-term: 

•  Develop  a  decision  tree  for  the  WoE  assessment  to  provide  guidance  on  when  additional  Non-
Animal Method-based information is sufficient to avoid subsequent in vivo testing (e.g. updating 
Agencies  Guidances  on  genotoxicity).  The  decision  tree  for  a  testing  strategy  for  cosmetic 
ingredients (SCCS, 2023) can serve as a starting point for further development and adoption in 
other regulatory areas. Non-animal approaches for ADME should where relevant be included. 
•  Develop  case  studies  integrating  (mechanistic)  in  vitro  test  methods  for  toxicokinetics  and 
genotoxicity into a defined approach / WoE approach (Possible actors: PARC, academia, industry). 
•  Develop  an  OECD  Test  Guideline  for  a  defined  approach  /  WoE  approach  for  genotoxicity 

assessment, including data interpretation procedure and guidance. 

•  Update respective EU-regulations to allow for hazard identification and classification based on in 

vitro data. 

Long-term: 

•  Define the information requirements for a WoE assessment that allows to overrule a positive 

result in the standard in vitro battery without follow-up in vivo testing. 

•  Modernize the standard in vitro battery for genotoxicity assessment. 

Updating/improving current testing strategies 
Current genotoxicity testing strategies are described in the revised REACH endpoint specific guidance for 
mutagenicity (IR&CSA Chapter R.7a part R.7.7), in the ICH guideline on genotoxicity testing (2008), the 
EFSA Scientific Opinion on genotoxicity testing strategies (2011), and the SCCS Notes of Guidance (2023). 
Further, work is ongoing under PARC for the development of an AOP-based IATA for genotoxicity. Actions 
listed in this document are expected to lead to changes of the above-mentioned testing strategy documents. 
Also, the evaluation of harmonisation across legislations as laid out in this document may inform changes 
to the respective guidance documents. 

A stepping stone on the way to full replacement of in vivo testing for genotoxicity is reduction of testing 
under the current regulatory paradigms. For example, in a somatic cell gene mutation study in transgenic 
rodents, the collection and storage of germ cells for potential further analysis  – should the study yield a 
positive result – can eliminate the need for a separate germ cell mutagenicity study.  

Integration of genotoxicity endpoints into regulatory mandated in vivo studies offers another possibility to 
reduce animal numbers and animal testing in the short- to medium-term. The combination of the in vivo 
comet assay with the in vivo micronucleus assay has been described in the respective OECD TGs 489 and 
474  to reduce  animal  numbers as  compared  to running  the two methods independently (Hamada  et al., 
2001; Pfuhler et al., 2009; Bowen et al., 2011). The Pig-a gene mutation assay can also be combined with 
those assays as described in OECD TG 470. Further, the integration of genotoxicity endpoints into repeated-
dose toxicity studies has been evaluated by an IWGT (International Workshop on Genotoxicity Testing) 
working group (Pfuhler et al., 2009; Rothfuss et al., 2011; EFSA 2011) and is mentioned in ECHA guidance 
in the revised IR&CSA Chapter 7.7. The previously issued guidance on combined protocols, with regards 
to treatment schedules, exposure duration and dose-level setting should be critically reviewed, as well as 
any published examples that support the guidance. The development and publication of new case studies 

121 

 
 
 
can provide robust evidence supporting the feasibility of meeting regulatory requirements with combined 
studies. 

Proposed Actions 

Short-term: 
• 

Identify  needs  to  further  work  on  integrated  in  vivo  studies  to  reduce  animal  numbers  while 
ensuring scientific robustness and regulatory acceptance of studies. 

•  Conduct case studies illustrating combined study protocols (Possible actors: PARC, HESI GTTC, 

Member States). 
Integrate actions and developments into an aligned testing strategy. 

• 
•  Update guidance documents for genotoxicity testing strategies and integrating in vivo study designs 

(Actors: DGs, ECHA, EFSA, SCCS). 

Harmonisation of in vitro and in vivo testing requirements under different 
legislations 
Genotoxicity assessment is an essential component of the safety assessment of all types of substances across 
sectors. Although the assessments have many elements in common (typically starting with testing in an in 
vitro battery followed up by in vivo testing in case of positive in vitro results) they also present several 
differences, which are due to various reasons (type of exposure, perceptions, history). Therefore, this is an 
area where there is room for harmonisation, which would allow for a common evaluation of substances 
under different legislative frameworks in line with the one-substance-one-assessment concept (OSOA). 

For instance, two evident differences in approaches include: a) the number of in vitro tests required in the 
basic battery under different regulated sectors and b) whether or not in vivo studies are required in case of 
negative in vitro battery results (e.g. they are mandatory for pharmaceuticals and plant protection products, 
but not for industrial chemicals). 

A suitable group to addressing such harmonisation issues could be the newly established EPAA working 
group  on  genotoxicity,  especially  due  to  its  cross-sector  composition  and  inclusion  of  EU  Agencies 
representatives.  

Proposed Actions 

Short-term: 
• 

Identify the main differences in approaches across sectors, understand the related reasons for such 
differences and evaluate the justifications for such differences (Possible actors: EPAA, PARC). 

Short- to medium-term: 

•  Analyse  the  identified  varying  elements  of  assessment  strategies  across  sectors  and  conclude 
whether these can be harmonised without reducing the current level of protection, else define what 
other lines of evidence will provide confident information (Possible actor: EPAA).  

Medium-term: 

•  Engage  with  EU  and  Member  State  regulators across  sectors  to amend  regulatory  requirements 
towards  a  harmonised  approach  and  the  implementation  of  the  OSOA  concept,  where  possible 
(Possible actors: Member States, COM, Agencies). 

Definitions 

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The term mutation applies both to permanent heritable and non-heritable genetic changes that may be 
manifested at the phenotypic level and to the underlying DNA modifications (including, for example, 
specific base pair changes and chromosomal translocations).  

The term gene mutation refers to permanent changes in the base sequences of a certain gene. The term 
clastogenicity  refers  to  structural  chromosome  aberrations.  A  clastogen  can  cause  the  loss  or 
rearrangements  of  chromosome  segments.  Aneugenicity  (aneuploidy  induction)  refers  to  a  change 
(loss or gain) in chromosome number in cells (numerical chromosome aberrations). 

The  term  genotoxicity  refers  to  permanent  (i.e.  mutation)  as  well  as  reversible  changes  in  genetic 
information. Both processes could be mediated by primary DNA damage. Primary DNA damage may 
be reversed by DNA repair or other cellular processes, and, thus, may or may not result in permanent 
alterations in  the  structure or  information  content  of the  genetic  material in  a  surviving  cell  or its 
progeny.  Genotoxicity  thus  encompasses  mutations,  including  gene  mutation,  clastogenicity  and 
aneugenicity, as well as primary DNA damage. 

Primary DNA damage can be identified using indicator tests to assess effects such as, DNA strand 
breaks and DNA adduct formation. Genotoxicity tests include mutagenicity tests and indicator tests. 

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Rothfuss A, Honma M, Czich A, Aardema MJ, Burlinson B, Galloway S, Hamada S, Kirkland D, Heflich RH, Howe 
J, Nakajima M, O'Donovan M, Plappert-Helbig U, Priestley C, Recio L, Schuler M, Uno Y and Martus HJ 
(2011)  Improvement  of  in  vivo  genotoxicity  assessment:  combination  of  acute  tests  and  integration  into 
standard toxicity testing. Mutat Res 723:108-20.  

Sachse  et  al.  (2026)  Quantitative  Evaluation  of  Genotoxicity  Data  for  Risk Assessment  and  Regulatory  Decision-

making: Time for a Paradigm Shift. Environment International 208: 110070.  

SCCS  (Scientific  Committee  on  Consumer  Safety)  2023.  SCCS  Notes  of  Guidance  for  the  testing  of  cosmetic 

ingredients and their safety evaluation 12th revision. SCCS/1647/22. 

White PA, Long AS,  Johnson GE. 2020. Quantitative  interpretation of genetic toxicity dose-response data for risk 
assessment  and  regulatory  decision-making:  Current  status  and  emerging  priorities.  Environmental  and 
molecular mutagenesis 61: 66-83. 

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4.3. Carcinogenicity 

Implementing change for carcinogenicity assessments 
Summary 

Carcinogenicity is considered an important toxicological effect in the safety evaluation of chemicals and 
pharmaceuticals. The rodent cancer bioassay, currently the standard method for assessing the carcinogenic 
potential of  chemicals,  has  several  limitations.  It  is  time-consuming, requires large  numbers  of  animals 
across  one  or  two  bioassays  (rats  and  mice),  has  poor  reproducibility  and  has  limited  ability  to  predict 
human carcinogenicity. To address these limitations, new approaches are being developed to modernize 
carcinogenicity assessment. 

A  new  approach  for  carcinogenicity  assessment  presents  an  excellent  case  example  for  regulatory 
implementation of alternatives to animal approaches for complex endpoints and implications for translation 
of novel approaches into practical solutions. Multiple initiatives are underway or already in use in a specific 
area  (see  ICHS1B  guidance  for  pharmaceuticals)  to  develop  frameworks  for  a  new  approach  to 
carcinogenicity assessment, incorporating weight of evidence (WoE)-based methodologies that utilize in 
silico, in vitro, and, while still necessary until non-animal approaches are considered robust, short-term in 
vivo (toxicity) tests. In the long term, the intention is to move towards an animal-free approach. 

To facilitate the development of a harmonized framework for a WoE-based carcinogenicity assessment, the 
European  Partnership  for Alternative Approaches  to Animal Testing  (EPAA)  has  established  a  working 
group that includes representatives from related initiatives, ensuring synergy and co-creation. The working 
group aims to identify opportunities for implementing a change for carcinogenicity assessments, with focus 
on achieving risk management goals that meet regulatory authority standards worldwide within a realistic 
timeframe, across all sectors. 

A review of ongoing initiatives across regulatory areas and jurisdictions revealed substantial commonalities, 
as they are all being built upon mechanistic approaches to identify potential carcinogenicity concerns earlier 
in  the  assessment  process,  i.e.  prior  to  or  instead  of  proceeding  to  a  bioassay.  Moreover,  they  share  a 
common rationale and justification for using alternative approaches, i.e. the need for a quicker assessment, 
higher human relevance and a reduction in the number of animals used. This led to the development of an 
overarching  carcinogenicity  assessment  framework,  comprising  various  components  that  experts  and 
stakeholders  must  consider  in  order  to  identify  current  and  future  information  needs  that  will  enable 
technological advances to keep pace with a changing regulatory paradigm. A harmonized approach, at least 
in  Europe  and  ideally  globally,  is  essential  for  successful  implementation.  However,  this  will  require 
addressing differences in regional legislation and information requirements, identifying commonalities, and 
determining what regulatory changes are needed to implement a new approach. Effective communication 
with  expert  committees  from  governmental  organizations,  stakeholders,  and  the  general  public  is  also 
crucial. 

Taking all this into consideration, a stepwise plan is proposed, outlining short-, medium-, and long-term 
actions designed to bridge scientific knowledge gaps and implement the necessary measures for a paradigm 
change. The goal is to inform and advance the roadmap for phasing out animal testing, ensuring a more 
efficient, innovative, effective and humane approach to carcinogenicity assessment. 

History of the approach: why changing carcinogenicity assessment? 

125 

 
 
The  rodent  cancer  bioassay  (OECD Test  Guidelines  451/453;  OECD,  2018a,  2018b),  is  currently  used 
worldwide  as  a  standard,  default  approach  for  assessing  the  carcinogenic  potential  of  most  chemicals68 
across various sectors. This bioassay (fundamentally unchanged since the 1960’s) is time intensive, uses 
large numbers of animals, and has limited ability to predict relevant human carcinogenic risk. To overcome 
these shortcomings, new approaches are being developed to facilitate the modernization of carcinogenicity 
assessment. A  carcinogenicity  assessment  includes  the  evaluation  of  both  genotoxic  and  non-genotoxic 
activity. Acknowledging that genotoxicity evaluation is an integral part of the carcinogenicity assessment, 
detailed actions for genotoxicity are discussed in a separate chapter of this SWD.  

Mapping Global Initiatives and Securing the Learnings 

Several  initiatives  are  currently  underway  to  develop  a  new  approach  for  carcinogenicity  assessment, 
incorporating WoE-based assessments that utilize in silico, in vitro, short-term in vivo tests and/or other 
available  data  with  the  final  aim  to  move  towards  an  animal-free  assessment.  Bringing  together  these 
initiatives is key given the challenges in evaluating carcinogenic potential of chemicals and pharmaceuticals 
and  ensuring  a  harmonized  scientific  approach.  To  foster  understanding,  the  EPAA  working  group  on 
carcinogenicity listed the efforts in different sectors and jurisdictions (see Table 7 for details). It should be 
acknowledged that the map might not necessarily be comprehensive. The primary objective of this exercise 
was to identify areas of commonality, differences, gaps and potential incompatibility, with the ultimate goal 
of developing a harmonized WoE-based approach for carcinogenicity assessment across industry sectors, 
including  industrial  chemicals,  agrochemicals,  biocidal  products,  food  contact  materials,  food  and  feed 
ingredients, cosmetic ingredients, pharmaceutical products and contaminants.  

The global scope of these initiatives is a crucial consideration when striving for synergies and concepts. 
The numerous efforts undertaken to date have yielded valuable insights that should be preserved and built 
upon  moving  forward.  An  analysis  of  the  various  initiatives  revealed  several  key  commonalities, 
particularly their mechanism-based approach, which enables the identification of potential carcinogenicity 
concerns. This mapping exercise aimed at facilitating the identification and alignment of scientific building 
blocks, ultimately contributing to the development of a unified approach for carcinogenicity assessment 
that can be applied consistently across different sectors. While some differences in approach were noted, 
no  significant  incompatibilities  were  identified,  suggesting  that  these  initiatives  can  be  aligned  and 
integrated to form a cohesive framework for carcinogenicity assessment69.  

Description  of  the  readiness,  applicability  domain  and  validation  status  of  single  alternatives  to  animal 
approaches that are possibly relevant for carcinogenicity assessments of chemicals and pharmaceuticals is 
not covered here as that is currently being addressed by the OECD non-genotoxic carcinogenicity Integrated 
Approach  to Testing  and Assessment  (IATA)  expert  group  (further  referred  to  as  OECD  NGTxC  IATA 
expert group; Jacobs et al., 2020).  

Table 7. EU and global initiatives considered in the mapping effort. 

Initiative 

Reference 

68 This no longer applies to pharmaceuticals since the addendum to the globally harmonised guidance ICHS13B which 
establishes a new carcinogenicity assessment framework for pharmaceuticals.   
69 A unified approach to carcinogenicity assessment is considered useful, even though the conclusions from it could differ in the 
end, as for pharmaceuticals, a benefit-risk assessment must be conducted while for industrial chemicals, no benefits are 
considered in the risk management decision.  

126 

 
 
 
 
EPAA Mechanistic prediction of carcinogenicity 
for agrochemicals 
ReCAAP (Rethinking chronic toxicity and 
Carcinogenicity Assessment for Agrochemicals 
Project) framework 

OECD IATA for Non-Genotoxic Carcinogens 

RISK-HUNT3R (RISK assessment of chemicals 
integrating HUman centric Next 
generation Testing strategies promoting the 3Rs) 
PARC (Partnership for the Assessment of Risks 
from Chemicals) 

HESI eSTAR Transcriptomic Data Files for 
Carcinogenicity 
ICHS1B Testing of Carcinogenicity of 
Pharmaceuticals - (R1) Addendum - WoE 
Approach 
OECD Guidance on Good Practices and 
Standardization of Sample Collection for Omics 
Analysis  

doi: 10.1080/10408444.2020.1841732 

for  Chronic  Toxicity 

OECD  (2024),  Case  Study  on  the  Use  of 
for  Testing  and 
Integrated  Approaches 
Assessment 
and 
Carcinogenicity 
of  Agrichemicals  with 
Exemplar  Case  Studies,  OECD  Series  on 
Testing  and  Assessment,  No.  402,  OECD 
Publishing, Paris. 
https://doi.org/10.1016/j.yrtph.2022.105160  
https://doi.org/10.3389/ftox.2024.1394361  
https://doi.org/10.1007/s00204-024-03753-y 
https://doi.org/10.1007/s00204-020-02784-5 

https://www.risk-hunt3r.eu/ 

IATA for genotoxicity; IATA for 
carcinogenicity 
https://www.eu-parc.eu/ 
https://doi.org/10.1093/toxsci/kfac041 

doi: 10.3389/ftox.2024.1353783 
doi/10.1111/bcp.15790 

Accepted by OECD WPHA (June session, to 
be declassified Q4 2025) 

Proposed Framework for Global Carcinogenicity Assessment of Chemicals 
The  summary  above  highlights  the  scientifically  agreed  importance  of  a  WoE-based  approach  for 
carcinogenicity assessment (including the CLP regulation; EC, 2025), clearly providing an opportunity for 
harmonization. By integrating the key elements from various initiatives, a proposed overarching framework 
for carcinogenicity assessment has been developed by the EPAA working group. This framework is depicted 
in Figure 8. The proposed framework might be applied uniformly across sectors and globally, although it is 
reasonable to expect that details regarding the tiering and decision criteria will vary across sectors, reflecting 
specific risk management approaches. Furthermore, the framework can be used for different contexts of 
use, addressing regulatory questions related to both cancer hazard (including the CLP regulation) and risk 
assessment.  To  achieve  a  globally  harmonized  chemical  safety  assessment,  it  is  essential  to  establish 
consistent methodologies for WoE-based evaluation of carcinogenic potential.  

The proposed global framework for carcinogenicity assessment (Figure 8) incorporates a number of tiered 
elements starting with problem formulation and understanding the external exposure or use scenarios in 
order to evaluate the exposure-based relevance for carcinogenic risk.   

127 

 
 
 
If no exposure potential is identified, or a clear carcinogenic hazard is already known, a justification may 
be  formulated  to  directly  address  these  risks,  and  no  further  data  generation  would  be  required.  The 
likelihood of a human carcinogenic potential can be defined and appropriate next steps followed, either to 
determine classification for labelling or derive a point of departure for risk assessment, as appropriate to 
the legislative requirements. 

If exposure potential is identified or cannot be appropriately characterized, the first step is to conduct a 
genotoxicity assessment, using Non-Animal Method-based approaches.  

If, based on the genotoxicity assessment (see chapter on Genotoxicity), a genotoxicity risk is identified, an 
(internal) Threshold of Toxicological Concern (iTTC) approach could be used to identify a threshold for 
risk assessment. If the TTC for potential DNA-reactive mutagens and/or carcinogens is exceeded or the 
TTC approach is not applicable, a WoE approach could be applied. If the potential Margin of Exposure 
(MoE)  is  ≥10,000 (based on  the current  EFSA  Guidance (EFSA,  2025); to  be updated  for  NAM-based 
approach), risk management may still be feasible under certain legislation. Based on the genotoxic Mode 
of Action (MoA) identified, classification for mutagenicity would also be determined where required (see 
chapter on Genotoxicity). For example, it is envisaged that classification for carcinogenicity may not be 
needed for substances classified as (germ cell) mutagens Category 1 (GHS/CLP), as these would in any 
case undergo severe risk management measures. 

If no genotoxicity risk is identified, a non-genotoxicity carcinogen assessment has to be conducted. First, 
internal exposure should be characterized to understand systemic risk, and this could be compared with an 
(intl) TTC  for  non-genotoxic  substances  for  risk  assessment.  If  the  (internal) TTC  is  exceeded  (or  this 
approach is not applicable), evaluation would continue using a screening toolbox to determine potential 
pathways leading to cancer, based on the OECD IATA for non-genotoxic carcinogens (Jacobs et al., 2020). 
This  toolbox  allows  NAM-based  identification  of  possible  human  relevant  MoAs  for  carcinogenicity 
(Heusinkveld  et  al.,  2020).  During  the  transition  phase  towards  only  using  non-animal  approaches,  the 
toolbox could be supplemented with in vivo subacute and/or subchronic repeated dose toxicity testing to 
provide further evidence for relevant key events. These in vivo studies will be phased out when NAM-based 
approaches have been demonstrated to be sufficiently reliable and relevant for regulatory decision-making.  
These various data will then be evaluated in a WoE assessment, using e.g. the ReCAAP framework (Hilton 
et al., 2022), the modular strategy (Louekari and Jacobs, 2024), and the OECD WoE assessment scheme 
(Figure  9),  to  define  the  likelihood  of  a  human  carcinogenic  potential.  These  data  will  then  inform 
classification determinations and derive a point of departure for relevant risk assessments, as appropriate to 
the legislative requirements. 

128 

 
 
 
Figure 8. Proposed global carcinogenicity assessment framework for chemicals. IATA: Integrated Approach to Testing and Assessment; MoA: 
mode of action; NGTXC: non-genotoxic carcinogen; TTC: threshold of toxicological concern. 

129 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Figure 9. Integration of evidence for non-genotoxic chemicals developed by the OECD NGTXC IATA expert group, with inputs from the OECD 
NGTXC  IATA,  ICH  S1B  (R1)  Guideline  and  ReCAAP  framework  (ref.  OECD  ENV/CBC/WRPR(2025)4). AOP:  adverse  outcome  pathway; 
DNEL: derived no-effect level; GHS: globally harmonized system; IATA: Integrated Approach to Testing and Assessment; MoA: mode of action; 
NGTXC:  non-genotoxic  carcinogen;  NR:  nuclear  receptor;  OEL:  occupational  exposure  limit;  PoD:  point  of  departure;  TTC:  threshold  of 
toxicological concern 

130 

 
 
 
 
Change Management Requirements for Implementation 
Although  science-based  considerations  are  the  basis  for  a  new  approach  to  carcinogenicity  assessment, 
change management requirements need to be defined in addition and put in place early on for an efficient 
implementation of such an approach. Elements for this, aside from the science-based initiatives thus far 
mentioned, 
instituting  best  practices  and  ensuring 
communication to all stakeholders, as depicted in Figure 10. A brainstorming exercise further elucidated 
the sub-elements shown in Figure 10 to help define relevant actions towards change management in support 
of the implementation of the new approach for carcinogenicity assessment. 

the  policy  roadmap, 

include  addressing 

Figure 10. Overview of the different elements that are required for the implementation of a new approach 
for carcinogenicity assessment. 

Summary Action Plan for Implementation of Framework 
Presented below are key actions for the short, medium and long-term, presenting requirements for a 
phased transition towards implementation of the new approach and framework for animal-free 
carcinogenicity assessment. 

Short-Term Actions  

•  Achieve buy-in from Member States authorities to work towards a new approach for carcinogenicity 

assessment  

131 

 
 
 
 
 
 
•  Define realistic protection goals for this endpoint  
•  Explore adaptation of CLP/GHS criteria using non-animal approaches for carcinogenicity assessment 
•  Achieve consensus on MOAs relevant for cancer in humans 
•  Build consensus on the IATA core evidence needed for (non-)carcinogenic substances 
•  Define  and  prioritize  combinations  of  non-animal  approaches  to  be  used  for  screening  of  pivotal, 

recognized key event perturbation of pathways to cancer 

•  Establish  and  test  using  case  studies WoE-based  approaches  combining  non-animal  approaches  and 

repeated-dose toxicity studies for different regulations and contexts of use  

•  Achieve general consensus for a framework for carcinogenicity assessment (see Figure 10) and human 

relevance across sectors 

•  Conduct read-across case studies, designed in co-creation with stakeholders, to increase the 

acceptance of read-across for carcinogenicity assessment 

•  Improve and refine internal TTC approach and perform case studies to demonstrate in what contexts it 

could be of added value 

•  (Further) develop more complex non-animal approaches for late key events of pathways to cancer to 

enable discrimination between adaptive and adverse responses 

Medium-Term Actions  

•  Perform uncertainty assessment of non-animal approaches for carcinogenicity assessment and compare 

to traditional approach 

•  Validate  and/or  qualify  combinations  of  non-animal  approaches  to  be  used  for  screening  of  pivotal, 

recognized key event perturbation of pathways to cancer 

•  Validate/or qualify more complex non-animal approaches for late key events of pathways to cancer to 

enable discriminating between adaptive and adverse responses 

•  Implement  new  WoE  based  non-animal  approaches  for  carcinogenicity  assessment  when  deemed 

acceptable for use in a given regulatory context  

•  Continue  exploring  adaptation  of  CLP/GHS  criteria  in  line  with  -methods  based  on  non-animal 

approaches - consider modification of hazard classes for mutagenicity and carcinogenicity 

•  Review  available  complex  non-animal  approaches  relevant  for  carcinogenesis  and  (further)  develop 
more complex non-animal approaches where needed, to allow for discrimination between adaptive and 
adverse responses 

•  Strengthen  the  gathering  of  biomonitoring  or  clinical  trial  data  to  enhance  exposure  knowledge  and 

ensure good quality and clear human data 

•  Discuss  sector-specific  case  studies  to  optimize  data  interpretation  and  methodologies  with  relevant 

stakeholders 

Long-Term Actions  

•  Implement new adapted regulatory process to an animal-free WoE-based carcinogenicity assessment 
when deemed acceptable for use in a given regulatory context; incorporate internal TTC approach where 
applicable  

•  Implement modified CLP/GHS criteria and possibly also modified hazard classes for mutagenicity and 

carcinogenicity 

132 

 
 
 
 
Conclusion 

Jacobs et al. (2016), Luijten et al. (2020) and Hilton et al. (2023) presented objectives, opportunities and 
challenges  with  the  current  regulatory  landscape  for  adopting  a WoE-based  animal-free  carcinogenicity 
assessment, and raised a call to experts from government, industry, academia and NGOs to convene as an 
international forum to develop a structured framework for regulatory decision-making that would enable 
the replacement of the rodent cancer bioassay. With this in mind, and a key part of consensus building, a 
collaborative approach was undertaken under the umbrella of the EPAA, available initiatives were mapped, 
a consensus framework was developed, and a plan of action is proposed towards a harmonized strategy for 
achieving a WoE-based carcinogenicity assessment without long-term rodent bioassays. 

The phases and completion of the actions summarized here rely on a continuum of progress from short-to 
mid-  to  long-term  achievable  milestones  for  policy,  science,  best  practices  and  communication.  The 
collective goal is to implement an animal-free approach successfully for carcinogenicity assessment at a 
global level across all sectors. This will be achieved in a stepwise manner. Removing the use of the rodent 
bioassay will initially necessitate  incorporating, where still necessary, short-term in vivo (toxicity) tests, 
while  in  the  long-term  the  intention  is  to  move  towards  an  animal-free  approach,  using  non-animal 
approaches with a transparent WoE assessment. 

133 

 
 
 
 
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Hayes AW, Papineni S, Peffer RC, Ramsingh D, Williamson Riffle B, Sanches da Rocha M, Ryan 
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4.4. Repeated dose toxicity 

4.4.1 EFSA chapter describing where the number of animals & species 
tested can be reduced 
Background  

Chemical  risk  assessment  has  been  built  based  mainly  on  animal  testing. These  studies  aim  to 
predict hazard identification and human safety protection. In several cases, such as in pesticide 
active substances, regulation sets data requirements to test more than one species, in studies with 
same duration. In other cases, animal studies are still required where safety information can already 
be retrieved through different assessment. With evolution of science and scientific knowledge, the 
strategies used for safety assessment might be revised and where possible updated.  

The  OECD  guidance  on  the  use  of  research  data70,  in  the  discussion  of  regulatory  relevance 
highlights that “Regulatory relevance changes as the regulatory framework develops over time. 
What is not relevant in the current framework may become relevant in the future, as new legislation 
or  scientific  guidance  is  established  and  vice  versa.”  Relevance  can  change  depending  on  the 
mandatory data requirements, but the later ones can also be adapted to the studies that are (over 
time) considered less relevant to ensure human safety protection.  

The collective knowledge and data along the years that the animal studies have built (requests dates 
back to 1960 (Box & Spielmann, 2005)) allow an informed retrospective analysis to evaluate where 
animal information might be redundant or unnecessary into human protection decisions. In 2022, 
EFSA started to explore the relevance of dog studies for the derivation of health-based guidance 
values for plant protection products (PPP) approval (Panzarea et al., 2022). The process involved 
the  building  of  a  dedicated  database,  retrospective  analysis  followed  by  experts’  judgment. A 
similar process can be applied for other animal studies. 

Although such process should be done continuously according to the Directive 2010/63/EU on the 
protection of animals, the European Commission roadmap is the opportunity to revise strategies 
that are mainly based on animals and might not be the best approach according with the current 
scientific knowledge and innovation. 

Ongoing actions 

Waiving of Sub-Chronic (90-day) dog studies with further aim to phase out  

For the approval of plant protection products (PPPs), the scientific rationale of using the dog as 
‘second’ species in the regulatory process has been debated for long time and culminated in the 
elimination of the one-year dog study (OECD TG 452; 2018) from the data requirements in the 
European Union (EU). 

Under  Regulation  (EU)  283/2013  on  setting  out  the  data  requirements  for  pesticides  active 
substance, short-term oral toxicity testing in rodents (90-day rat study) and non-rodents (90-day 
dog study) species are still required to address hazard identification and human safety of PPPs and 
to support the active substance approval in the EU. 

In  2022,  the  EFSA  PREV  Unit  and  Plant  Protection  Products  and  their  Residues  (PPR)  Panel 
members reviewed the available data on dog studies conducted with the pesticide active substances 
previously marketed in the EU and/or currently approved by the Union legislation. A retrospective 
analysis of the results on setting of health-based guidance values (HBGVs) i.e., acceptable daily 
intake (ADI), was performed.  

70OECD Guidance Document on the Generation, Reporting and Use of Research Data for Regulatory Assessments 
Series on Testing and Assessment No 417, 31 Oct 2025, pdf 

135 

 
 
 
 
In 101 out of 400 active substances registered in Europe, dog studies were used to set the ADI, 
96% of which experimental and biological variability, allometric body weight scaling and dose 
spacing were able to explain the differences observed between rodent and non-rodent species. Only 
for 5 active substances the dog 90-day study showed to be the most relevant for risk assessment 
without the  dismissal  of the  factors listed  previously. There  was,  however,  no  cases  where dog 
studies showed to include endpoints of toxicity based on physiological similarities with human. 
The expert analysis also concluded that for the cases where dog seemed to be the most sensitive 
species would need further additional evidence. 

As  a  follow  up,  a  new  project  is  ongoing  (following  a  new  mandate71)  that  aims  to  continue 
contributing  to  the  debate  by  reviewing/updating  existing  data  to  develop  a  tiered  assessment 
strategy, recommending the possible waiving of dog studies in the regulatory process when deemed 
scientifically justifiable. 

The  working  group  (WG)  constituted  of  EFSA  experts  is  revising  the  data  collected  with  the 
previous project to build the tiered approach. Before finalising, the group aims to: 

•  Test the tiered assessment strategy with proof-of-concept case studies with experimental 

data provided by industry; 

•  Consult with relevant stakeholders (through a workshop) to refine and/or further improve 

the strategy 

•  Harmonise with other ongoing projects (please see in next section, Recommendations from 

EPAA project team for phasing out the non-rodent species testing)  

Overall, the project can be the basis for a future removal of the 90-day dog study as second species 
from PPP data requirements. But most of all, can be used as an example of the type of analysis that 
can be done to reconsider the need of other (90-day) animal studies. 

EFSA recommends that the same exercise to be done for animal studies starting from the following 
questions: 

A.  What is the value of a by default ‘second’ species when all are a surrogate for humans? 

B.  What is unique of the study to be of any benefit in the chemical risk assessment and provide 

a protective ground for human population? 

C.  The  group  of  animals  (e.g.  number/sex/group)  treated  really  covering  the  intra-  and 

interspecies variability aspects? 

D.  Taking into consideration all 90-day studies available, how many play a pivotal role for 
hazard identification and/or setting protection levels? Could the information from these 
studies used to set protection levels, be retrieved from other studies? 

E.  What is  the  impact  of  the  study  on the  targeted  protection  level  to  be  achieved  for  the 
exposed human population, accepting that the study is specific enough or are there other 
approaches more suitable? 

90-day study for enzymes 

Following a similar principle as for the 90-day dog study, EFSA is carrying out a retrospective 
analysis regarding the use of 90-day rat study for enzyme testing. 

Under  regulation  (EU)  No  234-2011  setting  out  the  data  requirements  for  food  enzymes,  sub-
chronic oral toxicity testing in rodents (90-day study) is required for human safety evaluation of 
food enzymes and to support their approval in the European Union. 

71 https://open.efsa.europa.eu/questions/EFSA-Q-2024-00199  
136 

 
 
 
 
 
Under  the  current  EFSA  Guidance  (EFSA  CEP  PANEL,  2021),  food  enzymes  produced  from 
animals or plants commonly consumed as food, do not require additional in vivo toxicological data. 
Moreover,  waiving  of  90-day  tox  studies  for  food  enzymes  from  microbial  sources  is  already 
contemplated in the current guideline for the following cases: 

i) 

ii) 

iii) 

the  criteria  for  a  QPS  (Qualified  Presumption  of  Safety)  are  met  for  a  production 
microorganism and there is no concern from the manufacturing process,  
 there is no or negligible carry-over of the FE-Total Organic Solids (TOS) into the final 
product or  
 an appropriate substitute of toxicological data is available (EFSA CEP Panel, 2021).  

Following the approval of a self-mandate, a new WG of Experts has been established to review 
data available from food enzyme dossiers submitted to date. The aim of this retrospective review 
is to assess the hazard identification capacity of repeated 90-day oral studies in the food enzyme 
domain and to identify those cases where waiving of studies in the regulatory process would have 
been scientifically justifiable.  

Moreover, the use of non-animal approaches that could reliably and efficiently produce information 
that ensures robust safety assessment of food enzymes to human consumers while reducing animal 
use will be explored. 

Development and use of guidance documents that rely on tiered approaches to minimise the use 
of animals and maximising the use of Non-Animal Methods 

to  animal 

testing  being  strategically  enforced 

Despite  alternatives 
in  almost  every 
legislation/regulatory area, many of them still rely in animal studies. While the complete phase out 
or replacement of animal studies is still not scientifically (or regulatorily) possible, an approach 
that  prioritises  the  use  of non-animal  approaches  to retrieve the  relevant  information  should  be 
adopted.  A  sector-specific  tiered  approach  should  be  developed  that  relies  on  non-animal 
approaches in the lower tiers, that could distinguish chemicals with potential harm from non-toxic 
ones, understand its ADME and mechanistic properties of the chemical but also possible species 
differences. With this initial approach, specific and defined triggers could be incorporated into the 
lower tier that would help to decide where additional animal studies would be necessary or where 
animal studies would not be relevant. This approach gives more space to accommodate non-animal 
approaches and accompanied its development and availability, pushing animal studies to higher 
tiers and gradually reducing their need. 

The  implementation  of  a  tiered  approach  is  more  challenging  in  regulatory  areas  where  data 
requirements are specified in the legal text, which is a minority of the legislative areas under the 
EFSA remit (PPPs, GMO, Feed additives and Food enzymes). Nonetheless, in the Feed additives 
area, despite the Regulation (EC) No 429 establishes the requirements for the risk assessment, the 
EFSA FEEDAP Panel adopted guidance documents which are used in the assessments. With the 
years, waivers, use of existing data, extrapolations of conclusions between species, and use of in 
vitro tests have been applied. This has resulted in an effective reduction of the studies needed in 
our  assessments.  The  same  occurs  in  the  Food  enzyme  domain  where  data  requirements  are 
established in Regulations (EC) No 234/2011 and No 562/2012, and the EFSA CEP Panel Guidance 
is followed in the assessment process. 

For Food Enzymes and Food Additives and Flavourings, data requirements in all three areas are 
established in Regulation (EU) No 234 and specific Guidance documents have been developed by 
EFSA. In other sectors such as Novel Foods and Food Contact materials, the process is led by self-
mandated guidance’s coordinated and approved by EFSA panels that are renewed regularly. This 
process makes the update of risk assessment procedure easier, and more prone to accommodate the 
latest scientific technology, especially if relying into a tiered approach. 

137 

 
 
 
In  September  2024,  EFSA  has  published  the  updated  scientific  guidance  for  the  preparation  of 
applications for authorisation of novel foods72. In the revision process the Nutrition, Novel Foods 
and Food Allergens (NDA) updated the Tiered approach to conducting toxicology studies (Figure 
11) that reinforce the use on alternatives to animal testing and available data before conducting any 
animal study. The current approach, put more emphasis on ADME at lower tiers and allows the 
early identification and screening of species differences that would highlight the unnecessary use 
of animal studies (ex.: rapid and complete degradation in the GI tract into components with an 
established safety profile) 

Figure 11. Illustrative example of the tiered approach from EFSA guidance on the scientific 
requirements for an application for authorisation of a novel food in the context of Regulation 
(EU)  2015/2283  https://www.efsa.europa.eu/en/efsajournal/pub/8961  |  “Tiered  approach  for 
ADME & toxicological assessment of novel foods. The boxes in white represent triggers that will 
call for higher-tier studies as indicated by the arrows. Solid lines refer to the standardised way of 
conducting a tiered-based approach by moving from lower to higher tier assessments; dashed lines 
refer  to  a deviated tiered-based approach  where  the existing  data  require  Tier  III  studies,  from 
ADME to toxicity or from genotoxicity to repeated dose toxicity” 

EFSA’s Panel on Food Additives and Flavourings (FAF) has been working on the revision of the 
“Food Additive Guidance for submission for food additives evaluation” (EFSA ANS Panel, 2012) 
since October 2023, following a self-task mandate to update the content of the guidance in the light 
of the experience gained with the safety assessment of food additives applications submitted under 
Regulation (EC) No 1331/2008. 

A first draft of the revised guidance was endorsed by the FAF Panel in November 2024 which was 
published for public consultation on from December 2024 until February 2025. During the public 
consultation  several  comments  were  received  concerning  the  proposed  toxicological  data 
requirements and the revised tiered approach for toxicokinetic and toxicity testing. It was suggested 
to  consider  adapting  the  study  design  of  toxicity  studies  and  possibly  combining  multiple 
toxicokinetic and toxicity endpoints in one study to enhance the efficiency and ethical compliance. 
It was also suggested to apply a more flexible approach on the possible use of Non-Animal Method-

72 https://www.efsa.europa.eu/en/efsajournal/pub/8961  

138 

 
 
 
 
based methods in the risk assessment, even if not yet validated, to be considered as part of a weight 
of evidence approach. 

The Panel underwent a comprehensive revision of the tiered approach for toxicity studies, in view 
of  the  comments  received.  The  updated  version  of  the  tiered  approach  now  recommends 
prioritising the use of existing information and of non-animal testing (in silico and in vitro) in the 
first Tier to inform on the safety of the proposed food additive. When in vivo animal studies are 
recommended  in  higher  Tiers,  in  some  cases  study  designs  have  been  modified  with  the  aim 
of minimizing the use of experimental animals and maximizing the toxicological endpoints under 
investigation, in line with the 3Rs principle. The revised food additive guidance was adopted by 
the FAF Panel in November 2025 and was published in January 202673. 

The assessment of the safety and efficacy of the feed additive, done by the FEEDAP Panel, is done 
in line with the guidance documents produced by the FEEDAP Panel. The first set of guidance 
documents was released in 2008 and were in line with the implementing rules of Regulation (EC) 
No 1831/2003 available in Regulation (EC) No 429/2008. The FEEDAP Panel guidance documents 
have  been  updated  with  the  knowledge  gained  in  the  assessment  of  feed  additive  and  the  data 
requirements, especially regarding animal studies, have been reduced in comparison to the ones 
established  in  the  implementing  regulation.  At  this  regard,  the  newest  guidance  documents74 
establish  

i) 

ii) 
iii) 

the  possibility  to  use  available  data  (e.g.,  for  ADME,  toxicological  studies, 
safety/efficacy for the target species),  
 more waivers for data considering the type of substance,  
 purity and/or origin, and increased possibilities to extrapolate results between species, 
and iv) the use of in vitro tests.  

Examples in use already include (not exhaustive list): 

-  Assessment  of  flavouring  substances  using  already  existing  data  for  ADME  and/or 

NOAELs, read-across between substances,  

-  To establish the safety for the target species the applicants had to conduct tolerance trials 
in the different target species. Nowadays, one single study in a laboratory animal can be 
used to conclude on all animal species for most of the substances under evaluation. The 
applicants may also make use of already existing data75.  

-  The number of efficacy trials has been reduced considerably by increasing the number of 

- 

possibilities to extrapolate data76, 
In the case of the sub-chronic oral toxicity study (section 3.1 and 3.2.3 in the guidance on 
the safety for the consumer77), the waivers applied in the guidance on the safety for the 
consumer, have reduced considerably the number of dossiers in which the sub-chronic oral 
toxicity  study  (and  genotoxicity  testing)  are  needed.78  Major  impact  has  been  seen  in 
fermentation products (e.g. enzymes and amino acids).  

The guidance documents are revisited periodically to see further possibilities to reduce-refine-reuse 
data. 

Other possible actions/recommendations to be explored by EFSA 

73 Administrative guidance for the preparation of applications on food improvement agents (food enzymes, 
food additives and food flavourings) 
74 Available at https://www.efsa.europa.eu/en/applications/feedadditives/regulationsandguidance; safety for the target 
species, safety for the consumer, safety for the user, safety for the environment and efficacy guidance. 
75 https://www.efsa.europa.eu/en/efsajournal/pub/5021 
76 https://efsa.onlinelibrary.wiley.com/doi/10.2903/j.efsa.2024.8856 
77 https://efsa.onlinelibrary.wiley.com/doi/epdf/10.2903/j.efsa.2017.5022 
78 https://efsa.onlinelibrary.wiley.com/doi/epdf/10.2903/j.efsa.2017.5022 

139 

 
 
 
 
 
 
Refinement action 

Following the tiered approach, the requirement for animal studies is generally intrinsic to higher 
tiers  and  therefore, their  removal  is  often seen as  impossible.  In  such  cases,  it is  important the 
maximization of endpoints while maintaining or minimizing the number of animals. When in vivo 
animal studies are recommended in higher Tiers, study designs can be modified in some cases to 
allow for more information to be retrieved from the in vivo study. The aim is minimizing the use 
of experimental animals and at maximizing the toxicological endpoints under investigation, in line 
with the 3Rs principle. 

In the guidance on the assessment of the safety of feed additives for the target species79 the EFSA 
Panel on Additives and Products or Substances used in Animal Feed introduced the possibility of 
extrapolating  the  results  (NOAEL/BMD)  derived  from  toxicological  studies  with  laboratory 
animals to the different species/categories of animals based on simple calculations (conversion of 
mg/kg bw and day to mg/kg feed). This approach implied a considerable reduction in the number 
of tolerance studies required, especially for those substances in which 90-day toxicity studies were 
needed. 

Similar considerations and approaches can be adopted in other type of assessments. 

90-day rat and 28-day animal studies for Genetically Modified Organisms (GMO) assessment 

Under the current regulatory framework for GMOs (Regulation (EU) No 503/2013), applicants are 
mandated to submit a 90-day feeding study in rats using the whole food/feed derived from GM 
plants. 
However, the EFSA GMO Panel considers that such animal studies are not routinely necessary, as 
noted in its opinions on GM food and feed. Instead, they should only be considered on a case-by-
case basis. According to EFSA’s experience, the decision to conduct an animal study should be 
based  on  the  results  of  molecular  characterisation,  comparative  analysis,  and  toxicological 
assessment. If these evaluations do not reveal concerns, animal studies should not be required.8081 
Given the GMO Panel’s position on 90-day studies and EFSA’s ongoing work in related areas, this 
topic  could  benefit  from  expert  analysis  and  discussion.  It  could  contribute  to  a  reflection  on 
whether  to  waive  the  legal  requirement  for  90-day  oral  toxicity  studies  in  rats  for  whole  GM 
food/feed assessment. 

Furthermore,  the  EFSA  GMO  Panel  recently  published  a  scientific  opinion  addressing  current 
practice,  challenges  and  future  opportunities  in  the  safety  assessment  of  proteins  in  genetically 
modified organisms (GMOs) (EFSA GMO Panel, 2025)82. The Panel conducts safety assessments 
of proteins in GMOs based on its established guidance documents and relevant Codex Alimentarius 
guidelines,  including  the  use  of  animal  studies.  These  approaches  have  effectively  supported 
evaluations over the past two decades. However, the field is evolving. There is an increasing need 
to reduce reliance on animal testing, and the growing complexity of GMO applications calls for a 
modernised assessment strategy. Traditional toxicological in vivo studies were originally developed 
for  simple  chemicals,  and  their  relevance  for  assessing  proteins  remains  uncertain.  In  its  latest 
opinion, the GMO Panel recommends a stepwise, risk-based approach in which in vivo studies are 
conducted only when potential hazards are identified or when essential safety information cannot 
be obtained through alternative methods (see Figure 3 in EFSA GMO Panel, 2025). In short, an 

79 https://www.efsa.europa.eu/en/efsajournal/pub/5021 
80  EFSA  Panel  on  Genetically  Modified  Organisms  (GMO);  Scientific  Opinion  on  Guidance  for  risk 
assessment  of  food  and  feed  from  genetically  modified  plants.  EFSA  Journal2011;9(5):  2150.  [37  pp.] 
doi:10.2903/j.efsa.2011.2150. 
81  EFSA  (European  Food  Safety  Authority),  2014.  Explanatory  statement  for  the  applicability  of  the 
Guidance  of  the  EFSA  Scientific  Committee  on  conducting  repeated-dose  90-day  oral  toxicity  study  in 
rodents  on  whole  food/feed  for  GMO  risk  assessment.  EFSA  Journal2014;12(10):3871,  25  pp., 
doi:10.2903/j.efsa.2014.3871 
82 https://efsa.onlinelibrary.wiley.com/doi/full/10.2903/j.efsa.2025.9568 

140 

 
 
 
 
 
improved  strategy  for  protein  safety  assessment  underlined  in  this  document  includes:  (1) 
considering  history  of  safe  use  (HoSU),  read-across  and  phylogeny  defining  the  type  of  data 
required and remove the need for specific in vitro or in vivo studies; (2) applying advanced in silico 
tools, including  predictive computational models  and improved  phylogenetic analysis to  enable 
more accurate comparisons with known allergens, toxins or ‘safe’ proteins; (3) using standardised 
in vitro gastrointestinal models that replicate physiological conditions; (4) developing targeted in 
vivo  studies following  a  hypothesis  driven  approach;  (5)  evaluating  the  role  of exposure  in  the 
safety  assessment;  and,  where  necessary,  (6)  considering  post-market  monitoring  for  risk 
characterisation. The EFSA GMO Panel aims to integrate alternatives to animal testing into risk 
assessment to provide a scientiﬁc basis for waiving in vivo testing, aligning its approach with the 
3Rs principles (Replacement, Reduction, Reﬁnement) and the European Commission’s roadmap 
for phasing out animal testing. 

References 

Box, R.J., Spielmann, H. Use of the dog as non-rodent test species in the safety testing schedule associated 
with  the  registration  of  crop  and  plant  protection  products  (pesticides):  present  status. Arch 
Toxicol 79, 615–626 (2005). https://doi.org/10.1007/s00204-005-0678-0 

Panzarea, M., Terron, A., Coja, T., Pelkonen, O. Editorial: Relevance of dog studies for the derivation of 
(2022). 

protection 

approval 

products 

plant 

for 

health-based 
https://doi.org/10.2903/j.efsa.2022.e200923 

guidance 

values 

EFSA  Panel  on Additives  and  Products  or  Substances  used  in Animal  Feed  (FEEDAP);  Guido  Rychen, 
Gabriele Aquilina,  Giovanna Azimonti,  Vasileios  Bampidis,  Maria  de  Lourdes  Bastos,  Georges 
Bories,  Andrew  Chesson,  Pier  Sandro  Cocconcelli,  Gerhard  Flachowsky,  Jürgen  Gropp,  Boris 
Kolar,  Maryline  Kouba,  Marta  López-Alonso,  Secundino  López  Puente,  Alberto  Mantovani, 
Baltasar  Mayo,  Fernando  Ramos,  Maria  Saarela,  Roberto  Edoardo  Villa,  Robert  John  Wallace, 
Pieter  Wester,  Montserrat Anguita,  Jaume  Galobart,  Matteo  Lorenzo  Innocenti,  Laura  Martino. 
Guidance  on  the  assessment  of  the  safety  of  feed  additives  for  the  target  species  (2017). 
https://www.efsa.europa.eu/en/efsajournal/pub/5021 

141 

 
 
 
 
 
4.4.2 Chapter from EPAA project team with recommendations for phasing 
out the non-rodent species testing for (sub-) chronic toxicity testing with 
alternatives to animal testing across sectors 

Recommendations 

Basket 1: Short term 

•  Legal framework and regulatory use mapping:  

-  Conduct a comprehensive landscape mapping of the different regulatory requirements 
and uses of non-rodents in repeated-dose toxicity testing across the different sectors. 
Identify  where  it  would  be  possible  to  leverage  3Rs  approaches  that  are  already 
implemented to promote cross-sectorial alignment.  

- 

•  Tiered approach development: 

- 

Integrate international work in non-animal approaches and EFSA and US EPA 
evaluations of the dog study to develop a waiver framework and tiered strategy 
aimed at the elimination of dog testing for agrochemicals.  

-  Conduct an analysis to translate the process and outcome to other industry sectors 

- 

like feed additives, biocides and veterinary medicines.  
Introduce tiered approach to evaluate the need for two species (e.g. rodent and non-
rodent) chronic toxicity testing for human medicine. 

•  Animal reduction and refinement:  

-  Consider study design optimisation to reduce and refine the use of animals in the 

chronic and/or precursor dose-range finding studies in all sectors.  

-  EU-level and national legal framework mapping on refinement during in-life in the 

laboratory of large animals  

-  Conduct a review of EU-level and national policies for rehoming and map where 

• 

there would be opportunities for rehoming (e.g. non-terminal procedures). 
Integration of advanced new approach methodologies comparative systems:  
-  Explore the use of comparative rodent and non-rodent and/or human ADME/ 

toxicokinetic in silico and in vitro systems and toxicodynamic systems to enhance 
the understanding of species sensitivity, species translational capacity and human 
relevance which can guide the selection of appropriate representative species. 

•  Global collaboration:  

-  Strengthen existing partnerships and promote global stakeholder to achieve 

alignment on reducing the use of non-rodent as a second species. 

-  Facilitate international harmonisation through activities like the International 

Medicines Regulators’ Working Group on 3Rs. Create a multistakeholder 3Rs group, 
with representatives from each sector. Allow these to engage in agenda setting, 
research priorities where all stakeholders/sectors benefit (e.g. Regulatory Science 
Network Netherlands or HESI).  

 Basket 2: Medium term 

•  Standardisation and validation of in vitro methods:  

-  Promote standardisation and validation according OECD GD34 of in vitro assays to 
assess comparative ADME/toxicokinetic and toxicodynamic in rodent and non-
rodent species and/or human systems.  

142 

 
 
 
 
 
•  Regulatory endorsement and industry incentives:  

-  Promote regulatory acceptance and provide industry incentives via relevant European 

agency qualification advice on Non-Animal Method protocols and methods 
development for humand and veterinary medicinal products. 

•  Waiver framework expansion: 

-  Promote industry incentives for adoption of tiered approaches to industry to 

eliminate dog use in this sector.  

-  Promote broader international adoption of waiver framework in the agrochemical 
sector and tiered approach in other regulated products with regards to non-rodent 
testing. 

• 

Innovative research and data sharing:  
-  Foster the research and development of new approaches such as virtual control group 

(e.g. VICT3R), to further reduce non-rodent use.  

-  Use and reporting of historical control data (HCD) methodology (EFSA, 202583) 

which supports refinement and reduction through strengthening the statistical power 
of existing toxicity studies and avoiding additional confirmatory studies. This 
complements the above virtual control groups initiative, as the HCD methodology is 
relevant for assessing the adequacy and quality of control datasets under 
development. 

-  Promote cross-sector data sharing initiatives and link them to funding mechanisms to 

accelerate progress. 
•  Legislative adaptation:  

-  Explore and propose updates to EU legislation to accommodate tiered and alternative, 

non-animal testing approaches. 

Basket 3: Long term 

•  Advance research on non-animal alternatives: 

-  Further research and development of the non-animal approaches such as computational 
methods explored in the NC3Rs CRACK IT Challenge 'Virtual Second Species', to 
replace animal studies. 

•  Global regulatory adaptations: Substantial progress will require international legislation 
and guideline adaptations to enable tiered and alternative testing approaches and ultimately 
ensure removal of the non-rodent species. 

Background 

Testing a second, non-rodent species is a requirement in several industrial sectors in the EU and 
beyond. This testing is commonly conducted in dogs for (sub-) chronic toxicity and rabbits for 
developmental toxicity in addition to rodent studies. Nevertheless, other species such as mini-pigs 
and non-human primates could also be used for human medicines. The rationale for conducting 
non-rodent studies is to identify any potential health hazards that would not have been detected in 
rodent studies alone. However, alongside issues with translation of data from animals to humans, 
the  experimental  design  of  non-rodent  studies  has  several  limitations,  including  low  statistical 
power of the studies, leading to uncertainties in interpretation. As a consequence, the added value 
of these studies as part of a weight-of-evidence approach is questioned. In addition, recent analyses 
have shown that, for agrochemicals at least, testing in the dog as the second species, provided little 
added benefit to most risk assessments. (Bishop et al., 2023, Prior et al., 2020) 

83 Use and reporting of historical control data for regulatory studies - - 2025 - EFSA Journal - Wiley 
Online Library 

143 

 
 
 
 
 
In its current state, the recommendations focus on the non-rodent species testing for sub-chronic 
toxicity  and/or  chronic  toxicity  studies  which  are  typically  required  in  several  sectors  (e.g. 
agrochemical,  biocidal,  feed  additive,  human  and  veterinary  medicinal  products). The  different 
industry sectors are starting from different states of requirements and readiness for phasing out dog 
studies. An  overview  of  the  information  requirements  for  each  sector  is  presented  in  Table  8 
hereafter. In certain sectors, such as agrochemicals, comprehensive analyses of registered active 
substances have demonstrated that for the vast majority of pesticides the required 90-day dog study 
does  not  provide  additional  insights  relevant  to  human  health  protection  beyond  the  required 
toxicological  data  package  (e.g.  rodent  studies)  submitted  as  part  of  the  regulatory  package. 
Therefore, efforts should focus on identifying the limited number of substances for which dogs 
may exhibit greater sensitivity relevant to human. This can be achieved through integration of non-
animal approaches through comparative toxicokinetic and toxicodynamic assessments to evaluate 
any potential implications for human safety. In other sectors, where the relevance of dog studies to 
human  health  may  differ  (e.g.  first  in  human  clinical  trial  starting  dose),  the  development  and 
application of advance predictive tools and non-animal approaches is necessary. These should be 
tailored to the specific regulatory and scientific context of use of each sector. 

In order to phase out non-rodent species, extension of the scope to other sectors such as the food-
industry  (e.g.  food  additives)  and  to  other  non-rodent  second  species  uses  (e.g.  developmental 
toxicity) is needed in the future.

144 

 
 
Current information requirements 

Table 8: Summary of the regulatory requirements with respect to second non-rodent species for (sub-)chronic toxicity in the different sectors 

Industrial sectors   Non-rodent required as a second 
species for repeated dose toxicity 
testing Test guideline (TG)?  

Is dog always the 
non-rodent 
species? 

Human health protection goal 

Veterinary 
medicinalproducts   

Human medicinal 
products 

Non-food producing animals: no 
except if the dog is the target 
animal species (a 90-day dog 
toxicity study can be conducted 
instead of rodent) 
Food producing animals: Yes 
90-day (OECD TG 409): required 
Chronic (OECD TG 452): 
conditional 
OECD GD 116 as general 
guidance 
Yes, short-term and chronic 
(OECD TG 409, TG 452; OECD 
GD 116) 
Specific adjustments on study 
duration and one or two species 
approach depends on modalities 
and indications (e.g. biologicals, 
radiotherapeutics)  

Biocide products  Yes, conditionally requirement 

(OECD TG 409 if required) 

Dogs as the most 
commonly used 
species (e.g. 
Swine, mini pigs 
may also be used) 

Dog as the target animal species: 
- Target-animal safety  
- Secondary protection objective for human 
health 

Food producing animal: 
- User and consumer/human food hazard and 
risk assessment  

No, the most 
common species 
is the dog, but the 
most appropriate 
species should be 
used based on 
pharmacology, 
target homology, 
etc. (e.g. 
dog, NHP, mini 
pigs)  

Dogs as the 
commonly used 
species 

- Identification of potential target organs  
(on- and off-target toxicity | exaggerated 
pharmacology | chemically-mediated toxicity) 
- Assessment of reversibility of findings 
observed 
- Recommendations for clinical monitoring of 
findings  
- Exposure margins to intended human 
(therapeutic) exposure (MoE = margins of 
exposure) 
- Recommendation of starting dose for FiH 
(first-in-human) clinical trial 
- Product Labelling  
- Hazard identification (C&L) 
- Consumer (dietary) and non-dietary 
(industrial, professional, non-professional and 
secondary [e.g., indirect]) risk assessment 

145 

EU 
Actors  

EMA, 
EC 

Regulatory reference 
requirements for non-rodent 
species for repeat dose 
toxicity 
Reg. (EC) No 2019/6 
&Delegated Reg. (EC) No 
2021/805; .Reg. (EC) No 
470/2009. Reg. (EC) No 
2018/782   

VICH guidance 31, 37, 41, 
43, 44 

Dir. 2001/83/EC & EU reg. 
726/2004 

EMA, 
EC 

ICH guideline M3(R2), S4, 
S6(R1), S9 

Guideline on repeated dose 
chronic toxicity (CPMP) 
CPMP/SWP/1042/99 
Rev 1 Corr, and duration 
CPMP/ICH/300/9 

Reg. (EC) No 528/2012 

ECHA, 
EC 

 
 
 
 
  
 
 
 
 
 
 
 
 
 
 
 
- Hazard identification (C&L) 
- Consumer (dietary) and non-dietary (worker, 
operator, bystander, resident) safety (reference 
dose setting: ADI, AOEL) 
- Target species safety  
- Consumer safety (ADI, UL) 
- Users/workers safety 

Reg. (EC) No 283/2013 
specify the requirements for 
plant protection product 
active ingredients 
Reg. (EC) No 429/2008 

EFSA, 
ECHA, 
EC  

EFSA 

Guidance for submission for 
food additive evaluation 
(EFSA ANS Panel, 2012) 
N/A 

N/A 

N/A 

EFSA, 
EC 

EFSA, 
EC 
ECHA, 
EC 
SCCS, 
EC 

Plant protection 
products 

Feed additives 

Yes, for conventional active 
substances. 
(OECD TG 409; OECD GD 116) 
Exemption for Microbials. 
Yes, if necessary 
90-day (OECD 409) 
1-year (OECD 452) 

Food additives 

Food contact 
material 
Industrial 
chemicals 
Cosmetics 

No. One rodent species (e.g. rat) 
only. Non-rodent only if the 
rodent is not a relevant species 
No 

No 

No 

The dog is the 
requested species 
in the regulation 

Dogs as the most 
commonly used 
species (Swine, 
mini pigs may 
also be used) - 
OECD 409. 
N/A 

N/A 

N/A 

N/A 

N/A 

N/A 

N/A 

- Consumer safety  

TG: test guideline, C&L: classification and labelling; Reg.: regulation; UL: tolerable upper intake; ADI: acceptable daily intake 

146 

 
 
 
 
 
 
 
Activities related to the recommended actions 

Table 9: Activities identified related to the short:medium:long-term recommended actions 

Actions 

1st Basket – Improve the existing knowledge base 
- Short term activities (< ~5 yr) 

nd

 basket - Replacement after further 

2
method development activities 

rd

 basket - Transition to 

3
animal-free regulatory system 
activities 

Mapping Scientific 
Gaps  

(All  sectors)  Comprehensive  landscape  mapping  of  the 
different  regulatory  uses  of  non-rodents  in  repeated  dose 
toxicity testing across the different sectors and identification 
where it would be possible to leverage 3Rs approaches that are 
already implemented in other sectors. 
EPAA as a potential actor 
(AgChem) Evaluate results from independently conducted 
retrospective analyses from different stakeholders, in EU and 
beyond, on the additional value of the 90-day dog test for 
human health risk assessment for agrochemicals 
Ongoing projects: regulatory agencies (EFSA [6,9], US 
EPA), industry (e.g. Syngenta, Corteva)), review articles [1] 
(Agchem/biocide) Retrospective analysis of dog use for 
hazard classification and labelling  
No ongoing project identified.  
ECHA or Industry as a potential actor 
(Human medicine) Retrospective analysis of the use of two species in (sub-)chronic studies for human 
medicine 
Ongoing project: NC3Rs two species project phase 2 [10], review articles [2, 3, 4, 7] 
Other potential actor: EMA 
(Biocides, veterinary medicines) Retrospective analysis of 
the value of the dog study in risk assessment  
No ongoing project identified  
Potential actors: EMA, EFSA, ECHA, industry    

147 

 
 
 
 
 
 
 
 
 
 
 
 
 
Development and 
integration of 
innovative NAMs 

(All sectors) Landscape mapping of comparative 
toxicokinetic in vitro systems in rodent and non-rodent 
species (e.g. in vitro comparative metabolism study) and 
target organ systems to investigate comparative 
toxicodynamics and their application to support species 
sensitivity, human relevance and species translational 
capacity. 
Potential actors: industry, NAMs developers, CRO 
(All sectors) Accelerate validation and standardisation of comparative in vitro ADME methods that 
could inform PBPK modelling and OECD test guideline development. 
Example of ongoing projects: OECD test method development proposal for plasma protein binding 
studies; intrinsic clearance studies 
Potential actor: OECD members, ICAPO 
(Human medicine) Introduce tiered approach to evaluate the 
need for two species (e.g. rodent and non-rodent) chronic 
toxicity testing if conditions allow for human medicine 
Potential actors: animal protection NGOs, EMA 

Waiver 
framework and 
tiered approach 
strategy 
development  

(Agchem) Waiver framework development 
Ongoing project: EFSA [9]  
(Q4 2025/Q1 2026 Scientific opinion) 
Actors: EFSA, Industry 

(Agchem) Publish results of 
framework development; conduct 
outreach and promote international 
acceptance and use of waiver 
framework for 90-day dog study to 
ensure global harmonisation and avoid 
unnecessary testing should some 
markets still require the study. 

148 

(Human medicine) 
Application of advances in 
research into computational 
and mathematical modelling 
approaches (e.g. development 
of a suite of virtual dog tissues 
to model toxicological 
endpoints for New Chemical 
Entities NCE) 
Ongoing project: NC3Rs 
CRACK IT Challenge: virtual 
second species [11] 
Actors: NC3Rs, Industry data 
sharing 

 
 
 
 
 
 
 
Actors: trade associations (e.g Asia-
Pacific, Brazil); Authorities (e.g. 
ANVISA, Japan food safety 
committee) 

(Human and veterinary 
medicine/biocide)  
Further research to demonstrate the 
applicability of extending the Agchem 
framework to other product sectors 
where there is a similar context of use, 
avoiding unnecessary testing. 
Development of similar regulatory 
framework applicable to other product 
sectors/context of use. 
No ongoing project 
Potential actors: ECHA, EMA, EFSA 
(Human medicine)  
Developing and implementing virtual 
control groups to further reduce animal 
use in the in vivo toxicity studies that 
cannot be waived. 
Ongoing project: VICT3R on virtual 
control group in the human medicine 
sector [7] 
Actors: 

(Agchem) HESI joint TEA (Transforming the Evaluation of 
Agrochemicals) and PBPK waiver framework development 
for the 90-day Dog study for agrochemicals aiming to define 
the conditions under which this study may be necessary to 
inform human health risk assessment [12] 
Actor: HESI  

Consider study 
design 
optimisation and 
best-practices to 
reduce and refine 
the use of animals. 

(Human medicine) Toxicity study design optimisation in the 
(sub-)chronic studies required for safety assessment 
 Dose-range-finding optimisation 

-  Optimise study design, for example using 

microsampling to remove the need for toxicokinetic 
satellite animals (NC3Rs ongoing projects [13]; 
-  Encourage science-based case-by-case consideration 
of the use of recovery animals (NC3Rs ongoing 
project; Sewell et al., 2014; [8, 14]) 

-  Minimise NHP use in drug development (NC3Rs 

ongoing project; [5]). 

Actors: NC3Rs, industry 

149 

 
 
 
 
 
 
 
 
(Agrochemicals and veterinary 
medicine)  
Remove concurrent control groups 
and/or reduce the number of 
concurrent control animals with 
inclusion of some virtual control 
animals after further research and data 
sharing to develop virtual control 
group in these sectors 
Potential actors: NC3Rs, industry 
trade association 

Legislative 
changes84 and/or 
guideline updates 
in the different 
context of use 

EU-level and international legal framework mapping of 
refinement opportunities during in-life in the laboratory of 
large animals and rehoming policies following non-terminal 
procedures.  
No ongoing projects identified 
Potential actors: GRAAL [16], Beagle dogs of 
Burgundy[17]: White rabbit (for small animals) [18], 
Individuals (for farm animals) [19], animal protection 
NGOs, shelter/rescue partners, university  
(Human medicines) 
Example of ongoing activities (consultation): 
-Reflection paper on the current regulatory testing 
requirements for medicinal products for human use and 
opportunities for implementation of the 3Rs[20] 

-Concept paper on revision of the guideline on the principles 
of regulatory acceptance of 3Rs (replacement, reduction, 
refinement) testing approaches (EMA/CHMP/CVMP/JEG-
3Rs/450091/2012)[21]  
Actor: EMA 
(Veterinary medicines)  
Example of ongoing activities: 

84 The pharmaceutical legislation already allows flexibility and legislative changes are not foreseen or required there.  

150 

 
 
 
 
 
 
 
 
 
 
 
 
 
-Overview of the current regulatory testing requirements for 
veterinary medicinal products and opportunities for 
implementation of the 3Rs - Scientific guideline [22] 
Actor: EMA 
(Pesticides) 
Development of a Roadmap for Action on NAMs in Risk 
Assessment 
Actor: EFSA [23] 

(All sectors) Provide incentives to 
industry to encourage the waiver of 
non-rodent studies, especially when 
waiver framework is available for 
agrochemicals. Promote relevant 
European agency qualification advice 
team for human medicine on NAMs 
protocols and methods and relevant 
advisory European agency team on 
tiered approach 
Potential  Actor:  EFSA;  EMA  CHMP 
qualification [26]  
Legislative  adaptations  for  tiered  and 
alternative approaches in EU. 
 Actor: 
European 
member state forum discussion  

Commission, 

Removal of the OECD test 
guidelines 452, 409 after full 
replacement of the dog studies  
Actors: OECD members 

Reinforce existing 
partnership and 
promote 
collaboration 
between 

(Agrochem) HESI joint TEA and PBPK Committees (US 
EPA, industry, EFSA, animal protection NGOs) on the 
development of a waiver framework for the 90-day dog study 
for agrochemical risk assessment [25] 
Actor: HESI 

151 

 
 
 
 
  
 
 
 
 
 
 
 
international 
actors 

(Human and veterinary medicine) Facilitation of international 
harmonisation through the activities of the International 
Medicines Regulators Working Group on 3Rs (IMRWG3Rs) 
see Terms of Reference (ToR) for the International 
Medicines Regulators’ Working Group on 3Rs [24] 
Potential Actors: International medicines regulators WG on 
3Rs 

Legend: 
White box: activity not initiated 
Yellow box: ongoing activity 

152 

 
 
 
 
 
References 

Published papers 

1.  Bishop et al. 2023. Is the dog study necessary for pesticide toxicity testing? Crit Rev Toxicol 53(4): 

207-228.   

2.  Bower et al.2022. The Dog as a Second Species for Toxicology Testing Provides Value to Drug 

Development. Int J Toxicol. 2022 Dec;41(6):431-441 

3.  Chien. et al. 2023. Re-evaluating the need for chronic toxicity studies with therapeutic monoclonal 

antibodies, using a weight of evidence approach. Regulatory Toxicology and Pharmacology, 138, 
105329. 

4.  Galijatovic-Idrizbegovic et al. 2016. Role of chronic toxicology studies in revealing new toxicities. Reg 

Toxicol Pharm 82: 94-98 

5.  Guffroy et al. 2025. Scientific and Regulatory Policy Committee Points to Consider: Proposal and 
Recommendations to Reduce Euthanasia of Control Nonhuman Primates in Nonclinical Toxicity 
Studies. Toxicologic Pathology, 53(3):01926233241309905. 

6.  Panzarea et al. 2022. Editorial: Relevance of dog studies for the derivation of health-based guidance 

values for plant protection products approval. EFSA Journal 2022;20(9):e200923  

7.  Prior et al. 2020. Opportunities for use of one species for longer-term toxicology testing during drug 

development: A cross-industry evaluation. Reg Toxicol Pharm 113: 104624.  

8.  Sewell et al., 2014. Recommendations from a global cross-company data sharing initiative on the 

incorporation of recovery phase animals in safety assessment studies to support first-in-human clinical 
trials. Regulatory toxicology and pharmacology, 70(1)): 413-429.  

Hyperlinks 

9.  https://open.efsa.europa.eu/questions/EFSA-Q-2024-00199?search=2024-00199 
10.  https://www.nc3rs.org.uk/our-portfolio/review-use-two-species-regulatory-toxicology-studies 
11.  https://nc3rs.org.uk/crackit/virtual-second-species  
12.  www.Hesiglobal.org 
13.  https://nc3rs.org.uk/3rs-resource-library/study-designs-pharmaceutical-and-chemical-development; 
14.  https://nc3rs.org.uk/our-portfolio/reducing-use-recovery-animals 
15.  https://www.vict3r.eu/  
16.  https://www.graal-defenseanimale.org/english/ 
17.  https://beaglesofburgundy.org/ 
18.  https://www.white-rabbit.org/rehabilitation/ 
19.  https://www.facebook.com/Individus63  
20.  https://www.ema.europa.eu/en/overview-current-regulatory-testing-requirements-medicinal-products-

human-use-opportunities-implementation-3rs-scientific-guideline 

21.  https://www.ema.europa.eu/en/documents/scientific-guideline/draft-reflection-paper-current-regulatory-

testing-requirements-medicinal-products-human-use-opportunities-implementation-3rs-revision-
1_en.pdf  

22.  https://www.ema.europa.eu/en/documents/scientific-guideline/draft-reflection-paper-current-regulatory-

testing-requirements-veterinary-medicinal-products-opportunities-implementation-3rs-revision-
1_en.pdf 

23.  Development of a Roadmap for Action on New Approach Methodologies in Risk Assessment | EFSA 
24.  Terms of Reference (ToR) for the International Medicines Regulators’ Working Group on 3Rs 
25.  Transforming the Evaluation of Agrochemicals (TEA) - HESI - Health and Environmental Sciences 

Institut 

26.  Regulatory acceptance of new approach methodologies (NAMs) to reduce animal use testing | European 

Medicines Agency (EMA)

153 

 
 
Reducing and refining long-term toxicity testing  
ECETOC Thought starter: Reduction of animal numbers under the current EU regulatory schemes 

Note: ECETOC is a member of the Human Health working group developing actions for this 
roadmap. The recommendations described here have been endorsed by the Human Health working 
group. 

Key recommendations: 

- 

Implement  tiered  approach  into  EU  REACH Annex  XI  (in  silico,  in  vitro,  Smart  in  vivo, 
regulatory in vivo studies) 

-  Amend  REACH  Annexes  to  allow  replacing  90-day  repeated  dose  toxicity  information 

requirement by enhanced 28-day studies 

-  Develop guidance documents at OECD-level for the design of Smart in vivo studies 
-  Conduct further research to reduce study duration and/or animal numbers and to use add-ons in 

reproductive/developmental toxicity screening studies according to OECD TG422. 

-  Adopt  improved  use  of  kinetic  information  at  all  levels  (in  vitro  kinetics,  in  silico 
modeling/PBK/qIVIVE, in vivo (Smart) studies with integrated kinetic), to allow a more human 
relevant assessment and less reliance on animal studies.  

The action steps are laid out for inclusion in the EU COM roadmap to phase out animal testing 
enabling a reduction through refinement in current animal testing under the REACH regulation. 
Reduction of animal testing can already be implemented under the current regulatory paradigm. It is 
important to recognize that the transition to animal-free approaches may require longer lead times in 
certain cases, such as difficult-to-test substances. The work supporting the proposal below is currently 
ongoing  as  part  of  the  ECETOC  Human  Health  Transformational  Program  (HH  TP),  the  Staged 
Assessment and Smart studies Task Forces. 

Summary of the approach 

ECETOC developed a framework for the use of alternatives to animal approaches in assessing the safety 
of chemicals in Ball et al. (2022). The ECETOC framework is a tiered approach using the Threshold of 
Toxicological Concern TTC (Tier 0),  in silico assessment (Tier 1),  in vitro assessment (Tier 2) and 
targeted Smart in vivo studies (Tier 3) which includes hazard characterisation, exposure assessment and 
risk assessment. The framework can be applied to develop and test a hazard hypothesis and to support 
read-across  and  grouping.  If  a  decision  for  classification  or  safety  assessment  can  be  made  with 
confidence at one of the tiers, further testing might be omitted. Therefore, the ECETOC framework can 
support reducing and refining animal testing during the transition to phase out animal testing. 

The framework became one of the drivers behind the idea of the European Partnership for Alternative 
Approaches to Animal Testing (EPAA) to promote a Designathon which called for ideas and concepts 
on  how  non-animal  approaches  could  be  used  for  the  potentially  new  classification  system  of 
chemicals for “human systemic toxicity” (EPAA 2023). 

As part of the EPAA’s ‘NAM Designathon 2023’ challenge for human systemic toxicity (Worth et al. 
2025),  ECETOC  has  further  developed  an  exclusively  non-animal-based  classification  system  for 
“human systemic toxicity” (Doe et al. 2025). While both EPAA and ECETOC share the common goal 
of  advancing  alternative  methods,  ECETOC’s  proposal  adopts  a  different  approach.  Specifically,  it 
focuses on repeated dose toxicity, similar to the assessment carried out in the classification for ‘Specific 
Target Organ Toxicity – Repeated Exposure’ (STOT- RE) (Ball et al. 2022; Doe et al. 2025). 

During the transition period it would be prudent to retain the four major categories of the classification 
system: general toxicity, carcinogenicity, reproductive toxicity and mutagenicity, within any revisions. 

154 

 
 
The first area to be revised would be general toxicity, broadly corresponding to STOT-SE and STOT-
RE. 

Furthermore, a follow-up publication to Doe et al. (2025) is currently being drafted, where the last tier 
of the original framework for targeted Smart in vivo studies (Tier 3) is addressed. 

Extended  or  “Smart”  studies  can  be  defined  as  in  vivo  studies  that  employ  additional  or  different 
endpoints from conventional studies. We envision that these in vivo studies will be of short duration 
(e.g. 28 days). Smart 28-day studies will comply with current OECD test guidelines and be enhanced 
with modern technologies, such as ‘omics’, to obtain information on both the toxicodynamics and the 
kinetics of the substance studied (Henriquez et al. 2024). 

In addition to the above-mentioned paper focused on Smart in vivo studies, another paper is planned to 
address what the full framework would entail. How each of the parts would work combined and how 
the use of Smart in vivo studies can increase confidence in the characterisation of a hypothesized hazard 
for the whole assessment. 

Method readiness, applicability domain and validation status 

Smart studies85 

One aspect of the tiered testing strategy proposed by ECETOC is to conduct an extended or smart 28-
day in vivo study to close relevant data gaps regarding STOT-RE classification if the 90-day study is 
omitted. Therefore, an analysis of a database with substances tested in 28-day and 90-day repeated dose 
toxicity studies was conducted to identify potential information gaps by comparing the outcomes of 28-
day studies to those of 90-day studies. We evaluated the extent to which short-term studies with 28-day 
exposure can or cannot forecast the target organs and effects relevant for the classification of substances 
according to STOT-RE. 

Our  investigations  comparing  28-  and  90-day  studies  have  shown  that  information  of  relevance  to 
STOT-RE classification can be determined by either duration of study. Specific gaps (pathology effects 
only  observed  after  90  days  duration)  could  be  effectively  covered  by  the  additional  measurements 
(omics,  specific  markers  of  toxicity)  envisaged  in  Smart  in  vivo  studies.  The  comparison  further 
indicated  that  enhanced  blood  analysis  for  hormones  and  haematology  is  helpful  to  improve  the 
prediction of organ toxicity in 28-day studies for 90-day studies. Blood can also be used for genotoxicity 
assessment. The Smart study should also provide data for the transcriptional changes in some specific 
tissues as well as toxicokinetic assessment, to account for potential accumulation of parent compounds 
or metabolites. To achieve this, blood microsampling technologies will have to be introduced (Chapman 
et al. 2014). The study design and hence the additional investigations and technologies to be used serve 
two purposes: 

(1)  Enhance the sensitivity of the 28-day study to reduce the necessity for a 90-day study 

(default – to always include) and  

(2)  based on the results of the in silico (Tier 1, section 2.2) and in vitro (Tier 2, section 2.3) 
assessments to allow for a more in-depth investigation of the modes of action identified 
(bespoke study design), to allow for example the assessment of human relevance and 
sensitivities.  

Therefore,  in  addition  to  the  general  improvements  in  the  study  design,  it  is  also  envisioned  that 
compound specific parameters and/or intermediate time points could be included, based on the results 
of Tier-1 and -2 information. An overview of sample types and potential add-on techniques is provided 
in Table 10. Not all listed technologies can be added to a 28-day study in all combinations, there are 

85 ECETOC’s transformational program A framework to incorporate NAMs in regulatory toxicology. Bennard van 
Ravenzwaay, John Doe, and Lucy Wilmot. O-28, Pg 72, Abstracts of the 52nd EEMGS meeting – ECETOC Session Arh Hig 
Rada Toksikol 2024;75(Suppl. 1):22-209. https://eemgs.eu/wp-content/uploads/2024/10/Arhiv_75_Supp1-split.pdf   

155 

 
 
 
limitations  with  regards  to  sampling  time  points,  number  of  samples,  animal  welfare  as  well  as 
compatibility of methodological requirements. 

Overall, this would lead to a similar level of knowledge and confidence from the same or fewer animals 
studied, even more so for potential future waiving of high animal usage studies such as carcinogenicity, 
and multi or extended-one-generation and developmental toxicity studies. Work is currently ongoing, 
and we expect to publish our findings also related to these additional long-term endpoints in the first 
half of 2026. 

Table 10: Proposed add-ons for Smart in vivo studies 

Tissue/Types of samples 

Possible additional technology 

Biofluids 

• Blood Plasma 
• Blood Serum 
• Urine 
• Cerebrospinal fluid (CSF) 

Tissues 

• Fresh 
• Frozen 
• Fixed 

•  Toxicokinetic parameters 
•  Hormonal measurements 
•  Plasma-Metabolomics 

•  Toxicokinetic parameters 
• 
•  Metabolomics/Lipidomics (fresh and frozen 

Immunohistochemistry (IHC) 

only) 

•  Whole genome mRNA transcriptomics  
•  Single gene mRNA measurements e.g. 

hormones 

•  Gene panel mRNA measurements  

Examples of enhanced in vivo studies: 

Metabolomics  as  read-across  tool: An  example  with 3-aminopropanol  and  2-aminoethanol  - 
ScienceDirect (Sperber et al. 2019) 

The study aimed to use metabolomics to support a read-across approach from 2-aminoethanol (MEA) 
to 3-aminopropanol (3AP). Wistar rats were treated with MEA and 3AP at different dose levels. Blood 
samples  were  collected  for  metabolome  analysis  and  clinical  examinations  and  body  weight 
measurement  were  performed. The  study  demonstrated  the  high  structural  and  functional  similarity 
between  MEA  and  3AP,  supporting  the  use  of  read-across  to  fill  data  gaps  for  3AP's  REACH 
registration. 

Utility of in vivo metabolomics to support read-across for UVCB substances under REACH - 
PubMed (Kamp et al. 2024) 

The study aimed to use metabolomics to support a read-across approach for UVCB substances under 
REACH.  Researchers  analysed  15  steam-cracked  distillates  and  six  major  constituents,  including 
indene  and  dicyclopentadiene,  using  plasma  metabolomics  in  a  14-day  rat  oral  gavage  study. They 
identified metabolome patterns and grouped substances based on biological similarity, confirming target 
organs affected and supporting REACH registration. 

 Predicting  Drug-Induced  Hepatotoxicity  Using  QSAR  and  Toxicogenomics  Approaches  | 
Chemical Research in Toxicology (Low et al. 2011) 

The  study  aimed  to  develop  hybrid  models  combining  QSAR  and  toxicogenomics  to  predict 
hepatotoxicity.  Researchers  analysed  various  chemicals,  including  acetanilide,  thioamide,  alkyl 
chloride,  and  styrene  substructures,  using  chemical  descriptors  and  toxicogenomics  data.  They 
identified toxicophores and improved predictive accuracy for liver toxicity, demonstrating the value of 
hybrid modelling for identifying chemical alerts. 

PARC Additional Deliverable AD5.2 Guidance on animal studies in PARC, Work Package 5 – 
T5.1 (PARC 2023) 

156 

 
 
 
 
 
PARC has the option to conduct and / or subcontract test guideline (TG) conform animal studies to 
contract research organizations (CRO), if needed, to close regulatory data gaps of concern (task 5.1). 
To  benefit  from  such studies,  on top  of  the  test guideline  required endpoints like  histopathology  or 
clinical chemistry, additional parameters will be investigated (referred to as “TG+”) like toxicokinetics, 
toxicogenomics of target organs, other data based on alternatives to animal approaches or intra-organ 
concentrations. 

In silico methods 

The  use  of  Quantitative  Structure–Activity  Relationship  models  ((Q)SAR)  tools  for  predicting 
toxicological properties within the context of non-animal approaches has become a standard approach.  

The software and models commonly used consist of a mix of well-established commercial applications 
and  publicly  available,  free-to-use,  applications  that have  been  extensively  employed for  regulatory 
purposes, screening, prioritization, and research. Models include QSAR and SAR based models, and 
expert systems. Importantly, these models have undergone validation in accordance with the OECD 
principles  (2014)  and  they  are  accompanied  by  QMRF  (Quantitative  Model  Reporting  Format) 
documentation  that  adheres  to  guiding  principles  (OECD  Guidance  Document  on  the Validation  of 
(Quantitative) Structure-Activity Relationship (QSAR Models, 2014). Furthermore, the OECD (Q)SAR 
Assessment  Framework  (QAF)  (2023b;  2024)  offers  comprehensive  guidance  for  the  regulatory 
application  of  QSAR  models  and  ultimately  contribute  to  the  broader  acceptance  of  (Q)SARs  for 
regulatory purposes (OECD Series on Testing and Assessment No. 386 and No. 405). 

While no computation tool has been adopted as OECD test guideline, (Q)SAR can already be applied 
in various exploratory uses and support regulatory applications. For instance, in adapting information 
requirements  for  hazard  assessment  and  classification,  the  REACH  R.6  guideline  serves  as  a 
foundational reference for employing (Q)SAR approaches. 

In practice, (Q)SAR models are more frequently employed within a weight of evidence framework, 
particularly for assessing more complex endpoints. This approach allows for the integration of various 
types of information, which can help mitigate the uncertainties associated with (Q)SAR predictions. By 
combining (Q)SAR data with other relevant information, a more robust assessment can be achieved, 
ultimately supporting regulatory decision-making. 

What would make QSAR predictions more acceptable: 

•  Development of (Q)SAR models in accordance with regulatory set standards 
•  Application  of  models  validated  according  to  the  OECD  guiding  principles  for  (Q)SAR 

validations (2014) 

•  Assessment of predictions and reporting in accordance with the requirements set by QAF (2023, 

2024).  

•  Transparent reporting of uncertainties 
•  Harmonised acceptance criteria across legislations 

It would be beneficial if regulators could provide a comprehensive overview of accepted adaptations of 
standard  information  requirements  based  on  (Q)SAR  predictions,  specifying  the  relevant  endpoints. 
Additionally,  incorporating  transparent  reporting  on  any  rejected  adaptations  would  foster  trust  and 
clarity.  This  approach  would  not  only  outline  clear  directions  for  improving  the  use  of  (Q)SAR 
predictions in data gap filling but also encourage registrants to submit data generated through (Q)SAR 
methodologies. 

In vitro methods 

For the in vitro bioactivity assessment, Doe et al. (2025) used publicly available data from the ToxCast 
database (US EPA 2024) which contains the bioactivity data for over 9,500 chemicals from various 

157 

 
 
high-throughput  screening  assays  covering  a  range  of  endpoints  ranging  from  cell  viability  and 
proliferation assays to more specific hormone receptor assays.  

The  validation status  of the  assays varies,  they  are predominantly used  for  substance  screening  and 
prioritization. ToxCast and Tox21 represent a library of medium- and high-throughput in vitro assays 
sponsored by US EPA. In general, each assay assesses the effects of approximately 300-8000 chemicals 
against  a  defined  target,  which  ranges  from  cell  culture  systems  to  cell-free  assays.  Phase  I  of  the 
ToxCast program focused on a set of around 300 chemicals (primarily agrochemicals), with subsequent 
phases of the program expanding the list to up to 4000 chemicals. The Tox21 program represents an 
expanded assessment of 8000 chemicals against a smaller set of targets. Data generated by the ToxCast 
and Tox21 programs are freely available via the EPA Comptox dashboard as well as via the NTP ICE 
dashboard. 

When using ToxCast data for repeat-dose toxicity assessments, data on both potency (e.g., AC50 value) 
and severity (adverse effects associated with in vitro assay target) should be evaluated. For potency, it 
will be important to consider which point of departure value to select (e.g., AC50, ACC, BMDL) as well 
as defining potency criteria (i.e., what bioactive concentration ranges indicate high, intermediate and 
low potency). Moreover, when using any in vitro assay to understand in vivo hazards, it is important to 
understand the relevance of the dosimetry. For example, a xenobiotic that inhibits aromatase at high 
doses (>50 µM) may not be biologically relevant if the maximum attainable internal dose is far lower 
in vivo. As such, in vitro to in vivo extrapolation (IVIVE) assessments should be undertaken to better 
understand the likely maximum tolerated dose in vivo. 

Furthermore, consensus on the severity rating for assay endpoints will be needed to ensure consistency 
in  these  assessments.  Guidance  on  how  to  consider  negative  assay  data  also  will  be  needed.  When 
evaluating the use of ToxCast data, it is prudent to understand limitations in assay coverage such as 
cases where all molecular initiating events within an AOP may not be covered. One example is with 
skin sensitization where soft electrophilic xenobiotics may be identified in the ARE-NRF2 dissociation 
assay while no assays to identify hard electrophiles are currently available via the ToxCast database. 
Moreover, many assays lack metabolism and as such, are unlikely to identify compounds that need to 
be metabolized to their active toxic form. An example are the phosphothionates, such as malathion, 
which need to be oxidatively desulphonated to the active oxon in order to inhibit acetylcholinesterase.  
Furthermore, a more thorough understanding of the MIEs that drive repeat-dose toxicity and a better 
coverage of these by HTP assays would be needed to more fully predict  in vivo outcomes. A tiered 
assessment that incorporates other data streams may help to identify whether there are gaps in biological 
coverage during specific chemical assessments, an example of this has already been recorded in Doe et 
al. (2025).  

Commercial availability of methods 

It  is  beyond  the  scope  of  this  document  to  lay  out  the  commercial  availability  of  all  methods 
referenced. In general, the commercial availability of methods is key to allow method implementation. 
New method proposals need to be globally available and readily accessible at several CROs to properly 
cover user demands at sufficient quality. Regulatory certainty with regards to acceptance is needed for 
CROs to develop a business case for offering a new method.  

In the context of the proposed 28-day Smart study design the technologies which are being considered 
are not new (transcriptomics, metabolomics, measurement of parent and metabolites in blood, specific 
markers for organ toxicity) and can be implemented with common knowledge and expertise. Therefore, 
commercial availability will not be a hurdle for its implementation.  

158 

 
 
As sampling of tissues and fluids (in vivo) as well as intra- or extra-cellular matrixes has been shown to 
be a key component for interlaboratory comparability of results, guidance is needed. Therefore, ECHA 
has taken the lead of a project to develop the guidance and best practices for sample collection suitable 
for omics analyses, included under OECD WHPA workplan. This guidance aims to ensure sufficient 
quality samples, support harmonization through example protocols, provide clarity for all stakeholders 
on  principles  for  good  quality  samples  and  encourage  sampling  in  ongoing  studies.  The  guidance 
document is being developed by an ad hoc drafting group and supported by the OECD Expert Group 
on  Omics.  The  OECD  Omics  Reporting  Framework  (OECD  2023a)  is  intended  to  describe  the 
information that should be reported when an omics technology is applied in the context of regulatory 
decision-making, to enable assessment of the quality of the study from its design through the collection, 
analysis, and reporting of data.  

The  guidance,  however,  does  not  extend  to  the  interpretation  of  these  data,  which  is  an  ongoing 
ECETOC task force. These activities indicate that these ‘omics technologies have reached a state of 
maturity that would allow for their use in regulatory studies and decision-making processes.  

In  the  context  of  demonstrating  regulatory  applications,  a  case  study  using  the  phenoxy  herbicides 
MCPA, Mecprop-p and Dichlorprop-p showed that with the use of a metabolomics in a 28-day study 
in a read across approach a 90-day study could be waived without affecting the regulatory outcome (van 
Ravenzwaay et al. 2016). It is acknowledged that proper training of CROs and the development of SOPs 
will enhance quality and reliability of the obtained results. 

For the in silico models used by Doe et al., general hazard prediction is well-established, for example 
for predicting Ames mutagenicity. Additionally, there is a wide range of models available for routinely 
requested endpoints, such as acute oral toxicity, skin irritation, and eye irritation.  

Description of legislative areas that can make use of the approach, timing and recommended 
steps for regulatory acceptance 

The use of a tiered approach and Smart/enhanced in vivo studies is possible under the current REACH 
and CLP legislations but the regulatory acceptance of such an approach is unclear. Protection goals to 
be met by non-animal approaches need to be defined and this includes the definition of an acceptable 
protection level for tiered approaches. As laid out in the technical sections of this document, ECETOC 
believes  that  current  protection  goals  can  be  met  using  Smart  in  vivo  studies  while  allowing  for  a 
reduction and refinement of animal testing under the current regulatory paradigms. The implementation 
of  Smart  studies  should  require  only  limited  modification  of  the  current  REACH  Annex  VIII-X 
(Column 2). 

Further, regulatory compliance requires testing proposals for higher tier testing for human health effects 
to be submitted when developing and refining a REACH registration dossier. As current information 
requirements are largely based on in vivo testing via current OECD test guidelines, there is no time in 
the registration process to apply a tiered approach to develop a hazard hypothesis and adapt  in vivo 
testing protocols towards Smart study design.  

Therefore, REACH Annexes on standard information requirements need to be amended to include in 
silico and in vitro methods as well as adaptations for Smart study designs. This should not increase the 
listed methods required to run nor the mandatory requirements but allow flexibility to choose the most 
suitable approach while still reducing and refining animal testing where possible. REACH Annex XI 
should be amended to implement the use of tiered approaches similar to the use of Weight of Evidence 
(Annex  XI,  Section  1.2)  for  both  demonstrating  the  presence  and  absence  of  hazard  and  the 

159 

 
 
 
classification or non-classification of substances. Specifically for the case of classification, CLP text 
needs to be amended to lay out the principles for classification based on tiered approaches. 

REACH guidance on Information Requirements and Chemical Safety Assessment (ECHA 2025) needs 
to be amended to lay out the use of tiered approaches for the assessment of systemic toxicity endpoints. 

Dialogue between registrants and regulatory agencies is needed in the early implementation phase to 
refine  the  use  of  tiered  approaches.  Ideally,  a  new  review  process  for  tiered  approaches  to  support 
ongoing registrations will be implemented.  

Regulatory changes to other regulations for biocidal products, food and feed additives, plant protection 
products,  cosmetics,  detergents,  and  medicinal  products  have  been  discussed  but  no  conclusive 
recommendation  can  be  given.  It  is  evident  that  regulations  that  include  1:1  dossier  reviews  and 
flexibility on how to fulfil information requirements (e.g. cosmetics regulation) are amenable for the 
adoption of tiered approaches. 

Any additional recommendations for research in the area/Further technical work needed  

For  successful  implementation  of  tiered  approaches  and  enhanced/Smart  study  designs,  guidance  is 
required.  Also,  additional  scientific  work  can  be  envisioned  to  broaden  the  applicability  of  Smart 
studies. The following needs have been identified: 

1)  Development of Smart studies including reproductive and developmental toxicity endpoints 
The initial Smart study project focused on modifications to the 28-day study design as described in 
OECD  TG  407.  It  is  desirable  to  expand  the  concept  to  combined  reproductive  and  developmental 
toxicity, repeated dose toxicity screening studies (OECD TG 422) as this study type can already be used 
to  fill  the  Annex  VIII  information  requirement  and  inform  on  the  design  of  Annex  X  studies. 
Reproductive toxicity studies require special  considerations with regards to sampling time points as 
animals go through sensitive periods like mating, pregnancy and lactation which will have an impact 
on evaluation, for example for transcriptomics signatures or the metabolome (Henriquez et al. 2024). 

2)  Further reduction of study duration 

Opportunities to further reduce study durations should be explored. Published studies support the use 
of  earlier  time  points  for  detection  of  changes  to  the  transcriptomic  signatures  or  the  metabolome 
(Everett 2024; Sperber et al. 2019). Genotoxicity endpoints also may require a shorter exposure duration 
only. 

3)  Develop sampling guidance 

A Standard Project Submission Form was approved at the OECD WNT-37 meeting in April 2025 to 
include an update on sample cryopreservation suitable for omics in OECD TGs 407, 408, 422, 421, 
203, 210, 236. 

4)  Develop guidance documents to lay out the experimental design of enhanced/SMART in vivo 

studies 

Including but not limited to dose level setting, sampling time points and number of animals per dose 
group.  

5)  Develop or adapt guidance on data analysis and reporting  

160 

 
 
 
ECETOC  has  been  actively  involved  in  advancing  the  use  of  omics  technologies  in  regulatory 
toxicology.  The  Regulatory  Omics  Data  Analysis  Framework  (R-ODAF)  provides  guidelines  for 
analysing  omics  data  for  regulatory  purposes  and  helps  in  consistent  and  accurate  interpretation  of 
complex omics datasets (Verheijen et al. (2020); Verheijen et al. (2022)). Omics Data Reporting and 
Documentation (OORD) focuses on establishing standards for reporting and documenting omics data 
to ensure data used in regulatory assessments are of high quality and reproducible. Work is ongoing to 
develop an Omics Data Interpretation Framework for Regulatory Application (ODIFRA) (Gant et al. 
2023). These efforts will support the implementation of Smart studies. 

6)  Develop  case  studies  to  exemplify  the  use  of  enhanced  studies  to  demonstrate  biological 
similarity in read-across 

Enhanced studies have shown promise for the demonstration of biological similarity and to support 
grouping and read-across (OECD GD194, under revision). Further case studies should be conducted 
and published to support the evidence. Similarly, case studies exemplifying the use of Smart studies to 
waive higher tier testing should be published. 

7)  Further ECETOC work 

ECETOC  foresees  to  conduct  and  publish  a  fully  integrated  case  study  on  the  Staged  Assessment 
Framework (in silico/in vitro/Smart in vivo tiers). 

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4.5. Developmental neurotoxicity - the DNT in vitro battery  

This chapter was originally developed by one of the working group members (Swiss Centre for 
Applied Human Toxicology, SCAHT) for the human health working group, which reviewed its 
content before inclusion into the SWD. 

 Implementing change for developmental neurotoxicity (DNT) assessments 
Summary 

Developmental neurotoxicity (DNT) testing is undergoing a paradigm shift from animal-based studies 
to mechanistic, human-relevant alternatives to animal testing. Traditional in vivo assays are resource-
intensive, slow, and of limited translational value, leaving most chemicals untested for potential effects 
on brain development. The DNT In Vitro Battery (DNT IVB) addresses these limitations through 17 
complementary  human-relevant  assays  covering  key  neurodevelopmental  processes  such  as  neural 
proliferation, migration, differentiation, and synaptogenesis.  

Over the past 20 years, considerable advancements have been made to develop assays measuring KNPs. 
The early scientific workshops and the regulatory necessity led to a movement to implement NAMs 
into  chemical  DNT  assessment,  by  EFSA,  and  US  EPA,  overseen  by  OECD. This  multiyear  effort 
provided  the  foundation  for  an  OECD  project  that  started  in  2018,  which  described  DNT  assays, 
provided criteria that allow evaluation of the relevance of the data to DNT, and helped determine the 
degree of certainty of the results to inform the use of a DNT in vitro battery (DNT-IVB) in regulatory 
hazard determinations. In April of 2023, the OECD Working Party of National Coordinators of the Test 
Guideline program approved document 377 that described the DNT-IVB (OECD, 2023). 

 Currently multiple coordinated initiatives with several synergistic projects are underway to accelerate 
the development, standardization and regulatory use of NAMs for neurotoxicity. Within the European 
Union,  these  include  large  collaborative  efforts  such  as  PARC,  ONTOX,  RiskHunt3r,  CHIASMA, 
EFSA  Environmental  Neurotoxicants  activities,  and  EFSA  funded  Brain  Health  project,  (Tal  et  al., 
2024; EFSA., 2025). In parallel, there are various initiatives led by NIEHS, FDA and US EPA. OECD 
continues to support coordination and capacity building through guidance documents, webinars, and 
training materials, facilitating consistent interpretation and uptake across regulatory jurisdictions. As 
such, the DNT IVB can be used for hazard and risk assessment. A tiered testing framework is proposed, 
integrating computational (Tier 0), in vitro (Tier 1), confirmatory mechanistic (Tier 2), and complex 
model (Tier 3) approaches, enabling both hazard and risk assessment applications. Implementation for 
regulatory use requires legislative alignment and structured change management across sectors, guided 
by short-, medium-, and long-term actions encompassing policy adaptation, scientific refinement, best 
practices, and communication. 

The roadmap discusses a short-medium and long-term vision for a more globally harmonized, animal-
free strategy for DNT assessment that ensures high levels of health protection while advancing scientific 
innovation and regulatory efficiency. For a recent OECD review, please consult the recent publication 
“From Concept to Regulation: OECD’s Role in the development of the DNT In Vitro Battery” (Sachana 
et al., 2026) [34].  

History of the approach: why a change for DNT assessments? 

Prevalences  of  neurodevelopmental  disorders  like  autism  spectrum  disorder,  attention  deficit 
hyperactivity disorder or IQ loss have been on an international rise [1, 2, 3, 4, 5]. Such diseases pose an 
immense  burden  to  individuals,  families  and  society  [6].  While  differences  in  diagnosis  have  been 
identified as contributors to culmination of neurodevelopmental diseases, also specific chemicals have 
been strongly associated with adverse neurodevelopmental outcome in humans [7, 8, 9, 10]. Regulatory 
evaluation of the potential of compounds to alter the structure or function of the nervous system during 
pre-  and/or  postnatal  life  (developmental neurotoxicity,  DNT)  has  traditionally depended  on  in vivo 

163 

 
 
 
animal testing. Guideline studies conducted under U.S. EPA or OECD protocols are expensive, lengthy, 
and largely restricted to a narrow set of apical  behavioural endpoints. Their interpretation is further 
complicated  by  methodological  limitations  and,  as  with  many  animal-based  approaches,  substantial 
uncertainty when extrapolating results to humans [11]. Consequently, thousands of chemicals remain 
insufficiently assessed for their potential to interfere with nervous system development, leaving 
considerable  gaps  in  hazard  evaluation.  To  overcome  these  shortcomings,  new  approach 
methodologies  (NAMs)  are  being  advanced  to  generate  mechanism-based  and  human-relevant 
information on chemical hazards and risks while reducing reliance on animal use. Alternatives to animal 
testing integrate multiple complementary tools, including human-derived in vitro systems (e.g., neural 
cell  cultures),  alternative  species  models  (e.g.,  zebrafish),  advanced  chemical  analytics,  and 
computational in silico approaches. Over the past decade, coordinated international efforts have fostered 
the development and application of the DNT In Vitro Battery (IVB), a large panel of assays targeting 
key neurodevelopmental processes such as neural proliferation, migration, differentiation and neural 
network formation. The DNT IVB is intended to provide regulatory authorities with mechanistic data 
suitable for supporting chemical DNT hazard assessments [12]. 

The DNT IVB  

The DNT IVB was set up to cover a broad variety of key neurodevelopmental processes (Figure 12), 
which are fundamental for brain development.  

Figure 12: Schematic representation of key neurodevelopmental processes. These processes cover the basics 

of brain development. Figure from [13]. 

Seventeen assays currently cover this depicted biological applicability domain (Table 11). The assays 
have  been  intensively  characterized  and  described  [14]  and  also  used  for  mode-of-action  analyses, 
screening and AOP development (respective references can be found in [15, 12]).  

Table 11: Assays currently in the DNT In Vitro Battery [12]. 

Test Method 
(Assay) 

Proliferation 
NPC1 

Test System 
(Cell culture) 

Assay Duration/ 
Chem exposure 

DNT  
Endpoint 

Viability/Cytotoxicity 
Endpoint 

human NPC grown as 
proliferating 3D 
neurospheres 

72 h / 72 h 

neurosphere area, BrdU 
incorporation in 
dividing cells 

Resazurin reduction 
/LDH release 

164 

 
 
 
 
 
hNP1 Prolif 

human NPC 

24 h / 24 h 

BrdU incorporation in 
dividing cells 

ATP level 

human NPC 

24 h / 24 h 

apoptosis pathway 
(Caspase) activation 

ATP level 

Apoptosis 
hNP1 Apop 

Migration 
UKN2 

NPC2a 

human NSC-derived 
neural crest cells 

human NPC grown as 
differentiated 3D 
neurospheres 

72 h / 24 h 

72 h / 72 h 
120 h / 120 h 

number of cells moving 
into defined area 

mean distance of radial 
glia (nuclei negative for 
neuronal and 
oligodendrocyte 
markers) from edge of 
sphere 
mean distance of 
tubulin-positive 
neurons from edge of 
sphere 
mean distance of O4-
positive 
oligodendrocytes from 
edge of sphere 

Calcein-AM vital 
dye 
Resazurin 
reduction/LDH 
release 

Resazurin 
reduction/LDH 
release 

Resazurin 
reduction/LDH 
release 

Resazurin 
reduction/LDH 
release 

Resazurin reduction 
/LDH release 

Calcein-AM vital 
dye 
Calcein-AM vital 
dye 

NPC2b 

NPC2c 

human NPC grown as 
differentiated 3D 
neurospheres 

human NPC grown as 
differentiated 3D 
neurospheres 

120 h / 120 h 

120 h / 120 h 

Neuronal Differentiation 
NPC3 

human NPC grown as 
differentiated 3D 
neurospheres 

120 h / 120 h 

number of tubulin-
positive neurons 

Neurite outgrowth 
NPC4 

UKN4 

UKN5 

hN initiation 

Cortical 
initiation 

human NPC grown as 
differentiated 3D 
neurospheres 
human NSC-line 
(v-myc transformed) 

human iPSC-derived 
peripheral (sensory) 
neurons 
human NPC-derived 
neurons 
rat primary neocortex 

Neurite Maturation and Synaptogenesis 
rat primary neocortex 
Cortical 
maturation 
Cortical 
synapto 
Glial Differentiation 
NPC5 

rat primary neocortex  

human NPC grown as 
differentiated 3D 
neurospheres 

120 h / 120 h 

neurite length & area 

72 h / 24 h 

neurite area 

24 h / 24 h 

neurite area 

48 h / 48 h 

neurite length 

cell morphology 

48 h / 48 h 

neurite length 

cell morphology 

288 h / 120 h 

dendrite length 

cell morphology 

288 h / 120 h 

synapse number 

cell morphology 

120 h / 120 h 

number of O4-positive 
oligodendrocytes 

Resazurin reduction 
/LDH release 

Neural Network Formation 
Cortical MEA 

rat primary neocortex 

288 h / 288 h 

 Action potential spike 
and burst parameters 
related to network 
connectivity 

Resazurin 
reduction/total LDH 

165 

 
 
 
As the key neurodevelopmental processes covered by the DNT IVB are cellular key events (KE), a 
generic AOP  was  suggested  with  the  cellular  responses  representing  the  endpoints  covered  by  the 
battery (Figure 13). 

Figure 13: Diagram illustrating a selection of components of a generic Adverse Outcome Pathway (gAOP) 
for DNT. The gAOP shows the relationship between MIEs such as altered cell signaling, and key events 
(KEs)  at  the  cellular  level  (neurodevelopmental  processes)  and  organ  level  (nervous  system 
connectivity), which lead to an adverse outcome (AO) for the individual. Modified from [12].  

Mapping Initiatives and Securing the Learnings 

The DNT IVB was assembled in 2018 [16, 17] according to established readiness criteria [18]. Initially 
developed by two laboratories in the EU and one in the US, the battery was initially challenged with 
positive and negative compounds proving the test methods’ performance not only for mode-of-action 
analysis of few compounds, but also as screening tools with broad chemical and biological applicability 
domains  [19,  20].  During  the  process  of  OECD  recommendation,  OECD  member  states  and 
international agencies commented on the use, reporting and performance of the DNT IVB. While ready 
to  be  used  immediately  [12],  more  efforts  on  lab-to-lab  transfer,  glia  cell-mediated  DNT,  IATA 
(integrated approaches to testing and assessment) case studies, adverse outcome pathways (AOPs) as 
well as training were thought to benefit the overall trust in the DNT IVB. To accommodate these matters, 
there are a variety of initiatives on the way to support the DNT IVB on its path to regulatory application: 

•  EFSA Working Group on quantitative in vitro to in vivo extrapolation (QIVIVE) of the DNT IVB 

data (2024 - 2026). 

•  The EFSA DNT-RAP2 project is transferring all 17 assays (Table 11) of the DNT IVB from the 
developers`  laboratories  to  an  independent  laboratory  of  a  contract  research  organization 
(www.dntox.de; 2024 - 2027). 

•  The US Division of Translational Toxicology at the National Institute of Environmental Health 
Sciences  (NIEHS)  has  assigned  testing  of  >300  compounds  in  most  assays  of  the  DNT  IVB 
including 
(https://www.niehs.nih.gov/research/atniehs/dtt/strategic-
plan/health/developmental).  While  data  of  the  first  +100  compounds  are  already  available  in 
ToxCast, the second +100 dataset is produced, but not published yet and the third +100 substances 
are currently being tested (2022 - 2026). 

zebrafish 

•  The  EFSA  Brain  Health  project  (GP/EFSA/ED/2022/01)  aims  at  understanding  the  current 
coverage  of  glia-related  processes  in  the  DNT  IVB  by  focusing  on  astrocytes,  microglia  and 
myelinating  oligodendrocytes.  Single  cell  RNA  Seq  will  inform  on  molecular/cellular  aspects. 
Novel glia-focused AOPs are being set up (2023 - 2027). 

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•  EFSA assigned testing of +100 pesticides (substances with positive and negative outcomes in the 
DNT in vivo guideline study and compounds with no available DNT in vivo data) in the 17 assays 
of the DNT IVB (DNT-RAP1; 2024 - 2027). Results will allow drawing a conclusion of the DNT 
IVB to be suitable for compound classification within the CLP framework. 

•  The ASPIS cluster project ONTOX (https://ontox-project.eu) is creating ontologies for DNT based 

on physiological maps and AOP networks (2021 - 2026).  

•  The  ASPIS  cluster  project  RiskHun3r  (https://www.risk-hunt3r.eu)  produces  DNT  IATA  case 

studies (2021 - 2026). 

•  The public-public Partnership for the Assessment of Risk from Chemicals (PARC) is involved in 
several  DNT  activities.  Refinement  of  DNT  IVB  assays,  AOP  development  [21]  and  DNT 
regulatory case studies including application in a possible new hazard class for neurotoxicity as an 
ECHA initiative (https://youtu.be/Ts55QFMnor4; 2022 - 2029).  

•  The EFSA funded DNT-RAP4 project will provide four IATA case studies on selected compounds 

that diverge between DNT in vivo and in vitro results (2024 - 2027). 

•  The CHIASMA project will provide solutions for DNT and other  alternatives to animal testing  

applications in CLP regulation under REACH (2024 - 2027). 

•  The  OECD  is  publishing  a  series  of  webinars  as  training  material  for  the  DNT  IVB 
(https://www.youtube.com/playlist?list=PLJNHHjqEVIIfn6eDbh1N-tLAhxP45zBft; 2025-2026). 

These activities will further reduce the known main uncertainties, which are highlighted in the OECD 
initial recommendations from 2023 [12]. These include general uncertainties not specific for the DNT 
IVB and DNT IVB-specific uncertainties. Initiatives addressing respective uncertainties are given (in 
brackets in italics): 

1.  General uncertainties concerning in vitro testing: 

•  Toxicokinetics. ADME  (absorption,  distribution,  metabolism,  and  excretion)  of  the  DNT 
IVB assays are limited towards developing brain cells and do not contain e.g. maternal and 
fetal  liver  metabolism  or  barriers  like  the  placenta  or  the  developing  blood-brain-barrier 
(EFSA QIVIVE Working Group). 

•  Endocrine/immune  systems.  A  variety  of  systemic  impacts  of  hormones  and  immune 
signaling on brain development are not captured in the current DNT IVB (EFSA BrainHealth 
and PARC projects). 

•  Chemical domains. So far, there is a relatively limited number of tested compounds in the 
DNT IVB. Certain compound classes (for example volatile compounds) remain challenging 
in vitro (EFSA, NIEHS and PARC chemical testing).  

•  Inter-  and  intra-species  differences.  Some  assays  still  rely  on  animal-derived  neural  cell 
cultures. The human cell-systems have no full coverage of sex or human genetic diversity 
(PARC & EFSA Brain Health projects). 

2.  DNT IVB-specific uncertainties: 

•  Currently incomplete coverage of cell-to-cell interactions in some test systems (EFSA Brain 

Health project). 

•  Limited  availability  of  DNT AOPs  (EFSA  Brain  Health,  PARC,  ONTOX  and  CHIASMA 

projects, SCAHT AOP_HUB). 

•  Negatives/Sensitivity: False negative findings may occur due to a current lack of coverage 
of critical neurodevelopmental processes (EFSA Brain Health project, EFSA RAP4 project). 
•  Positives/Specificity:  False  positive  results  may  occur  based  on  the  uncertainties  listed 
above, mainly kinetics (e.g. no detoxification kinetics, no placental and blood-brain-barrier 

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modelled) or single cell type representation in some of the assays (e.g. neurons with lack of 
astrocytes)  (EFSA  QIVIVE  Working  Group,  EFSA  Brain  Health  project,  EFSA  RAP4 
project). 

•  Validation status/Inter-laboratory testing: While no formal OECD Guidance Document 34 
validation  procedure  was  conducted,  DNT  IVB  assays  were  scientifically  validated  for 
relevance and robustness. An inter-laboratory testing and transfer of the DNT IVB is needed 
(EFSA DNT-RAP2 project).  

While the current DNT IVB is ready-to-use, it will further evolve with scientific innovation that will 
feed into the lifecycle of DNT NAMs. 

This fits into the context of a six-step framework of the DNT IVB, from basic research to regulatory 
application  (Figure  14). It serves  as a  case  example  [22]  that might  be  utilized by  other  fields,  e.g. 
Developmental and Reproductive Toxicity (DART) [23]. The framework describes how the DNT IVB 
assays went from development through key steps towards regulatory acceptance. Scientific validation 
was core during this process ensuring biological relevance and description of applicability domain of 
the assays, toxicological domain covered and intra-laboratory reproducibility. 

Figure  14:The  DNT  New  Approach  Methodology  (NAM)  lifecycle:  a  six-step  framework  to  facilitate 
regulatory implementation. The DNT New-Approach Methodology lifecycle is divided into 6 key 
steps, that reflect the development of the DNT IVB from scientific innovation towards readiness for 
legal implementation. Figure from [22]. 

An ongoing step is the inter-laboratory transfer to a naive laboratory (EFSA RAP-2, see listed initiatives 
and  uncertainties  above).  In  making  the  DNT-IVB  publicly  available,  this  critical  step  will  further 
facilitate the application of the DNT IVB in regulatory frameworks. For this, the DNT IVB could be 
embedded in a defined tiered testing framework or a future defined approach. 

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Proposed DNT testing Framework  
The OECD Expert Group on DNT in vitro has been guiding the establishment, evaluation and regulatory 
use of the DNT IVB over many years. The OECD initial recommendations were a major step on the 
DNT IVB road to regulatory acceptance and application.  
One possible use of the DNT IVB is placing it into a framework like an IATA [24] or a defined approach 
(DA). The framework might be flexible depending on the regulatory question and is  currently under 
discussion at the OECD. There has been the unanimous agreement that the DNT IVB is one step of a 
potential tiered approach and does not contain tiers in itself. This agreement is based on the biological 
rationale of the battery: each key neurodevelopmental process represented by a battery assay is equally 
important, i.e. disturbances in proliferation of neural progenitor cells or alteration of differentiation or 
synaptogenesis will lead to an adverse outcome (Figure 13). Chemicals might affect different endpoints 
of the DNT IVB with different potencies rectifying the current need for running all assays of the battery 
for capturing the most sensitive endpoint as the point of departure (PoD) for risk assessment. 

Use  of  the  DNT  IVB  entails  two  major  applications,  (i)  hazard  assessment  for  e.g.  screening  and 
prioritization and (ii) risk assessment.  
(i) For hazard assessment purposes the battery is ready to be used and needs to be applied in its totality 
of the 17 assays. If available, an in silico QSAR approach might precede the in vitro testing, however 
at this point in time QSAR will not be a stand-alone approach for DNT hazard assessment. In addition, 
considerations of  the  impact  of metabolism  and the bioactivity  of metabolites within  the  DNT  IVB 
needs to be considered. After receiving benchmark concentrations (BMC) from the 17 assays, the most 
sensitive endpoint(s) can be identified. The most sensitive BMC can be used for calculating the effective 
from the nominal in vitro bioactive compound concentration. Such an approach will solely inform on 
hazard for studied key neurodevelopmental processes and could be followed up by a second hit assay. 
This might be advisable in case the hit occurred in an assay with sole neuronal cultures lacking glia or 
in rat cell-based assays. 
(ii) For risk assessment purposes a tiered approach around the DNT IVB is currently developed and a 
first outline of such an approach was recently discussed in the OECD `Workshop on critical innovations 
in pesticides safety testing and chemical risk assessment for developmental neurotoxicity (DNT)` [25] 
following previous considerations [26] resulting in a published proposal in the workshop proceedings 
[27].  This  tiered  testing  proposal  is  summarized  in  the  next  paragraph.  However,  there  are  various 
uncertainties in this proposal, among them, (1) a systematic analysis of all DNT available methods and 
sources of information has not been done; (2) the predictive capacity of each of the tiers has not been 
defined;  (3)  for  some  of  the  tiers  readiness  criteria  for  regulatory  use  have  not  been  discussed  or 
demonstrated (e.g., tier 1 TK, QSAR models or in vivo targeted studies); (4) the entire workflow in this 
strategy has never been employed for any case study. 

169 

 
 
 
 
 
 
Figure 15: A proposed tiered testing approach for DNT as discussed at the 2024 OECD workshop on critical 
innovations in pesticides safety testing and chemical risk assessment for developmental neurotoxicity (DNT) 
[25, 27]. 

The proposed tiered strategy consists of four tiers and aligns with the IATA concept (Figure 15).  

Tier 0 strongly relies on computational tools and modelling data (e.g., QSAR models, physiologically-
based  kinetic  (PBK)  modelling)  to  predict  DNT  potential  and  model  human  exposure  levels.  An 
acceptable margin of exposure might lead to an exit at Tier 0, whereas internal exposure or hazard alerts 
from Tier 0 will guide into Tier 1.  

Tier 1 then applies the DNT IVB [12, 19, 20,], running all 17 assays in parallel to identify effects on 
key neurodevelopmental processes and to derive benchmark concentrations. Importantly, these nominal 
concentrations  are  translated  to  in  vivo  equivalent  doses  using  quantitative  in  vitro  to  in  vivo 
extrapolation (qIVIVE). If a derived margin of exposure is considered safe at this level, there is an exit 
point at Tier 1.  

Tier  2  includes  confirmatory  or  orthogonal  assays  to  address  remaining  uncertainties  or  refine 
mechanistic  understanding.  This  can  be  achieved  via  available  or  by  building  new AOPs. Another 
approach is to include mechanism-based high-throughput assays [28], cell painting assays [29, 30] or 
by performing (high throughput) transcriptomics [31].  

Tier  3  is  thought  to  be  optional,  yet  preferred,  for  hit  confirmation.  For  this,  more  complex  model 
systems  like  3D  cultures,  human  BrainSpheres,  or  organoids  can  be  utilized.  At  this  stage,  also 
immunological  competence  may  be  implemented  in  BrainSpheres,  for  example,  by  adding  human 
induced  pluripotent  stem  cell-derived  microglia.  At  Tier  3,  testing  in  the  zebrafish  embryo  as  an 
alternative to mammalian testing may be applied. Its main advantages are biological complexity and 
early  behavioral  readouts  [21].  However,  species  differences,  mechanistic  relevance,  and  predictive 
value still need further investigation. Also, while considered a non-animal model in the EU, it remains 
a vertebrate, raising some ethical debate. Alternatively, targeted or as a very last resort DNT TG in vivo 
or behavioural tests using the rat might be performed as a Tier 3 confirmatory testing. 

170 

 
 
 
 
 
Change Management Requirements for Implementation 

The case of DNT differs from other testing endpoints concerning the implementation of non-animal 
approaches as in Union legislation, testing of chemicals for their adverse DNT effects is not mandatory, 
yet has to be performed under some regulations upon triggers of adult neurotoxicity or EATS (estrogen, 
androgen, thyroid, steroidogenesis)-mediated endocrine disruption. This legislative void has left a very 
large number substances untested for DNT. Therefore, effective change management for DNT hinges 
on two levers, which open the same door: (i) implementing mandatory DNT evaluation of chemicals 
into legislation across sectors and (ii) securing regulatory acceptance of the DNT in vitro battery as the 
only way how testing of large numbers of chemicals can be achieved.  

Science-based  considerations  have  been the  basis  for  a  new  approach to  DNT assessment  since the 
beginning of the journey [16, 17]. By forming the International STakeholder NETwork (ISTNET) for 
DNT, a testing roadmap as change management was agreed on by necessary stakeholders at that time: 
scientists  from  academia,  international  regulatory  agencies  and  industry  [32].  This  early  multi-
stakeholder effort was followed by placing the evolution of DNT testing using alternatives to animal 
testing under the OECD umbrella [33], which has led to the initial recommendations of the OECD [12] 
(phase 1 commitment). Now the phase 2 commitment needs further management and buy-in (Figure 
16). Relevant actions need definition towards supporting the implementation of the new approach for 
DNT assessment:  

•  Communication: Policy makers, regulators, public to align on need for more DNT assessments 

across regulations and where useful as well as means to conduct them. 

•  Policy roadmap: step-by-step implementation for plant protection products, biocidal products, 
REACH-chemicals, other chemicals in scope of the roadmap like UV filters, colorants, food 
and feed additives. 

•  Science-based initiatives: Harmonization of efforts towards the common goal 
•  Best practices: IATA, DA, AOP 

Figure  16:  Change  management  for  implementation  of  the  DNT  IVB.  Stakeholder  on-boarding  towards 
changes in regulation for DNT took place early [32]. Phase 1 commitment was achieved by the OECD 
initial recommendations for the DNT IVB [12]. Now the Phase 2 commitment phase will lead to changes 
in legislation and the acceptance of the DNT IVB for regulatory application. 

171 

 
 
 
 
 
Summary Action Plan for Implementation of Framework 
Presented below are the key actions for the short, medium and long-term, presenting requirements for 
a phased transition towards implementation of alternatives to animal testing for DNT assessment. 

Short-Term Actions – Use of the DNT IVB for the assessment of active substances use in 
plant protection products Policy  

•  Strongly prioritize DNT testing due to its fundamental societal significance. 
•   Achieve a revision of the data requirements for active substances used in plant protection products, 
introducing the option to consider use of the DNT-IVB where an assessment of DNT is required. 

•  Secure funding for case studies executing the proposed tiered testing approach.  
•  Establish safe spaces to stimulate dialogue between industry and regulators on data assessment. 

Science 

•  Improve computational methods for qIVIVE and hazard identification. 
•  (Further) develop and validate more complex alternatives to animal testing, like brain organoids 
and model organisms, for higher tier testing as a hit confirmation of tier 1 DNT IVB hazard 
assessment (see above) not using animals. 

•  Establish and test WoE-based IATAs and DA in a tiered testing format with the DNT IVB as a central 

tool.  

•  Continue refining the DNT IVB according to test method redundancy, human-specificity and costs 

as commenced within the PARC project. 

•  Perform  uncertainty  assessment  of  alternative  methods-based  approach  for  DNT  assessment  and 

compare to traditional approach and define level of protection. 

Best Practices 

•  Establish  a  coordinated  platform  for  collaboration  between  related  projects/initiatives,  ensuring 

synergy and co-creation.  

•  Develop guidance for WoE-based approaches for DNT assessment, based on e.g. lessons learned 

from (OECD) IATA case studies. 

•  Explore  together  with  ECHA  and  JRC  the  possibility  of  implementing  a  new  hazard  class  for 

neurotoxicity (including DNT). 

•  Support availability of DNT alternatives to animal testing by contract research organizations. 

Communication 

•  Develop a communication roadmap encompassing the needs identified for policy roadmap, science 

and best practices, differentiated where needed. 

•  Define and communicate benefits of the new paradigm especially concerning protection goals for 

consumers. 

•  Continue international stakeholder exchange to ensure alignment and success. 
•  Organize discussion and consultation fora. 

Medium-Term Actions – Consolidation and Confidence Achieved 

Policy 

•  Assess and align for which legislations DNT testing should be mandatory. 

172 

 
 
 
 
 
 
 
•  Adapt the regulatory processes and legislations for REACH-chemicals that enable a WoE alternative 

method-based approach for DNT assessment (possibly tiered by tonnage). 

•  Explore adapting CLP/GHS criteria for neurotoxicity including DNT alternatives to animal testing. 
•  Ensure dialogue between submitters and assessors via safe spaces during the assessment process.  
•  Secure funding for the development of missing DNT-IVB alternatives to animal testing as well as 

for translational activities. 

•  Monitor and review 3Rs impact of changed approach for DNT assessment; adapt long(er)-term goals 

of the roadmap where needed. 

Science 

•  Include  scientific  progress  on  alternatives  to  animal  testing  into  the  DNT  IVB  (e.g.  human 
neurodevelopmental diseases, immune system function, sensitive windows of exposure like puberty 
or old age) 

•  (Further) Develop guidance for WoE-based approach for DNT assessment 
•  Improve and refine internal TTC approach. 
•  Conduct read-across case studies on the basis of DNT in vitro alternatives to animal testing, designed 
in  co-creation  with  stakeholders,  to  increase  the  acceptance  of  read-across  for  carcinogenicity 
assessment. 

Best Practices 

•  Utilize the revised OECD GD34 for validation for the DNT alternatives to animal testing. 
•  Training for all relevant stakeholders, including application of WoE-based approach and hands-on 

lab training for validation and onboarding of new alternatives to animal testing. 

Communication 

•  Prepare materials and deliver outreach, communication and training on the WoE-based approach for 

DNT assessment. 

Long-Term Actions – Non-Animal Approach Implemented 

Policy 

•  Secure funds for applying best science in regulatory toxicology (translation of basic science methods 

into use for regulation). 

Science 

•  Ensure that the DNT alternative method-based approach follows the evolvement of science. Make 

use of best sciences. 

Best Practices 

•  Use sector-specific cases to optimize data interpretation and methodologies. 
•  Ensure all global guidance documents are aligned with one approach for DNT assessment. 
•  Review progress and learnings of newly implemented animal-free approach for DNT assessment. 
•  Implement  training  of  next  generation  professionals  (including  students,  regulators,  industry)  on 

new approaches for DNT assessment. 

Communication 

•  Communicate success. 

173 

 
 
 
 
 
 
 
 
 
 
Conclusion 

The phases and completion of the actions summarized here rely on a continuum of progress from short-
mid-long-term achievable for policy, science, best practices and communication. The collective goal is 
to implement the animal-free approach successfully for DNT assessment globally across all sectors to 
reach the highest protection level for the developing brains. 

References 

[1] UK: Russell et al., 2022. doi: 10.1111/jcpp.13505 
[2] DK: Jensen and Steinhausen 2015. doi: 10.4088/JCP.14m09094 
[3] US: Dvorak et al. 2023. doi: 10.1016/j.intell.2023.101734 
[4] Global meta-analysis: Talantseva et al., 2023. doi: 10.3389/fpsyt.2023.1071181 
[5] US: https://www. cdc.gov/mmwr/volumes/74/ss/ss7402a1.htm 
[6] EU: Bellanger et al., 2015. doi: 10.1210/jc.2014-4323 
[7] Grandjean and Landrigan 2014. doi: 10.1016/S1474-4422(13)70278-3 
[8] Miotto et al., 2024. doi: 10.1080/09603123.2024.2384963 
[9] EFSA 2019. doi: 10.2903/j.efsa.2019.5809 
[10] Bessems et al., 2023. Eionet Report – ETC HE 2023/14 
[11] Paparella et al., 2020. doi: 10.1016/j.reprotox.2020.08.002 
[12] OECD No 377, 2023. https://doi.org/10.1787/91964ef3-en 
[13] Aschner et al., 2017. doi: 10.14573/altex.1604201 
[14] Annex B of OECD 377, 2023. https://doi.org/10.1787/91964ef3-en 
[15] Crofton and Mundy 2021. https://doi.org/10.2903/sp.efsa.2021.EN-6924  
[16] Lein et al., 2005. doi: 10.1016/j.etap.2004.12.035 
[17] Fritsche et al., 2018. doi: 10.1093/toxsci/kfy175 
[18] Bal-Price et al., 2018. doi:10.14573/altex.1712081 
[19] Carstens et al., 2022. https://doi.org/10.1093/toxsci/kfac018 
[20] Blum et al., 2023. https://doi.org/10.1016/j.chemosphere.2022.137035 
[21] Tal et al., 2024. doi: 10.3389/ftox.2024.1359507 
[22] Blum et al., 2025. doi: 10.1016/j.cotox.2025.100528 
[23] Fritsche et al., 2024. doi:10.14573/altex.2410081 
[24] Hernandez et al., 2021. doi: 10.2903/j.efsa.2021.6599 
[25] OECD Workshop 2024. https://www.oecd.org/en/events/2024/10/workshop-critical-innovations-in-
pesticides-safety-testing-and-chemical-risk-assessment-for-developmental-neurotoxicity.html 
[26] Masjosthusmann et al., 2020.  https://doi.org/10.2903/sp.efsa.2020.EN-1938 
[27] OECD Workshop Proceedings 2025. https://one.oecd.org/document/ENV/CBC/MONO(2025)10/en/pdf 
[28] Jeong et al., 2022. doi: 10.1016/j.tiv.2022.105451 
[29] Cimini et al., 2023. doi: 10.1038/s41596-023-00840-9 
[30] Nyffeler et al., 2023. doi: 10.1016/j.taap.2023.116513. 
[31] Harrill et al., 2024. doi: 10.1093/toxsci/kfae108. 
[32] Bal-Price et al., 2015. doi: 10.1007/s00204-015-1464-2 
[33] Fritsche et al., 2018. doi: 10.1016/j.taap.2018.02.004 
[34] Sachana M et al., Frontiers in Toxicology, 2026 accepted in press 

174 

 
 
 
 
 
4.6. Developmental and reproductive toxicity (DART) assessments 

This chapter was originally developed by one of the working group members (Swiss Centre for 
Applied Human Toxicology, SCAHT) for the human health working group, which reviewed its 
content before inclusion into the SWD. 

Summary 

Developmental  and  Reproductive Toxicity  (DART)  assessment  currently  depends  largely  on  animal 
studies and is among the most animal-intensive domains of regulatory toxicology (Rovida & Hartung, 
2009). These studies are ethically debated, time- and resource-intensive, and their translation to humans 
is limited by inter-species differences. Important human-specific biology, such as the menstrual cycle, 
is not covered by existing animal test guidelines. This motivates the development and use of alternative 
methods that generate human-relevant, predictive information suitable for hazard and risk assessment.  

The current status of DART alternatives to animal testing shows comparatively strong coverage of early 
embryo-fetal development that capture early cell differentiation, gastrulation-like processes, and germ-
layer specification and differentiation. Within this domain, two human-based assays are already applied 
in tiered testing approaches in industrial and pharmaceutical development settings. By contrast, several 
domains remain under-developed and require targeted research and method development, notably early-
gestation placenta biology (first and early second trimester), male and female fertility, and postnatal 
windows  such  as  lactation,  mini-puberty,  and  puberty. At  present,  there  is  no  qualified  or  validated 
DART  in  vitro  battery  and  no  formally  agreed  structured  approach  specific  to  DART.  However, 
stepwise,  exposure-led  NGRA  frameworks  have  been  proposed  that  organize  DART-relevant 
alternatives  to  animal  testing  together  with  in  silico  tools  to  support  quantitative  decision-making 
(Mueller et al., 2025; Rajagopal et al., 2022). Ongoing efforts to advance human-relevant alternatives 
to animal testing for DART regulatory testing include dedicated DART initiatives and broader NGRA 
projects  that  bring  multiple  stakeholders  across  chemical,  pharmaceutical  and  food  risk  assessment 
together for consensus building and connect method development with fit-for-purpose use. 

On this basis, the chapter outlines short-, medium-, and long-term actions to address scientific gaps and 
implement  measures  needed  for  a  paradigm  change.  The  objective  is  to  support  and  advance  the 
roadmap to phase out animal testing, delivering DART assessment that is more efficient, effective, and 
humane while maintaining high levels of health protection. 

Why a change for DART assessments? 

Development  and  reproduction  are  major  public-health  concerns. According  to WHO,  about  6%  of 
pregnancies worldwide result in congenital birth defects, accounting for ~10% of deaths in children 
under the age of five (WHO, 2025a). Beyond known contributors, such as genetics, nutrition, infections, 
and  lifestyle,  environmental  chemicals  are  increasingly  linked  to  approximately  50%  of  congenital 
disorders  without  a  specifically  identified  cause  (WHO,  2025b). At  the  same  time,  many  countries 
report declining female and male fertility, and recent projections suggest that by the year 2100, ~97% 
of countries will have fertility rates below the level needed to sustain their populations (GBD, 2021).  

Pharmaceuticals 

Because  pregnant  women  are  considered  a  vulnerable  population,  they  are  typically  excluded  from 
clinical trials. Although limited guidance is available in ICHM3(R2), and additional considerations are 
provided in the Clinical Trial Coordination Group (CTCG) guideline, further guidance for the inclusion 
of  pregnant  women  in  clinical  trials  is  currently  being  prepared  (ICH  E21). As  of  now,  however, 

175 

 
 
 
 
 
 
regulatory assessment of DART endpoints for pharmaceuticals still relies primarily on animal studies 
defined in the ICHS5(R3) guideline. In general, DART is assessed in three separate studies covering 
the  reproductive  cycle:  the  fertility  and  early  embryonic  development  (FEED)  toxicity  study,  the 
embryo-fetal developmental (EFD) toxicity study (usually performed in both a rodent and a non-rodent 
species),  and  the  pre-and  postnatal  developmental  (PPND)  toxicity  study.  In  addition,  ICHS5(R3) 
provides qualification criteria for use of DART alternatives to animal testing under defined contexts of 
use, including a reference compound list of known human teratogenic pharmaceuticals. 

Chemicals 

For chemical risk assessment, mainly OECD guidance is followed (e.g. OECD TG 414, 421,422 and 
443). For a single substance registered under REACH at the highest tonnage band, DART studies can 
require up to ~4,500 animals (van den Heuvel et al., 2023). In particular, the EOGRTS (TG 443) is 
highly  animal-intensive  and,  while  ECHA  rates  it  as  a  credible  and  useful  tool  for  identifying 
reproductive  and  developmental  hazards,  it  has  also  faced  substantial  criticism  over  study  design 
choices (e.g. dose selection and cohort triggers), limited endocrine sensitivity, and overall animal use 
(ECHA,  2023).  Moreover,  the  translational  potential  of  animal  studies  to  humans  is  limited  by 
substantial species differences. This is especially evident in female reproduction and placenta biology: 
the human menstrual cycle is not mimicked in standard animal tests, and the placenta is among the most 
species-specific  organs  (Armstrong  et  al.,  2017).  Furthermore,  postnatal  periods  are  only  partially 
represented in current guidelines. In rats, TG 443 covers lactation and selected puberty markers, but 
mini  puberty,  which  is  a  transient  (postnatal  month  1-6)  reactivation  of  the  reproductive  hormonal 
system  that  mirrors  puberty  on  a  smaller  scale,  essential  for  imprinting  sexual  and  neuroendocrine 
development,  is  not  specifically  assessed.  In  rabbits,  standard  guidelines  do  not  include  postnatal 
endpoints  (TG  414).  Consequently,  thousands  of  chemicals  remain  inadequately  evaluated  for 
developmental and reproductive hazards. 

For  both  the  pharmaceutical  and  chemical  fields,  adopting  alternatives  to  animal  approaches  that 
generate  mechanistic,  human-relevant  evidence  for  DART  while  reducing  reliance  on  apical  animal 
endpoints is required. Complementary DART- alternatives to animal testing such as in silico methods 
and in vitro approaches for hazard identification, together with toxicokinetic and exposure modelling 
(PBPK/QIVIVE)  need  to  be  integrated  into  structured,  fit-for-purpose  testing  strategies  for  risk 
assessment. Over recent years, international efforts (Health and Environmental Sciences Institute, 2025; 
Schenk et al., 2010) have accelerated the development, evaluation, and alignment of DART-relevant 
alternatives  to  animal  testing  for  regulatory  use,  laying  the  groundwork  for  more  consistent 
frameworks. 
implementation 

across 

EU 

DART in vitro non-animal approaches 

DART  spans  female  and  male  reproduction,  placenta  development  and  function,  embryo-fetal 
development,  and  the  postnatal  periods  including  lactation,  and  puberty  (Figure  17),  which  are 
individual endpoints with very different molecular and cellular physiology. To support decision-making, 
coherent  batteries using non-animal approaches are needed to provide sufficient biological coverage 
across  these  phases,  processes,  and  crucial  pathways  to  ensure  protection,  potentially  with  domain-
specific batteries (e.g. male reproduction, female reproduction, and development). Once established, 
these batteries can serve as one tier within a broader tiered approach (see example DNT). Biological 
coverage  by  in  vitro  alternatives  to  animal  testing  has  advanced  markedly  for  early  embryo-fetal 
development  through  stem-cell-based  platforms,  enabled  by  human  iPSC/ESC  systems  that  can 
differentiate  towards  multiple  embryonic  lineages  (ectoderm,  mesoderm,  endoderm)  and  extra-
embryonic  lineages  (e.g.  trophoblast)  (Shahbazi  &  Pasque,  2024).  In  contrast,  other  areas  remain 
comparatively  under-developed  and  need  targeted  method  development,  notably  placenta  biology, 

176 

 
 
 
 
 
female reproduction, and the postnatal phases (lactation, postnatal and child development, puberty), as 
illustrated in Figure 17. 

Figure  17:  Schematic  representation  of  key  developmental  processes  and  corresponding  to 
alternative approaches (or new approach methodology (NAM)) coverage. 

177 

 
 
 
Female reproduction 

One area requiring further research and development of alternatives to animal testing is female fertility, 
which  is  particularly  important  given  substantial  inter-species  differences  in  oocyte  maturation, 
menstrual cycling, and menopause (Caligioni, 2009). Notably, neither rodents nor rabbits menstruate or 
exhibit a human-like menopause. Likewise, the placenta, central to healthy embryonic development, 
remains under-addressed. Beyond acting as a barrier, the placenta carries essential metabolic, endocrine, 
and immunological functions, and perturbations can trigger adverse effects on the developing child. 
Because  placenta  development,  morphology,  and  function  differ  markedly  across  species,  not  only 
aspects  of  female  reproduction,  but  also  human-specific  key  features  of  placenta  biology  are  not 
adequately  represented  in  traditional  animal  tests.  Although  some  in  vitro  models  for  female 
reproduction  and  placental  tissues  exist  (e.g.,  implantation,  menstrual-cycle  models,  placental 
organoids,  ex  vivo  placental  models),  they  are  primarily  used  for  basic  research  and  have  not  been 
developed for application in a regulatory context (Garcia-Alonso et al., 2021; Li et al., 2024). 

Male reproduction 

and 

germ 

focus 

testis, 

recent 

advances 

on Leydig,  Sertoli, 

The most common in vitro models for male reproductive systems target the testis and prostate. Within 
cell functionality, 
the 
emphasizing steroidogenesis, spermatogenesis,  and testicular  development.  While 
the  H295R 
steroidogenesis assay (OECD TG 456) is the only validated in vitro method for steroidogenesis testing, 
its suitability for assessing gonad steroidogenesis is frequently questioned due to its derivation from a 
cancerous, adrenal cell line of female origin (Botteri Principato et al., 2018). Human-based alternatives 
to  animal  testing  currently  in  the  refinement  and  optimization  stage  include  human  testis  organoids 
(Madureira Silva et al., 2025) that capture the structural and functional complexity of the testis, as well 
as  hiPSC-derived  Leydig-like  models  aimed  at  studying  steroidogenesis  (Sato  et  al.,  2025).  More 
complex alternatives to animal testing under development include early liver–testis multi-organ chips 
that  couple  biotransformation  with  germ-cell  toxicity,  and  ongoing  efforts  to  refine  robust 
differentiation  protocols  for  hiPSC-derived  spermatogonia  as  well  as  advanced  3D  cultures  that 
reconstruct an intact blood–testis barrier for transport studies. 

Development  

Currently, three DART alternatives to animal testing (Embryonic Stem Cell Test (EST), Whole Embryo 
Culture (WEC), and the Micromass assay) have been validated by ECVAM. These methods rely on 
animal-derived cells or ex vivo embryo culture. Many additional models are available and in various 
stages of development, however, a detailed landscaping of these approaches, while clearly needed, is 
beyond  the  scope  of  this  document.  Importantly,  human-based  systems  offer  clear  advantages  in 
biological  relevance,  but  their  qualification  or  validation  remains  challenging  due  to  the  limited 
availability of well-characterized human reference compounds. As a result, human and animal models 
need to be developed and used in parallel to ensure robust coverage of developmental processes. 

Two  commercially  available  but  not  yet  validated  assays  focus  on  early  developmental  phases  and 
processes:  ReproTracker  addresses  germ-layer  differentiation (Jamalpoor  et  al.,  2022),  and  devTOX 
quickPredict assesses stem-cell  proliferation (Palmer et al.,  2013).  However, they  do  not  cover later 
stages such as organogenesis and maturation. Ectodermal neurodevelopment is well supported by the 
DNT in vitro battery (DNT IVB; OECD, 2023) and other assays, like the UKN1 (Seidel et al., 2022; 
Krug et al., 2012), the RoFA assay (Dreser et al., 2020), or the Neurosetta assay (Lundin et al., 2024) 
studying neural tube formation, are being developed. Mesodermal and endodermal lineage coverage 
beyond early differentiation remains under-developed, and targeted method development is needed to 
address organogenesis and functional maturation in these domains. Prioritisation can happen through 
mapping with human developmental diseases and relevant pathways. 

Endocrine domain 

178 

 
 
 
Endocrine pathways play a central role in female and male reproduction as well as in pre- and postnatal 
development, making alternatives to animal testing that capture endocrine activity essential for DART 
assessment.  Compared  to  other  DART  domains,  endocrine  modalities,  particularly  the  estrogen, 
androgen, thyroid, and steroidogenesis (EATS) pathways, are relatively well served by existing in silico 
and  in  vitro  tools  (EFSA,  2018),  including  two  validated  assays:  the  H295R  steroidogenesis  assay 
(OECD TG 456) and the estrogen receptor binding assay (OECD TG 493). Non-EATS pathways are 
partially covered within the DNT-IVB (Koch et al., 2025), and important endocrine processes outside 
the established modalities, such as those along the hypothalamic-pituitary-gonadal (HPG) axis, still lack 
human-relevant  models.  Moreover,  current  approaches  often  focus  on  agonist  activity,  whereas 
antagonistic  mechanisms  and  more  complex  modes  of  endocrine  disruption  remain  challenging  to 
assess.  Continued  method  development  is  therefore  needed  to  ensure  more  complete  and 
mechanistically informative coverage of endocrine pathways relevant to DART.  

Overall,  the  biological  coverage  of  toxicity  endpoints  across  existing  DART  alternatives  to  animal 
testing  is  not  yet  fully  resolved.  Defining  the  context  of  use,  including  the  chemical  applicability 
domains and biological applicability domains of these test systems is crucial to understand their use in 
the regulatory  setting. Although many genes  and  pathways  relevant  to  fetal  development  have  been 
identified (Janowska-Sejda et al., 2022), the development of DART-relevant AOPs remains challenging 
and  substantial  effort  needs  to  be  spent  in  this  area.  Systematic  mapping  is  therefore  crucial  to  (i) 
delineate coverage of alternatives to animal testing across life stages, (ii) identify context of use and 
applicability domains of specific alternatives to animal testing and battery approaches, and (iii) establish 
best-fit combinations for first tier (screening) testing that can flag clear positives and low-concern cases 
before more refined higher-tier assessments are considered.  

Mapping Initiatives toward regulatory uptake of DART alternative to animal 
testing 
This  section  summarises  ongoing  initiatives  and  projects  that  support  the  uptake  of  DART-relevant 
methods  for  regulatory  decision-making.  Building  on  the  increasing  landscape  of  DART-relevant  
alternatives to animal testing , their emerging readiness, and the gaps identified earlier, several efforts 
have been set up to support and accelerate regulatory uptake. These efforts include dedicated DART 
groups and initiatives as well as broader next-generation risk assessment (NGRA) projects that include 
DART activities, such as the EU projects PARC and CHIASMA. Together, they aim to connect method 
development with fit-for-purpose use in structured assessment approaches. 

The initiatives listed below are among the major ongoing efforts: 

HESI DART 

The HESI Developmental and Reproductive Toxicology (DART) Committee brings together scientists 
from industry, government, and academia to exchange information, advance DART science, and build 
consensus  on  how  experimental  evidence  supports  human  health  risk  assessment  (Health  and 
Environmental  Sciences  Institute,  2025).  This  group  consists  of  industry,  regulatory  and  academic 
representatives  across  chemical,  pharmaceutical,  cosmetics  and  food  industries.  Typical  activities 
include workshops, consensus-building discussions, and publications aimed at improving how DART 
evidence is generated and used. 

Among several topic-focused working groups, the DART alternatives to animal testing /Alternatives 
working  group concentrates  on  creating  a Non-Animal  Method  toolbox and  clarifying contexts  of 
use for alternative assays to meet regulatory expectations and progress toward validation and use. The 
working group organised the hybrid workshop “NAMs in DevTox Testing: Moving the Needle Forward 
to Regulatory Use” held 30th September – 1st October 2025 (Washington, DC & online), focused on 
advancing regulatory application of DART  alternatives to animal testing . 

179 

 
 
 
 
In  addition,  the DARTable  Genome working  group  integrates  chemical–protein  target  information 
with  pharmacokinetics  and  developmental  biology  to  link  modes  of  action  to  DART  outcomes  and 
support data-driven IATAs. Ongoing work includes a conceptual framework and case studies with well-
characterised teratogens to illustrate how such pathway knowledge can inform PoDs and regulatory 
interpretation (Janowska-Sejda et al., 2022). 

Furthermore, this group aims to reduce animal testing by investigating the use of weight of evidence 
approaches to waive animal testing, by performing retrospective research, including investigating the 
need of testing in a second species for small molecules (Theunissen et al., 2016), and ways to reduce 
NHP (Bowman et al., 2026; Roos et al., 2025) testing for DART. 

ISTNET-DART 

The  International  STakeholder  NETwork  for  DART  (ISTNET-DART)  brings  together  regulators, 
international institutions, companies, and universities to advance mechanism-driven, human-relevant 
alternatives  to  animal  testing  for  DART  regulatory  testing.  Building  on  the  successful  example  of 
ISTNET-DNT, which led to an OECD recommendation for a DNT in vitro battery (DNT-IVB), DART 
assays are intended to be based on the underlying biology of human reproduction and development. The 
initiative began with an in-person workshop in Zurich (12–13 September 2024) with 61 participants 
from 12 countries, followed by online workshops in February and April 2025 (Fritsche et al., 2024). 
The Zurich meeting featured presentations on the biology of the reproductive and developmental cycles 
and  the  status  of  in  vitro  methods  for  DART  testing,  including  physiology-based  kinetic  (PBK) 
modelling,  and  was  followed  by  breakout  groups  on  female  and  male  reproductive  toxicity, 
developmental toxicity, and PBK modelling. Participants agreed that methods should be placed in the 
context of AOPs for DART or measured through adverse responses verified within the  Non-Animal 
Method  by  selected  benchmark  substances. A  meeting  report  has  been  published,  and  a  long  report 
including a roadmap is in preparation. 

Proposed Frameworks and Case Studies 
At present, there is no regulator-endorsed structured approach for DART alternatives to animal testing 
. However, some publications have proposed ways to organise DART-relevant alternatives to animal 
testing for decision-making. Approaches vary in maturity: some offer conceptual, tiered structures using 
AOP-guided integrations and decision trees structures (Burbank et al., 2023; Catlin et al., 2024; Hareng 
et al., 2005), while others implement and test stepwise approaches quantitatively.  

A recent case study demonstrated an adaptable approach for the assessment of chemicals with limited 
traditional toxicity data, leveraging a Non-Animal Method-based assessment workflow (Paul Friedman 
et  al.,  2025).  The  study  specifically  incorporated  DART  assessment  by  combining  in  silico 
(COMPARA, CERAPP, TEST DEV) and in vitro (ToxCast ER and AR models, devTOX quickPredict) 
alternatives  to  animal  testing  to  develop  DART  hazard  flags,  which  serve  as  qualitative,  putative 
indicators of hazard related to developmental toxicity and endocrine modulation. 

Quantitative, exposure-led assessments for DART utilize stepwise NGRA frameworks that integrate in 
silico  tools  and  human-relevant  alternatives  to  animal  testing.  These  frameworks  link  Non-Animal 
Method-derived hazard information (Point-of-Departure, or PoD) with exposure estimates to calculate 
the  Bioactivity:Exposure  Ratio  (BER),  which  serves  as  an  explicit  decision  criterion  for  risk 
prioritization.  Rajagopal  et  al.  (2022)  introduced  a  DART  Non-Animal  Method  "core  toolbox"  and 
evaluated its coverage against Developmental and Reproductive Signalling (DARS) markers related to 
human  development,  finding  nearly  80%  coverage  of  DARS  genes  by  the  mechanistic  tools  used. 
Subsequently, Mueller et al. (2025) evaluated such a framework using 37 benchmark compounds across 
49 exposure scenarios. This framework employed a Tier 0 in silico module (e.g., Derek Nexus, OECD 
QSAR Toolbox, VEGA, OPERA) and a Tier 1 PoD/BER module. The Tier 1 module combined broad 
screening  tools,  notably  High-Throughput  Transcriptomics  (HTTr)  and  Cell  Stress  Profiling  (CSP), 
with targeted DART Alternatives to animal testing, including In Vitro Pharmacological Profiling (IPP), 
the  hiPSC-based  ReproTracker  and  devTOX  quickPredict  assays,  and  endocrine  assays  (H295R 

180 

 
 
 
steroidogenesis  and  ER/AR  CALUX  pre-screens).  Using  a  conceptual  BER  threshold  of  1  to  flag 
high/uncertain  risk  scenarios,  the  approach  successfully  identified  17  of  the  18  high-risk  exposure 
scenarios  confirmed  by  existing  regulatory  opinions.  Key  limitations  identified  include  the  need  to 
establish an agreed-upon protective BER threshold and address remaining gaps in biological coverage 
necessary for comprehensive DART protection. 

Together, these studies illustrate how DART-relevant Alternatives to animal testing, exposure estimation 
(including pregnancy/fetal considerations), and explicit decision criteria (e.g. BER) can be combined 
and transparently tested with reference scenarios. 

Summary Action Plan 
The following short-, medium-, and long-term actions set out the requirements for a staged transition 
towards implementing alternatives to animal testing in DART assessment. 

Short-Term Actions 

Policy  

•  Raise political attention to the need to prioritise DART alternatives to animal testing due to the 

fundamental societal significance 

•  Acquire  targeted  funding  to  foster  innovation  in  DART  alternatives  to  animal  testing  and 
establish  frameworks  with  high  predictivity  (biology,  toxicology,  pathology)  and  feasibility 
(cost, time, efforts) 

•  Secure funding for case studies that apply available DART alternatives to animal testing in a 

WoE approach 

Science 

•  Perform a comprehensive landscaping of existing DART-relevant Alternatives to animal testing 
• 
Identify  gaps  across  female/male  reproduction,  placenta  biology,  embryo-fetal  development 
(EFD), and pre- and postnatal phases (lactation, mini-puberty, puberty) 

•  Establish  AOPs  and  KE-AO  relationships  to  support  integration  of  non-animal  method 

evidence  

•  Assess  readiness  of  existing  alternatives  to  animal  testing  and  describe  their  context  of  use 
(including  biological  and  chemical  applicability  domain).    Identify  methods  ready  for 
regulatory support, for instance to support WoE assessment for expected teratogens. 

•  Start qualifying (e.g. ICHS5(R3)) or validating (chemicals) advanced DART Alternatives to 

animal testing 

•  Develop and refine QSAR/read-across models for DART-relevant endpoints 

Best Practices 

•  Develop a strategic roadmap towards animal-free DART testing 
•  Establish exchange between DART initiatives and related projects to ensure synergies and joint 

development 

•  Adapt and operationalise DART-specific readiness criteria 
•  Expand  the  reference  compound  list  as  presented  in  ICHS5R3  Develop  guidance  for 
mechanistic WoE-based approach for DART assessment, based on e.g. lessons learned from 
(OECD) IATA case studies testing WoE-based approaches. 

Communication 

181 

 
 
 
 
 
 
•  Develop  communication  roadmap  encompassing  the  needs  identified  for  policy 

roadmap, science and best practices, differentiated where needed 

•  Engage more stakeholders and strengthen international stakeholder exchange to align 

goals 

•  Organise platforms for discussion and exchange 

Medium-Term Actions 

Policy  

•  Adapt  regulatory  processes  and  legislations  in  different  sectors  to  enable  integrated  Non-

Animal Method-based approach for DART assessment 

•  Secure targeted funding programmes to support development of missing Alternatives to animal 
testing, further development of existing alternatives to animal testing and translational activities 
•  Ensure  continuous  dialogue  between  submitters  and  assessors  via  safe  spaces  during  the 

assessment process. 

•  Establishment of an international expert group integrating different stakeholder perspectives on 

DART. 

Science 

•  Continue establishment, qualification, and validation of DART alternatives to animal testing to 
close applicability domains and reduce uncertainty to a level where negative results are reliable 
for regulatory decision-making 

•  Build an integrative framework that leverages existing Alternatives to animal testing, defining 

clear contexts of use and applicability domains 

•  Conduct  read-across  case  studies  on  the  basis  of  DART  in  vitro  non-animal  approaches, 
designed in co-creation with stakeholders, to increase the acceptance of read-across for DART 
assessment. 

Best Practices 

•  Create a living catalogue of ready-to-use DART alternatives to animal testing with contexts of 

use and applicability domains 

•  Organise inter-laboratory validation tests for alternatives to animal testing that are pivotal for 

IATA 

•  Define and establish key elements of quality (control) of Alternatives to animal testing 
•  Perform uncertainty assessment of Non-Animal Method-based approach for DART assessment 

and compare to traditional approach, and define level of protection 

•  Use sector-specific cases to optimize data interpretation and methodologies 
•  Ensure transferability and public availability of alternatives to animal testing through training 
for all relevant stakeholders for validation and onboarding of new Alternatives to animal testing 

Communication 

•  Ensure  continuous  communication  between  stakeholders  (industry,  regulators,  academia, 

politics) 

•  Communicate benefits and contexts of use of the initial framework 

Long-Term Actions 

Policy  

182 

 
 
 
 
 
 
 
•  Secure funding for applying best science in regulatory toxicology (translation of basic science 

methods into use for regulation) 
Implementation of IATA/battery tool that is ready for regulatory use 

• 

Science 

•  Achieve full replacement of DART assessments with alternatives to animal testing  
•  Ensure that the DART  Non-Animal Method approach follows the evolvement of science by 

including scientific progress on alternatives to animal testing into existing frameworks 

Best Practices 

•  Ensure alignment of global guidance documents within regulatory pillars (e.g., pharmaceuticals 

or chemicals) to support coherent approach for DART assessment. 

•  Review  progress  and  learnings  of  newly  implemented  animal-free  approach  for  DART 

• 

assessment. 
Implement training of next generation professionals (including students, regulators, industry) 
on new approaches for DART assessment. 

Communication 

• 

Integrate methods and concepts into the curriculum of schools and universities. 

Conclusion 

The  actions  outlined  here  represent  a  set  of  priorities  that  will  be  refined  as  methods,  data,  and 
evaluation criteria evolve. Their delivery depends on sustained, coordinated progress and continuous 
exchange among stakeholders including policy makers, regulators, method developers, and assay users. 
The unifying goal is to establish an animal-free framework for DART assessment that is usable across 
sectors and ensures a level of protection that is safe for human development and reproduction, spanning 
male and female fertility through prenatal and postnatal development to sexual maturation. 

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Cells 

11 

Neuroepithelial 
https://doi.org/10.3390/cells11213404 

and 

Shahbazi, M. & Pasque, V. Early human development and stem cell-based human embryo models. Cell stem cell 

31 (10/03/2024). https://doi.org/10.1016/j.stem.2024.09.002 

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Theunissen,  P.T.  et  al.  Comparison  of  rat  and  rabbit  embryo-fetal  developmental  toxicity  data  for  379 
pharmaceuticals: on the nature and severity of developmental effects. Crit Rev Toxicol 46 (2016/11). 
https://doi.org/10.1080/10408444.2016.1224807 

van den Heuvel, C., Klaver, N., Tonk, I., Coder, P. & Beekhuijzen, M. Is there any added value of the second 
generation in the Extended One-Generation Reproductive Toxicity Study (EOGRTS)? A retrospective 
(10/27/2023). 
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(2025b).  Congenital 
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(Elmsford,  N.Y.)  122 

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toxicology 

disorders 

disorders 

WHO 

WHO 

185 

 
 
 
 
4.7. Endocrine Disrupting Chemicals - Use of New Approach 
Methodologies for their assessment within European Union regulatory 
frameworks:  
Recommendations from the 2024 EPAA Partners’ Forum 
Introduction  

Objective of the Partners Forum  

This chapter describes the main conclusions of the European Partnership for Alternative Approaches to 
Animal Testing (EPAA) Partners’ Forum (PF) held on 14-15 November 2024 in Brussels on the use of 
non-animal approaches for endocrine disruption assessment.  

The importance of NAMs in regulatory Endocrine Disruption (ED) assessment 

In line with the regulatory frameworks implemented within the EU to reduce the levels of Endocrine 
Disrupting Chemicals (EDCs) for consumers, workers, and the environment, it was critical to establish 
validated  tests,  allowing  regulatory  authorities  to  conduct  hazard  and  risk  assessments  and  make 
relevant decisions (European Commission, Towards a comprehensive European Union framework on 
endocrine  disruptors,  2018;  EFSA  Scientific  Committee,  2013).  Many  of  these  methods  are  now 
available for the regulatory assessment of EDCs (Council Regulation (EC) No 440/2008). Regulatory 
measures were taken in the EU with Member States and the EC restricting the use of putative EDCs.86 

According to the WHO/International Programme on Chemical Safety (IPCS), an EDC is defined as “an 
exogenous substance or mixture that alters function(s) of the endocrine system and consequently causes 
adverse  health  effects  in  an  intact  organism,  or  its  progeny,  or  (sub)  populations”  (2002).  The 
identification of endocrine disrupting properties of a substance demands according to the definition to 
demonstrate adverse effects in an intact organism, requiring the use of animal studies. In order to shift 
away from this  paradigm  based on animal testing, further development and application of emerging 
technologies is needed to reduce time and better protect humans and the environment. 

EU regulatory frameworks 

The EU regulatory framework evolved over the years with specific provisions for the identification and 
regulation of ED in place in relevant pieces of legislation. Provisions for this purpose are included in 
the  EU  Regulation  on  Classification,  Labelling  and  Packaging  of  chemicals,87  the  regulation  on 
chemicals  in  general  ("REACH  Regulation"),  on  plant  protection  products,88  biocidal  products,89 
medical devices,90 water91 and in the revised legislation on toys.92 The requirements vary depending on 

86 Endocrine disruptors - Public Health - European Commission 
87 European Union. Amendment to Regulation (EC) No 1272/2008 as regards hazard classes and criteria for the 
classification, labelling and packaging of substances and mixtures. 2022. 2023/707 
88 European Parliament. Commission Regulation (EU) 2018/605 of 19 April 2018 amending Annex II to Regulation (EC) 
No 1107/2009 by setting out scientific criteria for the determination of endocrine disrupting properties. https://eur-
lex.europa.eu/eli/reg/2018/605/oj/eng July 23, 2025 
89 European Parliament. Commission Delegated Regulation (EU) 2017/2100 of 4 September 2017 setting out 
scientific criteria for the determination of endocrine-disrupting properties pursuant to Regulation (EU) No 528/2012 
of the European Parliament and Council. https://eur-lex.europa.eu/eli/reg_del/2017/2100/oj/eng July 23, 2025 
90 European Parliament. Regulation (EU) 2017/745 of the European Parliament and of the Council of 5 April 2017 on 
medical devices, amending Directive 2001/83/EC, Regulation (EC) No 178/2002 and Regulation (EC) No 1223/2009 
and repealing Council Directives 90/385/EEC and 93/42/EEC. https://eur-lex.europa.eu/eli/reg/2017/745/oj/eng July 
23, 2025 
91 European Parliament. Directive 2000/60/EC of the European Parliament and of the Council of 23 October 2000 
establishing a framework for Community action in the field of water policy. https://eur-
lex.europa.eu/eli/dir/2000/60/oj/eng July 23, 2025 
92 European Parliament. Regulation of the European Parliament and of the Council on the safety of toys and repealing 
Directive 2009/48/EC. https://eur-lex.europa.eu/legal-content/EN/TXT/HTML/?uri=CELEX:52023PC0462 July 23, 2025 

186 

 
 
 
the  specific  legislation.  Other  legislation,  such  as  that  on  food  contact  materials,93  cosmetics,94  or 
protecting workers at the workplace,95 while not containing specific provisions for ED, allow regulating 
such substances on a case-by-case assessment. All pieces of legislation make some use of non-animal 
approach data for the purpose of assessing EDCs. 

EU REACH  

Currently, REACH information requirements oblige registrants to generate only limited information 
specifically  on  endocrine  disruption.  Under  REACH,  EDCs  can  be  restricted  if  a  risk  has  been 
demonstrated from their use. EDCs can also be identified as substances of very high concern (SVHC) 
and be subjected to the authorisation procedure. In both procedures, non-animal approaches data can 
play a prominent role. With the introduction of the new ED hazard classes in classification, labelling 
and packaging of substances and mixtures (CLP) in 2023, all substances that have been identified before 
11  June  202596  as  SVHCs  under  REACH  due  to  endocrine  disrupting  properties  will  be  directly 
classified as ED Category 1 under CLP. 

CLP Regulation and Commission Delegated Regulation (EU) 2023/707 for CLP  
The  Classification,  Labelling  and  Packaging  of  chemicals  (CLP  regulation),  supports  the  increased 
integrative use of in vitro and in silico testing methods. In 2023, two new hazard classes for ED, for 
both  human  health  and  the  environment,  were  added  in  the  CLP  regulation,  in  line  with  the 
recommendations  in  the  Chemicals  strategy  for  Sustainability  (European  Commission  2020).  Two 
hazard categories for ED were established based on the level of evidence: known or presumed endocrine 
disruptors  (Category  1)  and  suspected  endocrine  disruptors  (Category  2),  applicable  to  both  human 
health and the environment. Classification relies heavily on evidence from human and animal data, but 
there is also a provision for the use of “non-animal data with equivalent predictive capacity”. Such data 
must  demonstrate  that  the  substance  meets  all  the  following  criteria:  (a)  endocrine  activity;  (b)  an 
adverse effect in an intact organism or its offspring or future generations; (c) a biologically plausible 
link  between  the  endocrine  activity  and  the adverse  effect (Figure  18).  If the  evidence  conclusively 
demonstrates that the adverse effects are not relevant to humans (or at the population or subpopulation 
level),  the  substance  shall  not  be  considered  an  endocrine  disruptor  for  human  health  (or  the 
environment). According  to  CLP,  ED  is  defined  as  the  alteration  of  one  or  more  functions  of  the 
endocrine  system  caused  by  an  endocrine  disruptor  (or  EDC).  Endocrine  activity  is  defined  as  the 
interaction with the endocrine system that may result in a response of that system, of target organs or 
target tissues, and that confers on a substance or the mixture the potential to alter one or more functions 
of  the  endocrine  system.  Adverse  effect  is  defined  as  a  change  in  morphology,  physiology,  growth, 
development, reproduction or lifespan of an organism, system, population or subpopulation that results 
in an impairment of functional capacity, an impairment of the capacity to compensate for additional 
stress or an increase in susceptibility to other influences. Biologically plausible link is defined as the 
correlation between an endocrine activity and an adverse effect, based on biological processes, where 
the correlation is consistent with existing scientific knowledge. 

93 European Commission. Commission Regulation (EU) 2023/915 of 25 April 2023 on maximum levels for certain 
contaminants in food and repealing Regulation (EC) No 1881/2006. https://eur-lex.europa.eu/eli/reg/2023/915/oj/eng July 
23, 2025 
94 European Parliament. Regulation (EC) No 1223/2009 of the European Parliament and of the Council of 30 November 
2009 on cosmetic products. https://eur-lex.europa.eu/legal-content/EN/ALL/?uri=celex%3A32009R1223 July 23, 2025 
95 European Council. Council Directive on the introduction of measures to encourage improvements in the safety and health 
of workers at work. https://eur-lex.europa.eu/legal-content/EN/TXT/?uri=CELEX%3A01989L0391-20081211 July 23, 2025 
96 The deadline extends to 11 June 2026 for substances for which the SVHC-assessment is ongoing, but for which a decision 
has not yet been adopted under REACH 

187 

 
 
 
Figure 18. Requirements to classify a chemical substance as endocrine disruptor. Adapted from 
the Chemical sector perspective presentation by Francesca Trivelloni and the NC3Rs presentation by 
Natalie Burden in the EPAA PF 2024. NAMs - New Approach Methodologies. 

Plant Protection Products and Biocidal Products Regulation. 

Commission Delegated Regulation (EU) No 2017/2100 for biocidal products (BPs) and Commission 
Regulation  (EU)  No  2018/605  for  plant  protection  products  (PPPs)  were  the  first  regulations  to 
introduce scientific criteria for the determination of endocrine-disrupting properties for both Human 
Health and the Environment. To assist the implementation of the criteria, the ECHA/EFSA/JRC 2018 
guidance document for the identification of EDs was developed, in the context of Regulations (EU) No 
528/2012 and (EC) No 1107/2009, which describes the stepwise process for the scientific assessment 
of ED properties of BPs and PPPs. The ED assessment includes the integration of regulatory studies as 
well as public literature studies and different types of evidence including in vivo, in vitro, in silico. More 
specifically,  the  ECHA/EFSA/JRC  ED  Guidance  describes  how  to  gather,  evaluate and  consider  all 
relevant information for the assessment, apply a weight of evidence (WoE) approach and conduct a 
mode of action (MoA) analysis, in order to help in establishing whether the substance meets (or does 
not) the ED criteria under the BP and PPP Regulations (ECHA, 2024). 

With  the  introduction  of  the  new  ED  Hazard  Classes  in  classification,  labelling  and  packaging  of 
substances and mixtures (CLP) in 2023, all substances that have already been identified as EDs under 
PPPR and BPR will be directly classified as ED Category 1 under CLP. An active substance concluded 
not to meet the ED criteria under PPPR or BPR, can be classified as Category 1, Category 2 or No 
classification under CLP, depending on data available when reassessed by the RAC committee. 

Organisation for Economic Co-operation and Development (OECD) Conceptual Framework and 
Guidance Document 150 

The OECD's work on EDs began in the 1990s, driven by the need of member countries to address the 
issue  of  EDCs.97 To  support  this  effort,  the  OECD  established  the  Endocrine  Disruptor Testing  and 
Assessment  Advisory  Group  (EDTA  AG)  and  other  expert  groups  focused  on  specific  topics  and 
endpoints,  such  as  thyroid  function.  One  significant  outcome  of  this  work  was  the  creation  of  the 
conceptual  framework  for  testing  and  assessing  EDCs.  This  conceptual  framework  organises 
information  across  different  biological  levels,  from  level  1  (biochemical  data)  to  level  5  (long-term 
testing on whole organisms) (Figure 19). Levels 1 and 2 do not differentiate between mammalian and 
non-mammalian tests. Level 4 has a similar number of tests for both mammalian and non-mammalian 
subjects.  Levels  3  and  5  include  more  non-mammalian  than  mammalian  methods.  In  total,  the 
conceptual framework includes 44 test guidelines (TGs) and three guidance documents (GDs).  

97 OECD. Endocrine disruptors. https://www.oecd.org/en/topics/sub-issues/testing-of-chemicals/endocrine-disrupters.html 
(Last accessed: 28 January 2025) 

188 

                                                                                                                                                                                 
 
 
 
 
Figure 19. OECD Conceptual Framework. Adapted from the OECD presentation by Elise Grignard 
in the EPAA PF 2024. QSAR - Quantitative Structure-Activity Relationships. * OECD states (2018): 
Level 3 - Some assays may also provide some evidence of adverse effects. 

The conceptual framework is linked to the Guidance Document 150 on Standardised Test Guidelines 
for  Evaluating  Chemicals  for  Endocrine  Disruption,  created  in  2012  and  updated  in  2018  (OECD, 
2018). This document provides detailed guidance on interpreting test outcomes and compiling evidence 
to determine if a substance is an EDC. It offers step-by-step instructions for analysing standard test 
results, weighing evidence for an endocrine activity, and identifying adverse effects in whole organisms. 
When  it  was  introduced,  this  guidance  was  the  first  comprehensive,  international  guidance  for 
identifying  EDCs.  It  supports  regulatory  authorities’  decisions  related  to  the  hazard  of  specific 
chemicals  evaluated  using  test  guidelines.  The  document  allows  for  flexible  interpretation  to 
accommodate different domestic legislation, policies, and practices. The document also highlights the 
importance  of  not  treating  the  conceptual  framework  as  a  linear  testing  strategy  but  more  as 
methodology for organising and interpreting data, such that the relative value of studies for different 
aspects of the assessment can be evaluated. 

Research challenges for advancing ED assessments while reducing the reliance on animal testing 

The main research challenges mentioned during the EPAA PF were the following:  

Endocrine activity vs adversity. Most currently used in vitro alternatives to animal testing address 
endocrine activity (i.e., mode of action) (Figure 18), corresponding to Level 2 of the OECD Conceptual 
Framework (Figure 19), but they are not yet sufficient on their own to identify endocrine adversity (or 
lack thereof) at Levels 4-5. Currently, there are no available NAMs that can conclusively determine 
adverse responses (or lack thereof) at the organism or population level following systemic (acute or 
repeated) exposure. Developing alternatives to animal testing to demonstrate adversity will take a long 
time.  

Therefore, short-term recommendations would be to 

(i) 

(ii) 

Develop  approaches  that  “predict  adversity”  (or  lack  thereof)  rather  than  “demonstrate 
adversity”. In this context, it is important to define what “adequately predictive” means.  

Explore combinations of alternatives to animal testing to help predict adversity, particularly in 
combination with PBK modelling and/or qIVIVE, as single alternatives to animal testing do 
not provide suitable data to be able to predict adversity. For example, some in vitro tests, such 
as cellular and organoid systems, may have the potential to characterise some adversity (e.g., 
nervous system cells not developing).  

189 

Level                                                                                    Level                                                           Level                                                         Level                                                                    Level                                                                                     
 
 
 
(iii) 

Explore  the  potential  of  the  extensive  data  generated  by  the  pharmaceutical  industry  on 
endocrine active pharmaceutical ingredients (APIs) and other compounds with potential side 
effects. This  could  be  instrumental  in  creating  and  completing  existing AOPs,  through  the 
identification of biomarkers for assessing adverse effects and causation for example.  

Uncertainties  around  negative  and  positive  results. A  substance  may  no  longer  be  considered  an 
EDC under a regulatory framework as if it shows negative results in assays for levels 2 and 3 of the 
OECD  conceptual  framework.  However,  this  requires  the  regulators  to  have  confidence  that  these 
results are truly negative. Such a conclusion, however, is only valid for the mechanistic information and 
mode of actions covered by these assays. 

On the other hand, if sufficient data on adversity are available, with the body of evidence indicating an 
ED-related concern, then the outcome could be “ED criteria are met” even if mechanistic studies are 
negative. It should also be noted that studies on adversity have limitations with regards to the endpoints 
covered,  which  may  mean  that  not  every  EDC  will  be  identified.  It  might  be  considered  that  the 
available  assays  are  not  sufficient  to  detect  all  possible  ED-related  modes  of  action,  and  that 
uncertainties, such as inadequate concentrations or doses, or unaccounted metabolic factors, can affect 
the interpretation of results.  

Considerations on certainty are similarly relevant for positive results observed in level 2 and 3 assays 
if such results are used for triggering further in vivo testing. For example, a positive in vitro assay may 
not manifest in vivo due to metabolism of the molecule to a less (or non-) active form, the dose might 
not be achievable in vivo, or the substance might not reach the target in vivo. Depending on the number 
of level 2 and 3 assays applied in parallel, potentially a high number of unnecessary follow-up testing 
might be triggered. 

Challenges to address:  

(i) 

(ii) 

(iii) 

Building  trust  in  alternatives  to  animal  testing  by  finding  consensus  on  acceptable 
uncertainty  and  predictive  capacity  to  prevent  assuming  inherent  superiority  of  in  vivo 
methods over alternatives to animal testing.  
Develop  a  solid  understanding  of  the  biological  mechanisms  that  allows  inter  alia  to 
determine  to  what  extent  mechanisms  are  covered  and  may  promote  widespread  use  of 
alternatives  to  animal  testing  for  the  identification  of  EDs  with  the  ultimate  goal  to 
minimise the need of animal testing.  
Build consensus on uncertainties of negative and positive results for both  in vivo and in 
vitro data. 

In conclusion, to prevent assuming inherent superiority of in vivo methods over alternatives to animal 
testing, it is crucial to build trust in the alternatives by finding consensus on acceptable uncertainty and 
predictive capacity. Widespread use of alternatives to animal testing for the identification of EDCs with 
the  goal  to  minimise  the  need  of  animal  testing  requires  a  solid  understanding  of  the  biological 
mechanisms  that  allows  inter  alia  to  determine  to  what  extent  mechanisms  are  covered,  but  also  a 
thoughtful consideration of the uncertainties of negative and positive results for both in vivo and in vitro 
data.  

Reduction vs replacement. While some in vitro methods can replace animal tests for relatively simple 
toxicities (e.g., the ToxCast ER pathway model to replace the Uterotrophic assay), in the short term, it 
may be challenging to replace all in vivo tests with alternatives to animal testing, due to the complexity 
of  the  whole  organism  and  testing  with  substances  not  suitable  for  culture  conditions.  To  start  the 
transition to alternatives to animal testing, the aim should be to reduce animal testing whilst ensuring 
equal protection levels. If a test (or battery of tests) is identified that can truly replace an in vivo test, 
efforts should be made to implement it. 

Knowledge gaps in alternative methods development  

There is a critical need for more knowledge on the impact of EDs at crucial life stages, inter-species 
differences, and the effects of mixtures. Additionally, only the alternatives to animal testing relevant to 

190 

 
 
EATS modalities which are validated are currently widely used, despite the existence of other EATS 
and non-EATS alternatives to animal testing that can address certain elements of other pathways (e.g. 
EURION Methods Table98).  

Challenges to address:  

(i) 

(ii) 

Increase knowledge on the impact of EDs at crucial life stages, inter-species differences, 
and the effects of mixtures.  
Address comprehensively non-EATS modalities which can have human and ecologically 
relevant impacts.  

(iii)  Map  the  available  alternatives  to  animal  testing  defining  applicability  domain  and  their 
potential  regulatory  context  of  use  to  identify  remaining  gaps  (e.g.,  through  designing 
specific  case  studies),  defining  a  testing  strategy  (e.g.,  using  Integrated Approaches  to 
Testing  and Assessment  (IATA)),  and  prioritising  the  development  of  those  relevant  to 
human health and the environment are essential steps.  
Ensure close collaboration between regulators, researchers and assay developers from the 
beginning of this process to ensure that alternatives to animal testing under development 
are scientifically robust and meet the current regulatory needs. 

(iv) 

Challenges to address:  

(i) 

(ii) 

(iii) 

(iv) 

(v) 

Need for predictive quantitative and toxicokinetic in silico approaches, such as those 
using AOPs  and  PBK  models,  to  extrapolate  in  vitro  results  to  species  of  interest-
relevant external doses used to set safety levels, to characterise the biological plausible 
link  between  the  mode  of  action  and  the  observe  adverse  effect  to  ultimately 
communicate hazard and assess risk.  
Implement test standardisation for in vitro and in silico methods to ensure relevance 
and reliability of results for hazard and risk assessments and build trust in existing and 
new alternatives to animal testing.  
Determine  non-animal  approaches  effect  concentrations  relevant  to  humans  or  the 
environment (i.e., molecular initiating events can be established as effect threshold).  
Investigate multi-organ, multi-endpoint, and multi-species issues in the extrapolation 
process;  not  unique  to  non-animal  approaches    but  relates  to  many  current  in  vivo 
approaches.  
Address the heterogeneity of environmental species sensitivity and the sensitivity of 
alternatives to animal testing for individual organisms and population effects. 

Validation, standardisation or qualification 

Challenges to address:  

(i) 

(ii) 

Clearly identify, prioritise and communicate regulatory needs for the development of test 
methods.  
Facilitate  the  transition  between  test  development  and  validation  (or  standardisation  or 
qualification) to accelerate potential uptake (e.g., PEPPER platform). 

Transition to holistic and mechanistic approaches. 

Challenges to address:  

(i) 

Building  stronger  links  with  the  pharmaceutical  industry  and  clinicians  by  defining 
mechanistic-based  panels  of  in  silico  and  in  vitro  assays  to  better  understand  disease 
mechanisms and facilitate the transition.  

98 EURION Methods table 

191 

 
 
 
 
 
 
(ii) 

(iii) 
(iv) 

Develop methods that provide information on multiple endpoints within the same test to 
allow multiple regulatory applications.  
Include biomarkers that can help predict or inform on adversity.  
Building a toolbox with holistic and mechanistic-driven assessments to focus resources and 
testing. 

Opportunities for cross-sector collaboration 

The  main  opportunities  for  cross-sector  collaboration  that  were  identified  by  EPAA  PF  forum 
participants to advance non-animal alternatives for ED-assessment were the following:  

Make use of internal exposure information or ADME/TK 

•  Refine the stepwise approach described in the ECHA-EFSA guidance for ED-assessment by 
including information on internal exposure, potency and life-stage applicability at earlier stages 
to prevent in vivo testing from occurring too early in the decision-making process. 

•  Proactively include all non-animal approach-based information and exposure information in 
regulatory decision-making, keeping in mind that hazard identification is required under CLP 
prior to any risk assessment/management. 

•  Utilise exposure information more effectively, considering use-dependent data. 

•  Explore  the  application  of  non-animal  approach-based  potency  data  for  the  effective 
classification and assessment of EDCs. It should be clarified that “potency” in in vitro studies 
is simply an indication of whether the substance is active or not, in order to anticipate activity 
in vivo. Potency is unrelated to the threshold of the adverse effect i.e. a dose over which the 
adverse  effect  is  observed.  This  includes  exploring  the  role  of  potency  data  in  regulatory 
decision-making, particularly in evaluating activity and redefining adversity by incorporating 
pathway-specific information. 

•  Address the challenge of using potency information across species, crucial for both human-

relevant and ecological Alternatives to animal testing. 

Prioritise non-animal approaches for validation 

• 

Identify promising non-animal approaches that could be validated to address regulatory gaps 
(e.g. PARC opportunity, linked to EPAA via PARC SYNet, EURION non-EATS Alternatives 
to animal testing). 

•  Facilitate  the  interface  between  researchers and  the  regulatory  community through  the AOP 

framework and assess the effectiveness of this mechanism. 

•  Develop case studies/IATA to create non-animal approach batteries, defined approaches, and 

testing strategies. 

•  Refine the current stepwise approach to ED assessment to increase flexibility and allow for 

“exit” steps where appropriate. 

Set up regulatory context for using alternatives to animal testing 

•  Describe  the  regulatory  context  of  use  for  available  non-animal  approaches  and  map  these 

against different EU regulatory frameworks to identify remaining gaps. 

•  Build  confidence  in  non-animal  approach  information  demonstrating  no  ED  activity  by 
identifying  regulatory  examples  where  non-animal  approaches  have  been  used  for  ED 
assessment to support the conclusion that ED criteria are not met (e.g., ToxCast ER pathway 
model) Examples of such confidence building in EFSA’s realm are the following: (i) experience 
from  pesticide  peer  review99,  (ii)  development  of  an  adverse  outcome  pathway  network  for 

99 Outcome of the pesticides peer review meeting on general recurring issues in mammalian toxicology and 
ecotoxicology related to endocrine disruptors 

192 

 
 
 
 
reproductive toxicity endpoints to support identification of endocrine disrupters100, (iii) EFSA 
database 
so 
far101.https://www.efsa.europa.eu/sites/default/files/documents/ed-report-overview-endocrine-
disrupting-assessment-pesticide-active-substances.xlsx 

assessments 

outcomes 

carried 

ED 

out 

the 

of 

of 

•  Ensure  that  complex  non-animal  approaches  (like  e.g.  organs-on-chip)  are  standardised, 
relevant and reliable and can be used in regulatory decision-making and are not ignored due to 
their complexity. 

•  Some participants of the EPAA PF recommended placing greater emphasis on external exposure 
data  when  assessing  whether  a substance may  pose  a  risk  for  specific  uses. This  evaluation 
should ideally occur before deciding to initiate animal testing to determine if the substance is 
an ED. Such an approach could also reduce the number of false positives from a non-animal 
approach battery. No consensus was reached on this point. There was a disagreement at the 
workshop on whether the EU approach for risk management of EDs should be based on hazard 
identification only or on information on exposure and risk as well. 

Collaboration across sectors 

•  Facilitate  ongoing  dialogue  between  researchers,  regulators,  and  industry  in  a  “safe  space” 
(trusted environment) to address scientific gaps and research challenges and build confidence 
without compromising the regulatory approval process. The creation of expert groups, such as 
the already established ECHA ED expert groups, could facilitate this dialogue. 

•  Create  a  common  platform  for  data  sharing,  e.g.  starting  from  EFSA’s  ED  database102  and 
especially  regarding  pharmaceutical  data,  to  better  demonstrate  the relevance  of  non-animal 
approaches, particularly in defining biologically meaningful responses and addressing multi-
organ  and  multi-endpoint  challenges  and  predicting  adversity.  This  platform  can  also  help 
inform  on  relevant AOPs,  review  testing  strategies,  revise  guidance,  analyse  negative  non-
animal approach results, and extrapolate results across species.  

•  Set  up  retrospective  case  studies  across  industries,  agencies,  and  researchers  to  better 
understand negative non-animal approach results and built confidence when "ED criteria were 
not  met".  Use  the  generated  data  and  insights  to  inform  a  revision  of  the  2018 
ECHA/EFSA/JRC ED guidance. 

•  Create  an  ED  NAM  User  Forum  organised  by  EPAA  to  facilitate  consensus-building  and 
support increased regulatory use of alternatives to animal testing for ED assessment using real-
life dossiers and case studies.  

Summary and conclusions 

The 2024 EPAA PF highlighted the increasing need in the use of alternatives to animal testing to assess 
EDs across various industrial sectors. While non-animal approaches can effectively identify endocrine 
activity, they are not yet sufficient on their own to identify chemicals as EDs since they do not provide 
the  necessary  information  for  assessing  adversity.  Standardised  predictive  models  and  integrating 
information  on  internal  exposure,  potency  and  life-stage  applicability  with  non-animal  approach 
findings  could  enhance  identification  of  EDs  and  risk  assessment  by  improving  the  link  between 
endocrine activity and adversity. Building trust in both existing and new in vitro and in silico methods 
is  crucial  for  the  effective  implementation  of  non-animal  approaches  for  EDs. Achieving  this  will 
require consensus on the acceptable level of uncertainty for evaluating endocrine activity and adverse 
effects as well as ensuring methods have predictive capacities equivalent to (or superior to) human, 
environment,  and  animal  data.Closer  dialogue  among  all  actors  involved,  e.g.,  regulatory  agencies, 

100 https://efsa.onlinelibrary.wiley.com/doi/abs/10.2903/sp.efsa.2026.EN-9974 
101 https://www.efsa.europa.eu/sites/default/files/documents/ed-report-overview-endocrine-disrupting-
assessment-pesticide-active-substances.xls 
102 Publicly available EFSA ED DB 

193 

 
 
 
 
 
academia  and  industry,  is needed to integrate  non-animal  approaches  into  safety  assessments across 
regulatory landscapes. 

References 

EFSA  Scientific  Committee.  Scientific  Opinion  on  the  hazard  assessment  of  endocrine  disruptors:  Scientific 
criteria  for  identification  of  endocrine  disruptors  and  appropriateness  of  existing  test  methods  for 
assessing effects mediated by these substances on human health and the environment. EFSA Journal. 
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European Chemicals Agency. Guidance on the application of the CLP Criteria – Guidance to Regulation (EC) No 
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5. Working together to design and implement non-
animal approaches in regulatory testing 

5.1. Role and composition of the roadmap steering team  
Overall description  
The core governance structures ensuring implementation of the roadmap will be a Roadmap Steering 
Team, based on an existing Commission inter-service group (ISG). The Roadmap Steering Team will 
oversee the implementation of the roadmap and ensure coordination between the other organisational 
structures  supporting the  roadmap  implementation.  Collaborative  structures  at  each  of the  three  EU 
agencies, ECHA, EFSA and EMA, will play an important role for implementing the roadmap and offer 
opportunities for stakeholders to get involved. An inter-agency working group within the EU Agency 
Network  (EUAN),  consisting  only  of  experts  from  the  three  agencies,  will  allow  an  efficient 
coordination between the EU agencies. 

These structures are expected to interlink as indicated in Figure 20 below: 

*RST action teams will be set up if needed with the option of stakeholder participation 

Figure 20: how the main organisational structures will interlink for the roadmap’s 
implementation 

5.2. Roadmap steering team 
A roadmap steering team will be set up, which will have the role to oversee and steer the actions set out 
in the roadmap in order to implement the roadmap successfully, provide feedback and suggestions, and 
activate  the  members’  own  networks  to  ensure  that  they  are  widely  and  actively  involved  in  the 
roadmap’s implementation. 

196 

 
 
 
 
 
 
 
 
 
 
Tasks of the roadmap steering team 

•  Driving forward implementation of the roadmap; 
•  Maintaining an overview of the progress of the roadmap’s actions; 
•  Facilitating exchange of views and communication between the different organisational 

• 

structures implementing the roadmap; 
Initiating and organising high-level communication activities to inform and engage 
stakeholders, in close collaboration with agencies or other established organisational 
structures (e.g. the EPAA, the European Reference Laboratory for Alternatives to Animal 
Testing (EURL ECVAM), etc.); 

•  Engaging with non-EU partner countries and multilateral organisations to promote phasing 

out animal testing for chemical safety assessments; 

•  Providing advice in line with the roadmap’s priorities, identifying initiatives that could feed 

into the roadmap’s actions and promoting the catalogue of transitional initiatives. 

Composition and working methods 
The roadmap steering team will be set up as a Commission ‘special group 103’. It will be composed of 
the Commission inter-service group members and represent all relevant Commission services, the three 
EU  agencies  (ECHA,  the  EMA  and  EFSA)  and  representatives  from  Member  States  and  different 
stakeholder organisations. To allow for fruitful discussions, the number of seats for Member States and 
stakeholders will be limited to a maximum of 12. The following composition is proposed: 

• 
• 
• 
• 
• 

two industry representatives; 
two NGO representatives; 
two representatives of the scientific community; 
three representatives from Member States representing different sectors ; 
further members will be determined by the ISG based on their expertise or function in other 
organisational structures. 

The representatives of the Member States and of the stakeholders will be selected by the ISG in response 
to a call for expressions of interest. Experience of animal-free approaches and connection with a wider 
community of stakeholders will be important selection criteria. Members will serve on the roadmap 
steering team for two years, with the possibility to renew their participation. The team’s mandate and 
working  procedures  will  be  determined  by  the  Commission  at  the  beginning  of  the  roadmap’s 
implementation phase. The roadmap steering team is expected to meet regularly (e.g. three to four times 
a year) to maintain the momentum of the roadmap’s implementation. 
Immediately after the publication of the roadmap, the Commission will draft a mandate and working 
procedures for the roadmap steering team and launch a call for participation. After the selection process, 
the Commission will set up the team and call for a first meeting. 
If needed, the Commission will set up action teams to assist the Roadmap Steering Team in advancing 
particular action points. Stakeholders can be invited to an action team and will be selected according 
to the Commission’s rules 104. 

103 Special groups are bodies that for various reasons do not fit into any existing framework, such as that on expert groups or 
comitology committees. Special groups do not qualify as ‘Commission expert groups’ or ‘other similar entities’ in the sense 
of Commission Decision C(2016)3301 establishing horizontal rules on expert groups. 
104 Commission Decision C(2016) 3301 establishing horizontal rules on the creation and operation of 
Commission expert groupshttps://eceuropaeu.sharepoint.com/sites/expert-groups/Shared 
Documents/Forms/AllItems.aspx?id=/sites/expert-groups/Shared Documents/guidelines-on-the-
implementation-of-the-horizontal-rules.pdf&parent=/sites/expert-groups/Shared Documents 

197 

 
 
 
 
 
 
5.3. Engaging Member-State and stakeholder experts in agency 
collaborative structures   
EU agency collaborative platforms and informal expert groups - context 
The European agencies (ECHA, EFSA and the EMA) have already been working for several years on 
identifying  alternatives  to  animal  methods  which  could  be  applied  in  the  regulatory  processes  they 
manage. Annex II describes in more detail the work of these three agencies in this area. Nevertheless, 
it may be necessary to adapt some of their internal organisational structures or working methods to 
improve  and  accelerate the  implementation  of  animal-free methods,  e.g.  setting up  or strengthening 
expert bodies in this specific field. 
Stakeholders were consulted on the need to have expert groups dedicated to animal-free approaches 
within the agencies. A large  majority  of  stakeholders consulted  share  the  view that scientific  expert 
groups 105 are necessary to provide advice on the development of alternative or animal-free approaches 
and on their uptake and use in a regulatory context 106. However, there are different views on their role 
and how these groups should be set up and organised 107. 
Some agencies such as the EMA and EFSA have already set up such groups on fostering the adoption 
of animal-free methods in some of the regulatory fields in their remits. However, these structures do not 
necessarily  cover  certain  tasks  which  would  be  desirable,  such  as  providing  non-binding  advice  on 
regulatory  case  studies  and  guidance  for  industry  to  implement  alternatives  to  animal  testing  108. 
Therefore, these structures might need to be adapted to cover the envisaged tasks described below. 
In  addition,  other  agencies  like  ECHA  did  not  have  these  types  of  structures  in  place  dedicated  to 
animal-free testing. During the roadmap development process, ECHA announced the creation of such 
a new structure  – the  ‘Collaborative Platform on Alternatives to Animal Testing (CP-AAT) – which 
would gather (i) experts from ECHA staff on alternatives to animal testing and the relevant legislation; 
(ii) experts from Member-State competent authorities on REACH, CLP and the BPR; and (iii) observers 
from the Commission and NGOs. 

Composition and tasks 
The collaborative organisational structures or expert groups will consist of experts from the authorities 
of the Member States and stakeholders. They will identify regulatory needs for animal-free test methods 
and contribute, where appropriate, to: 

• 

exchanging views and recommendations with the roadmap steering team on prioritisation of 
method development, validation, qualification and standardisation activities; 
•  providing input on the roadmap’s implementation to the roadmap steering team; 
• 

serving as a platform for exchange between the Member States, the agency, the Commission 
and stakeholders on regulatory acceptance of non-animal testing results within the remit of that 
agency, and as a forum for stakeholders from research, industry or NGOs to give input, while 
providing the opportunity for stakeholders such as the EPAA to present the outcome of their 
projects; 

105 The term ‘scientific expert group’ is used here in an unspecific meaning and should not be confused with a Commission 
expert group or a committee operated by an agency. Rather, it denotes a group or a collaborative platform with participation 
of experts in the field. 
106 See the second stakeholder survey report, Section 3.3, available at https://webgate.ec.europa.eu/circabc-
ewpp/d/d/workspace/SpacesStore/53fef874-5a58-45fa-b2ac-5e1450fecd9b/download. 
107 See the second stakeholder survey report, Section 3.3, available at https://webgate.ec.europa.eu/circabc-
ewpp/d/d/workspace/SpacesStore/53fef874-5a58-45fa-b2ac-5e1450fecd9b/download; the report on stakeholder interviews 
Section 3.2.1 available at https://webgate.ec.europa.eu/circabc-ewpp/d/d/workspace/SpacesStore/67861f4d-a282-493a-af96-
ec47557db0b1/download and the report on the need and feasibility of an advisory scientific committee on non-animal 
methods available at https://webgate.ec.europa.eu/circabc-ewpp/d/d/workspace/SpacesStore/6448df32-3864-4e0c-9fa7-
1b7e910b40e9/download. 
108 See descriptions of existing structures, gap analysis and conclusions in the following report: 
https://webgate.ec.europa.eu/circabc-ewpp/d/d/workspace/SpacesStore/6448df32-3864-4e0c-9fa7-1b7e910b40e9/download. 

198 

 
 
 
 
•  providing non-binding scientific advice on regulatory processes within the remit of the agency, 
e.g. for ECHA, as regards REACH in relation to (i) non-animal testing in compliance checks, 
(ii) testing proposals and (iii) substance evaluation or use of non-animal data for CLP-purposes. 

Implementation 
The  Commission  will  ensure  that  the  decentralised  EU  agencies  and  MS  can  effectively  support 
implementation  of  the  roadmap  through  the  new  collaborative  structures  or  that  already  existing 
structures are fit for purpose to boost the uptake of  alternatives to animal testing in their regulatory 
work.  Since  each  piece  of  Union  legislation  and  each  EU  agency  has  its  own  specificities,  the  EU 
agencies (ECHA, EFSA, EMA) should assess their current or new organisational structures taking into 
account  their  mandate,  working  methods  and  resources,  and  implement  measures,  if  needed,  to 
strengthen or adapt their existing structures. The composition of the current or proposed new groups 
dedicated to alternatives to animal testing, their tasks, working methods, and necessary resources should 
be described by the agencies and should serve as the basis for potential adaptations to current practices. 
In addition to fostering the uptake of alternatives to animal testing in their own regulatory fields, a key 
aspect will be for the agencies to put the mechanisms in place within these structures to ensure the cross-
fertilisation of best practice and knowledge with the other agencies via the EU Agency Network and 
with  the  roadmap  steering  team.  The  agencies  are  encouraged  to  ensure  good  communication  and 
efficient exchange of information within and between the agencies. 

5.4. EU Agency Network (EUAN) – working group on animal-free 
approaches 
The EU Agencies Network (EUAN) - context 
The  EU Agencies  Network  (EUAN)  supports  the  work  of  the  52  EU  decentralised  agencies,  joint 
undertakings (JU) and bodies. The Network provides its members with a  platform for exchange and 
cooperation on areas of common interest, enabling them to share their expertise at European level. It 
also serves as a single point of contact for the EU institutions when addressing cross-cutting issues of 
general interest to all agencies and JUs. The EUAN comprises sub-networks, working groups and task 
forces – fostering technical cooperation on specialised topics. They implement the EUAN strategy and 
annual work programmes and enhance inter-agency collaboration and communication 109. 
It is proposed to set up a EUAN working group on alternatives to animal testing to formalise and make 
visible the already ongoing inter-agency cooperation on alternative approaches and to provide a means 
for  agencies  to  speak  with  ‘one  voice’  on  phasing  out  animal  testing.  A  EUAN  working  group, 
consisting of experts from EFSA, ECHA and EMA, will facilitate the exchange of information on the 
development  and  implementation  of  alternatives  to  animal  testing  across  the  different  pieces  of 
legislation and bodies concerned, helping the EU agencies to keep abreast of the latest developments in 
regulatory science, and accelerate uptake across the different pieces of legislation. 

Proposed tasks of the EUAN working group on animal-free approaches 

•  Ensure that the EU agencies exchange best practice to foster the use of animal-free methods 

in their regulatory fields; 

•  Set priorities and develop actions addressing replacement (or reduction or refinement) in 

coordination with agency expert groups and the roadmap steering team; 

•  Provide a means to coordinate the participation of the EU agencies within EU and 

international fora. 

Implementation 

109 See description of EUAN at https://agencies-network.europa.eu/about-network/our-role-governance-and-strategy_en. 

199 

 
 
 
 
 
 
 
The  Commission  will  ask the  EU  agencies  to  set  up an  inter-agency  working  group  on  animal-free 
approaches within the EUAN. The new EUAN working group on animal-free approaches should meet 
on a regular basis. The input from the agencies’ dedicated structures on animal-free methods (e.g. expert 
groups) will be key for the work of the EUAN working group as hands-on regulatory knowledge will 
be shared for the benefit of all agencies and regulatory fields. 
A representative of the EUAN working group on animal-free approaches might become a member of 
the roadmap steering team to inform it about the developments within the agencies and participate in 
other relevant tasks for the overall implementation of the roadmap. 

200 

 
 
 
 
5.5. Translating innovative methods into regulatory applications – 
validation, standardisation, qualification 
Introduction 

Studies used for the regulatory assessment of chemicals in the EU often follow the test guidelines (TGs) 
of the Organisation for Economic Co-operation and Development (OECD).  ICH guidelines and  and 
VICH guidelines apply for human medicines and veterinary medicines, respectively. These guidelines 
support harmonised regulatory testing of safety, quality and efficacy in their respective domains. Other 
options  for  harmonising  the  acceptance  of  methods  include  the  standardisation  process  of  the 
International  Organization  for  Standardization  (ISO)  or  that  of  the  European  Committee  for 
Standardisation  (CEN),  which  cooperates  with  the  ISO.).  Standards  are  highly  relevant  for  medical 
devices, for example. 

Studies  for  the  regulatory  assessments  of  chemicals  are  usually  carried  out  by  CROs  on  behalf  of 
industry registrants/applicants for the purpose of satisfying standard information requirements set out 
in Union legislation and related (agency) guidance/guidelines. 

Validation in the context of OECD TG development 

Aim of validation 

Before a new test method can be accepted (e.g. for use in a regulatory context where the test method’s 
results are used to make regulatory-relevant decisions), the test method must be validated. Validation is 
a broadly accepted principle, which applies not only to regulatory testing but to all areas of experimental 
science.  Typically,  validation  consists  of  demonstrating  that  the  method’s  results  are  relevant  for  a 
specific purpose (i.e. the results provide us with information that is relevant for taking the decision) and 
reliable (i.e. there is consistency in the method’s results, when the same chemical is tested over time 
and/or in different places / laboratories). 

This  SWD  is  focused  on  the  assessment  of  the  safety  of  chemicals  in  a  regulatory  context.  In  the 
Communication and in this accompanying SWD, the term ‘validation study’ will be used for short for 
‘study to validate a method for the assessment of the safety of chemicals’. 

Over the past 25 years or so, most of the regulatory-relevant validation studies have been conducted 
with the aim of developing OECD TGs. As a result, ‘validation of method’ is – in the context of chemical 
safety testing – often wrongly understood as ‘development of an OECD TG’, even though this conflates 
the scientific process of validation with the more political one of acceptance among OECD members. 
Indeed, OECD Guidance Document 34 on method validation110 explicitly recognises these two parts of 
‘OECD’  validation:  1)  the  scientific  establishment  of  the  relevance  and  reliability  of  the  method’s 
results, and 2) the acceptance of the method as an OECD TG (by a consensus-searching process among 
the expert representatives of the regulatory authorities of the OECD member countries). This obviously 
is one of the great advantages of the validation process according to OECD GD34 and of the resulting 
TGs.  Likewise,  ‘formal  validation’  is  often  understood  as ‘validation  according to  OECD  Guidance 
Document  34’.  Consequently,  a  formal  validation  study  would  not  be  undertaken  unless  there  were 
sufficient international regulatory interest to develop a TG based on the method to be validated. The 
degree  of  international  regulatory  interest  is  assessed  through  the  annual  project  submission  and 
commenting  process  managed  by  the  OECD  working  party  of  national  coordinators  for  the  test 
guidelines programme (WNT). Prior to this assessment, within the EU, initial proposals for method 
validation and regulatory application can be submitted for a preliminary opinion on regulatory relevance 
to the Preliminary Assessment of Regulatory Relevance network (PARERE)111 – a network of cross-

110 https://www.oecd.org/content/dam/oecd/en/publications/reports/2005/08/guidance-document-on-the-
validation-and-international-acceptance-of-new-or-updated-test-methods-for-hazard-
assessment_2695fa12/e1f1244b-en.pdf 
111 https://joint-research-centre.ec.europa.eu/projects-and-activities/reference-and-measurement/european-union-
reference-laboratories/eu-reference-laboratory-alternatives-animal-testing-eurl-ecvam/alternative-methods-
toxicity-testing/advisory-and-consultation-bodies/parere-eurl-ecvam-network_en 

201 

 
 
 
sector  EU  regulators  coordinated  by  the  JRC  –  via  the  EURL  ECVAM  test  submission  process.  In 
addition,  the  International  Cooperation  on  Alternative  Testing  Methods  (ICATM)  often  discusses 
promising  methods  and  evaluates  their  relevance,  with  a  view  to  cooperating  on  validation  and 
supporting the OECD process. 

The system of validation in the context of OECD TG development has significant advantages for both 
regulators and industry, including: (i) the linking of standard information requirements with standard 
accepted tests; (ii) the quality assurance of data; (iii) business predictability; (iv) some degree of legal 
certainty; (v) efficiency in submission and assessment processes; and (vi) reuse of data between sectors 
and in different international jurisdictions. This reuse of data is possible due to the OECD agreement 
on mutual acceptance of data (MAD), and applies to data generated using TGs under the strict quality 
assurance conditions of good laboratory practice 112 (GLP). The development of an OECD TG requires 
consensus within the OECD WNT that there is enough regulatory need for the new TG across OECD 
member countries, as well as sufficient confidence in the proposed method. The system also has some 
disadvantages, including (i) the time taken to evaluate and adopt new methods; (ii) the need to gain 
consensus (unanimity) within the WNT; and (iii) the lack of accountability and transparency in cases 
where member countries delay or block the adoption process. 

Conducting validation 

Since the late 1990s, most formal validation studies for methods assessing the safety-relevant properties 
of chemicals conducted in the EU have been coordinated by the EURL ECVAM. Studies usually involve 
close  cooperation  among  method  developers  (e.g.  companies,  academic  labs,  research  institutes), 
industry  end  users  (e.g.  large  companies,  company  associations),  and  the  EU  network  of  over  30 
validation labs proposed by EU member countries (EU NETVAL), coordinated by the EURL ECVAM. 
Coordination of a validation study includes its overall design, establishing and facilitating a validation 
management  team,  ensuring  sufficient  resources,  reviewing  and  approving  study  plans  and  test 
protocols, providing day-to-day advice and troubleshooting, gathering and analysing data, producing a 
validation study report, and organising a peer review by ECVAM’s Scientific Advisory Committee 113. 
The outcome of the independent peer review marks the end of the formal validation process. The status 
of method validation and acceptance can be followed using the tracking system for alternative methods 
towards regulatory acceptance (TSAR). Usually, the EURL ECVAM – on behalf of the Commission – 
also supports subsequent TG development at the OECD. 

Over the past 20 years, EU framework programmes have invested close to EUR 1.5 billion directly into 
the  development  of  alternatives  to  animal  testing.  However,  the  translation  of  these  methods  into 
regulatory tests (e.g. OECD TGs) that satisfy standard information requirements has been extremely 
limited.  EU-funded  research  projects  invest  very  little  in  validation  activities  since  there  is  little 
incentive  to  do  so.  The  main  incentive  for  researchers,  in  particular  academic  researchers,  is  the 
publication  of  their  results  in  peer-reviewed  journals.  The  most  scientifically  rewarding  (and 
publishable)  part  of  a  validation  study  is  the  demonstration  of  the  relevance  of  a  newly  developed 
method, i.e. to show how the results bring information that is relevant for decision-makers, such as 
regulators. Similarly, a lot of attention has been given to the development of conceptual testing strategies 
and demonstration through case studies. This is illustrated by the (draft) position paper produced by the 
Partnership for the Assessment of Risks from Chemicals (PARC) on validation-related activities 114. 

The more costly part of a validation study – and much less popular among (academic) researchers – is 
the demonstration of the reliability of a method. This requires a significant number of repetitions of the 

112 OECD Principles on Good Laboratory Practice https://doi.org/10.1787/9789264078536-en. 
113 https://joint-research-centre.ec.europa.eu/projects-and-activities/reference-and-measurement/european-union-
reference-laboratories/eu-reference-laboratory-alternatives-animal-testing-eurl-ecvam/alternative-methods-
toxicity-testing/advisory-and-consultation-bodies/esac-eurl-ecvam-scientific_en. 
114 Partnership for the Assessment of Risks from Chemicals (PARC), ‘Validation-related activities within PARC to progress 
new methods and approaches for hazard and risk assessment of chemicals’, produced by PARC WP5 & WP6, Nov 2023 
(draft). 

202 

 
 
 
experiments,  preferably  in  multiple  laboratories  (called  interlaboratory  comparisons  or  studies,  or 
round-robin studies). Validation studies are being increasingly undertaken by companies (SMEs) that 
have developed methods that are perceived to have sufficient regulatory relevance, and that have gained 
support from their national coordinator for the OECD WNT group to submit a project to the OECD to 
develop a TG. Validation studies have also been carried out by industry associations that want to bring 
their methods up to the level of a TG, so that they can satisfy regulatory information requirements in 
their sector. Some years ago, in France, a public-private partnership called ‘Pepper’ was set up to carry 
out  validation  studies (primarily  laboratory ring trials  to assess  reproducibility)  on  in  vitro methods 
considered relevant for the identification of endocrine disrupting chemicals. 

In December 2023, the OECD WNT organised a workshop to discuss operational and financial aspects 
of validation, which clearly highlighted the organisational complexity and the specialist competences 
required, and underlined the need for appropriate funding mechanisms taking into consideration the 
high  costs  involved  (OECD  estimates  ~EUR 200 000-500 000  for  a  ‘simple’  validation  of  one 
method) 115. 

At  present,  the  validation  and  regulatory  acceptance  of  non-animal  approaches  is  a  long  and  time-
consuming  process,  which  is  not  considered  sufficient  for  addressing  wide-ranging  regulatory  and 
societal needs. There is a need to accelerate the availability of validated and regulatory accepted test 
methods for the safety assessment of chemicals. The European strategy for test method development 
and validation is a Member-State-born initiative aimed at prioritising and ensuring method development 
and validation, based on identified regulatory needs for chemical safety assessments and the deficit of 
validated methods to address those needs, taking into consideration the societal demand for animal-free 
methods. This initiative will be carried out under the One Substance, One Assessment (OSOA) Expert 
Group, ensuring a more comprehensive approach to the uptake of alternatives to animal testing. 

Standardisation 

Standards organisations such as CEN and ISO develop a large variety of standards, some of which are 
relevant  for  chemical  safety  assessments  and,  to  a  larger  extent,  for  the  assessment  of  products. 
Examples  include  testing  standards  for  medical  devices,  projects  to  develop  standards  for  the 
assessment of nanomaterials, or organ-on-a-chip approaches. 

The  development  of  CEN  or  ISO  standards  is  the  result  of  public-private  cooperation  involving 
industry,  service  providers,  public  authorities, etc.  The  European  Commission116  is  a  major  funding 
body for the development of CEN standards. Each year it publishes an annual work programme setting 
out its priorities, which may lead to mandates for standardisation. 

Standards adopted by CEN or ISO offer some of the advantages offered by OECD TGs. As with OECD 
TGs, once included in legislation or accepted by regulators, harmonised standards provide industry with 
certainty on which approaches can be used and how data should be generated. They also facilitate the 
authorities’ assessment of submitted data thanks to the standardisation of the data, and the presumption 
of  conformity  with  the  regulatory  requirements  covered  by  harmonised  standards  the  references  of 
which have been published in the Official Journal of the European Union. CEN standards are recognised 
in the EU and the European Free Trade Association, while ISO standards are intended to be applied 
worldwide.  For  this  reason,  data  generated  via  CEN  or  ISO  standards  support  the  international 
acceptance of data, which helps to prevent duplication of testing. 

At  European  (CEN)  level,  ‘harmonised  standards’,  support  the  application  of  the  ‘new  legislative 
framework’.  

Qualification 

115 OECD workshop report: Workshop report on operational and financial aspects of validation 
116 https://single-market-economy.ec.europa.eu/single-market/goods/european-standards/standardisation-policy_en. 

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‘Qualification’ is another relevant term and concept with a particular meaning in the context of this 
SWD.  It  is  understood  as a  voluntary  procedure to  evaluate  a  novel  method intended  for  a  specific 
context of use resulting in a ‘qualification opinion’ from an authority. 

The  EMA  offers  scientific  advice  to  support  the  qualification  of  innovative  methods  intended  for  a 
specific context of use in the research and development of new medicines, including the demonstration 
of their safety for a market authorisation. The advice is given to applicants by the EMA’s Committee 
for  Medicinal  Products  for  Human  Use  (CHMP)  on  the  basis  of  recommendations  made  by  the 
Scientific Advice Working Party. This qualification process leads to a CHMP qualification opinion or 
CHMP qualification advice. On the basis of the qualification advice, the EMA may propose a letter of 
support,  when  the  novel  methodology  under  evaluation  cannot  yet  be  qualified  but  is  shown  to  be 
promising on the basis of preliminary data. Qualification opinions and letters are made public on the 
EMA  website.  In  addition  to  seeking  qualification  advice  or  an  opinion,  developers  of  innovative 
medicines, technologies or methodologies can also engage in dialogue with the EMA’s Innovation Task 
Force  early  on  in  the  development  process,  to  help  decide  on  next  steps  in  their  development 
programmes.  In  recent  years,  developers  and  users  of  alternatives  to  animal  testing  have  been 
particularly  encouraged  to  make  use  of  these  innovation  support  measures.  Finally,  the  EMA’s  3Rs 
(replacement, reduction, refinement)  working  party is  currently  supporting the  revision  of  the  EMA 
guideline on the principles of regulatory acceptance of 3Rs testing approaches, which will include the 
specification  of  qualification  criteria  for  organ-on-chip  based  methods  (e.g.  for  addressing  safety 
questions associated with the heart and liver). 

Building on EMA experience, EFSA funded a project named NAMs4NANO with the aim of developing 
a  qualification  system  within  its  remit  to  facilitate  the  regulatory  implementation  of  non-guideline 
NAMs for a specific context of use (GP/EFSA/MESE/2022/01_Lot 1, see more information in Section 
2.2.4 ‘Fostering the use of NAMs for nanomaterials and nanoparticles risk assessment’). The project 
was  outsourced  to  a  consortium  of  EU  Member  States  and  non-EU  organisations,  with  the 
Bundesinstitut für Risikobewertung (BfR) (German Federal Institute for Risk Assessment) leading this 
task. A  first  proposal  for  a  qualification  system  developed  by  the  NAMs4NANO  consortium  was 
published to gather input from the scientific community (Haase et al., 2024). The proposal details a 
generic framework to speed up the regulatory use of suitable NAMs for chemical risk assessment in 
food and feed. It includes the establishment of an evaluation process and specific criteria to examine 
the  scientific  validity  of  non-guideline  NAMs.  The  framework  is  initially  proposed  for  the  risk 
assessment  of  nanomaterials  and  nanoparticles,  in  line  with  the  approach  defined  by  EFSA’s  nano 
guidance. Targeted discussions were held to further improve the qualification proposal and develop an 
implementation  plan,  involving  agencies  and  organisations  such  as  the  EMA,  ECHA,  the  Scientific 
Committee on Consumer Safety (SCCS), the JRC, the US Food and Drug Administration, the OECD, 
the  Malta  Initiative  Board,  and  the  EFSA  NanoNetwork,  along  with  scientific  and  industry 
representatives. From these exchanges, the need for an infographic to clarify the distinction between 
validation and qualification was identified. As a result, the figure below was collaboratively produced 
by the BfR, EFSA, OECD and JRC. All this work will be instrumental in refining the qualification 
proposal, which is expected to be finalised in 2027. 

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Figure 25: NAMs4NANO graphic analysis of validation vs qualification of alternatives 
to animal testing. This figure is also available in PDF at: 
https://www.efsa.europa.eu/it/topics/topic/nanotechnology 

For  EMA  qualification  purposes,  a  ‘context  of  use’  is  understood  to  describe  the  specific  intended 
application of a new approach methodology in medicines development or regulatory assessment, and 
the conditions under which it can inform regulatory decision-making. Similarly, in the context of the 
development of an EFSA qualification process, ‘context of use’ describes the precise circumstances, the 
intended  applications,  and  the  limits  under  which  a  NAM  could  be  used  for  a  specific  problem 
formulation (e.g. nanomaterial risk assessment in food and feed). 

Agencies may implement their own qualification frameworks to both non-standard methods and test 
guideline  methods  to  make  sure  they  work  in  their  specific  contexts  of  use.  The  requirements  for 
context-of-use-based qualification of a TG method may be lower than those for a non-standard method. 

OECD TGs include methods used for a particular regulatory purpose; however, this is usually broader 
than a context of use as applied in EMA and EFSA qualification processes. While a well-defined context 
of use is central to EMA and EFSA qualification processes, it plays a less critical role in the validation 
of methods under OECD TGs. 

Use of research data for regulatory purposes 

Next to  data  generated  with  validated  or  qualified test methods  or  according to regulatory  accepted 
standards,  criteria  have  recently  been  developed  for  the  acceptance  of  research  data  for  regulatory 
purposes. Examples are the OECD guidance on the use of research data, the pesticides guidance for 
literature search and the Critical appraisal of evidence for food and feed safety assessments. 

Key references and resources 

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2) 

1)  Validation, Qualification, and Regulatory Acceptance of New Approach Methodologies - A Report of 
the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM). March 
2024 (ICCVAM 2024 Report).  
International Cooperation on Alternative Test Methods, ICATM 
https://ntp.niehs.nih.gov/whatwestudy/niceatm/iccvam/international-partnerships/icatm 
https://joint-research-centre.ec.europa.eu/eu-reference-laboratory-alternatives-animal-testing-eurl-
ecvam/alternative-methods-toxicity-testing/advisory-and-consultation-bodies/international-cooperation-
alternative-test-methods-icatm_en. 

3)  The EU Reference Laboratory for alternatives to animal testing (EURL ECVAM) https://joint-research-
centre.ec.europa.eu/projects-and-activities/reference-and-measurement/european-union-reference-
laboratories/eu-reference-laboratory-alternatives-animal-testing-eurl-ecvam_en  

4)  Tracking system for alternative methods towards regulatory acceptance https://tsar.jrc.ec.europa.eu/. 
5)  The OECD Test Guidelines ProgrammeGuidelines for the Testing of Chemicals | OECD 
6)  The OECD IATA Case Studies Project https://www.oecd.org/chemicalsafety/risk-assessment/iata/. 
7)  OECD guidance document 34 on the validation and international acceptance of new or updated test 

methods for hazard assessment 
https://ntp.niehs.nih.gov/sites/default/files/iccvam/suppdocs/feddocs/oecd/oecd-gd34.pdf. 

8)  A framework for establishing scientific confidence in new approach methodologies. van der Zalm et al. 

(2022) https://link.springer.com/article/10.1007/s00204-022-03365-4. 

9)  New approach methodologies workshop: Towards an animal-free regulatory system for industrial 

chemicals (31 May–1 June 2023) https://echa.europa.eu/-/new-approach-methodologies-workshop-
towards-an-animal-free-regulatory-system-for-industrial-chemicals. 

10)  Building Confidence in New Evidence Streams for Human Health Risk Assessment, 

National Academies Press, 500 Fifth Street, NW, Keck 360, Washington, DC 20001;; 
http://www.nap.edu. 

11)  Readiness assessment online tools:  

https://readedtest.u-paris-sciences.fr/ 
https://scirap.org/Page/Index/ee9102de-4b17-4c3a-86b6-e3e70d6ca3d1/evaluate-reliability-and-
relevance. 

12)  EMA’s qualification system: 

https://www.ema.europa.eu/en/human-regulatory-overview/research-development/scientific-advice-
protocol-assistance/qualification-novel-methodologies-medicine-development 
https://www.efsa.europa.eu/en/news/nanotechnology-promoting-uses-new-assessment-methods 

13)  NAMs4NANO Proposal for a qualification system within EFSA’s remit: 

https://www.efsa.europa.eu/en/supporting/pub/en-9008. 

14)  Haase A, Barroso J, Bogni A, Bremer-Hoffmann S, Fessard V, Gutleb AC, Mast J, McVey E, 

Mertens B, Oomen AG, Ritz V, Serchi T, Siewert K, Stanco D, Usmani SM, Verleysen E, Vincentini O, 
van der Zande M, Cubadda F, 2024. Proposal for a fit for purpose qualification system for New 
Approach Methodologies (NAMs) in the food and feed sector. EFSA supporting 
publication 2024: 21(9):EN-9008. 96 pp. doi:10.2903/sp.efsa.2024.EN-9008. 

15)  OECD (2025), Guidance Document on the Generation, Reporting and Use of Research Data for 
Regulatory Assessments, OECD Series on Testing and Assessment, No. 417, OECD Publishing, 
Paris, https://doi.org/10.1787/8d49ec1d-en. 

16)  Submission of scientific peer-reviewed open literature for the approval of pesticide active substances 

under Regulation (EC) No 1107/2009, European Food Safety Authority, 
https://doi.org/10.2903/j.efsa.2011.2092Digital Object Identifier (DOI) (being revised)  
17) EFSA Draft guidance on critical appraisal of evidence for food and feed safety assessments 

https://connect.efsa.europa.eu/RM/s/consultations/publicconsultation2/a0lTk000007cNQz/pc1838 

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5.6. Safe spaces and regulatory exploration spaces for animal-free 
methodologies 
Context 

The European Commission’s roadmap towards the phasing out of animal testing calls for innovative 
regulatory  instruments  that  can  accelerate  the  uptake  of  animal-free  methodologies.  Among  these 
instruments, the concepts of a regulatory ‘safe space’ and a ‘regulatory exploration space’ (RES) have 
gained prominence. 

Safe  spaces  refer  to  platforms  that  allow  applicants  to  share  data  confidentially  with  regulators  to 
discuss the potential acceptability of specific methods for a given case. They provide opportunities for 
non-binding  consultation  and  exploratory  discussions  without  regulatory  consequences,  with  no 
obligations on either the submitter or the agency. 

Regulatory exploration spaces refer to fora where method developers, stakeholders, academia, NGOs, 
industry and regulators collaboratively discuss in an informal environment scientific-technical issues 
related to methods or approaches, their development or improvement, and their application (in different 
sectors), as well as problem formulations, in order to define new and creative solutions. 

A safe space is a bilateral exchange (applicant-regulator) while a regulatory exploration space can be 
multilateral, including different stakeholders. 

The term regulatory sandboxes was not used to avoid confusion with the pharma legislation, which 
defines  a  regulatory  sandbox  as  a  formal  framework  authorised  by  the  Commission,  which  allows 
innovative regulatory solutions to be tested under supervision and for a limited time. The startup and 
scaleup strategy also refers to regulatory sandboxes and states that a common EU legal definition will 
be introduced in the European Innovation Act. In this context, the term RES helps to clearly distinguish 
the roadmap’s informal, exploratory activities from legally formalised regulatory sandboxes. 
Stakeholder consultation 

Across multiple consultations and events, stakeholders have consistently voiced the need for structured, 
transparent and trusted mechanisms to support the development and regulatory exploration of animal-
free methodologies. 

A recurring request concerns the establishment of ‘safe spaces’: fora for early, non-binding dialogue 
between  industry  and regulators. These  would allow  the  confidential  discussion  of  innovative,  non-
traditional  approaches  before  formal  submission,  helping  to  build  mutual  understanding,  clarify 
regulatory expectations and de-risk innovation. Stakeholders note that existing mechanisms, such as 
helpdesks  or  informal  exchanges,  are  not  well  tailored  to  the  specific  challenges  of  animal-free 
methodologies and often lack the predictability and strategic value required. 

In  parallel,  many  stakeholders  have  also  expressed  strong  support  for  the  creation  of  regulatory 
exploration  spaces  (RESs).  RESs  would  enable  a  discussion  of  novel  approaches  under  regulatory 
oversight  without  immediate  consequences  for  authorisation  or  compliance  decisions.  Stakeholders 
consider  that  RESs  could  help  generate  case  studies,  inform  regulatory  guidance  updates  and  help 
identify acceptable use cases, thereby increasing confidence and transparency. 

Industry associations in particular have stressed that RESs should reflect real-world conditions, provide 
legal clarity and support the operational scalability of animal-free methodologies. 

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Stakeholders further highlight the need for coordination across sectors and agencies, suggesting that 
bodies such as the EPAA and the EURL ECVAM, and expert coordination groups at ECHA and the 
EMA could play pivotal roles in convening or supporting these mechanisms. 

According to stakeholders, at present, agencies do not operate dedicated RES mechanisms, although 
existing  advisory  services  and  scientific  fora  could  be  used  to  develop  and  test  RES  models.  The 
Commission  has  received  consistent  feedback  that  a  cross-sector  approach  to  both  safe  spaces  and 
RESs, possibly under a common EU coordination framework, would be highly beneficial. 

Agencies and safe spaces 

This section reviews existing support mechanisms at three EU agencies (EFSA, ECHA and the EMA) 
and explores how the safe‑space model could be advanced to foster the regulatory acceptance of animal-
free methodologies. 

EFSA has developed a comprehensive catalogue of support measures117 that guide applicants through 
the  entire  lifecycle  of  regulated  product  applications.  These  include  general  pre‑submission  advice 
(GPSA), renewal pre‑submission advice (RPSA), fast‑tracked services for SMEs, ad‑hoc clarification 
meetings and technical hearings, as well as training on the agency’s digital submission tools. 

EFSA  provides  advice  on  dossier  preparation  and  frequently  highlights  shortcomings  in  relation  to 
existing guidance. However, it does not engage in discussions on testing strategies; nor does it provide 
support  specific  to  non-animal  testing  approaches.  While  EFSA  is  not  permitted  to  offer  scientific 
advice directly to individual companies, it may participate in public discussions on broader scientific or 
regulatory issues. The  forthcoming  evaluation  of  EFSA  by  the  European  Commission,  covering  the 
period  from  2017  to  2024  and  expected  by  March  2026,  may  offer  an  opportunity  to  revisit  and 
potentially  revise  the  current  approach  to  pre-submission  advice.  Collectively,  these  services  boost 
transparency  and  procedural  clarity  and  are  valued  for  improving  the  quality  and  completeness  of 
dossiers. 

ECHA currently offers assistance in minimising the need for animal testing or applying animal-free 
methodologies  via  the  testing  proposal  evaluation  process.  ECHA  also  offers  general  regulatory 
assistance  through  its  Helpdesk.  The  high  volume  of  submissions  managed  by  ECHA  limits 
opportunities for additional informal dialogue of an exploratory nature.  

The EMA has the most developed structure for fostering early, iterative dialogue on innovative new 
approach methodologies. In this context the term ‘new approach methodology’ is used by the EMA to 
refer to methods that can not only replace but also reduce animal testing. The EMA’s Innovation Task 
Force provides a confidential forum for early-stage discussions on novel technologies, including new 
approach methodologies. Additionally, portfolio and technology meetings support strategic engagement 
on  large  product  pipelines,  offering  opportunities  to  discuss  innovative  approaches,  including  new 
approach methodologies. Through the scientific advice mechanism at the EMA, applicants can obtain 
formal  guidance  on  medicinal  product  development,  including  the  use  of  weight-of-evidence 
approaches that incorporate new approach methodologies Specifically, for regulatory acceptance of a 
new  approach  methodology,  applicants  may  request  qualification  advice,  which  offers  regulatory 
feedback on the method’s suitability for use in product development within a defined context of use. 
The  EMA  has  also  set  up  a  ‘safe  harbour’  pathway  for  the  voluntary  submission  of  new-approach-
methodology  data,  which  is  used  solely  for  the  purpose  of  collecting  information  on  new  approach 
methodologies for possible future regulatory acceptance with no consequences for regulatory decision-
making on medicinal products. This process is under review to further enhance the safe-space approach 
and facilitate data sharing. 

117 https://efsa.onlinelibrary.wiley.com/doi/10.2903/sp.efsa.2021.EN-6472 

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Future steps for safe spaces 

In both EFSA and ECHA there is no clearly defined safe-space mechanism for the early consideration 
and discussion of non-traditional approaches. Nor is there a dedicated structure in place to support their 
systematic integration into regulatory processes. While existing support measures have their uses, they 
do not constitute a true regulatory safe space as envisioned in this document. All advice must remain 
strictly within the boundaries of existing legislation and guidance. 

An additional layer of complexity stems from the requirement that all initiatives must operate in full 
compliance with sector-specific legislation and the broader governance and legal framework. Within 
these constraints, however, it remains unclear how more flexible and innovative processes, capable of 
functioning as genuine safe spaces, can be introduced. 

To  explore  the  feasibility  and  practical  implications  of  establishing  such  mechanisms,  it  is 
recommended  that  stakeholders,  the  Commission  and  the  agencies  discuss  possible  ways  of 
implementing  safe  spaces.  Further  discussions  on  practical  implementation  should  help  ensure  that 
future actions are grounded in evidence and informed by real-world experience. 

It is therefore proposed to organise a workshop, inviting stakeholders, Commission services and the 
agencies to discuss potential ways of implementing safe-space mechanisms and to identify practical 
challenges and enablers. The workshop could also be used to exchange views on the EMA’s ongoing 
safe-space experience, which may provide useful insights for similar approaches at other agencies, such 
as ECHA and EFSA. 

Depending on the outcome of these discussions, and if a need is identified, a continued dialogue on 
practical  implementation  could  be  pursued  before  committing  to  any  large-scale  initiative  requiring 
significant resources. This approach would ensure that future actions were evidence-based and informed 
by real-world experience. 

Given the level of interest in this area, the workshop and any potential follow-up activity should be 
conducted transparently and in close cooperation with relevant stakeholders. The EPAA could take the 
lead in organising the workshop, providing the necessary flexibility in both design and implementation, 
and involving agencies, stakeholders and Commission services. 

The Commission services will assess the outcomes of the workshop and subsequent discussions. On the 
basis  of  the  lessons  learned,  the  roadmap  steering  team  could  then  determine  whether  further  legal 
adaptations or additional resources were needed to scale up the safe-space initiative effectively. 

The regulatory exploration space concept 

The concept of a RES has emerged as a promising tool to support the structured implementation and 
evaluation  of  innovative  methodologies,  particularly  animal-free  methodologies,  in  real-world 
regulatory  contexts.  In  the  context  of  chemical  safety  assessments,  RESs  could  enable  method 
developers,  regulators, industry,  academia and  civil  society  organisations  to  work  collaboratively  in 
refining problem formulations, sharing relevant data, and assessing the performance and suitability of 
animal-free approaches before these are codified into regulatory guidance or legal frameworks. This 
would  support  mutual  learning,  de-risk  innovation  and  accelerate  confidence-building  in  new 
methodologies. A number of key principles underpin the effective operation of a RES. These include 
the need for clear governance rules, predefined criteria for evaluating outcomes, appropriate protections 
for  confidential  data,  and  mechanisms  to  ensure  transparency  and  the  dissemination  of  the  insights 
gained. Embedding such principles in the operating procedures of competent authorities would help 

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foster regulatory trust, reduce uncertainty for innovators and contribute to the EU’s broader ambition to 
lead in global non-animal safety science. 

Although the concept has not yet been fully formalised across EU regulatory agencies, several existing 
initiatives  already  display  key  features  of  RESs  and  can  serve  as  practical  models  for  further 
development.  At  ECHA,  the  Persistent,  Bioaccumulative  and  Toxic  Expert  Group  operates  as  an 
informal  advisory  body,  offering  scientific  input  on  complex  cases  involving  new  or  borderline 
methodologies. Although not a formal RES, it provides a protected space for early consideration of 
innovative scientific approaches. In addition, ECHA’s Expert Group on Endocrine Disruptors offers a 
dedicated forum for early, non-binding discussions on evolving methodologies relevant to endocrine 
disruption. Finally, the Alternative Methods Team is also involved in informal consultations, providing 
feedback  on  industry  proposals  and  scientific  advice  to  ECHA  processes,  Marie  Skłodowska-Curie 
Actions and industry. These informal groups enable regulators and stakeholders to explore complex 
scientific  issues  in  a  structured  yet  flexible  manner,  further  reinforcing  ECHA’s  capacity to  support 
RES-like engagement. EFSA, while not currently operating a formal RES, has developed stakeholder 
engagement  platforms  that  serve  many  of  the  same  purposes.  Through  public  consultations,  expert 
colloquia and technical stakeholder dialogues, EFSA provides structured opportunities for stakeholders 
to engage in the early discussion and exploration of new scientific approaches. These platforms enable 
multi-directional  learning  and  allow  for  the  transparent  exchange  of  knowledge  in  a  non-binding 
context, supporting the iterative development of regulatory thinking and scientific guidance. 

Other examples of similar activities are the Scientific Committee on Consumer Safety (SCCS) and the 
EPAA non-animal method (NAM) Designathon. 

The SCCS, operating under the Cosmetics Regulation that fully banned animal testing in the cosmetics 
sector, has implemented a dual-submission practice whereby both traditional and NAM-based data can 
be submitted for evaluation. This framework enables the informal consideration of NAMs alongside 
established  methods  and  serves  as  an  effective  RES  for  next-generation  risk  assessment  (NGRA) 
strategies. 

The  EPAA  NAM  Designathon,  for  example,  has  established  a  structured  co-creative  setting  where 
regulators, scientists, industry representatives and NGOs jointly develop and assess NAM-based testing 
strategies  using  real-world  case  studies. These  sessions  enable  structured  experimentation in  a  non-
binding format, promoting regulatory familiarity with novel approaches and facilitating alignment on 
scientific expectations. 

ECHA and EFSA are also active partners in the Accelerating the Pace of Chemical Risk Assessment 
(APCRA). The APCRA initiative serves as a practical example of a RES-like setting at international 
level. It enables regulators from multiple countries to collaboratively explore and evaluate the use of 
animal-free  methodologies  in  chemical  risk  assessments  through  case  studies,  without  regulatory 
consequences. APCRA demonstrates how structured, non-binding collaboration can build confidence 
in innovative approaches and support regulatory convergence. 

Future steps for RES 

While the agencies have not yet fully formalised RES mechanisms, stakeholders have called for them 
to be set up and coordinated under a common EU framework. Existing initiatives by platforms such as 
the  EPAA,  including  the  NAM  Designathon  and  User  Forum,  demonstrate  the  value  of  such 
collaborative  settings.  Building  on  these  models,  there  is  a  clear  recommendation  to  expand  and 
institutionalise  regulatory  RESs  across  sectors,  enabling  systematic,  low-risk  testing  of  innovative 
approaches and fostering convergence among EU agencies and different legislative sectors. 

The  EPAA  is  well  positioned  to  support the facilitation  or  coordination  of such  initiatives.  Existing 
formats, including the EPAA Designathon and User Forum, have demonstrated the feasibility of multi-
stakeholder  collaboration  and  could  serve  as  the  basis  for  more  structured  RES  activities.  Future 

210 

 
 
 
development should aim to institutionalise RESs across sectors, ensuring that agencies can test, adapt 
and build confidence in new methodologies in a controlled, transparent and legally secure manner. 

EFSA, taking into account the EMA’s experience, could explore the possible establishment of a RES 
for the qualification of non-animal approaches, with nanomaterials risk assessment as a first example 
of possible implementation. 

For  maximum  impact,  RES  activities  should  be  designed  to  complement  existing  initiatives  and  to 
enable coordination across agencies, Member States and legislative frameworks. A networked model, 
linking  agency-specific  structures  on  animal-free  methods  with  existing  advisory  processes,  would 
foster a coherent European ecosystem for innovation in chemical safety assessments. 

In conclusion, the development of RESs represents a necessary next step in supporting the transition to 
animal-free safety assessments. Such structures will enable the controlled and practical evaluation of 
innovative methods, promote cross-sector learning and provide the necessary evidence base for future 
guidance updates and regulatory reform. 

Conclusion and forward look 

RESs in synergy with safe spaces would strengthen the EU’s leadership in evidence-based, non-animal 
science. 

Setting up such mechanisms, anchored by collaborative structures that coordinate scientific evaluation, 
stakeholder dialogue and cross‑sector learning, would respond directly to recommendations emerging 
from the roadmap. It would also position the EU as a global leader in innovative, animal‑free safety 
assessments. 

All  the  different  activities  will  be  coordinated  by  the  roadmap  steering  team,  which  will  monitor 
progress and ensure alignment with wider EU policy objectives. 

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6. Indicators – managing change by measuring it 

Rationale and background 
In the context of the Commission’s roadmap, there is a need to establish indicators to monitor progress 
towards  the  final  goal  of  phasing  out  animal  testing  in  chemical  safety  assessments. The  aim  is  to 
develop a set of indicators that can be used to develop both cross-sector and sector-specific narratives. 
It is possible that some indicators will be applicable to all sectors, and that others will be sector-specific, 
depending  on  the  nature  of  the  available  underpinning  data.  It  is  also  expected  that  there  will  be 
methodological differences between sectors in terms of data availability and accessibility. Achieving a 
better overview could be the basis for future efforts to harmonise data collection. It is also considered 
that certain indicators might be more informative in the short and medium term, and others in the long 
term. In addition, when we start off with a first set of indicators to measure progress to better manage 
change, we might decide to add others or revise some in the first set to gain better knowledge on progress 
during implementation. Some indicators might also cease to be relevant if progress to an intermediate 
goal has been achieved, e.g. testing in a second species is no longer conducted. 

Crowd sourcing ideas from stakeholders 
During the Animal-Free Chemicals Safety Assessment Conference, 4-6 March 2025, co-organised by 
the EPAA and the Commission, two parallel break-out groups were set up to discuss indicators and how 
to best measure progress during implementation of the roadmap. About 40 participants representing 
different stakeholders took  part  in  these  discussions, and  there  was  also  a  reporting  back  session in 
plenary, collecting additional input from the full audience. The questions discussed were: 

•  What does success look like to you? 
•  What change is necessary to reach the successful outcome? 
•  Who should host and report on indicators? 

Many indicators were suggested, and after the conference a small core team made up of representatives 
of the Commission, industry and an animal welfare NGO worked further on the input and circulated a 
table of proposed indicators to the break-out groups’ participants for comments. The resulting list of 
indicators was presented at the third workshop to discuss the roadmap to phase out animal testing for 
chemical  safety  assessments,  16-17 June 2025  and  was  further  updated  based  on  the  feedback. The 
initial and broad collection of ideas from all stakeholders that will be further evaluated for relevance 
and feasibility is included in Annex 1. 

The indicators were classified in four categories to make sense of the underlying types of change: 

1.  engagement indicators 
2.  status indicators 
3.  counter-progress (weakness) indicators 
4.  progress (3Rs) indicators. 

Engagement indicators aim to capture public awareness and support, knowledge sharing, collaborations 
and partnerships. Status indicators are related to development and validation or qualification of non-
animal or alternative approaches, funding and investment, cost-effectiveness, industry adoption, and 
regulatory  acceptance.  The  counter-progress  or  weakness  indicators  aim  to  identify  barriers  to 
acceptance and regulatory uptake, efficiency and reliability. Progress indicators are directly related to 
the ultimate aim of the roadmap, i.e. the phasing out of animal testing. 

Composite indicators, i.e. several indicators used in combination, are defined as a separate group. 

Usefulness of indicators 

212 

 
 
 
 
 
Indicators should be relevant, reliable and feasible in order to measure change, understand progress and 
inform  priorities.  It  is  important  to  understand  that  an  indicator  is  not  only  a  number  to  be  used  in 
debates or to give the appearance of success. If used carefully and impartially, indicators will serve as 
a  means  to  better  monitor  the  transition  to  animal-free  chemical  safety  assessments  and  to  direct 
roadmap actions. For example, are the activities supported by the roadmap steering team and set out in 
the transitional initiatives leading to the expected progress? The indicators might tell a different story 
from that intended by those helping to implement the roadmap. For instance, the number of alternatives 
to animal testing developed does not have any impact if it is not regulatory relevant. Even though it was 
the intention for alternatives to animal testing to be used for regulatory testing, it might not be suitable 
for that purpose in the end. Cost-benefit considerations need to be measured to gain understanding in 
how to improve the ratio of alternatives to animal testing that can be used for regulatory testing and 
make it work in favour of progress. 

Indicator description 
To make further progress with the description of an indicator, we suggest that the following information 
must be available and identified for each indicator: 

1)  short title of indicator 
2)  owner 
3)  definition 
4)  expected usefulness 
5)  data source(s) including their availability/accessibility 
6)  methodology for finding, processing and interpreting the data 
7)  limitations in the interpretation of the indicator 
8)  frequency and timing of reporting. 

First suggested prototype indicators 
We  have  completed  the  indicator  description  for  the  indicators  listed  below  (Annex  2,  Example 
indicators) from the table in Annex 1 and suggest that these can be included in the first set of prototype 
indicators to be evaluated in a trial phase. 

Type of indicator 
Engagement 

Status 

Counter-progress 

Progress 

First set of prototypes to evaluate 

Awareness  of  non-animal  approaches  and  their  possible  application  in  chemical 
safety assessments (based on interviews with stakeholders) 
Use  of  positive  and  negative  language  in  public  discourse,  e.g.  political  speech, 
corporate speeches and publications (based on publicly available material) 
Follow-up of roadmap actions to assess the actionability and continued relevance 
of actions (in terms of inputs and relevant outputs) 
Relative cost of a chemical safety assessment based on non-animal approaches 
compared with the traditional animal test (both approaches providing equivalent 
information)  
Use  of  non-animal  approaches  to  trigger  additional  in  vivo  testing  (according  to 
regulatory information requirements and guidance) 
Bias in research funding towards the use of animal tests over non-animal approaches 
(number of grants fostering non-animal studies) 
Phasing  out  of  animal  testing  fulfilling  EU  regulatory  information  requirements 
(number of animals used to fulfil regulatory information requirements, horizontal 
and per legislative sector) 
Reduction  of  severity  in  animal  procedures  where  these  tests  are  still  performed 
(based on severity, horizontal and per-legislative-sector scores) 

Hosting an indicator and tell a story 

213 

 
 
 
 
 
 
 
 
 
 
It is envisaged that the set of indicators applied during roadmap implementation will not be fixed over 
time  and  will  have  distributed  ownership  (i.e.  responsibility  for  developing  and  reporting).  The 
Commission might not be the right developer and host for all indicators. For example, the EU agencies 
already report indicators that are relevant for the roadmap. Additional indicators are listed in the table 
in Annex  1,  some  of  which  other  stakeholders  might  have  an  interest  in  refining  and  reporting. An 
indicator can be sector specific and – even when only reported by a single company or organisation – 
still  be informative  for a  better  understanding  of  how  to tackle  different  issues to  achieve  progress. 
Therefore, specific stories might be of great interest and value, while the more horizontal indicators will 
tell a more general story. 
It is recommended that the roadmap steering team take on the task of evaluating selected indicators for 
their usefulness during the roadmap’s implementation. 

Next steps 
The Commission Communication sets out a few indicators, while the Commission may identify a longer 
and more detailed set of indicators when the implementation phase starts.  

References 
EPAA 

(2025)  Animal-Free  Chemical  Safety  Assessment  Conference,  Flash  Report, 
https://webgate.ec.europa.eu/circabc-ewpp/d/d/workspace/SpacesStore/4015d661-04da-440e-
96d9-a937e955d341/download. 

Conference Report, d7994cf5-4b38-9a8a-9cbc-0c89da0dcad8. 

EC  (2025) Third  workshop  to  discuss  the  roadmap  to  phase  out  animal  testing  for  chemical  safety 
assessments,  16-17 June  2025,  Helsinki,  hosted  by  ECHA,  https://echa.europa.eu/pt/-/third-
workshop-to-discuss-the-roadmap-to-phase-out-animal-testing-for-chemical-safety-
assessments. 

EC (2024) ALURES – ANIMAL USE REPORTING - EU SYSTEM, EU statistics database on the use 
of animals for scientific purposes under Directive 2010/63/EU. ALURES statistics - Section 1 
: Numbers of animals used for research, testing, routine production and education and training 
purposes - EU statistics database on the use of animals for scientific purposes

214 

 
 
 
 
Indicators - Annex 1 
Initial and broad collection of ideas from all stakeholders that will be further evaluated for relevance and feasibility. The eight prototype 
examples mentioned in the text and further described in Annex 2, are highlighted in green. 

1.  ENGAGEMENT INDICATORS  

(Public Awareness and Support, Knowledge Sharing, Collaborations and Partnerships) 
Definition 
Number of bilateral meetings between 
companies and agencies about testing 
strategies leading to a reduction of animals 
used or involving non-animal approaches  
Number of communications providing 
feedback from agencies to dossier 
submitters, made publicly available 

Must be relative to 
Each agency and substance 

Data holders 
Agencies 

Each agency and substance 

Agencies 

Title 
Create confidence in applying new testing 
strategies through bilateral discussion 
(Example 1) 

Create confidence through communication 
to better guide dossier submitters towards 
possible acceptance of information from 
non-animal approaches 
Knowledge Sharing supporting 
collective development towards more 
successful use of non-animal approaches 

Awareness of non-animal approaches and 
their application in chemical safety 
assessment 

Use of positive and negative language in 
public discourse, e.g. political speech, 
corporate speeches and publications 
(Example 2) 

Recognitions awarding faster progress 

Comments 
Transparent meeting records to be 
made available, in general without any 
substance specific details  

The records need to be transparent but 
can omit business-sensitive or 
confidential information.  

Sharing both successful and 
unsuccessful application of non-
animal approaches also as a summary 
of information and avoiding any 
confidential information 
Evaluate barriers for acceptance 
phasing out of animal testing, e.g. lack 
of training, upskilling, fear of losing 
jobs. 

This could be made also 
retrospectively to observe current 
trends and the development during the 
roadmap implementation.  

Sector and context 

Agencies, Research 
projects, Industrial 
associations 

Description of stakeholder 
interviewed and legal area(s) in 
scope of the roadmap 

Researchers 
performing 
interviews 

Specific communicative 
contexts 

Publicly available 

Description of context, 
frequency and popularity 

Collected by 3R 
centres, Member 
State authorities, 
industry 
associations, NGOs 

This could be made also 
retrospectively to observe current 
trends and the development during the 
roadmap implementation. 

Number of case studies on implementation 
of non-animal approaches with regulatory 
relevance and shared with broader 
audience 

Based on stakeholder interviews regarding 
acceptance of non-animal approaches in 
chemicals safety assessment (focusing on 
stakeholders not contributing directly to 
chemicals safety assessment) 

Sentiment analysis of specific 
communication, like comments from 
relevant regulatory committees, official 
communications and consultations by the 
Commission, MS authorities and other 
stakeholders 
Number of awards for accomplishments 
related to the phasing out of animal testing 
in chemical safety assessment 

215 

 
 
 
 
(Development and Validation of Alternatives, Funding and Investment, Cost-effectiveness, Industry Adoption, Regulatory Acceptance) 

2.  STATUS INDICATORS  

Title 
Follow-up of roadmap actions to 
assess the actionability and continued 
relevance of actions (Example 3) 

Definition 
Number of outputs related to each action 
presented in the Communication or Staff 
Working Document 

Must be relative to 
Each action 

Data holders 
Various - to be 
specified for each 
action 

Maintain innovation and build confidence 
by establishing non-animal methodology 
batteries 
Build capacity by dedicating a higher 
percentage of staff to animal-free safety 
assessment 

Number of new versions of non-animal 
methodology batteries accepted to ensure 
protection levels 
Number of FTEs working on issues related 
to animal-free and animal-based safety 
assessment in different sectors 

Per substance and decisions 
made  

Industry 

Total number of FTEs 
working on issues related to 
chemical safety assessment 
and sector 

Agencies, industry, 
CROs 

Maximise cost-benefit when replacing 
animal methods with in vitro testing 

Accelerate validation and acceptance of 
non-animal approaches   

Prioritise the development of new non-
animal approaches to fulfil a higher 
percentage of regulatory information 
requirements 

Cost of in vitro tests carried out for safety 
assessment compared to the cost of 
replaced animal methods 

Number of new non-animal approaches 
with successful validation acc. to OECD 
GD34and publication within the test 
guidelines programme or included in case 
study publications (IATA case studies 
project) 
% of regulatory standard information 
requirements for which OECD validated 
test methods and defined approaches are 
available  

Prioritise development of non-animal 
approaches to cover a higher number of 
chemicals and a higher number of 
endpoints  

% of chemicals and endpoints assessed in a 
regulatory framework relying on non-
animal approaches and without new animal 
testing 

Per substance assessed for 
regulatory safety assessment 

Agencies, industry, 
CROs 

OECD 

Agencies 

Agencies 

Time discussed at the OECD 
prior to acceptance or estimate 
of the research funding 
allocated per non-animal 
approach 

Time discussed at the OECD 
prior to acceptance or estimate 
of the research funding 
allocated per non-animal 
approach 

Time discussed at the OECD 
prior to acceptance or estimate 
of the research funding 
allocated per non-animal 
approach 

216 

Comments 
Over time, it is expected that 
roadmap actions will be refined, 
added, or discontinued, according to 
progress. 
Not as relevant for R&D pipeline 
screening methods, but rather 
regulatory application 
The combined information provides 
insight in whether the non-animal 
approaches are supporting the 
phasing out of animal use or is used 
for enhanced understanding of the 
animal studies. 
Other information might also be 
considered in assessment of a 
substance, but this indicator focuses 
on in vitro testing. 
To better understand how to make the 
process more efficient 

Similar to the one above but is 
relative to the breadth of regulatory 
needs.  
Non-animal approaches for a specific 
regulatory information requirement 
typical include several test methods 
Could also be considered whether 
there is a time frame for legal uptake 
within the EU 

 
 
 
 
 
Accelerate progress within the EU by 
regulatory acceptance prior to acceptance 
at OECD level, and encourage progress 
within other jurisdictions 

Number of non-animal approaches 
included in EU guidance and regulation 
prior to adoption by the OECD replacing 
animal testing 

Use of non-animal research data to build 
confidence and accelerate acceptance of 
non-animal approaches  
Resource allocation for confidence 
building and capability development 

Resource allocation for development of 
non-animal approaches to accelerate 
regulatory acceptance of non-animal safety 
assessment 
Development of guidance on reviewing 
dossiers including data from non-animal 
approaches 

Frequency of use of non-standard non-
animal research data in regulatory 
assessments 
Funding provided for case studies, 
trainings and workshops aiming at 
confidence building in non-animal 
approaches 
Funding provided for development non-
animal approaches that are eventually 
accepted for regulatory implementation 

Number of standard 
information requirements/ 
endpoints for which animal 
testing is required, or 
research funding and cost/ 
time of validation allocated 
per non-animal approach.  
Per endpoint or per substance 

Per use-sector and number of 
relevant chemistries 

Agencies 

Information on promotion of 
acceptance outside the EU could be 
collected. 

OECD guidance on better use of 
research data in regulatory 
assessments 

Agencies, Research 
authors, publishers 

Commission, MS 
authorities, agencies, 
industry 

Per use-sector, or 
percentage of overall research 
funding on NAM development  

Commission, MS 
authorities, agencies, 
industry 

Cost and time for reviewing non-animal 
approach-based dossiers or realistic case 
studies 

Cost and time used to review 
corresponding dossiers 
including in vivo studies 

Agencies 

Relative cost of a chemical safety 
assessment based on non-animal 
approaches compared with the traditional 
animal test (both approaches providing 
equivalent information) (Example 4) 
Affordability of manufacturers to replace 
animal testing with non-animal approaches  

Affordability of consumers NAM-based to 
replace animal testing with non-animal 
approaches 

Market price for running a substance 
through a non-animal battery/defined 
approach under GLP and market price for 
the corresponding animal test 

Manufacturers’ Willingness to Pay (WTP) 
for replacement of in vivo testing estimated 
through surveys or by observing real 
market choices 
Consumers’ Willingness to Pay (WTP) for 
ethical/cruelty-free products through 
surveys or by observing real market 
choices 

Per chemical and endpoint 

CROs, industry 

Cost per endpoint and per 
sector 

Industry, consultants 

Cost per product and per 
sector 

Consumers 

217 

Consider EPA metrics 
https://www.epa.gov/chemical-
research/new-approach-methods-
nams-animal-use-metrics-research-
and-development. Communication 
for regulators is important to help 
incentivise NAM use. 
Needed: guidance on what is included 
and excluded in the cost calculation 
Includes invertebrate testing when 
replacing vertebrate testing. 

Is there interest from manufactures to 
start ethical labelling on all consumer 
products? 

To inform the consumers, the cruelty-
free products need to be labeled as 
such, which could be of marketing 
interest to do also outside the 
cosmetics sector. 

 
 
 
 
 
Influence of consumer behaviour on the 
market 

Market share of ethical/cruelty-free 
products 

Other products in the same 
sector 

Industry 

Competitiveness of CROs and start-ups 
providing non-animal solutions 

Higher number of start-ups providing non-
animal solutions 
Adaptations/ derogations to animal testing 
mandated by legislation 

Added value (euros, % of GDP) generated 
by non-animal services sector compared to 
the loss of GDP in animal services sector 
Amount of venture capital given to start-
ups specialising in non-animal services 
Number of adaptations allowed to avoid 
animal testing when fulfilling information 
requirements  

Other sectors, 3rd country 
regions 

MS authorities 

Other sectors 

MS authorities 

Per substance and information 
requirement 

Agencies 

Regulatory guidance updates within the 
EU 

Number of revisions of guidance per year 
enabling phasing out of animal testing  

Legislative updates within the EU 

Number of legal text revisions enabling 
phasing out of animal testing  

Acceptance of non-animal approaches for 
regulatory submission (including use of 
invertebrates when replacing vertebrates) 

Number of endpoints covered solely by 
non-animal data in submissions  

Compare with baseline from 
publication of the roadmap 
and mapped to the causes 
triggering the update 
Compare with baseline from 
publication of the roadmap 
and mapped to the causes 
triggering the update 
Number of submissions AND 
reduction of animals used 

Agencies 

Commission 

Agencies 

Hazard classification based on non-animal 
approaches (including use of invertebrates 
when replacing vertebrates) 

Number of CLP hazard classes for which 
classification relies on non-animal 
approaches 

Risk assessment based on non-animal 
approaches (including use of invertebrates 
when replacing vertebrates) 

Number of substances/endpoints for which 
hazard characterisation relies entirely on 
non-animal approaches (REACH, PPPR, 
BPR, any other relevant piece of 
legislation) 

Total number of human health 
and environmental CLP 
hazard classes, relevant to 
animal testing 
Total number of risk assessed 
substances/endpoints, 
including updated assessments 

Commission, 
Agencies 

Commission, 
Agencies 

218 

To inform the consumers, the cruelty-
free products need to be labeled as 
such, which could be of marketing 
interest to do also outside the 
cosmetics sector.  
Link to EU start-up and scale-up 
strategy 

Link to EU start-up and scale-up 
strategy 
US EPA collects metrics on waivers 
applied with monthly meetings to 
discuss and update this data input. 
Could this approach work for EU 
agencies? 
Could be done for each of the 15 
identified legal sectors in the scope of 
the roadmap. 

Could be done for each of the 15 
identified legal sectors in the scope of 
the roadmap. 

Might be difficult for 
pharmaceuticals.  
The 117(3) report from ECHA 
provides a good summary under 
REACH, can this be replicated for 
other agencies?  

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Enable innovation despite animal testing 
ban for cosmetics 

Number of new cosmetic ingredients put 
on the market 

Compare with baseline since 
ban (2013) 

MSs, Commission, 
Industry 

Protection of human health and the 
environment 
while accelerating progress towards 
phasing out of animal testing 

Number of hazardous chemicals per 
endpoint with risk management decisions 
based on evidence from non-animal data 

Compare with baseline since 
publication of the roadmap 

Maintain or improve ability to innovate 
chemicals (REACH) 

Number of new chemicals registered 
and/or cost for registration dataset  

Phasing out of second species 
requirements 

Non-animal approaches used in self-
classification 

Tracking short, medium and long-term 
opportunities for replacement of animal tests 

Number of information requirements for 
second species deleted in legislation or 
regulatory guidance 
% of self-classifications based (partially or 
entirely) on non-animal approaches under 
CLP as reported by ECHA 
Broad scope tracking of all regulatory tests on 
animals in scope of the roadmap which 
can/can’t be phased out 

Compare with baseline since 
publication of the roadmap. 
Total number and/or cost of 
registered chemicals during 
certain time period  
Number of data requirements 
for second species at baseline 
(publication of the roadmap) 
% of harmonised classified 
substances 

ECHA 

Agencies 

ECHA 

Individual animal tests, performed 
across all legislation in scope of 
the roadmap 

Commission and 
agencies 

Need to differentiate between 
ingredients which need listing and 
others? 
How to prove that the decision was 
correct, not protective or over-
protective? On the other hand, how 
do we know whether we are doing the 
correct decisions based on animal 
testing? 
Number of new chemicals will also 
depend on many other factors such as 
global economy, energy cost, etc 

Phase out the less appropriate 
species… 

Changes in the population of 
short/medium/long-term "baskets" will 
illustrate progress. An important 
component is the collection of 
information and analysis of why animal 
testing persists. 

3.  COUNTER-PROGRESS (WEAKNESS) INDICATORS 

(Barriers to Adoption, Efficiency and Reliability) 

Title 
Use of non-animal approaches to trigger 
additional in vivo testing (Example 5) 

Encouraging more robust read-across 
arguments provided by submitter and more 
confidence in accepting read-across 
arguments 

Definition 
Number of animal tests carried out for 
endpoints triggered by the outcome from a 
non-animal approach in regulatory 
information requirements and guidance 
Number of read-across arguments for 
specific information requirements not 
accepted or rejected 

Must be relative to 
Number of substances 
assessed for these endpoints 

Data holders 
Agencies 

Comments 

Total number of substances 
with information submitted 
for the specific endpoint 

Agencies, SCCS 

Also include lessons learned 

219 

 
 
 
 
 
 
 
 
Tracking final, full replacement of animal 
tests 

Identification and tracking of the number of 
animal tests (conducted per year) which 
continue to be conducted despite appropriate 
non-animal approaches being broadly 
available 

The specific animal test in 
question, across all EU 
legislation in scope of the 
roadmap 

Agencies 

Bias in research funding towards the use of 
animal tests over non-animal approaches 
(Example 6)  

Number of grants fostering non-animal 
studies and number of grants fostering in 
vivo studies 

Total number of grants  

Research funders 

This would highlight very near term 
opportunities where the barriers to 
full replacement are relatively easily 
surmountable. An important 
component is the collection of 
information and analysis of why 
animal testing persists 

Title 
Phasing out animal testing fulfilling EU 
regulatory information requirement 
(Example 7) 

Definition 
Number of animals used for regulatory studies 
for regulatory studies, horizontal and per 
legislative sector 

Must be relative to 
To a baseline, also set prior to 
the publication of the roadmap 
when data is available 

4.  PROGRESS (THREE Rs) INDICATORS 

Phasing out animal testing carried out 
within the EU fulfilling non-EU regulatory 
information requirement only 
Reduction of severity in animal procedures 
where these tests are still performed 
(Example 8) 

Number of animals used for regulatory studies 
fulfilling non-EU requirements 

Severity scores, horizontal and per legislative 
sector 

To a baseline, also set prior to 
the publication of the roadmap 
if data is available 
Study type and endpoint 
including studies made to 
fulfil the same test guideline 

Data holders 
ALURES (EC, 
2024) – testing 
performed within 
the EU, 
Industry/Agencies 
– testing performed 
outside the EU 

Comments 
REACH A.117 report on 
alternatives from ECHA has 
previously provided useful data 
on numbers per OECD TG. 
Useful to indicate whether a 
submission is fulfilling more 
than one Information 
Requirement in EU legislation. 

ALURES, industry  Specify other jurisdictions 

requiring the testing 

ALURES 

Perhaps possible to map the 
studies currently used in safety 
assessment with highest 
severity, and then see whether 
they reduce, perhaps looking to 
a few specific regulatory uses 

Waiving of animal studies based on 
REACH, Annex XI provisions and gaining 
confidence in non-animal approaches  
Waiving of animal studies based on 
exposure under REACH (requires Annex 
XI/guidance update) 

Number and volume of chemicals with data 
requirements being fulfilled by Annex XI 
adaptations  
Number and volume of chemicals with data 
requirements being fulfilled by exposure-
based waiving  

Total number and volume of 
chemicals registered 

Total number and volume of 
chemicals registered 

ECHA 

ECHA 

220 

 
 
 
 
 
 
Use of a non-animal approach for a certain 
substance which is possible to apply to 
other substances  

Number of pharmaceutical ingredients to 
which the same assessment approach was 
applied 

Total number of 
pharmaceutical ingredients 
assessed for the same endpoint 

EMA 

Waiving of animal studies in veterinary 
medicines 

Number of waivers of animal tests accepted in 
vaccine batch testing  

Total number of waiver 
requests 

EU impact the phasing out of animal 
testing internationally 

Number of times when an EU initiative 
succeeded in phasing out an animal test in 
OECD guidance or replace GHS criteria based 
on animal testing etc 

Total number of EU initiatives 
to phase out animal testing in 
international context   

COMPOSITE INDICATORS 

EMA, Industry, 
AnimalHealthEuro
pe 
Commission 

Useful to also describe the 
initiatives 

Title 

Definition 

Must be relative to 

Data holders  Comments 

Summary of multiple indicators 
providing a clearer information 

A composite indicator is a useful tool for 
policy making and public communications in 
conveying information on performance 

Any information defined for the 
individual indicators being components 
of the composite indicator 

JRC 

The Competence Centre on 
Composite Indicators and 
Scoreboards at JRC could help 
to develop composite indicators 
that integrate and summarise 
multiple indicators.   

221 

 
 
 
 
 
 
 
 
Annex 2 – Example Indicators 

Example 1 

Awareness of stakeholders of non-animal approaches used/possible to use in chemicals safety assessment 

Owner 

Researchers performing interviews (possibly tasked by the JRC) 

Definition 

Stakeholders’ acceptance of non-animal approaches in chemicals safety assessment covering legislation under the 15 legal areas in scope of the roadmap 
(focusing on stakeholders not contributing directly to chemicals safety assessment) 

Expected utility 

Understanding barriers and perceptions of changing from animal to non-animal approaches, and thereby inform confidence building among these 
stakeholder groups  

Data source(s) including their availability/accessibility 

Interviews performed by researchers with different stakeholders, summary outcome made publicly available 

Methodology for finding, processing and interpreting the data 

The interviews will be carried out possibly by the same researchers, and in any case, following the same methodology and questionnaire on each occasion. 
If considered informative, some questions can be added to better understand development. Representatives of the same stakeholder groups must be 
selected for each occasion. It might be considered whether desired and feasible to interview additional stakeholders, if there is evidence that the coverage 
is not complete.  

Limitations in the interpretation of the indicator  

The number of interviews that can be performed, the selection of stakeholders. Risk for variation in methodology and interpretation of results, if carried out 
by different researchers. The indicator is qualitative rather than quantitative and therefore might be less specific when measuring change. However, it can 
be considered to rank the replies to the interviews, in for example five groups, by the researched, which would give some means of comparing interviews, 
as well as results from different years. 

Reporting (frequency and timing)  

Starting 2026, and to be repeated every 2 years. 

222 

 
 
 
Example 2 
Short title of indicator 

Use of positive and negative language in public discourse, e.g. political speech, corporate speeches and publications  

Owner 

Researcher collecting information (possibly tasked by the JRC) 

Definition 

Sentiment analysis or critical discourse analysis of specific communication, like comments from relevant regulatory committees, official communications 
and consultations by the Commission, MS authorities and other stakeholders 

Expected utility 

Understanding barriers and perceptions of changing from animal to non-animal approaches, and thereby improve communication and provide information 
mor efficiently 

Data source(s) including their availability/accessibility 

Interrogation of publicly available information in scope of the definition 

Methodology for finding, processing and interpreting the data 

AI informed research of publicly available information sources, evaluation of relevant content and classification in for example in five groups: (1) positive 
to phasing out of animal testing, (2) positive to phasing out of animal testing, but reluctant to feasibility, (3) neutral, (4) negative to phasing out of animal 
testing, but only in specific fields of application, (5) negative to phasing out of animal testing. If possible, specify context and owner of statements, to 
investigate how this information can be informative for any additional conclusions or suggested follow-up actions. This could be supplemented in cases 
with an in-depth analysis of specific texts using critical discourse analysis. 

Limitations in the interpretation of the indicator  

Misinterpretation of statement when taken out of its context. Discourse analysis is less subject to this bias. 

Reporting (frequency and timing)  

Starting retrospectively, for example 2020, and to be repeated every year. 

223 

 
 
 
 
Example 3 
Short title of indicator 

Follow-up of roadmap actions to assess the actionability and continued relevance of actions 
Owner 

Various - to be specified for each action 

Definition 

Number of outputs related to each action presented in the Communication or Staff Working Document 

Expected utility 

Over time, it is expected that roadmap actions will be refined, added, or discontinued, according to progress. 

Data source(s) including their availability/accessibility 

Collected by the Commission, and to be evaluated by the Roadmap Steering Team. The outputs are assumed to be publicly available, reported in context of 
specific projects, for example through the EPAA, or any registered transitional initiative. 

Methodology for finding, processing and interpreting the data 

The Commission with support of the Roadmap Steering Team, collect all relevant outputs to the roadmap actions, which is essential for evaluating progress 
towards phasing out of animal testing. The expected outputs will be mapped against the actions, and made publicly available, to allow for any stakeholder to 
complete the information. 
Limitations in the interpretation of the indicator  

The indicator is reporting on quantity rather than quality, and it could be evaluated whether it would be possible to include some qualitative element, which 
even could allow for a quality appraisal, to present together with the quantitative indicator. 

Reporting (frequency and timing)  

Starting 2027 and to be repeated every year 

224 

 
 
 
Example 4 

Short title of indicator 

Relative cost of a chemical safety assessment based on non-animal approaches compared with the traditional animal test  
Owner 

CROs, industry 

Definition 

Market price for running a substance through a non-animal battery/defined approach under GLP and market price for the corresponding animal test 

Expected utility 

Over time, the non-animal approach alternative might become more affordable, due to e.g. legal requirements and upscaled testing compared to the current 
animal testing for a specific endpoint. By following trends, from first introduction of new non-animal approaches to a broader use, we will learn how to 
tackle both increments in cost for new specific information requirements and how to efficiently lower overall costs (through smart testing strategies).  

Data source(s) including their availability/accessibility 

Data to be collected by Industrial Associations or directly from CROs on possible replacement approaches. 

Methodology for finding, processing and interpreting the data 

The Commission with support of the Roadmap Steering Team to list all animal methods in scope of the roadmap that could potentially be replaced by non-
animal approaches and publish this list. The Commission with support of, for example, EPAA, collect data on market prices both for animal methods and the 
non-animal approaches providing equivalent information. For those already in use in the EU, also retrospective information might be collected.  
Limitations in the interpretation of the indicator  

Variation in market prices (including fluctuations in exchange rates) for testing not performed within the EU, and difficulties to understand how much of the 
testing is done outside the EU, and what the real change in cost is. 

Reporting (frequency and timing)  

Starting 2027 and to be repeated every year 

225 

 
 
 
 
Example 5 

Short title of indicator 

Use of non-animal approaches  to trigger additional in vivo testing 
Owner 

Agencies 

Definition 

Number of animal tests carried out for endpoints triggered by the outcome from a non-animal approach in regulatory information requirements and guidance 

Expected utility 

The aim is to avoid triggering animal testing, and to enable a decision based on the information obtained from non-animal approaches. This can be achieved 
by better testing strategies, or enhancement of non-animal approaches, to further reduce uncertainty in the results obtained.   

Data source(s) including their availability/accessibility 

Agencies can collect this information in dossiers achieved within legal procedures, such as authorization. 

Methodology for finding, processing and interpreting the data 

Each agency can set up a list of non-animal data collected that often lead to further testing in the animal model, report on the number of animals used per 
substance assessed for each relevant endpoint or information requirement. As far as possible, there can also be provided information on the reasons the animal 
testing was considered necessary to perform, e.g. the substance was considered a difficult-to-test substance.  
Limitations in the interpretation of the indicator  

The best baseline for this indicator would be to understand the use of the animal methods prior to the introduction of the non-animal approach.  

Reporting (frequency and timing)  

Starting 2027 and to be repeated every year 

226 

 
 
 
 
 
Example 6 

Short title of indicator  

Bias in research funding towards the use of animal tests over non-animal approaches  

Owner  

Research funders  

Definition  

Number of grants fostering non-animal studies and number grants fostering in vivo studies  

Expected utility  

To understand when there is bias towards animal methods when funding a research project, to be able to for example provide guidance how to 
avoid such biases.    

Data source(s) including their availability/accessibility  

Investigating use of animal methods and non-animal approaches in funded projects, usually publicly available information.  

Methodology for finding, processing and interpreting the data  

All EU funding of relevant research that can be related to the development of test methods for chemical safety assessment must be listed, and the 
use of animal versus non-animal testing suggested in funded projects can be collected. It is useful also to collect information on endpoints, type 
of chemicals and whether non-animal approaches are used to enhance or replace animal testing, which could lead to classify the outcome in three 
categories: only animal methods, non-animal approaches to enhance animal testing, non-animal approaches to replace animal testing.    

Limitations in the interpretation of the indicator   

Full understanding for the selection of testing.  

Reporting (frequency and timing)   

Starting 2027 and to be repeated every year 

227 

 
 
 
 
Example 7 
Short title of indicator 

Phasing out animal testing carried out fulfilling EU regulatory information requirement 

Owner 

JRC - European Commission for testing performed with the EU; Industry/Agencies for testing performed outside the EU 

Definition 

Number of animals used for regulatory testing fulfilling requirements under EU legislation within the 15 legal areas in scope of the roadmap (each legal 
requirement can be specified and followed as such, why this indicator represents a group of sub-indicators) 

Expected utility 

Measuring progress towards the phasing out of animal testing and gaining confidence in non-animal approaches within specific legal areas and for specific 
requirements, that for example can inform other similar requirements in other legal areas, to use a similar approach to the phasing out of animal testing. 

Data source(s) including their availability/accessibility 

ALURES, publicly available with data from all Member States collected on a yearly basis; Industry/Agencies have information on tested substances 

Methodology for finding, processing and interpreting the data 

This data is already processed on a yearly basis by the JRC and presented in the European Commission Summary Report on the statistics on the use of 
animals for scientific purposes in the Member States of the European Union and Norway for each year (example the 2022 summary report: 
(https://circabc.europa.eu/ui/group/8ee3c69a-bccb-4f22-89ca-277e35de7c63/library/051e5787-7746-46cf-8a0d-310f84fd1900/details?download=true). It 
might be that Agencies or Industrial associations have some information on testing made outside the EU, or means to collect it. 
Limitations in the interpretation of the indicator  

Focusing only on statistics collected with the EU, this indicator is not providing any information whether the assumed phased out animal testing, was and is 
still performed outside the EU, why it is useful to combine this knowledge with available knowledge on testing performed outside the EU. 

Reporting (frequency and timing)  

Starting 2020 (including 27 Members States and Norway), and to be repeated every year 

228 

 
 
 
 
Example 8 
Short title of indicator 

Reduction of severity in animal procedures where these tests are still performed 

Owner 

JRC - European Commission 

Definition 

Severity scores, horizontal and per legislative sector, and type of test 

Expected utility 

Measuring progress towards the phasing out of the most severe animal testing by creating awareness, which are the most severe animal procedures used and to 
which extent in chemical safety assessment. 

Data source(s) including their availability/accessibility 

ALURES, publicly available with data from all Member States collected on a yearly basis 

Methodology for finding, processing and interpreting the data 

This data is already processed on a yearly basis by the JRC and presented in the European Commission Summary Report on the statistics on the use of animals 
for scientific purposes in the Member States of the European Union and Norway for each year (example the 2022 summary report: 
(https://circabc.europa.eu/ui/group/8ee3c69a-bccb-4f22-89ca-277e35de7c63/library/051e5787-7746-46cf-8a0d-310f84fd1900/details?download=true) 
Limitations in the interpretation of the indicator  

Focusing only on statistics collected within the EU, this indicator is not providing any information whether severe procedures are phased out within the EU and 
continued to be performed outside the EU. It could be considered to try to collect data from other sources on the most severe procedures, or the ones decreasing 
most within the EU. 

Reporting (frequency and timing)  

Starting 2020 (including 27 Members States and Norway), and to be repeated every year 

229 

 
 
 
Annexes 

Annex I – Chemical safety assessments under following 
legislations are in scope of the roadmap 

Overview list of regulatory areas and responsible agency 
1)  Chemicals registered under the REACH Regulation (ECHA)  

2)  Biocidal Products (ECHA)  

3)  Plant Protection Products and MRLs for active substances in plant protection products for 

food and feed (EFSA)  

4)  Food improvement agents (food additives, food enzymes and food flavourings) (EFSA)  

5)  Chemicals used in food contact materials (EFSA)  

6)  Feed additives (EFSA)  

7)  Human medicinal products (EMA)  

8)  Veterinary  medicinal  products  and  MRLs  for  active  substances  in  veterinary  medicinal 

products for food-producing animals (EMA)  

9) 

 Medical devices 

10) Chemicals used in materials/products in contact with drinking water (ECHA)  

11) Chemicals covered by the CAD and CMRD (ECHA)  

12) Chemicals used in human nutrition (EFSA)  

13) Detergents 

14) Classification, labelling and packaging of chemicals (ECHA)118 

15) Water and Waste legislation (identification of priority substances) (ECHA) 

118 The CLP Regulation is based on available data and does not trigger testing. However, it is considered to be in scope 
of the roadmap since classification relies on data generated under other pieces of legislation, with extensive guidance 
available that is providing information which data can be used and how. Furthermore, the CLP Regulation is 
considered to be key for switching to a regulatory hazard assessment based only on non-animal testing because of the 
classification criteria that it stipulates. 

230 

 
 
 
 
 
 
 
Legislations in more detail and sorted by agency responsibility 
(with requirements categorisation119) 
ECHA remit: 

•  Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH), 

Regulation (EC) No 1907/2006 [A] 

•  Classification, Labelling and Packaging (CLP) Regulation (EC) No 1272/2008 [C] 
•  Biocidal Products Regulation (BPR) (EC) No 528/2012 concerning the making available 

on the market and use of biocidal products [A] 

•  Drinking Water Directive (DWD) (EU) 2020/2184 on the quality of water intended for 

human consumption [B] 

•  Water Framework Directive 2000/60/EC [B] 
•  Environmental Quality Standards Directive 2008/105/EC (EQSD) [B] 
•  Groundwater Directive (2006/118/EC) [B] 
•  Occupational exposure limit (OEL) values under Chemical Agents Directive 98/24/EC 
(CAD) and Carcinogens, Mutagens or Reprotoxic Substances Directive 2004/37/EC 
(CMRD) [C] 

•  The Prior Informed Consent Regulation (EU) 649/2012 (PIC), governing the trade of 
certain hazardous chemicals that are banned or severely restricted in the EU [C?] 

•  Regulation (EU) No 2019/1021 on persistent organic pollutants (POPs) [C?] 
•  The Waste Framework Directive 2008/98/EC (WFD) [B] 
•  Toys Safety Regulation (in adoption process)  (currently Directive 2009/48/EC) [C] 

EFSA remit: 

•  Regulation  (EC)  No  178/2002  (the  General  Food  Law  Regulation),  amended  by 
Regulation  (EU)  2019/1381  on  the  transparency  and  sustainability  of  the  EU  risk 
assessment in the food chain [B] 

Feed and feed additives 

•  Regulation (EC) No 767/2009 on the placing on the market and use of feed [B] 
•  Feed Additive Regulation (EC) No 1831/2003 [B] 
•  Regulation (EC) No 429/2008 on the Rules for Implementing the Regulation (EC) No 

1831/2003 [A] 

•  Regulation (EU) 2019/4 on the manufacture, placing on the market and use of medicated 

feed [C] 

Food contact materials 

•  Regulation (EC) No 1935/2004 on materials and articles intended to come into contact 

with food [B] 

•  Commission Regulation (EU) No 10/2011 on plastic materials and articles intended to 

come into contact with food [B] 

•  Commission Regulation (EC) No 450/2009 on active and intelligent materials and 

articles [B] 

119 [A] legislation specifying concrete information requirements and distinct methods 
[B] legislation specifying general information requirements, where methods are specified in guidance or selected based 
on interaction with agency case by case 
[C] legislation not specifying specific nor general information requirements but requiring action on the basis of 
available data 

231 

 
 
 
Food improvement agents 

•  Regulation (EC) No 1331/2008 on authorisation procedure for food additives, food 

enzymes and food flavourings [C] 

•  Commission Regulation (EU) No 234/2011 implementing Regulation (EC) No 

1331/2008 [B] 

•  Regulation (EC) No 1332/2008 on food enzymes [B] 
•  Regulation (EC) No 1333/2008 on food additives [B] 
•  Regulation (EC) No 1334/2008 on flavourings and certain food ingredients with 

flavouring properties for use in and on foods [B] 

•  Regulation (EC) No 2065/2003 on smoke flavourings [B] 
•  Directive 2009/32/EC on the approximation of the laws of the Member States on 
extraction solvents used in the production of foodstuffs and food ingredients [B] 

Contaminants in food and feed 

•  Directive (EEC) 315/93 for contaminants in food 
•  Directive 2002/32/EC for undesirable substances in feed 

Nutrition applications 

•  Regulation (EC) No 1925/2006 on the addition of vitamins and minerals and of certain 

other substances to foods [C] 

•  Regulation (EC) No 108/2008 amending Regulation (EC) No 1925/2006 on the addition 

of vitamins and minerals and of certain other substances to foods [C] 

•  Directive 2002/46/EC on the approximation of the laws of the Member States relating to 

food supplements [C] 

•  Regulation (EU) No 609/2013 on food intended for infants and young children, food for 

special medical purposes, and total diet replacement for weight control [B] 

•  Regulation (EU) 2015/2283 on novel foods [B] 
•  Regulation (EC) 1829/2003 on genetically modified food and feed [B] 

Pesticides 

•  Regulation (EC) No 1107/2009 concerning the placing of plant protection products on 

the market [B] 

•  Commission Regulation (EU) No 283/2013 on the data requirements for active 

substances [A] 

•  Commission Regulation (EU) No 284/2013 on the data requirements for plant protection 

products [A] 

•  Commission Regulation (EU) No 546/2011 on uniform principles for evaluation and 

authorisation of plant protection products [C] 

•  Regulation (EC) No 396/2005 on maximum residue levels of pesticides in or on food and 

feed of plant and animal origin [B] 

•  Commission Implementing Regulation (EU) No 2020/1740 on the provisions necessary 

for the implementation of the renewal procedure for active substances [C] 

EMA remit:  

Authorisation 

232 

 
 
 
 
 
• 

 Directive 2001/83/EC on the Community Code relating to medicinal products for human 
use, as amended [B] 

•  Regulation (EC) No 726/2004 laying down Union procedures for the authorisation and 
supervision of medicinal products for human  use and establishing the European 
Medicines Agency. [B] 

•  Regulation (EC) No 2019/6 on veterinary medicinal products [B] 
•  Regulation (EC) No 470/2009 on the establishment of residue limits of 
pharmacologically active substances in foodstuffs of animal origin [B] 

•  Commission Implementing Regulation (EU) 2017/12 on form and content of the 

applications and requests for the establishment of maximum residue limits in accordance 
with Regulation (EC) No 470/2009 [B] 

•  Regulation (EU) No 2018/782 establishing the methodological principles for the risk 
assessment and risk management recommendations referred to in Regulation (EC) No 
470/2009 [A] 

•  Commission Regulation (EU) 2017/880 laying down rules on the use of a maximum 
residue limit established for a pharmacologically active substance in a particular 
foodstuff for another foodstuff derived from the same species and a maximum residue 
limit established for a pharmacologically active substance in one or more species for 
other species, in accordance with Regulation (EC) No 470/2009 of the European 
Parliament and of the Council [C] 

Scientific Committee on Consumer Safety (SCCS) remit: 

•  Cosmetic Product Regulation (EC) No 1223/2009 [B] 
•  Regulation (EU) 2023/988 on general product safety (non-food consumer products) [C] 

Not under any EU-agency 

•  Detergents Regulation (EU) 648/2004 and 2026/405 [A] 

Medical Devices  

•  Medical Devices Regulation (EC) No 2017/745 (MDR) [B] 

233 

 
 
 
 
 
 
 
 
Annex II - Ongoing agency activities (ECHA, EFSA and EMA) 
that support the phasing out of animal testing for chemical safety 
assessments 
This document outlines the work of the European Agencies (EMA, ECHA and EFSA) in relation 
to 3Rs120, with a focus on activities that are aligned with the European Commission’s ambition to 
phase out animal testing for chemical safety assessment. It gives some additional context to the 
legislative frameworks in  scope  of the  roadmap,  and the  distinct  competencies of  each  agency. 
Furthermore,  it  provides  information  the  ongoing  work  of  the  individual  agencies  and  their 
strategic visions, both in relation to the implementation of the roadmap and the integration of 3Rs 
methods and alternative to animal testing=more generally. Finally, it lists critical areas that are 
of  joint  interest  to  all  agencies  and  opportunities  for  collaborative  actions  across  sectors. 
Practically, such joint actions could be supported by the Interagency Working Group on Animal-
free Approaches which is proposed as part of the roadmap communication.  

EMA 
Legal Framework 

EMA, in cooperation with the European Medicines Regulatory Network (EMRN),  provides the 
scientific support for the authorisation of human and veterinary medicinal products in accordance 
with Directive 2001/83/EC and Regulation 2019/6, respectively. Medicinal products encompass 
chemical  pharmaceuticals, which are in scope of the roadmap, but also variety of other medicinal 
products including biologicals, vaccines, gene therapies, advanced therapy medicinal products and 
novel  therapy  veterinary  medicinal  products,  which  are  not.  It  is  important  to  note  that  EMAs 
overall strategy in relation to 3Rs covers all medicinal products, and not just those within the scope 
of the roadmap.  

Medicinal  products  may  be  centrally  authorised  by  the  Commission,  whereby  applications  are 
assessed by EMA and the authorisation is valid in all EU Members States. Medicinal products may 
also be authorised on a decentralised or national basis which is the responsibility of the national 
competent authorities (NCAs) in the relevant individual members states (i.e. the EMRN). The data 
to support marketing authorisation is submitted in a common digital format (electronic common 
technical document eCTD) and includes quality, non-clinical and clinical information. For human 
medicinal products (HMPs), animal testing is generally required in the non-clinical section as well 
as to address quality specifications. Animal testing is also required to generate  some safety and 
clinical data for target animal species in the case of veterinary medicinal products (VMPs). The 
same applies for consumer safety studies. Here, there is no scope for replacement or phasing out 
of  in  vivo  testing,  but  the  principles  of  reduction  and  refinement  are  addressed  as  part  of  the 
assessment.  Non-clinical  data  for  both  human  and  veterinary  medicinal  products  is  generally 
presented for pharmacology, pharmacokinetics and toxicology, and includes both animal and non-
animal  data.  Primary  pharmacology studies,  used  to demonstrate  efficacy  of  the  product  or  the 
proof-of-concept for its therapeutic use are not in scope of the  roadmap, which refers to safety 
assessment. However, application of the 3Rs is also pertinent to animal testing conducted for this 
purpose and forms part of the assessment.  

The availability of structured clinical data, generated in strictly regulated prospective clinical trials, 
is unique to the pharmaceutical sector. Furthermore, every medicinal product has a specified target 
population and dosing regimen and therefore an expected level of exposure. This means that every 
product must be assessed on a case-by-case basis, and the protection goals are variable in each 
case.  Consequently,  the  goals  and  outcomes  of  the  risk  assessment  differ  from  those  in  other 
chemical sectors. For HMPs, a holistic evaluation of the safety, exposure and efficacy of a chemical 
is conducted and leads to a benefit risk balance. Products can only be authorised on the basis of a 
positive benefit risk balance. For VMPs, the focus is also on a benefit risk balance,  which includes 
the health benefit for the treated animal against risks arising from its exposure to the product, for 

120 3Rs = Replace, Reduction and Refinement of animal testing 
234 

 
 
the human user and/or consumer, who may be exposed during its administration or to its residues 
via food of animal origin, and for the environment. 

Pharmaceutical legislation provides some minimal requirements for the performance of quality and 
non-clinical tests that should be performed and submitted in the dossier to support a marketing 
authorisation. However, these requirements should be read in conjunction with relevant European 
and international guidance documents including internationally harmonised guidelines specific to 
medicinal products; the ICH121 guidelines in the case of HMPs, and VICH122  in the case of VMPs. 
Medicines  regulators  like  EMA  and  FDA  may  also  develop  their  own  guidance  documents, 
including guidelines and reflection papers. Many existing guidances refer to the 3Rs principles and 
allow some flexibility to incorporate alternative methods where scientifically justified, as well as 
opportunities to reduce and refine animal use. These opportunities are collated in EMA’s Reflection 
papers  providing  an  overview  of  the  current  regulatory  testing  requirements  for  human  and 
veterinary medicinal products and  opportunities for the implementation of the 3Rs. In addition, 
Scientific Advice can be sought from EMA or NCAs to discuss deviations from guidance including 
on novel 3Rs approaches. 

For HMPs, an assessment of data (which may include animal studies) is also performed at an earlier 
stage of development to support the authorisation of clinical trials (Regulation 536/2014). Here, 
the  data  requirements  are  different  to  those  for  marketing  authorisation  given  the  restricted 
participants and controlled circumstances under which the investigational product is administered. 
However, the outcome of the evaluation is still based on a benefit risk balance. The evaluation and 
authorisation of clinical trials  is also the remit  of NCAs and not of EMA.  The same applies to 
clinical trials for VMPs. 

Finally, it should be noted that a significant proportion of applications for marketing authorisation 
of medicinal products are for generics or informed consent applications (and biosimilars, though 
not in the scope of the roadmap), where it is generally accepted that no animal testing is required.  

EMA activities on 3Rs 

EMA is committed to the application of the 3Rs across the lifecycle of medicinal products and to 
the  integration  of  alternative  to  animal  testing  into  the  medicines  regulatory  framework.  The 
agency’s strategic vision on 3Rs is underscored by a number of policy documents including the 
Ema Regulatory Science Strategy123  and the European Medicines Agencies Network Strategy to 
2028124 and it is supported by a standing 3Rs Working Party125 (3RsWP). The working party which 
is part of the non-clinical domain publishes its workplan on a 3 yearly basis, identifying annual 
priorities which include guidance development, stakeholder engagement and communication, pre-
authorisation support to innovative 3Rs method developers, oversight of expert groups and training 
activities. Some key ongoing activities demonstrating alignment with the roadmap objectives are 
outlined below. Readers are encouraged to consult the linked documents for further details, as well 

121 ICH: The International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use 
122 VICH: International Cooperation on Harmonisation of Technical Requirements for Registration of Veterinary 
Medicinal Products 
123 https://eceuropaeu.sharepoint.com/teams/GRP-Interservicegroup-roadmaponnon-animaltesting/Shared 
Documents/GROW ENV folder/SWD draft/Regulatory Science Strategy to 2025 
124https://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=&ved=2ahUKEwin1Jna8MmQ
AxUR2QIHHbqhPfYQFnoECA0QAQ&url=https%3A%2F%2Fwww.ema.europa.eu%2Fen%2Fdocument
s%2Fother%2Fseizing-opportunities-changing-medicines-landscape-european-medicines-agencies-
network-strategy-2028-final_en.pdf&usg=AOvVaw1o2XJmWqi6QeKzacAzcm09&opi=89978449 
125 https://www.ema.europa.eu/en/committees/working-parties-other-groups/chmp/3rs-working-party 

235 

 
 
 
as the EMA 3RsWP126, ethical use of animals in medicines testing127 and regulatory acceptance of 
NAMs128 webpages.  

Guidance activities: 

EMA  systematically  implements  the  3Rs  concept  and  refers  to  the  use  of  alternative  to  animal 
testing in all of its relevant guidance documents. The main goals of ongoing guidance activities are 
to  provide  an  inventory  of  regulatory  testing  requirements  involving  animal  use  and  describe 
opportunities for 3Rs implementation, and to provide a framework for acceptance of novel 3Rs 
approaches including a gradual phase in of alternative to animal testing.   

•  Cyclic revisions of the “Reflection papers providing an overview of the current regulatory 
testing  requirements  for  medicinal  products  for  human129  or  veterinary130  use  and 
opportunities for implementation of the 3Rs” 

•  Revision of the “Guideline on the principles of regulatory acceptance of 3Rs (replacement, 

reduction, refinement) testing approaches131” as outlined in the Concept Paper132  

Stakeholder engagement: 

Core  3RsWP  stakeholders  include  pharmaceutical  industry  associations,  NGOs,  EU  and 
international research consortia, ECHA, EFSA and OECD. Observers include EC services (DG 
SANTE, DG RTD, DG ENV, DG GROW, DG JRC (EURL ECVAM)) and EDQM. In recognition 
of the global context of medicines development and the need for international harmonisation on 
3Rs recommendations, EMA led in the establishment of an International Medicines Regulators’ 
Working Group on 3Rs (IMRWG3Rs).  

•  Annual Interested Parties meetings (reports available for 2023133, 2024134 and 2025135) 
• 

International  Medicines  Regulators’  Working  Group  on  3Rs  (terms  of  reference 
available136) 

Regulatory  acceptance  of  NAMs  –  supports  and  procedures  (safe  spaces  model,  overview 
available137): 

EMA fosters regulatory acceptance of alternative to animal testing by offering various mechanisms 
by which method developers can interact with EMA at different stages of development. 

Innovation Task Force briefing meetings 

• 
•  Qualification of novel methodologies 
•  Scientific advice 
•  Portfolio and technology meetings  

126 https://www.ema.europa.eu/en/committees/working-parties-other-groups/chmp/3rs-working-party 
127 https://www.ema.europa.eu/en/human-regulatory-overview/research-development/ethical-use-animals-
medicine-testing 
128 https://www.ema.europa.eu/en/human-regulatory-overview/research-development/ethical-use-animals-
medicine-testing/regulatory-acceptance-new-approach-methodologies-nams-reduce-animal-use-testing 
129 https://www.ema.europa.eu/en/overview-current-regulatory-testing-requirements-medicinal-products-
human-use-opportunities-implementation-3rs-scientific-guideline 
130 https://www.ema.europa.eu/en/overview-current-regulatory-testing-requirements-veterinary-medicinal-
products-opportunities-implementation-3rs-scientific-guideline 
131 https://www.ema.europa.eu/en/regulatory-acceptance-3r-replacement-reduction-refinement-testing-
approaches-scientific-guideline 
132 https://www.ema.europa.eu/en/documents/scientific-guideline/concept-paper-revision-guideline-
principles-regulatory-acceptance-3rs-replacement-reduction-refinement-testing-approaches_en.pdf 
133 https://www.ema.europa.eu/en/documents/report/report-3rswp-public-session_en.pdf 
134 https://www.ema.europa.eu/en/documents/report/report-3rswp-public-session-2024_en.pdf 
135 https://www.ema.europa.eu/en/documents/report/report-3rs-working-party-3rswp-plenary-meeting-
april-2025_en.pdf 
136 https://www.ema.europa.eu/en/documents/other/terms-reference-tor-international-medicines-regulators-
working-group-3rs_en.pdf 
137 https://www.ema.europa.eu/en/human-regulatory-overview/research-development/ethical-use-animals-
medicine-testing/regulatory-acceptance-new-approach-methodologies-nams-reduce-animal-use-testing 

236 

 
 
•  Voluntary submission of data (“safe harbour”) procedure 

Oversight of expert groups and training activities: 

The 3RsWP oversees the work of an operational expert group on the application of the 3Rs in batch 
release  and  quality  control  of  medicines  and  European  Specialised  Expert  Community  on  non-
clinical and NAMs which provides a platform for interaction between academic and regulatory 
experts. In addition, EMA provides training to regulators within the EMRN on 3Rs topics relevant 
to regulatory assessment of medicines via the EU-Network training centre.  

•  Batch release operational expert group 
•  Non-clinical and new approach methodologies ESEC138 
•  EU Network Training Centre139 

ECHA  
Legislative framework  

The primary objective of EU legislation regulating industrial chemicals is to provide a high level 
of  protection  of  human  health  and  the  environment.  It  is  underpinned  by  the  identification  of 
hazardous  properties  of  substances,  with  REACH  and  CLP  as  the  essential  horizontal  EU 
Regulations.  

Since  its  entry  into  force  in  2007,  REACH  is  the  regulatory  framework  producing  the  largest 
knowledge base on chemicals globally. REACH ensures that industry provides adequate data, using 
as a last resort tests on animals, to assess the hazardous properties of chemicals. CLP enables the 
classification of chemicals based on the adverse effects observed using standard testing methods, 
including tests on animals, and provides a framework for generic risk management. This system 
provides predictability and legal certainty for industry compliance and a framework for authorities 
to enforce.  

In  addition  to  REACH  and  CLP,  sectorial  legislation  complements  the  EU  framework  on  the 
identification of hazards, risk assessment and management of chemicals. Chemicals management 
relies on the effective interplay between REACH, CLP and the sectorial legislation. It depends on 
the cooperation of different actors (industry, authorities, third parties, etc.) according to the roles 
and  responsibilities  described  by  the  regulatory  frameworks,  providing  predictability  and  legal 
certainty for industry to comply and authorities to enforce. 

The generic system under REACH and CLP does not require the intervention of authorities by 
default – chemicals are not systematically assessed for safety by the regulators before they are put 
on the market; this assessment is done solely by industry (“the reversal of the burden of proof”). 
However,  authorities may scrutinise these  assessments  and  take regulatory  actions  (e.g. request 
further  information  or  initiate  regulatory  risk  management),  where  required.  In  addition,  this 
system allows adaptations to standard information requirements to be used for supporting hazards 
and risk assessment.  

ECHA activities on alternatives to animal testing 

ECHA is committed to promoting alternatives to animal testing and, within its mandate, has been 
actively addressing this topic since the agency’s establishment. 

The current work can is structured around the following main areas:  

138 https://www.ema.europa.eu/en/committees/working-parties-other-groups/chmp/non-clinical-new-
approach-methodologies-european-specialised-expert-community 
139 https://www.ema.europa.eu/en/about-us/how-we-work/european-medicines-regulatory-network/eu-
network-training-centre-eu-ntc 

237 

 
 
 
 
 
1.  Regulatory activities and use of alternative methods in ECHA’s processes 
2.  Translation of NAMs into regulatory applications 
3.  Input to harmonisation and reporting 
4.  Computational methods and data 
5.  Trainings and promotion of proper use in the regulatory context 
6.  Interactions with key stakeholders 

Regulatory activities and use of alternative methods in ECHA’s processes 

This  work  area  reflects  the  current  integration  of  New Approach  Methodologies  (NAMs)  into 
ECHA internal regulatory processes, such as evaluation, and outlines further implementation to 
support initiatives like chemical grouping. It also addresses legal obligations, including reporting 
on alternatives to animal testing. 

Key activities include: 

•  Assessment  of  QSAR  predictions  in  ECHA  processes,  primarily  through  expert 

consultations for REACH and Biocides 

•  Publication of the Article 117(3) report on the use of alternatives to animal testing under 

REACH 

•  Support for drafting and revising ECHA guidance involving alternatives to animal testing  

Translation of NAMs into regulatory applications 

This  work  area  aims  at  providing  regulatory  input  to  flagship  (research)  projects  aiming  at 
developing  alternatives to animal  testing  suitable for our  regulatory needs.  It  also  enables  joint 
efforts with authorities from Europe and other regions such as the US or Canada to work together 
towards the identification and acceptance of alternatives to animal testing in regulatory frameworks 

Key activities include: 

•  Implementation of Framework contracts for the development of NAM based tools and data 
for hazard identification and characterisation of industrial chemicals with an emphasis on 
omics  data  generation  and  methodological  developments  of  NAM  related  to  hazard 
assessment  of  industrial  chemicals  (including  both  toxicodynamic  and  toxicokinetic 
assessment)  

•  Participation in scientific  projects  which  address  key aspects  for regulatory  acceptance. 
Example  collaborations  include  APCRA  (Accelerating  the  Pace  of  Chemical  Risk 
Assessment), PARC (Partnership for the Assessment of Risks from Chemicals), and EU 
research consortium such as ASPIS 

•  Definition (and communication) of the critical needs to progress towards an animal-free 
regulatory  system  for  industrial  chemicals  and  expectations  for  new  approach  methods 
(NAMs) – e.g. within the ECHA KARC document (Key areas of regulatory needs) 

Input to harmonisation and reporting 

This  work  area  is  related  to  the  OECD  activities  important  for  the  further  development  and 
regulatory implementation of alternatives to animal testing. 

Key activities include: 

•  Collaboration with WPHA (Working Party on Hazard Assessment) to develop a Guidance 
on best practices and standardisation of omics samples and the WNT (Working Party for 
the Test Guideline Program) to include optional omics sampling in selected OECD Test 
Guideline studies to enable the analysis of additional molecular/mechanistic parameters. 

•  Active  participation  in  OECD  Expert  groups  such  as  the Advisory  group  on  Emerging 
Science in chemicals assessment (ESCA), the ‘omics Expert Group and the QSAR Expert 
Group. 

•  Development  of  the  OECD  QSAR  Assessment  Framework  (QAF)  and  example  case 

238 

 
 
 
studies. 

Computational methods and data 

This work area focuses on making ECHA registration data available for developers and scientists.  

Key activities include: 

•  Continuous development and maintenance of the OECD QSAR Toolbox. 
•  Promotion of the OECD QSAR Assessment Framework within academia and industry to 

enhance regulatory acceptance of QSAR predictions. 

•  Provision of toxicological data in the harmonised format IUCLID for further development 

of alternatives to animal testing. 

•  Provision  of  curated  datasets  with  experimental  data  for  selected  endpoints,  e.g.  acute 

toxicity; Adult neural toxicity, bioaccumulation and aquatic toxicity.  

•  Assessing  the  predictive  capacity  of  environmental  QSARs  and  predictive  models  to 

support prioritisation and scientific decision making further implemented. 

•  Assessment of QSARs reliability for selected regulatory endpoints. 
•  Horizon scanning of mature (computational) methods, technologies and tools of relevance 

to ECHA. 

Organisation of trainings and promotion of proper use of NAMs in the regulatory context  

This  work  area  is  aiming  at  sharing  ECHA  experience  and  knowledge  with  ECHA  staff, 
stakeholders and registrants. 

Key activities include: 

•  Trainings on NAMs for ECHA staff, Member States, committees, industry, and scientific 

community 

•  Public  webinars  on  application  of  QSARs,  QSAR  assessment  framework  and  PBK 

modelling  

•  Answering speaking requests and presentation of ECHA activities in dedicated fora (e.g. 

conferences, working groups) 

Interactions with stakeholders 

This work area is aiming at exchange of knowledge, experience and alignment in the areas of the 
key importance for the European institutions. In addition, dialogue with NGOs is also covered here. 

Key activities include: 

•  Contribution to the roadmap on phasing out animal testing for chemical safety assessments. 
•  Collaboration with Member States on activities related to alternatives to animal testing. 
•  Alignment with sister agencies, especially EFSA and EMA, and joint projects on guidance 

development 

•  Representing  ECHA  at  the  European  Partnership  for Alternative Approaches  to Animal 

Testing (EPAA). 

•  Dialogue with NGOs – presentation of plans and reply to questions. 
•  Dialogue  with  industrial  partners  and  CROs  involved  in  routine  alternatives  to  animal 

testing data generation. 

EFSA  
Legislative framework  

239 

 
 
 
 
The European Food Safety Authority (EFSA) has been established under the General Food Law 
Regulation (EC) No 178/2022140 that lays down the procedures in matters of food safety. The main 
aim of the regulation is to ensure high level of protection of human life, consumers' interests and 
environment in relation to food, while ensuring the effective functioning of the internal market.  

EFSA provides independent scientific advice on existing and emerging risks in the food chain to 
help protection of consumer and environment covering the remits of i) food and feed safety, ii) 
nutrition, iii) animal health and welfare, iv) plant protection and v) plant health.141 EFSA follows 
specific  regulations  under  the  different  legal  frameworks  covered  by  its  activities.  EFSA’s 
Scientific Committee142 has the task of supporting the work of the different Scientific Panels on 
cross-cutting  scientific  issues,  focusing  on  the  development  of  harmonised  risk  assessment 
methodologies. EFSA’s Scientific Panels143 are responsible for providing the scientific opinions of 
the Authority,  each  within their  own spheres  of competence. It is also  within  EFSA’s  remit  the 
independent  and  timely  risk  communication  regarding  food  and  feed  safety  as  well  as  the 
promotion of scientific cooperation.  

Since  March  2021,  EFSA  complies  with  Regulation  (EU)  2019/1381  on  transparency  and 
sustainability  of  the  EU  risk  assessment  in  the  food  chain,  keeping  the  highest  standards  of 
transparency.  Overall,  EFSA  publishes  around  500  outputs  on  a  yearly  basis  covering  generic 
mandates  as  well  as  applications  for  regulated  products,  which  are  governed  by  more  than  34 
different EU Directives and Regulations and follows 39 different workflows.  These outputs relate 
to  include  plant  protection  products,  plant  health,  food  and  feed  additives,  animal  health  and 
welfare, food flavourings, biological hazards, feed additives, food additives and flavourings, food 
enzymes, food contact materials, nutrition and novel food innovation foods, genetically modified 
organisms, and contaminants in the food and feed chain144.  

EFSA’s activities on alternatives to animal testing 

EFSA is committed to  implement  the principles of the 3R’s and integrating  alternatives to animal 
testing in food and feed  risk assessment, by working closely with the alternatives to animal testing 
community and exploring existing technology and legislative possibilities145 (EFSA SC, 2009146; 
EFSA, 2022a147; EFSA, 2022b148; EFSA, 2023149; EFSA, 2025150). 

EFSA has as a strategic objective, the development and integration of alternatives to animal testing 
and omics in regulatory risk assessment as a means to ensure preparedness for future risk analysis 
(EFSA Strategy 2027, 2021151). The possibility to use alternatives to animal testing is cited in a 
number  of  EFSA’s  guidance  documents  including  novel  foods,  food  additives  or  the  general 
applications of read-across in chemical risk assessment (EFSA NDA, 2024152; EFSA guidance on 
food  additives;  OECD,  2023,  EFSA  SC,  2025153).  Furthermore,  EFSA  has  worked  with  the 
alternatives to animal testing community to develop roadmaps as to increase the incorporation of 
NAMs  in  food  and  feed  risk  assessment,  making  their  use  when  relevant  and  possible  to 
complement the risk assessment). In some regulatory areas, within EFSA’s remit (such Pesticides; 
Feed and feed additives or Food contact material) there are established legal requirement that list 

140 https://eur-lex.europa.eu/legal-content/EN/ALL/?uri=CELEX:32002R0178 
141 https://www.efsa.europa.eu/en/topics 
142 https://www.efsa.europa.eu/en/science/scientific-committee-and-panels/scientific-committee 
143 https://www.efsa.europa.eu/en/science/scientific-committee-and-panels 
144More details are available in https://www.efsa.europa.eu/en/topics  
145 More information is available in https://www.efsa.europa.eu/en/topics/topic/alternatives-animal-testing and 
https://www.efsa.europa.eu/en/topics/topic/methodology. 
146 https://efsa.onlinelibrary.wiley.com/doi/abs/10.2903/j.efsa.2009.1052 
147 https://efsa.onlinelibrary.wiley.com/doi/pdf/10.2903/sp.efsa.2022.e200502 
148 https://efsa.onlinelibrary.wiley.com/doi/epdf/10.2903/sp.efsa.2022.e200506 
149 https://www.sciencedirect.com/science/article/pii/S0924224423000572 
150 https://efsa.onlinelibrary.wiley.com/doi/full/10.2903/j.efsa.2025.e220401 
151 https://www.efsa.europa.eu/sites/default/files/2021-07/efsa-strategy-
2027.pdfhttps:/www.efsa.europa.eu/sites/default/files/2021-07/efsa-strategy-2027.pdf 
152 https://efsa.onlinelibrary.wiley.com/doi/epdf/10.2903/j.efsa.2024.8961 
153 https://efsa.onlinelibrary.wiley.com/doi/epdf/10.2903/j.efsa.2025.9586 

240 

 
 
 
the  type  of  studies  to  be  used  in  the  assessment.  In  these  cases,  moving  to  different  type  of 
requirements,  including  the  move  to  a  complete  animal-free  assessment  relying  more  on 
alternatives  to  animal  testingmay  require  additional  work  for  both jurisdictions  (adjusting  legal 
frameworks) as well as risk assessors and academia (adjusting guidance documents and validating 
methodologies).  

The relevance of alternatives to animal testingin EFSA’s work is further highlighted in the EFSA 
2027  strategy154  o  “develop  and  integrate  new  scientific  developments  focusing  on  NAM-based 
methods and minimisation of animal testing, innovations in food systems, data, and technology, 
and  strive to  meet  One health  policy  needs”. This  has  triggered  the  development  of  a  series of 
NAMs’ activities in EFSA that can be grouped into the following major areas:  

•  Closing gaps and support scientific discovery on NAMs  

Regarding  the  challenge  of  closing  gaps  between  science  and  regulation,  EFSA  has  up  to  now 
invested  over  €  30  million  euros  in  projects  related  to  the  use  of  NAMs  in  food  and  feed  risk 
assessment. In 2022, EFSA established the Innovative Risk Assessment Methodologies programme 
(IRMA) and the Data. & Evidence programme (DEEP) s to ensure preparedness and knowledge of 
up-to-date risk assessment methodological developments. These programmes also aim to meet new 
scientific  challenges  that  EFSA  is  called  to  face  in  the  coming  five  years  in  food,  feed,  and 
environmental safety assessment155.  

EFSA’s funded project address NAMs’ tools for projects in topics such as inter-human variability 
in 
toxicodynamics,  kinetics  and  metabolism,  neurodevelopment,  endocrine  disruptors, 
environmental  risk  assessment,  pesticide  metabolites,  nanomaterials  in  food  and  feed,  protein 
safety, toxicogenomics and transcriptomics, artificial intelligence and in silico model development 
including  quantitative  structure–activity  relationship  (QSAR)  and  physiologically-based  kinetic 
models (PBK). These projects target unique needs of regulatory science within EFSA’s remit. They 
require ongoing updates and validation that connect theory with practice.  

•  Supporting NAMs inclusion and assessment in EFSA’s work  

A number of working groups (WGs) have been and/or are being established at EFSA to develop 
alternatives  to  animal  testing  strategies  such  as  benchmark  dose  modelling,  read-across, 
developmental  neurotoxicity  in  vitro  testing  battery,  use  of  quantitative  in  vitro  to  in  vivo 
extrapolation  (QIVIVE)/PBK  modelling,    phasing  out  dog  studies,  use  of  TK-TD  models  in 
environmental risk assessment, ADME testing and PBK modelling for human and environmental 
risk assessment. At the same time, EFSA has established several WGs that give support to different 
process and projects where the use of alternatives to animal testing is discussed such as the WG on 
Endocrine Disruptors, genotoxicity, Neurotoxicity and the Scientific Committee working group on 
NAMs. 

The  implementation  of  the  One  Substance,  One  Assessment  (OSOA)  approach  and  the 
development  of  an  EU-based  data  platform  will  significantly  support  the  reduction  of  animal 
testing. These initiatives support data sharing and harmonisation, preventing duplicate studies on 
the same substance being conducted under different regulatory remits.  

EFSA  collaborates  and  exchanges  data,  expertise  and  methodologies  with  sister  agencies  and 
relevant  stakeholders  such  as  the  Organisation  for  Economic  Co-operation  and  Development 
(OECD), in order to reach consensus on the applicable criteria/standards for alternatives to animal 
testing- data and their integration for use in feed and food risk assessment. For example, EFSA’s 
OpenFoodTox 3.0 project delivered a workflow within IUCLID that will make EFSA and ECHA 
data available under the same format, allowing stakeholders to consult such data and have a better 
understanding on how studies were used in the risk assessment. Bioaccumulation models have also 
been developed over the years and support assessment of persistence of chemicals in humans and 
terrestrial and aquatic compartments such as bioconcentration factors (BCF) and bioaccumulation 
factors (BAF). EFSA has also contributed to the development of an OECD Harmonised Template 

154 https://www.efsa.europa.eu/en/corporate-pubs/efsa-strategy-2027-science-safe-food-sustainability 
155 For more details, please see https://www.efsa.europa.eu/en/topics/topic/methodology 

241 

 
 
 
structure as to facilitate the reporting and structuring of QSAR data in the IUCLID database used 
by ECHA (OECD GD 386, 2023156). The aim of these activities is to improve the availability of 
standardised data within a harmonised framework for regulatory assessment. Finally, EFSA has 
actively  contributed  to  the  OECD  Initial  recommendations  on  the  Evaluation  of  Data  from 
Developmental Neurotoxicity (DNT) In-vitro Testing Battery (OECD 2023157). 

•  EFSA’s Knowledge and Innovation Community (KIC) on NAMs 

The  NAMs  Knowledge  and  Innovation  Community  (KIC)  aims  to  create  a  common  culture  to 
foster  cross-area  use  of  NAMs  within  EFSA  One  of  the  objectives  of  the  KIC  on  NAMs  is  to 
understand and share knowledge across EFSA community and beyond on the use of NAMs within 
EFSA’s regulatory and other regulatory frameworks, identify gaps in the EFSA strategy on NAMs 
and  how  these  NAMs  are  used/approached  by  the  Units  in  their  day-to-day  risk  assessments. 
Activities include contribution to define and prioritise future EFSA projects on NAMs, being the 
vehicle  to  articulate  collaboration  also  with  the  NAMs  ecosystem  outside  EFSA  including  the 
implementation of the roadmap of the European Commission. 

•  Strengthening NAMs cooperation beyond EFSA  

EFSA is involved in many OECD activities in relation to NAMs particularly with the Working 
Party on Hazard Assessment. These activities include supporting OECD working groups including 
harmonised  templates,  e-chem  portal,  QSAR  reporting  framework  (QSAR  RF),  developmental 
neurotoxicity in vitro batteries, AOP development and update of the guidance document of the use 
of PBK models for regulatory purposes. EFSA is also collaborating with the EPAA as members of 
the Steering Committee as well as the international consortiums ILMERAC (International Liaison 
Group on Methods for Risk Assessment of Chemicals in Food), Accelerating the Pace of Chemical 
Risk Assessment (ACPRA). In addition, over the last few years, EFSA has had active collaboration 
with the US-EPA and FDA particularly in the area of in silico modelling. In June 2025, EFSA has 
also participated to the Singapore WHO workshop on NAMs held from which a chapter on NAMs 
will be introduced in the Environmental Health Criteria 240 describing the principles of chemical 
risk assessment.  

Finally,  EFSA is  very active  in the  NAM  area  within  the PARC  and ASPIS  projects reviewing 
PARC projects and outputs and stimulating the use of PARC data and in silico models for EFSA ‘s 
ongoing risk assessments (e.g. mycotoxins, pesticides, contaminants, phthalates etc). 

•  Training and Guidance 

EFSA organises regular training courses on NAMs to EFSA staff and to EFSA’s experts from its 
Scientific  Panels  and  Scientific  Committees  (i.e.  NAMs,  TKPlate,  genotoxicity,  QSAR,  BMD, 
ED).  EFSA  also  provides  training  for  other  international  agencies  (US-EPA,  FDA,  NIEHS),  to 
Member States, Article 36 organisations and to research consortiums such as PARC. In addition, 
EFSA is developing several guidances to support applicants, Member States and the food and feed 
community on how to include alternatives to animal testingin risk assessment. This includes tiered 
approaches in area specific guidances, but also cross-area guidances such as weight of evidence 
and read-across.

156 https://www.oecd.org/content/dam/oecd/en/publications/reports/2023/11/q-sar-assessment-framework-
guidance-for-the-regulatory-assessment-of-quantitative-structure-activity-relationship-models-and-
predictions_9b064821/d96118f6-en.pdf 
157 https://www.oecd.org/en/publications/initial-recommendations-on-evaluation-of-data-from-the-
developmental-neurotoxicity-dnt-in-vitro-testing-battery_91964ef3-en.html 

242 

 
 
 
 
 
Critical areas and joint actions agreed between ECHA, EFSA and EMA 

Several key areas have been identified as being crucial for the transition to non-animal approaches 
and of common interest to EFSA, EMA, and ECHA. These include but are not limited to: 

•  Development of reliable ADME (absorption, distribution, metabolism and elimination) 

profiling and PBK (physiologically based kinetic) modelling tailored to regulatory needs. 
•  Establishing reliable (Q)IVIVE ((quantitative) in vitro to in vivo extrapolation) and robust 

dose-response modelling. 

•  Enhancing the use of in silico methods (primarily (Q)SAR ((quantitative) structure-activity 

• 

relationships) modelling) with more explicit criteria for the domain of applicability. 
Integration of novel methods, approaches or combinations of methods in (reproductive) 
toxicology assessment. 

•  Reduction of the number of species tested, where justified.  

Across these areas, the Agencies are committed to identifying commonalities, learning from each 
other as to method applicability in the different areas of work, and working together to ensure 
efficient and safe uptake of the models as appropriate in each of the different sectors. Each Agency 
will identify existing support, and new opportunities, for these developments within their sector. 

Example Actions 

Short-Term Replacements: 

•  Use of in vitro kinetic data and PBK models to refine testing strategies in chemical risk 

assessments, reducing or limiting the dependence on chronic and sub-chronic animal tests 
(while noting that non-clinical PK also informs clinical trials in the pharmaceutical sector, 
which may result in differing requirements).  

•  Supporting the implementation of the OSOA approach, keeping up to date the Openfoodtox 

database and the EU data platform to facilitate data sharing thus preventing the conduct of 
redundant studies, particularly when the same substances are being assessed under different 
frameworks. 

•  The use of bioaccumulation models and QSAR models. 
•  Establishing validation/qualification frameworks to standardise the evaluation of alternatives 

to animal testing, supporting global compliance/ trade harmonisation. 

Long-Term Developments/Replacements: 

•  Developing a substitution plan for invertebrate studies in ERA. 
•  Transitioning methodologies to focus on AOPs and KE rather than traditional animal 

endpoints, with the goal of combining multiple methods to address regulatory concerns. 

243 

 
 
 
Annex III - Engaging with the general public 
The importance of Public Engagement and More Inclusive Stakeholder Representation (perspective of 
the SAFE Consortium158)  

Rationale and Background  
The European Commission’s roadmap, inspired by the European Citizen Initiative (ECI) Save cruelty 
Free Cosmetics, and calls from the European Parliament to accelerate the transition gives a tremendous 
opportunity  to  strengthen  the  democratic  spirit  which  underpins  European  values  and  institutions. 
Engagement with European citizens is a strength for the roadmap implementation process. By involving 
citizens, including patient and consumer organisations, directly in the policy process a number of direct 
and indirect benefits can be gained.  

Effective implementation  
As a democratic institution, the EU fundamentally serves the interests of its citizens. Instead of solely 
relying on opinion polls, engagement with the general public will make sure that policy is more tailored 
to their desires and needs and will ensure that the resources spent towards developing and implementing 
the strategy of the roadmap will be implemented as efficiently and effectively as possible.  
Interests in society are diverse and cannot be easily generalized. Engagement is an effective way to 
shorten the transmission of ideas and increase the representation of various groups and their viewpoints 
whilst ensuring that the values of the people being represented are reflected in policy implementation. 
Ensuring broad participation can ensure that there is not undue influence from special interest groups, 
such as commercial interests. Broad participation also provides a means of representing the interests of 
non-human animals (Donaldson, 2020), see section 3 below. 

Strengthening EU institutions  
Consultation  with  citizens  can  inspire  a  sense  of  participation  in  the  implementation  process  of  the 
roadmap, but it also indirectly strengthens trust in EU institutions themselves. This helps address what 
has been referred to as a “democratic deficit” in EU institutions.  

Strengthening trust in science and evidence-informed policy 
Engagement will also help empower citizens in the science-policy process to become advocates against 
disinformation and misinformation and increase trust in the science-policy process (Grant, 2023; Janara 
and Donaldson, 2023; McGlacken and Hobson-West, 2022).  

Producing better science  
Opening  the  policy  process  to  public  engagement  and  consultation  can  help  guide  the  direction  of 
science inquiry which could help overcome possible biases in the existing scientific paradigm based on 
animal models. Involving citizens can help scientists formulate research questions which align with the 
needs and concerns of citizens. For example, in the healthcare domain, the James Lind Alliance (JLA) 
Priority Setting Partnerships (PSPs)159 are collaborative projects that bring together patients, carers, and 
clinicians to identify and prioritise the most important unanswered questions. 
Science  based  on  the  collaboration  and  engagement  of  citizens  can  also  be  used  to  facilitate  data 
collection in large-scale studies. For example, it could indirectly help human biomonitoring across the 
EU,  enabling  more  comprehensive  and  representative  datasets  to  drive  evidence-based  decision-
making. 

158 Accelerating the transition to animal-free NGRA: A transformative governance approach | NWO 
159 https://www.jla.nihr.ac.uk/about-priority-setting-partnerships 

244 

 
 
 
 
 
 
 
 
Inspiring Innovation 
Maintaining and strengthening public awareness of chemical safety assessment not only fosters a deeper 
understanding of its importance but also helps inspire and attract the next generation of scientists. By 
engaging communities in this critically important field, we can spark curiosity, drive innovation, and 
cultivate a diverse talent pool ready to tackle future challenges. 

Forms of Public Engagement 
 Multiple strategies exist to effectively engage with the general public (ESIC, 2025). Citizen assemblies 
(Patberg, 2024) and Citizen Juries (Setälä et al, 2023) are methods which see engagement with multiple 
people in a deliberative forum in order to openly discuss policy questions. This kind of engagement has 
been shown to enhance trust in institutions, despite polarizing opinions. It has for example been used in 
relation to the Conference on the future of Europe, and a report is available on how a European Citizen’s 
Assembly could be set up (Abels et al. 2022). Special care needs to be given in how these forms of 
public  engagement  are  constructed  to  ensure  that  the  full  diversity  of  citizens  is  represented.  The 
inclusion  of  citizens  who  reflect  the  diversity  of  a  given  country  or  region  as  well  as  patient  and 
consumer organisations should be considered.  
In addition to these participatory methods, more classic information campaigns can be used to engage 
citizens in safety assessment. Citizens are often not aware of the wide range of applications of animal 
testing  for  chemical  safety.  In  order  for  citizens  to  be  able  to  make  informed  decisions  and  play  a 
productive role in policy processes, a basic understanding of the policy and its implications is needed 
(Holmberg and Ideland, 2012). The EC and EU agencies could for example work together with civil 
society organizations to implement information campaigns.  
Also,  the  transition to  animal-free  safety  assessment  is  the  only transition  (as compared  to  e.g.,  the 
energy or food transition) in which the consumer has no apparent role. One way could be by providing 
consumers  information  on  which  products  have  been developed  using  animal testing  (e.g.  pesticide 
residues  in  food  products),  through  labelling.  This  market-based  approach  can  support  public 
engagement in the transition by providing animal-free options to European consumers. In this manner, 
consumers can support the acceleration of the transition by choosing animal-testing free options. 

Representation of non-human animals 
Many  (future)  non-human animals  obviously  have  high  stakes  in  the transition towards  animal  free 
safety assessment and should therefore, from a democratic perspective, be included in decision-making 
processes.  While  it  is  difficult  to  directly  include  non-human  animals  within  current  democratic 
processes  there  are  nevertheless  ways  to  include  non-human  animal  interests  through  indirect 
representation and through combinations of direct and indirect representation.   

Forms of Non-human Animal Representation  
Indirect representation by human proxies in citizens’ assemblies/juries or in expert groups can be  to the 
indirect  representation  of  future  generations  in  questions  regarding  e.g.  climate  change  (Hara  et  al, 
2019). This  means  regular  human  citizens  are  tasked  to  represent  the  perspective  and  interests  of  a 
specific group of non-human animals, or an individual animal, rather than their own (Magaña, 2022; 
van Veen and Helvoirt, 2024). They are given the time to understand the perspective and interests of 
this group or individual, including through meeting individuals of the group they represent. Thereafter 
they participate at meetings in the same way as the other representatives (see Van Veen & Van Helvoirt, 
2024). 
Another form of representation is through microboards, whereby a group of people that is close to an 
individual (human or non-human) forms a small group of people which is then involved in decision 
making processes (e.g. stakeholder consultations, deliberative processes) relating to a particular issue 
or task. In the case of animals, this could for example be human caretakers of an animal living in a 
sanctuary or in a human’s home (e.g. a mouse that lives in a sanctuary for mice, which formerly used 
to live in a laboratory, could be represented by human caretakers working in that sanctuary). 

245 

 
 
 
 
Expert indirect representation is also an alternative. This means appointing humans who are experts in 
animal welfare, biology or communication, to advocate on their behalf. These experts act as proxies, 
ensuring  that  animal  interests  are  considered in  decisions that  affect  them.  Examples  include:  a)  an 
Ombudsman for Animals, who investigates complaints and proposes reforms, ensuring that animals 
are treated ethically in society; b) a  Seat in Parliament for Animals held by an expert who speaks 
exclusively for animal concerns, without representing a political party or constituency; c) a  Labour 
Union for Animals (De Rooij, 2023) that would campaign for better working conditions for animals 
used in biomedical research and regulatory testing. 
The development of governance models for including the interests of non-human animals in decision 
making is a promising avenue of research. An example at the organisational level is the Zoöp Nieuwe 
Instituut in the Netherlands, which could be an inspiration for decision making in other organisations 
that carry out regulatory testing. 

Conclusions 
The  European  Commission’s  roadmap  presents  a  vital  opportunity  to  enhance  democracy,  trust  in 
science,  policy  effectiveness  and  efficiency  by  actively  engaging  citizens  and  by  considering  the 
representation  of  non-human  animals.  Through  inclusive  methods  like  citizens’  assemblies  and 
innovative representation models, the EU can foster better science, inspire innovation, and strengthen 
public trust in its institutions. Embracing these approaches ensures policies reflect diverse needs while 
advancing ethical and evidence-based decision-making.  

References 
Abels, G., Crum, B., Alemanno, A., Demidov, A., Hierlemann, D., Renkamp, A., & Trechsel, A. 
(2022). Next level citizen participation in the EU: Institutionalising European Citizens’ 
Assemblies. Bertelsmann Stiftung. 
https://research.vu.nl/ws/portalfiles/portal/158204609/Next_Level_Citizens_Participation_in_
the_EU.pdf 
De  Rooij,  M. 

een 
https://noordboek.nl/boek/hoeveel-vakantiedagen-heeft-een-varken/ 

(2023).  Hoeveel 

vakantiedagen 

varken?  Noordboek. 

heeft 

Donaldson, S. (2020). Animal Agora: Animal Citizens and the Democratic Challenge.  Social Theory 

and Practice, 46(4), 709–735. https://doi.org/10.5840/soctheorpract202061296 

ESIC (Evidence Synthesis Infrastructure Collaborative). 2025. ‘ESIC_citizen Call to Action_30 July 

2025’. 
https://evidencesynthesis.atlassian.net/wiki/spaces/ESE/pages/222167041/Citizen+call+to+act
ion 

Grant, W. J. (2023). The Knowledge Deficit Model and Science Communication. In Oxford Research 

Encyclopedia of Communication. https://doi.org/10.1093/acrefore/9780190228613.013.1396 

Hara, K., Yoshioka, R., Kuroda, M., Kurimoto, S., & Saijo, T. (2019). Reconciling intergenerational 
conflicts with imaginary future generations: Evidence from a participatory deliberation practice 
in 
1605–1619. 
https://doi.org/10.1007/s11625-019-00684-x 

a  municipality 

Sustainability 

Science, 

Japan. 

14(6), 

in 

Holmberg, T., & Ideland, M. (2012). Secrets and lies: “selective openness” in the apparatus of animal 
experimentation. Public Understanding of Science (Bristol, England), 21(3), 354–368. 
Janara,  L.,  &  Donaldson,  S.  (2023).  Animal  Ghosts  at  Canadian  Universities:  The  Politics  of 

Concealment and Transparency. Animals, 13(24), 3760. https://doi.org/10.3390/ani13243760 

Magaña,  P.  (2022). The  political  representation  of  nonhuman  animals.  Social  Theory  and  Practice, 

48(4), 665–690. 

McGlacken,  R.,  &  Hobson-West,  P.  (2022).  Critiquing  imaginaries  of  ‘the  public’  in  UK  dialogue 
around animal research: Insights from the Mass Observation Project.  Studies in History and 
Philosophy of Science, 91, 280–287. https://doi.org/10.1016/j.shpsa.2021.12.009 

Patberg, M. (2024). Farewell to constituent power? The Conference on the Future of Europe, citizens’ 
assemblies  and  the  democratic  minimum.  Global  Constitutionalism,  13(2),  411–428. 
https://doi.org/10.1017/S2045381723000333 

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Setälä,  M.,  Serup  Christensen,  H.,  Leino,  M.,  &  Strandberg,  K.  (2023).  Beyond  polarization  and 
selective trust: A Citizens’ Jury as a trusted source of information.  Politics, 43(4), 472–488. 
https://doi.org/10.1177/02633957211024474 

van Veen, A., & van Helvoirt, B. (2024). De-Mooo-cracy! A performative experiment with a cow-human 
citizen’s assembly: Evaluation report (p. 17). http://dx.doi.org/10.13140/RG.2.2.35140.44168 

Annex V Summaries of activities of the UK NC3Rs relevant to actions 
in this roadmap 

A) UK NC3Rs report - Refining and replacing acute toxicity tests in chemical 
safety assessments 

Introduction 
This  report  outlines  the  work  of  the  UK’s  National  Centre  for  the  Replacement,  Refinement  and 
Reduction of Animals in Research (NC3Rs) to avoid, reduce and refine the use of animals in acute 
toxicity testing for chemical safety assessment. It complements considerations in chapter 4.1. of this 
SWD.  

The UK’s NC3Rs is participating in the EPAA Acute Toxicity Taskforce, and in this function helping to 
shape international policy to promote the adoption of more refined approaches and support the broader 
integration of non-animal approaches into regulatory frameworks. By enabling scientifically justified 
reductions  in  animal  use,  NC3Rs  contribute  to  the  European  Commission’s  ambition  to  phase  out 
animal testing and advances global efforts to modernise chemical safety assessment while maintaining 
high standards of human health protection. Additional collaboration with the UK’s efforts to phase out 
animal testing is occurring at he level of the OECD. In November 2025, the UK published their national 
roadmap to phasing out animal testing, which has a very similar scope as the Commission roadmap. 

Background 
Acute toxicity studies are among the most severe animal tests still used in regulatory safety assessments. 
Typically conducted in rats, these studies aim to determine the dose or concentration of a substance that 
causes  death  in  50%  of  the  test  animals  (LD50/LC50).  Lethality  is  the  intended  endpoint,  and  the 
procedures are associated with high levels of animal suffering. 

While  acute  toxicity  testing  is  no  longer  required  for  pharmaceuticals  –  following  changes  to  ICH 
M3(R2) in 2009 as a result of previous NC3Rs work in this area (Chapman et al., 2010; Robinson & 
Chapman, 2009; Robinson et al., 2008) – it remains mandatory for chemicals and agrochemicals under 
various regulatory frameworks. Several OECD test guidelines continue to support these tests globally, 
including for hazard identification and classification and labelling (CLP). 

Validated non-animal approaches exist for predicting acute effects such as skin and eye irritation and 
sensitisation  but  replacing  oral  and  inhalation  acute  toxicity  tests  remains  more  complex.  While 
international efforts are underway to develop non-animal approaches, including in silico models for oral 
toxicity and in vitro systems for inhalation, these methods have not yet been accepted as stand-alone 
replacements in regulatory frameworks. Until full replacement is possible, the NC3Rs is leading efforts 
to refine these tests to reduce animal suffering. This includes promoting the use of “evident toxicity” – 

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clinical  signs  that  predict  lethality  –  instead  of  death  as  an  endpoint  and  influencing  international 
guideline revisions. 

NC3Rs Acute Toxicity Project: Progress and Impact  
The  NC3Rs  has  played  a  central  role  in  embedding  the  3Rs  into  acute  toxicity  testing  since  its 
establishment in 2004. Early work focused on removing the requirement for single-dose acute toxicity 
studies in pharmaceutical development, contributing to changes in ICH M3(R2) (Chapman et al., 2010; 
Robinson &  Chapman,  2009;  Robinson  et al.,  2008).  More recently, efforts  have  shifted  to  refining 
acute toxicity tests required for chemicals and agrochemicals, where they are still required. 

Currently, there are three OECD test guidelines for acute oral toxicity (TG 420, 423, and 425) and three 
for acute inhalation toxicity (TG 403, 433, and 436). Of these, only TG 420 and TG 433 allow the use 
of  evident toxicity  as  an endpoint. The  others  still  rely  on lethality,  despite the availability  of more 
humane alternatives. 

For  inhalation  studies,  the  NC3Rs  led  a  large  data-sharing  initiative  and  developed  guidance  that 
contributed to the adoption of TG 433 in 2017 (Sewell et al., 2015; 2018). This guideline uses evident 
toxicity  and  represents  a  significant  refinement  and  reduced  animal  numbers. Work  continues  with 
partners  including  the  EPAA Acute Toxicity Taskforce  and  the  UK’s  OECD  national  coordinator  to 
remove TG 403 (the LC50 method), following the precedent set by the deletion of oral and dermal 
equivalents (deleted TG 401 and revised TG 402). 

In 2024, OECD member countries accepted the NC3Rs proposal to revise TG 403 so that it is only used 
as  a  last resort.  However, some countries continue  to  justify  its  use  for  determining  acute  exposure 
guideline levels. To address this, work is underway to demonstrate that TG 433 can also meet these 
needs. 

For oral studies, TG 420 has permitted the use of evident toxicity since 1992, but uptake remains limited. 
This is due to a lack of clear guidance and the continued availability of lethality-based alternatives. In 
collaboration with the EPAA, the NC3Rs published new supporting data in 2024 to validate the use of 
evident toxicity in TG 420 (Sewell et al., 2024) and support its use for classification and labelling. 

In  May  2025,  the  NC3Rs  held  online  meetings  with  global  experts,  including  representatives  from 
industry and contract research organisations, to review current practices. These discussions revealed 
that  lethality-based  guidelines  are  still  widely  used,  with  regional  preferences  influencing  guideline 
selection. For oral studies, TG 423 is preferred for REACH and biocidal regulations, while TG 425 is 
used  for  agrochemicals.  For  inhalation, TG  403  remains  common  in  the  USA,  South America,  and 
Korea, while TG 436 is preferred in the EU. 

TG 403 also covers Concentration x Time (CxT) studies for gases, vapours, and aerosols, and aligns 
with  US  EPA  guideline  OPPTS  870.1300.  All  of  these  preferred  guidelines  still  use  death  as  the 
endpoint, underscoring the need for further progress. 

Challenges and Opportunities  
Despite progress, several barriers remain to wider adoption of evident toxicity, including concerns about 
regulatory  acceptance,  classification  cut-offs, the  need  for  point  estimates  of  LD50/LC50,  cost,  and 
perceived uncertainty compared to mortality endpoints.  

To address these challenges, the NC3Rs is working to: 

▪  Encourage global harmonisation of guideline use, ideally selecting a single guideline per route, 

using the most refined methods (i.e. evident toxicity) where possible. 
▪  Understand the drivers for the use of TG 403 and work towards deletion.  
▪  Provide further guidance on defining and applying evident toxicity within TG 420. A proposal 
for this work was submitted to the OECD in 2023. However, since some member  countries 
requested additional evidence, this is not yet on the OECD workplan. The NC3Rs is now doing 

248 

 
 
 
 
further  analysis  to  validate  the  use  of  evident  toxicity  for  retrospective  classification  and 
labelling, demonstrating that TG 420 does not result in misclassification. 

Intended Outcomes  
The NC3Rs aims to phase out mammalian acute toxicity tests that use lethality as an endpoint. This will 
be achieved by: 

▪  Promoting the use of evident toxicity across relevant sectors. 
▪  Supporting the development and regulatory acceptance of non-animal methods. 
▪ 

Influencing international guideline revisions to embed refinement and replacement approaches. 

Conclusion  
Through scientific leadership, collaboration, and regulatory engagement, the NC3Rs is building a robust 
evidence base to support the refinement of acute toxicity testing and is ultimately working towards the 
replacement  and/or  elimination  of  these  tests.  This  work  supports  a  transition  to  more  humane, 
scientifically robust approaches and contributes to international efforts to modernise chemical safety 
assessment frameworks while maintaining high standards of human health protection. The NC3Rs has 
led the development and validation of evident toxicity as a refined endpoint, helping to reduce animal 
suffering  while  maintaining  scientific  integrity,  and  laying  the  groundwork for updating  OECD  test 
guidelines.  

References 
Chapman  K  et al.  (2010) The  value  of  acute toxicity  studies to  support the  clinical  management  of 
overdose  and  poisoning:  a  cross-discipline  consensus.  Regulatory  Toxicology  and 
Pharmacology 58(3): 354-359. doi: 10.1016/j.yrtph.2010.07.003 

Robinson S and Chapman K (2009) Are acute toxicity studies required to support overdose for new 
doi: 

Pharmacology 

Toxicology 

55(1): 

110. 

and 

medicines? 
10.1016/j.yrtph.2009.06.012 

Regulatory 

Robinson  S  et  al.  (2008) A  European  pharmaceutical  company  initiative  challenging  the  regulatory 
requirement  for  acute  toxicity  studies  in  pharmaceutical  drug  development.  Regulatory 
Toxicology and Pharmacology 50(3): 345-352. doi: 10.1016/j.yrtph.2007.11.009 

Sewell et al. (2015). A global initiative to refine acute inhalation studies through the use of 'evident 
toxicity'  as  an  endpoint: Towards  adoption  of  the  fixed  concentration  procedure.  Regulatory 
Toxicology & Pharmacology. 73(3):770-9. doi: 10.1016/j.yrtph.2015.10.018. 

Sewell  et  al.  (2018).  An  evaluation  of  the  fixed  concentration  procedure  for  assessment 
toxicity.  Regulatory  Toxicology  &  Pharmacology.  94:22-32.  doi: 

of acute inhalation 
10.1016/j.yrtph.2018.01.001.  

Sewell et  al.  (2024).  New supporting  data  to  guide  the  use  of  evident  toxicity in acute oral  toxicity 
studies  (OECD  TG  420).  Regulatory  Toxicology  &  Pharmacology.  146:105517.  doi: 
10.1016/j.yrtph.2023.105517 

B) UK NC3Rs report – Reconsidering the use of two species for repeated 
dose toxicity testing in the development of pharmaceuticals 

Introduction 
Complementary to the recommendations given in chapters 4.4.1 and 4.4.2 on how to avoid repeated 
dose  toxicity  testing  in  a  second  species  for  plant  protection  products  and  others,  similar 
recommendations  have  been  worked  on  (and  are  still  ongoing)  for  repeated  dose  toxicity  testing  in 
pharmaceuticals by an international multi-stakeholder collaboration under the leadership of the UK’s 
National  Centre  for  the  Replacement,  Refinement  and  Reduction  of Animals  in  Research  (NC3Rs). 

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Insights from this project are relevant for all actions under the roadmap to avoid testing in a second 
species.  
This chapter outlines the work of the NC3Rs to reassess the regulatory requirement for two species in 
regulatory toxicology studies for the development of new pharmaceuticals. This reassessment is not 
only critical for reducing animal use, in line with the European Commission’s ambition to phase out 
animal  testing  where  scientifically  justified,  but  also  for  improving  the  efficiency  and  relevance  of 
safety testing by harnessing advances in science and technology. 

Background 
Regulatory repeated dose toxicology studies for pharmaceuticals have traditionally required testing in 
both a rodent (e.g. rat or mouse) and a non-rodent (e.g. dog, minipig or non-human primate) species to 
support first-in-human (FIH) trials and subsequent clinical development. This two-species requirement 
was established over 30 years ago in International Council for Harmonisation (ICH) guidelines such as 
ICH  M3(R2),  with  the  aim  of  ensuring  robust  hazard  identification  and  protecting  human  safety. 
However,  scientific  advances,  the  growing  availability  of  safety  data,  and  evolving  regulatory 
perspectives  now  support  a  critical  reassessment  of  this  default  requirement.  New  drug  modalities, 
human-specific  targets  and  the  development  of  advanced  in  vitro  and  in  silico  technologies  offer 
opportunities  to  reduce  animal  use  without  compromising  human  safety.  In  some  cases,  regulatory 
requirements have already evolved to reflect scientific progress, for example, ICH S6(R1) – the specific 
guideline for biotechnology-derived pharmaceuticals (i.e. biologics) – allows the use of a single species 
for later development  studies  if  "similar" toxicities are  identified in  two  species  in  First  In  Human-
enabling studies2. 
The NC3Rs, in collaboration with the ABPI (Association of the British Pharmaceutical Industry) and 
global stakeholders, has led two major data-sharing initiatives to evaluate the scientific justification for 
the  two-species  requirement  and  explore  opportunities  where  a  single  species  approach  might  be 
appropriate, in alignment with the principles of the 3Rs (Replacement, Reduction, Refinement). 

NC3Rs Two Species project: Phase I (2016-2020) 
The  first  phase  of  the  NC3Rs  Two  Species  project  brought  together  37  organisations,  including 
pharmaceutical and biotechnology companies, contract research organisations, academic institutions, 
and  regulatory  bodies. The  group  collected  and  reviewed  data  from  172  compounds,  covering  both 
biologics and small molecules at various stages of development (Prior et al., 2020). 
The analysis revealed that, for biologics, similar toxicities were often observed in both rodent and non-
rodent  species  during  short-term  studies.  This  finding  supports  the  use  of  a  single  species  in  later 
development, as permitted under ICH S6(R1). However, the option to reduce to one species was not 
consistently  applied  in  practice,  which  may  reflect  uncertainty  around  when  this  approach  is 
scientifically justified and whether it will be accepted by regulators. To address this, follow-on work 
focused on clarifying the definition of “similar” toxicities to encourage broader uptake of the single-
species approach (Prior et al., 2022). Supporting initiatives, such as the NC3Rs Virtual Second Species 
CRACK IT Challenge (see section 6), are also being developed to increase confidence in this approach 
by providing additional insight from predictive modelling of a second species alongside in vivo data 
from  one  species. This  could  help  bridge  the  gap  between  traditional  two-species  studies  and  more 
flexible, science-driven approaches to regulatory decision-making. 
The data also provided preliminary evidence that a single-species approach could be feasible for other 
types of drugs (e.g. small molecules), which are currently subject to the two-species requirement under 
ICH  M3(R2).  However,  the  evidence  base  was  not  yet  sufficient  to  support  regulatory  change, 
highlighting the need for further investigation. 

NC3Rs Two Species project: Phase II (2023-present) 

250 

 
 
 
 
 
 
Building on the findings of Phase I, a  second phase was launched in 2023 with a new international 
working group comprising 39 organisations. This phase focuses on generating a more robust evidence 
base to inform regulatory decision-making for small molecules developed under ICH M3(R2), aiming 
to  evaluate the feasibility and  scientific justification for using a  single species  in  long-term toxicity 
studies. Key questions include: 

▪  How often are similar toxicities identified in short-term studies across species? 
▪  How frequently do new or unexpected toxicities arise in long-term studies, and how do these 

differ between rodents and non-rodents? 

▪  What is the theoretical risk to humans if only one species is used for long-term studies? 

Data were collected from 17 companies consisting of 75 molecules across therapeutic areas 
such as neurology, anti-infectives, and oncology. For each molecule, information was gathered 
on  at  least  two  FIH-enabling  and  two  later  development  studies,  including  study  design, 
observed  toxicities  (severity,  monitorability,  reversibility),  and  their  impact  on  decision-
making. 
The analysis showed that toxicity findings occurred in approximately 90% of studies, but only 
around 30% were considered relevant to human safety, and fewer than 20% had an impact on 
the clinical programme. Retrospective review indicated that, for 75% of the molecules, the same 
development decisions could have been made using only one species.  
To explore the feasibility of prospective decision-making, a series of blinded exercises were 
conducted.  Experts  reviewed  early  and  FIH-enabling  data  without  access  to  later  findings, 
simulating real-world decision-making. These exercises suggested that, for approximately 70% 
of  the  molecules,  a  single-species  approach  would  have  been  appropriate,  with  no  human-
relevant toxicities missed in the absence of a second species. 
Further analysis is ongoing to identify common factors in molecules where the decisions were 
straightforward and to explore what additional data might be needed in more complex cases 
where  the  decision  was  more  challenging  or  where  expert  opinions  differed.  This  work  is 
intended  to  inform  future  regulatory  guidance  and  support  a  more  flexible,  evidence-based 
approach to species selection. 

NC3Rs Two Species project: Next steps 

Preliminary findings from Phase II suggest that a single-species approach may be sufficient in many 
contexts for small molecules, particularly when supported by mechanistic understanding and Non-
Animal Methods. The next phase of work will focus on two streams: 

1.  For molecules for which the decision to use a single species/two species was straightforward, the 
next phase will identify common contributing factors and explore how these could be evaluated in 
practice by developing tools – such as decision trees or weight of evidence frameworks – to support 
species selection. 

2.  For  more  complex  cases,  the  project  will  explore  what  additional  information,  such  as  prior 
experience  with  similar  molecules  or the  human relevance  of  observed toxicities,  could  support 
decision-making. 

A  workshop  in  Autumn  2025  focused  on  how  these  findings  could  inform  future 
recommendations, and what evidence would be required to support regulatory change, to work 
towards  a  more  flexible,  scientifically  justified  approach  to  species  selection,  that  reduces 
animal use without compromising human safety. The final project report will be published in 
2026, with updates available via the NC3Rs Two Species project page. 

NC3Rs CRACK IT Challenge: Virtual Second Species (2022-present) 

251 

 
 
 
 
 
To  complement  the  data-driven  approach,  the  NC3Rs  is  also  supporting  other  activities  to  promote 
alternative approaches that could increase confidence in the use of a single in vivo species as part of 
pharmaceutical development. For example, through its open-innovation scheme CRACK IT, the Virtual 
Second Species project aims to develop a machine-learning-aided, multi-scale modelling framework to 
predict toxicological endpoints in dogs. The goal is to ultimately replace the use of dogs in chronic 
toxicity studies, but such a model could be used in early discovery to screen for potential ‘show-stopper’ 
toxicities  in  the  dog,  helping  to  focus  investigations  on  target  organs  of  concern  (using  other in 
vitro and in silico models) before any animal testing is conducted. In future, this approach could also 
support the development of virtual models for other species. 
The  model  is  designed  to  provide  mechanistic  insight  and  predictive  toxicology  data,  supporting 
regulatory decision-making and reducing reliance on animal studies. This initiative complements the 
Two Species project by providing predictive tools that could further support single-species approaches 
in regulatory decision-making.  
Intended Outcomes  

The NC3Rs Two Species project aims to reduce the use of animals in regulatory toxicology 
studies for pharmaceuticals without compromising human safety. By identifying contexts where 
a single species may be sufficient, the project seeks to: 
Inform  updates  to  international  regulatory  guidelines,  including  ICH  M3,  and  support 
opportunities already allowed within ICH S6. 

▪ 

▪  Support the development of decision-making tools to guide species selection. 
▪  Promote broader acceptance of scientifically justified alternatives. 

Conclusion  
Through international collaboration, data sharing, and scientific innovation, the NC3Rs Two Species 
project  has  generated  a  robust  evidence  base  to  support  a  more  flexible,  risk-informed  approach  to 
species  selection  in  regulatory  toxicology.  The  findings  demonstrate  that,  in  many  cases,  a  single-
species  approach  can  provide  sufficient  information  to  ensure  human  safety,  particularly  when 
supported by mechanistic understanding and Non-Animal Methods. 
This work lays the groundwork for updating international regulatory guidelines, including ICH M3, and 
for broader implementation of existing flexibilities under ICH S6. It also highlights the importance of 
developing practical tools – such as decision trees and weight-of-evidence frameworks  – to support 
prospective decision-making. 
By enabling scientifically justified reductions in animal use, the project contributes to the European 
Commission’s ambition to phase out animal testing and advances global efforts to modernise regulatory 
science while maintaining high standards of human health protection. As the evidence base continues 
to grow, the NC3Rs remains committed to working with international partners to translate these findings 
into meaningful regulatory change. 

References 
[1] Prior et al., 2020. “Opportunities for use of one species for longer-term toxicology testing during 
drug  development:  A  cross-industry  evaluation,”  Regul.  Toxicol.  Pharmacol.,  vol.  113,  p. 
104624, Jun. 2020, doi: 10.1016/j.yrtph.2020.104624. 

[2]  Prior  et  al.,  2022.  “Exploring  the  Definition  of  ‘Similar  Toxicities’:  Case  Studies  Illustrating 
Industry and Regulatory Interpretation of ICH S6(R1) for Long-Term Toxicity Studies in One 
or  Two  Species,”  Int.  J.  Toxicol.,  vol.  41,  no.  3,  pp.  171–181,  May  2022,  doi: 
10.1177/10915818221081439. 

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