The document discusses the EU-ToxRisk project which aims to move toxicological assessment away from animal models towards human cell-based in vitro tests and better understanding of chemical adverse effects. It summarizes the EU-ToxRisk read-across strategy for filling data gaps, which involves characterizing target compounds, identifying similar source compounds, evaluating the source compounds, assessing uncertainties, and deriving no-observed adverse effect levels and derived no-effect levels. Main reasons for rejection of read-across justifications include unclear substance identity, lack of evidence supporting assumptions, inappropriate source data selection, and lack of scientific plausibility.

1. How to submit a successful
Read Across justification report
Costanza Rovida
CAAT-Europe
TEAM mastery S.r.l.
5th May 2020

2. EU-ToxRisk: An Integrated European ‘Flagship’ Programme driving
Mechanism-based Toxicity Testing and Risk Assessment for the 21st century
The aim of the EU-ToxRisk project is to move away from observational toxicology
based on animal models, progressing towards a toxicological assessment based on
in vitro test responses using human cells, together with better mechanistic
understanding of chemical adverse effects.
• H2020-supported collaborative project
• 30 million €
• 6 years starting 1st January 2016
• Coordinator: Leiden University, Bob Van de Water
• 39 partners
• International collaboration
• 14 Work Packages
www.eu-toxrisk.eu

3. Costanza Rovida, Webinar, 05.05.2020 3
Read across: what is it?
REACH (Registration Evaluation Authorisation and restriction of Chemicals)
Article 13(1):
Information on intrinsic properties of substances may be generated by means other than
tests, provided that the conditions set out in Annex XI are met. In particular for human
toxicity, information shall be generated whenever possible by means other than vertebrate
animal tests, through the use of alternative methods, for example, in vitro methods or
qualitative or quantitative structure-activity relationship models or from information from
structurally related substances (grouping or read-across). Testing in accordance with Annex
VIII, Sections 8.6 and 8.7, Annex IX and Annex X may be omitted where justified by
information on exposure and implemented risk management measures as specified in
Annex XI, section 3.
Annex XI
“Substances whose physicochemical, toxicological and ecotoxicological properties are
likely to be similar or follow a regular pattern as a result of structural similarity may be
considered as a group or ‘category’ of substances. Application of the group concept
requires that physicochemical properties, human health effects, and environmental effects
or environmental fate may be predicted from data for reference substance(s) within the
group by interpolation to other substances in the group (read-across approach). This
avoids the need to test every substance for every endpoint.”

4. “Read-across has been extensively used by registrants to avoid animal testing under REACH”
file:///C:/Users/Admin/Downloads/ED0217170ENN.en.pdf
“We are concerned that,
in many cases, the quality
of information on
alternatives in the
submitted dossiers is not
robust enough to replace
animal tests and
therefore we urge
registrants to update
their dossiers accordingly
before evaluation.”
ECHA Report, 2017:
The use of alternatives to testing on animals for the REACH Regulation

5. ECOPA asked about compliance decision letters that are sent by ECHA to registrants, in
particular, whether it would be possible to refine the letter to highlight even more clearly
the need to first apply any possible means to fulfil the requirement without vertebrate
animal tests, and only if not possible, to move to an in vivo test. ECHA responded that,
when a non-compliance is spotted, ECHA explains in full detail for example why a read-
across has failed, why a structural similarity may not be appropriately described, why
exclusion or inclusion criteria are missing, and why a final hypothesis has failed. All this
information is available for the registrant if they wish to improve their read across and to
come back with a new adaptation instead of performing a test. It was also highlighted
that ECHA has made lots of support material available on this topic. It was stressed that it
is the responsibility of the registrant to define their testing strategy and that ECHA is not in
a position to do that on behalf of the registrant. According to ECHA, a number of
registrants are in fact applying read-across and are coming back with improved
adaptations following the compliance process.

6. Main reasons for rejection:
→ Unclear substance identity, not possible to ascertain structural similarity
→ Lack of sufficient evidence to substantiate assumptions made within read-across
justifications
→ Read-across to inappropriate data
→ Lack of scientific plausibility
https://echa.europa.eu/support/registration/how-to-avoid-unnecessary-testing-on-animals/grouping-of-substances-and-read-across
http://doi.org/10.2823/619212

7. Main reasons for rejection:
→ Unclear substance identity, not possible to ascertain structural similarity
→ Lack of sufficient evidence to substantiate assumptions made within read-across
justifications
→ Read-across to inappropriate data
→ Lack of scientific plausibility
https://echa.europa.eu/support/registration/how-to-avoid-unnecessary-testing-on-animals/grouping-of-substances-and-read-across
http://doi.org/10.2823/619212

8. Main reasons for rejection:
→ Unclear substance identity, not possible to ascertain structural similarity
→ Lack of sufficient evidence to substantiate assumptions made within read-across
justifications
→ Read-across to inappropriate data
→ Lack of scientific plausibility
https://echa.europa.eu/support/registration/how-to-avoid-unnecessary-testing-on-animals/grouping-of-substances-and-read-across
http://doi.org/10.2823/619212

9. This project has received funding from the European Union’s Horizon 2020 Research
and Innovation programme under Grant Agreement no. 681002
Rovida, C., Barton-Maclaren, T., Benfenati, E., Caloni, F., Chandrasekera, C.,
Chesne, C., Cronin, M.T.D., De Knecht, J., Dietrich, D.R., Escher, S.E., Fitzpatrick,
S., Flannery, B., Herzler, M., Hougaard Bennekou, S., Hubesch B., Kamp, H.,
Kisitu, J., Kleinstreuer, N., Kovarich, S., Leist, L., Maertens, A., Nugent, K.,
Pallocca, G., Pastor, M., Patlewicz, G., Pavan, M., Presgrave, O.,
Smirnova, L., Schwarz, M., Yamada, T. and Hartung, T. (2020).
T4 Report - Internationalisation of read-across as a
validated new approach method (NAM) for regulatory
toxicology.
Accepted
https://caat.jhsph.edu/

10. Costanza Rovida, Webinar, 05.05.2020 10
EU-ToxRisk Read-Across Strategy
1. Problem Formulation
2. Target compound characterisation
3. Source compound identification
4. Source compound evaluation
5. Uncertainty assessment
6. Data Gap filling - Derive NOAEL and DNEL
RAx
hypothesis

11. Costanza Rovida, Webinar, 05.05.2020 11
EU-ToxRisk Read-Across Strategy
1. Problem Formulation
2. Target compound characterisation
3. Source compound identification
4. Source compound evaluation
5. Uncertainty assessment
6. Data Gap filling - Derive NOAEL and DNEL
RAx
hypothesis
1. Problem Formulation
→ What do I need? Definition of the final goal
→ Regulatory scope
→ Economical considerations

12. Costanza Rovida, Webinar, 05.05.2020 12
EU-ToxRisk Read-Across Strategy
1. Problem Formulation
2. Target compound characterisation
3. Source compound identification
4. Source compound evaluation
5. Uncertainty assessment
6. Data Gap filling - Derive NOAEL and DNEL
2. Target compound characterisation
→ Detailed analytical characterization
UVCB – all components
→ PhysChem properties (water solubility, Kow)
→Gather all existing information!
→QSAR assessment

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EU-ToxRisk Read-Across Strategy
1. Problem Formulation
2. Target compound characterisation
3. Source compound identification
4. Source compound evaluation
5. Uncertainty assessment
6. Data Gap filling - Derive NOAEL and DNEL
RAx hypothesis
Analogue (1:1)
Category (many:1, many:many)
Same type of effect(s)
(Bio) transformation to common compound(s)

14. Costanza Rovida, Webinar, 05.05.2020 14
EU-ToxRisk Read-Across Strategy
1. Problem Formulation
2. Target compound characterisation
3. Source compound identification
4. Source compound evaluation
5. Uncertainty assessment
6. Data Gap filling - Derive NOAEL and DNEL
Source compound identification
→ Chemical similarity (Tanimoto)
→Gather all existing information!
→Suitability

15. Costanza Rovida, Webinar, 05.05.2020 15
EU-ToxRisk Read-Across Strategy
1. Problem Formulation
2. Target compound characterisation
3. Source compound identification
4. Source compound evaluation
5. Uncertainty assessment
6. Data Gap filling - Derive NOAEL and DNEL
4. Source compound evaluation =
challenging the RAx hypothesis
→PhysChem properties (water solubility, Kow)
→Biological similarity
→ADME
→QSAR assessment
→Applicability Domain
→Definition of boundaries
→Identification of possible activity cliffs

16. Costanza Rovida, Webinar, 05.05.2020 16
EU-ToxRisk Read-Across Strategy
1. Problem Formulation
2. Target compound characterisation
3. Source compound identification
4. Source compound evaluation
5. Uncertainty assessment
6. Data Gap filling - Derive NOAEL and DNEL
RAx
hypothesis
5. Uncertainty assessment
EFSA: “all types of limitations in available knowledge
that affect the range and probability of possible
answers to an assessment question”
→Uncertainties associated with assessment inputs
→Uncertainties associated with assessment methodology
→(quantification of the uncertainty)
EFSA (2018)
Guidance on Uncertainty Analysis in Scientific Assessments

17. New Approach Methodologies (NAMs)
→ in silico prediction tools (QSAR, structural modelling)
→ any in vitro (validated) test systems
→ biochemical assays
→ transcriptomics (e.g. high throughput)
→ metabolomics
→ PBPK modelling
https://aopwiki.org/
AOP: Adverse Outcome Pathway
Molecular
properties
Molecular
initiating events
Cellular
responses
Organ
response
Organism
response

18. Costanza Rovida, Webinar, 05.05.2020 18
Integrated Approaches to Testing and Assessment (IATA)

19. Commercial Platform for New Approach Methodologies (NAMs) in Safety
Assessment – Launch at the EU-ToxRisk Stakeholder meeting , 12-02-2020
➢ In Silico Screening, Barry Hardy, Edelweiss Connect
➢ Safety Assessment, Costanza Rovida, CAAT - Europe & TEAM mastery S.r.l.
➢ High Throughput
In Vitro Testing,
Bart van der Burg, BDS
➢ DILI High Content
Screening, Bas ter Braak,
DESI
➢ Mechanistic investigations
in 3D InSight™ Liver
Microtissues,
Monika Kijanska, InSphero
➢ Renal Mitochondrial
Toxicity and Phase I/II
Metabolism, Paul Jennings, VU
➢ Transporter Modelling (Phase III Metabolism), Gerhard Ecker, Phenaris
➢ Neurotoxicity Assessment, Andras Dinnyes, Biotalentum

20. Costanza Rovida, Webinar, 05.05.2020 20
EU-ToxRisk Read-Across Strategy
1. Problem Formulation
2. Target compound characterisation
3. Source compound identification
4. Source compound evaluation
5. Uncertainty assessment
6. Data Gap filling - Derive NOAEL and DNEL
RAx
hypothesis

21. ALTEX 33(2), 2016
→ Robust justification explaining each step of the strategy
→ Demonstration that there is no bias in the selection of
source substances
→Honest management of discordant results
→Assessment of uncertainties
→ Final Conclusions
Final Read Across Justification Report

22. Thank you