An Integrated EUropean ‘Flagship’ Program Driving Mechanism-based Toxicity Testing and Risk Assessment for the 21st Century

EFSA/RIVM Symposium: The Future of Risk Assesment and Toxicity Testing for Chemical Mixtures, Utrecht 18th May 2016 Susanne Hougaard Bennekou, the Danish EPA

• Approaches to improve the efficiency of predictive toxicological testing to

address key areas of concern for human health

• Approaches to meet regulatory requirements (e.g. EU legislations on

REACH, cosmetics, biocides)

• To understand complex biological pathways of toxicological relevance

• To identify early markers predictive of toxicological effects in humans

• To develop and validate routine, non-animal approaches for toxicity testing

of chemical substances (excluding radio-chemicals).

• To involve confirmatory testing of mechanistic hypotheses

Horizon2020 Health Call PHC-33: New Approaches to Improve Predictive Human Safety Testing

Background of the call

• More effective, faster, cheaper toxicological testing to better predict

human risk and meet regulatory needs

• Improved toxicological knowledge to encourage ‘read across’ between

chemical substances for use in different research and regulatory domains

• Commercial exploitation of the developed toxicological testing methods

and assessment approaches, products and services

• Advancement of international co-operation in the field of predictive

toxicology and human safety testing

• Reduced use of laboratory animals in safety testing

Expected outcome of a future project

Horizon2020 Health Call PHC-33: New Approaches to Improve Predictive Human Safety Testing

US NRC 2007: Toxicity testing in the 21st Century: A vision and a strategy

• Key concept: most xenobiotic toxicities are related to effects on a limited number

of physiological pathways required for normal cellular maintenance, regulation or

adaptation.

• Defining AOPs allow toxicologists moving away from a ‘black box approach’,

investigating ‘apical endpoints’, towards an approach where effects are

mechanistically understood, allowing prevention and monitoring

Rationale

1. A rapid improvement of read across (RAX) approaches

2. A new “Adverse Outcome Pathway” (AOP)-based ab initio quantitative risk assessment approach

EU-ToxRisk will establish human-relevant testing strategies aligned along

validated knowledge of AOPs, and implemented in IATAs to meet risk

assessment purposes.

Expected outcome of EU-ToxRisk

Objective

• 38 European partners

• 1 US partner

• Academia & Research institutes

• SMEs

• Industry

• Regulators & other stakeholders

Collaborators in US, Japan, China,

India, Latin-America

EU-ToxRisk Partners

Partners Expertise

Omics

Test systems

Translation

Data

Modelling

High throughput

Stakeholders

Toxicokinetics

Integration of diverse expertise and disciplines

EU-ToxRisk: A Multidisciplinary Project

EU-ToxRisk Collaborations

• Active collaboration with JRC

• Direct interaction with U.S. EPA and NTP/NIEHS

• Interaction with industry and regulatory stakeholders

• Interaction other safety sciences EC funded research

• Interaction with Cosmetics Europe LRSS

• Invitation for cooperation with SEURAT-1 legacy holders

assay throughput

human relevance

coverage

toxicity en

dp

oin

ts

sys

tem

s

bio

logy

mo

del

ling

choice of EUToxRisk21 test systems and strategy

EU-ToxRisk Strategic Choices

assay throughput

human relevance

coverage

toxicity en

dp

oin

ts

sys

tem

s

bio

logy

mo

del

ling

choice of EU-ToxRisk

test systems and strategy

• 2D/3D human cells and tissue slices models

• Range from HCI to organ-on-a-chip

• Several hundred chemicals (not 10000+)

• Cmpd case studies with strategy optimisation

• Omics data connected to classical endpoints

• AOP as guidance for biological RAX

• Computational toxicology and data basing

• Biokinetics/experimental ADME data

• PBPK combined with multi-scale hazard

modelling

EU-ToxRisk Strategic Choices

assay throughput

human relevance

coverage

toxicity en

dp

oin

ts

sys

tem

s

bio

logy

mo

del

ling

choice of EU-ToxRisk

test systems and strategy

Integration of past experience in safety sciences

• SEURAT-1: DETECTIVE, NOTOX, HEMIBIO,

TOXBANK, COSMOS, SCREENTOX

• FP7 programs: Carcinogenomics, HeCaTos,

Chemscreen, OPENTOX, DiXa, ESNATS,

PredictIV

• IMI programs: eTOX, MIP-DILI, Safe-T,

OPENPHACTS

EU-ToxRisk Background Knowledge/Expertise

assay throughput

human relevance

coverage

toxicity en

dp

oin

ts

sys

tem

s

bio

logy

mo

del

ling

choice of EU-ToxRisk

test systems and strategy

Focus areas for improved risk assessment:

• Repeat Dose Toxicity (RDT)

• Liver

• Kidney

• Lung

• Neuro

• Developmental and Reprodictive Toxicity (DART)

EU-ToxRisk Focus Areas

Quality control + validation procedures exploitation

Human variability

Optimization spiral: ADME / exposure

Exposure scenarios

Risk Assessment (RA)

Optimization spiral: hazard

•sa

fety

th

resh

old

s •

un

cert

ain

ty

•ex

po

sure

sce

nar

ios

•

sub

po

pu

lati

on

s

Case 3 Case 4 (ab initio)

No YES

Case 1 Case 2

YES

N

o

Chemicals

I. Training cmpds

II. Test cmpds Industry case studies

Transport & Distribution

In vitro Chemical

Metabolism

Protein Binding &

Free Fraction

Physico-chemical

properties

qIVIVE: PBPK and PoD

for RA

Data bases +

Computational models

Data bases +

Computational models

In silico: QSAR

Target-docking

In vitro HT effects Cytotox

Stress response HT Omics

Time- and concentration-

dependency

Systems biology & quantitative

AOPs

Point-of-departure

(PoD) for RA 1.

2.

3.

1.

2.

3.

Toxicokinetics Hazard

2D HepG2 (GPF-reporter) ± CYP450s iPSC-derived hepatocytes (+reporters)

HepaRG + 3D HepG2

Throughput Complexity

2D/3D primary human hepatocytes + liver microtissues

multicellular and/or diseased

microtissues

precision cut human

liver slices

archived rodent liver material

Variabilities Human and in vivo anchoring Primary, long-term, differentiated Speed, cost

organ-on-a-chip microfluidics connection

to other tissues

Multi-organ integration

EU-ToxRisk Model Systems Rationale

Need addressed:

RDT

selection of test systems

case studies

biokinetics transcriptomics cytotoxicity

chemical selection

quantitative key event

IATA Strategies

Exp

loit

atio

n

Ris

k as

sess

men

t

in silico and experimental ADME

computational modelling

concentrations

Liver Kidney Lung Neuro DART

HepG2 + CYPs

HepaRG

semi3D sandw.

PHH

multicell 3D PHH

3D hRPTEC

iPSC sensory neurons

human EST-

cardiac neuro

zebra fish

embryo

3D air/liquid

hLEC

2D hRPTEC

primary hRPTECT

iPSC 3D

floating neurons

neurite growth assay

2D hLEC

primary A/L

3D hLEC

2D SH-SY5Y LUHMES

DART

2 2D and 3D high throughput and high content

pathway toxicity reporters

in silico prediciton of ADME and toxicity; biokinetics data

2 translation to fresh human tissue,

patient samples and human genetics

tie

red

te

stin

g

Matrix

1. Improved toxicological knowledge and quantitative understanding of concentration- and time-

response relationships for key RDT and DART adverse outcomes to be annotated in semi-qAOP

and some qAOP.

2. New robust RAX procedures incorporating toxicokinetics data and similarity evaluation on the

level of KE activation, and a complementary KE screen battery to easily fill data gaps of the RAX

procedure and meet regulatory needs.

3. A validated set of tiered IATAs for ab initio RDT and DART assessment for regulatory use.

4. Novel validated in silico tools and in vitro test systems ready for exploitation and industry uptake

allowing effective, faster and cheaper toxicological testing.

5. A sustainable EU-ToxRisk database open to the public domain and stakeholders worldwide.

6. An ultimate joint exploitation package for advanced in vitro assays and services.

7. A major reduction in the use of laboratory animals in safety testing across industries and

regulatory bodies.

8. Safety assessment guidelines based on animal-free approaches to bridge EU-ToxRisk proof-of-

concept to regulatory implementation.

Expected outcome

Mixture Risk Assessment NEEDS

In regard of grouping of pesticides for cumulative risk assessment and in particular of group refining, the current read-out from animal studies (apical endpoints) are not tailored for this purpose

Problem Formulation

Identification of priority chemicals

Exposure

Occurence data – priority chemicals/contaminants

Hazard

MoA information from

Toxicokinetic data

Toxicity studies

QSAR

OMICS

Research on characterising AOPs

Data collection: input to EU-ToxRisk

Mixture Risk Assessment NEEDS

1. A rapid improvement of read across (RAX) approaches

2. A new “Adverse Outcome Pathway” (AOP)-based ab initio quantitative risk assessment approach

Expected outcome of EU-ToxRisk

Thank you