Thomas Hartung

Doerenkamp-Zbinden Professor and Chair for Evidence-based Toxicology,

Director, Center for Alternatives to Animal Testing (CAAT)

Johns Hopkins University, Baltimore, US

Professor of pharmacology and toxicology, University of Konstanz, Germany

The implementation of the

Three Rs in the interest of

good and humane science

1

• Better science

• Less animals

• Human relevance

• Faster and cheaper results

• Refinement

• Information, Grants

• Think tank

• New tools, quality control

• EU branch, policy program

• Stakeholder consensus

Scientific American 2005

2

The Bernice Barbour

Foundation

Funding from industry, philanthropy and research funding agencies

…and individuals

3

• CAAT-Europe

• Four visiting scientists from Brazil (more

to come); collaboration with opinion

leaders; head validation management

group HET-CAM for BraCVAM

• Pan-American conference Apr 2016

• World Conference Seattle 2017; satellite

conferences?

• Collaborations Brazil, India, Korea, China,

Japan, Australia…

THE GLOBAL DIMENSION

4

32 articles / reports published

2 commissioned articles in

preparation

5 workshop reports pending

In vitro publication standards

5+ workshops planned

Ambassadors

Thomas Hartung

Marcel Leist

Bas Blaauboer

Alan Goldberg

5

Example

Two workshops 2011

Baltimore & Budapest

Covered in Nature 30 Jun 2011

7

Two sides of one coin…

Animal

Welfare

3Rs

Improved

Safety

Sciences

Shortcomings of animal tests

(1) Disparity of testing requirements and risk

acceptance for different products and geographical

areas,

(2) Throughput and costs of testing versus testing

needs,

(3) Limited predictivity for humans,

(4) Precautionary approaches from drug development

adapted to other areas,

(5) Animal use,

(6) New products not suitable for traditional tests,

(7) New hazards not covered,

(8) Mixtures of toxicants not addressed,

(9)I Idividual susceptibilities and vulnerable

subpopulations not covered

(10) poor basic research and publication standards

8

Genotoxic: sugarGenotoxic: salt

Protected against

TCDD in eggs

Same calculation

for alcohol:

One glass per

345 years

Protected

against minute

amounts of

pesticides

23 of 31

tested coffee

ingredients

carcinogenic

Natural

pesticides

10,000x more,

35 of 63

carcinogenic

Enjoy!!!

9

R22 harmful if swallowed

(LD50 = 150-200mg/kg in rats)

R 36 irritant to eyes

R 37 respiratory irritant

R 38 irritant to skin

Not carcinogenic,

but co-carcinogen (promotor)

Unclear mutagenicity

Embryonic malformations in

cat, dog, rat, mice, rabbit,

monkey

Unlikely to be brought to the

market today

10

There is some

good reason

for regulating new

products….

11

43 – 60% interspecies correlation3% well tested chemicals8% somewhat tested chemicals

100,000+ chemicals in consumer products12

All models are wrong, some are useful (G. Box)

60%

naive not less naive

14

QA of cell system is of critical importance

Good Cell Culture Practice (Coecke et al. 2005)

MCF-7 Karyotyping

Human Toxome

Extent of deviations from normal genome

Classification Kilobases Percentage of genome

Losses 4587603 51.2%

Deletions 667374 7.5%

Amplifications 26904 0.3%

Gains 2587093 28.9%

Normal 871166 9.7%

Centromeres 217339 2.4%

Total Abberations 7868974 87.8%

All Entries 8957479

SurePrint G3 ISCA CGH+SNP Microarray Kit, 4x180K

115234 CGH features.2440 CGH replicate probes, 59647 SNP features

reference mapping: caucasian female human reference DNA

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http://en.wikipedia.org/wiki/Organ-on-a-chip Stem cells &

Organo-typic

culture & High-

content

21st century

toxicology starts

with 21st century

cell culture

How to improve the predictive value of cell cultures

Good Cell Culture Practice

Functional

endpoints /

biomarker

= mechanism

High-content

= wholesome

characterization

High-throughput

= replicates & many

comparisons

Organo-typic

culture

Human (stem) cells

Integrated

Testing

Strategies

17

Human “mini-brain”

• All cell types but micro-glia

• 350um diameter

• 800 per batch

• Reproducible

• Electrophysiological active

• From patient cells:

gene/environment

interactions

developing from iPSC

18

Opportunities for human mini-

brain research

•Map the neurotoxic chemical universe

•Characterization of medical

countermeasures

•Neurotoxic and DNToxic side effects

•Brain trauma, infectious disease and

neurodegenerative disease research

•Individual susceptibility using patient

iPSC – genetic risk factors

•Long-term culture and co-culture with

other organs

Data processing Data acquisition

Generic workflow

Toxicant exposure

-omics

Bio-informatics:

information extraction POT identification and

validation

Rober Bachinsky

Marize

Valadares

Rita Sa

• Biologicals (biopharmaceutical products)

• Cell therapies

• Genetically modified and functional

food (nutraceuticals)

• Medical countermeasures to biological

and chemical terrorism and warfare

agents

• Medical devices

• Nanoparticles

Challenging new products

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• Atherosclerosis

• Male infertility and other possible

manifestations of endocrine disruption

• Autism and other developmental

behavioral disorders

• Immunotoxicity

• Childhood asthma and other

obstructive lung diseases,

• Obesity

• Diabetes

• Many diseases with an environmental

component (Parkinson, cancer…)

New hazards

22

‘Omics’ Image analysis

High content High through-put

Information rich

Bioinformatics &

Data-mining

Knowledge on

pathways

Systems Toxicology

Robotised / automated

testing

23

Why metabolomics & miRNomics?

DNA

RNA

Proteins

Genotype

Metabolites

Genomics(25,000 genes)

Transcriptomics(100,000 mRNAs)

Proteomics(1,000,000 proteins)

Metabolomics(5,000-7,000 metabolites)

Phenotype

Mammalian Species

miRNAsmiRNomics

(2,042 miRNAs)

ALTEX 2013

30, 209-225

METABOLOMICS

3 WORKSHOPS

2 INFODAYS

Mapping the Human Toxome by Systems Toxicology

Hewitt et al., 2005. Science, 307:1572-1573

Endocrine disruption• Use “omics” to map PoT for endocrine disruption

• Develop software tools

• Identify PoT

• Develop a process for PoT annotation, validation

• Establish public database on PoT.

www.humantoxome.com26

Need for evidence integration:• Test covers not all possible outcomes

of interest, classes of substances

(applicability domain), or severity

classes

• Positive test result rare (excessive

false-positives)

• Gold standard test is too costly/ too

many animals (need for prioritization)

• Human predictivity of a single test is

not satisfactory

• Existing data or Kinetic information

(QIVIVE) to be integrated.27

Many PoT = many tests

Need for data integration

Use of multiple information,

not stand-alone replacement

OECD: Integrated Approaches to

Testing and Assessment (IATA)

= ITS + kinetics + exposure + RA

Integrated Testing Strategies

Toxicology will make more use of

Integrated Testing Strategies

• ITS development sensitization &

eye irritation

• Commissioned whitepaper

Jaworska & Hoffmann

• WORKSHOP 2013

29

30

Vanessa Sa Rocha, Natura

$3 trillion $3 trillion

$3 trillion$200 billion

Animal testing

at $3 billion per year

Safety Testing only at the end of a costly

development process

Frontloading of toxicology / Green Toxicology

“fail early, fail cheap”

Anticipate human or

regulatory problems?

“test early, develop clean”

Green Toxicology

InfoDays

Green Toxicology

• Connecticut, Dec

2012

• Baltimore, Nov 2013

• Zurich, Switzerland

Oct 2014

• Frankfurt, Germany,

Mar 2015

• SoT 2015, San Diego

Regulation has to minimize mistakes

Difficulty to change traditional

approaches

Precaution and no compromises, thus

formal validation

Frontloading of toxicity allows

uncertainty

GreenTox reduces validation &

acceptance needs

Which R of the 3?

Read-across

Replace

Refine

The 4th R?

Reduce**pesticides

ALTEX 2014, 31:387-396

CAAT Read-across Initiative

International Steering Group & Whitepaper

Five working groups

“Good Read-across Practice”

Stakeholder Fora Brussels & Washington early 2016

Read-across-21c • Negative vs.

positive read-

across

• Support by

biological data not

only structure

• Expression of

uncertainty

• Local validity

• Application to

complex mixtures

“Test-across”, 2007

“Evidence-Based”

Evidence-based:

-- Tools

-- Approaches

E-B guiding principles:

-- Transparency ( understanding & reproducibility)

-- Objectivity ( limit bias)

-- Consistency ( structured approaches)39

Evidence

Studies

GuidelinesOr “shaming”

Enhancement:

-- Design

-- Conduct

--Reporting

Egs. of outcomes:

-- Data quality

-- Lit. review

RetrospectiveAssessment

2006-7: Publication / 1st conference

Mar 2011: US EBTC

Oct 2011: Secretariat at CAAT

www.ebtox.com

Jan 2012: First conference hosted by EPA

Jun 2012: EU EBTC

Diverse working groups

Jul 2013: IUTOX, Seoul, Korea

Sep 2013: EuroTox, Interlaken, Switzerland

Systematic reviews increasingly embraced

by EPA/IRIS, NTP and EFSA

Nov 2014: Forum Systematic Reviews

Feb 2015: FDA Training

Systematic review & related approaches:Gaining acceptance in toxicology

Application: Chemical Assessments

Feb. ‘15 Workshop

Global perspective

• Global economy

needs global

regulatory

standards

• Opportunities for

export and contract

testing

• No transition until

last important

market changes

• Investment into 21st

century technologies

for new economies

The difficulty lies, not in the new ideas,

but in escaping from the old ones.

John Maynard Keynes

(1883 - 1946)

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