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Thomas HartungDoerenkamp-Zbinden Professor and Chair for Evidence-based Toxicology, EHS
Director, Center for Alternatives to Animal Testing (CAAT)Joint appointment: Molecular Microbiology and Immunology
Bloomberg School of Public Health, Johns Hopkins University, Baltimore, US
Professor of pharmacology and toxicology, University of Konstanz, Germany
Opportunities for replacinganimals in toxicity testing
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We need all tools in the lifesciences, but all toolshave limitations
Animal welfare is not theonly reason foralternatives
The value of animal testresults is overestimated
3Rs (reduce, replace,refine) as societalcompromise
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Not toforget:We can alsodo a lot forreductionandrefinement
PharmaReduction &RefinementWork Group
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Traditional 3Rs methodswill not be the solution tothe problem
• Little perspective forcomplex endpoints• 2/3 fail validation• hardly solved thecosmetics 7th amendmentchallenge for 2009, no wayfor 2013
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EPAA 2008 Conference- 3 November 2008 Prof. Michael Schwarz
3Rs Science today: A solid basis for the future3Rs Science today: A solid basis for the future
2004-20092004-2009(www.reprotect.eu)(www.reprotect.eu)
LSHB-CT-2004-503257LSHB-CT-2004-503257Development of a novel approach in hazard and risk assessment ofDevelopment of a novel approach in hazard and risk assessment of
reproductive toxicity by a combination and application of reproductive toxicity by a combination and application of in vitroin vitro, tissue, tissueand sensor technologiesand sensor technologies
32 participating groups32 participating groups
Slides courtesy of M. Schwarz, Tuebingen
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ReProTectReProTect Reproductive cycleReproductive cycle
Late PrenatalDevelopment
Early PrenatalDevelopment Implantation
Fertilisation
Gameteproduction
andrelease
post-natalDevelopmen
t
3 tests
4 tests
7 tests
9 tests for endocrine
disrupters
4 tests
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The ReProTect Feasibility StudyChemical Female fertility Male fertility Developmen-
tal toxicity1
2
3
4
5
6
7
8
9
10
X
Correctly predicted
Not correctly predicted
Partly predicted
( )
Effect: Positive (red)or negative (green)
X X
X
X
( )( ) ( )
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An atmosphere of departure intoxicology
Lessons learned fromalternative methods andtheir validation
New technologies frombiotech and (bio-)informatics revolution
Mapping of pathways oftoxicity (PoT)
NAS vision report Tox-21c
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Friday 15th Feb 2008: Coalition of EPA, NTP andNIEHS-CGC to implement NRC vision
[Science 2008, 319:906-7]
• “We propose a shift from primarily in vivoanimal studies to in vitro assays, in vivo assayswith lower organisms, and computationalmodeling for toxicity assessments”
• “[toxicity testing] was expensive, time-consuming, used animals in large numbers anddidn’t always work” Francis Collin, now DirectorNIH
• “Animal testing won’t disappear overnight, butthe agencies’ work signals the beginning of anend.” Elias Zerhouni, at the time Director NIH
quotes: USA TODAY
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“We must bring 21st century approaches to 21st centuryproducts and problems. Toxicology is a prime example. Mostof the toxicology tools used for regulatory assessment rely onhigh-dose animal studies and default extrapolationprocedures and have remained relatively unchanged fordecades, despite the scientific revolutions of the past half-century. We need better predictive models to identifyconcerns earlier in the product development process toreduce time and costs. We also need to modernize the toolsused to assess emerging concerns about potential risks fromfood and other product exposures. … With an advanced fieldof regulatory science, new tools, including functionalgenomics, proteomics, metabolomics, high-throughputscreening, and systems biology, can replace currenttoxicology assays with tests that incorporate the mechanisticunderpinnings of disease and of underlying toxic side effects.This should allow the development, validation, andqualification of preclinical and clinical models that acceleratethe evaluation of toxicities during drug development.”
Hamburg, M.A.(2011).Advancingregulatoryscience.Science 331,987
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…and a couple of hundred ways to kill a cell
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Mapping the (finite number of)pathways of toxicity
Annotation to:-Hazard-Toxin (class)-Cell type-Species
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Most chemicals are nottoxic:•90% not acutely toxic•97% not skin corrosive•93% not skin irritant•97% not teratogenic•80-95% not carcinogenic•80% not eye irritating•65% not skin sensitizing
We need to find(concentrationsof) substances,which trigger noPoT
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Five signalling pathways are important during early development
for review see: Scientific frontiersin Developmental Toxicologyand Risk Assessment,Nat. Acad. Press, 2000
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The ReproGlo assay
Luciferase-reporter based detection system
R.E.
Luc-reporterR.E. R.E.
Stable transfection of reporters into mES cells
Fluorescence
0
Test chemical
Alamar blue(cytotoxicity)
(h)4624 48
In collaboration between ECVAM and Prof. M. Schwarz, Tübingen, Germany
Uibel et al. Reprod. Tox. 2010
Correctly identified human reproductive toxicants:
-Lithium chloride-Retinoic acid-Potency of different Valproic acid derivatives-(with metabolizing system) cyclophosphamide
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Office of Research and DevelopmentNational Center for Computational Toxicology
ToxCast Bioactivity Profiling
Bioinformatics/Machine Learning
in silico analysis
Cancer
ReproTox
DevTox
NeuroTox
PulmonaryTox
ImmunoTox
HTS -omics
in vitro testing
$Thousands
Dix et al, Tox Sci 95:5-12 (2007)www.epa.gov/ncct/toxcast
From R. Kavlock, EPA, 2010
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Office of Research and DevelopmentNational Center for Computational Toxicology
Deepwater Horizon
Oil Exploration Platform Explodes April 20, 2010• Estimated 4.9 million barrels of South Louisiana Crudereleased
1.8 million gallons of dispersant used• 1072K surface; 771K subsea• Corexit 9500A (9527 early in spill)
EPA Administrator call for less toxic alternative• Verification of toxicity information on NCP Product Schedule• ORD involvement in assessments of dispersant toxicity
From R. Kavlock, EPA, 2010
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Office of Research and DevelopmentNational Center for Computational Toxicology
Dispersant TranscriptionFactor Profiling
56
Judson et al , ES&T, 2010
Multi-assay pile-upIndication of generalized cell stressPrelude to cytotoxicity
Little specific activity seen except for PXR/ PPAR - consistent with xeno-sensingFrom R. Kavlock, EPA, 2010
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‘Omics’ Image analysis
High content High through-put
Information rich
Bioinformatics &Data-mining
Knowledge onpathways
Mapping ToxomeSystems Toxicology
Robotised / automatedtesting
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Proof of principle: Metabolic profilingand PoT identification in DNT
• DNT (autism, hyperactivity); animal test $1.4million, 1400 animals per substance
• Identify metabolite changes relevant forneurodevelopment with reference compounds
•Agilent thought leader award: LC/MS system
• Integrate with genomics
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!
Principal component analysis of metabolomics
!
lead
TCE
Rat “mini-brains”
PoT identification
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Lessons learned from
-Really took off when therewas private competition-Celera became“acceptable” only whenfeeding into public database
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Omics technologiesBioinformatics
Execution measurementsConcepts & outreach
Failed substancesPredictive modelsResources
Biological samplesPathway expertise
PoToMaC -The Pathways of ToxictyMapping Center
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Starting point: (Pre-)Validated Alternatives
Robust protocols, good cell modelsRegulatory acceptanceAvailable reference substancesThresholds of adversity defined$300 million of research & validation spent
a) 3T3 fibroblasts (acute toxicity, cancer, (phototoxicity)
b) Human artificial skin models (skin irritation and corrosion, genotoxicity, phototoxicity and skin penetration)
c) Human blood monocytes (inflammation, skin sensitization)
d) MCF-7 cells (endocrine disruption)e) HepaRG cells (liver toxicity)f) Human blood lymphocytes
(genotoxicity)
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PoT identification challenges
•Number, variants•Non-linear pathwyas•Combination of varioustechnologies beyondmetabolomics and genomics(e.g. phosphroylation of G-proteins)•Annotation•Validation•Governance of public database
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• Start into 21st CenturyToxicology
• Technological leadership• Accelerated paradigm shift• Substantial parts of the human
Toxome might be “mapable”with rather limited effort
• Scalable approach• Public database with EPA &
NTP under negotiation
• No concept for PoT mapping,validation
• No definition of adversity• Various additional components
needed for use of PoT• Transition from current system
Evidence-based toxicology
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Regulatorytoxicology
The evolution of toxicology
Mechanistictoxicology
In vitro & in silicotoxicology
Animal-basedtoxicology
Evidence-basedtoxicology
Systems toxicology
VAM
academia
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- Since 1974: „The Oxford Database of PerinatalTrials“ (3500 trials; 600 reviews)
- First Cochrane Center in 1992: Oxford, UK
- Cochrane Collaboration founded in 1993
- Today: a world-wide network of about 27.000scientists, physicians, ... About 5.000 reviews
- US Cochrane Center at Johns Hopkins
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Evidence-based Toxicology“Evidence-based medicine goes toxicology!”
Hoffmann and Hartung “Toward an evidence-based toxicology”,Human Exp. Tox., 2006
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Method
assess‐ment
Method
assess‐ment
Meta‐analysisof
studies
Meta‐analysisof
studies
EBM,mechanisticaltoxicology,biostatistics,validation
EBM,mechanisticaltoxicology,biostatistics,validation
EBTEBT
Causa‐tionofhealtheffects
Causa‐tionofhealtheffects
Clinicaltox.Clinicaltox.
What we lack:o Datao Information portalo Meta-analysis & WoE toolso Quality scoring toolso Probabilistic risk assessment
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Assessment tool for the quality of toxicological data Categorizes quality according to Klimisch scores Independent, but largely similar tools for in vivo and
in vitro data/studies Expert advisory group 2 rater experiments:
11 rater are applying the draft tool to 11 in vitro andin vivo studies
Tool now available on the ECVAM website published Schneider et al.
Tox Letters 2009, 189:138-144 Impact for existing data for REACH
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EBT Collaboration Thursday, March 10, 2011 Washington DC Convention CenterSteering Committee (and their organizations for identification)Melvin Andersen, The Hamner InstituteRichard Becker, Am. Chemical CouncilKim Boekelheide, Brown UniversityRobert Chapin, PfizerRodger Curren, IIVSSuzanne Fitzpatrick, US FDAJack Fowle, US EPAAlan Goldberg, JHU CAATThomas Hartung, JHU CAAT
Michael Holsapple, ILSI/HESIWendolyn Jones, CropLife AmericaRichard Judson, US EPAFran Kruszewski, American Cleaning InstituteMartin Stephens, Humane Society of the USBill Stokes, National Toxicology ProgramRaymond Tice, National Toxicology ProgramMark Vossenaar, AgilentNeil Wilcox, FDAJoanne Zurlo, JHU CAAT
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Definition of Validation
NewTEST METHOD
REFERENCE(TEST)
Reliability(reproducibility)
Relevance:scientific basis
Relevance:predictive capacity
✔!!!
✗?
ScientificKnowledge:- PoT- MoA
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The challenge to Tox-21c will be to steertoward quality control without the creationof obstacles by formal validation. A balancebetween precaution and innovation isnecessary, and this requires informeddecisions by the actors in the regulatoryarena. EBM has shown how the informeddecision process in clinical medicine can beserved. EBT promises to be its translationfor an informed decision process in riskassessment.
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Johns Hopkins is the right environment for EBT
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t4 - The Transatlantic Think-Tank of Toxicology
Konstanz
BaltimoreUtrecht
• Systematic reviews (evidence-based tox.)• Cost-benefit analyses• Workshops (reports)
ALTEXAltWeb
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NothingNothing isis asas powerfulpowerful asas an idea an idea
whosewhose time time hashas come. come.
Victor HugoVictor Hugo