The Use of New Toxicological Tools within a Chemical Risk...

NAS Green Chemistry Workshop, 20-21 September 2011, Washington DC

The Use of New Toxicological Tools within a Chemical Risk Assessment Framework: An EU

PerspectiveApplying 21st Century Toxicology to Green Chemical and Material Design

National Academy of Sciences Workshop 20

European Commission’s Joint Research Centre

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The Use of New Toxicological Tools within a Chemical Risk Assessment Framework: An EU

PerspectiveCentury Toxicology to Green Chemical and Material Design

National Academy of Sciences Workshop 20 -21 September 2011Sharon Munn

Systems ToxicologyInstitute of Health and Consumer Protection

European Commission’s Joint Research CentreIspra, Italy


OUTLINE• Introduction to EC’s Joint Research Centre

what we do • Regulatory landscape – setting the scene• Emerging technologies and application to more predictive risk

assessment• Use Cases

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OUTLINEIntroduction to EC’s Joint Research Centre - who we are and

setting the sceneEmerging technologies and application to more predictive risk


As a Directorate-General of the European Commission,the Joint Research Centre provides customer-driven scientific and technical support to Community policy making

Supporting citizen’s security, healthand environmental protection, safety of food and chemicals, alternative energies, nuclear safety, econometrics, prospective technologies…

JRC - Robust Science for Policy Making3

Robust Science for Policy Making


IHCP supporting EU legislation involving Risk Assessment- Chemicals, REACH&CLP (DG Enterprise & Industry, DG Environment, ECHA)

- Cosmetics (DG Health & Consumers, DG Enterprise & Industry )

- Biocides (DG Environment, DG Health & Consumers)

- Plant Protection Products (DG Environment,

- Food Safety, inc. GM, Novel (DG Health & Consumers, EFSA)

- General Consumer Product Safety

Cross-cutting legislation and issues- Protection of Animals used for Scientific Purposes. - Safety of Nanomaterials.

- Identification of Endocrine Active/Disrupting (ED) chemicals.- Combination effects of chemical mixtures (CEMC)

Policy Support4

IHCP supporting EU legislation involving Risk Assessment, REACH&CLP (DG Enterprise & Industry, DG Environment, ECHA)

(DG Health & Consumers, DG Enterprise & Industry )

(DG Environment, DG Health & Consumers)

(DG Environment, DG Health & Consumers)

, inc. GM, Novel (DG Health & Consumers, EFSA)

General Consumer Product Safety (DG Health & Consumers)

cutting legislation and issuesused for Scientific Purposes.

Endocrine Active/Disrupting (ED) chemicals.Combination effects of chemical mixtures (CEMC)

Policy Support


INTERNATIONAL COLLABORATIONS• Agreement signed between IHCP and NCCT of the US Environmental

Protection Agency, to facilitate exchange of research results and materials in relation to the ToxCast programme

• TOX21 Consortium• OECD QSAR Tool Box• OECD Molec Screening & Toxicogenomics• WHO/IPCS MoA framework • ILSI/HESI Risk 21• EPA NexGen• Global Risk Assessment dialogue

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INTERNATIONAL COLLABORATIONSAgreement signed between IHCP and NCCT of the US Environmental Protection Agency, to facilitate exchange of research results and materials in relation to the ToxCast programme

OECD Molec Screening & Toxicogenomics


Expert judgment of Weight of evidence

Animal model

DEFAULT: hazardous property relevant to humans (qualitative aspect) and humans at least as sensitive as animal (quantitative aspect),

Account for variability and uncertainty in animal model with additional factors

REGULATORY TOXICOLOGY 6

Expert judgment of Weight of evidence

Human

DEFAULT: hazardous property relevant to humans (qualitative aspect) and humans at least as sensitive as animal (quantitative aspect),

Account for variability and uncertainty in animal model with additional factors

REGULATORY TOXICOLOGY - CURRENT MODEL


WHY CHANGE?• Potential benefits?

– Ethical - reduce reliance on in vivo– Regulators need faster cheaper methods to screen many chemicals

already in the environment to identify the bad actors which require risk management –

– Product designers need {same}many chemicals to deselect bad actors from product development and prevent entry into the environment

– More predictive/accurate risk assessment from bettter understanding of why chemicals are toxic

Undesirable scenario

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WHY CHANGE?

in vivo testing in animals. Regulators need faster cheaper methods to screen many chemicals already in the environment to identify the bad actors which require

{same} faster cheaper methods to screen many chemicals to deselect bad actors from product development and prevent entry into the environment -More predictive/accurate risk assessment from bettter understanding

Undesirable scenario

Desirable scenario


Regulatory Drivers: Challenges and Opportunities• DIRECTIVE 2010/63/EU OF THE EUROPEAN PARLIAMENT

AND OF THE COUNCIL of 22 September 2010 on the protection of animals used for scientific purposes (replacing 86/609/EEC)– Chapter V. Avoidance of duplication and alternative approaches– 3Rs: replacement, reduction, refinement– EU Reference Lab (ECVAM) to coordinate and promote

development and use of alternatives

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Regulatory Drivers: Challenges and OpportunitiesDIRECTIVE 2010/63/EU OF THE EUROPEAN PARLIAMENT AND OF THE COUNCIL of 22 September 2010 on the protection of animals used for scientific purposes (replacing

Chapter V. Avoidance of duplication and alternative approaches3Rs: replacement, reduction, refinementEU Reference Lab (ECVAM) to coordinate and promote development and use of alternatives


Strong regulatory incentive in product legislation

Ø Directive on Cosmetic products 76/768marketing. Following provisions introducedØ A testing ban of finished cosmetic productsØ A testing ban on ingredients or combinationsØ A marketing ban since March 2009, it isand their ingredients which have been testedproducts.

ØExtension of deadline to MarchØRepeated dose toxicity (carcinogenicityØReproductive toxicityØToxicokinetics

Regulatory Drivers: Challenges and Opportunities 9

Strong regulatory incentive in product legislation

768/EEC introduces a ban on testing andintroduced by Dir. 2003/15/EC

products since September 2004combinations of ingredients since March 2009.

prohibited in the EU to market cosmetic productstested on animals, irrespective of the origin of these

March 2013 for complex endpoints(carcinogenicity and skin sensitisation)

Regulatory Drivers: Challenges and Opportunities - Cosmetics


2013 MARKETING BAN

State of the Art Review"Alternative (non-animal) methods for cosmetics testing: current status and future prospects—2010" in Archives of Toxicology: Vol 85, (5) (2011), p367 http://ec.europa.eu/consumers/sectors/cosmetics/files/pdf/animal_testing/final_report_at_en.pdf• MAIN FINDINGS

–Advanced methods and approaches hold a lot of promise for the future development of more predictive risk assessment, based on improved understanding of how toxic substances reach the target cells/organs (toxicokinetics) and perturb critical biological pathways.–The central importance of toxicokinetics is underlined

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2013 MARKETING BAN – COSMETICS

animal) methods for cosmetics testing: current status and 2010" in Archives of Toxicology: Vol 85, (5) (2011), p367

http://ec.europa.eu/consumers/sectors/cosmetics/files/pdf/animal_testing/final

Advanced methods and approaches hold a lot of promise for the future development of more predictive risk assessment, based on improved understanding of how toxic substances reach the target cells/organs (toxicokinetics) and perturb critical biological pathways.

The central importance of toxicokinetics is underlined




Regulatory Drivers: Challenges and Opportunities

Strong regulatory incentives introduced in product legislation• REACH (EC1907/2006) introduces the 3Rs principle by

avoidance of duplication of testingconcept of generating information by means other than the standard tests in the testing annexes (sostandard testing regime)– REACH Guidance on ITS and use of QSARs (2008)

http://guidance.echa.europa.eu/docs/guidance_document/information_requirements_en.htm

– The use of alternatives to testing on animals for the REACH Regulation 2011http://echa.europa.eu/publications/alternatives_test_animals_2011_en.asp

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Regulatory Drivers: Challenges and Opportunities - REACH

Strong regulatory incentives introduced in product legislationintroduces the 3Rs principle by

avoidance of duplication of testing and introducing the concept of generating information by means other than the standard tests in the testing annexes (so-called ‘adaptation’ of

REACH Guidance on ITS and use of QSARs (2008) http://guidance.echa.europa.eu/docs/guidance_document/information

The use of alternatives to testing on animals for the REACH

http://echa.europa.eu/publications/alternatives_test_animals_2011_e

http://guidance.echa.europa.eu/docs/guidance_document/information


http://guidance.echa.europa.eu/docs/guidance_document/information



Plant Protection Products (Regulation 1107/2009)• active substances only approved if free of ED properties or exposure negligible.• by end of 2013, the EC (DG ENV) must present criteria for evaluation of substances to

identify possible ED properties.

REACH (Regulation 1907/2006)• information on endocrine disrupting (ED) properties might be asked by the competent Authorities

during substance evaluation. • possible inclusion of substances in Annex XIV if weight of evidence shows that it has

properties (similar to CMR- and PB-type concerns)

Biocides (Regulation COM(2009)267, revision)• active substances with ED properties "on the basis of …. Community or internationally agreed

test guidelines" are possible candidates for substitution, may lose lowmore complete risk characterisation.

EDCs and legislation12

Plant Protection Products (Regulation 1107/2009)active substances only approved if free of ED properties or exposure negligible.by end of 2013, the EC (DG ENV) must present criteria for evaluation of substances to

information on endocrine disrupting (ED) properties might be asked by the competent Authorities

possible inclusion of substances in Annex XIV if weight of evidence shows that it has ED type concerns)

Biocides (Regulation COM(2009)267, revision)"on the basis of …. Community or internationally agreed

are possible candidates for substitution, may lose low-risk status, might require

EDCs and legislation


Political mandate to take action (2009)• Council of EU MS Environment Ministers invited the European Commission

(EC) "to consider appropriate modifications, guidelines and assessment methods, and report back to the Council by early 2012 at the latest".

State of the science• State of the Art Report on Mixture Toxicity presented in June 2010

conclude that sufficient information is available to start developing technical guidelines which could be applied across the different pieces of EU legislation.

Scientific opinion• Scientific Committees supporting DG Health and Consumers (DG SANCO)

expected to issue their views and recommendations shortly.

Political impetus • Danish Environment Minister - "Policy options for assessing the combined

effects of groups of substances [..] will be among Denmark's environmental policy priorities during its presidency of the EU in the first half of 2012".

CEMCs and legislation13

Political mandate to take action (2009)Council of EU MS Environment Ministers invited the European Commission (EC) "to consider appropriate modifications, guidelines and assessment methods, and report back to the Council by early 2012 at the latest".

State of the Art Report on Mixture Toxicity presented in June 2010 - DG ENV conclude that sufficient information is available to start developing technical guidelines which could be applied across the different pieces of EU legislation.

Scientific Committees supporting DG Health and Consumers (DG SANCO) expected to issue their views and recommendations shortly.

"Policy options for assessing the combined effects of groups of substances [..] will be among Denmark's environmental policy priorities during its presidency of the EU in the first half of 2012".

CEMCs and legislation


SCREENING AND PRIORITISATION • Situations where animal data not mandatory or impractical, need

predictive screening tools to identify potential bad actors [PBTs, vPvBs, CMRs, EDs]. – Prioritisation of water contaminants under Water Framework Directive– Asssessment of pesticide metabolites in food or feed– Prioritisation of lower tonnage substances for evaluation in REACH

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SCREENING AND PRIORITISATION –USE CASESSituations where animal data not mandatory or impractical, need predictive screening tools to identify potential bad actors [PBTs,

Prioritisation of water contaminants under Water Framework DirectiveAsssessment of pesticide metabolites in food or feedPrioritisation of lower tonnage substances for evaluation in REACH


WHAT’S NEW

Emerging technologies and application to more predictive risk assessment

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WHAT’S NEW

Emerging technologies and application to more predictive risk assessment


Computational Toxicology

Automation of in vitro methods

HTS/HCA

3D in vitrotissue models

PBBK Models

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EMERGING TECHNOLOGIES

Human stem cells

genomics, proteomics,

metabonomics

tissue models

PBBK Models


Strategy - Hypothesis-driven MoA– Purpose driven approach to design of test systems based on mode of

action of chemicals– Identify critical events in mode of action upstream from apical

endpoint – Develop methods (in vitro) for measuring such critical events– Form chemical categories based on structure

activity relationships– Provide data for modelling, grouping and prediction. – Use of in vitro/in silico/in vivo in combination – Extrapolate from in vitro dose concentration response to in vivo dose

response

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driven MoA-based approachPurpose driven approach to design of test systems based on mode of

Identify critical events in mode of action upstream from apical

Develop methods (in vitro) for measuring such critical eventsForm chemical categories based on structure-activity and activity-

Provide data for modelling, grouping and prediction. Use of in vitro/in silico/in vivo in combination – learning feedbackExtrapolate from in vitro dose concentration response to in vivo dose


Biochemical / Metabolic PathwaysCourtesy of Roche

Disturbance

Toxicological effect

Systemic toxicity deals with a complex system

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Systemic toxicity deals with a complex system


MechanismsS.

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Mechanisms in Drug-Induced Hepatotoxicity. Russmann, Current Med. Chemistry, 2009, 16, 3041-3053


Applying MOA-based approach in the laboratory

• An in vitro testing strategy to identifyhepatotoxic in humans and to associate(MoA) categories.– Using HepaRG metabolically competent– High Content Analysis (HCA) using

number of parameters at the same– Categorize chemicals according to– Comparison between in vitro and

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based approach in the laboratory

identify chemicals that are potentiallyassociate them with specific Mode-of-Action

competent cell lineusing automated cell imaging to measure

same timeto their responses

and in vivo data


Cell Count ROS Intensity

Nuclear Intensity

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9

4

8

11 4Not Toxic

Cell Count ROS Intensity

Nuclear Intensity

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9

4

8

11 4Not Toxic

Cell culturing and differentiation

Data AnalysisChemicals

categorization

In vitro Workflow21

Cell treatment: HTS (92 chemicals, qHTS format, 16 conc, 1:2 dil.)

Endpoints Measurement: HCS(cell loss, DNA content, steatosis, ROS, caspase3, cyt c)

In vitro Workflow


Neuronal Electrophysiology

• Microelectrode arrays– Measuring electrical activity in neuronal cells– Rat embryonic cortical neurons– Measure effects at sub-cytotoxic concentrations in specialised

cells– Functional assay based on a “living sample” which can be

monitored over time (low dose chronic toxicity studies).– Combinations of chemicals -– Network of communicating cells not amenable to automation

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Neuronal Electrophysiology - “the in vitro EEG”

Measuring electrical activity in neuronal cellsRat embryonic cortical neurons

cytotoxic concentrations in specialised

Functional assay based on a “living sample” which can be monitored over time (low dose chronic toxicity studies).

- Mixture effectsNetwork of communicating cells not amenable to automation


MEA chipNeuronal Electrophysiology- “the in vitro EEG”

Analysis

firing rate

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MEA chip

Signal acquisition

Signal processing



ChemicalReactivity

Mathematical Modelling

Complex Networks

( )

−⋅+

−⋅= i

ii

ii

tAtAtI21

expexp 21 ττ

StructureActivity/Prop.

Relations

Data & Info Management

Biophysics & PBBK

Biostatistics

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Structure-Activity/Prop.

Relations

Exploit existing data !

Read AcrossApplicability

Domains

Chemical Categories

Data & Info Management


Human bioconcentration factor

A. Tonnelier, S. Coecke, J-M Zaldívar, Screening of chemicals for human bioaccumulation potential with a physiologically based toxicokinetic model;

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Human bioconcentration factor

M Zaldívar, Screening of chemicals for human bioaccumulation potential with a physiologically based toxicokinetic model; submitted

NAS Green Chemistry Workshop, 20-21 September 2011, Washington DC 26


Repeated dose toxicity

Acrylamide, fibroblasts (3T3), NRU, repeat

“Towards the replacement of repeated dose systemic toxicity testing on animals”

6 projects, 70 institutions, 5 years,1 coord action, 50 MEuro, public

“Safety Assessment Ultimately Replacing Animal Testing”

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Repeated dose toxicity

Acrylamide, fibroblasts (3T3), NRU, repeat-dose, every 24h for 96h

“Towards the replacement of repeated dose systemic toxicity testing on animals”

6 projects, 70 institutions, 5 years,1 coord action, 50 MEuro, public-private partnership

“Safety Assessment Ultimately Replacing Animal Testing”


“Safety Evaluation Ultimately Replacing Animal Testing” SEURAT

The SEURAT strategy is to adopt a toxicological modeto describe how any substance may adversely affect human health, and to use this knowledge to develop complimentary theoretical, computational and experimental (in vitro) models that predict quantitative points of departure needed for safety assessment.

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“Safety Evaluation Ultimately Replacing Animal Testing” SEURAT

The SEURAT strategy is to adopt a toxicological mode-of-action framework to describe how any substance may adversely affect human health, and to use this knowledge to develop complimentary theoretical, computational and experimental (in vitro) models that predict quantitative points of departure needed for safety assessment.


The Building Blocks of SEURAT

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Stem cell differentiation for providing humanspecific target cells

Identification and investigation of human biomarkers

Delivery of computational tools to predict the effects of chemicals based on silico calculations and estimation techniques

Development of systems biological tools for organotypic human cell cultures

Supporting integrated data analysis and servicing of alternative testing methods in toxicologyCluster level Coordinating and Support Action

Development of a hepatic microfluidic bioreactor

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The Building Blocks of SEURAT-1

[email protected]

Stem cell differentiation for providing human-based organ

Identification and investigation of human biomarkers

Delivery of computational tools to predict the effects of chemicals based on in calculations and estimation techniques

Development of systems biological tools for organotypic human cell

Supporting integrated data analysis and servicing of alternative testing

Cluster level Coordinating and Support Action

Development of a hepatic microfluidic bioreactor

mailto:[email protected]


VALIDATION

• Validation– an intrinsic part of the scientific process– needs to be ‘fit for purpose’’ depending on how data will

ultimately be used - screening, weight of evidence, or critical data in regulatory decision

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VALIDATION

an intrinsic part of the scientific processneeds to be ‘fit for purpose’’ depending on how data will

screening, weight of evidence, or critical data in regulatory decision-making


Test definition

Within-lab reproducibility

Transferability

Between-lab reproducibility

Predictive capacity

Applicability domain

Performance standards

VALIDATION

ECVAM Modular Approach31

Reliability

Relevance

Validation Managem

ent

Independent Peer Review

VALIDATION

ECVAM Modular Approach


Use of automation in validation

NRU 3T3 cytotoxicity assay- first example of formal ECVAM validation activity involving an automated assay

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Use of automation in validation


ICCVAM/NICEATM Coordinated Study• Reporter gene assay - ER binding.• Agonist and antagonist formats.• 78 chemicals in test set.• 3 test labs (manual) inc. ECVAM.• Peer review March 2011.

“LUMI-CELL” Automation

• qHTS format with 96-well plates.• 16 plates/concentrations, DL of 2. • 44 chemicals per plate, 2 replicates.• 3 biological repeats.• pos/neg control, fixed conc, 4 replicates.• Acceptance and normalisation criteria needed adapting.

Automation at IHCP

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ICCVAM/NICEATM Coordinated Study

C1

C16

C2

CELL” Automation

Acceptance and normalisation criteria needed adapting.


HARMONISED PLATFORM FOR DATA EXCHANGE

OECD harmonised template 201• IHCP in collaboration with OECD and ECHA are now

developing a new harmonised template to capture in vitro/in silico data as ‘íntermediate effects’ within a toxicity pathway/mode of action framework

• Building a taxonomy of intermediate effects which may ultimately be identified as key events in a mode of action/adverse outcome pathway

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HARMONISED PLATFORM FOR DATA EXCHANGE

IHCP in collaboration with OECD and ECHA are now developing a new harmonised template to capture in vitro/in silico data as ‘íntermediate effects’ within a toxicity pathway/mode of action frameworkBuilding a taxonomy of intermediate effects which may ultimately be identified as key events in a mode of action/adverse outcome pathway


USE CASES IN HAZARD ASSESSMENT• A model-based prioritisation exercise for the European Water Framework

Directive. • Computational models for prediction of nanoparticle toxicity.

– Development of computational models to predict the behaviour of nanomaterials in biological systems to allow researchers to streamline and prioritise the toxicological testing of nanomaterials. http://ihcp.jrc.ec.europa.eu/our_labs/computational_toxicology/computationalpredict-nanoparticle-toxicity

• Prospects for the use of computational methods in the toxicological assessment of chemicals in food. – JRC update of results obtained in the PESTISAR project, performed during 2009

the EFSA.• A framework for assessing in silico toxicity predictions: Case studies with

selected pesticides. http://ihcp.jrc.ec.europa.eu/our_labs/computational_toxicology/publications

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USE CASES IN HAZARD ASSESSMENTbased prioritisation exercise for the European Water Framework

Computational models for prediction of nanoparticle toxicity. Development of computational models to predict the behaviour of nanomaterials in biological systems to allow researchers to streamline and prioritise the toxicological testing of

http://ihcp.jrc.ec.europa.eu/our_labs/computational_toxicology/computational-models-

Prospects for the use of computational methods in the toxicological assessment

JRC update of results obtained in the PESTISAR project, performed during 2009-2010 for

toxicity predictions: Case studies with

http://ihcp.jrc.ec.europa.eu/our_labs/computational_toxicology/publications

http://ihcp.jrc.ec.europa.eu/our_labs/computational_toxicology/computational


http://ihcp.jrc.ec.europa.eu/our_labs/computational_toxicology/computational



Hazard:• Persistence(BIOWIN, BIOHCWIN and OECD Povand LRTP screening tool)• Bioaccumulation(EPI BCFBAF, CAESARBioaccumulation, JRC QSAR BCF)• Toxicity(3 ADMET QSAR Toxicity models)

Risk Ratio =

RISK

Daginnus, K. et al, Int. J. Environ. Res. Public Health

Use of computational methods to evaluate over 2000 substances for the European Water Framework Directive

Identification of Priority Hazardous Substances36

Exposure:• EU production level(IUCLID and SPIN databases)• Use(IUCLID database)•Environmental data/Multimedia modelling(ECETOC TRA tool and OECD Pov and LRTP screening tool)

Risk Ratio = PECPNEC

RISK

Daginnus, K. et al, Int. J. Environ. Res. Public Health 2011, 8, 435-455

Use of computational methods to evaluate over 2000 substances for the European Water Framework Directive

Identification of Priority Hazardous Substances


European Partnership for Alternative Approaches to Animal Testing• Public/Private partnership between the European Commission,

European trade associations, and companies from seven industry sectors. – The partners are committed to pooling knowledge and resources to accelerate the

development, validation and acceptance of alternative approaches to further the replacement, reduction and refinement (3Rs) of animal use in regulatory testing.

Lead theme for 2011 "Integrated Testing Strategies and their impact on the 3Rs"

• Integrated Testing Strategies for more scienceanimal-reduction approaches.

http://ec.europa.eu/enterprise/epaa/index_en.htm

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European Partnership for Alternative Approaches to Animal TestingPublic/Private partnership between the European Commission, European trade associations, and companies from seven industry

The partners are committed to pooling knowledge and resources to accelerate the development, validation and acceptance of alternative approaches to further the replacement, reduction and refinement (3Rs) of animal use in regulatory testing.

Lead theme for 2011 "Integrated Testing Strategies and their impact on the

Integrated Testing Strategies for more science-based, faster throughput and





CONCLUDING REMARKS• Strong regulatory drivers to find alternative approaches less

reliant on animals• Adopt toxicological mode of action framework as a strategy

within which to apply new knowledge and tools• Recognise importance of reliability and relevance of results• Apply in vitro/in silico/in vivo in integrated manner• Increased interaction between disciplines but also industry cross

sector sharing of ideas and data• Establishment of in vitro to in vivo extrapolation models

importance of toxicokinetics

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CONCLUDING REMARKSStrong regulatory drivers to find alternative approaches less

Adopt toxicological mode of action framework as a strategy within which to apply new knowledge and toolsRecognise importance of reliability and relevance of resultsApply in vitro/in silico/in vivo in integrated mannerIncreased interaction between disciplines but also industry cross-sector sharing of ideas and dataEstablishment of in vitro to in vivo extrapolation models –


Thank you

[email protected]

http://ihcp.jrc.ec.europa.eu/

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mailto:[email protected]

http://ihcp.jrc.ec.europa.eu/

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