Adverse Outcome Pathways: A Framework for Organizing Mechanistic Information to

Improve Chemical Assessment

Kristie Sullivan, MPH

Director, Toxicology and Regulatory Testing Issues

OUTLINE

• About Physicians Committee

• Why AOPs?

• OECD AOP Programme

• Case Examples

• Application to Tobacco Chemicals

• How to Get Involved

• Physician-founded in 1985

• Aims to increase scientific and ethical standards for medical

education and research, and improve the public health

• Toxicology: modernize and improve

TOXICITY TESTING REVOLUTION

Toxicity Testing Revolution

5

TOXICITY TESTING REVOLUTION

Evolution of the Revolution

7

AOP: Organizational Framework

How are AOPs Used?

• Organize available evidence into context

• Link mechanistic data to adverse outcomes

• Highlight research needs

• Mechanistic support for chemical grouping and read-

across to fill data gaps

• Put in vitro assays into regulatory context

• Create testing strategies/frameworks (IATA)

• Identify assay gaps/needs

9

• Harmonized test guidelines and docs by >30 member countries

• Foundation for chemical characterization & tool support

– QSARs

– Test Guidelines

– Integrated Approaches to Testing and Assessment

• Expert group to develop, guide, and promote: Extended Advisory

Group for Molecular Screening and Toxicogenomics (EAGMST)

10

AOP Elements

ADVERSE OUTCOME

KEY EVENTS

11

KEY EVENT RELATIONSHIPS

• AOP-Knowledgebase to collect information and allow

collaboration

• International platform for review and publication

• Guidance Document and User’s Handbook

http://www.oecd.org/chemicalsafety/testing/adverse-

outcome-pathways-molecular-screening-and-

toxicogenomics.htm12

Third party Applications,

plugins

AOP-KBHub

Shared chemical, biological and toxicological ontologies

AOP-KB

Intermediate Effects DBPut

chemical-related AOP components

in a regulatory context

AOP XplorerVisualize attribute networks to

discover & explore AOPs in a broader

context

EffectopediaDetailed development of

structured & computational AOPs

AOP WikiCollaborative development of AOP

descriptions & evidence

Slide: Clemens Whittwher, JRC

AOPKB.orgAOPWIKI.org

AOP Page

Section 1 - Title

Section 4 – Abstract

Section 5a – Summary of the AOP

MIE

KEs

AO

Key Event Relationships

Section 6 – Scientific evidence supporting the linkages in the AOP

Applicability domain(s) of the AOP

Life-stage Taxonomic

Sex

Section 7 – Overall Assessment of the AOP

Modified Bradford Hill Considerations

KE Pages

KER Pages

MIE Page

AO Page

• Description • Measurement/

detection • Taxonomic

applicability

• Description • Measurement/

detection • Taxonomic

applicability • Evidence for

chemical initiation

Chemical initiator(s)

• Description • Measurement/

detection • Taxonomic

applicability • Regulatory relevance

Section 5b – MIE, KE, and AO descriptions Figure 2. Overview of the organization of content pages in the AOP-wiki relative to sections of the AOP template. Sections 1, 4, 5a, and 7 are found on the main page for an individual AOP. Information related to sections 5b and section 6 are entered into separate content pages that can be linked to multiple individual AOP pages.

• Title • Description • Biological plausibility • Empirical support • Inconsistencies and

uncertainties • Quantitative

understanding

Linkage table

AOP Wiki 14

Slide courtesy of Steve Edwards, EPA

15Slide: Anne Gourmelon, OECD

PROPOSAL

CREATION

REVIEW

STATIC VERSION

16Slide: Anne Gourmelon, OECD

ENDORSEMENT AND PUBLICATION

APPLICATION

How Are AOPs Developed?

Open, stakeholder-inclusive effort

• Expert workshop setting

• By leading experts over time*

• One main team or lab

• “Crowd-sourced”

• Computationally-predicted17

How Are AOPs Developed?

18

MIEAdverse

OutcomeKey Event Key Event Key Event

MIEAdverse

Outcome

How Are AOPs Developed?

AOP elements are interoperable and form networks

Chemical 1 MIE1 KE KE AO1

Pathway networks

• Vulnerability analysis• Toxicity of mixtures• Alternative test prioritisation

KE

Chemical 2 MIE2 KE AO2

KE

Chemical 3 MIE3

KEX

KE AO3

Chemical 4 MIE4 KEY AO4KE

Chemical 5 MIE5 KE KE AO5KE

Chemical2

Chemical4

Chemical5

concentration Slide: Hristo Aladjov and Joop DeKnecht, OECD

Authors: Malgorzata Nepelska, Sharon Munn, Brigitte Landesmann; Systems Toxicology Unit, Joint Research Centre, European Commission

AOP Wiki

AOP Wiki

AOP Wiki

AOP Wiki

How are AOPs Developed?

• Computationally-predicted

– Automated literature searches

– Data-base mining

– High-content or high-throughput data sets

• Project 1.29: A catalog of putative AOPs that will enhance the

utility of US EPA Toxcast high throughput screening data for

hazard identification

How Are AOPs Developed?

• OECD Guidance:

– Guidance document on developing and assessing adverse

outcome pathways (#184) + Handbook

• Villeneuve et al, Adverse Outcome Pathway (AOP)

Development I: Strategies and Principles, Tox Sci 142(2),

2014, 312–320

• Villeneuve et al, Adverse Outcome Pathway Development

II: Best Practices, Tox Sci, 142(2), 2014, 321–33027

Sensitization of the Respiratory Tract (Project 1.20)

• Occupational Asthma

– Acute and chronic symptoms of upper and lower

airways

– Sensitization and elicitation phase

– Sensitization can occur via dermal exposure

• Focus: LMW organic compounds28

Sensitization of the Respiratory Tract AOP Project

Kent Carlson, CPSC

Stella Cochrane, Unilever

Steve Enoch, LJMU

Janine Ezendam, RIVM

Ian Kimber, U of Manchester

Grace Patlewicz, EPA

Erwin Roggen, Novozymes

Katherina Sewald, Fraun. ITEM

Kristie Sullivan, PCRM

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Sensitization of the Respiratory Tract AOP Project

• Motivations

– Regulatory need for assays to detect and

distinguish respiratory sensitizers

– Mechanisms not as well understood

– Support development of in silico and in vitro TM

30

Initial Issues

• Sparse literature for LMW RS-chemicals

• Literature available for related, but separate

pathways

– Skin Sensitization

– Metal complexes (e.g., chloroplatinates)

– Proteins

• Defining the Adverse Outcome

• Defining elements cohesively with skin AOP31

Cellular Danger Signals: Activation

of Inflammatory Cytokines and

Chemokines and Cytoprotective

Gene Pathways (Th2)

MIE: Covalent Binding to Lysine Residues on Proteins

T-cell Activation-Proliferation-Polarization (Th2)

LMW Organic Chemical Exposure

AO: Sensitisation of the Respiratory Tract and Allergic Asthma upon Challenge

Dendritic Cell Activation (Th2

Skewed) and Migration

33

Naive dendritic cell

Matured dendritic cell

RecognitionUptake

Processing of Antigen

SIGNAL 1

Naive T-cell

DC-Th2 cellinteraction

SIGNAL 2DANGER SIGNAL

Matured Thcell

SIGNAL 3

T-cell proliferation

B-cell activation

IL-4, IL-5, IL-13

IgE

Mast cells

Protein

LMW agent

EAR

Local antigen presentation to

effector cellsEosinophils

Effector T-cells

LAR

Histamine

Protein

LMW agent

Hapten-carriercomplex MIE

KE 2KE 3a

KE 3b

KE 4

Adverse Outcome

Slide: Katherina Sewald and Janine Ezendam

Evaluating the AOP• Biological Plausibility of KERs: Moderate

• Essentiality of KEs: Moderate

– Some blocking experiments for KEs 2 and 3

• Empirical Support for KERs: Moderate

– Preponderance of evidence supportive

– Mechanistic studies in the literature are confined to one or

a few hallmark sensitizers

– Temporality and quantitative information has been

pursued in only a few studies34

Research Gaps

• Dendritic cell polarization and T-cell effector

response differs from skin sensitization—why?

• There is some indication that binding site and

protein preference sets Th2 response into motion

• Quantitative/Potency considerations

• Individual variation in attainment of AO

35

36

Cellular Danger

Signals

Covalent

Binding to

ProteinsT-cell

Activation-

Proliferation-

Polarization

Chemical

Exposure

AO:

Sensitisation

of the

Respiratory

Tract

Dendritic Cell

Activation and

Migration

QSAR &

structural alerts

Epithelial cells, Tissues

[cytokine release, gene

expression, oxidative stress]

Peptide

Reactivity

[Lys/Cys

depletion ratio]

Dendritic cell-

based assays

[surface

markers,

cytokines,

gene

expression/pr

ofiling]

In vivo assays: T-

cell proliferation,

IgE,

hypersensitivity

response

Cellular

Danger

Signals:

Nrf2-ARE

pathway

vs. Th2

correlation

Covalent

Binding to

Lysine

residues

Th1 T-

cell

Activatio

n

Skin

sensitiz

-ation

Th2-

skewed

DC

activation

Covalent

Binding to

Cysteine

residues

Resp.

sensitiz

-ation

DC

Migra-

tion

DC

activation

Th2 T-

cell

Activatio

n

MIE KE 2 KE 3 KE 4 AO

Skin and Respiratory AOPs

AOPs for Tobacco Assessment• Project 1.25: The Adverse Outcome Pathway from Induction

of Secretion of Inflammatory Cytokines Leading to Lung

Emphysema

• Smoke / Ingredient exposure-outcome (KERs) information

• Complex adverse outcomes amenable to AOP networks

w/shared key events

• Systems toxicology evidence—and sbv process—can

strengthen existing AOPs and connections

• Highlight research needs

– Illuminate species or genetic differences

– Identify potential susceptible populations

– Generate hypotheses

– Prioritize future research based on essential

missing information

39

AOPs for Tobacco Assessment

• Mechanistic support for “alternative”

approaches to assess products

– Read-across with data from structurally similar

constituents

– Rank constituents based on weight of evidence

– Rank products to support modified risk

– Target constituents for replacement

40

AOPs for Tobacco Assessment

AOPs for Tobacco Assessment

• Create testing strategies/frameworks

• Put in vitro assays into regulatory context

Inflammation &

Oxidative stress

Tissue Destruction

& Remodeling

CiliaryDysfunction

&Ion

Transport Irregularity

Goblet Cell Hyperplasi

a &Mucus

Production

“Adversity” vis a visCOPD

Chemical Stressor(s)

41

Non-governmental roles

• Scientific AOP Development

• Tool development AOP KB

• Education, outreach, and training

• Prizes!

Kristie Sullivan, MPH

ksullivan@pcrm.org

• YouTube: AOP Learning Channel

• http://www.oecd.org/chemicalsafety/testing/adverse-outcome-

pathways-molecular-screening-and-toxicogenomics.htm

• http://aopwiki.org

• www.ASCCTOX.org

Thank you for your attention!

• Willett et al 2014. Pathway-based toxicity: history, current approaches and liver fibrosis and

steatosis as prototypes. ALTEX. doi: 10.14573/altex.1401283.

• Villeneuve et al 2014a. Adverse Outcome Pathway Development I: Strategies and Principles.

Tox Sci. doi: 10.1093/toxsci/kfu199.

• Villeneuve et al 2014b. Adverse Outcome Pathway Development II: Best Practices. Tox Sci.

doi: 10.1093/toxsci/kfu200.

• Adeleye et al 2014. Implementing Toxicity Testing in the 21st Century (TT21C): Making

safety decisions using toxicity pathways, and progress in a prototype risk assessment.

Toxicology. doi: 10.1016/j.tox.2014.02.007.

• Tollefsen et al 2014. Applying Adverse Outcome Pathways to support Integrated

Approaches to Testing and Assessment. Reg Tox Pharm. doi: 10.1016/j.yrtph.2014.09.009.

• Bal-Price et al. 2015. International STakeholder NETwork (ISTNET): creating a

developmental neurotoxicity (DNT) testing road map for regulatory purposes. Arch

Toxicol. doi: 10.1007/s00204-015-1464-2.

• Patlewicz et al 2015. Proposing a scientific confidence framework to help support the

application of adverse outcome pathways for regulatory purposes. Reg Tox Pharm. doi:

10.1016/j.yrtph.2015.02.011.