Progress Made on Tox21:

A Framework for the

Next Generation of Risk Science

Daniel Krewski, PhD, MHA

Professor and Director

McLaughlin Centre for

Population Heath Risk Assessment

&

Risk Sciences International

October 1, 2014

Outline

• NexGen: The next generation of risk science • TT21C: Toxicity testing in the 21st century (NRC, 2007)

• New Risk Assessment Methodologies (NRC, 2009)

• Population heath risk assessment (Krewski et al., 2007;

Chiu et al., 2011)

• Principles of risk management (Krewski et al., 2014)

• Case study prototypes (Krewski et al., 2014)

• Exposure assessment in the 21st Century (NRC,

2013)

• Future perspectives

McLaughlin Centre for Population Health Risk Assessment

McLaughlin Centre for Population Health Risk Assessment

Next generation risk assessment

McLaughlin Centre for Population Health Risk Assessment

Three Cornerstones

• New paradigm for toxicity

testing (TT21C), based on

perturbation of toxicity

pathways

• Advanced risk assessment

methodologies, including

those addressed in Science

and Decisions

• Population health approach:

multiple health determinants

and multiple interventions

McLaughlin Centre for Population Health Risk Assessment

Three Cornerstones

• New paradigm for toxicity

testing (TT21C), based on

perturbation of toxicity

pathways

• Advanced risk assessment

methodologies, including

those addressed in Science

and Decisions

• Population health approach:

multiple health determinants

and multiple interventions

Toxicity Testing in the 21st Century

www.nas.edu

Building the Scientific Toolbox

(Andersen & Krewski, 2009, Tox. Sci)

McLaughlin Centre for Population Health Risk Assessment

Human Toxicology Project Consortium

Hamburg, M. (2011), Science (Editorial). Vol. 335. p. 987

Collins, F.S., Gray, G.M. & Bucher, J.R. (2008),

Science (Policy Forum). Vol. 319. pp. 906 - 907

McLaughlin Centre for Population Health Risk Assessment

Reaction from Experts in Risk Assessment

8 +1 Invited Commentaries 2009-2010

6 Invited Commentaries 2009

Reaction from Experts in Toxicology

International Perspectives

Expert Panel on

the Integrated

Testing of

Pesticides

McLaughlin Centre for Population Health Risk Assessment

Canada-China collaboration

in toxicological risk assessment . . .

McLaughlin Centre for Population Health Risk Assessment Krewski et al. (2011), ARPH

Mapping Toxicity Pathways

McLaughlin Centre for Population Health Risk Assessment

Using Case Studies to implement NRC- TT21C report

1. Select a group of well-studied prototype compounds/pathways

2. Design & ‘validate’ human/rodent cell or tissue surrogate based

toxicity pathway assays

3. Examine results from the panel of assays to assess adversity

4. Refine quantitative risk assessment tools, i.e., computational systems biology pathway (CSBP) models and in vitro-in vivo extrapolation (IVIVE).

5. Integrate results into proposed health safety/risk assessments. Show the entire process in practice.

6. Take a look at results and modify steps to insure the process assist quantitative safety assessment

TT21C: Toxicity Pathway and Network Biology Program at The Hamner http://www.thehamner.org/institutes-centers/institute-for-chemical-safety-sciences/toxicity-testing-in-the-21st-century/

Specific TT21C Pathway Projects at Hamner

Receptor mediated pathways

PPARa – liver responses Estrogen Pathway – uterine and breast tissue responses Aryl-hydrocarbon receptor in liver

Stress Pathways involving chemical reactivity

DNA damage (p53-mdm2) – creating a computational model Oxidative stress (Nrf2-Keap1) Mitochondrial damage

See Mid-year report:

(http://www.thehamner.org/content/hamner_tt21c_midyear_report_2014.pdf)

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.

NIH Transformative Research Project

McLaughlin Centre for Population Health Risk Assessment

Three Cornerstones

• New paradigm for toxicity

testing (TT21C), based on

perturbation of toxicity

pathways

• Advanced risk assessment

methodologies, including

those addressed in Science

and Decisions

• Population health approach:

multiple health determinants

and multiple interventions

KEY MESSAGES

Enhanced framework

Formative focus

Four steps still core

Matching analysis to decisions

Clearer estimates of population risk

Advancing cumulative assessments

People and capacity building

Phase I Formulating and

Scoping Problem

For environmental

condition:

• What’s the

problem?

• What are the

options for altering?

• What assessments

are needed to

evaluate options?

Phase III Risk Management

• Relative benefits of

proposed options?

• How are other factors

(e.g., costs) affected by

options?

• Which option is chosen?

What’s the uncertainty

and justification?

• How to communicate it?

• Should decision

effectiveness be

evaluated? If so, how?

Phase II Planning and

Risk Assessing

Stage 1: Planning for:

• Options Assessment

• Uncertainty and

Variability Analysis

Stage 2: Assessing

Stage 3: Confirming

Utility of Assessment

No

t O

K

OK

Stakeholder involvement at each phase

“Risk-Based Decision-Making” Framework

Methodological Issues

• Adversity

• Variability

• Susceptible populations

• Dose and species extrapolation

• Mixtures and multiple stressors

• Uncertainty analysis

McLaughlin Centre for Population Health Risk Assessment

Issue Current Approach NexGen Approach

Adverse outcomes

Apical outcomes in mammalian systems, or precursors to these outcomes, serve as the basis for risk assessment.

In vitro assays identify critical toxicity pathway perturbations, which serve as the basis for risk assessment, even in the absence of a direct link with an apical outcome.

Methodological Issues: (1) Adversity

.

McLaughlin Centre for Population Health Risk Assessment

Issue Current Approach NexGen Approach

Dose-response

assessment

Empirical or biologically-based models describe apical endpoints, and determine an appropriate point of departure (such as the benchmark dose) for establishing a reference dose.

Computational systems biology pathway models describe dose-response relationships for pathway perturbations, reflecting dose-dependent transitions throughout the dose range of interest.

Methodological Issues: (2) Dose-response

McLaughlin Centre for Population Health Risk Assessment

A WHO/PAHO Collaborating Centre

Signal-to-Noise Crossover Dose (SNCD) Sand, Portier & Krewski (2011), EHP

Issue Current Approach NexGen Approach

Inter-individual variability

Adjustment factors used in establishing reference doses account for inter-individual variability in PK and PD. Variability in exposure is also taken into account.

Variability in biological response is characterized through the use of a diverse range of human cell lines. Dosimetry models link variation in human exposure with corresponding in vitro doses.

Methodological Issues: (3) Variability

From Zeise et al. (2012): “Assessing Human Variability in the

Next Generation Health Assessments of Environmental Chemicals”

Issue Current Approach NexGen Approach

Susceptible populations

Life-stage, genetics, and socioeconomic and lifestyle factors determine susceptible population groups.

Molecular and genetic epidemiology defines susceptible populations in terms of critical pathway perturbations.

Methodological Issues: (4) Susceptibility

How susceptibility arises from variability (from Zeise et al., 2012)

Issue Current Approach NexGen Approach

Dose and

species extrapolation

Dose and species extrapolation translate animal test results to humans.

Cellular assays provide direct measures of toxicity pathway perturbations in humans. IVIVE techniques and pathway modeling calibrate in vitro and in vivo exposures. Sensitive in vitro tests are used to evaluate risk directly at environmental exposure levels.

Methodological Issues: (5) Extrapolation

McLaughlin Centre for Population Health Risk Assessment

Rotroff DM, Wetmore BA, Dix DJ, Ferguson SS, Clewell HJ, Houck KA, Lecluyse EL, Andersen ME, Judson RS, Smith CM, Sochaski MA, Kavlock RJ, Boellmann F, Martin MT, Reif DM, Wambaugh JF, Thomas RS (2010) Incorporating human dosimetry and exposure into high-throughput in vitro toxicity screening. Toxicol Sci 117: 348-358

Issue Current Approach NexGen Approach

Mixtures and

multiple stressors

Common experimental protocols include testing of mixtures and factorial experiments with joint exposures. However, there are only a limited number of such studies because of cost and complexity of experimental design.

Cost-effective high throughput technologies permit expanded testing of mixtures and multiple stressors.

Methodological Issues: (6) Mixtures

Issue Current Approach NexGen Approach

Uncertainty analysis

Uncertainty considerations include species differences in susceptibility, low-dose and route-to-route extrapolation, and exposure ascertainment.

Probabilistic risk assessments characterize overall uncertainty, and identify the most important sources of uncertainty that guide value-of-information decisions.

Methodological Issues: (7) Uncertainty

Adverse Effect

Toxicity Pathway

Key Events

MOA

HTS Assays

Intrinsic

Clearance

Plasma Protein

Binding

PopulationsPK Model

Biological Pathway Activating

Concentration (BPAC)

Probability Distribution

Dose-to-Concentration

Scaling Function (Css/DR)

Probability Distribution

Probability Distribution

for Dose

that Activates

Biological Pathway

BPADL

Pharmacodynamics Pharmacokinetics

Reverse Toxicokinetics (rTK):

in vitro concentration to in vivo dose

McLaughlin Centre for Population Health Risk Assessment

Three Cornerstones

• New paradigm for toxicity

testing (TT21C), based on

perturbation of toxicity

pathways

• Advanced risk assessment

methodologies, including

those addressed in Science

and Decisions

• Population health approach:

multiple health determinants

and multiple interventions

Health Risk Science Determinants and Interactions

Health Risk Policy Analysis Evidence Based Policy

Biology

and

Genetics

Social

and

Behavioural

Biology-social interactions

Environment

and

Occupation

Biology-environment

interactions

Population Health

Environment-social

interactions

Multiple Interventions

Advisory

Regulatory

Economic

Community

Technological

Chiu, W.A., et al., Approaches to advancing quantitative human health risk assessment of environmental chemicals

in the post-genomic era, Toxicol. Appl. Pharmacol. (2010), doi:10.1016/j.taap.2010.03.019

McLaughlin Centre for Population Health Risk Assessment

Social-Environment Interaction

Darby et al. (2005), Radon in homes and risk of lung cancer: collaborative analysis

of individual data from 13 European case-control studies. BMJ 330: 223-226

McLaughlin Centre for Population Health Risk Assessment

Social-Genetic Interaction

McLaughlin Centre for Population Health Risk Assessment

Risk

Management

Risk Management Principles (1/2)

• Beneficence and non-maleficence (do more

good than harm)

• Natural justice (a fair process of decision

making)

• Equity (ensure an equitable distribution of risk)

• Utility (seek optimal use of limited risk

management resources)

• Honesty (be clear on what can and cannot be

done to reduce risk)

McLaughlin Centre for Population Health Risk Assessment

Risk Management Principles (2/2)

• Acceptability of risk (do not impose risks that

are unacceptable to society)

• Precaution (be cautious in the face of

uncertainty)

• Autonomy (foster informed risk decision-making

for all stakeholders)

• Flexibility (continually adapt to new knowledge

and understanding)

• Practicality (the complete elimination of risk is

not possible)

McLaughlin Centre for Population Health Risk Assessment

McLaughlin Centre for Population Health Risk Assessment

Case

Study

Prototypes

NexGen Tiered Approach to Risk Assessment www.epa.gov/risk/nexgen/docs/NexGen-Program-Synopsis.pdf

Tier 1: Screening and Ranking

Judson et al. (2010), EST

Tier 2: Limited Scope Assessment

Traditional versus Toxicogenomics Determination of BMD

Thomas et al. (2012), Mutation Research

Thomas et al. (2012) found a strong correlation between

transcriptional BMDs for specific pathways and traditional BMDs

McLaughlin Centre for Population Health Risk Assessment

• To identify toxicity pathways using

‘omics’ data

• To determine accuracy of

predicting in vivo responses from

in vitro toxicity pathway induction

from toxicants

• To develop a biologically-based

dose-response (BBDR) based on

the integration of diverse data sets

Lung Injury and Ozone

Tier 3: Major Assessment

0.5

1

1.5

2

2.5

3

-4 0 4 8 12 16 20 24

IL8

Fo

ld-C

han

ge

Time (hrs)

IL8 RNA Expression Time Course Relative to Mean Air Value

1 ppm O3

0.75 ppm O3

0.5 ppm O3

0.25 ppm O3

Air

Response time course of IL8 RNA expression, relative to mean air

value. Peak response occurs 3 hours post exposure cessation.

McLaughlin Centre for Population Health Risk Assessment

Lung Injury and Ozone

Conclusion

NF-κB signaling is seen early

during ozone exposure,

and there is a clear dose-response

with the cytokine IL-8.

McLaughlin Centre for Population Health Risk Assessment

Three Cornerstones

• New paradigm for toxicity

testing (TT21C), based on

perturbation of toxicity

pathways

• Advanced risk assessment

methodologies, including

those addressed in Science

and Decisions

• Population health approach:

multiple health determinants

and multiple interventions

Exposure Assessment

McLaughlin Centre for Population Health Risk Assessment

Hubal et al., 2010, JTEH 13:299

Exposomics

McLaughlin Centre for Population Health Risk Assessment

``The exposome represents

the combined exposures

from all sources that reach

the internal chemical

environment. Toxicologically

important classes of

exposome chemicals are

shown. Signatures and

biomarkers can detect these

agents in blood or serum.``

Two Approaches to Exposomics

Rappaport et al., 2011 J.Exp. Sc. & Envir. Epi. 21:5

McLaughlin Centre for Population Health Risk Assessment

Biomarkers of Exposure

McLaughlin Centre for Population

Health Risk Assessment

NRC Report

McLaughlin Centre for Population Health Risk Assessment

National Health and Nutrition Examination Survey

Human Biomonitoring Programs

NHANES

Canadian Health Measures

Survey (CHMS)

Toxicity Pathways at Multiple Levels of Biological Organization

Mapping toxicity pathways

is key to identifying biomarkers

Balshaw, NIH

Biomonitoring Equivalents

McLaughlin Centre for Population Health Risk Assessment

McLaughlin Centre for Population Health Risk Assessment

Establishing Biomonitoring Equivalents

Hayes et al., 2008

Biomonitoring Equivalents (BEs)

Biomonitoring Equivalents (BEs) are defined as the concentration of a chemical (or

metabolite) in a biological medium (blood, urine, human milk, etc.) consistent with

defined exposure guidance values or toxicity criteria including reference doses and

reference concentrations (RfD and RfCs), minimal risk levels (MRLs), or tolerable

daily intakes (TDIs) [Hays, 2007. Regul. Toxicol. Pharmacol. 47(1), 96–109].

McLaughlin Centre for Population Health Risk Assessment

http://www.biomonitoringequivalents.net/html/chemical_specific_bes.html

• 2,4-D

• Acrylamide

• Cadmium

• Cyfluthrin

• Di(2-ethylhexyl)phthalate

• Phthalate Esters: Diethyl phthalate Di-n-butyl phthalate Benzylbutyl phthalate

• Polychlorinated dibenzo-p-dioxins and dibenzofurans

• Toluene

• Trihalomethanes: Chloroform Dibromochloromethane Bromodichloromethane Bromoform

Reverse Toxicokinetics (rTK)

McLaughlin Centre for Population Health Risk Assessment

Rotroff DM, Wetmore BA, Dix DJ, Ferguson SS, Clewell HJ, Houck KA, Lecluyse EL, Andersen ME, Judson RS, Smith CM, Sochaski MA, Kavlock RJ, Boellmann F, Martin MT, Reif DM, Wambaugh JF, Thomas RS (2010) Incorporating human dosimetry and exposure into high-throughput in vitro toxicity screening. Toxicol Sci 117: 348-358

Incorporating Human Dosimetry and Exposure

Into High-Throughput In Vitro Toxicity Screening

Adverse Effect

Toxicity Pathway

Key Events

MOA

HTS Assays

Intrinsic

Clearance

Plasma Protein

Binding

PopulationsPK Model

Biological Pathway Activating

Concentration (BPAC)

Probability Distribution

Dose-to-Concentration

Scaling Function (Css/DR)

Probability Distribution

Probability Distribution

for Dose

that Activates

Biological Pathway

BPADL

Pharmacodynamics Pharmacokinetics

Reverse Toxicokinetics (rTK):

in vitro concentration to in vivo dose (Dix, 2011)

Example: Bisphenol A

Estrogenicity In Vitro

vs.

In Vivo Reproductive Toxicity

• Rat reproduction tests resulted in a NOEL of 50 mg/kg/day,

with a corresponding RfD of 0.5 mg/kg/day

• HTRA lower limit BPADL99 is 0.16 mg/kg/day, derived from six

ToxCast estrogen receptor assays

• Actual human exposure is estimated to be 0.00008 mg/kg/day

McLaughlin Centre for Population Health Risk Assessment

McLaughlin Centre for Population Health Risk Assessment

Rotroff DM, Wetmore BA, Dix DJ, Ferguson SS, Clewell HJ, Houck KA, Lecluyse EL, Andersen ME, Judson RS, Smith CM, Sochaski MA, Kavlock RJ, Boellmann F, Martin MT, Reif DM, Wambaugh JF, Thomas RS (2010) Incorporating human dosimetry and exposure into high-throughput in vitro toxicity screening. Toxicol Sci 117: 348-358

Exposure Science in the 21st

Century

“An NRC committee will develop a long-range vision for exposure science . . . . It will include development of a unifying conceptual framework for advancement of exposure science to study and assess human and ecological contact with chemical, biological, and physical stressors in their environments. concern.”

http://www.nap.edu/openbook.php?record_id=13507

Conclusions and Future Directions

• TT21C (NRC, 2007) has received widespread support, both in the

United States and internationally: progress towards the implementation

of TT21C ahead of projected timetable

• NexGen (EPA, 2014) integrates three cornerstones of the future of risk

science: TT21C, new assessment methodologies, and a population

health perspective

• High-throughput exposomics: assess toxicity pathway perturbations

measured directly in humans

• RS21C: a coordinated international approach needed to chart the future

of risk science, building on the powerful new tools, technologies, and

methodologies now available to the scientific community

McLaughlin Centre for Population Health Risk Assessment