ADVERSE OUTCOME PATHWAYS -APPLICATION IN CONSUMER [AND ENVIRONMENTAL] SAFETY RISK ASSESSMENTS

CARL WESTMORELAND

SAFETY & ENVIRONMENTAL ASSURANCE CENTRE (SEAC)UNILEVER R&D

AOPS

• Safety assessment of consumer products

• Exploring AOPS in consumer safety assessment

» Skin sensitisation

» Systemic toxicity

• Challenges

Can we use x% of ingredient yin product z?

CAN WE USE A NEW INGREDIENT SAFELY?

CURRENT SAFETY ASSESSMENT PROCESS FOR INGREDIENTS IN CONSUMER PRODUCTS

Consider product type and consumer habits

Determine route and amount of exposure

Identify toxicological endpoints of potential

concern

Identify critical end point(s) for risk

assessment

Identify available toxicology data

Identify supporting safety data (e.g. QSAR,

HoSU)

Evaluate required vs. available support

Conduct risk assessment for each critical endpoint

Conduct toxicology testing as required

Overall safety evaluation for product – define acceptability

and risk management measures

Source Environmental

Containment

Exposure Molecular

Initiating

Event

Organelle

and

Molecular

Assemblies

Effects

Cellular

Effects

Tissue

Effects

Organ

Effects

Organ

Systems

Effects

Individual

Effects

Population

Effects

Community

Effects

Toxicity Pathway

Mode of Action

Adverse Outcome Pathway

Source to Outcome Pathway

Adapted from Kevin Crofton 2010, OECD AOP Meeting Definitions

FROM TOXICITY PATHWAYS TO ADVERSE OUTCOME PATHWAYS (AOPS)

EXAMPLE 1: SKIN SENSITISATION

We risk assess to prevent skin sensitisation in consumers:

» What risk does ingredient X at conc. Y in product Z pose to the consumer?

Focus should be on prevention of human adverse outcome, not replacing animal test(s)

1. Identify pathways driving human adverse response = allergic contact dermatitis.

2. Develop test methods to predict key toxicity pathways.

3. Will response be adverse for a relevant exposure scenario?

Risk ?

Pro

du

ct

X

HazardExposure

Historical Non-animal In Vivo

Induction of skin allergy is a multi-stage process driven by toxicity pathways:

- Mechanistic understanding is captured in Adverse Outcome Pathway (AOP)

- How can we make risk assessment decisions by integrating this evidence?

Modified from ‘Adverse Outcome Pathway (AOP) for Skin Sensitisation’, OECD report

1. Skin Penetration

2. Electrophilicsubstance:

directly or via auto-oxidation or metabolism

3-4. Haptenation:covalent

modification of epidermal proteins

5-6. Activation of epidermal

keratinocytes & Dendritic cells

7. Presentation of haptenated protein by Dendritic cell resulting

in activation & proliferation of specific

T cells

8-10. Allergic Contact Dermatitis: Epidermal

inflammation following re-exposure to

substance due to T cell-mediated cell death

Key Event 1 Key Event 2 + 3 Key Event 4 Adverse Outcome

OECD AOP FOR SKIN SENSITISATION

Pro

du

ct

hazard in vivo response

Adverse

Non-Adverse

exposure

1. Skin Penetration

2. Electrophilicsubstance:

directly or via auto-oxidation or metabolism

3-4. Haptenation:covalent

modification of epidermal proteins

5-6. Activation of epidermal

keratinocytes & Dendritic cells

7. Presentation of haptenated protein by Dendritic cell resulting

in activation & proliferation of specific

T cells

8-10. Allergic Contact Dermatitis: Epidermal

inflammation following re-exposure to

substance due to T cell-mediated cell death

X

OUR CHALLENGE: WHAT RISK DOES INGREDIENT X AT CONC. Y IN PRODUCT Z POSE TO CONSUMERS?

1. Generate relevant non-animal data for the chemical and the exposure scenario

2. Use data as inputs for two linked mathematical models capable of predicting magnitude of human sensitiser-specific immune response

3. Invest in clinical research to establish adverse threshold in human immune response →allows model prediction(s) to underpin risk assessment decisions

Adverse

Non-Adverse

allergic immune response

time

No

. CD

8+

T c

ells

dose Y

dose X

haptenated skin protein prediction

1. Skin Penetration

3-4. Haptenation:covalent

modification of epidermal proteins

5-6. Activation of epidermal

keratinocytes & Dendritic cells

7. Presentation of haptenated protein by Dendritic cell resulting

in activation & proliferation of specific

T cells

8-10. Allergic Contact Dermatitis: Epidermal

inflammation following re-exposure to

substance due to T cell-mediated cell death

2. Electrophilicsubstance:

directly or via auto-oxidation or metabolism

Maxwell et al (http://axlr8.eu/assets/axlr8-progress-report-2012.pdf) pp219-226

APPLYING THE AOP TO RISK ASSESSMENT

SEAC

For foreseeable future

Traditional

Toxicology

Our Unilever stated aim

No Unilever animal tests

Tox pathway activation in vitro

Modelling networks & exposure

Applying TT21C principles

NEW TOXICOLOGY PATHS

WHICH PATHWAYS?

• Pro-oxidant + Free-radical based• Mitochondrial targets + energy production disruption• Specific receptor agonist/antagonist targets» Functional receptors + cell surface receptors» Nuclear receptor mediated + promoter regions» Hormonal including ED

• Electrophiles covalently binding» DNA and protein dysfunction (incl. haptens)

• Immuno-specific mechanisms + cytokine effects• Disruption of calcium homeostasis + signalling• Direct necrosis or apoptosis induction• Impairment of cell proliferation and tissue repair• Interfering with ion transporters + metabolism

Exposure & Consumer Use Assessment

High-content information in vitroassays in human cells & models

Dose-response assessments

Computational models of the circuitry of the relevant toxicity pathways

PBPK models supporting in vitro to in vivo extrapolations

Risk assessment based on exposures below the levels of significant pathway perturbations

Chemistry-led

alerts & in vitro

screening

CHARACTERISING THE CELLULAR RESPONSE TO DNA DAMAGE

0.5

1

1.5

2

2.5

3

0.0

10.0

20.0

30.0

40.0

50.0

60.0

0.001 0.201 0.401 0.601 0.801 1.001 1.201 1.401 1.601 1.801 2.001

PP53 TK6

Total p53

H2AX

pp53 RC

HT Total P53

HT H2AX

Mn data

FACs MN data HT1080

INTEGRATE DATA INTO MODELS/NETWORKS

▪ Basal function

▪ Response to small perturbations

▪ Response to larger perturbations

▪ Assessing mechanism of underlying threshold giving rise to increased mutation

Computational modeling

of dose response for DNA

damage pathway

activation

IN VITRO IN VIVO (HUMAN EXPOSURE) EXTRAPOLATION

Exposure mg/kg/day

Target site concentration (µM)

Acceptable in vitro concentration (µM)

THOUGHTS/CHALLENGES (1)

Case Studies» Encourage pragmatism

» What is the key information to allow decision-making?

Multidisciplinary teams » Key importance of modelling in the process

» Interactions between modellers, chemists, biologists and toxicologists accelerates pathway characterisation.

» Multi-disciplinary teams are difficult to steer but necessary.

THOUGHTS/CHALLENGES (2)

Risk Assessment: Source to Outcome Pathway» Full integration of exposure to develop quantitative AOPs to go from hazard

identification to risk assessment for both human health and the environment

» Effective integration of expertise.

Relevance of in vitro concentrations to in vivo» MEASURING free concentrations vs nominal

» Importance of understanding kinetics

Defining adversity» What are we trying to prevent in our consumers / environment?

» Need to consider up-front how adversity will manifest:• Is there a defined clinical outcome (eg skin allergy)?• Can we define ‘adversity’ in the pathway in vitro? (e.g. DNA damage)• Irreversible disruption of homeostasis ?

THANK YOU

Yeyejide Adeleye, Maja Aleksic, Nora Aptula, Mahesh Batakurki, Emma Butler, Paul Carmichael, Kristina Castle, Catherine Clapp,

Richard Cubberley, Claire Davies, Michael Davies, Eliot Deag, Matthew Dent, Sue Edwards, Julia Fentem, Paul Fowler, Antonio

Franco, Nichola Gellatly, Nicola Gilmour, Stephen Glavin, Dave Gore, Todd Gouin, Steve Gutsell, Colin Hastie, Juliette Hodges, Geoff Hodges, Sandrine Jacquoilleot, Gaurav Jain, Penny Jones, Kevin

Lam, Heidi Langley, Yvan Le Marc, Moira Ledbetter, Jin Li, Cameron MacKay, Ian Malcomber, Sophie Malcomber, Stuart Marshall, David

Mason, Gavin Maxwell, Claire Moore, Craig Moore, Beate Nicol, Sean O’Connor, Ruth Pendlington, Juliette Pickles, Oliver Price,

Fiona Reynolds, Jayne Roberts, Nicola Roche, Paul Russell, Ouarda Saib, David Sanders, Andrew Scott, Sharon Scott, David Sheffield,

Wendy Simpson, Ilias Soumpasis, Chris Sparham, Vicki Summerfield, Dawei Tang, Sivaram TK, Roger van Egmond, Andrew

White, Sam Windebank