APPLICABILITY OF

TOXICOGENOMICS-BASED ASSAYS

WITHIN THE INDUSTRIAL CONTEXT

DR CARL WESTMORELAND

Slides available at www.TT21C.org

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Unilever is one of the world’s leading suppliers of fast-moving consumer goods. Our products are sold in over 190 countries and used by 2 billion consumers every day.

CAN WE USE A NEW INGREDIENT SAFELY?

Will it be safe • For our consumers? • For our workers? • For the environment?

Can we use x% of ingredient y in product z?

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TOXICITY ENDPOINTS (HUMAN HEALTH)

e.g. Relevant toxicity endpoints based on the Scientific Committee

on Consumer Products guidance document “Notes of Guidance for

the Testing of Cosmetic Substances and their Safety Evaluation”

• Acute toxicity

• Corrosivity and irritation

• Skin sensitisation

• Dermal/percutaneous absoprtion

• Repeated dose toxicity

• Reproductive toxicity

• Mutagenicity/genotoxicity

• Carcinogenicity

• Toxicokinetic studies

• Photo-induced toxicity

http://ec.europa.eu/health/scientific_committees/consumer_safety/docs/sccs_s_006.pdf

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 5

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 6

EU: SCCS OPINION 2010

‘…. majority of the

existing alternative

methods [are] only

suitable for hazard

identification of

cosmetic ingredients

and do not give

information on

potency. Thus, a full

human health risk

assessment cannot be

performed’

http://ec.europa.eu/health/scientific_committees/consumer_safety/statements/index_en.htm 7

CURRENT SCIENTIFIC REALITY: NON-ANIMAL APPROACHES FOR SAFETY DECISIONS

Human Health

Toxicology Endpoint

Timeline for Replacement of Animal

Testing

[Note: Regulatory Acceptance would require

an additional 4-8 years]

Comments

Repeated dose toxicity No timeline for full replacement could

be foreseen Ongoing work still at research stage

Carcinogenicity No timeline for full replacement could

be foreseen

Current in vitro test methods are

inadequate for generating the dose-

response information required for safety

assessment

Skin Sensitisation 2017 – 2019 for full replacement

Several non-animal test methods under

development & evaluation; data

integration approaches for safety

assessment required

Reproductive Toxicity No timeline for full replacement could

be foreseen

Ongoing work still at research stage

>2020 to identify key biological

pathways

Toxicokinetics No timeline for full replacement could

be foreseen

Ongoing work still at research stage

2015 – 2017: prediction of renal &

biliary excretion and lung absorption

Compiled from Adler et al (2011) Adler et al (2011), Archives in Toxicology, 85 (5) 367-485

Past:

• hazard focus

• emphasis on tests for classification and labelling (‘positives/negatives’)

• direct replacement of a specific animal test

NEW APPROACHES TO RISK ASSESSMENT WITHOUT ANIMALS

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NEW APPROACHES TO RISK ASSESSMENT WITHOUT ANIMALS

Now

» focus on non-animal approaches for consumer safety risk assessment

» data required for safety decision should be driver

» dose response information is essential

» understanding the underpinning human biology

» we are not looking for a way to do the animal test without the animal

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US NRC REPORT JUNE 2007

“Advances in toxicogenomics,

bioinformatics, systems

biology, epigenetics, and

computational toxicology could

transform toxicity testing from a

system based on whole-animal

testing to one founded primarily

on in vitro methods that

evaluate changes in biologic

processes using cells, cell

lines, or cellular components,

preferably of human origin.”

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ADVERSE OUTCOME PATHWAYS (AOP) SOURCE TO OUTCOME PATHWAYS (S2OP)

• Source to Outcome Pathways (Crofton et al, 2011)

Adapted from OECD (2012)

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• Proposal for a template and guidance on developing and assessing the Completeness of Adverse Outcome Pathways

Source Environmental Containment

Exposure Molecular Initiating

Event

Organelle Effects

Cellular Effects

Tissue Effects

Organ Effects

Organ Systems Effects

Individual Effects

Population Effects

Community Effects

PATHWAYS-BASED APPROACHES TO SAFETY ASSESSMENT

At the heart of all pathways-based approaches to safety assessment is the need for:

• Detailed mechanistic understanding of the biology which can lead to adverse effects in patients/consumers and workers

• Tools to interrogate these pathways with chemicals of interest

• Risk assessment approaches to combine dose response relationships with exposure information in humans

• The ability to be able to make a robust safety decision

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PATHWAYS-BASED APPROACHES TO SAFETY ASSESSMENT

At the heart of all pathways-based approaches to safety assessment is the need for:

• Detailed mechanistic understanding of the biology which can lead to adverse effects in patients/consumers and workers

• Tools to interrogate these pathways with chemicals of interest

• Risk assessment approaches to combined dose response relationships with exposure information in humans

• The ability to be able to make a robust safety decision

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A JOINT AMBITION

“Toxicogenomics - the application of genomics-based technologies in toxicological research - may provide tools to tackle these uncertainties. It also offers the opportunity to develop tests that require fewer laboratory animals or cause them less inconvenience, and may eventually replace animal tests completely by in vitro assays using animal or human cells”

This website represents the efforts Unilever and partners are taking to progress the science of toxicity pathways: to put in place the tools and novel thinking needed to implement TT21C/AOP- based risk assessments. In so doing, we aim to ultimately remove our dependence on apical endpoint toxicological studies and bring novel science to the decisions we make on the safe use of chemicals within consumer products www.tt21c.org 15

EXPLORING PATHWAYS-BASED RISK ASSESSMENT AT UNILEVER

• Systemic Toxicology Risk Assessment

- DNA damage

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A TT21C PROTOTYPE TOX PATHWAY (AOP): GENOTOXICITY/DNA DAMAGE

• Joint research program with Hamner Institutes • Develop tools to assess DNA-damage stress

pathways • Examine dose-dependent transitions for case-

study mutagenic compounds • Apply data to develop a computational systems

biology model of the p53-mdm2 network • Q: Can we use genotoxicity tox-pathway in TT21C

paradigm to provide a prototype proof of principle for TT21C/AOP?

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TIME/DOSE: DNA DAMAGE & P53 ACTIVATION

Time

Dose

ETP

QUE

MMS

p-p53 p53 p-H2AX

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MODELLING ULTRASENSITIVITY IN P53 ACTIVATION

10-4

10-3

10-2

10-1

0

10

20

30

10-4

10-2

100

102

1

2

3

10-4

10-2

100

102

1

3

5

10-4

10-2

100

102

1

3

5

7

9

ATM γH2AX

p53 Micronuclei F

old

ch

an

ge

Etoposide (μM)

Fo

ld c

ha

ng

e

Etoposide (μM)

Etoposide (μM) Etoposide (μM)

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IN VITRO TO IN VIVO (HUMAN EXPOSURE) EXTRAPOLATION

Exposure

mg/kg/day Target site

concentration

(µM)

In vitro

adaptive/adverse

threshold

concentration (µM)

– measuring &

modelling FREE

CONCENTRATIONS

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Exposure & Consumer Use Assessment

High-content information in vitro assays 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 21

A LONG-TERM VISION: SOURCE TO OUTCOME PATHWAY-BASED SAFETY RISK ASSESSMENT

• fully integrated exposure and hazard assessment at different levels of biological organisation

To reduce uncertainty

within our risk assessments…

...we will focus on

characterising the key

impacts…

…and replace our current reliance on

apical endpoint studies…

...of marketing any new

ingredient via:

• greater mechanistic understanding of ingredient properties to allow extrapolation from Molecular Initiating Events (MIEs) to an adverse outcome

• better communication of acceptable risk using defined protection goals (consumer, environmental)

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ACKNOWLEDGEMENTS

Yeyejide Adeleye, Maja Aleksic, Nora Aptula, Mahesh Batakurki, Emma Butler, Paul Carmichael, Stella Cochrane, Sarah Cooper, Carol Courage, René Crevel,

Richard Cubberley, Tom Cull, Claire Davies, Michael Davies, Eliot Deag, Matthew Dent, Sue Edwards, Julia Fentem, Chris Finnegan, 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, Sarah Kang-King-Yu, Anja Lalljie, Yvan Le Marc, Moira Ledbetter, Jin Li, Cameron MacKay, Ian Malcomber, Sophie Malcomber, Stuart

Marshall, Gavin Maxwell, Helen Minter, Craig Moore, Beate Nicol, Sean O’Connor, Deborah Parkin, Ruth Pendlington, Juliette Pickles, Mike Pleasants,

Oliver Price, Fiona Reynolds, Jayne Roberts, Nicola Roche, Paul Russell, Ouarda Saib, David Sanders, Paul Sanderson, Gary Sassano, Andrew Scott, Sharon Scott, David Sheffield, Nikol Simecek, Wendy Simpson, Ilias Soumpasis, Chris Sparham,

Richard Stark, Vicki Summerfield, Diana Suárez-Rodriguez, Dawei Tang, Jeff Temblay, Sivaram TK, Roger van Egmond, Carl Westmoreland, Andrew White &

Sam Windebank 23

WORKING WITH SCIENTIFIC PARTNERS GLOBALLY

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