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1 UNILEVER, SEAC AND SAFETY ASSESSMENT WITHOUT ANIMALS CURRENT APPROACHES AND RESEARCH NEEDS The funders’ perspective – The remit of the joint awards Andrew Scott NC3Rs/Unilever PhD Studentship Launch event 2 nd April 2019

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Page 1: UNILEVER, SEAC AND SAFETY ASSESSMENT WITHOUT ANIMALS. Scott - Unilever... · UNILEVER, SEAC AND SAFETY ASSESSMENT WITHOUT ANIMALS ... Cell Health: LDH, Phospholipidosis, Steatosis,

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UNILEVER, SEAC AND SAFETY ASSESSMENT WITHOUT ANIMALSCURRENT APPROACHES AND RESEARCH NEEDS

The funders’ perspective – The remit of the joint awards

Andrew Scott

NC3Rs/Unilever PhD Studentship Launch event2nd April 2019

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CONTENT OVERVIEW

• Unilever

• SEAC

• How we assure safety - Next Generation Risk Assessment

• Research activities and needs

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UNILEVER IS A GLOBAL COMPANY AND WE MAKE MANY OF THE WORLD’S FAVOURITE BRANDS

2018 TURNOVER €51BN

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“Building brands through benefit-led innovations,unlocked by science &

technology”

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Protecting Consumers, Workers & our Environment by ensuring Unilever’s Products & Processes are Safe & Sustainable by Design

SEACSAFETY & ENVIRONMENTAL ASSURANCE CENTRE

Scientific Excellence And Collaboration

➢ Centre of Excellence in Safety & Sustainability Sciences

▪ Deploy expertise on higher risk business projects

▪ Collaborate with leading external research teams to develop new capability

▪ Leverage science & global networks for consumer trust & freedom to operate

➢ Unilever Safety Governance▪ Provide scientific evidence to manage

safety risks & environmental impacts

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SEAC

SEAC’S CAPABILITYChemistry, Environmental Safety, Environmental Sustainability, Exposure Science, Informatics, Life-Cycle Impact Assessment, Microbiology, Modelling, Process & Occupational Safety,Risk Assessment, Toxicology

70%DEGREES OR HIGHER

30%PhDs

>600PUBLICATIONS SINCE 2005

>15LANGUAGES

4HONORARYPROFESSORS

>20NATIONALITIES

FUTURE TALENTworking with Universities to supportdevelopment of future scientists

LEADING EDGE SAFETY & ENVIRONMENTAL SCIENCES CAPABILITY

PARTNERSHIPSour innovation ecosystems involve >50 collaborators & service providers across the world

17 PhDs10 POST-DOCS

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SCIENTIFIC PARTNERSHIP

www.tt21c.org

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8https://www.unilever.com/news/press-releases/2018/unilever-supports-calls-for-a-worldwide-animal-testing-ban-on-cosmetics.html

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OUR APPROACH TO SAFETY SCIENCEASSURING SAFETY WITHOUT THE USE OF ANIMALS

30+ Years Investment

50+ Collaborations

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CAN WE USE AN INGREDIENT SAFELY?

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

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MAXIMISING USE OF EXISTING INFORMATION AND NON-ANIMAL APPROACHES

• All available safety data

• in silico predictions

• Exposure-based waiving approaches

• History of safe use

• Read across

• Use of existing OECD in vitro approaches

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• Using new tools and approaches to build a risk assessment to enable decisions to be made (without animal tests)

• An exposure-led risk assessment solution to biological pathway-indicated hazard concerns

Exposure led Mechanistic Hypothesis driven

NEXT GENERATION RISK ASSESSMENT (NGRA)

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ICCR PRINCIPLES OF RISK ASSESSMENT WITHOUT ANIMAL TESTING

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ICCR NINE PRINCIPLES OF NEXT GENERATION RISK ASSESSMENT (NGRA)

• Main overriding principles: • The overall goal is a human safety risk assessment • The assessment is exposure led • The assessment is hypothesis driven• The assessment is designed to prevent harm

• Principles describe how a NGRA should be conducted: • Following an appropriate appraisal of existing information• Using a tiered and iterative approach• Using robust and relevant methods and strategies•

• Principles for documenting NGRA: • Sources of uncertainty should be characterized and documented• The logic of the approach should be transparently and documented

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Plasma concentration

Uncertainty

Uncertainty

10x -

100x -

10x -

100x -

SYSTEMIC & BIOLOGICAL EFFECTS – WHAT IT MIGHT LOOK LIKE

Low

High

Ex

po

su

re /

Do

se

POD from various in vitro assays(range characters multiple cell lines)

Toxicity markers from Cell-based assays

Marker 3 2.9-3.3µM

Marker 2 2-3µMMarker 1

1.97µM

Tissueconc

1.5µM

0.05µM withclearance

without clearance

Uncertainty

Refinement of internal exposure

do ADME studies

PBK prediction0.0001µM

20µM

BioSpyderTranscription profile

In vitro assays

Oxidative Stress NOAEC

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NGRA CORE ELEMENTS

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IN VIVO KINETICS

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UNDERSTANDING CONSUMER EXPOSURE

Systemic Exposure In vitro Assays:Kinetic SolubilityThermodynamic SolubilityMetabolic Stability-Human Hepatocytes-Human CYP450 Isoforms-Human Hepatic MicrosomesStability in Human PlasmaPlasma Protein BindingPartitioning in Human Blood

• Predicting systemic exposure• Enabling us to select and test relevant doses• Increased role for clinical work to confirm systemic exposure levels

PBK Modelling

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INPUTS: DERMAL PENETRATION

Dermal Kinetics

Sk

in B

ioa

vail

ab

ity

ex vivo human

skin

Understanding delivery to the systemic circulation following dermal application

Understanding the kinetics of an ingredient in the skin to allow risk assessments for local endpoints

Davies et al (2011) Toxicol Sci 119, 308-18

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INPUTS: IN VITRO SYSTEMS FOR METABOLISM STUDY

In vitro model

HL fraction

S9

2D

suspensionsSandwich

culturePlated 2D

HepatoPac

3D

spheroids

Advanced

2DCo-cultured hepatocyteheterologously-

expressed system

microsomes

Typical Incubation period

~ 2 hours~ 4 hours

~ 24 hours ~ 7 days 1 ~ 2 (3) days~ 24 hours

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OutputPopulationPBK

ModelPrediction

methodInput

Uncertainty

PBK MODELLING CHALLENGES

In silico

In vitro

Physico-chemicalCL/metabolismProtein binding

Dermal penetration

TransportersPhysiologyExposure

No established in silico method for predicting Clearance rate

How to account for population variability, e.g. differences metabolism?

Several options for in vitro clearance. What is best for a compound?

Need to understand uncertainty through process, e.g. parameter & model uncertainty.

Unclear when transporters important.

Assumption that plasma concentration is the most relevant metric for QIVIVE. Is this always true?

Commercial? Open source?Many tissues or simple compartmental?What are the domains of

applicability/confidence?

Clinical PK data

Need to account for limited population coverage.

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IN VITRO KINETICS

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IN VITRO ASSAYS IN RISK ASSESSMENT

0-10% serum

single cells / microtissues

high concentrations

short exposure time

no bioaccumulation

limited metabolism

100% serum

connected cells / tissues

low concentrations

long / pulsed exposure

tissue bioaccumulation over time

full metabolic pathways

What metrics can we use to compare effects

in vivo to effects in vitro to improve QIVIVE?

effect in vitro effect in vivotarget site

intracellular

free concentration

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MECHANISTIC UNDERSTANDING, DOSE RESPONSE AND DECISION MAKING

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PATHWAY CHARACTERISATION(TARGETS)

EXAMPLES:3D and organotypic cell modelsMolecular dynamic simulations

Integrated in vitro systems

IN SILICO-FIRST

EXAMPLES:MIE in silico Atlas & QSARsSkin haptenation modellingIn silico receptor screening

Tier 1

In silico-first approaches for identifying pathways of

concern, building weight of evidence and formulating

hypotheses for testing

PATHWAY IDENTIFICATION(TARGETS AND OFF-TARGETS)

EXAMPLES:HT-Transcriptomics

In vitro screening panelsHigh content imaging

SPME free concentration

Tier 2

Identifying/characterising lead MIEs and pathways

through experimental data generation, informatics data

mining and computational modelling

Tier 3

Characterisation of response in biologically

relevant in vitro systems or complex computational

models for decision making

A TIERED AND ITERATIVE APPROACH

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UNILEVER/US EPA 2015-2020: JOINT CRADA

CASE STUDY CHEMICALS

1. Caffeine

2. Curcumin

3. Bisdemethoxycurcumin

4. Tetrahydrocurcumin

5. 6-Gingerol

6. Coumarin

7. Hydroquinone

8. Doxorubicin

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CELL STRESS PANEL

• Range of biomarkers covering ~10 cell stress pathways:

Mitochondrial Toxicity: MitoSOX, PGC1α, MMP, ATP, Glu/Gal

Oxidative Stress: GSH, ROS, SRXN1, NRF2

DNA damage: pH2AX, p53

Inflammation: TNFAIP3, ICAM1, NFkB p65, IL-1β, IL-8, HMGB1

ER Stress: PERK, ATF4, CHOP, XBP1, BiP, ER Tracker

Metal Stress: MTF-1, Metallothionein

Osmotic Stress (NFAT5); Heat Shock (HSP70); Hypoxia (HIF1α)

Cell Health: LDH, Phospholipidosis, Steatosis, pHrodo indicator, apoptosis (caspase-3/7) & necrosis (ToPro-3)

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NOTEL* is the derived concentration of a compound that does not elicit a meaningful change in gene expression (i.e. the threshold of the concentration that elicits minimal mechanistic activity).

HIGH THROUGHPUT TRANSCRIPTOMICS

Recommended approaches in the application of toxicogenomics to derive points of departure for chemical risk assessmentFarmahin et al (2017) Arch Tox 91, 2045-65

Thomas et al (2013). Toxicol Sci, 134, 180-94

*NOTEL = No observed transcriptional effect level

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HIGH THROUGHPUT TRANSCRIPTOMICS

Fold change >1.5Genes in pathway >3Fishers exact test >0.1(3 independent experiments)

HepG2BMD minimum – 295BMD medium– 450BMD maximum 962

MCF7BMD minimum – 117 BMD medium – 167BMD maximum 290

HepaRGBMD minimum –237BMD medium – 344BMD maximum 913

POD - NOTEL value

Conc. Phenoxyethanol (µM)

Acc

um

ula

tio

n

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NGRA – EXPOSURE-DRIVEN CASE STUDIES

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NGRA – EXPOSURE-DRIVEN CASE STUDIES

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In-vitro cell culture

Characterise stress response

Low-risk exposure

• Which cell model? 2D or 3D? • Primary or cell line?• Which pathways/biomarkers?• How many time points/dose points?

• How do we calculate the tipping point?• Number of pathways (coverage)• Duration of response

• Cells in media vs tissue?

• Chronic vs acute exposure

Characterise uncertainties to facilitate decision making

Prof B. van de Water, U. Leiden

UNCERTAINTY AND DECISION MAKING

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RESEARCH NEEDS

The remit of the joint awards

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RESEARCH NEEDS – POTENTIAL AREAS FOR PhDs

Exposure

• What is the impact of clearance mechanisms (other than skin and liver) on systemic bioavailability, e.g. renal clearance?

• What is the impact of active transport mechanisms (intracellular) to enable IVIVE?

• What is the most relevant metric of exposure in vitro for IVIVE?

Inhalation

• What pathways should we be concerned about for respiratory toxicity? Beyond Fibrosis?

• What is the impact of receptors on respiratory toxicity?

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RESEARCH NEEDS – POTENTIAL AREAS FOR PhDs(CONTINUED)

Immune Effects

• What is the influence of the microbiome on Oral Immunity?

• Local Exposure - Skin, Lung & Oral cavity – can we challenge the assumption that our risk assessment should be based on 100% bioavailability

Uncertainties

• Biological coverage - Right choice of Cells /Cell Model?

• Repeat dosing – considerations for local exposure and IVIVE?

• Influence/relevance of clearance in our models?

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THANK YOU…

www.tt21c.org