DEVELOPMENT OF A SCREEN TO

IDENTIFY STRESS PATHWAYS OF

CONCERN FOR USE IN NON-ANIMAL

SAFETY ASSESSMENT

SAFETY & ENVIRONMENTAL ASSURANCE CENTRE

Sarah Hatherell

Cambridge ADME-Tox Workshop, 16th May 2019

WE MAKE MANY OF THE WORLD’S FAVOURITE BRANDS

EVERY DAY, 2.5 BILLION PEOPLE USE UNILEVER PRODUCTS

SEAC – SAFETY & ENVIRONMENTAL ASSURANCE CENTRE

PROTECTING CONSUMERS, WORKERS & OUR ENVIRONMENT BY ENSURING UNILEVER’S PRODUCTS & PROCESSES ARE SAFE & SUSTAINABLE BY DESIGN

COLLABORATIONWe partner with leading

scientists from around the globe

SHARING SCIENCE

GOVERNANCEWe provide scientific evidence

to manage safety risks & environmental impacts for

new technologies

APPLYING SCIENCE ADVANCING SCIENCE

NEW CAPABILITYWe harness the latest science to create new tools to assess

innovations of the future

CENTRE OF EXCELLENCE – SAFETY &ENVIRONMENTAL SUSTAINABILITY SCIENCES

SCIENTIFIC PARTNERSHIP

www.tt21c.org

OUR APPROACH TO SAFETY SCIENCEASSURING SAFETY WITHOUT THE USE OF ANIMALS

30+ Years Investment

50+ Collaborations

CAN WE USE A NEW INGREDIENT SAFELY?

Can we safely use x% of ingredient yin product z?

NEXT GENERATION RISK ASSESSMENT (NGRA)

• Using new tools and approaches to build risk assessments without animal tests.

Exposure led Mechanistic Hypothesis driven

• Base decisions on exposure and biological pathway-indicated hazard concerns (using human in-vitro cell models).

EXPOSURE-LED RISK ASSESSMENTS

Many relevant compounds will likely have a non-specific toxicity leading to cellular stress.

New chemical ingredient

Applied e.g. skin/hair

Food/beverage

Inhaled

Systemic Exposure

Local exposure

Exposure-led

Determine biological

pathway-level effects

Specific (receptor mediated)

Non-specific (stress-

response)Above TTC?

CELLULAR STRESS WORKSHOP

DEVELOPING A TIERED STRATEGY

UncertaintyMechanistic understanding

Hazard Identification

• Publications• In-silico alerts• MIE atlas• AOP wiki

Pathway identification

• Transcriptomics• Proteomics• Receptor screens• Stress Panel

Pathway characterisation

• Live cell imaging• Systems toxicology

models• Repeat dose• Organotypic models

Tier I Tier II Tier III

CELL STRESS PANEL V1 – DATA GENERATED AT

*Technology exceptions include ELISAs for inflammation markers; promega kits for LDH; Glu/Gal assay.**Timepoint exceptions include Glu/Gal assay (6, 24 & 48hrs) and PLD/Steatosis assay (6, 24 & 72hrs)

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-8

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)

Technology: High Content Imaging*

Cell line: HepG2 (human liver carcinoma) – 2D, 96-well plate format

Time Points: 1, 6 & 24 hrs**

Platform

Use typical exposure scenarios:

PBPK models:

CELL STRESS PANEL V1 – DATA GENERATED AT

15 chemicals tested including:

Compounds known to induce one or more stress pathways: CDDO-Me, tBHQ, DEM

“Low-risk”compounds with known human exposures: Phenoxyethanol, Niacinamide, Caffeine

“High risk” compounds with known human exposures: Doxorubicin, Diclofenac, Troglitazone

Choosing the dosing range:

Calculate ‘free concentration’:

Use in-vitro exposure models:

Dose range:

Chemical- specific

8 doses plus a solvent control

Determined using models & cytotoxicity data

LINKING EXPOSURE AND POINT OF DEPARTURE

Generate data

PBPK models of systemic exposure

In-vitro exposure model

Quantify evidence of a response, calculate PoD

Summarise data

GRAPHICAL SUMMARY

Blood plasma FREE concentration (shaded region indicates uncertainty)

Positive biomarkers (i.e. ‘hits’)

Mean FREE concentration PoD (*) and 95% uncertainty

range (o’s)

Log10 scale

Colours indicate pathway

For each timepoint

Oxidative stress Mitotox Inflammation

Cell health/other DNA damage ER stress

6 hours1 hour24 hours

Doxorubicin

6 hours1 hour 24 hours

Phenoxyethanol

MARGIN OF EXPOSURE AND SAFETY DECISIONS

Concentration

10x fold difference?

Concentration

10 fold difference?

Exposure Tipping point

Safety decision

Safety decision

Refine uncertainties

MARGIN OF EXPOSURE AND SAFETY DECISIONS

6 hours1 hour 24 hours

6 hours1 hour 24 hours

Scenario 1

Scenario 2

TROGLITAZONE > PIOGLITAZONE > ROSIGLITAZONE

• Drugs used in the management of Type II Diabetes.

• Induce idiosyncratic hepatotoxicity, potentially through mitochondrial & oxidative stress. Troglitazone withdrawn from the market.

• Main aim was to see if the order of hepatotoxic incident rates (Troglitazone > Pioglitazone > Rosiglitazone) was reflected in the order of in vitro potency detected as part of this cell stress panel.

PIOGLITAZONE, ROSIGLITAZONE & TROGLITAZONE

Rosiglitazone

Troglitazone

Pioglitazone

1 hour

1 hour

1 hour

6 hour

6 hour

6 hour

24 hour

24 hour

24 hour

CELL STRESS PANEL: THE STORY SO FAR…

We are still refining, but…• We observe that for well-known low-risk compounds there is little or no

stress-pathway activity (at relevant exposures). • Known modes of toxicity are detected for compounds such as Doxorubicin or

Troglitazone. • Gaining confidence in it’s use as a Tier 2 screen for pathway identification.• Further characterisation required where exposure & response overlap.

Questions remain, including…• What happens with other cell types/more organotypic models?• What happens under chronic/repeat exposure conditions?

ACKNOWLEDGEMENTS

Unilever• Alistair Middleton

• Andrew White

• Maria Baltazar

• Joe Reynolds

• Beate Nicol

• Hequn Li

• Sharon Scott

• Sophie Malcomber

• Sophie Cable

Cyprotex• Paul Walker

• Caroline Bauch

• Laura Purdie

• Tim Smith

• Helen Gill