Development of a screen to identify stress pathways of concern...
Transcript of Development of a screen to identify stress pathways of concern...
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