IMPLEMENTATION OF NEXT GENERATION SAFETY

ASSESSMENTS (NGSA): A CONSUMER PRODUCT INDUSTRY VIEW

PAUL CARMICHAEL

CHANGING GLOBAL ENVIRONMENT

• Animal testing of cosmetic products and ingredients (and sale of) prohibited throughout the European Union

• Bans in Norway, India and Israel• Partial bans in New Zealand, South Korea, Turkey• A shift in attitudes to testing in the United States,

Canada, Brazil, Australia and other countries

SUCCESSES IN NON-ANIMAL ALTERNATIVES

• Internationally accepted toxicity tests that do not use animals

• Guidelines published by OECD

USA NRC REPORT JUNE 2007: TT21C

“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.”

PERTURBATION OF TOXICITY PATHWAYS

BiologicInputs

NormalBiologicFunction

Adaptive StressResponses

Early CellularChanges

Exposure

Tissue Dose

Biologic Interaction

Perturbation

Low DoseHigher Dose

Morbidityand

Mortality

Cell Injury

Higher yet

(From Andersen & Krewski, 2009, Tox Sci, 107, 324)

PERSONAL CARE CONSUMER PRODUCTS INDUSTRY CAN BE SUCCESSFUL IN THIS

1. Chemical ingredients not generally intended to be pharmacologically active (compare Pharmaceutical Co.)

2. Topical exposure with low bioavailability

3. Receptive regulatory environment

Making an exposure-led safety decision based on confidence that the safe level is within or below the adaptive homeostasis response, captured by appropriate in vitro systems and complemented with network computational models

UNDERSTANDING CONSUMER EXPOSURE

Dermal kinetics

Sk

in B

ioa

vail

ab

ity

ex vivo human skin

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

• Understanding delivery to the systemic circulation following dermal application

• Enabling us to test relevant doses

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

UNDERSTANDING CONSUMER EXPOSURE

Systemic exposureIn 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

PBPK Modelling

AOP

IMPLEMENTATION OF NAS TT21C

TT21C

High-throughput screening

Focused pathways approach

Chemical ingredient

Post-translational modification vs Transcription

In food/beverageApplied to skin/hair Inhaled

Penetrates skin

Bioavailable

Chemical stability/Metabolism/QSAR alerts/HTS Bioassays/for MIEs

Specific targets (receptor pharmacology) Non-specific effects

Stress networks (~10)

Resolution vs Persistence vs Progression over timeCharacterise and relate dose response to actual human exposure (dose/time)

Adaption vs Adversity

Chemical ingredient

Local effects

Skin/eye irritation

Skin allergy

Lung immuno

Skin mutagenicity

Exposure base waiving/TTC/HoSU/Read Across

Post-translational modification vs Transcription

In food/beverageApplied to skin/hair Inhaled

Penetrates skin

Bioavailable

Chemical stability/Metabolism/QSAR alerts/HTS Bioassays/for MIEs

Specific targets (receptor pharmacology) Non-specific effects

Stress networks (~10)

Resolution vs Persistence vs Progression over timeCharacterise and relate dose response to actual human exposure (dose/time)

Adaption vs Adversity

PROFESSOR KIM BOEKELHEIDE, BROWN UNIVERSITY

DR IMRAN SHAH, EPA

EXAMPLES OF NGSA SCIENCE

DNA DAMAGE PROJECT

Traditionally “risk assessment” of ‘genotoxins’ have been based on linear models

Jenkins et al 2010

Batchelor et al 2009

Understand how safety may be assured for complex toxicological endpoints using data derived from a toxicity pathways-based approach that is rooted in mechanistic understanding of the underlying biology

• HCI: Cellular response to DNA damage (p53 pathway + case study chems)

• Localization of Mn & DNA damage response proteins in single cells

• phos-p53, total-p53, p21, MDM2, Chk2, p-ATM, H2AX

• High throughput flow cytometry (FACS)

• Alterations in gene expression following DNA damage

BIODYNAMICS OF DNA DAMAGE

Con

Overlay

ETP

p-H2AX p53 BP1

DNA REPAIR CENTERS CAN BE COUNTED USING HIGH CONTENT IMAGING (HCI)

24h

Efficiently repaired, short-lived Poorly repaired, long-lived

QUANTITATION OF DNA REPAIR CENTERS BY HCI

NEOCARZINOSTATIN ETOPOSIDE

• Homeostasis likely requires perfect adaptation of both rapidly acting pathways (post-translational modification) and slower acting pathways (transcriptional)

• Lower doses: rapid post translational modification

• Higher doses: At some point (depletion of p53 reserves or other post-translational modification), pathway moves to transcriptional control

Repair centre quantification, sensor kinase, PTM using phosphoproteomics

Dose and “Energy”

CHARACTERISATION OF P53 PATHWAY AT LOW DOSES (HCI INSIGHTS)

EXAMPLES OF NGSA SCIENCE

BIODYNAMICS OF OXIDATIVE STRESS

Reactive oxygen species(ROS)

Production Removal

Determining the tipping-point when homeostatic regulatory mechanisms become saturated and shift from an adaptive to an adverse state

NRF2-KEAP1 PATHWAY

ROS

NRF2 NRF2

Keap1Keap1

NRF2

NRF2

NRF2

Keap1ox

Anti-oxidative stress response genes

ROS

NRF2

?

De-novo synthesis

Proteolysis

Nucleus

Cytosol

GSH

GSSG

reduction

nuclear export

nuclear import

Homeostasis of oxidative stress

SRXN1

NRF2

Fyn

tBHQ

DEM

Oxidative StressReporter

DNA DamageReporter

Mitochondrial MorphologicalReporter

Unfolded ProteinStress ResponseReporter

>50 different candidate reporter cell lines

Image analysis

Safety Assessment: Classification of compounds

into type & severity of toxicity

384 well plateslive or fixed imaging

… and more

ER MorphologicalReporter

CytoskeletalIntegrityReporter

Towards a Pathways of Toxicity HCI Platform

Image acquisition

HCI LIVE CELL IMAGING OF NRF2

Red: nucleus Green: NRF2 in the cytoplasm Yellow: NRF2 in the nucleus

BEFORE TREATMENT AFTER TREATMENT

DEM: low dose medium dose high dose

EXAMPLES OF NGSA SCIENCE

MITOCHONDRIAL TOX PROJECT

Understand how safety may be assured for complex toxicological endpoints using data derived from a toxicity pathways-based approach that is rooted in mechanistic understanding of the underlying biology

PGC-1α pathway perturbation by

doxorubicin induces the

adaptive/adverse response in

human cardiomyocytes

TM

RM

Mit

oS

OX

** *

** **

***

Exposure time: 12h

Mitochondrial superoxide was indicated by MitoSOX

Mitochondrial membrane potential (MMP) was indicated by TMRM

DOX INCREASES MITOCHONDRIAL SUPEROXIDE GENERATION AND DECREASES MMP

26

DOSE RESPONSE OF DOX-INDUCED CARDIAC MITOCHONDRIAL TOXICITY PROFILE

100%

DOX Concentration

AdversityNo

effect

Adaptation

27

Co-ordination is apparent across the stress pathways – How do we utilise this for decision making?

– How to extrapolate this to the individual level with exposure and temporal aspects?

CHALLENGES IN USING CELL STRESS PATHWAYS

Judson RS, Kavlock RJ, Setzer RW, Hubal EA, Martin MT, Knudsen TB, Houck KA, Thomas RS, Wetmore BA, Dix DJ.

Three Year Collaborative Research Agreement:

Further development of:

1. ToxCast technologies for i.d. of MIEs2. High throughput transcriptomics3. Integration of metabolic competence4. Translation of results into next

generation safety assessments (BPAD/RD/IVIVE) for case study chemicals

Assessing health risks of chemical ingredients without animal studies

Better reflecting the actual risk associated with intended human exposure

Office of Research and DevelopmentNational Center for Computational Toxicology

High-throughput Risk Assessment for ER bioactivity

Slide from Dr Richard Judson, EPA,

with thanks.

0.005% 0.1%Level in Product

Bioactivity (In vitro IC50)/ Predicted Plasma Conc)

IC50 90nM

Predicted Plasma Conc 33nM

Predicted Plasma Conc

IC50 90nM

COMPARISON OF PREDICTED EXPOSURE AND IN VITRO IC50 (EPA PRINCIPLES):UNILEVER INGREDIENT

MOE

NGSA STRATEGY TO GET TO MARKET: ON-TARGET (PATHWAY) DISRUPTION

Systemic Exposure

Dose0.005% 0.02% 0.1% 0.2%

EBW?

In Vitro IC50

Systemic Dose From PBPK Model

In Vitro Kinetics

PKPD ModelClinical Confirmation of Exposure

Clinical Safety Biomarkers

In Vitro PKPD Model Parametrisation

EBW

In Vitro IC50

Systemic Dose From PBPK Model

In Vitro Kinetics

Clinical Confirmation of Exposure

Clinical Safety Biomarkers

In Vitro PKPD Model Parametrisation

Full PKPD Model with Simulations for Ingredient

EBW = Exposure based waiving

WOE FOR OFF-TARGET EFFECTS (BESPOKE APPROACH FOR INGREDIENT)

Dose0.005% 0.02% 0.1% 0.2%

Transcriptomics in multiple cell types

Cerep “Safety Screen 44”

BioSeekTranscriptomics in Multiple Cell Types

Extended Cerep Screen

In Vitro liver enzyme induction assay Proteomics?

Metabolomics?ToxCast data

Clinical Safety Data

BIG CHANGES IN SCIENCE BEING USED IN TOXICOLOGY: MULTIDISCIPLINARY RESEARCH

Non-animal approaches to assure safety rely on a new network of scientific disciplines working together• Exposure science

• Computational/mathematical modelling

• Informatics

• Complex 3D cell/tissue culture/imaging

• Molecular and high content biology

• Transcriptomics and proteomics

• Mechanistic chemistry

New challenges around standards and quality

Reynolds et al (2014), Biochemist, 36, 19-25

CONCLUSIONS

Changing global environment for toxicology –demands next generation safety assessments

Multi-disciplined, creative, bespoke solutions –characterising pathway perturbations in context with actual exposures

Chinese toxicologist need not wait for EU/USA to provide ‘validated tests’ where they are not already available – need to establish robust, quality science to provide solutions here and now

Chinese Centre of Excellence for NGSA?

ACKNOWLEDGEMENTS

Hamner/ScitoVation

Rebecca Clewell

Bowen Huang

Salil Pendse

Bin Sun

Sean Rowley

Patrick McMullen

Pergentino Balbuena

Joe Trask

Susan Ross

Linda Pluta

Qiang Zhang (Emory)

Melvin Andersen

Unilever

Andy White

Yeyejide Adeleye

Alistair Middleton

Kristina Castle

Sarah Cooper

Carol Courage

Penny Jones

Gaurav Jain

Stephen Glavin

Jaya Vethamanickam

Jin Li

Paul Fowler

Matt Dent

Sophie Malcomber

Beate Nicol

EPA

Rusty Thomas

Imran Shah

Richard Judson

Strand Life Science

Kas Subramanian

Narasimha M.K

Nalina R

Sonali Das

Leiden University

Bob van de Water

Stephen Winks

AMMS

Shuangqing Peng

Jiabin Guo

Haitao Yuan

Tingfen Zhang

Lan Cui

Minyue Hou

Jian Yin

Xu Ping

谢谢Xièxiè

www.TT21C.org