PATHWAYS-BASED CHEMICAL TOXICITY

ASSESSMENT – UNILEVER PERSPECTIVES

CARL WESTMORELAND AND PAUL CARMICHAEL

WEDNESDAY, MAY 11TH, 2016

1

THE REASONWE’RE DOING IT

2

NON-ANIMAL APPROACHES TO ASSURING CONSUMER SAFETY

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

3

SAFETY ASSESSMENT PROCESS FORINGREDIENTS 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

EXPOSURE HAZARD

4

THE CURRENT APPROACH

NOAEL*

NOAEL ÷10, 100 or

1000

5

* NOAEL = No Observed Adverse Effect Level

‘SAFE’ DOSE

CONFIDENCE IN THE CURRENT APPROACH

NOAEL*

NOAEL ÷10, 100 or

1000

“animals are intact biological systems -a good model of the

human system” “not the ideal model of uncertainty but it

is pragmatic”

“there’s a broad level of acceptance in the approach (scientific

& regulatory)”

“we've done it this way for decades and it seems to

work”

6

* NOAEL = No Observed Adverse Effect Level

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.”

7

AOP

IMPLEMENTATION OF NAS TT21C

TT21C

High-throughput screening

Focused pathways approach

8

EXPOSURE

HAZARD

10

THE CENTRAL ROLE OF EXPOSURE SCIENCE

Start with exposure rather than toxicity

‘ ……. Human safety depends on exposure and toxicity. Indeed, the 2012 European Commission report on addressing the NewChallenges for Risk Assessment states, “ A paradigm shift is likely from a hazard-driven process to one that is exposure-driven” ….’

Pastoor et al (2014), Crit Rev Toxicol, 44(S3): 1–5

9

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

11

Particle size distribution in liquid or particulate aerosols will determine penetration depth of materials into the respiratory tract.

HUMAN INHALATION EXPOSURE

12

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 13

SUCCESSES IN NON-ANIMAL ALTERNATIVES

• Internationally accepted toxicity tests that do not use animals.

• Guidelines published by OECD

14

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)

ADVERSE OUTCOME PATHWAYS (AOP)

Adapted from OECD (2012)

16

ADVERSE OUTCOME PATHWAYS (AOP)

Adapted from OECD (2012)

17

Dr T Allen

Dr P Russell

BIG CHANGES IN SCIENCE BEING USED IN TOXICOLOGY:MULTIDISCIPLINARY RESEARCH

Non-animal approaches to assuring consumer safety rely on a new network of scientific disciplines working together

• Exposure science

• Computational/mathematical modelling

• Informatics

• Complex 3D cell/tissue culture

• Molecular and high content biology

• Transcriptomics, proteomics and metabolomics

• Mechanistic chemistry

New challenges around standards and quality

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

19

Global Market

Test Markets

Absolute Risk Assessmentvs.

Benchmarking Risk Management

Clinical Studies

Biomarkers

20

Case studies where we bring all of the relevant information together for a specific question

Identifies where the uncertainties lie in the process as it evolves

• Which areas to do further work on?

BUILDING CONFIDENCE IN PATHWAYS APPROACHES

21

AOP

IMPLEMENTATION OF NAS TT21C

TT21C

High-throughput screening

Focused pathways approach

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

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

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

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

EVALUATING DOSE RESPONSE FOR DNA DAMAGE

0.0001 0.001 0.01 0.1 1 10 1000

20

40

60

80

Concentration (M)

To

tal p

53 %

Resp

on

der

Total p53

Clewell et al., 2014. Toxicol. Sci. 142(1)

Con

Overlay

ETP

p-H2AX p53 BP1

DNA REPAIR CENTERS CAN BE COUNTED USING HCI

24h

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

QUANTITATION OF DNA REPAIR CENTERS BY HCI

Fold

ch

ange

Fold

ch

ange

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

Etoposide (μM)Etoposide (μM)

Etoposide (μM)Etoposide (μM)

Li et al, 2014

BIODYNAMIC MODEL (FED BY HCI)

Basal

ETP

DSBH2AX

DSBγ-H2AX

ATM

ATMp

p53

p53

p

Φ

MN

Mass conversion

Activation

Inhibition

Positive Feedback Loop

Degradation

Micronuclei

p53 (Inactive)

Phospho-p53 (Active)

Basal DSB production

Etoposide

Free ATM

ATM recruited to break site

Phosphorylated ATM (active)

Complex of DSB & H2AX

Complex of DSB & γH2AX

Complex of TopII, DSB&H2AXTopIIDSBH2AX

ETP

DSBH2AXrepair repair

ATMp

p53

p

p53

MN

Φ Φ

Φ

DSBγH2AX

ATM

Basal

TopIIDSBH2AXrepair

Basal

ETP

ETP

4

6

7810

17

22

2120

24 25

12

15

23

26

11

1

14

52

3

9

13

191618

ATMfree

ATMfree

ATMfree

• 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)

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

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

BUILD A TOOL BOX WITH HCI AND COMPLEMENTARY TECHNIQUES

HCI FEEDING A COMPUTATIONAL MODEL

• Can capture behaviour of NRF2-KEAP1 pathway for specific chemicals

• System of coupled nonlinear ODEs• 15 variables, including: NRF2, KEAP1, GSH, ROS and

SRXN1• 30 parameters representing

• Association and dissociation rates• Translation rates• Transcription rates• Turnover/removal rates• Concentration thresholds

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

HCI CYTOTOXICITY AND MITOCHONDRIAL TOXICITY BY DOX

Nuclei Mito Tracker MitoSOX 39

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

40

DOSE RESPONSE OF DOX-INDUCED CARDIAC MITOCHONDRIAL TOXICITY PROFILE

100%

DOX Concentration

AdversityNo

effect

Adaptation

41

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?

HCI: A POWERFUL TOOL FOR TT21C SAFETY ASSESSMENTCHALLENGES IN USING CELL STRESS PATHWAYS

WORKING WITH SCIENTIFIC PARTNERS GLOBALLY

THANK YOU …

Slides available at www.tt21c.org

谢谢Xièxiè

www.TT21C.org