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
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