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Cindy A. Ryan The Procter & Gamble Company

2014 PCPC Science Symposium

Skin Sensitization: Development of Alternative Methods

The “3 Rs” of Alternatives

Refinement – alleviate or minimize pain and distress and enhance well-being

Reduction – comparable information with fewer or more information with same

Replacement – achieve information without the use of animals

Strategy for Development of Test Methods for Skin Sensitization

NH2

NH2

T

DC

NH2

NH2

GP Tests, HRIPT

LLNA

Skin penetration

Protein binding

DC activation

1° T reaction

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Adverse Outcome Pathway and Predictive Testing

Chemical Structure & Properties

Organism Response

Organ Response

Cellular Response

Molecular Initiating

Event

1. Skin Penetration

2. Electrophilic substance:

directly or via auto-oxidation or metabolism

3-4. Haptenation: covalent

modification of epidermal proteins

5-6. Activation of epidermal

keratinocytes & Dendritic cells

7-8. Presentation of haptenated protein by Dendritic cell resulting

in activation & proliferation of specific

T cells

9-11. Allergic Contact Dermatitis: Epidermal

inflammation following re-exposure to

substance due to T cell-mediated cell death

Key Event 1 Key Events 2 + 3 Key Event 4 Adverse Outcome

In chemico models

In silico models SAR/ QSAR

In vitro cell-based models

Reactivity Assays Modified version of flow diagram from ‘The Adverse

Outcome Pathway for Skin Sensitisation initiated by

Covalent Binding to Proteins’, OECD report

In Silico Methods: Structure Activity Relationships (SAR/QSAR)

SAR are useful for estimating the toxicity of a chemical when actual data are lacking

Find structurally similar chemicals (structural analogs) for which data exist, and then to relate those data to the chemical of interest

Examples

DEREK

ToxTree

TIMES-SS

OECD Toolbox

In Silico Methods: Skin Penetration

Epidermal bioavailability is a pre-requisite for skin sensitization

Amount in epidermis rather than total systemic uptake

Depending on phys/chem properties of the chemical

Lipophilicity/ hydrophilicity, MW, etc.

Example

Kasting Toxicokinetic model (Adv. Drug Deliv. Rev.2013, 65: 221–236.)

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Adverse Outcome Pathway and Predictive Testing

Chemical Structure & Properties

Organism Response

Organ Response

Cellular Response

Molecular Initiating

Event

1. Skin Penetration

2. Electrophilic substance:

directly or via auto-oxidation or metabolism

3-4. Haptenation: covalent

modification of epidermal proteins

5-6. Activation of epidermal

keratinocytes & Dendritic cells

7-8. Presentation of haptenated protein by Dendritic cell resulting

in activation & proliferation of specific

T cells

9-11. Allergic Contact Dermatitis: Epidermal

inflammation following re-exposure to

substance due to T cell-mediated cell death

Key Event 1 Key Events 2 + 3 Key Event 4 Adverse Outcome

In chemico models

In silico models SAR/ QSAR

In vitro cell-based models

Modified version of flow diagram from ‘The Adverse

Outcome Pathway for Skin Sensitisation initiated by

Covalent Binding to Proteins’, OECD report

AREc32 [CXR Bio.]

DPRA [P&G]

PPRA [P&G]

Chemical reactivity

Chemical-Protein Reactivity: Skin Sensitization

Nucleophilic-electrophilic interaction:

Hapten

E

:Nu

The correlation of skin protein reactivity and skin sensitization is well established and has been known for many years. (Landsteiner and Jacobs, 1936; Dupuis and Benezra, 1982; Lepoittevin et al, 1998)

Hapten Pro/Pre-Hapten

Protein

Protein

O

O

F

F

F

O

O

F

F

F

HAPTENIZATION

Covalent Protein Modification: Key step in the induction of skin sensitization

Mechanism-based classification for skin sensitizers (modified by Aynur et al. 2007)

Sensitizing chemical applied to the skin

Electrophiles (hapten)

Michael acceptor domain

SNAr domain

SN2 domain

Schiff base

domain

Acylating agent

domain

Proelectrophile (non-electrophile)

Via abiotic transformation (pre-haptens)

Via metabolic transformation (pro-haptens)

Chemical is not reactive Chemical is reactive

Mechanism of reaction Allergen

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Screening method for evaluation skin sensitization potential (haptens, prehaptens)

Direct Peptide Reactivity Assay (DPRA)

• The reactivity is quantified based on the percentage of peptide depletion (HPLC/PDA)

Incubation for 24 h, 25°C (dark)

Cysteine (Ac-RFAACAA-COOH)

Lysine (Ac-RFAAKAA-COOH)

1:10 at pH 7.4

1:50 at pH 10.2

Synthetic model peptides in buffer

Test chemical in solvent

O

N

N+

O

O-

O

N

N+ O

O-

O

O

N

N+

O

-O

O

In Chemico: Direct Peptide Reactivity Assay (DPRA) Method

HPLC/PDA

Calculation of peptide depletion

In Chemico: Direct Peptide Reactivity Assay (DPRA) Method

Prediction Model based on Cys 1:10 and Lys 1:50 (n=81)

NS/W/M/S

Test (29 / 11 / 3 / 0)

Total Sample (29 / 15 / 20 / 17)

Minimal Reactivity (26 / 5 / 1 / 0)

Low Reactivity (3 / 6 / 2 / 0)

Avg Score < 6.376%

Avg Score < 22.62% Avg Score > 22.62%

Test (0 / 4 / 17 / 17)

Moderate Reactivity (0 / 1 / 6 / 3)

High Reactivity (0 / 3 / 11 / 14)

Avg Score < 42.47%

Avg Score > 6.376% Avg Score > 42.47%

Non-sensitizing Sensitizing

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ECVAM endorsement DPRA

• ECVAM Pre-validation started in 2009 (after successful inter-laboratory evaluation).

• Development of SOP and testing 2010-2011.

• ECVAM acceptance December 2013

Principle of the Assay

Objective: Develop a modified version of the DPRA that includes metabolic activation and LC/MS/MS detection methods.

Next Generation Peptide Reactivity Assay: Peroxidase Peptide Reactivity Assay

Test Chemical

Peptide-chemical ADDUCT

Readout = Non-adducted peptide monomer measured by LC/MS/MS

Monitored but not quantified vs monomer

X reversed by DTT

Auto-oxidation

Not quantified but can be observed using MALDI

Reactive Metabolite (electrophilic hapten)

P e r o x i d a s e ( O ) P e r o x i d a s e ( R )

H 2 O 2 H 2 O

Fe2+ + H2O2 Oxidant

Blocked by desferroxamine

X Fenton Chemistry

Peptide (nucleophile)

Peptide Dimer

Test Chemical

Peroxidase Peptide Reactivity Assay

Lysine (- HRP/P) Pot. Phos. (pH 7.4) 25% final organic Conc. range 0.01-25 mM

Enzymatic reactivity Non-enzymatic reactivity

Cysteine (- HRP/P) Pot. Phos. (pH 7.4) 1% final organic Conc. range 0.04-5 mM

Cysteine (+ HRP/P) Pot. Phos. (pH 7.4) 1% final organic Conc range 0.04-5 mM

Sample processing and analysis by LC/MS/MS

Data reduction, hazard prediction and reactivity potency estimation

In Chemico: Peroxidase Peptide Reactivity Assay (PPRA) Method

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Quantify and Classify ‘positives’

Minimally Reactive (MR)

(reactivity is not quantified by EC25)

Analyze peptide depletion data (Cysteine + HRP/P)

DPMAX*

>24.9% <15.1%

1. Analyze depletion data for hazard prediction: (yes/no) 2. Quantify reactivity: EC25 3. Classify chemicals: Minimally Reactive (MR), Reactive (R), Highly Reactive (HR)

For hazard prediction, chemicals that were classified as ‘MR’ were considered non-sensitizers and ‘R’ or ‘HR’ classified chemicals were considered sensitizers.

Highly Reactive (HR) Reactive (R)

Lowest EC25 <0.1 mM

Lowest EC25 >0.1 mM

*DPMAX - highest depletion value observed across the concentration range examined.

Interpret data

(analyze & classify)

15.1% < DPMAX < 24.9%

In Chemico: PPRA Data Analysis and Prediction Model

Non-sensitizing

Sensitizing

• Phase A: Training and preliminary transferability assessment – 12 non-blinded chemicals tested once

– Completed

• Phase B: Definitive method transfer assessment – 24 coded chemicals tested once (BLR)

– 12 coded chemicals tested in 3 independent runs (WLR)

– Expected completion by end of 2014

In Chemico: PPRA Inter-Laboratory Study 3 Participating Labs (1 US, 2 European)

Adverse Outcome Pathway and Predictive Testing

Chemical Structure & Properties

Organism Response

Organ Response

Cellular Response

Molecular Initiating

Event

1. Skin Penetration

2. Electrophilic substance:

directly or via auto-oxidation or metabolism

3-4. Haptenation: covalent

modification of epidermal proteins

5-6. Activation of epidermal

keratinocytes & Dendritic cells

7-8. Presentation of haptenated protein by Dendritic cell resulting

in activation & proliferation of specific

T cells

9-11. Allergic Contact Dermatitis: Epidermal

inflammation following re-exposure to

substance due to T cell-mediated cell death

Key Event 1 Key Events 2 + 3 Key Event 4 Adverse Outcome

In chemico models

In silico models SAR/ QSAR

In vitro cell-based models

Modified version of flow diagram from ‘The Adverse

Outcome Pathway for Skin Sensitisation initiated by

Covalent Binding to Proteins’, OECD report

KeratinoSensTM [Givaudan]

LuSens [BASF]

Keratinocytes

PBMDC [Beiersdorf]

h-CLAT [KAO/Shiseido]

MUSST [L’Oreal]

Dendritic Cells

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Cell-Based In Vitro Test Methods

KeratinoSensTM Protocol developed by A. Natsch

(Givaudan)

The Keap1 – Nrf2 – ARE signaling pathway of cells specifically

responds to electrophiles

Nrf2

Keap1

SH SH SH

ARE-regulated gene ARE DNA Antioxidant response element

In Vitro Cell-Based: Human Cell Line Activation Test (hCLAT)

• Cell-based method with THP-1 cells as surrogate DCs – Developed by scientists at Kao and Shiseido (2003) – Cosmetics Europe Ring Trials (2004-2006) – Series of Japanese Inter-laboratory trials (AATEX, 2008,

13(1):27-35; 13(2): 55-62,63-69,70-82)

• Formally evaluated in a EURL ECVAM-coordinated validation study in collaboration with JaCVAM – Submission completed in December 2008 – Expected completion by end of 2014

In Vitro Cell-Based: Human Cell Line Activation Test (hCLAT)

Plate (1x106 cells/well) in 24-well plate, treat with test chemical for 24 hours

Pre-culture cells for 48-72 hours (0.2-0.4 x 106 cells/mL).

Harvest cells, wash and block FcR (0.01% Globulins) for 15 min.

Divide cells into 3 aliquots, stain with FITC-conjugated monoclonal antibodies

(isotype control, CD86, CD54) for 30 min.

Analyze by flow cytometry - mean fluorescence intensity of CD86 and CD54,

cell viability by propidium iodide exclusion.

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Alternatives for Skin Sensitization: The Challenge – Data Integration

Hazard ID and Potency

(NESIL) and QRA

Bioavailability

SAR

Peptide Reactivity

T cell Activation

Metabolism

DC Activation

Modeling Simulation

Data Integration / ITS / WoE / IATA

Bayesian Network (P&G)

Artificial Neural Network (Shiseido)

Weight of Evidence (BASF)

IC50

KEC1.5

7%

6%

KEC3

Cysteine

59%

55%

DPRACys

39%

CD86

LLNA

DPRALys

24% 20% 8%

16%

59%

Bioavailability

57% 5% 20%

20% 36%

TIMES logKow

Cfree

AUC120

Jaworska et al. J. Appl. Tox. 2013, 33: 1353–1364; Dataset published Natsch et al. J. Appl. Tox. 2013, 33:1337-1352

Data set n=145 : training set n=124; test set n=21

In the external validation ( n=21) ITS-2 predictions were 86% correct for potency, 95% for hazard.

• Graphical Model - Integrate Data - Decision Tool

• Target Variable - Potency class

- Non - Weak - Moderate - Strong

- derived from the LLNA

• Input Variables - Skin bioavailability

- Kastings model - Phys/chem info

- TIMES prediction - Peptide reactivity

- DPRA - Keratinosens

- Dendritic cell activation - U937 CD86

Bayesian Network Integrated Testing Strategy (BN-ITS)

Open source ITS Pirone et al. (2014) developed R version of the original BN ITS-2 and produced consistent results. The data

and a fully documented version of the code are publicly available http://ntp.niehs.nih.gov/go/its.

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Risk =

Pro

du

ct

X

Hazard X Exposure

Historical In Silico In Vivo

Skin Sensitization Risk Assessment

In Vitro

? A single generic set of tests as in vivo replacement strategy is unlikely to be the most effective.

Combinations of tests batteries, covering relevant mechanistic steps, organized in a logical way are needed: Bayesian Network

IC50

KEC1.5

7%

6%

KEC3

Cysteine

59%

55%

DPRACys

39%

CD86

LLNA

DPRALys

24% 20% 8%

16%

59%

Bioavailability

57% 5% 20%

20% 36%

TIMES logKow

Cfree

AUC120

Jaworska et al. J. Appl. Tox. 2013

Thank you!