10/31/2014. Skin Sensitization: Development of Alternative Methods. The 3 Rs of Alternatives

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1 Skin Sensitization: Development of Alternative Methods Cindy A. Ryan The Procter & Gamble Company 2014 PCPC Science Symposium The 3 Rs of Alternatives Refinement alleviate or minimize pain and distress and enhance wellbeing Reduction comparable information with fewer or more information with same Replacement achieve information without the use of animals NH 2 Strategy for Development of Test Methods for Skin Sensitization GP Tests, HRIPT NH 2 Skin penetration Protein binding NH 2 DC DC activation T 1 T reaction LLNA NH 2 1

2 Adverse utcome Pathway and Predictive Testing Chemical Structure & Properties Molecular Initiating Event Cellular rgan rganism 1. Skin Penetration 2. Electrophilic substance: directly or via auto-oxidation or metabolism 7-8. Presentation of Allergic Contact 3-4. Haptenation: 5-6. Activation of haptenated protein by Dermatitis: Epidermal covalent epidermal Dendritic cell resulting inflammation following modification of keratinocytes & in activation & re-exposure to epidermal proteins Dendritic cells proliferation of specific substance due to T cellmediated cell death T cells Key Event 1 Key Events Key Event 4 Adverse utcome In silico SAR/ QSAR In chemico In vitro cell-based Reactivity Assays Modified version of flow diagram from The Adverse utcome Pathway for Skin Sensitisation initiated by Covalent Binding to Proteins, ECD report In Silico Methods: Structure Activity Relationships (SAR/QSAR) SAR are useful for estimating the toxicity of a chemical when actual data are lacking ind 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 ECD 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: ) 2

3 Adverse utcome Pathway and Predictive Testing Chemical Structure & Properties Molecular Initiating Event Cellular rgan rganism 1. Skin Penetration 2. Electrophilic substance: directly or via auto-oxidation or metabolism 7-8. Presentation of Allergic Contact 3-4. Haptenation: 5-6. Activation of haptenated protein by Dermatitis: Epidermal covalent epidermal Dendritic cell resulting inflammation following modification of keratinocytes & in activation & re-exposure to epidermal proteins Dendritic cells proliferation of specific substance due to T cellmediated cell death T cells Key Event 1 Key Events Key Event 4 Adverse utcome In silico SAR/ QSAR In chemico DPRA [P&G] PPRA [P&G] AREc32 [CXR Bio.] Chemical reactivity In vitro cell-based Modified version of flow diagram from The Adverse utcome Pathway for Skin Sensitisation initiated by Covalent Binding to Proteins, ECD report Chemical-Protein Reactivity: Skin Sensitization Nucleophilic-electrophilic interaction: Pro/Pre-Hapten :Nu Hapten E Hapten Protein Protein HAPTENIZATIN 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) Covalent Protein Modification: Key step in the induction of skin sensitization Chemical is reactive Sensitizing chemical applied to the skin Chemical is not reactive Proelectrophile (non-electrophile) Electrophiles (hapten) Mechanism of reaction Via abiotic transformation (pre-haptens) Allergen Via metabolic transformation (pro-haptens) Michael acceptor S NAr S N2 Schiff base Acylating agent Mechanism-based classification for skin sensitizers (modified by Aynur et al. 2007) 3

4 In Chemico: Direct Peptide Reactivity Assay (DPRA) Method 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) Test chemical in solvent N + - N Synthetic model peptides in buffer Lysine (Ac-RAAKAA-CH) 1:50 at ph 10.2 Incubation for 24 h, 25 C (dark) N N N + N Cysteine (Ac-RAACAA-CH) 1:10 at ph 7.4 In Chemico: Direct Peptide Reactivity Assay (DPRA) Method Calculation of peptide depletion HPLC/PDA Prediction Model based on Cys 1:10 and Lys 1:50 (n=81) Total Sample (29 / 15 / 20 / 17) NS/W/M/S Avg Score < 22.62% Avg Score > 22.62% Test (29 / 11 / 3 / 0) Test (0 / 4 / 17 / 17) Avg Score < 6.376% Avg Score > 6.376% Avg Score < 42.47% Avg Score > 42.47% Minimal Reactivity (26 / 5 / 1 / 0) Low Reactivity (3 / 6 / 2 / 0) Moderate Reactivity (0 / 1 / 6 / 3) High Reactivity (0 / 3 / 11 / 14) Non-sensitizing Sensitizing 4

5 ECVAM endorsement DPRA ECVAM Pre-validation started in 2009 (after successful inter-laboratory evaluation). Development of SP and testing ECVAM acceptance December 2013 Next Generation Peptide Reactivity Assay: Peroxidase Peptide Reactivity Assay bjective: Develop a modified version of the DPRA that includes metabolic activation and LC/MS/MS detection methods. Principle of the Assay H 2 H 2 2 Test Chemical enton Chemistry e 2+ + H 2 2 X xidant Blocked by desferroxamine Not quantified but can be observed using MALDI P e r o x i d a s e ( ) P e r o x i d a s e ( R ) Auto-oxidation Peptide (nucleophile) Peptide-chemical ADDUCT Reactive Metabolite (electrophilic hapten) Readout = Non-adducted peptide monomer measured by LC/MS/MS reversed by DTT X Peptide Dimer Monitored but not quantified vs monomer In Chemico: Peroxidase Peptide Reactivity Assay (PPRA) Method Test Chemical Peroxidase Peptide Reactivity Assay Non-enzymatic reactivity Enzymatic reactivity Lysine (- HRP/P) Pot. Phos. (ph 7.4) 25% final organic Conc. range mm Cysteine (- HRP/P) Pot. Phos. (ph 7.4) 1% final organic Conc. range mm Cysteine (+ HRP/P) Pot. Phos. (ph 7.4) 1% final organic Conc range mm Sample processing and analysis by LC/MS/MS Data reduction, hazard prediction and reactivity potency estimation 5

6 In Chemico: PPRA Data Analysis and Prediction Model Analyze peptide depletion data (Cysteine + HRP/P) <15.1% DP MAX* >24.9% Minimally Reactive (MR) (reactivity is not quantified by EC25) Non-sensitizing 15.1% < DPMAX < 24.9% Interpret data Quantify and Classify (analyze & positives classify) Lowest Lowest EC25 >0.1 mm EC25 <0.1 mm Reactive (R) Highly Reactive (HR) *DP MAX - highest depletion value observed across the concentration range examined. Sensitizing 1. Analyze depletion data for hazard prediction: (yes/no) 2. Quantify reactivity: EC25 3. Classify chemicals: Minimally Reactive (MR), Reactive (R), Highly Reactive (HR) or hazard prediction, chemicals that were classified as MR were considered non-sensitizers and R or HR classified chemicals were considered sensitizers. In Chemico: PPRA Inter-Laboratory Study 3 Participating Labs (1 US, 2 European) 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 Adverse utcome Pathway and Predictive Testing Chemical Structure & Properties Molecular Initiating Event Cellular rgan rganism 1. Skin Penetration 2. Electrophilic substance: directly or via auto-oxidation or metabolism In silico SAR/ QSAR 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 Allergic Contact Dermatitis: Epidermal inflammation following re-exposure to substance due to T cellmediated cell death Key Event 1 Key Events Key Event 4 Adverse utcome Keratinocytes In chemico Modified version of flow diagram from The Adverse utcome Pathway for Skin Sensitisation initiated by Covalent Binding to Proteins, ECD report In vitro cell-based KeratinoSens TM [Givaudan] LuSens [BAS] Dendritic Cells h-clat [KA/Shiseido] MUSST [L real] PBMDC [Beiersdorf] 6

7 Cell-Based In Vitro Test Methods KeratinoSens TM Protocol developed by A. Natsch (Givaudan) The Keap1 Nrf2 ARE signaling pathway of cells specifically responds to electrophiles SH SH Keap1 SH Nrf2 DNA ARE Antioxidant response element ARE-regulated gene 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 ( ) Series of Japanese Inter-laboratory trials (AATEX, 2008, 13(1):27-35; 13(2): 55-62,63-69,70-82) ormally 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) Pre-culture cells for hours ( x 10 6 cells/ml). Plate (1x10 6 cells/well) in 24-well plate, treat with test chemical for 24 hours Harvest cells, wash and block cr (0.01% Globulins) for 15 min. Divide cells into 3 aliquots, stain with ITC-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. 7

8 Alternatives for Skin Sensitization: The Challenge Data Integration Bioavailability DC Activation T cell Activation Metabolism Peptide Reactivity SAR Modeling Simulation Hazard ID and Potency (NESIL) and QRA Data Integration / ITS / WoE / IATA Bayesian Network (P&G) Artificial Neural Network (Shiseido) Weight of Evidence (BAS) Bayesian Network Integrated Testing Strategy (BN-ITS) Graphical Model Target Variable Input Variables - Integrate KEC1.5 Data - Potency TIMES class - Skin logkow bioavailability - Decision Tool - Non - Kastings model - Phys/chem info 7% IC50 Cysteine 6% KEC3 59% 55% 39% DPRACys 16% 20% - Weak 36% - Moderate LLNA Bioavailability - TIMES prediction Cfree -20% Strong 5% - Peptide 57% reactivity - derived from the LLNA 24% 20% 8% CD86 DPRALys Data set n=145 : training set n=124; test set n=21 Jaworska et al. J. Appl. Tox. 2013, 33: ; Dataset published Natsch et al. J. Appl. Tox. 2013, 33: DPRA 59% - Keratinosens - Dendritic cell activation - U937 CD86 AUC120 In the external validation ( n=21) ITS-2 predictions were 86% correct for potency, 95% for hazard. pen 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 8

9 Product 10/31/2014 Skin Sensitization Risk Assessment Risk = Hazard X Exposure? Historical In Silico In Vitro Vivo X A single generic set of tests as in vivo replacement strategy is unlikely to be the most effective. KEC1.5 TIMES logkow Combinations of tests batteries, covering relevant mechanistic steps, organized in a logical way are needed: Bayesian Network 16% 20% 36% 7% 59% LLNA Bioavailability Cfree IC50 Cysteine 20% 5% 57% 6% 55% 24% 20% 8% 59% 39% DPRALys AUC120 KEC3 CD86 DPRACys Jaworska et al. J. Appl. Tox Thank you! 9