Global Harmonization Efforts for Skin Sensitization IATA

Nicole C. Kleinstreuer, PhDActing DirectorNational Toxicology Program Interagency Center for the Evaluation of Alternative Toxicological Methods (NICEATM)Nicole.Kleinstreuer@nih.gov

Conflict of Interest Statement

No conflict of interest to declare. This content does not represent official US government policy.

Overview/Objectives

The role of ICCVAM and the National Strategic Roadmap OECD and the AOP Framework for IATA Development Understanding Defined Approaches (DAs) Skin Sensitization as a Case Study DAs for Hazard and Potency Characterization Progress toward Regulatory Implementation

Interagency Coordinating Committee for the Validation of Alternative Methods H.R. 4281 (106th): ICCVAM Authorization Act of 2000 To establish, wherever feasible, guidelines, recommendations, and regulations that

promote the regulatory acceptance of new and revised toxicological tests that protect human and animal health and the environment while reducing, refining, or replacing animal tests and ensuring human safety and product effectiveness.

7 Regulatory AgenciesConsumer Product Safety CommissionDepartment of AgricultureDepartment of the InteriorDepartment of TransportationEnvironmental Protection AgencyFood and Drug Administration Occupational Safety and Health Administration

9 Research AgenciesAgency for Toxic Substances and Disease Registry National Institute for Occupational Safety and HealthNational Cancer InstituteNational Institute of Environmental Health SciencesNational Library of MedicineNational Institutes of HealthDepartment of DefenseDepartment of EnergyNational Institute of Standards and Technology

• Other participants include: NCATS, Tox21 Representatives

ICCVAM

https://ntp.niehs.nih.gov/go/natl-strategy

US National Strategic Roadmap

The AOP Framework for IATA Development

Gather existing information

Problem formulation

Weight of Evidence Assessment: Adequate information for decision-making?

Generate additional information

Weight of Evidence assessment: Adequate information for decision-making?

Regulatoryconclusion

YES

NO

YES

NO

AOP

Multiple strategies e.g. in house data, mining of relevant data bases,

literature search

Expert Judgement

Patience Browne, OECD

Guidance on the AOP Framework

http://www.oecd.org/chemicalsafety/risk-assessment/iata-integrated-approaches-to-testing-and-assessment.htm

Defined approaches:– Remove expert judgement– Are not flexible and are suitable for harmonisation

OECD has the first proposal for Defined Approach that would be covered by mutual acceptance of data (MAD)

From IATA to DAsIATA Defined Approaches

Designed in response to problem formulation

Designed to address pre-defined endpoint/prediction

Inputs are defined by user Defined information sources

Sequence of input, next steps, decision context defined by user

Sequence defined and next steps are rule-based

Expert judgement for weighting data, interpreting data

Fixed data interpretation procedure

Conclusion may be open to interpretation

Regulatory conclusion is clear

IATA

Designed in response to problem formulation

Inputs are defined by user

Sequence of input, next steps, decision context defined by user

Expert judgement for weighting data, interpreting data

Conclusion may be open to interpretation

DA Evaluation Framework

• Structure• DA Elements • Information Provided

• Relevance• Mechanistic Coverage

• Predictive Capacity• Performance Compared

to Reference Data • Reliability

• Reproducibility

• Applicability

• Technical Limitations

• Chemical Space

• Complexity

• Data Interpretation Procedure

• Transparency

• Availability of Elements

AOP for Skin Sensitization

Chemical Structure & Properties

Molecular Initiating Event

Cellular Response

Organ Response Organism Response

MetabolismPenetration

Electrophilicsubstance

Covalent interaction with skin proteins

Key Event 1

• Activation of inflammatory cytokines

• Induction of cytoprotective genes

Keratinocytes responsesKey Event 2

• Induction of inflammatory cytokines and surface molecules

• Mobilisation of DCs

Dendritic Cells (DCs)Key Event 3

• Histocompatibility complexes presentation by DCs

• Activation of T cells• Proliferation of

activated T-cells

• Inflammation upon challenge with allergen

Key Event 4 Adverse OutcomeT-cell proliferation

DENDRITIC CELLs

MIGRATION TO LOCAL LYMPH NODE

T-CELL

PROLIFERATION

*Adapted from illustration by D. Sailstad

KERATINOCYTES

OECD (2014)

Test Methods Mapped to AOP

Chemical Structure & Properties

Molecular Initiating Event

Cellular Response

Organ Response Organism Response

MetabolismPenetration

Electrophilicsubstance

Covalent interaction with skin proteins

• Induction of inflammatory cytokines and surface molecules

• Mobilisation of DCs

• Activation of inflammatory cytokines

• Induction of cytoprotective genes

• Histocompatibility complexes presentation by DCs

• Activation of T cells• Proliferation of

activated T-cells

• Inflammation upon challenge with allergen

Dendritic Cells (DCs)

Keratinocytes responses

Key Event 1

Key Event 2

Key Event 3Key Event 4 Adverse

OutcomeT-cell proliferation

TG442C

TG442E

TG442D

In Vitro In Vivo

GPMT

LLNA

DPRAADRA

KeratinoSensLuSens

hCLAT, USENS, IL-8

Objective: analysis of available non-animal DAs for skin sensitization NICEATM collaboration with Cosmetics Europe

– Curation/generation of in vivo LLNA and human data in vitro cell-based data that maps to AOP in silico computer predictions, chemical

structural features & properties

Qualitative and quantitative evaluation of DAs used in OECD guidance documents as case studies

Fully transparent approach (i.e., build open-source code packages) Evaluate performance against LLNA and human hazard/potency categories Develop international harmonized OECD test guideline

Hoffmann et al. 2018 Crit Rev Tox Kleinstreuer et al. 2018 Crit Rev Tox

Global Skin Sensitization Project

Meta models

Support vector

machine

Artificial Neural

Networks

Sequential Testing Strategy

Integrated TestingStrategy

Regression Model

2 out of 3 Consensus

PREDICTION

Bayesian Networks

Defined Approaches

2 out of 3

Kao KE 3/1 STSTotal Score

Prediction

6-7 Strongsensitiser

2-5 Weaksensitiser

0-1 Not classified

Kao ITSv2

TB

Defined Approaches

Hazard and Potency

Categorization

Artificial Neural Network Models

Continuous EC3 prediction Can be translated into potency classes:

NS, Weak/Moderate, Strong/Extreme Built using proprietary software

(QwikNet), reproduced in R Two models:

DPRA, hCLATDPRA, hCLAT, KeratinoSens

Run over multiple iterations and averaged

Hirota et al. 2015

Quantitative EC3 Prediction

Historical Accuracy of Animal Tests

Hazard

72%-82%

Potency (GHS)

54% - 60%

Hazard

~72%

Potency (GHS)

~60%

GPMT / BuehlerLLNAAgainst Human Clinical Patch Test Data

DAs 77%-85% 63%-69%

Hazard Potency (GHS)

Non-Animal Defined Approach Evaluation

Most non-animal testing strategies evaluated so far perform better than the LLNA at

predicting human skin sensitization hazard and potency.

(And when compared to the LLNA, are equivalent in performance to the LLNA at

predicting itself.)Hoffmann et al. 2018 Crit Rev Tox

Kleinstreuer et al. 2018 Crit Rev Tox

US EPA Release of Draft Interim Science Policy: Use of Alternative Approaches for Skin Sensitization as a Replacement for Laboratory Animal Testing (10 April 2018)

– Joint policy between Office of Pesticide Programs (OPP) and Office of Pollution Prevention and Toxics (OPPT)

– Applies to pesticide active ingredients, inerts, and single chemicals regulated under amended TSCA

– Two DAs currently accepted: “AOP 2 out of 3” and “KE 3/1 STS”– Includes assays covered by the respective KE-based OECD TGs – Policy will be updated to accept more DAs as the OECD GL work develops

US EPA Regulatory Progress

https://www.epa.gov/pesticides/epa-releases-draft-policy-reduce-animal-testing-skin-sensitization

In February 2020, the U.S. Food and Drug Administration (FDA) issued draft guidance on “Nonclinical Safety Evaluation of the Immunotoxic Potential of Drugs and Biologics.”

– FDA no longer recommends that sponsors conduct the murine local lymph node assay to assess the sensitization potential of topical drug products due to the limitations of the assay.

– As an alternative to accepted guinea pig tests, FDA will consider a battery of in silico, in chemico, and in vitro studies that have been shown to adequately predict human skin sensitization with an accuracy similar to existing in vivo methods

US FDA Regulatory Progress

https://www.fda.gov/regulatory-information/search-fda-guidance-documents/nonclinical-safety-evaluation-immunotoxic-potential-drugs-and-biologics-guidance-industry

OECD DA SS Guideline Project

2017 work plan: JRC/US/Canada co-leads– Aims to substitute the need for animal testing for skin sensitisation

based on a combination of methods which, individually, predict key endpoint responses on the AOP

– Resulting guideline will be amenable to the agreement on Mutual Acceptance of Data (MAD)

To meet regulatory requirements, need: – DAs that discriminate skin sensitisers from non sensitisers, – DAs that discriminate strong from moderate/weak sensitisers (GHS

potency categories).

Future work will cover DAS that address regulatory needs of quantitative risk assessment

Expert Group on DA SS

68 members covering regulatory authorities, OECD national coordinators, validation experts, animal welfare and industry stakeholders, method developers, etc.

Focusing on resolution of scientific issues:1. Finalization of curated reference data2. Performance comparison3. Applicability Domain 4. Confidence and Uncertainty

Update, discussion, and feedback received at April 2020 WNT (virtual) meeting Virtual 2-day F2F meeting in June 2020 to discuss outstanding issues and working towards

finalizing draft DA guideline Planning for final OECD DA guideline to be submitted for written approval by late 2020

NTP is supporting testing of a broad range of chemicals in:DPRA, KeratinoSens, hCLAT

– Pesticide actives, agrochemical formulations, dermal excipients, personal care product ingredients, “challenge” chemicals

Chemical nominations from multiple agencies/stakeholders– US FDA– EPA: OPP, OPPT, ORD– CPSC– NTP– ICATM partners

Testing began in late 2017 and will be completed in 2020– Assays are ongoing with approximately 125 chemicals completed to date

Expanding Coverage of Chemical Space

AcknowledgementsOECD Patience Browne Anne GourmelonUS EPA Anna Lowit Evisabel CraigEURL ECVAM Silvia Casati David AsturiolHealth Canada Michele Regimbald-Krnel Cameron Bowes Pierre Therriault

NICEATM GroupUS FDA Paul Brown Suzy FitzpatrickOECD EG DA SSICCVAM EG Skin Sens

Extra Slides

Direct Peptide Reactivity Assay (DPRA)

Assesses protein reactivity of a test substance

Uses two heptapeptides– One with cysteine (Cys) and one with lysine

(Lys) as the reactive center– Incubate with test substance and measure

disappearance of peptides with HPLC Average depletion > 6.38% = sensitizer

– OECD Test Guideline 442C (2019)

Unreacted peptide

Test chemical

Reaction mixture

Measurements%Cys peptide depletion%Lys peptide depletion%Ave.Lys.Cys depletion

Assesses the activation of an ARE element as an indication of keratinocyte activation– Uses KeratinoSens cells (derived from HaCaT

keratinocytes), which contain a luciferase reporter

– Electrophilic agents, which tend to be skin sensitizers, activate the ARE

– Measures fold-induction of luciferase activity; induction >1.5-fold = sensitizer

– OECD Test Guideline 442D (2018) MeasurementsFold inductionEC1.5, EC3.0% viability

KeratinoSens™

Measures 2 cell surface markers, CD86 and CD54, on dendritic cell surrogates (THP-1 cells)

– Assesses the maturation process of dendritic cells as they transform from antigen processing cells to antigen presenting cells

– CD86 relative fluorescence intensity (RFI) ≥150% and/or CD54 RFI ≥ 200% at any dose indicates a sensitizer

– OECD Test Guideline 442E (2017)

h-CLAT

THP-11x106 cells /mL

24 h

Chemical exposure at8 doses based on CV75 Cell staining (CD86 & CD54)

Flow cytometric analysis

MeasurementsEC200 (CD54), EC150 (CD86)% viability (CV75)

FcR blocking27

OECD QSAR Toolbox v4.3 used for in silico read-across skin sensitization hazard predictions

– Based on chemical grouping with structurally and mechanistically similar analogues using LLNA and guinea pig data

– Used automated workflow for skin sensitization

– When automated workflow failed to make a prediction (due to insufficient number of analogues), Skin Sensitization Profiler for DASS is applied to parent chemical, auto-oxidation products, and skin metabolites

OECD Toolbox Protocol and Results