Finding the right skin sensitization testing strategy for “difficult-to-test” substances

A practical guide for regulatory safety assessment and product development


difficult-to-test substances

Why selecting the right testing strategy matters

As regulatory safety assessment increasingly relies on non-animal methods, evaluating the skin sensitization potential of difficult-to-test substances remains a significant challenge. Materials such as pre- and pro-haptens, hydrophobic substances, UVCBs, botanical extracts, formulations, metals, and medical device extracts may not fit neatly within the applicability domains of conventional in vitro assays.

Choosing an appropriate testing strategy is therefore critical. Selecting a testing strategy that is not well suited to the substance and testing objective can lead to inconclusive results, unnecessary repeat testing, regulatory uncertainty, increased costs, and delayed development timelines. The most effective strategy depends on three factors:
• The characteristics of the test substance
• The purpose of the assessment
• The applicability domains of available test methods

This guide outlines how to select fit-for-purpose non-animal testing strategies for difficult-to-test substances, with a particular focus on OECD Guideline 497: Defined Approaches for Skin Sensitization (DASS) and the role of GARD®skin (OECD TG 442E).

Key takeaways

  • Difficult-to-test substances commonly include pre- and pro-haptens, hydrophobic substances, complex mixtures such as UVCBs and botanical extracts, and materials with unique biological behaviour, including metals and medical device extracts.
  • These substances may challenge the applicability domains of conventional skin sensitization methods.
  • OECD Guideline 497: Defined Approaches for Skin Sensitization (DASS) provide standardized frameworks for integrating data from multiple NAMs.
  • Successful strategy selection depends on substance characteristics, testing objectives, and method applicability domains.
  • GARD®skin demonstrates broad applicability across several difficult-to-test substance categories and can help address data gaps where other methods face limitations.
  • For product development, combining hazard identification and potency assessment can support safer formulation decisions and candidate prioritization.
Author: Andy Forreryd, PhD
  • In vitro toxicology expert with extensive experience in non-animal skin sensitization assessment and difficult-to-test substances.
  • Member of ISO/TC 194/WG 8 on irritation and sensitization and the OECD expert group on respiratory sensitization.

What is the best testing strategy for difficult-to-test substances?

There is no single testing strategy suitable for all difficult-to-test substances. The optimal approach depends on:

  • The type and composition of the substance
  • Solubility and other physicochemical properties
  • Whether metabolic activation may be required
  • Regulatory or product development objectives
  • The applicability domains of the selected methods
  • Whether the goal is hazard identification, potency assessment, or both

For many challenging materials, robust non-animal assessment can be achieved using OECD TG 497 Defined Approaches that incorporate methods such as GARD®skin, EpiSensA, and ADRA, provided that method selection is aligned with the substance characteristics, applicability domains, and testing objectives.

Who is this guide for?

This guide is intended for:

  • Toxicologists
  • Regulatory affairs professionals
  • Cosmetic safety assessors
  • Medical device product safety managers
  • Product development scientists
  • Safety and risk assessment professionals

What are difficult-to-test substances?

“Difficult-to-test substances” are materials whose physicochemical properties, composition, or biological behaviour make them challenging to evaluate using conventional in vitro methods.

In skin sensitization assessment, difficult-to-test substances typically fall into one or more of the following categories:

  • Pre- and pro-haptens, which require chemical or metabolic activation before they can induce a sensitization response.
  • Hydrophobic or poorly soluble substances, which can be difficult to evaluate in aqueous test systems.
  • Complex mixtures, formulations, and UVCBs (Unknown or Variable Composition, Complex Reaction Products, or Biological Materials), which may contain numerous constituents with different chemical properties and biological activities.
  • Materials with unique biological behaviour, including metals, polymers, and medical device extracts.

These materials are encountered across a wide range of industries, including chemicals, cosmetics, fragrances, personal care products, medical devices, and specialty materials. While they differ considerably in composition and use, they often share characteristics that can challenge the performance or applicability of conventional in vitro methods used for skin sensitization assessment.

New to skin sensitization assessment? Start with our Quick Guide to Skin Sensitization Testing, which provides an overview of sensitization mechanisms, key regulatory concepts, and the most commonly used non-animal methods.

Why are some substances difficult to test for skin sensitization?

The challenges associated with difficult-to-test substances typically fall into four broad categories:

Metabolic activation requirements

Some sensitizers require activation before they can trigger an immune response.

  • Pre-haptens become sensitizers through abiotic transformation, such as oxidation.
  • Pro-haptens require enzymatic metabolism to form sensitizing compounds.

Because not all in vitro methods replicate these activation processes, pre- and pro-haptens may be more challenging to detect using certain assays.

Low solubility and high hydrophobicity

Hydrophobic and highly lipophilic substances can be difficult to evaluate in aqueous test systems because of limited solubility. Poor solubility may reduce interaction between the test material and key assay components, potentially affecting assay performance and result interpretation.

Complex or poorly characterized composition

Complex substances such as UVCBs, botanical extracts, essential oils, formulations, and medical device extracts may contain numerous constituents with different chemical properties and sensitization mechanisms.

In addition to creating challenges for testing, incomplete compositional information can limit the applicability of methods that rely on defined chemical structures or physicochemical parameters. For example, molecular weight is often required for concentration calculations in assays such as DPRA and ADRA, while structural information is essential for many in silico predictions and read-across approaches.

Material-specific behaviour

Certain materials, including metals, polymers, and medical device extracts, may interact with biological systems differently than conventional chemicals. These characteristics may require additional consideration when selecting appropriate test methods and interpreting results.
Understanding these challenges is a critical first step in selecting a testing strategy that is appropriate for both the substance being evaluated and the intended use of the data.

Start with the testing objective

The most effective testing strategy starts with a simple question: What decision do you need the data to support?

Regulatory assessment

Typical regulatory objectives include:

  • GHS/CLP hazard classification
  • Skin sensitization hazard identification
  • Potency sub-categorization (Category 1A/1B)
  • Cosmetic safety assessments
  • Product Information File (PIF) support
  • Safety Data Sheet (SDS) preparation
  • Weight-of-evidence assessments under ISO 10993-10 for medical devices

For regulatory purposes, testing strategies should prioritize OECD-recognized methods and Defined Approaches while ensuring that the selected methods are appropriate for the test substance and fall within their applicability domains.

Product Development

Product development teams often need answers much earlier in the innovation process.

Typical questions include:

  • Is this candidate likely to be a skin sensitizer?
  • Which formulation has the lowest sensitization potential?
  • What concentration levels may be appropriate for further development?
  • Which candidates should be prioritized?
  • How can late-stage safety risks be reduced?

In these situations, quantitative potency information may be more valuable than a simple sensitizer/non-sensitizer classification.

OECD TG 497 and non-animal skin sensitization testing

Modern skin sensitization assessment is increasingly built around the skin sensitization Adverse Outcome Pathway (AOP).

Key Events in the Skin Sensitization AOP

  • KE1: Covalent binding to skin proteins
  • KE2: Keratinocyte activation
  • KE3: Dendritic cell activation
  • KE4: T-cell activation and allergic response

To support regulatory decision-making without animal testing, OECD TG 497 introduced Defined Approaches (DAs) that combine results from multiple New Approach Methodologies (NAMs).

The two most commonly used approaches are:

2 out of 3 Defined Approach (2o3 DA)

Primarily used for:

  • Hazard identification
  • Skin sensitizer versus non-sensitizer classification

Integrated Testing Strategy (ITS)

Primarily used for:

  • Hazard identification
  • GHS potency sub-categorization

For a broader discussion of non-animal skin sensitization testing strategies and how OECD Defined Approaches fit into the current regulatory landscape, see our article Non-Animal Skin Sensitization Testing Strategies. 

OECD TG 497 strategies for difficult-to-test substances

For difficult-to-test substances, one of the key challenges in applying OECD TG 497 Defined Approaches is identifying suitable assays to generate the data required for the relevant key events within the skin sensitization AOP.

This is especially relevant for Key Event 1 assessment, where protein reactivity methods such as DPRA and ADRA may not be suitable for all challenging materials. While ADRA extends the applicability domain compared with DPRA, highly complex mixtures, poorly soluble substances, and certain challenging materials may still present difficulties.

When potency sub-categorization is required, the availability of suitable KE1 data can become a limiting factor. Within the Integrated Testing Strategy (ITS), KE1 data are often needed alongside KE3 and in silico inputs to support potency predictions.

Hazard identification

Recent updates to OECD TG 497 have expanded the opportunities to assess difficult-to-test substances by allowing additional assay combinations within the 2 out of 3 Defined Approach (2o3 DA).

For challenging materials, one of the primary considerations is selecting assays with applicability domains that are compatible with the test substance. In practice, combinations based on KE2 and KE3 methods often provide broader applicability than strategies relying heavily on KE1 assays, particularly for poorly soluble substances, UVCBs, and complex mixtures.

GARD®skin + EpiSensA

Among the available options, the combination of EpiSensA (KE2) and  GARD®skin (KE3) has demonstrated broad applicability across highly challenging substance categories, including:

  • UVCBs
  • Hydrophobic and highly lipophilic substances
  • Complex mixtures and formulations
  • Difficult petroleum-derived materials
  • Other poorly soluble substances

This combination benefits from the broad applicability domains of both assays, making it particularly valuable when conventional KE1 methods are not technically suitable for the test material. Recent industry experience has shown that KE2/KE3-based approaches can provide robust hazard identification for many materials that are challenging to evaluate using traditional assay combinations.

GARD®skin + ADRA

Another useful strategy combinesGARD®skin (KE3) with ADRA (KE1).

Compared with DPRA, ADRA offers improved applicability for some poorly soluble and challenging substances because it can be performed at substantially lower test concentrations. However, as a KE1 assay, ADRA still has applicability limitations and may not be suitable for all difficult-to-test materials.

For substances that fall within its applicability domain, this combination can provide an efficient route to hazard identification and may facilitate subsequent potency sub-categorization using an OECD TG 497 Integrated Testing Strategy.

Hazard identification and potency categorization

Integrated Testing Strategy (ITS)

When potency sub-categorization is required, the situation becomes more challenging.

Unlike hazard identification, ITS approaches require multiple information sources, including:

  • A KE1 assay (e.g., ADRA)
  • A KE3 assay ( e.g., GARD®skin)
  • In silico predictions

For difficult-to-test substances, each of these inputs may present limitations. UVCBs, formulations, extracts, and poorly characterized mixtures frequently fall outside the applicability domains of QSAR models because constituent identities, molecular structures, or constituent concentrations may be unknown. Similarly, obtaining reliable KE1 data can be difficult when protein reactivity assays are affected by solubility or compatibility issues.

As a result, potency categorization according to OECD TG 497 remains one of the most significant challenges in skin sensitization assessment of difficult-to-test substances. While ADRA has expanded the applicability of KE1 testing relative to DPRA, finding suitable inputs for all components of an ITS is still often the limiting factor.

Why GARD®skin has broad applicability for difficult-to-test substances

Many established in vitro methods were originally developed and validated using well-characterized chemicals. As a result, challenging materials such as pre- and pro-haptens, hydrophobic substances, UVCBs, natural extracts, and medical device extracts may fall outside their optimal applicability domains.

GARD®skin has demonstrated broad applicability across many of these challenging substance categories. This is largely due to three characteristics of the assay:

Supports metabolic activation

Pre- and pro-haptens require chemical or metabolic activation before they can induce a sensitization response. GARD®skin utilizes a dendritic-like cell system that expresses several metabolizing enzymes involved in the activation of sensitizers, including enzymes from the ALDH, CYP, and NAT pathways. This capability is considered an important contributor to the assay’s demonstrated applicability to pre- and pro-haptens.

Compatible with a broad range of solvents

Hydrophobic and poorly soluble substances are often challenging to evaluate in aqueous test systems due to limited solubility. To improve compatibility with these materials, GARD®skin supports an extended panel of solvent systems, including non-polar solvents that are compatible with the assay. This helps expand testing possibilities for highly lipophilic and poorly water-soluble substances.

Highly sensitive biological response

Complex mixtures, extracts, and poorly characterized materials may contain sensitizing constituents at relatively low concentrations. GARD®skin measures a highly sensitive genomic response in dendritic-like cells and can detect biological activity from small amounts of dissolved test material. This sensitivity can be particularly valuable when evaluating complex substances and other challenging test materials.

Demonstrated applicability across challenging substance categories

Published studies and testing experience have demonstrated applicability across a range of difficult-to-test materials, including:

  • Pre- and pro-haptens
  • Hydrophobic substances
  • UVCBs
  • Natural extracts and essential oils
  • Agrochemical formulations
  • Metals
  • Medical device extracts

As a result, GARD®skin can help generate decision-relevant data for materials that may be difficult to evaluate using methods with more restrictive applicability domains.

GARD® stand-alone testing strategies for product development decisions

While OECD TG 497 Defined Approaches are primarily used to support regulatory decision-making, product development teams often have different objectives. Early-stage candidate selection, formulation optimization, and risk management may benefit from GARD®skin stand-alone testing strategies that provide actionable hazard and potency information throughout the development process.

GARD®skin measures 196 genomic biomarkers linked to multiple key events in the skin sensitization Adverse Outcome Pathway (AOP), providing human-relevant mechanistic information beyond what many early-stage testing approaches typically offer. In addition to its strong predictive performance, the assay has demonstrated broad applicability across multiple difficult-to-test substance categories, including hydrophobic substances, natural extracts, formulations, UVCBs, and other challenging materials.

The GARD®skin platform can support several testing strategies depending on the development stage and information needed.

Hazard identification

GARD®skin or GARD®skin Medical Device

For substances with unknown sensitization potential, GARD® assays can be used as stand-alone methods for hazard identification. The choice of assay depends on the material being evaluated:

  • GARD®skin for chemicals, mixtures, formulations, botanical extracts, UVCBs, and other difficult-to-test substances.
  • GARD®skin Medical Device for medical device extracts and solid materials, supporting skin sensitization assessment in accordance with ISO 10993-10.

This strategy provides early insight into sensitization hazard and can support candidate screening and go/no-go decisions during development.

Hazard identification and quantitative potency assessment

GARD®skin  → GARD®skin Dose-Response

When both hazard and potency information are needed, a tiered testing strategy can be applied. GARD®skin is first used to determine whether a substance has skin sensitizing potential, followed by GARD®skin Dose-Response to generate quantitative potency information.

This approach can support:

  • Candidate prioritization
  • Ingredient selection
  • Formulation optimization
  • Risk management decisions

By combining hazard identification with quantitative potency information, development teams can make more informed decisions earlier in the development process.

Quantitative potency assessment

GARD®skin Dose-Response

For substances that are already known or expected to be sensitizers, GARD®skin Dose-Response can be used directly to generate quantitative potency information.

Potency data can support:

  • Ingredient selection
  • Formulation optimization
  • Candidate prioritization
  • Product design
  • Risk evaluation

By providing quantitative potency insights early in development, teams can compare alternatives, identify safer options, and reduce downstream development risks.

Interested in how potency information can be used for risk assessment, candidate prioritization, and formulation development? Read our detailed guide to Skin Sensitization Potency Assessment. 

A five-step checklist for selecting the right strategy

Before initiating any skin sensitization program, consider the following questions:

Step 1. What decision am I trying to make?

Regulatory classification, risk assessment, candidate selection, or formulation optimization?

Step 2. What type of substance am I testing?

Neat chemical, pre- or pro-hapten, UVCB, natural extract, formulation, metal, or medical device extract?

Step 3. Are there known applicability limitations?

Assess solubility, composition, metabolic activation requirements, and technical feasibility before selecting methods.

Step 4. Which key events should be covered?

Choose methods that collectively provide robust biological coverage across the skin sensitization AOP.

Step 5. Which strategy best matches the substance characteristics?

Select methods based on physicochemical properties, applicability domains, and the intended use of the data.

Conclusion

There is no universal solution for skin sensitization assessment of difficult-to-test substances. Successful programs begin with a clear understanding of the testing objective, the characteristics of the substance, and the strengths and limitations of available methodologies.

For regulatory applications, this means building scientifically justified strategies based on OECD-recognized Defined Approaches and fit-for-purpose method selection.

For product development, it means generating human-relevant information early enough to support safer, smarter, and more efficient decision-making.

By combining regulatory expertise, mechanistic understanding, and fit-for-purpose methods such as GARD®skin and GARD®skin Dose-Response, organizations can confidently address challenging skin sensitization questions without relying on animal testing.

Further reading

To learn more about skin sensitization testing and non-animal assessment strategies, explore these related resources:

Frequently Asked Questions (FAQs)

What are difficult-to-test substances in skin sensitization assessment?

Difficult-to-test substances are materials whose physicochemical properties, composition, or biological behaviour challenge the performance or applicability of conventional in vitro methods. Examples include pre- and pro-haptens, hydrophobic substances, UVCBs, natural extracts, formulations, metals, and medical device extracts.

Why are pre- and pro-haptens challenging to test?

Pre-haptens and pro-haptens require chemical or metabolic activation before they can induce sensitization. Not all in vitro methods replicate these activation processes, which can affect assay performance.

Why are hydrophobic substances difficult to test?

Highly hydrophobic substances often have poor aqueous solubility, which can create challenges for some assay technologies and affect method applicability.

Are UVCBs considered difficult-to-test substances?

Yes. Their variable or partially unknown composition can make them difficult to evaluate using methods that rely on defined chemical structures, molecular weight information, or constituent-specific data.

What non-animal methods are available for skin sensitization testing?

Common NAMs used within OECD frameworks include GARD®skin, EpiSensA, ADRA, DPRA, KeratinoSens™, h-CLAT, and relevant in silico approaches.

Which OECD TG 497 approach is most suitable for difficult-to-test substances?

The optimal approach depends on the characteristics of the test material and the assessment objective. For hazard identification, combinations based on EpiSensA and GARD®skin have demonstrated broad applicability to several challenging substance categories, including UVCBs and highly hydrophobic substances. For potency sub-categorization, additional data inputs from a KE1  assay and in silico methods are required, and applicability limitations should be carefully evaluated.

Why is GARD®skin useful for difficult-to-test substances?

GARD®skin combines human-cell-based biology, genomic biomarkers, and machine-learning analysis, and has demonstrated applicability across multiple challenging substance categories, including pre- and pro-haptens, hydrophobic materials, UVCBs, natural extracts, metals, and medical device extracts.

For regulatory testing, GARD®skin extends the overall applicability of DASS, helping to fill data gaps in regulatory skin sensitization testing.

Beyond regulatory use, GARD® stand‑alone testing provides human-relevant, mechanistic insights that support confident product development decisions, especially helpful when you need to prioritize candidates, refine formulations, or understand material behavior early.

Can skin sensitization potency be assessed without animal testing?

Yes. Quantitative approaches such as GARD®skin Dose-Response can provide potency information that supports product development, candidate evaluation, and formulation decisions.

Need help selecting a testing strategy?

Our scientific experts can help identify a fit-for-purpose skin sensitization testing strategy for difficult-to-test substances, including hydrophobic materials, UVCBs, formulations, natural extracts, metals, and medical device extracts.

Connect with our scientific experts to discuss your projects.

Contact our experts

DISCOVER NAM SERVICE PORTFOLIO
CONNECT WITH A SCIENTIFIC EXPERT

About the Author

Andy Forreryd, PhD

In vitro toxicology expert, member of the OECD expert group on respiratory sensitization, member of ISO/TC 194/WG 8

Dr Andy Forreryd is an in vitro toxicology expert with extensive experience in non-animal skin sensitization assessment and difficult-to-test substances. He collaborates closely with industry leaders to advance novel applications of New Approach Methodologies (NAMs) for sensitization testing, with the aim to replace animal studies.

Dr Forreryd is a co-developer of the GARDskin assay (OECD TG 442E) for the assessment of chemical sensitizers. He is an active member of the OECD expert group on respiratory sensitization, and ISO/TC 194/WG 8 for skin sensitization testing of medical devices.

References

  1. OECD Test Guidline No. 442C (2026): In Chemico Skin Sensitisation
  2. OECD Test Guidline No. 442D (2026): In Vitro Skin Sensitisation
  3. OECD Test Guidline 442E (2024): In Vitro Skin Sensitisation
  4. OECD Test Guideline 497 (2026): Defined Approaches on Skin Sensitisation.