The complex pathway of skin sensitisation has been considered difficult to model in vitro: how could such an intricate, multi-step, physiological pathway be accurately represented in a single test? Thanks to the significant efforts of multiple scientific groups over more than a decade, we now have a portfolio of three in vitro skin sensitisation tests with direct relevance to humans.
The complex pathway of skin sensitisation was only recently mapped out in detail, using a system known as an Adverse Outcome Pathway (AOP). This was a critical development as it paved the way for a whole animal test system to be replaced with a combination of mechanistic, human-based in vitro methods. The three tests measure human responses to a chemical at three points in the AOP – these are described as key events.
• KEY EVENT 1. The first key event is contact between the chemical and skin proteins. Protein binding increases the likelihood of the chemical being a skin sensitiser. This is measured by the Direct Peptide Reactivity Assay (DPRA).
• KEY EVENTS 2 AND 3. These are measured using human cell culture based tests that assess the activation of skin epidermal cells (keratinocytes) and immune cells (dendritic cells) – these are the KeratinoSens™ and h-CLAT methods respectively.
When should I use in vitro skin sensitisation tests?
A variety of global regulations now cite the three in vitro tests as the default approach for skin sensitisation assessment, including the European Chemicals Agency (ECHA) Guidance on the applicability of the methods to REACH (we recommend this as a user-friendly read.)
As with any testing strategy, the known capabilities and limitations of the methods must be taken into account. Current OECD guidance is provided in each of the relevant Test Guidelines. Current regulatory guidance, including IATA (Integrated Approaches to Testing and Assessment) and the guide by ECHA, describes a “two out of three” approach, whereby two positive results would lead to classification of a chemical as a skin sensitiser.
Ideally, all three tests should be performed to build the best possible picture of the human skin sensitisation potential of the chemical or mixture. However, in practice, many labs are first performing two tests (usually the DPRA and KeratinoSens™), only following up with a third (h-CLAT) if confirmation is needed to obtain the two out of three classification. This strategy is often cost-driven as the h-CLAT is the most expensive of the three tests, and commercial factors drive resistance to replacing the outdated animal tests. While this remains the mainstream approach, there are some current views in favour of using the DPRA and h-CLAT combination (Roberts and Patlewicz, 2016), as well as a school of thought that the DPRA is the single most predictive test, since it looks at the molecular initiating event of the AOP (Benigni et al, 2016). Test chemicals should be considered on a case-by-case basis to take scientific and commercial factors into account, while prioritising a comprehensive safety assessment.
Difficulties with benchmarking against historic animal test data
It’s worth keeping in mind that the in vitro skin sensitisation tests were all originally validated against historical data from the Local Lymph Node Assay (LLNA). There is strong performance data for all three in vitro methods, using the LLNA as a benchmark. However, the LLNA has many drawbacks including a high incidence of false positive results, variation with dose vehicle and predictivity issues (eg inconsistencies with human patch test data) (Anderson et al, 2011). All three of the in vitro TGs state “Furthermore when evaluating non-animal methods for skin sensitisation, it should be kept in mind that the LLNA, as well as other animal tests, may not fully reflect the situation in the species of interest. i.e. humans.”
The validation of in vitro methods against less-than-ideal benchmarks is an ongoing challenge, and more creative approaches to validation are needed.
Download your copy of our guide to in vitro skin sensitisation testing
If you are interested in learning more about in vitro skin sensitisation testing, download our free ebook which includes latest OECD guidance, notes on assessing potency and using skin sensitisation tests for mixtures.