In the last artlcle of this series on our core regulatory tests we’re covering KeratinoSens™ (OECD TG442d).
The Adverse Outcome Pathway for skin sensitisation, published by the OECD in 2012, broke allergic contact dermatitis into a sequence of Key Events. A chemical binds to skin proteins, keratinocytes respond, dendritic cells activate, and on repeat exposure the immune system produces an allergic reaction. Mapping that sequence was what made replacing the animal tests possible, because each Key Event could be addressed by a separate in vitro method rather than trying to model the whole response at once.
The KeratinoSens™ assay sits in OECD Test Guideline 442D and covers Key Event 2. It works well, it’s accepted internationally, but for years like a lot of other cell-based assays, it carried an awkward inconsistency: the standard protocol used bovine serum in the cell culture medium. That’s the part of the story we ended up involved in, and the version of the protocol we developed is now in the guideline.
How the test works
The KeratinoSens™ cell line comes from HaCaT, an immortalised human keratinocyte line. The cells are transfected with a plasmid carrying a luciferase gene under the control of an Antioxidant Response Element (ARE) taken from a gene known to be upregulated by contact sensitisers.
The consequence is elegant. When a sensitiser is applied, it activates the Nrf2-dependent pathway the ARE responds to, which switches on the luciferase gene alongside it. The cells produce luciferase, and luciferase produces light. Measure the luminescence and you have a quantitative readout of whether the pathway has been triggered.
A test item inducing luciferase activity at or above 1.5-fold over the untreated control is classified as a sensitiser. Below that, non-sensitiser. A parallel cytotoxicity test runs alongside, so the prediction is made from healthy, viable cells rather than dying ones.
The serum problem
Cell culture media conventionally contain foetal bovine serum, an undefined mixture of growth factors and proteins. It became the standard because it is cheap, widely available, and supports a broad range of cell lines without needing to be tailored to each one. This leaves an animal-derived material at the centre of a method intended to replace animal testing.
There’s a scientific argument alongside the ethical one. Serum composition varies from batch to batch, which introduces variability into the system. And for a test predicting human responses, a bovine product is not the most relevant thing to have in the culture medium.
XCellR8 adapted the KeratinoSens™ method to animal-product-free conditions and validated the adapted protocol against the OECD proficiency chemicals and Performance Standards. The work was published in ALTEX in 2017.
The regulatory acceptance followed. ECHA confirmed in 2016 that the adapted method could be used in REACH registration dossiers, and the protocol was subsequently published in the new edition of OECD Test Guideline 442D, adopted in June 2018.
That last point is worth stating plainly. The animal-product-free protocol isn’t a variant we run alongside the official method. It is in the guideline.
Why one test isn’t enough
KeratinoSens™ addresses Key Event 2 only, and skin sensitisation is complex enough that no single assay covers the endpoint. The guideline says as much: data should be considered within an integrated approach.
In practice that means combining methods addressing different Key Events. TG 442C covers Key Event 1, protein binding, through in chemico methods including the DPRA. TG 442D covers Key Event 2, keratinocyte activation, which is where KeratinoSens™ sits. TG 442E covers Key Event 3, dendritic cell activation, through methods including h-CLAT and GARDskin.
OECD Test Guideline 497, published in June 2021 and updated in 2025, formalised how those results get combined. It describes three defined approaches. The best known is “2 out of 3”: two concordant results from DPRA, KeratinoSens™ and h-CLAT give a hazard classification without needing the third. The ITS combines Key Events 1 and 3 with an in silico prediction from Derek Nexus or the OECD QSAR Toolbox, and reaches potency categorisation rather than hazard identification alone. SARA-ICE, added in the 2025 update, derives a point of departure for risk assessment.
That distinction matters under REACH, where a positive hazard call from the 2 out of 3 approach isn’t the end of the process, because potency sub-categorisation still has to be addressed.
The significance of TG 497 is that these are defined approaches with fixed data interpretation procedures. They don’t require expert judgement to reach a conclusion, which is what makes them usable in a regulatory submission.
Where it sits
TG 442D is accepted for hazard identification under the UN GHS and is appropriate for compliance with the EU Cosmetics Regulation 1223/2009 and the CLP Regulation 1272/2008. It was incorporated into REACH in 2016, when Annex VII was amended to make non-animal methods the default requirement.
The animal method it replaces, the Local Lymph Node Assay, is now permitted only as a last resort. ECHA’s position is that in vivo methods can be used only where the in chemico and in vitro methods are not adequate for the substance, or cannot be used for classification and risk assessment.
We run KeratinoSens™ to GLP under our MHRA accreditation, in the animal-product-free conditions we developed, and data is recognised across all territories participating in the OECD’s Mutual Acceptance of Data arrangement.
If you need skin sensitisation data and want to work out which combination of methods suits your substance, get in touch.
Email info@x-cellr8.com or call +44 (0)1925 607 134.
———
Contact us if you’d like to learn how you can support our mission, become a corporate sponsor or donate to our crowdfunder.
Email info@x-cellr8.com
Call +44 (0)1925 607 134.
Follow Us!