In June 2016, the European Chemicals Agency (ECHA) released updated draft guidance for skin sensitisation testing (Information Requirements and Chemical Safety Assessment Chapter R.7a, section.R.7.3 Skin sensitisation). The draft guidance highlights the need to consider existing information and non-animal methods (e.g., in silico, in chemico and in vitro) as the primary and prevailing means to assess dermal sensitisation risks. Furthermore, the use of in vivo testing is restricted to implementation only if the existing data in combination with in silico, in vitro and in chemico tests are proven to be ineffective, inconclusive or otherwise insufficient.
The recommended in chemico and in vitro methods used to assess skin sensitisation potential include tests to cover 3 of the key stages of the Adverse Outcome Pathway (AOP) for skin sensitisation. These include:
- peptide/protein binding (Direct Peptide Reactivity Assay – DPRA)
- activation of the Nrf2-Keap1-ARE toxicity pathway (KeratinoSensTM assay)
- induction of CD54 and/or CD86 cell surface markers (h-CLAT assay).
Dermal bioavailability should also be an initial consideration because if a chemical cannot penetrate the skin then it is unlikely to bind to peptides/proteins to activate the subsequent steps of the AOP.
The REACH Regulation states that new data from tests on vertebrate animals should only be generated as a last resort. This very much puts the responsibility on the registrant to avoid animal testing unless there is good reason. However, there are some circumstances when animal testing (typically the Local Lymph Node Assay – LLNA) is considered unavoidable. These include where:
- existing information on similar substances and /or QSAR’s suggest the chemical is a strong or extreme sensitiser, yet in vitro / in chemico tests are unable to conclude on the risk for classification and labelling purposes.
- the chemical does not fall into the applicability domain for the in chemico / in vitro tests for skin sensitization.
- the in chemico / in vitro tests are inconsistent and this inconsistency cannot be explained scientifically.
- skin sensitisation potency i.e., Cat 1A (extreme and strong sensitisers) vs 1B (moderate sensitisers) cannot be determined based on non-animal testing approaches.
Although it is possible to categorise into sensitisers (Cat 1) or non-sensitisers (non-classified) using the validated in chemico / in vitro test methods, these methods are not currently used on their own to predict potency or sub-categorisation (Cat 1A vs Cat 1B). However, combining additional information for similar substances with data generated in the in chemico / in vitro tests using a Weight of Evidence approach may assist in defining potency. Several non-animal test methods are also currently under development, and these methods are expected to have enhanced capability for predicting skin sensitisation potency.
One of these promising test methods is SenCeeTox® (referred to on page 307 of the ECHA guidance). SenCeeTox® covers a number of different stages of the AOP and categorises into 4 different potency classes. Both 2D and 3D skin models can be assessed allowing formulations and insoluble preparations to be evaluated as well as standard solutions. Cyprotex is currently working alongside a European association to validate SenCeeTox® as an internationally accepted method.
The current draft EHCA guidance is available to the public and open for comment. Adoption and enforcement is expected in autumn 2016.
Learn more about in vitro skin sensitisation testing options.
Download the poster we presented on in vitro methods for determining skin sensitisation potency.