March 3, 2015 by Owen Green

REACH Annex VII requires assessment of available human animal and alternative data followed by an in vivo test (usually OECD429 mouse LLNA and variations OECD442A and B or the less popular OECD406 guinea pig maximisation test). The latter can be avoided if the substance can be classified for skin sensitisation or corrosivity from the other tests or if it is a strong acid or is flammable at room temperature.

The examination of the Adverse Outcome Pathway (AOP) for skin sensitisation shows four key events:

  1. Interaction with cellular protein;
  2. Activation of inflammatory cytokines and induction of cyto-protective gene pathways;
  3. Induction of inflammatory cytokines and surface molecules and mobilisation of dendritic cells;
  4. Presentation of histocompatibility complexes by dendritic cells, activation of T-cells and proliferation of activated T-cells.

There are three tests that have been developed which cover three different sectors of the AOP:

Direct Peptide Reactivity Assay (DPRA) – addresses the mechanism of haptenation, the molecular initiating event of skin sensitisation (AOP1) and measures cysteine and lysine peptide percentage depletion in synthetic heptapeptides containing either cysteine or lysine. This test is validated by EURL ECVAM for transferability and reliability. OECD accepted (TG 442c).

KeratinoSens™ – addresses the responses in keratinocytes (AOP2) by measuring activation of the antioxidant/electrophile response element-dependent pathway and measures luciferase gene fold induction and cytotoxicity in a human keratinocyte-derived cell line. OECD accepted (TG 442d).

Human Cell Line Activation Test (h-CLAT) – addresses responses in dendritic cells (AOP3) by measuring modulation of the expression of co-stimulatory and adhesion molecules in human monocytic leukaemia cell line (THP-1) and measures relative fluorescence intensity (RFI) of CD86 and CD54 and cytotoxicity. Not currently accepted by OECD but validated by EURL ECVAM.

There are some limitations to the substances that can be used in these tests. The tests cannot be used for highly cytotoxic substances, those of low solubility in aqueous media or those which are not stable at high pH.

It is proposed that an integrated testing strategy for skin sensitisation potential can be developed for a chemical using these in vitro tests exclusively leading to normal classification for the end point. It is envisaged that a minimum of two of the three tests should be conducted. For example the test chemical would be subject to the h-CLAT test which, if positive, could be indicative of strong or weak sensitiser depending on whether the minimum induction threshold (MIT) was determined as the smaller of either the EC150 or EC200. If the test is negative then the DPRA test could be initiated and a positive result in this test would indicate a weak sensitiser whereas a negative result would confirm a non-sensitiser. The percentage success of the combination of two in vitro tests compared with the LLNA has been very high in a database of 213 substances (approximately 90% accuracy with known sensitisers or non-sensitisers.

On the commercial side, it is estimated that the cost of two in vitro tests would be equal to or less than the cost of an in vivo LLNA.

A draft revised version of the ECHA guideline Section R.7.3 on skin and respiratory sensitisation is currently under preparation to take the new developments into account and the public release is foreseen before summer 2016.