Further improvements to 3T3-NRU assay
One limiting factor of the in vitro 3T3 NRU phototoxicity test is the requirement of aqueous solubility of the test substance.
Reconstituted 3D human skin models could offer a number of advantages in comparison to the 3T3-NRU-PT:
- these 3D models have both viable primary skin cells and skin barrier, therefore they are directly relevant to the target tissue
- a wide selection of chemicals, even in complex mixtures or in dermatological patches, can be applied, simulating more closely a topical application to the skin
- materials with extreme pH values can be tested
- histological comparison between control and exposed samples can be conducted
- depending on the barrier function of the stratum corneum, the absorption and penetration of the original chemicals or molecules created during exposure could provide more relevant results than tests performed on simpler systems (giving fewer false-positive results)
Prevalidation of the EpiDerm phototoxicity test
In 1999 ECVAM funded a prevalidation study on the EpiDerm phototoxicity test, with promising outcome. This model is based on the in vitro skin model Skin2 (Edwards et al., 1994) which had been discontinued in October 1996. It was further proposed by industry to apply the model in a tiered strategy to identify those chemicals that are predicted to be likely to be phototoxic in the 3T3-NRU-PT but are negative in vivo.
In response, the European Medicines Agency (EMA) has suggested in a Draft Guidance Document on Photosafety Testing (EMEA, 2002), that confirmatory testing can be performed on such a skin model.
In feasibility study on the prevalidated human 3D model EpiDerm-PT, successfully applied phototoxic potency testing, showing the usefulness of reconstructed human tissue models for prediction of phototoxicity of topically applied substances and formulations.
In certain cases, this study demonstrated that the human condition may be underpredicted and that a precautionary factor of about 10 should be considered for extrapolation (Kejlová et al., 2007).
Joint ECVAM-EFPIA workshop on phototoxicity testing 2010 in Somma Lombardo, Italy
During this workshop (Ceridono et al., 2012 and Bouvier d'Yvoire et al., 2012), experts from academia, pharmaceutical industry and regulatory authorities presented 'hands-on' experience with the 3T3-NRU-PT in order to discuss why results differ from the original validation exercise and to review technical issues and to reflect on strategies how to improve the usability of the assay for non-topical pharmaceuticals.
It became apparent, that changes to the protocol as outlined in the OECD Test Guideline 432 and the use of different sets of test chemicals are the main causes for the different test outcomes in comparison to the original validation study.
Furthermore, it was suggested that a reduction in the high number of positive results could be achieved:
- by testing only relevant chemicals that have a Molar Extinction Coefficient (MEC) larger than 1000 L mol-1 cm-1,
- by limiting the maximum concentration under irradiation to 100 mg/mL and
- by considering higher concentrations without irradiation to assess IC50 values for PIF concentrations, if necessary.
In 2013 the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use published a Safety Guideline (ICH S10) which followed up on workshop proposals to better define how data based on OECD TG 432 can be used for risk assessment of pharmaceuticals.