Last updated: January 18, 2016
Non-animal Test Methods for Phototoxic Evaluation
In the early 2000s, regulatory agencies in the US and EU published guidelines for photosafety assessments of drug candidates. The European Medicines Agency (EMEA) Committee for Proprietary Medicinal Products (CPMP) issued guidance on photosafety testing in 2002 (EMEA/CPMP, 2002), and the Food and Drug Administration (FDA) Center for Drug Evaluation and Research (CDER) also published guidance for industry photosafety testing in 2002 (FDA/CDER, 2002). The EMEA also published a concept paper in 2008 (EMEA/CPMP, 2008), which proposes a testing strategy that attempts to merge the testing proposals recommended by the FDA and the EMEA. On the other hand, the Organisation for Economic Co-operation and Development (OECD) approved a test guideline on phototoxic assessment; the in vitro 3T3 Neutral Red Uptake (NRU) Phototoxicity Assay (PT) as a validated methodology for evaluating the phototoxic potential of chemicals (OECD, 2004). Considering these documents, the International Conference of Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) published ICH S10 guideline, “Photosafety Evaluation of Pharmaceuticals” in 2014 (ICH, 2014).
In these guidelines, chemicals or drug candidates, administered to the skin and/or eyes and distributed to the skin and/or eyes after administration, need to be examined for their phototoxic potential. The Grotthuss-Draper law (Radiation, 2009), also known as the first law of photochemistry, states that light must be absorbed by a compound in order for photochemical reactions to take place. On the basis of this principle, all the guidelines have suggested that the phototoxic potential of chemicals is related to the photochemical properties of compounds, especially light absorption properties within 290–700 nm, and they have described the need for measurement of the light absorption properties of chemicals as a first screening. Henry, et al. (2009) measured UV absorption in the 290-to-700-nm range of 35 phototoxic compounds, and proposed that photosafety testing of compounds might not be needed if the molar extinction coefficient (MEC) of a compound is estimated to be less than 1,000 M-1⋅cm-1 per UV absorption spectral analysis. The ICH S10 guideline recommends UV absorption spectral analysis as a criterion for evaluating the phototoxic potentials of drugs; however, UV absorption of chemicals would not always correlate directly with their phototoxic potential, so a combination of MEC with other appropriate screening systems might be advantageous in avoiding false predictions.
In addition to light absorption and distribution to light-exposed tissue, the generation of a reactive species from drug candidates following absorption of UV-visible light is described as a key characteristic of chemicals for causing phototoxic reactions in the ICH S10 guideline. On the basis of the key characteristic, the ROS (Reactive Oxygen Species) assay (Onoue and Tsuda, 2006; Onoue et al., 2008) has been also recommended by ICH S10 guideline as an in vitro tool for evaluating the photosafety of drug candidates. This assay is a well-validated methodology for photosafety evaluation of pharmaceuticals (Onoue et al., 2013b; Onoue et al., 2014). Recent attention has been drawn to the strategic use of ROS assay for photosafety assessment on cosmetic and food additives (Onoue et al., 2008; Onoue et al., 2011; Onoue et al., 2013a, Nishida et al., 2015), as well as pharmaceutical substances.
The cosmetics industry has been directly affected by the 7th Amendment (Directive 2003/15/EC) to the Cosmetic Directive (Directive 76/768/EEC), which called for a ban on marketing of cosmetic products containing ingredients that have been tested in animals for toxicity as of March 2013. Therefore, a reliable and comprehensive non-animal photosafety screening strategy is urgently needed. Currently, Personal Care Products Council (PCPC) guidance recommends the use of the ROS assay for photosafety evaluation of cosmetic ingredients (PCPC, 2014). Given this background, Japan submitted the Standard Project Submission Form (SPSF) on the ROS assay to OECD, because the publication of a standardized Test Guideline for the ROS assay should be of great value.
Currently, the OECD guideline (TG 432) recommends only the in vitro 3T3 NRU PT, which sets specific criteria for evaluating phototoxic risk. The assay was drafted as an alternative method for in vivo phototoxicity testing and submitted to the OECD by the European Centre for the Validation of Alternative Methods (ECVAM) and the European Cosmetics, Toiletry and Perfumery Association (COLIPA) (Spielmann et al., 1994). The ICH S10 guideline also recommends that the in vitro 3T3 NRU PT be used for evaluating the photosafety of drugs. However, the in vitro 3T3 NRU PT often provides false-positive results and the results from the assessments would not always reflect other types of in vitro phototoxic risk, including photogenotoxicity and photoallergy as well as in vivo phototoxicity. Thus, inclusive in vitro screening methodologies and strategies are also needed for more complete and reliable photosafety evaluation.
Furthermore, the ICH S10 guideline also recommends that reconstructed human skin models can be used to assess the phototoxicity potential of clinical formulations via the dermal route. Reconstructed human skin models, with the presence of a stratum corneum, permit testing of various types of topically applied materials ranging from neat chemicals to final clinical formulations. The assays developed with reconstructed human skin to date measure cell viability with and without irradiation. These assays appear to be capable of detecting known human acute dermal phototoxicants. Under adequate test conditions, a negative result in a reconstructed human skin assay indicates that the direct phototoxicity potential of the formulation can be regarded as low. In this case, generally no further phototoxicity testing is recommended. However, the sensitivity of some assays can be less than that of human skin in vivo, wherein the lowest concentration eliciting a positive response can be higher than in human skin in vivo. Consequently, it is important to understand the sensitivity of any assay selected and, if appropriate and feasible, to adjust the assay conditions accordingly (e.g., testing higher strength formulations, increasing exposure time).
There are no in vitro models that specifically assess ocular phototoxicity, regardless of the route of administration. While negative results in the 3T3 NRU-PT or a reconstructed human skin assay might suggest a low risk, the predictive value of these assays for ocular phototoxicity is unknown. If photochemical reactivity is observed in a test chemical, further phototoxicity tests for photoirritation, photoallergy, and photogenotoxicity should be conducted. However, the EMEA and International Workshop on Genotoxicity Tests (IWGT) no longer recommend photogenotoxicity testing as part of standard photosafety evaluation (Lynch et al., 2011).
Hajime Kojima, PhD
National Institute of Health Sciences (NIHS), Japan
Satomi Onoue, PhD
School of Pharmaceutical Sciences, University of Shizuoka, Japan
AltTox Editorial Board reviewer(s):
Kristie Sullivan, MPH