Last updated: May 25, 2016
“Phototoxicity is defined as a toxic response from a substance applied to the body which is either elicited or increased (apparent at lower dose levels) after subsequent exposure to light, or that is induced by skin irradiation after systemic administration of a substance” (OECD TG 432, 2004).
The interaction of an exogenous substance in the skin (systemic drug or topically applied substance) with ultraviolet radiation can result in a phototoxic reaction that typically resembles sunburn. “Acute phototoxic reactions are characterized by erythema and edema followed by hyperpigmentation. Long-term ultraviolet phototoxicity results in chronic sun damage and skin cancer formation. Also, certain chemicals such as psoralen molecules and coal tar are photocarcinogenic” (Epstein, 1999).
Kim, Park, and Lim (2015) explain the mechanisms of phototoxicity as either a direct mode of action where “unstable species from excited state directly react with the endogenous molecules” or, an indirect mode of action where “endogeneous molecules react with secondary photoproducts.”
The Animal Test(s)
Testing on guinea pigs, rabbits, or other rodents were accepted methods prior to the development and validation of cell-based test systems for assessing potential phototoxicity. An explanation of species selection and animal test procedures can be found in the historical EPA document, Dermatotoxicity (EPA, 1982).
Human and animal photosafety tests are also described in more recent regulatory guidance, including the report from the Second ECVAM Workshop on Phototoxicity Testing (Spielmann et al., 2000), and the ICH S10 Guidance for Industry, Photosafety Evaluation of Pharmaceuticals (ICH, 2013).
Validated Non-animal Methods
|3T3 Neutral Red Uptake Phototoxicity Test (3T3 NRU PT)||Phototoxicity|
OECD TG 432 (2004)
|3T3 Neutral Red Uptake Phototoxicity Test: Application to UV filter chemicals||Phototoxicity|
OECD TG 432 (2004)
|Reactive Oxygen Species (ROS) Photosafety Assay||Photosafety evaluation of pharmaceutical products|
ICH S10 (2013)
As the initial assessment, phototoxic substances can be identified, in most cases, on the basis of their photochemical properties – “whether a compound absorbs photons at any wavelength between 290 and 700 nm” (ICH, 2013). Testing has confirmed that phototoxins usually “exhibit potent UV/VIS absorption with molar extinction coefficients [MEC] of over 1000M(-1)cm(-1)” (Bauer et al., 2014; Onoue et al., 2013). Furthermore, “metabolism does not typically result in chromophores that are substantially different from those in the parent molecule” (ICH, 2013).
An ECVAM Scientific Advisory Committee (ESAC) endorsed the 3T3 NRU phototoxicity test method in 1997, stating that “the 3T3 NRU PT is a scientifically validated test which is ready to be considered for regulatory acceptance” (ESAC Statement, 1997). The Organisation for Economic Cooperation and Development’s guidance, OECD TG 432, describes the protocol for conducting the 3T3 NRU PT. The assay measures the viability of mouse Balb/c 3T3 cells following their exposure to a chemical in the presence and absence of light. Cytotoxicity (cell death) causes a reduction in the uptake of Neutral Red (a dye) (NRU) by the cells. The concentration-dependent reduction in dye uptake 24 hours following treatment is used as the assay endpoint, typically the IC50 value (the concentration of test substance that reduces cell viability to 50% of the untreated control value).
ECVAM identified the advantages of using 3-dimensional (3D) skin models rather than monolayer fibroblast cells (the 3T3 assay) to assess phototoxic potential, and funded a prevalidation study of the EpiDerm™ phototoxicity test in 1999. There appears to be continued interest in the potential of the 3D skin test method (ICH S10 guidance described below), but further validation has not been completed. The MatTek Corporation provides additional information, and a protocol, for using the EpiDerm™ model for phototoxicity testing.
A test battery consisting of the yeast growth inhibition phototoxicity assay and the red blood cell photohemolysis assay underwent an independent peer review by the Japanese Center for the Validation of Alternative Methods (JaCVAM) in 2010. The JaCVAM Regulatory Acceptance Board postponed endorsing the regulatory use of this method until further results are obtained.
An independent JaCVAM peer review panel endorsed the validity of the Reactive Oxygen Species (ROS) Photosafety Assay in 2013. “The panel concluded that the reproducibility and predictivity of the ROS assay is sufficient to support its use in an integrated photosafety testing and decision strategy for drug research and development. In this integrated strategy, negative results in the ROS assay would not require further testing in animals or other tests, while positive, weakly positive, and inconclusive results would proceed to the next level of testing in an in vitro test system such as the 3T3 Phototoxicity Assay (OECD Test Guideline 432)” (p. 10). The validation study reports and proposed protocol for the ROS assay are available on the JaCVAM website.
The International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) is the international body that “brings together the drug regulatory authorities and the pharmaceutical industry of Europe, Japan, and the United States to make recommendations towards achieving greater harmonisation in the interpretation and application of technical guidelines and requirements for pharmaceutical product registration.” The purpose of the ICH Guideline, ICH S10, Photosafety Evaluation of Pharmaceuticals, “is to recommend international standards for photosafety assessment, and to harmonise such assessments supporting human clinical trials and marketing authorizations for pharmaceuticals. It includes factors for initiation of and triggers for additional photosafety assessment and should be read in conjunction with ICH M3(R2).”
Testing strategies proposed for systemic drugs versus dermally applied substances in ICH S10 guidance have some minor differences, so the correct section needs to be consulted. Examination of effective testing strategies for similar chemicals in the same class is also recommended. ICH S10 recommends, as noted above, that determination of the molar extinction coefficient (MEC) should be the first assessment: [for] “MEC values greater than 1000 L mol-1 cm-1 (between 290 and 700 nm), no further photosafety testing is recommended and no phototoxicity is anticipated in humans” (p. 10). When further testing is warranted, a negative result in the ROS assay also indicates a low probability that the substance would be phototoxic in humans. The in vitro assays, 3T3 NRU PT or reconstructed 3D human skin model, can be used for further assessment, and negative results again indicate phototoxicity potential “can be regarded as low” (p. 10). Human clinical photosafety testing approaches, including photoallergy testing for dermal products, are also outlined in ICH S10.
Methods for detecting ocular phototoxicity (both in vitro and in vivo) are not sufficiently developed (ICH S10). Although this information is not required in the US or Japan, it is required in the EU, and the further assessment/development of in vitro methods for ocular phototoxicity could have substantial animal welfare benefits.
Additional information on in vitro methods for phototoxicity testing:
Sherry L. Ward, PhD, MBA
AltTox Contributing Editor
AltTox Editorial Board reviewer(s):
The information provided here is intended only as an overview, and is neither guidance or a comprehensive review of the laws and regulations on phototoxicity testing. Individual countries/regions and their regulatory authorities usually provide specific guidance on hazard/toxicity testing requirements.