In Vitro Phototoxicity

Phototoxicity is defined as a toxic response elicited by topically or systemically administered photoreactive chemicals after the exposure of the body to environmental light.

In vitro tests such as the 3T3 Neutral Red Uptake (NRU) phototoxicity test (OECD 432) or the RHE 3D skin phototox test (OECD 498) are used to identify the phototoxic potential of a test chemical activated by exposure to light. These tests evaluate photo-cytotoxicity by the relative reduction in viability of cells exposed to the test chemical in the presence versus absence of light using either 2D or 3D models.

Photosafety assessment aims to determine whether risk minimization measures are warranted to prevent adverse events in humans.

Four different effects have been discussed in connection with photosafety testing: phototoxicity, photoallergy, photogenotoxicity and photocarcinogenicity. Photosensitization is a general term occasionally used to describe all light-induced tissue reactions.

For a test chemical to demonstrate phototoxicity and/or photoallergy, the following characteristics are critical:

  • Absorbs light within the range of natural sunlight (290-700 nm);
  • Generates a reactive species following absorption of UV-visible light;
  • Distributes sufficiently to light-exposed tissues (e.g., skin, eye).

If one or more of these conditions is not met, a compound will usually not present a concern for direct phototoxicity. However, increased sensitivity of skin to light can also occur through indirect mechanisms.

In vitro 2D models

The In Vitro Phototoxicity test was first published by Spielmann in 1994 (Toxicol In Vitro 8:793-796) as a report and recommendation to ECVAM for the establishment of the in vitro model for the evaluation of phototoxicity. Before the end of that decade, further evaluation were published (Spielmann et al (1998) Toxicol In Vitro 12:305-327) culminating in the development and publication of the OECD Test Guideline 432 for the assay.

The principle of the test is that Balb/c 3T3 cells are treated with the substance under investigation and subsequently irradiated with 5 Joules of UV-A light to assess the effect of the UV light on the toxicity profile of the test substance. An identical treatment is performed in parallel that is not exposed to light thus enabling the toxicity profile without the influence of the light to be established. The toxicity profiles of both treatments are then assessed via Neutral Red Uptake and an IC50 is calculated where possible. By direct comparison of the UV-A treatment and the non UV-A treatment, a Photo-Irritation Factor (PIF) may be calculated from the corresponding IC50 values each treatment has yielded. Should the substance not yield an IC50 when not exposed to light, an alternative calculation (Mean Photo Effect, MPE) can be used to estimate the phototoxic potential of the substance instead.

OECD Test Guideline 432

In vitro 3D models

The phototoxicity assay using a 3D human reconstructed skin model was first published by Liebsch et al. in 1997 (ALTEX 4:165) and was followed up by Jones et al in 2001 (Toxicol Methods 11:1-19). Over the following 8-10 years or so, more data were published using 3D skin models however it took until 2017 for the validation of the test system to be completed and the OECD Test Guideline 498 was published two years later in 2019.

OECD Test Guideline 498

In silico

To be completed