How is Phototoxicity defined?
Phototoxicity is defined as an acute reaction induced by a single treatment with a chemical in combination with ultraviolet or visible radiation. In vitro, the reaction can be induced by an appropriate concentration of a chemical and dose of light. The term “acute” includes immediate and delayed (e.g. after 48h) reactions (1).
Which methods are validated and accepted?
In Europe the in vitro 3T3 Neutral Red Uptake (NRU) phototoxicity test (PT) was accepted in 2000 as validated alternative method to identify the phototoxic potential of test substances. Since then in vivo testing in animals for acute phototoxic potential of chemicals is no longer permitted in Europe (2). So far the 3T3-NRU-PT is the core test required for phototoxicity and the only validated in vitro test for assessment of phototoxicity, but it has some limitations:
- Requires aqueous solubility of test substances
- Does not provide specific information on mechanisms of phototoxicity (2)
- Does not allow to assess the bio-availability of a test compound in the human skin (2)
The use of reconstructed human epidermis models can overcome these limitations due to their organotypic structure with a functional stratum corneum which allows testing of topically applied chemicals. This Human 3-D Skin Model In Vitro Phototoxicity Test (H3D PT) can handle solutions and suspensions as well as dermatological patches and therefore simulates the in vivo situation more closely than a monolayer cell culture. Due to the functional barrier of the epidermis models the H3D PT may miss the correct identification of phototoxins that cannot enter the skin (2). Nevertheless, several studies were performed and have shown the capacity of reconstructed epidermis models to predict phototoxicity (3, 4, 5, 6, 7). So far, the use of reconstructed epidermis models for identification of the phototoxic potential of test substances is not officially validated. However, some cosmetic companies already use the H3D PT as a second tier phototoxicity test (2).
How does the test work?
Phototoxicity test using epiCS
Skin equivalents are exposed to different concentrations of a test compound for 18 - 24 hours. Afterwards, the skin equivalents are irradiated with UVA light at an intensity of 6 J/cm2 whereas identically exposed equivalents are kept in the dark. After irradiation the test compound is washed off and skin equivalents are incubated overnight. For evaluation of the phototoxic potential of the test compound the MTT assay is performed (modified from: 4).
Interpretation of results
A test compound is classified (considered) phototoxic if exposure of the substance (in at least one concentration) plus irradiation reduces tissue viability to at least 70% compared to control tissues (exposed but non-irradiated).
Fig. 1: Phototoxicity test of Chlorpromazine (CPZ) using the three-dimensional human skin model epiCS (former EST 1000). (A) Relevant reduction in viability of irradiated equivalents in comparison with the non irradiated equivalents after treatment with CPZ. (B) Skin equivalents after MTT extraction. The first two rows (top) represent irradiated skin equivalents after treatment with CPZ. The lower two rows contain non irradiated skin equivalents after treatment with CPZ. (modified from: 4)
General procedure for test substances
statistical reasons, the
assay has to be carried out in triplicates. Substances are applied onto skin models followed by UVA-irradiation (n=3)
and the same substances are applied onto skin models without irradiation (n=3).
Controls are carried out in the same way.
For each substance and for each control six skin models are required.
Apply 50 µl of the test substance (under sterile conditions) topically onto the centre of the skin model by using a sterile micro-pipette.
- Positive control: Apply 50 µl of the positive control (e.g. chlorpromazine 0.01 %) onto the centre of the skin model.
- Negative control: Apply 50 µl of the negative control (1 x PBS) onto the centre of the skin model
Incubate all skin
models for 24 hrs at 37
°C, 5 % CO
2, 95 % relative humidity (RH).
Irradiate three skin models (one triplicate of test substance or control) with 6 J/cm 2 UVA. In parallel store the three non-irradiated controls in the dark at room temperature.
To remove the test substance from the surface of the skin models, rinse the tissue with PBS to remove any residual test material. Fill and empty inserts 20 times in a constant soft stream of PBS using a squeeze bottle.
Remove excess PBS by gently shaking inserts and blotting the bottom with a piece of paper towel.
Incubate all skin models in culture medium at 37 °C, 5 % CO 2and 95 % RH for 18 hrs.
Perform a standard MTT-assay to determine the viability of the models.
For further Information, please check the epiCS manual:
- Spielmann H., Lovell WW., Hoelzle E., et al. (1994). In vitro phototoxicity testing. The report and recommendations of ECVAM workshop 2. Alternatives to Laboratory Animals (ATLA) 22, 314-348.
- Liebsch M., Spielmann H., Pape W., Krul C., Deguercy A., Eskes C. (2005). UV-induced effects. Alternatives to Laboratory Animals 33 (1), 131-146.
- Edwards SM., Donnelly TA., Sayre RM., Rheins LA., Spielmann H., Liebsch M. (1994) Quantitative in vitro assessment of phototoxicity using a human skin model: Skin2. Photodermatology, Photoimmunology & Photomedicine 10, 111–117.
- Heppenheimer A., Poth A., Hoffmann J. (2011). Comparative Investigation of Phototoxicity of Chemical Compounds by using the Standard Balb 3T3 NRU Phototoxicity Test and a Three-Dimensional Human Skin Model (EST1000).
- Liebsch M., Döring B., Donelly T.A., Logemann P., Rheins LA., Spielmann H. (1995). Application of the human dermal model Skin2 ZK 1350 to phototoxicity and skin corrosivity testing. Toxic in Vitro 9, 557-562.
- Liebsch M., Barrabas C., Traue T., Spielmann H. (1997) Entwicklung eines in vitro Tests auf dermale Phototoxizität in einem Modell menschlicher Epidermis (EpiDermTM). ALTEX 14, 165-174.
- Augustin C., Collombel C., Damour O. (1997) Use of dermal equivalent and skin equivalent models for identifying phototoxic compounds in vitro. Photodermatology Photoimmunology Photomedecine 13(1-2), 27-36.
- Lelièvre D., Justine P., Christiaens F., Bonaventure N., Coutet J., Marrot L., Tinois-Tessonneaud E. and Cotovio J. (2007). The EPISKIN Assay (EPA): Development of an in vitro tiered strategy to predict phototoxic potential. ALTEX 14, Special Issue, 389-396.