Phototoxicity

 Cells plate

    1. ECVAM validated test methods

    2. Test methods under validation by ECVAM

    3. Development and optimisation of alternative methods

 

Background

 

Phototoxicity (photoirritation) is defined as a toxic response that is elicited after the initial exposure of skin to certain chemicals and subsequent exposure to light, or that is induced by skin irradiation after systemic administration (oral, intravenous) of a chemical substance [1]. If a chemical absorbs UV or visible light, it needs to be determined if it is likely to cause adverse phototoxic effects when intended for human use.

The outcome of an international EU/ECVAM/COLIPA validation exercise [2] documented that the phototoxic potential of chemicals can be predicted by applying the 3T3-NRU in vitro phototoxicity test (3T3-NRU-PT).

This assay consists of the immortalized mouse fibroblast cell line, Balb/c 3T3 and is based on a comparison of the cytotoxicity of a chemical when tested in the presence and in the absence of exposure to a non-cytotoxic dose of simulated solar light. Cytotoxicity in this test is expressed as a concentration-dependent reduction of the uptake of the vital dye Neutral Red when measured 24 hours after treatment with the test chemical and irradiation [3]. The test chemical together with the irradiation may alter the cell surface and in effect may result in a decreased uptake and binding of the Neutral Red Dye. Differences in this uptake can be measured with a spectrophotometer, which allows in essence the distinction and quantification between viable, damaged or dead cells.

The 3T3-NRU-PT-assay gained regulatory acceptance in all EU Member States in 2000 and in the OECD Member States in 2004 as Test Guideline (TG) 432. It is now widely used in the chemical and cosmetics industries.

 

Alternatives test methods and approaches

 

1. ECVAM Validated test methods


The International EU/COLIPA In Vitro Phototoxicity Validation Study [2]

Regulatory accepted: OECD Test Guideline 432

 


2. Test methods under validation by ECVAM


Currently there are not test methods under validation.


3. Development/ optimisation / improvement of alternatives methods


One limiting factor of the 3T3-NRU in vitro 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 [4]

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 [5] 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 [6].

In response, the European Medicines Agency (EMA) has suggested in a Draft Guidance Document on Photosafety Testing [7], 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 [8].

 

JOINT ECVAM-EFPIA WORKSHOP ON PHOTOTOXICITY TESTING 2010 IN SOMMA LOMBARDO, ITALY [9,10]

During this workshop, 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.

The newly formed pdf icon ICH S10 will follow-up on workshop proposals to better define how data based on OECD TG 432 can be used for risk assessment of pharmaceuticals.

   

Relevant publications

[1] 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.

[2] Spielmann H, Balls M, Dupuis J, Pape WJ, Pechovitch G, de Silva O, Holzhütter  HG, Clothier R, Desolle P, Gerberick F, Liebsch M, Lovell WW, Maurer T, Pfannenbecker U, Potthast JM, Csato M, Sladowski D, Steiling W, Brantom P. The International EU/COLIPA In Vitro Phototoxicity Validation Study: Results of Phase II (Blind Trial). Part 1: The 3T3 NRU Phototoxicity Test. Toxicol In Vitro. 1998 Jun 1;12(3):305-27.

[3] Borenfreund E and Puerner JA (1985). Toxicity determination in vitro by morphological alterations and Neutral Red absorption. Toxicology Letters 24, 119-124.

[4] Liebsch M, Traue D, Barrabas C et al. Prevalidation of the EpiDerm phototoxicity test. In: Clark D, Lisansky S, Macmillan R, eds. Alternatives to Animal Testing II: Proceedings of the Second International Scientific Conference Organised by the European Cosmetic Industry. Brussels/Newbury: CPL Press, 1999: 160-166.

[5] Edwards SM, Donnelly TA, Sayre RM et al. Quantitative in vitro assessment of phototoxicity using a human skin model, Skin2. Photodermatol Photoimmunol Photomed 1994; 10:111-117.

[6] Jones PA, King AV, Lovell W et al. Phototoxicity testing using 3D reconstructed human skin models. In: Clark D, Lisansky S, Macmillan R, eds. Alternatives to Animal Testing II: Proceedings of the Second International Scientific Conference Organised by the European Cosmetic Industry. Brussels/Newbury: CPL Press, 1999: 160-166.

[7] EMEA. Note for guidance on photosafety testing (CPMP/SWP/398/01). London: European Agency for the Evaluation of Medicinal Products, Committee for Proprietary Medicinal Products, 2002. Available at: EMA.europa.eu

[8] Kejlová K, Jírová D, Bendová H et al. Phototoxicity of bergamot oil assessed by in vitro techniques in combination with human patch tests. Toxicol In Vitro 2007; 21(7):1298-303.

[9] Ceridono M, Tellner P, Bauer D et al. The 3T3 neutral red uptake phototoxicity test: practical experience and implications for phototoxicity testing-the report of an ECVAM-EFPIA workshop. Regul Toxicol Pharmacol. 2012 Aug;63(3):480-8. Epub 2012 Jun 9.

[10] Bouvier d'Yvoire M, Bremer S, Casati S et al. ECVAM and new technologies for toxicity testing. In: Balls M, Combes RD, Bhogal N, eds. New Technologies for Toxicity Testing. Landes Bioscience and Springer Science+Business Media, New York, NY and Austin TX, 2012: 154-180.


Links

Regulation:

  • Commission Regulation (EC) No 440/2008: Method B.41 of Annex to 440/2008/EC (EU Test Methods Regulation), the method has originally been adopted in April 2000
  • OECD Test Guideline 432: adopted in April 2004
  • ICH S10 EWG: International Conference on Harmonisation of technical Requirements for Registration of Pharmaceuticals for human use, S10: Photosafety Evaluation of Pharmaceuticals:

to be adopted by June 2013.

 

ESAC Statement:

 

See also:

 

       

      Photo: Microscopic observation of cells in a 24-well cell culture plate. Copyright EU 2012.