In vitro phototoxicity testing

In Vitro Phototoxicity Testing a Procedure Involving Multiple Endpoints... 

Spielmann, H., Lowell, W.W., Holzle, E. et at (1994) In vitro phototoxicity testing. The report and recommendations of ECVAM workshop 2. Alternatives to Laboratory Animals, 22, 314—348. 

C O LI P A international in vitro phototoxicity test validation study and the ECVAM/... 

Phototoxicity or chemical phototoxicity is the term used for an acute reaction that can be induced by a single treatment with a chemical and UV or visible radiation. The basic mechanism of phototoxicity can be described as an increase in toxicity of a chemical induced by exposure to UV or visible radiation. Therefore the phototoxic potential of a chemical can be measured as an increase in cytotoxicity after exposure to UV or visible light. The 3T3 NRU PT test is a validated and robust in vitro phototoxicity test according to the criteria laid down by the ECVAM Workshop on practical aspects of the validation of toxicity test procedures and the conclusion of the ECVAM/COLIPA validation study [72], Owing to the convincing performance of the 3T3 NRU PT test, the test is now established and in use in industry laboratories to screen... 

Two additional tests, the red blood cell phototoxicity test (RBC PT) (Pape et al., 1994) and the Human 3-D Skin Model In Vitro phototoxicity test (H3D PT) (Roguet et al., 1994 Bernard et al, 1999 Liebsch et al, 1999 Jones et al, 2003), are regarded useful and important adjunct tests to overcome some limitations of the 3T3-NRU-PT, like the fairly low UVB tolerance of 3T3 fibroblasts. Moreover, the RBC PT enables evaluation of the phototoxic mechanisms involved (Okamoto et al, 1999) and the H3D PT model is qualified as an adjunct test to further investigate chemicals with (probably false) positive outcomes in the 3T3 NRU PT. 

Spielmann, H.. Balls, M., Dupuis, J., Pape, W.J.W., Pechovitch, G., De Sdva, O., Holzhiitter, H.G., Clothier, R. et al. (1998) EU/COLIPA In vitro phototoxicity validation study, results of phase 11 (bHnd trial), part 1 the 3T3 NRU phototoxicity test. Toxicology In Vitro An International Journal Published in Association with BIBRA 12, 305-327. 

Spielmann H, Balls M, Dupuis J, Pape WJ, Pechovitch G, De Silva O, Holzhutter HG, Clothier R, Desolle P, Gerberick F, Liebsch M, Lovell WW, Maurer T, Pfan-nenbecker U, Potthast JM, Csato M, Sladowski D, Steiling W, Brantom P. The international EU/COLIPA in vitro phototoxicity validation study Results of phase II (bhnd trial) Part 1. The 3T3 NRU phototoxicity test. Toxicol In Vitro 1998 12(3) 305-27. 

A number of in vitro models have been developed for assessing the phototoxic potential of chemicals. The most widely used in vitro phototoxicity assay is the in vitro 3T3 Neutral Red Uptake Phototoxicity Test (3T3 NRU PT). This assay was validated by ECVAM and subsequently adopted by the Organization for Economic Co-operation Development (OECD) as Test Guideline (TG) 432 (OECD,... 

Because the 3T3 NRU PT has a high sensitivity but low specificity for predicting clinical phototoxicity, a negative result in the 3T3 NRU PT indicates that no further testing is required. However, a positive result should be confirmed by further in vivo or clinical testing. In vitro 3D RhE tests can be performed when solubility issues prevent use of the 3T3 NRU PT or for topical formulations. However, the sensitivity of the in vitro 3D tests has in some cases been found to be lower than in vivo tests, so assay conditions should consider the use of higher strength formulations or increased... 

The only in vitro studies fully validated and included in the EU test methods (a.2) are for skin corrosivity and phototoxicity, but efforts are underway to speed up validation for other endpoints. An original aim was that there would be no animal studies for the basic level of registration, but as there is currently no suitable in vitro test for skin sensitisation, the mouse local lymph node assay is included in Annex V of the Regulation (Table 9). The in vitro tests for skin and eye irritation are not fully validated, so as a compromise they are accepted for registration at up to 10 tonnes per annum (i.e., they are in Annex V), but if the results are negative they have to be confirmed by a standard animal test at 10 tonnes per annum (i.e., the Annex VI data, as shown in Table 10). 

The test is based on an in vitro assay of the uptake of the dye, neutral red (NR), in Balb/c 3T3 fibroblasts. It was developed to detect the phototoxicity induced by the combined interaction of the test substance and light of the wavelength range from 315 to 400 nm, the so-called UVA. The cytotoxicity is evaluated in the presence (+UVA) or absence (-UVA) of UVA light exposure, after application of a nontoxic dose of the compound. The cytotoxicological impact is assessed via the inhibition of the fibroblasts to take up the vital dye NR (NR is a weak cationic dye, penetrating easily into the cell membrane by a nonionic diffusion and accumulates in the lysosomes) one day after the initial treatment. Normally, healthy cells may incorporate and bind NR. Alterations of the cell surface or the lysosomal membranes, however, lead to a decreased uptake and binding of the dye. 

M. Liebsch, C. Barrabas, D. Traue, and H. Spielmann. Development of a new in vitro test for dermal phototoxicity using a model of reconstituted human epidermis. ALTEX 14 165-174 (1997). 

In Vitro 3T3 NRU Phototoxicity Test (Original Guideline, adopted 13 April 2004) (Software Phototox Version 2.0)... 

EU test method B 41, Phototoxicity - in vitro 3T3 NRU phototoxicity test... 

The phototoxicity test 3T3 NRU was proposed in 1994 and is so far the only in vitro method that has been validated by European regulatory authorities for predicting the photoirritant potential of substances [5,40,41]. In this test, the mouse fibroblasts cell line Balb/c 3T3 is exposed to simulated solar UV (or, more frequently, solar UVA) in the presence of the test compound after an incubation of 1 h in the dark. Evaluation of cytotoxicity is performed 24h post-exposure using the neutral red uptake (NRU) method. N RU permits to distinguish live and dead cells, since intact cells retain this dye (detailed method in INVITOX protocol 78). The validation was performed with substances selected on the basis of their in vivo photoirritant or phototoxic properties. Some of these structures are shown in Table 19.1. 

Organization for Economic Co-operation and Development (OECD), OECD Guidelines for the Testing of Chemicals No. 432 In vitro 3T3 NRU Phototoxicity, Paris, France, 2002c. 

PrzybiUa B, Schwab-Przybilla U, Ruzicka T, Ring J. Phototoxicity of non-steroidal anti-inflammatory drugs demonstrated in vitro by a photo-basophil-histamine-release test. Photodermatol 1987 4(2) 73-8. 

An important method using fungi is Daniels test for phototoxicity, which utilizes the yeast Candida albicans as the test organism. A 1988 study compared favorably the results of this test with the results of photo-patch testing in volunteers for samples from six furocoumarin-containing plants. Many test materials which produce an erythemic response in the photoirritant test are not analyzed as positive in this test. A new test method, Solatex-pi, has demonstrated capability to predict the potential for photoirritation of materials in this class as well as that of other well-known photoirritants. Solatex-pi utilizes the two compartment physicochemical model of Skintex to predict the interactive effects of specific chemicals and UV radiation. Solatex-pi is being validated by Frame and the BGA (Zebet) as an in vitro test to predict photoirritants. 

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