A General Measure of In Vitro Phototoxicity Derived from Pairs of Dose–Response Curves and its Use for Predicting the In Vivo Phototoxicity of Chemicals

Hermann-Georg Holzhütter

In pharmacology, it is common to evaluate the influence of external effectors (for example, temperature, pH, and presence of a second drug) on dose–response relations by the potency factor (PF50): PF50 = ED50 (– effector) ED50 (+ effector) where ED50 (± effector) denotes the 50% effective dose in the presence and in the absence of the effector, respectively. In this paper, the external effector is ultraviolet (UV) light, and PF50 is referred to as the photoirritancy factor (PIF). There are two parameters which limit the applicability and toxicological reliability of the PIF. Firstly, the physical properties (for example, water solubility) of the chemical tested and the constraints of the biological test system may make it difficult, or even impossible, to achieve sufficiently high doses to observe 50% of the maximal response. In such cases, no numeric value of the potency factor can be computed. Secondly, the potency factor does not take into account the absolute change in response induced by UV light, i.e. depending on the shape of the ±UV dose–response curves, the absolute change in response may be small although the PIF is large, and vice versa. This paper proposes a more general measure of phototoxicity, the mean photo effect (MPE), which can be assessed from pairs of dose–response curves, even if the 50% response level is not reached in one curve or in both. The MPE is a weighted average of PIFd values across different dose levels (d being common to both dose–response curves). The absolute response changes, ΔRd, i.e. the differences between the –UV curve and the +UV curve are used as weighting factors. The numerical computation of the MPE is based on theoretical curves obtained by fitting a mathematical model to the experimental dose–response data. Plotting PIFd and ΔRd versus the corresponding doses permits differences in the shapes of the two curves to be assessed, and possible alterations in the toxic mechanisms induced by UV light to be revealed. The variance of MPE is estimated by a bootstrap procedure. The use of the MPE is illustrated by its application to dose–response data obtained with a human keratinocyte assay of fibroblasts in the EU/COLIPA international validation project on photoirritancy.
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A Study on UV Filter Chemicals from Annex VII of European Union Directive 76/768/EEC, in the In Vitro 3T3 NRU Phototoxicity Test

Horst Spielmann, Michael Balls, Jack Dupuis, Wolfgang J. W. Pape, Odile de Silva, Hermann-Georg Holzhütter, Frank Gerberick, Manfred Liebsch, Will W. Lovell and Uwe Pfannenbecker

In 1996, the Scientific Committee on Cosmetology of DGXXIV of the European Commission asked the European Centre for the Validation of Alternative Methods to test eight UV filter chemicals from the 1995 edition of Annex VII of Directive 76/768/EEC in a blind trial in the in vitro 3T3 cell neutral red uptake phototoxicity (3T3 NRU PT) test, which had been scientifically validated between 1992 and 1996. Since all the UV filter chemicals on the positive list of EU Directive 76/768/EEC have been shown not to be phototoxic in vivo in humans under use conditions, only negative effects would be expected in the 3T3 NRU PT test. To balance the number of positive and negative chemicals, ten phototoxic and ten non-phototoxic chemicals were tested under blind conditions in four laboratories. Moreover, to assess the optimum concentration range for testing, information was provided on appropriate solvents and on the solubility of the coded chemicals. In this study, the phototoxic potential of test chemicals was evaluated in a prediction model in which either the Photoirritation Factor (PIF) or the Mean Photo Effect (MPE) were determined. The results obtained with both PIF and MPE were highly reproducible in the four laboratories, and the correlation between in vitro and in vivo data was almost perfect. All the phototoxic test chemicals provided a positive result at concentrations of 1µg/ml, while nine of the ten non-phototoxic chemicals gave clear negative results, even at the highest test concentrations. One of the UV filter chemicals gave positive results in three of the four laboratories only at concentrations greater than 100µg/ml; the other laboratory correctly identified all 20 of the test chemicals. An analysis of the impact that exposure concentrations had on the performance of the test revealed that the optimum concentration range in the 3T3 NRU PT test for determining the phototoxic potential of chemicals is between 0.1µg/ml and 10µg/ml, and that false positive results can be obtained at concentrations greater than 100µg/ml. Therefore, the positive results obtained with some of the UV filter chemicals only at concentrations greater than 100µg/ml do not indicate a phototoxic potential in vivo. When this information was taken into account during calculation of the overall predictivity of the 3T3 NRU PT test in the present study, an almost perfect correlation of in vitro versus in vivo results was obtained (between 95% and 100%), when either PIF or MPE were used to predict the phototoxic potential. The management team and participants therefore conclude that the 3T3 NRU PT test is a valid test for correctly assessing the phototoxic potential of UV filter chemicals, if the defined concentration limits are taken into account.
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The Use of Human Keratinocytes in the EU/COLIPA International In Vitro Phototoxicity Test Validation Study and the ECVAM/COLIPA Study on UV Filter Chemicals

Richard Clothier, Angie Willshaw, Helen Cox, Michael Garle, Helen Bowler and Robert Combes

The EU/COLIPA in vitro phototoxicity study involved the testing of 30 chemicals in Phase II, and the ECVAM/COLIPA study on UV filter chemicals involved the testing of 20 chemicals, for which in vivo human and/or animal data were available. Primary human keratinocytes, from four separate male donors, were not found to be sensitive to the 5J/cm2 UVA produced by the SOL500 lamp when assayed by using the neutral red uptake endpoint, as employed with the 3T3 cells used in these international interlaboratory validation studies. The primary human keratinocytes tested in one laboratory alongside the 3T3 fibroblasts gave consistent indications of phototoxicity with all the phototoxicants tested in the Phase II and UV filter studies. The one exception was bithionol, which was predicted to be non-phototoxic in both studies. None of the non-phototoxic chemicals resulted in a positive reaction with the Photoirritation Factor (PIF) version of the prediction model. However, when the Mean Photo Effect (MPE) prediction model version was applied (with a cut-off point of 0.1), one sunscreen agent, octyl salicylate, was deemed to have phototoxic potential. The entire set of negative rated chemicals included in Phase II and in the UV filter study were also rated as non-phototoxic by the MPE prediction model.
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In Vitro Phototoxicity Testing: Development and Validation of a New Concentration Response Analysis Software and Biostatistical Analyses Related to the Use of Various Prediction Models

Björn Peters and Hermann-Georg Holzhütter

As demonstrated in several validation studies, the dermal phototoxic potential of chemicals in humans can be effectively assessed by in vitro methods. The core of these methods is to monitor dose-response curves of a chemical in the absence and presence of light, to quantify the difference between these two curves by appropriate measures (either the photo-irritancy factor [PIF], or the mean photo effect [MPE]), and to use these measures as predictors of in vivo phototoxicity. We present new concentration-response analysis software for in vitro phototoxicity testing, which runs on current personal computers, and takes into account all the limitations identified when using a former program. We also demonstrate the validity and robustness of this new software by applying it retrospectively to all data available from two phases of the EU/COLIPA validation trial for the 3T3 neutral red update in vitro phototoxicity test. Some frequently raised questions pertaining to the use of prediction models in phototoxicity testing are addressed, including: the necessity of using prediction models based on a cut-off; whether it is justifiable to use sharp prediction cut-off values; whether there is a biostatistical justification for the highest concentration of the test chemical; and whether repeated testing of a chemical is required.
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ECVAM’s Research and Validation Activities in the Fields of Topical Toxicity and Human Studies

Valérie Zuang

This paper outlines the research, prevalidation and validation activities that ECVAM has undertaken in collaboration with its partners in the field of topical toxicity testing and human volunteer studies, from its creation until now (1994-2002).
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The Contributions of the European Cosmetics Industry to the Development of Alternatives to Animal Testing: Dialogue with ECVAM and Future Challenges

Odile de Silva

COLIPA (the European Federation of the Cosmetics Industry) represents 24 international companies and 2000 small and medium-sized enterprises. Together with ECVAM, COLIPA has been involved in the development and validation of alternative methods since the beginning of the validation efforts. The work of the Steering Committee on Alternatives to Animal Testing (SCAAT) is based on collaboration between companies, but also with academia, trade associations, the Scientific Committee on Cosmetics and Non-Food Products (SCCNFP), European Commission Directorates General, and ECVAM. Some success has been achieved, but some validation efforts have failed. One lesson is that the search for alternatives requires a lot of humility.
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Phototoxicity and Acute Toxicity Studies Conducted by the FRAME Alternatives Laboratory: A Brief Review

Richard H. Clothier

FRAME and the University of Nottingham have been in association for the past 25 years. During this time, the research in the FRAME Alternatives Laboratory (FAL) at the University of Nottingham, which is partly funded by FRAME and also, more recently, by ECVAM, has involved participation in a number of international validation studies. Validation has become a pre-requisite for the regulatory acceptance of in vitro alternative test procedures, and a number of key lessons have been learned from these studies. The directors of validation studies need to ensure that standard operating procedures (SOPs) are fully complied with, and that the equipment used is certified to be of an acceptable standard. Database managers need to be able to check the original data, and to ensure adherence to procedures agreed before the study began. When the validation study is part of an integrated EU Framework Project, such as ACute-Tox, the Workpackage Leader must have the ability to understand and evaluate the data to be presented for inclusion in the study analysis, and to check that it complies with acceptance criteria. The potential to relate observed cellular biochemical changes to morphological endpoints also increases the level of understanding of the relevance and/or limitations of an assay. For example, exposure to a surfactant can induce the temporary loss of adhesion junctions between adjacent epithelial cells, resulting in the loss of barrier integrity and other effects on cell culture activity, which can potentially be restored over time. Unexpected results from the NRU phototoxicity assay with human keratinocytes instead of 3T3 cells, stimulated research into the ability of the in vitro assay, not only to identify phototoxins, but also to identify their possible mechanisms of action and mechanisms underlying the protective capacity within human primary keratinocytes in vitro. The protective effects of UV-filters can also be used to ascertain their effects on the photoactivation of drugs.
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An Evaluation of the Effects of Photoactivation of Bithionol, Amiodarone and Chlorpromazine on Human Keratinocytes In Vitro

Linzi Reid, Nancy Khammo and Richard H. Clothier

Human skin is a continual target for chemical toxicity, due to its constant exposure to xenobiotics. The skin possesses a number of protective antioxidant systems, including glutathione and enzymic pathways, which are capable of neutralising reactive oxygen species (ROS). In combination with certain chemicals, the presence of ROS might augment the levels of toxicity, due to photoactivation of the chemical or, alternatively, due to an oxidatively-stressed state in the skin which exisited prior to exposure to the chemical. Bithionol is a phototoxic anti-parasitic compound. The mechanism of its toxicity and the possible methods of protection from its damaging effects have been explored. The capacity of keratinocytes to protect themselves from bithionol and other phototoxic chemicals has been investigated. In addition, the potential of endogenous antioxidants, such as vitamin C and E, to afford protection to the cells, has been evaluated. The intracellular glutathione stores of HaCaT keratinocytes were reduced following treatment with biothionol. Following photoactivation, both bithionol and chlorpromazine had similar effects, which suggests that glutathione is important in the detoxification pathway of these chemicals. This was confirmed by means of the visual identification of fluorescently-labelled glutathione. Endogenous antioxidants were unable to protect the HaCaT keratinocytes from bithionol toxicity or chlorpromazine phototoxicity. Amiodarone was shown to have no effect on cellular glutathione levels, which suggests that an alternative mechanism of detoxification was occurring in this case. This was supported by evidence of the protection of HaCaT cells from amiodarone phototoxicity via endogenous antioxidants. Thus, it appears that amiodarone toxicity is dependent on the levels of non-gluathione antioxidants present, whilst bithionol and chlorpromazine detoxification relies on the glutathione antioxidant system. This type of approach could indicate the likely mechanisms of phototoxicity of chemicals in vitro, with relevance to potential effects in vivo.
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FRAME and the Validation Process

Michael Balls and Richard Clothier

FRAME’s historical involvement in the development of the principles of validation, whereby the reliability and relevance of a procedure are established for a specific purpose, and in the practical application of the process, is summarised, and examples of participation in various validation studies on in vitro tests are reviewed. Emphasis is placed on the need for a parallel invalidation process, and on the role of ATLA as a forum for objective reporting and discussion on all aspects of the validation process.
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