in vitro tests

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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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MEIC Evaluation of Acute Systemic Toxicity

Björn Ekwall, Barbro Ekwall and Michael Sjöström

The Multicenter Evaluation of In vitro Cytotoxicity (MEIC) programme was set up to evaluate the relevance for human acute toxicity of in vitro cytotoxicity tests. A total of 61 assays were used to test all 50 reference chemicals. The results of all the tests and the human database were presented in the first five papers of this series. An evaluation of the relevance for human acute toxicity of all submitted test results with use of hard linear regression modelling was presented in the next two papers, and demonstrated a high relevance of in vitro tests, notably tests involving human cell lines. In the present study, multivariate partial least square (PLS) modelling with latent variables analysis has been used to reach two objectives. The first objective was to study the prediction of human acute toxicity by the 61 assays. The second objective
was to select a practical battery from the 61 assays, with an optimal prediction of lethal blood concentrations from human acute poisonings of the chemicals. A two-component PLS model of all 61 assays predicted three sets of lethal blood concentrations (clinical, forensic and peak concentrations) very well (R2 = 0.77, 0.76 and 0.83, Q2 = 0.74, 0.72 and 0.81, respectively), providing correlative evidence for a high relevance for human acute toxicity of most of the assays. The assays with human cells were highly predictive, whereas assays with very short incubation times and non-fish ecotoxicological assays were least predictive. These findings confirm the previous results from linear regression analysis. To select an optimal battery, 24 successive PLS models of in vitro data were compared with lethal peak concentrations. The battery selection was based on 38 chemicals with reliable and relevant lethal peak concentrations. An initial PLS model of all 61 assays was used to select the 15 most predictive and most distinct assays. Subsequent PLS models were used to measure the decrease in prediction when assays were deleted from the 15-test battery, as well as the increase in prediction when some extrapredictive assays (as identified by the deletion process) were added later to an optimal two-test battery. The most predictive three-test battery (R2 = 0.79 and Q2 = 0.78 for all 50 chemicals) included two circumstantial assays. The most predictive and most cost-effective battery consisted of three human cell line assays, with four endpoints and two exposure times, i.e. protein content (24 hours), ATP content (24 hours), inhibition of elongation of cells (24 hours), and pHchange (7 days). This 1, 5, 9, 16 battery exclusively measures basal cytotoxicity, and is highly predictive (R2 = 0.77 and Q2 = 0.76 for 50 chemicals) of the actual lethal peak blood concentrations from acute poisonings in humans. The battery prediction compares favourably with the prediction of human lethal dose by a PLS model of rat and mouse 50% lethal dose (LD50) values for the 50 chemicals (R2 = 0.65 and Q2 = 0.64). The three assays of the battery and other promising MEIC assays should be formally validated as soon as possible. The battery can be used immediately for several non-
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Biostatistical Methods for the Validation of Alternative Methods for In Vitro Toxicity Testing

Lutz Edler and Carina Ittrich

Statistical methods for the validation of toxicological in vitro test assays are developed and applied. Validation is performed either in comparison with in vivo assays or in comparison with other in vitro assays of established validity. Biostatistical methods are presented which are of potential use and benefit for the validation of alternative methods for the risk assessment of chemicals, providing at least an equivalent level of protection through in vitro toxicity testing to that obtained through the use of current in vivo methods. Characteristic indices are developed and determined. Qualitative outcomes are characterised by the rates of false-positive and false-negative predictions, sensitivity and specificity, and predictive values. Quantitative outcomes are characterised by regression coefficients derived from predictive models. The receiver operating characteristics (ROC) technique, applicable when a continuum of cut-off values is considered, is discussed in detail, in relation to its use for statistical modelling and statistical inference. The methods presented are examined for their use for the proof of safety and for toxicity detection and testing. We emphasise that the final validation of toxicity testing is human toxicity, and that the in vivo test itself is only a predictor with an inherent uncertainty. Therefore, the validation of the in vitro test has to account for the vagueness and uncertainty of the “gold standard” in vivo test. We address model selection and model validation, and a four-step scheme is proposed for the conduct of validation studies. Gaps and research needs are formulated to improve the validation of alternative methods for in vitro toxicity testing.
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The Need for a Formal Invalidation Process for Animal and Non-animal Tests

Michael Balls and Robert Combes

A plethora of regulations require that many chemicals and chemical products are tested for efficacy and/or toxicity. When permitted to operate effectively and without bias, the ECVAM/ICCVAM/OECD validation process can be used to independently establish that new animal and non-animal test procedures are sufficiently relevant and reliable for their stated purposes and should be considered for regulatory use. However, the validation process is under threat because of vested interests of various kinds, and it is clear that many currently-accepted animal tests and candidate animal and non-animal tests do not, and could never, meet the agreed criteria for necessity, test development, prevalidation, validation and acceptance. We therefore need an invalidation process to parallel and protect the validation process, so that such methods could be independently reviewed and declared irrelevant and/or unreliable for their claimed purposes. An additional advantage of such a process would be that valuable resources would no longer be wasted in attempts to secure the acceptance of inherently inadequate tests.
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