Cytotoxicity Evaluation of the First Ten MEIC Chemicals: Acute Lethal Toxicity in Man Predicted by Cytotoxicity in Five Cellular Assays and by Oral LD50 Tests in Rodents
Determination of the Starting Dose for Acute Oral Toxicity (LD50) Testing in the Up and Down Procedure (UDP) From Cytotoxicity Data
Horst Spielmann, Elke Genschow, Manfred Liebsch and Willi Halle
To reduce the number of animals used in acute oral toxicity testing, cytotoxicity data (IC50) can be used to determine the starting dose for in vivo testing by applying the standard regression between IC50 and acute oral LD50 values in the Register of Cytotoxicity (RC). In the RC, the correlation between cytotoxicity, represented by the mean IC50 (IC50x), and the acute oral LD50 of rats and/or mice has been determined for 347 chemicals by applying the linear regression model for log-transformed pairs of IC50 versus oral LD50. The standard regression line of the two toxicity parameters is characterised by an intercept a = 0.625 and regression coefficient b = 0.435, and 252 of 347 chemicals (72.6 %) are located within a dose-range differing by not more than 0.699 (factor FG ≤ log 5) from the standard regression line. In the present study, we have used the RC and its IC50/LD50 regression model to predict the LD50 values from cytotoxicity data for nine chemicals which were tested in an evaluation study of the Up and Down Procedure (UDP). For seven of the nine chemicals, LD50 values (mg/kg) predicted from the RC were in the same doserange as LD50 values determined in vivo, while the dose-range differed by more than one order of magnitude for the two remaining chemicals. Thus, the prediction of LD50 values from cytotoxicity data was promising in this limited data set. It is proposed that a tiered in vitro/in vivo testing approach will reduce animal use in the UDP method. As the first step, the in vitro cytotoxicity of a new chemical is determined. By applying the RC regression and adapting it to the sensitivity of a specific cell line, the LD50 value (mg/kg) can be predicted from the IC50 value. The predicted LD50 dose is then used as the starting dose in the UDP. In the RC model, the precision of the prediction increases with decreasing toxic potential, and the majority of industrial chemicals (around 90%) are not toxic according to EU classification criteria.
Tarja Toimela, Hanna Mäenpää and Hanna Tähti
A mini-review is presented of the current techniques for maintaining Müller cells in a culture. Within the retina, Müller cells are the predominant glial cells. These highly specialised cells extend over the entire neural retina. One of the most important of the various physiological functions of Müller cells is to regulate the balance of ions and neurotransmitters in the retina. Disturbance of these regulatory functions may lead to toxic effects on receptor and other neural cells in the neuroretina, and may be a common mechanism of clinical retinal neuropathy. The main excitatory neurotransmitter in the retina is glutamate. Müller cells regulate the amount of glutamate in the synaptic regions of the neural network in the retina. Accumulation of extra glutamate seems to be an important mechanism for initiating pathological changes leading to retinal damage. Many previous in vitro studies on the role of Müller cells in retinal toxicology have been based on the use of morphological and histochemical methods. In cell toxicology studies, it is important to develop culture techniques able to provide more cells for biochemical determinations.
Assessment of Ocular Irritation by Image Processed Quantification of Cell Injury in Human Corneal Cell Cultures and in Corneal Constructs
Maria Engelke, Jens Patzke, Svitlana Tykhonova and Michaela Zorn-Kruppa
Currently, there are no accepted alternative tests for the replacement of animals in ocular irritation testing. This study focused on the quantification of cellular viability as a measure of toxic events in immortalised human corneal cell cultures and a three-dimensional corneal construct. Simultaneous vital dye staining by calcein AM and ethidium homodimer-1 was used to provide “live” and “dead” probes, respectively. For further quantification, we have developed image processing tools to evaluate digital images obtained from confocal fluorescence scanning microscopy measurements. Based on the finding that ocular irritation can be related to the extent of cell injury at the various cell layers of the cornea, we extended our studies from corneal cell cultures to an in vitro human corneal equivalent system comprising epithelial, stromal keratocyte and endothelial layers. Our results showed that the microscopic measurement of cellular injury by using either cell cultures or in vitro corneal constructs, combined with image processed quantification, can provide insight into the extent of the toxic effects.
Emanuela Corsini and Erwin L. Roggen
At present, several animal-based assays are used to assess immunotoxic effects such as immunosuppression and sensitisation. The use of whole animals, however, presents several secondary issues, including expense, ethical concerns and relevance to human risk assessment. There is a growing belief that non-animal approaches can eliminate these issues without impairing human safety, provided that biological markers are available to identify the immunotoxic potentials of new chemicals to which humans may be exposed. Driven by the 7th Amendment to the EU Cosmetics Directive, the new EU policy on chemicals (the REACH system), proposals to update the European legislation on the protection of animals used in research, and emerging visions and strategies for predicting toxicity, such in vitro methods are likely to play a major role in the near future. The realisation that the immune system can be the target of many chemicals, resulting in a range of adverse effects on the host’s health, has raised serious concerns from the public and within the regulatory agencies. Hypersensitivity and immunosuppression are considered the primary focus for developing in vitro methods in immunotoxicology. However, in vitro assays to detect immunostimulation and autoimmunity are also needed. This review of the state-of-the-art in the field of in vitro immunotoxicity, reveals a lack of cell-based immunotoxicity assays for predicting the toxicity of xenobiotics toward the immune system in a simple, fast, economical and reliable way.