The ACuteTox Testing Strategy: The Way Forward to Replace the Animal Tests Used Today in the Field of Acute Systemic Toxicity

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Acute Systemic Toxicity

The ACuteTox Testing Strategy: The Way Forward to Replace the Animal Tests Used Today in the Field of Acute Systemic Toxicity

By Cecilia Clemedson, Expertrådet ECB Environmental Competence Ltd

Published: December 6, 2007

About the Author(s)
Cecilia Clemedson is the Scientific Coordinator of the ACuteTox project (www.acutetox.org) and the Coordinator of the ForInViTox project. She has extensive experience in pre-validation of in vitro methods for acute systemic toxicity testing. Between 1992 and 1999, she worked with both the in vitro-in vitro and in vitro-in vivo evaluations of the MEIC (Multicentre Evaluation of in vitro cytotoxicity tests) programme. She was also the Scientific Secretary of the MEIC program involving 97 laboratories and involved in the compilation of human and animal toxicity data within this project. She has a Ph.D. in in vitro neurotoxicology (1992) and has published more than 35 articles in peer reviewed journals on in vitro neurotoxicology and pre-validation of in vitro methods for acute systemic toxicity testing. She is one of the founders of Expertrådet ECB Environmental Competence Ltd (www.expertradet.se), which is a consultant company with special expertise in in vitro toxicology.

Dr. Cecilia Clemedson
Expertrådet ECB Environmental Competence Ltd
Rådan 3
SE-191 40 Sollentuna
Sweden
E-mail: c.clemedson@cctoxconsulting.a.se;

While animal tests for topical toxicity have been successfully replaced one by one by alternative methods, for systemic toxicity only some reduction or refinement methods have been developed and accepted so far. This despite the fact that an extensive amount of work has been performed since the 70s leading to a great number of existing in vitro models for acute toxicity. Several studies have also shown good correlation between in vitro basal cytotoxicity data and rodent LD50 values (for example the Willi Halle’s Register of Cytotoxicity) and even better correlation of around 70% between the in vitro IC50 values and human lethal blood concentrations (the MEIC study). However, even though these validation studies show a surprisingly good correlation, general cytotoxicity does not always predict acute systemic toxicity in satisfactory way.

Necessity of new test strategies for estimating acute toxicity

Most of the previous studies assumed that the fundamental basis for acute toxicity is the interference of compounds with basal functions of all cell types. Thus, the concentration of a chemical causing 50% of the cells in a cell culture to suffer basal cytotoxicity had been assumed to be indicative of its LD50 in vivo, irrespective of the cell type involved. For such a relationship to hold, it is implicit that the concentration of the test chemical in a cell culture is comparable to the concentration that the target cells in vivo would be exposed to. This assumes that the chemical is subjected to rapid oral absorption and even distribution in the organism, and that there is no rapid elimination of the chemical and no major formation of more toxic metabolites.

The concept of using basal cytotoxicity to estimate acute toxicity also assumes that in vivo toxicity is not primarily due to an impairment of specific functions of differentiated cells, i.e. organ- or tissue-specific effect is not a major determinant of acute effects.

Thus, the reasons why the acute systemic toxicity is relatively poorly predicted by basal cytotoxicity tests for some chemicals are either that the chemicals interact with organ-specific targets at low tissue concentrations, have restricted accessibility to the target tissues, are dependent on bioactivation or are quickly eliminated or detoxified through metabolism. These issues were considered in the ERGATT/CFN. Integrated Toxicity Test Scheme (ECITTS) project and it was shown that relevant toxic doses can be estimated on the basis of biokinetic models and relevant in vitro-derived toxicity data.

In conclusion, it is clear that the correlation between basal cell cytotoxicity and acute lethal potency does not hold for all chemicals and that there are outliers and such a phenomenon is to be expected. From the results of previous studies it has been estimated that, if IC50 values for basal cell cytotoxicity are used for classification purposes, the data for some 30% of chemicals would result in either an underestimation or an overestimation of the predicted LD50s. Thus, in vitro IC50 values are not always reliable indicators of in vivo acute toxicity. The results of the MEIC study clearly indicated that the basal cytotoxicity test should be complemented by two types of tests in order to improve the predictability: in vitro tests for kinetics and organ-specific toxicity. In the ACuteTox project, which is an EU-funded project with 35 partners that started in January 2005, the aim is to identify the main parameters (such as absorption, distribution, metabolism, excretion, and/or organ specificity) that could improve the in vitroin vivo correlation for acute systemic toxicity.

The ACuteTox testing strategy for acute systemic toxicity

The modern toxicology strategy applied in ACuteTox is to include all crucial information about a test compound. This could include, for example, its physical and chemical properties (e.g. pKa values, lipophilicity, molecular weight, reactive groups), structural similarities to compounds with known biological activity (SAR), bioavailiability and biological activity already available. The approach will be used to estimate how a compound interacts with molecular targets that are important for a certain biological effect in humans. This is done by using an integrated approach. The main challenge is to identify chemical properties of toxicity alerts and to compose a test battery with relevant endpoints.

Within the ACuteTox project, it is suggested that an ideal in vitro/in silico test battery should consist of a basal cytotoxicity high throughput test (for example the 3T3 Neutral red uptake test) that assess cell viability after an exposure time longer than one cell cycle. Furthermore, the probability that the chemical, or its metabolites, will reach crucial target tissues has to be estimated. Together with estimates of uptake and distribution in the body, data about metabolism should be integrated into the toxicity evaluation. For estimation of uptake and distribution it would be preferable to use in silico models, which could limit the costs and time and still give sufficient reliable information.

It is also necessary to take into account of the possibility that a chemical will be active against specific cells leading to neurotoxicity, nephrotoxicity, haematopoietic toxicity and/or hepatotoxicity. Ideally high throughput in vitro systems capable of measuring crucial neurotoxic, nephrotoxic, hepatoxic and haematopoietic endpoints should be included in a ACuteTox testing battery. The intention is to propose an ideal testing strategy consisting of 5-7 in vitro/in silico tests that has the capacity to correctly predict at least 90% of compounds. However, we should have in mind that such a testing strategy probably would be far too complex and expensive in order to replace the current LD50 tests. The big challenge is therefore to reduce the ideal testing strategy to an acceptable number (approx. 3) of simple and robust tests and still keep a high predictability. Results from the MEIC study showed that tests for neurotoxicity are very important to include in such as test battery, whereas tests for hepatotoxicity and biotransformation may not have the same importance for acute toxicity as for long-term toxicity.

The results of ACuteTox show that a broad collection of neurotoxicity assays could in a very good way predict the neurotoxic compounds which are not picked up by the basal cytotoxicity tests. The challenge is now to find a more general neurotoxicity assay that could pick up several different neurotoxic mechanisms of action. Furthermore, it appears important to include assays to estimate uptake and distribution of chemicals in the body. The future results of the ACuteTox project will tell if a testing strategy based just a few tests will be reliable enough, or if a more complex testing strategy is needed in order make a sufficiently good prediction of acute systemic toxicity.

1 ERGATT is the European Research Group for Alternatives in Toxicity Testing and CFN, the Swedish National Board for Laboratory Animals
©2007 Cecilia Clemedson