Assaying embryotoxicity in the test tube: Current limitations of the embryonic stem cell test (EST) challenging its applicability domain

Abstract

Testing for embryotoxicity in vitro is an attractive alternative to animal experimentation. The embryonic stem cell test (EST) is such a method, and it has been formally validated by the European Centre for the Validation of Alternative Methods. A number of recent studies have underscored the potential of this method. However, the EST performed well below the 78% accuracy expected from the validation study using a new set of chemicals and pharmaceutical compounds, and also of toxicity criteria, tested to enlarge the database of the validated EST as part of the Work Package III of the ReProTect Project funded within the 6th Framework Programme of the European Union. To assess the performance and applicability domain of the EST we present a detailed review of the substances and their effects in the EST being nitrofen, ochratoxin A, D-penicillamine, methylazoxymethanol, lovastatin, papaverine, warfarin, β-aminopropionitrile, dinoseb, furosemide, doxylamine, pravastatin, and metoclopramide. By delineation of the molecular mechanisms of the substances we identify six categories of reasons for misclassifications. Some of these limitations might also affect other in vitro methods assessing embryotoxicity. Substances that fall into these categories need to be included in future validation sets and in validation guidelines for embryotoxicity testing. Most importantly, we suggest conceivable improvements and additions to the EST which will resolve most of the limitations.

Keywords::

• Reproductive toxicology
• teratogenicity
• embryotoxicity
• applicability domain
• in vitro methods
• alternatives to animal testing
• embryonic stem cell test (EST)

Acknowledgments

We thank Katharina Schlechter and Birgitta Slawik for expert help in performing the embryonic stem cell test.

Declaration of interest

The work described in the current manuscript was funded within the 6th Framework Programme of the European Union (Contract No: LSHB-CT-2004-503257). The author’s affiliation is as shown on the cover page. The authors have sole responsibility for the writing and content of the paper.