Assessment of the Embryonic Stem Cell Test and application and use in the pharmaceutical industry

Abstract

BACKGROUND:The European Centre for the Validation of Alternative Methods (ECVAM) designed the Embryonic Stem Cell Test (EST) as a tool for classifying developmentally toxic compounds. An in vitro tool to assess developmental toxicity would be of great value to the pharmaceutical industry to help with toxicity-associated attrition. METHODS: ECVAM's EST protocol was used, but employing a different mouse embryonic stem cell (ESC) line and an alternative differentiation medium. A subset of the compounds used to validate the EST assay along with a number of in-house pharmaceutical compounds plus marketed pharmaceutical compounds were used to assess the EST performance with receptor-mediated compounds. RESULTS: Our results with ECVAM compounds mirrored ECVAM's. Compounds that were developmentally toxic in vivo were classified by the EST as moderate risk. Overall, the accuracy was 75% with the current set of data and the predictivity of low-, moderate-, and high-risk compounds was 90, 71, and 60% while the precision was 59, 86, and 100%, respectively. Interestingly, a number of the non-developmentally toxic compounds had values for the 3T3 IC 50 values, which were lower than the ESC IC 50 and ID 50, a situation not taken into account by ECVAM when designing the EST algorithm. CONCLUSIONS: The assay as currently constructed has a significant falsepositive rate (B40%), but a very low false-negative rate (B7%). Additional moderate-and high-risk compounds need to be assessed to increase confidence, accuracy, and understanding in the EST's predictivity. Birth Defects Res (Part B) 83: 104-111, 2008.

Mouse and Human Embryonic Stem Cells: Can They Improve Human Health by Preventing Disease?

“…Other lines have since been used with success. For example, a DBA/1lacj line derived at Pfizer gave results that mirrored those obtained in the original validation study by ECVAM [15]. As was seen in the original study, the EST, when used with DBA/1lacj cells, predicted high risk compounds better than low risk counterparts.…”

Assessment of technical protocols for novel embryonic stem cell tests with molecular markers (Hand1- and Cmya1-ESTs): a preliminary cross-laboratory performance analysis

“…cytotoxicity with ESCs and 3T3 fibroblasts in addition to ESC cardiac differentiation inhibition (Spielmann et al, 1997). Recently, embryotoxicity of a variety of compounds like pharmaceuticals (Paquette et al, 2008;Eckardt and Stahlmann, 2010), cosmetics (Chen et al, 2010), industrial chemicals (de Jong et al, 2009) and nanoparticles (di Guglielmo et al, 2010), has been assessed with the EST. Although it is considered a promising in vitro test for developmental toxicity, there are also increasing demands for in vitro alternatives that can provide a less expensive and higher through-put means for testing.…”

Alternative models in developmental toxicology

“…In a study by Marx-Stoelting et al [2009], only 2 of 13 drugs were correctly categorized. In a study by Paquette et al [2008], 19 Pfizer compounds that were receptormediated were tested with an overall accuracy in a modified version of the mEST of 53%. Compounds that were non-or weakly teratogenic scored especially poorly in this assay.…”

“…For these assays, the compounds need to be soluble in aqueous media. Small amounts of ethanol or DMSO can be added to the cultures to increase solubility; Paquette et al [2008] used 0.25% (v/v) for the upper limit of DMSO and 0.5% (v/v) for 70% ethanol in the mEST; higher concentrations were toxic to the cells. In the zebrafish assays, DMSO is the most commonly used solvent; concentrations used were 0.5% [Brannen et al 2010] or 0.2% .…”