Prevalidation of the Embryonic Stem Cell Test (EST)—A New In Vitro Embryotoxicity Test

Abstract

Pluripotent embryonic stem cells (ES cells) of the mouse (cell-line D3) can be maintained in the undifferentiated state in the presence of LIF (Leukaemia Inhibitory Factor). Upon withdrawal of LIF, these cells differentiate into various cell types under appropriate conditions. This property of ES cells allowed us to develop an in vitro embryotoxicity test, the Embryonic Stem Cell Test (EST; In Vitro Toxicology 1997, 10, 119–127), which does not require taking embryonic cells or tissues from pregnant animals. In the EST, the effect of test chemicals on three endpoints is assessed: inhibition of the differentiation of ES cells into contracting myocard, cytotoxicity in ES cells and cytotoxicity in mouse 3T3 fibroblasts, which are serving as differentiated cells in the test. The results of a prevalidation study of the EST are described, which was conducted according to the ECVAM prevalidation scheme. In the first stage of the study (Phase I), a standard operating procedure (SOP) was elaborated. In the second phase (Phase II), the interlaboratory transferability of the EST was assessed using three test chemicals representing three classes of embryotoxicity (a strong, a weak and a non-embryotoxic chemical) in two European laboratories (ZEBET at the BgVV in Berlin, Germany; ECVAM at the JRC in Ispra, Italy) and one US laboratory (Institute for In Vitro Sciences (IIVS) in Gaithersburgh, MA, USA). In the final stage of prevalidation (Phase III), nine test chemicals and a positive control were tested under blind conditions at ZEBET and ECVAM. The statistical evaluation of the results led to the development of an improved prediction model for the EST.