Prevalidation of the embryonic stem cell test (EST) - A new in vitro embryotoxicity test

Pluripotent embryonic stem cells (ES cells) of the mouse (cell-line D3) can be maintained in the undifferentiated stare in the presence of LIF (Leukae mia Inhibitory Factor). Upon withdrawal of LIF, these cells differentiate i nto various cell types under appropriate conditions. This property of ES ce lls allowed us to develop an in vitro embryotoxicity test, the Embryonic St em Cell Test (EST; In Vitro Toxicology 1997, 10, 119-127), which does not r equire 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 di fferentiated cells in the test. The results of a prevalidation study of the EST are described, which was conducted according to the ECVAM prevalidatio n scheme. In the first stags of the study (Phase I), a standard operating p rocedure (SOP) was elaborated. In the second phase (Phase IT), the interlab oratory transferability of the EST was assessed using three test chemicals representing three classes of embryotoxicity (a strong, a weak and a non-em bryotoxic chemical) in two European laboratories (ZEBET at the BgVV in Berl in, Germany: ECVAM? at the JRC in Ispra, Italy) and one US laboratory (Inst itute for In Vitro Sciences (IIVS) in Gaithersburgh, MA, USA). In the final stage of prevalidation (Phase III), nine test chemicals and a positive con trol were tested under blind conditions at ZEBET and ECVAM. The statistical evaluation of the results led to the develop ment of an improved predictio n model for the EST. (C) 1999 Elsevier Science Ltd. All rights reserved.