The ECVAM International Validation Study on In Vitro Embryotoxicity Tests: Results of the Definitive Phase and Evaluation of Prediction Models

Abstract

From 1996 to 2000, ZEBET (Centre for Documentation and Evaluation of Alternative Methods to Animal Experiments at the BgVV, Berlin, Germany) coordinated the European Centre for the Validation of Alternative Methods (ECVAM) prevalidation and validation study on three embryotoxicity tests: a) a test employing embryonic stem cell lines (EST); b) the micromass (MM) test; and c) the postimplantation rat whole-embryo culture assay (WEC test). The main objectives of the study were to assess the performance of these three in vitro tests in discriminating between non-embryotoxic, weakly embryotoxic and strongly embryotoxic compounds. Phase I of the study (1997) was designed as a prevalidation phase, for test protocol optimisation, and for the establishment of a comprehensive database of in vivo and in vitro data on embryotoxic compounds. Phase II (1998–2000) involved a formal validation trial, conducted under blind conditions on 20 test compounds selected from the database, which were coded and distributed to the participating laboratories. In the preliminary phase of the validation study, six chemicals out of the 20, which showed embryotoxic potential, were tested. These results were used to define new biostatistically based prediction models (PMs) for the MM and WEC tests, and to evaluate those developed previously for the EST. As a next step, the PMs were evaluated by using the results for the remaining 14 chemicals of the definitive phase of the validation study. The three in vitro embryotoxicity tests proved to be applicable to testing a diverse group of chemicals with different embryotoxic potentials (non-embryotoxic, weakly embryotoxic, and strongly embryotoxic). The reproducibility of the three in vitro embryotoxicity tests were acceptable according to the acceptance criteria defined by the Management Team. The concordances between the embryotoxic potentials derived from the in vitro data and from the in vivo data were good for the EST and the WEC (PM2) test, and sufficient for the MM test and the WEC (PM1) tests according to the performance criteria defined by the Management Team before the formal validation study. When applying the PM of the EST to the in vitro data obtained in the definitive phase of the formal validation study, chemicals were classified correctly in 78% of the experiments. For the MM and the WEC tests, the PMs provided 70% and 80% (PM2) correct classifications, respectively. And, very importantly, an excellent predictivity (100%, except for PM1 of the WEC test, with 79%, considered as good) was obtained with strongly embryotoxic chemicals in each of the three in vitro tests.

Developmental exposure to ethanol increases the neuronal vulnerability to oxygen–glucose deprivation in cerebellar granule cell cultures

“…Whole embryo cell cultures show gene expression patterns very similar, over time, to in utero embryos; and, in the case of rat, cultures are matching to a great extent the gene expression profile in human embryos undergoing neurulation and early embryogenesis (Robinson et al, 2012b). Therefore, given the high in vivo translation potential of whole embryo cell cultures (Genschow et al, 2002), the results can benefit of better support for a possible in vivo extrapolation. However, whole embryos display tissue heterogeneity, which may mask some changes in specific tissues or cells (Zhou et al, 2011).…”

“…In this experiment whole embryo mouse cultures were performed on four control embryos and four ethanol-treated ones, the latter characterized by a phenotype of open neural tubes (Wang et al, 2008). Transcriptomic studies suggest that teratogenic effects observed in whole embryo cultures are relevant to previously identified mechanisms of toxicity in vivo (Genschow et al, 2002;Luijten et al, 2010;Zhou et al, 2011).…”

Review of current and “omics” methods for assessing the toxicity (genotoxicity, teratogenicity and nephrotoxicity) of herbal medicines and mushrooms

“…Overall accuracy of 70% (Genschow et al, 2002) Foeniculum vulgare L. (no teratogenicity) (Ostad et al, 2004) Mammalian whole embryo culture test (WEC) Embryos are isolated on day 10 of gestation and cultured for 48 h; morphological, developmental, functional and growth parameters are measured…”

“…Overall accuracy of 80% (Genschow et al, 2002) Dihydroartemisinin primarily affects primitive red blood cells, causing subsequent tissue damage and dysmorphogenesis (Longo et al, 2006) Methods based on embryos of lower order species Frog embryo teratogenesis assay -Xenopus (FETAX) During the first 96 h Xenopus embryos development parallels many of the major processes of human organogenesis (Measurement of mortality, malformation, and growth inhibition)…”

Cellular Reprogramming: A New Technology Frontier in Pharmaceutical Research

“…Despite validation studies reporting a relatively promising predictive efficiency of this assay compared to other in vitro models (i.e. correct classification of 78% of well-characterized compounds into the categories of non-embryotoxic, weakly embryotoxic and strongly embryotoxic compounds) (80), this test is limited in its ability to predict drug toxicity profiles for human diseases. Species-specific pharmacokinetic profiles frequently do not address the metabolism or the genetic diversity of patients living in heterogeneous conditions, thereby giving rise to drug-induced responses that may be clinically irrelevant in humans.…”