Embryonic stem cells developmental toxicity tests

In Europe, the developmental toxicity testing (including teratogenicity) of new cosmetic ingredients is performed according to the Cosmetics Directive 76/768/EEC only alternatives leading to full replacement of animal experiments should be used. This chapter presents the three scientifically validated animal alternative methods for the assessment of embryotoxicity the embryonic stem cell test (EST), the micromass (MM) assay, and the whole embryo culture (WEC) assay. 

Van Dartel DAM, Piersma AH (2011) The embryonic stem cell test combined with toxi-cogenomics as an alternative testing model for the assessment of developmental toxicity. Reprod Toxicol 32 235-244... 

Therefore, a validation exercise with a variety of compounds with unknown mechanisms of developmental toxicity has only limited value if only used to derive an overall predictability rate of a single assay. It is more useful to elucidate the applicability domain of the assay in terms of the mechanisms of development covered, and to validate that aspect by testing compounds that do or do not affect the applicability domain. For single end point assays such as specific receptor activation assays, this exercise is relatively straightforward. For more complex assays such as those involving embryonic cell differentiation, the understanding of the applicability domain is more complex, as extensive research with the embryonic stem cell test has learned (27, 47). Whole embryo cultures are probably more straightforward in terms of applicability domain as they involve the entire embryo in a limited window of development, but such assays are complex and not animal free. 

Gene set assembly for quantitative prediction of developmental toxicity in the embryonic stem cell test. Toxicology 284(l-3) 63-71... 

The embryonic stem cell test is an animal-free alternative test method for developmental toxicity. Mouse embryonic stem cells are cultured in a hanging drop method to form embryoid bodies. These embryoid bodies, when plated on tissue culture dishes, differentiate to form contracting myocardial cell foci within 10 days. Inhibition of cardiomyocyte differentiation by test compounds serves as the end point of the assay, as monitored by cormting contracting muscle foci under the microscope. 

Key words Embryonic stem cell test. Embryonic stem cells. Alternative test method, Cardiomyocyte differentiation. Developmental toxicity... 

Due to the limited applicability of in silico SAR approaches for developmental toxicity, there is more reliance on in vitro screening. From what has been publicly disclosed, it is evident that the four in vitro tests used for industrial screening are chick embryonic neural retina (CENR) micromass culture, whole embryo culture (WEC, rodent or rabbit), and mouse embryonic stem cells (EST). Recently, there has been significant interest within the pharmaceutical industry in the use of zebrafish for developmental toxicity testing,30 but because this aspect is in its infancy, there is little that has been publicly disclosed except limited abstracts and slide decks at several workshops.31 Although reviewed in considerable detail elsewhere,30-32 36 each test will be briefly compared and contrasted here. 

Recent advances in embryonic stem cell technology have made these cells available for a variety of toxicity models.48 50 The use of murine embryonic stem cells for developmental toxicity testing is based upon the observation that, in... 

Figure 9-5 The sensitivity of the conceptus to a theoretical teratogen during rat gestation (modified from 161). The most susceptible window is organogenesis with low levels of vulnerability at the time of implantation and the period of functional maturation. Superimposed are the approximations of when the developmental landmarks that are represented in the four in vitro tests occur. The chick embryo neural retina model (CENR) represents events around GD 10-13. The mouse embryonic stem cell test (EST) corresponds roughly to the period of GD 6-10 in the rat, near the peak of sensitivity. Whole embryo culture (WEC) recapitulates the window at the peak of sensitivity, between GD 9-11 or GD 10-12 depending upon the window within which the culture is conducted. Rabbit cultures are also done between GD 10-12. Represented by the single ( ) and double asterisk ( ), respectively, are the initiation and termination of the dosing period in regulatory compliant preclinical embryo/fetal toxicity studies. Thus, the zebrafish is the only model that permits exposure to test article during this important period.

Figure 9-6 A generic strategy integrating the three facets of developmental toxicity risk assessment namely (a) the risk of pharmacologic modulation of the therapeutic target during gestation, (b) in silico, SAR and (c) in vitro screening. Abbreviations The chick embryo neural retina (CENR) embryonic stem cell test (EST), whole embryo culture (WEC), Good Laboratory Practice (GLP), Embryo/Fetal Developmental Toxicity (EFD) study. "Front-loading" is the conduct of the EFD study prior to Phase lib.

Seiler A, Visan A, Buesen R, et al. Improvement of an in vitro stem cell assay for developmental toxicity The use of molecular endpoints in the embryonic stem cell test. Reprod Toxicol. 2004 18(2) 231-240. 

Adler S, Lindqvist J, Uddenberg K, et al. Testing potential developmental toxicants with a cytotoxicity assay based on human embryonic stem cells. Altern Lab Anim. 2008 36(2) 129-140. 

Buesen R, Genschow E, Slawik B, Visan A, Spielmann H, Luch A, Seiler A (2009) Embryonic stem cell test remastered comparison between the validated EST and the new molecular FACS-EST for assessing developmental toxicity in vitro. Toxicol Sci 108 389-400... 

Bremer S, Hartung T (2004). The use of embryonic stem cells for regulatory developmental toxicity testing in vitro—the current status of test development. Curr. Pharmac. Design. 10 2733-2747. 

Kuske, B., Pulyanina, P. Y., Zur Nieden, N. 1. 2012. Embryonic stem cell test stem cell use in predicting developmental cardio-toxicity and osteotoxicity. Methods in Molecular Biology, 889, 147-79. 

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