Embryonic stem cell test

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. 

Genschow E et al (2004) Validation of the embryonic stem cell test in the international... 

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... 

The most extensive formal validation study in this area addressed whole embryo culmre (WEC), micromass (MM), and the embryonic stem cell test (EST) (26). This validation study proved a great learning experience in view of understanding the value of a study with a limited amount of diverse compounds in terms of extrapolation to the universe of chemicals. Subsequent application of the validated EST taught us that the 80% predictability was not reproduced with additional compounds (27). One of the issues underlying this discrepancy was in the mathematical prediction model used, which did not always appear to match the biology of the assay in terms of observed differentiation inhibition. 

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. 

Marx-Stoelting P, Adriaens E, Ahr HJ et al (2009) A review of the implementation of the embryonic stem cell test (EST). The report and recommendations of an ECVAM/ReProTect Workshop. Altern Lab Anim 37(3) 313-328... 

Paquette JA, RumpfSW, Streck RD et al (2008) Assessment of the embryonic stem cell test and application and use in the pharmaceutical industry. Birth Defects Res B Dev Reprod Toxicol 83(2) 104-111... 

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

A modified version of the ECVAM-approved embryonic stem cell test (EST) (52) was recently developed by Hunter and coworkers in NHEERL (31). This assay is capable of quantitatively assessing cytotoxicity and cardiomyocyte differentiation and was used to test the ToxCast Phase I chemical library (32). Briefly, male murine J1 mES cells were seeded onto gelatin-coated 96-well plates at a known density in differentiation media on day 0. On day 1, cells were treated with chemicals ranging from 0.0125 to 12.5 pM, and on day 9 In-Cell Western (Li-Cor Biosciences) assays were assayed for a- and P-cardiac Myosin Heavy Chain (MYH6/MYH7)... 

Genschow E, Spiehnann H, Scholz G, Pohl I, Seiler A, Clemann N, Bremer S, Becker K (2004) Validation of the embryonic stem cell test in the international ECVAM validation study on three in vitro embryotoxicity tests. Altern Lab Anim 32 209-244... 

Spielmann H, Pohl I, Doering B, Liebsch M, Moldenhauer E (1997) The embryonic stem cell test, an in vitro embryotoxicity test using two permanent mouse cell lines, 3 T3 fibroblasts and embryonic stem cells. In Vitro Toxicol 10 119-127... 

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... 

Fig. 2. The embryonic stem cell test. Droplets with 750 ES cells per 20 l complete medium are pipetted on the inner side of a cover lid of a 94 x 16 mm bacterial Petri dish (a). 56 droplets are cultured in a Flanging drop method.The lid is placed on a Petri dish, containing 5 ml of PBS (b).The cells will form Embryoid bodies (EB) (c) by proliferation and clustering. After 3 days the EB are transferred to a Petri dish containing 5 ml suspension medium (d). The EB are cultured for 2 days and then transferred to a 24-well plate (e) where they will further differentiate into contracting cardiomyocytes. The scoring is pertormed at day 10 by examining the EB under a light microscope (f).

Seiler AE, Spieknann H (2011). The validated embryonic stem cell test to predict embryotoxicity in vitro. Nat Protoc 6(7) 961-978... 

Marx-Stoelting P, Adriaens E, Ahr Hf, Bremer S, GarthoffB, Gelbke HP, Piersma A, Pellizzer C, Reuter U, Rogiers V, Schenk B, Schwengberg S, Seiler A, Spielmann H, Steemans M, Stedman DB, Vanparys P, Vericat fA, Verwei M, van der Water E, Weimer M, Schwarz M (2009) A review of the implementation of the embryonic stem cell test (EST). The report and recommendations of an ECVAM/ ReProTect Workshop. Altern Lab Anim 37 313-328... 

Theimissen PT et al (2012) Dose-response toxicogenomic evaluation of valproic acid, cyproconazole and hexaconazole in the neural embryonic stem cell test (ESTn). Toxicol Sci 125(2) 430 38... 

In 2007, the DART committee held a workshop on alternative assays, which was followed up by a workshop held at the European Teratology Society Annual Meeting in 2009. These workshops focused on three alternative assays (1) whole embryo culture (WEC), (2) mouse embryonic stem cell tests (mESC), and (3) zebrafish. Each assay was presented and data from users were shared, and strengths and limitations were discussed. It should be noted that the WEC and mESC are validated by ECVAM as alternative embryotoxicity assays. Still, there are numerous research needs before even validated tests can achieve regulatory acceptance. The discussions, conclusions, and recommendations of the 2007 workshop were published by Chapin et al. (14). Bullet lists of next steps to move forward were defined for each assay (14) and are briefly summarized here ... 

In the 1990s, ECVAM held a forum to vet and evaluate new alternative assays, and developed a list of compounds for testing (24). The key driver for this activity was the fact that DART studies require large numbers of animals. The primary focus of this activity was embryo-fetal toxicity. The list generated from this forum was tested in three assays (later validated by ECVAM) (1) the micromass assay, (2) the rat WEC assay, and (3) the embryonic stem cell test (25). Compounds on the Brown list were classified as either strong, weak, or non-teratogens. The three assays successfully predicted the compound classification about 80% of the time. However, the embryonic stem cell test later performed poorly on a different group of chemicals with known in vivo activities (26). 

The embryonic stem cell test is an in vitro embryotoxicity test that uses permanent mouse cell lines that can differentiate into a variety of distinct cell types in vitro (Spielmann et al., 1997). The test has been validated in different laboratories utilizing known test chemicals with different embryotoxic properties (Scholz et al., 1999 Genschow et al.,... 

Although some SAR models have claimed reasonable success,20 due to the factors described previously, we have not found SAR approaches particularly useful however, in the one instance DEREK identified a "hit," the chemical in question did test positive for embryotoxicity in vitro (whole embryo culture and the embryonic stem cell test). 

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.

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