Micromass assay

Wise, L.D., Clark, R.L., Rundell, J.O. and Robertson, R.T. (1990a). Examination of a rodent limb bud micromass assay as a prescreen for developmental toxicity. Teratology 41 341-351. 

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). 

Fig. 13.8 Ti me-dependent MS response for pseudoephedrine when 0.1% Tween 80 was used in the formulation that was dosed in the rats via either intravenous (IV) or per os (PO) routes. The HPLC-MS/MS assay was performed in the ESI mode using (a) Thermo-Finnigan Quantum MS (b) AB Sdex 3000 MS (c) Waters-Micromass Quattro Ultima MS. The PK samples were spiked with...

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. 

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. 

All major mass spectrometer manufacturers produce suitable instruments for steroid analyses. Typical instruments used in steroid assays are the Applied Biosystems API 2000-5000 series (www.appliedbiosytems.com), the Finnigan TSQ quantum (www. thermo.com), Agilent QQQ or QTof instruments (www.Agilent.com), and Micromass Quattro instruments (www.waters.com). 

Reprotoxicity and teratogenicity—micromass and whole-embryo assays/stem cell assay... 

Another important source of information on the status of alternative test development, with particular emphasis on the requirements for cosmetics testing, is a review paper published in 2011 by Adler and coauthors [9], Table 1 summarizes those relevant for reproductive toxicity. Several assays refer to the detection of endocrine effects on steroidogenesis based on a variety of cell types, and, as already mentioned, they will be dealt with in another chapter of this book. The other tests can be subdivided in placental toxicity/transport, preimplantation toxicity, female and male toxicity, and developmental toxicity. The tests that are suitable for detecting developmental toxicity include the EST, the whole-embryo assay, the micromass test (all three already described above), the zebrafish embryo teratogenicity assay, and the frog embryo teratogenesis assay (FETAX). 

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