Micromass test

Spielmann H et al (2004) Validation of the rat Kmb bud micromass test in the international ECVAM validation study on three in vitro embryotoxicity tests. Altern Lab Anim 32 245-274... 

The micromass test is based on the use of limb buds from D14 rat embryos [3], Following trypsinization the cell suspension is plated in microwells and allowed to differentiate for 5 days in the presence of the testing chemicals. Then cell viability is measured by neutral red staining, and differentiation is evaluated by alcian blue staining. Cytotoxity and inhibition of differentiation are the two toxicological endpoints [2],... 

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). 

Figure 13. Changes in 6 Fe values of HPS Fe standard as a function of contaminate elements Al, Mg, or La (12.5 to 75 ppb). All solutions were 400 2 ppb Fe. The Fe isotope compositions of the solutions are shifted from those in the pure Fe standard (6 Fe = +0.49 0.05%o) as a function of the impurity concentration. The magnitude of the this shift with impurity concentration is variable, as shown by data collected during three analytical sessions (parts A, B, and C). These impurity matrix elements do not produce molecular isobars, as evidenced by the fact that 5 Fe and 5 Fe values plot along a mass-dependent array (part D). Note that an important conclusion of these tests is that accuracy of Fe isotope measurements cannot be demonstrated by preservation of mass-dependent trends in Fe/ Fe and Fe/ Fe. Data were collected using the Univ. of Wisconsin-Madison Micromass IsoProbe.

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 implementation of animal test protocols in the 1980s has been accompanied by the development of a host of alternative methods to study adverse effects of chemicals on reproductive and developmental parameters. For example, rat whole embryo culture stems from the seventies (16), as does the rat limb bud organ culture (17) and rat limb bud and brain micromass was developed in the eighties (18). An elegant nonvertebrate alternative model used regeneration of polyps of Hydra atUnuata from dissociated cells (19). Animal-free in vitro alternatives include those employing the proliferation of a human embryonic palatal mesenchymal cell line (20), the attachment of a mouse ovarian tumor cell line (21), and the differentiation of a neuroblastoma cell line (22) and a embryonal carcinoma cell line (23). Various overviews of methods have been published over the years (24). The predictability of... 

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. 

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). 

Tissue culture has been used to a limited extent for developmental toxicity studies of the studies that have used tissue culture systems, those using chick neural crest cells (Greenberg, 1982) and human embryonic palatal mesenchyme (Pratt et al., 1980, 1982) have been especially useful. In addition, the micromass teratogen test, first described by Flint Orton (1984), has been used as a screening tool, and this test has been used successfully for many years. This method uses cultures of limb and CNS cells. Rat cells are normally used, but mouse and chick cells have also been studied. Many different endpoints can be assessed. For a detailed description of the method, the validation studies and discussion of its predictive value, see Flint... 

Chick embryo neural retina cells have also been grown in culture (Daston et al., 1995), and studies indicate positive responses to chemicals at concentrations similar to those active in in vivo tests. Reinhardt (1993) has reviewed the use of organ slices, aggregate cell cultures and the micromass techniques for the study of neurodev-elopmental toxicity. Two recent reviews of these in vitro systems, especially the micromass and the chick embryo neural retinal cell system, have been published (Daston, 1996a Mirkes, 1996). 

Flint OP (1993) In vitro tests for teratogens Desirable endpoints, test batteries and current status of the micromass teratogen test. Reprod Toxicol, 7(Suppl 1) 103-111. 

Figure 13.10. Plot of the mass difference between reference spectrum and acquired spectrum. MSI static calibration, 28 matches of 28 tested references, SD = 0.0465. (Courtesy of Micromass Quattro LC.)...

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. 

Finally, in order to replace the OECD (2001) guideline 414 about developmental toxicity testing, the ESCT, the whole embryo culture and the micromass culture have been formally validated (ECVAM). 

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