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Protocol no. 114
IN VITRO MICROMASS TERATOGEN ASSAY

The effect of a test compound, in the presence and absence of S-9 mix, on the differentiation and growth of rat limb bud and CNS cells in vitro indicates whether it is potentially a teratogen in vivo.

CONTACT

Mrs P. Uphill Department of Cellular Toxicology Huntingdon Life Sciences P.O.B. 2 Huntingdon Cambridgeshire, PE18 6ES England

RATIONALE

Rat embryo midbrain and limb mesenchyme cells, when cultured at high cell densities, differentiate to form foci of neurones and chondrocytes, respectively. The cultures retain some aspects of cell behaviour that are most critical to embryogenesis, namely adhesion, movement, communication, division and differentiation. Thus, these cultures can be used as a short-term assay system for the prediction of teratogenic potential.

BASIC PROCEDURE

The mesencephalon (CNS) and forelimb buds (LB) are dissected out of rat embryos on gestation day 13. The LB and CNS tissues are transferred to separate test tubes and disaggregated by trypsinization into single cell suspensions which are cultured in 96-well microtitre plates in the presence and absence of dilutions of test compound, with or without S-9 mix. After 5 days, the cultures are assessed for cytotoxicity by means of neutral red uptake and foci of differentiated cells (neurones and chondrocytes) are enumerated. The IC50 for inhibition of differentiation and for cytotoxicity are compared. The compound is potentially teratogenic if it inhibits differentiation at non-cytotoxic concentrations. Specific effects on either LB or CNS cultures and the level of toxicity to each type of culture are also assessed.

TEST STATUS

The micromass assay was originally subjected to an intralaboratory blind-trial validation with 46 model compounds (Flint and Orton, 1984). Subsequently, it has undergone validation in an interlaboratory blind trial (Parsons et al., 1990; Uphill et al., 1990). The European Federation of Pharmaceutical Industries has recognized this system as being of value in the identification of teratogens (Whittaker and Faustman, 1994).

REFERENCES

  1. Brown N.A., Spielmann H., Bechter R., Flint O.P., Freeman S.J., Jelinek R.J., Koch E., Nau H., Newall D.R., Palmer A.K., Renault J.-Y., Repetto M.F., Vogel R., and Wiger R. (1995) Screening chemicals for reproductive toxicity: the current alternatives. The report and recommendations of an ECVAM/ETS workshop (ECVAM Workshop 12) ATLA 23: 869-882.
  2. Flint O.P. (1980) The effects of sodium salicylate, cytosine arabinoside and eserine sulphate on rat limb buds in culture. in Teratology of the Limb (eds, H.J. Merker, H.Nau and D. Neubert) Walter de Gruyter and Co.; Berlin, pp. 325-338.
  3. Flint O.P. (1983) A micromass culture method for rat embryonic neural cells. J. Cell. Sci. 61: 247-262.
  4. Flint O.P. (1987) An in vitro test for teratogens using cultures of rat embryo cells. in In Vitro Methods in Toxicology (eds. C.K. Atterwill and C.E. Steele) Cambridge University Press; Cambridge England, pp. 339-363.
  5. Flint O.P., and Orton T.C. (1984) An in vitro assay for teratogens with cultures of rat embryo midbrain and limb bud cells. Toxicol. Appl. Pharmacol. 76: 383-395.
  6. Freeman S.J., and Brown N.A. (1987) Sub-mammalian and sub-vertebrate models in teratogenicity screening. in In Vitro Methods in Toxicology (eds. C.K. Atterwill and C.E. Steele) Cambridge University Press; Cambridge England, pp. 391-409.
  7. Heuer J., Graeber I.M., Pohl I., and Spielmann H. (1994) An in vitro embryotoxicity assay using the differentiation of embryonic mouse stem cells into haematopoietic cells. Toxicol. inVitro 8: 558-587.
  8. Keller S.J., and Smith M.K. (1982) Animal virus screens for potential teratogens. I. Poxvirus morphogenesis. Teratogenesis Carcinogenesis Mutagenesis 2:361-374.
  9. Laschinski G., Vogel R. and Spielmann H. (1991) Cytotoxicity test using blastocyst-derived euploid embryonal stem cells: a new approach to in vitro teratogenesis screening. Reproductive Toxicol. 5: 57-64.
  10. Newall D.R., and Beedles K.E. (1994) The stem-cell test - A novel in vitro assay for teratogenic potential. Toxicol. in Vitro 8: 697-701. Parsons J.F., Rockley J., and Richold M. (1990) In vitro micromass teratogen test: interpretation of results from a blind trial of 25 compounds using three separate criteria. Toxicol In Vitro 4: 609-611.
  11. Peters P.W.J., and Piersma A.H. (1990) In vitro embryotoxicity and teratogenicity studies. Toxicol In Vitro 4: 570-576. Spielmann H., Pohl I., Dçring B., Moldenhauer F. (1995) In vitro embryotoxicity assay using two permanent cell lines: mouse embryonic stem cells and 3T3 fibroblasts Abstracts of the 23rd ETS Conference 1995, Dublin Teratology in press Steele V E, Morrisey R E, Elmore E.L., et al. (1988) Evaluation of two in vitro assays to screen for potential developmental toxicants. Fund. Appl. Toxicol. 11: 673-684.
  12. Uphill P.F., Wilkins S., and Allen J.A. (1990) In vitro micromass teratogen test: results from a blind trial of 25 compounds. Toxicol In Vitro 4: 623-626.
  13. Whittaker S.G., and Faustman E.M. (1994) In vitro assays for developmental toxicity. in In Vitro Toxicology (ed. S. Cox Gad) Raven Press; New York, pp. 97-122. IP-114 July/96