In vitro micromass teratogen test: Interpretation of results from a blind trial of 25 compounds using three separate criteria.

The results are presented on an interlaboratory study to validate the Micromass assay by testing compounds under code using a similar protocol, with an assessment of results for 25 compounds tested without S-9 mix. Four of these were co-tested with S-9 mix. Three separate sets of criteria have been proposed (Flint, 1986 and 1987; Flint and Orton, 1984) for interpreting the results for teratogenic hazard from in vitro data using IC(50) values: (i) the '< 500 mug/ml rule', (ii) the '< 50 mug/ml rule' and (iii) the 'specific inhibition of cell differentiation 2-fold rule'. The data were decoded and assessed using the criteria for their sensitivity, specificity and accuracy. Using the '< 500 mug/ml rule' a higher sensitivity was obtained but at the expense of a high false positive frequency (50%). Conversely, the '2-fold rule' gave a high specificity with only 10% false positives. Diphenhydramine and furazolidone gave false positive responses; the teratogens beta-aminopropionitrile and methotrexate were not detected. Selective inhibition of cell differentiation was related to high potential teratogenicity. In the application of the test, it is suggested that if there was an indication of teratogenic hazard using the '2-fold rule' the compound should be rejected without recourse to animal testing. In its present form the assay cannot be used to unequivocally identify non-teratogens.