The cell transformation assay using Bhas 42 cells (Bhas 42 CTA) was developed as an in vitro test method for predicting the carcinogenicity of chemicals. The assay can detect tumor promoters as well as tumor initiators. The usefulness of Bhas 42 CTA has been previously verified using 6-well culture plates (6-well method). Meanwhile, we have developed an alternative method using 96-well culture plates (96-well method) in anticipation of its potential utility for high-throughput automated applications. The assay procedures are fundamentally the same between the 6-well and 96-well methods. This study has been carried out to confirm the comparability of the 6-well and 96-well methods for chemically induced transformation. 3-Methylcholanthrene and 12-O-tetradecanoylphorbol 13-acetate were found to yield similar dose-dependent responses between the 6-well and 96-well methods. The 96-well method was subsequently tested using 32 chemicals that had already been tested by the 6-well method. The positive and negative judgments determined from the 96-well method were concordant with those determined from the 6-well method, except for sodium arsenite. Overall, it was concluded that the results obtained using the 6-well method and the 96-well method were analogous.

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The animal-free prediction of inhalation toxicities in the lungs is very important concerning various low-volatile organic carbons such as phthalate. Phthalate are contained in plastics as plastisizer, easily released into environment as plastic ages, and ingested through dust. We therefore investigated benzylbutyl phthalate (BBP) permeation using an A549 cell-based lung alveolus model, in which the cell monolayers were formed on semipermeable membranes between two chambers filled with cell culture medium. With kinetic parameters obtained via these experiments, the model largely described the concentration changes in the three compartments (the apical, A549 cell, and basolateral layers) but revealed very high BBP accumulation in the alveolus cell layer at equilibrium, which did not likely reflect the in vivo situation. We therefore changed the parameter of thickness of the cell layer from 10 (cultured A549 cells) to 0.5 μm (alveoli) and the parameter of the concentration in basolateral compartment to be always zero because of the continuous perfusion of blood in vivo. After changing these parameters, the accumulation of BBP remarkably decreased, and the total permeated amount significantly increased. These results indicated that various parameters and assumptions should be changed to overcome the limitations and/or properties of existing culture models to improve the predictive accuracy of the system when using in vitro cell-based tissue models and numerical simulations to predict health hazards in humans.

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On evaluating the effects of dental biomaterials on embryotoxicity in humans, the effects of human metabolic activation factors cannot be ignored. However, the routine the Embryonic Stem Cell Test (EST) method does not reflect the effects of metabolic activation in humans, because of the use of mouse ES-D3 cells. Thus, a TEST LIVER^TM-human (Toyobo, Osaka) culture with teratogenic thalidomide was exposed to mouse ES-D3 cells to evaluate cell differentiation of contracting myocardial cells. Also, standard reagents for atomic absorption spectroscopy, i.e., component elements of dental alloys (Ag, Cu, Pd, Sn and Zn), were used. As a result, thalidomide, Ag, and Sb significantly reduced the contraction rate compared with the control group. A preliminary culture using TEST LIVER^TM-human is likely to facilitate evaluation of the effects of human metabolism in mouse ES cells.

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