A Study on the Dose Setting of Test Chemicals for the Promotion Assay in Bhas 42 Cell Transformation Assay

Abstract

We have proposed a cell transformation assay using the Bhas 42 cells (Bhas 42 CTA) as an in vitro method for predicting the carcinogenicity of chemicals. The Bhas 42 CTA consists of two assays: one is the initiation assay and the other is the promotion assay. An in-house study on Bhas 42 CTA had been performed using approximately a hundred test chemicals. In that study, seven chemicals induced the severe cell killing in the promotion assays and their promoting activities were unable to be evaluated. The aim of this study was to find the cause of severe cell killing that occasionally occurred in the promotion assay. We presumed that the severe cell killing was attributed to the failure of dose setting caused by the difference of treatment periods between the cell growth assay (for 3 days) and the promotion assay (for 10days). In this study, we compared the inhibition rates in the cell growth assays between the chemical treatments for 3 days and 10 days. For seven chemicals that had induced the severe cell killing in the promotion assays, a larger inhibition was caused by the treatment for 10 days than for 3 days. For the chemicals whose promotion assays had succeeded, the growth inhibition was similar between two treatment conditions. These results demonstrated that the severe cell killing in the promotion assays was attributed to the failure of dose setting arising from the difference of the period of chemical treatment between the cell growth assay for dose setting and the promotion assay.