Studies on the standardization of cytotoxicity tests and new standard reference materials useful for evaluating the safety of biomaterials.

Standard reference materials (SRM) made of polyurethane films containing various amounts of cytotoxic compounds such as zinc diethyldithiocarbamate (ZDEC) and zinc dibutyldithiocarbamate (ZDBC) are proposed. The cytotoxicity indices of five dithiocarbamates and the SRM films obtained by colony assay were compared with those by published standards, i.e., neutral red assay (NF S 90-702), agar diffusion assay (USP XXII), and millipore filter diffusion assay (DIN-V 13 930). Among them, colony assay using 3 cell lines of Balb 3T3, L929 and V79 was found to be most sensitive. The extraction method and direct contact method were proposed as two test methods of colony assay. In the former, culture media after incubation with test materials were used as test solution, and in the latter, cells were directly cultured on test materials. The extraction method gave a linear relationship between the cytotoxic potentials of the SRM and its concentration of ZDEC or ZDBC. The cytotoxic potentials of the SRM correlated well with the thickness of inflammatory layer in rabbit muscle implantation tests of the SRM. The direct contact method was found to be useful to detect weak cytotoxicity because of its high sensitivity. Based on these data, we propose the colony assay and the two kinds of new SRMs as Japanese standard test method for evaluating the cytotoxicity of biomaterials.