Characterization of Candidate Chemical Reference Substances for In Vitro Cytotoxicity Testing (ISO 10993-5) Using the L929 Mouse Fibroblast Cell Line
Keywords:
Cytotoxicity, ISO 10993-5, Positive Control, Negative Control, L929 Cells, MTT AssayAbstract
Reliable reference materials and appropriate working concentrations are essential for ensuring the performance of in vitro cytotoxicity assays conducted according to ISO 10993-5. Although the standard recommends reference materials for assay verification, it does not specify working concentrations for soluble control substances that consistently produce defined cytotoxic responses in L929 cells. This study characterized the dose–response profiles of five reference substances, including zinc acetate, zinc chloride, Triton X-100, zinc diethyldithiocarbamate (ZDEC), and high-density polyethylene (HDPE), to identify laboratory-specific working concentrations suitable for routine in vitro cytotoxicity testing. L929 mouse fibroblasts were exposed to serial concentrations of the test substances, and cell viability was determined using the MTT assay following ISO 10993-5. All soluble control substances produced concentration-dependent reductions in cell viability, with ZDEC exhibiting the greatest cytotoxic potency, followed by Triton X-100, zinc chloride, and zinc acetate. HDPE extract also exhibited concentration-dependent effects; however, only the 5% extract maintained cell viability above the 70% cell viability threshold used for cytotoxicity classification, whereas higher extract concentrations reduced cell viability below this threshold. Based on the observed dose–response relationships, candidate laboratory-specific working concentrations were identified for each candidate control substance under the experimental conditions employed in this study. These findings provide a practical basis for selecting positive and negative control concentrations for routine ISO 10993-5 cytotoxicity testing using L929 cells under comparable laboratory conditions but should not be interpreted as constituting a formal validation of the assay method.
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