Development of a tool for measuring uncertainty of acute cytotoxicity results using the methodology of OECD Guidance Document 129

• This study marks a significant metrological advancement in the OECD protocols. • An open-access tool was developed for acute cytotoxicity uncertainty measurement. • Measuring uncertainty of results can help the Chemical Safety Assessment. • The combined IC50 and uncertainty values can be obtained, improving reliability. • The integration of metrology can improve the reproducibility of non-animal methods. Despite the use of strict acceptance criteria in validated methods for in vitro toxicology safety non-animal methods, the evaluation of statistical uncertainties in the results and their impact on the reproducibility and reliability of these methods is limited. Technical and operational variations in intra- and inter-laboratory settings can interfere with the results, and measurement uncertainty estimations are crucial for a better understanding of the potential sources of errors and ensuring that the assays are under control. The measurement uncertainty characterizes the dispersion of values, which can be attributed to the measurement results. This study applied the Monte Carlo statistical model to calculate the measurement uncertainty for the half-maximal inhibitory concentration (IC50) of sodium dodecyl sulfate (SDS) using OECD Guidance Document 129. We focused on acute oral cytotoxicity in vitro and developed a specific tool for this purpose. The Monte Carlo method enabled the identification of the contributions to the measurement uncertainty of IC50 and their confidence intervals for the in vitro cytotoxicity assay recommended by OECD GD 129. Furthermore, the statistical tool developed in this study, although specifically applied to OECD GD 129, has the potential to be adapted and extended to other in vitro toxicological assays based on dose-response curves, thus supporting broader applications in New Approach Methodologies (NAMs) by enhancing reproducibility and confidence in alternative methods validation processes. In conclusion, this study demonstrates the effectiveness of the Monte Carlo statistical model in quantifying the measurement uncertainty of sodium dodecyl sulfate (SDS) IC50 values, with a comprehensive analysis yielding a combined IC50 of 34.6 μg/mL with a combined uncertainty of 1.21 μg/mL. These findings highlight the importance of incorporating metrological evaluations to increase the reliability of in vitro methods. By enabling more informed decision-making, this study can significantly contribute to the advancement of toxicological research and NAMs.