Transferability and reproducibility of IMMUNOTOX-T assay, an in vitro method for evaluation of chemical-mediated immunotoxicity.

In vivo evaluation of the immunotoxicity of xenobiotics is mainly carried out in rodents. Recent trends following the 3R (reduction, refinement, replacement) principle have driven development of in vitro methods for screening immunotoxic chemicals. The recently reported IMMUNOTOX-T immunotoxicity assay uses the human monocytic leukemia cell line THP-1, and evaluates potential immunotoxicity by profiling production of various cytokines by cells treated with test chemicals. Development of the assay involved small-scale evaluation of transferability, within-laboratory repeatability (WLR), and between-laboratory reproducibility (BLR). The IMMUNOTOX-T assay was transferred to, and tested by, a Participating laboratory in compliance with Good Laboratory Practice. All test substances were tested under blinded conditions. Selected vehicles, 75% cell viability (CV75), and relative cytokine production levels (RCPL, %) of IL-6, IL-8, and TNF-α at three concentrations (0.01x, 0.1x, and 0.5 × CV75) of each test compound were compared with levels induced by respective vehicle controls. The following chemicals were tested by the Lead and Participating laboratory: dexamethasone, chlorambucil, and glycerol. There was no statistically significant difference in the data generated by the two laboratories. Regarding WLR, eight chemical substances (two immunosuppressants, three non-immunotoxicants and three immune-modulatory chemicals) were tested twice (independently), and RCPLs for 24 cytokines calculated, by the Participating laboratory. The duplicate experiments showed 93.8% similarity in mean RCPLs (i.e., no significant differences between the duplicate values for 180/192 cytokine tests for all eight substances). The BLR for the eight test substances was evaluated by comparing the mean RCPLs of 24 cytokines calculated by the two laboratories. Combining all of the results for the eight tests revealed that the magnitude of similarity between the data from the Lead and Participating laboratory was 88.5% (there were no significant differences in mean RCPLs for 170/192 cytokine tests). Thus, the IMMUNOTOX-T assay is transferable, with good WLR and BLR. The online version contains supplementary material available at 10.1007/s43188-025-00333-9.