Detection of Microbial Contamination in Nanomaterials Using LAL, rFC and Cell-Based Assays: Implications for Nanotoxicological Hazard Assessment

Accurate detection of microbial contamination is essential in the assessment of toxicological and immunological responses to various materials, as low-level contaminants can lead to confounding results. Traditional endotoxin testing relies on the Limulus Amebocyte Lysate (LAL) assay, which depends on horseshoe crab blood and raises both ecological and ethical concerns. Sustainable alternatives such as recombinant Factor C (rFC) provide a promising solution, yet validation for the detection of endotoxin in nanomaterials remains incomplete. In this study, we have used rFC alongside Toll-like receptor (TLR) reporter assays to detect both endotoxin and broader microbial contaminants in 31 nanomaterials from diverse classes. Special attention was given to assay interference by nanomaterials to ensure reliable detection. The rFC assay demonstrated a sensitive detection limit of 0.005 EU/mL, equivalent to the LAL assay, and showed that more than 50% of tested nanomaterials contained low-level endotoxin contamination. Additionally, several nanomaterials activated the TLR2 reporter, indicative of microbial contaminants beyond endotoxin. These results suggest that rFC can serve as a sustainable and reliable replacement for LAL in nanomaterial endotoxin testing but also emphasize the limitations of relying solely on endotoxin-specific assays. We recommend that future nanotoxicological evaluations integrate rFC with complementary methods, such as TLR-based approaches, and include thorough interference controls to ensure robust and comprehensive microbial contamination assessment.