Bronopol and benzisothiazolinone exhibit synergistic toxicity with enhanced cell stress responses exceeding the impacts of individual compounds.

Bronopol and benzisothiazolinone (BIT) constitute antimicrobial-preservative co-formulations in consumer products, creating mixture exposure scenarios. Current risk assessments evaluate individual compounds, potentially underestimating mixture toxicity. This study investigated individual and combined toxicological effects using stress granule formation as an early biomarker. A549 lung epithelial cells were exposed to Bronopol, BIT, or combinations, assessed through real-time stress granule monitoring, viability assays, oxidative stress measurements, and integrated stress response pathway analysis. Mixture interactions were evaluated using the Bliss Independence Model. Both preservatives induced concentration-dependent stress granule formation and PERK-eIF2α activation. Critically, co-exposure produced pronounced synergism exceeding additive predictions. Sub-threshold concentrations potentiated toxicity, enhancing stress responses at lower concentrations. Combined treatments accelerated signaling kinetics and uniquely activated JNK phosphorylation. Most notably, co-exposure amplified reactive oxygen species 9-18-fold above individual compounds, demonstrating substantial synergy. These findings indicate that mixture exposures pose greater health risks than individual compound predictions, supporting mixture-based safety evaluation frameworks.