From cellular toxicity and inflammation to human safety: An integrated in vitro–in silico assessment of 5-hydroxymethylfurfural (HMF)

5-Hydroxymethylfurfural (HMF) is a heat-induced food contaminant whose toxicological relevance remains under debate. This study integrates in vitro assays, in silico modelling, and probabilistic exposure assessment to characterize the risk associated with HMF. Differentiated 3T3-L1 cells, a metabolic and inflammation-responsive model, were exposed to concentrations of HMF (1–1000 µg/mL). Cell viability remained above 95% up to 16 µg/mL, which was defined as the in vitro NOAEL. Acute exposure (150 µg/mL, 12 h) markedly upregulated IL-6, TNF-α, IFN-γ, and TGF-β, indicating activation of pro-inflammatory pathways likely mediated by oxidative stress. In vitro–in vivo extrapolation (IVIVE) converted the cellular NOAEL into oral equivalent doses, ranging from 1.067 to 12.935 µg/kg bw/day, depending on the pharmacokinetic assumptions. Monte Carlo simulation (100,000 iterations) predicted mean dietary exposures (µg/kg bw/day) of 541 for adults, 605 for adolescents, and 223 for infants. Margin of exposure (MOE) estimates placed 50–56% of adults and adolescents in an intermediate-risk category, while 43–49% fell within the high-risk range (MOE < 10), and Infants within the low-risk range. These findings highlight the pro-inflammatory potential of HMF in adipocytes and support the value of combining in vitro data with probabilistic modelling to refine human dietary risk assessment. • HMF triggered pro-inflammatory responses in differentiated adipocytes. • Adipocyte inflammation supports HMF as a biologically relevant dietary contaminant. • In vitro data were translated to human exposure using IVIVE approaches. • Probabilistic modelling identified population subgroups with elevated dietary risk. • Data gaps in human bioavailability and kinetics remain critical for risk refinement.