Two-stage prediction (TSP) model had been developed to predict toxicities of mixtures containing complex components, but its prediction power need to be further validated. Six phenolic compounds and six heavy metals were selected as mixture components. One mixture (M1) was built with equivalent-effect concentration ratio and four mixtures (M2-M5) were designed with fixed concentration ratio. In M1-M5, the toxicities were well predicted by TSP model, while CA overestimated and IA underestimated the toxicities. In M1-M5, compared with the actual mixture EC50 value, the prediction errors of TSP model (13.9%, 17.9%, 19.2%, and 17.3% and 15.8%, respectively) were significantly lower than those in the CA (higher than 30%) and IA models (20.9%, 33.0%, 20.6%, 21.8% and 12.5%, respectively). Thus, the TSP model performed better than the CA and IA model.
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