Supplementary Material for: Simultaneous evaluation of three alternative models to animal caries testing for assessing the anticaries efficacy of monofluorophosphate dentifrices

Introduction: To simultaneously assess three alternative models to animal caries testing – an in situ remineralization model, an in vivo salivary fluoride clearance model, and a modified in vitro pH cycling model – for measuring the anticaries efficacy of dentifrices containing monofluorophosphate (MFP) up to 2800 ppm F. Methods: A series of MFP dentifrices were prepared at 250, 1100 and 2800 ppm F as MFP in a silica abrasive base with a placebo toothpaste (fluoride-free). Model #1: In a randomized crossover trial, in situ remineralization (% surface microhardness recovery of partially demineralized enamel) and enamel fluoride uptake (microbiopsy) were assessed over 7, 14, and 21 days in 15 participants. Model #2: In vivo salivary fluoride clearance was evaluated in 26 participants using a randomized, crossover design by measuring salivary fluoride concentration over 30 minutes following 2 minutes of brushing. Model #3: A modified in vitro pH cycling model with pre-hydrolyzed (2N HCl) MFP dentifrice supernatants was used to evaluate lesion progression over 19 days with 6 hours demineralization and 17 hours remineralization daily (Monday-Friday with weekend remineralization) and twice daily morning and afternoon 2-minute dentifrice slurry treatments on 10 teeth per dentifrice. Results: There was a statistically significant impact of dose (p<0.001) observed in all three models, and the three models are highly correlated with one another and with animal caries testing results (Spearman’s ρ > 0.77, p < 0.0001). In situ hardening, fluoride uptake, and fluoride AUC clearance showed linear relationships with MFP dose. Acid hydrolysis successfully liberated ionic fluoride and lesion progression in pH cycling was exponentially suppressed with fluoride dose. Conclusion: The in situ, in vivo, and modified in vitro models each provided dose-responsive and sensitive measures of MFP activity. Importantly, the modification of pH cycling for MFP hydrolysis enabled a well-developed laboratory protocol to be adapted to an important fluoride source. These approaches appear to offer viable alternatives to animal caries models for evaluating the efficacy of MFP-containing dentifrices and each could be chosen in different settings to understand the impact of monofluorophosphate-containing dentifrices on caries prevention.