Examining Uncertainty in In Vitro-In Vivo Extrapolation Applied in Fish Bioconcentration Models

In vitro biotransformation rates were determined for 30 chemicals, mostly fragrance ingredients, using trout liver S9 fractions (RT-S9) and incorporated into in vitro-in vivo extrapolation (IVIVE) models to predict bioconcentration factors (BCFs). Predicted BCFs were compared against empirical BCFs to explore potential major uncertainties involved in the in vitro methods and IVIVE models: (i) in vitro chemical test concentrations; (ii) different gill uptake rate constant calculations (k₁); (iii) protein binding (different calculations and measurement of the fraction of unbound chemical, f_U); (iv) species differences; and (v) extrahepatic biotransformation. Predicted BCFs were within 0.5 log units for 44% of the chemicals compared to empirical BCFs, whereas 56% were overpredicted by >0.5 log units. This trend of overprediction was reduced by alternative k₁ calculations to 32% of chemicals being overpredicted. Moreover, hepatic in vitro rates scaled to whole body biotransformation rates (k_B) were compared against in vivo k_B estimates. In vivo k_B was underestimated for 79% of the chemicals. Neither lowering the test concentration, nor incorporation of new measured f_U values, nor species matching avoided the tendency to overpredict BCFs indicating that further improvements to the IVIVE models are needed or extrahepatic biotransformation plays an underestimated role.