In vitro-in silico study on the influence of dose, fraction bioactivated and endpoint used on the relative potency value of pyrrolizidine alkaloid N-oxides compared to parent pyrrolizidine alkaloids.

Pyrrolizidine alkaloids (PAs) and their N-oxides (PA-N-oxides) are phytotoxins found in food, feed and the environment. Yet, limited data exist from which the relative potency of a PA-N-oxide relative to its corresponding PA (REP) can be defined. This study aims to investigate the influence of dose, fraction bioactivated and endpoint on the REP of a series of pyrrolizidine N-oxides using in vitro-in silico data and physiologically based kinetic (PBK) modeling. The first endpoint used to calculate the REP was the ratio of the area under the concentration-time curve of PA resulting from an oral dose of PA-N-oxide divided by that from an equimolar dose of PA (Method 1). The second endpoint was the ratio of the amount of pyrrole-protein adducts formed under these conditions (Method 2). REP values appeared to decrease with increasing dose, with the decrease for Method 2 already starting at lower dose level than for Method 1. At dose levels as low as estimated daily human intakes, REP values amounted to 0.92, 0.81, 0.78, and 0.68 for retrorsine N-oxide, seneciphylline N-oxide, riddelliine N-oxide and senecivernine N-oxide, respectively, and became independent of the dose or fraction bioactivated, because no GSH depletion, saturation of PA clearance or PA-N-oxide reduction occurs. Overall, the results demonstrate the strength of using PBK modeling in defining REP values, thereby substantiating the use of the same approach for other PA-N-oxides for which in vivo data are lacking.