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 (REPPANO to PA) can be defined. This study aims to investigate the influence of dose, fraction bioactivated and endpoint on the REPPANO to PA 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 REPPANO to PA 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). REPPANO to PA 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, REPPANO to PA 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 REPPANO to PA values, thereby substantiating the use of the same approach for other PA-N-oxides for which in vivo data are lacking.