Physiologically based biokinetic (PBBK) modeling and validation of dose-, species-, interindividual- and matrix dependent effects on the bioactivation and detoxification of safrole

<strong>Keywords: safrole, PBBK model, DNA adduct, mace </strong> Safrole has been demonstrated to be carcinogenic in rodent studies at high doses of the pure compound. The use of pure safrole in foodshas already been prohibited. As a result, the main exposure to safrole occurs through the use of herbs and spices containing low levels of safrole, such as nutmeg, mace, star anise, pimento, cinnamon, and black pepper, and food products containing these herbs and spices or their essential oils. The Scientific Committee on Food of the European Union concluded in their evaluation that safrole is genotoxic and carcinogenic and that reductions in the exposure and restriction in the use levels are indicated. This opinion is based on carcinogenicity data from rodent studies as adequate human data were not available. Therefore, translation from animal bioassays at high dose levels of the pure compound to the risk for the human population exposed to safrole at relatively low levels via dietary intake within the complex food matrix is obviously needed. The aim of this thesis was to obtain insight into the dose-, species-, interindividual- and matrix dependent effects on the bioactivation and detoxification of safrole using physiologically based biokinetic (PBBK) modeling. PBBK models for safrole in male rats and humans were developed based on in vitro metabolic parameters determined, in silico derived partition coefficients, and physiological parameter values taken from literature. The performance of the PBBK model for rats was evaluated by comparison of predicted levels of 1,2-dihydroxy-4-allylbenzene, 1′-hydroxysafrole glucuronide and total urinary safrole metabolites to the reported levels of these metabolites in urine of rats exposed to safrole. This evaluation revealed that the predictions adequately matched observed experimental values. The PBBK model for humans was evaluated by comparison of the PBBK predicted and the reported experimental data on the level of total safrole metabolites detected in the urine of human volunteers exposed to safrole whichshowed an adequately match. The comparison of the PBBK model for rats and humans revealed that the predicted level of formation of 1ʹ-hydroxysafrole in human liver is fourfold higher than that for rat liver and the predicted formation of 1ʹ-sulfooxysafrole is about fivefold higher than that for rat liver. This indicates that the interspecies differences in toxicokinetics for bioactivation of safrole between rat an human are in line with the uncertainty factor normally taken into account for interspecies differences in toxicokinetics of 4. Species differences between humans and rats in the nature of the detoxification pathways of 1ʹ-hydroxysafrole were larger, with the formation of 1ʹ-oxosafrole being the main detoxification pathway in humans but a minor pathway in rats and glucuronidation of 1ʹ-hydroxysafrole being less important in humans than in rats. Monte Carlo simulations revealed that the formation of 1′-sulfooxysafrole was predicted to vary 4- to 17-fold in the population (fold-difference between the 95^th and median, and 95^th and 5^th percentile, respectively). Risk assessment of safrole resulting from consumption of herbs and spices containing safrole should be performed taking into account the possible modulating effect of other compounds present in these herbs or spices. In this study, mace was chosen as the model spice of interest because it contains significant levels of safrole. Mace fraction with the highest SULT inhibiting activity was identified as malabaricone C. Studies using human HepG2 cells exposed to 1ʹ-hydroxysafrole and in the presence of mace extract showed that formation of the DNA adduct <em>N</em>²-(<em>trans</em>-isosafrol-3′-yl)-2′-deoxyguanosine was inhibited. To investigate the possible effects on safrole bioactivation to 1′-sulfooxysafrole by malabaricone C-containing mace extract could also be expected in vivo, the SULT inhibition was integrated into the PBBK model. The PBBK models predicted that at a dose of 50 mg/kg bw safrole and a ratio of malabaricone C-containing mace extract to safrole similar to the level of these constituents in mace, inhibition of 1′-sulfooxysafrole formation by malabaricone C-containing mace extract for rats and humans amounts to 90 and 100%, respectively. To see whether the inhibition of safrole DNA adduct formation by malabaricone C-containing mace extract is also observed in in vivo, to the end, Sprague-Dawley rats were orally exposed to mace extract and safrole. The results demonstrated that safrole DNA adduct formation in the liver of Sprague-Dawley rats by the mace extract was reduced by 55%. The results of the in vitro and in vivo studies that demonstrated inhibition of the formation of safrole DNA adducts by mace extract, support that combination effects should be taken into account in the risk assessment when safrole is tested in the presence of a relevant food matrix. To integrate thefood matrix dependent modulation of safrole bioactivation in the risk assessment of safrole, the so-called Margin of Exposure (MOE) approach can be used. This revealed that when safrole would be tested in rodent bioassays in the presence of a matrix containing SULT inhibitors the MOE values would be higher and the need for risk management actions would be lower.