Alternative testing strategy to quantify inter-species, inter-ethnic, and inter-individual variation in bioactivation and toxicity of food-borne alkenylbenzenes and pyrrolizidine alkaloids

The aim of the present thesis was to perform the risk and safety assessment of two groups of natural food-borne toxins, namely alkenylbenzenes and pyrrolizidine alkaloids (PAs) for the Chinese population as compared to the overall Caucasian population using methods including the Margin of Exposure (MOE) approach, physiologically based kinetic (PBK) modelling-based reverse dosimetry and the combination of PBK modelling and Monte Carlo simulation. Chapter 1 introduced the model compounds studied in the thesis, namely alkenylbenzenes and 1,2-unsaturated PAs, the aim of the thesis and the strategies applied to perform a mode of action based safety and risk assessment. The regulations and consumer behaviour towards botanical preparations in China and in several EU countries were also presented. In chapter 2, a risk assessment of 10 plant food supplements (PFS), 23 traditional Chinese medicines (TCM) and 38 herbal teas containing alkenylbenzenes obtained at the Chinese market was performed using the Margin of Exposure (MOE) approach. Use of about half of the samples would result in estimated intakes upon daily life-time exposure that give rise to MOE values lower than 10 000, suggesting a potential priority for risk management. For short-term exposure such as two weeks consumption, applying Haber’s rule, only one TCM (TCM 6) still had a MOE value below 10 000. It is concluded that consumption of Chinese botanical preparations raise a concern because of exposure to alkenylbenzenes, especially when exposure is for longer periods of time. Chapter 3 studied the differences in bioactivation and detoxification of the food-borne genotoxic carcinogen estragole between Chinese and Caucasians using a mode of action based PBK modelling approach. The outcomes of the model predictions showed that the detoxification of the proximate carcinogenic metabolite 1’-hydroxyestragole, mainly by conversion to 1’-oxoestragole, was similar in both ethnic groups. For the bioactivation pathway, at realistic daily intakes of estragole, in the Chinese population only 0.02 % of the dose was converted to the ultimate carcinogenic metabolite 1’-sulfooxyestragole, while this value amounted to 0.09 % of the dose in Caucasian subjects. This 4.5-fold difference accompanied by similar rates of detoxification may indicate a lower risk of estragole for the Chinese population at similar levels of exposure. In chapter 4, it was demonstrated that combining in vitro cytotoxicity data obtained with primary rat hepatocytes with PBK modelling-based reverse dosimetry could adequately predict in vivo acute liver toxicity of lasiocarpine and riddelliine for rats. The PBK models for rats for lasiocarpine and riddelliine were developed based on data derived from in silico approaches, incubation experiments using subcellular tissue fractions and literature to define the PBK model parameters. Concentration-response curves obtained from in vitro cytotoxicity assays in primary rat hepatocytes were converted to in vivo dose-response curves for acute liver toxicity by PBK-modelling based reverse dosimetry. From the in vivo dose-response curves thus obtained points of departure (PoDs) were derived that were compared to available literature data on in vivo liver toxicity of lasiocarpine. The predicted PoDs appeared to fall well within the range of PoDs obtained from the available oral single dose in vivo studies. In conclusion, this chapter showed that PBK modelling based-reverse dosimetry can translate in vitro concentration-response curves to in vivo dose-response curves to predict the acute liver toxicity of lasiocarpine and riddelliine in rats. Chapter 5 investigated the inter-species and inter-ethnic human differences in acute liver toxicity of lasiocarpine and riddelliine using the approach developed in chapter 4 for rats. Thus, PBK models for lasiocarpine and riddelliine for the average Chinese and Caucasian were developed, based on the PBK models defined and validated for these PAs in rats (chapter 4). Subsequently, the models were used to convert in vitro toxicity data obtained in pooled Caucasian primary hepatocytes to predict in vivo dose-response curves for acute liver toxicity of lasiocarpine and riddelliine in humans, from which PoDs were derived that were compared to PoDs derived in a similar manner for rats in chapter 4 to obtain insight in inter-species differences. Similarly, the PoDs obtained for the average Chinese and Caucasian were compared to provide insights in inter-ethnic differences. The inter-species differences amounted to 2-fold for lasiocarpine and 8.2-fold for riddelliine with humans being more sensitive than rats. The inter-ethnic human differences varied 2-fold for lasiocarpine and 5-fold for riddelliine with the average Caucasian being more sensitive than the average Chinese. Altogether, this chapter shows a proof-of-principle for a method to predict inter-species and inter-ethnic differences in in vivo liver toxicity for PAs by an alternative testing strategy integrating in vitro cytotoxicity assays with PBK modelling-based reverse dosimetry. Chapter 6 investigated the effect of inter-individual and inter-ethnic (Chinese and Caucasian) kinetic differences in bioactivation of lasiocarpine, and definition of chemical specific adjustment factors (CSAFs) by applying PBK modelling and Monte Carlo simulation. The results revealed an inter-ethnic variation of 2.1-, 3.3- and 4.3-fold when comparing the predicted 7-GS-DHP formation at the GM, 90th and 99th percentile of the Chinese and Caucasian population, respectively, at a dose of level of 8 ng/kg bw lasiocarpine, indicating that the Caucasian population was more sensitive. The CSAFs obtained based on the GM and 90th percentile individuals of the Chinese, Caucasian and the two populations combined were 3.3, 5.2 and 5.7, respectively. The CSAFs obtained based on the GM and the 99th percentile were 8.3, 17.0 and 19.5 for inter-individual variations in the Chinese, the Caucasian population and the two populations combined, respectively. The CSAF values obtained in this chapter indicate that the default safety factor of 3.16 for inter-individual human kinetic differences may not be sufficiently protective for any of the populations. Chapter 7 presents the general discussion based on the results of the present thesis placing the results in a wider perspective while also presenting future perspectives. It is concluded that the present thesis showed an alternative to animal testing approach to define in vivo dose-response curves for risk assessment, based on in silico and in vitro data. It was demonstrated that combining in vitro toxicity data with the kinetic processes integrated into a PBK model adequately predicts in vivo acute liver toxicity of PAs. The obtained results show the possibility to use the method to provide a PoD to perform risk assessment or to define CSAFs for inter-species and intra-species differences. Additionally, this thesis provided insight in the possibilities to build ethnic-specific PBK models that can be used to further refine the risk assessment of compounds that are genotoxic and carcinogenic and also have non-genotoxic endpoints. Moreover, this thesis illustrated that there is a potential priority for risk management for several botanical preparations containing alkenylbenzenes or PAs for the Chinese population when considering daily life-long exposure. Altogether, this thesis provided a proof-of-principle to perform a risk and safety assessment for food-borne toxins based on only in vitro and in silico data.