Development of a fed version of the in vitro gastrointestinal test

According to the different parameters influencing PAHs mobilization inside the gastrointestinal tract, it appears that food plays a major role. As described before, PAHs are easily mobilized in the fat constituents of the food components due to their hydrophobic properties, and they can be transported in the lumen and be potentially absorbed into the cell walls of the intestine, involving potential hazards for human health (Gron et al., 2003). Moreover, including food in in vitro digestion models seems very essential for the development of realistic simulated gastrointestinal extraction procedures. Several studies have shown the dramatic increase in PAHs bioaccessible fractions when using a fed version of a physiologically-based extraction test (Hack et al., 1996; Versantvoort et al., 2004), which is important towards the evaluation of the risk to human health when ingesting PAHs via soils.

Several in vitro gastrointestinal tests involving food or food and soil have been used and developed (Rotard et al., 1995; Hack et al., 1996; Holman et al., 2002; Versantvoort et al., 2004; Oomen et al., 2006) to assess bioaccessibility of several contaminants such metals or PAHs. However, there is still not a procedure that has

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proven satisfactory robustness as with the Unified BARGE Method. In order to realize a common and robust approach, several members of BARGE, which have developed the UBM, have developed a fed version of the Unified BARGE Method, based principally on the work from the RIVM (Cave et al., 2010). At the present stage, the method needs to be compared in different laboratories to estimate robustness, in order to be available in commercial laboratories, on a routine basis. A first evaluation has shown good performance of the analytical method and comparable results with another fed in vitro gastrointestinal test, the SHIME (Simulator of Human Intestinal Microbial Ecosystems of Infants) (Cave et al., 2010). This new method was called FORES(h)t (Fed ORganic Estimation Human Simulation Test) by the BGS and was developed to analyse PAHs bioaccessibilities from soils (Cave et al., 2010). It will be a fed version of the UBM, and more focused on the evaluation of PAHs bioaccessibilities, using a fed state of the digestion.

The development of the FORES(h)t method needed an understanding of the fundamental changes that occur when a digestion model includes food. The FORES(h)t method was based on a method developed by the RIVM, which justify those modifications (Cave et al., 2010). Indeed, the RIVM has developed an in vitro gastrointestinal test including food and considering digestion of ―average children‖ in the Netherlands. This new model was firstly based on the fact that an adult or a child is half of the day in a fed state and the other half on a fasted state (Oomen et al., 2006). Therefore, applying a fed state in the simulation of soil ingestion will give a more realistic and ―non-conservative‖ value of bioaccessibility (Versantvoort et al., 2004). Addition of food involves many modifications to the gastrointestinal tract such as changes on the secretion of gastric, bile and pancreatic fluids, differences of gastric and intestinal motility patterns, and modifications in visceral blood and lymph flow (Versantvoort et al., 2004). As the human physiology is significantly modified when eating food, the fed version of the in vitro gastro-intestinal test will involve numerous modifications. In the RIVM method, the food intake was based on food consumption during a meal from men and women aged 19-65 years old in the

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Netherlands (Versantvoort et al., 2004). The food constituent was chosen in order to comply with the mean intake of adults during a cooked meal, constituted with a known proportion of calories, proteins, carbohydrates and fat. Two infant formulas were chosen because the proportion of energy and nutrients are very close to those of a cooked meal. Oil was added with the infant formula to reach as nearly as possible the same amount of fat and calories, contained in a cooked meal. The amount of soil added in the RIVM test is based on the involuntary ingestion of 100 mg/day of soil considering the ―average behaviour of a child‖ (cf. chapter 1). Therefore, the amount of soil added in the process was based on these previous assumptions. After several studies on the soil-to-solution ratio, the RIVM concluded that quantities of soil between 0.2 and 0.6 g were a good option because lower quantity of soils could lead to heterogeneity of the contaminant concentration in a soil and also give difficulties to detect some of the compounds at low levels (Oomen et al., 2006). However, the FORES(h)t method is employed in England for the moment, therefore the mean intake of energy and nutrients was based on the daily food consumptions of a children aged 4-6 years old and living in the UK (Cave et al., 2010).

The main changes that occur in human digestion when eating food are the residence times, the pH, the bile and pancreatic juices secretions, and the volume of food and digestive fluids (Versantvoort et al., 2004): (a) in the mouth, the saliva secretion is increased. (b) Emptying the stomach when food is ingested can take more time than with a fasted state, whereas no differences are observed in the small intestine. (c) In the small intestine, the bile secretion is increased, until food is removed from the stomach. (d) The pancreatic secretion also increases significantly in the duodenum when food is part of the digestion. The pH gradually increases also after eating, with a pH increasing from 1.5-2 to 3-7 in the stomach and from 5.5 to 7.5 in the duodenum and ileum, contrary to the jejunum where there are no differences. (e) Finally, the volume of digested fluids will depend on the amount of food and liquid ingested during a meal, and therefore will vary between a fasted and a fed state. Based on these

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assumptions the RIVM has developed a fed physiologically-based extraction test for the analysis of metals and lipophilic compounds (e.g : benzo(a)pyrene) in various matrices such as soil or food (Versantvoort et al., 2004). When applying the fed version of a gastrointestinal model the scientists from the RIVM demonstrated that the food was clearly increasing the bioaccessibility (from 5 to 43 %) of benzo(a)pyrene, but not of the metals Pb, As and Cd due mainly to the lipophilic character of benzo(a)pyrene (Versantvoort et al., 2004).

When developing this model, four important considerations were made, as it is in general for the development of other in vitro gastrointestinal tests: absorption (1) will occur in the intestine, so a model involving only the stomach is not sensible. This test (2) should represent a worst case scenario, however the model should be as realistic as possible. The scenario (3) involved will depend on the contaminant and on the matrix studied. Finally (4), the test needs to be easily applicable, robust and reproducible (Versantvoort et al., 2004).