Human in vitro digestion models

1. Human in vitro digestion models powerful tools to predict maximum oral (relative) bioavailability Esther F.A. Brandon Centre for Substances and Integrated Risk Assessment National Institute for Public Health and the Environment (RIVM)

2. Humans are exposed to many compounds environment food, medicines air water soil consumer products work place inhalatory dermal oral

3. Outline of presentation • bioaccessibility and bioavailability • in vitro digestion models • examples • lead from paint in top • folic acid from dietary supplements • validation • conclusions

4. Externalexposure Exposure to contaminant in a matrix Ingestion of matrix + contaminant mouth oesophagus, stomach, small intestine FB= Fraction released from matrix = bioaccessible fraction small intestine portal vein FA= Fraction of FB absorbed by small intestine FH= Fraction of FA after the liver without being metabolised liver systemic circulation F= Fraction reaching systemic circulation = bioavailable fraction F = FB x FA x FH Internal exposure Oral exposure: bioaccessibility and bioavailability

5. Oral exposure • release depends on type of oral contact • release depends on type of matrix • release from matrix  exposure • release from matrix can be measured by sampling • one way to study release after oral exposure is using in vitro digestion models

6. In vitro digestion model • principle • various compartments of the human gastrointestinal tract (mouth to small intestine) are simulated • digestive juices are prepared artificially based on human physiology • matrix is introduced in mouth compartment, then transferred to the stomach and finally to the small intestine • transit times depend on the input of the risk assessor and human physiology • sampling compartment based on site of absorption

7. In vitro digestion models

8. Developed in vitro digestion models • for application of compounds in food and supplements • fasted conditions • fed conditions • for application of consumer products • sucking • sucking and then swallowing • direct swallowing under fasted conditions • direct swallowing under fed conditions • for application of soil • fasted conditions • fed conditions

9. Different products and compounds tested mycotoxins from food folic acid from dietary supplements and enriched food products folate from natural food sources azo dyes in textile lead in street chalk and paint scraped from tops benzoic acid in finger paint lead and arsenic from contaminated soils lead from house dust www.greenpeace.org.uk

10. example - lead in paint scraped from top • paint: lead level 14.4-15.2 mg/g • situation simulated: ingestion of scraped of paint • bioaccessibility under fasted conditions ~9.5% • bioaccessibility under fed conditions ~4% • large difference between external and internal exposure • based on risk assessment this top is not safe for children (11 mg paint leads to exceeding the TDI)

11. Validation • for lead and arsenic from soil (Oomen et al,. 2006) • the mycotoxins aflatoxin B1 and ochratoxin A investigating different adsorbents (Versantvoort et al., 2004) Although relevant in vivo data are scarce, we succeeded to preliminary validate the model for some cases These cases showed good correlation and never underestimated the bioavailability

12. Scientific conclusions • internal exposure can be considerably less than external exposure • bioaccessibility/bioavailability is highly dependent on matrix and compound • bioaccessibility can easily be measured experimentally • the outcome should be interpreted as indicative

13. Relevancy for industry, policy makers and upholders • more accurate risk assessment of ingested contaminants • more accurate exposure assessment for other compounds, e.g. vitamins

14. Acknowledgment • Agnes Oomen (Centre for Substances and Integrated Risk Assessment, RIVM) • Adrienne Sips (Centre for Substances and Integrated Risk Assessment, RIVM) • Carolien Versantvoort (Centre for Substances and Integrated Risk Assessment, RIVM) • Cathy Rompelberg (Centre for Nutrition and Health, RIVM) • Marco Blokland and co-workers (Laboratory for Food and Residue Analyses , RIVM) • Peter Bragt and Martien Spanjer (Food and Consumer Product Safety Authority) • Bülent Kabak (University of Cukurova, Turkey) • Paula Alvito (Food Safety and Nutrition Centre, Portugal) • Karin Ljung (Swedish University of Agricultural Sciences, Sweden) • Rawad Massoud (Utrecht University, The Netherlands)

15. RIVM reports and articles • Kabak B, Brandon EFA, Vara I, Sizoo EA, Blokland MH, van Egmond HP, Sips AJAM. Effects of probiotic bacteria on the bioaccessibility of aflatoxin B1 and ochratoxin A using an in vitro digestion model under fed conditions. In preparation. • Oomen AG, Brandon EFA, Swartjes FA, Sips AJAM (2006). How can information on oral bioavailability improve human health risk assessment for lead-contaminated soils? Implementation and scientific basis. RIVM report 711701042, Bilthoven, the Netherlands. Available at http://www.rivm.nl/bibliotheek/rapporten/711701042.pdf • Brandon EFA, Oomen AG, Rompelberg CJM, Versantvoort CHM, van Engelen JGM, Sips AJAM (2006). Consumer product in vitro digestion model: bioaccessibility of contaminants and its application in risk assessment. Reg Toxicol Pharmacol 44: 161-171. • Versantvoort CHM, Oomen AG, van de Kamp E, Rompelberg CJM, Sips AJAM (2005). Applicability of an in vitro digestion model in assessing the bioaccessibility of mycotoxins from food. Food Chem Toxicol 43: 31-40. • Versantvoort CHM, van de Kamp E, Rompelberg CJM. Development and applicability of an in vitro digestion model in assessing the bioaccessibility of contaminants from food (2004). RIVM report 320102002, Bilthoven, the Netherlands. Available at http://www.rivm.nl/bibliotheek/rapporten/320102002.pdf • Oomen AG, Rompelberg CJM, Bruil MA, Dobbe CJG, Pereboom DPKH, Sips AJAM (2003). Development of an in vitro digestion model for estimating the bioaccessibility of soil contaminants. Arch Environ Contam Toxicol 44: 281-287.