1ISSX 2006 | Esther Brandon

Human in vitro digestion modelspowerful tools to predict maximum oral (relative) bioavailability

Esther F.A. BrandonCentre for Substances and Integrated Risk AssessmentNational Institute for Public Health and the Environment (RIVM)

2ISSX 2006 | Esther Brandon

consumerproducts

work place

environment

air water soil

oraldermal

inhalatory

food, medicines

Humans are exposed to many compounds

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Outline of presentation

• bioaccessibility and bioavailability

• in vitro digestion models

• examples – lead from paint in top– folic acid from dietary supplements

• validation

• conclusions

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Oral exposure: bioaccessibility and bioavailability

External exposure

small intestineportal vein

liver

mouth

oesophagus, stomach,

small intestine

systemic circulation

Internal exposure

Exposure to contaminant in a matrix

Ingestion of matrix + contaminant

FB = Fraction released from matrix = bioaccessible fraction

FA= Fraction of FB absorbed by small intestine

FH = Fraction of FA after the liver without being metabolised

F = Fraction reaching systemic circulation = bioavailable fraction

F = FB x FA x FH

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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

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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

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In vitro digestion models

+ saliva + gastric juice

rotate5 min at 37 C

rotate2 h at37 C

+ duodenal juice+ bile(+ NaHCO3)

rotate2 h at37 C

centrifuge (5 min 2750 g)separate chyme and pellet

chyme pellet

+

+ matrix

emptytest tube

step 1: “mouth”

step 2:“stomach”

step 3:“small intestine”

analysis of compound

+ saliva + gastric juice

rotate5 min at 37 C

rotate2 h at37 C

+ duodenal juice+ bile(+ NaHCO3)

rotate2 h at37 C

centrifuge (5 min 2750 g)separate chyme and pellet

chyme pellet

+

+ matrix

emptytest tube

step 1: “mouth”

step 2:“stomach”

step 3:“small intestine”

analysis of compound

8ISSX 2006 | Esther Brandon

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

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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

10ISSX 2006 | Esther Brandon

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)

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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

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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

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Relevancy for industry, policy makers and upholders

• more accurate risk assessment of ingested contaminants

• more accurate exposure assessment for other compounds, e.g. vitamins

random sample survey or for ad hoc situations

Dutch Food and Consumer Product Safety Authority

in vitro digestion model for relevant exposure scenario

internal exposure value for realistic worst case scenario

risk assessment

product safe?

yes or no

industry

new product or sample survey from a batch

14ISSX 2006 | Esther Brandon

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)

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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.