A generic cross-chemical predictive PBTK model with multiple entry routes running as application in MS Excel; design of the model and comparison of predictions with experimental results.
Aim: Physiologically based toxicokinetic (PBTK) models are computational tools, which simulate the absorption, distribution, metabolism, and excretion of chemicals. The purpose of this study was to develop a physiologically based pharmacokinetic (PBPK) model with a high level of transparency. The model should be able to predict blood and urine concentrations of environmental chemicals and metabolites, given a certain environmental or occupational exposure scenario. Model: The model refers to a reference human of 70 kg. The partition coefficients of the parent compound and its metabolites (blood:air and tissue:blood partition coefficients of 11 organs) are estimated by means of quantitative structure-property relationship, in which five easily available physicochemical properties of the compound are the independent parameters. The model gives a prediction of the fate of the compound, based on easily available chemical properties; therefore, it can be applied as a generic model applicable to multiple compounds. Three routes of uptake are considered (inhalation, dermal, and/or oral) as well as two built-in exercise levels (at rest and at light work). Dermal uptake is estimated by the use of a dermal diffusion-based module that considers dermal deposition rate and duration of deposition. Moreover, evaporation during skin contact is fully accounted for and related to the volatility of the substance. Saturable metabolism according to Michaelis-Menten kinetics can be modelled in any of 11 organs/tissues or in liver only. Renal tubular resorption is based on a built-in algorithm, dependent on the (log) octanol:water partition coefficient. Enterohepatic circulation is optional at a user-defined rate. The generic PBTK model is available as a spreadsheet application in MS Excel. The differential equations of the model are programmed in Visual Basic. Output is presented as numerical listing over time in tabular form and in graphs. The MS Excel application of the PBTK model is available as freeware. Experimental: The accuracy of the model prediction is illustrated by simulating experimental observations. Published experimental inhalation and dermal exposure studies on a series of different chemicals (pyrene, N-methyl-pyrrolidone, methyl-tert-butylether, heptane, 2-butoxyethanol, and ethanol) were selected to compare the observed data with the model-simulated data. The examples show that the model-predicted concentrations in blood and/or urine after inhalation and/or transdermal uptake have an accuracy of within an order of magnitude. Conclusions: It is advocated that this PBTK model, called IndusChemFate, is suitable for 'first tier assessments' and for early explorations of the fate of chemicals and/or metabolites in the human body. The availability of a simple model with a minimum burden of input information on the parent compound and its metabolites might be a stimulation to apply PBTK modelling more often in the field of biomonitoring and exposure science. Keywords: biomarker of exposure; blood; body burden; internal exposure; PBTK-model; prediction, urine *Author to whom correspondence should be addressed. INTRODUCTION A physiologically based toxicokinetic (PBTK) or physiologically based pharmacokinetic (PBPK) model is an structural mathematical model, comprising the tissues and organs of the body with each perfused by, and connected via, the blood circulatory system. Such models are computational tools that can refine the assessment of the fate of chemicals in the body by simulation. In PBTK models, the body is subdivided into anatomical compartments representing individual organs or tissue groups. The transport of chemical in the body is described by mass balance differential equations that incorporate blood flows, partitioning into compartments and tissue volumes. After incorporation of elimination processes like metabolism and excretion, the fate and disposition of the parent chemical and metabolites can be predicted and extrapolated. Most PBPK models are chemical specific. Often, they are built for very specific purposes, for example, the estimation of disposition of a certain drug prior to in vivo studies Several initiatives were taken to develop PBPK models that can be used for industrial compounds Partitioning between blood:air and between tissue:blood is related to easy available physicalchemical properties. The relationships were worked out into QSPRs, which algorithms have been incorporated into the PBTK model. A novel dermal uptake module has been added to the PBTK model. Also, serial metabolism and urinary excretion have been incorporated in the model. The human physiological parameters such as organ volumes, blood flows, cardiac output, and alveolar ventilation are adopted from Technical Guidance Documents of REACH (ECHA, 2008a , b) and are presented in Stepwise numerical integration routine according to Euler can be entered. The integration intervals can be set. Minimum is an integration interval of 1000 steps h -1 , best results are found at 10 000 steps h -1 . Prediction of partitioning of chemicals A partition coefficient is the ratio of the concentration of a chemical between two phases in thermodynamic equilibrium. The tissue:blood partition coefficients are relevant for simulation of the distribution in the body. The blood:air partition coefficient controls the uptake of a compound in the alveoli. A novel QSPR to estimate the blood:air partition coefficient has been derived. A wide range of VOCs with measured blood:-air values for humans from many sources were reported in the paper of In case of substance with Blood : air partition a vapour pressure .4000 coefficient Pa and a dimensionless 5 0:8417=HenryDL Other substances Blood : air partition coefficient 5 0:4445=HenryÀDL þ 0:005189  K oa : ðN 5 57 ; R 2 5 0:99Þ ð2Þ A plot of experimental human partition coefficients blood:air and QSPR estimated values is presented in HenryÀDL 5 Vapour pressure  molecular weight=ðwater solubility  gas constant  temperature°KÞ ð3Þ logðK oa Þ 5 logðK ow Þ À logðHenryÀDLÞ ð4Þ For the blood:tissue partitioning, the QSPR algorithm as described by As an example, the algorithm for the brain:blood partition coefficient is shown as formula 5. Application of this equation to adipose tissue results into negative partition coefficients in case of log(K ow ) , 0.4. This has no scientific meaning. So if the partition coefficient adipose tissue:blood is estimated to be ,0.1, the adipose tissue:blood partition coefficient is fixed to 0.1. Modelling of uptake of chemicals Tissue concentrations for each of the chemicals and metabolites can be simulated for either acute, occupational, or environmental exposure regimes with its typical duration, routes, concentrations, or dose rate. The impact of exercise that may influence uptake, distribution, metabolism, and excretion is accounted for by two levels of exercise (at rest and at light exercise, with a heart rate of, respectively, 78 and 114 beats min -1 ) with corresponding physiology parameters (cardiac output and pulmonary ventilation) according to Inhalation in the IndusChemFate PBTK model is controlled by the concentration of the compound in the inhaled air, the alveolar ventilation, and the blood:air partition coefficient. In the model, the maximum concentration in inhaled air is limited at the level of saturated vapour pressure. The actual concentration in inhaled air can be lower than the environmental air concentration due to wearing of respiratory protective equipment (5 RPE). The reduction factor of the RPE can be entered. The default respiratory reduction factor 1 (5 no RPE). In the last two decades, the awareness has grown that dermal absorption of chemicals after environmental and/or occupational exposure can be very significant. This has lead to the development of PBPK models with an integrated dermal compartment (1) Dermal deposition of a substance (liquid) on the skin, (2) Diffusion to the stratum corneum (SC), and (3) Absorption to the dermis/blood flow. After or during deposition of a liquid or solid substance on the skin, evaporation of the substance and dermal absorption will start simultaneously (see scheme in The dermal absorption from the vapour phase is also considered (see Oral intake of compounds is considered as a bolus dose that is applied to the intestinal lumen (via the stomach) and then absorbed into the intestinal tissue at a first order rate. From the intestines, the compound is released to the blood stream towards the liver (portal vein). The first order absorption rate is defined as the velocity at which the oral dose is absorbed by the intestinal tissue (as a fraction of the dose in the lumen per hour). Stomach and intestines are lumped in the model. The oral dose [in milligrams per kilogram body weight (BW)] and the absorption rate are the required input parameters for oral uptake in the model. Enterohepatic circulation Phase II metabolism with conjugation of metabolites generally increases the solubility. Enzymes produced by intestinal bacteria-such as b-glucuronidase, sulfatase, and various glycosidases-deconjugate these compounds in the intestines, releasing the parent compounds after which these are readily reabsorbed across the intestinal wall to the blood. This results in enterohepatic circulation (of conjugated phase II metabolites). Few published PBPK models consider enterohepatic circulation If, for example, the removal ratio of a nonconjugated metabolite from the liver by enterohepatic circulation is set 0, there is no enterohepatic circulation. If in the case of a conjugated metabolite, the removal ratio is set to 1, 50% of the total amount that leaves the liver per unit of time is excreted to blood, and 50% to the intestinal lumen via bile, available for reabsorption with a fixed rate of 0.3 h -1 . Elimination The chemical in the human body is eliminated in the model by two processes: metabolism (or biotransformation) and direct excretion in air or urine. Biotransformation is described by Michaelis-Menten saturable metabolism following the mathematical algorithms as described by Contrary to many PBPK models, the occurrence of metabolism is not limited to the liver compartment but can be considered in any of the 11 model compartments. However, the default setting is metabolism in the liver only. Metabolic kinetic parameters are the maximum velocity of metabolism [5 V max in lmol/(kg tissue  h)] and the MichaelisMenten constant (5k M in lM). Preferably, these values are taken form experimental data with human tissue. Conversion of reported experimental V max to the proper units is given in the addendum. When parallel metabolic pathways are involved, the V max and k M values for a specific metabolite production can be set as different from those the parent compound. That is possible because the model considers both removal of the parent compound and production of metabolite as separate steps. That means the biotransformation of the parent compound occurs for only x% into the metabolite of interest and for (100Àx)% into other (unknown) metabolites. V max and k M metabolism constants of a series of VOC have recently been summarized (Aylward et al., 2010). Substances can be excreted via urine, either unchanged as parent compound or as a metabolite. DeWoskin and Thompson (2008) published a paper in which renal clearance is modelled in great detail; however, the required input data transcends application in a generic model. Urinary excretion is mainly based on the lipophilicity of substances, assuming that lipophilic substances are less water soluble and therefore excreted via urine to a lower extent. The QSPR as developed by The total volume of excreted urine in 24 h is set to 1.44 l. Generic PBTK-model in MS Excel 847 by guest on October 14, 2011 annhyg.oxfordjournals.org Downloaded from When the volatility is high, chemicals (and in a few cases a metabolite) will be exhaled. The exhaled concentration is a mixture of the inhaled air concentration (air that has not reached the alveoli) and alveolar air. The concentration of a compound in the alveolar space of the lungs is controlled by the blood concentration in the (arterial) lung blood and the blood: air partition coefficient. The amount of a compound that is exhaled is calculated by multiplying the alveolar concentration by the alveolar ventilation rate. Mass balance After every model simulation, a mass balance is calculated. Absorbed amounts per route are summarized and compared with the total of excreted amounts, amounts in tissues, and amounts to undefined metabolites, not assigned to the metabolic route considered. The Excel application of the PBTK model The generic PBTK model is available as a spreadsheet application in MS Excel. The differential equations of the model are programmed in Visual Basic. The spreadsheet template can be operated after a few instructions. The numerical integration is fast. A typical simulation of 24 h after exposure takes a few seconds on a standard personal computer, including the plotting of the results. The full mathematical description of the PBTK model is presented in Supplementary 1 (available at Annals of Occupational Hygiene online) in the online edition. The mathematical description of dermal absorption of chemicals is presented in supplement 2 in the online edition. Output is presented as numerical listing over time in tabular form and in graphs. The program does provide the amounts in micromoles and concentrations in micromoles per litre. After each run, amounts and concentrations in compartments and fluids are listed together with the estimated partition coefficients of the chemical and metabolites under study and the data of the mass balance. Also, graphs of the concentrations in alveolar air, blood, and urine are presented. The IndusChemFate PBTK model is available free of charge from the CEFIC-LRI website as a Visual Basic application in Microsoft Excel (available at http://www.cefic-lri.org/lri-toolbox/induschemfate last accessed on 7 September, 2011). EXPERIMENTAL Studies with exposure to chemicals (inhaled concentration or dermal dose rate) and with repeated measurements of concentration of the chemical and metabolites in blood and/or urine were searched for. Six experimental or observational studies with six different compounds were selected, e.g. the compounds pyrene The time course of the blood and urine concentrations of the parent compound and/or metabolites were simulated with the PBTK model IndusChemFate following the reported exposure scenario of the selected study. The physical--chemical input parameters of the compounds-molecular weight, density, vapour pressure, log(octanol:water) partition coefficient at pH 5.5 and at 7.4, and water solubility-were taken from the EPI suite database of US-EPA (2009) , the Chemspider database RESULTS Comparison 1: excretion of hydroxylated metabolite of pyrene in creosote impregnating worker The concentration of a 1-hydroxypyrene (1-OHP, a hydroxylated metabolite of pyrene) was measured in urine of an operator of a creosote impregnating site The excretion of 1-OHP was simulated with the PBTK model IndusChemFate. Reported exposure data of creosote plant operators of other studies were used to make an estimate of the representative exposure scenario. Airborne exposure of creosote plant operators to pyrene might be up to 3 lg m measurements of pyrene ranges from ND-90 ng cm -2 skin in 8-h work shift; the exposed dermal surface area is large: neck, wrist, and jaw/neck of creosoting workers were clearly exposed The PBTK model requires input data of three compounds: the parent compound pyrene, 1-OHP, and 1-OHP-gluc. The physical-chemical input data and the kinetic input data are presented in The simulation showed that 1-OHP-gluc is the dominant metabolite in urine. Free 1-OHP was predicted to be is ,0.1% of 1-OHP-gluc. The simulated excretion pattern of 1-OHP-gluc of the last 4 days of a working week is shown in Comparison 2: blood and urine concentrations of MTBE and metabolites after inhalation Six volunteers (three males and three females) were exposed to a concentration of 40 p.p.m. MTBE (methyl-tertiary-butylether) (5 144 mg m -3 ) for 4 h in a dynamic exposure chamber In the body, MTBE is metabolized to tert-butanol (Metabolite 1). This metabolite is further metabolized, mainly to MPD (Metabolite 2), which is further oxidized to HiBA (Metabolite 3). The fate of MTBE and three metabolites in blood and urine was simulated with PBPK model IndusChemFate with the given scenario of exposure: 4-h exposure to a concentration of 140 mg m -3 . The entry data of MTBE and the three metabolites that were used for the simulation are presented in Furthermore, blood levels were reported. The concentration of MTBE and t-butanol in blood was determined at the end of the 4-h exposure period. The mass balance showed that exhalation of MTBE was the preferred route of elimination; the exhaled amount of excreted MTBE and metabolites in 48 h was 2-3 fold larger than the excreted amount in urine (respectively, 34 lmol kg -1 BW and 13.2 lmol kg -1 BW). Comparison 3: urine concentrations of NMP after transdermal vapour absorption Dermal vapour phase absorption is an important route of uptake of the solvent N-methyl-2-pyrrolidone (NMP). This particular aspect was investigated in an experimental study with 16 volunteers exposed to 80 mg m -3 NMP for 8 h under either whole body, i.e. inhalation plus dermal vapour exposure, or dermal only conditions The fate of NMP and metabolites after 8-h exposure to a concentration of 80 mg m À3 was simulated with PBPK model IndusChemFate. The chemicalspecific data of NMP and metabolites that were used for the simulation are presented in I
