Molecular Biosciences Department, Fundamental Science Division, Battelle, Pacific Northwest Laboratory, 902 Battelle Boulevard, Mail Stop P7-59, Richland, Washington 99352
Received January 18, 2002; accepted March 26, 2002
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ABSTRACT |
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Key Words: dermal; toluene; human; aqueous; PBPK; breath analysis.
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INTRODUCTION |
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From the 1950s through the 1970s, the rate of uptake of a chemical through the skin was generally estimated based on studies of humans (Paustenbach and Leung, 1993). More recently, evaluations of dermal uptake of a chemical are made using animal skin (in vivo or in vitro) or human skin in vitro. In vivo, the rate of uptake of a chemical through the skin has been estimated using radiolabeled compounds and tracking the radioactivity in blood and excreta following topical application (Paustenbach and Leung, 1993
). While this method for determining percutaneous absorption provides an estimate of the total absorbed dose, it often fails to reveal information on the uptake, distribution, and clearance phases of dermal absorption kinetics. Further, since blood levels may be very low in these situations, this practice is often restricted by sensitivity limits of the assay or analysis. An additional drawback with this methodology is that the nature of the radioactivity, whether it represents the parent or metabolites, is often undefined; thus kinetic interpretation is limited.
Recent studies have illustrated that exhaled breath presents a useful alternative to radiotracer studies by providing a non-invasive methodology for assessing bioavailability of volatile compounds (Corley et al., 2000; Thrall et al., 2000
). Breath measurements are particularly useful in studies where repeated sample collection in real time allows for the tracking of absorption and elimination trends. Since breath concentrations reflect blood concentrations, continual analysis of exhaled breath provides a unique opportunity to evaluate differences in the rapid exponential emptying of the blood compartment that occurs immediately following peak exposure. Furthermore, the non-invasive nature of breath analysis improves the participation rate in controlled human exposure and environmental or occupational biomonitoring studies.
For exhaled breath measurements to be useful, they must be evaluated using some form of a kinetic model. Physiologically based pharmacokinetic (PBPK) models are particularly useful for integrating a variety of data, including breath analysis, to determine the penetration rates of chemicals through the skin. A PBPK model is exceptionally well suited for assessing dermal exposures under non-steady state conditions, where the transdermal flux is a function of the permeability coefficient (Kp), the area exposed, and the changing concentration gradient across the skin (Jepson and McDougal, 1997). The integration of real-time, exhaled-breath measurements with a PBPK model to determine dermal absorption has been successfully used for a number of compounds, including methyl chloroform, trichloroethylene, and benzene (Thrall et al., 2000
).
The objective of the study presented here was to evaluate the dermal absorption of aqueous toluene in humans under realistic exposure conditions using exhaled breath analysis and PBPK modeling. To compare the significance of dermal absorption with inhalation exposures, each human volunteer participated in 2 phases of study: one with dermal exposure only, and one with combined dermal and inhalation exposure. A PBPK model developed to describe dermal exposures to aqueous toluene in F344 male rats (Thrall and Woodstock, in press) was modified to describe each human volunteer and used to estimate permeability coefficient values.
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MATERIALS AND METHODS |
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The study protocol entailed monitoring volunteer exhaled breath for background toluene levels for 5 min prior to entry into the tub. Throughout the study, the water was continually mixed using an 0.5 horsepower turbine agitator. During the exposure phase, each volunteer was submerged to neck level in the tub for 2030 min, while continually providing exhaled breath for real-time analysis. A weighed quantity of neat toluene, prepared in a separate room, was added to the water near the agitator after the volunteer was comfortably situated in the hydrotherapy tub, and during the on-going analysis of exhaled breath. The addition of the toluene to the bathwater was designated as time zero for water analysis. At the end of the monitoring period, the volunteer left the tub, briefly towel dried, and provided 1530 min of post-exposure exhaled breath for real-time analysis. Volunteers participated in 2 separate study phases, with at least 1 week between each phase. In the first phase, exposure to toluene was by the dermal route only; volunteers were provided purified breathing air (Certified Grade D) throughout the exposure period, while immersed in the hydrotherapy tub. In the second phase, the volunteer was allowed to breathe the room air during the exposure phase, and thus exposures were by both dermal and inhalation routes. Regardless of study phase, breathing air was provided to the volunteer to isolate any volatilized compound in the room during the 5-min pre-exposure background check and for the post-exposure period.
Real-time breath analysis system.
The exhaled-breath monitoring system utilized during the human studies consisted of a breath inlet device connected to a Discovery II (LGC Inc., Los Gatos, CA) ion-trap tandem-mass spectrometer (MS/MS) equipped with an atmospheric sampling glow-discharge ionization source (ASGDI) and has been described previously (Gordon et al., 1998). Volunteers wore a Hans Rudolph facemask (Kansas City, MO) containing two 1-way, non-rebreathing valves. Purified breathing air was supplied by a gas cylinder to a 15-l foil-lined buffer bag to provide air to the volunteer on demand. Expired air was passed through a large-diameter Teflon tube into a heated-glass mixing chamber (1.3-l volume). Breath samples entered the mixing chamber through a tube that bent off to one side, and exited the mixing chamber via a tube bent in the opposite direction, thus ensuring that samples were well mixed by turbulent flow. The ASGDI-MS/MS system continually drew air samples from the center of the mixing chamber at a calibrated rate of 12 l/h to provide a new data point every 4.6 s. Excess exhaled air was vented from the mixing chamber via a large-bore hole exit tube with negligible flow restriction.
Intensity data from the mass spectrometer were converted to concentration (ppb) using external gas standards prepared in Tedlar bags (Supelco Inc., Bellefonte, PA) and a calibration curve. A new calibration curve was generated on each day of operation. The ASGDI-MS/MS methodology had detection limits in the 210-ppb range for toluene and calibrations were linear to 57 ppm (57,000 ppb).
A pilot study was conducted to evaluate the volatilized loss of toluene from the hydrotherapy tub following addition of toluene to the water. In this case, the hydrotherapy tub was filled with water as described for the human volunteer studies. The breath inlet device was positioned over the water at the level roughly corresponding to the breathing zone of the volunteers and used to continually sample the toluene air concentration prior to, and for 30 min after, addition of the toluene to the water, as described previously.
Human blood to air partition coefficient.
Heparinized human blood (single donor) was obtained from Golden West Biologicals, Inc. (Temecula, CA). The human blood-to-air partition coefficient was determined using a vial equilibration method, as described by Sato and Nakajima (1979) and modified by Gargas et al. (1989). Briefly, 2 ml of heparinized blood was weighed into a 25-ml scintillation vial and sealed airtight with caps that were modified by drilling a 4-mm-diameter hole in the center and replacing the cap liner with a Teflon-coated rubber septum (Supelco Inc., Bellefonte, PA). Toluene (1 ml) was introduced into the sealed vial as a vapor from a Tedlar gas-sampling bag (Supelco Inc., Bellefonte, PA) containing an air concentration of 5000 ppm. Sealed vials were incubated at 37°C with shaking in a vortex evaporator for 1 and 3 h. At the end of the incubation period, the toluene headspace concentration was determined by gas chromatography (GC), using a Hewlett Packard model 5890 Series II system (Avondale, PA) as described above. Tissue to blood partition coefficients were determined by dividing tissue to air values determined in the F344 rat as reported by Gargas et al. (1989) by the human blood to air value determined here.
PBPK model.
The dermal toluene PBPK model (Fig. 1) has been used to successfully describe the dermal absorption of aqueous toluene in the rat (Thrall and Woodstock, in press). Anatomical compartments in the model were used to describe the distribution of toluene into the rapidly perfused, slowly perfused, fat, liver, and skin compartments. The skin compartment in the model represented exposed skin; non-exposed skin was lumped into the slowly perfused compartment. Total skin, with a volume of 10% of the bw, was assumed to receive 5% of the cardiac output. The exposed skin volume and blood-flow rate were calculated as described by Jepson and McDougal (1997). For toluene, metabolism has been shown to occur primarily in the liver, and to be independent of the route of exposure (Cohr and Stokholm, 1979
). Although metabolism at the site of entry is considered possible, this loss was assumed to be insignificant in comparison to liver metabolism. Therefore, metabolism was described as occurring only in the liver. Model parameters specific to human physiology, partition coefficients, and metabolism were taken from the literature or determined here (Table 2
). To simulate dermal exposures, the rate of change in the concentration of toluene in the skin compartment (Csk, µg/ml) was related to the rate of penetration through the skin (the flux) and the rate of delivery due to blood flow and arterial concentration (the perfusion) as described by Thrall et al. (2000). In the PBPK model, this is written as:
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RESULTS |
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Human subjects participated in 2 separate studies: a dermal-only exposure and a dermal-plus-inhalation exposure. Target water toluene initial concentrations were 500 µg/l, or approximately 1.9 mg/kg bw for a 70-kg volunteer in 265 l of water. The actual water concentrations were verified for each volunteer by GC analysis of water samples collected immediately following addition of the toluene to the hydrotherapy tub, and at 5-min intervals thereafter. Actual initial exposure concentrations were found to range from 455 to 550 µg/ml (Table 3). A comparison of the triplicate water samples collected at different locations within the tub at the same time interval indicated that the toluene was well mixed within the hydrotherapy tub (data not shown). Tap water samples collected prior to the addition of toluene had no measurable toluene present.
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DISCUSSION |
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Due to differences in exposure conditions, it is difficult to directly compare the permeability coefficients determined here to literature estimates. For example, Kezic et al. (2001) exposed human volunteers to neat toluene for brief (3-min) periods of time; however, permeability may have been altered due to reported skin irritation. The U.S. EPA (1992) human Kp value for aqueous toluene was estimated to be 1 cm/h based on flux data from Dutkiewicz and Tyras (1968). In the human studies described by Dutkiewicz and Tyras (1968), the amount of toluene absorbed was quantified by measuring the loss of the compound from the donor solution, and steady-state conditions were not verified. However, permeability may be overestimated by assuming that the rate of chemical loss from the exposure solution represents the average flux into the skin (Jepson and McDougal, 1997). In addition, erroneous estimates of percutaneous absorption may be determined when standard Ficks-law calculations of dermal flux are used without verifying that steady state was achieved (Jepson and McDougal, 1997
). Therefore, it is particularly important for dermal absorption estimation methods to be able to account for unsteady-state dermal absorption (Roy et al., 1996
). A PBPK model is ideally suited for estimating the actual permeability when the exposure concentration changes with time.
In the studies reported here, each volunteer, with the exception of one, participated in two study phases: a dermal only, and an inhalation-plus-dermal exposure phase. Thus, these studies provided a unique opportunity to compare the relative impact of each route of exposure on total body burden. It has been suggested that, for a given concentration of some volatile organic compounds, the daily dose absorbed during a 20-min bath may be similar in magnitude to the daily inhalation dose from volatilization of these same organic compounds (Shatkin and Szejnwald-Brown, 1991). However, the data presented here suggests that inhalation exposure to volatilized toluene from tap water is transient and contributes little to the overall total body burden over the course of the 30-min bath. This was illustrated by the rapid loss of toluene in the room air following addition of toluene to the hydrotherapy tub (Fig. 2), and by comparison of the exhaled breath profiles for a single volunteer exposed to toluene by dermal versus dermal-plus-inhalation routes (Fig. 7
).
To estimate the relative importance of dermal and oral exposures, the total amount of toluene absorbed through each route of exposure was calculated using the PBPK model. For a 70-kg male taking a 30-min bath in municipal water containing an initial concentration of 500 µg/l of toluene, the dermal route will result in an estimated 0.41 µg toluene absorbed/kg bw (ignoring the inhalation contribution). In comparison, the oral-equivalent dose, calculated by assuming 100% oral bioavailability and the consumption of 2 liters of water/70 kg bw will contribute 14 µg/kg when toluene is present at the same concentration. Therefore, for a total (dermal + oral) body burden of 14.41 µg toluene absorbed/kg bw, the dermal route contributes approximately 2.8% of the daily body burden. It bears mentioning that these calculations of the relative contribution by route of exposure to total body burdens are exposure-specific as well as chemical-specific. The role of dermal uptake may be of greater or lesser importance, depending upon the actual exposure scenario. For example, while these studies were conducted at a water temperature of approximately 38°C, Corley et al. (2000) observed significant increases in exhaled chloroform and thus skin Kp values in volunteers exposed in bathwater studies as water temperature was increased from 30 to 40°C. Further, a sensitivity analysis of the PBPK model parameters used here indicated that the estimation of the Kp value was most sensitive to the breathing rate. Perhaps future studies could employ human plethysmography to individually measure breathing rates and thus reduce the variability in estimated Kp values.
In summary, the experiments on volunteers reported here provided definitive data for assessing the absorption of aqueous toluene through human skin. These whole-body experiments using human volunteers avoided interspecies extrapolations and variations in absorption based on the site of application. Furthermore, the utilization of sensitive, real-time instrumentation allowed for controlled exposures to be conducted under a realistic bath-water scenario. Analysis of the resultant exhaled breath data, using a PBPK model, estimated an average whole-body Kp for dermal absorption of aqueous toluene of 0.012 ± 0.007 cm/h. This value is more than 80 times lower than the U.S. EPA estimate based on a prior study (U.S. EPA, 1992).
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ACKNOWLEDGMENTS |
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NOTES |
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1 To whom correspondence should be addressed. Fax: (509) 376-9064. E-mail: karla.thrall{at}pnl.gov.
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