* Chemical Dosimetry, Battelle, Pacific Northwest Division, Post Office Box 999, MSIN P7-59, Richland, Washington 99352; and
Department of Dermatology, University of California, San Francisco, California 94143.
Received January 2, 2000; accepted April 4, 2000
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ABSTRACT |
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Key Words: percutaneous absorption; PBPK modeling; real-time breath analysis; permeability coefficient.
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INTRODUCTION |
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In general, previous investigations of dermal absorption have employed conditions that do not mimic actual exposure situations and may significantly overestimate potential human body burdens. For example, dermal penetration methods using an impermeable occlusion technique can drastically alter skin hydration and temperature and create an artificially high exposure concentration due to the suppression of normal loss by volatilization (U.S. EPA, 1992). As a result, the use of occlusive techniques can affect bioavailability and will generally provide a worst-case dermal penetration scenario (highest uptake), which may be completely unrelated to realistic environmental exposure situations.
Erroneous estimates of percutaneous absorption may also be determined when standard Fick's law calculations of dermal flux are used without verifying that steady state was achieved. In comparison, a physiologically based pharmacokinetic (PBPK) model can be used to assess transdermal flux as a function of the permeability coefficient (KP), the area exposed (SA), and the concentration gradient across the skin (C) during nonsteady-state conditions. In addition, PBPK models can include mathematical descriptions of the test exposure system that account for loss of chemical through volatilization and the resulting changes in matrix concentration. Due to their fundamental biological basis (i.e., tissue characteristics, blood flow, metabolism rates, etc.), PBPK models have become increasingly popular for estimating kinetic parameters following dermal exposure as a function of the exposure matrix and duration (Corley et al., 1997
; Corley et al., 2000
; Jepson and McDougal, 1997
; Jepson and McDougal, 1999
; McDougal et al., 1986
).
Percutaneous absorption of TCE has been investigated in rodents and in human skin in vitro (Bogen et al., 1992; Morgan et al., 1991
; Nakai et al., 1999
). Bogen et al. (1992) reported a Kp of 0.21 cm/h for trichloroethylene in hairless guinea pigs following exposures of most of the body surface in a water matrix. In partial thickness human skin in vitro, the KP was reported to be 0.12 cm/h (Nakai et al, 1999
). Although some comparisons of the contributions of different routes of exposures have been made for humans while showering (Weisel and Jo, 1996
), a human in vivo KP for TCE has not previously been reported.
The objective of this research was, therefore, to investigate the percutaneous absorption of TCE in rats and humans under realistic exposure conditions using a noninvasive technique (Poet et al. 2000). To evaluate the significance of dermal exposures to TCE in water and soil, the TCE PBPK model of Fisher et al. (1998) was modified by adding a skin compartment based on the design of Jepson and McDougal (1997) and used to define and compare skin permeability (KP) under various exposure scenarios. Occluded and nonoccluded dermal exposures were conducted in rats and humans using soil and water containing low levels of TCE. Exhaled breath was analyzed using an atmospheric sampling glow discharge ionization (ASGDI) source mass spectrometer (MS/MS) to follow the kinetics of TCE continuously throughout the absorption, distribution, and elimination phases according to Poet et al. (2000). Rats, which are commonly used in in vivo dermal absorption studies, were included for comparison with humans and for assessing multiple exposure concentrations.
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MATERIALS AND METHODS |
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HPLC-grade (99.5% pure) trichloroethylene (CAS #79-01-6) was obtained from Sigma Chemical Co. (St. Louis, MO.). All other chemicals were reagent grade or better and were obtained from Sigma Chemical Co. The soil sample was collected in Yolo County, California, and was prepared by passing it through a 40-mesh sieve and retaining it on an 80-mesh sieve. The soil was analyzed by an independent lab (DANR Analytical Laboratory, University of California) and consisted of 30% sand, 18% clay, 52% silt, an organic content of 1.3%, and a pH of 6.8. No volatile contaminants were detected in the soil using the MS/MS system described below.
Dermal Exposures in Rats
Application techniques.
Dermal exposures to TCE were conducted in both soil and water matrices using methods reported previously (Poet et al., 2000). Male F344 rats were anesthetized and the hair on the lower back clipper shaved the day prior to exposure. Groups of three rats were used at each concentration in occluded or nonoccluded water and soil exposures. An outline of the experimental design is given in Table 1
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For soil exposures, 1 g of soil at TCE target concentrations of 5000, 20,000, and 40,000 mg/kg was placed in the nonoccluded exposure system for 3 h. Preliminary studies with a nonoccluded system, patterned after the occluded glass cell but covered with a vapor permeable ceramic frit, were not successful at preventing leakage of TCE into the off-gassing chamber. Therefore, the nonoccluded exposure system used in previous work (Poet et al., 2000) was used for TCE soil exposures. Briefly, a ring of self-adhesive DuoDerm® (Bristol-Myers Squibb, Princeton, NJ) was attached to the clipped area on the back of each rat. The topical dose of TCE-spiked soil was applied to the 8-cm2 area bounded by the DuoDerm®, and covered with a transparent dressing (Bioclusive®, Johnson & Johnson, Arlington, TX) that allowed free passage of vapor. The patch was covered with an aerated weighboat and a muslin patch containing activated charcoal. The entire patch system was secured with self-adherent wrap. An 8-cm2 patch area representing approximately 3% of the total skin surface area was chosen to mimic limited exposure situations. For occluded soil samples, target concentrations of 5000 and 15,000 mg/kg TCE in 5 g of soil were placed in the occluded glass cell system as described for water exposures.
Dermal exposure conditions.
To quantify total absorbed dose, the exact weight of administered dose was recorded and samples of exposure media (soil or water), the amount remaining at the end of the exposure, and the amount volatilized to the charcoal were analyzed using gas chromatography as described previously (Poet et al., 2000).
Immediately following dermal application, rats were individually placed in 2-liter off-gassing chambers as described by Gargas (1990), and TCE was quantified in the chamber, representing exhaled breath. A Teledyne Discovery II MS/MS equipped with an ASGDI source sampled from the off-gassing chamber approximately every 5 s as described previously (Poet et al., 2000). TCE was quantified by selective ion monitoring of the m/z ratios 127131. For occluded soil and water exposures, exhaled breath was monitored for 5 h. For nonoccluded soil exposures, exhaled breath levels declined more rapidly and the studies were terminated after 3 h.
Dermal Exposures in Human Subjects
Study participants.
Nine healthy volunteers participated in the study; medical histories and demographic data including, gender, race, age, body weight, and height are given in Table 2. The studies were conducted under approval from both the University of California at San Francisco human subjects Institutional Review Board (IRB), and the Pacific Northwest National Laboratory IRB in compliance with multiple project assurance number DOE.MPA.PNNL96-2000. Written consent was obtained from each subject prior to participation. Subjects reported no chronic conditions, significant cardiovascular, hepatic, central nervous system, renal, hematological, or gastrointestinal diseases, and no dermatological problems. As TCE is highly lipophilic and the pharmacokinetics of TCE in exhaled breath are sensitive to the amount of body fat, the percent body fat for each subject was determined using a hand-held near-infrared body fat analyzer (Futrex®, Gaithersburg, MD).
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Subjects were provided clean breathing air via a face mask with a two-way nonrebreathing valve to prevent inhalation exposure. The exhaled breath was passed through a heated mixing chamber (1.3-l volume) from which the MS/MS withdrew a sample for analysis approximately every 5 s as described previously (Poet et al., 2000). Excess exhaled air was vented from the mixing chamber to a hood with negligible flow restriction via a large bore hole exit tube. Background exhaled breath measurements were taken for 4 min before each exposure.
PBPK Model
A previously established PBPK model for TCE (Fisher et al., 1998) was modified to include a separate skin compartment to describe the uptake of TCE following dermal exposure as described by Jepson and McDougal (1997). For rat exposures, an equation was also added to describe the amount of chemical in the off-gassing chamber in terms of input from the exhaled breath and removal from the chamber either by rebreathing or to the MS/MS:
![]() | (1) |
where ACH is the amount of TCE in the chamber (mg), QALV is the alveolar ventilation rate (l/h), CEXH is the concentration of TCE in the exhaled breath (mg/L), CCH is the concentration of TCE in the chamber (mg/L), and FCH is the air flow through the chamber (l/h).
The dermal PBPK model incorporated specific descriptions for richly and poorly perfused tissues, fat, liver, lung, and skin (Fig.1). TCE-specific parameters, including partition coefficients and metabolism rates, were taken from the literature (Fisher et al., 1998
; Fisher et al., 1991
; Mattie et al., 1994
). Media (soil and water) partition coefficients were determined using the method of Gargas et al. (1989). Briefly, quadruplicate samples of 1 g soil or 2 ml water were placed in sealed vials and incubated for 2 h with TCE. The amount of TCE in the headspace of vials containing soil or water was compared to empty reference vials containing only TCE. The skin:soil and skin:water partition coefficients were calculated by dividing the skin:air partition coefficient by the soil:air or water:air partition coefficient, respectively. Parameters for the rat and human PBPK models are given in Tables 3 and 4
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For human subjects, the observed delay before the appearance of TCE in exhaled breath was estimated in an iterative manner from the exhaled breath data upon optimization of Kloss and Kp. The delay was estimated for each individual in each exposure by manual adjustment of the time before exposure onset until the best visual fit to experimental exhaled breath concentrations was achieved. The permeability coefficient and Kloss were estimated for each individual animal or human subject by a least-squares fit of the model to the data using the SimusolvTM (Dow Chemical Co.) optimization subroutine. The percent variability explained for the optimized values using a simple well-stirred skin compartment in the PBPK model was always 80%.
Data Analysis
Values are presented as the mean ± SD of n = 34 exposures, with the exception of human soil hand immersion, which was preformed using two volunteers. A Student's t-test or an ANOVA followed by the Student-Newman-Keuls test was used for statistical comparisons of parameters for different exposure conditions, where appropriate, at p < 0.01.
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RESULTS |
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A cyclical pattern in the exhaled breath profile was noted for most rat exposures (see especially Fig. 3). This wave in the exhaled breath levels was likely associated with patterns of activity and was not compensated for by the PBPK model. In the model, the pulmonary rate was held constant over the exposure period, whereas breathing rate would be expected to change with activity level and cause the variations in exhaled breath levels that were observed.
Dermal Exposures of Humans to TCE
Three human volunteers were exposed to TCE for 2 h by placing a hand in 4 liters of water containing actual exposure concentrations ranging from 810 to 1300 mg/l, as measured by GC analysis. A 0.1- to 0.55-h delay in the appearance of TCE in exhaled breath occurred in all human volunteers. The delay in the dermal absorption of chemicals in humans is commonly reported and likely due to stratum corneum loading of the chemical (Roberts et al., 1999). No delay was observed in any of the rat exposures. The delay was estimated for each individual exposure by manual adjustment of the time for the onset of exposure in the PBPK model. Peak exhaled breath TCE levels associated with the whole-hand water exposures ranged from 750 to 1200 ppb for the three volunteers over these exposure concentrations. A representative exhaled breath profile is given in Fig. 6
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The exhaled breath data from each individual volunteer was analyzed using the PBPK model with physiological parameters set for humans (Table 3). The body weight, total body surface area, body surface area exposed, and percent body fat were individually set for each subject, as given in Table 2
. For human exposures, a KP of 0.017 ± 0.003 cm/h provided a good fit to all of the data sets for water exposures. The estimated KP for both occluded and nonoccluded soil exposures averaged over 3-fold lower (0.005 ± 0.002 cm/h) than the KP values estimated for water exposures, a difference equivalent to that observed in the rat exposures (Table 6
).
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DISCUSSION |
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Exposures via the aqueous matrix lead to higher dermal bioavailabilities in both rats and humans. In the studies described here, the KP for TCE in water was 3.5-fold higher than for soil in both rats and humans. A comparison of soil and water patch exposures showed that in humans, six times more TCE was absorbed from water than soil. A similar finding was observed in rats, where twice as much applied TCE was absorbed from water exposures compared to soil.
A patch system that permitted the volatilization of TCE was used for some of the rat exposures, and a loss term was included in the PBPK model to facilitate the assessment of dermal uptake during changing exposure concentrations as a result of evaporation. The volatility of TCE played an important role in the amount available to be absorbed through skin. Preliminary studies in humans confirmed that exhaled breath TCE concentrations were below the MS/MS level of detection with nonoccluded patch exposure conditions due to volatilization of the TCE.
In rats, there was no difference in KP between occluded and nonoccluded soil exposures once volatilization (Kloss) was accounted for in the PBPK model. Although the percentage absorbed in occluded and nonoccluded exposures was the same, TCE was still present in the soil in the occluded exposure system at the end of exposure, but not in the nonoccluded system. According to these results, nonocclusion (allowing the chemical to volatilize) will have little effect on total absorption over short exposure periods, depending on loss of chemical from the exposure site, but could have significant impact over longer exposures. Although the percent absorbed was the same between occluded and nonoccluded soil exposures following these 35 h exposures, longer exposures to nonoccluded soil would not be likely to result in a higher percentage absorbed.
Studies have shown that the dermal absorption of numerous compounds is greater in rats than in humans (Bronaugh, 1998; Jepson and McDougal, 1997
; McDougal et al., 1990
; U.S. EPA, 1992
; Wester and Maibach, 1986
). However, the vast majority of pharmacokinetic studies are conducted using rats, and the impact of multiple factors can be more easily and economically investigated using nonhuman models. In this study, both rat and human dermal exposures were conducted to allow direct comparison of TCE dermal absorption between rats and human subjects and to validate the noninvasive technique for use in human studies. Using the methods described here, the KP for the percutaneous absorption of TCE from a water matrix was 0.31 cm/h in rats and 0.015 cm/h in humans following hand immersion studies. This value for KP in rats is simular to those reported in other rodents. The value estimated for humans is lower than rats and in vitro human skin (Nakai et al., 1999
). Although the human KP was expected to be less than the rat KP, the magnitude of the difference was likely due to site-specific anatomical differences in percutaneous absorption, which are well known (U.S. EPA, 1992
). The stratum corneum is thicker on the hand than on the back (Scheuplein and Blank, 1971
), and Maibach et al. (1971) found that the percutaneous absorption of pesticides varied with site of application in humans.
A number of predictive models have been proposed for estimating the rate of chemical transport across the skin. The U.S. Environmental Protection Agency (U.S. EPA) generic dermal bioavailability equation, based on Fick's Law (U.S. EPA, 1992) is
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where SA is the surface area (cm2), C is the starting concentration (mg), and T is the time (h). Equation 4 is used when total absorption can be determined and steady state is either achieved or assumed not to be a factor (Bogen et al., 1992; Kezic et al., 1997
; Nakai, et al., 1999
; U.S. EPA, 1992
). In the work described here, a PBPK model was used to represent dermal absorption characteristics, describe experimental data, and determine dermal absorption parameters. A PBPK model offers several advantages, including the ability to estimate permeability constants, describe dosimetry, and integrate factors such as chemical volatility, and the ability to incorporate nonsteady-state exposure conditions. The rapid collection of kinetic data such as those obtained using the noninvasive real-time breath analysis system provided the opportunity to assess the uptake, distribution, and elimination phases of dermal absorption in both rats and humans.In exposure conditions where the dose is finite (or a volatile chemical is allowed to evaporate from the exposure matrix), the simple Equation 4 will rarely provide an accurate prediction of Kp. The generic Fick's Law Equation does not take into account the change in the exposure matrix due to volatilization or uptake. At later time points, the generic equation will underpredict KP values as exposure concentration declines due to volatilization or uptake. In contrast, shorter exposures that do not reach steady state would result in estimated KP values that are higher than the actual value. A comparison of KP values estimated using the PBPK model and calculated based on Equation 4 for an occluded rat exposure is shown in Fig. 11
. In the occluded system, there is no evaporative loss of compound, and rats show no delay in the appearance of compound in the exhaled breath. Under these conditions, KP is overpredicted using Equation 4 until peak exhaled breath concentrations are reached (around 1 h), then underpredicted as exhaled breath concentrations decline as the exposure concentration decreases solely due to uptake. Equation 4 results in permeability values that are estimated from absorption averages, whereas the PBPK model-estimated KP is instantaneous. Thus, the PBPK model provides a consistent KP value by considering the loss of TCE from the exposure system by integrating concentration over time (dC/dt).For human water hand immersion exposures, there is essentially an inexhaustible amount of TCE to be absorbed, and thus Equation 4 does not result in a substantial underprediction of KP at later time points (Fig. 12
). Equation 4, however, fails to adjust time for the apparent delay observed in absorption of compounds through human skin. Thus, the average KP determined using Fick's law equation underpredicts for short exposures and slowly rises, whereas the PBPK model estimates a consistent KP value. Using Equation 4, the KP for TCE in humans would be predicted to range from 0.0062 to 0.028 cm/h. This range results in a 4.5-fold difference in the amount of TCE expected to be absorbed.In conclusion, breath analysis and PBPK modeling were used to ascertain percutaneous permeability coefficients for TCE absorption in rats and humans. The breath analysis provided real-time insight into the lag time in absorption of TCE in human subjects and a simple noninvasive way of quantifying absorption. Furthermore, PBPK modeling proved to be a reliable method of determining KP, relative to the generic Fick's law equation, under various exposure conditions. The KP values reported for TCE absorption in hairless guinea pigs (Bogen et al., 1992
) and human skin in vitro (Nakai, et al., 1999
) are in the same range as the rat value determined here, whereas the human in vivo KP was much lower. Differences in the estimation of Kp can have a significant impact on predictions of dermal bioavalability. A PBPK model can be used to correct for nonsteady-state exposure conditions and other factors, including volatility and individual physiological variations, and thus provide an accurate estimation of Kp.
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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: torka.poet{at}pnl.gov.
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