* Battelle, Pacific Northwest Division, PO Box 999, Richland, Washington 99352; and
Department of Dermatology, PO Box 0989, University of California, San Francisco, California 94143
Received May 17, 1999; accepted November 1, 1999
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ABSTRACT |
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Key Words: methyl chloroform; percutaneous absorption; permeability coefficients; physiologically based pharmacokinetic model.
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INTRODUCTION |
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The extent to which a chemical is absorbed through the skin can vary greatly depending on multiple factors, including: chemical properties and concentration at the skin surface, temperature, location and surface area exposed, duration of exposure, exposure matrix, skin hydration, and whether or not the exposures are occluded (U.S. EPA, 1992). Quantitating the dermal penetration rate, or flux, is an integral part of dermal exposure risk assessment. Generally, the skin is assumed to act like a simple membrane, and Fick's law is often used to describe transdermal flux at steady state:
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In this study, methods were developed to study the percutaneous absorption of volatile chemicals in humans. Our approach employed exhaled-breath elimination profiles coupled with an established PBPK model to assess the bioavailability of methyl chloroform (1,1,1-trichloroethane, MC) following dermal exposures in aqueous or soil matrices. MC was chosen as a pilot chemical to develop the exposure methodology for the following reasons: (1) MC is not extensively metabolized (Schumann et al., 1982), and thus the detection of parent compound is not complicated by competing liver metabolism, and (2) a validated PBPK model was already available (Reitz et al., 1988
). Methods were also developed to assess the percutaneous absorption of MC in rats to directly compare the percutaneous absorption between humans and rats, a species that is commonly used in toxicity testing and dermal absorption studies. To conduct these studies under realistic environmental exposure conditions, a non-occluded patch system was developed that allowed for volatilization of MC from the soil without contamination of inhaled or exhaled breath.
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MATERIALS AND METHODS |
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HPLC grade (99.9%) 1,1,1-trichloroethane (methyl chloroform, CAS #71556) was obtained from Sigma Chemical Co. (St. Louis, MO.). All other chemicals were reagent grade or better and were obtained from Sigma Chemical Co.
Human Subjects
Six healthy Caucasian male volunteers participated in the study; medical histories and demographic data, including age, body weight, and height, are given in Table 1. The studies were conducted under approval from both the Pacific Northwest National Laboratory human subjects Institutional Review Board (IRB), in compliance with multiple project assurance number DOE.MPA.PNNL96-2000, and the University of California San Francisco IRB. Formal, written consent was obtained from all subjects prior to their participation. Subjects reported no chronic conditions, significant cardiovascular, hepatic, central nervous system, renal, hematological, or gastrointestinal diseases, and no dermatological problems. Since MC is highly lipophilic, MC pharmacokinetics are sensitive to the amount of body fat. Therefore, the percen body fat for each subject was determined using a hand-held, near-infrared body-fat analyzer (Futrex®, Gaithersburg, MD).
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Rats were exposed to MC in 600 mg of soil at target concentrations ranging from 0.050.5% MC. The soil sample used in these studies was collected in California (Yolo County). Soil was prepared by passing through a 40-mesh sieve and retaining on an 80-mesh sieve. The soil consisted of 30% sand, 18% clay, and 52% silt, with an organic content of 1.3%, and a pH of 6.8. A ring of DuoDerm® (Bristol-Myers Squibb, Princeton, NJ) was attached to theclipped area on the back of the rats using a cyanoacrylate adhesive. The topical dose of MC-spiked soil was applied to the 8-cm2 area bounded by the DuoDerm® and covered with Bioclusive® transparent dressing (Johnson & Johnson, Arlington, TX). The transparent dressing allowed free passage of moisture, thereby mimicking realistic exposure scenarios. To prevent the rats from breathing the MC volatilized from the patch, a weighboat with holes drilled in the center was placed over the transparent dressing to permit the movement of air, and was covered with a muslin patch containing activated charcoal. The entire patch system was secured with self-adherent wrap (Fig. 1). Preliminary studies confirmed that the patch system efficiently trapped volatilized MC and prevented contamination of the chamber air. An 8-cm2-patch area, representing approximately 3% of the total skin surface area, was chosen to mimic limited exposure situations.
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To quantitate total absorbed dose, the amount of MC in the original exposure media (soil or water), the amount remaining at the end of the exposure, and the amount volatilized to the charcoal patch were analyzed. Headspace gas chromatography was used for the media and patch components. Aliquots of dosing solution (100 µl water or 100 µg of soil) and the remaining solution after exposure were placed in 20-ml headspace vials along with 0.1% trichloroethylene used as an internal standard, sealed with Teflon-lined septa, and heated at 80°C for 60 min. MC was analyzed using a Headspace Autosampler (Perkin-Elmer 40XL) linked to a Hewlett-Packard 5890 Series II GC (Avondale, PA). A Restek Rtx-Volatiles column was used (30m x 0.32mm x 1.5 µm) cross-bonded with phenylmethyl polysiloxane. The oven temperature was set at 90°C and the injection and FID detector temperatures were set at 100°C. Helium was the carrier gas at 8 psi. Charcoal from the non-occluded patch system was extracted using toluene and MC concentrations measured using similar GC conditions, except for 2 µl of toluene that was injected (splitless) onto the GC. The retention times of MC and trichloroethylene internal standard were approximately 3 and 3.7 min, respectively.
Human Exposure Conditions
Each human volunteer immersed their left hand in a container of either water (4 L) at a target concentration of 0.1%, or soil (4 kg) with a target concentration of 0.75% for 4 min after beginning to breathe into the MS/MS system. The containers were prepared in a separate room and were covered with plastic wrap prior to use. One volunteer immersed his hand in 4 L of water without MC for 2 h prior to exposure to determine the effect of pre-hydration on dermal bioavailability. Samples (water or soil) were collected from the container every 0.5 h and measured by GC headspace analysis as before, to determine the concentration of MC remaining in the exposure media over time.
Subjects were provided clean breathing air via a facemask with a 2-way non-rebreathing valve so as to eliminate inhalation exposure. The exhaled breath was passed through a heated mixing chamber (1.3 L volume) from which the MS drew a sample for analysis approximately every 5 sec. Excess exhaled air was vented from the mixing chamber to a hood with negligible flow restriction, via a large borehole exit tube.
PBPK Model
A previously established PBPK model for MC (Reitz et al., 1988) was modified to include a separate skin compartment to describe the uptake of MC following dermal exposure as described by Jepson and McDougal (1997). In addition, equations were added to describe the off-gassing chamber (for rat exposures), according to Thrall and Kenny (1996) and Gargas (1990). Equation 2 describes the amount of chemical in the off-gassing chamber in terms of input from the exhaled breath and removal from the chamber, either by re-breathing or to the MS/MS,
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The dermal PBPK model incorporated specific descriptions for richly and slowly perfused tissues, fat, liver, lung, and skin (Fig. 2). MC-specific parameters, including blood:tissue and skin:air partition coefficients and metabolism rates were taken from the literature (Mattie et al. 1994
; Reitz et al., 1988
). Media (soil and water) partition coefficients were determined using the method of Gargas et al. (1989). Briefly, quadruplicate samples of 1 g of soil or 2 ml of water were placed in sealed vials and incubated for 2 h prior to sampling the headspace for MC. The amount of MC in the headspace of vials containing soil or water was compared to empty reference vials. The soil:skin and water:skin partition coefficients were calculated by dividing the soil:air or water:air partition coefficient by the skin:air partition coefficient, respectively. Parameters for the rat and human PBPK models are given in Tables 2 and 3
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For human subjects, the observed delay before the appearance of MC in exhaled breath was estimated in an iterative manner from the exhaled breath data upon optimization of Kloss and Kp. Kloss and Kp were estimated for each individual animal or human subject using the 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 >86%.
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RESULTS |
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Human Subjects
MC was found in the exhaled breath of human subjects with peak levels ranging from approximately 1100 ppb to 1500 ppb following exposures to 0.0850.14% MC in water (Fig. 6). The peak exhaled breath concentration from three male subjects exposed to 0.610.77% MC in soil was more variable and ranged from 8002000 ppb (Fig. 7
). A lag time in the appearance of MC in exhaled breath occurred in all subjects and ranged from 0.31.3 h. The delay was estimated for each individual exposure by manual adjustment of the time before the PBPK model initiated the exposure. The average delay time before appearance of MC was similar for both soil (0.80 ± 0.40 h) and water (0.83 ± 0.43 h) exposures (Table 5
). The termination of exposure was artificially adjusted to account for the lag time.
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DISCUSSION |
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The octanol:water partition coefficient is often used to estimate Kp (Guy and Potts, 1993; U.S. EPA, 1992
). The estimated Kp for MC based on the octanol:water partition coefficient is 0.018 cm/h (U.S. EPA, 1992
), almost 3 times greater than the value predicted here for humans. The uncertainty in Kp predicted from the octanol:water partition coefficient is expected to be within an order of magnitude (U.S. EPA, 1992
). Reifenrath et al. (1984) reported that penetration rates were more directly related to octanol:water partition coefficients for water-soluble compounds than for lipid soluble compounds in human skin grafted nude mice.
In this study, a simple, well-stirred skin compartment was used in the PBPK model to describe the dermal flux. Previous investigators have used models that included dual skin compartments to represent the stratum corneum and epidermis (Chinery and Gleason, 1993; Shatkin and Brown, 1991
). However, the single compartment has the advantage of having fewer variables that need to be independently determined. Furthermore, models using a single skin compartment have been found to adequately describe the dermal absorption of similar test chemicals, such as; chloroform, dibromomethane, and bromochloromethane (Corley et al., 1999; Jepson and McDougal 1997
). The simple, single-compartment model used here sufficiently described the exhaled breath data after a delay function was included to describe each individual human exposure.
The estimated permeability coefficients in rats were higher than those estimated in humans. This is consistent with previous studies that showed that animal skin is more permeable than human skin (Bronaugh, 1998; Jepson and McDougal, 1997
; McDougal et al., 1990
). Studies have shown that the percentage of percutaneous absorption can range from 1- to 20-fold higher in rats compared to humans (U.S. EPA, 1992
; Bartnik et al., 1987
; Bronaugh, 1998
). In the present study, the human hand-water immersion exposure is essentially an occluded exposure and most closely mimics the rat occluded-water patch exposure. Under these exposure conditions, the rat Kp of 0.25 cm/h is roughly 40 x higher than observed for the human (Kp 0.0063 cm/h). Additionally, studies in human volunteers with 4 dermal exposure patches similar to the single patch used with rats, containing either water or soil matrices, resulted in no quantifiable MC in the exhaled breath. This is likely due to the small percent surface of area exposed in humans and the rapid volatilization of MC. However, these differences may also reflect variations in the permeability due to exposure site-specific factors.
In rats, the permeability coefficients were constant over the exposure range (0.040.6% MC) in non-occluded soil. In both rats and humans, the permeability coefficients for exposures to MC in water were greater than those estimated for exposures to MC in soil. Human volunteer subject #1 participated in both the water and soil exposure trials, and provided an opportunity to compare soil versus water matrix effects. For MC dermal uptake from a soil matrix, the KP for this volunteer was 0.002 cm/h whereas the KP for the aqueous matrix was 0.0064 cm/h, indicating that exposure via the aqueous matrix leads to a higher bioavailability. The apparent Kps that best fit the data for all 3 subjects exposed to MC in soil were approximately 4 times lower than predicted based on the absorption for MC in water.
The hand of one male subject was pre-hydrated by soaking in clean water for 2 h prior to a 1-h exposure to aqueous MC (subject #4). In this subject, peak exhaled-breath concentrations were more than 7 times greater than peak concentrations observed in the other subjects (Fig. 8). Pre-hydration of the single subject resulted in a higher estimated Kp, shorter lag time, and greater percentage absorbed than expected based on data from non-prehydrated subjects (Table 5
). The high Kp in this subject is consistent with the assumption that hydration results in an increased permeability to many compounds (U.S. EPA, 1992
).
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The percent of dose absorbed was highly dependent on hydration, length of exposure, and whether the exposure was occluded or not. Thus, a simple percent absorbed dose, or "bioavailability factor" is experiment- and exposure-specific and is difficult to generalize for use in risk assessments. These data demonstrate that appropriately defined Kps coupled with PBPK modeling should be used when available for dermal risk assessment.
For example, the dermal PBPK model for MC was used to simulate the dermal bioavailability for adults and children exposed to MC-contaminated water and soil in a residential setting, using EPA standard default factors for adult and child body weights, surface area exposed, soil burden (U.S. EPA, 1992), and parameters determined in this study (KP and Kloss) (Table 6
). For these simulations, the media MC concentration was fixed at 0.01%. To demonstrate the impact of the exposure matrix (soil versus water) and occlusion on the total absorbed dose, simulations were limited to 2-h and 24-h exposures.
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ACKNOWLEDGMENTS |
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NOTES |
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1 To whom correspondence should be addressed at Chemical Dosimetry, Battelle, Pacific Northwest Division, PO Box 999 MSIN P7-59, Richland, WA 99352. Fax: (509) 3769064. E-mail: torka.poet{at}pnl.gov.
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