* University of California, San Francisco, Dermatology Department, PO Box 0989, San Francisco, California 94143-0989; University of California,San Francisco, California 94143-0989;
Exponent, Boulder, Colorado 80301;
Integral Consulting, Inc., Mercer Island, Washington; and ¶ Georgia-Pacific Corporation, Atlanta, Georgia 30303-1847
Received September 30, 2003; accepted February 5, 2004
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ABSTRACT |
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Key Words: dermal arsenic absorption; CCA; arsenic exposure; environmental arsenic.
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INTRODUCTION |
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Among several challenges associated with studying exposure to arsenic from environmental media is the large degree of exposure to background levels of arsenic from the diet (Schoof, 1999a,b
; Yost et al., 2004
). Typical daily urinary arsenic excretion for Rhesus monkeys consuming the standard diet of Purina monkey chow is 515 µg As/day. In the Wester et al. (1993)
research, the use of a radiolabeled arsenic source circumvented the confounding effects of concomitant dietary exposures and associated difficulties in data interpretation. For study of environmental samples (e.g., contaminated soils or treated wood), it is not practicable to use a radiolabeled source. Therefore, a new research protocol was designed, incorporating a low-arsenic diet. Urine samples were analyzed using inductively coupled plasma/mass spectrometry, which provided an adequately low detection limit for total arsenic in urine. This alteration in the study design allows for a sensitive evaluation of dermal arsenic absorption from natural environmental media.
The research reported herein describes the use of the Rhesus monkey model to measure the dermal absorption of arsenic from water and from residues collected from the surface of CCA-treated wood. The Rhesus monkey is a relevant animal model for in vivo human percutaneous absorption (Wester and Maibach, 1975, 1989
).
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MATERIALS AND METHODS |
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The iv dose (1060 µg arsenic/monkey) was administered as a solution of sodium arsenate heptahydrate in deionized (DI) water (2120 mg/l arsenic). For the iv dose, each monkey received 0.5 ml of the dosing solution injected into the saphenous vein. The iv dose was given while the monkeys were in their metabolic cages, so the monkeys did not spend any time in the metabolic restraint chairs, as they did with the topical doses.
For the soluble arsenic dose, arsenic was administered in water onto the monkey's skin at an application rate of 5 µl/cm2 evenly applied across 100 cm2 of skin, to achieve a total dermal dose of 1430 µg arsenic (Table 1). The solution was prepared from sodium arsenate heptahydrate in DI water, which was acidified with 1% nitric acid (trace-metal grade). The soluble arsenic dose was designed to match the arsenic dose applied in the CCA-treated wood residue.
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The residue, in the form of a fine particulate, was supplied by the American Chemistry Council (ACC, 2003), and represents the material present on the surface of CCA-treated wood, which an individual might contact during use of, or play on, structures made of treated wood. In collecting the "residue" from the surface of the wood, efforts were made to collect the material on the surface of the wood that might be dislodged during direct human contact with the wood. Specifically, CCA-treated boards consisting of either Southern Yellow Pine or Ponderosa Pine were removed from in-service residential decks in Michigan and Georgia. Deck structures ranged from one to four years of age and had no coatings applied. Aged structures were selected, because they were believed to best represent the material that an individual might contact over time. As described below, recent chemical characterization work indicates that the chemical structure of the arsenic in the residue collected from the surface of decks is indistinguishable from the form of arsenic in newly treated or aged CCA-treated wood structures. A total of 1456 board sections (each 2 ft. long) were collected and shipped to Michigan State University, where the residue was collected as a single composite from multiple boards. The residue was collected by wiping the boards with a soft-bristle test-tube brush while rinsing with DI water. The rinsate and residue collected in this manner were filtered through glass wool, concentrated by rotary evaporation under vacuum at 46°C, and then air dried in a fume hood at 22°C and 65% humidity. The dried residue was irradiated using Cobalt-60 irradiation for 3 h, to eliminate possible microbial contamination of the sample.
Duplicate aliquots of the residue material used in the dermal dosing studies were analyzed for arsenic, chromium, copper, iron, and manganese concentrations, which involved digestion in refluxing nitric acid and analysis by inductively coupled plasma mass spectroscopy (ICP-MS; EPA Method 6010B; U.S. EPA, 1997). This analytical method was used to ensure adequate sensitivity for all metals of interest. As a means of comparing the composition of the CCA residue with the composition of treated wood, samples (a 1-cm2 wood chip from the top 0.2 cm of wood surface) of newly treated wood and a sample of weathered wood from a five-year-old CCA-treated residential deck were subjected to identical digestion and analyses.
For very fine soil (i.e., silty clay), a loading of 5.4 mg/cm2 of skin results in a monolayer (U.S. EPA, 2001b). Because the residue appears similar in particle size distribution to silty clay, and a loading rate of 4 mg/cm2 of the residue provides complete coverage on a flat surface, a loading rate of 4 mg/cm2 was selected for this study. Application of 4 mg/cm2 on 100 cm2 of skin area resulted in a total dose of 1422 µg arsenic (Table 1). The residue was applied as a dry powder, and spread in an even layer across the exposure area.
In Vivo Model.
Female Rhesus monkeys were selected for this research because of their ability to duplicate the biodynamics of percutaneous absorption in humans, and because previous studies of percutaneous arsenic absorption have used this same model. Prior research indicates that percutaneous absorption in the Rhesus monkey is similar to absorption in humans across a variety of chemicals and range of dermal penetration characteristics (Wester and Maibach, 1975). This research indicates that measurements from the monkey are just slightly higher than their counterparts in the human. Results from other species (pig, rat, rabbit) are not nearly as close to the values measured in humans, and indicate that, of the species tested, absorption in the monkey is closest to that in the human.
The monkeys were approximately 20 years old, which is the same approximate age as the monkeys used in the previous dermal arsenic absorption research (Wester et al., 1993). The animals reside within the monkey colony maintained by the University of California, San Francisco, and have not been used for active research for 18 months. Prior to the beginning of the current series of studies, no topical doses had been applied to the skin of these animals for more than four years.
Each topical dose was applied to a pre-measured 100-cm area of abdominal skin of three monkeys. The dosing area was demarked by "masking" the boundaries with a single layer of Tegaderm (a water-vapor-permeable adhesive membrane available from 3M Health Care, St. Paul, MN) and then was dosed by spreading the fluid (5 µl/cm2) or residue (4 mg/cm2) evenly across the 100-cm2 dosing area. The dosing area was then covered with a layer of Tegaderm to ensure that the material remained in contact with the skin. The Tegaderm patch over the dosing area extended well beyond the boundaries of the exposure area. In addition to the Tegaderm patch, the abdomen of each monkey was wrapped with Spandage Instant Stretch Bandage (MEDI-TECH International Corp., Brooklyn, NY) to ensure that the applied dose was kept in direct contact with the skin throughout the dosing period. This bandage is of a web construction; most of the Tegaderm was exposed to the open air for moisture and air exchange. Following application of the topical doses, the monkeys were placed in metabolic restraint chairs for the duration of the eight-h dosing period. The eight-h dosing period was selected to represent an upper bound of time that an individual might remain in contact with residues, and is also the upper limit of time that the monkey can remain in the metabolic restraint chair. During this time, the monkeys had free access to water, but were restricted from touching their abdominal area. Researchers remained in the room and interacted with the monkeys, and the monkeys were hand fed bananas and liquid diet during this stage.
Urine was collected during the 8-h dosing period in a pan under the metabolic chair. After 8 h, the monkeys were removed from the chairs, the Spandage bandage and Tegaderm patch were removed, and the applied doses were removed using a soap and water wash (50/50 v/v, soap and water, followed by water, soap, and two final water washes). The monkeys were then transferred to metabolic cages for continued urine collection over the following seven days.
As with humans, significant exposure to arsenic occurs from the normal diet (Schoof, 1999a,b
; Yost, 2004
). Urinary excretion of total arsenic for Rhesus monkeys on the standard diet of Purina Monkey Chow falls in the range of 5 to 15 µg/daylevels that would obscure accurate detection of the arsenic that might be absorbed following topical application of arsenic. Therefore, the monkeys were provided a low-arsenic diet (Primate Liquidiet from BioServe, Inc.) for seven days prior to each dose. The powdered Liquidiet formulation also was prepared into meal bars, which were provided ad libitum to the monkeys during the research period (seven days prior to dosing through seven days after dosing). The diet was supplemented with pieces of banana and apple, which are both known to be low in total arsenic (Schoof et al., 1999a
). DI water was provided ad libitum. The liquid diet was provided as both liquid and solid forms. Preference was for the solid form. The monkeys maintained their body weight during the study.
The monkey urine samples were preserved with nitric acid (2%) at the time of collection, and shipped to Battelle Pacific Northwest Laboratories in Sequim, Washington, for analysis. At Battelle, the urine samples were acidified with an additional 2% (by volume) of concentrated nitric acid and analyzed for total arsenic by ICP/MS (Method 1638, U.S. EPA, 2002). This method provides a method detection limit (MDL) of approximately 0.1 µg/l arsenic in monkey urine. Quality assurance and quality control (QA/QC) samples included a method blank, duplicates, matrix spikes, and a laboratory control sample at a 5% frequency of analysis.
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RESULTS |
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For the soluble dose, absorption rates were 3.4, 0.62, and 4.4% for the three monkeys in the study (Table 3). Dosing levels used in our earlier research on the dermal absorption of arsenic are compared to those used in this study in Table 1. Despite the nearly seven-fold difference in the dermal loading rate between the two studies, the average absorption rate for the group dosed with soluble arsenic (2.8%) is consistent with results from Wester et al. (1993) (Table 6). These results are consistent with the previous study, wherein absorption rates were relatively consistent (range of 26.4%) despite a five-orders-of-magnitude change in the dose levels (i.e., an applied dose range of 0.000024 to 2.1 µg/cm2). These data strongly support the suggestion that the difference in the measured absorption rates in the Wester et al. (1993)
research reflects experimental variability rather than dose-related differences in absorption (U.S. EPA, 2001b
). This is consistent with our understanding of individual variability in percutaneous absorption in humans and animals (Wester and Maibach, 1991
, 1997
).
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The time profiles for urinary arsenic excretion by each monkey are provided in Figure 2. These charts show a consistent time course for the three monkeys; peak excretion of arsenic occurs within 24 h of the dermal application of the soluble dose, with a rapid return to near-background levels of excretion within 48 to 72 h. Peak 24-h urinary arsenic excretion following the soluble dose ranged up to a maximum value of 41.6 µg. The time profile for arsenic excretion following dermal application of the CCA residue is also consistent across all three monkeys. Figure 2 depicts that, following application of the CCA residue, there is no increase in urinary arsenic excretion, followed out through time.
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DISCUSSION |
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Although the results indicate that the urinary arsenic levels following topical administration of arsenic in CCA residues are not distinguishable from background, the non-zero values for background urinary arsenic excretion, and the variability of the measured background values, impose some limits regarding the sensitivity of the model to detect an absorbed dose. A statistical evaluation using a comparison of means (t-test) for our data indicates that the absorbed dose would need to be in the range of 0.10 to 0.16% of the applied dose, at the dosing levels used in this study, for daily arsenic excretion levels to be detectable above background. Thus, while these data suggest that there may not be any dermal absorption of arsenic from CCA residue (no monkey demonstrated urinary arsenic excretion that was statistically different from background), the uncertainty associated with this research model tells us that dermal absorption of arsenic from CCA residues is at least an order of magnitude lower than absorption of soluble arsenic from solution.
Extensive chemical analyses indicate that the arsenic present in the CCA residue used in this study is structurally and chemically identical to the arsenic present on the surface of newly treated or aged CCA-treated wood (Nico et al., 2003), thus making it an appropriate study substrate for understanding the potential dermal absorption of arsenic following contact with CCA-treated wood. The negligible absorption of arsenic from the CCA residues derives from the fact that this arsenic is chemically bound with other metals (particularly chromium) and ultimately to the wood structure (Bull, 2001
; Nico et al., 2003
). The physico-chemical conditions on the surface of the skin do not result in the liberation of arsenic from the residue, thus precluding absorption. These results indicate that percutaneous absorption of arsenic from environmental media can be significantly different from soluble arsenic or even soluble arsenic mixed with environmental media (Table 6). Therefore, it is not appropriate to apply generic assumptions regarding dermal absorption to these unique matrices, and medium-specific analysis may be required to understand the dermal absorption from them (and potential associated risks). This appears to be true for arsenic, and may be true for other metals that form similarly stable complexes in the environment. The latter point should not be overgeneralized until additional metals have been thoroughly studied.
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ACKNOWLEDGMENTS |
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NOTES |
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1 To whom correspondence should be addressed. Fax: (415) 753-5304. E-mail: rcwgx{at}itsa.ucsf.edu
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REFERENCES |
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