* Geo-Centers, Inc., 2856 G Street (AFRL/HEST), Wright-Patterson Air Force Base, Ohio 454337400;
Mantech, P.O. Box 31009, Dayton, Ohio 45437-0009; and
Operational Toxicology Branch, Air Force Research Laboratory (AFRL/HEST), 2856 G Street, Building 79, Wright-Patterson Air Force Base, Ohio 454337400.
Received October 25, 1999; accepted January 4, 2000
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ABSTRACT |
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Key Words: jet fuel; JP-8; mixture; nonane; decane; undecane; dodecane; tridecane; tetradecane; pentadecane; naphthalene; methyl naphthalene; dimethyl naphthalene; methyl benzene (toluene); dimethyl benzene (xylene); trimethyl benzene; ethyl benzene; diethylene glycol monomethyl ether; dermal absorption; skin penetration; Fischer 344 rat; dermatomed skin; static diffusion cell; flux; permeability coefficient; skin concentration.
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INTRODUCTION |
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Human exposures to JP-8 in vapor, aerosol, and liquid forms all have the potential to be harmful. Reduced volatility of JP-8 compared to JP-4 decreases inhalation exposure because air concentrations are lower. Situations where fuel becomes aerosolized have the greatest potential to be hazardous via inhalation. Both aerosol and liquid forms of JP-8 have the potential to cause local and systemic effects with prolonged or repeated skin contact. JP-8 aerosol has been shown to result from aircraft engine starts at low ambient temperatures. This aerosol, from incomplete combustion, may be inhaled, irritate the eyes, or soak clothing and come into prolonged contact with the skin of ground personnel. Other sources of potential dermal exposure to JP-8 are: (1) splashes during refueling or fuel handling, (2) handling of engine parts that are coated with fuel, (3) contact with sides of fuel tanks during fuel-tank maintenance operations, and (4) contact with fuel leaks on the undersides of aircraft or on ramps.
Jet fuels are composed of hundreds and perhaps thousands of individual hydrocarbon chemicals and their isomers. The composition of JP-8 batches is continuously variable because the specification is based primarily on performance characteristics rather than chemical composition. The primary specifications related to composition are: aromatics are limited to 22% and total sulfur is limited to 0.3% by weight (U.S. Air Force, 1992). JP-8 generally contains about 18% aromatic hydrocarbons, and the rest is aliphatic hydrocarbons (9% C8-C9, 65% C10-C14, and 7% C15-C17) with an average molecular weight of 180 (Committee on Toxicology, 1996
). Table 1
shows the relative proportions of the major hydrocarbon components of one batch of JP-8 (#3509). JP-8 differs from hydrodesulphurized kerosene only by the additives to inhibit icing, corrosion, and static electricity.
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JP-8, when tested on rabbits and guinea pigs, was slightly irritating to the skin and a weak skin sensitizer (Kinkead et al.,1992). JP-8 is more irritating to rats than the JP-4 it replaced (Baker et al., 1999
). In general, related petroleum middle distillates cause chronic irritation and inflammation with repeated applications (Freeman et al., 1990
; Grasso et al., 1988
). Petroleum middle distillates have also been shown to increase the incidence of skin cancer in mice treated for 24 months to a lifetime (Broddle et al., 1996
; Freeman et al., 1993
). Nessel and coworkers (1999) suggest that prolonged irritation is necessary for tumor formation. There is not enough information available on skin absorption to be able to determine the duration and mass of dermal exposure that would cause irritation.
Absorption of any chemical into the skin reflects the potential for irritation. The mechanism of chemical-induced irritation is not completely understood, but the chemical must enter the skin to cause irritation. Chemicals may irritate by a nonspecific structural effect on lipids of the skin or by a direct toxic action on the living cells of the skin (Bowman, 1985). Patrick and coworkers (1985) suggested different mechanisms of irritation for different chemicals in their study because blood flow, vascular permeability, skin thickness, and infiltration of white blood cells showed a differential response depending on the irritant. They also clearly showed that the same amount of irritants in different vehicles caused different degrees of irritation, presumably by changing the concentration of the irritant that is absorbed into the skin.
Chemicals enter into (absorption) and pass through (penetration) the skin based on their chemical characteristics (Dugard and Scott, 1984a; Flynn, 1990
; Scheuplein and Blank, 1971
). Large molecular weight chemicals tend to move more slowly though the skin. Polarity and lipid solubility also have important effects. Charged chemicals do not passively cross membranes, including the skin, very well, and chemicals that have an affinity for lipids can often enter the primary skin barrier, the stratum corneum. The vehicle or the other components that make up a mixture of chemicals can also have a great effect on the rate of penetration. If a chemical is applied to the skin in a vehicle, the relative affinity of the chemical for the skin versus the affinity of the chemical for the vehicle will determine whether the chemical will have a tendency to stay in the vehicle or be driven into the skin by the thermodynamics of the situation (Barry et al., 1985
; Jepson and McDougal, 1997
). Diffusion cells, using isolated skin from laboratory animals or humans, are often used to measure the rate of absorption of chemicals (Bronaugh, 1982). These in vitro tests have many assumptions but can be useful estimates of potential fluxes in human exposure situations if they are carefully accomplished. The purpose of this investigation was to measure the absorption and penetration of JP-8 and its major constituents, with rodent skin, in order to assess the potential for deleterious effects with human exposures.
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MATERIALS AND METHODS |
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Skin and receptor solution samples containing fuel components were analyzed on a Gas Chromatograph with FID. Samples were injected at 140°C with a Tekmar 7000 headspace sampler (Cincinnati OH) onto a 0.53 mm x 30 m SPB-1 column in a Varian 3700 (Palo Alto, CA) GC. The column oven temperature was programmed to hold at 50°C for 5 min and then increase at 5°C/min to 190°C. Results were processed with Perkin-Elmer Nelson integration software (Norwalk CT). Total integrated peak areas after subtraction of controls were used to determine total JP-8 absorption. Detection limit in the skin was 0.05 µg per mg skin and, in the receptor solution, was 0.1 µg per 20 µl sample. For the analysis of individual components, major individual peak identities were confirmed by the retention time check with known standards. Isomers of methyl naphthalene and dimethyl naphthalene were integrated separately and their masses were combined. Diethylene glycol monomethyl ether (DIEGME), the only substituted hydrocarbon, gave 37% of the FID response of JP-8 and the DIEGME response was corrected appropriately.
Identities of components in skin and receptor solution were confirmed on another analytical system. A Hewlett-Packard (Wilmington DE) 5890 gas chromatograph with a 5971 series mass spectrometric detector (MSD) and a 0.20mm x 30m SPB-1 (Supelco, Bellefont PA) column was used. The temperature program was an initial temp of 50°C, increased at 2°C per minute, to 180°C final temperature. Fuel and receptor solution samples were analyzed on the GC/MSD. The identities of components in JP-8 were confirmed on the same gas chromatograph using total ion current with a 0.2 mm x 60 m SPB-1 column with a 60/1 split. The oven temperature was 80°C initially, and was increased at 3°C per minute to 230°C.
Skin Preparation
Male rats (CDF® F-344/CrlBr, Charles River Breeding Laboratories), weighing 267363 g, were sacrificed using CO2 asphyxiation. The back of the animal was closely clipped of fur with Oster® animal clippers (McMinnville, TN) and a #40 blade, taking care not to damage the skin. An Oster® finishing clipper (0.22 mm) was used to carefully remove the fur stubble. A thin cardboard circle the diameter of the outside edge of the diffusion cell was used as a template to mark a circle on the midscapular area of the rat's back with a waterproof marker. The skin from a different rat was used for each diffusion cell. The marked skin containing the future exposure site was gently excised from the back using scissors and blunt dissection. The skin was placed, stratum-corneum side up, on a 5 x 30 cm oak board and dermatomed to 560 micrometers using a Padgett dermatome (Kansas City, MO). This skin thickness was chosen because it was determined to be the average capillary depth in Fischer-344 rats (Grabau et al., 1995). The skin was trimmed with scissors to match the size of the circular mark and placed on the glass receptor chamber that was previously filled with receptor solution.
Diffusion Cell Methods
Static diffusion cells with 4.9 cm2 skin exposure area (Fig. 1) were used to determine flux and skin concentrations of JP-8 and its components. These brown glass cells (Crown Glass Company, Somerville NJ), fit 9 to a countertop console, provided magnetic stirring of the receptor solution and fluid flow to the water jackets (not shown in Fig. 1
) around the receptor cells. These diffusion cells have a 12.5-ml stirred receptor compartment right under the skin with a 7-cm-long sampling port. The receptor compartment was filled with a solution of 6% Volpo 20 (polyethylene glycol-20 oleyl ether, Croda, Mill Hill, PA) in physiological saline. A Volpo/saline receptor solution was chosen to assure that the solubility of JP-8 components in the receptor solution would not be a limiting factor in determining penetration. Solubility of JP-8 in Volpo/saline was 2.05 ± 0.22 mg/ml and 0.033 ± 0.011 mg/ml in saline alone at 37°C. Skin temperature in the cells was controlled at 32°C with a Haake DC3 circulating water bath (Karlsruhe, Germany). The donor chamber was placed on top of the skin and secured by screw clamps. Two milliliters of JP-8 was placed in the donor chamber and the donor cell was sealed with a glass stopper.
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Skin concentrations were sampled in a different set of 8 identically prepared diffusion cells at half-hour intervals for 3.5 h. Control skin concentrations were determined by immediately sampling the skin after applying the JP-8. At the sampling point, each diffusion cell was taken apart, and the skin was rapidly sampled. The skin sampling procedure was as follows: (1) quickly but thoroughly wiping the JP-8 off the skin surface with gauze; (2) placing the skin on a prefrozen polypropylene cutting board; (3) pouring liquid nitrogen over the top of the skin; and (4) taking 4-mm biopsy punches. Three biopsy punches were taken from each skin sample, placed into individual tared headspace sampling vials, and reweighed. Triplicate samples were averaged for each time point. Concentrations of control-skin samples were subtracted from each sample to account for the amount of JP-8 that could not be removed from the surface of the skin. The skin concentration experiment was repeated on 7 different days and the results were pooled.
Flux and Permeability Determinations
Flux (mass/area time) was determined from the slope of the plot of cumulative chemical mass per unit area in the receptor solution over time. Time points before chemical was detected in the receptor solution were not used in the determination of slope. Flux was determined for each diffusion cell and reported with standard deviation. The first two h of the experiment were used to determine the flux for DIEGME because, after 2 h, the flux slowed down due to depletion of the DIEGME in the JP-8. Permeability coefficients (distance/time) from JP-8 were estimated according to Bond and Barry (1988) for each component by dividing individual fluxes by the concentration of the component in JP-8.
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RESULTS |
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JP-8 Skin Penetration
After JP-8 was placed on the skin in the diffusion cell, many hydrocarbon peaks were detected in the receptor solution samples. Most of the peaks could not be identified because they were very small and not resolved from other peaks. All of the peaks that could be identified showed a linear absorption rate except the deicer additive, DIEGME, which decreased with time. The time course of appearance of identified and unidentified hydrocarbon peaks, excluding DIEGME, in the receptor solution during 2-hour experiments, is shown in Figure 2. Some diffusion cells had some detectable peaks at 0.5 h, but a reliable average of total peak area could only be attained at 1 h. The shape of this plot of the hydrocarbon components was linear and the flux calculated from the slope of the linear regression (R2 = 0.97) through the points in Figure 2
is 20.3 micrograms per cm2 per h. Figure 3
appears linear because 2 ml of JP-8 was enough to provide a dose of the JP-8 components that was not diminished by the penetration of the components through the skin, and the receptor solution was not saturated. The maximum solubility of JP-8 in volpo/saline receptor solution is 2.05 ± 0.22 mg/ml at 37°C and the maximum concentration achieved in the receptor solution during any of our experiments was 52.4 µg/ml. In contrast to the refined hydrocarbon components, DIEGME penetration was steep at first and then nearly plateaued, as shown in Figure 3
. The rate of penetration decreased with time because the concentration of DIEGME in the JP-8 was diminished during the 4-hour experiment. Two mls of JP-8 contain 1.28 mg of DIEGME (0.08%) and 0.118 mg were in the receptor solution at the end of the 4-h experiment. For this reason the flux of DIEGME was estimated from the first 2 h of the experiment.
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DISCUSSION |
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In our static diffusion cells the flux of JP-8 was measured to be 20.3 µg/cm2/h based on the sum of the individual (identified and unidentified) GC integration areas (excluding the additive, DIEGME). Penetration through the skin in vivo might result in different overall and individual fluxes as well as different relative permeabilities. The diffusion pathway for in vitro and in vivo studies is necessarily different. With in vitro studies, the chemical has to diffuse all the way through the skin to reach the receptor solution, but with in vivo studies the chemical probably enters the blood stream at the capillaries right below the epidermis. For most chemicals, the primary barrier is thought to be the stratum corneum, and the resistance to diffusion caused by the dermis in an in vitro study is thought to be negligible. The relatively aqueous dermis below the epidermis might, however, provide a barrier to the very lipophilic chemicals that are present in JP-8. Another difference between our results and the human situation is a species difference. Rat skin has been suggested to be 2 to 3 times more permeable than human skin (McDougal, 1990; Vecchia, 1997).
If we can assume our experimental situation is a reasonable, but conservative, approximation of a human exposure situation, we can then use the principles related to Fick's Law to estimate the total amount of chemical that might penetrate in any particular human exposure scenario (Leung and Paustenbach, 1994). If we know the flux (J), the surface area exposed (A), and the exposure time (t), we can estimate the total amount of chemical penetrated through human skin according to:
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It is useful to be able to compare the estimated absorbed dose dermally with existing standards for inhalation or oral exposures. The current interim NRC recommended standard equivalent to an occupational exposure limit (OEL) for JP-8 vapor is 350 mg/m3 (Committee on Toxicology, 1996). Calculations could be made to compare the total amount of chemical absorbed from inhalation with the dermal route (Walker et al., 1996
), but in the case of JP-8, an inhalation exposure would be primarily to the volatile components of the mixture. The dermal exposure would be to volatile and non-volatile components, and so the inhalation comparison would not be valid. Reference doses (RfD's) for the oral route of exposure would be more representative of the mixture composition for dermal exposures. The Total Petroleum Hydrocarbon Criteria Working Group has recommended RfDs for several hydrocarbon fractions found in fuels (Total Petroleum Hydrocarbon Criteria Working Group, 1997
). These RfDs are shown in Table 5
for chemicals that penetrated the skin. Using the Hazard Index approach (U.S. EPA, 1989) to weight the RfD based on proportion of each fraction which penetrates the skin results in a composite RfD for the mixture of 0.091 mg/kg/day (Table 5
). This approach assures that the appropriate chemical fractions are considered in the assessment of the risk to dermal exposure. If we assume that all of the composite RfD would be absorbed, we can compare an internal oral dose with an internal dermal dose estimated from this study. A 70-kg person could orally absorb 6.37 mg/day (0.091 mg/kg/day x 70 kg) every day for a lifetime without appreciable risk. From the absorption rate measured above, it would take about 22 min to absorb 6.37 mg.
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ACKNOWLEDGMENTS |
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NOTES |
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REFERENCES |
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