metabolism of phenanthrene in the hairless guinea pig: Comparison of in vitro and in vivo results

FUNDAMENTAL

AND

APPLIED

TOXICOLOGY

16, 5 17-524

( 199 1)

Percutaneous Absorption/Metabolism of Phenanthrene in the Hairless Guinea Pig: Comparison of in Vitro and in Vivo Results K . M . E . NG,* I. CHU,*~’ R. L. BRONAUGH,~

C. A. FRANKLIN,*

AND D. A. SOMERS*

*Environmental and Occupational Toxicology Division, Bureau of Chemical Hazards, Environmental Health Directorate, Ottawa, Ontario, Canada; and TDermal and Ocular Toxicology Branch, Food and Drug Administration, Washington, D.C.

Received June 15, 1990: accepted December 13, 1990 Percutaneous Absorption/Metabolism of Phenanthrene in the Hairless Guinea Pig: Comparison of in Vitro and in Viva Results. NG, K. M. E., CHU, I., BRONAUGH, R. L., FRANKLIN, C. A., AND &3MERS, D. A. (1991). Fundam. Appl. Toxicol. 16, 517-524. The in vitro and in vivo percutaneous absorption/metabolism of phenanthrene was investigated in hairless guinea pigs. Flowthrough diffusion cells and Hepes-buffered Hanks’ balanced salt solution (HHBSS) as receptor fluid were used in the in vitro system. When phenanthrene was applied to excised guinea pig skin mounted on the cells at dose levels of 6.6 and 15.2 rcg/cm2, 89.7 and 79.1% of the administered doses were respectively absorbed into the skin and receptor fluids during a 24-hr perfusion period. These results are consistent with the in vivo data which showed approximately 80% absorption over the same period of time. Phenanthrene was metabolized in vitro into phenanthrene 9, lodihydrodiol, 3,4dihydrodiol, 1,2-dihydrodiol, and traces of hydroxy phenanthrenes. Of the materials absorbed in vitro, 92% was the parent compound and 7% the dihydrodiol metabolites. When a nonviable in vitro system was used, 68% of the applied 15.2 &/cm2 dose was absorbed. Data from the present study demonstrate that the in vitro system is a good model for predicting in vivo percutaneous absorption of phenanthrene, and that penetration of phenanthrene through the skin is controlled more by the passive rate of diffusion than by metabolism.

Dermal penetration is the primary route of occupational exposure to many industrial chemicals and pesticides. The amount absorbed is dependent on many factors including site of application, animal species, temperature and hydration of the skin, and the solubility of the chemical. Recent emphasis on quantifying percutaneous absorption has arisen because of the need to better estimate systemic dose following exposure. An estimate of the systemic dose can be made by multiplying the administered dose with the percentage of absorption. This systemic dose can then be compared to the estimated systemic dose following oral exposure of animals for risk assessment ’ To whom requests for reprints should be addressed.

purposes. Although this approach does not take into account the pharmacokinetics of dermal absorption versus oral absorption, it does facilitate risk assessment processes for regulatory purposes. There are two general approaches to estimating percutaneous absorption. One approach uses in vivo methods in a variety of animal species, including humans. Many chemicals have been studied in vivo in rats, rabbits, pigs, monkeys, and man, Penetration is determined by measurements of excreted radiolabeled chemicals and/or residues remaining in the tissues. The other approach uses animal or human skin preparations in static (Franz, 1975) or flow-through diffusion (Bronaugh and Stewart, 1985) cells. From a 517

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regulatory perspective, there are many advantages of using an in vitro method that can accurately predict in vivo percutaneous absorption. One distinct advantage is the capability of testing compounds on human skin, thus avoiding the problems inherent in animal to human extrapolation. Other benefits include cost effectiveness, reduced use of animals, and rapidity of testing. Despite these advantages, there are numerous methodological and scientific issues which must be resolved for in vitro techniques before the method can be used for regulatory purposes (Franklin et al., 1989). In brief, areas requiring further study and/or standardization of methods include refinement of skin preparation techniques, choice of appropriate receptor fluid and in vitro cells, optimal perfusion times, skin binding, and metabolic activity. Comparative in vivolin vitro studies suggest reasonable concordance between in vitro and in vivo systems for hydrophilic substances. Lipophilic compounds have not been extensively studied but discrepant data are not uncommon. Accordingly, we have instituted a research program for in vitro/in vivo validation work with chemicals which span the range of lipophilicity. Phenanthrene was selected as the first test compound in our in vitro/in vivo validation program. The testing compound and the system were selected based on the following considerations. Phenanthrene and its alkylated derivatives account for more than 26% w/w of coal liquefaction products and constitute a potential source of occupational exposure (Chu et al., 1988). Dermal absorption studies on this compound would not only provide comparative data for in viva/in vitro validation but also provide absorption information that is required for the risk assessment of coal liquefaction products. Hairless guinea pig skin can be easily dermatomed to thin layers of 250 pm without leaving holes of hair shafts which would result in the leakage of the test compound across the skin preparation, and the data generated from this species of animal can be compared with those of other compounds

ET

AL

obtained from the same laboratory (Collier al., 1989). MATERIALS

AND

et

METHODS

Chemicals. Phenanthrene and 9-hydroxyphenanthrene. purchased from Aldrich Chemical (Milwaukee, WI), were purified by recrystallization to a purity of greater than 99% as confirmed by TLC and GLC techniques. [9%]Phenanthrene (10.4 mCi/mmol) was procured from Sigma Chemical (St. Louis, MO) and had a stated radiochemical purity of greater than 99%. The radiochemical purity was confirmed by GLC and radio-TLC. Phenanthrene 9, IO-dihydrodiol was synthesized by reduction of the corresponding quinone using a method previously described (Booth et al., 1950). Hepes-buffered Hanks’ balanced salt solution (HHBSS) was obtained from Flow Laboratories (McLean, VA). Other chemicals and solvents were of reagent grade and procured commercially. Instrumental methods. GC/MS analysis was performed in a Finnigan mass spectrometer (Model 4000 operated at 70eV electron impact) equipped with an SPB-5 capillary column (0.32 mm i.d. X 30 m. Supelco, Bellefonte, PA) and an INCOS data system. The initial oven temperature was held at 50°C for 0. I min and was increased at a rate of lS”C/min to 100°C followed by S”C/min to 250°C. Helium was used as a carrier gas at a head pressure of 14 psi. Measurements of radioactivity were made with a Searle Mark III iiquid scintillation counter, and quench was corrected by use of external standards. Radioactivity on the TLC plates was determined in a Berthold Beta camera (Model LB 292) or a Bioscan System 200 (Bioscan, Washington, DC). In vitro experiments. Female hairless guinea pigs [strain Crl:IAF/HA (hr/hr) BR] were purchased from Charles River Laboratories (Wilmington, MA) and were used at 20-30 weeks of age. Animals were housed at the FDA animal care unit with free access to food (Purina Chow) and water. A 12-hr alternated light and dark cycle was maintained. Guinea pigs were caged singly in plastic cages and supplied with standard heat-treated hardwood bedding. Animals were euthanized by CO* asphyxiation. The skin was quickly removed, cleaned with 1% detergent solution. and immediately prepared for use in penetration/metabolism studies. A 250~pm layer of skin was obtained using a Padgett dermatome (Electra-Dermatome Model B, Padgett Dennatome Division, Kansas City, MO), punched into circles of 1.7 cm’ in area and mounted into Teflon flow-through diffusion cells with an exposed area of 0.64 cm2 as previously described (Bronaugh and Stewart, 1985). The receptor fluid was a Hepes-buffered (25 mM) Hanks’ balanced salt solution containing gentamicin sulfate (50 mgjI) prepared as previously described (Collier et ai., 1989). In most experiments, 4% bovine serum albumin (BSA) was added to increase the solubility of phenanthrene. For control experiments using nonviable skin, the receptor fluid

ABSORPTION/METABOLISM was distilled water (adjusted to pH 7.4) with 4% BSA and gentamicin sulfate. The receptor solutions were filtered through sterile Nalgene 0.2~pm-pore cellulose acetate filters (Nalge Co., Rochester, NY) and aerated with 99% O2 throughout the experiments. The diffusion cells were heated to maintain a skin surface temperature of 32°C. [“ClPhenanthrene was applied to the stratum corneum surface in 10 ~1 of acetone vehicle at doses of 6.6 and 15.2 &cm2. Perfusion was continued at a rate of 1.5 ml/hr with fractions collected at 6-hr intervals for 24 hr. At the end of the 24-hr period, skin sample surfaceswere washed three times with 0.3 ml of a 1% aqueous detergent solution and three times with water to remove unabsorbed material. The exposed skin was homogenized with a Polytron homogenizer in a 50:50 phosphate buffer (25 mM, pH 7.4): methanol solution and the homogenates were extracted twice with 2 vol of ethyl acetate. The extracts were dried with 10 g anhydrous sodium sulfate, reduced in volume under nitrogen and spotted on Whatman silica gel TLC plates. The samples were chromatographed using 0.1% triethylamine in cyclohexane-ethyl acetate (1: 1). To determine total radioactivity in receptor fluids, a 100-~1 aliquot was removed from each fraction of receptor fluid collected and counted for radioactivity. Each fraction was then extracted twice with 20 ml of ethyl acetate, dried, reduced in volume, spotted on TLC plates, and determined using a Bioscan System 200 imaging plate scanner. Idenrification g/ metabolites. The radioactive spots as detected by a Beta camera or Bioscan scanner were removed from the plates and extracted with ethyl acetate. After the solvent was evaporated, the material that remained was silylated with a mixture of equal amounts of Tri-Sil-Z and BSTFA (Pierce, Rockford, IL) and analyzed by GC/MS. Synthesized standards were analyzed in a similar manner to provide reference GC/MS data. In vivo experiments. The in vivo method developed by Feldman and Maibach (1969) was used in the present study. The dorsal skin of female hairless guinea pigs was washed with a 1% aqueous detergent solution, rinsed with water, and dried. Single doses of [“‘Clphenanthrene (25 fig) in 50 ~1 of acetone were applied to a 4-cm* area of the dorsal region of each of five guinea pigs. Nonocclusive foam cups, covered with gauze, were placed around the 4-cm2 area and wrapped with Vetrap (3M, Minneapolis, MN) tape in order to prevent animals from ingesting the dosed chemical. The animals were kept in glassmetabolism cages(Jencons, England) with free accessto food and water. Collection of urine and feces was made for each animal at 6 and 12 hr on the first day then at daily intervals for 5 days post-treatment. The skin surface was washed with soap and water at 24 hr to remove unabsorbed material. Radioactivity in the urine and feceswere determined using a liquid scintillation counter. To correct for incomplete excretion a group of five guinea pigs was given intramuscularly single doses of [14C]phenanthrene (25 pg) in 100

OF PHENANTHRENE

519

~1 of an aqueous vehicle (Emulphoti/ethanol/normal saline: I / l/8). The animals were similarly kept in metabolism cages for collection and determination of 14Ccontent in excreted materials. Urine samples (1 ml) were mixed with 10 ml Dimulume 30 (Packards, Downers Grove, IL) and counted for the radioactive content. A I-g fecal sample was thoroughly mixed with 30% aqueous isopropyl alcohol (IO ml) to produce a 10% homogenate, of which 1 ml was digested with Soluene-350 (0.5 ml, Packards) at 50°C for 2 hr, and decolorized with 30% hydrogen peroxide (0.5 ml). The decolorized sample was mixed with 10 ml Dimulume and its radioactivity was determined in a liquid scintillation counter. The equation (Feldman and Maibach, 1969) used to determine percutaneous absorption is percentage of dose absorbed = total urinary radioactivitv following dermal administration x looo/ 0. total urinarv radioactivitv followingim administ&ion

RESULTS Permeation of Phenanthrene Flow-Through Cells

in the in Vitro

Percutaneous penetration of phenanthrene was assessed using an HHBSS receptor fluid with or without BSA (Table 1). In the absence of BSA, the percentages of the applied dose found in the receptor fluid for the low-dose group (6.6 &cm’) were 2.4% at 6 hr and 8.7% at 24 hr. In the presence of BSA, the first 6-hr receptor fluid fraction contained 38.9% of the applied dose representing a 15-fold increase over the amount absorbed during the same period without using BSA. When BSA was utilized, 78% of the applied dose was found in the receptor fluid in 24 hr. The doses retained in the skin were 11.8 and 59.7% for the groups with and without BSA, respectively. Absorption was also studied in the high-dose group (15.2 wg/cm’) using only HHBSS and BSA as perfusate (Table 1). The amount of absorption measured every 6 hr was comparable with the data from the corresponding low-dosed group 2 Emulphor was a surfactant obtained from Domtar, Montreal.

520

NC ET AL. TABLE I PERMEATIONOFPHENANTHRENEININ

VITRO FLOW-THROUGH

6.6 pg Phenanthrene/cm2

Receptor fluid 6 hr 12 hr 18 hr 24 hr Dose remaining in skin Total dose Skin wash Total dose recovered

HHBSS

HHBSS + BSAb

2.4 + 0.5 6.4 + 1.2 8.3 i 1.3 8.7 -+ 2.0 59.7 f 2.0 68.5 f 2.0 10.3 + 2.0 78.8 k 2.1

38.9 AZ8.1 63.6 + 8.8 73.4 f 8.5 78.0 + 8.6 11.8 + 0.1 89.7 + 8.7 3.5 * 1.2 93.3 f 8.4

CELLS"

15.2 pg Phenanthrene/cm’ Water + BSA 20.8 41.4 52.1 57.4 8.8 66.2 2.2 68.4

f 0.5 f 0.9 -+ 2.5 f 2.7 * 1.0 f 3.3 2 0.2 + 3.5

HHBSS + BSA 33.7 + 58.1 f 67.2 t 71.3 + 7.8 + 79.1 + 1.6 f 80.7 f

7.3 7.7 6.5 5.7 2.8 3.6 0.4 3.2

’ Data are means + SEM and represent percentage of applied dose obtained from three to four determinations. b HHBSS, Hepes-buffered Hanks’ balanced salt solution; BSA, bovine serum albumin.

although the percentages of absorption at each time period were slightly lower (Table 1). Data also indicated that most of the dose penetrated through the skin preparation within the first 12 hr postadministration. The parent compound and metabolites contained in the 6-hr fraction of HHBSS + 4% BSA receptor fluid were determined (Fig. 1). Of the dose absorbed, only a small fraction was due to the metabolites. The ratios of the parent compound to metabolites at each time period were approximately 10: 1 for the lowdose group and 20: 1 for the high-dose group. To ascertain whether the observed metabolites were due to enzymatic metabolism by the dermal tissues rather than nonenzymatic reactions or artifacts, two receptor fluid systems with varying metabolism capacity were used, and the metabolites were quantitated in each system (Fig. 2). These systems were: (1) distilled water + BSA and (2) HHBSS + BSA. The use of distilled water as perfusion fluid has previously been shown to result in a loss of skin viability during the first 6 hr of perfusion because it deprived the skin of essential minerals and nutrients required for the maintenance of metabolic activity (Collier et al., 1989). Enzymatic formation of metabolites was twofold greater with HHBSS at the 12-,

18-, and 24-hr periods, although during the 6hr period both systems resulted in approximately the same amounts of metabolites. However, the percentages of the absorbed dose at each of the time points were comparable for both systems (Table 1). Identification

of Phenanthrene

Metabolites

Metabolites were determined in the 6.6 pg/ cm2 group. Analysis of receptor fluid fractions collected at 6-hr intervals showed that approximately 8% of phenanthrene was metabolized in the in vitro preparations to phenanthrene 9, IO-dihydrodiol, phenanthrene 1,2dihydrodiol, phenanthrene 3,4-dihydrodiol, and hydroxyphenanthrenes (Table 2). The chemical structures were assigned by comparing the chromatographic and mass spectrometric data of the isolated metabolites with pertinent data derived from reference standards and/or from the literature. Identification of phenanthrene and phenanthrene 9,10-dihydrodiol was made by comparing their TLC Rf values, GC retention times, and mass spectral fragmentation patterns with the standards (Table 2). The unknowns and reference standards had identical

ABSORPTION/METABOLlSM

521

OF PHENANTHRENE

Holder, 1978; Boyland and Sims, 1962) were also detected, but occurred in very small quantities that precluded the assignment of the position of hydroxylation. In Vivo Absorption of Phenanthrene

_ ._ 0.0

6.0

12.0 TIME (HOUR]

18.0

24.0

FIG. 1. In vitro penetration of phenanthrene and metabohtes in hairless guinea pig skin. Results are expressed as mean f SEM (nr = 3) of the cumulative percentage of applied dose found in the receptor fluids. Phenanthrene at 15.2 &cm3 (A); metabohtes at 15.2 &g/cm2 (A); phenanthrene at 6.6 &cm* (0); metabolites at 6.6 pg/cm* (0). The perfusion fluid for both dose groups is HHBSS with BSA.

chemical characteristics. Assignment of chemical structures for metabolites 1,Zdihydrodiol and 3,4-dihydrodiol was based on the observations described below. First, the Rf values of the two metabolites on TLC plates were comparable to data reported for the two isomers in the literature (Chaturapit and Holder, 1978). Second, the molecuIar ions of both metabolites had m/z values of 356 and fragments which were characteristic of trimethylsilyl derivatives of phenanthrene dihydrodiol, but the 1,Zisomer had retention times significantly longer than that of the 3,4isomer found in the present study as well as the one reported by Goksoyr et al. (1986). Thus the GC peak with a longer retention time was assigned as the 1,2-isomer. All three dihydrodiols are tram isomers as the cleavage of epoxide intermediates by a water molecule yields the sterically unhindered trans-configuration. When the radioactive spots corresponding to the three diols were isolated and counted by a liquid scintillation counter, the quantity of each isomer was found as follows: 9, IO-isomer, 3.5%; 1,2-isomer, 2.0%; and 3,4isomer 1.4% (Table 2). Traces of radioactive material corresponding to the reported Rrvalues for hydroxy derivatives (Chaturapit and

In vivo data are given in Fig. 3. Absorption was expressed as cumulative dose absorbed and excreted in urine following dermal application. Results were corrected for incomplete excretion using the urinary data obtained from guinea pigs dosed intramuscularly with phenanthrene at the same dose levels. Approximately 80% of the administered dose was absorbed through the skin. Of the dose absorbed, 25% was found in 6-hr samples, and 69% in 12-hr samples. The amount of the dose recovered in the 24-hr skin wash varied from 0.13 to 1.04% of the administered dose. No significant absorption was observed after 24 hr. DISCUSSION For lipophilic compounds such as phenanthrene, in vitro absorption rates cannot be accurately measured from aqueous receptor fluid data, since most of the absorbed material

5.01

0.0 0.0

6.0

12.0 18.0 TIME (HOUR)

24.0

FIG. 2. Dermal penetration of phenanthrene metabolites. Data denote mean i SEM of the cumulative percentage of dose determined in receptor fluids from two individual samples: (1) bovine serum albumin + water as receptor fluid (0); (2) bovine serum albumin f Hepes-buffered Hanks’ salt solution (0).

522

NG ET AL.

remains in the skin. Use of a thin dermatomed membrane and a more lipophilic receptor fluid can overcome this problem. A nonionic surfactant such as PEG 20 oleyl ether in the receptor fluid can facilitate partitioning from skin (Bronaugh and Stewart, 1984) but viability of the skin is lost. A Hepes-buffered Hanks’ balanced salt solution has been demonstrated to maintain the viability of skin for 24 hr in flow-through diffusion cells (Collier et al., 1989). Addition of serum protein (either 10% fetal bovine serum or 4% bovine serum albumin) to a physiological buffer was shown to simulate in vivo absorption when the amount of chemical in the receptor and skin is totaled (Bronaugh et al., 1989a,b). For phenanthrene, the addition of BSA to HHBSS increased the partitioning from skin into the 24-hr receptor fluid ninefold (Table I), enabling accurate rates of absorption to be determined. Phenanthrene was readily absorbed through hairless guinea pig skin by both in vitro and in vivo methods (Table I, Fig. 3).

0.0 y 0.0

I 1.0

2.0

3.0 4.0 TINE (DAY)

5.0

6.0

FIG. 3. In vivo percutaneous absorption of phenanthrene. Data are expressedas mean f SEM of cumulative excretion data in urine of five animals, dosed topically with 25 pg [‘%]phenanthrene to a 4-cm* dorsal area.

The percentages of absorbed dose, measured every 6 hr, were approximately the same, suggesting that the in vitro system used here can simulate the percutaneous absorption of phenanthrene in the whole-animal experiments. The in vitro and in vivo comparison

TABLE 2 (6.6 pgPHENANTHRENE/Crn2)

METABOLITESANDTHEIRCHROMATOGRAPHICANDMASSSPECTRAL

% of Administered dose

Rf values on TLC system

GC retention time (min)

Mass spectralc fragments m/z (relative abundance)

92.2

0.94 (0.94)”

23.05 (23.05)=

3.5

0.53 (0.53)”

28.43 (28.43)’

1,2-Dihydrodiol

2.0

0.34 (0.39)b

32.85

truns-3,4-Dihydrodiol

1.4

0.43 (0.50)b

28.73

0.84 (SF)b

30.32-3 1.02

178 (M+, 100) 177 (IO), 176 (17) 152 (IO), 151 (7), 150 (6), 89 (17), 88 (13), 76 (21), 75 (9X 63 (8) 356 (M+, 25) 341 (19), 325 (8), 267 (8), 266 (13) 253 (6), 25 1 (7) 235 (6), 180 (II), 166 (13), 147 (67), 73 (100) 356 (M+, 9), 267 (6), 266 (5) 253 (9). 193 (57) 180 (6) 166 (9), 148 (IO), 73 (100) 356 (M+, 13), 267 (9), 266 (8), 253 (12), 251 (6), 235 (8) 193 (44) 180(12), 166 (lo), 147 (7), 73 (loo) 266 (M+, 100)

Phenanthrene

truns-9, IO-Dihydrodiol

tram-

Hydroxyphenanthrenes

Trace

’ Data derived from authentic samples. b From Chaturapit and Holder (1978); SF, solvent front. ’ GC and GC/MS data are obtained from trimethyl silyl derivatives; values in parentheses represent relative abundance.

ABSORPTION/METABOLISM

studies have also been successfully conducted with other PAHs in this laboratory. At the two dose levels studied (6.6 and 15.2 &cm2), the rates of penetration (percentage of applied dose) into the 6-hr fractions were approximately the same, indicating first-order kinetics. Skin metabolism of phenanthrene could also be studied in the in vitro diffusion cell system. As seen in Fig. 2, significantly greater metabolism was observed in viable skin than in the nonviable controls (water + BSA). The control radioactivity may be due to either metabolism in the initial few hours prior to cell death or nonenzymatic biotransformation of phenanthrene. Approximately 7% of the absorbed phenanthrene was biotransformed to the three diol metabolites (Table 2). These results are consistent with previous findings of between 1 and 10% metabolism of other xenobiotics (butylated hydroxytoluene, acetyl ethyl tetramethyltetralin, benzo[a]pyrene, and 7 ethoxycoumarin) metabolized by the skin P450 enzyme system (Bronaugh et al., 1989a; Storm et al., 1990). Phenanthrene is neither mutagenic in Salmonella typhimurium assays, nor tumorigenic in mouse skin studies (IARC Monographs, 1983). The lack of these toxicities was reflected in our dermal metabolism data. In our studies, the predominate metabolite was found to be K-region phenanthrene 9,10-dihydrodiol, and no Bay-region 3,4-dihydrodiol epoxide or its rearranged product was detected. The inability of phenanthrene to form the active carcinogenic intermediates is therefore consistent with its negative cancer bioassay data reported in the literature. The percutaneous absorption of phenanthrene was shown to be primarily a passive diffusion process since only a small percentage of the absorbed compound was metabolized. Phenanthrene absorption increased in a linear manner when the topical dose applied was more than doubled. However, the observed metabolism of the compound during absorption at the higher dose did not increase, resulting in decreased metabolism on a percentage basis (Fig. 1). An understanding of skin metabolism of PAH

OF

PHENANTHRENE

523

compounds remains extremely important from a toxicological standpoint. Phenanthrene absorption has previously been studied by in vitro methods with nonviable Sprague-Dawley female rat skin (Roy et al., 1987). A number of polynuclear aromatic compounds was applied to skin and percutaneous absorption was determined by comparing mass spectrometry chromatograms in the donor and receptor fluid. By this method, 43% of applied phenanthrene was absorbed through rat skin-a value substantially lower than that in the current guinea pig experiments. Since the barrier properties of guinea pig skin would be expected to compare more favorably to rat skin, we measured the in vivo absorption of phenanthrene in rats and obtained a value of 82% (data not shown). Differences in experimental methodology may explain, at least in part, this discrepancy. The doses applied to skin were not reported by Roy and co-workers and may have been much higher than in our study in order to achieve sufficient sensitivity for mass spectral analysis of the donor and receptor fluid. The value reported for anthracene absorption (43% of applied dose) using mass spectral chromatograms of test compounds (Roy et al., 1987) was lower than a value reported by the same group (Yang et al., 1986) for anthracene (55.9%) when absorption was determined by measuring radioactivity in the receptor fluid. The in vitro method has been demonstrated to give accurate in vivo skin absorption values for phenanthrene. The features which are considered to be essential in the in vitro/in vivo validations of phenanthrene include maintenance of skin viability and metabolic activity, use of physiologically conducive receptor fluid, flow-through cells, and inclusion of the amount of chemicals in the skin as part of absorbed dose. Similar in vitro/in vivo studies have been completed with other PAHs in this laboratory. Results of these studies also demonstrated the predictive capability of the in vitro method. Further in vitro studies with chemicals of diverse physicochemical prop-

524

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erties are required to test the predictive ability of this in vitro method. ACKNOWLEDGMENTS This study is funded in part by the Panel on Energy Research and Development (PERD). The authors are indebted to J. Kelly for technical assistance.

REFERENCES E., AND TURNER, E. E. (1950). The reduction of oquinone with lithium aluminium hydride. J. Chem. Sot. 64, 1188- 1190. BOYLAND, E., AND SIMS, P. (1962). Metabolism of polycyclic compounds. Biochem. J. 84,571-582. BRONAUGH, R. L., AND STEWART, R. F. (1984). Methods for in vitro percutaneous absorption studies. III. Hydrophobic compounds. J. Pharm. Ski. 73, 1255-1258. BRONAUGH, R. L., AND STEWART, R. F. (1985). Methods for percutaneous absorption studies. IV. The flowthrough diffusion cells. J. Pharm. Ski. 74, 64-67. BRONAUGH, R. L., STEWART, R. F., AND STORM, J. E. (1989a). Extent of cutaneous metabolism during percutaneous absorption of xenobiotics. Toxicol. Appl. Pharmacol. 99,534-543. BRONAUGH, R. L., COLLIER, S. W., AND STEWART, R. F. (1989b). In vitro percutaneous absorption of a hydrophobic compound through viable hairless guinea pig skin. Toxicologist 9, 6 1. CHATURAPIT, S., AND HOLDER, G. M. (1978). Studies on the hepatic microsomal metabolism of [“Clphenanthrene. Biochem. Pharmacol. 27, 1865- 187 1. CHU, I., VILLENEUVE, D. C.. C&E, M., VALLI, V. E., AND OTSON, R. (1988). Dermal toxicity of a mediumboiling (154-378°C) coal liquefaction product in the rat-Part 1. J. Toxicol. Environ. Health 23, 193-206. BOOTH, J., BOYLAND,

COLLIER, S. W., SHEIKH, N. M., SAKR, A., LICHTIN, J. L., STEWART, R. F., AND BRONAUGH, R. L. (1989). Maintenance of skin viability during in vitro percutaneous absorption/metabolism studies. Toxicol. Appl. Pharmacol. 99, 522-533. FELDMANN, R. J., AND MAIBACH, H. I. (1969). Absorption of some organic compounds through the skin in man. J. Invest. Dermatol. 54, 339-404. FRANKLIN, C. A., SOMERS, D. A., AND CHU, I. (1989). Use ofpercutaneous absorption data in risk assessment. J. Am. CON. Toxicol. 8, 8 15-827. FRANZ, T. J. ( 1975). Percutaneous absorption. On the relevance of in vitro data. J. Invest. Dermatol. 64, 190195. GOKS@YR, A., SOLBAKKEN, J. E., AND KLUNGS~YR, J. (1986). Regioselective metabolism of phenanthrene in Atlantic cod (Gadus morhua): Studies on the effects of monooxygenase inducers and role of cytochrome p-450. Chem. Biol. Interact. 60, 247-263. IARC Monographs (1983). International Agency for Research on Cancer Monographs on the evaluation of the carcinogenic risk of chemicals to humans. In Polyaromatic Compounds Part 1. Vol. 32, pp. 419-430. ROY, T. A., YANG, J. J.. AND CZERWINSKI,M. H. (1987). Evaluating the percutaneous absorption of polynuclear aromatics using in vivo and in vitro techniques and structure activity relationships. In Alternative Methods in Toxicology, Approaches to Validation (A. M. Goldberg, Ed.), Vol. 5, pp. 471-482. Liebert. New York. STORM, J. E., COLLIER, S. W., STEWART, R. F., AND BRONAUGH, R. L. (1990). Metabolism of xenobiotics during percutaneous penetration: Role of absorption rate and cutaneous enzyme activity. Fundam. Appl. Toxicol. 15, 132-141.

YANG, J. J., ROY, T. A., AND MACKERER, C. R. (1986). Percutaneous absorption of anthracene in the rat: Comparison of in vivo and in vitro results. Toxicol. Znd. Health 2, 79-84.