Percutaneous absorption and excretion of xenobiotics after topical and intravenous administration to pigs

FUNDAMENTAL

AND APPLIED

TOXICOLOGY

l&7

14-722 ( 1989)

Percutaneous Absorption and Excretion of Xenobiotics after Topical and Intravenous Administration to Pigs MICHAEL P. CARVER' AND J. EDMOND

RIVIERE~

Cutaneous Pharmacology and Toxicology Center, College of Veterinary Medicine and Interdepartmental Toxicology Program, North Carolina State University, 4700 Hillsborough Street, Raleigh, North Carolina 27606

Received September 12.1988; accepted June &I989 Percutaneous Absorption and Excretion of Xenobiotics after Topical and Intravenous Administration to Pigs. CARVER, M. P., AND RIVIERE, J. E. (1989). Fundam. Appl. Toxicol. 13, 7 14-722. Interspecies comparisons suggest that the weaning pig is a suitable surrogate for man in percutaneous absorption studies. Despite known anatomical and physiological similarities between porcine and human skin, very few investigations of percutaneous absorption phenomena have been conducted in pigs. This study examined radiolahel excretion patterns after intravenous (iv) and topical administration of six “C-radiolabeled compounds in weanling Yorkshire sows. Radiolabel recovery from excrement collected over 6 days following iv doses in physiological saline (200 fig, 10 pCi) showed that malathion (M), parathion (P), caffeine (C), and benzoic acid (B) were primarily excreted into urine (>80%), while greater fractions of testosterone (T, 72%) and progesterone (R, 35%) were fecally eliminated. Percutaneous absorption was determined from total urine and fecal excretion of radiolabel after topical application, corrected for incomplete excretion following iv administration. Topical doses in ethanol (200 rg, 10 &i) were applied at a surface concentration of 40 pg cm-’ and penetrated in the following rank order (percentage dose): B (25.7%) > R (16.2%) > C (11.8%) > T (8.8%) > P (6.7%) > M (5.2%). Fecal clearances of radiolabel, expressed as a percentage of total excretion, were greater after topical administration for four of the six compounds (B, C, P, and T, p < 0.05). Calculations based on urinary excretion alone underestimated percutaneous absorption determined from total excmtion by 5-30%, although the difference between the two estimates was statistically significant only for C (p < 0.05). These results suggest that percutaneous absorption estimates based on urinary radiolabel excretion alone should be interpreted with caution whenever compounds with unknown penetration characteristics are used. Factors known to affect human skin absorption, such as applied dose, anatomical region, sex, age, various vehicles and solvents, and differences in cutaneous metabolism, should be more closely examined in ah animal species used to model percutaneous absorption phenomena in man. 0 1989 Society ofToxicology.

In order to fully characterize the interaction of skin with topical agents, a better understanding of the mechanisms of percutaneous absorption is necessary, particularly with regard to the animals used as surrogates for hu-

man percutaneous absorption studies. Comparative anatomy of animal and human skin suggests that certain white-skinned porcine species have many dermatological characteristics in common with man. Similarities between human and pig skin in pellage density and thickness of the various dermal layers (Yardley, 1983; Bronaugh et al., 1982; Kligman, 1964), cutaneous vasculature and microcirculation (Pavletic, 1980; Ingram and Weaver, 1969; Forbes, 1967), biochemistry

’ Present address: Drug Metabolism Department, Smith Kline and French Research & Development Laboratories, Mail Stop L-720, P.O. Box 1539, King of&ssia, PA 19406. ‘To whom reprint requests and correspondence should be addressed. 0272-0590/89$3.00 Copyright 0 1989 by the Society ofToxicology. All rights of reproduction in any form reserved.

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and cellular metabolism (Riviere et al., 1986a,b; Klain et al., 1986; Weinstein, 1966), histochemistry and enzyme distribution (Meyer et al., 1978; Meyer and Neurand, 1976), epidermal and surface lipid content (Yardley, 1983; Elias, 1981,1983; Gray et al., 1978; Meyer et al., 1978; Nicolaides et al., 1968), and ultrastructure (Monteiro-Riviere, 1986; Monteiro-Riviere and Stromberg, 1985) are well documented. Furthermore, percutaneous absorption rates and penetration characteristics for a wide range of compounds in pigs and man are comparable both in vivo (Reifenmth and Hawkins, 1986; Reifenmth et al., 1984a,b; Chow et al., 1978; Bartek and Labudde, 1975; Bartek et al., 1972), and in vitro (Hawkins and Reifenrath, 1984, 1986; Marzulh et al., 1969). Despite the fact that pigs may provide an appropriate surrogate for human skin, surprisingly few studies have been conducted using these animals. Factors known to affect percutaneous absorption in other animals and man, such as applied surface concentration, anatomic location of the application site, vehicle and solvent effects, and cutaneous biotransformation, have not been examined in pigs. The present study was undertaken to examine in greater detail the disposition of topically applied compounds in pigs, with particular emphasis on describing the excretion patterns following intravenous (iv) and topical administration. In addition, a database for topical absorption of several chemical classes is needed to validate the isolated perfused porcine skin flap (IPPSF) as a useful in vitro model for examining the mechanisms of percutaneous absorption and dermal toxicity (Carver et al., 1989; Monteiro-Riviere et al., 1987; Riviere et al., 1986a,b). METHODS Materials. [ I-methyl-“‘C]Calfeine (sp act 47.5 mCi/ mmol) and [7-r4C]bcnzoic acid (19.3 mCi/mmol) were purchased from New England Nuclear (Boston, MA). [2,3-i4C]malathion (37 mCi/mmol), [ring 2,6-‘4C]pam thion (2 1 mCi/mmol), [ring 4-‘4C]progesterone (56 mCi/

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715

mmol), and [ring 4-‘4C]testosterone (56.9 mCi/mmol) were purchased from Amersham (Arlington Heights, IL). Unlabeled (cold) caffeine (C), benzoic acid (B), testosterone (T), and progesterone (R) were purchased Born Sigma (St. Louis, MO). Cold malathion (M) and parathion (P) were purchased from Chem Service, Inc. (West Chester, PA). The radiochemical purity of all “C-labeled compounds was certified by manufacturer’s thin-layer chromatographic analyses to be 297%. Dosing solutions were prepared for iv injection by addition of cold compound in ethanol solution to the radiolabeled material. This was further diluted in sterile physiological (pH 7.4) saline to a final concentration of 0.02 mg/ml and a radioactive concentration of 1 &i/ml. For topical administration, dosing solutions were prepared by diluting cold and radiolabeled compound in 100% ethanol, to a final concentration of 1 mg/ml and 50 &i/ml. Both topical and iv dosing solutions were prepared within approximately 1 week of use and were stored refrigerated until needed. Experimentalprocedures. Female weanling Yorkshire swine, weighing approximately 20 kg each (17.8 + 0.4, n = 64), were used in all studies. Pigs were acclimated for at least 1 week before entering into the study, housed two to four animals per pen at standard temperature (72’F) and light-dark cycle ( 12: 12 hr), and fed 15% pig and sow pellets (2 lb/day, Wayne Feed Div., Chicago, IL) and water ad Iibitum. Water intake was limited to approximately 2 liters/day during excretion studies to prevent dilution of the radiolabel below the sensitivity of the assay used. Pigs (n = 4 per compound) were weighed and anesthetized by intramuscular injection of 4 ms/ke xylazine (Rompun; Miles/Bayvet, Shawnee, KS) and 16 mg/kg ketamine (Ketaset; Bristol Laboratories, Syracuse, NY). Ten milliliters of the iv dosing solution was administered to each pig by bolus injection into an ear vein. Each pig was individually placed into a stainlesssteel metabolism cage immediately following the iv injection, so that total urine and feces could be collected separately (at room temperature). Urine was collected at 6, 12 (where available), and 24 hr and daily thereafter for a total of 6 days. The volume of each collection was measured and an aliquot was stored in a polypropylene vial at -20°C until analysis. Feces were collected daily for 6 days and the total sample, placed in plastic bags, was also stored at -20°C. At the end of the study, pigs were euthanized with 60-80 m&kg (iv) pentobarbital solution (Uthol; Butler, Columbus, OH). Liver, both kidneys, and spleen were removed and weighed. Small samples ofeach organ, along with samples of lung, colon, skeletal muscle, and skin were stored as described above. Another group of pigs (n = 4 per compound) were weighed and anesthetized as described for the iv studies. A procedure similar to that described previously for the porcine back (Bartek et al, 1972) was modified slightly to allow ventral application of each compound in this study. The abdominal area on each pig was lightly shaved with electric clippers; care was taken not to damage the

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skin surface (Wester and Maibach, 1975a,b). The intended area to be dosed (5 X 1 cm) was marked and a small foam rubber border ( 10 X 5 X 2 cm) with the center cut out was glued to the skin immediately adjacent to this area. Neither the glue nor the border touched the area to be dosed. Using a micropipettor (Hamilton; Reno, NV), 200 pl ofthe ethanolic dosing solution was applied to the site, to provide an applied surface concentration of 40 pg cm-‘. A nylon screen and a nonocclusive gauze pad were used to cover the foam border and were held in place by wrapping the pig’s midsection with tape (Elasticon; Johnson & Johnson, New Brunswick, NJ). Pigs were then placed in the metabolism cages as described above. Excrement collections and tissue samples at termination of the study were the same as those for the iv pigs, with the added collection and storage of the foam border, dosed site, and adjacent skin. Except for a slight and transient erythema reaction following topical T application, no toxicologic or pharmacologic responses were noted for either route of administration and internal organs appeared normal upon visual examination at time of necropsy. Analytical methods. Each daily fecal collection from both iv and topically dosed pigs was individually weighed and ground into a paste in a Waring commercial blender, with addition of 100 to 300 ml physiological saline (pH 7.4) to facilitate grinding. An aliquot of the homogenate was collected in a polypropylene vial and stored until assay. Aliquots of urine (500-800 PI), plasma (500 Al), red blood cells (200 rl), tissue samples from the internal organs (0.3-0.8 g), and fecal homogenate samples (0.6-0.8 g) were oxidized without further preparation in an openflame tissue oxidizer (Model 306; Packard Instrument Co., Downers Grove, IL). The trapped radiolabel was measured using a liquid scintillation counter (LSC) equipped with automatic external standard quench and color correction ( 12 19 Rackbeta; LBK Wallac, Turku, Finland). Skin from the application sites and the skin under the foam border (adjacent to application sites) were individually weighed and immersed in a flask containing tissue solubihzer (BTS-450; Beckman Instruments, Fullerton, CA), in amounts of 5-10 ml/g wet tissue wt. The flask was placed in a water bath overnight, maintained at 42’C with shaking (80 osc./min). Aliquots of the solubiIized skin (100 ~1) were added to 10 ml of scintillation cocktail (Scintiverse Bio-HP, Fisher Scientific, Fair Lawn, NJ) and counted by LSC as described above. Foam rubber borders were extracted in an 80~20 (v/v) ethanokmethanol mixture. Samples of the extract (500 ~1) were added to 10 ml of scintillation cocktail and counted as described above. Calculations and data anaiysis. Urine and fecal excre tion totals for each pig were calculated by multiplying radiolabel concentrations in each sample aliquot by the total volume (weight, for feces) collected. Carcass residues were calculated by multiplying radiolabel concentrations in sampled tissues by total organ weights for

liver, kidneys, and spleen, or by estimated organ weights for lung, colon, skin, and muscle. The estimated organ weights were determined using previously published organ weight-to-body weight ratios in pigs (McMeekan, 1940). For each iv pig, individual values of urine and fecal recovery, total excretion (urine + feces), and total recovery (excretion + carcass totals) were summed to provide means and standard errors within each compound. Total recoveries in topically dosed pigs included the additional radiolabel amounts found in the foam rubber border, dosed skin and from the skin adjacent to the dosed area out to the perimeter of the foam border. Percutaneous absorption, expressed as a percentage of total dose, was corrected for incomplete excretion using the following formula (Feldmann and Maibach, 1965): Corrected absorption =

Topical excretion x looI iv excretion 0

(1)

Percutaneous absorption estimates based on urine alone or urine + feces for each route were determined to compare the two methods for calculating fractional absorption (Andersen et al., 1980). Elimination rates for iv and topical excretion were estimated from simple linear regression of a sigma-minus plot of log [amount remaining to be excreted] vs time (Gibaldi and Perrier, 1982). AII data in the tables and figures are reported as means + SE and inferences were based on Student’s t statistic.

RESULTS The overall disposition of the radiolabel for the two routes of administration is shown in Tables 1 and 2. Total recoveries following the iv doses were high for B and C (near 90%), somewhat lower for the steroids (60-80%), and lowest for the more volatile organophosphates (50-60%). As can be seen in Table 1, carcass totals were negligible for all but the organophosphates. Radiolabel concentrations in individual organs (not shown) were near the lower limits of detection in most cases, although small amounts of C were found in the liver (0.3%), Pin the skin (0.7%), and R in the lungs (0.2%). Significant residues of M were found in all organs assayed, particularly the skeletal muscle which contained over 11% of the injected M. For the nonsteroidal compounds, urinary excretion was much greater than fecal excretion, averaging over 80% of the total amounts excreted during the 6&y collection period. In contrast,

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TABLE 1 RADIOLABEL RECOVERY (I DOSE) FOLLOWING IV ADMINISTRATION OF %-LABELED COMPOUNDS TO PIGS” Compound

Urine

Feces

Ct3XWb

Benzoic acid Caffeine Malathion ’ Parathion Progesterone Testosterone

84.5 -e 9.0d 68.2 +2.9 32.2 f 3.3 57.8 22.3 22.1 + 4.0 54.4+ 3.6

4.6 k 1.2 16.6 k4.1 4.9 to.7 2.020.4 40.0 k4.2 21.2 k2.7

0.1 j, 0.05 1.5 kO.5 13.1 k2.7 1.2+0.6 0.6kO.l 0.7 kO.3

Total excretionc

Total recovery

89.1 f 9.3 84.8 + 3.2 37.1 k2.6 59.9 i 2.1 62.1 f 4.4 75.7 + 2.2

89.3 + 9.3 86.3 + 3.2 50.2 + 3.5 61.1 + 2.0 62.7 k4.4 76.3 + 2.4

’ Total dose = 200 pg, 10 PCi per pig (N = 4). b Whole body residues obtained by multiplying radiolabel concentrations by measured organ weights for liver, kidneys, and spleen or by estimated organ weights (McMeekan, 1940) for lung, colon, skin, and muscle. ’ Correction factor for topical excretion studies. d Means t- SE. 'N=3.

25 to 70% of the total steroid excretion occurred by the fecal route. Following topical administration (Table 2) most of the recovered radiolabel was found in the border surrounding the application site and in the skin sample constituting the site itself. A small percentage of the dose was occasionally found in the skin surrounding this site. In addition, total recoveries were improved compared to iv administration for both the organophosphates and the steroids. As suggested by the low carcass totals, indi-

vidual organs retained levels which were barely detectable for any of the compounds. Although urinary excretion again predominated for all except the steroids, fecal clearance represented a greater fraction of total elimination after topical administration. This latter finding is illustrated in Fig. 1, in which the fecal clearances, expressed as a percentage of total for each route, are compared. As can be seen, for four out of six compounds studied (B, C, I’, and T), the fraction of total radiolabel excreted by the fecal route was

TABLE 2 RADIOLABEL RECOVERY (46 DOSE) FOLLOWING TOPICAL ADMINISTRATION OF ‘%-LABELED COMPOUNDSTO PIGS~ Dosed Compound Benzoic acidd Caffeine Malathiond

Urine 2O.Ok2.3' 5.8 + 0.4

1.7kO.3 f 0.6

Feces

Carcassb

f 0.3

0.8 k 0.4

4.2 + 1.0 0.2 + 0. I

2.7 f 1.0 0

2.9

1.0

Parathion

3.1

0.9 kO.2

1.3 +

Progesteroned Testosterone

2.6 + 0.6 3.8 f 1.7

7.4 f 1.3 2.8 f 0.8

0.3kO.l 0.7 2 0.2

Border 40.2 2 0.3 75.3 + 4.9 86.2 + 7.6 77.2 k 1.8 58.2 + 0.8 81.2k9.0

Skill

12.2 k 1.0 2.8 + 0.8 2.1 kO.1 0.6+0.1 8.6 2 1.0 17.6 3~ 4.9

Adjacent skin’

Total excretion

Total recovery

9.1 A2.1 1.9kO.2 2.0 f 1.2 0.6 + 0.3 7.2 + 0.2 4.7 + 1.7

22.9 + 2. I 10.0 * 1.3 1.9+0.2 4.0 + 0.8 10.1 f 1.9 6.6 + 2.4

85.4 k 4.2 92.7 + 3.3 92.2 z!z 8.6 83.6 2 1.2 84.4 + 2.3 111.0~4.5

’ Total dose = 200 pg, 10 FCi per pig (N = 4). ’ See footnote to Table 1 for description of how this parameter was calculated. c Skin taken from area immediately adjacent to dosed site out to external diameter of foam border. dN=3.

e Means + SE.

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CARVER

B

C

M

P

R

T

FIG. 1. Comparison of fractional fecal excretion after iv (untilled bars) and topical (crosshatched bars) administration. Bars represent means + 1 SE of three to four pigs per compound and route. Asterisks denote significant differences between routes (p < 0.05).

greater after topical administration than by iv dosage (p < 0.05). Only M, for which the fecal clearance declined slightly following topical application, had almost identical excretion patterns for both routes of administration. As expected, elimination rates were generally greater following iv administration than from topical application, with statistically significant differences (p < 0.05) seen for four out of the six compounds (C, P, R, and T). Most of the radiolabel appeared in the urine and feces during the first 24 hr post-iv injection, with the highest rates during the first collection interval (O-6 hr, urine alone) for all compounds. Intravenous elimination was generally biphasic, as exemplified by B, with a rapid elimination during the first day (Ti = 2.5 hr) and a much slower rate over the next 5 days (T$ = 19.6 hr). Terminal elimination half-lives (Ti), calculated from the mean elimination rates, ranged from 15 to 20 hr following iv administration. Biphasic elimination patterns were not seen in the sigma-minus plots of topical excretion data, indicating that the elimination rates (Tj = 18-35 hr) were masked kinetically by the much slower absorption rate processes. The period of peak skin absorption occurred during the second collection interval (6-24 hr) for all compounds except R (48-72 hr). Percutaneous absorption estimates for each compound applied topically are shown

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in Table 3. The following rank order was obtained:B>R>C>T>P>M.Withthe exception of the reversal of C and R, skin absorption was highest for the organic acid/base compounds (B, C), intermediate for the steroids (R, T), and lowest for the volatile organophosphates (M; P). Calculations using urinary excretion data alone in Eq. ( 1) (Table 3) usually resulted in lower estimates of percutaneous absorption, by 5-30%. The difference in the two estimates was statistically significant only for C (p < 0.05), however, and the rank order of skin penetration was not affected by excluding fecal excretion from the calculation of corrected absorption in Eq. ( 1). DISCUSSION Percutaneous absorption studies in both man and experimental animals have traditionally been conducted using urinary excretion alone to measure topical bioavailability. Although it is widely assumed that the route of excretion is independent of the route of administration in these studies, this hypothesis

TABLE 3

Corrected absorption (96 dose) Compound Benzoic acid b Caffeine Malathion b Parathion Progesterone b Testosterone

Total excrement

Urine alone

25.7 + 2.4 11.8+ 1.6 5.2 +0..5 6.72 1.3 16.2 f 3.1 8.8 k3.1

23.1? 2.7 8.5 + 0.6' 5.4 +0.!3 5.4+ 1.1 11.7e2.9 7.Ok 3.1

a Means -t SE (N = 4). Corrected absorption following topical application in ethanol (40 pg cm?), calculated using either urine + feces (total excrement) or urine data alone and Eq. (1) (Methods). bN=3. ’ Signiticantly less than estimate based on total excrement (p < 0.05).

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has rarely been tested. Differences in topical vs parer&ml excretion profiles have been reported for both T (Andersen et al., 1980) and polychlorinated biphenyls (Wester et al., 1983) in guinea pigs, 1,3diphenylguanidine in rats (Shah et al., 1985), and dimethylbenzanthracene in mice (Sanders et al., 1986). These findings, in addition to the present results, suggest that the underlying assumptions inherent in using urinary excretion data alone to assesspercutaneous absorption may not always be valid. Percutaneous absorption was significantly underestimated for only one compound (C) in this study by using urine alone. However, the finding of altered urinary-fecal excretion ratios for four of six compounds indicates that further work is needed to criticahy evaluate this hypothesis. In addition, the mechanism by which these altered excretion patterns occur is not known. Although iv doses are much more rapidly and completely bioavailable than after topical application, previous studies in pigs, in which parenteral and topical doses of these same compounds differed by several orders of magnitude, did not produce similar results (Reifenrath et al., 1984a,b). Thus, it appears that our findings cannot be explained as merely a dose-dependent pharmacokinetic effect. Because the metabolic disposition of topical agents can be dramatically altered from that following systemic administration (Shah et al., 1985; Nacht et al., 1981; Noonan and Wester, 1980; Greaves, 197 I), it is intriguing to consider whether first-pass cutaneous metabolism during percutaneous absorption could have occurred in the present study, followed by preferential excretion of some metabolites in the feces. There is no direct evidence that either B or C is metabolized by skin, although the relatively obscure finding that C is more toxic when administered topically (Zesch, 1986) might be related to differences in systemic vs cutaneous biotransformation pathways. No bioactivation of P to its more toxic oxygenated derivative, paraoxon, was seen when skin slices were incubated in

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vitro (Fred&sson et al., 196 1). However, tissue viability plays an important role in in vitro studies of skin function and was not adequately addressed in that investigation. Preliminary results in our laboratory using the IPPSF, an in vitro model for which cutaneous biochemical function, morphology, and their relationship to tissue viability have been well characterized (Riviere et al., 1986a,b, 1987a; Monteiro-Riviere et al., 1987), demonstrated’ in recirculating experiments a substantial capacity for P metabolism during percutaneous absorption in pig skin. The majority of the ethyl acetate-extractable radiolabel which penetrated during the 8-hr studies comigrated with paraoxon (69%) upon thin-layer chromatographic separation. Unmetabolized P (24%), the paraoxon hydrolysis product pnitrophenol (5%), and a small aqueous residue (~2%) accounted for the remainder of the 14C-radiolabel recovered in the perfusate (Carver et al., 1988; Riviere et al., 1987b). Moreover, there have been several reports of T metabolism by both human and animal skin (Kao and Hall, 1987; Kao et a/., 1985; Gomez and Hsia, 1968). Further work will be necessary to determine to what extent, if any, these and other compounds are metabolized during percutaneous absorption, It is not clear why previous investigations, employing approximately the same methods (Reifenrath et al., 1984a,b), did not demonstrate similar urinary-fecal excretion ratios. A possible explanation is that the lower dose applied in earlier studies (4 pg crnm2 vs our 40 fig cme2) may produce insufficient chemical concentrations in the skin layers in which metabolism takes place. Since little, if anything, is known about the kinetics of metabolic pathways in skin, these questions cannot yet be resolved. We chose to apply 40 kg cme2 merely to provide absorption data in pigs at a different surface concentration, since the percutaneous absorption and excretion of these compounds have already been thoroughly examined at lower concentrations in man and other species.

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Percutaneous absorption totals in the present study were generally lower than values reported by others in pigs at the lower applied surface concentration (Reifenrath and Hawkins, 1986; Reifenrath et al., 1984a,b; Bartek and Labudde, 1975; Bartek et al., 1972). Because overall rank orders of absorption, both by compound and by chemical class, are in agreement, these findings are consistent with the inverse relationship between fractional (%) absorption and applied surface concentration which has been reported to occur in both humans and rhesus monkeys (Wester et al., 1977a,b; Wester and Maibach, 1976; Maibach and Feldmann, 1969). A similar relationship for finite doses applied in vitro has also been documented (Scheuplein and Ross, 1974). The application site may also have altered absorption in this study, since an area on the back has traditionally been the site of choice for animal studies. The effect of dosing site on percutaneous absorption is reasonably well known in humans (Rougier et al., 1986, 1987; Wester and Maibach, 1985; Maibach et al,, 197 1; Feldmann and Maibach, 1967). However, except for the rhesus monkey which appears to be very similar to man (Wester et al., 1980; Britz et al., 1980; Noonan and Wester, 1980) and the rat (Rougier et al., 1987; Horhota and Fung, 1978), little is known regarding regional variation in experimental animals. The ventral abdomen was chosen in the present study because it is the location from which the IPPSF is obtained. Experimental conditions were duplicated as closely as possible to permit valid in vivo-in vitro comparisons, which are published elsewhere (Carver etal., 1989). Finally, if animal data are to have any relevance in human dermal risk assessment, the confounding effects of various anatomic regions and applied doses, vehicles and solvents, sex, age, and species differences in cutaneous biotransformation pathways must be systematically evaluated in the animal models proposed as surrogates for man.

ACKNOWLEDGMENTS This work represents partial fulfillment of the requirements for the degree of Doctor of Philosophy (M.P.C.) in the Interdepartmental Toxicology Program, North Carolina State University. Funding was provided by the U.S. Army Medical Research and Development Command, Contract No. DAMDl7-84C-4103. Portions of this work were presented at the Society of Toxicology (SOT) 25th Annual Meeting, New Orleans, Louisiana, March 3-7,1986; the SOT 26th Annual Meeting, Washington, DC., February 24-27,1987; and the Sixth Medical Chemical Defense Bioscience Review, Columbia, Maryland, August 4-6, 1987. The authors thank Mr. Richard A. Rogers for technical assistance.

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effect of stripping and occlusion. Arch. Dermatol. 91, 661-666. FELDMAN, R. J., AND MAIBACH, H. I. (1967). Regional variation in percutaneous penetration of “C-cortisol inman. J. Invest. Dermatol. 48, 181-183. FORBES, P. D. (1967). Vascular supply of the skin and heir in swine. In Advances in Biologyofskn (W. Montagna and R. Dobson, Eds.), Vol. 9, pp. 419-432. Pergamon, Oxford, NY. FREDFUKSSON, T., FARRIOR, W. L., AND WITTER, R. F. (1961). Studies on the percutaneous absorption of parathion and paraoxon. I. Hydrolysis and metabolism within the skin. Acta Derm. Venereal. 41, 335343. GIBALDI, M., AND PERRIER, D. (1982). Pharmacokinetits, 2nd ed., pp. 53-59,438-440. Dekker, New York. GOMEZ, E. C., AND HSIA, S. L. (1968). In vitro metabolism of testosterone-4-‘4C and 4-Androstene-3,17-dione+‘% in human skin. Biochemistry 7,24-32. GRAY, G. M., WHITE, R. J., AND MAJER, J. R. (1978). l-(3’-O’acyl)-&glucosyl-N-dihydroxypentatriacontadienoylsphingosine, a major component of the glucosylceramides of pig and human epidermis. Biochim.

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