Percutaneous Penetration and Skin Retention of Topically Applied Compounds: An In Vitro-In Vivo Study WILLIAM
G. REIFENRATH~, GEORGEs. HAWKINS,AND MICHAEL s. KURTZ
Received Jul 23,1990,from the Letterman Army Institute of Research, Presidio of San Francisco, CA 94729. August 21, 1 l90.
Abstract 0 Radiolabeled compounds with varying partition coefficients (paraoxon, benzoic acid, parathion, and DDT) were chosen to study the percutaneous penetration and extent of dermal retention in pig skin both in vitro and in vivo. Radiolabel distributions within the skin were determined from 1 min to 24 h after applicationin ethanol. The distribution of radioactivityin the skin during the first 4 h was comparable between in vitro and in vivo experiments. At 24 h, radioactive residues in the dermis were significantly higher in vitro than in vivo for DDT, the most lipophilic compound. Increasingair flow over the skin surface significantly increased evaporative loss for volatile compounds (benzoic acid, N,Ndiethyl-mtoluamide, malathion, parathion, and DDT), significantly decreased the residues in the upper skin layer for N,N-diethyl-mtoluamide, malathion, parathion, and DDT, significantly decreased the dermal residue for malathion, and significantly decreased the penetration of N,Ndiethyl-mtoluamide, malathion, and parathion. On a percentage basis, increasing the dose of parathion and paraoxon from 4 to 1000 pg/cm2 resulted in significantly lower residues in the dermis. When applied to the dermis, the more hydrophilic benzoic acid and paraoxon better penetrated the dermis than the more hydrophobic parathion and DDT. An ethanol vehicle facilitated the penetration of parathion into the dermis and receptor fluid. These results indicate that the dermis interacted with the penetrant during both in vitro and in vivo percutaneous absorption. Factors such as partition coefficient and dose of the penetrant, air flow over the skin, and vehicle changed the distribution of penetrants in the skin and percutaneous penetration. The dermis may exhibit a depot for lipophilic compounds during in vitro percutaneous absorption that is not observed in vivo. Formation of this depot was related to diffusabilityof penetrant through the dermis rather than limited solubility in the receptor fluid.
The evolution of integuments which limited loss of body water and constituents was a requirement for the appearance of land-based animals. The epidermis and, in particular, the stratum corneum have long been recognized as a human’s principal barrier to water loss through skin.1 The stratum corneum also provides some protection against hazardous environmental chemicals.* The proximity of the blood circulation to the skin surface was also likely an evolutionary requirement for thermoregulation of homeotherms. Capillaries form loops in the papillary layer between the ridges of the dermal-epidermal junction.3 From the foregoing, one might conceptualize percutaneous absorption as a process of diffusion through the stratum corneum, followed by systemic uptake at the epidermal-dermal junction. While this hypothesis may be true for some compounds, it has been suggested that “insoluble penetrants may be deposited” in the dermis.4 Scheuplein has calculated that a tissue-water partition coefficient of >400 was required before the diffusional resistance of a 200-pm layer of dermis had comparable diffusional resistance to a stratum corneum 10-pm thick.5 Assuming that octanol-water partition coefficients reflect tissue-water partition coefficients, many drugs and pesticides would fall into this category. Finally, it would appear that some compounds can evade capillary uptake and dermal deposition and diffuse into subcutaneous tissue to achieve a local therapeutic effect.6.7 526 / Journal of PharmaceuticalSciences Vol. SO, No. 6, June 7997
Accepted for publication
To further study the role of the dermis in the absorption process, the skin distribution and percutaneous penetration were determined for compounds spanning a large range of partition coefficients and molecular weights. The effects of dose, time, and velocity of air over the skin surface on the disposition of the topically applied compounds were determined. In vitro measurements of percutaneous penetration and dermal accumulation were compared with those conducted in vivo.
Experimental Section Chemi~als-[7-’~C]Benzoicacid, [4-14C]caffeine, [carbonyl14CWJV-diethyl-rn-toluamide, y-[U-14C11,2,3,4,5,6-hexachlorocyclohexane (lindane), [4-’4Cltestosterone, and [4-’4Clprogesterone were obtained from New England Nuclear, Boston, MA. Diethyl p-nitro [ringU-14C]phenylphosphate (paraoxon), 0,O-diethyl-0-p-nitro [ring-2,6-’4Clphenylphosphorothioate(parathion), 0,O-dimethyl S-(l,2-diethoxycarbonyl[1,2-’4Clethylphosphorodithioate (malathion), and p,p’-dichlorodi[U-’4Clphenyltrichloroethane (DDT) were obtained from Amersham Corp., Arlington Heights, IL. Fluocinolone [2-14Clacetonide was obtained from Syntex Corporation, Palo Alto, CA. Labeled compounds had radiochemical purities of 98% or greater as determined by TLC. Unlabeled compounds were obtained with a reported purity of 97% or greater and were found homogeneous by TLC. The DDT, lindane, and malathion were obtained from Chem Service, Westchester, PA. Benzoic acid was obtained from Matheson, Coleman and Bell, Los Angeles, CA. Parathion was obtained from Pfaltz and Bauer Inc., Stamford, CT. Paraoxon, caffeine, testosterone, and progesterone were obtained from Sigma Chemical Company, St. Louis, MO. NJV-Diethyl-rntoluamide was obtained from Aldrich Chemical Company, Milwaukee, WI. Fluocinolone acetonide was obtained from Syntex Corp., Palo Alto, CA. Animals. Yorkshire female pigs (Boswell Laboratory Animals, Corcoran, CA) weighing 15-25 kg were utilized in this study. In Vitro Skin Penetration and Retention MeasurementsApplication to theEpidermis-In vitro measurements of percutaneous penetration were conducted with a 1-mm split-thickness pig skin preparation.8 For one experiment, a n epidermal layer -100 ym in thickness was removed from the skin with a dermatome (Brown model 9Ol,Zimmer-USA, Warsaw, IN) and then immediately repositioned. This preparation was referenced as “presplit”. Flow diffusion cells (Laboratory Glass Apparatus, Berkeley, CA) were used according to published procedures.* Benzoic acid, parathion, paraoxon, and DDT were applied (0.8 cm2 of skin) at a dose of 4 pg/cm2 and a radioactive dose of -0.1 yCi in a 5-pL volume of ethanol. Measurements were made over a 1-min to 48-h period. Evaporation cells containing Tenax GC powder (Alltech Associates, Arlington Heights, IL) as a vapor adsorbent were used with air flow a t a rate of 60 or 600 mL/min. At the end of a given time interval, the area of skin application was cut from the perimeter portion using a No. 9 cork borer. The perimeter portion was assayed for radioactivity to determine if peripheral transfer of applied compound had occurred. The inner area was frozen to a microtome stage (model 880, American Optical, Buffalo, NY) with embedding solution (Tissue Tek I1 O.C.T., Miles Laboratories, Naperville, IL). An upper 100-gm layer containing epidermis and some dermis was cut from the dermis. Radioactivity in the two layers was recovered by combustion (Packard model 306 sample oxidizer, Downers Grove, IL) to determine skin retention. 0022-3549/97/0600-0526$07 .OO/O 0 7997,American PharmaceuticalAssociation
Radioactivity adsorbed to the tenax powder represents evaporative loss, and radioactivity in the receptor fluid determines percutaneous penetration (Packard model CA 1900 scintillation counter). To determine the extent of possible cross contamination of skin layers between specimens, an excised skin specimen (sample A) was mounted on a penetration cell and dosed with a small amount of radiolabeled paraoxon (10 pg/cm2, 13 000 dpm). After 1 min, the skin surface was blotted wich a dry cotton ball, and the sample was microtomed. The microtome and blade were decontaminated in a standard fashion. A second skin specimen (Sample B) was dosed with paraoxon (1 mg/cm2, 316 000 dpm) and was processed as detailed for sample A. Following decontamination of the equipment, a third skin specimen (Sample C) was dosed and processed as per Sample A. This sequence was repeated with skin samples D (1 mgicm' of paraoxon) and E ( 10 pg/cm2 of paraoxon). Radioactive residues blotted from the skin surface and recovered from the epidermal and dermal sections for Sample A were subtracted from those found in samples C and E. The difference could be attributed to cross contamination. For sample C, the skin surface residue increased to 35% versus 10% for Sample A (percent of 13 000 DPM); the epidermal layer had a small decrease 65% versus 70% for Sample A; and the dermis residue increased to 4.8% versus 3.5% for Sample A. Residues in Sample E were 11% for the skin surface, 68% for the epidermal layer, and 4.7% for dermis. The difference in radioactive dose between samples exaggerated the error in this example. For similar doses of radioactivity, as used in this study, the error would be 1/25 of that found in this example or < 5 4 of the value obtained. Application to the Dermis-Split-thickness pig skin was attached to skin penetration cells filled with tissue culture media and was allowed to equilibrate. After 30 min, skin samples were removed from the cells and visible water was blotted from the dermal surface. After 10 min, radiolabeled compounds were applied to the dermal surface a t a dose of4 pg/cm2 in 5 p L ofethanol. Ten minutes after application, the skin samples were repositioned on the penetration cells. The dermal side contacted sf.atic tissue culture media for 1 h or flowing media (3 mL/h) for 24 h after application of radioactive compounds. Skin samples were removed from the cells and the epidermal layer was cut from the dermis with the microtome. The two skin layers and the receptor fluid were assayed for radioactivity. In Vivo Skin Penetration and Skin Retention Studies-One Minute to One Hour Post Appltcation-Weanling Yorkshire pigs were premedicated by an im injection of atropine (-3 mL of a 400-pg/mL solution, Elkins-Sinn, Cherry Hill, NJ), ketamine (-1 mL of a 100-mgimL solution, Vetalar, Parke-Davis, Morris Plains, NJ), and xylazine ( - 1.5 mL of a 2O-mg/mL solution, Rompum, Mobay, Shawnee, KS). The pigs were anesthetized with halothane, intubated, and placed in a sling for the duration of the experiment. A catheter was placed in an ear vein and an iv drip of normal saline (Travenol Laboratories, Deerfield, IL) was started. The pigs were initially given -2 mL of nembutal (Nembutal-Sodium, 50 mg/mL, Abbot, Chicago) and then maintained on nembutal during the following hour (-450 mg o r 30 mgikg total dose for a 15-kg pig). The animals were sacrificed with an overdose of nenibutal a t the end of the experiment. The compounds were applied to three 0.8-cm2 sites on the dorsal surface of the pig a t a chemical dose of 4 pg/cm2and a radioactive dose of -0.1 pCi in 5 pL of ethanol. Skin was harvested using a No. 9 cork borer and a scalpel. A 100-pm layer was removed from the skin plugs as described in the in vitro experiments. The two layers were assayed for radiolabel by sample combustion and scintillation counting. One Hour to 24 Hours Post Application-Female weanling Yorkshire pigs weighing 30-510 Ibs were prepared for study by clipping the hair t Oster model A2, Milwaukee, WI) from their backs and marking up to four rectangular application sites (2 x 4 cm) on their backs. A nonocclusive protective patch9 was placed over the sites. At 1,2,4,24, or 120 h prior to sacrifice, test chemicals were applied (4 pgicm' or 1 mgicm') to the sites wi:h a blunt-tipped syringe (Hamilton, Reno, NV). The animals were housed in metabolism cages during the experiment. Following sedation (xylazine and ketamine) and sacrifice (T-61 solution, Fort Dodge Laboratories, Las Vegas, NV), the protective patch was removed and the skin surface was decontaminated with cotton swabs dry or lightly dampened with ethanol. A 6 x 12-cm area, which contained the application site, was excised. A 100-pm layer, consisting of epidermis and a portion of the dermis, was removed with the dermatome. Two layers of dermis were removed from subcutaneous tissue with the dermatome set to cut a 1-mm thickness of skin. The dermatome cutting surface was wiped with
alcohol-moistened cotton after each layer was cut; residual compound or radioactivity was added to that found in the layer. A control experiment determined that radioactivity remaining on the dermatome cutting head following cleaning was only a small percentage (< 10%)of the radioactivity recovered in subsequent layers.10 Penetrant was assayed by radiometric or chromatographic procedures. Gas Chromatographic Assay of Parathion and Paraoxon in the Epidermal, Dermal. and Subcutaneous Fat Layers of SkinFollowing excision, the epidermal layer was placed in -3 mL of 2-butanone. Dermal and subcutaneous fat layers were first homogenized with dry ice in a blender (Waring, with a 110-mL cup) and placed in 2-butanone (3-5 mL). The butanone:tissue mixture was allowed to stand for 1 h prior to centrifugation a t 2000 rpm (IEC clinical centrifuge, Needham Heights, MA). The supernatant was decanted from the tissue pellet. For subcutaneous fat, the butanone was passed through a column of florisil. The extract was concentrated (1.5-2.0 mL) prior to analysis. A gas chromatograph (HP model 5880) equipped with a flame ionization detector, autoinjector, and integrator was fitted with a glass column (2 mm x 2.4 m, packed with 5% QF-1 on 100/120 Supelcoport, Supelco, Bellefonte, PA). The flow rate of nitrogen carrier gas was 60 mumin. The injector temperature was 220 "C, the oven temperature was 200 "C, and the detector temperature was 230 "C. Radiometric Analyses-The Tenax GC powder from vapor traps, receptor fluid (tissue culture media), and 2-butanone extracts of skin were placed directly into counting solution (Optifluor, Packard Instruments) and counted on a Packard model 460 scintillation counter. Radioactivity in the skin layers was recovered from 200-mg portions with a sample oxidizer. Statistical A n a l y s e e I n vitro and in vivo measurements of skin surface/epidermal residues (Tables I and 11) were compared using a two-way analysis of variance. If a significant F occurred, Tukey's test was employed to determine which measurements differed. Dermal residues were rank ordered and analysis of variance with Tukey's test was performed on the rankings. The effect of dose on the disposition of compounds on the skin (Table 111 versus Table 11)was evaluated by two-way analysis of variance and Tukey's test. The effects of air flow (Table IV) and vehicle (Tables VII and VIII) on skin disposition were determined by a t test. The effects of compound and time on the distribution of radioactivity after dermal application (Table V) and the effect of using pre-split skin (Table IX)on skin penetration and residues in the skin were evaluated by two-way analysis of variance and Tukey's test. All evaluations of significance were carried out at the 0.05 level of confidence.
ResuIt s The disposition of radioactivity versus time following topical application (4 pg/cm2) of labeled compounds to splitthickness pig skin mounted in diffusion cells is given in Table I. One minute after application, 3 4 % of the applied dose could be found in the dermis. Dermal levels remained relatively constant during 4 h after topical application. Penetration into the receptor fluid was
G. REIFENRATH~, GEORGEs. HAWKINS,AND MICHAEL s. KURTZ
Received Jul 23,1990,from the Letterman Army Institute of Research, Presidio of San Francisco, CA 94729. August 21, 1 l90.
Abstract 0 Radiolabeled compounds with varying partition coefficients (paraoxon, benzoic acid, parathion, and DDT) were chosen to study the percutaneous penetration and extent of dermal retention in pig skin both in vitro and in vivo. Radiolabel distributions within the skin were determined from 1 min to 24 h after applicationin ethanol. The distribution of radioactivityin the skin during the first 4 h was comparable between in vitro and in vivo experiments. At 24 h, radioactive residues in the dermis were significantly higher in vitro than in vivo for DDT, the most lipophilic compound. Increasingair flow over the skin surface significantly increased evaporative loss for volatile compounds (benzoic acid, N,Ndiethyl-mtoluamide, malathion, parathion, and DDT), significantly decreased the residues in the upper skin layer for N,N-diethyl-mtoluamide, malathion, parathion, and DDT, significantly decreased the dermal residue for malathion, and significantly decreased the penetration of N,Ndiethyl-mtoluamide, malathion, and parathion. On a percentage basis, increasing the dose of parathion and paraoxon from 4 to 1000 pg/cm2 resulted in significantly lower residues in the dermis. When applied to the dermis, the more hydrophilic benzoic acid and paraoxon better penetrated the dermis than the more hydrophobic parathion and DDT. An ethanol vehicle facilitated the penetration of parathion into the dermis and receptor fluid. These results indicate that the dermis interacted with the penetrant during both in vitro and in vivo percutaneous absorption. Factors such as partition coefficient and dose of the penetrant, air flow over the skin, and vehicle changed the distribution of penetrants in the skin and percutaneous penetration. The dermis may exhibit a depot for lipophilic compounds during in vitro percutaneous absorption that is not observed in vivo. Formation of this depot was related to diffusabilityof penetrant through the dermis rather than limited solubility in the receptor fluid.
The evolution of integuments which limited loss of body water and constituents was a requirement for the appearance of land-based animals. The epidermis and, in particular, the stratum corneum have long been recognized as a human’s principal barrier to water loss through skin.1 The stratum corneum also provides some protection against hazardous environmental chemicals.* The proximity of the blood circulation to the skin surface was also likely an evolutionary requirement for thermoregulation of homeotherms. Capillaries form loops in the papillary layer between the ridges of the dermal-epidermal junction.3 From the foregoing, one might conceptualize percutaneous absorption as a process of diffusion through the stratum corneum, followed by systemic uptake at the epidermal-dermal junction. While this hypothesis may be true for some compounds, it has been suggested that “insoluble penetrants may be deposited” in the dermis.4 Scheuplein has calculated that a tissue-water partition coefficient of >400 was required before the diffusional resistance of a 200-pm layer of dermis had comparable diffusional resistance to a stratum corneum 10-pm thick.5 Assuming that octanol-water partition coefficients reflect tissue-water partition coefficients, many drugs and pesticides would fall into this category. Finally, it would appear that some compounds can evade capillary uptake and dermal deposition and diffuse into subcutaneous tissue to achieve a local therapeutic effect.6.7 526 / Journal of PharmaceuticalSciences Vol. SO, No. 6, June 7997
Accepted for publication
To further study the role of the dermis in the absorption process, the skin distribution and percutaneous penetration were determined for compounds spanning a large range of partition coefficients and molecular weights. The effects of dose, time, and velocity of air over the skin surface on the disposition of the topically applied compounds were determined. In vitro measurements of percutaneous penetration and dermal accumulation were compared with those conducted in vivo.
Experimental Section Chemi~als-[7-’~C]Benzoicacid, [4-14C]caffeine, [carbonyl14CWJV-diethyl-rn-toluamide, y-[U-14C11,2,3,4,5,6-hexachlorocyclohexane (lindane), [4-’4Cltestosterone, and [4-’4Clprogesterone were obtained from New England Nuclear, Boston, MA. Diethyl p-nitro [ringU-14C]phenylphosphate (paraoxon), 0,O-diethyl-0-p-nitro [ring-2,6-’4Clphenylphosphorothioate(parathion), 0,O-dimethyl S-(l,2-diethoxycarbonyl[1,2-’4Clethylphosphorodithioate (malathion), and p,p’-dichlorodi[U-’4Clphenyltrichloroethane (DDT) were obtained from Amersham Corp., Arlington Heights, IL. Fluocinolone [2-14Clacetonide was obtained from Syntex Corporation, Palo Alto, CA. Labeled compounds had radiochemical purities of 98% or greater as determined by TLC. Unlabeled compounds were obtained with a reported purity of 97% or greater and were found homogeneous by TLC. The DDT, lindane, and malathion were obtained from Chem Service, Westchester, PA. Benzoic acid was obtained from Matheson, Coleman and Bell, Los Angeles, CA. Parathion was obtained from Pfaltz and Bauer Inc., Stamford, CT. Paraoxon, caffeine, testosterone, and progesterone were obtained from Sigma Chemical Company, St. Louis, MO. NJV-Diethyl-rntoluamide was obtained from Aldrich Chemical Company, Milwaukee, WI. Fluocinolone acetonide was obtained from Syntex Corp., Palo Alto, CA. Animals. Yorkshire female pigs (Boswell Laboratory Animals, Corcoran, CA) weighing 15-25 kg were utilized in this study. In Vitro Skin Penetration and Retention MeasurementsApplication to theEpidermis-In vitro measurements of percutaneous penetration were conducted with a 1-mm split-thickness pig skin preparation.8 For one experiment, a n epidermal layer -100 ym in thickness was removed from the skin with a dermatome (Brown model 9Ol,Zimmer-USA, Warsaw, IN) and then immediately repositioned. This preparation was referenced as “presplit”. Flow diffusion cells (Laboratory Glass Apparatus, Berkeley, CA) were used according to published procedures.* Benzoic acid, parathion, paraoxon, and DDT were applied (0.8 cm2 of skin) at a dose of 4 pg/cm2 and a radioactive dose of -0.1 yCi in a 5-pL volume of ethanol. Measurements were made over a 1-min to 48-h period. Evaporation cells containing Tenax GC powder (Alltech Associates, Arlington Heights, IL) as a vapor adsorbent were used with air flow a t a rate of 60 or 600 mL/min. At the end of a given time interval, the area of skin application was cut from the perimeter portion using a No. 9 cork borer. The perimeter portion was assayed for radioactivity to determine if peripheral transfer of applied compound had occurred. The inner area was frozen to a microtome stage (model 880, American Optical, Buffalo, NY) with embedding solution (Tissue Tek I1 O.C.T., Miles Laboratories, Naperville, IL). An upper 100-gm layer containing epidermis and some dermis was cut from the dermis. Radioactivity in the two layers was recovered by combustion (Packard model 306 sample oxidizer, Downers Grove, IL) to determine skin retention. 0022-3549/97/0600-0526$07 .OO/O 0 7997,American PharmaceuticalAssociation
Radioactivity adsorbed to the tenax powder represents evaporative loss, and radioactivity in the receptor fluid determines percutaneous penetration (Packard model CA 1900 scintillation counter). To determine the extent of possible cross contamination of skin layers between specimens, an excised skin specimen (sample A) was mounted on a penetration cell and dosed with a small amount of radiolabeled paraoxon (10 pg/cm2, 13 000 dpm). After 1 min, the skin surface was blotted wich a dry cotton ball, and the sample was microtomed. The microtome and blade were decontaminated in a standard fashion. A second skin specimen (Sample B) was dosed with paraoxon (1 mg/cm2, 316 000 dpm) and was processed as detailed for sample A. Following decontamination of the equipment, a third skin specimen (Sample C) was dosed and processed as per Sample A. This sequence was repeated with skin samples D (1 mgicm' of paraoxon) and E ( 10 pg/cm2 of paraoxon). Radioactive residues blotted from the skin surface and recovered from the epidermal and dermal sections for Sample A were subtracted from those found in samples C and E. The difference could be attributed to cross contamination. For sample C, the skin surface residue increased to 35% versus 10% for Sample A (percent of 13 000 DPM); the epidermal layer had a small decrease 65% versus 70% for Sample A; and the dermis residue increased to 4.8% versus 3.5% for Sample A. Residues in Sample E were 11% for the skin surface, 68% for the epidermal layer, and 4.7% for dermis. The difference in radioactive dose between samples exaggerated the error in this example. For similar doses of radioactivity, as used in this study, the error would be 1/25 of that found in this example or < 5 4 of the value obtained. Application to the Dermis-Split-thickness pig skin was attached to skin penetration cells filled with tissue culture media and was allowed to equilibrate. After 30 min, skin samples were removed from the cells and visible water was blotted from the dermal surface. After 10 min, radiolabeled compounds were applied to the dermal surface a t a dose of4 pg/cm2 in 5 p L ofethanol. Ten minutes after application, the skin samples were repositioned on the penetration cells. The dermal side contacted sf.atic tissue culture media for 1 h or flowing media (3 mL/h) for 24 h after application of radioactive compounds. Skin samples were removed from the cells and the epidermal layer was cut from the dermis with the microtome. The two skin layers and the receptor fluid were assayed for radioactivity. In Vivo Skin Penetration and Skin Retention Studies-One Minute to One Hour Post Appltcation-Weanling Yorkshire pigs were premedicated by an im injection of atropine (-3 mL of a 400-pg/mL solution, Elkins-Sinn, Cherry Hill, NJ), ketamine (-1 mL of a 100-mgimL solution, Vetalar, Parke-Davis, Morris Plains, NJ), and xylazine ( - 1.5 mL of a 2O-mg/mL solution, Rompum, Mobay, Shawnee, KS). The pigs were anesthetized with halothane, intubated, and placed in a sling for the duration of the experiment. A catheter was placed in an ear vein and an iv drip of normal saline (Travenol Laboratories, Deerfield, IL) was started. The pigs were initially given -2 mL of nembutal (Nembutal-Sodium, 50 mg/mL, Abbot, Chicago) and then maintained on nembutal during the following hour (-450 mg o r 30 mgikg total dose for a 15-kg pig). The animals were sacrificed with an overdose of nenibutal a t the end of the experiment. The compounds were applied to three 0.8-cm2 sites on the dorsal surface of the pig a t a chemical dose of 4 pg/cm2and a radioactive dose of -0.1 pCi in 5 pL of ethanol. Skin was harvested using a No. 9 cork borer and a scalpel. A 100-pm layer was removed from the skin plugs as described in the in vitro experiments. The two layers were assayed for radiolabel by sample combustion and scintillation counting. One Hour to 24 Hours Post Application-Female weanling Yorkshire pigs weighing 30-510 Ibs were prepared for study by clipping the hair t Oster model A2, Milwaukee, WI) from their backs and marking up to four rectangular application sites (2 x 4 cm) on their backs. A nonocclusive protective patch9 was placed over the sites. At 1,2,4,24, or 120 h prior to sacrifice, test chemicals were applied (4 pgicm' or 1 mgicm') to the sites wi:h a blunt-tipped syringe (Hamilton, Reno, NV). The animals were housed in metabolism cages during the experiment. Following sedation (xylazine and ketamine) and sacrifice (T-61 solution, Fort Dodge Laboratories, Las Vegas, NV), the protective patch was removed and the skin surface was decontaminated with cotton swabs dry or lightly dampened with ethanol. A 6 x 12-cm area, which contained the application site, was excised. A 100-pm layer, consisting of epidermis and a portion of the dermis, was removed with the dermatome. Two layers of dermis were removed from subcutaneous tissue with the dermatome set to cut a 1-mm thickness of skin. The dermatome cutting surface was wiped with
alcohol-moistened cotton after each layer was cut; residual compound or radioactivity was added to that found in the layer. A control experiment determined that radioactivity remaining on the dermatome cutting head following cleaning was only a small percentage (< 10%)of the radioactivity recovered in subsequent layers.10 Penetrant was assayed by radiometric or chromatographic procedures. Gas Chromatographic Assay of Parathion and Paraoxon in the Epidermal, Dermal. and Subcutaneous Fat Layers of SkinFollowing excision, the epidermal layer was placed in -3 mL of 2-butanone. Dermal and subcutaneous fat layers were first homogenized with dry ice in a blender (Waring, with a 110-mL cup) and placed in 2-butanone (3-5 mL). The butanone:tissue mixture was allowed to stand for 1 h prior to centrifugation a t 2000 rpm (IEC clinical centrifuge, Needham Heights, MA). The supernatant was decanted from the tissue pellet. For subcutaneous fat, the butanone was passed through a column of florisil. The extract was concentrated (1.5-2.0 mL) prior to analysis. A gas chromatograph (HP model 5880) equipped with a flame ionization detector, autoinjector, and integrator was fitted with a glass column (2 mm x 2.4 m, packed with 5% QF-1 on 100/120 Supelcoport, Supelco, Bellefonte, PA). The flow rate of nitrogen carrier gas was 60 mumin. The injector temperature was 220 "C, the oven temperature was 200 "C, and the detector temperature was 230 "C. Radiometric Analyses-The Tenax GC powder from vapor traps, receptor fluid (tissue culture media), and 2-butanone extracts of skin were placed directly into counting solution (Optifluor, Packard Instruments) and counted on a Packard model 460 scintillation counter. Radioactivity in the skin layers was recovered from 200-mg portions with a sample oxidizer. Statistical A n a l y s e e I n vitro and in vivo measurements of skin surface/epidermal residues (Tables I and 11) were compared using a two-way analysis of variance. If a significant F occurred, Tukey's test was employed to determine which measurements differed. Dermal residues were rank ordered and analysis of variance with Tukey's test was performed on the rankings. The effect of dose on the disposition of compounds on the skin (Table 111 versus Table 11)was evaluated by two-way analysis of variance and Tukey's test. The effects of air flow (Table IV) and vehicle (Tables VII and VIII) on skin disposition were determined by a t test. The effects of compound and time on the distribution of radioactivity after dermal application (Table V) and the effect of using pre-split skin (Table IX)on skin penetration and residues in the skin were evaluated by two-way analysis of variance and Tukey's test. All evaluations of significance were carried out at the 0.05 level of confidence.
ResuIt s The disposition of radioactivity versus time following topical application (4 pg/cm2) of labeled compounds to splitthickness pig skin mounted in diffusion cells is given in Table I. One minute after application, 3 4 % of the applied dose could be found in the dermis. Dermal levels remained relatively constant during 4 h after topical application. Penetration into the receptor fluid was
