In vitro permeation of repellent DEET and sunscreen oxybenzone across three artificial membranes

DEET and oxybenzone are two essential active ingredients in repellent and sunscreen products. We performed a series of in vitro diffusion studies to evaluate the transmembrane permeation of DEET and oxybenzone across three artificial membranes, low-density polyethylene (LDPE), low fouling composite (LFC) and mixed cellulose esters (MCE), from concurrent use of commercial repellent and sunscreen preparations. Permeation of DEET and oxybenzone across the test membranes was synergistically increased when both the repellent and the sunscreen formulations were applied simultaneously. Different application sequences and formulation types also resulted in variable permeation profiles of DEET and oxybenzone. Compared to biological piglet epidermis under the identical experimental conditions, transmembrane permeation of DEET was suppressed in LDPE and LFC membranes, but enhanced in MCE membrane; transmembrane permeation of oxybenzone was reduced in LFC membrane, but increased in LDPE and MCE membranes. Permeability coefficients of DEET and oxybenzone in all three artificial membranes were significantly different from those in piglet skin. It was concluded that the permeation profiles of the compounds were dependent upon physicochemical characteristics of the membranes and the formulations.     

Tissue deposition of the insect repellent DEET and the sunscreen oxybenzone from repeated topical skin applications in rats

Insect repellent N,N-diethyl-m-toluamide (DEET) and sunscreen oxybenzone are capable of enhancing skin permeation of each other when applied simultaneously. We carried out a cellular study in rat astrocytes and neurons to assess cell toxicity of DEET and oxybenzone and a 30-day study in Sprague-Dawley rats to characterize skin permeation and tissue disposition of the compounds. Cellular toxicity occurred at 1 μg/mL for neurons and 7-day treatment for astrocytes and neurons. DEET and oxybenzone permeated across the skin to accumulate in blood, liver, and brain after repeated topical applications. DEET disappeared from the application site faster than oxybenzone. Combined application enhanced the disposition of DEET in liver. No overt sign of behavioral toxicity was observed from several behavioral testing protocols. It was concluded that despite measurable disposition of the study compounds in vivo, there was no evidence of neurotoxicological deficits from repeated topical applications of DEET, oxybenzone, or both.     

Percutaneous permeation comparison of repellents picaridin and DEET in concurrent use with sunscreen oxybenzone from commercially available preparations

Concurrent application of insect repellent picaridin or DEET with sunscreens has become prevalent due to concerns on West Nile virus and skin cancer. The objectives of this study were to characterize the percutaneous permeation of picaridin and sunscreen oxybenzone from commercially available preparations and to compare the differences in permeability between picaridin and DEET in association with oxybenzone. In vitro diffusion studies were carried out to measure transdermal permeation of picaridin and oxybenzone from four different products, using various application concentrations and sequences. Results were then compared to those of repellent DEET and sunscreen oxybenzone under identical conditions. Transdermal permeation of picaridin across human epidermis was significantly lower than that of DEET, both alone and in combination with oxybenzone. Concurrent use resulted in either no changes or suppression of transdermal permeation of picaridin and oxybenzone. This finding was different from concurrent use of DEET and oxybenzone in which a synergistic permeation enhancement was observed. In addition, permeation of picaridin, DEET and oxybenzone across human epidermis was dependent on application concentration, use sequence, and preparation type. It was concluded from this comparative study that picaridin would be a better candidate for concurrent use with sunscreen preparations in terms of minimizing percutaneous permeation of the chemicals.     

Percutaneous characterization of the insect repellent DEET and the sunscreen oxybenzone from topical skin application

The synergistic percutaneous enhancement between insect repellent DEET and sunscreen oxybenzone has been proven in our laboratory using a series of in vitro diffusion studies. In this study, we carried out an in vivo study to characterize skin permeation profiles from topical skin application of three commercially available repellent and sunscreen preparations. The correlation between skin disposition and drug metabolism was attempted by using data collected. Both DEET and oxybenzone permeated across the skin after the application and achieved substantial systemic absorption. Combined use of DEET and oxybenzone significantly enhanced the percutaneous penetration percentages (ranging 36-108%) due to mutual enhancement effects. Skin disposition indicated that DEET produced a faster transdermal permeation rate and higher systemic absorption extent, but oxybenzone formed a concentrated depot within the skin and delivered the content slowly over the time. In vivo AUCP/MRT of DEET and oxybenzone was increased by 37%/17% and 63%/10% when the two compounds were used together. No DEET was detected from the urine samples 48 h after the application. Tape stripping seemed to be a satisfactory approach for quantitative assessment of DEET and oxybenzone penetration into the stratum corneum. It was also concluded that pharmacological and toxicological perspectives from concurrent application of insect repellent and sunscreen products require further evaluation to ensure use efficacy and safety of these common consumer healthcare products.     

In-vitro permeation of the insect repellent N,N-diethyl-m-toluamide (DEET) and the sunscreen oxybenzone

The permeation behaviours of the insect repellent N,N-diethyl-m-toluamide (DEET) and the sunscreen oxybenzone were assessed in a series of in-vitro diffusion studies, using piglet skin and poly (dimethylsiloxane) (PDMS) membrane. The transmembrane permeability of DEET and oxybenzone across piglet skin and PDMS membrane was dependent on dissolving vehicles and test concentrations. An enhanced permeation increase across piglet skin was found for DEET and oxybenzone when both compounds were present in the same medium (DEET: 289% in propylene glycol, 243% in ethanol and 112% in poly(ethylene glycol) (PEG-400); oxybenzone: 139% in PEG-400, 120% in propylene glycol and 112% in ethanol). Permeation enhancement was also observed in PDMS membrane (DEET: 207% in ethanol, 124% in PEG-400 and 107% in propylene glycol; oxybenzone: 254% in PEG-400, 154% in ethanol and 105% in propylene glycol). PDMS membrane was found to be a suitable candidate for in-vitro diffusion evaluations. This study shows that the permeations of the insect repellent DEET and the sunscreen oxybenzone were synergistically enhanced when they were applied simultaneously.     

In vitro permeation characterization of repellent picaridin and sunscreen oxybenzone

Picaridin and oxybenzone are two active ingredients found in repellent and sunscreen preparations, respectively. We performed a series of in vitro diffusion studies to evaluate the transmembrane permeation of picaridin and oxybenzone across human epidermis and poly(dimethylsiloxane) (PDMS) membrane. Permeation of picaridin (PCR) and oxybenzone (OBZ) across human epidermis was suppressed when both active ingredients were used concurrently; increasing concentration of the test compounds further reduced the permeation percentage of picaridin and oxybenzone. While permeation characteristics were correlative between human epidermis and PDMS membrane, permeability of PDMS membrane was significantly larger than that of human epidermis. The findings were different from concurrent use of repellent DEET and sunscreen oxybenzone in which a synergistic permeation enhancement was observed. Further comparative studies are therefore needed to understand permeation mechanisms and interactions between picaridin and oxybenzone.     

Tissue disposition of the insect repellent DEET and the sunscreen oxybenzone following intravenous and topical administration in rats

The insect repellent N,N‐diethyl‐m‐toluamide (DEET) and sunscreen oxybenzone (OBZ) have been shown to produce synergistic permeation enhancement when applied concurrently in vitro and in vivo. The disposition of both compounds following intravenous administration (2 mg/kg of DEET or OBZ) and topical skin application (100 mg/kg of DEET and 40 mg/kg of OBZ) was determined in male Sprague‐Dawley rats. Pharmacokinetic analysis was also conducted using compartmental and non‐compartmental methods. A two‐compartment model was deemed the best fit for intravenous administration. The DEET and oxybenzone permeated across the skin to accumulate in blood, liver and kidney following topical skin application. Combined use of DEET and oxybenzone accelerated the disappearance of both compounds from the application site, increased their distribution in the liver and significantly decreased the apparent elimination half‐lives of both compounds (p < 0.05). Hepatoma cell studies revealed toxicity from exposure to all treatment concentrations, most notably at 72 h. Although DEET and oxybenzone were capable of mutually enhancing their percutaneous permeation and systemic distribution from topical skin application, there was no evidence of increased hepatotoxic deficits from concurrent application. Copyright © 2011 John Wiley & Sons, Ltd.     

DEET-loaded solid lipid particles for skin delivery: in vitro release and skin permeation characteristics in different vehicles

DEET (N,N-diethyl m-toluamide) is a lipophilic compound which has a common use as an insect repellent and causes not only skin irritation but also systemic side effects at high concentrations in long-term skin application. In this study, DEET is incorporated into solid lipid particles, a colloidal drug delivery system, in order to reduce the percutaneous permeation and avoid toxic effects and also maintain drug effectiveness on the skin surface for a long duration of insect repellence. Solid lipid particles were prepared based on emulsion systems at different concentrations and after the characterization studies, the formulation with 20% lipid phase and 1:1 drug:lipid ratio was carried to in vitro release and skin permeation studies. Solid lipid particles with DEET were compared to free DEET using cream and hydrophilic gel vehicles. Results showed that incorporation of DEET into solid lipid particles reduced the release rate and skin permeation of DEET. Imaging studies using scanning electron microscopy showed that there were still solid lipid particles on skin surface after 2 h indicating that DEET could be present for a longer time on the application site.     

Microencapsulation decreases the skin absorption of N,N-diethyl-m-toluamide (DEET).

The insect repellent N,N-diethyl-3-methylbenzamide (DEET) is widely used and is generally regarded as safe when used according to label instructions. Yet many studies have shown it to be absorbed through the skin. The objective of this study was to determine whether the skin absorption rate of DEET could be decreased while maintaining an evaporation rate consistent with effective repellency. To this end, an aqueous suspension containing 14C-DEET (15%w/w) entrapped in walled polysaccharide microcapsules was prepared and tested for skin absorption in vitro using modified Franz cells maintained in a fume hood. The control formulation was 15%w/w DEET in ethanol. Two doses (3 microL and 5 microL per 0.79 cm2 cell) of each formulation were applied to split-thickness human cadaver skin (n=8/dose), and permeation was monitored for 24h. The microencapsulated DEET formulation lead to a 25-35% reduction of radiolabel permeation compared to the ethanolic DEET formulation. Skin levels of radioactivity at 24h were comparable, indicating that DEET evaporation from the microencapsulated formulation was comparable to or greater than that from ethanol. Hence microencapsulation increased the ratio of DEET evaporation rate to skin penetration rate relative to unencapsulated control in this in vitro study.     

A bioassay for mosquito repellency against Aedes aegypti: method validation and bioactivities of DEET analogues

Objectives Vector‐borne diseases are still a major mortality factor in Africa and South‐east Asia and effective mosquito repellents are therefore needed. An efficient and safe in‐vitro assay system using artificial blood and skin substitute could facilitate the development of novel repellents, as most assays currently rely on human subjects or vertebrate whole blood. Moreover, examining the skin permeation profiles could provide safer mosquito repellents. The new assay system could serve as an initial system for testing new repellent candidates upon validation with DEET and its analogues. Methods N,N‐Diethyl‐meta‐toluamide (DEET) and five analogues were synthesised and used to validate a novel in‐vitro bioassay using artificial blood and collagen membrane. Repellency against Aedes aegypti was correlated with lipophilicity and skin permeation. Key findings The new in‐vitro assay showed good reproducibility (interday relative standard deviation <10% at high concentrations). Four of the five DEET analogues showed repellency similar or superior to that of DEET. Repellency correlated linearly with lipophilicity but stronger repellents tended to permeate skin better. Conclusions The new in‐vitro assay using blood substitute and collagen membrane significantly simplifies screening of possible mosquito repellents. Lipophilicity as well as skin permeation profiles should be considered before testing of compounds that are candidates for mosquito repellents.     

The Potential of a Site-Specific Delivery of Thiamine Hydrochloride as a Novel Insect Repellent Exerting Long-Term Protection on Human Skin: In-vitro,Ex-vivo Study and Clinical Assessment

Thiamine hydrochloride (TH) was thought to exert a good insect repellent activity. The purpose of this work was to develop a formulation that releases TH in sustained regimen on human skin. Long lasting protection against mosquito bites was achieved. Pullulan acetate (PA) was used to prepare TH nanospheres. Optimal system was incorporated in Pluronic® hydrogel. Formulae were tested for in-vitro release and ex-vivo permeation. Complete protection time (CPT) was done adopting Kaplan-Meier survival function for the synthetic repellent (DEET), TH solution and nanospheres in hydrogel. Release profile of TH solution, nanospheres and nanosphere-loaded hydrogel (DG) demonstrated an added effect of DG, where t _1/2 was 11.2 ± 1.4 h. SEM for DG showed homogenous dispersion of nanospheres inside the matrix of the gel. Ex-vivo permeation showed only 0.761 ± 0.04% of TH in hydrogel permeated the skin after 12 h, while 44.98 ± 3.2% permeated when TH solution was applied. Clinical study revealed a significant difference in CPT between TH solution with either DEET or (DG) (p<0.05), and no significant difference between DEET and DG with CPT 400 ± 31 and 360 ± 18 min, respectively (P > 0.05). The high efficacy of TH-loaded hydrogel rendered it a successful alternative for DEET, offering long protection against mosquito bites.     

Synergic enhancing-effect of DEET and dodecylamine on the skin permeation of testosterone from a matrix-type transdermal delivery system

The synergic in vitro skin permeation enhancing-effect of N,N-diethyl-m-toluamide (DEET) and dodecylamine was investigated in order to develop a novel non-scrotal matrix-type transdermal delivery system of testosterone (TS). When DEET was loaded in DuroTak^® 87-2510 together with 2% TS and 3% dodecylamine, the in vitro rat skin permeation rate of TS synergistically increased as DEET concentration increased up to 0.5%. No further increase in permeation was observed thereafter and a plateau was observed up to 3.8% DEET. Moreover, compared to 0.5% DEET concentration, the addition of 3.8% of DEET in combination with 3% dodecylamine and 6% TS further increased the permeation rate of TS, and the maximum permeation rate of 11.21 μg/cm²/h was achieved. The in vitro skin permeation rates of TS from a transdermal delivery system of DuroTak^® 87-2510 containing 6% TS, 3% dodecyamine, and 3.8% DEET were in the following order: hairless mouse skin > rat skin > human cadaver skin. Assuming that a system with a surface area of 60 cm² is applied, the human cadaver skin permeation rate of 5.74 μg/cm²/h achieved in this study can be interpreted as being equivalent to delivering ～ 8.?27?mg of TS per day. Considering that the commercially available product (Testoderm^®TTS) for non-scrotal skin of the same surface area is designed to administer 5?mg of TS per day, the new formulation could maintain therapeutic plasma concentration of TS at a smaller surface area of 40 cm².     

Development and characterization of micellar systems for application as insect repellents

N,N-diethyl-meta-toluamide (DEET) is a widely used insect repellent due to its high efficacy. In this work, micellar systems based on poly(ethylene oxide)–poly(propylene oxide)–poly(ethylene oxide) triblock copolymer were developed and studied for the purpose of controlling the release and cutaneous permeation of DEET, using concentrated solutions of the copolymer Pluronic F127 to form thermoreversible gels. The formulations presented thermoreversible gelation above 5 °C and altered rheological behavior at 15 and 25 °C. The presence of the drug drastically changed the sol–gel transition temperatures. The micrographs suggest that DEET induced the formation of anisotropic structures, and Maltese Crosses were observed. The formulation containing 10 wt% DEET and 15 wt% Pluronic F127 presented sustained drug release for up to 7 h. DEET release profile followed the Higuchi kinetics model. There was a reduction of approximately 35% in the amount of DEET absorbed through the skin after 6 h. About 62% of DEET from the formulation consisting of Pluronic F127 and DEET remain retained on the skin. The anisotropic structure may constitute a barrier to diffusion and thereby controlling the drug release effectively. These tests suggest that the tested samples exhibit safety profile greater than some commercially available products.     

Self-double-emulsifying drug delivery system incorporated in natural hydrogels: a new way for topical application of vitamin C

This study aimed at the in vitro evaluation of topical hydrogels containing vitamin C-loaded self-double-emulsifying drug delivery system (SDEDDS). The liquid SDEDDS was converted into suitable unmodified xanthan gum hydrogels formulation and the prepared semi-solid hydrogels provided appropriate gel characteristics like pH, viscosity, spreadability, etc. The 5-week storage test displayed that the prepared hydrogels possessed good physicochemical stability. In addition, no significant cytotoxicity to L929 cells was observed for the vitamin C-loaded SDEDDS-based hydrogels, and the prepared hydrogels depicted a sustained drug release over an 8-h period. In vitro permeation study showed that the vitamin C-loaded SDEDDS-based hydrogels could significantly enhance vitamin C retention in the skin and permeation through the skin. The overall results demonstrated that the hydrogels containing vitamin C-loaded SDEDDS could be considered as a promising formulation for topical application.     

Influence of Transcutol CG on the skin accumulation and transdermal permeation of ultraviolet absorbers.

The objective of this study was to determine the influence of Transcutol CG concentration on the transdermal permeation and skin accumulation of two ultraviolet (UV) absorbers, 2-hydroxy-4-methoxybenzophenone (oxybenzone) and 2-octyl-4-methoxycinnamate (cinnamate). The concentration of the UV absorber was held constant at 6% (w/w) for all vehicle systems while the concentration of Transcutol CG was varied from 0 to 50% (w/w). Data showed that both UV absorbers exhibited increases in skin accumulation with increasing concentrations of Transcutol CG. Skin accumulation of oxybenzone was significantly (P<0.05) greater than that of cinnamate for all formulations investigated. Oxybenzone skin accumulation ranged from 22.9+/-2.8 microg/mg (0% Transcutol CG) to 80.8+/-27.2 microg/mg (50% Transcutol CG). Cinnamate skin accumulation ranged from 9.0+/-0.9 microg/mg to 39.8+/-12.2 microg/mg at 0 and 50% Transcutol CG, respectively. No significant differences were found in the transdermal permeation of oxybenzone or cinnamate for any of the formulations tested. The results of this study demonstrate that the inclusion of Transcutol CG in sunscreen formulations increases the skin accumulation of the UV absorbers oxybenzone and cinnamate without a concomitant increase in transdermal permeation.     

Active ingredients in sunscreens act as topical penetration enhancers for the herbicide 2,4-dichlorophenoxyacetic acid

Agricultural workers are encouraged to use sunscreen to decrease the risk of UV-related skin cancer. Our previous studies have shown certain commercial sunscreens to be penetration enhancers. The focus of this project is to determine whether active ingredients in sunscreen formulations (i.e., the UV absorbing components and insect repellants for the sunscreen/bug repellant combinations) also act as dermal penetration enhancers for herbicides in vitro. The total percentages of 2,4-dichlorophenoxyacetic acid (2,4-D) penetrating through hairless mouse skin in 24 h ranged from 54.9 +/- 4.7 for the no sunscreen control to 86.9 +/- 2.5 for padimate-o. Of the active ingredients tested (7.5% octyl methoxycinnamate, 7% octocrylene, 0.6% oxybenzone, 5% homosalate, 5% octyl salicylate, 8% padimate-o, 10% sulisobenzone, and 9.5% and 19% N,N-diethyl-m-toluamide [DEET]), all but octocrylene led to a significant increase in total 2,4-D penetration as compared to the control (P < 0.05), and only octocrylene and oxybenzone did not significantly decrease the corresponding lag time. Octyl salicylate (P < 0.01) and octyl methoxycinnimate (P < 0.05) significantly increased the 3H2O penetration across mouse skin, indicating physical damage to the stratum corneum. Additional studies demonstrated that the penetration enhancement seen across hairless mouse skin also occurred with human skin. Thus, the active ingredients of sunscreen formulations enhance dermal penetration of the moderately lipophilic herbicide 2,4-D.     

Review of the biodistribution and toxicity of the insect repellent N,N-diethyl-m-toluamide (DEET)

A review of the biodistribution and toxicity of the insect repellent N,N-diethyl-m-toluamide (DEET) is presented. Workers using repellent containing this compound may be exposed to greater than 442 g in 6 mo. In human studies, variable penetration into the skin of from 9 to 56% of a topically applied dose and absorption into the circulatory system of approximately 17% have been reported. Excretion of DEET by humans was initially rapid but not as complete as in animal models. Only about one-half of the absorbed DEET was excreted by humans over 5 d. Depot storage of DEET in the skin was also documented. Skin irritant effects, including scarring bullous dermatitis in humans, were reported. One animal study that reported embryotoxicity could not be confirmed by other investigators. The limited testing for mutagenicity and carcinogenicity provided negative results. Neurotoxic effects were observed in workers exposed to 4 g or more per week. Six young girls developed encephalopathies after exposure to unspecified amounts of DEET ranging from small to massive doses. Three of these girls later died. The cause of their death has not been resolved. Because of the lack of information, further research into the absorption, carcinogenicity, and neurotoxic effects is needed.     

Effect of penetration modifiers on the dermal and transdermal delivery of drugs and cosmetic active ingredients

In this study the effect of 2 penetration modifiers, dimethyl isosorbide (DMI) and diethylene glycol monoethyl ether (DGME) on the skin delivery of hydroquinone (HQ), salicylic acid (SA) and octadecenedioic acid (DIOIC) was investigated. Ten percent DMI and DGME were separately formulated into oil-in-water emulsions containing 1.8% HQ, SA and DIOIC, respectively. Skin delivery and the flux across split-thickness human skin of the active ingredients were determined using Franz diffusion cells. An emulsion with 10% water incorporated instead of the water-soluble penetration modifiers served as a control. The study showed that neither 10% DMI nor 10% DGME significantly enhanced the skin permeation of the various lipophilic active ingredients or the uptake into the skin. It was hypothesized that the addition of the penetration modifiers to the emulsions not only enhanced the solubility of the various active ingredients in the skin but also in the formulation, resulting in a reduced thermodynamic activity and hence a weaker driving force for penetration. Therefore, the effect of DMI and DGME on the solubility of the active ingredients in the skin was counteracted by a simultaneous reduction in the thermodynamic activity in the formulation.     

Antipsoriatic microemulsion gel formulations for topical drug delivery of babchi oil (Psoralea corylifolia)

The aim of this study was to investigate and evaluate a microemulsion gel-based system of babchi oil (Psoralea corylifolia) for the treatment of psoriasis, which could provide improved permeation of the drug through the skin and increased patient compliance. Babchi oil is used because its chief constituent psoralen is a photoactive furocoumarin that binds to DNA when exposed to UV light to form photoproducts with pyrimidine base. This action inhibits DNA synthesis and causes decrease in cell proliferation. Moreover, babchi oil, in addition to providing psoralen, also acts as an oily phase for microemulsion system. The presence of surfactant and cosurfactant increases the permeation. On the basis of qualitative and quantitative estimation of all eight brands of babchi oil, Bakuchi Tail was selected for microemulsion formulation. Microemulsions were prepared by aqueous phase-titration method. Pseudoternary phase diagrams were constructed for the identification of microemulsion existence zones. Prepared microemulsions were subjected to different thermodynamic stability tests and characterized for droplet size, viscosity and refractive index. In vitro skin permeation of babchi oil through rat abdominal skin was determined by the Franz diffusion cell. The in vitro skin permeation profile of formulation F2, which consisted of 1.67% v/v of babchi oil, 8.33% v/v of oleic acid, S(mix) 55% v/v of Tween 80 Transcutol-P (S/Co ratio 1:1) and 35% v/v of distilled water, was significant when compared with other microemulsion formulations (p < 0.05). Formulation F2 was converted into microemulsion gel by adding 1% Carbopol-940 and coded as MGF2. Formulation MGF2 was selected for its in vivo antiinflammatory effects determined by footpad edema. The results suggested that microemulsion gel is a potential vehicle for improved topical delivery of psoralen and that microemulsion gels are potential vehicles for improved topical delivery of babchi oil.     

Preparation of griseofulvin for topical application using N-methyl-2-pyrrolidone

We attempted to prepare a new griseofulvin formulation for topical application using N-methyl-2-pyrrolidone (NMP). Griseofulvin dissolves poorly in both water and oil, but dissolves in NMP to a concentration of about 100 mg/ml. A soybean oil-water emulsion with soybean lecithin and NMP as emulsifier and co-solvent, respectively, was prepared using a Microfluidizer, a high-pressure homogenizer. The size of the droplets in emulsion was about 200 nm, and the emulsion was stable for over 3 months. The skin permeation of griseofulvin through Yucatan micropig skin was studied in vitro using vertical type cells under donor phase open conditions. The permeation of griseofulvin from the NMP-water mixture (0-40%) into the skin tended to increase with increasing NMP concentration, although this finding was not statistically significant. Permeation from emulsion (oil phase, 20%; NMP 10-40%) was significantly higher than that from the water-NMP mixture. Permeation from the oil-NMP mixture was highest among the formulations investigated, and permeation from emulsion under donor phase closed conditions was significantly lower than that under open conditions. We believe that the evaporation of water from the emulsion after application to the skin was an important factor in skin permeation enhancement. When the emulsion containing 3% l-menthol was applied, a sufficient skin concentration (47 microg/cm3 in dermis) was obtained.