Effects of glyceryl glucoside on AQP3 expression, barrier function and hydration of human skin.

BACKGROUND/AIM: Aquaporins (AQPs) present in the epidermis are essential hydration-regulating elements controlling cellular water and glycerol transport. In this study, the potential of glyceryl glucoside [GG; alpha-D-glucopyranosyl-alpha-(1->2)-glycerol], an enhanced glycerol derivative, to increase the expression of AQP3 in vitro and ex vivo was evaluated. METHODS: In vitro studies with real-time RT-PCR and FACS measurements were performed to test the induction by GG (3% w/v) of AQP3 mRNA and protein in cultured human keratinocytes. GG-containing formulations were applied topically to volunteer subjects and suction blister biopsies were analyzed to assess whether GG (5%) could penetrate the epidermis of intact skin, and subsequently upregulate AQP3 mRNA expression and improve barrier function. RESULTS: AQP3 mRNA and protein levels were significantly increased in cultured human keratinocytes. In the studies on volunteer subjects, GG significantly increased AQP3 mRNA levels in the skin and reduced transepidermal water loss compared with vehicle-controlled areas. CONCLUSION: GG promotes AQP3 mRNA and protein upregulation and improves skin barrier function, and may thus offer an effective treatment option for dehydrated skin.

Design and application of a screening and training protocol for odour testers in the field of personal care products.

The assessment of odours and in particular of human axillary odour is an integral part of the research and development of deodorant and anti-perspirant products. One method to perform odour assessment is the odour evaluation that is carried out by experts, designated as odour testers or sniffers. Product development decisions are therefore based on human assessment. As for every scientific measurement, the influencing factors need to be standardized or regularly calibrated as effectively as possible for reasons of quality assurance. We therefore developed a screening and training concept aiming to examine the general suitability of odour testers by determining the individual odour sensitivity for relevant odours. This newly developed method is based on the national and international standards and guidelines EN 13725:2003, VDI 3882 sheet 1 and ASTM-1207. Suitable odour testers are subsequently trained to correlate their individual odour intensity perception with an intensity calibration scale in order to achieve reproducible results. Training sessions held on a regular basis help to achieve a greater homology in the response of an existing panel. Our established screening and training protocol has already been successfully put into practice and is also subject to permanent improvement with regard to practical requirements.

Aging skin is functionally anaerobic: importance of coenzyme Q10 for anti aging skin care.

The functional loss of mitochondria represents an inherent part in modern theories trying to explain the cutaneous aging process. The present study shows significant age-dependent differences in mitochondrial function of keratinocytes isolated from skin biopsies of young and old donors. Our data let us postulate that energy metabolism shifts to a predominantly non-mitochondrial pathway and is therefore functionally anaerobic with advancing age. CoQ10 positively influences the age-affected cellular metabolism and enables to combat signs of aging starting at the cellular level. As a consequence topical application of CoQ10 is beneficial for human skin as it rapidly improves mitochondrial function in skin in vivo.

Contact angle measurement - a reliable supportive method for screening water-resistance of ultraviolet-protecting products in vivo.

Substantivity of sunscreen formulations is affected by the wash-out rate of ultraviolet-absorber and -reflector compounds in water. Water-resistance of sunscreen formulations is currently determined according to a standardized European Cosmetic Toiletry and Perfumery Association (COLIPA) protocol, encompassing the determination of a minimal erythemal dose before and after a defined immersion step in water. It can be supposed that the higher the wettability of a treated skin area, the higher is the wash-out rate of sunscreen compounds. This present report addresses the validity of determining the wettability of treated skin alone as a measure for the water-resistance of sunscreen products. The report addresses the robustness, accuracy and congruence of a recently developed wettability test, based on the measurement of the contact angle (CA) of a sessile water drop on treated skin areas. Contact angle data of 66 sunscreen formulations are compared with the corresponding results of 81 water-resistance tests, using the sun protection factor (SPF)/immersion/SPF method. Sunscreen products tested by the CA method were applied to the skin of the volar forearm of test subjects at a defined dose and drying-time, using a standardized application and recording device. Contact angles between a sessile water drop and skin were recorded by a Charge-Coupled Device (CCD) camera and subjected to automatic contour analysis. Taking the SPF/immersion/SPF method as gold standard, accuracy parameters of the CA method were determined. By using an appropriate cut-off level of CAs, the CA method has a specificity and positive-predictive value of 100%, and turns out to be a reliable screening method to identify water-resistant formulations. Based on our findings, those formulations that give CAs above 30 degrees may be categorized water-proof without further testing by the COLIPA water-resistance method.

Stimulation of skin's energy metabolism provides multiple benefits for mature human skin.

As an organism ages, there is a decline in mitochondrial function and cellular energy balance. This decline is both accelerated by and can cause the formation of reactive oxygen species (ROS) that damage nuclear and mitochondrial DNA, lipid membranes as well as structural and catalytic proteins, especially those involved in energetic pathways of cells. Further, ROS have also been linked to some of the detrimental skin changes that occur as a result of photoaging. We have previously shown that levels of Coenzyme Q10 (CoQ10), a component of the respiratory chain in mitochondria, are reduced in skin cells from aging donors, and that topical supplementation can ameliorate processes involved in skin aging. Creatine is another important component of the cellular energy system and phosphocreatine, its phosphorylated form, functions as a reservoir for high energy phosphates. Unfortunately the creatine system and thus the energy storage mechanism in skin are negatively affected by aging and conditions of oxidative stress. This article reviews some of our in vivo data about the synergistic effects of combining a stabilized form of Creatine with CoQ10 and clearly depicts their beneficial effects as active ingredients in topical formulations.

Distribution of sunscreens on skin.

The effectiveness of sunscreens was originally achieved by incorporation of soluble organic UV absorbers such as cinnamates and others into cosmetic formulations. Determinations of the sun protection factor (SPF) of emulsions containing different organic UV absorbers clearly indicate that the efficacy depends on the absorption characteristics of each single UV filter substance. Nowadays, micronised pigments such as titanium dioxide or zinc oxide have also been found to be protective against harmful UV rays. Our investigations using optical and electron microscopy proved that neither surface characteristics, particle size nor shape of the micronised pigments result in any dermal absorption of this substance. Micronised titanium dioxide is solely deposited on the outermost surface of the stratum corneum and cannot be detected in deeper stratum corneum layers, the human epidermis and dermis.

In human keratinocytes the Common Deletion reflects donor variabilities rather than chronologic aging and can be induced by ultraviolet A irradiation.

Mitochondrial DNA mutations play a major role in human aging processes and degenerative diseases. The most frequently reported marker for mutations of the mitochondrial DNA in human skin is a 4977 bp large-scale deletion, called the Common Deletion. Although this deletion is rarely detectable and constitutes only one example of the multitude of about 50,000 known mutations in mitochondrial DNA, it can represent "the tip of the iceberg" of all types of mitochondrial DNA mutations. We established a quantitative real-time polymerase chain reaction assay to detect the Common Deletion in vitro as well as in vivo/ex vivo. In contrast to previous studies, we were able to demonstrate that the Common Deletion is frequently abundant in keratinocytes isolated from various donors. Quantitative analysis of the mutation indicated interperson variations but obviously no relation to the donors' ages. Prolonged proliferation of keratinocytes led to a distinct reduction in the amount of the Common Deletion. Single ultraviolet A irradiation (12 J per cm2 and 15 J per cm2) neither in vitro nor in vivo increased the incidence of the mutation in keratinocytes, whereas repetitive irradiation resulted in a clear increase in vitro. Again, prolonged cultivation of these irradiated cells caused a significant reduction in the amounts of the deletion. In view of these results, the Common Deletion appears to be a useful marker rather for ultraviolet-A-induced alterations than for chronologic aging in human skin keratinocytes.

High-pressure freezing provides new information on human epidermis: simultaneous protein antigen and lamellar lipid structure preservation. Study on human epidermis by cryoimmobilization.

Current transmission electron microscopy techniques do not permit simultaneous visualization of skin ultrastructure and stratum corneum extracellular lipids. We developed a new procedure, which entails application of high-pressure freezing followed by freeze-substitution with acetone containing uranyl acetate, followed by low temperature embedding in HM20. Electrospray ionization mass spectrometry showed that the amount of lipids lost during preparation was minimal. The ultrastructure of cryoprocessed skin was compared with that of conventionally prepared skin samples. Cryoprocessing, but not conventional processing, enabled visualization of lipid stacks within epidermal lamellar bodies, as well as the extracellular lipid domains of the stratum corneum and the ultrastructure within keratinocytes. Anti-filaggrin immunocytochemistry also showed, e.g., excellent preservation of filaggrin on cryoprocessed samples. Additionally, the cytosol of keratinocytes appeared to be organized in "microdomain"-like areas. Finally, the stratum corneum appeared more compact with smaller intercellular spaces and hence tighter cell-cell interactions, after cryoprocessing, than after conventional tissue preparation for transmission electron microscopy. We conclude here that only cryoprocessing preserves skin in a close to native state.

Regulation of HMG-CoA synthase and HMG-CoA reductase by insulin and epidermal growth factor in HaCaT keratinocytes.

Synthesis of cholesterol, via the isoprenoid/mevalonate pathway, is required for keratinocyte growth and differentiation, and maintenance of the stratum corneum lipid lamellae. 3-hydroxy-3-methylglutaryl coenzyme A synthase catalyzes the first step in isoprenoid/mevalonate synthesis and under some conditions controls the flux into the pathway. We have investigated whether selected growth factors and hormones could increase 3-hydroxy-3-methylglutaryl coenzyme A synthase mRNA in keratinocytes. Northern blotting was used to demonstrate that 10 microg per ml insulin and 0.1 microg per ml epidermal growth factor both increased steady-state levels of 3-hydroxy-3-methylglutaryl coenzyme A synthase mRNA by 2.5 and 6-fold, respectively. Epidermal growth factor and insulin also increased 3-hydroxy-3-methylglutaryl coenzyme A reductase enzyme activity. 3-hydroxy-3-methylglutaryl coenzyme A synthase promoter activity in a luciferase reporter construct was increased 2-fold by insulin and 2.9-fold by epidermal growth factor. When a mutation in the sterol regulatory element was introduced into the 3-hydroxy-3-methylglutaryl coenzyme A synthase promoter, activity was not increased by insulin, but was increased by epidermal growth factor. Mutation of an AP-1 site in the 3-hydroxy-3-methylglutaryl coenzyme A synthase promoter did not affect the increase in activity following treatment with insulin or epidermal growth factor. Therefore, 3-hydroxy-3-methylglutaryl coenzyme A synthase expression in keratinocytes is regulated by insulin and epidermal growth factor by different mechanisms. These results suggest a role for hormones and growth factors in the control of epidermal cholesterol synthesis.

Localization of ceramide and glucosylceramide in human epidermis by immunogold electron microscopy.

Ceramides and glucosylceramides are pivotal molecules in multiple biologic processes such as apoptosis, signal transduction, and mitogenesis. In addition, ceramides are major structural components of the epidermal permeability barrier. The barrier ceramides derive mainly from the enzymatic hydrolysis of glucosylceramides. Recently, anti-ceramide and anti-glucosylceramide anti-sera have become available that react specifically with several epidermal ceramides and glucosylceramides, respectively. Here we demonstrate the detection of two epidermal covalently bound omega-hydroxy ceramides and one covalently bound omega-hydroxy glucosylceramide species by thin-layer chromatography immunostaining. Moreover, we show the ultrastructural distribution of ceramides and glucosylceramides in human epidermis by immunoelectron microscopy on cryoprocessed skin samples. In basal epidermal cells and dermal fibroblasts ceramide was found: (i) at the nuclear envelope; (ii) at the inner and outer mitochondrial membrane; (iii) at the Golgi apparatus and the endoplasmic reticulum; and (iv) at the plasma membrane. The labeling density was highest in mitochondria and at the inner nuclear membrane, suggesting an important role for ceramides at these sites. In the upper epidermis, ceramides were localized: (i) in lamellar bodies; (ii) in trans-Golgi network-like structures; (iii) at the cornified envelope; and (viii) within the intercellular space of the stratum corneum, which is in line with the known analytical data. Glucosylceramides were detected within lamellar bodies and in trans-Golgi network-like structures of the stratum granulosum. The localization of glucosylceramides at the cornified envelope of the first corneocyte layer provides further proof for the existence of covalently bound glucosylceramides in normal human epidermis.

Induction of mRNA for matrix metalloproteinase 1 and tissue inhibitor of metalloproteinases 1 in human skin in vivo by solar simulated radiation.

Repeated exposure to solar ultraviolet radiation results in premature skin aging due, in part, to the degradation of dermal collagen by fibroblast collagenase (matrix metalloproteinase 1 [MMP-1]). We have established TaqMan reverse transcription (RT) polymerase chain reaction (PCR) systems to quantify the messenger RNA (mRNA) expression of MMP-1 and its specific inhibitor TIMP-1 in human buttock skin exposed in vivo to solar simulated radiation (SSR). A time-course study (n = 6) with two minimal erythema doses (MED) of SSR showed maximal induction of MMP-1 and TIMP-1 at 24 h. A dose-response study (n = 6) sampled at 24 h revealed that doses of about 1 MED were necessary to induce expression of MMP-1 mRNA, and our data suggest that the response is saturated at about 2 MED. We also investigated SSR-induced gene expression in the dermis and epidermis separately (n = 5). MMP-1 was present in both tissues, but TIMP-1 was only detected in the dermis. In general, we could only measure MMP-1 mRNA in the nonirradiated control skin of volunteers who were smokers. We hypothesize very large interpersonal variation with MMP-1 induction compared with TIMP-1 which was detected in all the control sites. This suggests a lack of relationship between MMP-1 and TIMP-1 mRNA expression. The large donor variability for MMP-1 in all the studies demonstrates that it is important to analyze gene expression individually.

P53-dependent UVB responsiveness of human keratinocytes can be altered by cultivation on cell cycle-arrested dermal fibroblasts.

In cultured human keratinocytes, the tumor suppressor p53 acts as a control element in the protective response to UVB radiation and is affected by a variety of factors linked to cellular adhesion and differentiation. Because keratinocytes within the epidermis are not a homogeneous population but differ in their proliferative capacity and differentiation status, we compared the UVB responsiveness of primary keratinocyte populations isolated from various skin biopsies using p53 expression as a marker for their sensitivity to UVB. Besides keratinocytes exhibiting a UVB dose- and time-dependent upregulation of p53, keratinocyte populations were detected with high p53 expression levels even without irradiation. Such keratinocytes did not regulate p53 expression in response to UVB. Furthermore their p53-mediated UVB response was influenced by cocultivation with human dermal fibroblasts (HDF) but not with cell cycle-arrested human normal keratinocytes or HaCaT keratinocytes. When these cells were cultivated together with arrested HDF, they did not only reveal increased p53 expression levels after UVB treatment but also a more pronounced transcriptional activation of the p53 downstream target gene p21. These findings indicate that the UVB response of keratinocytes, specifically the activation of the tumor suppressor p53, is heterogeneous and can be affected by growth conditions.

Analysis of UV-B-induced DNA damage and its repair in heat-shocked skin cells.

The heat-shock response is a cellular defence mechanism against environmental stresses that is evolutionarily conserved from bacteria to man. Numerous reports demonstrate the beneficial effects of heat-shock protein induction on cell survival under toxic or oxidative stress, e.g., in cardiac and cerebral ischemia or prior to organ transplantation. However, there is little data on the effects of heat treatment on damage caused by UV irradiation. Applying three independent techniques, we have tested the influence of thermal pretreatment of skin cells (1 h, 43 degrees C) on the initial extent of UV-B-induced DNA damage and its subsequent repair. For cultured human epidermal keratinocytes and dermal fibroblasts we can show reduced levels of nucleotide-excision-repair-associated DNA strand incision in the comet assay. Moreover, immunostaining and flow cytometric quantitation of thymidine dimers immediately and one day after irradiation, respectively, reveal that the initial DNA damage is not (keratinocytes) or only moderately (fibroblasts) lower in heat-shocked cells as compared to untreated controls. However, excision repair of dimers is significantly attenuated during the first 24 h in both cell types. Furthermore, using a modified host-cell reactivation assay, we are able to demonstrate that repair of UV-B-damaged plasmid DNA is lower if the transfected cells are previously heat shocked. In summary, heat treatment (1 h, 43 degrees C) inducing heat-shock proteins reduces nucleotide excision repair of UV-B-mediated DNA lesions in fibroblasts and keratinocytes during the following 24 h. This is not necessarily caused by elevated heat-shock protein levels themselves. Possibly the direct thermal damage of repair enzymes is more severe than the potential protective effects of heat-shock proteins.

Hydration dynamics of human fingernails: an ellipsometric study.

We use spectroscopic ellipsometry to obtain the complex refractive index, ñ=n+ik, of human fingernails. By studying the change of ñ upon hydration and dehydration, we reveal three different time domains with typical time constants of 4, 150, and 3200 min. A simple model that takes into account the presence of one fast and one slow process is fully consistent with the observed hydration and dehydration dynamics. We attribute these processes to "free" water incorporated between the keratin filaments and water more tightly "bound" in keratin complexes, respectively. From our model we determine the hydration profiles of "free" and "bound" water during, both, hydration and dehydration.

Adsorption kinetics of surfactant mixtures from micellar solutions as studied by maximum bubble pressure technique.

The adsorption kinetics of micellar solutions of anionic/cationic SDS/DATB mixtures with mixing ratios of 10/1 and 10/2, respectively, are studied experimentally by means of the maximum bubble pressure method. For long adsorption times the adsorption of the highly surface-active anionic/cationic complex leads to a decrease of dynamic surface tension in comparison to the single SDS system. However, the situation is the reverse for short adsorption times where the dynamic surface tension is increased by addition of the cationic surfactant, although the overall concentration is increased. This unexpected behavior is explained by partial solubilization of free SDS molecules into micelles formed by SDS/DTAB complexes. With increasing overall concentration, when eventually the CMC of SDS is reached, the anionic/cationic complex itself is solubilized by SDS micelles. Finally, no complex micelles, which for their part can solubilize an excess of SDS molecules, are present. Hence, the dynamic properties of the solution are no longer influenced by the depletion of SDS molecules and the mixture tends to behave like a pure SDS solution.

Dielectric spectroscopy of concentrated cosmetic W/O-emulsions: possibilities to distinguish product changes caused by coalescence, sedimentation and variation of ingredients.

A quick result whether a newly developed cosmetic water-in-oil (W/O)-emulsion shows constant sensory behavior (stable) or whether it changes its behavior over time (instable) is an important aspect in cosmetic research. In order to observe changes as quickly as possible, analytic methods are used. An established method is rheology, a sensitive method that gives direct information on sensory aspects. Additional information concerning the kind of instability allows a more focused improvement of formulation in the case of instabilities. In order to gain this information, an additional analytic method with a more ingredient specific focus is needed. In this article, the possibilities of using dielectric spectroscopy in order to get additional information are discussed. For concentrated W/O-emulsions a dependence of emulsion behavior from volume median droplet diameter d(v50) is visible by both methods: rheology and dielectric spectroscopy. A variation of droplet size distribution at constant volume median droplet diameter d(v50) does not change the results for the examined emulsions. Quantitative information about mean droplet size is possible with calibration. Because of their different physical forci, rheology and dielectric spectroscopy complement each other in high-sensitive detection of coalescence. In contrast to the mechanical properties of W/O-emulsion, dielectric spectroscopy gives additional information concerning some reasons of change in emulsion structure. Mechanism like sedimentation and coalescence can be distinguished and the phase where changes take place (oil phase or water phase) can be located. The latter is possible by a new parameter - the correlation between maximum of dielectric loss epsilon''(f(R)) and the relaxation frequency f(R). Their correlation can be described by a simple power law. By coupling rheology and dielectric spectroscopy, an improvement in fast emulsion development and production control may be achieved without losing the advantage of a quick and easy measurement procedure.

A quick, practical test procedure to evaluate the performance of instruments used for in vitro UV protection measurements.

The in vitro determination of the UV protection of sunscreens is usually performed by means of transmission measurements with special photometers. Many different instruments are used. Besides numerous commercially available instruments, which are equipped by the manufacturer for the specific measurement, other modular instruments are used. We present here a quick and practical method to evaluate the performance of these instruments with respect to their measuring ranges and to compare the uniformity and reliability of the results obtained with these instruments.

Influence of cleansing on stratum corneum tryptic enzyme in human skin.

Desquamation in human skin is a well-balanced process of de novo production of corneocytes and their shedding from the skin surface. The proteolysis of corneodesmosomes is an important step in the final desquamation process. In the degradation of these adhesion molecules, the stratum corneum tryptic enzyme (SCTE) plays a key role. In initial studies with extracts of porcine epidermis, SCTE was shown to be inactivated by low concentrations of sodium lauryl ether sulphate (SLES). These in vitro findings were supported by in situ results obtained by measuring the release of fluorescent dyes coupled to trypsin-specific substrates incubated on human skin cross-sections. Moreover, in further studies, it could be demonstrated that the SCTE activity in the human horny layer decreases after in vivo application of cleansing products containing SLES. After repeated washing of human volunteers with tap water, a standard market cleansing product (SLES/betaine system) or a new improved cleansing product (SLES/betaine/disodium cocoyl glutamate system), the specific SCTE activity was determined in extracts from the uppermost layers of the stratum corneum. It could be shown that after application of the new formula the remaining SCTE activity was significantly higher than after use of the standard market formula. This ex vivo approach has proven to be very helpful for measuring surfactant effects on human skin enzymes. Using this assay, we developed an improved shower gel formula, which leads to a significantly higher skin enzyme activity after application, compared to a standard market formula.

Pros and cons: cryo-electron microscopic evaluation of block faces versus cryo-sections from frozen-hydrated skin specimens prepared by different techniques.

Over the last two decades, several different preparative techniques have been developed to investigate frozen-hydrated biological samples by electron microscopy. In this article, we describe an alternative approach that allows either ultrastructural investigations of frozen human skin at a resolution better than 15 nm or sample throughput that is sufficiently high enough for quantitative morphological analysis. The specimen preparation method we describe is fast, reproducible, does not require much user experience or elaborate equipment. We compare high-pressure freezing with plunge freezing, and block faces with frozen-hydrated slices (sections), to study variations in cell thickness upon hydration changes. Plunge freezing is optimal for morphological and stereological investigations of structures with low water content. By contrast, high-pressure freezing proved optimal for high-resolution studies and provided the best ultrastructural preservation. A combination of these fast-freezing techniques with cryo-ultramicrotomy yielded well-preserved block faces of the original biological material. Here we show that these block faces did not exhibit any of the artefacts normally associated with cryo-sections, and--after evaporating a heavy metal and carbon onto the surface--are stable enough in the electron beam to provide high-resolution images of large surface areas for statistical analysis in a cryo-SEM (scanning electron microscope). Because the individual preparation steps use only standard equipment and do not require much experience from the experimenter, they are generally more usable, making this approach an interesting alternative to other methods for the ultrastructural investigation of frozen-hydrated material.

Field evaluation of the efficacy of proprietary repellent formulations with IR3535 and picaridin against Aedes aegypti.

Seven proprietary repellent formulations (3 hydro-alcoholic spray solutions and 4 skin lotions) with active ingredient IR3,535 (ethyl butylacetylaminopropionate, EBAAP) or Picaridin (hydroxyethyl isobutyl piperidine carboxylate, KBR 3,023, Bayrepel) were tested in a field study on 10 test persons over a period of 10 h for their efficacy at preventing bites. The tests were conducted in Belo Horizonte, Brazil on field populations of the yellow fever mosquito Aedes aegypti. The concentration of the active substances ranged from 10% to 20%. All the tested samples provided lasting protection (time to first bite) over several hours: ranging from 5 h 20 min to 6 h 50 min with a mean of approximately 6 h. The longest protection until the second bite (=first confirmation bite) was approximately 7 h 40 min, whereas the shortest protection was 6 h 50 min. The longest protection until the third bite (=second confirmation bite) was 8 h 35 min, whereas the shortest protection was 7 h 40 min. In the control tests in which none of the samples were applied, the mean times until the first, second and third bites were 26, 46 and 59 min, respectively. The basis for this field study was provided by two American guidelines, which have the greatest international acceptance. The first is a draft guideline from the Environmental Protection Agency (EPA (United States Environmental Protection Agency), Product performance test guidelines. OPPTS 810.3700. Insect repellents for human skin and outdoor premises. Public Draft, 1999) and the second is a standard from the American Society for Testing and Materials (ASTM (American Society for Testing and Materials International), E 939-94 (reapproved 2,000): standard test method of field testing topical applications of compounds as repellents for medically important and pest arthropods (including insects, ticks, and mites): I. Mosquitoes, 2,000). Both guidelines recommend measuring the duration of protection until the first and second bites and also determining the relative protection efficacy in terms of a 95% protection level. The ASTM standard permits different repellents to be applied, whereas the EPA guidelines only permit the use of a single repellent (in different concentrations) on the extremities (forearms or lower leg). In the study presented here, to exclude any possibility of different repellents or concentrations of a single repellent having a reciprocal effect on each other, each test person had repellent samples applied to only one of their forearms. The other forearm was used as a control for making comparative checks every hour and for determining the biting pressure. There was no significant difference in protection times between the two active substances.