Multiple parameter assessment of skin irritancy

In a previously developed guinea pig model for the study of skin irritancy, the irritant skin reactions caused by repeated open applications of low concentrations of sodium lauryl sulphate (SLS) have been studied macroscopically and microscopically. 2 new assessment methods, evaporimetry, which reflects the water barrier function of stratum corneum, and laser Doppler flowmetry to measure the cutaneous blood flow, have been added and compared with the existing methods of assessment in the model. In the present study of the irritant reaction caused by 1% SLS in 9 test animals, the 5 assessment parameters all showed values which, compared to control untested skin, increased progressively over the 3 days of application. In the assessment of skin irritancy, both evaporimetry and laser Doppler flowmetry have been shown to be useful non-invasive tools which can be quickly and reproducibly performed.     

Assessment of skin irritancy in man by laser Doppler flowmetry

It is desirable to use more objective methods than visual scoring for the assessment of skin irritancy reactions. In the present study the blood flow in skin sites exposed to sodium lauryl sulfate (SLS) was recorded by a laser Doppler flowmeter. The irritant was applied to the volar forearm of healthy volunteers in concentrations ranging from 0.001% to 5% under occlusion for 24 h. The test sites were scored visually and the blood flow was recorded at 3 different times: 26 h, 48 h and 72 h after the application of SLS. Approximately 950 recordings were performed and a clear relationship was observed between the applied doses of SLS, the recorded blood flow values and the corresponding scores. In a few cases a deviation from the general trend was observed, implying that the naked eye might be unreliable for the assessment of skin irritancy reactions.     

Animal model for assessment of skin irritancy

Irritant skin reactions from repeated open applications of low concentrations of sodium lauryl sulphate (SLS) have been studied macroscopically and microscopically in guinea pigs. After 3 applications daily for 3 days, 2% SLS aqueous solutions gave a naked eye assessment, increase in epidermal thickness and total dermal inflammatory cell response, which was greater than for a 1% SLS solution. The dermal inflammatory cell response was mainly mononuclear (lymphocytic) in nature. With the SLS reactions as control, various organic solvents were studied and ranked against the SLS reactions and internally. Trichlorethylene was the most irritant solvent, ranking as high as 2% SLS. Other chlorinated hydrocarbons and aromatic hydrocarbons tested caused irritant reactions. The alcohols and acetone gave no reaction. White spirit was as irritant as trichlorethylene. Thinners were less irritant, around the level of the 1% SLS control reaction. The 4-day experimental design is a convenient and suitable animal model for screening irritant potential, and provides information relevant to the pathogenesis of irritant contact reactions.     

Acute irritation thresholds in subjects with Type I – Type VI skin

It has long been recognized that human skin can be subdivided into simple categories based on their sensitivity to sunlight - from Type I, never tans, always burns, to Type VI, marked constitutive pigmentation. There is also evidence that the more readily sunburnt type of skin is also more susceptible to the effect of irritants. In the present work, the irritancy threshold for sodium lauryl sulfate (SLS) has been assessed using a recently described 4-h acute skin irritation patch test. A total of 110 subjects covering all 6 skin types were examined and their threshold for acute irritancy defined as the lowest concentration of SLS, applied under 4-h occlusion, which would induce a clinically detectable irritant response. The SLS dose response generated using a range of concentrations (0.1%-20%) demonstrated that there was no significant difference between the groups under these test conditions. Even for Type VI skin ( n =25), the dose-response curve fell within the general pattern. These results reinforce the general applicability of predictions of acute irritant potential made in groups of human volunteers.     

Efficacy of skin barrier creams

2 barrier creams (BC) were evaluated against the anionic detergent sodium lauryl sulphate (SLS) using a new human test model. In the repetitive irritation test (RIT) on human skin, the irritant SLS is applied to the ventral forearm of Healthy volunteers daily for 2 weeks. 1%, 5%, and 10% SLS is exposed to the skin for 30 min. using a glass cup 2.5 cm in diameter. The BC is applied 30 min before the irritant. Cutaneous irritation is assessed on a score for erythema (0 to 5+), and quantified by various biophysical techniques: transepidermal water loss (TEWL) by evaporimetry, skin blood flow volume (BFV) by laser-Doppler velocimetry, and -skill colour bi colorimetry (La^* value). 10 subjects were tested with SLS on one forearm without pretreatment (control) and with Taktosan Salbe as BC on the other forearm. A 2nd panel of 10 subjects was tested in the same way with SLS and Marly skin as BC. Taktosan Salbe was extremely effective in reducing the irritation by SLS: there were significant differences regarding all lest parameters for 10% SLS in the 2nd week. The most differentiating parameter was TEWL, revealing statistical differences as early as the 1st week for 10%. SLS and Taktosan Salbe, while the least differentiating sensitivity was found for La^*. In contrast, there was no significant suppression of irritancy in any parameter with Marly skin, either in the 1st week or in the 2nd week with any concentration of SLS. The results show the differentiating potential of the model developed. Results obtained with the previously described animal model are confirmed- Noninvasive biophysical techniques, particularly TEWL measurements, might be extremely valuable in identifying new active ingredients of BC.     

Effect of skin barrier competence on SLS and water-induced IL-1alpha expression

For screening of a potential irritant it is essential that an early marker for irritation should be chosen which could be detected before the physiological signs of irritation occur. Interleukin 1 alpha (IL-1alpha) is widely accepted as such a marker in both in vivo and in vitro test systems. In this study, we have determined the mRNA levels of IL-1alpha in the epidermis after topical application of sodium dodecyl sulphate (SLS) in both a commercially available epidermal kit (EpiDerm) and in excised skin. Furthermore, we have determined the effect of water, the vehicle for SLS, on IL-1alpha mRNA levels. Topical application of water to excised skin increases IL-1alpha mRNA levels sixfold in the epidermis whereas topical application of water to EpiDerm cultures did not alter IL-1alpha mRNA levels. This is explained by the finding that EpiDerm cultures have a sub-optimal barrier function when compared with excised skin - topical application of SLS was clearly toxic at much lower concentrations in EpiDerm cultures (0.2% SLS) than in excised skin (5% SLS). Also caffeine penetration was 10-fold higher through EpiDerm cultures than through the excised skin. Therefore, incubation of control EpiDerm cultures at 100% humidity effectively mimics topical exposure to water. An additional increase in IL-1alpha mRNA levels observed between topical application of water and SLS is similar (about threefold) in both experimental systems. In conclusion, in vitro reconstructed epidermis models, such as EpiDerm, can be used as a predictive model for irritancy screening. However, great care should be taken when interpreting the results due to the fact that EpiDerm cultures do not have a competent barrier function and therefore lower irritant concentrations are required than in in vivo or ex vivo studies in order to induce cytotoxic effects. Furthermore, the irritant effects of the vehicle should not be neglected. Our results show clearly that the topical application of water to excised skin results in increased levels of IL-1alpha mRNA in the epidermis. This is a cytokine that is widely used as an early marker for skin irritation.     

Artificial disruption of skin barrier prior to irritant patch testing does not improve test design

BACKGROUND: Irritant patch testing is often performed as a 24- or 48-h occlusive patch test with low concentrations of sodium lauryl sulphate (SLS). OBJECTIVES: The aim of this study was to investigate potential ways to shorten this test procedure and obtain precise test results. PATIENTS AND METHODS: Thirty-six healthy volunteers underwent irritant patch testing with different pretreatments (PT) of the test fields. Occlusive test chambers were applied on the upper back with SLS 0.5%, 1%, 2% and 5% in large Finn Chambers(R). The patches were removed after 4 and 24 h, respectively, depending on the concentration used. Test fields were pretreated as follows: PT 0, field without any PT (control); PT 1, prick with lancet; PT 2, prick with test stamp; PT 3, scratch with lancet; PT 4, incision with standardized incision instrument (0.1-0.2 mm depth). Skin reactions were evaluated by transepidermal water loss (TEWL), skin erythema and skin hydration and as well by a visual score (VS) at 4, 24 and 72 h. RESULTS: Our data show an obvious distinction between PT 0-2 and PT 3-4 at all measurement methods. The average TEWL values with PT 3-4 were higher than those with PT 0-2, especially on the 4-h course. This distinction may derive from the shape and size of the skin impairment achieved by PT 3-4, leading to a mechanical barrier disruption. However, SLS may infiltrate directly into deeper skin layers supported by capillarity. Consequently, no or little penetration through the epidermis and interaction with its structures occurs, which is responsible for irritant skin reactions. The SLS dose in the upper skin layers is therefore lower at these PTs. The lower remaining dose of SLS also explains this distinction, especially for the VS. Additionally, there are presumed reactions in deeper layers of the epidermis and dermis at PT 3-4. CONCLUSIONS: In summary, all data suggest a different reaction pattern from the classical irritant response. Therefore, application without any PT seems to be best suited for irritancy skin testing, especially for visual assessment. PTs prior to irritant patch testing have been shown to be unjustifiable.     

In vitro evaluation of skin sensitivity of povidone-iodine and other antiseptics using a three-dimensional human skin model

Povidone-iodine (PVP-I) is an antiseptic which has been widely used in various fields. It was reported to have a weaker skin irritancy than other antiseptics in the Draize skin irritation test using rabbits. Recent increased concern for animal welfare requires us to use skin models in the tests as an alternative to animal testing. Actually, there are some skin models already commercialized, which are available to evaluate skin irritancy caused by e.g. chemical reagents, cosmetics or medicines. In this study, we evaluated the potential of a PVP-I solution and other antiseptics to cause irritation using a cultured human skin model (three-dimensional skin model) under conditions similar to clinical use. This skin model has two layers like a real skin, such as the dermis and epidermis which includes the cornified layer. For the evaluation of skin irritancy in this model, cell viability was evaluated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay as an endpoint. Antiseptic formulations such as benzalkonium chloride (BAC), benzethonium chloride (BEC), chlorhexidine gluconate (CHG) and alkyldiaminoethylglycine hydrochloride (AEG) were used in this study. As a result, PVP-I showed a significantly weaker skin irritancy compared to the other antiseptics. The present in vitro study results revealed a correlation with the results of previously conducted in vivo skin irritancy tests using rabbits.     

Patterns of activation and inhibition of fibrinolysis in the normal skin of rat, guinea pig, and rabbit.

The distribution and intensity of fibrinolytic activity and of inhibitors of fibrinolysis in the normal skin of the rat, guinea pig and rabbit were studied with histochemical techniques. Rat skin exhibited the highest overall fibrinolytic activity and rabbit skin the lowest, with guinea-pig intermediate. The distribution of fibrinolytic areas differed in the different species. The fibrinolytic activity was caused by an activator of plasminogen related to the blood vessels or in some instances (mainly in the rabbit) to the epidermis. The ability to inhibit plasmin was highest in guinea pig skin and lowest in rat skin, with rabbit skin intermediate. In all 3 species the inhibition was related chiefly to the muscular layer. Epidermis was an additional source of inhibition.     

A novel in vivo model in guinea pigs for dry skin syndrome

Background/aims: The lack of a suitable, validated animal model for the comparison of the pharmacological effectiveness of known and potential moisturizers in the treatment of “dry skin syndrome” led us to develop such an in vivo model. Methods: “Dry skin syndrome” was induced in guinea pigs by daily application of 2% sodium lauryl sulphate (SLS) in deionized water on one of the two shaved flanks for three consecutive days. After ascertaining skin dryness, that side was treated with an agent for 6 days. The in vivo humectant effect was measured by a Corneometer CM 825^®, erythema was measured by a Mexameter MX 16^®. In some cases histological studies were carried out. Results: The treatment with the 2% SLS led to a consistent “dry skin syndrome” for 2 weeks. Glycerol, Vaseline, urea and ammonium lactate treatments validated the model, since the Corneometer CM 825^® readings of the treated dry side was equal to that of the control untreated side after 1 week of treatment. Mexameter MX 16^® measurements showed abolishment of the erythema by glycerol only. Histological study showed that SLS treatment creates acanthosis that is partially reversed by Vaseline and fully reversed by glycerol treatment. Conclusion: The guinea pig dry skin model is a relevant model of the human “dry skin syndrome”. The instrumental results combined with the histological findings indicate that erythema measurements are relevant for the determination of curative effect.     

Evaluation of skin irritancy of sodium lauryl sulphate: a comparative study between the replica method and visual evaluation

For 20 years, using the replica method, we have evaluated the Stun irritancy of about 10.0UO commercial products which come into contact with the skin. In this method, test substances are usually applied on the flexor side of the upper arm for 24 h by semi-open patch test. Subsequently, skin replicas are taken and skin irritancy is evaluated microscopically. In the semi-open patch test, test substances are not completely occluded as in the closed patch test. Thus, this method is less invasive than the closed patch test method to the tested subjects, In this study, we investigated the sensitivity of microscopic scoring (MS) of the replica and visual scoring (VS) of the skin. Sodium lauryl sulphate (SLS) at 0.02%, 0.05% and 0.2% was applied on 20 subjects' upper arms., using closed and semi-open methods. In both the closed patch test and the semi-open patch test, the value of MS correlated with the concentration of SLS, while VS did not show such a clear correlation. In addition, we compared 2 clinical tests for skin irritancy which are commonly performed in Japan: VS of 48 h closed patch test reaction on the subjects' upper bucks and MS of 24 h semi-open patch test reaction on the subjects' upper arms (replica method). MS on the upper arms resulted in a constant score, regardless of the location of application, while VS on the upper back produced results which differed widely depending on the location. Thus, the replica method is B useful clinical test for skin irritancy. because it is sensitive, reproducible and non-invasive.     

Topical D-vitamins: multiparametric comparison of the irritant potential of calcipotriol, tacalcitol and calcitriol in a hairless guinea pig model

The irritant potential of calcipotriol. 1α.24-diliydroxy vitamin D₃, (tacalcitol) and 1α, 25-dihyd rosy-vitamin- D₃ (calcitriol) was compared in a hairless guinea pig model. Randomized, occlusive patch testing for 2 days was used. Each group of X animals was tested simultaneously with the 3 substances and a placebo vehicle. 3 dose levels i.e. 500 μg/ml, 50 μg/ml and 5 μg/ml were used, Test sites were evaluated at day 2 (2 h after removal of the patches) and again at day 3. Evaluation was blinded and based on a multiple parameter assessment of skin irritancy. comparing clinical scoring. skin perfusion using high resolution laser Doppler image scanning, skin colour (a*, Minolta ChromaMeter) and skin thickening (20 MHz ultrasound) indicating oedema. Skin biopsies were taken for histological preparation and assessment of epidermal hyperplasia. No difference was observed between the irritant potential of calcipotriol, tacalcitol and calcitriol bused on clinical scoring as well as objective non-invasive measuring techniques. All 3 substances showed a dose-dependent and equal increase in clinical irritation score. cutaneous blood flow, skin colour and epidermal hyperplasia. The cutaneous inflammatory reaction was dominated by vasodilation and increased cutaneous perfusion. Oedema formation was only seen ill the highest dosages tested. Skin barrier damage was not induced as TEWL remained unaffected. The hairless guinea pig appears a valid model to test irritancy of topical D-vitamins since the same profile of irritancy was previously established in humans for 2 of the compounds tested. calcitriol and calcipotriol.     

The efficiency of humectants as skin moisturizers in the presence of oil

Background/aims: The research on the treatment of “dry skin syndrome” is hampered by the lack of a suitable animal model. Formerly, we developed a validated guinea pig in vivo model in which the dry skin syndrome persists at least for 1 week. We can, therefore, compare the pharmacological effectiveness of known and potential moisturizers for the treatment of dry skin syndrome. Our aim is to study whether the moisturizing efficiency of humectants depends on the solvents in which they are dissolved. Methods: “Dry skin syndrome” was induced on the shaved skin on one side of guinea pigs by daily application of 2% sodium lauryl sulphate in deionized water (SLS) for 3 days. The other shaved side was used as control. After ascertaining skin dryness, that side was treated for 6 days with glycerol or 1,2‐hexanediol in different solvents: water, or medium chain triglycerides (MCT) or mixtures of MCT with isopropyl alcohol in different proportions. Measurement of the in vivo moisturizing effect was carried out by a Corneometer CM 825^®; erythema was measured by a Mexameter MX 16^®. Results: Treatments with glycerol (1M) in water reversed the skin dryness shown by both instruments. When dissolving glycerol in MCT, no moisturizing effect was found, probably because glycerol does not dissolve in the oil. No moisturizing effect was found with different combinations of glycerol in the mixtures of MCT and isopropyl alcohol. No moisturizing effect was found using another polyol moisturizer: 1,2 hexanediol (1M) dissolved in MCT oil. Glycerol or 1,2‐hexanediol abolished the erythema only when they were dissolved in water alone. Conclusion: Polyol moisturizers such as glycerol or 1,2‐hexanediol do not act in the presence of oils against the sodium lauryl sulphate‐induced dry skin in our guinea pig model. Since in an oil‐in‐water (O/W) emulsion, the water evaporates within several minutes, one has to question the ability of moisturizing emulsions to treat dry skin. In such instances, one cannot draw conclusions about the moisturizing efficiency of the preparation merely from the presence of the humectant. One has to study the effect of the finished preparation.     

Bullous pemphigoid antibodies. Human skin as a substrate for indirect immunofluorescence assay

Human skin, the target organ for bullous pemphigoid (BP) antibodies, is thought to be a less sensitive substrate for the indirect immunofluorescence assay of BP antibodies than monkey or guinea pig esophagus. To examine the reasons for this puzzling phenomenon, we compared the titers of BP antibodies obtained when human skin, monkey, and guinea pig esophagus were used as substrates. We found the titers of BP antibodies obtained with human skin from sites commonly involved in BP (flexor arm, flexor thigh, popliteal fossa) were as high and usually higher than those obtained with monkey and guinea pig esophagus. In contrast, much lower titers were obtained with human skin from sites rarely involved in the disease (scalp, face, extensor arm). These findings suggest that human skin as a substrate is at least as sensitive as monkey or guinea pig esophagus for the indirect immunofluorescence assay of BP antibodies when the skin is obtained from regions on the body commonly involved in BP.     

8-甲氧补骨脂素乳膏皮肤刺激性和致敏性实验研究

目的观察8-甲氧补骨脂素乳膏对动物皮肤的毒性作用。方法用健康新西兰兔进行皮肤刺激性试验;用健康豚鼠进行皮肤刺激和致敏试验。结果8-甲氧补骨脂素乳膏对新西兰兔完整皮肤和破损皮肤无刺激性,对豚鼠完整皮肤无致敏作用。结论8-甲氧补骨脂素乳膏皮肤局部用药对实验动物具有较好的安全性。     

Basal transepidermal water loss, skin thickness, skin blood flow and skin colour in relation to sodium-lauryl-sulphate- induced irritation in normal skin

The influence of basal transepidermal water loss (TEWL), skin thickness, blood flow and skin colour on susceptibility to sodium-lauryl-sulphate(SLS)-induced irritant contact dermatitis was studied in 70 healthy volunteers. SLS 0.5% was applied as a patch test. For assessment of basal values and skin response to SLS, bioengineering methods were used: TEWL was measured by an evaporimeter, skin thickness by ultrasound A-scan, blood flow by laser Doppler flowmetry, and skin colour by a colorimeter, using the L*a*b* system of the Commission Internationale de l'Eclairage (CIE). By use of multiple regression analysis, it was demonstrated that basal TEWL was substantially related to skin susceptibility to SLS, high basal TEWL predicting an increased susceptibility to SLS. Also increased light reflection from the skin, indicating a 'fair' skin, was found to be associated with increased susceptibility to SLS.     

Response of skin to ammonium persulphate.

In order to investigate the histamine liberating actions of ammonium persulphate, skin slices from three species (guinea pig, rat and monkey) were incubated in vitro with concentrations of ammonium persulphate ranging from 1 to 1000 microgram/ml. None of these concentrations released significant amounts of histamine in guinea pig or monkey skin. In the rat the highest concentration (1000 microgram/ml) released 20-24% of the histamine content of the skin, but the intensitivity of this response to cooling indicates a non-specific "toxic" action on mast cells. By contrast a known chemical histamine liberator, compound 48/80, released significant amounts of histamine from skin at much lower concentrations in all three species. Ammonium persulphate is clearly not a potent histamine liberator. Ammonium persulphate dermatitis is presumably a result of increased sensitivity of skin mast cells, due to immunological or other factors, in susceptible individuals.     

Changes in skin barrier function following long-term treatment with moisturizers, a randomized controlled trial

BACKGROUND: Moisturizers are commonly used by patients with dry skin conditions as well as people with healthy skin. Previous studies on short-term treatment have shown that moisturizers can weaken or strengthen skin barrier function and also influence skin barrier recovery. However, knowledge of the effects on skin barrier function of long-term treatment with moisturizers is still scarce. OBJECTIVES: To investigate the impact of long-term treatment with moisturizers on the barrier function of normal skin, as measured by transepidermal water loss (TEWL) and susceptibility to an irritant, and to relate those effects to the composition of the designed experimental moisturizers. METHODS: Volunteers (n = 78) were randomized into five groups. Each group treated one volar forearm for 7 weeks with one of the following preparations: (i) one of three simplified creams, containing only a few ingredients in order to minimize the complexity of the system; (ii) a lipid-free gel; (iii) one ordinary cream, containing 5% urea, which has previously been shown to decrease TEWL. The lipids in the simplified creams were either hydrocarbons or vegetable triglyceride oil, and one of them also contained 5% urea. After 7 weeks, treated and control forearms were exposed for 24 h to sodium lauryl sulfate (SLS) using a patch test. TEWL, blood flow and skin capacitance of both SLS-exposed and undamaged skin were evaluated 24 h after removal of patches. Additionally, a 24-h irritancy patch test of all test preparations was performed on 11 volunteers in order to check their possible acute irritancy potential. RESULTS: Changes were found in the barrier function of normal skin after 7 weeks of treatment with the test preparations. The simplified creams and the lipid-free gel increased TEWL and skin response to SLS, while the ordinary cream had the opposite effect. One of the simplified creams also decreased skin capacitance. All test preparations were shown to be nonirritant, both by short-term irritancy patch test and by measurement of blood flow after long-term treatment. CONCLUSIONS: Moisturizers influence the skin barrier function of normal skin, as measured by TEWL and susceptibility to SLS. Moreover, the effect on skin barrier function is determined by the composition of the moisturizer. The ingredients which influence the skin barrier function need to be identified, and the mechanism clarified at the molecular level.     

Nonanoic acid--an experimental irritant

Irritant contact dermatitis is defined as a non-immunological skin reaction following exposure to various chemical, mechanical and physical factors. It is known that the skin response to irritants depends on the irritant applied and differs between chemically different irritants. Sodium lauryl sulfate (SLS) is an anionic detergent and the most frequently used substance in experimental irritant contact dermatitis. In 1980, it was suggested that nonanoic acid (NNA) could be used as a positive control when patch testing. Since then, NNA has been used as an experimental irritant in several studies and has been used as a chemically different substance compared to SLS. The present article presents a review of the application of NNA in studies on skin irritancy and experimental irritant contact dermatitis.     

An interlaboratory evaluation of the Buehler test for the identification and classification of skin sensitizers

The correct identification of potential skin sensitizers is an essential first step in enabling a proper risk assessment to be made and to permit the implementation of appropriate risk management practices designed to avoid the induction of sensitization, Consequently, regulatory guidelines around the world demand that new substances are evaluated to assess their skin sensitization potential. There are two guinea pig test methods which are generally recognised, the guinea pig maximisation test (GPMT) and the occluded patch test described by Buehler. In different countries, one procedure seems to be more prevalent and acceptable to regulatory authorities than the other. Notably, in the European Union, the latest revision of the Annex V (Directive 92/32/EC) Test Method for skin sensitization asks that justification should be given in the situation where the notifi this paper, the validity of the Buehler protocol in the context of European legislation is critically examined. Results from two laboratories are collated. showing that the method can identify significant contact allergens, particularly those which would he registered formally as such according to European legislation. It is demonstrated that minor methodological variations can he tolerated without compromising tesi sensitivity, hut it is recommended that suitable positive control testing is the best way to ensure proper test conduct.