The tandem repeated irritation test: a new method to assess prevention of irritant combination damage to the skin

The effect of a protective cream was tested in a new tandem repeated irritation test with tandem application of 0.5% sodium lauryl sulphate (SLS) and undiluted toluene. The irritants were applied twice daily for 30 min to the ventral forearms of 20 volunteers. Irritant cutaneous reactions were quantified by a visual score, transepidermal water loss, chromametry and skin capacitance. Concurrent application of SLS/toluene induced stronger reactions than those caused by twice daily application of each irritant on its own. A protective effect of the protective cream was obtained against all treatment combinations and was significant for SLS/SLS (p < or = 0.01) and SLS/ toluene (p < or = 0.05). Our results indicate that the tandem repetitive irritation test has great potential in the evaluation of skin care products to prevent irritant contact dermatitis.     

Comparison of human back versus arm skin region for its suitability to test weak irritants

Background/aims: Large-scale comparative screening of der-matological and cosmetic products require non-invasive bioengineering methods which give reproducible results that can be statistically analyzed. To determine the most suitable test region for testing irritating substances, the response of the skin to the weak irritant sodium lauryl sulphate (SLS) was measured on standardized locations on the forearm and back. Furthermore, using 3 bioengineering methods, we aimed to establish the most sensitive method to measure skin response to SLS.
Methods: The baseline measurements of transepidermal water loss (TEWL), cutaneous blood flow (CBF) and skin colour a*-value were performed at 6 standardized locations on the volar forearms and at 12 locations on the back on 2 consecutive days, day 1 and 2, in 9 healthy volunteers. On day 2, 9 out of the 18 sites were exposed to 0.5% SLS by occlusive 24-h patches and the skin response was evaluated on days 3 and 4.
Results: For TEWL, CBF and a*-value, no significant regional variations were found within individuals at any time on the arm locations and on the back locations. However, significant inter-individual variation was found. At the back on day 4, a slightly higher skin response to SLS was found compared to that found on the forearms as measured by TEWL and the a*-value. Of the 3 bioengineering methods used, TEWL was the most sensitive method to detect cutaneous response to the weak irritant used. TEWL measurements on days 1 and 2 performed at the 6 standardized locations on the volar forearms inside and outside a specially constructed air convection protection box, showed no relevant differences in measured TEWL values.
Conclusion: Both the forearms and the back were found to be suitable sites for irritancy testing. Since considerable differences in mean reactivity between subjects have been observed the use of large numbers of subjects in predictive irritant patch testing is required.     

Acute irritant contact dermatitis: recovery time in man

Our understanding of the details of the recovery time of acute irritant contact dermatitis (ICD) is limited. We examined skin reactivity to a model surfactant, sodium lauryl sulfate (SLS). on previous acute ICD and normal sites over time with visual grading and noninvasive instruments. Acute ICD was induced on the upper arms of 18 volunteers (aged 30 to 51 years) by occluded application of 1% SLS for 24 h. Previous ICD and normal sites were provoked by occluded application of 2% or 7.5% SLS 30 min daily 4 consecutive days. Skin reactivity was assessed daily by visual erythema scoring (VES), transepidermal water loss (TEWL), skin color reflectance (SCR) and electrical capacitance (EC). Skin function of previous ICD sites assessed, by VES, TEWL. SCR, and EC did not normalize until 2 weeks later: all parameters of previous ICD returned to normal after 3 weeks. While skin reactivity to 2% und 7.5% SLS showed no differences between previous ICD and normal sites at 4 weeks, differences of irritant reactivity especially 7,5% SLS between previous ICD and normal sites were significant at 3 weeks post-provocation. Our results demonstrate that irritation evaluated with irritant provocation was long-lasting, even though skin functional parameters assessed by various bioengineering instruments returned to normal. Complete recovery of skin function including irritability after acute ICD induced by 1% SLS was achieved approximately 4 weeks later. The date were generated with a model surfactant: it remains to be determined whether similar responses will be noted with chemicals of different physicochemical properties.     

Assessment of anti-inflammatory activity of Poria cocos in sodium lauryl sulphate-induced irritant contact dermatitis

OBJECTIVE: In the present study, we evaluated the anti-inflammatory activity of Poria cocos (PoCo) on experimentally induced irritant contact dermatitis (ICD) in a repeated sodium lauryl sulphate (SLS) irritation model. METHODS: The anti-irritative effect of PoCo was evaluated with a visual score and quantified by non-invasive bioengineering methods, namely chromametry and transepidermal water loss. Three concentrations of PoCo in base cream DAC (amphiphilic emollient; German pharmacopoeia) were tested in a 4-day repetitive irritation test using SLS. RESULTS: A statistically significant anti-inflammatory activity was observed for PoCo by all three methods when applied in parallel to the induction period of ICD. Application of PoCo after induction of ICD once a day for 5 days, starting just at the end of 4 days, was without any effect. CONCLUSION: An anti-inflammatory efficacy of PoCo on the elicitation phase of the ICD induced by repeated SLS test could be observed and quantified by three independent, non-invasive biophysical assessment parameters. This effect can be explained by its influence on pro-inflammatory enzymes, namely phospholipase A2.     

Effect of different moisturizers on SLS-irritated human skin

Moisturizers are widely used to treat irritant contact dermatitis (ICD). Their use is, however, not well-documented and standardized models for testing skin care products are needed to acquire documentation of their efficacy. The present study was undertaken to evaluate the effect of 6 commonly-used moisturizers on the recovery of irritated human skin. No commercial interests were involved in the study. 36 healthy volunteers had patch tests with SLS 0.5% applied on their forearms/upper arms for 24 h. After irritation of the skin, all volunteers had a moisturizer applied on one forearm/upper arm, respectively, 3 x daily for the following 5 days. The other forearm/upper arm served as an untreated control. Each moisturizer was tested on 12 volunteers and each volunteer tested 2 moisturizers at the same time. Evaluation was done on days 1, 3 and 8 by transepidermal water loss, electrical capacitance, laser Doppler flowmetry, DermaSpectrometry and clinical scoring. All 6 moisturizers were found to accelerate regeneration of the skin barrier function when compared to irritated non-treated skin. The most lipid-rich moisturizers improved barrier restoration more rapidly than the less lipid-rich moisturizers. We suggest this experimental model for further moisturizer efficacy testing.     

The hairless guinea-pig as a model for treatment of cumulative irritation in humans

BACKGROUND: The effect of six skin-care formulations (SCFs) on experimentally induced cumulative irritation was studied in hairless guinea-pigs (HLGPs) and in human volunteers (HVs). The formulations were a basic cream, a carbomer cream and four modifications of the carbomer cream, containing either 10% isopropyl palmitate (IPP cream), 10% glycerol (glycerol cream), 19.5% canola oil (canola oil cream) or 0.5% (-)-alpha-bisabolol (bisabolol cream). METHODS: In HLGP, irritant dermatitis was induced with 30 min daily exposure for 4 days to 0.5% sodium lauryl sulfate aq. (SLS). In HVs, irritant dermatitis was induced with 10 min daily exposure for 5+4 days (no irritation on weekends) to 3% SLS aq. on the right and 30% nonanoic acid (NON) in n-propanol on the left volar forearm. Clinical scoring was performed daily; evaporimetry (total epidermal water loss (TEWL)), hydration and colorimetry were measured at baseline (day 0) in the middle and at the end of treatment. Treatments were applied twice daily. The basic cream and the IPP cream were excluded from testing in HLGP because they were known from previous studies to be irritant in HLGP, while all formulations were known to be equally and well tolerated locally in humans. RESULTS: All formulations worsened the skin irritation in HLGP: the glycerol cream the least, the canola oil cream the most, while the bisabolol cream and the carbomer cream were indistinguishable. In humans, the glycerol cream was better than 'No Treatment' after cumulative irritation with both SLS and NON. The basic cream was better tolerated in humans than was expected from previous testing in HLGPs. CONCLUSION: In conclusion, the results from the studies in HLGPs and HVs are in agreement with regard to ranking of the SCFs. Further, the glycerol cream showed a positive treatment effect on both SLS- and NON-irritated skin in HVs.     

Barrier recovery and influence of irritant stimuli in skin treated with a moisturizing cream

Moisturizers are used daily by many people to alleviate symptoms of clinically and subjectively dry skin. Recent studies suggest that certain ingredients in creams may accelerate the recovery of a disrupted barrier and decrease the skin susceptibility to irritant stimuli. In the present single-blind study, a moisturizing cream was tested for its influence both on barrier recovery in surfuctant-damaged skin and on the susceptibility of normal skin to exposure to the irritant sodium lauryl sulphate (SLS). Parameters measured were transepidermal water loss (TEWL) and skin corneometer values, indicating degree of hydration. Treatment of surfactant-damaged skin with the test cream for 14 days promoted barrier recovery, as observed as a decrease in TEWL. Skin corneometer values also normalized more rapidly during the treatment. In normal skin, use or the test cream significantly reduced TFWL after 14 days of treatment, and irritant reactions to SLS were, significantly decreased. Skin corneometer values increased after only 1 application and remained elevated after 14 days. In conclusion, the accelerated rate of recovery of surfactant-damaged skin and the lower degree of SLS-induced irritation in normal skin treated with the test cream may be of clinical relevance in attempts to reduce contact dermatitis due to irritant stimuli.     

Chronic irritant contact dermatitis: recovery time in man

Chronic irritant contact dermatitis (ICD) is a common skin disease, especially in the workplace, but determining the recovery time of chronic ICD is not easy. To measure the recovery time of chronic ICD, we examined the skin reactivity to a model surfactant, sodium lauryl sulfate (SLS), on previous chronic ICD and normal sites by visual grade and non-invasive instruments. Chronic ICD was induced on the forearms of 10 volunteers (aged 23 to 43 years) by occluded application of 1% SLS for 30 min on 5 consecutive days each week for 3 weeks. Previous ICD and normal sites were provoked by the occluded application of 7.5% SLS for 30 min daily on 4 consecutive days, 2, 5 and 10 weeks after induction. Skin reactivity was assessed daily by awarding visual erythema scores, visual scale scores and measuring transepidermal water loss, skin color reflectance, and electrical capacitance. Skin reactivity of previous chronic ICD sites to SLS showed hyperreactivity compared to normal sites even after the 10th week post-induction.     

Beneficial effects of a skin tolerance-tested moisturizing cream on the barrier function in experimentally-elicited irritant and allergic contact dermatitis

In experimentally-induced irritant (ICD) and allergic (ACD) contact dermatitis, an oil-in-water (o/w) cream was applied to investigate its effects on a disturbed barrier function compared to untreated physiological barrier repair. Transepidermal water loss (TEWL) measurements were performed. Before the start of the experiments, the skin tolerance of the cream was examined, revealing the non-irritating characteristics of the ingredients and the absence of any contact allergic patch test reaction. In the ICD study, sodium lauryl sulfate (SLS) patches were applied to the forearms of young female volunteers. Consequently, it was observed that repeated cream application (14 days, 2x/day) significantly improved the TEWL of SLS-damaged skin, leading to a complete recovery on day 15. In the ACD study, disruption of skin barrier function was obtained by a nickel-mediated contact allergy patch (CAP) test. The cream was then applied 2x/day for 4 consecutive days. Assessment of TEWL clearly showed that recovery of the disrupted skin significantly improved after cream application in comparison to untreated barrier repair.     

Cutaneous sodium lauryl sulphate irritation potential: age and regional variability

To investigate differences in response to irritation according to age and site seven young-adult and eight elderly females were exposed to 0.25% sodium lauryl sulphate (SLS) under patch-test occlusion for 24 h. Ten anatomical regions were tested: forehead, upper arm, volar and dorsal forearm, postauricular, palm, abdomen, upper back, thigh, and ankle. The skin responses were evaluated 24 h post-patch removal by visual scoring and by transepidermal water loss measurements (TEWL). Non-treated symmetrical anatomical regions served as controls. SLS induced a mild erythematous reaction on most anatomical regions except the palm and TEWL was significantly increased as compared with controls. The elderly group demonstrated significantly less susceptibility to SLS-induced irritation for most regions of the body as indicated by visual scores and TEWL measurements. In addition, evaluation of the stratum corneum water content following SLS irritation demonstrated lower responses in the old age group for most regions. The thigh had the highest reactivity and the palm the lowest, in both age groups. These data suggest that young adult skin is more sensitive to SLS than old skin and that SLS irritation varies considerably with respect to region. Moreover, objective TEWL measurements seem to be a better indicator of irritant susceptibility, especially in the elderly, than clinical evaluation by visual scoring.     

Experimental irritant contact dermatitis due to cumulative epicutaneous exposure to sodium lauryl sulphate and toluene: single and concurrent application

In clinical practice, cutaneous exposure to a variety of irritants such as surfactants and solvents is frequent. Although the induction of irritant dermatitis by single irritants has been extensively studied in recent years, our knowledge of the effects of simultaneous application of different irritants is limited. Using non-invasive techniques for measurements of transepidermal water loss (TEWL) and skin colour reflectance, we quantified the irritant effects of single and concurrent application of 0.5% sodium lauryl sulphate (SLS) and undiluted toluene (TOL) in vivo. The irritants were applied twice daily for 30 min to the volar forearms of 20 volunteers. Repeated application of SLS and TOL induced an irritant reaction, as indicated by an increase in TEWL and skin redness. In contrast to SLS alone, the application of TOL alone induced only a moderate increase in TEWL, confirming previous results. Concurrent application of SLS/TOL and TOL/SLS induced significantly stronger reactions than those caused by twice daily application of each irritant on its own. Our results demonstrate that a mixed application of an anionic detergent and an organic solvent has an additive effect on skin irritation. It is suggested that pretreatment with SLS causes an increased susceptibility to TOL irritation and vice versa. Thus, the necessity for special precautions against skin absorption of TOL when handling detergents such as SLS is emphasized.     

Anti-inflammatory effect of pimecrolimus in the sodium lauryl sulphate test

Background Pimecrolimus is a calcineurin inhibitor used for the topical treatment of inflammatory skin diseases. We have shown previously that pimecrolimus cream is not effective on intact skin in the ultraviolet erythema test. Objective To test the anti‐inflammatory effect of pimecrolimus cream after damage of the skin barrier by sodium lauryl sulphate (SLS) in a randomised, placebo‐controlled, observer‐blinded study. Methods SLS (3% v/v) was applied under occlusion on the back of 36 healthy volunteers for 24 h. Subsequently, the test areas were treated for 24 h with pimecrolimus cream, 1% hydrocortisone in a hydrophilic ointment, and the vehicle alone over three consecutive days. One control area remained untreated. The erythema index and the transepidermal water loss (TEWL) served as readout parameters to assess the SLS‐induced skin irritation. Results Pimecrolimus cream and 1% hydrocortisone cream significantly reduced the SLS‐induced erythema. The two test preparations did not have a significant effect on the TEWL. Conclusion After damage to the skin barrier by SLS, pimecrolimus seems to penetrate into the skin as shown by a reduction of the irritation‐induced erythma. These data further support the notion that pimecrolimus is selectively effective in the treatment of skin disorders with an impaired function of the epidermal barrier.     

Air flow at different temperatures increases sodium lauryl sulphate-induced barrier disruption and irritation in vivo

BACKGROUND: Combined exposure to dry climatic conditions and local heat sources together with detergents represents a common workplace situation. These conditions may support the induction of chronic barrier disruption leading subsequently to irritant contact dermatitis (ICD). OBJECTIVES: To test the irritant and barrier disrupting properties of air flow at different temperatures and velocities. METHODS: Using noninvasive biophysical measurements such as transepidermal water loss (TEWL) (TM 210; Courage & Khazaka, Cologne, Germany) we assessed the effects of short-term exposure to air flow at different temperatures (24 degrees C and 43 degrees C) in combination with sodium lauryl sulphate (SLS) 0.5% on the skin of 20 healthy volunteers in a tandem repeated irritation test. Chromametry was used to control the accuracy of the SLS irritation model. RESULTS: In our study air flow alone did not lead to a significant increase in TEWL values. Sequential treatment with air flow and SLS led to an impairment of barrier function and irritation stronger than that produced by SLS alone. The two different air flow temperatures led to different skin temperatures but had no influence on permeability barrier function. CONCLUSIONS: Warm air flow has an additional effect on the SLS-induced barrier disruption in a tandem irritation test with sequential exposure to SLS/air flow. This combination is suspected to promote ICD in workplace and household situations, especially in short-term applications as tested in our model.     

Sequential application of cold and sodium lauryl sulphate decreases irritation and barrier disruption in vivo in humans

BACKGROUND: Irritant contact dermatitis (ICD) is one of the most frequent types of occupational dermatitis. Different factors are involved in the development of contact dermatitis. In the food-processing industry, the combined exposure to different irritants may be involved in the development of ICD. Few data have been published regarding the irritant potential of sodium lauryl sulphate (SLS) in combination with cold. OBJECTIVES: The present study was intended to analyse whether cold exposure and low skin temperature influence the development of ICD. METHODS: Twenty (part I) and 12 (part II) healthy volunteers were exposed twice daily for 4 days to SLS alone, different low temperatures alone (4 degrees C six times for 90 s with an interval of 20 s or 15 degrees C for 10 min) or a combination of cold and SLS (19.6 microL SLS 1% cm(-2), part I; or 52.6 microL SLS 0.5% cm(-2), part II) using the tandem repetitive irritation test. Irritant cutaneous reactions were measured by noninvasive biophysical methods with transepidermal water loss as a parameter for permeability barrier function and skin colour reflectance together with visual scoring as parameters for inflammatory reactions. RESULTS: Cold alone caused no significant skin reaction compared with untreated control. Exposure to SLS alone and SLS together with cold (independent of the applied temperature of 4 or 15 degrees C) twice daily induced a clear irritant reaction and barrier disturbance. Reactions did not differ whether SLS was applied before or after cold. Furthermore, 'tandem application' of cold and SLS diminished the barrier disruption and irritant reaction compared with SLS alone. CONCLUSIONS: We conclude that the application of cold may have a protective effect on the development of ICD, at least in our short-term model.     

Skin irritant reactivity following experimental cumulative irritant contact dermatitis

Despite the frequency of irritant contact dermatitis, very little is known about the duration of barrier function impairment following cumulative irritant contact dermatitis. We studied post-irritation irritant reactivity by assessing the response to SLS irritation in previously irritated sites. Cumulative irritant contact dermatitis was induced on the forearms of 15 volunteers aged 18 to 50 years by repeated occluded application of 0.5% SLS I h per day over 3 weeks. 3, 6 and 9 weeks later, previously irritated and unirritated control sites were challenged with 2% SLS under occlusion for 23 h. Irritation was assessed by visual scoring, transepidermal water loss (TEWL) as an indicator of epidermal barrier function, and capacitance as a parameter of epidermal water content. While no difference in irritant reactivity between pre-irritated and unirritated sites was observed 3 weeks following irritant contact dermatitis, there was a significant hyporeactivity of previously irritated skin as expressed by clinical scores, TEWL and capacitance at 6 and 9 weeks. Our results indicate that epidermal barrier function remains altered even 9 weeks after cumulative irritant contact dermatitis. With regard to patch testing, post-irritation hyporeactivity might be a cause of false-negative tests on previously irritated sites.     

Evaluation of skin susceptibility to irritancy by routine patch testing with sodium lauryl sulfate

Irritant patch testing with sodium lauryl sulfate (SLS) will become more and more a routine test determining skin susceptibility in men. Recently, it has been shown that for practical reasons, irritant SLS patch testing can take place on the back simultaneously with a routine allergic patch test to other contact allergens. However, SLS patch testing has mostly been performed on the forearm in studying experimental skin irritation so far. The aim of this study was to determine whether there is a relationship in skin response to aqueous SLS (0.125%; 0.25%; 0.5% and 1.0%) between the forearm and the back assessed by visual scoring and measurement of transepidermal water loss (TEWL). We found a pronounced reaction of the forearm compared to the back. TEWL values as well as visual scores correlated well with SLS concentration. There was also a high correlation in visual scoring between the forearm and the back. Based on test sensitivity and specificity we suggest a 48 hrs patch test for routine screening with 0.5% SLS on the forearm evaluated by TEWL measurement or visual scoring 24 hrs after patch removal. A mild erythema (scored as < or =1) is considered to be normal. If for practical reasons, the SLS patch is placed on the back simultaneously with the allergic patch test, 0.5% SLS may be sufficient, too. TEWL measurement so far provides a reliable method and will certainly be necessary for experimental studies on irritant skin reactions, particularly when different SLS concentrations are used. After a 48 hrs patch test with SLS 0.5% TEWL measurement should be performed at 72 hrs. A value of < or =31.6 g/m(2)hr seems to follow the normal distribution.     

Efficacy of corticosteroids in acute experimental irritant contact dermatitis?

Background/aims: Topical corticoids are used to treat irritant contact dermatitis (ICD) in humans. However, their clinical efficacy remains sub judice. This study was designed to assess the efficacy of low- and medium-potency corticosteroids on irritant dermatitis.
Methods: We induced an acute ICD via open application of sodium lauryl sulphate (SLS) on the hands of subjects. The dorsal side of hands was irritated with 10% SLS five times in one day. Once on day 1 and twice daily on days 2–5, 1% hydrocortisone, 0.1% betamethasone-17-valerate and vehicle cream (petrolatum) were applied subsequently. Visual grading, bioengineering techniques and squamometry were used to quantify skin response.
Results: Corticosteroids were found ineffective in treating the surfactant-induced irritant dermatitis when compared with the vehicle and with the untreated control.
Conclusion: The counterintuitive result (in a relatively realistic and robust model) should be interpreted with caution until verified with other irritants of varying physicochemical properties.     

Sodium lauryl sulfate irritant patch tests. II. Variations of test responses among subjects and comparison to variations of allergic responses elicited by Toxicodendron extract

Inflammation was induced on the forearms of volunteers by twenty-four closed patch tests to either the irritant 10% sodium lauryl sulfate (SLS) or Toxicodendron extract. Each chemical was tested at eight sites on the ventral forearms of each volunteer in order to assess the variability of response among test sites in individual subjects. Inflammation was assessed about 10 minutes after patch tests were removed. The degree of inflammation elicited by both Toxicodendron and SLS was variable among subjects, but variation among individual test sites was much more marked in subjects tested with SLS (p less than 0.002). The marked variability of responses to irritation that occur in any single subject may explain why irritant patch test responses do not reliably identify the irritation-prone individual.     

Applicability of an exaggerated forearm wash test for efficacy testing of two corticosteroids,tacrolimus and glycerol,in topical formulations against skin irritation induced by two different irritants

Background/Purpose: Alternatives to corticosteroids in the treatment of irritant contact dermatitis (ICD) are needed and may include glycerol and topical immunomodulators like tacrolimus. Because the efficacy of different treatments in experimentally induced ICD may vary depending on the irritant applied, we tested the efficacy of four anti‐irritant compounds using the two different irritants sodium lauryl sulfate (SLS) and nonanoic acid (NON). Methods: In a randomized, double‐blind, controlled trial, healthy volunteers were exposed to 5% SLS and 50% NON (the right and the left forearm, respectively) in a cumulative wash test. Induction of ICD was obtained by three daily washings for 7 days, followed by a maintenance phase with two daily washings for 12 days. Treatment (triamcinolone acetonide, clobetasol propionate, tacrolimus and glycerol ointment) was started at day 7 and applied immediately after washing. Vehicle and no treatment served as the control. Reactions were evaluated clinically and instrumentally. Results: No treatments were significantly better than the other treatments and controls. There was a tendency toward a dose‐dependent response to corticoid treatment, and a trend toward worsened irritancy by tacrolimus on SLS‐irritated skin. Explained variance in the experiment by anova revealed a very small effect of treatments compared with an immense and significant subject effect. Conclusion: No claims of effective anti‐irritant properties for any of the ointments can be maintained. Application of the present wash test as a tool for anti‐irritant efficacy testing may be complicated by the small observed variance explained by treatment.     

Multicentre study for the development of an in vivo model to evaluate the influence of topical formulations on irritation

Although skin protective products to prevent irritant skin reactions are in wide use, neither standardized test models to prove differences in efficacy exist, nor has the quality or the reproducibility of results been evaluated in a multicentre approach. This should be mandatory when developing or testing skin care products. Therefore, we have designed a multicentre study in an approach to find a standardized test procedure for the evaluation of skin protective products. In this irritation study, a repeated short-time occlusive irritation test (ROIT) with a standardized protocol has been evaluated in 2 phases (12 days and 5 days protocol) in 4 (n=20) respectively 6 (n=33) skilled centres. The skin reaction was induced by 2 irritants (0.5% aq. SLS and toluene, 2x a day for 30 min). Its modification by 3 different cream bases with different hydrophilicity was analyzed. The irritation was monitored by bioengineering methods (TEWL measurement, colorimetry) and by clinical scoring. The evaluation showed that significant results could already be achieved with the 5-day protocol. Furthermore, in spite of the expected inter-centre variations due to heterogeneity of the individual threshold of irritation, interpretation of clinical score, and inter-instrumental variability, the ranking of the vehicles regarding reduction of the irritant reaction was consistent in all centres.