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Skin Research and Technology 2001; 7: 49–55 Printed in Denmark. All rights reserved Copyright C Munksgaard 2001 Skin Research and Technology ISSN 0909-752X Surfactant irritation: in vitro corneosurfametry and in vivo bioengineering Bernard Gabard, Eric Chatelain, Elsbeth Bieli and Stephan Haas Biopharmacy Department, Spirig Pharma Ltd., Egerkingen, Switzerland Background/aims: Irritant reactions to surfactants, cleansing products, soaps and detergents are common in clinical and occupational dermatology. Mildness has become a major benefit claimed, and testing for mildness now ranks among the first concerns of the manufacturing industry. A wealth of publications deals with this problem, trying to improve the methodology, reduce the costs of testing and facilitate decision-making. Differences in vivo can be measured clinically and/or instrumentally. This is difficult, as commercially available products are generally safe to use and none are harsh in the absolute sense. Methods: Nineteen different products (syndets, shampoos, personal cleansers), all claiming to be mild, were tested in vitro by a newly introduced method, corneosurfametry. For evaluating the aggressiveness of the products, the calculation of an index of irritation (IOI) was proposed. A concentration-effect curve of sodium lauryl sulfate (SLS) as standard and model surfactant was obtained. Some of the products were further tested in vivo with a flex wash test and with a soap chamber test and compared to SLS. Bioengineering methods (transepidermal water loss TEWL, skin color) were used to evaluate the results. Results and Conclusions: The results of the corneosurfametry allowed us to classify the products in three categories, with increasing aggressiveness towards the stratum corneum, according to their IOIs. The in vivo tests were not able to discriminate between the products, but ranks from the results of the bioengineering measurements showed a good correlation between TEWL changes, but not between colour changes, and IOIs from corneosurfametry. Corneosurfametry emerged as a simple, low-cost and fast method for ranking commercial products according to their mildness. However, the skin bioengineering techniques showed that some products could lead to skin reactions, such as erythema, that could not be detected by the in vitro technique. I irritation and to detect differences possibly not apparent to the eye (4–7). Ideally, an irritation test for evaluating the mildness of cleansing products should be representative of the conditions of every day use (2). Thus, exposure should be open and repeated (usually during relatively short time periods). That is not the case for the soap-chamber test conducted under occlusive conditions for a prolonged period of time. For these reasons, repeated wash or immersion tests should be preferred (2, 8–11). However, repeated wash or immersion tests are time-consuming, only a few products can be tested simultaneously and, in addition, they are influenced by factors such as season, environmental conditions, age and race, and thus require many subjects, given the known variability of the individual responses. In comparison, in vitro tests can be better standardised. Bovine red blood cell hemolysis, pH rise of bovine serum albumin, collagen swelling and zein solu- reactions are common and important in clinical and occupational dermatology. Detergents, surfactants, soaps, cleansing products and dish-washing products can damage the skin, and are considered an important risk factor in the development of irritant contact dermatitis. Mildness has become a major benefit claimed, especially for surfactants and soaps, and testing for mildness now ranks among the first concerns of the manufacturing industry (1). A vast amount of literature deals with the problems of cleansing product skin irritation potential on human skin (2). A major step was the introduction, by Frosch & Kligman, of the so-called soap chamber test, in which occlusive daily exposure for 5 days made it possible to separate soaps into classes of mild, moderate and severe irritancy potential (3). Further development included the use of non-invasive bioengineering measurement methods, such as electrical conductance, transepidermal water loss, colorimetry and Doppler velocimetry, to quantify the damage due to RRITANT Key words: cleansing products – detergents – skin surface biopsy – mildness testing c Munksgaard, 2001 Accepted for publication 28 July 2000 49 Gabard et al. bilization are used for evaluating surfactant mildness (12–14). However, data obtained from these tests may not always correlate with in vivo observations (13, 14). Thus the need remains for a reliable, practical, low-cost and fast laboratory test that can assess and predict the irritation potential of cleansing agents. The first step, and a key feature in the irritation process, is the interaction of the surfactant(s) with the stratum corneum. It is now believed that this constitutes a significant contribution to the ensuing barrier damage (15–17). Corneosurfametry (18) was introduced in 1994 by Piérard as a predictive bioassay for evaluating the alterations induced by surfactants on human stratum corneum. A good correlation between the results of this in vitro assay and evaluation of mildness by a modified soap-chamber in vivo test could already be demonstrated (19). It was the purpose of our experiments to investigate in more detail the interaction of cleansing products with human stratum corneum using this in vitro technique and to relate these results to those of two other well-known repeated in vivo assays. Material and Methods Corneosurfametry This assay was conducted exactly as described by Piérard et al. (18). Briefly, skin surface biopsies (SSBs) (19) were taken with a microscopic slide and cyanoacrylate glue. Aqueous solutions of the test products (0.5/1/5/10%) or sodium lauryl sulfate (SLS; 0.01– 4%) were sprayed uniformly on the SSBs. After incubation at 25 æC for 2 h, the SSBs were thoroughly rinsed with tap water, dried, and stained for 3 min with basic fuchsin–toluidine blue dye solution. The colour changes were evaluated with a tristimulus Chromameter CR 300 (Minolta, Osaka, Japan) after placing the SSBs on a white reference tile. The corneosurfametric index of mildness (CIMΩL* – Chroma C*) was calculated as described elsewhere (17). To investigate a possible dose dependency and to eliminate the small but always present variations in CIMs between repeated experiments, an index of irritation (IOI) was calculated: IOIΩ1- (CIMtest product/CIMwater) The IOI varies between 0 for water (or very mild test products: CIMtest productΩCIMwater) and a maximum of 1 for very harsh products (low CIMtest product). Flex wash test The flex wash test was conducted using the published methodology (9) with slight modifications and additional instrumental evaluation of the skin surface. Six healthy volunteers with intact skin in the antecubital fossae participated in the test. Products (0.5 ml) were pipetted onto the flex area of one arm. This area was washed by gently rubbing the product on the skin for exactly 60 s with a fleecy sponge-like towel previously moistened in warm tap water. The washing procedure was standardised as an elliptical motion with approximately 120 strokes per min. The washed area was then rinsed under running tap water until the lather was removed and gently patted dry with a soft paper towel. The same procedure was repeated on the contralateral arm with another product to be tested or with water for control purposes. These 1-min washing procedures were repeated 3 times daily (9 a.m.; 1 and 4 p.m.) for 4 days. SLS (2%) was used as standard, and water as a control substance. Bioengineering measurements were conducted daily 1 h before the first washing procedure and at 8 a.m. on day 5. Repeated closed patch test (modified soap chamber test) The original Frosch & Kligman test (3) was modified slightly: test products were used undiluted and the TABLE 1. Test products and their claims Test products Code Claims Shampoos Commercial Experimental CSH1-CSH6 ESH1, ESH2 Mild, extra-mild, very mild, for frequent use, perfectly tolerated, care of sensitive hair Specially designed for dry and fragile hairs Antidandruff shampoos Commercial Experimental CASH1-CASH4 EASH1-EASH3 (No particular claim) (No particular claim) Syndets Commercial Experimental CSY1, CSY2 ESY1, ESY2 Mild, cleans the skin naturally Specially designed as baby syndets Experimental: proprietary products from Spirig Pharma Ltd. 50 Surfactant irritation chambers were left on the skin for 6 h from day 1 to day 5. Briefly, 100 ml of the test products were pipetted on filter paper disks fitted in 12 mm aluminium Finn-chambers. Five chambers were positioned on each forearm of six healthy volunteers and fixed with surgical wrap. After 6 h, the chambers were removed and the skin was gently cleaned with a soft paper towel to remove product residues. This procedure was repeated for 5 days. Every other day bioengineering measurements were performed before patch application and 1 h after removal of the patch. SLS solutions (0.05% and 0.5%) were used as standards, and water was used as a control substance. Fig. 1. Corneosurfametry: concentration-effect curve of SLS. Test products Bioengineering measurements The following bioengineeering techniques were used: transepidermal water loss (TEWL; Evaporimeter EP2; Servomed, Kinna, Sweden) and skin colour (Chromameter CR300, Minolta, Osaka, Japan; a* – hue). Measurements were performed in a controlled environment (T: 22∫0.1 æC; RH: 45∫5%) after acclimatisation of the volunteers for 15 min and following published guidelines (21, 22). Test products were 19 shampoos and syndets available on the Swiss and/or German market, and/or proprietary developments from Spirig Company (Table 1). Most of the products were claimed to be mild, very mild, extra mild or for babies. Sodium lauryl sulfate (SLS; biochemical grade ⬎98% purity; Fluka, Buchs, Switzerland) was used as standard and model substance. Distilled water was used throughout as control. Fig. 2. Corneosurfametry: concentration-effect curves of SLS (H) and of 19 different test products. Classification in Group 1 (mild), Group 2 (intermediate) and Group 3 (aggressive). For classification details see Table 2. 51 Gabard et al. Statistical analysis The results of corneosurfametry (IOIs) were analysed by the Kruskal-Wallis test. In case of statistical significance, an analysis of variance (ANOVA) with a Student-Newman-Keuls post-hoc multiple comparison test followed. The statistical analysis (ANOVA) of the results of the in vivo tests was performed on the sums of the changes in baseline TEWL and a* hue between days 1 and 5 after logarithmical transformation (data were not normally distributed). Correlations between product ranking from IOIs and from in vivo data were analysed by Spearman’s rank correlation procedure. All statistical procedures were performed with SPSS for Windows V9.0 software. Results Corneosurfametry SLS-IOIs were linearly dependent on log[concentration] in the range investigated (Fig. 1). This dose-effect curve may be handled as a pharmacological dose-response curve, with an Emax (Emax: maximum possible effect, height of the plateau of the curve) of 0.670∫0.189 at 2% SLS and a D50% (D50%: dose which gives EΩ1/2 Emax), calculated at about 0.17%, just under the critical micelle concentration (0.2%) (2, 16). The test products could be classified in three categories (Fig. 2): Group 1 or mild products showed maximal IOIs at about 0.1 and no dose-effect curve. Group 2 or intermediate products were characterised by IOIs from 0.1 to 0.2 at 1% and more pronounced dose-effect curves. Group 3 products, the most aggressive products under our test conditions, showed IOIs ⬎0.2 at 1% and significant doseeffect curves (Table 2). In vivo tests In both tests, the well-known irritation reaction to SLS was noticed (Figs. 3 & 4). In the repeated patch test, one volunteer interrupted the test on day 3, two further attained a score of 3 on day 4 with SLS 0.5% and no reapplication was done. With SLS (0.05%), all volunteers completed the trial. This was also the case in the the flex wash test. Eight different products belonging to the three categories defined from the corneosurfametry results were tested in both in vivo tests (Table 3). In the flex wash test, a statistically significant deterioration of the skin barrier (as TEWL changes) and erythemal reaction (a* hue) was obvious with product no. 5. Sporadic erythemal reactions were noticed with other products. In the repeated patch test, no statistically significant changes were seen. Correlation between corneosurfametry and in vivo tests The test products were ranked on the basis of the sums of changes from baseline between days 1 and 5 (data used for the statistical analysis). This ranking TABLE 2. Classification of the test products from IOIs Test product class Mean IOI (95% CI) Group 1: Mild Group 2: Intermediate Group 3: Aggressive Dose-effect curve Concentration 1% Concentration 5% ⬍0.09 0.14 (0.10–0.17) 0.22 (0.20–0.23) ⬍0.09 0.22 (0.16–0.27) 0.36 (0.33–0.38) Absent Sometimes flat Significant TABLE 3. Statistical evaluation of the sums of the changes from baseline between first and last measurements Test product rank (group) Flex wash 1: SLS (conc. %) S* (2%) 2: 3: 4: 5: 6: 7: 8: 9: NS NS NS S* NS NS NS NS CSH4 (Gr. 3) CSH1 (Gr. 2) CSY1 (Gr. 2) CASH3 (Gr. 2) CSH3 (Gr. 2) EASH3 (Gr. 1) ESH1 (Gr. 1) ESY2 (Gr. 1) Colour a* TEWL Repeated patch S* (0.5%) NS (0.05%) NS NS NS NS NS NS NS NS Flex wash Repeated patch S* (2%) S* (0.5%) NS (0.05%) NS NS NS NS NS S* NS NS S* NS NS S* S* S* NS NS NS: not statistically different from water-induced changes; S*: statistically different from water-induced changes (P⬍0.05). 52 Surfactant irritation Fig. 3. Repeated closed patch test: bioengineering measurements (transepidermal water loss, left and skin colour a*-hue, right) after application of distilled water (P), SLS 0.05% (h) and SLS 0.5% (g). (Means∫standard deviation) Fig. 4. Flex wash test: bioengineering measurements [transepidermal water loss (left) and skin colour a*-hue (right)] after application of distilled water (P) and SLS 2.0% (g). (Means∫standard deviations) was compared with the one obtained from the corneosurfametry (IOIs, Table 4). High, statistically significant rank-correlation coefficients were found between the changes in TEWL in both in vivo tests and the corneosurfametry. Lower or statistically not significant correlations were observed between color changes and corneosurfametry. Discussion Claims of mild cleansing products are a major concern of industry, research laboratories and, last but not least, dermatologists. Consumers also have become concerned about this particular point, and claims of mildness sometimes override the primary purpose of 53 Gabard et al. TABLE 4. Correlation (Spearman rank correlation) between corneosurfametry ranking (IOI at 1% test concentration) and ranking from TEWL and/or colour a* (sums of the baseline between first and last measurements) Corneosurfametry rank (group) 1: 2: 3: 4: 5: 6: 7: 8: 9: SLS (conc. %) CSH4 (Gr. 3) CSH1 (Gr. 2) CSY1 (Gr. 2) CASH3 (Gr. 2) CSH3 (Gr. 2) EASH3 (Gr. 1) ESH1 (Gr. 1) ESY2 (Gr. 1) Spearman rank sum IOI (1%) 0.59 0.25 0.20 0.19 0.16 0.15 0.018 0.014 0.011 Colour a* TEWL Flex wash Repeated patch Flex wash Repeated patch 1: 5: 6: 2: 4: 9: 8: 3: 7: 1: 2: 5: 4: 6: 3: 7: 9: 8: 1: 5: 2: 6: 7: 8: 4: 3: 9: 1: 7: 5: 3: 8: 2: 6: 9: 4: SLS CASH3 CSH3 CSH4 CSY1 ESY2 ESH1 CSH1 EASH3 0.867 (S*) SLS CSH4 CASH3 CSY1 CSH3 CSH1 EASH3 ESY2 ESH1 0.867 (S*) SLS CASH3 CSH4 CSH3 EASH3 ESH1 CSY1 CSH1 ESY2 0.767 (S*) SLS EASH3 CASH3 CSH1 ESH1 CSH4 CSH3 ESY2 CSY1 0.317 (NS) NS: not statistically significant; S*: statistically significant (P⬍0.05). the products, the cleansing benefit. A wealth of publications are dealing with this problem, trying to improve the methodology, reduce the costs and facilitate and speed up decision-making, for example (1, 2, 11, 14, 16). Among the main problems the tests deal with are the influence of the test conditions on product mildness classification, the use of human volunteers with different skin types, the necessity of using a huge number of volunteers due to the variations of the results, and the difficulties of classification due to the general relationship between aggressiveness of the test method and its ability to discriminate among the products (1, 2). This last point was adressed, in particular, in our experiments, as the products tested were all claimed to be mild. Mildness was confirmed throughout in two in vivo tests. It is known that mild methodologies tend to distinguish harsh materials from the remainder unless experimental conditions are adapted (e.g., self-assessment and bioengineering measurements) or the sample size is greatly increased (1). On the other side, aggressive procedures such as the repeated patch test tend to distinguish mild products from the rest. Thus, SLS was easily recognized as harsh, but as the cleansers were of low irritancy, they could not be properly classified by a classical analysis of the test results. Moving the test procedure down the aggressiveness axis to the flex wash test allowed, besides the higher SLS-concentration, one product (no. 5, Table 3) to be separated from the others. But this test, as well as the repeated patch test, is not easy to perform. Both are time-consuming, and the flex wash test allows testing of only two products at one time. Both, in addition, require the use of human volunteers with their cohort of constraints, such as standardised measurement conditions, influence of season and skin type. 54 On the other hand corneosurfametry has emerged as a simple, fast, low-cost in vitro test for ranking mildness of cleansing products. The concentration-effect curve using IOIs as the effect measurement showed a D50% at the critical micelle concentration (CMC). Rhein already discussed the relationship between the CMC and the interactions of surfactants with the stratum corneum, with relevance to skin irritation (16). She pointed out that SLS irritation still increases at doses above the CMC, a fact that was already known but that appears to contradict the hypothesis that the surfactant monomer is the irritative species. Our findings are in accordance with her statements. Moreover, the concentration-effect curve of the IOIs showed a maximum at about 2% SLS, which is the maximal concentration recommended by the recently published guidelines on SLS irritation testing (23). This shows that the concentration-effect curve of SLS obtained by the technique of corneosurfametry is meaningful for the in vivo effects of surfactants. Using corneosurfametry, we were able to rank the products tested and to propose a classification. The test was simple to perform, fast and reproducible. We also could show that the classification found in this test ranking corresponded to the one obtained from the changes in the barrier function in both in vivo tests, although these changes in vivo were not significantly different statistically from those of distilled water. However, a limit of the corneosurfametry is that dermal irritation noticed as erythema could not be foreseen. The complex relationship between erythema and barrier changes has been the subject of several publications and is not fully understood (2). A recent publication by Tupker deals with the problem of ranking detergents using several tests and of the correlation between the results of these different Surfactant irritation tests (2). As he pointed out, literature on the comparison of the irritancy of detergents tested by several techniques, such as the patch test and the wash test, is sparse. Corneosurfametry could be a valuable addtion to help solve this problem. References 1. Muharata RI, Nicoll GA. Mildness testing for personal washing products. In: Augst LB, ed. Cosmetic claims substantiation. New York: Marcel Dekker, Inc., 1997: 153–169. 2. Tupker RA, Bunte EE, Wiechers JW, Coenraads PJ. Irritancy ranking of anionic detergents using one time occlusive, repeated occlusive and repeated open tests. Contact Dermatitis 1999: 40: 316–322. 3. Frosch PJ, Kligman AM. The soap chamber test. J Am Acad Dermatol 1979: 1: 35–41. 4. Freeman S, Maibach HI. Study of irritant contact dermatitis produced by repeat patch test with sodium lauryl sulfate and assessed by visual methods, transepidermal water loss, and laser Doppler velocimetry. J Am Acad Dermatol 1988: 19: 496–502. 5. Berardesca E, Maibach HI. Transepidermal water loss and skin surface hydration in the noninvasive assessment of stratum corneum function. Dermatosen 1990: 38: 50–53. 6. Agner T, Serup J. Sodium lauryl sulfate for irritant patch testing – a dose-response study using bioengineering methods for determination of skin irritation. J Invest Dermatol 1990: 95: 543–547. 7. Zhou J, Mark R, Stoudemayer T, Sakr A, Lichtin JL, Gabriel KL. The value of multiple instrumental and clinical methods, repeated patch applications, and daily evaluations for assessing stratum corneum changes induced by surfactants. J Soc Cosmet Chem 1991: 42: 105–128. 8. Lukacovic MF, Dunlap FE, Michaels SE, Visscher MO, Watson DD. Forearm wash test to evaluate the clinical mildness of cleansing products. J Soc Cosmet Chem 1988: 39: 355–366. 9. Duke Strube D, Koontz SW, Muharata RI, Theiler RF. The flex wash test: a method for evaluating the mildness of personal washing products. J Soc Cosmet Chem 1989: 40: 297– 306. 10. Clarys P, Barel AO. Comparison of three detergents using the patch test and the hand/forearm immersion test as measurements of irritancy. J Soc Cosmet Chem 1997: 48: 141– 149. 11. Paye M, Gomes G, Zerweck CR, Piérard GE, Grove GL. A hand immersion test under laboratory-controlled usage conditions: the need for sensitive and controlled assessment methods. Contact Dermatitis 1999: 40: 133–138. 12. Pape WJW, Hoppe U. Standardization of an in vitro red blood cell test for evaluating the acute cytotoxic potential of tensides. Drug Res 1990: 40: 498–502. 13. Morrison BM, Paye M. A comparison of three in vitro screening tests with an in vivo clinical test to evaluate the irritation potential of antibacterial liquid soaps. J Soc Cosmet Chem 1995: 46: 291–299. 14. Goffin V, Paye M, Piérard GE. Comparison of in vitro predictive tests for irritation induced by anionic surfactants. Contact Dermatitis 1995: 33: 38–41. 15. Simion FA, Rhein LD, Grove GL, Wojtkowski JM, Cagan RH, Scala DD. Sequential order of skin responses to surfactants during a soap chamber test. Contact Dermatitis 1991: 25: 242–249. 16. Rhein LD. Review of properties of surfactants that determine their interactions with stratum corneum. J Soc Cosmet Chem 1997: 48: 253–274. 17. Shukuwa T, Kligman AM, Stoudemayer TJ. A new model for assessing the damaging effects of soaps and surfactants on human stratum corneum. Acta Derm Venereol 1997: 77: 29–34. 18. Piérard GE, Goffin V, Piérard-Franchimont C. Corneosurfametry: a predictive assessment of the interaction of personalcare cleansing products with human stratum corneum. Dermatology 1994: 189: 152–156. 19. Piérard GE, Goffin V, Hermanns-Lê T, Arrese JE, PiérardFranchimont C. Surfactant-induced dermatitis: comparison of corneosurfametry with predictive testing on human and reconstructed skin. J Am Acad Dermatol 1995: 33: 462–469. 20. Marks R, Dawber RPR. Skin surface biopsy: an improved technique for the examination of the horny layer. Br J Dermatol 1971: 84: 117–123. 21. Pinnagoda J, Tupker RA, Agner T, Serup J. Guidelines for transepidermal water loss (TEWL) measurement. Contact Dermatitis 1990: 22: 164–178. 22. Fullerton A, Fischer T, Lahti A, Wilhelm KP, Takiwaki H, Serup J. Guidelines for measurement of skin colour and erythema. Contact Dermatitis 1996: 35: 1–10. 23. Tupker RA, Willis C, Berardesca E, et al. Guidelines on sodium lauryl sulfate (SLS) exposure tests. Contact Dermatitis 1997: 37: 53–69. Address: Bernard Gabard Biopharmacy Department Spirig Pharma Ltd. CH-4622 Egerkingen Switzerland Tel: π41 62 387 87 26 Fax: π41 62 398 24 68 e-mail: bernard.gabard/spirig.ch 55