comparative studies on the ocular and dermal irritation

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Food and Chemical Toxicology xxx (2006) xxx–xxx

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Comparative studies on the ocular and dermal irritationpotential of surfactants

A. Mehling *, M. Kleber, H. Hensen

Cognis Deutschland GmbH and Co.KG, Product Safety and Regulations, Henkelstr. 56, 40551 Duesseldorf, NRW, Germany

Received 14 March 2006; accepted 27 October 2006
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Comparative studies on the irritation potential of 18 surfactants were performed using the same stock solution of surfactant for eachstudy. The ocular irritation potential of surfactants was studied using the red blood cell test (RBC), the hen’s egg test-chorioallantoicmembrane (HET-CAM) and the Skinethic ocular tissue model. The skin irritation potential was assessed based on data obtained fromhuman studies using a 24 h epicutaneous patch test (ECT) and a soap chamber test (SCT). The same pH and active substance (AS) con-tent for all surfactants tested was used depending on the test conducted. In general, clusters of substances with varying irritation potentialwere identified similarly by most tests. These results show that when using standardized test conditions in which pH and % AS are thesame for each surfactant tested, there is a good correlation between the in vitro ocular irritation assays themselves as well as between thedermal and ocular irritation assays. In particular the RBC test seems to be not only highly predictive for ocular irritation (H50/DI) butalso for dermal irritation and changes in barrier function induced by surfactants (DI).� 2006 Elsevier Ltd. All rights reserved.

Keywords: Alternative methods; Irritation; Dermal; Ocular; Surfactant(s)

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R1. Introduction

Cleanliness is an age-old need and surfactants have beenused by humankind for this purpose since ancient times. Inthe last decades, surfactant molecules have been in thefocus of scientific and technological developments for thepurpose of creating new surfactants as well as to modifyand optimize their performance. This is particularly evidentin respect to applications in industrial and household deter-

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Abbreviations: APG, alkyl polyglucoside; AS, active substance; DI,denaturation index; GCP, good clinical practice; ECT, epicutaneous patchtest; H50, half maximal effective concentration of hemolysis; HET-CAM,hen’s egg test-chorioallantoic membrane; MTT, 3-[4.5-dimetyl-thiazol-2-yl]-2,5-diphenyl tetrazolium bromide (methyltetrathiazolium); OD, opticaldensity; PBS, phosphate-buffered saline; Q, irritation quotient; RBC, redblood cell test; SCT, soap chamber test; SDS, sodium lauryl sulfate; SOP,standard operational procedure; TEWL, transepidermal water loss.

* Corresponding author. Tel.: +49 211 7940 9209; fax: +49 211 200619209.

E-mail address: [email protected] (A. Mehling).

Please cite this article in press as: Mehling, A. et al., Comparative stu(2006), doi:10.1016/j.fct.2006.10.024

gent market as well as in cosmetics. Today surfactants arewidely used in many daily-used products ranging fromhousehold and laundry detergents, from fabric condition-ers to shampoos, body washes, hand soaps, etc.

Surfactants are amphipathic molecules and contain ahydrophilic and a lipophilic moiety allowing them to inter-act with both polar and nonpolar molecules, respectively.Surfactants are generally classified according to these prop-erties or based on their chemical constitution. Based ontheir charge, they are classified as anionic, nonionic, cat-ionic or amphoteric surfactants. Due to the amphipathicproperties of surfactants they can therefore be used ascleansers, emulsifiers, foaming and wetting agents, etc.When applied to the skin, they can interact with skin struc-tures, in particular the lipid and protein components(Cooper and Berner, 1985). Although surfactants used inrinse-off products are generally well tolerated, they are arisk for the development of irritant contact dermatitis(Dihoum et al., 1996; Dykes, 1998). A prerequisite forproducts designed for topical application is therefore that

dies on the ocular and dermal irritation ..., Food Chem. Toxicol.

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they need to have a low ocular/mucous membrane and der-mal irritation potential. The irritation potential is extre-mely dependent on the concentration of the substance, aswell as on the composition and pH of the formulation(Rhein et al., 1990; Turkoglu and Pekmezci, 1999; Barandaet al., 2002). Due to the ability of surfactants to emulsifyand to reduce the surface tension of water, surfactantscan remove lipids from the skin during the washing pro-cess. Therefore, the major dermal irritant effects manifestthemselves particularly in dryness and roughness of theskin although in rare cases more severe damage to the skincan be elicited (Bartnik and Kunstler, 1987; Loffler et al.,2000). Ocular irritation reactions to surfactants range fromreddening and tearing of the eyes to severe damage to theocular tissue, once again depending on the surfactant, thepH and the concentrations tested (Bartnik and Kunstler,1987).

There are various methods regularly used to discrimi-nate between detergents in respect to their potential irri-tancy and occupational hazard classifications. The Draizeeye irritation test has been the gold standard for testingeye irritation for many years. Due to the concern for ani-mal welfare and the limitations of the method, alternativemethods such as the red blood cell test (RBC; Pape et al.,1999; INVITTOX Protocol Nr. 37), the hen’s egg test-cho-rioallantoic membrane (HET-CAM) test (Steiling et al.,1999; INVITTOX Protocol Nr. 96) and tests using oculartissue models have been developed to assess the mucousmembrane/ocular irritation potential of surfactants andother substances. Dermal irritation is often assessed inhumans using epicutaneous patch testing (ECT; one-timeocclusive patch test), whereas soap chamber tests (SCT)can be used to assess irritation following repeated occlusiveexposure to the surfactant.

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RRTable 1

Description of the surfactants tested in this study

Product name INCI

Plantacare 818 Coco glucosidePlantacare 1200 Lauryl glucosidePlantacare 2000 Decyl glucosidePlantapon LGC sorb Sodium lauryl glucose carboxylate and

lauryl glucosideGluadin WK Sodium cocoyl hydrolyzed wheat proteinPlantapon ACG 50 Disodium cocoyl glutamatePlantapon LC 7 Laureth-7 citratePGFAC-S Sodium cocoyl hydrolyzed wheat

protein glutamateTexapon ALS benz Ammonium lauryl sulfateTexapon K 12 G Sodium lauryl sulfateTexapon K14 S special 70% Sodium myreth sulfateTexapon N 70 Sodium laureth sulfateTexapon SB 3 KC Disodium laureth sulfosuccinateDehyton DC Disodium cocoamodiacetateDehyton MC Sodium cocoamphoacetateDehyton PK 45 Cocamidopropyl betaineDehyton ML Sodium lauroamphoacetateGluadin WQ Laurdimonium hydroxypropyl

hydrolyzed wheat protein


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The aim of this study was to assess the comparability ofthe test results of various methods. In order to avoid lot-to-lot inconsistencies, the irritation potential of 18 surfactantswas evaluated using the same stock solution of surfactantfor each study. When labeling according to occupationalhazard classification, substances are generally tested undi-luted and at the pH of the stored raw material. This doesnot reflect the conditions found in the personal hygieneproducts, such as body-washes, in which the pH usuallyranges from pH 5 to 8. Therefore, in this study the compat-ibility of the surfactant solutions at a pH that is relevant forthis type of consumer product was assessed. Except wherethe limitations of the method made a different pH necessary,testing was carried out with products adjusted to a pH of6.5. As compatibility is also dependent on the concentrationtested, equivalent concentrations based on the active sub-stance content (AS, w/w) of each substance were tested.The ocular irritation potential of surfactants was studiedusing three in vitro test systems currently being used asalternatives to animal studies, namely the RBC test, theHET-CAM assay and the acute eye irritation assay basedon the commercially available ocular tissue model manufac-tured by Skinethic Laboratories (France). The skin irrita-tion potential was assessed based on data obtained from a24 h ECT. The SCT was used to assess the cumulative der-mal irritation potential of products after repeated exposure.
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2. Materials and Methods

2.1. Test products and reagents

All surfactants used, with the exception of the sodium lauryl sulfate(SDS) controls, were obtained from Cognis Deutschland GmbH &Co.KG (Germany). A classification of these surfactants can be found inTable 1. The same stock solutions were used for all tests (12% AS; pH 6.5).

Surfactant type Chemical description

Nonionic Alkyl-polyglucosideNonionic Alkyl-polyglucosideNonionic Alkyl-polyglucosideAnionic/nonionic Alkyl-glucoside carboxylate

Anionic Protein fatty acid condensateAnionic Acyl-glutamateAnionic Alkyl-ether citrateAnionic Protein-glutamic fatty acid condensate

Anionic Alkyl-sulfateAnionic Alkyl-sulfateAnionic Alkyl-ether-sulfateanionic Alkyl-ether-sulfateAnionic Alkyl-ether-sulfosuccinateAmphoteric Alkyl-amphoacetateAmphoteric Alkyl-amphoacetateAmphoteric Alkyl-amidobetaineAmphoteric Alkyl-amphoacetateCationic Quaternized protein hydolysate


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Table 2The pH and concentration of the surfactant solutions tested (adjustedfrom identical stock solutions with 12% AS concentrations and a pH of6.5)

Test pH Concentration tested(AS%, w/w)

RBC 7.4 1.0% (initial concentration)HET-CAM 6.5 3.0%Skinethic 7.0 0.6%ECT 6.5 2.0%SCT 6.5 1.0%

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All solutions used for one type of test were adjusted to the same pH andAS% depending on the test used (Table 2). The aqueous dilutions wereadjusted to the AS concentration and pH (NaOH or citric acid orhydrochloric acid) needed for each test prior to testing. Tests were con-ducted using coded samples (‘‘double-blind’’). Regression curves andcoefficients were calculated using the Microsoft Excel 2003 program(trendline functions) after omitting the values for the positive and negativecontrols. No corrections for outliers were made.

2.2. RBC test

The RBC test was carried out in accordance with INVITTOX ProtocolNr. 37 using porcine erythrocytes obtained from one animal. Briefly,membrane damage was assessed by hemoglobin leakage and proteininteractions were assessed by denaturation of hemoglobin. Each surfactantwas tested in triplicate. Test samples were diluted to an initial concen-tration of 1% AS in PBS and the pH adjusted to 7.4. Various concen-trations of the test substance were aliquoted into 1.5 mL reaction vials and25 lL of the RBC suspension (8 · 109 cells/mL) added. The vials weregently agitated for 10 min at room temperature and centrifuged for 1 min.The OD530 of the supernatant was measured and the half-maximal effec-tive concentration for hemolysis determined from the dose–response curve(H50). At least eight equidistant concentrations of each surfactant weremonitored to assess the concentration response curve for hemolysis. Thehypotonic release of hemoglobin resulting from the addition of theerythrocytes to distilled water was set to 100% hemolysis. In order todetermine the denaturation index, 975 lL of a 0.1% dilution of the samplewas added to 25 lL of the RBC suspension and gently agitated for 10 minat room temperature. Following centrifugation for 10 min, the extinctionof the supernatant was measured at 575 nm to obtain the denaturationindex (DI). The denaturation index is calculated using 0.1% of the testsubstance relative to the internal standard SDS (3.47 mmol/L). The H50/DI ratio is then used as a measure of the eye irritation potential accordingto the following classification: >100: Not irritating; >10: Slightly irritating;>1: Moderately irritating; >0.1: Irritating; <0.1: Very irritating.

2.3. HET-CAM assay

The HET-CAM assay was carried out according to the standard ofoperational procedure (SOP) of the COLIPA project: MethodologyAlternatives: The HET-CAM SOP 2nd edition (INVITTOX protocol No.96; Steiling et al., 1999) using the reaction-point method. In brief, fertilizedeggs were incubated 9 day prior to use. Six eggs were used for each testsubstance. After exposing the CAM and rinsing it with warm PBS, 300 lLof the test solution (3% AS; pH 6.5) diluted in water were applied to theCAM. The intensity of the reactions hemorrhage, lysis and coagulationwere semi-quantitatively assessed on a scale of 0 (no reaction) to 3 (strongreaction). The time-point of appearance and the intensity of any reactionsoccurring within 5 min were documented. The irritation quotient [Q] wasthen calculated and expressed relative to the standard Texapon ASV [5%AS; special fatty alcohol ether-sulfate]. The following classifications basedon Q were made: 60.8: slightly irritating; >0.8 to <1.2: moderately irri-tating; P 1.2 to <2.0: irritating; P 2.0 severely irritating.


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2.4. Acute eye irritation assay

Prior to testing, the solution was diluted in water to a concentration of0.6% AS and the pH was adjusted to pH 7.0. Tests were performedaccording to the protocol proposed by Skinethic laboratories(www.skinethic.com). In brief, 30 lL of the samples were topically appliedto triplicate samples of reconstituted human corneal epithelial tissues(Skinethic Laboratories, size: 0.5 cm2) and incubated at 37 �C and 5% CO2

for 10 min, 1 h and 3 h. PBS was used as a negative control, SDS (0.5%and 1.0%) as a positive control. In order to assess viability, two culturesfor each tested time-point were placed on 300 lL of 0.5 mg/mL MTT for30 min at room temperature and subjected to visual assessment. After 3 h,the cultures were placed in 1.5 mL of isopropanol for 2 h to quantitativelyextract the MTT. Optical density (OD) was measured at 570 nm and theresults expressed relative to the negative control (PBS). One culture treatedwith a test substance was fixed in formalin and embedded in paraffin.Histological assessments were carried out on sections stained with hema-toxylin and eosin (H and E). The classification of the irritation potentialwas based on viability as assessed by the MTT-test and visual assessments.Products are classified according to the following classes: Nonirritating,very slightly irritating, slightly irritating, irritating and very irritating.

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PR2.5. Epicutaneous patch test

Test products were diluted to 2% AS in water and the pH adjusted topH 6.5 prior to testing. Tests were carried out on 20 healthy and informedvolunteers using the principles of good clinical practice (GCP) for guid-ance. In principle, the test was performed as previously described (Forsteret al., 2000). Seventy five microlitres of the aqueous solutions were appliedocclusively to the backs of the volunteers using large Finn chambers withfilter discs backed on ScanPore�. After 24 h, the patches were removed.Visual assessments of each individual parameter (erythema, edema,squamation and fissure) were carried out 6 h, 24 h, 48 h and 72 h afterpatch removal. Reactions were scored according to predefined gradingparameters on a scale of 0 (no reaction) – 4 (strong reaction). The totalirritation score was calculated and the values expressed relative to SDS.The classification was defined in this study following a non-validatedcategorization based on the total irritation scores (as % SDS): >75:severely irritating; >50: irritating; >25: moderately irritating; >10: slightly(610 very slightly or not irritating).

2.6. Soap chamber test

Test materials were diluted to 1% AS and the pH adjusted to pH 6.5prior to testing. Tests were carried out on healthy and informed volunteers(SCT 1:21 volunteers; SCT 2:22 volunteers) using a protocol derived fromFrosch and Kligman (1979) and the principles of good clinical practice(GCP) for guidance. 100 lL of each sample was occlusively applied to theventral forearm using small Hilltop Chambers backed on Scanpor�.Repeated patching was performed for 24 h (1st application), followed by6 h each day for the next 4 days (days 2–5). The first visual assessment ofreactions (erythema, dryness and fissure) was performed 15 min afterpatch removal on day 2; further visual assessments were carried out priorto reapplication of the patch on days 3–5 as well as on day 8. Results wereexpressed as total irritation scores relative to SDS. Transepidermal waterloss (TEWL) was measured on day 1, 5 and on day 8 following acclima-tization for at least 20 min at 20 �C (±1�C) and 50% relative humidity(±5%). SCT irritation scores were calculated by adding the scores ofedema, dryness and fissure on day 5 and expressed relative to SDS. Theclassification of the irritation potential was defined in this study followinga non-validated categorization based on the total irritation scores (as %SDS): >100: severely irritating; >25 irritating; >15: moderately irritating;>10: slightly irritating (610 very slightly or not irritating). Althoughirritation classification is rarely done using TEWL, the following a non-validated grading system was used to evaluate the irritation potential: >30:severely irritating; >20 irritating; >15: moderately irritating; <15: slightlyirritating (610 very slightly or not irritating).


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3. Results

Identical stock solutions (12% AS; pH 6.5) were used forall tests in order to avoid variations in the test results dueto differences in the inherent properties of the test product.As the concentration of active substance (AS) and the pHplay a major role in eliciting irritant reactions, the pH and% AS were adjusted according to Table 2 depending on themethod used prior to testing. With the exception of theECT, all tests were carried out within the same month.Some of the samples were tested in the ECT in the samemonth whereas some of the samples were tested twomonths later. Tests were carried out ‘‘double-blind’’ andwhere appropriate randomized.

3.1. RBC test

The red blood cell test is a cell-based photometric testand was developed to assess the initial cellular reactionstaking place in ocular irritation processes elicited by surfac-tants and surfactant-based products (Pape et al., 1999;Pape and Hoppe, 1990). The principle of the RBC test isthe assessment of membrane damage and protein denatur-ation caused by surfactants. To this aim, the damage to thecell membrane is assessed by the amount of hemoglobinleakage into the supernatant and the changes in proteinconformation are assessed by hemoglobin denaturation.As the physiological pH of the blood is �7.4, all solutionswere adjusted to this pH in order to avoid shifts in the irri-tation potential due to the pH alone. The H50/DI quotientis used as a measure for the classification of ocular irritancyin analogy to the Draize eye irritation test. The ocular irri-tation potential of the surfactants as assessed by using theRBC test can be found in Table 3. Of the five similar sur-factants also tested by Pape et al. (1999) a good correlationwas obtained with results from this study (Table 4). Thisdemonstrates the reproducibility and robustness of themethod for assessing the irritation potential of surfactants.

The surfactants Dehyton MC, Gluadin WQ, DehytonML, Plantacare 2000, Plantacare 818, Gluadin WK, andPlantapon LC7 were classified as not irritating wherebythe surfactants Plantapon LC7, Gluadin WK and Planta-pon 818 were the least irritating. The surfactantsPGFAC-S, Plantapon ACG 50, Plantapon LGC, and Plan-tacare 1200 were classified as slightly irritating, the surfac-tants Plantapon SB3, Dehyton DC and Dehyton PK45were classified as moderately irritating, and the surfactantsPlantapon K14S, Texapon ALS, Texapon K12 G and Tex-apon N70 were classified as irritating.

3.2. HET-CAM assay

The HET-CAM assay is an organotypic model used toassess ocular irritation by taking advantage of the effectsa product has when applied to the fragile blood vessel net-work of the CAM. The method used in this study adheredto the protocol described by Steiling et al. (1999). As the


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products were all transparent, the reaction-time methodwas used, in which the time-point and severity of the occur-rence of the parameters hemorrhage, lysis and coagulationare examined during a maximum time period of 5 min. Thecalculated irritation index is then expressed relative to thebenchmark substance Texapon ASV (special alkyl-ether-sulfate Na/Mg salt; 5% AS) which is set to Q = 1.0 (mod-erately irritating). The pH of the products was adjusted to6.5 prior to application to the CAM. The products weretested with an AS content of 3%. The results are depictedin Table 3.

The surfactants Dehyton DC, Dehyton MC, DehytonML, Gluadin WQ, Gluadin WK, Plantacare 818, Planta-care 1200, Plantacare 2000, and Plantapon LC7, PlantaponLGC, PGFAC-S, Texapon SB3 were classified as slightlyirritating (there is no classification ‘‘not irritating’’ accord-ing to this protocol) whereby the surfactants PlantaponLC7, Plantacare 1200 and PGFAC-S were the least irritat-ing under these test conditions. The surfactants PlantaponACG 50 and Texapon K14S were classified as moderatelyirritating, Texapon N70 was classified as irritating and thesurfactants Dehyton PK45, Texapon ALS and TexaponK12 G were classified as severely irritating.

3.3. Acute eye irritation assay

In contrast to the other two models, the Skinethicin vitro reconstituted human corneal epithelial tissue modelconsists of airlift culture of differentiated keratinocytesresulting in a three-dimensional structure resembling theouter cornea of the human eye. The principle of this testsystem is the evaluation of cytotoxicity of the topicallyapplied product via the MTT reduction assay and visualassessment of the tissues. Histological evaluations furthercharacterize possible damage and/or cytotoxicity. Theproducts were tested at a pH of 7.0 and an AS content of0.6%. Due to the lack of skin samples available for testing,Dehyton ML and Gluadin WQ were not included in thistest. The results can be found in Table 3.

The surfactants Dehyton DC, Gluadin WK, Plantacare818, Plantacare 1200, Plantacare 2000, Plantapon ACG 50,Plantapon LC7 and PGFAC-S and were graded as not irri-tating. The surfactant Plantapon LGC was classified asvery slightly irritating; the surfactants Dehyton MC, Texa-pon K14 S and Plantapon SB3 were judged to be slightlyirritating. Dehyton PK45, Texapon ALS, Texapon K12G and Texapon N70 were assessed as being irritating.Dehyton ML and Gluadin WQ were not tested in thisassay.

Histological assessment of the H and E stained tissuesafter topical application of the surfactants for up to 3 hrevealed the following histological assessments: Absenceof significant histological alterations were found when test-ing Gluadin WK, Plantapon LC7 and PGFAC-S thus cat-egorizing these 3 surfactants as being the least irritating;very slight cellular alterations in the superficial layer wasfound when testing the products Dehyton DC, Plantacare


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Table 3Scores and classifications obtained by the ocular irritation methods used in this study

Surfactant RBC HET-CAM Skinethic ocular

H50

(lg/mL)DI (%) H50/DI Classification Irrit. Pot.

(Q)Classification 10 min (%)* 1 h (%)* 3 h (%)* Classification

Plantacare 818 95.38 0.17 552.46 Not irritating 0.40 Slightlyirritating

100.82 ± 6.14 92.68 ± 1.37 77.5 ± 3.03 Not irritating

Plantacare 1200 61.41 0.66 92.86 Slightlyirritating

0.24 Slightlyirritating

102.26 ± 3.20 76.59 ± 11.29 88.03 ± 1.87 Not irritating

Plantacare 2000 128.15 0.37 342.76 Not irritating 0.59 Slightlyirritating

100.57 ± 5.07 91.89 ± 3.17 71.27 ± 13.28 Not irritating

Plantapon LGC sorb 80.79 2.08 38.86 Slightlyirritating


108.36 ± 0.98 86.75 ± 11.81 67.8 ± 0.53 Very slightlyirritating

Gluadin WK 87.47 0.07 1337.88 Not irritating 0.72 Slightlyirritating

98.93 ± 4.18 75.56 ± 3.51 77.88 ± 5.52 Not irritating

Plantapon ACG 50 323.34 13.65 23.68 Slightlyirritating

1.16 Moderatelyirritating

95.79± 3.82 86.09 ± 4.88 92.63 ± 20.32 Not irritating

Plantapon LC 7 – – >10,000 Not irritating 0.21 Slightlyirritating

100.94 ± 0.09 91.11 ± 5.48 85.26 ± 4.54 Not irritating

PGFAC-S 128.23 6.77 18.94 Slightlyirritating


106.16 ± 1.78 96.43 ± 4.28 95.9 ± 4.63 Not irritating

Texapon ALS 13.72 98.48 0.14 Irritating 3.02 Severelyirritating

83.96 ± 0.27 46.7 ± 11.29 29.17 ± 0.80 Irritating

Texapon K 12 G 21.85 99.80 0.22 Irritating 2.59 Severelyirritating

68.68 ± 6.23 31.28 ± 5.05 16.19 ± 2.23 Irritating

Texapon K14 S special70% S

13.62 34.03 0.40 Irritating 1.07 Moderatelyirritating

97.8 ± 0.09 48.09 ± 8.30 39.32 ± 5.52 Slightly irritating

Texapon N 70 10.32 59.20 0.17 Irritating 1.56 Irritating 65.09 ± 23.93 39.99 ± 3.68 24.7 ± 2.67 IrritatingTexapon SB 3 KC 74.44 27.95 2.66 Moderately

irritating0.72 Slightly

irritating97.8 ± 5.60 66.85 ± 1.11 78.26 ± 6.42 Slightly irritating

Dehyton DC 39.16 5.04 7.77 Moderatelyirritating


105.09 ± 5.96 91.35 ± 8.73 85.38 ± 6.86 Not irritating

Dehyton MC 28.07 0.27 102.40 Not irritating 0.42 Slightlyirritating

100.57 ± 4.00 75.62 ± 9.07 58.92 ± 6.15 Slightly irritating

Dehyton ML 183.34 0.83 222.13 Not irritating 0.79 Slightlyirritating

nd nd nd nd

Dehyton PK 45 18.26 2.32 7.87 Moderatelyirritating

2.05 Severelyirritating

80.13 ± 2.13 41.38 ± 2.4 36.29 ± 3.56 Irritating

Gluadin WQ 58.39 0.24 220.80 Not irritating 0.72 Slightlyirritating

nd nd nd nd

nd, not determined.* Percentage of viability compared to the negative control (MTT test).

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Table 4Comparison of the H50/DI values obtained using the RBC-test in this study and the study of Pape et al. (1999)

Surfactant nomenclature This study Study of Pape et al. (1999)

This study Study Pape et al. (1999) H50 /DI Classification H50/DI Classification

Texapon N70 Na-laureth-sulfate 0.17 Irritating 0.28 IrritatingTexapon ALS NH4-laurylsulfate 0.14 Irritating 0.31 IrritatingTexapon K12 G Na-laurylsulfate 0.22 Irritating 0.28 IrritatingTexapon SB3 Laureth-sulfosuccinate 2.66 Moderately irritating 3.73 Moderately irritatingDehyton PK 45 Cocoamidopropylbetaine 7.87 Moderately irritating 2.83 Moderately irritating




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818, Plantacare 1200, Plantacare 2000 and Plantapon ACG50. Cellular alterations in the upper layers were evident at3 h for Dehyton MC, Dehyton PK45, Plantapon LGC,Texapon K14 S and Texapon SB3. Tissue disintegrationin the upper cell layers was observed following applicationof Texapon ALS, Texapon K12 G and Texapon N70. Tis-sue disintegration was already found 10 min after applica-tion of Texapon ALS and Texapon K12 G identifyingthese two surfactants as those with the highest irritationpotential in this assay.

3.4. Epicutaneous patch test

The ECT is a method to ascertain the dermal irritationpotential of a substance following a single application ofthe test substance onto the back of a volunteer. It allowsskin compatibility to be assessed under the exaggeratedconditions of occlusivity. The parameters erythema,edema, squamation and fissure are assessed 6 h, 24 h,48 h and 72 h after patch removal (Walker et al., 1996).The scores are then used to calculate the total irritation

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ETable 5Comparison of the values obtained in the dermal compatibility tests relative t

Surfactant Epicutaneous patch test Soap chamber

Total irritation score Total irritation

ECT1 ECT2 Average SCT1 SCT2

Plantacare 818 16.31 11.1 13.69 5.12Plantacare 2000 5.44 6.81 6.13 4.65Plantacare 1200 16.31 17 16.67 4.42Plantapon LGC Sorb 2.13 2.13 1.16Gluadin WK 2.13 2.13 2.62Plantapon ACG 50 9.36 9.36 4.88Plantapon LC 7 2.55 2.55 1.63PGFAC-S 16.31 16.31 2.33Texapon ALS 81.56 81.56 176.82Texapon N 70 58.21 48.79 33.26Texapon K 12 G 104.3 104.34 192.90Texapon K 14 S special 70% 27.2 27.23 17.76Texapon SB 3 KC 5.11 5.11 4.67Dehyton DC 14.14 14.14 4.42Dehyton MC 8.51 8.51 13.46Dehyton ML 27.19 27.19 20.93Dehyton PK 45 16.31 10.6 13.48 11.78 10.00Gluadin WQ 15.22 15.22 2.99Water 2.17 3.83 3 4.11 2.33SDS 0.2% 100 100SDS 0.5% 100


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Fscore. Prior to testing, the pH of the products was adjustedto pH 6.5 and the products tested with 2% AS. The resultscan be found in Table 5.

A clear-cut grading scheme for the irritation potential inhuman tests has not yet been established as the protocolshave not been standardized, assessment of the irritationeffects and controls differ depending on the laboratory con-ducting the test and the reactions of volunteers beingtested. Therefore, classification of the irritation potentialwas defined following a non-validated grading based onthe total irritation scores (as % SDS): >75: severely irritat-ing; >50: irritating; >25: moderately irritating; >10: slightly(610: very slightly irritating). According to this scheme, thesurfactants Dehyton MC, Gluadin WK, Plantapon ACG50, Plantacare 2000, Plantapon LC7, Plantapon LGCand Texapon SB3 were classified as very slightly irritatingwith the surfactants Plantapon LC7, Plantapon LGC andGluadin WK being the least irritating. The surfactantsDehyton DC, Dehyton PK45, Gluadin WQ, Plantacare818, PGFAC-S and Plantacare 1200 were judged tobe slightly irritating The surfactants Dehyton ML and

o SDS (=100%)

test (values on day 5)

score Irritation score erythema TEWL (d5)

Average SCT1 SCT2 Average SCT1 SCT2 Average

5.12 5.94 5.94 34.1 34.134.65 4.95 4.95 33.6 33.584.42 6.93 6.93 36.6 36.561.16 2.48 2.48 31.3 31.312.62 1.95 1.95 26.78 26.784.88 3.47 3.47 34.9 34.881.63 0.99 0.99 26.7 26.742.33 2.48 2.48 31.1 31.12176.82 137 136.96 78.21 78.2141.02 45.91 49.5 47.71 49.82 65 57.42192.90 148.6 148.64 115.1 115.1117.76 17.51 17.51 35.52 35.524.67 4.28 4.28 26.52 26.524.42 5.94 5.94 31.2 31.1513.46 14.01 14.01 32.04 32.0420.93 22.96 22.96 38.48 38.4810.89 10.51 11.9 11.20 30.78 39 34.912.99 4.28 4.28 28.3 28.33.22 4.28 4.28 �3.89 0 �1.945100 100 100 100 100 100 100


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Texapon K14 S were classified as being moderately irritat-ing, Texapon N70 as irritating. Texapon ALS and TexaponK12 G were the most irritating and being evaluated asbeing very irritating.

3.5. Soap chamber test

The SCT is used to assess the cumulative irritationpotential of a product. The substances are occlusivelyapplied to the ventral forearm for 24 h on the first dayand for 6 h on the next 4 days. The parameters erythema,dryness and fissure are rated visually and the scores onday 5 used to calculate the total irritation score. Changesin skin barrier function are assessed by transepidermalwater loss (TEWL). The scores obtained in this study canbe found in Table 5. Once again, there is no internationalstandardized method or classification scheme. The classifi-cation was therefore defined following a non-validated def-inition based on the total irritation scores (as % SDS):>100: severely irritating; >25 irritating; >15: moderatelyirritating; >10: slightly irritating (610 very slightly or notirritating). Although irritation classification is rarely doneusing TEWL, the following a non-validated grading systemwas used to evaluate the irritation potential: >30: severelyirritating; >20 irritating; >15: moderately irritating; 615:slightly irritating (610 very slightly or not irritating).

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Fig. 1. Comparison of the irritation classifications of the various surfactants baslightly irritating, these two classes were combined into one class. The Skinethfollows: Not irritating = not or slightly irritating; very slightly irritatingirritating = irritating; severely irritating = severely irritating. For the comparisovalidated definitions were used: ECT irritation scores (as % SDS): >75: severelySCT irritation scores (as % SDS): >100: severely irritating; >25: irritating;classification is rarely done using TEWL, the following a non-validated grairritating; >20 irritating; >15: moderately irritating; <15: slightly irritating (610of the classification between the methods used.


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Under the test conditions used, Dehyton DC, DehytonMC, Dehyton PK45, Gluadin WQ, Gluadin WK, Planta-care ACG 50, Plantacare 818, Plantacare 1200, Plantacare2000, Plantapon LC7, Plantapon LGC, PGFAC-S andTexapon SB3 were classified as being slightly irritating,whereby Gluadin WK, Gluadin WQ, Plantapon LC7,Plantapon LGC and PGFAC-S had the lowest irritationpotentials. Dehyton ML and Texapon K14 S were judgedto be moderately irritating. Texapon N70 was classifiedas irritating whereas Texapon ALS and Texapon K12 Gwere severely irritating. The same classification was foundwhen assessing TEWL whereby the lowest irritation poten-tials were found for Plantapon LC7, PGFAC-S, DehytonDC and Plantapon LGC. The highest irritation potentialswere found for Texapon ALS and Texapon K12 G.

DPR

O3.6. Comparison of the irritation potential found in the

different tests

A schematic representation of the classification of theirritation potentials as described in this study is depictedin Fig. 1. In this context, it is important to note, that theclassification of the irritation potential found in the dermaltests according to the scores defined in this study is not val-idated. Furthermore, this classification is based on theresults obtained by surfactants only. There is a good corre-
E
sed on the methods used. As not all methods differentiate between not andic protocol exhibits slightly different classifications which were adjusted as= not- or slightly-irritating; slightly irritating = moderately irritating;n of the irritation classes in the dermal irritation studies the following non-irritating; >50: irritating; >25 moderately irritating; 625 slightly irritating;>15: moderately irritating; 615: slightly irritating. Although irritation

ding system was used to evaluate the irritation potential: >30: severelyvery slightly or not irritating). In general, there was a good comparability


UNCORRECTEDPROOF

Table 6Correlation of the results (R2 coefficients of determination (bold) as well as the equations are given; both were calculated using the trendline option of the Microsoft Excel 2003 program)

Method RBC (H50/DI) HET-CAM (Q) Skinethic average ECT total irritationscore

SCT total irritationscore

SCT irritation scoreerythema

SCT TEWL (d5)

SCT total

irritation

score

0.5234 0.7289 0.7606* 0.9023 1.0 0.9926 0.9185

y = 22.886x � 0.3231 y = 0.0118x

+ 0.6639y = 1E + 09x � 4.3632 y = 1.9166x � 16.227 – y = 0.7666x + 2.76 y = 2.4789x � 71.998

SCT

irritation

score

erythema

0.5705 0.734 0.7502* 0.9266 0.9926 1.0 0.9964

y = 23.728x � 0.3223 y = 0.0154x

+ 0.6257y = 5E + 08x � 4.1576 y = 1.4911x � 10.525 y = 0.7666x + 2.76 – y = 1.3003x � 3.0642

SCT TEWL

(d5)

0.4679 0.6446 0.6706 0.9314 0.9185 0.9964 1.0

y = 48.24x � 0.0816 y = x22.425x

+ 18.159y = 4393.8x � 1.106 y = 0.7529x + 22.977 y = x2.4789 x � 71.998 y = 1.3003x � 3.0642 –

ECT total

irritation

score

0.4588* 0.6726 0.6319 1.0 0.9023 0.9266 0.9314

y = 28.978x � 0.2497 y = 29.366x

� 5.9773y = � 1.2258x + 117.71 – y = 1.9166x � 16.227 y = 1.4911x � 10.525 y = 0.7529x

+ 22.977

HET-CAM

(Q)

0.546 1.0 0.7061 0.6726 0.7289 0.734 0.6446

y = � 0.4184x + 1.5831 – y = � 19.583x + 96.885 y = 29.366x � 5.9773 y = 0.0118x + 0.6639 y = 0.0154 x + 0.6257 y = 22.425x + 18.159

RBC

(H50/DI)

1.0 0.546 0.5528* 0.4588* 0.5234 0.5705 0.4679

– y = � 0.4184x

+ 1.5831y = 60.736x0.0667 y = 28.978x � 0.2497 y = 22.886x � 0.3231 y = 23.728x � 0.3223 y = 48.24x � 0.0816

RBC/DI 0.9107* 0.7147 0.6068** 0.8737 0.8539 0.8763 0.8052

y = 64.455x � 1.1958 y = 0.0201x

+ 0.6356y = � 1E � 05x4 +0.0023x3 � 0.1476 x2 +2.2971x + 82.686

y = 0.8088x + 8.0451 y = 1.6261x � 3.602 y = 0.6928x + 2.7007 y = 1.3233x � 34.254

Skinethic

average

0.5528* 0.7061 1.0 0.6319 0.7606* 0.7502* 0.6706

y = 60.736x0.0667 y = � 19.583x

+ 96.885– y = � 1.2258x + 117.71 y = 1E + 09x � 4.3632 y= 5E+08x � 4.1576 y = 4393.8 x � 1.106

* Correlation according to the ‘‘power’’ function.** Polynomial; all others linear.

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lation between the classifications obtained by the varioustests evaluated in this study although the methods usedare either validated to assess ocular irritation or used toevaluate dermal irritation and not both. There were no dif-ferences in the classification according to the irritationscores obtained in the epicutaneous patch test, the soapchamber test or the TEWL measurements. Slight variationscan be found within the ocular irritation tests. TexaponSB3 exhibited a lower irritation potential in the HET-CAM than in the other two tests. The irritation potentialof Plantapon ACG 50, Dehyton PK45, Texapon ALSand Texapon K12 G was higher when assessed via theHET-CAM assay compared to the other two ocular testsbut, in the case of Texapon ALS and Texapon K12 G,the classification had a better correlation to the dermaltests. The amphoteric surfactants and Dehyton PK45 var-ied depending on the ocular test used. Being amphotericmolecules this may in part be due to the change in chargedue to the pH.

To compare the variability between the tests, the scoresobtained in each method was entered into a MicrosoftExcel spreadsheet and plotted against each other to createa scatter graph. Regression curves and correlation coeffi-cients were calculated using the Microsoft Excel 2003 pro-gram after omitting the values for the positive and negativecontrols as these were not the same for all methods. Nocorrections for outliers were made. Correlations betweenthe test systems was calculated using the trend line functionof the Microsoft Excel 2003 program. If more than onevalue was available, averages were used. In order to obtaina single value, the averages of the values obtained at thethree time-points was calculated and used for the compar-isons. The r2 values (coefficients of determination) alongwith the corresponding equations are depicted in Table 6.In particular the different parameters assessed in the der-mal tests showed an excellent correlation to one another.Interestingly, although the H50/DI scores obtained usingthe RBC showed only a moderate correlation to the othertest scores, the DI value of the RBC test showed an excel-lent correlation to the irritation scores of both the ECT(r2 = 0.8737) and the SCT (r2 = 0.8539) as well as to theTEWL (r2 = 0.8052) and erythema scores (r2 = 0.8763)obtained in the SCT. The Skinethic model and the HET-CAMs also correlated well with the dermal tests, althoughto a lesser extent than the RBC (DI).

4. Discussion

Assessment of ocular and dermal irritation plays a rolein both occupational hazard assessment and classificationas well as in consumer product development. In the former,the product is tested undiluted and the pH is not adjusted.It reflects the situation of some types of industrial use and/or transport and/or storage of the raw materials takesplace. The pH of certain products are often adjusted tovery high or low pHs, or very high concentrations of sur-factants for storage or transport purposes are used in order


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to ensure microbial contamination will not take place inthe absence of preservatives. In contrast, during productdevelopment of cosmetics, other aspects and approachesare taken into account and mildness of the formulation isindispensable. To this aim, the concentration of surfactantsin personal care formulations and their pH are modified toensure compatibility. Therefore, the surfactants tested inthis study can be found in cosmetic formulations, such asbody washes and shampoos, and, as a rule, have low irrita-tion potentials in these applications.

As mentioned above, the concentration of a surfactantplays a major role in the irritation potential of a productand surfactants as cosmetic formulation ingredients areusually used at much lower concentrations than for bulkproduct purposes. Results obtained from ECTs previouslycarried out as described above reveal the direct dependenceof the irritation score on the AS concentration, or in otherwords the dose dependency, of the surfactant tested. Thishas been reported for SDS by Brasch et al. (1999). Studiesinitiated by our department in which Texapon N70 wastested at: 0.25%; 0.5%; 0.75% and 1.0% AS also showeda dose-dependency resulting in the following irritationscores relative to those elicited by 0.5% SDS: 31%; 47%;51% and 60%, respectively (data not shown). Therefore,the concentrations according to AS% should also be takeninto account when testing for irritation in particular whencomparing similar substances. The concentrations used inthis study were chosen depending on the requirements ofthe test system, the ability to differentiate between the sur-factants and consumer use conditions. For example, a 3%AS dilution would represent a 1:4 to 1:3 dilution of a typ-ical rinse-off product. Therefore, this concentration wasused in the HET-CAM assays. Testing a single test concen-tration for the RBC test is not feasible as serial dilutionsand concentrations are needed to obtain the H50 values.The irritation potential is also dependent on the applica-tion time of the product (Bartnik and Kunstler, 1987;Loffler et al., 2000). The application times and the occlusiveconditions used in ECTs and SCTs create highly exagger-ated conditions. As the SCT uses repetitive occlusive appli-cations and the ECT only used one application, lowerconcentrations of surfactants are used in the SCT. Inrinse-off products there is only short non-occlusive contactwith the skin and dilution of the product during rinsing ishigh thus lowering irritation responses under useconditions.

The pH of cosmetic preparations is generally adjusted toa more skin compatible range than is necessary to avoidmicrobial contamination during transport and storage ofthe undiluted product. Binding of surfactants to proteins,which in turn can play a role in irritation, is in part pH-dependent (Bartnik and Kunstler, 1987; Rhein et al.,1990). A correlation between the pH and irritant effecthas been reported for skin cleaners (Baranda et al.,2002). The pH of the stratum corneum of healthy skinhas been reported to lie in the range of 4.0–6.8 dependingon the location on the body, gender, chronobiological


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effects and test method used (Yosipovitch et al., 1998;Ehlers et al., 2001; Matousek and Campbell, 2002; Wil-liams et al., 2005; Mehling and Fluhr, in press). HET-CAM tests performed using APGs also reveal that reac-tions are very dependent on the pH tested. An irritationscore Q of 0.55 was obtained at a pH of 11.5 whereas theirritation score at a pH of 7.0 was 0.19 (Aulmann and Ster-zel, 1997). In general, personal hygiene products are there-fore usually adjusted to a pH somewhere within the rangeof pH 5 to pH 8. In this study, with the exception of theRBC test (pH 7.4; physiological pH of blood) and the acuteeye irritation test (pH 7.0), the surfactant formulationswere tested at a pH of 6.5.

As the pH and the concentration of a product cangreatly influence the outcome of skin and ocular irritationstudies, the approach used in this study was to furtherstandardize the different test method by using the sameconcentrations of active substance and pH within the testmethod being used in order to obtain a better comparabil-ity of the results. Although methods used to evaluate bothocular and dermal irritation were compared, in generalclusters of substances with varying irritation potentialwere identified similarly by most tests, e.g. surfactantswith the highest irritation potentials such Texapon ALS,Texapon K12 G and surfactants with a more moderateirritating potential, e.g. Texapon N70, as well as the verymild surfactants, e.g. Texapon LC7, the non-ionic APG-type surfactants tested, and Gluadin WK were found inthe same clusters. Results from Loffler and Happle(2003b) also reported clear differences in the irritationpotential of three surfactants with sodium lauryl sulfateto be higher than that of sodium laureth sulfate withthe non-ionic APG having the lowest number of irritationresponses thus further confirming in the results obtainedin this study. The grading used to assess the dermal irrita-tion potential of the surfactants correlates well with theocular tests although much more data is needed to vali-date the classification. The similar clustering may in partbe due to interactions with proteins and the breakdownof epithelial integrity typical of both ocular and dermalirritation (Xu et al., 2000). These results show that whenusing standardized test conditions in which surfactantsare tested with the same pH and % AS within a testmethod, there is a good correlation between the in vitroocular irritation assays themselves and between the der-mal and ocular irritation assays. Therefore, an initialassessment of surfactants in respect to both their ocularand dermal irritation potential, e.g. for screening pur-poses, can be achieved by using one or more of the abovementioned standardized test conditions to test the prod-uct. One point that should be kept in mind when testingis that intraindividual and interindividual responses aswell as population differences in skin reactivity differdepending on the volunteer, chronobiological rhythms,season, etc. (Basketter et al., 1996; Robinson, 2001; Rob-inson, 2002; Loffler and Happle, 2003b; Mehling andFluhr, in press). Therefore, in particular when testing sur-


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factants and surfactant-based formulations in ECTs con-ducted according to the protocol used in this study, ourexperience over the years has shown that using 0.5%SDS and 1.0% (AS) Texapon N70 as positive controlsallows a good comparison of the data as 1% (AS) Texa-pon N70 elicits approximately 50% (±10%) of the reac-tions elicited by 0.5% SDS when comparing the totalirritation scores.

The values obtained when using the red blood cell testseem to be particularly good in assessing both ocular(H50/DI) and dermal (DI) irritation potential of surfac-tants. This test was developed to assess the initial cellularreactions elicited by irritating chemicals. The RBC testhas been shown to be particularly amenable to the evalua-tion of the ocular irritation potential of surfactants and hasbeen shown to produce reliable results in both intra- andinter-laboratory reproducibility as well as a good fit to pre-diction models (Pape et al., 1999). Although the H50/DIcorrelations were not particularly good when comparingthe RBC to other tests, probably due to the high degreeof variation based on the logarithmic scale used for gradingand still leading to the same classification, a very good cor-relation between the RBC denaturation indices and thedermal tests was found. Pfannenbecker et al. (2005) alsoreported an excellent correlation between erythema in a24 h ECT and the DI (R2 = 0.974; y = 28.996x). Dermalirritation and in particular erythema has been ascribed tothe interactions between substances and the proteins ofthe skin (Ananthapadmanabhan et al., 2004). As the dena-turation index is a measure of protein denaturation elicitedby the test substance, this would be consistent with theeffects found on skin and gives a plausible explanationfor the excellent correlation. This is also in line with thegood correlation found when using the zein test, a testbased on protein interactions with the test substance, toscreen for dermal irritation potentials (Deo et al., 2003).Therefore, this test seems particularly well suited as ascreening test for both the ocular and dermal irritationpotential of surfactants using the H50/DI for the ocular irri-tation potential and the DI as a measure for the dermalirritation potential of the substance.

In conclusion, when assessing irritation and sensitiza-tion, it is essential that dermatologists and cosmetic scien-tists bear in mind that pH and concentration (% AS) effectscan play a major role in provoking skin irritation. Irrita-tion can in turn facilitate the induction or elicitation ofallergic contact dermatitis. Furthermore, irritation can leadto clinically confusing results thus leading to potentiallyfalse classifications of irritants as allergens (Basketteret al., 2004). Furthermore, often product dilutions aretested which do not necessarily result in equivalent concen-trations in respect to active substance in the product. Thisis of particular relevance when products are available indifferent AS concentrations or pH, e.g sodium laureth sul-fate is sold at different AS concentrations, e.g. at 28% or70% AS; the pH of Plantacare 818 is adjusted to �12 forstorage purposes whereas Dehyton PK45 has a pH of


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4.5–5.5, thus making product dilutions differ substantiallyin both pH and AS%. Therefore, these two parameters,as well as the vehicle, should always be noted in studieson irritation potential and sensitization in order to allowa better comparison of study results. Furthermore, irritat-ing substances such as SDS are recommended as controlswhen assessing contact dermatitis following patch testingwith allergens in order to differentiate between the allergicand irritant reactions (Brasch et al., 1999; Loffler et al.,2005). It is also of importance to design testing proceduresbased on whether hazard assessments or development ofconsumer products are the focus of the study. In this study,emphasis was laid on personal care product developmentand therefore the pH and concentration of the productswere adjusted to relevant values. In general, clusters of sub-stances with varying irritation potential were identifiedsimilarly by most tests. These results show that when usingstandardized test conditions described in this study, there isa good correlation between the results obtained with thein vitro ocular irritation assays themselves, and betweenthe dermal and ocular irritation assays. In particular theRBC test seems to be not only highly predictive for ocularirritation (H50/DI) but also for dermal irritation andchanges in barrier function induced by surfactants (DI),making this test an ideal screening tool to assess the com-patibility of surfactant and surfactant formulations duringproduct development.

5. Uncited reference

Loffler and Happle (2003a).

Conflict of interest

The authors are not aware of any conflicts of interest.
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comparative studies on the ocular and dermal irritation

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