Comparative studies on the ocular and dermal irritation potential of surfactants

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1

2

Comparative studies on the ocular and dermal irritation

3

potential of surfactants

4

A. Mehling

^*

, M. Kleber, H. Hensen

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

6 Received 14 March 2006; accepted 27 October 2006

7

8 Abstract

9 Comparative studies on the irritation potential of 18 surfactants were performed using the same stock solution of surfactant for each 10 study. The ocular irritation potential of surfactants was studied using the red blood cell test (RBC), the hen’s egg test-chorioallantoic 11 membrane (HET-CAM) and the Skinethic ocular tissue model. The skin irritation potential was assessed based on data obtained from 12 human studies using a 24 h epicutaneous patch test (ECT) and a soap chamber test (SCT). The same pH and active substance (AS) con- 13 tent for all surfactants tested was used depending on the test conducted. In general, clusters of substances with varying irritation potential 14 were identiﬁed similarly by most tests. These results show that when using standardized test conditions in which pH and % AS are the 15 same for each surfactant tested, there is a good correlation between the in vitro ocular irritation assays themselves as well as between the 16 dermal and ocular irritation assays. In particular the RBC test seems to be not only highly predictive for ocular irritation (H50/DI) but 17 also for dermal irritation and changes in barrier function induced by surfactants (DI).

19

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

21

22 1. Introduction

23 Cleanliness is an age-old need and surfactants have been 24 used by humankind for this purpose since ancient times. In 25 the last decades, surfactant molecules have been in the 26 focus of scientiﬁc and technological developments for the 27 purpose of creating new surfactants as well as to modify 28 and optimize their performance. This is particularly evident 29 in respect to applications in industrial and household deter-

30 gent market as well as in cosmetics. Today surfactants are

31 widely used in many daily-used products ranging from

32 household and laundry detergents, from fabric condition-

33 ers to shampoos, body washes, hand soaps, etc.

34 Surfactants are amphipathic molecules and contain a

35 hydrophilic and a lipophilic moiety allowing them to inter-

36 act with both polar and nonpolar molecules, respectively.

37 Surfactants are generally classiﬁed according to these prop-

38 erties or based on their chemical constitution. Based on

39 their charge, they are classiﬁed as anionic, nonionic, cat-

40 ionic or amphoteric surfactants. Due to the amphipathic

41 properties of surfactants they can therefore be used as

42 cleansers, emulsiﬁers, foaming and wetting agents, etc.

43 When applied to the skin, they can interact with skin struc-

44 tures, in particular the lipid and protein components

45 (Cooper and Berner, 1985). Although surfactants used in

46 rinse-oﬀ products are generally well tolerated, they are a

47 risk for the development of irritant contact dermatitis

48 (Dihoum et al., 1996; Dykes, 1998). A prerequisite for

49 products designed for topical application is therefore that

doi:10.1016/j.fct.2006.10.024

Abbreviations: APG, alkyl polyglucoside; AS, active substance; DI, denaturation index; GCP, good clinical practice; ECT, epicutaneous patch test; 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, optical density; PBS, phosphate-buﬀered saline; Q, irritation quotient; RBC, red blood 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 2006 19209.

E-mail address:Annette.Mehling@Cognis.com(A. Mehling).

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50 they need to have a low ocular/mucous membrane and der- 51 mal irritation potential. The irritation potential is extre- 52 mely dependent on the concentration of the substance, as 53 well as on the composition and pH of the formulation 54 (Rhein et al., 1990; Turkoglu and Pekmezci, 1999; Baranda 55 et al., 2002). Due to the ability of surfactants to emulsify 56 and to reduce the surface tension of water, surfactants 57 can remove lipids from the skin during the washing pro- 58 cess. Therefore, the major dermal irritant eﬀects manifest 59 themselves particularly in dryness and roughness of the 60 skin although in rare cases more severe damage to the skin 61 can be elicited (Bartnik and Ku¨nstler, 1987; Loﬄer et al., 62 2000). Ocular irritation reactions to surfactants range from 63 reddening and tearing of the eyes to severe damage to the 64 ocular tissue, once again depending on the surfactant, the 65 pH and the concentrations tested (Bartnik and Ku¨nstler, 66 1987).

67 There are various methods regularly used to discrimi- 68 nate between detergents in respect to their potential irri- 69 tancy and occupational hazard classiﬁcations. The Draize 70 eye irritation test has been the gold standard for testing 71 eye irritation for many years. Due to the concern for ani- 72 mal welfare and the limitations of the method, alternative 73 methods such as the red blood cell test (RBC;Pape et al., 74 1999; INVITTOX Protocol Nr. 37), the hen’s egg test-cho- 75 rioallantoic membrane (HET-CAM) test (Steiling et al., 76 1999; INVITTOX Protocol Nr. 96) and tests using ocular 77 tissue models have been developed to assess the mucous 78 membrane/ocular irritation potential of surfactants and 79 other substances. Dermal irritation is often assessed in 80 humans using epicutaneous patch testing (ECT; one-time 81 occlusive patch test), whereas soap chamber tests (SCT) 82 can be used to assess irritation following repeated occlusive 83 exposure to the surfactant.

84 The aim of this study was to assess the comparability of

85 the test results of various methods. In order to avoid lot-to-

86 lot inconsistencies, the irritation potential of 18 surfactants

87 was evaluated using the same stock solution of surfactant

88 for each study. When labeling according to occupational

89 hazard classiﬁcation, substances are generally tested undi-

90 luted and at the pH of the stored raw material. This does

91 not reﬂect the conditions found in the personal hygiene

92 products, such as body-washes, in which the pH usually

93 ranges from pH 5 to 8. Therefore, in this study the compat-

94 ibility of the surfactant solutions at a pH that is relevant for

95 this type of consumer product was assessed. Except where

96 the limitations of the method made a diﬀerent pH necessary,

97 testing was carried out with products adjusted to a pH of

98 6.5. As compatibility is also dependent on the concentration

99 tested, equivalent concentrations based on the active sub-

100 stance content (AS, w/w) of each substance were tested.

101 The ocular irritation potential of surfactants was studied

102 using three in vitro test systems currently being used as

103 alternatives to animal studies, namely the RBC test, the

104 HET-CAM assay and the acute eye irritation assay based

105 on the commercially available ocular tissue model manufac-

106 tured by Skinethic Laboratories (France). The skin irrita-

107 tion potential was assessed based on data obtained from a

108 24 h ECT. The SCT was used to assess the cumulative der-

109 mal irritation potential of products after repeated exposure.

110 2. Materials and Methods

111 2.1. Test products and reagents

112

All surfactants used, with the exception of the sodium lauryl sulfate

113

(SDS) controls, were obtained from Cognis Deutschland GmbH &

114

Co.KG (Germany). A classiﬁcation of these surfactants can be found in

115

Table 1. The same stock solutions were used for all tests (12% AS; pH 6.5).

Table 1

Description of the surfactants tested in this study

Product name INCI Surfactant type Chemical description

Plantacare 818 Coco glucoside Nonionic Alkyl-polyglucoside

Plantacare 1200 Lauryl glucoside Nonionic Alkyl-polyglucoside

Plantacare 2000 Decyl glucoside Nonionic Alkyl-polyglucoside

Plantapon LGC sorb Sodium lauryl glucose carboxylate and lauryl glucoside

Anionic/nonionic Alkyl-glucoside carboxylate

Gluadin WK Sodium cocoyl hydrolyzed wheat protein Anionic Protein fatty acid condensate

Plantapon ACG 50 Disodium cocoyl glutamate Anionic Acyl-glutamate

Plantapon LC 7 Laureth-7 citrate Anionic Alkyl-ether citrate

PGFAC-S Sodium cocoyl hydrolyzed wheat

protein glutamate

Anionic Protein-glutamic fatty acid condensate

Texapon ALS benz Ammonium lauryl sulfate Anionic Alkyl-sulfate

Texapon K 12 G Sodium lauryl sulfate Anionic Alkyl-sulfate

Texapon K14 S special 70% Sodium myreth sulfate Anionic Alkyl-ether-sulfate

Texapon N 70 Sodium laureth sulfate anionic Alkyl-ether-sulfate

Texapon SB 3 KC Disodium laureth sulfosuccinate Anionic Alkyl-ether-sulfosuccinate

Dehyton DC Disodium cocoamodiacetate Amphoteric Alkyl-amphoacetate

Dehyton MC Sodium cocoamphoacetate Amphoteric Alkyl-amphoacetate

Dehyton PK 45 Cocamidopropyl betaine Amphoteric Alkyl-amidobetaine

Dehyton ML Sodium lauroamphoacetate Amphoteric Alkyl-amphoacetate

Gluadin WQ Laurdimonium hydroxypropyl

hydrolyzed wheat protein

Cationic Quaternized protein hydolysate

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116 All solutions used for one type of test were adjusted to the same pH and

117 AS% depending on the test used (Table 2). The aqueous dilutions were

118 adjusted to the AS concentration and pH (NaOH or citric acid or

119 hydrochloric acid) needed for each test prior to testing. Tests were con-

120 ducted using coded samples (‘‘double-blind’’). Regression curves and

121 coeﬃcients were calculated using the Microsoft Excel 2003 program

122 (trendline functions) after omitting the values for the positive and negative

123 controls. No corrections for outliers were made.

124 2.2. RBC test

125 The RBC test was carried out in accordance withINVITTOX Protocol

126 Nr. 37 using porcine erythrocytes obtained from one animal. Brieﬂy,

127 membrane damage was assessed by hemoglobin leakage and protein

128 interactions were assessed by denaturation of hemoglobin. Each surfactant

129 was tested in triplicate. Test samples were diluted to an initial concen-

130 tration of 1% AS in PBS and the pH adjusted to 7.4. Various concen-

131 trations of the test substance were aliquoted into 1.5 mL reaction vials and

132 25lL of the RBC suspension (8·10⁹cells/mL) added. The vials were

133 gently agitated for 10 min at room temperature and centrifuged for 1 min.

134 The OD530of the supernatant was measured and the half-maximal eﬀec-

135 tive concentration for hemolysis determined from the dose–response curve

136 (H50). At least eight equidistant concentrations of each surfactant were

137 monitored to assess the concentration response curve for hemolysis. The

138 hypotonic release of hemoglobin resulting from the addition of the

139 erythrocytes to distilled water was set to 100% hemolysis. In order to

140 determine the denaturation index, 975lL of a 0.1% dilution of the sample

141 was added to 25lL of the RBC suspension and gently agitated for 10 min

142 at room temperature. Following centrifugation for 10 min, the extinction

143 of the supernatant was measured at 575 nm to obtain the denaturation

144 index (DI). The denaturation index is calculated using 0.1% of the test

145 substance relative to the internal standard SDS (3.47 mmol/L). The H50/

146 DI ratio is then used as a measure of the eye irritation potential according

147 to the following classiﬁcation: >100: Not irritating; >10: Slightly irritating;

148 >1: Moderately irritating; >0.1: Irritating; <0.1: Very irritating.

149 2.3. HET-CAM assay

150 The HET-CAM assay was carried out according to the standard of

151 operational procedure (SOP) of the COLIPA project: Methodology

152 Alternatives: The HET-CAM SOP 2nd edition (INVITTOX protocol No.

153 96; Steiling et al., 1999) using the reaction-point method. In brief, fertilized

154 eggs were incubated 9 day prior to use. Six eggs were used for each test

155 substance. After exposing the CAM and rinsing it with warm PBS, 300lL

156 of the test solution (3% AS; pH 6.5) diluted in water were applied to the

157 CAM. The intensity of the reactions hemorrhage, lysis and coagulation

158 were semi-quantitatively assessed on a scale of 0 (no reaction) to 3 (strong

159 reaction). The time-point of appearance and the intensity of any reactions

160 occurring within 5 min were documented. The irritation quotient [Q] was

161 then calculated and expressed relative to the standard Texapon ASV [5%

162 AS; special fatty alcohol ether-sulfate]. The following classiﬁcations based

163 on Q were made:60.8: slightly irritating; >0.8 to <1.2: moderately irri-

164 tating;P1.2 to <2.0: irritating;P2.0 severely irritating.

165 2.4. Acute eye irritation assay

166

Prior to testing, the solution was diluted in water to a concentration of

167

0.6% AS and the pH was adjusted to pH 7.0. Tests were performed

168

according to the protocol proposed by Skinethic laboratories

169

(www.skinethic.com). In brief, 30lL of the samples were topically applied

170

to triplicate samples of reconstituted human corneal epithelial tissues

171

(Skinethic Laboratories, size: 0.5 cm²) and incubated at 37C and 5% CO2

172

for 10 min, 1 h and 3 h. PBS was used as a negative control, SDS (0.5%

173

and 1.0%) as a positive control. In order to assess viability, two cultures

174

for each tested time-point were placed on 300lL of 0.5 mg/mL MTT for

175

30 min at room temperature and subjected to visual assessment. After 3 h,

176

the cultures were placed in 1.5 mL of isopropanol for 2 h to quantitatively

177

extract the MTT. Optical density (OD) was measured at 570 nm and the

178

results expressed relative to the negative control (PBS). One culture treated

179

with a test substance was ﬁxed in formalin and embedded in paraﬃn.

180

Histological assessments were carried out on sections stained with hema-

181

toxylin and eosin (H and E). The classiﬁcation of the irritation potential

182

was based on viability as assessed by the MTT-test and visual assessments.

183

Products are classiﬁed according to the following classes: Nonirritating,

184

very slightly irritating, slightly irritating, irritating and very irritating.

185 2.5. Epicutaneous patch test

186

Test products were diluted to 2% AS in water and the pH adjusted to

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pH 6.5 prior to testing. Tests were carried out on 20 healthy and informed

188

volunteers using the principles of good clinical practice (GCP) for guid-

189

ance. In principle, the test was performed as previously described (Fo¨rster

190

et al., 2000). Seventy ﬁve microlitres of the aqueous solutions were applied

191

occlusively to the backs of the volunteers using large Finn chambers with

192

ﬁlter discs backed on ScanPore. After 24 h, the patches were removed.

193

Visual assessments of each individual parameter (erythema, edema,

194

squamation and ﬁssure) were carried out 6 h, 24 h, 48 h and 72 h after

195

patch removal. Reactions were scored according to predeﬁned grading

196

parameters on a scale of 0 (no reaction) – 4 (strong reaction). The total

197

irritation score was calculated and the values expressed relative to SDS.

198

The classiﬁcation was deﬁned in this study following a non-validated

199

categorization based on the total irritation scores (as % SDS): >75:

200

severely irritating; >50: irritating; >25: moderately irritating; >10: slightly

201

(610 very slightly or not irritating).

202 2.6. Soap chamber test

203

Test materials were diluted to 1% AS and the pH adjusted to pH 6.5

204

prior to testing. Tests were carried out on healthy and informed volunteers

205

(SCT 1:21 volunteers; SCT 2:22 volunteers) using a protocol derived from

206

Frosch and Kligman (1979)and the principles of good clinical practice

207

(GCP) for guidance. 100lL of each sample was occlusively applied to the

208

ventral forearm using small Hilltop Chambers backed on Scanpor.

209

Repeated patching was performed for 24 h (1st application), followed by

210

6 h each day for the next 4 days (days 2–5). The ﬁrst visual assessment of

211

reactions (erythema, dryness and ﬁssure) was performed 15 min after

212

patch removal on day 2; further visual assessments were carried out prior

213

to reapplication of the patch on days 3–5 as well as on day 8. Results were

214

expressed as total irritation scores relative to SDS. Transepidermal water

215

loss (TEWL) was measured on day 1, 5 and on day 8 following acclima-

216

tization for at least 20 min at 20C (±1C) and 50% relative humidity

217

(±5%). SCT irritation scores were calculated by adding the scores of

218

edema, dryness and ﬁssure on day 5 and expressed relative to SDS. The

219

classiﬁcation of the irritation potential was deﬁned in this study following

220

a non-validated categorization based on the total irritation scores (as %

221

SDS): >100: severely irritating; >25 irritating; >15: moderately irritating;

222

>10: slightly irritating (610 very slightly or not irritating). Although

223

irritation classiﬁcation is rarely done using TEWL, the following a non-

224

validated grading system was used to evaluate the irritation potential: >30:

225

severely irritating; >20 irritating; >15: moderately irritating; <15: slightly

226

irritating (610 very slightly or not irritating).

Table 2

The pH and concentration of the surfactant solutions tested (adjusted from identical stock solutions with 12% AS concentrations and a pH of 6.5)

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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227 3. Results

228 Identical stock solutions (12% AS; pH 6.5) were used for 229 all tests in order to avoid variations in the test results due 230 to diﬀerences in the inherent properties of the test product.

231 As the concentration of active substance (AS) and the pH 232 play a major role in eliciting irritant reactions, the pH and 233 % AS were adjusted according toTable 2depending on the 234 method used prior to testing. With the exception of the 235 ECT, all tests were carried out within the same month.

236 Some of the samples were tested in the ECT in the same 237 month whereas some of the samples were tested two 238 months later. Tests were carried out ‘‘double-blind’’ and 239 where appropriate randomized.

240 3.1. RBC test

241 The red blood cell test is a cell-based photometric test 242 and was developed to assess the initial cellular reactions 243 taking place in ocular irritation processes elicited by surfac- 244 tants and surfactant-based products (Pape et al., 1999;

245 Pape and Hoppe, 1990). The principle of the RBC test is 246 the assessment of membrane damage and protein denatur- 247 ation caused by surfactants. To this aim, the damage to the 248 cell membrane is assessed by the amount of hemoglobin 249 leakage into the supernatant and the changes in protein 250 conformation are assessed by hemoglobin denaturation.

251 As the physiological pH of the blood is7.4, all solutions 252 were adjusted to this pH in order to avoid shifts in the irri- 253 tation potential due to the pH alone. The H50/DI quotient 254 is used as a measure for the classiﬁcation of ocular irritancy 255 in analogy to the Draize eye irritation test. The ocular irri- 256 tation potential of the surfactants as assessed by using the 257 RBC test can be found inTable 3. Of the ﬁve similar sur- 258 factants also tested byPape et al. (1999)a good correlation 259 was obtained with results from this study (Table 4). This 260 demonstrates the reproducibility and robustness of the 261 method for assessing the irritation potential of surfactants.

262 The surfactants Dehyton MC, Gluadin WQ, Dehyton 263 ML, Plantacare 2000, Plantacare 818, Gluadin WK, and 264 Plantapon LC7 were classified as not irritating whereby 265 the surfactants Plantapon LC7, Gluadin WK and Planta- 266 pon 818 were the least irritating. The surfactants 267 PGFAC-S, Plantapon ACG 50, Plantapon LGC, and Plan- 268 tacare 1200 were classified as slightly irritating, the surfac- 269 tants Plantapon SB3, Dehyton DC and Dehyton PK45 270 were classified as moderately irritating, and the surfactants 271 Plantapon K14S, Texapon ALS, Texapon K12 G and Tex- 272 apon N70 were classified as irritating.

273 3.2. HET-CAM assay

274 The HET-CAM assay is an organotypic model used to 275 assess ocular irritation by taking advantage of the eﬀects 276 a product has when applied to the fragile blood vessel net- 277 work of the CAM. The method used in this study adhered 278 to the protocol described by Steiling et al. (1999). As the

279 products were all transparent, the reaction-time method

280 was used, in which the time-point and severity of the occur-

281 rence of the parameters hemorrhage, lysis and coagulation

282 are examined during a maximum time period of 5 min. The

283 calculated irritation index is then expressed relative to the

284 benchmark substance Texapon ASV (special alkyl-ether-

285 sulfate Na/Mg salt; 5% AS) which is set toQ= 1.0 (mod-

286 erately irritating). The pH of the products was adjusted to

287 6.5 prior to application to the CAM. The products were

288 tested with an AS content of 3%. The results are depicted

289 inTable 3.

290 The surfactants Dehyton DC, Dehyton MC, Dehyton

291 ML, Gluadin WQ, Gluadin WK, Plantacare 818, Planta-

292 care 1200, Plantacare 2000, and Plantapon LC7, Plantapon

293 LGC, PGFAC-S, Texapon SB3 were classiﬁed as slightly

294 irritating (there is no classiﬁcation ‘‘not irritating’’ accord-

295 ing to this protocol) whereby the surfactants Plantapon

296 LC7, Plantacare 1200 and PGFAC-S were the least irritat-

297 ing under these test conditions. The surfactants Plantapon

298 ACG 50 and Texapon K14S were classiﬁed as moderately

299 irritating, Texapon N70 was classiﬁed as irritating and the

300 surfactants Dehyton PK45, Texapon ALS and Texapon

301 K12 G were classiﬁed as severely irritating.

302 3.3. Acute eye irritation assay

303 In contrast to the other two models, the Skinethic

304 in vitro reconstituted human corneal epithelial tissue model

305 consists of airlift culture of diﬀerentiated keratinocytes

306 resulting in a three-dimensional structure resembling the

307 outer cornea of the human eye. The principle of this test

308 system is the evaluation of cytotoxicity of the topically

309 applied product via the MTT reduction assay and visual

310 assessment of the tissues. Histological evaluations further

311 characterize possible damage and/or cytotoxicity. The

312 products were tested at a pH of 7.0 and an AS content of

313 0.6%. Due to the lack of skin samples available for testing,

314 Dehyton ML and Gluadin WQ were not included in this

315 test. The results can be found inTable 3.

316 The surfactants Dehyton DC, Gluadin WK, Plantacare

317 818, Plantacare 1200, Plantacare 2000, Plantapon ACG 50,

318 Plantapon LC7 and PGFAC-S and were graded as not irri-

319 tating. The surfactant Plantapon LGC was classiﬁed as

320 very slightly irritating; the surfactants Dehyton MC, Texa-

321 pon K14 S and Plantapon SB3 were judged to be slightly

322 irritating. Dehyton PK45, Texapon ALS, Texapon K12

323 G and Texapon N70 were assessed as being irritating.

324 Dehyton ML and Gluadin WQ were not tested in this

325 assay.

326 Histological assessment of the H and E stained tissues

327 after topical application of the surfactants for up to 3 h

328 revealed the following histological assessments: Absence

329 of signiﬁcant histological alterations were found when test-

330 ing Gluadin WK, Plantapon LC7 and PGFAC-S thus cat-

331 egorizing these 3 surfactants as being the least irritating;

332 very slight cellular alterations in the superﬁcial layer was

333 found when testing the products Dehyton DC, Plantacare

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Table 3

Scores 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 Slightly

irritating

100.82 ± 6.14 92.68 ± 1.37 77.5 ± 3.03 Not irritating

Plantacare 1200 61.41 0.66 92.86 Slightly

irritating

0.24 Slightly

irritating

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 Slightly

irritating

100.57 ± 5.07 91.89 ± 3.17 71.27 ± 13.28 Not irritating

Plantapon LGC sorb 80.79 2.08 38.86 Slightly

irritating

0.80 Slightly

irritating

108.36 ± 0.98 86.75 ± 11.81 67.8 ± 0.53 Very slightly irritating

Gluadin WK 87.47 0.07 1337.88 Not irritating 0.72 Slightly

irritating

98.93 ± 4.18 75.56 ± 3.51 77.88 ± 5.52 Not irritating

Plantapon ACG 50 323.34 13.65 23.68 Slightly

irritating

1.16 Moderately

irritating

95.79± 3.82 86.09 ± 4.88 92.63 ± 20.32 Not irritating

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

irritating

100.94 ± 0.09 91.11 ± 5.48 85.26 ± 4.54 Not irritating

PGFAC-S 128.23 6.77 18.94 Slightly

irritating

0.41 Slightly

irritating

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 Severely

irritating

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 Severely

irritating

68.68 ± 6.23 31.28 ± 5.05 16.19 ± 2.23 Irritating Texapon K14 S special

70% S

13.62 34.03 0.40 Irritating 1.07 Moderately

irritating

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 Irritating

Texapon SB 3 KC 74.44 27.95 2.66 Moderately

irritating

0.72 Slightly

irritating

Dehyton DC 39.16 5.04 7.77 Moderately

irritating

0.63 Slightly

irritating

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 Slightly

irritating

Dehyton ML 183.34 0.83 222.13 Not irritating 0.79 Slightly

irritating

nd nd nd nd

Dehyton PK 45 18.26 2.32 7.87 Moderately

irritating

2.05 Severely

irritating

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 Slightly

irritating

nd nd nd nd

nd, not determined.

* Percentage of viability compared to the negative control (MTT test).

A.Mehlingetal./FoodandChemicalToxicologyxxx(2006)xxx–xxx5 FCT3713No.ofPages12,Model5+

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334 818, Plantacare 1200, Plantacare 2000 and Plantapon ACG 335 50. Cellular alterations in the upper layers were evident at 336 3 h for Dehyton MC, Dehyton PK45, Plantapon LGC, 337 Texapon K14 S and Texapon SB3. Tissue disintegration 338 in the upper cell layers was observed following application 339 of Texapon ALS, Texapon K12 G and Texapon N70. Tis- 340 sue disintegration was already found 10 min after applica- 341 tion of Texapon ALS and Texapon K12 G identifying 342 these two surfactants as those with the highest irritation 343 potential in this assay.

344 3.4. Epicutaneous patch test

345 The ECT is a method to ascertain the dermal irritation 346 potential of a substance following a single application of 347 the test substance onto the back of a volunteer. It allows 348 skin compatibility to be assessed under the exaggerated 349 conditions of occlusivity. The parameters erythema, 350 edema, squamation and ﬁssure are assessed 6 h, 24 h, 351 48 h and 72 h after patch removal (Walker et al., 1996).

352 The scores are then used to calculate the total irritation

353 score. Prior to testing, the pH of the products was adjusted

354 to pH 6.5 and the products tested with 2% AS. The results

355 can be found inTable 5.

356 A clear-cut grading scheme for the irritation potential in

357 human tests has not yet been established as the protocols

358 have not been standardized, assessment of the irritation

359 eﬀects and controls diﬀer depending on the laboratory con-

360 ducting the test and the reactions of volunteers being

361 tested. Therefore, classiﬁcation of the irritation potential

362 was deﬁned following a non-validated grading based on

363 the total irritation scores (as % SDS): >75: severely irritat-

364 ing; >50: irritating; >25: moderately irritating; >10: slightly

365 (610: very slightly irritating). According to this scheme, the

366 surfactants Dehyton MC, Gluadin WK, Plantapon ACG

367 50, Plantacare 2000, Plantapon LC7, Plantapon LGC

368 and Texapon SB3 were classiﬁed as very slightly irritating

369 with the surfactants Plantapon LC7, Plantapon LGC and

370 Gluadin WK being the least irritating. The surfactants

371 Dehyton DC, Dehyton PK45, Gluadin WQ, Plantacare

372 818, PGFAC-S and Plantacare 1200 were judged to

373 be slightly irritating The surfactants Dehyton ML and

Table 4

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

Surfactant nomenclature This study Study ofPape et al. (1999)

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

Texapon N70 Na-laureth-sulfate 0.17 Irritating 0.28 Irritating

Texapon ALS NH4-laurylsulfate 0.14 Irritating 0.31 Irritating

Texapon K12 G Na-laurylsulfate 0.22 Irritating 0.28 Irritating

Texapon SB3 Laureth-sulfosuccinate 2.66 Moderately irritating 3.73 Moderately irritating

Dehyton PK 45 Cocoamidopropylbetaine 7.87 Moderately irritating 2.83 Moderately irritating

Table 5

Comparison of the values obtained in the dermal compatibility tests relative to SDS (=100%) Surfactant Epicutaneous patch test Soap chamber test (values on day 5)

Total irritation score Total irritation score Irritation score erythema TEWL (d5)

ECT1 ECT2 Average SCT1 SCT2 Average SCT1 SCT2 Average SCT1 SCT2 Average

Plantacare 818 16.31 11.1 13.69 5.12 5.12 5.94 5.94 34.1 34.13

Plantacare 2000 5.44 6.81 6.13 4.65 4.65 4.95 4.95 33.6 33.58

Plantacare 1200 16.31 17 16.67 4.42 4.42 6.93 6.93 36.6 36.56

Plantapon LGC Sorb 2.13 2.13 1.16 1.16 2.48 2.48 31.3 31.31

Gluadin WK 2.13 2.13 2.62 2.62 1.95 1.95 26.78 26.78

Plantapon ACG 50 9.36 9.36 4.88 4.88 3.47 3.47 34.9 34.88

Plantapon LC 7 2.55 2.55 1.63 1.63 0.99 0.99 26.7 26.74

PGFAC-S 16.31 16.31 2.33 2.33 2.48 2.48 31.1 31.12

Texapon ALS 81.56 81.56 176.82 176.82 137 136.96 78.21 78.21

Texapon N 70 58.21 48.79 33.26 41.02 45.91 49.5 47.71 49.82 65 57.42

Texapon K 12 G 104.3 104.34 192.90 192.90 148.6 148.64 115.1 115.11

Texapon K 14 S special 70% 27.2 27.23 17.76 17.76 17.51 17.51 35.52 35.52

Texapon SB 3 KC 5.11 5.11 4.67 4.67 4.28 4.28 26.52 26.52

Dehyton DC 14.14 14.14 4.42 4.42 5.94 5.94 31.2 31.15

Dehyton MC 8.51 8.51 13.46 13.46 14.01 14.01 32.04 32.04

Dehyton ML 27.19 27.19 20.93 20.93 22.96 22.96 38.48 38.48

Dehyton PK 45 16.31 10.6 13.48 11.78 10.00 10.89 10.51 11.9 11.20 30.78 39 34.91

Gluadin WQ 15.22 15.22 2.99 2.99 4.28 4.28 28.3 28.3

Water 2.17 3.83 3 4.11 2.33 3.22 4.28 4.28 3.89 0 1.945

SDS 0.2% 100 100 100 100 100 100 100 100 100

SDS 0.5% 100

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374 Texapon K14 S were classiﬁed as being moderately irritat- 375 ing, Texapon N70 as irritating. Texapon ALS and Texapon 376 K12 G were the most irritating and being evaluated as 377 being very irritating.

378 3.5. Soap chamber test

379 The SCT is used to assess the cumulative irritation 380 potential of a product. The substances are occlusively 381 applied to the ventral forearm for 24 h on the first day 382 and for 6 h on the next 4 days. The parameters erythema, 383 dryness and fissure are rated visually and the scores on 384 day 5 used to calculate the total irritation score. Changes 385 in skin barrier function are assessed by transepidermal 386 water loss (TEWL). The scores obtained in this study can 387 be found inTable 5. Once again, there is no international 388 standardized method or classification scheme. The classifi- 389 cation was therefore defined following a non-validated def- 390 inition based on the total irritation scores (as % SDS):

391 >100: severely irritating; >25 irritating; >15: moderately 392 irritating; >10: slightly irritating (610 very slightly or not 393 irritating). Although irritation classiﬁcation is rarely done 394 using TEWL, the following a non-validated grading system 395 was used to evaluate the irritation potential: >30: severely 396 irritating; >20 irritating; >15: moderately irritating; 615:

397 slightly irritating (610 very slightly or not irritating).

398 Under the test conditions used, Dehyton DC, Dehyton

399 MC, Dehyton PK45, Gluadin WQ, Gluadin WK, Planta-

400 care ACG 50, Plantacare 818, Plantacare 1200, Plantacare

401 2000, Plantapon LC7, Plantapon LGC, PGFAC-S and

402 Texapon SB3 were classiﬁed as being slightly irritating,

403 whereby Gluadin WK, Gluadin WQ, Plantapon LC7,

404 Plantapon LGC and PGFAC-S had the lowest irritation

405 potentials. Dehyton ML and Texapon K14 S were judged

406 to be moderately irritating. Texapon N70 was classiﬁed

407 as irritating whereas Texapon ALS and Texapon K12 G

408 were severely irritating. The same classiﬁcation was found

409 when assessing TEWL whereby the lowest irritation poten-

410 tials were found for Plantapon LC7, PGFAC-S, Dehyton

411 DC and Plantapon LGC. The highest irritation potentials

412 were found for Texapon ALS and Texapon K12 G.

413 3.6. Comparison of the irritation potential found in the

414 diﬀerent tests

415 A schematic representation of the classiﬁcation of the

416 irritation potentials as described in this study is depicted

417 in Fig. 1. In this context, it is important to note, that the

418 classiﬁcation of the irritation potential found in the dermal

419 tests according to the scores deﬁned in this study is not val-

420 idated. Furthermore, this classiﬁcation is based on the

421 results obtained by surfactants only. There is a good corre-

Fig. 1. Comparison of the irritation classifications of the various surfactants based on the methods used. As not all methods differentiate between not and slightly irritating, these two classes were combined into one class. The Skinethic protocol exhibits slightly different classifications which were adjusted as follows: Not irritating = not or slightly irritating; very slightly irritating = not- or slightly-irritating; slightly irritating = moderately irritating;

irritating = irritating; severely irritating = severely irritating. For the comparison of the irritation classes in the dermal irritation studies the following non- validated deﬁnitions were used: ECT irritation scores (as % SDS): >75: severely irritating; >50: irritating; >25 moderately irritating;625 slightly irritating;

SCT irritation scores (as % SDS): >100: severely irritating; >25: irritating; >15: moderately irritating; 615: slightly irritating. Although irritation 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: slightly irritating (610 very slightly or not irritating). In general, there was a good comparability of the classiﬁcation between the methods used.

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Table 6

Correlation of the results (R²coeﬃcients 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 irritation

score

SCT total irritation score

SCT irritation score erythema

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 + 0.6639

y= 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 + 0.6257

y= 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 + 18.159

y= 4393.8x 1.106 y= 0.7529x+ 22.977 y=x2.4789x 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 5.9773

y= 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= 0.0154x+ 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

+ 1.5831

y= 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 + 0.6356

y= 1E 05x4 + 0.0023x3 0.1476x2 + 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= 4393.8x 1.106

* Correlation according to the ‘‘power’’ function.

**Polynomial; all others linear.

8A.Mehlingetal./FoodandChemicalToxicologyxxx(2006)xxx–xxx FCT3713No.ofPages12,Model5+

21November2006DiskUsed

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422 lation between the classifications obtained by the various 423 tests evaluated in this study although the methods used 424 are either validated to assess ocular irritation or used to 425 evaluate dermal irritation and not both. There were no dif- 426 ferences in the classification according to the irritation 427 scores obtained in the epicutaneous patch test, the soap 428 chamber test or the TEWL measurements. Slight variations 429 can be found within the ocular irritation tests. Texapon 430 SB3 exhibited a lower irritation potential in the HET- 431 CAM than in the other two tests. The irritation potential 432 of Plantapon ACG 50, Dehyton PK45, Texapon ALS 433 and Texapon K12 G was higher when assessed via the 434 HET-CAM assay compared to the other two ocular tests 435 but, in the case of Texapon ALS and Texapon K12 G, 436 the classification had a better correlation to the dermal 437 tests. The amphoteric surfactants and Dehyton PK45 var- 438 ied depending on the ocular test used. Being amphoteric 439 molecules this may in part be due to the change in charge 440 due to the pH.

441 To compare the variability between the tests, the scores 442 obtained in each method was entered into a Microsoft 443 Excel spreadsheet and plotted against each other to create 444 a scatter graph. Regression curves and correlation coeﬃ- 445 cients were calculated using the Microsoft Excel 2003 pro- 446 gram after omitting the values for the positive and negative 447 controls as these were not the same for all methods. No 448 corrections for outliers were made. Correlations between 449 the test systems was calculated using the trend line function 450 of the Microsoft Excel 2003 program. If more than one 451 value was available, averages were used. In order to obtain 452 a single value, the averages of the values obtained at the 453 three time-points was calculated and used for the compar- 454 isons. The r² values (coeﬃcients of determination) along 455 with the corresponding equations are depicted inTable 6.

456 In particular the diﬀerent parameters assessed in the der- 457 mal tests showed an excellent correlation to one another.

458 Interestingly, although the H50/DI scores obtained using 459 the RBC showed only a moderate correlation to the other 460 test scores, the DI value of the RBC test showed an excel- 461 lent correlation to the irritation scores of both the ECT 462 (r²= 0.8737) and the SCT (r²= 0.8539) as well as to the 463 TEWL (r²= 0.8052) and erythema scores (r²= 0.8763) 464 obtained in the SCT. The Skinethic model and the HET- 465 CAMs also correlated well with the dermal tests, although 466 to a lesser extent than the RBC (DI).

467 4. Discussion

468 Assessment of ocular and dermal irritation plays a role 469 in both occupational hazard assessment and classiﬁcation 470 as well as in consumer product development. In the former, 471 the product is tested undiluted and the pH is not adjusted.

472 It reﬂects the situation of some types of industrial use and/

473 or transport and/or storage of the raw materials takes 474 place. The pH of certain products are often adjusted to 475 very high or low pHs, or very high concentrations of sur- 476 factants for storage or transport purposes are used in order

477 to ensure microbial contamination will not take place in

478 the absence of preservatives. In contrast, during product

479 development of cosmetics, other aspects and approaches

480 are taken into account and mildness of the formulation is

481 indispensable. To this aim, the concentration of surfactants

482 in personal care formulations and their pH are modiﬁed to

483 ensure compatibility. Therefore, the surfactants tested in

484 this study can be found in cosmetic formulations, such as

485 body washes and shampoos, and, as a rule, have low irrita-

486 tion potentials in these applications.

487 As mentioned above, the concentration of a surfactant

488 plays a major role in the irritation potential of a product

489 and surfactants as cosmetic formulation ingredients are

490 usually used at much lower concentrations than for bulk

491 product purposes. Results obtained from ECTs previously

492 carried out as described above reveal the direct dependence

493 of the irritation score on the AS concentration, or in other

494 words the dose dependency, of the surfactant tested. This

495 has been reported for SDS byBrasch et al. (1999). Studies

496 initiated by our department in which Texapon N70 was

497 tested at: 0.25%; 0.5%; 0.75% and 1.0% AS also showed

498 a dose-dependency resulting in the following irritation

499 scores relative to those elicited by 0.5% SDS: 31%; 47%;

500 51% and 60%, respectively (data not shown). Therefore,

501 the concentrations according to AS% should also be taken

502 into account when testing for irritation in particular when

503 comparing similar substances. The concentrations used in

504 this study were chosen depending on the requirements of

505 the test system, the ability to diﬀerentiate between the sur-

506 factants and consumer use conditions. For example, a 3%

507 AS dilution would represent a 1:4 to 1:3 dilution of a typ-

508 ical rinse-oﬀ product. Therefore, this concentration was

509 used in the HET-CAM assays. Testing a single test concen-

510 tration for the RBC test is not feasible as serial dilutions

511 and concentrations are needed to obtain the H50 values.

512 The irritation potential is also dependent on the applica-

513 tion time of the product (Bartnik and Ku¨nstler, 1987;

514 Loﬄer et al., 2000). The application times and the occlusive

515 conditions used in ECTs and SCTs create highly exagger-

516 ated conditions. As the SCT uses repetitive occlusive appli-

517 cations and the ECT only used one application, lower

518 concentrations of surfactants are used in the SCT. In

519 rinse-oﬀ products there is only short non-occlusive contact

520 with the skin and dilution of the product during rinsing is

521 high thus lowering irritation responses under use

522 conditions.

523 The pH of cosmetic preparations is generally adjusted to

524 a more skin compatible range than is necessary to avoid

525 microbial contamination during transport and storage of

526 the undiluted product. Binding of surfactants to proteins,

527 which in turn can play a role in irritation, is in part pH-

528 dependent (Bartnik and Ku¨nstler, 1987; Rhein et al.,

529 1990). A correlation between the pH and irritant eﬀect

530 has been reported for skin cleaners (Baranda et al.,

531 2002). The pH of the stratum corneum of healthy skin

532 has been reported to lie in the range of 4.0–6.8 depending

533 on the location on the body, gender, chronobiological

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