Toxicology Letters 225 (2014) 185–191
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Method for detecting the reactivity of chemicals towards peptides as an alternative test method for assessing skin sensitization potential Sun-A. Cho a,1 , Yun Hyeok Jeong a,1 , Ji Hoon Kim a , Seoyoung Kim a , Jun-Cheol Cho a,c , Young Heo b , Kyung-Do Suh c , Susun An a,∗∗ , Kyeho Shin a,∗ a
AmorePacific Corporation R&D Unit, Yongin-si, Republic of Korea College of Natural Sciences, Catholic University of Daegu, Kyongsan-si, Republic of Korea c Division of Chemical Engineering, College of Engineering, Hanyang University, Seoul, Republic of Korea b
h i g h l i g h t s • The skin sensitization potential evaluated using spectrophotometric-monitoring methods reactivity of chemicals toward peptides. • The combination results of cysteine and lysine peptide depletion showed good positive correlations. • New in vitro model is a good alternative evaluation model for the prediction of the skin sensitization potential.
a r t i c l e
i n f o
Article history: Received 11 June 2013 Received in revised form 10 December 2013 Accepted 11 December 2013 Available online 19 December 2013 Keywords: Skin sensitization Peptide assay Spectrophotometric method Cosmetic ingredient
a b s t r a c t Cosmetics are normally composed of various ingredients. Some cosmetic ingredients can act as chemical haptens reacting toward proteins or peptides of human skin and they can provoke an immunologic reaction, called as skin sensitization. This haptenation process is very important step of inducing skin sensitization and evaluating the sensitizing potentials of cosmetic ingredients is very important for consumer safety. Therefore, animal alternative methods focusing on monitoring haptenation potential are undergoing vigorous research. To examine the further usefulness of spectrophotometric methods to monitor reactivity of chemicals toward peptides for cosmetic ingredients. Forty chemicals (25 sensitizers and 15 non-sensitizers) were reacted with 2 synthetic peptides, e.g., the cysteine peptides (Ac-RFAACAA-COOH) with free thiol group and the lysine peptides (Ac-RFAAKAA-COOH) with free amine group. Unreacted peptides can be detected after incubating with 5,5 -dithiobis-2-nitrobenzoic acid or fluorescamineTM as detection reagents for free thiol and amine group, respectively. Chemicals were categorized as sensitizers when they induced more than 10% depletion of cysteine peptides or more than 30% depletion of lysine peptides. The sensitivity, specificity, and accuracy were 80.0%, 86.7% and 82.5%, respectively. These results demonstrate that spectrophotometric methods can be an easy, fast, and high-throughput screening tools predicting the skin sensitization potential of chemical including cosmetic ingredient. © 2013 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Some chemicals can elicit allergic contact dermatitis (ACD) via skin sensitization. Estimation of the skin sensitization potential for new ingredients is an important part of the safety assessment in cosmetics and topical drugs. For many decades, identification of
∗ Corresponding author at: AmorePacific Corporation R&D Unit, 314-1, Bora-dong, Giheung-gu, Yongin-si, Gyeonggi-do 446-729, Republic of Korea. Tel.: +82 31 280 5710; fax: +82 31 284 8478. ∗∗ Corresponding author at: AmorePacific Corporation R&D Unit, 314-1, Bora-dong, Giheung-gu, Yongin-si, Gyeonggi-do 446-729, Republic of Korea. Tel.: +86 21 5910 0685; fax: +86 21 5910 0817. E-mail addresses: ssan@amorepacific.com (S. An), rsj001@amorepacific.com, [email protected] (K. Shin). 1 These two authors have contributed equally to this work. 0378-4274/$ – see front matter © 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.toxlet.2013.12.007
potential skin sensitization hazards for cosmetic ingredients have evaluated by guinea-pig maximization test (GPMT) (Magnusson and Kligman, 1969). However, GPMT determined the skin sensitization potential by evaluating eczema and edema in elicitation phase so cannot provide quantitative data (Nukada et al., 2012). Moreover, concerning the animal welfare, the animal experiment is restricted for cosmetics and cosmetic ingredient since 2009 (EC, 2003). For these reason, the murine local lymph node assay (LLNA) was developed and adapted by OCED Test Guideline 429 (OECD, 2002). In comparison with the guinea pig methods, LLNA can provide quantitative data of skin sensitization potential by the measurement of lymphocyte proliferation in draining lymph nodes (EC3: Estimated concentration that produces a stimulation index of 3 in the murine local lymph node assay) (Kimber and Basketter, 1992). And LLNA showed a similar degree of sensitivity and specificity in terms of hazard identification compare to guinea pig methods (Dean et al.,
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2001) by use of reduced number of animals and shorter test period. However, this method still requires animals for testing and cannot satisfy the demand for full replacement of animals. In the cosmetic industry, alternative methods to animal experiments are undergoing a vigorous progress. Cosmetics Europe (Cosmetics Europe – The Personal Care Association) is involved in researching alternative studies and risk assessment methodologies, and the following 3 test methods are in the ECVAM (European Centre for the Validation of Alternative Methods) pre-validation stage: human cell line activation test (h-CLAT), myeloid U937 skin sensitization test (MUSST), and direct peptide reactivity assay (DPRA) (Adler et al., 2011). Skin sensitizers itself cannot directly induce skin sensitization. In skin sensitization process, the formation of adducts between chemical and endogenous protein and/or peptides in skin is essential (Dupuis et al., 1980; Landsteiner and Jacobs, 1935; Patlewicz et al., 2001). In skin proteins, the side chains of many amino acids contain electron-rich groups capable of reacting with nucleophilic allergens. Lysine and cysteine are those most often cited as electron-rich amino acids that can react with electrophiles strongly. Haptens (generally small molecules with a molecular weight less than 1000 Da) can interact with biological macromolecules in skin by formation of bonds of various strengths and these reactions called hapten–protein conjugation in mechanism of skin sensitization. The strength and bond stability of interaction between chemical and protein can critical point of hapten–protein conjugation that is first step of skin sensitization (Gerberick et al., 2008). Almost chemicals can form a hapten–protein conjugate and can induce a skin sensitization, thus the evaluation of the reactivity between chemicals and proteins and/or peptides can serve as an animal alternative for skin sensitization (Divkovic et al., 2005; Gerberick et al., 2008). In previous studies, the measurement of the reactivity between chemicals with protein and/or peptides has been formed by two methods. One is by monitoring the depletion of a selected peptide in the presence of chemicals using liquid chromatography with ultraviolet (UV) detection (Gerberick et al., 2004, 2007a), the other is by monitoring adduct formation between the peptide and the chemical using liquid chromatography mass spectrometry (LC/MS) or nuclear magnetic resonance (NMR) (Ahlfors et al., 2005; Aleksic et al., 2007, 2008; Alvarez-Sanchez et al., 2004a,b). The former chromatography-based methods have an advantage, that a very small quantity of sample is needed for the analysis (AlvarezSanchez et al., 2004a,b; Gerberick et al., 2007a; Natsch et al., 2007). The analysis using LC/MS- and NMR provide more detailed information than methods based on liquid chromatography. Although HPLC-, LC/MS-, and NMR-based analysis methods have the abovementioned advantages, these methods are rather expensive, time-consuming, and require skilled operators. In contrast, spectrophotometry is a conventional method used to detect changes in coloured samples and can be used even when very small quantities are available for analysis. Previously, a rapid and inexpensive spectrophotometric assay was developed for determining the reactivity of chemicals towards glutathione (GSH) (Schultz et al., 2005). The free thiol group of GSH was monitored by derivatization of GSH with 5,5 -dithiobis-2-nitrobenzoic acid (DTNB) and subsequent spectrophotometric detection and used to determine the reactivity of unsaturated carbonyl-containing compounds towards GSH. In previous our study, we established spectrophotometric assay methods to determine the reactivity of chemicals towards 2 chemical groups, i.e., the thiol group of a cysteine-containing peptide (cysteine peptide) and the amino group of a lysine-containing peptide (lysine peptide). We used DTNB as the detection reagent for the free thiol group and fluorescamine as the detection reagent for the free amine group. We also investigated the possibility of using this method as an in vitro sensitization test. By combination of the 2 methods using each type of peptide, our new method achieved
a high degree of sensitivity, specificity, and accuracy (Jeong et al., 2013). In this study, we reconfirm the estimation for skin sensitization using previous our established spectrophotometric assay methods. In addition, we ensure the possibility of application for cosmetic ingredients by use of our previous in vitro alternative for skin sensitization. 2. Material and methods 2.1. Peptides and test chemicals We used 2 model peptides, a cysteine peptide (Ac-RFAACAACOOH) and a lysine peptide (Ac-RFAAKAA-COOH), which have already been used in a previous study (Gerberick et al., 2004). Two synthetic model peptides were purchased from Peptron Co. (Daejeon, Korea) with >95% purity. The chemical name, class, chemical abstract system (CAS) number, and LLNA data of 40 test materials are presented in Table 1. We selected 30 chemicals that have been well categorized by their sensitization potential obtained from LLNA results and clinical data. Another 10 chemicals were selected from common ingredients, which are used in cosmetics. The test chemicals included 25 known sensitizers (S) and 15 nonsensitizers (NS). The peptide-to-chemical ratios used were 1:5 for cysteine and 1:10 for lysine. 2.2. Measurement of cysteine peptide depletion The cysteine peptide reaction solution was prepared by diluting the cysteine peptide stock solution to 400 M with 100 mM sodium phosphate buffer (pH 8.0, 1 mM EDTA) before the experiment. Chemical reaction solutions were prepared by diluting chemical stock solutions to 2 mM with 100 mM sodium phosphate buffer (pH 8.0, 1 mM EDTA). Then, 90 L of peptide reaction solution and 90 L of chemical reaction solution were reacted in the 96-well ELISA plate for 24 h at room temperature. After 24 h, 20 L of a 10 mM DTNB solution solubilized in sodium phosphate buffer (pH 8.0) was added to each well of the plate. The mixture was incubated for 3 min to achieve complete reaction between unreacted peptide and DTNB. Both, before the addition of the DTNB solution and after the reaction between unreacted peptide and DTNB, the optical density (OD) was measured using an UV–vis spectrophotometer (SpectraMAX 190TM , Molecular Devices, CA, USA; absorption wavelength: 412 nm). 2.3. Measurement of lysine peptide depletion The lysine peptide reaction solution was prepared by diluting the lysine peptide stock solution to 200 M with 100 mM sodium phosphate buffer (pH 10.0, 1 mM EDTA) before the experiment and chemical reaction solutions were prepared by diluting chemical stock solutions to 2 mM with isopropanol. Then, 100 L of peptide reaction solution and 100 L of chemical reaction solution were reacted in the 96-well ELISA plate for 24 h at room temperature. After incubation, 180 L of the reaction mixtures were transferred to a light-proof black clear-bottom 96-well plate (Greiner Bio-One, Frickenhausen, Germany). Next, 20 L of a 20 mM fluorescamine solution (prepared in DMSO) was added to each well of the plate and incubated for 3 min to achieve complete reaction between unreacted peptide and fluorescamine. Fluorescence intensity was measured using a fluorometer before the addition of fluorescamine solution and after the reaction between unreacted peptide and fluorescamine (Flexstation 3, Molecular Devices; excitation: 390 nm, emission: 465 nm, 475 nm).
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Table 1 List of tested chemicals. Group
S S S S S S S S S S S S S S S S S S S NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS
Test material
Oxazolonea 2-4 Dinitrochlorobenzene (DNCB)a Glutaric dialdehydea Lauryl gallatea Isoeugenola Diethyl maleatea Hexylcinnamic aldehyde (HCA)a Diphenylcyclopropenoneb 5-Chloro-2-methyl-4-isothiazolin-3-on (MCI)a 1,4-Dihydroquinonea 2-Aminophenola 4-Phenylenediamine (PPD)a Cobalt chloridec 2-mercaptobenzothiazolea Propyl gallatea Liliala Benzocainea Methyldibromoglutaronitrila Eugenola Dimethyl sulfoxidea Sodium lauryl sulfate (SLS)a Glycerola Citrala Phenyl benzoatea Cinnamic alcohola Imidazolidinyl ureaa Methyl metacrylatec Isopropanola Methyl salicylatea Lactic acida Chlorobenzenea Benzalkonium chlorided Tween 80e Methyl parabenb Resorcinola Salicylic acida Sodium benzoate Dehydroacetic acid Phenoxyethanol Ethylhexyl Methoxycinnamate
CAS No.
LLNA
15646-46-5 97-00-7 111-30-8 1166-52-5 97-54-1 141-05-9 101-86-0 886-38-4 26172-55-4 123-31-9 95-55-6 106-50-3 7646-79-9 149-30-4 121-79-9 80-54-6 1994-09-07 35691-65-7 97-53-0 67-68-5 151-21-3 56-81-5 53912-40-5 93-99-2 104-54-1 39236-46-9 80-62-6 67-63-0 119-36-8 50-21-5 108-90-7 8001-54-5 9005-65-6 99-76-3 108-46-3 69-72-7 532-32-1 520-45-6 9004-78-8 5466-77-3
Supplier
EC3 (%)
Category
0.003 0.05 0.1 0.3 1.2 5.8 11 0.003 0.009 0.11 0.4 0.16 0.8 1.7 0.32 19 22 1.3 13 72 14 N.C. 13 20 21 24 90 N.C. >20 C N.C. N.C. N.C. N.C. N.C. N.C. W.U. W.U. W.U. W.U.
Extreme Extreme Strong Strong Moderate Moderate Weak Extreme Extreme Strong Strong Strong Strong Moderate Strong Weak NS Moderate Weak Weak Weak (False-positive) NS Weak Weak Weak Weak Weak NS NS NS NS NS (False negative) NS NS NS NS NS NS NS NS
Sigma–Aldrich Sigma–Aldrich Sigma–Aldrich Sigma–Aldrich Sigma–Aldrich Sigma–Aldrich Sigma–Aldrich Sigma–Aldrich Fluka Sigma–Aldrich Fluka Sigma–Aldrich Sigma–Aldrich Sigma–Aldrich Sigma–Aldrich KIMEX Sigma–Aldrich Sigma–Aldrich Sigma–Aldrich Sigma–Aldrich Sigma–Aldrich Sigma–Aldrich Sigma–Aldrich Sigma–Aldrich Sigma–Aldrich Sigma–Aldrich Sigma–Aldrich Sigma–Aldrich Sigma–Aldrich Sigma–Aldrich Sigma–Aldrich Sigma–Aldrich Sigma–Aldrich Sigma–Aldrich Sigma–Aldrich Sigma–Aldrich Sigma–Aldrich Nippon Cohsei Sigma–Aldrich Sigma–Aldrich
% EC3 data obtained from Gerberick et al. (2005) (a), Gerberick et al. (2007b) (b), ICCVAM (2009) (c), Ashikaga et al. (2010) (d), Nukada et al. (2012) (e). N.C., not calculate. S/NS, sensitizer/non-sensitizer. W.U., widely used in cosmetics.
2.4. Calculation of peptide depletion ratio The reactivity of a chemical towards a peptide was expressed as the peptide depletion ratio after incubation of the peptide with the chemical, and the peptide depletion ratio was calculated as follows (Fig. 1): peptide depletion ratio (%) = (1 − Punreacted /Ptotal ) × 100. Some chemicals have a unique colour or may interfere in spectrophotometric property changes upon reaction with the peptide and/or react with the detection reagent. Background signals due to this interference must be considered for an exact evaluation. We used 3 different controls and measured the spectrophotometric values before and after addition of the detection reagents to avoid this kind of background signal (Fig. 1) (Jeong et al., 2013). The calculated peptide depletion ratios are presented as means ± SD and were obtained from 3 independent experiments, which were carried out in triplicate. 2.5. Comparison of skin sensitization potential results from peptide depletion and in vivo data The skin senstitization potentials of the 40 chemicals determined using peptide depletion were compared with the in vivo
data from the EC3 of LLNA (Ashikaga et al., 2010; ICCVAM, 2009; Gerberick et al., 2005, 2007b; Nukada et al., 2012). We evaluated the new alternative method by calculating the accuracy, sensitivity, and specificity of the results based on the modified classification of the in vivo data of the 40 chemicals as follows: sensitivity (%) = TP/(TP + FN) × 100, specificity (%) = TN/(TN + FP) × 100, and accuracy (%) = (TP + TN)/(TP + TN + FP + FN) × 100, where TP, true positive; TN, true negative; FN, false negative and FP, false positive. 3. Results 3.1. Data on chemical reactivity towards cysteine and lysine peptides The reactivity of the 40 test chemicals towards the peptides (peptide depletion ratio) is shown in Table 2. The results indicated a strong correlation between sensitizing potency and depletion of model peptide. The classification presented in Table 3 is based on
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Table 2 Percent depletion of the cysteine peptide or lysine peptide with regard to test chemicals. Test material
Oxazolone 2-4-Dinitrochlorobenzene (DNCB) Glutaric dialdehyde Lauryl gallate Isoeugenol Diethyl maleate Hexylcinnamic aldehyde (HCA) Diphenylcyclopropenone 5-Chloro-2-methyl-4-isothiazolin-3-on (MCI) 1,4-Dihydroquinone 2-Aminophenol 4-Phenylenediamine (PPD) Cobalt chloride 2-mercaptobenzothiazole Propyl gallate Lilial Benzocaine Methyldibromoglutaronitril Eugenol Dimethylsulfoxide (DMSO) Sodium lauryl sulfate (SLS) Glycerol Citral Phenyl benzoate Cinnamic alcohol Imidazolidinyl urea Methyl metacrylate Isopropanol Methyl salicylate Lactic acid Chlorobenzene Benzalkonium chloride Tween 80 Methyl paraben Resorcinol Salicylic acid
% depletion of cysteine peptide
% depletion of lysine peptide
Run 1
Run 2
Run 3
Average
SD
Run 1
10.88 99.42 0.84 24.58 98.75 98.57 86.29 89.09 23.00 100.93 91.50 97.42 −0.15 86.26 101.71 17.80 1.18 97.10 6.14 1.64 8.61 −0.14 19.69 21.35 1.70 −10.95 14.48 −2.26 9.49 10.13 1.41 19.19 13.95 −3.48 −1.54 −1.32
12.19 99.45 0.93 16.56 96.53 100.00 76.29 88.39 25.34 103.38 96.14 100.74 −1.93 89.46 102.83 9.25 0.96 94.29 7.09 0.13 5.64 1.63 20.40 24.75 2.64 −11.02 7.38 −1.97 12.40 9.51 0.41 38.21 15.72 0.41 −0.84 0.88
12.38 99.02 4.15 9.21 96.02 92.39 110.56 89.62 18.10 100.94 97.84 98.43 −2.39 90.17 100.27 12.28 −0.31 98.57 8.92 −0.62 7.83 0.36 11.94 18.92 1.25 −13.90 11.29 −4.82 7.48 7.75 −2.58 13.50 17.91 2.21 0.89 1.86
11.82 99.29 1.97 16.79 97.10 96.99 91.05 89.03 22.14 101.75 95.16 98.87 −1.49 88.63 101.60 13.11 0.61 96.65 7.38 0.38 7.36 0.62 17.35 21.67 1.86 −11.95 11.05 −3.02 9.79 9.13 −0.25 23.63 15.86 −0.29 −0.50 0.48
0.82 0.24 1.89 7.69 1.45 4.04 17.62 0.62 3.69 1.41 3.28 1.70 1.18 2.09 1.28 4.34 0.80 2.17 1.41 1.15 1.54 0.91 4.69 2.93 0.71 1.69 3.56 1.56 2.47 1.23 2.08 12.94 1.98 2.91 1.25 1.63
99.80 3.67 99.18 48.67 5.72 3.21 1.09 10.60 10.39 80.13 79.39 85.15 −4.35 −4.57 52.90 17.10 50.72 7.94 0.78 −5.40 6.54 13.42 12.22 6.13 12.13 15.18 9.54 11.81 0.03 −13.76 −2.77 −12.05 15.57 14.65 6.49 −12.13
Run 2 99.76 −1.84 99.75 48.63 4.18 −1.10 −3.78 9.62 5.40 82.26 54.95 87.45 −8.47 13.97 57.54 7.49 24.84 7.18 13.05 −5.24 −2.02 2.81 11.88 10.26 3.00 5.97 17.77 10.79 3.95 −2.77 −0.03 0.23 14.78 16.44 6.57 −0.48
Run 3
Average
SD
99.69 16.68 99.58 50.10 15.68 14.71 5.21 −4.14 6.35 80.69 79.79 85.69 5.43 −4.45 54.19 3.19 55.64 3.75 11.97 1.15 8.10 13.43 8.77 4.52 7.41 9.87 9.22 6.13 3.13 −0.62 6.71 12.30 15.25 13.89 0.35 −14.49
99.75 6.17 99.51 49.13 8.53 5.61 0.84 5.36 7.38 81.03 71.38 86.10 −2.46 1.65 54.87 9.26 43.73 6.29 8.60 −3.16 4.21 9.89 10.96 6.97 7.51 10.34 12.18 9.58 2.37 −5.72 1.30 0.16 15.20 14.99 4.47 −9.03
0.05 9.51 0.29 0.83 6.24 8.17 4.50 8.24 2.65 1.10 14.23 1.20 7.14 10.67 2.39 7.12 16.55 2.23 6.80 3.74 5.45 6.13 1.90 2.96 4.56 4.63 4.85 3.03 2.07 7.05 4.88 12.18 0.40 1.30 3.57 7.50
LLNA results and clinical data. For the majority of the extreme and strong allergens, depletion of >70% was observed for both peptides. Interestingly, DNCB and diphenylcyclopropenone demonstrated significant depletion of the cysteine peptide but not the lysine peptide. On the contrary, oxazolone and glutaric dialdehyde demonstrated significant depletion of only the lysine peptide. Finally, only a few non-sensitizers (e.g., benzalkonium chloride, Tween 80) demonstrated peptide depletion values similar to those observed with the sensitizers. Several weak sensitizers (e.g., eugenol, cinnamic alcohol) showed low reactivity towards the peptides.
(93.3% or 100%), but lower sensitivity (32.0%) and accuracy (70.0% or 57.5%). Combination of the 10% cut-off for the cysteine peptide with the 30% cut-off for the lysine peptide increased the prediction for sensitivity (80.0%) and accuracy (82.5%). For the combination of the 10% cut-off for the cysteine peptide with the 20% cut-off for the lysine peptide, the sensitivity, specificity, and accuracy were also sufficiently high (80.0% for sensitivity and specificity. 75% for accuracy). Compare to the other alternative for sensitization, the prediction of skin sensitization potential showed relatively high (Table 4).
3.2. Classification of chemicals based on their reactivity towards the cysteine and lysine peptides and combination of the classification cut-offs for the 2 peptide methods
4. Discussion
In this study, various cut-off criteria were tested to determine the appropriate cut-off criteria that showed highest accuracy for discriminating sensitizers and non-sensitizers. In addition, we tried to combine the results of the 2 peptide models. When we combined the best cut-off criteria for each peptide, we could obtain better results. A chemical was classified as a sensitizer when the accuracy of the combined cut-offs was higher than the classification cut-off of either the cysteine peptide method or the lysine peptide method. The results of the study are illustrated in Table 3, where the chemicals are classified according to the combination of the classification cut-offs of each peptide method. The sensitivity, specificity, and accuracy when a 10% cut-off was applied for the cysteine peptide method were 76.0%, 86.7%, and 77.5.0%, respectively. The cut-off for the lysine peptide method (20% or 30%) showed higher specificity
We tested 25 sensitizers and 15 non-sensitizers by measuring the reactivity of chemicals towards peptides using spectrophotometric analysis. Among these 40 chemicals, 18 chemicals were LLNA performance standards. We included 10 chemicals that were widely used in cosmetics and considered as non-sensitizer because we did not have any LLNA results, animal clinical data and report of skin sensitization for human. Test chemicals were classified mainly according to LLNA results. The spectrophotometric assay method using a cysteine peptide was based on the method developed by Schultz et al. (2005). Due to the characteristics of the spectrophotometric method used in this prediction model, care should be taken when making a prediction for chemicals that have a unique colour or may interfere in spectrophotometric property changes upon reaction with peptides and/or react with detection reagents. In a previous study, we established spectrophotometric assay methods by determining the reactivity
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Table 3 Classification of chemicals using different cut offs of peptide depletion ratios. Test material
Local lymph node assay (LLNA) grade
Oxazolone 2-4-Dinitrochlorobenzene (DNCB) Diphenylcyclopropenone 5-Chloro-2-methyl-4isothiazolin-3-on (MCI) Hydroquinone 2-Aminophenol Glutaric dialdehyde Lauryl gallate 4-Phenylenediamine (PPD) Cobalt chloride Propyl gallate Isoeugenol Diethyl maleate 2-Mercaptobenzothiazole Citral Methyldibromoglutaronitril Resorcinol Hexylcinnamic aldehyde (HCA) Phenyl benzoate Cinnamic alcohol Imidazolidinyl urea Lilial Methyl methacrylate Eugenol Benzocaine Dimethylsulfoxide (DMSO) Sodium lauryl sulfate (SLS) Glycerol Isopropanol Methyl salicylate Lactic acid Chlorobenzene Benzalkonium chloride Tween 80 Sodium benzoate Methyl paraben Dehydroacetic acid Phenoxyethanol Salicylic acid Ethylhexyl methoxycinnamate
Group
Peptide depletion
Criteria of classification
Cysteine
Lysine
Cysteine > 10 Lysine > 20
Lysine > 30
Cysteine > 10 or lysine > 20
Cysteine > 10 or lysine > 30
Extreme Extreme
S S
11.82 99.29
99.75 6.17
S S
S NS
S NS
S S
S S
Extreme Extreme
S S
89.03 22.14
5.36 7.38
S S
NS NS
NS NS
S S
S S
Extreme Extreme Strong Strong Strong Strong Strong Moderate Moderate Moderate Moderate Moderate Moderate Weak Weak Weak Weak Weak Weak Weak S (equivocal) NS NS NS NS NS/very weak NS/very weak NS NS (false negative) NS NS NS NS NS NS NS
S S S S S S S S S S S S S S S S S S S S S NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS
101.75 95.16 1.97 16.79 98.87 −1.49 101.60 97.10 96.99 88.63 17.35 96.65 −0.50 91.05 21.67 1.86 −11.95 13.11 11.05 7.38 0.61 0.38 7.36 0.62 −3.02 9.79 9.13 −0.25 23.63 15.86 −1.07 −0.29 −1.07 −1.67 0.48 −5.08
81.03 71.38 99.51 49.13 86.10 −2.46 54.87 8.53 5.61 1.65 10.96 6.29 4.47 0.84 6.97 7.51 10.34 9.26 12.18 8.60 43.73 −3.16 4.21 9.89 9.58 2.37 −5.72 1.30 0.16 15.20 21.03 14.99 10.19 15.35 −9.03 10.64
S S NS S S NS S S S S S S NS S S NS NS S S NS NS NS NS NS NS NS NS NS S S NS NS NS NS NS NS
S S S S S NS S NS NS NS NS NS NS NS NS NS NS NS NS NS S NS NS NS NS NS NS NS NS NS S NS NS NS NS NS
S S S S S NS S NS NS NS NS NS NS NS NS NS NS NS NS NS S NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS
S S S S S NS S S S S S S NS S S NS NS S S NS S NS NS NS NS NS NS NS S S S NS NS NS NS NS
S S S S S NS S S S S S S NS S S NS NS S S NS S NS NS NS NS NS NS NS S S NS NS NS NS NS NS
76.0 86.7 77.5
32.0 93.3 70
32.0 100.0 57.5
80.0 80.0 75
80.0 86.7 82.5
Sensitivity (%) Specificity (%) Accuracy (%)
of chemicals towards the thiol group of a cysteine peptide and the amino group of a lysine peptide using DTNB and fluorescamine as detecting reagents of the free thiol and amine groups, respectively (Jeong et al., 2013). Our new spectrophotometric-based method has several advantages, it is easy, fast, inexpensive and need not expert. To assure the usefulness of this method, it is essential to assess its sensitivity, specificity, and accuracy. We obtained good results when we set the classification cut-off for cysteine peptide depletion at >10% and for lysine peptide depletion at >30%. Benzalkonium chloride, sodium benzoate, and Tween
Table 4 The prediction of each animal alternative method for skin sensitization potentials.
Sensitivity (%) Specificity (%) Accuracy (%) a
Spectrophotometric DPRA (this study)
DPRAa
h-CLATa
DEREKa
80 86.7 82.5
79 80 79
88 72 84
83 56 76
The prediction data obtained from Nukada et al. (2013).
80 were classified as sensitizer in this study, while they were categorized as non-sensitizer previously. Eugenol, resorcinol, cobalt chloride, cinnamyl alcohol, and imidazolidinyl urea were classified as non-sensitizers, while they were previously categorized as weak sensitizers. Benzalkonium chloride was categorized as sensitizer according to the LLNA result. However, this chemical has been considered as an irritant in many articles; thus, LLNA results were occasionally believed to be false-positive. In this study, we selected benzalkonium chloride as a non-sensitizer, but it was classified as sensitizer on the basis of its reactivity towards peptides. It was suggested that the sensitizing potential of this chemical is controversial. In contrast, benzocaine was classified as a sensitizer on the basis of animal test results, although the results were equivocal. In summary, the prediction models, in which we combined the classification cut-offs of the 2 peptide methods, achieved a high degree of sensitivity, specificity, and accuracy. The combination of a 10% cut-off for the cysteine peptide method and a 30% cut-off for the lysine peptide method resulted in over 80% for each criterion. Although these methods need further improvement and optimization, these results demonstrate that spectrophotometric methods
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Fig. 1. Scheme of the peptide reactivity assay and formula for calculating the depletion ratio. Every test set was composed of 4 wells, including 3 control groups. Optical density (OD) or fluorescence intensity (FI) was measured just before and after adding the detection reagent. These OD or FI values were used to calibrate and calculate the peptide depletion rate. Ptotal is the total peptide added to each well. SVADP and SVBDP are the spectrophotometric values after and before addition of the detection reagent to the peptide-only control, respectively. SVADS and SVBDS are the spectrophotometric values after and before addition of the detection reagent to the solvent-only control, respectively. SVADR and SVBDR are the spectrophotometric values after and before addition of the detection reagent to reaction solutions, respectively. SVADC and SVBDC are the spectrophotometric values after and before addition of the detection reagent to the chemical-only control, respectively.
could serve as easy, fast, and high-throughput screening tools for the prediction of the skin sensitization potential of chemicals such as haptens and provide a high degree of sensitivity, specificity, and accuracy compared with traditional methods. Conflict of interest None. Acknowledgement This research was supported by a grant (11182MFDS575) from Ministry of Food and Drug safety in 2011. References Adler, S., Basketter, D., Creton, S., Pelkonen, O., van Benthem, J., Zuang, V., Andersen, K., Angers-Loustau, A., Aptula, A., Bal-Price, A., Benfenati, E., Bernauer, U., Bessems, J., Bois, F., Boobis, A., Brandon, E., Bremer, S., Broschard, T., Casati, S., Coecke, S., Corvi, R., Cronin, M., Daston, G., Dekant, W., Felter, S., Grignard, E., Gundert-Remy, U., Heinonen, T., Kimber, I., Kleinjans, J., Komulainen, H., Kreiling, R., Kreysa, J., Leite, S.B., Loizou, G., Maxwell, G., Mazzatorta, P., Munn, S., Pfuhler, S., Phrakonkham, P., Piersma, A., Poth, A., Prieto, P., Repetto, G., Rogiers, V., Schoeters, G., Schwarz, M., Serafimova, R., Tähti, H., Testai, E., van Delft, J., van Loveren, H., Vinken, M., Worth, A., Zaldivar, J.M., 2011. Alternative (non-animal) methods for cosmetics testing: current status and future prospects-2010. Arch. Toxicol. 85, 367–485. Ahlfors, S.R., Kristiansson, M.H., Lindh, C.H., Jonsson, B.A., Hansson, C., 2005. Adducts between nucleophilic amino acids and hexahydrophthalic anhydride, a structure inducing both types I and IV allergy. Biomarkers 10, 321–335. Aleksic, M., Pease, C.K., Basketter, D.A., Panico, M., Morris, H.R., Dell, A., 2007. Investigating protein haptenation mechanisms of skin sensitisers using human serum albumin as a model protein. Toxicol. In Vitro 21, 723–733. Aleksic, M., Pease, C.K., Basketter, D.A., Panico, M., Morris, H.R., Dell, A., 2008. Mass spectrometric identification of covalent adducts of the skin allergen 2,4-dinitro-1-chlorobenzene and model skin proteins. Toxicol. In Vitro 22, 1169–1176.
Alvarez-Sanchez, R., Basketter, D.A., Pease, C., Lepoittevin, J.P., 2004a. Covalent binding of the 13C-labeled skin sensitizers 5-chloro-2-methylisothiazol-3-one (MCI) and 2-methylisothiazol-3-one (MI) to a model peptide and glutathione. Bioorg. Med. Chem. Lett. 14, 365–368. Alvarez-Sanchez, R., Divkovic, M., Basketter, D., Pease, C., Panico, M., Dell, A., Morris, H., Lepoittevin, J.P., 2004b. Effect of glutathione on the covalent binding of the 13C-labeled skin sensitizer 5-chloro-2-methylisothiazol-3-one to human serum albumin: identification of adducts by nuclear magnetic resonance, matrixassisted laser desorption/ionization mass spectrometry, and nanoelectrospray tandem mass spectrometry. Chem. Res. Toxicol. 17, 1280–1288. Ashikaga, T., Sakaguchi, H., Sono, S., Kosaka, N., Ishikawa, M., Nukada, Y., Miyazawa, M., Ito, Y., Nishiyama, N., Itagaki, H., 2010. A comparative evaluation of in vitro skin sensitisation tests: the human cell-line activation test (h-CLAT) versus the local lymph node assay (LLNA). Altern. Lab. Anim. 38 (4), 275–284. Dean, J., Twerdok, L., Tice, R., Sailstad, D., Hattan, D., Stokes, W., 2001. ICCVAM evaluation of the murine local lymph node assay. Conclusions and recommendations of an independent scientific peer review panel. Regul. Toxicol. Pharmacol. 34, 258–273. Divkovic, M., Pease, C.K., Gerberick, G.F., Basketter, D.A., 2005. Hapten-protein binding: from theory to practical application in the in vitro prediction of skin sensitization. Contact Dermatitis 53, 189–200. Dupuis, G., Benezra, C., Schlewer, G., Stampf, J.L., 1980. Allergic contact dermatitis to alpha-methylene-gamma-butyrolactones. Preparation of alantolactone-protein conjugates and induction of contact sensitivity in the guinea pig by an alantolactone-skin protein conjugate. Mol. Immunol. 17, 1045–1051. EC, 2003. Directive 2003/15/EC of the European Parliament and of the Council of 27 February 2003. Off. J. Eur. Union L66, 26–35. Gerberick, G.F., Ryan, C.A., Kern, P.S., Dearman, R.J., Kimber, I., Patlewicz, G.Y., Basketter, D.A., 2004. A chemical dataset for evaluation of alternative approaches to skin-sensitization testing. Contact Dermatitis 50, 274–288. Gerberick, G.F., Ryan, C.A., Kern, P.S., Schlatter, H., Dearman, R.J., Kimber, I., Patlewicz, G.Y., Basketter, D.A., 2005. Compilation of historical local lymph node data for evaluation of skin sensitization alternative methods. Dermatitis 16 (4), 157–202. Gerberick, G.F., Vassallo, J.D., Foertsch, L.M., Price, B.B., Chaney, J.G., Lepoittevin, J.P., 2007a. Quantification of chemical peptide reactivity for screening contact allergens: a classification tree model approach. Toxicol. Sci. 97 (2), 417–427. Gerberick, G.F., Ryan, C.A., Dearman, R.J., Kimber, I., 2007b. Local lymph node assay (LLNA) for detection of sensitization capacity of chemicals. Methods 41, 54–60. Gerberick, F., Aleksic, M., Basketter, D., Casati, S., Karlberg, A.T., Kern, P., Kimber, I., Lepoittevin, J.P., Natsch, A., Ovigne, J.M., Rovida, C., Sakaguchi, H., Schultz, T., 2008. Chemical reactivity measurement and the predicitve identification of skin
S.-A. Cho et al. / Toxicology Letters 225 (2014) 185–191 sensitisers. The report and recommendations of ECVAM Workshop 64. Altern. Lab. Anim. 36, 215–242. ICCVAM, 2009. Recommended Performance Standards: Murine Local Lymph Node Assay, NIH Publication Number 09-7357. Jeong, Y.H., An, S., Shin, K., Lee, T.R., 2013. Peptide reactivity assay using spectrophotometric method for high-throughput screening of skin sensitization potential of chemical haptens. Toxicol. In Vitro 27 (1), 264–271. Kimber, I., Basketter, D., 1992. The murine local lymph node assay: a commentary on collaborative studies and new directions. Food Chem. Toxicol. 30, 165–169. Landsteiner, K., Jacobs, J., 1935. Studies on the sensitization of animals with simple chemical compounds. J. Exp. Med. 61, 643–656. Magnusson, B., Kligman, A., 1969. The identification of contact allergens by animal assay. The guinea pig maximization test. J. Invest. Dermatol. 52, 268–276. Natsch, A., Gfeller, H., Rothaupt, M., Ellis, G., 2007. Utility and limitations of a peptide reactivity assay to predict fragrance allergens in vitro. Toxicol. In Vitro 21, 1220–1226.
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Nukada, Y., Ashikaga, T., Miyazawa, M., Hirota, M., Sakaguchi, H., Sasa, H., Nishiyama, N., 2012. Prediction of skin sensitization potency of chemicals by human Cell Line Activation Test (h-CLAT) and an attempt at classifying skin sensitization potency. Toxicol. In Vitro 26 (7), 1150–1160. Nukada, Y., Miyazawa, M., Kazutoshi, S., Sakaguchi, H., Nishiyama, N., 2013. Data integration of non-animal tests for the development of a test battery to predict the skin sensitizing potential and potency of chemicals. Toxicol. In Vitro 27 (2), 609–618. OECD, 2002. Guidelines for Testing of Chemicals. No. 429: Skin Sensitisation: The Local Lymph Node Assay. 4. Organisation for Economic Co-operation and Development (OECD), Paris, France. Patlewicz, G., Basketter, D.A., Smith, C.K., Hotchkiss, S.A., Roberts, D.W., 2001. Skinsensitization structure-activity relationships for aldehydes. Contact Dermatitis 44, 331–336. Schultz, T.W., Yarbrough, J.W., Johnson, E.L., 2005. Structure-activity relationships for reactivity of carbonyl-containing compounds with glutathione. SAR QSAR Environ. Res. 16, 313–322.
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Toxicology Letters journal homepage: www.elsevier.com/locate/toxlet
Method for detecting the reactivity of chemicals towards peptides as an alternative test method for assessing skin sensitization potential Sun-A. Cho a,1 , Yun Hyeok Jeong a,1 , Ji Hoon Kim a , Seoyoung Kim a , Jun-Cheol Cho a,c , Young Heo b , Kyung-Do Suh c , Susun An a,∗∗ , Kyeho Shin a,∗ a
AmorePacific Corporation R&D Unit, Yongin-si, Republic of Korea College of Natural Sciences, Catholic University of Daegu, Kyongsan-si, Republic of Korea c Division of Chemical Engineering, College of Engineering, Hanyang University, Seoul, Republic of Korea b
h i g h l i g h t s • The skin sensitization potential evaluated using spectrophotometric-monitoring methods reactivity of chemicals toward peptides. • The combination results of cysteine and lysine peptide depletion showed good positive correlations. • New in vitro model is a good alternative evaluation model for the prediction of the skin sensitization potential.
a r t i c l e
i n f o
Article history: Received 11 June 2013 Received in revised form 10 December 2013 Accepted 11 December 2013 Available online 19 December 2013 Keywords: Skin sensitization Peptide assay Spectrophotometric method Cosmetic ingredient
a b s t r a c t Cosmetics are normally composed of various ingredients. Some cosmetic ingredients can act as chemical haptens reacting toward proteins or peptides of human skin and they can provoke an immunologic reaction, called as skin sensitization. This haptenation process is very important step of inducing skin sensitization and evaluating the sensitizing potentials of cosmetic ingredients is very important for consumer safety. Therefore, animal alternative methods focusing on monitoring haptenation potential are undergoing vigorous research. To examine the further usefulness of spectrophotometric methods to monitor reactivity of chemicals toward peptides for cosmetic ingredients. Forty chemicals (25 sensitizers and 15 non-sensitizers) were reacted with 2 synthetic peptides, e.g., the cysteine peptides (Ac-RFAACAA-COOH) with free thiol group and the lysine peptides (Ac-RFAAKAA-COOH) with free amine group. Unreacted peptides can be detected after incubating with 5,5 -dithiobis-2-nitrobenzoic acid or fluorescamineTM as detection reagents for free thiol and amine group, respectively. Chemicals were categorized as sensitizers when they induced more than 10% depletion of cysteine peptides or more than 30% depletion of lysine peptides. The sensitivity, specificity, and accuracy were 80.0%, 86.7% and 82.5%, respectively. These results demonstrate that spectrophotometric methods can be an easy, fast, and high-throughput screening tools predicting the skin sensitization potential of chemical including cosmetic ingredient. © 2013 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Some chemicals can elicit allergic contact dermatitis (ACD) via skin sensitization. Estimation of the skin sensitization potential for new ingredients is an important part of the safety assessment in cosmetics and topical drugs. For many decades, identification of
∗ Corresponding author at: AmorePacific Corporation R&D Unit, 314-1, Bora-dong, Giheung-gu, Yongin-si, Gyeonggi-do 446-729, Republic of Korea. Tel.: +82 31 280 5710; fax: +82 31 284 8478. ∗∗ Corresponding author at: AmorePacific Corporation R&D Unit, 314-1, Bora-dong, Giheung-gu, Yongin-si, Gyeonggi-do 446-729, Republic of Korea. Tel.: +86 21 5910 0685; fax: +86 21 5910 0817. E-mail addresses: ssan@amorepacific.com (S. An), rsj001@amorepacific.com, [email protected] (K. Shin). 1 These two authors have contributed equally to this work. 0378-4274/$ – see front matter © 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.toxlet.2013.12.007
potential skin sensitization hazards for cosmetic ingredients have evaluated by guinea-pig maximization test (GPMT) (Magnusson and Kligman, 1969). However, GPMT determined the skin sensitization potential by evaluating eczema and edema in elicitation phase so cannot provide quantitative data (Nukada et al., 2012). Moreover, concerning the animal welfare, the animal experiment is restricted for cosmetics and cosmetic ingredient since 2009 (EC, 2003). For these reason, the murine local lymph node assay (LLNA) was developed and adapted by OCED Test Guideline 429 (OECD, 2002). In comparison with the guinea pig methods, LLNA can provide quantitative data of skin sensitization potential by the measurement of lymphocyte proliferation in draining lymph nodes (EC3: Estimated concentration that produces a stimulation index of 3 in the murine local lymph node assay) (Kimber and Basketter, 1992). And LLNA showed a similar degree of sensitivity and specificity in terms of hazard identification compare to guinea pig methods (Dean et al.,
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2001) by use of reduced number of animals and shorter test period. However, this method still requires animals for testing and cannot satisfy the demand for full replacement of animals. In the cosmetic industry, alternative methods to animal experiments are undergoing a vigorous progress. Cosmetics Europe (Cosmetics Europe – The Personal Care Association) is involved in researching alternative studies and risk assessment methodologies, and the following 3 test methods are in the ECVAM (European Centre for the Validation of Alternative Methods) pre-validation stage: human cell line activation test (h-CLAT), myeloid U937 skin sensitization test (MUSST), and direct peptide reactivity assay (DPRA) (Adler et al., 2011). Skin sensitizers itself cannot directly induce skin sensitization. In skin sensitization process, the formation of adducts between chemical and endogenous protein and/or peptides in skin is essential (Dupuis et al., 1980; Landsteiner and Jacobs, 1935; Patlewicz et al., 2001). In skin proteins, the side chains of many amino acids contain electron-rich groups capable of reacting with nucleophilic allergens. Lysine and cysteine are those most often cited as electron-rich amino acids that can react with electrophiles strongly. Haptens (generally small molecules with a molecular weight less than 1000 Da) can interact with biological macromolecules in skin by formation of bonds of various strengths and these reactions called hapten–protein conjugation in mechanism of skin sensitization. The strength and bond stability of interaction between chemical and protein can critical point of hapten–protein conjugation that is first step of skin sensitization (Gerberick et al., 2008). Almost chemicals can form a hapten–protein conjugate and can induce a skin sensitization, thus the evaluation of the reactivity between chemicals and proteins and/or peptides can serve as an animal alternative for skin sensitization (Divkovic et al., 2005; Gerberick et al., 2008). In previous studies, the measurement of the reactivity between chemicals with protein and/or peptides has been formed by two methods. One is by monitoring the depletion of a selected peptide in the presence of chemicals using liquid chromatography with ultraviolet (UV) detection (Gerberick et al., 2004, 2007a), the other is by monitoring adduct formation between the peptide and the chemical using liquid chromatography mass spectrometry (LC/MS) or nuclear magnetic resonance (NMR) (Ahlfors et al., 2005; Aleksic et al., 2007, 2008; Alvarez-Sanchez et al., 2004a,b). The former chromatography-based methods have an advantage, that a very small quantity of sample is needed for the analysis (AlvarezSanchez et al., 2004a,b; Gerberick et al., 2007a; Natsch et al., 2007). The analysis using LC/MS- and NMR provide more detailed information than methods based on liquid chromatography. Although HPLC-, LC/MS-, and NMR-based analysis methods have the abovementioned advantages, these methods are rather expensive, time-consuming, and require skilled operators. In contrast, spectrophotometry is a conventional method used to detect changes in coloured samples and can be used even when very small quantities are available for analysis. Previously, a rapid and inexpensive spectrophotometric assay was developed for determining the reactivity of chemicals towards glutathione (GSH) (Schultz et al., 2005). The free thiol group of GSH was monitored by derivatization of GSH with 5,5 -dithiobis-2-nitrobenzoic acid (DTNB) and subsequent spectrophotometric detection and used to determine the reactivity of unsaturated carbonyl-containing compounds towards GSH. In previous our study, we established spectrophotometric assay methods to determine the reactivity of chemicals towards 2 chemical groups, i.e., the thiol group of a cysteine-containing peptide (cysteine peptide) and the amino group of a lysine-containing peptide (lysine peptide). We used DTNB as the detection reagent for the free thiol group and fluorescamine as the detection reagent for the free amine group. We also investigated the possibility of using this method as an in vitro sensitization test. By combination of the 2 methods using each type of peptide, our new method achieved
a high degree of sensitivity, specificity, and accuracy (Jeong et al., 2013). In this study, we reconfirm the estimation for skin sensitization using previous our established spectrophotometric assay methods. In addition, we ensure the possibility of application for cosmetic ingredients by use of our previous in vitro alternative for skin sensitization. 2. Material and methods 2.1. Peptides and test chemicals We used 2 model peptides, a cysteine peptide (Ac-RFAACAACOOH) and a lysine peptide (Ac-RFAAKAA-COOH), which have already been used in a previous study (Gerberick et al., 2004). Two synthetic model peptides were purchased from Peptron Co. (Daejeon, Korea) with >95% purity. The chemical name, class, chemical abstract system (CAS) number, and LLNA data of 40 test materials are presented in Table 1. We selected 30 chemicals that have been well categorized by their sensitization potential obtained from LLNA results and clinical data. Another 10 chemicals were selected from common ingredients, which are used in cosmetics. The test chemicals included 25 known sensitizers (S) and 15 nonsensitizers (NS). The peptide-to-chemical ratios used were 1:5 for cysteine and 1:10 for lysine. 2.2. Measurement of cysteine peptide depletion The cysteine peptide reaction solution was prepared by diluting the cysteine peptide stock solution to 400 M with 100 mM sodium phosphate buffer (pH 8.0, 1 mM EDTA) before the experiment. Chemical reaction solutions were prepared by diluting chemical stock solutions to 2 mM with 100 mM sodium phosphate buffer (pH 8.0, 1 mM EDTA). Then, 90 L of peptide reaction solution and 90 L of chemical reaction solution were reacted in the 96-well ELISA plate for 24 h at room temperature. After 24 h, 20 L of a 10 mM DTNB solution solubilized in sodium phosphate buffer (pH 8.0) was added to each well of the plate. The mixture was incubated for 3 min to achieve complete reaction between unreacted peptide and DTNB. Both, before the addition of the DTNB solution and after the reaction between unreacted peptide and DTNB, the optical density (OD) was measured using an UV–vis spectrophotometer (SpectraMAX 190TM , Molecular Devices, CA, USA; absorption wavelength: 412 nm). 2.3. Measurement of lysine peptide depletion The lysine peptide reaction solution was prepared by diluting the lysine peptide stock solution to 200 M with 100 mM sodium phosphate buffer (pH 10.0, 1 mM EDTA) before the experiment and chemical reaction solutions were prepared by diluting chemical stock solutions to 2 mM with isopropanol. Then, 100 L of peptide reaction solution and 100 L of chemical reaction solution were reacted in the 96-well ELISA plate for 24 h at room temperature. After incubation, 180 L of the reaction mixtures were transferred to a light-proof black clear-bottom 96-well plate (Greiner Bio-One, Frickenhausen, Germany). Next, 20 L of a 20 mM fluorescamine solution (prepared in DMSO) was added to each well of the plate and incubated for 3 min to achieve complete reaction between unreacted peptide and fluorescamine. Fluorescence intensity was measured using a fluorometer before the addition of fluorescamine solution and after the reaction between unreacted peptide and fluorescamine (Flexstation 3, Molecular Devices; excitation: 390 nm, emission: 465 nm, 475 nm).
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Table 1 List of tested chemicals. Group
S S S S S S S S S S S S S S S S S S S NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS
Test material
Oxazolonea 2-4 Dinitrochlorobenzene (DNCB)a Glutaric dialdehydea Lauryl gallatea Isoeugenola Diethyl maleatea Hexylcinnamic aldehyde (HCA)a Diphenylcyclopropenoneb 5-Chloro-2-methyl-4-isothiazolin-3-on (MCI)a 1,4-Dihydroquinonea 2-Aminophenola 4-Phenylenediamine (PPD)a Cobalt chloridec 2-mercaptobenzothiazolea Propyl gallatea Liliala Benzocainea Methyldibromoglutaronitrila Eugenola Dimethyl sulfoxidea Sodium lauryl sulfate (SLS)a Glycerola Citrala Phenyl benzoatea Cinnamic alcohola Imidazolidinyl ureaa Methyl metacrylatec Isopropanola Methyl salicylatea Lactic acida Chlorobenzenea Benzalkonium chlorided Tween 80e Methyl parabenb Resorcinola Salicylic acida Sodium benzoate Dehydroacetic acid Phenoxyethanol Ethylhexyl Methoxycinnamate
CAS No.
LLNA
15646-46-5 97-00-7 111-30-8 1166-52-5 97-54-1 141-05-9 101-86-0 886-38-4 26172-55-4 123-31-9 95-55-6 106-50-3 7646-79-9 149-30-4 121-79-9 80-54-6 1994-09-07 35691-65-7 97-53-0 67-68-5 151-21-3 56-81-5 53912-40-5 93-99-2 104-54-1 39236-46-9 80-62-6 67-63-0 119-36-8 50-21-5 108-90-7 8001-54-5 9005-65-6 99-76-3 108-46-3 69-72-7 532-32-1 520-45-6 9004-78-8 5466-77-3
Supplier
EC3 (%)
Category
0.003 0.05 0.1 0.3 1.2 5.8 11 0.003 0.009 0.11 0.4 0.16 0.8 1.7 0.32 19 22 1.3 13 72 14 N.C. 13 20 21 24 90 N.C. >20 C N.C. N.C. N.C. N.C. N.C. N.C. W.U. W.U. W.U. W.U.
Extreme Extreme Strong Strong Moderate Moderate Weak Extreme Extreme Strong Strong Strong Strong Moderate Strong Weak NS Moderate Weak Weak Weak (False-positive) NS Weak Weak Weak Weak Weak NS NS NS NS NS (False negative) NS NS NS NS NS NS NS NS
Sigma–Aldrich Sigma–Aldrich Sigma–Aldrich Sigma–Aldrich Sigma–Aldrich Sigma–Aldrich Sigma–Aldrich Sigma–Aldrich Fluka Sigma–Aldrich Fluka Sigma–Aldrich Sigma–Aldrich Sigma–Aldrich Sigma–Aldrich KIMEX Sigma–Aldrich Sigma–Aldrich Sigma–Aldrich Sigma–Aldrich Sigma–Aldrich Sigma–Aldrich Sigma–Aldrich Sigma–Aldrich Sigma–Aldrich Sigma–Aldrich Sigma–Aldrich Sigma–Aldrich Sigma–Aldrich Sigma–Aldrich Sigma–Aldrich Sigma–Aldrich Sigma–Aldrich Sigma–Aldrich Sigma–Aldrich Sigma–Aldrich Sigma–Aldrich Nippon Cohsei Sigma–Aldrich Sigma–Aldrich
% EC3 data obtained from Gerberick et al. (2005) (a), Gerberick et al. (2007b) (b), ICCVAM (2009) (c), Ashikaga et al. (2010) (d), Nukada et al. (2012) (e). N.C., not calculate. S/NS, sensitizer/non-sensitizer. W.U., widely used in cosmetics.
2.4. Calculation of peptide depletion ratio The reactivity of a chemical towards a peptide was expressed as the peptide depletion ratio after incubation of the peptide with the chemical, and the peptide depletion ratio was calculated as follows (Fig. 1): peptide depletion ratio (%) = (1 − Punreacted /Ptotal ) × 100. Some chemicals have a unique colour or may interfere in spectrophotometric property changes upon reaction with the peptide and/or react with the detection reagent. Background signals due to this interference must be considered for an exact evaluation. We used 3 different controls and measured the spectrophotometric values before and after addition of the detection reagents to avoid this kind of background signal (Fig. 1) (Jeong et al., 2013). The calculated peptide depletion ratios are presented as means ± SD and were obtained from 3 independent experiments, which were carried out in triplicate. 2.5. Comparison of skin sensitization potential results from peptide depletion and in vivo data The skin senstitization potentials of the 40 chemicals determined using peptide depletion were compared with the in vivo
data from the EC3 of LLNA (Ashikaga et al., 2010; ICCVAM, 2009; Gerberick et al., 2005, 2007b; Nukada et al., 2012). We evaluated the new alternative method by calculating the accuracy, sensitivity, and specificity of the results based on the modified classification of the in vivo data of the 40 chemicals as follows: sensitivity (%) = TP/(TP + FN) × 100, specificity (%) = TN/(TN + FP) × 100, and accuracy (%) = (TP + TN)/(TP + TN + FP + FN) × 100, where TP, true positive; TN, true negative; FN, false negative and FP, false positive. 3. Results 3.1. Data on chemical reactivity towards cysteine and lysine peptides The reactivity of the 40 test chemicals towards the peptides (peptide depletion ratio) is shown in Table 2. The results indicated a strong correlation between sensitizing potency and depletion of model peptide. The classification presented in Table 3 is based on
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Table 2 Percent depletion of the cysteine peptide or lysine peptide with regard to test chemicals. Test material
Oxazolone 2-4-Dinitrochlorobenzene (DNCB) Glutaric dialdehyde Lauryl gallate Isoeugenol Diethyl maleate Hexylcinnamic aldehyde (HCA) Diphenylcyclopropenone 5-Chloro-2-methyl-4-isothiazolin-3-on (MCI) 1,4-Dihydroquinone 2-Aminophenol 4-Phenylenediamine (PPD) Cobalt chloride 2-mercaptobenzothiazole Propyl gallate Lilial Benzocaine Methyldibromoglutaronitril Eugenol Dimethylsulfoxide (DMSO) Sodium lauryl sulfate (SLS) Glycerol Citral Phenyl benzoate Cinnamic alcohol Imidazolidinyl urea Methyl metacrylate Isopropanol Methyl salicylate Lactic acid Chlorobenzene Benzalkonium chloride Tween 80 Methyl paraben Resorcinol Salicylic acid
% depletion of cysteine peptide
% depletion of lysine peptide
Run 1
Run 2
Run 3
Average
SD
Run 1
10.88 99.42 0.84 24.58 98.75 98.57 86.29 89.09 23.00 100.93 91.50 97.42 −0.15 86.26 101.71 17.80 1.18 97.10 6.14 1.64 8.61 −0.14 19.69 21.35 1.70 −10.95 14.48 −2.26 9.49 10.13 1.41 19.19 13.95 −3.48 −1.54 −1.32
12.19 99.45 0.93 16.56 96.53 100.00 76.29 88.39 25.34 103.38 96.14 100.74 −1.93 89.46 102.83 9.25 0.96 94.29 7.09 0.13 5.64 1.63 20.40 24.75 2.64 −11.02 7.38 −1.97 12.40 9.51 0.41 38.21 15.72 0.41 −0.84 0.88
12.38 99.02 4.15 9.21 96.02 92.39 110.56 89.62 18.10 100.94 97.84 98.43 −2.39 90.17 100.27 12.28 −0.31 98.57 8.92 −0.62 7.83 0.36 11.94 18.92 1.25 −13.90 11.29 −4.82 7.48 7.75 −2.58 13.50 17.91 2.21 0.89 1.86
11.82 99.29 1.97 16.79 97.10 96.99 91.05 89.03 22.14 101.75 95.16 98.87 −1.49 88.63 101.60 13.11 0.61 96.65 7.38 0.38 7.36 0.62 17.35 21.67 1.86 −11.95 11.05 −3.02 9.79 9.13 −0.25 23.63 15.86 −0.29 −0.50 0.48
0.82 0.24 1.89 7.69 1.45 4.04 17.62 0.62 3.69 1.41 3.28 1.70 1.18 2.09 1.28 4.34 0.80 2.17 1.41 1.15 1.54 0.91 4.69 2.93 0.71 1.69 3.56 1.56 2.47 1.23 2.08 12.94 1.98 2.91 1.25 1.63
99.80 3.67 99.18 48.67 5.72 3.21 1.09 10.60 10.39 80.13 79.39 85.15 −4.35 −4.57 52.90 17.10 50.72 7.94 0.78 −5.40 6.54 13.42 12.22 6.13 12.13 15.18 9.54 11.81 0.03 −13.76 −2.77 −12.05 15.57 14.65 6.49 −12.13
Run 2 99.76 −1.84 99.75 48.63 4.18 −1.10 −3.78 9.62 5.40 82.26 54.95 87.45 −8.47 13.97 57.54 7.49 24.84 7.18 13.05 −5.24 −2.02 2.81 11.88 10.26 3.00 5.97 17.77 10.79 3.95 −2.77 −0.03 0.23 14.78 16.44 6.57 −0.48
Run 3
Average
SD
99.69 16.68 99.58 50.10 15.68 14.71 5.21 −4.14 6.35 80.69 79.79 85.69 5.43 −4.45 54.19 3.19 55.64 3.75 11.97 1.15 8.10 13.43 8.77 4.52 7.41 9.87 9.22 6.13 3.13 −0.62 6.71 12.30 15.25 13.89 0.35 −14.49
99.75 6.17 99.51 49.13 8.53 5.61 0.84 5.36 7.38 81.03 71.38 86.10 −2.46 1.65 54.87 9.26 43.73 6.29 8.60 −3.16 4.21 9.89 10.96 6.97 7.51 10.34 12.18 9.58 2.37 −5.72 1.30 0.16 15.20 14.99 4.47 −9.03
0.05 9.51 0.29 0.83 6.24 8.17 4.50 8.24 2.65 1.10 14.23 1.20 7.14 10.67 2.39 7.12 16.55 2.23 6.80 3.74 5.45 6.13 1.90 2.96 4.56 4.63 4.85 3.03 2.07 7.05 4.88 12.18 0.40 1.30 3.57 7.50
LLNA results and clinical data. For the majority of the extreme and strong allergens, depletion of >70% was observed for both peptides. Interestingly, DNCB and diphenylcyclopropenone demonstrated significant depletion of the cysteine peptide but not the lysine peptide. On the contrary, oxazolone and glutaric dialdehyde demonstrated significant depletion of only the lysine peptide. Finally, only a few non-sensitizers (e.g., benzalkonium chloride, Tween 80) demonstrated peptide depletion values similar to those observed with the sensitizers. Several weak sensitizers (e.g., eugenol, cinnamic alcohol) showed low reactivity towards the peptides.
(93.3% or 100%), but lower sensitivity (32.0%) and accuracy (70.0% or 57.5%). Combination of the 10% cut-off for the cysteine peptide with the 30% cut-off for the lysine peptide increased the prediction for sensitivity (80.0%) and accuracy (82.5%). For the combination of the 10% cut-off for the cysteine peptide with the 20% cut-off for the lysine peptide, the sensitivity, specificity, and accuracy were also sufficiently high (80.0% for sensitivity and specificity. 75% for accuracy). Compare to the other alternative for sensitization, the prediction of skin sensitization potential showed relatively high (Table 4).
3.2. Classification of chemicals based on their reactivity towards the cysteine and lysine peptides and combination of the classification cut-offs for the 2 peptide methods
4. Discussion
In this study, various cut-off criteria were tested to determine the appropriate cut-off criteria that showed highest accuracy for discriminating sensitizers and non-sensitizers. In addition, we tried to combine the results of the 2 peptide models. When we combined the best cut-off criteria for each peptide, we could obtain better results. A chemical was classified as a sensitizer when the accuracy of the combined cut-offs was higher than the classification cut-off of either the cysteine peptide method or the lysine peptide method. The results of the study are illustrated in Table 3, where the chemicals are classified according to the combination of the classification cut-offs of each peptide method. The sensitivity, specificity, and accuracy when a 10% cut-off was applied for the cysteine peptide method were 76.0%, 86.7%, and 77.5.0%, respectively. The cut-off for the lysine peptide method (20% or 30%) showed higher specificity
We tested 25 sensitizers and 15 non-sensitizers by measuring the reactivity of chemicals towards peptides using spectrophotometric analysis. Among these 40 chemicals, 18 chemicals were LLNA performance standards. We included 10 chemicals that were widely used in cosmetics and considered as non-sensitizer because we did not have any LLNA results, animal clinical data and report of skin sensitization for human. Test chemicals were classified mainly according to LLNA results. The spectrophotometric assay method using a cysteine peptide was based on the method developed by Schultz et al. (2005). Due to the characteristics of the spectrophotometric method used in this prediction model, care should be taken when making a prediction for chemicals that have a unique colour or may interfere in spectrophotometric property changes upon reaction with peptides and/or react with detection reagents. In a previous study, we established spectrophotometric assay methods by determining the reactivity
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Table 3 Classification of chemicals using different cut offs of peptide depletion ratios. Test material
Local lymph node assay (LLNA) grade
Oxazolone 2-4-Dinitrochlorobenzene (DNCB) Diphenylcyclopropenone 5-Chloro-2-methyl-4isothiazolin-3-on (MCI) Hydroquinone 2-Aminophenol Glutaric dialdehyde Lauryl gallate 4-Phenylenediamine (PPD) Cobalt chloride Propyl gallate Isoeugenol Diethyl maleate 2-Mercaptobenzothiazole Citral Methyldibromoglutaronitril Resorcinol Hexylcinnamic aldehyde (HCA) Phenyl benzoate Cinnamic alcohol Imidazolidinyl urea Lilial Methyl methacrylate Eugenol Benzocaine Dimethylsulfoxide (DMSO) Sodium lauryl sulfate (SLS) Glycerol Isopropanol Methyl salicylate Lactic acid Chlorobenzene Benzalkonium chloride Tween 80 Sodium benzoate Methyl paraben Dehydroacetic acid Phenoxyethanol Salicylic acid Ethylhexyl methoxycinnamate
Group
Peptide depletion
Criteria of classification
Cysteine
Lysine
Cysteine > 10 Lysine > 20
Lysine > 30
Cysteine > 10 or lysine > 20
Cysteine > 10 or lysine > 30
Extreme Extreme
S S
11.82 99.29
99.75 6.17
S S
S NS
S NS
S S
S S
Extreme Extreme
S S
89.03 22.14
5.36 7.38
S S
NS NS
NS NS
S S
S S
Extreme Extreme Strong Strong Strong Strong Strong Moderate Moderate Moderate Moderate Moderate Moderate Weak Weak Weak Weak Weak Weak Weak S (equivocal) NS NS NS NS NS/very weak NS/very weak NS NS (false negative) NS NS NS NS NS NS NS
S S S S S S S S S S S S S S S S S S S S S NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS
101.75 95.16 1.97 16.79 98.87 −1.49 101.60 97.10 96.99 88.63 17.35 96.65 −0.50 91.05 21.67 1.86 −11.95 13.11 11.05 7.38 0.61 0.38 7.36 0.62 −3.02 9.79 9.13 −0.25 23.63 15.86 −1.07 −0.29 −1.07 −1.67 0.48 −5.08
81.03 71.38 99.51 49.13 86.10 −2.46 54.87 8.53 5.61 1.65 10.96 6.29 4.47 0.84 6.97 7.51 10.34 9.26 12.18 8.60 43.73 −3.16 4.21 9.89 9.58 2.37 −5.72 1.30 0.16 15.20 21.03 14.99 10.19 15.35 −9.03 10.64
S S NS S S NS S S S S S S NS S S NS NS S S NS NS NS NS NS NS NS NS NS S S NS NS NS NS NS NS
S S S S S NS S NS NS NS NS NS NS NS NS NS NS NS NS NS S NS NS NS NS NS NS NS NS NS S NS NS NS NS NS
S S S S S NS S NS NS NS NS NS NS NS NS NS NS NS NS NS S NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS
S S S S S NS S S S S S S NS S S NS NS S S NS S NS NS NS NS NS NS NS S S S NS NS NS NS NS
S S S S S NS S S S S S S NS S S NS NS S S NS S NS NS NS NS NS NS NS S S NS NS NS NS NS NS
76.0 86.7 77.5
32.0 93.3 70
32.0 100.0 57.5
80.0 80.0 75
80.0 86.7 82.5
Sensitivity (%) Specificity (%) Accuracy (%)
of chemicals towards the thiol group of a cysteine peptide and the amino group of a lysine peptide using DTNB and fluorescamine as detecting reagents of the free thiol and amine groups, respectively (Jeong et al., 2013). Our new spectrophotometric-based method has several advantages, it is easy, fast, inexpensive and need not expert. To assure the usefulness of this method, it is essential to assess its sensitivity, specificity, and accuracy. We obtained good results when we set the classification cut-off for cysteine peptide depletion at >10% and for lysine peptide depletion at >30%. Benzalkonium chloride, sodium benzoate, and Tween
Table 4 The prediction of each animal alternative method for skin sensitization potentials.
Sensitivity (%) Specificity (%) Accuracy (%) a
Spectrophotometric DPRA (this study)
DPRAa
h-CLATa
DEREKa
80 86.7 82.5
79 80 79
88 72 84
83 56 76
The prediction data obtained from Nukada et al. (2013).
80 were classified as sensitizer in this study, while they were categorized as non-sensitizer previously. Eugenol, resorcinol, cobalt chloride, cinnamyl alcohol, and imidazolidinyl urea were classified as non-sensitizers, while they were previously categorized as weak sensitizers. Benzalkonium chloride was categorized as sensitizer according to the LLNA result. However, this chemical has been considered as an irritant in many articles; thus, LLNA results were occasionally believed to be false-positive. In this study, we selected benzalkonium chloride as a non-sensitizer, but it was classified as sensitizer on the basis of its reactivity towards peptides. It was suggested that the sensitizing potential of this chemical is controversial. In contrast, benzocaine was classified as a sensitizer on the basis of animal test results, although the results were equivocal. In summary, the prediction models, in which we combined the classification cut-offs of the 2 peptide methods, achieved a high degree of sensitivity, specificity, and accuracy. The combination of a 10% cut-off for the cysteine peptide method and a 30% cut-off for the lysine peptide method resulted in over 80% for each criterion. Although these methods need further improvement and optimization, these results demonstrate that spectrophotometric methods
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Fig. 1. Scheme of the peptide reactivity assay and formula for calculating the depletion ratio. Every test set was composed of 4 wells, including 3 control groups. Optical density (OD) or fluorescence intensity (FI) was measured just before and after adding the detection reagent. These OD or FI values were used to calibrate and calculate the peptide depletion rate. Ptotal is the total peptide added to each well. SVADP and SVBDP are the spectrophotometric values after and before addition of the detection reagent to the peptide-only control, respectively. SVADS and SVBDS are the spectrophotometric values after and before addition of the detection reagent to the solvent-only control, respectively. SVADR and SVBDR are the spectrophotometric values after and before addition of the detection reagent to reaction solutions, respectively. SVADC and SVBDC are the spectrophotometric values after and before addition of the detection reagent to the chemical-only control, respectively.
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