Development of an In Vitro Method to Estimate the Sensitization Induction Level of Contact Allergens

Valentina Galbiati1, Sue Gibbs2, Erwin Roggen3, Emanuela Corsini1

1 Department of Environmental Science and Policy, Università degli Studi di Milano, Milan, 2 Department of Oral Cell Biology, ACTA, Amsterdam, 3 3Rs Management and Consultancy, Lyngby
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 20.15
DOI:  10.1002/cptx.44
Online Posting Date:  February, 2018
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Recently, an in vitro procedure, which combines the epidermal equivalent potency assay with assessment of IL‐18 to provide a single test for identification and classification of skin sensitizers, was developed and validated. This unit will describe a simple in vitro method for estimation of the expected sensitization induction level interpolating in vitro EC50 and IL‐18 SI2 values to predict LLNA EC3 and/or human NOEL from standards curves generated using reference contact allergens, based on the use of Reconstituted human Epidermis (RhE).© 2018 by John Wiley & Sons, Inc.

Keywords: reconstituted human epidermis; 3D model; in vitro; contact allergens; potency; contact hypersensitivity

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Table of Contents

  • Introduction
  • Basic Protocol 1: RhE IL‐18 Potency Test
  • Support Protocol 1: Guideline for Making Dilutions of Test Substances
  • Support Protocol 2: Preparation of Dose Response Experiments
  • Basic Protocol 2: MTT Assay for Assessment of EC50
  • Support Protocol 3: MTT Assay
  • Support Protocol 4: Test for Interference of Test Substances with MTT Endpoint
  • Basic Protocol 3: IL‐18 Determination
  • Basic Protocol 4: RhE and Potency Estimation
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: RhE IL‐18 Potency Test

  • EpiDerm™ (MatTek In Vitro Life Science Laboratories)
  • Epiderm culture medium (EPI‐100‐MM)
  • 6‐well plates
  • 37°C, 5% CO 2, 95% humidity incubator
NOTE: The protocol provided with the EpiDermTM is considered applicable to any commercially available as well as in house developed RhE (dos Santos et al., ). However, qualitative differences between the RhEs from different providers, as well as between RhE batches from the same provider, require careful assessment and, if required, adaptation of this protocol to assure reproducibility of the binary decisions as well as relative potency ranking across different RhE models and batches.NOTE: Upon arrival, the RhE‐containing package should be checked for breaches. The tissues and components should be free of contamination. Any abnormalities related to the color of the medium, shipping agar, and the presence of unusual features on the tissue surface (like blisters) should trigger caution. If any of these irregularities are observed before or during the assay, the tissue supplier should be immediately contacted, providing photo‐documentation of the effect observed. Only properly transported and entirely intact tissue without any visual evidence of contamination and defects should be used in the experiments.

Support Protocol 1: Guideline for Making Dilutions of Test Substances

  • 2,4‐Dinitrochlorobenzene (DNCB)
  • Acetone: olive oil (AOO)
  • Dulbecco's modified phosphate‐buffered saline without calcium and magnesium (d‐PBS)
  • Dimethyl sulfoxide (DMSO)
  • Epiderm culture medium (EPI‐100‐MM)
  • Laboratory balance (accuracy 0.1 mg)
  • Solid and liquid compounds (what does this mean)
NOTE: All solutions, including stock solutions, must be made on the day of exposure.Stock solutions should ideally be prepared as 20% weight/volume (w/v) for solid substances, and 20% volume/volume (v/v) in case of liquid substances. In this way, test is set to accommodate for test substances without available information on MW and density. For solid compounds: 200 mg of a test substance is weighed down, and the volume is adjusted to 1 ml using an appropriate vehicle. In case of solubility issues at 200 mg/ml, the highest possible concentration should be prepared. For liquid compounds: 200 μl of a test substance is pipetted and the volume is adjusted to 1 ml using an appropriate vehicle. In case of solubility issues at 200 mg/ml, the highest possible concentration should be prepared.IMPORTANT NOTE: Treat viscous compounds as a solid compound.All solutions should be prepared at room temperature (20° to 26°C) or max warmed to 37°C if solubility issues make it necessary.Vortexing is required after the preparation and also shortly before the application to the RhE to assure homogeneity.Final concentrations higher than 200 mg/ml will not be tested. This will enable all unknown sensitizers from weak to extreme to be tested. Depending on solubility, dissolve chemicals in appropriate vehicle. Vehicle selection may have a significant impact on the effect of a substance on cell viability and IL‐18 release. The preferred order of choice is listed below:
  • dPBS
  • 1% DMSO: 50 μl DMSO + 4950 μl dPBS
  • AOO (4:1): 8 ml acetone + 2 ml olive oil
Never filter the chemical solutions.The vehicle/solvent with the highest dissolving capacity and the lowest cytotoxicity to the selected RhE should be chosen for the in vitro skin sensitization assay. Choose the vehicle that results in highest maximum solubility. When maximum solubility is the same for all vehicles, select dPBS over 1% DMSO or AOOVortex mixing, sonication and/or warming to max. 37°C may be used to aid solubilization.Poor solubility of a solvent is indicated by:
  • Formation of two layers or separation into two layers shortly after vortexing;
  • Formation of a milky suspension;
  • Visible precipitation of the test substance (e.g., after 10 min at room temperature).
In order to select the most appropriate vehicle for a given test substance the use of hierarchical procedure of solvent selection is recommended (Fig.  ). In order to accurate determine the RhE‐specific EC50 values, test substances are tested using two independent RhE batches in a dose response, starting with 200 mg/ml (for solid) or 200 μl/ml (for liquids) or next highest soluble concentration.The start concentration is diluted (2 fold dilution step) until 0.10 mg/ml (solids) or 0.10 μl/ml (for liquids) is reached.Dilutions are made in the following order: 200, 100, 50, 25, 12.5, 6.25, 3.12, 1.56, 0.78, 0.39, 0.20, and 0.10 mg/ml until a clear solution is reached. The vehicle with the highest dissolving capacity will be chosen. Dilution should be performed as described in Table 20.15.3.
Table 0.5.3   MaterialsPreparation of Solutions and Dilutions

Dilution to be tested μl of previous dilution μl of vehicle
1× dilution Master stock concentration in vehicle (e.g., 200 mg/ml)
2× dilution 300 μl (1× dilution) 300 μl
4× dilution 300 μl (2× dilution) 300 μl
8× dilution 300 μl (4× dilution) 300 μl
16× dilution 300 μl (8× dilution) 300 μl
32× dilution 300 μl (16× dilution) 300 μl
64× dilution 300 μl (32× dilution) 300 μl
128× dilution 300 μl (64× dilution) 300 μl
256× dilution 300 μl (128× dilution) 300 μl
512× dilution 300 μl (256× dilution) 300 μl
1024× dilution 300 μl (512× dilution) 300 μl
2048× dilution 300 μl (1024× dilution) 300 μl

Prepare fresh DNCB as positive control for each experiment. Make a 3 mg/ml stock solution in AOO.IMPORTANT NOTE: It is known that AOO dissolves some plastic laboratory devices. It is therefore recommended to use polypropylene tubes to prepare the chemical solution dissolved in AOO and impregnate the paper filters for the chemical exposure on devices composed of glass (e.g., glass slides or tissue culture dishes made of glass).

Support Protocol 2: Preparation of Dose Response Experiments

  • Test chemicals
  • 2,4‐Dinitrochlorobenzene (DNCB)
  • Acetone: olive oil (AOO)
  • RhE inserts
  • EpiDerm™ (MatTek In Vitro Life Science Laboratories)
  • Finn Chamber filter paper discs (8 mm)
  • 100‐mm tissue culture dish or on a glass
  • Tweezers
  • Pencil

Basic Protocol 2: MTT Assay for Assessment of EC50

  • EpiDerm™ (MatTek In Vitro Life Science Laboratories)
  • Tweezers
  • 24‐well plates
  • 1.5‐ml microcentrifuge

Support Protocol 3: MTT Assay

  • MTT solution
  • 3‐(4,5‐dimethylthiazol‐2yl)‐2,5‐diphenyltetrazolyum bromide powder
  • Isopropanol
  • EpiDerm™ (MatTek In Vitro Life Science Laboratories)
  • Dulbecco's modified phospahate‐buffered saline without calcium and magnesium (dPBS)
  • 24‐well plates
  • Tweezers
  • 37°C, 5% CO 2, 90% relative humidity incubator
  • Paper towels
  • Parafilm
  • Spectrophotometer

Support Protocol 4: Test for Interference of Test Substances with MTT Endpoint

  Materials list
  • Test substance(s)
  • Deionized water
  • 37°C, 5% CO 2, 90% relative humidity incubator
  • ‐20°C freezer

Basic Protocol 3: IL‐18 Determination

  • Mouse anti‐human IL‐18‐UNLB (Southern Biotech, cat. no. 15800‐01)
  • Coating buffer: d‐PBS
  • Recombinant human IL‐18 (MBL International, cat. no. B001‐5)
  • Ice
  • Wash buffer: d‐PBS + 0.01% Tween 20
  • Blocking and dilution buffer: d‐PBS + 1% bovine serum albumin (BSA)
  • Rabbit anti IL‐18 (MyBioSource, cat. no. MBS20206561)
  • Goat anti‐rabbit IgG‐horseradish peroxidase conjugate (Bio‐Rad/AbD Serotec cat. no. 170‐6515 lot # RRID:AB_11125142)
  • TMB liquid substrate supersensitive for ELISA
  • 96‐well flat‐bottom ELISA plates
  • Plate sealers
  • 4°C incubator
  • Paper towels
  • Multi‐well pipettor
  • Adjustable micropipets: 2‐20 μl, 20‐200 μl, 200‐1000 μl
  • ELISA plate reader equipped with 595 nm
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Literature Cited

Literature Cited
  Andres, E., Barry, M., Hundt, A., Dini, C., Corsini, E., Gibbs, S., … Ferret, P. J. (2016). Preliminary performance data of the RHE/IL‐18 assay performed on SkinEthicTM RHE for the identification of contact sensitizers. International Journal of Cosmetic Science, doi:
  Antonopoulos, C., Cumberbatch, M., Mee, J. B., Dearman, R. J., Wei, X. Q., Liew, F. Y., … Groves, R. W. (2008). IL‐18 is a key proximal mediator of contact hypersensitivity and allergen‐induced Langerhans cell migration in murine epidermis. Journal of Leukocyte Biology, 83, 361–367. doi:
  Basketter, D. A., Alépée, N., Ashikaga, T., Barroso, J., Gilmour, N., Goebel, C., … Templier, M. (2014). Categorization of chemicals according to their relative human skin sensitizing potency. Dermatitis, 25, 11–21. doi:
  Basketter, D. A., Clapp, C., Jefferies, D., Safford, B., Ryan, C. A., Gerberick, F., … Kimber, I. (2005). Predictive identification of human skin sensitization thresholds. Contact Dermatitis, 53, 260–267. doi:‐1873.2005.00707.x.
  Basketter, D. A., Lea, L. J., Cooper, K., Stocks, J., Dickens, A., Pate, I., … Kimber, I. (1999). Threshold for classification as a skin sensitizer in the local lymph node assay: A statistical evaluation. Food and Chemical Toxicology, 37, 1167–1174. doi:‐6915(99)00112‐X.
  Boeniger, M. F., & Ahlers, H. W. (2003). Federal government regulation of occupational skin exposure in the USA. International Archives of Occupational and Environmental Health, 76, 387–399. doi:‐002‐0425‐2.
  Corsini, E., Galbiati, V., Mitjans, M., Galli, C. L., & Marinovich, M. (2013). NCTC 2544 and IL‐18 production: A tool for the identification of contact allergens. Toxicology in Vitro, 27, 1127–1134. doi:
  Corsini, E., Mitjans, M., Galbiati, V., Lucchi, L., Galli, C. L., & Marinovich, M. (2009). Use of IL‐18 production in a human keratinocyte cell line to discriminate contact sensitizers from irritants and low molecular weight respiratory allergens. Toxicology in Vitro, 23, 789–796. doi:
  Corsini, E., Papale, A., Galbiati, V., & Roggen, E. L. (2014). Safety evaluation of cosmetics ingredients: In vitro opportunities for the identification of contact allergens. Cosmetics, 1, 61–74. doi:
  Corsini, E., Roggen, E. L., Galbiati, V., & Gibbs, S. (2016). Alternative approach for potency assessment: In vitro methods. Cosmetics, 3, 7. doi:
  Cumberbatch, M., Dearman, R. J., Antonopoulos, C., Groves, R. W., & Kimber, I. (2001). Interleukine (IL)‐18 induces Langerhans cell migration by a tumor necrosis factor‐alpha‐and IL‐1beta‐dependent mechanism. Immunology, 102, 323–330. doi: 10.1046/j.1365‐2567.2001.01187.x.
  De Groot, A. C. 1994. Patch testing: Test concentrations and vehicles for 3700 chemicals, (2nd ed.). Amsterdam: Elsevier.
  dos Santos, G. G., Spiektra, S. W., Sampt‐Sardjoepersad, S. C., Reinders, J., Scheper, R. J., & Gibbs, S. (2011). A potential in vitro epidermal equivalent assay to determine sensitizer potency. Toxicology in Vitro, 25(1), 347–357. doi: 10.1016/j.tiv.2010.10.008.
  Galbiati, V., Mitjans, M., Lucchi, L., Viviani, B., Galli, C. L., Marinovich, M., & Corsini, E. (2011). Further development of the NCTC 2544 IL‐18 assay to identify in vitro contact allergens. Toxicology in Vitro, 25, 724–732. doi:
  Galbiati, V., Papale, A., Marinovich, M., Gibbs, S., Roggen, E., & Corsini, E. (2017). Development of an in vitro method to estimate the sensitization induction level of contact allergens. Toxicology Letters, 271, 1–11. doi:
  Gibbs, S., Corsini, E., Spiekstra, S. W., Galbiati, V., Fuchs, H. W., Degeorge, G., … Roggen, E. (2013). An epidermal equivalent assay for identification and ranking potency of contact sensitizers. Toxicology and Applied Pharmacology, 272, 529–541. doi:
  Griem, P., Goebel, C., & Scheffler, H. (2003). Proposal for a risk assessment methodology for skin sensitization based on sensitization potency data. Regulatory Toxicology and Pharmacology, 38, 269–290. doi:
  ICCVAM Test Method Evaluation Report. 2011. Usefulness and Limitations of the Murine Local Lymph Node Assay for Potency Categorization of Chemicals Causing Allergic Contact Dermatitis in Humans. NIH Publication No. 11‐7709.
  Kimber, I., Basketter, D. A., Gerberick, G. F., Ryan, C. A., & Dearman, R. J. (2011). Chemical allergy: Translating biology into hazard characterization. Toxicological Sciences, 120, S238–S268. doi:
  McLelland, J., Shuster, S., & Matthews, J. N. (1991). ‘Irritants’ increase the response to an allergne in allergic contact dermatitis. Archives of Dermatology, 127, 1016–1019. doi: 10.1001/archderm.1991.01680060090010.
  Mosmann, T. (1983). Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. Journal of Immunological Methods, 65, 55–63. doi:‐1759(83)90303‐4.
  Naik, S. M., Canno, G., Burbach, G. J., Singh, S. R., Swerlick, R. A., Wilcox, J. N., … Caughman, S. W. (1999). Human keratinocytes constituvely express interleukine 18 and secrete biologically active interleukine‐18 after treatment with proinflammatory mediators and dinitrochlorobenzene. Journal of Investigative Dermatology, 113, 766–772. doi: 10.1046/j.1523‐1747.1999.00750.x.
  Okamura, H., Tsutsi, H., Komatsu, T., Yutsudo, M., Hakura, A., Tanimoto, T., … Hattori, K. (1995). Cloning of a new cytokine that induces IFN‐gamma production by T cells. Nature, 378(6552), 88–91.
  Peiser, M., Tralau, T., Heidler, J., Api, A. M., Arts, J. H. B., Basketter, D. A., … Luch, A. (2012). Allergic contact dermatitis: Epidemiology, molecular mechanisms, in vitro methods and regulatory aspects. Cellular and Molecular Life Sciences, 69, 763–781. doi:‐011‐0846‐8.
  Rustemeyer, T., van Hoogstraten, I. M. W., von Blomberg, B. M. E., & Scheper, R. J. 2006. Mechanisms in allergic contact dermatitis. In: P. J. Frosch, T. Menne, & J.‐ P. Lepoittevin (Eds.), Contact Dermatitis (pp. 11–33). Berlin Heidelberg: Springer.
  Schneider, K., & Akkan, Z. (2004). Quantitative relationship between the local lymph node assay and human skin sensitization assays. Regulatory Toxicology and Pharmacology, 39, 245–255. doi:
  Spiektra, S. W., dos Santos, G. G., Scheper, R. J., & Gibbs, S. (2009). Potential method to determine irritant potency in vitro – Comparison of two reconstructed epidermal culture models with different barrier competency. Toxicology In Vitro, 23, 349–355. doi: 10.1016/j.tiv.2008.12.010.
  Teunis, M. A., Spiekstra, S. W., Smits, M., Adriaens, E., Eltze, T., Galbiati, V., … Gibbs, S. (2014). International ring trial of the epidermal equivalent sensitizer potency assay: Reproducibility and predictive‐capacity. Altex, 31, 251–268. doi:
  Thyssen, J. P., Linneberg, A., Menne, T., & Johansen, J. D. (2007). The epidemiology of contact allergy in the general population‐prevalence and main findings. Contact Dermatitis, 57, 287–299. doi:‐0536.2007.01220.x.
  Van der Veen, J. W., Soeteman‐Hernández, L. G., Ezendam, J., Stierum, R., Kuper, F. C., & van Loveren, H. (2014). Anchoring molecular mechanisms to the adverse outcome pathway for skin sensitization: Analysis of existing data. Critical Reviews in Toxicology, 44, 590–599. doi:
  Van Och, F. M., van Loveren, H., van Wolfswinkel, J. C., Machielsen, A. J., & Vandebriel, R. J. (2005). Assessment of potency of allergenic activity of low molecular weight compounds based on IL‐1alpha and IL‐18 production by a murine and human keratinocyte cell line. Toxicology, 210, 95–109. doi: 10.1016/j.tox.2005.01.004.
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