An epidermal equivalent assay for identification and ranking potency of contact sensitizers.

The purpose of this study was to explore the possibility of combining the epidermal equivalent (EE) potency assay with the assay which assesses release of interleukin-18 (IL-18) to provide a single test for identification and classification of skin sensitizing chemicals, including chemicals of low water solubility or stability. A protocol was developed using different 3D-epidermal models including in house VUMC model, epiCS® (previously EST1000™), MatTek EpiDerm™ and SkinEthic™ RHE and also the impact of different vehicles (acetone:olive oil 4:1, 1% DMSO, ethanol, water) was investigated. Following topical exposure for 24h to 17 contact allergens and 13 non-sensitizers a robust increase in IL-18 release was observed only after exposure to contact allergens. A putative prediction model is proposed from data obtained from two laboratories yielding 95% accuracy. Correlating the in vitro EE sensitizer potency data, which assesses the chemical concentration which results in 50% cytotoxicity (EE-EC50) with human and animal data showed a superior correlation with human DSA05 (μg/cm(2)) data (Spearman r=0.8500; P value (two-tailed)=0.0061) compared to LLNA data (Spearman r=0.5968; P value (two-tailed)=0.0542). DSA05=induction dose per skin area that produces a positive response in 5% of the tested population Also a good correlation was observed for release of IL-18 (SI-2) into culture supernatants with human DSA05 data (Spearman r=0.8333; P value (two-tailed)=0.0154). This easily transferable human in vitro assay appears to be very promising, but additional testing of a larger chemical set with the different EE models is required to fully evaluate the utility of this assay and to establish a definitive prediction model.

[1]  E. Corsini,et al.  Immunotoxicology: Opportunities for Non-animal Test Development , 2009, Alternatives to laboratory animals : ATLA.

[2]  H. Kandárová,et al.  In Vitro Skin Irritation Testing: Improving the Sensitivity of the EpiDerm Skin Irritation Test Protocol , 2009, Alternatives to laboratory animals : ATLA.

[3]  T. Mosmann.  Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. , 1983, Journal of immunological methods.

[4]  J. Nicolas,et al.  Skin contact irritation conditions the development and severity of allergic contact dermatitis. , 2007, The Journal of investigative dermatology.

[5]  Marina Marinovich,et al.  NCTC 2544 and IL-18 production: a tool for the identification of contact allergens. , 2013, Toxicology in vitro : an international journal published in association with BIBRA.

[6]  T. Luger,et al.  Dissection of antigenic and irritative effects of epicutaneously applied haptens in mice. Evidence that not the antigenic component but nonspecific proinflammatory effects of haptens determine the concentration-dependent elicitation of allergic contact dermatitis. , 1996, The Journal of clinical investigation.

[7]  D. Basketter,et al.  Does Irritation Potency Contribute to the Skin Sensitization Potency of Contact Allergens? , 2007, Cutaneous and ocular toxicology.

[8]  M Liebsch,et al.  The ECVAM Prevalidation Study on the Use of EpiDerm for Skin Corrosivity Testing , 2000, Alternatives to laboratory animals : ATLA.

[9]  Emanuela Corsini,et al.  Transfer of a two-tiered keratinocyte assay: IL-18 production by NCTC2544 to determine the skin sensitizing capacity and epidermal equivalent assay to determine sensitizer potency. , 2013, Toxicology in vitro : an international journal published in association with BIBRA.

[10]  I Kimber,et al.  Threshold for classification as a skin sensitizer in the local lymph node assay: a statistical evaluation. , 1999, Food and chemical toxicology : an international journal published for the British Industrial Biological Research Association.

[11]  C L Galli,et al.  Further development of the NCTC 2544 IL-18 assay to identify in vitro contact allergens. , 2011, Toxicology in vitro : an international journal published in association with BIBRA.

[12]  I. Kimber,et al.  Interleukin (IL)‐18 induces Langerhans cell migration by a tumour necrosis factor‐α‐ and IL‐1β‐dependent mechanism , 2001, Immunology.

[13]  Henk Van Loveren,et al.  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. , 2005, Toxicology.

[14]  I Kimber,et al.  Skin sensitisation, vehicle effects and the local lymph node assay. , 2001, Food and chemical toxicology : an international journal published for the British Industrial Biological Research Association.

[15]  E. Corsini,et al.  Present and future of in vitro immunotoxicology in drug development , 2010, Journal of immunotoxicology.

[16]  E. Epstein,et al.  New cell formation in human sebaceous glands. , 1966, The Journal of investigative dermatology.

[17]  A M Kligman,et al.  The identification of contact allergens by human assay. 3. The maximization test: a procedure for screening and rating contact sensitizers. , 1989, The Journal of investigative dermatology.

[18]  A. Price,et al.  (32) Extensive cutaneous Crohn's disease , 1983 .

[19]  S. Shuster,et al.  Quantitation of sensitization and responsiveness to dinitrochlorobenzene in normal subjects. , 1983, The British journal of dermatology.

[20]  D. Basketter,et al.  The impact of vehicle on assessment of relative skin sensitization potency of 1,4-dihydroquinone in the local lymph node assay. , 1999, American journal of contact dermatitis : official journal of the American Contact Dermatitis Society.

[21]  Valérie Zuang,et al.  Alternative (non-animal) methods for cosmetics testing: current status and future prospects—2010 , 2011, Archives of Toxicology.

[22]  K Schneider,et al.  Quantitative relationship between the local lymph node assay and human skin sensitization assays. , 2004, Regulatory toxicology and pharmacology : RTP.

[23]  R. Luebke.  Immunotoxicant Screening and Prioritization in the Twenty-first Century , 2012, Toxicologic pathology.

[24]  M Liebsch,et al.  The ECVAM International Validation Study on In Vitro Tests for Skin Corrosivity. 2. Results and Evaluation by the Management Team. , 1998, Toxicology in vitro : an international journal published in association with BIBRA.

[25]  R. Winkelmann.  Some unusual histochemical properties of kangaroo skin. , 1966, The Journal of investigative dermatology.

[26]  P. Friedmann,et al.  Sex differences in susceptibility to development of contact hypersensitivity to dinitrochlorobenzene (DNCB) , 1989, The British journal of dermatology.

[27]  Marina Marinovich,et al.  Use of IL-18 production in a human keratinocyte cell line to discriminate contact sensitizers from irritants and low molecular weight respiratory allergens. , 2009, Toxicology in vitro : an international journal published in association with BIBRA.

[28]  S. Gibbs,et al.  Progress on the development of human in vitro dendritic cell based assays for assessment of the sensitizing potential of a compound. , 2009, Toxicology and applied pharmacology.

[29]  Valérie Zuang,et al.  The ECVAM International Validation Study on in Vitro Tests for Acute Skin Irritation: Report on the Validity of the EPISKIN and EpiDerm Assays and on the Skin Integrity Function Test a , 2007, Alternatives to laboratory animals : ATLA.

[30]  J. Clements,et al.  Development of an in vitro corrosion/irritation prediction assay using the EpiDerm skin model. , 2007, Toxicology in vitro : an international journal published in association with BIBRA.

[31]  S. E. King,et al.  The development of allergic contact dermatitis in females during the comparison of two predictive patch tests , 1977, Contact dermatitis.

[32]  Carsten Goebel,et al.  Proposal for a risk assessment methodology for skin sensitization based on sensitization potency data. , 2003, Regulatory toxicology and pharmacology : RTP.

[33]  A. Kligman.  The identification of contact allergens by human assay. I. A critique of standard methods. , 1966, The Journal of investigative dermatology.

[34]  Francis N. Marzulli,et al.  Contact allergy: Predictive testing in man , 1976, Contact dermatitis.

[35]  A. Cavani,et al.  New aspects of the molecular basis of contact allergy , 2007, Current opinion in allergy and clinical immunology.

[36]  Ian Kimber,et al.  Compilation of Historical Local Lymph Node Data for Evaluation of Skin Sensitization Alternative Methods , 2005, Dermatitis : contact, atopic, occupational, drug.

[37]  Andreas Luch,et al.  T-cell recognition of chemicals, protein allergens and drugs: towards the development of in vitro assays , 2010, Cellular and Molecular Life Sciences.

[38]  Nathalie Alépée,et al.  A catch-up validation study on reconstructed human epidermis (SkinEthic RHE) for full replacement of the Draize skin irritation test. , 2010, Toxicology in vitro : an international journal published in association with BIBRA.

[39]  S. Spiekstra,et al.  A potential in vitro epidermal equivalent assay to determine sensitizer potency. , 2011, Toxicology in vitro : an international journal published in association with BIBRA.

[40]  G. R. Thompson,et al.  Cinnamic aldehyde: a survey of consumer patch-test sensitization. , 1983, Food and chemical toxicology : an international journal published for the British Industrial Biological Research Association.

[41]  H. Okamura,et al.  Cloning of a new cytokine that induces IFN-γ production by T cells , 1995, Nature.

[42]  Helen F McGarry,et al.  The murine local lymph node assay: regulatory and potency considerations under REACH. , 2007, Toxicology.

[43]  Ian Kimber,et al.  IL‐18 is a key proximal mediator of contact hypersensitivity and allergen‐induced Langerhans cell migration in murine epidermis , 2008, Journal of leukocyte biology.

[44]  J. Wilcox,et al.  Human keratinocytes constitutively express interleukin-18 and secrete biologically active interleukin-18 after treatment with pro-inflammatory mediators and dinitrochlorobenzene. , 1999, The Journal of investigative dermatology.

[45]  T. Agner,et al.  Combined effects of irritants and allergens , 2002, Contact dermatitis.

[46]  S. Spiekstra,et al.  Potential method to determine irritant potency in vitro - Comparison of two reconstructed epidermal culture models with different barrier competency. , 2009, Toxicology in vitro : an international journal published in association with BIBRA.

[47]  I Kimber,et al.  Vehicle effects on skin sensitizing potency of four chemicals: assessment using the local lymph node assay , 2001, International journal of cosmetic science.

[48]  S. Shuster,et al.  'Irritants' increase the response to an allergen in allergic contact dermatitis. , 1991, Archives of dermatology.