Assessing skin allergic potential of compounds with in vitro methods

Related article: 3R methods

Project acronym: Two-tiered assay

Objectives:  
To pre-validate a two-step in vitro method that assesses capacity and potency of chemicals to induce skin-snesitization.

Key collaborations:
Novozymes, Denmark  
VU Medical Center, The Netherlands  University of Milan, Italy
Danish Technical University, Denmark
National Institute for Public Health and the Environment, The Netherlands  BASF, Germany

Funding:
ZonMw, Dierproeven Begrensd III, 114011015
EU Sixth Framework Programme (FP6) Integrated Project – Sens-it-iv, LSHBCT-2005-018681)

Relevant publications:
1: Teunis MA, Spiekstra SW, Smits M, Adriaens E, Eltze T, Galbiati V, Krul C,
Landsiedel R, Pieters R, Reinders J, Roggen E, Corsini E, Gibbs S. International ring trial of the epidermal equivalent sensitizer potency assay: reproducibility and predictive-capacity. ALTEX. 2014 Feb 17.

2: Teunis M, Corsini E, Smits M, Madsen CB, Eltze T, Ezendam J, Galbiati V, Gremmer E, Krul C, Landin A, Landsiedel R, Pieters R, Rasmussen TF, Reinders J, Roggen E, Spiekstra S, Gibbs S. 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. Toxicol In Vitro.
2013 Apr;27(3):1135-50.

3: Gibbs S, Corsini E, Spiekstra SW, Galbiati V, Fuchs HW, Degeorge G, Troese M, Hayden P, Deng W, Roggen E. An epidermal equivalent assay for identification and ranking potency of contact sensitizers. Toxicol Appl Pharmacol. 2013 Oct
15;272(2):529-41.

4: dos Santos GG, Spiekstra SW, Sampat-Sardjoepersad SC, Reinders J, Scheper RJ, Gibbs S. A potential in vitro epidermal equivalent assay to determine sensitizer potency. Toxicol In Vitro. 2011 Feb;25(1):347-57.

5: Galbiati V, Corsini E. The NCTC 2544 IL-18 assay for the in vitro
identification of contact allergens. Curr Protoc Toxicol. 2012 Nov;Chapter
20:Unit 20.8
.

General aims
Two of the assays from the Sens-it-iv toolbox were selected to be used in a two-tiered approach. Previously, a number of sensitizing chemicals had been tested separately with these assays. The aim of this project was to gather enough evidence to show the reliability of this approach; thus constituting a pre-validation study. With respect to assessing the sensitizing capacity and potency of chemicals, no formally validated and accepted in vitro method is currently available. The integrated EC project Sens-it-iv, which ended with a scientific conference in November 2011, aimed to develop promising methods to replace current animal models for chemical allergen testing.

Benefits and applications of this research
The EU Cosmetics Regulation, the REACH legislation and the new EU Regulation for the use of animals in Life Sciences are major drivers behind the development of 3R methods. Animal-free test systems are increasingly accepted by regulatory authorities as bricks in integrated safety assessment strategies, provided that the methods are scientifically validated. Selected assays providing evidence about the capacity of chemicals to haptenize or trigger epidermal inflammation and DC activation/maturation are currently being driven towards formal validation, but the process of acceptance is slow and can take up to 15 years. This prevalidation study contributes to the toolbox available for animal-free testing of chemicals and could be one of the accepted and implemented alternatives.

Materials and methods
It is commonly accepted that no single assay can fully mimic all of the different in vivo aspects of sensitization. One would need a set of specific, mechanistically driven assays to identify potential skin sensitizers with a non-animal approach. Another advantage of tiered testing is that, depending on the physicochemical properties of the chemical, it may not have to pass through the whole testing strategy. Sometimes a chemical will be labeled as a potential hazard after the first experiment, which saves time and money. Unequivocal results could lead to the testing of that particular chemical in animals in order to classify it. In order for a tiered approach to work, the applicability domain should be clearly defined in each tier.

Innovative aspects in replacing animal testing
Within the EU Sixth Framework Programme (FP6), a project called Sens-it-iv was designed to establish a non-animal testing strategy that could be used to replace current animal-based assays. Sens-it-iv centred on human biological markers for identifying allergic reactions and skin sensitization, or dermatitis, and for classifying sensitisers according to their potency. A further project was then staged under the auspices of the Netherlands Organisation for Health Research and Development (ZonMw) to evaluate the use and portability of two human cell-based assays that had been developed within Sens-it-iv and held promise as a substitute for the LLNA test in a combined two-tiered approach: the NCTC2544 assay to determine skin sensitization capacity of a chemical and the epidermal equivalent potency assay which, as the name suggests, assesses the level of its potency.

Collaborations and expertises
Confidence and trust in sharing views, comments and difficult issues are of the utmost importance in order to succeed. The project was initiated in 2009 and the participants remained involved over the complete duration – the participants collaborated very well together to make this project successful. Organising a formal end meeting has helped to evaluate the progress made and to make plans for future enterprises. The project also received substantial financial contributions from the partners, on top of the contributions from ZonMw and Sens-it-iv.

Main challenges and limitations
Skin sensitization is a complex multistep immunological process involving multiple cell types and biological mechanisms. The tests required for the in vitro identification of skin sensitizers are a matter of debate. Six key mechanisms have been proposed to cover the essential steps of sensitization induction – haptenation, epidermal inflammation, dendritic cell (DC) activation, DC maturation, DC migration and T-cell priming – but such an approach would increase the expense of testing eight- to ten-fold. The data seems to indicate that a haptenation test combined with a test addressing epidermal inflammation, and another determining dendritic cell (DC) activation, would provide more than 95% predictive accuracy.

Animal-free test systems are increasingly accepted by regulatory authorities as bricks in integrated safety assessment strategies, provided that the methods are scientifically validated. But animal-free approaches are often squeezed between regulatory authorities demanding sufficient real-life data from industry demonstrating the strengths and limitations of the new methods, and industry hesitating to implement new approaches unless these methods are accepted by the authorities. Selected assays providing evidence about the capacity of chemicals to haptenize or to trigger epidermal inflammation and DC activation/maturation are currently being driven towards formal validation, but the process of acceptance is slow and can take up to 15 years.

Educational programs and student participation
Several students from the Life Sciences program have participated in this project. Students from the second and third year of this program have performed experiments using the different techniques presented above. Several projects derived from this work were presented on posters and during oral presentations. Also students in the Minor ‘Food & Pharma’ have contributed to this research. One particular nice result was the poster prize that was won by Ms. Deliah Goilo, at the Domain Applied Sciences Symposium in 2012. Scientific elements of this research have been incorporated into the course ‘General Immunology’.

Results of the project
The project has yielded several interesting end-products: We published the data of the first phase of the project in Teunis et al. 2013, and another manuscript is in preparation. The results of the project lead to another publication in the open source journal International Innovation in April 2013. Furthermore, the results have been submitted to ECVAM for review. They will consider whether formal validation is feasible on the basis of these and other results on the tested methods.

Looking to the future
The project has formally ended but we are exploring the possibility to perform some experiments to repeat unequivocal results. Furthermore, we will be able to use the technology that was validated in other projects, concerned with human safety of chemicals.

Authors: Dr. Marc A.T. Teunis, Mieke Smits