In the screening of new chemicals, it should be very important for ethical, safety and economic reasons to have methods to identify immunotoxic compounds without the use of animals. There is a pressing need for alternative non-animal methods to reduce and ultimately replace animal tests for this endpoint as also required by some European regulations (i.e., Cosmetics Regulation and REACH (registration, evaluation, authorization and restriction of chemicals)). In particular, adopted in 2007, the European chemicals policy, commonly known as REACH, demands the safety assessment of thousands of marketed chemical substances. As stated in the first article of the REACH regulation: “This Regulation should also promote the development of alternative methods for the assessment of hazards of substances.” Furthermore, in compliance with the 7th Amendment to Directive 76/768/EEC, animal testing and marketing of cosmetic ingredients and their products within Europe are fully forbidden since March 2013. These clear-cut regulatory developments collectively illustrate the need for alternative methods.
In Vitro Assessment of Contact Sensitizers
Several in vivo
methods exist that have been proven to be very accurate in terms of predictive identification and potency classification of sensitizers [19
]. The challenge is now to obtain the same quality of information using in silico
or in vitro
methods. Four goals have been identified for a full replacement of skin sensitization animal data [20
Hazard identification: prediction of potential sensitizer (yes/no answer);
Classification and labeling (i.e., GHS, EU-CLP (European Union regulation-Classification, Labelling and Packaging)): besides yes/no answer, some potency determination is required;
Hazard characterization: prediction of potency of the sensitizer, i.e., non-sensitizer, weak, moderate, strong, extreme (dose-response information);
Risk assessment: accurate evaluation of relative skin sensitizing potency to support effective risk assessment.
Chemical allergens are low molecular weight compounds (<1000 Dalton) too small to be seen by our immune system (hapten), therefore, they must bind to self-macromolecules to form a complete antigen. The complex formation is related to electrophilic reactivity and hydrophobicity of the allergen. Chemical allergens can be divided in three classes: (1) haptens; (2) pro-haptens, which require metabolic activation; and (3) pre-haptens, which spontaneously oxidize to form haptens. The metabolic competence of the in vitro system must be therefore carefully evaluated, as the lack of it may lead to false negative results.
ACD is a delayed type hypersensitivity reaction caused mainly by reactive T helper 1 and interferon (IFN)-γ producing CD8+ T cells (Tc1), which requires previous sensitization by the same chemicals [21
]. In order for a chemical to induce skin sensitization, several key steps must be taken. Briefly, following skin absorption and the formation of the complete antigen, the development of ACD then requires the activation of innate immune cells, including keratinocyes (KC) required for maturation and migration of dendritic cells (DC), and DC, required for the activation of T cells. The acquisition of specific immune response will then take place at the level of draining lymph nodes, where DC migrate and stimulate the activation of hapten-specific responsive T-cells and the generation of Tc1 effector cells.
In Table 1
, these key passages and the in vitro
opportunities are reported. Some of these tests have been or are currently in prevalidation or have been submitted for consideration. In particular, the direct peptide reactivity assay (DPRA), which measures depletion of a cysteine- and a lysine-containing peptide in the presence of the test chemical [23
], and the KeratinoSens™ assay [24
], which is based on a luciferase reporter gene under the control of an anti-oxidant response element of the human AKR1C2 gene stably inserted into HaCaT keratinocytes, have successfully passed the prevalidation phase at EURL/ECVAM (European Union Reference Laboratory for alternatives to animal testing) , and recommendations have been published [25
]. Soon, the human cell line activation assay (h-CLAT) [26
], addressing CD86 and CD54 upregulation in THP-1 cells, will be available as well. The documents available online summarize the assayʼs mechanistic relevance, the performance as well as the applicability and limitations and makes recommendations for further work.
A number of assays for the in vitro
identification of contact and respiratory sensitization have been developed within the integrated European Framework Program 6 Project Sens-it-iv, (LSHB-CT-2005-018681; 2005–2011) [27
]. The Sens-it-iv Toolbox is the major deliverable of this project. In Table 2
, the most advanced and promising assays developed within, or with contribution of Sens-it-iv are reported. Many of the tests listed in Table 2
have been submitted to the EURL-ECVAM for prevalidation.
One unifying characteristic of chemical allergens is the reactivity with proteins for the effective induction of skin sensitization. Most of chemical allergens are electrophilic and react with nucleophilic amino acids. One potential alternative approach to skin sensitization hazard identification is the use of (Quantitative) structure activity relationships ((Q)SARs) coupled with appropriate documentation and performance characteristics. A number of (Q)SARs and expert systems have been developed and are described in the literature, i.e.
, OECD (Organization for Economic Cooperation and Development) toolbox, ToxTree, TOPKAT, Derek, TOPS-MODE, etc
. None of the systems appear to perform sufficiently well to act as a standalone tool for hazard identification, but they may be very useful within a structured decision support system as part of a safety assessment strategy [28
]. Goebel et al.
] offer a nice overview of published reactivity-based QSAR models and their use in non-animal safety assessment of skin sensitization.
Concerning chemical reactivity, in the DPRA, generally, non-allergens and weak allergens demonstrated minimal to low peptide reactivity, whereas moderate to extremely potent allergens displayed moderate to high peptide reactivity. Classifying minimal reactivity as non-sensitizers and low, moderate, and high reactivity as sensitizers, a prediction accuracy of 89% was reported [23
Besides its barrier function, the skin has been recognized as an immunologically active tissue. KC may convert nonspecific exogenous stimuli into the production of cytokines, adhesion molecules and chemotactic factors [29
]. After KC, Langerhans cells (LC) represent the second most prominent cell type in the epidermis (2%–5% of the epidermal population). LC are the principal antigen-presenting cells (APC) in the skin [30
]. Due to their anatomical location and their significant role in the development of ACD, the use of both of these cell types to evaluate sensitizing potency in vitro
is amply justifiable. In addition to chemical processing, LC activation and migration requires the binding of cytokines produced by KC as a result of initial chemical exposure. Furthermore, the irritant capacity of allergens might present an additional risk factor so that irritant allergens may be stronger allergens than non-irritant ones [31
Starting from the in vivo
observation that in mice, IL-1α expression by KC was selectively up-regulated after application of contact sensitizers but not tolerogen or irritant [32
], similar results were reproduced in vitro
using the murine KC cell line HEL30 [33
]. Other authors [34
] obtained similar results and, furthermore observed that the rank of potency was similar to the ranking established using the LLNA. Similarly, using human KC it has been demonstrated that allergens, but not irritants or tolerogens, induced IL-12 [35
]. Among other cytokines produced by KC, IL-18 has been demonstrated to favor TH
1 type immune responses by enhancing the secretion of pro-inflammatory mediators such as TNFα, IL-8, and IFNγ, and to play a key proximal role in the induction of allergic contact dermatitis. The increase in intracellular IL-18 content was used to discriminate contact allergens from low molecular weight respiratory allergens and irritants [37
]. At non cytotoxic concentrations (cell viability >80% as assessed by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) reduction assay), all contact sensitizers tested induced a dose-dependent increase in IL-18, whereas both irritants and respiratory allergens failed, indicating that cell-associated IL-18 may provide an in vitro
tool for identification and discrimination of contact vs.
respiratory allergens and/or irritants [38
]. A total of 33 chemicals were tested, with an overall accuracy of 89.7%.
In line with this, we recently published a multicenter study, involving different laboratories located in Europe and in the US, on the possibility to use the release of IL-18 in epidermal equivalent 3D models (EE) for the in vitro
identification and ranking potency of contact allergens [41
]. The assay provides a single test for the identification and classification of skin sensitizing chemicals, including chemicals with low water solubility or stability, which are problematic to use in traditional cell culture systems. A protocol was developed using different 3D-epidermal models including in house VUMC (VU University Medical Centre) model, epiCS®
(previously EST1000TM), MatTek EpiDermTM and SkinEthicTM RHE. Following topical exposure for 24 h 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 has been 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/cm2
) data (Spearman correlation r
= 0.8500; p
= 0.0061) compared to LLNA data (Spearman correlation r
= 0.5968; p
= 0.0542), where DSA05 represents the 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 correlation r
= 0.8333; p
= 0.0154). The advantage of the proposed assay would be the possibility to identify in the same test the allergenic potential of a chemical (by the amount of IL-18 release) and its potency (by its irritant potential). The in vitro
reconstructed 3D epidermis model allows a chemical exposure that mimics human exposure (topical application), and overcomes all drawbacks of traditional submerged culture. Furthermore, 3D organotypic epidermis models possess in vivo
-like barrier properties and metabolizing capabilities, providing significant benefits over monolayer culture models for the assessment of contact sensitization potential. This easily transferable human in vitro
assay appears to be very promising, but additional testing of a larger chemical set is required to fully evaluate the usefulness of this assay and to establish a definitive prediction model.
Besides the EE model, it is important to note that other in vitro
methods can also be used for potency classification [26
]. A first indication of potency may come, for example, from the concentration required to induce a threshold of positive response (CD86 ≥ 150) in the h-CLAT system. A good correlation (r
= 0.839, p
< 0.01) was indeed found between the h-CLAT thresholds and LLNA EC3 values [42
], if a chemical has a Minimum Induction Threshold >10 it should be ranked as strong sensitizer, if <10 as a weak.
The key passage 4 in chemical-induced skin sensitization involves DC maturation and migration. Upon antigen capture, the DC undergoes a maturation process leading to the up-regulation of co-stimulatory molecules (CD86, CD80, and CD40), MHC Class II molecules and the CD83 protein [43
]. The establishment of human in vitro
models of DC had offered the possibility to demonstrate that haptens were able to directly activate cultured DC [44
]. A recent ECVAM workshop has reviewed the state of the art of the use of DC and human myeloid cell lines for the predictive identification of skin sensitization hazard [47
]. Among the several endpoints investigated in different experimental models, CD86, IL-8, and p38 MAP kinase appear to be the most promising and robust biomarkers described to date in DC based assays [18
Activated dendritic cells then migrate to the T-lymphocyte regions of lymphoid organs, where they lose antigen-processing activity and become potent immunostimulatory cells. Triggering of T cell responses to chemicals is the key event (key passage 5) that decides whether initial sensitization that results in transient inflammation in the target organ will lead to manifest disease upon subsequent contact with the chemical. Since T cells are the pathogenic effector in chemical-induced allergic contact dermatitis, respiratory allergy and adverse drug reaction, T cell-based in vitro
assay are important to identify contact and respiratory allergens. In this context, the T cell priming assay represents a promising tool to analyze the human naïve T cell repertoire [50
Although it should not be ruled out a priori that skin sensitization testing may, in the future, be addressed by one single method only, all methods currently under evaluation or evaluated for skin sensitization at EURL-ECVAM are not intended as a stand-alone methods, but for use within integrated approaches in combination with other information to predict the skin sensitization potential of chemicals and, where possible, the potency assessment of skin sensitizers. It is expected that in vitro
data will be integrated via statistical correlation into a testing strategy along with the peptide reactivity data, bioavailability data, and some informed rating of structural alerts in order to establish an acceptable exposure level [51
]. This will need to build experience in how to apply/integrate non-animal data to different exposure scenario for risk assessment decision-making. A range of integrated approaches to testing and assessment solutions may also be foreseen to cover different regulatory goals (i.e.
, hazard identification, classification, and potency assessment). The testing strategy will largely depend on information at hand, and it is expected to be chemical-specific [52
It has been anticipated that it will take at least another 7–9 years for the full replacement of the in vivo
animal models currently used to assess sensitization [20
]. Nevertheless, these in vitro
methods can already be used for hazard identification, i.e.
, to discriminate between sensitizers and non-sensitizers, and in the near future, potency classification for labeling is a realistic possibility.
Key passages in chemical-induced skin sensitization and in vitro opportunities.
Key passages in chemical-induced skin sensitization and in vitro opportunities.
|Key passage number||In vitro opportunities||References|
|1. Skin penetration and access to viable epidermis||Human skin biopsis, pig skin, reconstituted human epidermis||[53,54]|
|2. Binding to macromolecules (haptenation)||QSAR/Expert systems; Peptide binding assay (DPRA); allergen-peptide/protein interaction assay||[23,28,54,55]|
|3. Local trauma: epidermal inflammation (danger signals)||Keratinosens™; KC activation; NCTC2544 IL-18 assay; KC gene expression profile ||[24,33,34,35,36,37,38,39,40,56,57]|
|4. Antigen processing: dendritic cells maturation and migration||DC-like up-regulation of class II antigens and costimulatory molecules, i.e., CD54, CD86; Cytokine release, i.e., IL-8; LC-like MUTZ-3 cells migration assay; DC-like gene expression profile||[26,41,42,43,44,45,46,47,58,59,60,61,62,63,64]|
|5. Antigen presentation to TH cells and memory T-cell generation (immunogenicity)||In vitro T-cell activation||[50,65,66]|
The Sens-it-iv Toolbox for skin sensitization.
The Sens-it-iv Toolbox for skin sensitization.
|Target cells||Name of the assay||N° of chemicals||TSF||SOP|
|Keratinocytes||NCTC2544 IL-18 assay||33||yes||yes|
|Human Epidermal Equivalent||30||yes||yes|
|Maturation #1 (CD86, CD54, IL-8)||Stopped||-||-|
|Maturation # 2 (DotScan)||20||yes||yes|
|T cells||Primary T-cell stimulation||6||yes||E|
|Others||Neutrophils-THP-1 metabolization Proteomic marker profile||12||yes||E|