Building confidence in skin sensitisation potency assessment using new approach methodologies: report of the 3rd EPAA Partners Forum, Brussels, 28th October 2019.

Skin sensitising substances that induce contact allergy and consequently risk elicitation of allergic contact dermatitis (ACD) remain an important focus regarding the replacement of animal experimentation. Current in vivo methods, notably the local lymph node assay (LLNA) refined and reduced animal usage and led to a marked improvement in hazard identification, characterisation and risk assessment. Since validation, regulatory confidence in the LLNA approach has evolved until it became the first choice assay in most regulated sectors. Currently, hazard identification using the LLNA is being actively replaced by a toolbox of non-animal approaches. However, there remains a need to increase confidence in the use of new approach methodologies (NAMs) as replacements for LLNA sensitiser potency estimation. The EPAA Partners Forum exchanged the current state of knowledge on use of NAMs in various industry sectors and regulatory environments. They then debated current challenges in this area and noted several ongoing needs. These included a requirement for reference standards for potency, better characterisation of applicability domains/technical limitations of NAMs, development of a framework for weight of evidence assessments, and an increased confidence in the characterisation of non-sensitisers. Finally, exploration of an industry/regulator cross-sector user-forum on skin sensitisation was recommended.

In vitro RHE skin sensitisation assays: Applicability to challenging substances.

In the last 20 years, alternative approaches to the identification of skin sensitisation hazards have been at the forefront of the 3Rs and have helped refine the validation and acceptance processes. However, experience with the local lymph node assay showed that, post-validation, challenges still occurred, particularly when a wider diversity of chemical substances was addressed, a situation which will arise with validated in vitro alternatives. In the present work, a range of substances potentially challenging to assess in current nonanimal OECD test guidelines were evaluated in several of the emerging in vitro alternatives. Twelve such substances (of which just over half were known skin sensitisers) were assessed in 4 assays, all based on reconstructed human epidermis (RHE) models. For hazard identification, the overall predictive accuracy ranged around 70% for three assays, although for one (SensCeeTox), it fell below 50% when human data was used as the benchmark. In most cases, sensitivity was high, such that sensitisation was overpredicted. As the substances were challenging to assess in other nonanimal methods, the results indicate that the 3D RHE models may be a useful tool for assessing skin sensitisation potentials without needing to revert to animal use.

Interspecies assessment factors and skin sensitization risk assessment.

For many endpoints in toxicology, an interspecies safety factor remains a standard requirement. However, for skin sensitization, the hazard and potency predictions, notably from the local lymph node assay (LLNA) have been shown to correlate well with human data. Despite this, there are always exceptions, both over and under predictions. For this reason it has been suggested that an interspecies factor of 15 would accommodate potential "errors". An alternative approach is suggested in which an evidence-based strategy is taken: the large majority of the information indicates a human:LLNA ratio of 1, therefore a corrective factor would best be applied where our knowledge of the underlying chemistry of sensitization indicates that it is necessary.

Skin sensitization: Implications for integration of clinical data into hazard identification and risk assessment.

Skin sensitization associated with allergic contact dermatitis is a common health problem and is an important consideration for toxicologists in safety assessment. Historically, in vivo predictive tests have been used with good success to identify substances that have the potential to induce skin sensitization, and these tests formed the basis of safety evaluation. These original tests are now being replaced gradually either by in vitro assays or by further refinements of in vivo methods such as the local lymph node assay. Human data have also been available to inform classification decisions for some substances and have been used by risk managers to introduce measures for exposure reduction. However, humans encounter hazards in the context of exposure rather than in the form of intrinsic hazards per se, and so in this article, we have examined critically the extent to which human data have been used to refine classification decisions and safety evaluations. We have also evaluated information on the burden of human allergic skin disease and used this to address the question of whether, and to what extent, the identification and evaluation of skin sensitization hazards has led to an improvement of public and/or occupational health.

T helper cell 2 immune skewing in pregnancy/early life: chemical exposure and the development of atopic disease and allergy.

During the last 50 years there has been a significant increase in Western societies of atopic disease and associated allergy. The balance between functional subpopulations of T helper cells (Th) determines the quality of the immune response provoked by antigen. One such subpopulation - Th2 cells - is associated with the production of IgE antibody and atopic allergy, whereas, Th1 cells antagonize IgE responses and the development of allergic disease. In seeking to provide a mechanistic basis for this increased prevalence of allergic disease, one proposal has been the 'hygiene hypothesis', which argues that in Westernized societies reduced exposure during early childhood to pathogenic microorganisms favours the development of atopic allergy. Pregnancy is normally associated with Th2 skewing, which persists for some months in the neonate before Th1/Th2 realignment occurs. In this review, we consider the immunophysiology of Th2 immune skewing during pregnancy. In particular, we explore the possibility that altered and increased patterns of exposure to certain chemicals have served to accentuate this normal Th2 skewing and therefore further promote the persistence of a Th2 bias in neonates. Furthermore, we propose that the more marked Th2 skewing observed in first pregnancy may, at least in part, explain the higher prevalence of atopic disease and allergy in the first born.

The hapten-atopy hypothesis III: the potential role of airborne chemicals.

One explanation for the large increase in the prevalence of atopic disease in developed countries during the last 50 years is the 'hygiene hypothesis'. This proposes that a reduced exposure to pathogenic microorganisms at a key period(s) during development results in the maintenance or acquisition of an atopic phenotype. Alternatively, or additionally, we have postulated that increased exposure to chemicals generally, and to irritant/haptenic chemicals in particular, during critical windows of maternal pregnancy/early life have also contributed to changes in the prevalence of atopic disease. Having previously reviewed the potential roles of oral and cutaneous exposure to chemicals on the subsequent diagnosis of atopic disease, we here consider possible evidence of a role for exposure to airborne chemicals as a contributory factor in acquired susceptibility to atopic allergy. After controlling for known confounders, five specific maternal occupations during pregnancy have been implicated as being associated with subsequent atopic disease in the offspring. Each of these occupations is characterized by high and persistent exposure to airborne chemicals. High-level exposure to volatile organic compounds in the domestic environment, either during pregnancy or in early life, is also associated with development of childhood atopic disease. Similarly, sustained exposure to airborne chlorinated chemicals from swimming pools during childhood has been associated with the development of atopic allergy. A possible immunological basis for these associations is that exposure to certain airborne chemicals, even at low levels, can result in the delivery of 'danger' signals that, in turn, bias the immune response towards the selective induction or maintenance of preferential T helper 2-type immune responses consistent with the acquisition of allergic sensitization.

Why does allergic contact dermatitis exist?

The skin immune system's propensity to produce allergic contact dermatitis (ACD) to harmless chemicals, while otherwise being an efficient defence system, represents a dermatological paradox. We postulate that a major role in signalling in ACD is played by Toll-like receptor (TLR)2 and TLR4, and arises from their activation by extracellular danger-associated molecular patterns (DAMPs). Ligand activation of TLR4/2 results in the expression of interleukins (ILs) IL-1ß, IL-6, IL-12, IL-18 and IL-23, tumour necrosis factor-α and interferon-α. These cytokines promote acquisition of sensitization, and facilitate elicitation of contact allergy via multiple mechanisms, including the recruitment of CD4+ Th1 and Th17 cells. As Th1 cells secrete large amounts of DAMPs, a DAMP immune circuit (positive-feedback loop) is created. This is an important driver of skin sensitization and skin inflammation. Pathogenic extracellular bacteria, but not commensal bacteria, produce pathogen-associated molecular pattern molecules, which stimulate the expression of Th1- and Th17-promoting cytokines via TLR2 and TLR4. This also induces an immune circuit. The ability of the skin immune system to activate host defence mechanisms and to distinguish between pathogenic bacteria and commensals provides an explanation for why skin sensitization and ACD develop, as they are processes that rely on the same biological pathways. These pathways may also shed light on the pathogenesis of chronic pustular inflammatory dermatoses (e.g. acne vulgaris). The existence of safety signals from commensal bacteria, which prevent initiation of these pathways, may provide opportunities for novel therapeutic approaches to the treatment of inflammatory skin diseases.

Inter-relationships between different classes of chemical allergens.

Although allergic sensitization of the respiratory tract induced by chemicals is not as common as skin sensitization, it is nevertheless an important occupational health issue. Respiratory allergy to chemicals, characterized typically by rhinitis and asthma, is associated with considerable morbidity and with related socioeconomic costs. Several experimental approaches have been proposed for the prospective identification of chemical respiratory allergens, but none of these has yet been validated formally. In the absence of a widely accepted method for respiratory allergen identification, it is appropriate and relevant to explore their relationship with skin-sensitizing chemicals. A series of chemicals known to cause immune-mediated respiratory allergy in humans has been examined. The majority of the respiratory allergens tested were found to elicit positive responses in one or more standard tests used for the identification of skin-sensitizing potential (guinea pig maximization test, the Buehler test and/or the local lymph node assay). We suggest that this observation might form the basis of a potentially useful paradigm for initial characterization of the respiratory-sensitizing potential of chemicals. Specifically, chemicals that fail to elicit positive responses in accepted skin-sensitization test methods might also be regarded as lacking the inherent potential to cause allergic sensitization of the respiratory tract.

Allergic contact dermatitis: epidemiology, molecular mechanisms, in vitro methods and regulatory aspects. Current knowledge assembled at an international workshop at BfR, Germany.

Contact allergies are complex diseases, and one of the important challenges for public health and immunology. The German 'Federal Institute for Risk Assessment' hosted an 'International Workshop on Contact Dermatitis'. The scope of the workshop was to discuss new discoveries and developments in the field of contact dermatitis. This included the epidemiology and molecular biology of contact allergy, as well as the development of new in vitro methods. Furthermore, it considered regulatory aspects aiming to reduce exposure to contact sensitisers. An estimated 15-20% of the general population suffers from contact allergy. Workplace exposure, age, sex, use of consumer products and genetic predispositions were identified as the most important risk factors. Research highlights included: advances in understanding of immune responses to contact sensitisers, the importance of autoxidation or enzyme-mediated oxidation for the activation of chemicals, the mechanisms through which hapten-protein conjugates are formed and the development of novel in vitro strategies for the identification of skin-sensitising chemicals. Dendritic cell cultures and structure-activity relationships are being developed to identify potential contact allergens. However, the local lymph node assay (LLNA) presently remains the validated method of choice for hazard identification and characterisation. At the workshop the use of the LLNA for regulatory purposes and for quantitative risk assessment was also discussed.

The Hapten-Atopy hypothesis II: the 'cutaneous hapten paradox'.

One explanation for the striking increase in atopic disease in developed countries over the last 50 years has been the 'Hygiene Hypothesis'; a reduced exposure to pathogenic microorganisms. We have postulated previously that oral and cutaneous exposure to chemicals generally and to haptens in particular, may have also contributed to the increased prevalence of atopic disease; the 'Hapten-Atopy Hypothesis'. The purpose here is to extend further that hypothesis by consideration of the impact interplay between the innate and adaptive immune systems may have on the development of atopic allergy. It is clear that experimental cutaneous exposure to hapten can generate immune responses of different types with regard to T-helper (Th) cell phenotype. Allergic contact dermatitis is frequently associated with a selective Th1 (and Tc1)-driven inflammation, whereas atopic dermatitis is characterized by preferential Th2 cell responses. We postulate here that initial innate immune responses to chemical haptens result in the promotion of Th1 cell responses secondary to stimulation of Toll-like receptor. However, we argue also that under conditions where there is prolonged skin exposure to hapten there will be a shift of Th cell phenotype to selective Th2-type responses. The significance of such interactions is the possibility that repeated low-level skin exposure to certain types of hapten may result in the creation of an immunological environment in which the development of Th2 immune responses to third party antigens is favoured. The hypothesis is advanced that the nature and conditions of skin exposure to common haptens may impact on the quality of cutaneous immune responses such that in some circumstances the development atopic disease is favoured.

Defining occupational and consumer exposure limits for enzyme protein respiratory allergens under REACH.

A wide range of substances have been recognized as sensitizing, either to the skin and/or to the respiratory tract. Many of these are useful materials, so to ensure that they can be used safely it is necessary to characterize the hazards and establish appropriate exposure limits. Under new EU legislation (REACH), there is a requirement to define a derived no effect level (DNEL). Where a DNEL cannot be established, e.g. for sensitizing substances, then a derived minimal effect level (DMEL) is recommended. For the bacterial and fungal enzymes which are well recognized respiratory sensitizers and have widespread use industrially as well as in a range of consumer products, a DMEL can be established by thorough retrospective review of occupational and consumer experience. In particular, setting the validated employee medical surveillance data against exposure records generated over an extended period of time is vital in informing the occupational DMEL. This experience shows that a long established limit of 60 ng/m(3) for pure enzyme protein has been a successful starting point for the definition of occupational health limits for sensitization in the detergent industry. Application to this of adjustment factors has limited sensitization induction, avoided any meaningful risk of the elicitation of symptoms with known enzymes and provided an appropriate level of security for new enzymes whose potency has not been fully characterized. For example, in the detergent industry, this has led to general use of occupational exposure limits 3-10 times lower than the 60 ng/m(3) starting point. In contrast, consumer exposure limits vary because the types of exposure themselves cover a wide range. The highest levels shown to be safe in use, 15 ng/m(3), are associated with laundry trigger sprays, but very much lower levels (e.g. 0.01 ng/m(3)) are commonly associated with other types of safe exposure. Consumer limits typically will lie between these values and depend on the actual exposure associated with product use.

Does hapten exposure predispose to atopic disease? The hapten-atopy hypothesis.

Contact allergy data indicates that atopics have heightened oral tolerance to haptens (chemical allergens). We speculate here, that artificially increased oral exposure to chemicals compete with dietary proteins for the development of oral tolerance, predisposing to the acquisition of food protein allergy and representing one driver for the increasing prevalence of protein allergy and/or atopy. Hapten exposure via other surfaces such as the skin and airways might also be important in promoting atopic disease. Consistent with this hypothesis it is notable that over 40 years, with the huge increase in atopic disease, there has also been an increase in dietary hapten exposure through processed food, formula milk and oral antibiotic and drug use.

Atopic dermatitis and allergic reactions to individual fragrance chemicals.

BACKGROUND: Allergic contact dermatitis prevalence is reported as equal in atopic and nonatopic dermatitis. Atopic dermatitis is under-represented in those with allergic contact dermatitis to agents having cutaneous and dietary exposure. We compared rates of atopic dermatitis between patients with allergic contact dermatitis arising out of individual fragrance chemicals with known oral/cutaneous exposure against exclusively cutaneous exposure. METHODS: Between 1982 and 2007, 37 065 dermatitis patients were tested with Fragrance mix I. Those who were positive were tested for individual fragrance allergy. Chemicals were categorized according to whether their exposure pattern was solely cutaneous, oral or mixed. Current and past atopic dermatitis rates were compared between the whole population and groups allergic to individual fragrances. Age and gender were controlled. RESULTS: Cinnamic alcohol and cinnamal allergy groups had reduced rates of both 'current' [24/266 (9.0%) P = 0.0008, 38/364 (10.4%) P = 0.0005] and 'past' atopic dermatitis [44/266 (16.5%) P = 0.009, 70/346 (19.2%) P = 0.037]. Atopic dermatitis rates in groups allergic to Evernia prunastri and hydroxycitronellal (cutaneous exposure only) were not reduced [120/597 (20.1%) and 41/153 (26.8%)]. Groups allergic to cinnamic alcohol (P < 0.0001, P < 0.0001) and cinnamal (P < 0.0001, P < 0.004) had reductions in 'current' and 'past' atopic dermatitis, compared with Evernia prunastri. CONCLUSIONS: Patients allergic to individual fragrances with dietary exposure have reduced rates of atopic dermatitis. This suggests that patients with atopic dermatitis have heightened oral tolerance to dietary haptens, in contrast to the known close association of atopic dermatitis with food-protein allergy. Haptens may interfere with food protein tolerance by binding to soluble protein to alter its configuration and immunogenic profile.

Skin irritation and sensitization: mechanisms and new approaches for risk assessment. 1. Skin irritation.

Cutaneous irritation presents a major health problem with serious social and occupational impact. The interaction between an irritant and the human skin depends on multiple factors: the intrinsic properties and the nature of the irritant itself, and specific individual- and environment-related variables. The main pathological mechanisms of irritancy include skin barrier disruption, induction of a cytokine cascade and involvement of the oxidative stress network; all of them resulting in a visible or subclinical inflammatory reaction. In vivo, different non-invasive parameters for the evaluation of skin irritation and irritant potential of compounds and their specific formulations have been introduced, such as epidermal barrier function, skin hydration, surface pH, lipid composition, skin colour and skin blood flow. The diverse physiological changes caused by irritating agents require implementation of a multiparametric approach in the evaluation of cutaneous irritancy.

Skin irritation and sensitization: mechanisms and new approaches for risk assessment.

Allergic contact dermatitis (ACD) is a common skin disease with a significant social and economic impact. In contrast to irritation, skin sensitization is a response of the adaptive immune system, in which there is a delayed T-cell-mediated allergic response to chemically modified skin proteins. The chemicals that can covalently modify the skin proteins and trigger an allergic reaction are referred to as haptens or sensitizers. Attempts have been made in many countries to reduce the problems of ACD by the implementation of legislations related to skin-sensitizing chemicals, as well as by the early detection and risk assessment of substances with sensitizing properties. For many years, the simple identification of sensitizing chemicals was performed in guinea pig tests. A murine test, the local lymph node assay (LLNA), has been validated as a replacement for the guinea pig tests. Despite the recent introduction of in vitro methods for the identification of sensitizing chemicals, the LLNA results (when coupled with good exposure data) can be used as the starting point for a quantitative risk assessment. The quantitative risk assessment is aimed to identify the safe use thresholds for any potential skin sensitizer.

Skin sensitization: strategies for the assessment and management of risk.

Allergic contact dermatitis (ACD) is to a considerable extent a preventable disease. Limitation can be achieved by correct identification of skin sensitizers, characterization of their potency, understanding human skin exposure and application of good risk assessment/management strategies. Various methods exist which are accurate for the predictive identification of chemicals that possess skin-sensitizing properties. These are enshrined in regulations that aim to provide a harmonized approach to hazard identification. One of the methods, the local lymph node assay, also delivers information on the relative potency of sensitizers. Efforts are continuing in the European Union and at the Organization for Economic Cooperation and Development to use elements of this information for regulatory categorization of skin sensitizers. However, greater use can be made of this potency information in the application of quantitative risk assessments. Such assessments depend also on the availability of accurate data on human skin exposure, one aspect where legislation has little role to play. Management of risks by restriction of skin exposure is, in contrast, a key point where legislation can play an important role, helping to establish a level playing field for industry and setting good standards based on the legislator's ability to access all data. Ultimately, the combination of accurate hazard identification, potency measurement, risk assessment and management, underpinned by enabling legislation, will lead to reduction of ACD. For individuals who do still develop contact allergy, avoidance of ACD should continue to be a goal, based on raising awareness of skin protection, allergen labelling and other skincare strategies.

Dendritic cells from control but not atopic donors respond to contact and respiratory sensitizer treatment in vitro with differential cytokine production and altered stimulatory capacity.

BACKGROUND: Chemical haptens induce both contact and allergic respiratory disease with dendritic cells (DCs) controlling and directing immune responses in vivo. Contact and respiratory haptens may promote differential cytokine production yet distinguishing these effects in vitro remains difficult due to human donor variability. Objective We sought to determine the effect of atopic status on the ability of DC to respond to contact and respiratory sensitizer treatment in vitro as DC from atopic donors are believed to promote Th2-type responses. METHODS: Enriched DC from control or atopic donors were treated for 4 h with levels of the contact sensitizer 2,4-dinitrochlorobenzene (DNCB) or the respiratory sensitizer trimellitic anhydride (TMA) that did not reduce cell viability. A sensitive intracellular detection technique was used to measure cytokine production, while T cell responses were assessed in a mixed leucocyte reaction. RESULTS: DC from control, non-atopic, donors produced cytokines differentially in response to sensitizer treatment; DNCB treatment significantly increased the production of Th1 cytokines IL-12 and IFN-gamma while TMA induced the production of IL-13. Control donor DC treated with TMA stimulated less in a mixed leucocyte reaction than untreated cells with any response reduced further by blocking IL-13 in culture. However, DC from atopic donors showed no significant alteration in either cytokine production or T cell stimulatory capacity after sensitizer treatment. CONCLUSION: Haptens modulate DC by changing the production of cytokines that may play a role in T cell stimulation and subsequent polarization of the immune response. DC from atopic donors were unresponsive to chemical sensitizer treatment, and may be deficient in inducing divergent T cell responses.