Development of an In Vitro Sensitisation Test Using a Coculture System of Human Bronchial Epithelium and Immune Cells.

Chemical respiratory sensitisation is a serious health problem. However, to date, there are no validated test methods available for identifying respiratory sensitisers. The aim of this study was to develop an in vitro sensitisation test by modifying the human cell line activation test (h-CLAT) to detect respiratory sensitisers and distinguish them from skin sensitisers. THP-1 cells were exposed to the test chemicals (two skin sensitisers and six respiratory sensitisers), either as monocultures or as cocultures with air-liquid interface-cultured reconstructed human bronchial epithelium. The responses were analysed by measuring the expression levels of surface markers on THP-1 cells (CD86, CD54 and OX40L) and the concentrations of cytokines in the culture media (interleukin (IL)-8, IL-33 and thymic stromal lymphopoietin (TSLP)). The cocultures exhibited increased CD54 expression on THP-1 cells; moreover, in the cocultures but not in the monocultures, exposure to two uronium salts (i.e. respiratory sensitisers) increased CD54 expression on THP-1 cells to levels above the criteria for a positive h-CLAT result. Additionally, exposure to the respiratory sensitiser abietic acid, significantly increased IL-8 concentration in the culture medium, but only in the cocultures. Although further optimisation of the method is needed to distinguish respiratory from skin sensitisers by using these potential markers (OX40L, IL-33 and TSLP), the coculture of THP-1 cells with bronchial epithelial cells offers a potentially useful approach for the detection of respiratory sensitisers.

Risk assessment of the asthma-induction potential of substances in spray products for car cabin detailing - based on EU's Chemical Agents Directive, using harmonised classifications and quantitative structure-activity relationship (QSAR).

Exposure to spray-formulated products for car cabin detailing is a potential risk for asthma induction. With a focus on the asthma-related endpoints sensitisation and irritation of the lungs, we performed an occupational risk assessment based on requirements in the EU Chemical Agents Directive. We identified 71 such spray products available in Denmark. We identified ingredient substances in safety data sheets and screened for harmonised classifications of respiratory sensitisation and airway irritation. For respiratory sensitisation, we also applied quantitative structure-activity relationship (QSAR). We modelled the exposure during 15 min of work inside a car cabin, and determined the risk ratio of the products by further applying occupational exposure limits - mainly derived no-effect levels (DNELs) from the European Chemicals Agency (ECHA) set on respiratory irritation. Four substances had a harmonised classification for respiratory irritation (bronopol, 2-phenoxyethanol, 2-methoxypropanol, and butan-1-ol). Seven substances were positive in the QSAR model for respiratory sensitisation (monoethanolamine, bronopol, glycerol, methyl salicylate, benzoic acid, ammonium benzoate, and sodium benzoate). Two vinyl treatment products had a risk ratio > 1 based on the level of sodium benzoate and its DNEL set on respiratory irritation. Two products had risk ratios of 0.69 and 0.73, respectively, based on 2-methyl-2 H-isothiazol-3-one and its acute DNEL set on respiratory irritation. In conclusion, 10 substances that may pose a risk for asthma induction were identified in the products. Two of the 71 products had a risk ratio > 1, meaning they may pose an asthma-induction risk in the modelled exposure scenario and using respiratory irritation DNELs from ECHA.

Evidential requirements for the regulatory hazard and risk assessment of respiratory sensitisers: methyl methacrylate as an example.

This review addresses the need for a framework to increase the consistency, objectivity and transparency in the regulatory assessment of respiratory sensitisers and associated uncertainties. Principal issues are considered and illustrated through a case study (with methyl methacrylate). In the absence of test methods validated for regulatory use, formal documentation of the weight-of-evidence for hazard classification both at the level of integration of individual studies within lines of evidence and across a broad range of data streams was agreed to be critical for such a framework. An integrated approach is proposed to include not only occupational studies and clinical evidence for the regulatory assessment of respiratory sensitisers, but also information on structure and physical and chemical factors, predictive approaches such as structure activity analysis and in vitro and in vivo mechanistic and toxicokinetic findings. A weight-of-evidence protocol, incorporating integration of these sources of data based on predefined considerations, would contribute to transparency and consistency in the outcome of the assessment. In those cases where a decision may need to be taken on the basis of occupational findings alone, conclusions should be based on transparent weighting of relevant data on the observed prevalence of occupational asthma in various studies taking into account all relevant information including the range and nature of workplace exposures to the substance of interest, co-exposure to other chemicals and study quality.

"In Litero" Screening: Retrospective Evaluation of Clinical Evidence to Establish a Reference List of Human Chemical Respiratory Sensitizers.

Despite decades of investigation, test methods to identify respiratory sensitizers remain an unmet regulatory need. In order to support the evaluation of New Approach Methodologies in development, we sought to establish a reference set of low molecular weight respiratory sensitizers based on case reports of occupational asthma. In this context, we have developed an "in litero" approach to identify cases of low molecular weight chemical exposures leading to respiratory sensitization in clinical literature. We utilized the EPA-developed Abstract Sifter literature review tool to maximize the retrieval of publications relevant to respiratory effects in humans for each chemical in a list of chemicals suspected of inducing respiratory sensitization. The literature retrieved for each of these candidate chemicals was sifted to identify relevant case reports and studies, and then evaluated by applying defined selection criteria. Clinical diagnostic criteria were defined around exposure history, respiratory effects, and specific immune response to conclusively demonstrate occupational asthma as a result of sensitization, rather than irritation. This approach successfully identified 28 chemicals that can be considered as human respiratory sensitizers and used to evaluate the performance of NAMs as part of a weight of evidence approach to identify novel respiratory sensitizers. Further, these results have immediate implications for the development and refinement of predictive tools to distinguish between skin and respiratory sensitizers. A comparison of the protein binding mechanisms of our identified "in litero" clinical respiratory sensitizers shows that acylation is a prevalent protein binding mechanism, in contrast to Michael addition and Schiff base formation common to skin sensitizers. Overall, this approach provides an exemplary method to evaluate and apply human data as part of the weight of evidence when establishing reference chemical lists.

The use of Genomic Allergen Rapid Detection (GARD) assays to predict the respiratory and skin sensitising potential of e-liquids.

Electronic cigarettes (e-cigarettes) are an increasingly popular alternative to combustible tobacco cigarettes among smokers worldwide. A growing body of research indicates that flavours play a critical role in attracting and retaining smokers into the e-cigarette category, directly contributing to declining smoking rates and tobacco harm reduction. The responsible selection and inclusion levels of flavourings in e-liquids must be guided by toxicological principles. Some flavour ingredients, whether natural extracts or synthetic, are known allergens. In this study, we used the Genomic Allergen Rapid Detection (GARD) testing strategy to predict and compare the respiratory and skin sensitising potential of three experimental and two commercial e-liquids. These novel, myeloid cell-based assays use changes in the transcriptional profiles of genomic biomarkers that are collectively relevant for respiratory and skin sensitisation. Our initial results indicate that the GARD assays were able to differentiate and broadly classify e-liquids based on their sensitisation potential, which are defined mixtures. Further studies need to be conducted to assess whether and how these assays could be used for the screening and toxicological assessment of e-liquids to support product development and commercialisation.

Immunological Processes Driving IgE Sensitisation and Disease Development in Males and Females.

IgE sensitisation has increased significantly over the last decades and is a crucial factor in the development of allergic diseases. IgE antibodies are produced by B cells through the process of antigen presentation by dendritic cells, subsequent differentiation of CD4⁺ Th2 cells, and class switching in B cells. However, many of the factors regulating these processes remain unclear. These processes affect males and females differently, resulting in a significantly higher prevalence of IgE sensitisation in males compared to females from an early age. Before the onset of puberty, this increased prevalence of IgE sensitisation is also associated with a higher prevalence of clinical symptoms in males; however, after puberty, females experience a surge in the incidence of allergic symptoms. This is particularly apparent in allergic asthma, but also in other allergic diseases such as food and contact allergies. This has been partly attributed to the pro- versus anti-allergic effects of female versus male sex hormones; however, it remains unclear how the expression of sex hormones translates IgE sensitisation into clinical symptoms. In this review, we describe the recent epidemiological findings on IgE sensitisation in male and females and discuss recent mechanistic studies casting further light on how the expression of sex hormones may influence the innate and adaptive immune system at mucosal surfaces and how sex hormones may be involved in translating IgE sensitisation into clinical manifestations.

Thresholds in chemical respiratory sensitisation.

There is a continuing interest in determining whether it is possible to identify thresholds for chemical allergy. Here allergic sensitisation of the respiratory tract by chemicals is considered in this context. This is an important occupational health problem, being associated with rhinitis and asthma, and in addition provides toxicologists and risk assessors with a number of challenges. In common with all forms of allergic disease chemical respiratory allergy develops in two phases. In the first (induction) phase exposure to a chemical allergen (by an appropriate route of exposure) causes immunological priming and sensitisation of the respiratory tract. The second (elicitation) phase is triggered if a sensitised subject is exposed subsequently to the same chemical allergen via inhalation. A secondary immune response will be provoked in the respiratory tract resulting in inflammation and the signs and symptoms of a respiratory hypersensitivity reaction. In this article attention has focused on the identification of threshold values during the acquisition of sensitisation. Current mechanistic understanding of allergy is such that it can be assumed that the development of sensitisation (and also the elicitation of an allergic reaction) is a threshold phenomenon; there will be levels of exposure below which sensitisation will not be acquired. That is, all immune responses, including allergic sensitisation, have threshold requirement for the availability of antigen/allergen, below which a response will fail to develop. The issue addressed here is whether there are methods available or clinical/epidemiological data that permit the identification of such thresholds. This document reviews briefly relevant human studies of occupational asthma, and experimental models that have been developed (or are being developed) for the identification and characterisation of chemical respiratory allergens. The main conclusion drawn is that although there is evidence that the acquisition of sensitisation to chemical respiratory allergens is a dose-related phenomenon, and that thresholds exist, it is frequently difficult to define accurate numerical values for threshold exposure levels. Nevertheless, based on occupational exposure data it may sometimes be possible to derive levels of exposure in the workplace, which are safe. An additional observation is the lack currently of suitable experimental methods for both routine hazard characterisation and the measurement of thresholds, and that such methods are still some way off. Given the current trajectory of toxicology, and the move towards the use of non-animal in vitro and/or in silico) methods, there is a need to consider the development of alternative approaches for the identification and characterisation of respiratory sensitisation hazards, and for risk assessment.

Toluene diisocyanate (TDI) airway effects and dose-responses in different animal models.

Many inhalation exposure studies have been performed with toluene diisocyanate (TDI) in different animal species. Many were targeted at respiratory irritation and/or sensitisation. As there is still no broadly accepted guideline for the performance of respiratory sensitisation tests, protocols used and endpoints investigated are numerous. In this review we collected data from those respiratory sensitisation and/or irritation studies that provided threshold or dose-response information. Against this aim, and as TDI is a model substance for a respiratory sensitiser, a great number of mechanistic studies are not cited in this paper, although they were checked for relevant information. The literature data available allow the conclusion that both respiratory irritation and sensitisation may be interdependent, and both irritation and sensitisation by TDI is a threshold phenomenon. Across species, the majority of NOAECs for respiratory sensitisation are in the range of 0.005 to 0.03 ppm, whereas the LOAEC is about 0.02 to 0.4 ppm.