This report explores the potential of novel 6-aryloxy-2-aminopyrimidine-benzonitrile scaffolds as promising anti-infective agents in the face of the increasing threat of infectious diseases. Starting from 2-amino-4,6-dichloropyrimidine, a series of 24 compounds inspired from the antiviral drugs dapivirine, etravirine, and rilpivirine were designed and synthesized via a two-step reaction sequence in good yields. Biological testing of synthetic analogs revealed potent inhibition against both viral and tuberculosis targets. Notably, compounds 5p (2,4-dimethyl substitution; IC50 = 44 ± 4.9 µM; selectivity index [SI] = 20) and 5 s (3-thiophenphenyl; IC50 = 6 ± 1 µM; SI = 120) showed significant antiviral activity against pandemic influenza virus A/Puerto Rico/8/34 (H1N1) with positive toxicity profiles and also exhibited good IC50 values (5p, IC50 = 10 ± 2 µM; SI = 9 and 5 s, IC50 = 16 ± 2 µM; SI = 60) against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (Wuhan strain) compared with favipiravir. In addition, analogs 5a, 5r, 5t, and 5u showed good antitubercular activity against Mycobacterium tuberculosis H37Rv strain and compounds 3, 5f, 5n, and 5q showed moderate antibacterial activity against gram+ve and gram-ve bacterial strains, suggesting that this scaffold has a broad spectrum of therapeutic effects.

T cell activation is the final key event (KE4) in the adverse outcome pathway (AOP) of skin sensitization. However, validated new approach methodologies (NAMs) for evaluating this step are missing. Accordingly, chemicals that activate an unusually high frequency of T cells, as does the most prevalent metal allergen nickel, are not yet identified in a regulatory context. T cell reactivity to chemical sensitizers might be especially relevant in real-life scenarios, where skin injury, co-exposure to irritants in chemical mixtures, or infections may trigger the heterologous innate immune stimulation necessary to induce adaptive T cell responses. Additionally, cross-reactivity, which underlies cross-allergies, can only be assessed by T cell tests. To date, several experimental T cell tests are available that use primary naïve and memory CD4+ and CD8+ T cells from human blood. These include priming and lymphocyte proliferation tests and, most recently, activation-induced marker (AIM) assays. All approaches are challenged by chemical-mediated toxicity, inefficient or unknown generation of T cell epitopes, and a low throughput. Here, we summarize solutions and strategies to confirm in vitro T cell signals. Broader application and standardization are necessary to possibly define chemical applicability domains and to strengthen the role of T cell tests in regulatory risk assessment.

Substituted saturated N-heterocycles have gained momentum as effective scaffolds for the development of new drugs. In this study, we coupled partly saturated benzothiazoles with substituted piperazines and evaluated their antimicrobial activity. Following a three-step reaction sequence from commercially available cyclic 1,3-diones, a series of novel 2-[4-substituted-1-piperazinyl]-N-(7-oxo-4,5,6,7-tetrahydrobenzo[d]thiazol-2-yl)acetamides (7a-af) were synthesised. 2-Amino-5,6-dihydro-benzo[d]thiazol-7(4H)-ones, obtained through the condensation of cyclohexane-1,3-diones with thiourea, were acetylated with chloroacetic chloride and then reacted with N-substituted piperazines 6a-p to give the desired products 7a-af in excellent yields. All 32 new compounds were fully characterised by their 1 H-nuclear magnetic resonance (NMR), 13 C-NMR and high-resolution mass spectrometry spectra. The synthetic compounds 7a-af were tested in vitro for their efficacy as antimicrobials against pathogenic strains of Gram-positive and Gram-negative bacteria, Streptococcus mutans and Salmonella typhi, respectively, as well as against fungal strains, including Candida albicans 3018 and C. albicans 4748. Ciprofloxacin and fluconazole served as the reference drugs. While compounds 7c and 7l showed inhibition against fungal strains with zones of inhibition of 11 and 1 mm, respectively, four analogues (7d, 7l, 7n, and 7r) demonstrated strong antibacterial action (zone of inhibition in the range of 10-15 mm). Three compounds (7j, 7l, and 7w) also exhibited moderate antitubercular activity (MIC: 6.25 µg/mL) against Mycobacterium tuberculosis H37Rv. Molecular docking investigations and the predicted physicochemical and ADMET (absorption, distribution, metabolism, excretion, and toxicity) properties for the potent compounds made this scaffold useful as a pharmacologically active framework for the development of potential antimicrobial hits.

For over a decade, the skin sensitization Adverse Outcome Pathway (AOP) has served as a useful framework for development of novel in chemico and in vitro assays for use in skin sensitization hazard and risk assessment. Since its establishment, the AOP framework further fueled the existing efforts in new assay development and stimulated a plethora of activities with particular focus on validation, reproducibility and interpretation of individual assays and combination of assay outputs for use in hazard/risk assessment. In parallel, research efforts have also accelerated in pace, providing new molecular and dynamic insight into key events leading to sensitization. In light of novel hypotheses emerging from over a decade of focused research effort, mechanistic evidence relating to the key events in the skin sensitization AOP may complement the tools currently used in risk assessment. We reviewed the recent advances unraveling the complexity of molecular events in sensitization and signpost the most promising avenues for further exploration and development of useful assays.

Environmental chemicals and contaminants cause a wide array of harmful implications to terrestrial and aquatic life which ranges from skin sensitization to acute oral toxicity. The current study aims to assess the quantitative skin sensitization potential of a large set of industrial and environmental chemicals acting through different mechanisms using the novel quantitative Read-Across Structure-Activity Relationship (q-RASAR) approach. Based on the identified important set of structural and physicochemical features, Read-Across-based hyperparameters were optimized using the training set compounds followed by the calculation of similarity and error-based RASAR descriptors. Data fusion, further feature selection, and removal of prediction confidence outliers were performed to generate a partial least squares (PLS) q-RASAR model, followed by the application of various Machine Learning (ML) tools to check the quality of predictions. The PLS model was found to be the best among different models. A simple user-friendly Java-based software tool was developed based on the PLS model, which efficiently predicts the toxicity value(s) of query compound(s) along with their status of Applicability Domain (AD) in terms of leverage values. This model has been developed using structurally diverse compounds and is expected to predict efficiently and quantitatively the skin sensitization potential of environmental chemicals to estimate their occupational and health hazards.

Researchers active in the field of inflammatory skin diseases from the spectrum of dermatitis and eczema are well aware of a considerable overlap in the clinical pictures and proposed sets of diagnostic criteria for these diseases, which can hardly be overcome through the clinical or epidemiological research. In effect, patients are included in studies based on vague and overlapping criteria, while heterogeneous study populations may, in turn, lead to non-representative outcomes and continued confusion. In this narrative review, a systematics of diseases from the spectrum of dermatitis and eczema is proposed based on the origins of causative factors and the pathomechanisms involved. Difficulties in differentiating between these diseases are discussed, and the extent to which advances in the "omics" sciences might help to overcome them is considered. Of all the "omics" research in this field, more than 90% of the published papers were devoted to atopic dermatitis, with a striking underrepresentation of other diseases from the spectrum of dermatitis and eczema, conditions which collectively exceed the rates of atopic dermatitis by far. A greater "omics" research effort is urgently needed to tackle other dermatitides, like allergic, irritant and protein contact dermatitis, as well as radiation, seborrheic, stasis or autoimmune dermatitis. Atopic dermatitis findings should be validated not only against healthy donors but also other dermatitides. A clinic-oriented approach is proposed for future "omics" studies in the field of dermatitis and eczema.

Exposure to skin sensitizers is common and regulated in many industry sectors. For cosmetics, a risk-based approach has been implemented, focused on preventing the induction of sensitization. First, a No Expected Sensitization Induction Level (NESIL) is derived, then modified by Sensitization Assessment Factors (SAFs) to derive an Acceptable Exposure Level (AEL). The AEL is used in risk assessment, being compared with an estimated exposure dose, specific to the exposure scenario. Since in Europe there is increased concern regarding exposure towards potentially sensitizing pesticides via spray drift, we explore how existing practice can be modified to allow Quantitative Risk Assessment (QRA) of pesticides for bystanders and residents. NESIL derivation by the Local Lymph Node Assay (LLNA), the globally required in vivo assay for this endpoint, is reviewed alongside consideration of appropriate SAFs. Using a case study, the principle that the NESIL in μg/cm² can be derived by multiplying LLNA EC3% figure by a factor of 250 is adopted. The NESIL is then reduced by an overall SAF of 25 to establish an exposure level below which there is minimal bystander and resident risk. Whilst this paper focuses on European risk assessment and management, the approach is generic and universally applicable.

Skin sensitization is a term used to refer to the regulatory hazard known as allergic contact dermatitis (ACD) in humans or contact hypersensitivity in rodents, an important health endpoint considered in chemical hazard and risk assessments. Information on skin sensitization potential is required in various regulatory frameworks, such as the Directive of the European Parliament and the Council on Registration, Evaluation and Authorization of Chemicals (REACH). The identification of skin-sensitizing chemicals previously required the use of animal testing, which is now being replaced by alternative methods. Alternative methods in the field of skin sensitization are based on the measurement or prediction of key events (KE), i.e., (i) the molecular triggering event, i.e., the covalent binding of electrophilic substances to nucleophilic centers in skin proteins; (ii) the activation of keratinocytes; (iii) the activation of dendritic cells; (iv) the proliferation of T cells. This review article focuses on the current state of knowledge regarding the methods corresponding to each of the key events in skin sensitization and considers the latest trends in the development and modification of these methods.

Our aim is to develop and apply next generation approaches to skin allergy risk assessment that do not require new animal test data and better quantify uncertainties. Quantitative risk assessment for skin sensitisation uses safety assessment factors to extrapolate from the point-of-departure to an acceptable human exposure level. It is currently unclear whether these safety assessment factors are appropriate when using non-animal test data to derive a point-of departure. Our skin allergy risk assessment model Defined Approach uses Bayesian statistics to infer a human-relevant metric of sensitiser potency with explicit quantification of uncertainty, using any combination of human repeat insult patch test, local lymph node assay, direct peptide reactivity assay, KeratinoSens™, h-CLAT or U-SENS™ data. Here we describe the incorporation of benchmark exposures pertaining to use of consumer products with clinical data supporting a high/low risk categorisation for skin sensitisation. Margins-of-exposure (potency estimate to consumer exposure level ratio) are regressed against the benchmark risk classifications, enabling derivation of a risk metric defined as the probability that an exposure is low risk. This approach circumvents the use of safety assessment factors and provides a simple and transparent mechanism whereby clinical experience can directly feed-back into risk assessment decisions.

Skin sensitization following the covalent modification of proteins by low molecular weight chemicals (haptenation) is mediated by cytotoxic T lymphocyte (CTL) recognition of human leukocyte antigen (HLA) molecules presented on the surface of almost all nucleated cells. There exist 3 nonmutually exclusive hypotheses for how haptens mediate CTL recognition: direct stimulation by haptenated peptides, hapten modification of HLA leading to an altered HLA-peptide repertoire, or a hapten altered proteome leading to an altered HLA-peptide repertoire. To shed light on the mechanism underpinning skin sensitization, we set out to utilize proteomic analysis of keratinocyte presented antigens following exposure to 2,4-dinitrochlorobenzene (DNCB). We show that the following DNCB exposure, cultured keratinocytes present cysteine haptenated (dinitrophenylated) peptides in multiple HLA molecules. In addition, we find that one of the DNCB modified peptides derives from the active site of cytosolic glutathione-S transferase-ω. These results support the current view that a key mechanism of skin sensitization is stimulation of CTLs by haptenated peptides. Data are available via ProteomeXchange with identifier PXD021373.

Dermal contact is the main route of exposure for most cosmetics; however, inhalation exposure could be significant for some formulations (e.g., aerosols, powders). Current cosmetic regulations do not require specific tests addressing respiratory irritation and sensitisation, and despite the prohibition of animal testing for cosmetics, no alternative methods have been validated to assess these endpoints to date. Inhalation hazard is mainly determined based on existing human and animal evidence, read-across, and extrapolation of data from different target organs or tissues, such as the skin. However, because of mechanistic differences, effects on the skin cannot predict effects on the respiratory tract, which indicates a substantial need for the development of new approach methodologies addressing respiratory endpoints for inhalable chemicals in general. Cosmetics might present a particularly significant need for risk assessments of inhalation exposure to provide a more accurate toxicological evaluation and ensure consumer safety. This review describes the differences in the mechanisms of irritation and sensitisation between the skin and the respiratory tract, the progress that has already been made, and what still needs to be done to fill the gap in the inhalation risk assessment of cosmetic ingredients.

In 2008, a proposal for assessing the risk of induction of skin sensitization to fragrance materials Quantitative Risk Assessment 1 (QRA1) was published. This was implemented for setting maximum limits for fragrance materials in consumer products. However, there was no formal validation or empirical verification after implementation. Additionally, concerns remained that QRA1 did not incorporate aggregate exposure from multiple product use and included assumptions, e.g. safety assessment factors (SAFs), that had not been critically reviewed. Accordingly, a review was undertaken, including detailed re-evaluation of each SAF together with development of an approach for estimating aggregate exposure of the skin to a potential fragrance allergen. This revision of QRA1, termed QRA2, provides an improved method for establishing safe levels for sensitizing fragrance materials in multiple products to limit the risk of induction of contact allergy. The use of alternative non-animal methods is not within the scope of this paper. Ultimately, only longitudinal clinical studies can verify the utility of QRA2 as a tool for the prevention of contact allergy to fragrance materials.

Historically skin sensitisation risk assessment for cosmetic ingredients was based on animal models, however regulatory demands have led to Next Generation Risk Assessment (NGRA), using data from New Approach Methodologies (NAM) and Defined Approaches (DA). This case study was meant to investigate if the use of resorcinol at 0.2% in a face cream was safe and a maximum use concentration could be defined. The NAM data and DA predictions could not provide sufficient confidence to determine a point of departure (POD). Therefore, the application of read-across was explored to increase the level of confidence. Analogue searches in various tools and databases using "mode of action" and "chemical structural features" retrieved 535 analogues. After refinement by excluding analogues without a defined structure, similar reactivity profile and skin sensitisation data, 39 analogues remained. A final selection was made based on three approaches: expert judgment, chemical similarity or Local Lymph Node Assay data (LLNA). All read-across approaches supported a moderate potency. A POD derived from the LLNA EC3 of 3.6% was determined leading to a favourable NGRA conclusion and a maximum use concentration of 0.36%. This was supported by a traditional risk assessment based on the available animal data for resorcinol.

Recently, several non-animal approaches contributing to the identification of skin sensitisation hazard have been introduced. Their validation and acceptance has largely been directed towards regulatory classification. Considering the driving force for replacement of in vivo tests centred on cosmetics, it is reasonable to ask how well the new approaches perform in this respect. In the present study, 219 substances, largely cosmetic raw materials (including dyes, preservatives and fragrances), have been evaluated in our Defined Approach integrating a stacking meta model (version 5), incorporating the individual outcomes of 3 in vitro validated methods (Direct Peptide Reactivity Assay, Keratinosens™, U-SENS™), 2 in silico tools (TIMES SS, TOXTREE) and physicochemical parameters (volatility, pH). Stacking meta model outcomes were compared with existing local lymph node assay (LLNA) data. Non-sensitisers comprised 68/219; 86 were weak/moderate and 65 were stronger sensitisers. The model version revision demonstrate the gain to discriminate sensitizers to non-sensitiser when the in silico TIMES model is incorporated as input parameter. The 85% to 91% accuracy for the cosmetics categories, indicates the stacking meta model offers value for the next generation risk assessment framework. These results pinpoint the power of the stacking meta model relying on a confidence based on the probability given in any individual prediction.

At the ESCD congress held in Manchester in 2016, a session was organized to encourage more dialogue between clinicians with expertise in skin sensitization and toxicologists seeking to provide effective risk assessment to prevent human health issues. That session focused on the remaining uncertainties regarding the induction and regulation of skin sensitization in humans, and the opportunities and challenges associated with the refinement and improvement of risk assessment methodologies. This short article, prompted by those discussions, debates what the authors regard as being among the most important and most intriguing uncertainties about skin sensitization and allergic contact dermatitis in humans, and the most significant opportunities for improving risk assessment. The aim has been to provide a basis for mapping out the areas that might benefit from a closer alignment between the relevant clinical community and toxicologists charged with the responsibility of ensuring that skin sensitization risks are understood and managed.