Human Cell Line Activation Test Research Articles

Predicting points of departure and potency categories for fragrance ingredients by integrating OECD in vitro models

Continuous potency assessment is crucial for conducting quantitative risk assessment (QRA) of sensitizers. Quantitative regression models, based on in vitro methods, have been developed to calculate points of departure for use in skin sensitization QRA. These models calculate a point of departure as a predicted value for Local Lymph Node Assay (LLNA) EC3 or potency value (PV), integrating data from the kinetic Direct Peptide Reactivity Assay (kDPRA), KeratinoSens (KS) assay, and human Cell Line Activation Test (h-CLAT). The goal of this study was to determine how in vitro predicted EC3s and PVs compare to published reference data. In vitro data were combined in point of departure regression models to predict EC3s and PVs. These points of departure were then grouped into sensitization potency categories, such as extreme, strong, moderate, weak, very weak, or non-sensitizer, as previously described. Trends in potency distribution and high concordance between predicted EC3 and PV categories and published potency categories were observed. Furthermore, the median absolute fold-misprediction ranged between 1.8 and 2.5 for models predicting EC3 and between 1.7 and 3.4 for those predicting PVs. These regression models are a promising animal alternative for determining sensitization quantitative potency for fragrance ingredients, thereby facilitating QRA.

Evaluation of the immunotoxicity potential of nanomaterials using THP-1 cells.

With the expansion of nanomaterials (NMs) usage, concerns about their toxicity are increasing, and the wide variety of NMs makes it difficult to assess their toxicity. Therefore, the development of a high-throughput, accurate, and certified method to evaluate the immunotoxicity of NMs is required. In this study, we assessed the immunotoxicity potential of various NMs, such as nanoparticles of silver, silica, and titanium dioxide, using the human Cell Line Activation Test (h-CLAT) at the cellular level. After exposure to silver nanoparticle dispersions, the expression levels of CD86 and CD54 increased, suggesting the activation of antigen-presenting cells (APCs) by silver nanoparticles. Quantification of silver ions eluted from silver nanoparticles and the activation of APCs by silver ions suggested that it was due to the release of silver ions. Silica nanoparticles also increased the expression of CD86 and/or CD54, and their activation ability correlated with the synthesis methods and hydrodynamic diameters. The ability of titanium dioxide to activate APCs differed depending on the crystal type and hydrodynamic diameter. These results suggest a potential method to evaluate the immunotoxicity potential of various NMs based on their ability to activate APCs using human monocytic THP-1 cells. This method will be valuable in assessing the immunotoxicity potential and elucidating the immunotoxic mechanisms of NMs.

Proof of concept testing of a positive reference material for in vivo and in vitro sensitization testing of medical devices.

In vivo skin sensitization tests are required to evaluate the biological safety of medical devices in contact with living organisms to provide safe medical care to patients. Negative and positive reference materials have been developed for biological tests of cytotoxicity, implantation, hemolysis, and in vitro skin irritation. However, skin sensitization tests are lacking. In this study, polyurethane sheets containing 1 wt/wt % 2,4-dinitrochlorobenzene (DNCB-PU) were developed and evaluated as a positive reference material for skin sensitization tests. DNCB-PU sheet extracts prepared with sesame oil elicited positive sensitization responses for in vivo sensitization potential in the guinea pig maximization test and the local lymph node assay. Furthermore, DNCB-PU sheet extracts prepared with water and acetonitrile, 10% fetal bovine serum-containing medium, or sesame oil elicited positive sensitization responses as alternatives to animal testing based on the amino acid derivative reactivity assay, human cell line activation test, and epidermal sensitization assay, respectively. These data suggest that the DNCB-PU sheet is an effective extractable positive reference material for in vivo and in vitro skin sensitization testing in medical devices. The formulation of this reference material will lead to the development of safer medical devices that contribute to patient safety.

In Vitro and In Vivo Methods for Analysis of Nanoparticles' Potential toInduce Delayed-Type Hypersensitivity Reactions.

Delayed-type hypersensitivity (DTH) reactions are among the common reasons for drug withdrawal from clinical use during the post-marketing stage. Several in vivo methods have been developed to test DTH responses in animal models. They include the local lymph node assay (LLNA) and local lymph node proliferation assay (LLNP). While LLNA is instrumental in testing topically administered formulations (e.g., creams), the LLNP was proven to be predictive of drug-mediated DTH in response to small molecule pharmaceuticals. Global efforts in reducing the use of research animals lead to the development of in vitro models to predict test-materials' mediated DTH. Two such models include theanalysis of surface marker expression in human cell lines THP-1 and U-937. These tests are known as the human cell line activation test (hCLAT) and myeloid U937 skin sensitization test (MUSST or U-SENS), respectively. Here we describe experimental procedures for all these methods, discuss their in vitro-in vivo correlation, and suggest a strategy for applying these tests to analyze engineered nanomaterials and nanotechnology-formulated drug products.

Screening Allergenic Potencies of Skin-Contact Products Using the Human-Derived THP-1 Cell Activation Test.

With the prevalence of allergic contact dermatitis (ACD) from the usage of skin-contact products, like wearable, skin care, and hair care products, screening their skin sensitizing potential is necessary, for the sake of alleviating the consequent public health impact. In the present study, a total of 77 skin-contact products classified by four categories, watch bands (WBs), skin care products (SCPs), hair care products (HCPs), and rubber gloves (RGs), were investigated, using an optimized in vitro assay of human cell line activation test (h-CLAT). Extracting the products using neutral artificial sweat simulated well the practical usage scenarios, and testing the extracts showed that 26 of them were allergy test positive, including nine WBs, six SCPs, two HCPs, and nine RGs. The allergenic response was mainly characterized by the induction of CD54 expression, and diverse paradigms of CD54 and CD86 levels were observed by analyzing dose-response curves, which could also be influenced by the compromised viability of the THP-1 cells. The data implicated the intricate regulation by different contributors to suspicious ingredients in the test samples. Altogether, a promising methodology for testing skin allergy potential was well established for commonly used commodities by neutral artificial sweat extraction coupled with h-CLAT screening. The findings would be of great help in tracing the potential allergens in practical products and improving their qualities.

Potency classification of isothiazolinone compounds based on defined approaches of skin sensitization in OECD GL 497.

Regulatory decisions for skin sensitization are now based on adverse outcome pathway (AOP) and integrated approaches to testing and assessment (IATA). Based on these, Organisation for Economic Co-operation and Development (OECD) guidelines on defined approaches for skin sensitization were adopted with a fixed data interpretation procedure (DIP). In the guidelines, "Defined Approaches" (DA) on skin sensitization uses the results from multiple information sources of in chemico, in vitro, and in silico data to achieve an equivalent predictive capacity as those of the animal tests. In this review, we evaluated the skin sensitization of eleven isothiazolinone compounds including 4,5-Dichloro-2-octyl-3(2H)-isothiazolone (DCOIT), 2-n-Octyl-4-isothiazolin-3-one (OIT), 2-Methyl-4-isothiazolin-3-one (MIT), 1,2-Benzisothiazolin-3-one (BIT), 1,2-Benzisothiazolin-3-one, 2-butyl (BBIT), 5-Chloro-2-methyl-4-isothiazolin-3-one (CMIT), 2-methyl-4,5-trimethylene-4-isothiazolin-3-one (MTMIT), 2-methyl-1,2-benzothiazol-3-one (MBIT), 2-methyl-1,2-benzothiazole-3-thione (MBIT-S), 1,2-benzisothiazolin-3-one, 2-methyl-, 1,1-dioxide (BBIT-O), and a mixture of CMIT/MIT. Data from direct peptide reactivity assay (DPRA), human cell line activation (h-CLAT) test, and quantitative structure activity relationship (QSAR) Toolbox were evaluated and were applied to the DIP to derive a prediction of hazard identification and a potency classification. Among the evaluated chemicals, six isothiazolinone compounds were classified to be UN GHS 1A, one compound to be UN GHS 1, and four compounds could not be classified due to lack of data. The results of sensitizer chemicals were found to coincide well with those of in vivo test.

Reconstructed human epidermis-based testing strategy of skin sensitization potential and potency classification using epidermal sensitization assay and in silico data.

The hazards and potency of skin sensitizers are traditionally determined using animal tests such as the local lymph node assay (LLNA); however, significant progress has been made in the development of non-animal test methods addressing the first three mechanistic key events of adverse outcome pathway in skin sensitization. We developed the epidermal sensitization assay (EpiSensA), which is a reconstructed human epidermis-based assay, by measuring four genes related to critical keratinocyte responses during skin sensitization. Four in vitro skin sensitization test methods (EpiSensA, direct peptide reactivity assay [DPRA], KeratinoSens™, and human cell line activation test [h-CLAT]) were systematically evaluated using 136 chemicals including lipophilic chemicals and pre/pro-haptens, which may be related to assay-specific limitations. The constructed database included existing and newly generated data. The EpiSensA showed a broader applicability domain and predicted the hazards with 82.4% and 78.8% accuracy than LLNA and human data. The EpiSensA could detect 76 out of 88 sensitizers at lower concentrations than the LLNA, indicating that the EpiSensA has higher sensitivity for the detection of minor sensitizing constituents. These results confirmed the potential use of the EpiSensA in evaluating a mixture of unknown compositions that can be evaluated by animal tests. To combine different information sources, the reconstructed human epidermis-based testing strategy (RTS) was developed based on weighted multiple information from the EpiSensA and TImes MEtabolism Simulator platform for predicting Skin Sensitization (TIMES-SS; RTSv1) or Organization for Economic Cooperation and Development (OECD) QSAR Toolbox automated workflow (RTSv2). The predictivities of the hazards and Globally Harmonized System (GHS) subcategories were equal to or better than the defined approaches (2 out of 3, integrated testing strategy [ITS]v1, and ITSv2) adopted as OECD Guideline 497.

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.

Application of Defined Approaches to Assess Skin Sensitization Potency of Isothiazolinone Compounds.

Isothiazolinones (ITs) are widely used as antimicrobial preservatives in cosmetics and as additives for preservation of consumer and industrial products to control bacteria, fungi, and algae. Although they are effective biocides, they have the potential to produce skin irritation and sensitization, which poses a human health hazard. In this project, we evaluated nonanimal defined approaches (DAs) for skin sensitization that can provide point-of-departure estimates for use in quantitative risk assessment for ITs. The skin sensitization potential of six ITs was evaluated using three internationally harmonized nonanimal test methods: the direct peptide reactivity assay, KeratinoSens™, and the human cell line activation test. Results from these test methods were then applied to two versions of the Shiseido Artificial Neural Network DA. Sensitization hazard or potency predictions were compared with those of the in vivo murine local lymph node assay (LLNA). The nonanimal methods produced skin sensitization hazard and potency classifications concordant with those of the LLNA. EC3 values (the estimated concentration needed to produce a stimulation index of three, the threshold positive response) generated by the DAs had less variability than LLNA EC3 values, and confidence limits from the DAs overlapped those of the LLNA EC3 for most substances. The application of in silico models to in chemico and in vitro skin sensitization data is a promising data integration procedure for DAs to support hazard and potency classification and quantitative risk assessment.

Upregulation of CD86 and IL-12 by rhododendrol in THP-1 cells cocultured with melanocytes through ROS and ATP

BackgroundThe tyrosinase inhibitor rhododendrol (RD), used as a skin whitening agent, reportedly has the potential to induce leukoderma. ObjectiveAlthough an immune response toward melanocytes was demonstrated to be involved in leukoderma, the molecular mechanism is not fully understood. MethodsWe hypothesized that if RD is a pro-hapten and tyrosinase-oxidized RD metabolites are melanocyte-specific sensitizers, the sensitizing process could be reproduced by the human cell line activation test (h-CLAT) cocultured with melanocytes (h-CLATw/M) composed of human DC THP-1 cells and melanoma SK-MEL-37 cells. Cell surface expression, ROS generation and ATP release, mRNA expression, and the effects of several inhibitors were examined. ResultsWhen RD was added to the h-CLATw/M, the expression of cell-surface CD86 and IL-12 mRNA was greatly enhanced in THP-1 cells compared with those in the h-CLAT. The rapid death of melanoma cells was induced, with ROS generation and ATP release subsequently being greatly enhanced, resulting in the cooperative upregulation of CD86 and IL-12. Consistent with those observations, an ROS inhibitor, ATP receptor P2X7 antagonist, or PERK inhibitor antagonized the upregulation. CD86 upregulation was similarly observed with another leukoderma-inducible tyrosinase inhibitor, raspberry ketone, but not with the leukoderma noninducible skin-whitening agents ascorbic acid and tranexamic acid. ConclusionRD is a pro-hapten sensitizer dependent on tyrosinase that induces ROS generation and ATP release from melanocytes for CD86 and IL-12 upregulation in DCs, possibly leading to the generation of tyrosinase-specific cytotoxic T lymphocytes. The coculture system h-CLATw/M may be useful for predicting the sensitizing potential to induce leukoderma.

In-Vitro, In-Vivo, Molecular Docking and ADMET Studies of 2-Substituted 3,7-Dihydroxy-4H-chromen-4-one for Oxidative Stress, Inflammation and Alzheimer's Disease.

Plants' bioactives are well-known safe drugs for vital diseases. Flavones and Flavonoid-rich dietary supplements are known to exhibit neuroprotective potential. In this study, we isolated a flavone 2-(3,4-dimethoxyphenyl)-3,7-dihydroxy-4H-chromen-4-one from Notholirion thomsonianum and it was evaluated against various targets of the oxidative stress-related neurological disorders. The compound showed excellent acetyl and butyrylcholinesterase inhibitions in its profile, giving IC50 values of 1.37 and 0.95 μM, respectively. Similarly, in in-vitro MAO-B assay, our flavone exhibited an IC50 value of 0.14 μM in comparison to the standard safinamide (IC50 0.025 μM). In in-vitro anti-inflammatory assay, our isolated compound exhibited IC50 values of 7.09, 0.38 and 0.84 μM against COX-1, COX-2 and 5-LOX, respectively. The COX-2 selectivity (SI) of the compound was 18.70. The compound was found safe in animals and was very effective in carrageenan-induced inflammation. Due to the polar groups in the structure, a very excellent antioxidant profile was observed in both in-vitro and in-vivo models. The compound was docked into the target proteins of the respective activities and the binding energies confirmed the potency of our compound. Furthermore, absorption, distribution, metabolism, excretion, and toxicity (ADMET) results showed that the isolated flavone has a good GIT absorption ability and comes with no hepatic and cardiotoxicity. In addition, the skin sensitization test, in-vitro human cell line activation test (h-CLAT) and KeratinoSens have revealed that isolated flavone is not skin sensitive with a confidence score of 59.6% and 91.6%. Herein, we have isolated a natural flavone with an effective profile against Alzheimer's, inflammation and oxidative stress. The exploration of this natural flavone will provide a baseline for future research in the field of drug development.

Sensitization properties of acetophenone azine, a new skin sensitizer identified in textile.

Acetophenone azine (CAS no. 729-43-1) present in sports equipment (shoes, socks and shin pads) has been suspected to induce skin allergies. Twelve case reports of allergy in children and adults from Europe and North America were published between 2016 and 2021. The objective of this study was to confirm that acetophenone azine is indeed a skin sensitizer based on in vitro/ in vivo testings derived from the Adverse Outcome Pathway (AOP) built for skin sensitization by OECD in 2012. Acetophenone azine was tested in vitro according to the human cell line activation test (h-CLAT) and the ARE-Nrf2 Luciferase Test (KeratinoSens) and in vivo using the Local Lymph Nodes Assay (LLNA). Both the h-CLAT and the KeratinoSens were positive whereas the LLNA performed at 5, 2.5 and 1% (wt/vol) of acetophenone azine, was negative. Based on these results, acetophenone azine was considered as a skin sensitizer. This was recently confirmed by its classification under the CLP regulation.

Tumor necrosis factor-alpha, prostaglandin-E2 and interleukin-1β targeted anti-arthritic potential of fluvoxamine: drug repurposing.

Fluvoxamine, a selective serotonin re uptake inhibitor, is used to treat depression. The aim of present study was to evaluate fluvoxamine in acute (egg albumin-induced inflammation) and chronic inflammatory rat models (formaldehyde and complete Freund's adjuvant (CFA)-induced arthritis). Fluvoxamine showed highly significant (p<0.001) protective effect at dose of 50 mg/kg orally with percentage suppression 21.3% as compared to disease control group in acute model. Likewise, formaldehyde-induced arthritic experiment confirmed the significant (p<0.001) anti-arthritic behavior, showed by fluvoxamine (50 mg/kg orally) throughout the study. Moreover, In CFA-induced model, the higher dose (fluvoxamine 50 mg/kg) exhibited highly significant (p<0.001) decrease in paw thickness and arthritic score with significant increase in weight of animals from 123.8± 1.934 g to 130.2± 1.655 g, significantly decreased the level of RF and CRP to level of 12.0±0.707 and 11.40±0.50 respectively and restoration of SOD, CAT (69.8±1.5, 72.0±1.4 respectively). Furthermore, the level of TNF-α, PGE2, and IL-1β (147.0±2.0, 406.8±2.5, and 93.8±1.3 respectively) in arthritic animals was reduced to significant (p<0.001) level (53.8±1.3, 205±3.6, and 42.0±1.4 respectively) after treatment with fluvoxamine. Furthermore, molecular docking of fluvoxamine against TNF-α, PGE2, and IL-1β protein targets showed good binding energies which hereby from computational studies proves our compound anti-inflammatory potential. In addition, absorption, distribution, metabolism, excretion, and toxicity (ADMET) studies reveled that fluvoxamine has very good pharmacokinetic profile with no specific hepatic toxicity and good absorption level. In addition, the skin sensitization test in vitro human cell line activation test (h-CLAT) and KeratinoSens have revealed that isolated flavone is not skin sensitive with confidence score of 59.6% and 91.6%. The current findings validated the anti-arthritic potential of fluvoxamine but it should be recommended for clinical investigation in future research.

A development of a graph-based ensemble machine learning model for skin sensitization hazard and potency assessment.

Many defined approaches (DAs) for skin sensitization assessment based on the adverse outcome pathway (AOP) have been developed to replace animal testing because the European Union has banned animal testing for cosmetic ingredients. Several DAs have demonstrated that machine learning models are beneficial. In this study, we have developed an ensemble prediction model utilizing the graph convolutional network (GCN) and machine learning approach to assess skin sensitization. The model integrates in silico parameters and data from alternatives to animal testing of well-defined AOP to improve DA predictivity. Multiple ensemble models were created using the probability produced by the GCN with six physicochemical properties, direct peptide reactivity assay, KeratinoSens™, and human cell line activation test (h-CLAT), using a multilayer perceptron approach. Models were evaluated by predicting the testing set's human hazard class and three potency classes (strong, weak, and non-sensitizer). When the GCN feature was used, 11 models out of 16 candidates showed the same or improved accuracy in the testing set. The ensemble model with the feature set of GCN, KeratinoSens™, and h-CLAT produced the best results with an accuracy of 88% for assessing human hazards. The best three-class potency model was created with the feature set of GCN and all three assays, resulting in 64% accuracy. These results from the ensemble approach indicate that the addition of the GCN feature could provide an improved predictivity of skin sensitization hazard and potency assessment.

Co-Culture of THP-1 Cells and Normal Human Epidermal Keratinocytes (NHEK) for Modified Human Cell Line Activation Test (h-CLAT)

To improve the accuracy of skin sensitization prediction of chemicals by conventional alternative methods using cells, it is important to reproduce the environment of skin in vitro, such as the crosstalk between keratinocytes and dendritic cells (DCs). We developed a skin sensitization test system based on the markers and criteria of the human cell line activation test (h-CLAT), which combines THP-1 cells as a surrogate for DCs and keratinized normal human epidermal keratinocytes (NHEK). After exposure to chemicals via keratinized NHEK, the cell surface expression of CD54 and CD86 on THP-1 was measured by flow cytometry. This co-culture system evaluated 2,4-dinitrochlorobenzene (DNCB), a typical sensitizer, as positive, lactic acid (LA), a non-sensitizer, as negative, and isoeugenol (IE), a prohapten that requires biological activation to acquire skin sensitization, as positive. However, the expression levels of CD54 and CD86 in DNCB-treated THP-1 were lower than those in normal h-CLAT. Therefore, we investigated the effects of the medium and secretion by NHEK cells on THP-1 cells. CD54 and CD86 expression was enhanced in monocultured THP-1 in the medium for keratinized NHEK and in the conditioned medium of keratinized NHEK. The increase in CD54 and CD86 by changes in the medium type was higher than that by the NHEK secretion; therefore, it was found that the medium composition has a large effect on the evaluation index among the experimental parameters in the co-culture system. It is necessary to find the optimal medium for immunotoxicity assessment in the co-culture system.

Application of Defined Approaches for Skin Sensitization to Agrochemical Products

Skin sensitization testing is a regulatory requirement for safety evaluations of pesticides in multiple countries. Globally harmonized test guidelines that include in chemico and in vitro methods reduce animal use, but no single assay is recommended as a complete replacement for animal tests. Defined approaches (DAs) that integrate data from multiple non-animal methods are accepted; however, the methods that comprise them have been evaluated using monoconstituent substances rather than mixtures or formulations. To address this data gap, we tested 27 agrochemical formulations in the direct peptide reactivity assay (DPRA), the KeratinoSens™ assay, and the human cell line activation test (h-CLAT). These data were used as inputs to evaluate three DAs for hazard classification of skin sensitization potential and two DAs for potency categorization. When compared to historical animal results, balanced accuracy for the DAs for predicting in vivo skin sensitization hazard (i.e., sensitizer vs. nonsensitizer) ranged from 56 to 78%. The best performing DA was the “2 out of 3 (2o3)” DA, in which the hazard classification was based on two concordant results from the DPRA, KeratinoSens, or h-CLAT. The KE 3/1 sequential testing strategy (STS), which uses h-CLAT and DPRA results, and the integrated testing strategy (ITSv2), which uses h-CLAT, DPRA, and an in silico hazard prediction from OECD QSAR Toolbox, had balanced accuracies of 56–57% for hazard classification. Of the individual test methods, KeratinoSens had the best performance for predicting in vivo hazard outcomes. Its balanced accuracy of 81% was similar to that of the 2o3 DA (78%). For predicting potency categories defined by the United Nations Globally Harmonized System of Classification and Labelling of Chemicals (GHS), the correct classification rate of the STS was 52% and that of the ITSv2 was 43%. These results demonstrate that non-animal test methods have utility for evaluating the skin sensitization potential of agrochemical formulations as compared to animal reference data. While additional data generation is needed, testing strategies such as DAs anchored to human biology and mechanistic information provide a promising approach for agrochemical formulation testing.

Expansion of the Cosmetics Europe skin sensitisation database with new substances and PPRA data

The assessment of skin sensitisation is a key requirement in all regulated sectors, with the European Union's regulation of cosmetic ingredients being most challenging, since it requires quantitative skin sensitisation assessment based on new approach methodologies (NAMs).To address this challenge, an in-depth and harmonised understanding of NAMs is fundamental to inform the assessment. Therefore, we compiled a database of NAMs, and in vivo (human and local lymph node assay) reference data. Here, we expanded this database with 41 substances highly relevant for cosmetic industry. These structurally different substances were tested in six NAMs (Direct Peptide Reactivity Assay, KeratinoSens™, human Cell Line Activation Test, U-SENS™, SENS-IS, Peroxidase Peptide Reactivity Assay).Our analysis revealed that the substances could be tested without technical limitations, but were generally overpredicted when compared to reference results. Reasons for this reduced predictivity were explored through pairwise NAM comparisons and association of overprediction with hydrophobicity.We conclude that more detailed understanding of how NAMs apply to a wider range of substances is needed. This would support a flexible and informed choice of NAMs to be optimally applied in the context of a next generation risk assessment framework, ultimately contributing to the characterisation and reduction of uncertainty.

In vitro assessment of skin sensitization, irritability and toxicity of bacteriocins and reuterin for possible topical applications

Bacteriocins and reuterin are promising antimicrobials for application in food, veterinary, and medical sectors. In the light of their high potential for application in hand sanitizer, we investigated the skin toxicity of reuterin, microcin J25, pediocin PA-1, bactofencin A, and nisin Z in vitro using neutral red and LDH release assays on NHEK cells. We determined their skin sensitization potential using the human cell line activation test (h-CLAT). Their skin irritation potential was measured on human epidermal model EpiDerm™. We showed that the viability and membrane integrity of NHEK cells remained unaltered after exposure to bacteriocins and reuterin at concentrations up to 400 µg/mL and 80 mg/mL, respectively. Furthermore, microcin J25 and reuterin showed no skin sensitization at concentrations up to 100 µg/mL and 40 mg/mL, respectively, while pediocin PA-1, bactofencin A, and nisin Z caused sensitization at concentrations higher than 100 µg/mL. Tissue viability was unaffected in presence of bacteriocins and reuterin at concentrations up to 200 µg/mL and 40 mg/mL, respectively, which was confirmed by measuring cytokine IL-1α and IL-8 levels and by histological analysis. In conclusion, the current study provides scientific evidence that some bacteriocins and reuterin, could be safely applied topically as sanitizers at recommended concentrations.

Integrated skin sensitization assessment based on OECD methods (I): Deriving a point of departure for risk assessment_suppl2

Three guidelines covering key events in the skin sensitization adverse outcome pathway are endorsed by the Organisation for Economic Co-operation and Development (OECD). A recent guideline covers defined approaches (DA) to combine data from these tests for regulatory (sub)classification. The guideline methods provide continuous data that could characterize the sensitization potency on a more granular scale beyond (sub)classifications. We assembled a comprehensive database of in vitro and in vivo results from OECD guideline tests. Building on a previous approach using regression models, we provide quantitative models using input data from the kinetic direct peptide reactivity assay (kDPRA), the KeratinoSens™ (KS) assay, and the human cell line activation test (h-CLAT) to calculate a point of departure (PoD) in the form of a predicted local lymph node assay (LLNA) EC3 value for use in risk assessment. Predictive models include results from either two or all three assays. Detailed analysis versus in vivo data estimates redundancy between different tests and helps guide model selection. All models were tested on a set of case studies selected for their availability of multiple LLNA reference data in the OECD database. The predicted PoDs were within or close to the range of the variation of the historical LLNA data for most of these cases studies, and overall, the models predicted the in vivo value with a median fold-misprediction factor of around 2.5. The robustness of the models was characterized by comparing a comprehensive historical database versus the curated dataset provided by the OECD working group on DA.

Integrated skin sensitization assessment based on OECD methods (I): Deriving a point of departure for risk assessment_suppl4

Three guidelines covering key events in the skin sensitization adverse outcome pathway are endorsed by the Organisation for Economic Co-operation and Development (OECD). A recent guideline covers defined approaches (DA) to combine data from these tests for regulatory (sub)classification. The guideline methods provide continuous data that could characterize the sensitization potency on a more granular scale beyond (sub)classifications. We assembled a comprehensive database of in vitro and in vivo results from OECD guideline tests. Building on a previous approach using regression models, we provide quantitative models using input data from the kinetic direct peptide reactivity assay (kDPRA), the KeratinoSens™ (KS) assay, and the human cell line activation test (h-CLAT) to calculate a point of departure (PoD) in the form of a predicted local lymph node assay (LLNA) EC3 value for use in risk assessment. Predictive models include results from either two or all three assays. Detailed analysis versus in vivo data estimates redundancy between different tests and helps guide model selection. All models were tested on a set of case studies selected for their availability of multiple LLNA reference data in the OECD database. The predicted PoDs were within or close to the range of the variation of the historical LLNA data for most of these cases studies, and overall, the models predicted the in vivo value with a median fold-misprediction factor of around 2.5. The robustness of the models was characterized by comparing a comprehensive historical database versus the curated dataset provided by the OECD working group on DA.