Development of an in vitro skin sensitization test using human cell lines: the human Cell Line Activation Test (h-CLAT). I. Optimization of the h-CLAT protocol.

The aim of this study is to optimize the experimental conditions for an in vitro skin sensitization test using the human cell lines THP-1 and U-937. As regards pre-culturing time, the expression of CD86 on DNCB-treated THP-1 cells tended to be higher after 48h and 72h pre-culture compared with other time points evaluated. Next, we investigated the effect of chemical treatment time, and found that induction of CD86 expression on THP-1 cells by DNCB reached a plateau after 24h. Augmentation of CD86 expression is often observed when cells are treated with a subtoxic dose of allergens. To determine the appropriate dose of test samples, the cytotoxicity of test samples to THP-1 and U-937 cells was assessed with MTT assay, and the 50% inhibitory concentration (IC50) of each test sample was calculated. Based on the cytotoxicity assay data, four concentrations in the range between toxic and non-toxic were selected (0.1x, 0.5x, 1x and 2x IC50). Several kinds of antibodies were tested for staining THP-1 and U-937 cells treated with allergens/non-allergens (e.g., DNCB, Ni/SLS), and suitable antibodies for staining CD86 and CD54 were selected. We confirmed that the working dilutions of the selected CD86 and CD54 antibodies were appropriate for use in our method. The effect of an FcR blocking procedure was also evaluated. The mean fluorescence intensity (MFI value) was decreased by the FcR blocking procedure, which indicated that non-specific staining was blocked. Therefore, this procedure should be included in the method. Based on our findings, the protocol for this assay was optimized and the experimental conditions to be used in a future validation study were identified. We propose to call this kind of in vitro skin sensitization test h-CLAT, which is short for human Cell Line Activation Test.     

Development of an in vitro skin sensitization test using human cell lines; human Cell Line Activation Test (h-CLAT). II. An inter-laboratory study of the h-CLAT.

Recent regulatory changes have placed a major emphasis on in vitro safety testing and alternative models. In regard to skin sensitization tests, dendritic cells (DCs) derived from human peripheral blood have been considered in the development of new in vitro alternatives. Human cell lines have been also reported recently. In our previous study, we suggested that measuring CD86 and/or CD54 expression on THP-1 cells (human monocytic leukemia cell line) could be used as an in vitro skin sensitization method. An inter-laboratory study among two laboratories was undertaken in Japan in order to further develop an in vitro skin sensitization model. In the present study, we used two human cell lines: THP-1 and U-937 (human histiocytic lymphoma cell line). First we optimized our test protocol (refer to the related paper entitled "optimization of the h-CLAT protocol" within this journal) and then we did an inter-laboratory validation with nine chemicals using the optimized protocol. We measured the expression of CD86 and CD54 on the above cells using flow cytometry after a 24h and 48h exposure to six known allergens (e.g., DNCB, pPD, NiSO(4)) and three non-allergens (e.g., SLS, tween 80). For the sample test concentration, four doses (0.1x, 0.5x, 1x, and 2x of the 50% inhibitory concentration (IC(50))) were evaluated. IC(50) was calculated using MTT assay. We found that allergens/non-allergens were better predicted using THP-1 cells compared to U-937 cells following a 24 h and a 48 h exposure. We also found that the 24h treatment time tended to have a better accuracy than the 48 h treatment time for THP-1 cells. Expression of CD86 and CD54 were good predictive markers for THP-1 cells, but for U-937 cells, expression of CD86 was a better predictor than CD54, at the 24h and the 48 h treatment time. The accuracy also improved when both markers (CD86 and CD54) were used as compared with a single marker for THP-1 cells. Both laboratories gave a good prediction of allergen/non-allergen, especially using THP-1 cells. These results suggest that our method, human Cell Line Activation Test (h-CLAT), using human cell lines THP-1 and U-937, but especially THP-1 cells at 24h treatment, may be a useful in vitro skin sensitization model to predict various contact allergens.     

Predicting skin sensitization potential and inter-laboratory reproducibility of a human Cell Line Activation Test (h-CLAT) in the European Cosmetics Association (COLIPA) ring trials

Regulatory policies in Europe prohibited the testing of cosmetic ingredients in animals for a number of toxicological endpoints. Currently no validated non-animal test methods exist for skin sensitization. Evaluation of changes in cell surface marker expression in dendritic cell (DC)-surrogate cell lines represents one non-animal approach. The human Cell Line Activation Test (h-CLAT) examines the level of CD86 and CD54 expression on the surface of THP-1 cells, a human monocytic leukemia cell line, following 24 h of chemical exposure. To examine protocol transferability, between-lab reproducibility, and predictive capacity, the h-CLAT has been evaluated by five independent laboratories in several ring trials (RTs) coordinated by the European Cosmetics Association (COLIPA). The results of the first and second RTs demonstrated that the protocol was transferable and basically had good between-lab reproducibility and predictivity, but there were some false negative data. To improve performance, protocol and prediction model were modified. Using the modified prediction model in the first and second RT, accuracy was improved. However, about 15% of the outcomes were not correctly identified, which exposes some of the limitations of the assay. For the chemicals evaluated, the limitation may due to chemical being a weak allergen or having low solubility (ex. α-hexylcinnamaldehyde). The third RT evaluated the modified prediction model and satisfactory results were obtained. From the RT data, the feasibility of utilizing cell lines as surrogate DC in development of in vitro skin sensitization methods shows promise. The data also support initiating formal pre-validation of the h-CLAT in order to fully understand the capabilities and limitations of the assay.     

Prediction of skin sensitization potency using machine learning approaches

The replacement of animal use in testing for regulatory classification of skin sensitizers is a priority for US federal agencies that use data from such testing. Machine learning models that classify substances as sensitizers or non‐sensitizers without using animal data have been developed and evaluated. Because some regulatory agencies require that sensitizers be further classified into potency categories, we developed statistical models to predict skin sensitization potency for murine local lymph node assay (LLNA) and human outcomes. Input variables for our models included six physicochemical properties and data from three non‐animal test methods: direct peptide reactivity assay; human cell line activation test; and KeratinoSens™ assay. Models were built to predict three potency categories using four machine learning approaches and were validated using external test sets and leave‐one‐out cross‐validation. A one‐tiered strategy modeled all three categories of response together while a two‐tiered strategy modeled sensitizer/non‐sensitizer responses and then classified the sensitizers as strong or weak sensitizers. The two‐tiered model using the support vector machine with all assay and physicochemical data inputs provided the best performance, yielding accuracy of 88% for prediction of LLNA outcomes (120 substances) and 81% for prediction of human test outcomes (87 substances). The best one‐tiered model predicted LLNA outcomes with 78% accuracy and human outcomes with 75% accuracy. By comparison, the LLNA predicts human potency categories with 69% accuracy (60 of 87 substances correctly categorized). These results suggest that computational models using non‐animal methods may provide valuable information for assessing skin sensitization potency. Copyright © 2017 John Wiley & Sons, Ltd.     

Novel approach for classifying chemicals according to skin sensitizing potency by non-radioisotopic modification of the local lymph node assay

The murine local lymph node assay (LLNA) is currently recognized as a stand-alone sensitization test for determining the sensitizing potential of chemicals, and it has the advantage of yielding a quantitative endpoint that can be used to predict the sensitization potency of chemicals. The EC3 has been proposed as a parameter for classifying chemicals according to the sensitization potency. We previously developed a non-radioisotopic endpoint for the LLNA based on 5-bromo-2'-deoxyuridine (BrdU) incorporation (non-RI LLNA), and we are proposing a new procedure to predict the sensitization potency of chemicals based on comparisons with known human contact allergens. Nine chemicals (i.e. diphencyclopropenone, p-phenylenediamine, glutaraldehyde, cinnamicaldehyde, citral, eugenol, isopropyl myristate, propyleneglycol and hexane) categorized as human contact allergen classes 1-5 were tested by the non-RI LLNA with the following reference allergens: 2,4-dinitrochlorobenzene (DNCB) as a class 1 human contact allergen, isoeugenol as a class 2 human contact allergen and alpha-hexylcinnamic aldehyde (HCA) as a class 3 human contact allergen. Consequently, nine test chemicals were almost assigned to their correct allergen class. The results suggested that the new procedure for non-RI LLNA can provide correct sensitization potency data. Sensitization potency data are useful for evaluating the sensitization risk to humans of exposure to new chemical products. Accordingly, this approach would be an effective modification of LLNA with regard to its experimental design. Moreover, this procedure can be applied also to the standard LLNA with radioisotopes and to other modifications of the LLNA.     

Method for detecting the reactivity of chemicals towards peptides as an alternative test method for assessing skin sensitization potential

Cosmetics are normally composed of various ingredients. Some cosmetic ingredients can act as chemical haptens reacting toward proteins or peptides of human skin and they can provoke an immunologic reaction, called as skin sensitization. This haptenation process is very important step of inducing skin sensitization and evaluating the sensitizing potentials of cosmetic ingredients is very important for consumer safety. Therefore, animal alternative methods focusing on monitoring haptenation potential are undergoing vigorous research. To examine the further usefulness of spectrophotometric methods to monitor reactivity of chemicals toward peptides for cosmetic ingredients. Forty chemicals (25 sensitizers and 15 non-sensitizers) were reacted with 2 synthetic peptides, e.g., the cysteine peptides (Ac-RFAACAA-COOH) with free thiol group and the lysine peptides (Ac-RFAAKAA-COOH) with free amine group. Unreacted peptides can be detected after incubating with 5,5′-dithiobis-2-nitrobenzoic acid or fluorescamine™ as detection reagents for free thiol and amine group, respectively. Chemicals were categorized as sensitizers when they induced more than 10% depletion of cysteine peptides or more than 30% depletion of lysine peptides. The sensitivity, specificity, and accuracy were 80.0%, 86.7% and 82.5%, respectively. These results demonstrate that spectrophotometric methods can be an easy, fast, and high-throughput screening tools predicting the skin sensitization potential of chemical including cosmetic ingredient.     

Non-animal methods to predict skin sensitization (II): an assessment of defined approaches**

Skin sensitization is a toxicity endpoint of widespread concern, for which the mechanistic understanding and concurrent necessity for non-animal testing approaches have evolved to a critical juncture, with many available options for predicting sensitization without using animals. Cosmetics Europe and the National Toxicology Program Interagency Center for the Evaluation of Alternative Toxicological Methods collaborated to analyze the performance of multiple non-animal data integration approaches for the skin sensitization safety assessment of cosmetics ingredients. The Cosmetics Europe Skin Tolerance Task Force (STTF) collected and generated data on 128 substances in multiple in vitro and in chemico skin sensitization assays selected based on a systematic assessment by the STTF. These assays, together with certain in silico predictions, are key components of various non-animal testing strategies that have been submitted to the Organization for Economic Cooperation and Development as case studies for skin sensitization. Curated murine local lymph node assay (LLNA) and human skin sensitization data were used to evaluate the performance of six defined approaches, comprising eight non-animal testing strategies, for both hazard and potency characterization. Defined approaches examined included consensus methods, artificial neural networks, support vector machine models, Bayesian networks, and decision trees, most of which were reproduced using open source software tools. Multiple non-animal testing strategies incorporating in vitro, in chemico, and in silico inputs demonstrated equivalent or superior performance to the LLNA when compared to both animal and human data for skin sensitization.     

Development of quantitative structure-activity relationship (QSAR) models to predict the carcinogenic potency of chemicals I. Alternative toxicity measures as an estimator of carcinogenic potency

Determining the carcinogenicity and carcinogenic potency of new chemicals is both a labor-intensive and time-consuming process. In order to expedite the screening process, there is a need to identify alternative toxicity measures that may be used as surrogates for carcinogenic potency. Alternative toxicity measures for carcinogenic potency currently being used in the literature include lethal dose (dose that kills 50% of a study population [LD(50)]), lowest-observed-adverse-effect-level (LOAEL) and maximum tolerated dose (MTD). The purpose of this study was to investigate the correlation between tumor dose (TD(50)) and three alternative toxicity measures as an estimator of carcinogenic potency. A second aim of this study was to develop a Classification and Regression Tree (CART) between TD(50) and estimated/experimental predictor variables to predict the carcinogenic potency of new chemicals. Rat TD(50)s of 590 structurally diverse chemicals were obtained from the Cancer Potency Database, and the three alternative toxicity measures considered in this study were estimated using TOPKAT, a toxicity estimation software. Though poor correlations were obtained between carcinogenic potency and the three alternative toxicity (both experimental and TOPKAT) measures for the CPDB chemicals, a CART developed using experimental data with no missing values as predictor variables provided reasonable estimates of TD(50) for nine chemicals that were part of an external validation set. However, if experimental values for the three alternative measures, mutagenicity and logP are not available in the literature, then either the CART developed using missing experimental values or estimated values may be used for making a prediction.     

Analysis of the Local Lymph Node Assay (LLNA) variability for assessing the prediction of skin sensitisation potential and potency of chemicals with non-animal approaches

The knowledge of the biological mechanisms leading to the induction of skin sensitisation has favoured in recent years the development of alternative non-animal methods. During the formal validation process, results from the Local Lymph Node Assay (LLNA) are generally used as reference data to assess the predictive capacity of the non-animal tests. This study reports an analysis of the variability of the LLNA for a set of chemicals for which multiple studies are available and considers three hazard classification schemes: POS/NEG, GHS/CLP and ECETOC. As the type of vehicle used in a LLNA study is known to influence to some extent the results, two analyses were performed: considering the solvent used to test the chemicals and without considering the solvent. The results show that the number of discordant classifications increases when a chemical is tested in more than one solvent. Moreover, it can be concluded that study results leading to classification in the strongest classes (1A and EXT) seem to be more reliable than those in the weakest classes. This study highlights the importance of considering the variability of the reference data when evaluating non-animal tests.     

体外人源细胞激活试验法应用于4种染发剂的皮肤变态反应研究

目的 研究体外人源细胞激活试验h-CLAT法应用在染发剂的皮肤变态反应检测中的可行性,通过和现行化妆品标准中体内皮肤变态反应法的比较,评价体外人源细胞激活试验应用在染发剂皮肤变态反应试验上的优劣。方法 参考OECD指导原则442E的体外人源细胞系激活试验方法,应用流式细胞仪技术在类树突样的人源白血病细胞系THP-1上对4种染发剂进行体外皮肤变态反应检查,并与体内皮肤变态反应法的检测结果相比较。结果 体外h-CLAT试验表明,4种染发剂中#2,#3和#4染发剂的CD54RFI都 < 200且CD86RFI < 150,即体外试验结果为阴性与体内皮肤变态反应试验比较,结果一致;而#1染发剂为体外结果阳性,且有效致敏浓度为315 μg·mL^-1,与体内变态反应阴性结果不同。结论 与体内皮肤变态反应试验相比,体外人源细胞系激活试验在对染发剂的检验上有周期短、测试方法简便、评价结果客观等优点。缺点是无法反映整个过敏反应的过程,存在假阳性的可能。     

Test battery with the human cell line activation test,direct peptide reactivity assay and DEREK based on a 139 chemical data set for predicting skin sensitizing potential and potency of chemicals

To develop a testing strategy incorporating the human cell line activation test (h‐CLAT), direct peptide reactivity assay (DPRA) and DEREK, we created an expanded data set of 139 chemicals (102 sensitizers and 37 non‐sensitizers) by combining the existing data set of 101 chemicals through the collaborative projects of Japan Cosmetic Industry Association. Of the additional 38 chemicals, 15 chemicals with relatively low water solubility (log Kow > 3.5) were selected to clarify the limitation of testing strategies regarding the lipophilic chemicals. Predictivities of the h‐CLAT, DPRA and DEREK, and the combinations thereof were evaluated by comparison to results of the local lymph node assay. When evaluating 139 chemicals using combinations of three methods based on integrated testing strategy (ITS) concept (ITS‐based test battery) and a sequential testing strategy (STS) weighing the predictive performance of the h‐CLAT and DPRA, overall similar predictivities were found as before on the 101 chemical data set. An analysis of false negative chemicals suggested a major limitation of our strategies was the testing of low water‐soluble chemicals. When excluded the negative results for chemicals with log Kow > 3.5, the sensitivity and accuracy of ITS improved to 97% (91 of 94 chemicals) and 89% (114 of 128). Likewise, the sensitivity and accuracy of STS to 98% (92 of 94) and 85% (111 of 129). Moreover, the ITS and STS also showed good correlation with local lymph node assay on three potency classifications, yielding accuracies of 74% (ITS) and 73% (STS). Thus, the inclusion of log Kow in analysis could give both strategies a higher predictive performance. Copyright © 2015 John Wiley & Sons, Ltd.     

Activation of human nuclear receptors by perfluoroalkylated substances (PFAS)

Perfluoralkylated substances (PFAS) such as perfluorooctanoic acid (PFOA) or perfluorooctanesulfonic acid (PFOS) are used to produce, e.g., surface coatings with water- and dirt-repellent properties. These substances have been shown to be hepatotoxic in rodents, and the mechanism of action is mostly attributed to the PFAS-mediated activation of the peroxisome proliferator-activated receptor alpha (PPARα). In the present study, we investigated by using luciferase-based reporter gene assays whether PFOA, PFOS and six alternative PFAS can activate, in addition to PPARα, eight other human nuclear receptors. All tested PFAS except for perfluorobutanesulfonic acid (PFBS) were able to activate human PPARα. Perfluoro-2-methyl-3-oxahexanoic acid (PMOH) and 3H-perfluoro-3-[(3-methoxypropoxy) propanoic acid] (PMPP) were weak agonists of human PPARγ. The other human nuclear receptors (PPARδ, CAR, PXR, FXR, LXRα, RXRα and RARα) were not affected by any PFAS tested in this study. Although PMOH was more effective than PFOA in stimulating PPARα in the transactivation assay, it was less effective in stimulating PPARα-dependent target gene expression in human HepG2 hepatocarcinoma cells. Notably, any effect observed in this in vitro study only occurred at concentrations higher than 10 μM of the respective PFAS which is in all cases several magnitudes above the average blood concentration in the Western population. Thus, the results suggest that nuclear receptor activation may only play a minor role in potential PFAS-mediated adverse effects in humans.     

Pathways and Kinetics of Deslorelin Degradation in an Airway Epithelial Cell Line (Calu-1)

Purpose. The objective of this study is to investigate the pathways and kinetics of degradation of deslorelin, pGlu¹-His²-Trp³-Ser⁴-Tyr⁵-D-Trp⁶-Leu⁷-Arg⁸-ProNHEt⁹ (Des1-9), in a human airway epithelial cell line (Calu-1). Methods. The degradation of deslorelin in membrane and cytosolic fractions of Calu-1 cells was studied at 37°C up to 24 h. The degradation products were separated using HPLC and identified by amino acid analysis, sequencing, and mass spectrometry. The rate constants for deslorelin degradation and the formation of degradation products were determined by fitting the concentration vs. time data to pharmacokinetic models using WinNonlin^TM. The effect of enzyme inhibitors, captopril, phosphoramidon, and disodium EDTA on deslorelin degradation was also assessed. Results. Des1-3, Des4-9, and Des5-9 were the deslorelin fragments detected in the membrane fraction. Apart from these degradation products, Des5-7 was also detected in the cytosolic fraction. The deslorelin degradation was 8.5 times faster in the cytosolic fraction compared to the membrane fraction. The disappearance of deslorelin and the kinetics of degradation products could be explained by simple 2 compartment iv bolus model and 1 compartment absorption model, respectively. EDTA and captopril decreased deslorelin degradation in the membrane and cytosolic fractions. Conclusions. Deslorelin is initially cleaved at the 3-4 bond in the membrane and cytosolic fractions, possibly by a metalloendopeptidase and/or angiotensin converting enzyme, with the degradation being more rapid in the cytosol.     

A review of the Buehler guinea pig skin sensitization test and its use in a risk assessment process for human skin sensitization

The Buehler test is a valuable procedure for screening the sensitization potential of chemicals prior to human exposure. Our experience of over 20 years has shown it to be effective in detecting strong, moderate, and most weak sensitizers. The topical exposure inherent in the Buehler test allows it to be utilized to investigate dose responses, cross reactivity between structurally related chemicals, and the sensitization potential of contaminants in raw material mixtures. For safety assessment purposes, Buehler test results provide an initial indication of the sensitization potential of the material in question under relevant, but exaggerated, exposure conditions. These results can be compared to results on benchmark chemicals to assess sensitization risk for subsequent human exposure. Optimizing the sensitivity of the Buehler test requires adherence to the published methodology and proper interpretation of the challenge and rechallenge data obtained. Adjuvant-type test methods are generally considered to be more sensitive than topical methods. However, when done properly, topical test procedures such as the Buehler test or the open epicutaneous test can accurately detect most chemicals with any realistic potential for sensitizing humans by the topical route. Moreover, from a risk assessment perspective, these topical tests avoid the problems of overestimating the weak sensitization potential of many topically applied materials or underestimating the sensitization potential of very strong sensitizers; both are potential concerns with invasive adjuvant-type test methods. The Buehler test or other topical test methods are particularly valuable for comparative sensitization risk assessment since human sensitization data on benchmark materials are all derived from topical exposure. The risk assessment is developed by comparing the guinea pig data on the new material versus relevant benchmark chemicals or formulations and also by evaluating the existing human sensitization data on the benchmark material. These data are then utilized to predict human sensitization risk from topical exposure to the new material. Confirmation of human safety can be derived from human repeat insult patch testing (HRIPT) and other clinical tests such as the product use test and the diagnostic patch test. Utilized in this manner, the Buehler test is an integral component of an overall skin sensitization safety assessment program for a new chemical or product formulation.     

Assessment of immunotoxicity induced by chemicals in human precision-cut lung slices (PCLS)

Occupational asthma can be induced by a number of chemicals at the workplace. Risk assessment of potential sensitizers is mostly performed in animal experiments. With increasing public demand for alternative methods, human precision-cut lung slices (PCLS) have been developed as an ex vivo model.Human PCLS were exposed to increasing concentrations of 20 industrial chemicals including 4 respiratory allergens, 11 contact allergens, and 5 non-sensitizing irritants. Local respiratory irritation was characterized and expressed as 75% (EC₂₅) and 50% (EC₅₀) cell viability with respect to controls. Dose–response curves of all chemicals except for phenol were generated. Local respiratory inflammation was quantified by measuring the production of cytokines and chemokines. TNF-α and IL-1α were increased significantly in human PCLS after exposure to the respiratory sensitizers trimellitic anhydride (TMA) and ammonium hexachloroplatinate (HClPt) at subtoxic concentrations, while contact sensitizers and non-sensitizing irritants failed to induce the release of these cytokines to the same extent. Interestingly, significant increases in T_H1/T_H2 cytokines could be detected only after exposure to HClPt at a subtoxic concentration.In conclusion, allergen-induced cytokines were observed but not considered as biomarkers for the differentiation between respiratory and contact sensitizers. Our preliminary results show an ex vivo model which might be used for prediction of chemical-induced toxicity, but is due to its complex three-dimensional structure not applicable for a simple screening of functional and behavior changes of certain cell populations such as dendritic cells and T-cells in response to allergens.     

Activation of human neutrophils by titanium dioxide (TiO2) nanoparticles

This paper describes the in vitro effects of titanium dioxide (TiO₂) nanoparticles (NPs) upon human neutrophils. Kinetic experiments revealed no cell necrosis after 24 h of treatment with TiO₂ (0–100 μg/ml). In contrast, TiO₂-induced change in cellular morphology in a concentration-dependent manner in neutrophils over time, indicating its potential to activate these cells. To further support this, we demonstrated that TiO₂ markedly and rapidly induced tyrosine phosphorylation events, including phosphorylation of two key enzymes, p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinases-1/2 (Erk-1/2). We also determined the effects of TiO₂ on two neutrophil functions requiring a longer exposure period between NPs and cells: apoptosis and cytokine production. Interestingly, at concentrations ⩾20 μg/ml, TiO₂ inhibited neutrophil apoptosis in a concentration-dependent manner after 24 h of treatment. Supernatants from TiO₂-induced neutrophils were harvested after 24 h and tested for the presence of 36 different analytes (cytokines, chemokines) using an antibody array assay. TiO₂ treatment increased production of 13 (36%) analytes, including IL-8, which exhibited the greatest increase (∼16 × control cell levels). The increased production of IL-8 was confirmed by ELISA. We conclude that TiO₂ exerts important neutrophil agonistic properties in vitro.