RESUMEN
The Epidermal Sensitization Assay (EpiSensA) is a reconstructed human epidermis (RhE)-based gene expression assay for predicting the skin sensitization potential of chemicals. Since the RhE model is covered by a stratified stratum corneum, various kinds of test chemicals, including lipophilic ones and pre-/pro-haptens, can be tested with a route of exposure akin to an in vivo assay and human exposure. This article presents the results of a formally managed validation study of the EpiSensA that was carried out by three participating laboratories. The purpose of this validation study was to assess transferability of the EpiSensA to new laboratories along with its within- (WLR) and between-laboratory reproducibility (BLR). The validation study was organized into two independent stages. As demonstrated during the first stage, where three sensitizers and one non-sensitizer were correctly predicted by all participating laboratories, the EpiSensA was successfully transferred to all three participating laboratories. For Phase I of the second stage, each participating laboratory performed three experiments with an identical set of 15 coded test chemicals resulting in WLR of 93.3%, 93.3%, and 86.7%, respectively. Furthermore, when the results from the 15 test chemicals were combined with those of the additional 12 chemicals tested in Phase II of the second stage, the BLR for 27 test chemicals was 88.9%. Moreover, the predictive capacity among the three laboratories showed 92.6% sensitivity, 63.0% specificity, 82.7% accuracy, and 77.8% balanced accuracy based on murine local lymph node assay (LLNA) results. Overall, this validation study concluded that EpiSensA is easily transferable and sufficiently robust for assessing the skin sensitization potential of chemicals.
Asunto(s)
Alérgenos , Dermatitis Alérgica por Contacto , Humanos , Animales , Ratones , Reproducibilidad de los Resultados , Alérgenos/toxicidad , Epidermis , Piel , Haptenos/toxicidad , Ensayo del Nódulo Linfático Local , Alternativas a las Pruebas en AnimalesRESUMEN
The aim of this study is to validate an in vitro skin irritation test (SIT) using three-dimensional reconstructed human epidermal (RhE) skin equivalents prepared by layer-by-layer (LbL) method (LbL-3D Skin) in a series of interlaboratory studies. The goal of these validation studies is to evaluate the ability of this in vitro test to reliably discriminate skin irritant from nonirritant chemicals, as defined by OECD and UN GHS. This me-too validation study is to assess the within- and between-laboratory reproducibility, as well as the predictive capacity, of the LbL-3D Skin SIT in accordance with performance standards for OECD TG 439. The developed skin model, LbL-3D Skin had a highly differentiated epidermis and dermis, similar to the validated reference methods (VRM) and native human skin. The quality parameters (cell survival in controls, tissue integrity, and barrier function) were similar to VRM and in accordance with OECD TG 439. The LbL-3D Skin SIT validation study was performed by three participating laboratories and consisted of three independent tests using 20 reference chemicals. The results obtained with the LbL-3D Skin demonstrated high within-laboratory and between-laboratory reproducibility, as well as high accuracy for use as a stand-alone assay to distinguish skin irritants from nonirritants. The predictive potency of LbL-3D Skin SIT using total 54 test chemicals were comparable to those in other RhE models in OECD TG 439. The validation study demonstrated that LbL-3D Skin has proven to be a robust and reliable method for predicting skin irritation.
Asunto(s)
Irritantes , Pruebas de Irritación de la Piel , Humanos , Animales , Reproducibilidad de los Resultados , Pruebas de Irritación de la Piel/métodos , Irritantes/toxicidad , Piel , Epidermis , Técnicas In Vitro , Alternativas a las Pruebas en AnimalesRESUMEN
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.
Asunto(s)
Alérgenos , Interleucina-33 , Humanos , Técnicas de Cocultivo , Piel , Epitelio , CitocinasRESUMEN
The estimated concentrations for a stimulation index of 3 (EC3) in murine local lymph node assay (LLNA) is an important quantitative value for determining the strength of skin sensitization to chemicals, including cosmetic ingredients. However, animal testing bans on cosmetics in Europe necessitate the development of alternative testing methods to LLNA. A machine learning-based prediction method can predict complex toxicity risks from multiple variables. Therefore, we developed an LLNA EC3 regression model using CatBoost, a new gradient boosting decision tree, based on the reliable Cosmetics Europe database which included data for 119 substances. We found that a model using in chemico/in vitro tests, physical properties, and chemical information associated with key events of skin sensitization adverse outcome pathway as variables showed the best performance with a coefficient of determination (R2) of 0.75. In addition, this model can indicate the variable importance as the interpretation of the model, and the most important variable was associated with the human cell line activation test that evaluate dendritic cell activation. The good performance and interpretability of our LLNA EC3 predictable regression model suggests that it could serve as a useful approach for quantitative assessment of skin sensitization.
Asunto(s)
Dermatitis Alérgica por Contacto/diagnóstico , Ensayo del Nódulo Linfático Local , Aprendizaje Automático , Alternativas a las Pruebas en Animales , Animales , Línea Celular , Bases de Datos Factuales , Células Dendríticas/efectos de los fármacos , Europa (Continente) , Humanos , Queratinocitos/efectos de los fármacos , Ratones , Linfocitos T/efectos de los fármacos , Naciones Unidas/normasRESUMEN
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.
Asunto(s)
Alternativas a las Pruebas en Animales/métodos , Biología Computacional/métodos , Simulación por Computador , Cosméticos/efectos adversos , Dermatitis Alérgica por Contacto/inmunología , Piel/inmunología , Animales , Cosméticos/farmacología , Dermatitis Alérgica por Contacto/etiología , Humanos , Ratones , Piel/efectos de los fármacosRESUMEN
Cosmetics Europe, the European Trade Association for the cosmetics and personal care industry, is conducting a multi-phase program to develop regulatory accepted, animal-free testing strategies enabling the cosmetics industry to conduct safety assessments. Based on a systematic evaluation of test methods for skin sensitization, five non-animal test methods (DPRA (Direct Peptide Reactivity Assay), KeratinoSensTM, h-CLAT (human cell line activation test), U-SENSTM, SENS-IS) were selected for inclusion in a comprehensive database of 128 substances. Existing data were compiled and completed with newly generated data, the latter amounting to one-third of all data. The database was complemented with human and local lymph node assay (LLNA) reference data, physicochemical properties and use categories, and thoroughly curated. Focused on the availability of human data, the substance selection resulted nevertheless resulted in a high diversity of chemistries in terms of physico-chemical property ranges and use categories. Predictivities of skin sensitization potential and potency, where applicable, were calculated for the LLNA as compared to human data and for the individual test methods compared to both human and LLNA reference data. In addition, various aspects of applicability of the test methods were analyzed. Due to its high level of curation, comprehensiveness, and completeness, we propose our database as a point of reference for the evaluation and development of testing strategies, as done for example in the associated work of Kleinstreuer et al. We encourage the community to use it to meet the challenge of conducting skin sensitization safety assessment without generating new animal data.
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Cosméticos/efectos adversos , Bases de Datos Factuales , Dermatitis Alérgica por Contacto/inmunología , Piel/inmunología , Alternativas a las Pruebas en Animales/métodos , Cosméticos/farmacología , Dermatitis Alérgica por Contacto/etiología , Humanos , Piel/efectos de los fármacosRESUMEN
It is important to predict the potential of cosmetic ingredients to cause skin sensitization, and in accordance with the European Union cosmetic directive for the replacement of animal tests, several in vitro tests based on the adverse outcome pathway have been developed for hazard identification, such as the direct peptide reactivity assay, KeratinoSens™ and the human cell line activation test. Here, we describe the development of an artificial neural network (ANN) prediction model for skin sensitization risk assessment based on the integrated testing strategy concept, using direct peptide reactivity assay, KeratinoSens™, human cell line activation test and an in silico or structure alert parameter. We first investigated the relationship between published murine local lymph node assay EC3 values, which represent skin sensitization potency, and in vitro test results using a panel of about 134 chemicals for which all the required data were available. Predictions based on ANN analysis using combinations of parameters from all three in vitro tests showed a good correlation with local lymph node assay EC3 values. However, when the ANN model was applied to a testing set of 28 chemicals that had not been included in the training set, predicted EC3s were overestimated for some chemicals. Incorporation of an additional in silico or structure alert descriptor (obtained with TIMES-M or Toxtree software) in the ANN model improved the results. Our findings suggest that the ANN model based on the integrated testing strategy concept could be useful for evaluating the skin sensitization potential.
Asunto(s)
Dermatitis Alérgica por Contacto/etiología , Redes Neurales de la Computación , Pruebas de Irritación de la Piel/métodos , Animales , Línea Celular , Simulación por Computador , Humanos , Ganglios Linfáticos/efectos de los fármacos , Ratones , Medición de Riesgo , Piel/citología , Piel/efectos de los fármacosRESUMEN
Although photoallergens require UV energy for antigen formation, the subsequent immune response is considered to be the same as in ordinary skin sensitization. Therefore, in vitro tests for skin sensitization should also be applicable for photoallergy testing. In this study, we examined whether activation of the Keap1 (Kelch-like ECH-associated protein 1)-Nrf2 (nuclear factor-erythroid 2-related factor 2)-ARE (antioxidant response element) pathway could be used to assess the photoallergenic potential of chemicals, using the reporter cell line AREc32 or KeratinoSens(TM) . First, we identified an appropriate UVA irradiation dose [5 J cm(-2) irradiation in phosphate-buffered saline (PBS)] by investigating the effect of UV irradiation on ARE-dependent gene induction using untreated or 6-methylcoumarin (6-MC)-treated cells. Irradiation of well-known photoallergens under this condition increased ARE-dependent gene expression by more than 50% compared with both vehicle and non-irradiated controls. When the cut-off value for detecting photoallergens was set at 50% induction, the accuracy of predicting photoallergenic/phototoxic chemicals was 70% in AREc32 cells and 67% in KeratinoSens(TM) cells, and the specificity was 100% in each case. We designate these assays as a photo-ARE assay and photo-KeratinoSens(TM) , respectively. Our results suggest that activation of the Keap1-Nrf2-ARE pathway is an effective biomarker for evaluating both photoallergenic and phototoxic potentials. Either of the above tests might be a useful component of a battery of in vitro tests/in silico methods for predicting the photoallergenicity and phototoxicity of chemicals. Copyright © 2015 John Wiley & Sons, Ltd.
Asunto(s)
Alérgenos/toxicidad , Elementos de Respuesta Antioxidante , Dermatitis Fototóxica/metabolismo , Proteína 1 Asociada A ECH Tipo Kelch/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , Rayos Ultravioleta/efectos adversos , Línea Celular Tumoral , Cumarinas/toxicidad , Relación Dosis-Respuesta en la Radiación , Regulación de la Expresión Génica , Marcadores Genéticos , Humanos , Proteína 1 Asociada A ECH Tipo Kelch/genética , Factor 2 Relacionado con NF-E2/genética , Sensibilidad y Especificidad , Transducción de SeñalRESUMEN
The presented Bayesian network Integrated Testing Strategy (ITS-3) for skin sensitization potency assessment is a decision support system for a risk assessor that provides quantitative weight of evidence, leading to a mechanistically interpretable potency hypothesis, and formulates adaptive testing strategy for a chemical. The system was constructed with an aim to improve precision and accuracy for predicting LLNA potency beyond ITS-2 (Jaworska et al., J Appl Toxicol 33(11):1353-1364, 2013) by improving representation of chemistry and biology. Among novel elements are corrections for bioavailability both in vivo and in vitro as well as consideration of the individual assays' applicability domains in the prediction process. In ITS-3 structure, three validated alternative assays, DPRA, KeratinoSens and h-CLAT, represent first three key events of the adverse outcome pathway for skin sensitization. The skin sensitization potency prediction is provided as a probability distribution over four potency classes. The probability distribution is converted to Bayes factors to: 1) remove prediction bias introduced by the training set potency distribution and 2) express uncertainty in a quantitative manner, allowing transparent and consistent criteria to accept a prediction. The novel ITS-3 database includes 207 chemicals with a full set of in vivo and in vitro data. The accuracy for predicting LLNA outcomes on the external test set (n = 60) was as follows: hazard (two classes)-100 %, GHS potency classification (three classes)-96 %, potency (four classes)-89 %. This work demonstrates that skin sensitization potency prediction based on data from three key events, and often less, is possible, reliable over broad chemical classes and ready for practical applications.
Asunto(s)
Teorema de Bayes , Técnicas de Apoyo para la Decisión , Dermatitis Alérgica por Contacto/etiología , Pruebas Cutáneas/métodos , Alternativas a las Pruebas en Animales/métodos , Animales , Sesgo , Bases de Datos de Compuestos Químicos/estadística & datos numéricos , Humanos , Ensayo del Nódulo Linfático Local , Reproducibilidad de los Resultados , Medición de Riesgo/métodosRESUMEN
In the two years since the last workshop report, the environment surrounding the prediction of skin sensitisation hazards has experienced major change. Validated non-animal tests are now OECD Test Guidelines. Accordingly, the recent cross sector workshop focused on how to use in vitro data for regulatory decision-making. After a review of general approaches and six case studies, there was broad consensus that a simple, transparent stepwise process involving non-animal methods was an opportunity waiting to be seized. There was also strong feeling the approach should not be so rigidly defined that assay variations/additional tests are locked out. Neither should it preclude more complex integrated approaches being used for other purposes, e.g. potency estimation. All agreed the ultimate goal is a high level of protection of human health. Thus, experience in the population will be the final arbiter of whether toxicological predictions are fit for purpose. Central to this is the reflection that none of the existing animal assays is perfect; the non-animal methods should not be expected to be so either, but by integrated use of methods and all other relevant information, including clinical feedback, we have the opportunity to continue to improve toxicology whilst avoiding animal use.
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Alternativas a las Pruebas en Animales/métodos , Cosméticos/toxicidad , Educación/métodos , Informe de Investigación , Piel/efectos de los fármacos , Alternativas a las Pruebas en Animales/tendencias , Animales , Cosméticos/administración & dosificación , Cosméticos/farmacocinética , Dermatitis Alérgica por Contacto/metabolismo , Dermatitis Alérgica por Contacto/patología , Educación/tendencias , Europa (Continente) , Finlandia , Humanos , Informe de Investigación/tendencias , Medición de Riesgo/métodos , Medición de Riesgo/tendencias , Piel/metabolismo , Piel/patologíaRESUMEN
Sensitization, the prerequisite event in the development of allergic contact dermatitis, is a key parameter in both hazard and risk assessments. The pathways involved have recently been formally described in the OECD adverse outcome pathway (AOP) for skin sensitization. One single non-animal test method will not be sufficient to fully address this AOP and in many cases the use of a battery of tests will be necessary. A number of methods are now fully developed and validated. In order to facilitate acceptance of these methods by both the regulatory and scientific communities, results of the single test methods (DPRA, KeratinoSens, LuSens, h-CLAT, (m)MUSST) as well for a the simple '2 out of 3' ITS for 213 substances have been compiled and qualitatively compared to both animal and human data. The dataset was also used to define different mechanistic domains by probable protein-binding mechanisms. In general, the non-animal test methods exhibited good predictivities when compared to local lymph node assay (LLNA) data and even better predictivities when compared to human data. The '2 out of 3' prediction model achieved accuracies of 90% or 79% when compared to human or LLNA data, respectively and thereby even slightly exceeded that of the LLNA.
Asunto(s)
Alternativas a las Pruebas en Animales/métodos , Bases de Datos Factuales , Fármacos Dermatológicos/toxicidad , Piel/efectos de los fármacos , Animales , Línea Celular , Humanos , Ratones , Piel/patología , Células U937RESUMEN
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.
Asunto(s)
Alternativas a las Pruebas en Animales/métodos , Cosméticos/toxicidad , Piel/efectos de los fármacos , Línea Celular , Cosméticos/química , Determinación de Punto Final , Humanos , Ensayo del Nódulo Linfático Local , Péptidos/química , Valor Predictivo de las Pruebas , Medición de Riesgo , Sensibilidad y Especificidad , Pruebas Cutáneas , Solubilidad , Pruebas de Toxicidad , Agua/químicaRESUMEN
The skin sensitization potential of chemicals has been determined with the use of the murine local lymph node assay (LLNA). However, in recent years public concern about animal welfare has led to a requirement for non-animal risk assessment systems for the prediction of skin sensitization potential, to replace LLNA. Selection of an appropriate in vitro test or in silico model descriptors is critical to obtain good predictive performance. Here, we investigated the utility of artificial neural network (ANN) prediction models using various combinations of descriptors from several in vitro sensitization tests. The dataset, collected from published data and from experiments carried out in collaboration with the Japan Cosmetic Industry Association (JCIA), consisted of values from the human cell line activation test (h-CLAT), direct peptide reactivity assay (DPRA), SH test and antioxidant response element (ARE) assay for chemicals whose LLNA thresholds have been reported. After confirming the relationship between individual in vitro test descriptors and the LLNA threshold (e.g. EC3 value), we used the subsets of chemicals for which the requisite test values were available to evaluate the predictive performance of ANN models using combinations of h-CLAT/DPRA (N = 139 chemicals), the DPRA/ARE assay (N = 69), the SH test/ARE assay (N = 73), the h-CLAT/DPRA/ARE assay (N = 69) and the h-CLAT/SH test/ARE assay (N = 73). The h-CLAT/DPRA, h-CLAT/DPRA/ARE assay and h-CLAT/SH test/ARE assay combinations showed a better predictive performance than the DPRA/ARE assay and the SH test/ARE assay. Our data indicates that the descriptors evaluated in this study were all useful for predicting human skin sensitization potential, although combinations containing h-CLAT (reflecting dendritic cell-activating ability) were most effective for ANN-based prediction.
Asunto(s)
Alternativas a las Pruebas en Animales/métodos , Redes Neurales de la Computación , Pruebas Cutáneas/métodos , Piel/metabolismo , Línea Celular , Células Dendríticas/metabolismo , Humanos , Ensayo del Nódulo Linfático Local , Medición de Riesgo , Sensibilidad y EspecificidadRESUMEN
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.
RESUMEN
We have ongoing projects that are developing New Approach Methods (NAMs) for systematic toxicology. One NAM is to develop the immunotoxicity evaluation with non-animal test methods for the Organisation for Economic Co-operation and Development (OECD)Test Guideline (TG). The development of this evaluation includes the following steps: 1) adverse outcome pathway (AOP), 2) detailed review paper, 3) test methods based on AOP, 4) validation study of test methods for developing TGs, and 5) integrated approaches to testing and assessment (IATA). I believe that the NAMs developed on these steps may enable risk assessment of a chemical with non-animal test methods in near future.
Asunto(s)
Organización para la Cooperación y el Desarrollo Económico , Medición de RiesgoRESUMEN
BACKGROUND: Recent changes in regulatory restrictions and social opposition to animal toxicology experiments have driven the need for reliable in vitro tests for predicting the skin sensitizing potentials of a wide variety of industrial chemicals. Previously, we developed the human cell line activation test (h-CLAT) as a cell-based assay to predict the skin sensitizing potential of chemicals, and showed the correspondence between the h-CLAT and the murine local lymph node assay results. OBJECTIVES: This study was conducted to investigate the predictive performance of the h-CLAT for human skin sensitizing potential. MATERIALS/METHODS: We selected a total of 66 test chemicals with known human sensitizing potential, and tested all chemicals with the h-CLAT. We then evaluated the performance of the h-CLAT in predicting human sensitizing potential. RESULTS AND CONCLUSION: Forty-five of 51 tested sensitizers were positive in the h-CLAT, indicating relatively high sensitivity. Also, 10 of 15 non-sensitizers were correctly detected as negative. The overall agreement between human data and h-CLAT outcome was 83%. Furthermore, the h-CLAT could accurately predict the human sensitizing potential of 23 tested chemicals that were amines, heterocyclic compounds, or sulfur compounds. Our data indicate the utility of the h-CLAT for predicting the human skin sensitizing potential of a variety of chemicals.
Asunto(s)
Alérgenos/farmacología , Dermatitis Alérgica por Contacto/etiología , Monocitos/efectos de los fármacos , Compuestos Orgánicos/farmacología , Alérgenos/química , Alérgenos/toxicidad , Alternativas a las Pruebas en Animales , Antígeno B7-2/metabolismo , Línea Celular Tumoral , Dermatitis Alérgica por Contacto/inmunología , Humanos , Molécula 1 de Adhesión Intercelular/metabolismo , Monocitos/inmunología , Monocitos/metabolismo , Compuestos Orgánicos/química , Compuestos Orgánicos/toxicidad , Valor Predictivo de las Pruebas , Piel/efectos de los fármacos , Piel/inmunologíaRESUMEN
Several non-animal testing methods to assess photoallergic potential have been developed so far, while none of them have yet to be validated and regulatory accepted. Currently, some photoreactivity assays such as UV-VIS spectral analysis and ROS assay are generally used for initial photosafety assessments because of their high sensitivity. However, they have a low specificity, generating a high percentage of false positive results, and the development of a follow-up assessment method is desired. Therefore, this study aimed to develop an in chemico photoallergy testing method, photo-direct peptide reactivity assay (photo-DPRA). Based on photosafety information, 34 photoallergens and 16 non-photoallergens were selected and subjected to UV-VIS spectral analysis, ROS/micellar ROS assays, photo-DPRA, sequential testing strategy (STS) consisting of all three methods, and 3T3 neutral red uptake phototoxicity testing (3T3 NRU PT). Combination of the methods addressing the key events of photoallergy exhibited high prediction performance. Our results showed the proposed strategy would be useful to predict the photoallergic potential of chemicals as the follow-up assessment for false positive chemicals by UV/VIS spectral analysis and ROS assay.
Asunto(s)
Dermatitis Fotoalérgica/diagnóstico , Hipersensibilidad a las Drogas/diagnóstico , Dermatitis Fotoalérgica/etiología , Relación Dosis-Respuesta a Droga , Hipersensibilidad a las Drogas/etiología , Humanos , Luz/efectos adversos , Especies Reactivas de Oxígeno , Sensibilidad y Especificidad , Espectrofotometría UltravioletaRESUMEN
Phototoxicity is a toxic response elicited by topically applied or systemically administered photoreactive chemicals after exposure to light and can be broadly categorized into photoirritation, photoallergy, photogenotoxicity, and photocarcinogenicity. The need in the 21st century for accurate evaluation of photosafety has led to the publication of a number of guidelines from government agencies in Europe and the U.S.A. as well as the Organisation for Economic Co-operation and Development (OECD). In this review, we first discuss the mechanisms of phototoxicity and how they can be evaluated. We then discuss the state of the art and challenges now faced in photosafety evaluation of pharmaceuticals and cosmetics. Additionally, we describe the latest developments in OECD test guidelines (TG) for assessing photosafety, including revisions to the in vitro 3T3 neutral red uptake (NRU) phototoxicity test (TG 432) and the newly adopted reactive oxigen species (ROS) assay (TG 495). We will emphasize the importance of selecting the most appropriate means of evaluation with reference to the latest guidelines and other legal criteria for conducting photosafety evaluation.
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Dermatitis Fototóxica/diagnóstico , Dermatitis Fototóxica/etiología , Luz/efectos adversos , Rojo Neutro/toxicidad , Células 3T3 , Animales , Células Cultivadas , Humanos , Ratones , Especies Reactivas de Oxígeno/análisis , Seguridad , Pruebas de Toxicidad/métodosRESUMEN
We previously developed the human cell-line activation test (h-CLAT) in vitro skin sensitisation test, based on our reported finding that a 24-hour exposure of THP-1 cells (a human monocytic leukaemia cell line) to sensitisers is sufficient to induce the augmented expression of CD86 and CD54. The aim of this study is to confirm the predictive value of h-CLAT for skin sensitisation activity by employing a larger number of test chemicals. One hundred chemicals were selected, according to their categorisation in the local lymph node assay (LLNA), as being: extreme, strong, moderate and weak sensitisers, and non-sensitisers. The correlation of the h-CLAT results with the LLNA results was 84%. There were some false negatives (e.g. benzoyl peroxide, hexyl cinnamic aldehyde) and some false positives (e.g. 1-bromobutane, diethylphthalate). Eight out of the 9 false negatives (89%) were water-insoluble chemicals. The h-CLAT could positively predict not only extreme and strong sensitisers, but also moderate and weak sensitisers, though the detection rates of weak sensitisers and non-sensitisers were comparatively low. Some sensitisers enhanced both CD86 and CD54 levels, and some enhanced the level of only one of them. The use of the combination of CD86 and CD54 induction as a positive indicator, improved the accuracy of the test. In conclusion, the h-CLAT is expected to be a useful cell-based in vitro method for predicting skin sensitisation potential.