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U937 cell line activation test (U-SENS™): An OECD adopted in vitro skin sensitisation assay addressing the activation of dendritic cells
S128
Abstracts / Toxicology Letters 280S (2017) S127–S130
P-04-02-03 U937 cell line activation test (U-SENSTM ): An OECD adopted in vitro skin sensitisation assay addressing the activation of dendritic cells Cécile Piroird, Laurent Nardelli, José Cotovio, Nathalie Alépée L’Oreal R&I, Aulnay sous Bois, France The current knowledge of the mechanisms associated with skin sensitisation has been summarised as an Adverse Outcome Pathway (AOP), starting with the molecular initiating event through intermediate events to the adverse effect, namely allergic contact dermatitis. U937 cell line activation Test (U-SENSTM ) addresses this 3rd Key Event on this AOP by quantifying the change in the expression of a cell surface marker associated with the process of activation of monocytes and DC (CD86) in the human histiocytic lymphoma cell line, following exposure to sensitisers. The measured expression level of CD86 cell surface marker in the cell line U937 was then used for supporting the discrimination between skin sensitisers and non-sensitisers. The transferability and reliability of the U-SENSTM in 4 laboratories (38 tested chemicals) and the predictivity on 175 chemicals using the adapted prediction model resulting of the EURL-ECVAM independently peer review and acceptance. Compared with LLNA results, the accuracy in distinguishing skin sensitisers (UN GHS Cat.1) from non-sensitisers is 86% (N = 166) with a sensitivity of 91% (118/129) and a specificity of 65% (24/37). Considering all available evidence and input from regulators and stakeholders, the U-SENSTM was recommended by EURL ECVAM and adopted in an OECD Test Guideline to be used as part of an IATA to support the discrimination between sensitisers and non-sensitisers for the purpose of hazard classification and labelling. http://dx.doi.org/10.1016/j.toxlet.2017.07.353 P-04-02-04 Determination of contact sensitization potential of chemicals using in vitro reconstructed normal human epidermal model EpiDerm: Impact of the modality of application Silvia Letasiova 1 , Emanuela Corsini 2 , Valentina Galbiati 2 , Helena Kandarova 1 , Dagmar Lehmeier 3 , Helge Gehrke 3
the surface of EpiDerm. Acetone: olive oil (4:1) was used as vehicle in all cases. A total of five chemicals from 3 different sources were tested. The testing set included 3 senzitizers, namely 2,4dinitrochlorobenzene, cinnamaldehyde and isoeugenol/eugenol, and 2 non senzitizers, lactic acid and salicylic acid. Four independent dose–response experiments were conducted in 3 laboratories, resulting in correct prediction of the sensitizing potency of test chemicals. The assessment of IL-18 release using in vitro reconstructed normal human epidermal model EpiDerm appears to be a promising tool for in vitro determination of contact sensitization potential. http://dx.doi.org/10.1016/j.toxlet.2017.07.354 P-04-02-05 Online Integrated Testing Strategy (ITS) for quantitative skin sensitization potency assessment using Bayesian networks and accounting for bioavailability Ahmed Abdelaziz Sayed, Maja Brajnik, Daniel Bachler, Thomas Exner, Barry Hardy Douglas Connect GmbH, Basel, Switzerland The public health problem of skin sensitization is attributed with high direct and indirect costs. Many in vitro tests are available for evaluating the hazard of chemical skin sensitization aiming to replace and reduce the in vivo animal test (Local Lymph Node Assay – LLNA). Strategies for integrating such tests, together with in silico calculations of chemical bioavailability, have been proposed in literature. The integrated testing strategy for skin sensitization (ITS-3) [1] is one such approach. It utilizes Bayesian networks for predicting skin sensitization hazard and guides the testing process. In this work, we reproduce the results from the ITS-3 article and develop a web-application (DC SkinSens) that guides the user through the application of the ITS-3 strategy to assess chemical potency of skin sensitization potency (LLNA pEC3). Open and free tools were favoured during the course of this work to ensure reproducibility and encourage adoption by regulators and industry. The Bayesian network was built and trained using the statistical package R. The overall network accuracy for 4-category classification (non-, weak, moderate or strong sensitizers) was 80% while accuracy of the most confident predictions was 98%. The web application is freely accessible online on https://its.douglasconnect.com.
1
MatTek IVLSL, Bratislava, Slovakia Laboratory of Toxicology, Department of Pharmacological and Biomolecular Sciences, Università degli Studi di Milano, Milan, Italy 3 Eurofins BioPharma Product Testing Munich GmbH, Munich, Germany 2
Assessment of skin sensitization potential has traditionally been conducted in animal models, such as the Mouse Local Lymph Node Assay (LLNA) and the Guinea Pig Maximisation Test (GPMT). However, a growing focus and consensus for minimizing animal use have stimulated the development of in vitro methods to assess skin sensitization. Interleukin-18 (IL-18) release in reconstructed human epidermal models has been identified as a potentially useful endpoint for the identification and classification of skin sensitizing chemicals, including chemicals of low water solubility or stability (Gibbs et al, Toxicol Appl Pharmacol, 2013). The purpose of this study was to investigate the impact of the modality of chemical exposure on the predictive capacity of the assay. EpiDerm tissue viability assessed by MTT assay and IL-18 release assessed by ELISA were evaluated after 24 h topical exposure to test chemicals either impregnated in 8 mm diameter paper filters or directly applied to
Reference Jaworska, J.S., Natsch, A., Ryan, C., Strickland, J., Ashikaga, T., Miyazawa, M., 2015. Bayesian integrated testing strategy (ITS) for skin sensitization potency assessment: a decision support system for quantitative weight of evidence and adaptive testing strategy. Arch. Toxicol. 89, 2355–2383.
http://dx.doi.org/10.1016/j.toxlet.2017.07.355 This abstract has been withdrawn
http://dx.doi.org/10.1016/j.toxlet.2017.07.356
Abstracts / Toxicology Letters 280S (2017) S127–S130
P-04-02-03 U937 cell line activation test (U-SENSTM ): An OECD adopted in vitro skin sensitisation assay addressing the activation of dendritic cells Cécile Piroird, Laurent Nardelli, José Cotovio, Nathalie Alépée L’Oreal R&I, Aulnay sous Bois, France The current knowledge of the mechanisms associated with skin sensitisation has been summarised as an Adverse Outcome Pathway (AOP), starting with the molecular initiating event through intermediate events to the adverse effect, namely allergic contact dermatitis. U937 cell line activation Test (U-SENSTM ) addresses this 3rd Key Event on this AOP by quantifying the change in the expression of a cell surface marker associated with the process of activation of monocytes and DC (CD86) in the human histiocytic lymphoma cell line, following exposure to sensitisers. The measured expression level of CD86 cell surface marker in the cell line U937 was then used for supporting the discrimination between skin sensitisers and non-sensitisers. The transferability and reliability of the U-SENSTM in 4 laboratories (38 tested chemicals) and the predictivity on 175 chemicals using the adapted prediction model resulting of the EURL-ECVAM independently peer review and acceptance. Compared with LLNA results, the accuracy in distinguishing skin sensitisers (UN GHS Cat.1) from non-sensitisers is 86% (N = 166) with a sensitivity of 91% (118/129) and a specificity of 65% (24/37). Considering all available evidence and input from regulators and stakeholders, the U-SENSTM was recommended by EURL ECVAM and adopted in an OECD Test Guideline to be used as part of an IATA to support the discrimination between sensitisers and non-sensitisers for the purpose of hazard classification and labelling. http://dx.doi.org/10.1016/j.toxlet.2017.07.353 P-04-02-04 Determination of contact sensitization potential of chemicals using in vitro reconstructed normal human epidermal model EpiDerm: Impact of the modality of application Silvia Letasiova 1 , Emanuela Corsini 2 , Valentina Galbiati 2 , Helena Kandarova 1 , Dagmar Lehmeier 3 , Helge Gehrke 3
the surface of EpiDerm. Acetone: olive oil (4:1) was used as vehicle in all cases. A total of five chemicals from 3 different sources were tested. The testing set included 3 senzitizers, namely 2,4dinitrochlorobenzene, cinnamaldehyde and isoeugenol/eugenol, and 2 non senzitizers, lactic acid and salicylic acid. Four independent dose–response experiments were conducted in 3 laboratories, resulting in correct prediction of the sensitizing potency of test chemicals. The assessment of IL-18 release using in vitro reconstructed normal human epidermal model EpiDerm appears to be a promising tool for in vitro determination of contact sensitization potential. http://dx.doi.org/10.1016/j.toxlet.2017.07.354 P-04-02-05 Online Integrated Testing Strategy (ITS) for quantitative skin sensitization potency assessment using Bayesian networks and accounting for bioavailability Ahmed Abdelaziz Sayed, Maja Brajnik, Daniel Bachler, Thomas Exner, Barry Hardy Douglas Connect GmbH, Basel, Switzerland The public health problem of skin sensitization is attributed with high direct and indirect costs. Many in vitro tests are available for evaluating the hazard of chemical skin sensitization aiming to replace and reduce the in vivo animal test (Local Lymph Node Assay – LLNA). Strategies for integrating such tests, together with in silico calculations of chemical bioavailability, have been proposed in literature. The integrated testing strategy for skin sensitization (ITS-3) [1] is one such approach. It utilizes Bayesian networks for predicting skin sensitization hazard and guides the testing process. In this work, we reproduce the results from the ITS-3 article and develop a web-application (DC SkinSens) that guides the user through the application of the ITS-3 strategy to assess chemical potency of skin sensitization potency (LLNA pEC3). Open and free tools were favoured during the course of this work to ensure reproducibility and encourage adoption by regulators and industry. The Bayesian network was built and trained using the statistical package R. The overall network accuracy for 4-category classification (non-, weak, moderate or strong sensitizers) was 80% while accuracy of the most confident predictions was 98%. The web application is freely accessible online on https://its.douglasconnect.com.
1
MatTek IVLSL, Bratislava, Slovakia Laboratory of Toxicology, Department of Pharmacological and Biomolecular Sciences, Università degli Studi di Milano, Milan, Italy 3 Eurofins BioPharma Product Testing Munich GmbH, Munich, Germany 2
Assessment of skin sensitization potential has traditionally been conducted in animal models, such as the Mouse Local Lymph Node Assay (LLNA) and the Guinea Pig Maximisation Test (GPMT). However, a growing focus and consensus for minimizing animal use have stimulated the development of in vitro methods to assess skin sensitization. Interleukin-18 (IL-18) release in reconstructed human epidermal models has been identified as a potentially useful endpoint for the identification and classification of skin sensitizing chemicals, including chemicals of low water solubility or stability (Gibbs et al, Toxicol Appl Pharmacol, 2013). The purpose of this study was to investigate the impact of the modality of chemical exposure on the predictive capacity of the assay. EpiDerm tissue viability assessed by MTT assay and IL-18 release assessed by ELISA were evaluated after 24 h topical exposure to test chemicals either impregnated in 8 mm diameter paper filters or directly applied to
Reference Jaworska, J.S., Natsch, A., Ryan, C., Strickland, J., Ashikaga, T., Miyazawa, M., 2015. Bayesian integrated testing strategy (ITS) for skin sensitization potency assessment: a decision support system for quantitative weight of evidence and adaptive testing strategy. Arch. Toxicol. 89, 2355–2383.
http://dx.doi.org/10.1016/j.toxlet.2017.07.355 This abstract has been withdrawn
http://dx.doi.org/10.1016/j.toxlet.2017.07.356