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Automated INVITTOX protocol # 130
S154
Abstracts / Toxicology Letters 258S (2016) S62–S324
P08-045 Comparative systems toxicology assessment of the Tobacco Heating System 2.2 and reference cigarettes (3R4F), on human organotypic respiratory tissue cultures P. Leroy ∗ , C. Mathis, A. Iskandar, S. Frentzel, A. Elamin, T. Keyur, E. Garcia, A. Knorr, N. Ivanov, J. Hoeng, M. Peitsch Philip Morris International R&D, Philip Morris Products S.A., Quai Jeanrenaud 5, 2000 Neuchâtel, Switzerland With the development of new tobacco products that may have the potential to reduce individual risk and population harm in comparison to smoking cigarettes, a careful and robust assessment strategy is required. We developed and performed large scale in vitro toxicological studies using human organotypic respiratory epithelial cultures allowing direct aerosol exposure at the air-liquid interface. Various endpoints were measured to investigate the impact of an acute exposure to different dilutions of the aerosol generated by a Tobacco Heating System (THS2.2) versus mainstream smoke from the University of Kentucky standard reference cigarette, 3R4F. These comprehensive studies comprised on average 5 experimental repetitions, using different production batches of tissue cultures (from the same cell donor) and different generation of aerosol/smoke. Numerous endpoints were measured ranging from nicotine/carbonyls content in the aerosol/smoke, adenylate kinase, CYP1A1/1B1 activity, release of inflammatory markers, determination of cilia beating activity, and assessment of morphological features by histology. By using appropriate statistical models and by considering various sources of variability (e.g. aerosol/smoke generation, batch of cells), consistent results were obtained between experimental repetitions for the same product. Nicotine and carbonyls data also confirmed the inherent products variability during aerosol/smoke generation and support the need of multiple exposure runs to extend the generalizability and the confidence in the study results. For all endpoints, the results support a reduced impact of THS2.2 aerosol exposure on respiratory epithelial tissue cultures compared to exposure to cigarette smoke at similar nicotine concentration, with inherent product, test system and methods variabilities taken into account. http://dx.doi.org/10.1016/j.toxlet.2016.06.1588 P08-046 An in vitro model of dry eye on conjunctival cells combining exposure to low humidity, airflow, and formaldehyde gas M. Vitoux 1,∗ , S. Achard 2 , K. Kessal 1 , S. Melik Parsadaniantz 1 , C. Baudouin 3 , F. Brignole Baudouin 1 1
UMR S 968 INSERM, UMR 7210 CNRS, Vision Institute, UPMC, Paris, France 2 EA 4064, Faculté de Pharmacie, Université Paris-Descartes, Sorbonne Paris Cité, Paris, France 3 Quinze-Vingts National Ophtalmology Hospital, Paris, France Dry eye is a multifactorial disease whose incidence is continuously rising. Chronic environmental stress conditions are known to be involved in epithelial alterations linked to ocular discomfort. Formaldehyde is the major pollutant of indoors where we spend 80% of our lives. However, there has never been any experimental study assessing the combined effects of dry and polluted atmo-
spheres. We evaluated in vitro the inflammatory responses and cellular death of conjunctival cells exposed using the Vitrocell® system to various controlled atmospheres combining low humidity (LH), airflow (AF) or formaldehyde gas (FG). Cells were exposed at the air–liquid interface to LH conditions without AF, with AF or with FG flow at 100 or 1200 g/m3 for 15–30 mn, followed by a recovery period of 1 h for gene expression or 24 h for protein secretion. Cell viability decreased in relation with the growing stress conditions, ranging from LH alone, LH + AF to LH + AF + FG in a FG concentration dependent manner, starting for LH alone after 30 mn-exposure, and for FG after 15 mn-exposure. Cells responded by stimulating gene expression of pro-inflammatory cytokines from 15 mn-exposure for IL-6 and IL-8, and from 30 mn for CCL2, but no gene stimulation was observed for MIF. The release of cytokines into the extracellular medium was observed after 15 mn for IL-8 and 30 mn for IL-6, MIF and CCL2. Formaldehyde aggravates dry condition-induced cytotoxic and pro-inflammatory effects. This model seems to be a relevant tool to study the inflammatory responses observed in some dry eye diseases caused by combined environmental disturbances like low humidity, airflow, and pollutants. http://dx.doi.org/10.1016/j.toxlet.2016.06.1589 P08-047 Automated INVITTOX protocol # 130 J. Wiest cellasys GmbH – R&D, Munich, Germany The IMOLA-IVD technology monitors the extracellular acidification, cellular respiration and changes in impedance of living cells. In combination with a standard peristaltic pump, proprietary fluidic modules and control software it was possible to set up different cell based assays or organ-on-chip models. In this work we set up a configuration to transfer the cytosensor microphysiometer test method for identification of eye irritation which was validated by the European commission for Validation of Alternative Methods (ECVAM). It measures the extracellular acidification rate (EAR) of L929 mouse fibroblasts and the influence of e.g. the detergent sodium dodecyl sulfat (SDS) toward their EAR. The results show that the determination of the metabolic rate decrement by 50% (MRD50) value can be automated with the proposed set-up. Furthermore it was possible to further develop the INVITTOX protocol toward a fetal bovine serum (FBS) free assay. This simplifies the assay (no difference between seeding and low-buffered treatment medium) and excludes ethical issues related to FBS. Furthermore an increase in reproducibility is expected since inter-laboratory differences due to the problem of different personnel and different lots of FBS are overcome. The necessary adaptations of the INVITTOX protocol # 130 are described and measurements using FBS-free L929 fibroblasts are presented. http://dx.doi.org/10.1016/j.toxlet.2016.06.1590
Abstracts / Toxicology Letters 258S (2016) S62–S324
P08-045 Comparative systems toxicology assessment of the Tobacco Heating System 2.2 and reference cigarettes (3R4F), on human organotypic respiratory tissue cultures P. Leroy ∗ , C. Mathis, A. Iskandar, S. Frentzel, A. Elamin, T. Keyur, E. Garcia, A. Knorr, N. Ivanov, J. Hoeng, M. Peitsch Philip Morris International R&D, Philip Morris Products S.A., Quai Jeanrenaud 5, 2000 Neuchâtel, Switzerland With the development of new tobacco products that may have the potential to reduce individual risk and population harm in comparison to smoking cigarettes, a careful and robust assessment strategy is required. We developed and performed large scale in vitro toxicological studies using human organotypic respiratory epithelial cultures allowing direct aerosol exposure at the air-liquid interface. Various endpoints were measured to investigate the impact of an acute exposure to different dilutions of the aerosol generated by a Tobacco Heating System (THS2.2) versus mainstream smoke from the University of Kentucky standard reference cigarette, 3R4F. These comprehensive studies comprised on average 5 experimental repetitions, using different production batches of tissue cultures (from the same cell donor) and different generation of aerosol/smoke. Numerous endpoints were measured ranging from nicotine/carbonyls content in the aerosol/smoke, adenylate kinase, CYP1A1/1B1 activity, release of inflammatory markers, determination of cilia beating activity, and assessment of morphological features by histology. By using appropriate statistical models and by considering various sources of variability (e.g. aerosol/smoke generation, batch of cells), consistent results were obtained between experimental repetitions for the same product. Nicotine and carbonyls data also confirmed the inherent products variability during aerosol/smoke generation and support the need of multiple exposure runs to extend the generalizability and the confidence in the study results. For all endpoints, the results support a reduced impact of THS2.2 aerosol exposure on respiratory epithelial tissue cultures compared to exposure to cigarette smoke at similar nicotine concentration, with inherent product, test system and methods variabilities taken into account. http://dx.doi.org/10.1016/j.toxlet.2016.06.1588 P08-046 An in vitro model of dry eye on conjunctival cells combining exposure to low humidity, airflow, and formaldehyde gas M. Vitoux 1,∗ , S. Achard 2 , K. Kessal 1 , S. Melik Parsadaniantz 1 , C. Baudouin 3 , F. Brignole Baudouin 1 1
UMR S 968 INSERM, UMR 7210 CNRS, Vision Institute, UPMC, Paris, France 2 EA 4064, Faculté de Pharmacie, Université Paris-Descartes, Sorbonne Paris Cité, Paris, France 3 Quinze-Vingts National Ophtalmology Hospital, Paris, France Dry eye is a multifactorial disease whose incidence is continuously rising. Chronic environmental stress conditions are known to be involved in epithelial alterations linked to ocular discomfort. Formaldehyde is the major pollutant of indoors where we spend 80% of our lives. However, there has never been any experimental study assessing the combined effects of dry and polluted atmo-
spheres. We evaluated in vitro the inflammatory responses and cellular death of conjunctival cells exposed using the Vitrocell® system to various controlled atmospheres combining low humidity (LH), airflow (AF) or formaldehyde gas (FG). Cells were exposed at the air–liquid interface to LH conditions without AF, with AF or with FG flow at 100 or 1200 g/m3 for 15–30 mn, followed by a recovery period of 1 h for gene expression or 24 h for protein secretion. Cell viability decreased in relation with the growing stress conditions, ranging from LH alone, LH + AF to LH + AF + FG in a FG concentration dependent manner, starting for LH alone after 30 mn-exposure, and for FG after 15 mn-exposure. Cells responded by stimulating gene expression of pro-inflammatory cytokines from 15 mn-exposure for IL-6 and IL-8, and from 30 mn for CCL2, but no gene stimulation was observed for MIF. The release of cytokines into the extracellular medium was observed after 15 mn for IL-8 and 30 mn for IL-6, MIF and CCL2. Formaldehyde aggravates dry condition-induced cytotoxic and pro-inflammatory effects. This model seems to be a relevant tool to study the inflammatory responses observed in some dry eye diseases caused by combined environmental disturbances like low humidity, airflow, and pollutants. http://dx.doi.org/10.1016/j.toxlet.2016.06.1589 P08-047 Automated INVITTOX protocol # 130 J. Wiest cellasys GmbH – R&D, Munich, Germany The IMOLA-IVD technology monitors the extracellular acidification, cellular respiration and changes in impedance of living cells. In combination with a standard peristaltic pump, proprietary fluidic modules and control software it was possible to set up different cell based assays or organ-on-chip models. In this work we set up a configuration to transfer the cytosensor microphysiometer test method for identification of eye irritation which was validated by the European commission for Validation of Alternative Methods (ECVAM). It measures the extracellular acidification rate (EAR) of L929 mouse fibroblasts and the influence of e.g. the detergent sodium dodecyl sulfat (SDS) toward their EAR. The results show that the determination of the metabolic rate decrement by 50% (MRD50) value can be automated with the proposed set-up. Furthermore it was possible to further develop the INVITTOX protocol toward a fetal bovine serum (FBS) free assay. This simplifies the assay (no difference between seeding and low-buffered treatment medium) and excludes ethical issues related to FBS. Furthermore an increase in reproducibility is expected since inter-laboratory differences due to the problem of different personnel and different lots of FBS are overcome. The necessary adaptations of the INVITTOX protocol # 130 are described and measurements using FBS-free L929 fibroblasts are presented. http://dx.doi.org/10.1016/j.toxlet.2016.06.1590