- Email: [email protected]
The CULTEX® Radial Flow System as in vivo model for the assessment of lung toxicity
S256
P-09-02 / Toxicology Letters 280S (2017) S254–S279
measurement as a metabolic parameter and impedance measurement as a morphological parameter yields better prediction capability to determine the eye irritation potential of new chemicals. First multi-parametric measurements show comparable MRD50 (metabolic rate decrement by 50%) values of sodium dodecyl sulfate (SDS). However, an effect of SDS toward the cellular impedance can only be detected at a higher concentration. The expanded protocol will be used to create a new prediction model and it will be evaluated if it is possible to address the whole range of classification in the field of eye irritation. References Hartung, T., Bruner, L., Curren, R., et al., 2010. First alternative method validated by a retrospective weight-of-evidence approach to replace the Draize eye test for the identification of non-irritant substances for a defined application domain. ALTEX 27, 43–51. Wiest, J., 2016. Automated INVITTOX protocol # 130. Toxicol. Lett. 258 (Suppl.), S154.
http://dx.doi.org/10.1016/j.toxlet.2017.07.910
P-09-02-07 Development of an in-vitro testing battery to assess biocompatibility of medical devices Elisabeth Mertl, Christian Kirchnawy OFI, Vienna, Austria In order to guarantee safety for the end-users medical devices and other solid products have to be tested for adverse reactions on the skin before market authorization. Animal testing is still state of the art, but ethically questionable. One of the key aspects of our recent research is to establish an in-vitro testing battery to examine the biocompatibility of medical devices. The first task was the development of an appropriate extraction method. With the help of skin models, cell based testing methods (ARE assay, dendritic cell assay) and a chromatographic method (DPRA), such extracts can be assed for cytotoxicity, irritation and sensitisation. To identify a possible sensitisation potential, we are aiming to develop a screening method to cover the entire spectrum of the skin sensitization process, from the molecular initiation (DPRA), to the keratinocyte-response (ARE) and activation of dendritic cells (h-Clat). So far, various samples have been tested in the different in-vitro assays and additionally samples were examined with animal testing in order to compare the results showing more sensitive responses in the in-vitro assays. These assays are developed not only with a sufficient sensitivity, but also to be robust, simple to use, ethically correct and inexpensive. http://dx.doi.org/10.1016/j.toxlet.2017.07.911
P-09-02-08 Biological effects of whole-aerosol exposure of human bronchial tissues to cigarette smoke and nicotine-containing vapor Shinkichi Ishikawa, Kazushi Matsumura, Kanae Ishimori, Nobumasa Kitamura, Satoru Munakata, Yuichiro Takanami, Shigeaki Ito Scientific Product Assessment Center, Japan Tobacco Inc., Yokohama, Kanagawa, Japan Novel tobacco- and nicotine-containing vapor products are gaining popularity worldwide. Since most of these products generate
vapor without combustion, the vapor is expected to contain lower levels of potentially harmful constituents and elicit fewer biological effects than combustible cigarettes. The aim of this study is to examine the biological effects of our novel tobacco vapor product (NTV), which is designed to generate nicotine- and flavor-containing vapor without combustion. We used an in vitro whole-smoke exposure system that mimics exposure in the human respiratory tract with a commercially available human bronchial tissue model (MucilAir). Tissues were exposed to aerosols generated from the reference cigarette K3R4F and NTV every other day for 20 days. Following repeated exposure, we analyzed histological changes and gene expression profiles using microarray technologies. K3R4F smoke exposure induced histological changes, such as hyperplasia, which was indicated by an increase in CK5- and CK14-expressing basal cells. These changes were not induced in control and NTV vapor exposures. Gene expression was more perturbed in tissues exposed to K3R4F smoke than in those exposed to NTV vapor. Microarray analysis indicated that several biological events were significantly induced in K3R4F-exposed tissues. Overall, our findings indicate that the in vitro bronchial epithelial tissue model MucilAir can be induced into a hyperplastic phenotype by smoke exposure, and microarray data suggests possible mechanisms underlying these histological changes. Whole-aerosol exposure of bronchial tissue cultures is thus useful for studying the biological effects of both combustible cigarettes and vapor products. http://dx.doi.org/10.1016/j.toxlet.2017.07.912
P-09-02-09 ® The CULTEX Radial Flow System as in vivo model for the assessment of lung toxicity Amelie Tsoutsoulopoulos 1 , Niklas Möhle 2 , Katrin Gohlsch 3 , Andreas Breit 3 , Harald Mückter 3 , Thomas Gudermann 3 , Olaf Krischenowski 2 , Sebastian Hoffmann 4 , Horst Thiermann 1 , Dirk Steinritz 1,3 , Michaela Aufderheide 2 1
Bundeswehr Institute of Pharmacology and Toxicology, Munich, Germany, 2 Cultex Laboratories GmbH, Hannover, Germany, 3 Walther-Straub-Institute of Pharmacology and Toxicology, Ludwig-Maximilians-Universität, Munich, Germany, 4 seh Consulting + Services, Paderborn, Germany Introduction: Toxicological risk assessment of airborne particles represents a major task due to the increasing number of chemicals on the market. Animal experiments, which appear as best suitable at first glance, are difficult and cost intensive. Moreover, the EU REACH program demands the reduction of animal experiments by using alternative methods. However, replacement of in vivo ® acute toxicity studies remains still a challenge. The CULTEX RFS in vitro method, which exposes human lung epithelial cells at the air-liquid-interface (ALI), has been developed to address that issue. Funded by the German BMBF, the methodology was standardized, optimized and transferred to participating laboratories for further validation. Objective: Purpose of the project is to validate, improve and ® standardize the CULTEX RFS methodology to meet regulatory requirements. Materials and methods: For experiments, human A549 cells were grown on microporous membranes and exposed to clean air or to 12 coded substances at the ALI for 15, 30 and 60 min. Cell viability was determined 24 h after exposure by WST-assay. Using in vivo acute inhalation data as reference, the preliminary predic® tivity of the CULTEX RFS method was evaluated.
P-09-02 / Toxicology Letters 280S (2017) S254–S279
S257
Results: After initial methodological refinements, results improved, remained stable and were comparable with those of the former project (BMBF 0315710, 2013). Our results correlate well with acute inhalation toxicity classification obtained from the standard in vivo test. Conclusion: We propose this test method as a tool to address the acute inhalation toxicity of dust, for which often no hazard data are available.
P-09-02-11 Searching for an alternative to BALB/3T3 cells to develop an effective method for simultaneous phototoxic and photogenotoxic screening system
http://dx.doi.org/10.1016/j.toxlet.2017.07.913
Department of Environmental Health Sciences, Medical University of Warsaw, Warsaw, Poland
P-09-02-10 Development of an innovative bioreactor to simulate cutaneous absorption and metabolism for risk assessment purposes
Photosafety is an important issue that should be evaluated for pharmaceuticals or ingredients of personal care products. Many of chemicals after solar irradiation generate photoproducts that could be significantly more toxic and genotoxic than the parent compounds. One of the techniques proposed by the OECD and EMA to evaluate the phototoxicity of chemicals is the in vitro neutral red uptake (NRU) assay with the BALB/3T3 cells. However, to verify the photogenotoxicity of tested compounds an additional test should be performed. According to reports one of the techniques suitable for this purpose is the micronucleus test. The mammalian V79 fibroblasts are among cell lines recommended for this assay instead of the BALB/3T3 cells. Conducting micronucleus assay requires additional evaluation of the toxicity and phototoxicity of tested substance for V79 cells. Assessment of the phototoxicity and photogenotoxicity could be more effective if performed simultaneously with only one cell line. In this project we compared the results of the NRU assay obtained for V79 cells and BALB/3T3 cells. The cell lines were exposed to a number of compounds with documented different phototoxic abilities and irradiated in the sunlight simulator. The NRU assay was prepared on the basis of the OECD 432. The statistical analysis of obtained data allowed to verify the hypothesis of comparable sensitivity of both cell lines. The project was financed by the Medical University of Warsaw, Faculty of Pharmacy from the Grant for Young Scientists managed by Anna Zgadzaj in years 2016-2017 (FW14/PM2/16).
Paul Quantin 1,2 , Sophie Catoire 2 , Herve Ficheux 2 1
BMBI CNRS 7338, Université de Technologie de Compiègne, Compiègne, France, 2 Department of Toxicology, THOR Personal Care, Compiègne, France The bioavailability of a substance administered on the skin depends on many factors. Skin penetration is assessed, most of the time, by using Franz cells fitted with different kinds of skin membrane: human skin, reconstructed skin, animal skin. All these methods are very useful in generating relevant penetration parameters however there are many drawbacks: • animal skin can no longer be used for cosmetic testing; • human skin is relatively difficult to handle; • reconstructed epidermis offers a good alternative but some metabolic functions are lost, leading to incomplete information when used for risk assessment purposes. Recently, artificial membranes (i.e. Strat-MTM ) have been developed to offer an alternative able to overcome some of these issues. As anticipated, reproducibility of penetration data is improved compared with the conventional method but metabolic information is still lacking. In order to fill this last gap we have developed a bioreactor to mimic both skin absorption and skin metabolism. It consists of a Franz cell diffusion chamber fitted with an artificial membrane on the top with CYPs immobilised in the bottom of the receptor chamber. The artificial membrane is STRAT-MTM and the CYPs are bacterial membrane fractions expressing selective CYP, in this case CYP1A2. This enzyme has been selected because, according to a previous study, it is highly inducible into skin models and because its metabolism against caffeine is well characterised. We are able to mimic the whole process of absorption and metabolism; both caffeine and its main metabolite, paraxanthine are monitored. http://dx.doi.org/10.1016/j.toxlet.2017.07.914
Anna Zgadzaj, Zuzanna Czacharowska, Ramona Figat, Grzegorz Nałecz-Jawecki
http://dx.doi.org/10.1016/j.toxlet.2017.07.915
P-09-02-12 Multiparametric assessment of the effects of chemotherapeutic drugs on the ® (electro)physiology of Pluricyte Cardiomyocytes Farbod Famili, Peter Nacken, Tessa De Korte, Celine Hechard, Marijn Vlaming, Stefan Braam Pluriomics, Leiden, Netherlands Cardiotoxicity is a major cause for drug attrition during pharmaceutical drug development and remains a challenge due to the difficulties in obtaining human heart tissues, and to propagate them in vitro. With the introduction of human induced pluripotent stem cell (hiPSC) and cardiac differentiation technologies, hiPSC-derived cardiomyocytes open new paths for heart diseases modelling, new drugs screening, and drug candidates testing for cardiotoxicity. We have developed fully functional hiPSC-derived ventricular ® cardiomyocytes (Pluricyte Cardiomyocytes) which resembles to mature human cardiac cells in many aspects. This was confirmed by an increased contraction profile, a highly organized sarcomere organization, as well as improved electrophysiological properties (negative resting membrane potential, well defined action potential plateau and rapid depolarization).
P-09-02 / Toxicology Letters 280S (2017) S254–S279
measurement as a metabolic parameter and impedance measurement as a morphological parameter yields better prediction capability to determine the eye irritation potential of new chemicals. First multi-parametric measurements show comparable MRD50 (metabolic rate decrement by 50%) values of sodium dodecyl sulfate (SDS). However, an effect of SDS toward the cellular impedance can only be detected at a higher concentration. The expanded protocol will be used to create a new prediction model and it will be evaluated if it is possible to address the whole range of classification in the field of eye irritation. References Hartung, T., Bruner, L., Curren, R., et al., 2010. First alternative method validated by a retrospective weight-of-evidence approach to replace the Draize eye test for the identification of non-irritant substances for a defined application domain. ALTEX 27, 43–51. Wiest, J., 2016. Automated INVITTOX protocol # 130. Toxicol. Lett. 258 (Suppl.), S154.
http://dx.doi.org/10.1016/j.toxlet.2017.07.910
P-09-02-07 Development of an in-vitro testing battery to assess biocompatibility of medical devices Elisabeth Mertl, Christian Kirchnawy OFI, Vienna, Austria In order to guarantee safety for the end-users medical devices and other solid products have to be tested for adverse reactions on the skin before market authorization. Animal testing is still state of the art, but ethically questionable. One of the key aspects of our recent research is to establish an in-vitro testing battery to examine the biocompatibility of medical devices. The first task was the development of an appropriate extraction method. With the help of skin models, cell based testing methods (ARE assay, dendritic cell assay) and a chromatographic method (DPRA), such extracts can be assed for cytotoxicity, irritation and sensitisation. To identify a possible sensitisation potential, we are aiming to develop a screening method to cover the entire spectrum of the skin sensitization process, from the molecular initiation (DPRA), to the keratinocyte-response (ARE) and activation of dendritic cells (h-Clat). So far, various samples have been tested in the different in-vitro assays and additionally samples were examined with animal testing in order to compare the results showing more sensitive responses in the in-vitro assays. These assays are developed not only with a sufficient sensitivity, but also to be robust, simple to use, ethically correct and inexpensive. http://dx.doi.org/10.1016/j.toxlet.2017.07.911
P-09-02-08 Biological effects of whole-aerosol exposure of human bronchial tissues to cigarette smoke and nicotine-containing vapor Shinkichi Ishikawa, Kazushi Matsumura, Kanae Ishimori, Nobumasa Kitamura, Satoru Munakata, Yuichiro Takanami, Shigeaki Ito Scientific Product Assessment Center, Japan Tobacco Inc., Yokohama, Kanagawa, Japan Novel tobacco- and nicotine-containing vapor products are gaining popularity worldwide. Since most of these products generate
vapor without combustion, the vapor is expected to contain lower levels of potentially harmful constituents and elicit fewer biological effects than combustible cigarettes. The aim of this study is to examine the biological effects of our novel tobacco vapor product (NTV), which is designed to generate nicotine- and flavor-containing vapor without combustion. We used an in vitro whole-smoke exposure system that mimics exposure in the human respiratory tract with a commercially available human bronchial tissue model (MucilAir). Tissues were exposed to aerosols generated from the reference cigarette K3R4F and NTV every other day for 20 days. Following repeated exposure, we analyzed histological changes and gene expression profiles using microarray technologies. K3R4F smoke exposure induced histological changes, such as hyperplasia, which was indicated by an increase in CK5- and CK14-expressing basal cells. These changes were not induced in control and NTV vapor exposures. Gene expression was more perturbed in tissues exposed to K3R4F smoke than in those exposed to NTV vapor. Microarray analysis indicated that several biological events were significantly induced in K3R4F-exposed tissues. Overall, our findings indicate that the in vitro bronchial epithelial tissue model MucilAir can be induced into a hyperplastic phenotype by smoke exposure, and microarray data suggests possible mechanisms underlying these histological changes. Whole-aerosol exposure of bronchial tissue cultures is thus useful for studying the biological effects of both combustible cigarettes and vapor products. http://dx.doi.org/10.1016/j.toxlet.2017.07.912
P-09-02-09 ® The CULTEX Radial Flow System as in vivo model for the assessment of lung toxicity Amelie Tsoutsoulopoulos 1 , Niklas Möhle 2 , Katrin Gohlsch 3 , Andreas Breit 3 , Harald Mückter 3 , Thomas Gudermann 3 , Olaf Krischenowski 2 , Sebastian Hoffmann 4 , Horst Thiermann 1 , Dirk Steinritz 1,3 , Michaela Aufderheide 2 1
Bundeswehr Institute of Pharmacology and Toxicology, Munich, Germany, 2 Cultex Laboratories GmbH, Hannover, Germany, 3 Walther-Straub-Institute of Pharmacology and Toxicology, Ludwig-Maximilians-Universität, Munich, Germany, 4 seh Consulting + Services, Paderborn, Germany Introduction: Toxicological risk assessment of airborne particles represents a major task due to the increasing number of chemicals on the market. Animal experiments, which appear as best suitable at first glance, are difficult and cost intensive. Moreover, the EU REACH program demands the reduction of animal experiments by using alternative methods. However, replacement of in vivo ® acute toxicity studies remains still a challenge. The CULTEX RFS in vitro method, which exposes human lung epithelial cells at the air-liquid-interface (ALI), has been developed to address that issue. Funded by the German BMBF, the methodology was standardized, optimized and transferred to participating laboratories for further validation. Objective: Purpose of the project is to validate, improve and ® standardize the CULTEX RFS methodology to meet regulatory requirements. Materials and methods: For experiments, human A549 cells were grown on microporous membranes and exposed to clean air or to 12 coded substances at the ALI for 15, 30 and 60 min. Cell viability was determined 24 h after exposure by WST-assay. Using in vivo acute inhalation data as reference, the preliminary predic® tivity of the CULTEX RFS method was evaluated.
P-09-02 / Toxicology Letters 280S (2017) S254–S279
S257
Results: After initial methodological refinements, results improved, remained stable and were comparable with those of the former project (BMBF 0315710, 2013). Our results correlate well with acute inhalation toxicity classification obtained from the standard in vivo test. Conclusion: We propose this test method as a tool to address the acute inhalation toxicity of dust, for which often no hazard data are available.
P-09-02-11 Searching for an alternative to BALB/3T3 cells to develop an effective method for simultaneous phototoxic and photogenotoxic screening system
http://dx.doi.org/10.1016/j.toxlet.2017.07.913
Department of Environmental Health Sciences, Medical University of Warsaw, Warsaw, Poland
P-09-02-10 Development of an innovative bioreactor to simulate cutaneous absorption and metabolism for risk assessment purposes
Photosafety is an important issue that should be evaluated for pharmaceuticals or ingredients of personal care products. Many of chemicals after solar irradiation generate photoproducts that could be significantly more toxic and genotoxic than the parent compounds. One of the techniques proposed by the OECD and EMA to evaluate the phototoxicity of chemicals is the in vitro neutral red uptake (NRU) assay with the BALB/3T3 cells. However, to verify the photogenotoxicity of tested compounds an additional test should be performed. According to reports one of the techniques suitable for this purpose is the micronucleus test. The mammalian V79 fibroblasts are among cell lines recommended for this assay instead of the BALB/3T3 cells. Conducting micronucleus assay requires additional evaluation of the toxicity and phototoxicity of tested substance for V79 cells. Assessment of the phototoxicity and photogenotoxicity could be more effective if performed simultaneously with only one cell line. In this project we compared the results of the NRU assay obtained for V79 cells and BALB/3T3 cells. The cell lines were exposed to a number of compounds with documented different phototoxic abilities and irradiated in the sunlight simulator. The NRU assay was prepared on the basis of the OECD 432. The statistical analysis of obtained data allowed to verify the hypothesis of comparable sensitivity of both cell lines. The project was financed by the Medical University of Warsaw, Faculty of Pharmacy from the Grant for Young Scientists managed by Anna Zgadzaj in years 2016-2017 (FW14/PM2/16).
Paul Quantin 1,2 , Sophie Catoire 2 , Herve Ficheux 2 1
BMBI CNRS 7338, Université de Technologie de Compiègne, Compiègne, France, 2 Department of Toxicology, THOR Personal Care, Compiègne, France The bioavailability of a substance administered on the skin depends on many factors. Skin penetration is assessed, most of the time, by using Franz cells fitted with different kinds of skin membrane: human skin, reconstructed skin, animal skin. All these methods are very useful in generating relevant penetration parameters however there are many drawbacks: • animal skin can no longer be used for cosmetic testing; • human skin is relatively difficult to handle; • reconstructed epidermis offers a good alternative but some metabolic functions are lost, leading to incomplete information when used for risk assessment purposes. Recently, artificial membranes (i.e. Strat-MTM ) have been developed to offer an alternative able to overcome some of these issues. As anticipated, reproducibility of penetration data is improved compared with the conventional method but metabolic information is still lacking. In order to fill this last gap we have developed a bioreactor to mimic both skin absorption and skin metabolism. It consists of a Franz cell diffusion chamber fitted with an artificial membrane on the top with CYPs immobilised in the bottom of the receptor chamber. The artificial membrane is STRAT-MTM and the CYPs are bacterial membrane fractions expressing selective CYP, in this case CYP1A2. This enzyme has been selected because, according to a previous study, it is highly inducible into skin models and because its metabolism against caffeine is well characterised. We are able to mimic the whole process of absorption and metabolism; both caffeine and its main metabolite, paraxanthine are monitored. http://dx.doi.org/10.1016/j.toxlet.2017.07.914
Anna Zgadzaj, Zuzanna Czacharowska, Ramona Figat, Grzegorz Nałecz-Jawecki
http://dx.doi.org/10.1016/j.toxlet.2017.07.915
P-09-02-12 Multiparametric assessment of the effects of chemotherapeutic drugs on the ® (electro)physiology of Pluricyte Cardiomyocytes Farbod Famili, Peter Nacken, Tessa De Korte, Celine Hechard, Marijn Vlaming, Stefan Braam Pluriomics, Leiden, Netherlands Cardiotoxicity is a major cause for drug attrition during pharmaceutical drug development and remains a challenge due to the difficulties in obtaining human heart tissues, and to propagate them in vitro. With the introduction of human induced pluripotent stem cell (hiPSC) and cardiac differentiation technologies, hiPSC-derived cardiomyocytes open new paths for heart diseases modelling, new drugs screening, and drug candidates testing for cardiotoxicity. We have developed fully functional hiPSC-derived ventricular ® cardiomyocytes (Pluricyte Cardiomyocytes) which resembles to mature human cardiac cells in many aspects. This was confirmed by an increased contraction profile, a highly organized sarcomere organization, as well as improved electrophysiological properties (negative resting membrane potential, well defined action potential plateau and rapid depolarization).