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Epidermal equivalent assay: A useful tool to predict toxicity and sensitization potential of ingredients used in permanent hair dyes
S180
Abstracts / Toxicology Letters 238S (2015) S56–S383
the real time assay reagents. The results indicate the real time assay reagents do not adversely affect viability of cells during the periods measured and enabled multiplexing with a variety of other assays. We conclude the novel real time assay methods will enable more efficient measurement of cytotoxicity in combination with several other in vitro endpoints. http://dx.doi.org/10.1016/j.toxlet.2015.08.521
P07-023 Epidermal equivalent assay: A useful tool to predict toxicity and sensitization potential of ingredients used in permanent hair dyes T.B. Zanoni ∗ , T. Pedrosa, S.B.M. Barros, S.S. Maria-Engler University, Department of Clinical Chemistry & Toxicology, Sao Paulo, Brazil Nowadays billions of people use hair dyes, among them, the most representative class are the permanent hair dyes reaching up to 80% of worldwide consumption. Permanent hair dyes are formed after successive reactions between a primary intermediate (ex. p-phenylenediamine) and a coupler (ex. Resorcinol) that after oxidative reactions with hydrogen peroxide (H2 O2 ) results in permanent color changes inside the hair shaft. In 2001, the European Union began re-evaluating the toxicity of hair dyes. Since 2007, 85 hair dyes have been banned for not being considered safe for consumers. The purpose of this study was to evaluate the toxic and the sensitization potential of some ingredients and their mixtures used in permanent hair dyes, using and in house epidermal equivalent. The tested concentration was below the maximum allowance recommended by the European Union. Our results regarding MTT reduction assay show that 2000 g/mL of p-phenylenediamine (PPD), 2000 g/mL of Resorcinol and 2% of H2 O2 did not significantly reduce epidermis viability. Although, the combination of PPD/H2 O2 and PPD/Resorcinol/H2 O2 reduced cell viability to around 20%. Moreover, we observed that the high decrease on epidermis viability resulted on morphological alterations on epidermal constitution. Thus, it is known that PPD and Resorcinol are both extreme and moderate skin sensitizers, respectively. Next, we attempted to predict the sensitization potential of the mixture of the ingredients by measuring IL-18 production by ELISA. This cytokine is a useful biomarker potentially able to predict sensitization in human keratinocytes. For all ingredients and mixtures, the levels of IL-18 increased when compared to negative control, although there were no significant statistical variation among them. In conclusion, by measuring intracellular IL-18 production, we suggest that the mixtures do not vary the sensitization potential of each ingredient. Considering that epidermis is the first route of exposure to hair dyes, we suggest that similar effects could be induced in human skin after exposure to hair dyes. In addition, we conclude that epidermal equivalents could be used to identify the toxic potential of permanent hair dyes and recommend that they should be used for screening tests. http://dx.doi.org/10.1016/j.toxlet.2015.08.522
P07-024 Using AOPs to transition to alternative approaches to predict toxicity S. Munn ∗ , B. Landesmann, M. Nepelska, A. Price, A. Rolaki, M. Sachana, C. Wittwehr, M. Whelan European Commission, Ispra, Italy An Adverse Outcome Pathway (AOP) is a conceptual framework portraying existing knowledge of the linkage between the Molecular Initiating Event (MIE), and an Adverse Outcome (AO), connected by a chain of Key Events (KE) and the relationships between them (KER). A sufficiently detailed description of an AOP might support chemical risk assessment by indicating the relationships between perturbations at molecular and cellular level with perturbations and ultimately adverse effects occurring at tissue, organ and organism level. Once relationships are established it may be possible to develop an in vitro testing strategy to be predictive of adverse outcomes. The OECD-launched project “Adverse Outcome Pathway Knowledge Base” (AOP KB) enables the scientific community to share and discuss their AOP related knowledge. In particular, the AOP Wiki allows all stakeholders to build AOPs by entering and then linking information about MIEs, KEs, AOs and Chemical Initiators. The authors are developing a number of AOPs within the AOP Wiki, including (1) hepatocyte injury via protein alkylation to liver fibrosis; (2) inhibition of the Na+ /I− symporter decreasing thyroid hormone synthesis leading to learning and memory deficit in children; (3) interaction with the N-methyl-d-aspartate receptor (NMDAR) mediating neuronal cell death in the adult brain (over activation) or decreased synaptogenesis in the developing brain (acute or long term blockage of NMDAR), (4) PPAR activation leading to decreased fertility in adult female rodents and malformations of the reproductive tract in prenatal male rodents. Evaluation of the evidence supporting the AOPs considers biological plausibility, essentiality of KEs as well as consistency of supporting data. However, often quantitative data on the response–response relationships between the key events is lacking. A collaborative effort is currently underway to devise appropriate experimental in vitro models to further elucidate the key events and if possible, to establish the quantitative relationships between them. This research effort could then be used to inform in vitro test systems which, when coupled with toxicokinetic data, should allow the toxic potential of exogenous chemicals to be predicted from early key events, preferably including a point of departure for risk assessment. http://dx.doi.org/10.1016/j.toxlet.2015.08.523
P07-025 The time course of particle-induced cell migration correlates with up-regulation of pro-inflammatory cytokines and chemokines G. Westphal ∗ , I. Schremmer, A. Bryk, N. Rosenkranz, D. Weber, G. Johnen, T. Brüning, J. Bünger Ruhr-University Bochum, IPA, Bochum, Germany Introduction: Particle-induced chronic inflammation of the lung is characterized by the accumulation of macrophages and neutrophils. This study investigates the migration of inflammatory cells in response to particle challenge and correlates the time course of the corresponding cell signaling in vitro. Methods: NR8383 rat alveolar macrophages were challenged with coarse quartz, BaSO4 (inert control), and nanosized SiO2 in concentrations ranging from
Abstracts / Toxicology Letters 238S (2015) S56–S383
the real time assay reagents. The results indicate the real time assay reagents do not adversely affect viability of cells during the periods measured and enabled multiplexing with a variety of other assays. We conclude the novel real time assay methods will enable more efficient measurement of cytotoxicity in combination with several other in vitro endpoints. http://dx.doi.org/10.1016/j.toxlet.2015.08.521
P07-023 Epidermal equivalent assay: A useful tool to predict toxicity and sensitization potential of ingredients used in permanent hair dyes T.B. Zanoni ∗ , T. Pedrosa, S.B.M. Barros, S.S. Maria-Engler University, Department of Clinical Chemistry & Toxicology, Sao Paulo, Brazil Nowadays billions of people use hair dyes, among them, the most representative class are the permanent hair dyes reaching up to 80% of worldwide consumption. Permanent hair dyes are formed after successive reactions between a primary intermediate (ex. p-phenylenediamine) and a coupler (ex. Resorcinol) that after oxidative reactions with hydrogen peroxide (H2 O2 ) results in permanent color changes inside the hair shaft. In 2001, the European Union began re-evaluating the toxicity of hair dyes. Since 2007, 85 hair dyes have been banned for not being considered safe for consumers. The purpose of this study was to evaluate the toxic and the sensitization potential of some ingredients and their mixtures used in permanent hair dyes, using and in house epidermal equivalent. The tested concentration was below the maximum allowance recommended by the European Union. Our results regarding MTT reduction assay show that 2000 g/mL of p-phenylenediamine (PPD), 2000 g/mL of Resorcinol and 2% of H2 O2 did not significantly reduce epidermis viability. Although, the combination of PPD/H2 O2 and PPD/Resorcinol/H2 O2 reduced cell viability to around 20%. Moreover, we observed that the high decrease on epidermis viability resulted on morphological alterations on epidermal constitution. Thus, it is known that PPD and Resorcinol are both extreme and moderate skin sensitizers, respectively. Next, we attempted to predict the sensitization potential of the mixture of the ingredients by measuring IL-18 production by ELISA. This cytokine is a useful biomarker potentially able to predict sensitization in human keratinocytes. For all ingredients and mixtures, the levels of IL-18 increased when compared to negative control, although there were no significant statistical variation among them. In conclusion, by measuring intracellular IL-18 production, we suggest that the mixtures do not vary the sensitization potential of each ingredient. Considering that epidermis is the first route of exposure to hair dyes, we suggest that similar effects could be induced in human skin after exposure to hair dyes. In addition, we conclude that epidermal equivalents could be used to identify the toxic potential of permanent hair dyes and recommend that they should be used for screening tests. http://dx.doi.org/10.1016/j.toxlet.2015.08.522
P07-024 Using AOPs to transition to alternative approaches to predict toxicity S. Munn ∗ , B. Landesmann, M. Nepelska, A. Price, A. Rolaki, M. Sachana, C. Wittwehr, M. Whelan European Commission, Ispra, Italy An Adverse Outcome Pathway (AOP) is a conceptual framework portraying existing knowledge of the linkage between the Molecular Initiating Event (MIE), and an Adverse Outcome (AO), connected by a chain of Key Events (KE) and the relationships between them (KER). A sufficiently detailed description of an AOP might support chemical risk assessment by indicating the relationships between perturbations at molecular and cellular level with perturbations and ultimately adverse effects occurring at tissue, organ and organism level. Once relationships are established it may be possible to develop an in vitro testing strategy to be predictive of adverse outcomes. The OECD-launched project “Adverse Outcome Pathway Knowledge Base” (AOP KB) enables the scientific community to share and discuss their AOP related knowledge. In particular, the AOP Wiki allows all stakeholders to build AOPs by entering and then linking information about MIEs, KEs, AOs and Chemical Initiators. The authors are developing a number of AOPs within the AOP Wiki, including (1) hepatocyte injury via protein alkylation to liver fibrosis; (2) inhibition of the Na+ /I− symporter decreasing thyroid hormone synthesis leading to learning and memory deficit in children; (3) interaction with the N-methyl-d-aspartate receptor (NMDAR) mediating neuronal cell death in the adult brain (over activation) or decreased synaptogenesis in the developing brain (acute or long term blockage of NMDAR), (4) PPAR activation leading to decreased fertility in adult female rodents and malformations of the reproductive tract in prenatal male rodents. Evaluation of the evidence supporting the AOPs considers biological plausibility, essentiality of KEs as well as consistency of supporting data. However, often quantitative data on the response–response relationships between the key events is lacking. A collaborative effort is currently underway to devise appropriate experimental in vitro models to further elucidate the key events and if possible, to establish the quantitative relationships between them. This research effort could then be used to inform in vitro test systems which, when coupled with toxicokinetic data, should allow the toxic potential of exogenous chemicals to be predicted from early key events, preferably including a point of departure for risk assessment. http://dx.doi.org/10.1016/j.toxlet.2015.08.523
P07-025 The time course of particle-induced cell migration correlates with up-regulation of pro-inflammatory cytokines and chemokines G. Westphal ∗ , I. Schremmer, A. Bryk, N. Rosenkranz, D. Weber, G. Johnen, T. Brüning, J. Bünger Ruhr-University Bochum, IPA, Bochum, Germany Introduction: Particle-induced chronic inflammation of the lung is characterized by the accumulation of macrophages and neutrophils. This study investigates the migration of inflammatory cells in response to particle challenge and correlates the time course of the corresponding cell signaling in vitro. Methods: NR8383 rat alveolar macrophages were challenged with coarse quartz, BaSO4 (inert control), and nanosized SiO2 in concentrations ranging from