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Standardization of in vitro skin irritation test using reconstructed human epidermis tissues (RhE model)
Abstracts / Toxicology Letters 238S (2015) S56–S383
expression in hepatocytes, which may explain the corresponding deficiency of HLC differentiation. Conclusions: The present gene regulatory network approach identifies key transcription factors for interventions to improve HLC differentiation.
S189
P07-048 Epidermal equivalent assay: A useful tool to predict toxicity and sensitization potential of ingredients used in permanent hair dyes
http://dx.doi.org/10.1016/j.toxlet.2015.08.545
T.B. Zanoni ∗ , T. do Nasciemento Pedrosa, S.B.M. Barros, S.S. Maria-Engler
P07-047 Standardization of in vitro skin irritation test using reconstructed human epidermis tissues (RhE model)
University of Sao Paulo, Department of Clinical Chemistry & Toxicology, School of Pharmaceutical Sciences, University of São Paulo, São Paulo, Brazi (FCF/USP), Av. Lineu Prestes, 580, CEP 05508-900, São Paulo, Brazil
T. Desai, N. Patel ∗ , K. Tendulkar, R. Nagane, M. Patel
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 were 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.
Jai Research Foundation, Toxicology, Valvada, India Skin irritation is the production of reversible tissue damage, manifested by erythema and oedema of the skin. An in vitro skin irritation test is validated as alternatives to Draize skin irritation test which avoid pain and suffering of animals and addresses the human health endpoint skin irritation. In the present study, an in vitro skin irritation test has been validated using SkinEthic RhE Model and will be used as a potential replacement for in vivo Draize skin irritation test. Reconstructed human epidermis (obtained from human derived non-transformed epidermal keratinocytes) closely mimics the histological, morphological, biochemical and physiological properties of the upper parts of the human skin, i.e., the epidermis. Present study was conducted to validate this technique at our laboratory under GLP requirements. Various 10 irritant and non irritant chemicals with their known skin irritation property were used to validate the study. Each chemical was applied topically in triplicates for the period of 42 min. Concurrent positive control (sodium dodecyl sulfate 5% aqueous) and negative controls (Dulbecco’s Phosphate Buffered Saline) were used to check the response of the tissues and acceptance of the study. At the end of the treatment period, cytotoxicity was determined by the conversion of the vital dye MTT [3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide, Thiazolyl blue] into a blue formazan salt, which was quantitatively measured after extraction from tissues. The results obtained in this study were in accordance with the irritant properties of the respective chemicals as defined by the United Nations Globally Harmonized System of Classification and Labeling of Chemicals as indicated in OECD Guideline N◦ 439 under the present experimental conditions. Naphthalene aceticacid, Isopropanol, Methyl stearate, Heptyl butyrate, Hexyl salicylate were classified in UN GHS No Category and Cyclamen aldehyde, 1-bromohexane, Potassium hydroxide (5% aq), 1-methyl-3-phenyl-1-piperazine, Heptanal were classified in UN GHS Category 2. This shows suitability of the test system and procedures used in JRF. This shows suitability of the test system and procedures under the present experimental conditions for performing the in vitro skin irritation studies in reconstructed human epidermis (RhE). http://dx.doi.org/10.1016/j.toxlet.2015.08.546
http://dx.doi.org/10.1016/j.toxlet.2015.08.547
expression in hepatocytes, which may explain the corresponding deficiency of HLC differentiation. Conclusions: The present gene regulatory network approach identifies key transcription factors for interventions to improve HLC differentiation.
S189
P07-048 Epidermal equivalent assay: A useful tool to predict toxicity and sensitization potential of ingredients used in permanent hair dyes
http://dx.doi.org/10.1016/j.toxlet.2015.08.545
T.B. Zanoni ∗ , T. do Nasciemento Pedrosa, S.B.M. Barros, S.S. Maria-Engler
P07-047 Standardization of in vitro skin irritation test using reconstructed human epidermis tissues (RhE model)
University of Sao Paulo, Department of Clinical Chemistry & Toxicology, School of Pharmaceutical Sciences, University of São Paulo, São Paulo, Brazi (FCF/USP), Av. Lineu Prestes, 580, CEP 05508-900, São Paulo, Brazil
T. Desai, N. Patel ∗ , K. Tendulkar, R. Nagane, M. Patel
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 were 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.
Jai Research Foundation, Toxicology, Valvada, India Skin irritation is the production of reversible tissue damage, manifested by erythema and oedema of the skin. An in vitro skin irritation test is validated as alternatives to Draize skin irritation test which avoid pain and suffering of animals and addresses the human health endpoint skin irritation. In the present study, an in vitro skin irritation test has been validated using SkinEthic RhE Model and will be used as a potential replacement for in vivo Draize skin irritation test. Reconstructed human epidermis (obtained from human derived non-transformed epidermal keratinocytes) closely mimics the histological, morphological, biochemical and physiological properties of the upper parts of the human skin, i.e., the epidermis. Present study was conducted to validate this technique at our laboratory under GLP requirements. Various 10 irritant and non irritant chemicals with their known skin irritation property were used to validate the study. Each chemical was applied topically in triplicates for the period of 42 min. Concurrent positive control (sodium dodecyl sulfate 5% aqueous) and negative controls (Dulbecco’s Phosphate Buffered Saline) were used to check the response of the tissues and acceptance of the study. At the end of the treatment period, cytotoxicity was determined by the conversion of the vital dye MTT [3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide, Thiazolyl blue] into a blue formazan salt, which was quantitatively measured after extraction from tissues. The results obtained in this study were in accordance with the irritant properties of the respective chemicals as defined by the United Nations Globally Harmonized System of Classification and Labeling of Chemicals as indicated in OECD Guideline N◦ 439 under the present experimental conditions. Naphthalene aceticacid, Isopropanol, Methyl stearate, Heptyl butyrate, Hexyl salicylate were classified in UN GHS No Category and Cyclamen aldehyde, 1-bromohexane, Potassium hydroxide (5% aq), 1-methyl-3-phenyl-1-piperazine, Heptanal were classified in UN GHS Category 2. This shows suitability of the test system and procedures used in JRF. This shows suitability of the test system and procedures under the present experimental conditions for performing the in vitro skin irritation studies in reconstructed human epidermis (RhE). http://dx.doi.org/10.1016/j.toxlet.2015.08.546
http://dx.doi.org/10.1016/j.toxlet.2015.08.547