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The utility of QSARs in predicting acute fish toxicity of pesticide metabolites: A retrospective validation approach
S322
Abstracts / Toxicology Letters 238S (2015) S56–S383
P14-003 The utility of QSARs in predicting acute fish toxicity of pesticide metabolites: A retrospective validation approach N. Burden 1,∗ , S.K. Maynard 2 , L. Weltje 3 , J.R. Wheeler 4 1
NC3Rs, London, United Kingdom Syngenta, Bracknell, United Kingdom 3 BASF SE, Limbergerhof, Germany 4 Dow AgroSciences, Abingdon, United Kingdom 2
Under EC Regulation 1107/2009 there is a requirement for registrants to establish whether pesticide metabolites are potentially harmful to the environment, and as such fish acute toxicity assessments may be carried out. The number of metabolites can be considerable; thus this area of testing may use many vertebrates. EFSA’s recent “Guidance on tiered risk assessment for plant protection products for aquatic organisms in edge-of-field surface waters” outlines opportunities to apply non-testing methods such as Quantitative Structure Activity Relationship (QSAR) models. Provision of a scientific evidence base to support the use of QSARs in the prediction of the acute fish toxicity of pesticide metabolites, and subsequent regulatory acceptance, could reduce the numbers of animals used. This work examines the potential for QSARs to be used for pesticide metabolites through a retrospective validation approach. Ecotoxicity data for 679 metabolites contained within the Pesticide Properties Database (http://sitem.herts.ac.uk/ aeru/ppdb/en/atoz.htm) were extracted and filtered to restrict the dataset to metabolites with experimental fish LC50 values. QSAR calculations were performed for 190 metabolites using the US EPA’s ECOSAR software. The most conservative LC50 value generated by ECOSAR was selected for comparison to experimental LC50 values. The relationship between the predicted and experimental values was assessed. Preliminary analysis revealed a significant correlation between predicted and experimental fish LC50 values (r = 0.60, p < 0.001, Spearman correlation). For the majority of the metabolites the predicted values were equal to or lower than their experimental values. For the remaining values the mean ratio of experimental:predicted LC50 was 0.24 (SEM 0.03), i.e. QSAR predicted values were on average only 4.1 times higher than those derived experimentally. Considering normal experimental variability, and the risk assessment procedure, these estimates may have regulatory utility. This initial conservative analysis indicates that there is value in further refining the QSAR approach to improve the prediction of acute fish toxicity of pesticide metabolites. Refinements to this analysis will be undertaken before this method can be considered for incorporation into regulatory guidance. http://dx.doi.org/10.1016/j.toxlet.2015.08.917
P14-004 Differences between cytotoxicity of chemicals determined in vitro on young and old cells of a senescent cell line K. Miranowicz-Dzierzawska ∗ , L. Zapor, J. Skowron Central Institute for Labour Protection – National Research Institute, Department of Chemical, Aerosol and Biological Hazards, Laboratory of Toxicology, Warsaw, Poland Question: The aim of the study was to assess whether there are differences between the results of determination of chemicals cytotoxicity obtained on senescent cells of different age.
Methods: The tests were conducted on CCD-1136Sk (ATCC® CRL-2697TM ), a senescent cell line of human diploid skin fibroblasts. Experiments were conducted on passages from 4 to 21. Experiments were conducted on passages from 4 to 21. The cells were exposed to acetone, 2-propanol, 2-methoxyethanol, 2-ethoxyethanol, 1,4-dioxane, or tetrahydrofuran. Cytotoxicity of xenobiotics was evaluated using two cell viability assays: MTT assay, which determines the metabolic activity of cells, and NRU assay, which assesses the integrity of cell membranes. In addition, the toxicity of acetone for CCD-1136Sk cells and for a permanent cell line (the human epidermoid carcinoma cell line, A431) (passages 31 ÷ 88) was also compared. Results: Acetone proved to be the least toxic for fibroblasts CCD-1136Sk, whereas 2-ethoxyethanol was the most toxic. The older skin fibroblasts CCD-1136Sk (passage 16 and 21) were more susceptible to the cytotoxic effect of almost all tested organic solvents, with the exception of 2-methoxyethanol, in comparison with younger cells, although not for all tested concentrations of the compounds. There were no differences in the toxicity of acetone on early passages of fibroblasts CCD-1136Sk and A431 cells, but late passages of CCD-1136Sk cells were more susceptible to the cytotoxic effect of acetone than cells of the permanent cell line A431. Conclusions: The results showed that the passage number of human diploid skin fibroblasts had an important impact on the susceptibility of cells to chemicals. The concentration of the tested compounds may also play a key role in the influence of organic solvents on successive passages of human diploid fibroblasts. Funding This paper has been based on the results of a research task I.P.07 carried out within the scope of the third stage of the National Programme “Improvement of safety and working conditions” partly supported in 2014–2016 – within the scope of research and development – by the Ministry of Science and Higher Education/National Centre for Research and Development. The Central Institute for Labour Protection – National Research Institute is the Programme’s main co-ordinator. http://dx.doi.org/10.1016/j.toxlet.2015.08.918
P14-006 In vitro developmental neurotoxicity testing models using hiPSC-derived neural stem cells H. Yi ∗ , J.-H. Seo, Y.-I. Park, J.-Y. Song, H.-G. Kang Animal and plant quarantine agency, Anyangsi, Republic of Korea Developing brain is particularly sensitive to chemical disturbances such as methyl mercury (MeHg). Because of the high cost of maintaining laboratory animals and animal welfare issue, standardized and predictive in vitro developmental neurotoxicity test system is critically required. HiPSC-derived neural precursor cells (iNPCs) can be used as a model for early brain development. We evaluated several in vitro test methods using iNPCs to predict adverse effects of low dose of MeHg, CPX/BPAA on neural development. HiPSC-derived neural precursor cells (hNSC) purchased from Gibco (Carlsbad, CA) were maintained in specific media for neural stem cell maintenance. Differentiation of hNSC was induced to twenty-days. MeHg were treated in 100, 200, 400 nM. A veterinary drug CPX (25–100 M) was treated with BPAA (50–200 M). As quantitative evaluation methods for functional neurons, neurite outgrowth, fluorescence intensity of TUJ1 and membrane potential of neurons were measured by ArrayScan VTI HCS reader (Thermo Scientific). After 20 days of differentiation, hNSCs were turned into neurons with branching axons (neurites). Treatment of low dose of MeHg, CPX/BPAA during 20 days of differentiation did not
Abstracts / Toxicology Letters 238S (2015) S56–S383
P14-003 The utility of QSARs in predicting acute fish toxicity of pesticide metabolites: A retrospective validation approach N. Burden 1,∗ , S.K. Maynard 2 , L. Weltje 3 , J.R. Wheeler 4 1
NC3Rs, London, United Kingdom Syngenta, Bracknell, United Kingdom 3 BASF SE, Limbergerhof, Germany 4 Dow AgroSciences, Abingdon, United Kingdom 2
Under EC Regulation 1107/2009 there is a requirement for registrants to establish whether pesticide metabolites are potentially harmful to the environment, and as such fish acute toxicity assessments may be carried out. The number of metabolites can be considerable; thus this area of testing may use many vertebrates. EFSA’s recent “Guidance on tiered risk assessment for plant protection products for aquatic organisms in edge-of-field surface waters” outlines opportunities to apply non-testing methods such as Quantitative Structure Activity Relationship (QSAR) models. Provision of a scientific evidence base to support the use of QSARs in the prediction of the acute fish toxicity of pesticide metabolites, and subsequent regulatory acceptance, could reduce the numbers of animals used. This work examines the potential for QSARs to be used for pesticide metabolites through a retrospective validation approach. Ecotoxicity data for 679 metabolites contained within the Pesticide Properties Database (http://sitem.herts.ac.uk/ aeru/ppdb/en/atoz.htm) were extracted and filtered to restrict the dataset to metabolites with experimental fish LC50 values. QSAR calculations were performed for 190 metabolites using the US EPA’s ECOSAR software. The most conservative LC50 value generated by ECOSAR was selected for comparison to experimental LC50 values. The relationship between the predicted and experimental values was assessed. Preliminary analysis revealed a significant correlation between predicted and experimental fish LC50 values (r = 0.60, p < 0.001, Spearman correlation). For the majority of the metabolites the predicted values were equal to or lower than their experimental values. For the remaining values the mean ratio of experimental:predicted LC50 was 0.24 (SEM 0.03), i.e. QSAR predicted values were on average only 4.1 times higher than those derived experimentally. Considering normal experimental variability, and the risk assessment procedure, these estimates may have regulatory utility. This initial conservative analysis indicates that there is value in further refining the QSAR approach to improve the prediction of acute fish toxicity of pesticide metabolites. Refinements to this analysis will be undertaken before this method can be considered for incorporation into regulatory guidance. http://dx.doi.org/10.1016/j.toxlet.2015.08.917
P14-004 Differences between cytotoxicity of chemicals determined in vitro on young and old cells of a senescent cell line K. Miranowicz-Dzierzawska ∗ , L. Zapor, J. Skowron Central Institute for Labour Protection – National Research Institute, Department of Chemical, Aerosol and Biological Hazards, Laboratory of Toxicology, Warsaw, Poland Question: The aim of the study was to assess whether there are differences between the results of determination of chemicals cytotoxicity obtained on senescent cells of different age.
Methods: The tests were conducted on CCD-1136Sk (ATCC® CRL-2697TM ), a senescent cell line of human diploid skin fibroblasts. Experiments were conducted on passages from 4 to 21. Experiments were conducted on passages from 4 to 21. The cells were exposed to acetone, 2-propanol, 2-methoxyethanol, 2-ethoxyethanol, 1,4-dioxane, or tetrahydrofuran. Cytotoxicity of xenobiotics was evaluated using two cell viability assays: MTT assay, which determines the metabolic activity of cells, and NRU assay, which assesses the integrity of cell membranes. In addition, the toxicity of acetone for CCD-1136Sk cells and for a permanent cell line (the human epidermoid carcinoma cell line, A431) (passages 31 ÷ 88) was also compared. Results: Acetone proved to be the least toxic for fibroblasts CCD-1136Sk, whereas 2-ethoxyethanol was the most toxic. The older skin fibroblasts CCD-1136Sk (passage 16 and 21) were more susceptible to the cytotoxic effect of almost all tested organic solvents, with the exception of 2-methoxyethanol, in comparison with younger cells, although not for all tested concentrations of the compounds. There were no differences in the toxicity of acetone on early passages of fibroblasts CCD-1136Sk and A431 cells, but late passages of CCD-1136Sk cells were more susceptible to the cytotoxic effect of acetone than cells of the permanent cell line A431. Conclusions: The results showed that the passage number of human diploid skin fibroblasts had an important impact on the susceptibility of cells to chemicals. The concentration of the tested compounds may also play a key role in the influence of organic solvents on successive passages of human diploid fibroblasts. Funding This paper has been based on the results of a research task I.P.07 carried out within the scope of the third stage of the National Programme “Improvement of safety and working conditions” partly supported in 2014–2016 – within the scope of research and development – by the Ministry of Science and Higher Education/National Centre for Research and Development. The Central Institute for Labour Protection – National Research Institute is the Programme’s main co-ordinator. http://dx.doi.org/10.1016/j.toxlet.2015.08.918
P14-006 In vitro developmental neurotoxicity testing models using hiPSC-derived neural stem cells H. Yi ∗ , J.-H. Seo, Y.-I. Park, J.-Y. Song, H.-G. Kang Animal and plant quarantine agency, Anyangsi, Republic of Korea Developing brain is particularly sensitive to chemical disturbances such as methyl mercury (MeHg). Because of the high cost of maintaining laboratory animals and animal welfare issue, standardized and predictive in vitro developmental neurotoxicity test system is critically required. HiPSC-derived neural precursor cells (iNPCs) can be used as a model for early brain development. We evaluated several in vitro test methods using iNPCs to predict adverse effects of low dose of MeHg, CPX/BPAA on neural development. HiPSC-derived neural precursor cells (hNSC) purchased from Gibco (Carlsbad, CA) were maintained in specific media for neural stem cell maintenance. Differentiation of hNSC was induced to twenty-days. MeHg were treated in 100, 200, 400 nM. A veterinary drug CPX (25–100 M) was treated with BPAA (50–200 M). As quantitative evaluation methods for functional neurons, neurite outgrowth, fluorescence intensity of TUJ1 and membrane potential of neurons were measured by ArrayScan VTI HCS reader (Thermo Scientific). After 20 days of differentiation, hNSCs were turned into neurons with branching axons (neurites). Treatment of low dose of MeHg, CPX/BPAA during 20 days of differentiation did not