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ZFminus1: A strategy to reduce animal tests for developmental toxicity testing by a combined use of mammalian models and the zebrafish embryotoxicity test (ZFET or ZETA)
Reproductive Toxicology 64 (2016) 22–26
Contents lists available at ScienceDirect
Reproductive Toxicology journal homepage: www.elsevier.com/locate/reprotox
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F-1 ZFminus1: A strategy to reduce animal tests for developmental toxicity testing by a combined use of mammalian models and the zebrafish embryotoxicity test (ZFET or ZETA) Elisabet Teixidó 1,∗ , David Leuthold 1 , Alexander Amberg 2 , Andreas Czich 2 , Eckart Krupp 2 , Stefan Scholz 1 1
UFZ – Helmholtz Centre for Environmental Research, Department Bioanalytical Ecotoxicology, Leipzig, Germany 2 Sanofi-Aventis Deutschland GmbH, R&D DSAR/Preclinical Safety FF, Frankfurt am Main, Germany Developmental toxicological studies are usually required in both a rodent and a non-rodent species. The need of a developmental study in a non-rodent species has been repeatedly questioned. On the other hand, the zebrafish embryo test has received increasing attention for predicting teratogenic effects and is currently used by several pharmaceutical and chemical companies for screening purposes. Efforts are needed to optimize the protocols and further improve its performance. The aim of this study is the application of the zebrafish embryo test with one rodent test and finally also in silico tests to achieve the same level of predictivity in developmental safety testing when tests are conducted in two mammalian test species. A set of model compounds were evaluated using the zebrafish embryo test with additional endpoints such as behavior (embryonic movement, locomotor response) in order to increase the concordance level to in vivo results. Automatic methods for handling, positioning and orientation were established to analyse phenotypes [1]. Furthermore, we implemented first automatic image-based analyses of zebrafish morphological features using the KNIME® analytical platform [2]. For each endpoint concentration-response curves were derived and compared to the lethal concentration. This was used to derive global response patterns resulting in feature signatures to group chemicals and estimate the hazard potential. The preliminary results showed that compound solubility is a limiting factor of the zebrafish embryotoxicity test. We also observed that polar compounds like lamivudine showed a reduced or no effect. It should be noted that a frequently observed effect in exposed embryos was non-inflation of swim bladder and early mortality (before 24 hpf). The approach 0890-6238/
described in this study contributes to understand the predictive capacity of the zebrafish embryo assay and strategies to improve it. Moreover, the use of automatic and quantitative methods increase experimental reproducibility and could be applied to large scale screenings of the influence of various chemicals on zebrafish development. References [1] R. Pulak, Tools for automating the imaging of zebrafish larvae, Methods 96 (2016) 118–126. [2] M.R. Berthold, N. Cebron, F. Dill, T.R. Gabriel, T. Kötter, T. Meinl, P. Ohl, C. Sieb, K. Thiel, B. Wiswedel, KNIME: The Konstanz Information Miner, in: C. Preisach, H. Burkhardt, L. Schmidt-Thieme, R. Decker (Eds.), Data Anal. Mach. Learn. Appl., Springer, Berlin, Heidelberg, 2008, pp. 319–326.
http://dx.doi.org/10.1016/j.reprotox.2016.06.048 F-2 In vitro developmental toxicity potencies of petroleum substances in the embryonic stem cell test as compared to their in vivo developmental toxicity potencies Lenny Kamelia 1,∗ , Jochem Louisse 1 , Ivonne M.C.M. Rietjens 1 , Peter J. Boogaard 2 1 Division of Toxicology, Wageningen University, The Netherlands 2 Shell Health, Shell International bv, The Hague, The Netherlands
The REACH legislation requires substances that are registered in the EU at a volume of >100 tonnes/year to be tested for prenatal developmental toxicity. This will involve a huge number of experimental animals, if the data requirements are fulfilled following the current OECD testing guidelines. The application of in vitro assays, such as the embryonic stem cell test (EST), may reduce animal experimentation and resources needed to study the developmental toxicity potencies of petroleum substances. Petroleum substances are UVCBs (substances of Unknown or Variable composition, Complex reaction products and Biological materials) comprising different types of hydrocarbons. The ability of some petroleum substances to induce developmental toxicity has been associated with particular constituents, i.e. the polycyclic aromatic hydrocarbons (PAHs). Heavy petroleum substances like heavy fuel oil (HFO) and untreated lubricating oils contain relatively high concentrations
Contents lists available at ScienceDirect
Reproductive Toxicology journal homepage: www.elsevier.com/locate/reprotox
Free Communications
F-1 ZFminus1: A strategy to reduce animal tests for developmental toxicity testing by a combined use of mammalian models and the zebrafish embryotoxicity test (ZFET or ZETA) Elisabet Teixidó 1,∗ , David Leuthold 1 , Alexander Amberg 2 , Andreas Czich 2 , Eckart Krupp 2 , Stefan Scholz 1 1
UFZ – Helmholtz Centre for Environmental Research, Department Bioanalytical Ecotoxicology, Leipzig, Germany 2 Sanofi-Aventis Deutschland GmbH, R&D DSAR/Preclinical Safety FF, Frankfurt am Main, Germany Developmental toxicological studies are usually required in both a rodent and a non-rodent species. The need of a developmental study in a non-rodent species has been repeatedly questioned. On the other hand, the zebrafish embryo test has received increasing attention for predicting teratogenic effects and is currently used by several pharmaceutical and chemical companies for screening purposes. Efforts are needed to optimize the protocols and further improve its performance. The aim of this study is the application of the zebrafish embryo test with one rodent test and finally also in silico tests to achieve the same level of predictivity in developmental safety testing when tests are conducted in two mammalian test species. A set of model compounds were evaluated using the zebrafish embryo test with additional endpoints such as behavior (embryonic movement, locomotor response) in order to increase the concordance level to in vivo results. Automatic methods for handling, positioning and orientation were established to analyse phenotypes [1]. Furthermore, we implemented first automatic image-based analyses of zebrafish morphological features using the KNIME® analytical platform [2]. For each endpoint concentration-response curves were derived and compared to the lethal concentration. This was used to derive global response patterns resulting in feature signatures to group chemicals and estimate the hazard potential. The preliminary results showed that compound solubility is a limiting factor of the zebrafish embryotoxicity test. We also observed that polar compounds like lamivudine showed a reduced or no effect. It should be noted that a frequently observed effect in exposed embryos was non-inflation of swim bladder and early mortality (before 24 hpf). The approach 0890-6238/
described in this study contributes to understand the predictive capacity of the zebrafish embryo assay and strategies to improve it. Moreover, the use of automatic and quantitative methods increase experimental reproducibility and could be applied to large scale screenings of the influence of various chemicals on zebrafish development. References [1] R. Pulak, Tools for automating the imaging of zebrafish larvae, Methods 96 (2016) 118–126. [2] M.R. Berthold, N. Cebron, F. Dill, T.R. Gabriel, T. Kötter, T. Meinl, P. Ohl, C. Sieb, K. Thiel, B. Wiswedel, KNIME: The Konstanz Information Miner, in: C. Preisach, H. Burkhardt, L. Schmidt-Thieme, R. Decker (Eds.), Data Anal. Mach. Learn. Appl., Springer, Berlin, Heidelberg, 2008, pp. 319–326.
http://dx.doi.org/10.1016/j.reprotox.2016.06.048 F-2 In vitro developmental toxicity potencies of petroleum substances in the embryonic stem cell test as compared to their in vivo developmental toxicity potencies Lenny Kamelia 1,∗ , Jochem Louisse 1 , Ivonne M.C.M. Rietjens 1 , Peter J. Boogaard 2 1 Division of Toxicology, Wageningen University, The Netherlands 2 Shell Health, Shell International bv, The Hague, The Netherlands
The REACH legislation requires substances that are registered in the EU at a volume of >100 tonnes/year to be tested for prenatal developmental toxicity. This will involve a huge number of experimental animals, if the data requirements are fulfilled following the current OECD testing guidelines. The application of in vitro assays, such as the embryonic stem cell test (EST), may reduce animal experimentation and resources needed to study the developmental toxicity potencies of petroleum substances. Petroleum substances are UVCBs (substances of Unknown or Variable composition, Complex reaction products and Biological materials) comprising different types of hydrocarbons. The ability of some petroleum substances to induce developmental toxicity has been associated with particular constituents, i.e. the polycyclic aromatic hydrocarbons (PAHs). Heavy petroleum substances like heavy fuel oil (HFO) and untreated lubricating oils contain relatively high concentrations