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Toxic effects of glyphosate in the embryonic development of the zebrafish (Danio rerio)
Abstracts / Toxicology Letters 258S (2016) S62–S324
were integrated with exposure considerations and physiologicallybased kinetic (PBK) modelling. These play a fundamental role in identifying potential target organs and the related internal concentrations to build a mode-of action hypothesis anchored on adverse outcome pathways (AOPs), where possible. Furthermore, the applicability of threshold of toxicological concern (TTC) and read-across approaches was examined. In vitro to in vivo extrapolation (IVIVE) modelling and refinement supported the final overall conclusion of the safe use, regarding repeated dose toxicity, for the specific chemical and use scenario. The funding from the European Community’s 7th Framework program (FP7/2007-2013) and from Cosmetics Europe is gratefully acknowledged. The abstract does not reflect US EPA policy. Reference Gocht, T., et al., 2015. ALTEX 32, 9–24.
http://dx.doi.org/10.1016/j.toxlet.2016.06.1807 P12-062 Exposure to endocrine disruptors 17alpha-ethinylestradiol and estradiol affect genotoxicity and expression of cytochrome P450 1A1 mediated by a carcinogenic pollutant benzo[a]pyrene in rats “in vivo” Dohalská 1,∗ ,
1, ˇ Klusonová
Mrízová 1 ,
L. Boˇrek Z. I. Draˇcínská 1 , T. Cajthaml 2 , M. Stiborová 1
J.
Holecová 1 ,
H.
1
Department of Biochemistry, Faculty of Science, Charles University in Prague, , Prague 2, Czech Republic 2 Institute of Microbiology, Academy of Science of the Czech Republic, , Prague 4, Czech Republic
Endocrine disruptors (ED) are compounds mimicking or antagonizing the effects of endogenous hormones. Synthetic estrogen 17␣-ethinylestradiol and carcinogen benzo[a]pyrene (BaP) are assigned as exogenous ED. Here, we examined the effect of 17␣ethinylestradiol and estradiol on induction potency of BaP on expression of selected cytochromes P450 and on its genotoxicity in rats “in vivo”. BaP acted as a strong inducer of CYP1A1, but not of the other cytochromes P450 examined. On the contrary, no induction of the tested enzymes by 17␣-ethinylestradiol and estradiol was found. But, when these disruptors were co-administered with BaP, they increased its potency to induce CYP1A1. Further, while estradiol and 17␣-ethinylestradiol did not form covalent adducts with DNA, they influence the formation of two BaP-derived covalent DNA adducts in rat organs. The major adduct, was formed in all tested organs of rats treated with BaP alone or together with 17␣ethinylestradiol or estradiol. In contrast, the minor adduct was detectable only in rat lung. Exposure of rats to BaP with 17␣ethinylestradiol led to a decrease in levels of the major adduct in all rat organs, but had no effect on formation of minor adduct. A decrease in levels of the major adduct in liver and kidney was also caused by co-treatment of rats with BaP and estradiol, but this exposure increased formation of both adducts in lung. The results found indicate that genotoxic activity of BaP in rats “in vivo” is modulated by estradiol and its synthetic derivative 17␣ethinylestradiol. “Supported by GACR (15-02328S) and UNCE (204025/20)”. http://dx.doi.org/10.1016/j.toxlet.2016.06.1808
S225
P12-063 Relevance of particle overload rat lung tumor responses for human risk assessment D.B. Warheit 1,∗ , R. Kreiling 2 , L. Levy 3 1
Chemours Company Clariant Produckte (DE) GmbH 3 Cranfield University 2
The relevance of particle-overload related lung tumors in rats for human risk assessment following chronic inhalation exposures to poorly soluble particulates (PSP) has been a controversial issue for >30 years. In 1998, an ILSI Working Group addressed this topic and agreed that the findings of PSP overload lung tumors were unique to rats and there was no evidence of human lung cancers in PSP workers; but reached no full consensus on regulatory impacts. Since the publication of the ILSI report, there have been important new data which impact decisions on the human relevance issue. Accordingly, we focused on five significant and interrelated factors which provide important insights on fundamental differences in pulmonary responses between particle overload-exposed rats and occupationally-exposed humans. The 5 factors are described by the following issues: (1) Interspecies differences in lung responses of rats vs. other rodents, triggering different adverse outcome pathways (AOPs); (2) Interspecies differences in particle kinetics of rats vs nonhuman primates and humans triggering differential particle-related pulmonary responses. (3) Advanced and updated human respiratory tract models allowing more informed particle translocation/retention estimates. (4) Differences in morphologies and characterization of rat vs. human pulmonary tumor types and locations within the respiratory tract. (5) Comprehensive in-depth analysis of available epidemiological data from PSP production workers demonstrating no correlation between particle exposures and lung cancers or other non-malignant respiratory diseases. Accordingly, the only plausible conclusion is that results from chronic particle-overload inhalation studies with PSPs in rats have no relevance for lung cancer risks in humans. http://dx.doi.org/10.1016/j.toxlet.2016.06.1810 P12-064 Toxic effects of glyphosate in the embryonic development of the zebrafish (Danio rerio) N. Ayala Soldado ∗ , A.J. Lora Benítez, A.M. Molina López, R. Jiménez Guerrero, A. De Frías Medina, M. Moyano Salvago Department of Pharmacology, Toxicology and Forensic Medicine, Córdoba University, Córdoba, Spain Glyphosate is a water-soluble, non-selective and broadspectrum systemic herbicide whose active component is N(phosphonomethyl) glycine. It has been largely used to control weeds in wetlands. On March 2015, the International Agency for Research on Cancer (IARC) assessed the carcinogenicity of the herbicide glyphosate. As a result, glyphosate has been classified as probably carcinogenic to humans (Group 2A). The aim of this work was the evaluation of the effects of glyphosate exposure in zebrafish (Danio rerio) embryo as experimental model. Eggs
S226
Abstracts / Toxicology Letters 258S (2016) S62–S324
production and Zebrafish embryo toxicity assay (ZETA) test was conducted in accordance with OECD specific guidelines. Zebrafish embryos were exposed to the test substance with semistatic technique (2.5, 5.5, 12, 25 and 50 ppm). Apical sublethal and endpoints of acute toxicity in zebrafish embryos were checked at 5, 24, 36, 48, 72 and 96 h post-fertilization. Mortality of embryos was found to be around 10% at concentrations levels of 5.5, 12 and 25 ppm of glyphosate, whereas the highest mortality levels were found at the concentration of 50 ppm of glyphosate, with around 95% of embryos’ mortality, therefore exceeding the maximum rates established by the OECD. In sublethal effect studies, eggs exposed to ≥5.5 ppm of glyphosate presented a premature hatching rate at 36 h post-fertilization, with the highest hatching rate (40%) corresponding to 25 ppm of glyphosate. Completion of morula and gastrula was found in all concentrations assayed, but mainly in 5.5 and 12 ppm concentrations with around 15% of embryos affected. http://dx.doi.org/10.1016/j.toxlet.2016.06.1811 P12-065 Are inhaled iron oxides lung carcinogens? S. Hubbard 1,∗ , C. Pease 2 , T. Rücker 3 , T. Birk 4 1
SAHCo Ltd, Regulatory Toxicology Consultancy, Chester, UK Ramboll ENVIRON UK Limited, Leeds, UK 3 Ramboll ENVIRON Germany GmbH, München, Germany 4 Ramboll ENVIRON Germany GmbH, Essen, Germany 2
A body of evidence will be presented regarding epidemiology, toxicity and lung bioavailability as to whether iron oxides are human lung carcinogens. Observed lung tumours on rats result from a generic particle overload effect and local inflammation that is rat-specific under the dosing conditions of intratracheal instillation. This proposed mode of action is not relevant to real-life human exposure. However emerging differences are seen between ‘bulk’ iron oxides (particles where 70% are >100 nm) and ‘nano’ iron oxides (.95% fall on the range 1–100 nm). Evidence suggests ‘bulk’ iron oxides are not genotoxic/mutagenic, whereas the data for ‘nano’ iron oxide is conflicting. Genotoxicity was not observed in an in vivo genotoxicity study on ‘nano’ iron oxides via inhalation. Hypothetically, with the larger surface area of ‘nano’ iron oxide particulates, toxicity could be exerted via the generation of reactive oxygen species (ROS) in the cell. However, ROS generation as a basis for explaining rodent lung tumourigenicity is only apparent if free iron from intracellular ‘nano’ scale iron oxide becomes bioavailable at significant levels inside the cell. This would not be expected from ‘bulk’ iron oxide particles. Furthermore, human epidemiological evidence from a number of studies suggests that iron oxide is not a human carcinogen, and therefore based upon the complete weight of evidence, we conclude that ‘bulk’ iron oxides are not human carcinogens. http://dx.doi.org/10.1016/j.toxlet.2016.06.1812
P12-066 Use of toxicokinetic data in bioaccumulation assessment J. Peltola Thies 1 , T.A. Lantto 1 , S. Lapenna 1,∗ , G. Muller 1 , Z. Kloslova 1 , J. Caley 1 , S. Andersen 2 , M. Gudbrandsen 2 , T. Hofer 3 , O. Myhre 3 , E. Verbruggen 4 , P. Lepper 1 1
European Chemicals Agency, Helsinki, Finland Norwegian Environment Agency, Trondheim, Norway 3 Norwegian Institute of Public Health, Oslo, Norway 4 National Institute for Public Health and the Environment, Bilthoven, The Netherlands 2
ECHA has observed that a high number of substances are more likely to bioaccumulate in air-breathing organisms than in aquatic species based on screening data (Koa and Kow). The numerical bioaccumulation (B) criteria defined in the REACH regulation do not sufficiently address the bioaccumulation potential of such substances. Elimination half-lives have been proposed as a potential alternative or complementary metric to assess bioaccumulative properties of substances in air-breathing organisms. With a view to explore the potential of using this type of data, we hereby report the generation and preliminary analyses of a data compilation of mammalian elimination half-life data complemented with other available toxicokinetic data. The data compilation consists of both known bioaccumulative substances such as those identified as PBT/vPvB under REACH, and/or POPs under the Stockholm Convention and substances with various bioaccumulation potential such as substances used in veterinary medicines, pesticides and biocides. Elimination half-life data from OECD Toolbox and toxicokinetic data from REACH registration dossiers were also utilised when applicable. Further work is in progress. The data compilation information has been used to analyse the relationships between the chemical structures and elimination half-lives, taking into account the variation in study conditions. The presentation will give an overview of the current work and will discuss the findings from the perspective of assessment approach development. http://dx.doi.org/10.1016/j.toxlet.2016.06.1813 P12-067 Oxytetracycline effects in aquatic and terrestrial biotic systems A. Anadón 1,∗ , J.L. Rodriguez 1 , C. Fernández 2 , G. Carbonell 2 , J. Pro 2 1 Department of Toxicology and Pharmacology, Faculty of Veterinary Medicine, Universidad Complutense de Madrid, 28040 Madrid, Spain 2 Department of Environment, Laboratory for Ecotoxicology, INIA, 28040 Madrid, Spain
The presence of antibiotics in the environment and their wide dissemination at low concentrations, mainly in the aquatic environment has been detected into surface waters (rivers, lakes, streams, and estuaries). The result from antibiotics release into environment is antibiotic-resistant bacteria apparition. Antibiotics on the biosphere may have impact not only for human health but also on the structure and activity of aquatic and terrestrial systems. Because oxytetracycline (OTC), antibiotic widely used in human and veterinary medicine against Gram-positive, Gram-negative, “Spirochetes”, “Mycoplasmas”, “Chlamydiae” and “Rickettsiae” microorganisms, could cause an environmental impact, this work tested OTC in aquatic and terrestrial environments, through the use of ecotoxicological assays proposed by the OECD assessing
were integrated with exposure considerations and physiologicallybased kinetic (PBK) modelling. These play a fundamental role in identifying potential target organs and the related internal concentrations to build a mode-of action hypothesis anchored on adverse outcome pathways (AOPs), where possible. Furthermore, the applicability of threshold of toxicological concern (TTC) and read-across approaches was examined. In vitro to in vivo extrapolation (IVIVE) modelling and refinement supported the final overall conclusion of the safe use, regarding repeated dose toxicity, for the specific chemical and use scenario. The funding from the European Community’s 7th Framework program (FP7/2007-2013) and from Cosmetics Europe is gratefully acknowledged. The abstract does not reflect US EPA policy. Reference Gocht, T., et al., 2015. ALTEX 32, 9–24.
http://dx.doi.org/10.1016/j.toxlet.2016.06.1807 P12-062 Exposure to endocrine disruptors 17alpha-ethinylestradiol and estradiol affect genotoxicity and expression of cytochrome P450 1A1 mediated by a carcinogenic pollutant benzo[a]pyrene in rats “in vivo” Dohalská 1,∗ ,
1, ˇ Klusonová
Mrízová 1 ,
L. Boˇrek Z. I. Draˇcínská 1 , T. Cajthaml 2 , M. Stiborová 1
J.
Holecová 1 ,
H.
1
Department of Biochemistry, Faculty of Science, Charles University in Prague, , Prague 2, Czech Republic 2 Institute of Microbiology, Academy of Science of the Czech Republic, , Prague 4, Czech Republic
Endocrine disruptors (ED) are compounds mimicking or antagonizing the effects of endogenous hormones. Synthetic estrogen 17␣-ethinylestradiol and carcinogen benzo[a]pyrene (BaP) are assigned as exogenous ED. Here, we examined the effect of 17␣ethinylestradiol and estradiol on induction potency of BaP on expression of selected cytochromes P450 and on its genotoxicity in rats “in vivo”. BaP acted as a strong inducer of CYP1A1, but not of the other cytochromes P450 examined. On the contrary, no induction of the tested enzymes by 17␣-ethinylestradiol and estradiol was found. But, when these disruptors were co-administered with BaP, they increased its potency to induce CYP1A1. Further, while estradiol and 17␣-ethinylestradiol did not form covalent adducts with DNA, they influence the formation of two BaP-derived covalent DNA adducts in rat organs. The major adduct, was formed in all tested organs of rats treated with BaP alone or together with 17␣ethinylestradiol or estradiol. In contrast, the minor adduct was detectable only in rat lung. Exposure of rats to BaP with 17␣ethinylestradiol led to a decrease in levels of the major adduct in all rat organs, but had no effect on formation of minor adduct. A decrease in levels of the major adduct in liver and kidney was also caused by co-treatment of rats with BaP and estradiol, but this exposure increased formation of both adducts in lung. The results found indicate that genotoxic activity of BaP in rats “in vivo” is modulated by estradiol and its synthetic derivative 17␣ethinylestradiol. “Supported by GACR (15-02328S) and UNCE (204025/20)”. http://dx.doi.org/10.1016/j.toxlet.2016.06.1808
S225
P12-063 Relevance of particle overload rat lung tumor responses for human risk assessment D.B. Warheit 1,∗ , R. Kreiling 2 , L. Levy 3 1
Chemours Company Clariant Produckte (DE) GmbH 3 Cranfield University 2
The relevance of particle-overload related lung tumors in rats for human risk assessment following chronic inhalation exposures to poorly soluble particulates (PSP) has been a controversial issue for >30 years. In 1998, an ILSI Working Group addressed this topic and agreed that the findings of PSP overload lung tumors were unique to rats and there was no evidence of human lung cancers in PSP workers; but reached no full consensus on regulatory impacts. Since the publication of the ILSI report, there have been important new data which impact decisions on the human relevance issue. Accordingly, we focused on five significant and interrelated factors which provide important insights on fundamental differences in pulmonary responses between particle overload-exposed rats and occupationally-exposed humans. The 5 factors are described by the following issues: (1) Interspecies differences in lung responses of rats vs. other rodents, triggering different adverse outcome pathways (AOPs); (2) Interspecies differences in particle kinetics of rats vs nonhuman primates and humans triggering differential particle-related pulmonary responses. (3) Advanced and updated human respiratory tract models allowing more informed particle translocation/retention estimates. (4) Differences in morphologies and characterization of rat vs. human pulmonary tumor types and locations within the respiratory tract. (5) Comprehensive in-depth analysis of available epidemiological data from PSP production workers demonstrating no correlation between particle exposures and lung cancers or other non-malignant respiratory diseases. Accordingly, the only plausible conclusion is that results from chronic particle-overload inhalation studies with PSPs in rats have no relevance for lung cancer risks in humans. http://dx.doi.org/10.1016/j.toxlet.2016.06.1810 P12-064 Toxic effects of glyphosate in the embryonic development of the zebrafish (Danio rerio) N. Ayala Soldado ∗ , A.J. Lora Benítez, A.M. Molina López, R. Jiménez Guerrero, A. De Frías Medina, M. Moyano Salvago Department of Pharmacology, Toxicology and Forensic Medicine, Córdoba University, Córdoba, Spain Glyphosate is a water-soluble, non-selective and broadspectrum systemic herbicide whose active component is N(phosphonomethyl) glycine. It has been largely used to control weeds in wetlands. On March 2015, the International Agency for Research on Cancer (IARC) assessed the carcinogenicity of the herbicide glyphosate. As a result, glyphosate has been classified as probably carcinogenic to humans (Group 2A). The aim of this work was the evaluation of the effects of glyphosate exposure in zebrafish (Danio rerio) embryo as experimental model. Eggs
S226
Abstracts / Toxicology Letters 258S (2016) S62–S324
production and Zebrafish embryo toxicity assay (ZETA) test was conducted in accordance with OECD specific guidelines. Zebrafish embryos were exposed to the test substance with semistatic technique (2.5, 5.5, 12, 25 and 50 ppm). Apical sublethal and endpoints of acute toxicity in zebrafish embryos were checked at 5, 24, 36, 48, 72 and 96 h post-fertilization. Mortality of embryos was found to be around 10% at concentrations levels of 5.5, 12 and 25 ppm of glyphosate, whereas the highest mortality levels were found at the concentration of 50 ppm of glyphosate, with around 95% of embryos’ mortality, therefore exceeding the maximum rates established by the OECD. In sublethal effect studies, eggs exposed to ≥5.5 ppm of glyphosate presented a premature hatching rate at 36 h post-fertilization, with the highest hatching rate (40%) corresponding to 25 ppm of glyphosate. Completion of morula and gastrula was found in all concentrations assayed, but mainly in 5.5 and 12 ppm concentrations with around 15% of embryos affected. http://dx.doi.org/10.1016/j.toxlet.2016.06.1811 P12-065 Are inhaled iron oxides lung carcinogens? S. Hubbard 1,∗ , C. Pease 2 , T. Rücker 3 , T. Birk 4 1
SAHCo Ltd, Regulatory Toxicology Consultancy, Chester, UK Ramboll ENVIRON UK Limited, Leeds, UK 3 Ramboll ENVIRON Germany GmbH, München, Germany 4 Ramboll ENVIRON Germany GmbH, Essen, Germany 2
A body of evidence will be presented regarding epidemiology, toxicity and lung bioavailability as to whether iron oxides are human lung carcinogens. Observed lung tumours on rats result from a generic particle overload effect and local inflammation that is rat-specific under the dosing conditions of intratracheal instillation. This proposed mode of action is not relevant to real-life human exposure. However emerging differences are seen between ‘bulk’ iron oxides (particles where 70% are >100 nm) and ‘nano’ iron oxides (.95% fall on the range 1–100 nm). Evidence suggests ‘bulk’ iron oxides are not genotoxic/mutagenic, whereas the data for ‘nano’ iron oxide is conflicting. Genotoxicity was not observed in an in vivo genotoxicity study on ‘nano’ iron oxides via inhalation. Hypothetically, with the larger surface area of ‘nano’ iron oxide particulates, toxicity could be exerted via the generation of reactive oxygen species (ROS) in the cell. However, ROS generation as a basis for explaining rodent lung tumourigenicity is only apparent if free iron from intracellular ‘nano’ scale iron oxide becomes bioavailable at significant levels inside the cell. This would not be expected from ‘bulk’ iron oxide particles. Furthermore, human epidemiological evidence from a number of studies suggests that iron oxide is not a human carcinogen, and therefore based upon the complete weight of evidence, we conclude that ‘bulk’ iron oxides are not human carcinogens. http://dx.doi.org/10.1016/j.toxlet.2016.06.1812
P12-066 Use of toxicokinetic data in bioaccumulation assessment J. Peltola Thies 1 , T.A. Lantto 1 , S. Lapenna 1,∗ , G. Muller 1 , Z. Kloslova 1 , J. Caley 1 , S. Andersen 2 , M. Gudbrandsen 2 , T. Hofer 3 , O. Myhre 3 , E. Verbruggen 4 , P. Lepper 1 1
European Chemicals Agency, Helsinki, Finland Norwegian Environment Agency, Trondheim, Norway 3 Norwegian Institute of Public Health, Oslo, Norway 4 National Institute for Public Health and the Environment, Bilthoven, The Netherlands 2
ECHA has observed that a high number of substances are more likely to bioaccumulate in air-breathing organisms than in aquatic species based on screening data (Koa and Kow). The numerical bioaccumulation (B) criteria defined in the REACH regulation do not sufficiently address the bioaccumulation potential of such substances. Elimination half-lives have been proposed as a potential alternative or complementary metric to assess bioaccumulative properties of substances in air-breathing organisms. With a view to explore the potential of using this type of data, we hereby report the generation and preliminary analyses of a data compilation of mammalian elimination half-life data complemented with other available toxicokinetic data. The data compilation consists of both known bioaccumulative substances such as those identified as PBT/vPvB under REACH, and/or POPs under the Stockholm Convention and substances with various bioaccumulation potential such as substances used in veterinary medicines, pesticides and biocides. Elimination half-life data from OECD Toolbox and toxicokinetic data from REACH registration dossiers were also utilised when applicable. Further work is in progress. The data compilation information has been used to analyse the relationships between the chemical structures and elimination half-lives, taking into account the variation in study conditions. The presentation will give an overview of the current work and will discuss the findings from the perspective of assessment approach development. http://dx.doi.org/10.1016/j.toxlet.2016.06.1813 P12-067 Oxytetracycline effects in aquatic and terrestrial biotic systems A. Anadón 1,∗ , J.L. Rodriguez 1 , C. Fernández 2 , G. Carbonell 2 , J. Pro 2 1 Department of Toxicology and Pharmacology, Faculty of Veterinary Medicine, Universidad Complutense de Madrid, 28040 Madrid, Spain 2 Department of Environment, Laboratory for Ecotoxicology, INIA, 28040 Madrid, Spain
The presence of antibiotics in the environment and their wide dissemination at low concentrations, mainly in the aquatic environment has been detected into surface waters (rivers, lakes, streams, and estuaries). The result from antibiotics release into environment is antibiotic-resistant bacteria apparition. Antibiotics on the biosphere may have impact not only for human health but also on the structure and activity of aquatic and terrestrial systems. Because oxytetracycline (OTC), antibiotic widely used in human and veterinary medicine against Gram-positive, Gram-negative, “Spirochetes”, “Mycoplasmas”, “Chlamydiae” and “Rickettsiae” microorganisms, could cause an environmental impact, this work tested OTC in aquatic and terrestrial environments, through the use of ecotoxicological assays proposed by the OECD assessing