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GreenScreen® for Safer Chemicals, as a support tool for the EU reach regulation: Identifying chemicals of very high concern (SVHC) and safer alternatives
S124
Abstracts / Toxicology Letters 229S (2014) S40–S252
assessment of chemicals. As data are inherently variable, there is always a risk to select a model that defines a lower confidence bound of the BMD (BMDL) that, contrary to expected, exceeds the true BMD. The aim of this study was to investigate how often and under what circumstances such anomalies occur under current modeling practice. Continuous data were generated from a realistic dose-effect curve by Monte Carlo simulations using four dose groups and a set of five different dose placement scenarios, group sizes between 5 and 50 animals and coefficients of variations of 5–15%. The BMD calculations were conducted using nested exponential models, as most BMD software use nested approaches. “Non-protective” BMDLs (higher than true BMD) were frequently observed, in some scenarios reaching 80%. The phenomenon was mainly related to the selection of the non-sigmoidal exponential model. In conclusion, non-sigmoid models should be used with caution as it may underestimate the risk, illustrating that awareness of the model selection process and sound identification of the pointof-departure is vital for health risk assessment. This observation calls for a revision of the current dose-response model selection paradigm, e.g. as expressed in the expert opinions from European Food Safety Agency (EFSA) and in the current modeling softwares, in which dose–response/effect models are selected based on principle of the Occham’s razor. http://dx.doi.org/10.1016/j.toxlet.2014.06.442 P-2.97 The inhibitory effect of salinity on the photosynthetic machinery of mustard plant and the alleviation of this effect by ethylene Mehar Fatma ∗ , Nafees Ahmad khan Aligarh Muslim University, Aligarh, India Our flora ecosystem is under constant stress from non-optimal soil or surrounding conditions. The mustard plant (Brassica juncea L. cv. Varuna) which remains an important source of edible oil and other nutritional values for the tropical countries and worldwide has a special cultivation requirement for optimal output (relative humidity 62–70%, average day/night temperature of 22/14 ± 3 ◦ C and relatively moist soil). Out of the numerous abiotic stresses, the stress caused by salinity on mustard plant remains a major concern owing to different soil condition in different geographical regions. This salinity adversely affects the plant productivity by exerting inhibitory effects on metabolism of the plant. Excessive amount of salt when assimilated by plant roots and accumulated to toxic levels in the leaves causes premature senescence and reduced photosynthetic capacity of the plant to a level at which the growth of the plant is completely arrested. In order to understand the course of action that give rise to tolerance of salt, the role of ethylene in the alleviation of salinity by S application was evaluated in mustard and was compared the effectiveness with reduced glutathione (GSH) formation in mustard. The contribution of ethylene (200 L/L) in the alleviation of salinity mediated by 200 S (200 mg/kg soil) was worked out. Ethephon (ethylene source) treatment more conspicuously alleviated the effects of 100 mM NaCl when applied to plants grown with 200 S. The present findings can provide the opportunity to develop agronomic practice to reduce the toxic level of salinity. http://dx.doi.org/10.1016/j.toxlet.2014.06.443
P-2.98 Significance of nail mercury concentrations as a biomarker of prenatal methylmercury exposure in relation to segmental hair mercury concentrations at parturition Mineshi Sakamoto 1,∗ , Hing M. Chan 2 , José L. Domingo 3 , Ricardo Oliveira 4 , Katsuyuki Murata 5 1
National Institute for Minamata Disease, Minamata, Japan, University of Ottawa, Ottawa, Canada, 3 Universitat Rovira i Virgili, Reus, Spain, 4 Universidade Federal do Para, Belem, Brazil, 5 Akita University, Akita, Japan
2
We investigated how mercury concentrations in finger- and toenails relate to those in umbilical cord blood and hair of pregnant women at parturition. Mercury concentrations were measured in maternal hair, maternal and umbilical cord blood, finger- and toenail sample pairs collected at parturition. Mercury concentrations in hair were measured in 1-cm incremental segments from the scalp end towards the tip. Among the segmental hair, finger- and toenails Mercury concentration showed the strongest correlation with third segmental hair 3–4 cm from the scalp (r = 0.89 for fingernails and r = 0.85 for toenails). The fingernail usually grows approximately 3 mm per month and is replaced by new one in about 6 months. The growth speed of the toenail is generally 50–80% of that of the fingernail. The growth rate of hair is usually 1 cm per month and hair Mercury concentrations measured at the first 1 cm-segment from the scalp reflect the blood methylmercury concentration or body burden of methylmercury during the last month. Therefore, we can be assume that maternal blood and umbilical cord blood at parturition reflect the methylmercury exposure level at parturition, while the mercury concentrations in the first 1-cm hair segment from scalp reflect methylmercury exposure level at late gestation, and the finger- and toenails reflect the methylmercury exposure level at the early stage of the 3rd trimester of pregnancy. These results show that finger- and toenails can be used as biomarkers for prenatal methylmercury exposure, especially at the early stage of the 3rd trimester of pregnancy. http://dx.doi.org/10.1016/j.toxlet.2014.06.444 P-2.99 GreenScreen® for Safer Chemicals, as a support tool for the EU reach regulation: Identifying chemicals of very high concern (SVHC) and safer alternatives Mouna Zachary ∗ , Ann Marie Gebhart, Margaret Whittaker Toxservice LLCs, Washington, DC, USA In the EU, under REACH regulation, companies are faced with the need to phase out chemicals of very high concern (SVHC) and are challenged to identify and demonstrate the safety of chemical alternatives. These substances are mainly identified by their harmonised classification and labelling according to the CLP Regulation based on their intrinsic hazard properties. GreenScreen® for Safer Chemicals (GS) is proposed here as a tool that can help companies assess and avoid chemicals of high concern under REACH. The GreenScreen® method is transparent and freely available. It includes 18 human and environmental health hazard endpoints for the chemical and its environmental transformation products. The GS criteria are in line with international standards and regulations including the Globally Harmonized System of Classification and Labelling of Chemicals (GHS), OECD testing protocols and the Euro-
Abstracts / Toxicology Letters 229S (2014) S40–S252
pean REACH Regulation. For example, GreenScreen® ’s Benchmark 1 criteria align with the definition of SVHC under REACH. In the US, leading companies, driven by the desire to be competitive and stay ahead of future regulations, are using GreenScreen® . In the EU, the method is included as part of the training programme offered by (SUBSPORT) project. Although GreenScreen® is freely available, technical expertise is required to complete its assessment. ToxServices conducts third-party GreenScreen® assessments as a qualified profiler. To this end, the poster will provide an overview of the GS methodology, its application, uses and associated challenges using examples drawn from GreenScreen® assessments conducted by ToxServices. http://dx.doi.org/10.1016/j.toxlet.2014.06.445 P-2.100 Highly selective MRM3 identification and quantitation of cortisol and cortisone in human hair using an online SPE LC-ESI/MS3 method in negative mode Natalia Soares Quinete ∗ , Jens Bertram, Jessica Lang, Thomas Kraus Institute for Occupational and Social Medicine, Medical Faculty, RWTH Aachen University, Aachen, Germany Hair analysis has been increasingly used to establish longterm biomarkers of exposure to both endogenous and exogenous substances, with a special emphasis on steroidal hormones. Natural corticosteroids, hydrocortisone (cortisol) and cortisone, in human hair have been associated to physiological and psychological strains, anxiety and depression. Hair is a very complex matrix, which might jeopardize analyte detection at low concentrations. A new, highly selective and sensitive method based on fragments of second order, MS3 (MS/MS/MS), was developed and validated on an API 5500 QTrap mass spectrometer for the analysis of hair cortisol and cortisone. An online solid phase extraction (SPE) was performed on a C8 RAM phase following by a reversed-phase C18 column with methanol and 0.02% ammonium hydroxide as mobile phase. The developed method required minimal sample preparation and the injection of only 50 L of sample leading to a LOQ of 2 pg/mg. Good linear responses were observed in the range 2–200 pg/mg (R2 > 0.99) and extraction recoveries ranged between 84–108% and 74–105% for cortisol and cortisone, respectively. Intra- and inter-assay coefficients of variation were between 3.6 and 14.5%. In order to evaluate the applicability of the method, preliminary tests (N = 10) were conducted in 3 cm hair samples (close to scalp) of healthy volunteers from the local university hospital. Average concentrations in hair were 5.5 ± 3.2 and 14.2 ± 9.0 pg/mg for cortisol and cortisone, respectively. Further investigations on cortisol and cortisone as biomarkers for chronic psychological strain will be assessed as a next step. http://dx.doi.org/10.1016/j.toxlet.2014.06.446 P-2.101 Tetrahydroxylated-benzo[a]pyrene isomer analysis after hydrolysis of DNA-adducts isolated from white blood cells Nathalie Grova 1,∗ , Emilie M. Hardy 1 , Guillaume Salquèbre 1 , Henri Schroeder 2 , Brice M.R. Appenzeller 1 1 Laboratory of Analytical Human Biomonitoring-CRP-Santé, Luxembourg, France, 2 Unité de Recherche Animal et Fonctionnalités des Produits Animaux, INRA UC340, Vandoeuvre-lès-Nancy, France
S125
On the assumption of associations between exposure to benzo[a]pyrene and certain health disorders, several recent research studies have been dedicated to the development of strategies aimed at assessing human exposure to this ubiquitous pollutant. As part of these research efforts, a method was designed for the analysis of four tetrahydroxylated-benzo[a]pyrene isomers resulting from the hydrolysis of their respective diol-epoxide precursors which are involved in DNA-adduct formation. The analytical sensitivity necessary to reach environmental levels of concentration was obtained by means of gas chromatography–tandem mass spectrometry. The suitability of the method was firstly evaluated by the analysis of DNA isolated from white blood cells of human volunteers. The results obtained confirm that this method was sufficiently sensitive to detect environmental exposure levels as all the samples measured were above the limit of quantification for benzo[a]pyrene-r-7,t-8,t-9,c-10-tetrahydrotetrol(±) and two of them tested positive for benzo[a]pyrene-r-7,t-8,c-9,c-10tetrahydrotetrol(±). The method was subsequently applied to the analysis of DNA isolated from white blood cells of rats submitted to benzo[a]pyrene-controlled exposure. The study led to the detection of the four targeted tetra-OH-benzo[a]pyrenes as well as of two unknown isomers in all benzo[a]pyrene-treated animals. Interestingly, benzo[a]pyrene-r-7,t-8,c-9,c-10-tetrahydrotetrol(±) proved to be the most abundant isomer in both treated and control animals, followed by benzo[a]pyrene-r-7,t-8,t-9,c-10tetrahydrotetrol(±); which points out to interspecies differences in the nature of the tetrahydroxylated-benzo[a]pyrene isomers formed. These results highlight the need to focus on all the tetrahydroxylated-benzo[a]pyrene isomers, which could be indicators of benzo[a]pyrene-associated toxicity related to an individual’s own metabolism, rather than limit to a single form. http://dx.doi.org/10.1016/j.toxlet.2014.06.447 P-2.102 A novel toolkit for appraising the methodological quality of literature reviews in the toxicological sciences Paul Whaley ∗ , Crispin Halsall Lancaster Environment Centre, Lancaster University, Lancaster, UK Objective: Many conflicting literature reviews have been published about the safety of chemical compounds such as bisphenol-A (BPA). As yet, no guidelines have been published to aid readers in evaluating the methodological quality of these reviews, increasing the challenge of determining which of these reviews are the more credible. We developed a literature review appraisal toolkit to aid production of structured critiques of the validity of literature reviews in the toxicological sciences. Method: We reviewed the literature on best practices in literature review in evidence-based medicine and identified 10 toolkits currently used in medicine for appraising the methodological quality of literature reviews of clinical trials. This allows us to identify a series of domains and signalling questions relevant to review of toxicological literature reviews. We tested the toolkit on three European Food Safety Authority (EFSA) Scientific Opinions on BPA. Results: Nine domains were identified under which a toxicological literature review can be appraised. Within each domain, the reader’s judgments of the utility and validity of methods used in the literature review are elicited via explanatory text and signalling questions. The toolkit is most appropriate to appraisal of hypothesis-driven literature reviews. The EFSA Scientific Opinions were found to have important methodological flaws.
Abstracts / Toxicology Letters 229S (2014) S40–S252
assessment of chemicals. As data are inherently variable, there is always a risk to select a model that defines a lower confidence bound of the BMD (BMDL) that, contrary to expected, exceeds the true BMD. The aim of this study was to investigate how often and under what circumstances such anomalies occur under current modeling practice. Continuous data were generated from a realistic dose-effect curve by Monte Carlo simulations using four dose groups and a set of five different dose placement scenarios, group sizes between 5 and 50 animals and coefficients of variations of 5–15%. The BMD calculations were conducted using nested exponential models, as most BMD software use nested approaches. “Non-protective” BMDLs (higher than true BMD) were frequently observed, in some scenarios reaching 80%. The phenomenon was mainly related to the selection of the non-sigmoidal exponential model. In conclusion, non-sigmoid models should be used with caution as it may underestimate the risk, illustrating that awareness of the model selection process and sound identification of the pointof-departure is vital for health risk assessment. This observation calls for a revision of the current dose-response model selection paradigm, e.g. as expressed in the expert opinions from European Food Safety Agency (EFSA) and in the current modeling softwares, in which dose–response/effect models are selected based on principle of the Occham’s razor. http://dx.doi.org/10.1016/j.toxlet.2014.06.442 P-2.97 The inhibitory effect of salinity on the photosynthetic machinery of mustard plant and the alleviation of this effect by ethylene Mehar Fatma ∗ , Nafees Ahmad khan Aligarh Muslim University, Aligarh, India Our flora ecosystem is under constant stress from non-optimal soil or surrounding conditions. The mustard plant (Brassica juncea L. cv. Varuna) which remains an important source of edible oil and other nutritional values for the tropical countries and worldwide has a special cultivation requirement for optimal output (relative humidity 62–70%, average day/night temperature of 22/14 ± 3 ◦ C and relatively moist soil). Out of the numerous abiotic stresses, the stress caused by salinity on mustard plant remains a major concern owing to different soil condition in different geographical regions. This salinity adversely affects the plant productivity by exerting inhibitory effects on metabolism of the plant. Excessive amount of salt when assimilated by plant roots and accumulated to toxic levels in the leaves causes premature senescence and reduced photosynthetic capacity of the plant to a level at which the growth of the plant is completely arrested. In order to understand the course of action that give rise to tolerance of salt, the role of ethylene in the alleviation of salinity by S application was evaluated in mustard and was compared the effectiveness with reduced glutathione (GSH) formation in mustard. The contribution of ethylene (200 L/L) in the alleviation of salinity mediated by 200 S (200 mg/kg soil) was worked out. Ethephon (ethylene source) treatment more conspicuously alleviated the effects of 100 mM NaCl when applied to plants grown with 200 S. The present findings can provide the opportunity to develop agronomic practice to reduce the toxic level of salinity. http://dx.doi.org/10.1016/j.toxlet.2014.06.443
P-2.98 Significance of nail mercury concentrations as a biomarker of prenatal methylmercury exposure in relation to segmental hair mercury concentrations at parturition Mineshi Sakamoto 1,∗ , Hing M. Chan 2 , José L. Domingo 3 , Ricardo Oliveira 4 , Katsuyuki Murata 5 1
National Institute for Minamata Disease, Minamata, Japan, University of Ottawa, Ottawa, Canada, 3 Universitat Rovira i Virgili, Reus, Spain, 4 Universidade Federal do Para, Belem, Brazil, 5 Akita University, Akita, Japan
2
We investigated how mercury concentrations in finger- and toenails relate to those in umbilical cord blood and hair of pregnant women at parturition. Mercury concentrations were measured in maternal hair, maternal and umbilical cord blood, finger- and toenail sample pairs collected at parturition. Mercury concentrations in hair were measured in 1-cm incremental segments from the scalp end towards the tip. Among the segmental hair, finger- and toenails Mercury concentration showed the strongest correlation with third segmental hair 3–4 cm from the scalp (r = 0.89 for fingernails and r = 0.85 for toenails). The fingernail usually grows approximately 3 mm per month and is replaced by new one in about 6 months. The growth speed of the toenail is generally 50–80% of that of the fingernail. The growth rate of hair is usually 1 cm per month and hair Mercury concentrations measured at the first 1 cm-segment from the scalp reflect the blood methylmercury concentration or body burden of methylmercury during the last month. Therefore, we can be assume that maternal blood and umbilical cord blood at parturition reflect the methylmercury exposure level at parturition, while the mercury concentrations in the first 1-cm hair segment from scalp reflect methylmercury exposure level at late gestation, and the finger- and toenails reflect the methylmercury exposure level at the early stage of the 3rd trimester of pregnancy. These results show that finger- and toenails can be used as biomarkers for prenatal methylmercury exposure, especially at the early stage of the 3rd trimester of pregnancy. http://dx.doi.org/10.1016/j.toxlet.2014.06.444 P-2.99 GreenScreen® for Safer Chemicals, as a support tool for the EU reach regulation: Identifying chemicals of very high concern (SVHC) and safer alternatives Mouna Zachary ∗ , Ann Marie Gebhart, Margaret Whittaker Toxservice LLCs, Washington, DC, USA In the EU, under REACH regulation, companies are faced with the need to phase out chemicals of very high concern (SVHC) and are challenged to identify and demonstrate the safety of chemical alternatives. These substances are mainly identified by their harmonised classification and labelling according to the CLP Regulation based on their intrinsic hazard properties. GreenScreen® for Safer Chemicals (GS) is proposed here as a tool that can help companies assess and avoid chemicals of high concern under REACH. The GreenScreen® method is transparent and freely available. It includes 18 human and environmental health hazard endpoints for the chemical and its environmental transformation products. The GS criteria are in line with international standards and regulations including the Globally Harmonized System of Classification and Labelling of Chemicals (GHS), OECD testing protocols and the Euro-
Abstracts / Toxicology Letters 229S (2014) S40–S252
pean REACH Regulation. For example, GreenScreen® ’s Benchmark 1 criteria align with the definition of SVHC under REACH. In the US, leading companies, driven by the desire to be competitive and stay ahead of future regulations, are using GreenScreen® . In the EU, the method is included as part of the training programme offered by (SUBSPORT) project. Although GreenScreen® is freely available, technical expertise is required to complete its assessment. ToxServices conducts third-party GreenScreen® assessments as a qualified profiler. To this end, the poster will provide an overview of the GS methodology, its application, uses and associated challenges using examples drawn from GreenScreen® assessments conducted by ToxServices. http://dx.doi.org/10.1016/j.toxlet.2014.06.445 P-2.100 Highly selective MRM3 identification and quantitation of cortisol and cortisone in human hair using an online SPE LC-ESI/MS3 method in negative mode Natalia Soares Quinete ∗ , Jens Bertram, Jessica Lang, Thomas Kraus Institute for Occupational and Social Medicine, Medical Faculty, RWTH Aachen University, Aachen, Germany Hair analysis has been increasingly used to establish longterm biomarkers of exposure to both endogenous and exogenous substances, with a special emphasis on steroidal hormones. Natural corticosteroids, hydrocortisone (cortisol) and cortisone, in human hair have been associated to physiological and psychological strains, anxiety and depression. Hair is a very complex matrix, which might jeopardize analyte detection at low concentrations. A new, highly selective and sensitive method based on fragments of second order, MS3 (MS/MS/MS), was developed and validated on an API 5500 QTrap mass spectrometer for the analysis of hair cortisol and cortisone. An online solid phase extraction (SPE) was performed on a C8 RAM phase following by a reversed-phase C18 column with methanol and 0.02% ammonium hydroxide as mobile phase. The developed method required minimal sample preparation and the injection of only 50 L of sample leading to a LOQ of 2 pg/mg. Good linear responses were observed in the range 2–200 pg/mg (R2 > 0.99) and extraction recoveries ranged between 84–108% and 74–105% for cortisol and cortisone, respectively. Intra- and inter-assay coefficients of variation were between 3.6 and 14.5%. In order to evaluate the applicability of the method, preliminary tests (N = 10) were conducted in 3 cm hair samples (close to scalp) of healthy volunteers from the local university hospital. Average concentrations in hair were 5.5 ± 3.2 and 14.2 ± 9.0 pg/mg for cortisol and cortisone, respectively. Further investigations on cortisol and cortisone as biomarkers for chronic psychological strain will be assessed as a next step. http://dx.doi.org/10.1016/j.toxlet.2014.06.446 P-2.101 Tetrahydroxylated-benzo[a]pyrene isomer analysis after hydrolysis of DNA-adducts isolated from white blood cells Nathalie Grova 1,∗ , Emilie M. Hardy 1 , Guillaume Salquèbre 1 , Henri Schroeder 2 , Brice M.R. Appenzeller 1 1 Laboratory of Analytical Human Biomonitoring-CRP-Santé, Luxembourg, France, 2 Unité de Recherche Animal et Fonctionnalités des Produits Animaux, INRA UC340, Vandoeuvre-lès-Nancy, France
S125
On the assumption of associations between exposure to benzo[a]pyrene and certain health disorders, several recent research studies have been dedicated to the development of strategies aimed at assessing human exposure to this ubiquitous pollutant. As part of these research efforts, a method was designed for the analysis of four tetrahydroxylated-benzo[a]pyrene isomers resulting from the hydrolysis of their respective diol-epoxide precursors which are involved in DNA-adduct formation. The analytical sensitivity necessary to reach environmental levels of concentration was obtained by means of gas chromatography–tandem mass spectrometry. The suitability of the method was firstly evaluated by the analysis of DNA isolated from white blood cells of human volunteers. The results obtained confirm that this method was sufficiently sensitive to detect environmental exposure levels as all the samples measured were above the limit of quantification for benzo[a]pyrene-r-7,t-8,t-9,c-10-tetrahydrotetrol(±) and two of them tested positive for benzo[a]pyrene-r-7,t-8,c-9,c-10tetrahydrotetrol(±). The method was subsequently applied to the analysis of DNA isolated from white blood cells of rats submitted to benzo[a]pyrene-controlled exposure. The study led to the detection of the four targeted tetra-OH-benzo[a]pyrenes as well as of two unknown isomers in all benzo[a]pyrene-treated animals. Interestingly, benzo[a]pyrene-r-7,t-8,c-9,c-10-tetrahydrotetrol(±) proved to be the most abundant isomer in both treated and control animals, followed by benzo[a]pyrene-r-7,t-8,t-9,c-10tetrahydrotetrol(±); which points out to interspecies differences in the nature of the tetrahydroxylated-benzo[a]pyrene isomers formed. These results highlight the need to focus on all the tetrahydroxylated-benzo[a]pyrene isomers, which could be indicators of benzo[a]pyrene-associated toxicity related to an individual’s own metabolism, rather than limit to a single form. http://dx.doi.org/10.1016/j.toxlet.2014.06.447 P-2.102 A novel toolkit for appraising the methodological quality of literature reviews in the toxicological sciences Paul Whaley ∗ , Crispin Halsall Lancaster Environment Centre, Lancaster University, Lancaster, UK Objective: Many conflicting literature reviews have been published about the safety of chemical compounds such as bisphenol-A (BPA). As yet, no guidelines have been published to aid readers in evaluating the methodological quality of these reviews, increasing the challenge of determining which of these reviews are the more credible. We developed a literature review appraisal toolkit to aid production of structured critiques of the validity of literature reviews in the toxicological sciences. Method: We reviewed the literature on best practices in literature review in evidence-based medicine and identified 10 toolkits currently used in medicine for appraising the methodological quality of literature reviews of clinical trials. This allows us to identify a series of domains and signalling questions relevant to review of toxicological literature reviews. We tested the toolkit on three European Food Safety Authority (EFSA) Scientific Opinions on BPA. Results: Nine domains were identified under which a toxicological literature review can be appraised. Within each domain, the reader’s judgments of the utility and validity of methods used in the literature review are elicited via explanatory text and signalling questions. The toolkit is most appropriate to appraisal of hypothesis-driven literature reviews. The EFSA Scientific Opinions were found to have important methodological flaws.