- Email: [email protected]
Different HBCD stereoisomers are metabolized differently
Abstracts / Toxicology Letters 196S (2010) S1–S36
relating between the effects of single substances and their mixtures. In experimental rat tests, different biochemical parameters were used as criteria for assessment of mixture toxicity, while in Daphnia magna and Stenocypris werneri we used immobilization following exposure to toxicants. In both cases, interaction was expressed in terms of “additive”, “potentiatian” and “antagonism”; based on the numerical value of the estimated “Interaction Indices”. In mixtures contained pesticides and heavy metals, their interactive toxicity based on alteration in some biochemical parameters in rat’s sera, was accounted to “antagonism” for the majority of the tested combinations. On the other side, the results of joint action estimated by cotoxicity factor (TF) method, in Daphnia and Stenocypris bioassays, showed an agreement with the results of “concentration addition (CA) method” accounting to 93–100% and 79% with “independent action (IA) method”. Therefore, the TF model, as a simple method, was suggested to assess the interactive toxicity of binary mixtures having either similar or dissimilar mode of action, as well as to be used in conjunction with the methods based on CA and IA models. Also, by using a novel Daphnia—bioassay method, it was possible to assess the potential toxicity of mixtures of contaminants (e.g., pesticides and heavy metals) in vegetables, and to categorize toxic hazards in six definite ratings. doi:10.1016/j.toxlet.2010.03.147 HBCD as a Replacement Flame Retardant; Another POP S27-1 Hexabromocyclododecane (HBCD) complex chemistry: Detection and analytical methods A. Covaci 1 , M. Abdallah 2 , L. Roosens 1 , S. Harrad 2 University of Antwerp, Belgium, 2 University of Birmingham, United Kingdom
1
An overview of the analytical methodologies for the determination of HBCD isomers, including sample preparation and instrumental approaches, is presented. HBCD isomers have been measured recently in a variety of matrices including biological tissues, environmental samples, and textiles. Traditionally, HBCD has been determined via GC-ECNI/MS. However, GC techniques have a number of serious limitations, such as interconversion of the HBCD isomers above 160 °C, degradation in dirty injection systems or above 240 °C, and weaker structural identification in ECNI/MS. In contrast, alpha-, beta- and gamma-HBCD isomers can be separated easily using reversed-phase liquid chromatography and determined by mass spectrometry (LC–MS/MS). Furthermore, HBCD stereoisomers can be resolved on an enantioselective LC column. Isomer-specific data are useful to establish profiles and to understand the sources, distribution and fate of individual isomers. Two additional isomers (delta-HBCD and epsilon-HBCD) have been isolated from technical products with delta-HBCD identified recently in fish. However, LC–MS analysis is prone to ion suppression resulting in decreased sensitivity. This problem is avoided by thorough sample clean-up to remove interfering components and by using 13C-labelled and 2Hlabelled HBCDs as internal standards to compensate for potential variations in sensitivity during and between sample runs. The comparability of results obtained using GC–MS or LC–MS has been investigated in a few studies, but results are inconclusive due to substantial variation in the methodologies employed and in the type of samples analysed (fish, dust). Until now, only a limited number of intercalibration studies have been performed for HBCD. Laboratories were able to determine satisfactorily total
S33
HBCD in marine samples (RSD < 35%). However, determination of low HBCD concentrations should be improved. There is also a need for environmentally relevant reference materials certified for HBCDs that can be used for method validation. doi:10.1016/j.toxlet.2010.03.149
S27-2 Hexabromocyclododecane (HBCD) flame retardant in the environment, biota and humans: Sterioisomeric paradox R. Letcher Government of Canada, Canada 1,2,5,6,9,10-Hexabromocyclododecane (HBCD) is a brominated flame retardant (BFR) that is incorporated into a wide range of consumer products to delay the ignition of combustion and thus suppress flammability. A main application is the incorporation of HBCD in polystyrene foam boards used for thermal insulation by the construction industry. HBCD ranks among the largest massproduced BFRs worldwide, with a recent global demand reported to be ∼17,000 metric tons/year. Four technical mixtures of HBCD are currently available for commercial use and are comprised of three major sterioisomers, alpha-HBCD (∼10%), beta-HBCD (∼9%) and gamma-HBCD (∼81%), and minor impurities of other stereoisomers. These structural isomers of HBCD can also possess chirality and thus the number of isomers is even larger. Since HBCD is an additive flame retardant, it is thus pre-disposed to leaching out of commercial products, which leads to contamination and subsequent HBCD accumulation and persistence in the environment. The potential for HBCD to accumulate in organisms and biomagnify within a food web was initially observed in fish and lower aquatic organisms, with subsequent evidence of bioaccumulation in top feeding fish, birds and mammals including humans. Stereoisomer-specific processes have been shown to result in, e.g., the preferential accumulation of the alpha-isomer in biotic samples relative to the dominance of the gamma-isomer in commercial HBCD mixtures. Recent studies have demonstrated that the betaand gamma-HBCD are significantly metabolized in vitro and in vivo assay while the alpha-isomer was not, where isomer-specific (metabolic) biotransformation is the most likely explanation for almost exclusive accumulation of alpha-HBCD in biota. The present study will review and summarize the current state-of-knowledge with respect to stereoisomer-specific presence, bioaccumulation, fate and toxicokinetics of HBCDs in biota and their food webs including in humans, mammals, birds and fish, as well as results from captive studies with HBCD-dosed animals. doi:10.1016/j.toxlet.2010.03.150
S27-3 Different HBCD stereoisomers are metabolized differently H. Hakk Biosciences Research Laboratory, United States The requirement of flame retardancy in most consumer products has resulted in a vast array of chemical compounds that unfortunately have toxic effects and are environmental pollutants. Hexabromocyclododecane (HBCD) is an additive brominated flame retardant applied to extruded and high-impact polystyrene foams (<2.5% by weight) used as thermal insulation in buildings, and HBCD is the only suitable flame retardant for these appli-
S34
Abstracts / Toxicology Letters 196S (2010) S1–S36
cations. HBCD the second highest-volume BFR used in Europe. HBCD was first detected in fish and sediment samples in Sweden in 1998, and have been detected in a variety of environmental samples. Chemically HBCD is a complex mixture of stereoisomers, and the commercial mixture contains a majority of the gammaHBCD (∼81%) with minor amounts of alpha-HBCD (∼10%) and beta-HBCD (∼9%); however, alpha-HBCD is the most abundant isomer reported in biota. Two possible interpretations of these results would be that alpha-HBCD is the most bioavailable stereoisomer, or that metabolic biotransformations favor beta- and gamma-HBCD over alpha-HBCD. This report will present recent laboratory results that lend support to the later hypothesis. Stereoisomer-specific biotransformations have been observed in mice in which gammaHBCD is readily converted to both beta- and alpha-HBCD, but where alpha-HBCD is resistant to such isomerization. The high molecular weight and aliphatic chemical structure of HBCD lend themselves to an intriguing metabolic pathway, and this will be discussed in context of the known metabolic pathways for PCDD/Fs, PBDEs, or PCBs. Possible conjugation of HBCD, urinary volatiles, and involvement of carrier protein systems will be discussed, as well as observed debromination, and oxidative and reductive metabolic events. doi:10.1016/j.toxlet.2010.03.151
S27-4 Toxicity and toxicokinetics of hexabromocylododecane D. Szabo University of North Carolina, United States Hexabromocyclododecane (HBCD) is a brominated flame retardant added to consumer products for over 40 years. Advances in the understanding of HBCD complex chemistry and methods of detection have identified the HBCD commercial mixture to be composed of three main diastereomers; alpha, beta and gamma. We know these stereoisomers to have different chemical and physical properties but differences in biological effects are still yet to be determined. Toxicity studies on HBCD are limited. Furthermore, no stereoisomer toxicity or toxicokinetic study has been reported in any mammalian organism. Toxicity studies performed to date are after exposure to the commercial mixture. Characteristic end points in animal toxicity have focused on hepatotoxicity, thyroid disruption, developmental neurotoxicity and carcinogenisis. Behavioral effects have been observed in mice after administration during a critical period after birth. As the scientific knowledge of today is based on the commercial HBCD mixture, the possible consumer health risk from HBCD appears concerning. However, the presence of many and important data gaps, including those in stereoisomer profiles focusing on carcinogenicity, reproduction, and developmental toxicity, as well as additional routes of exposure, may further heighten this concern as we may currently be underestimating the toxicity effects of HBCD. This report aims to review the current toxicity studies of HBCD and present recent in vivo laboratory results in the kinetics of individual stereoisomers.
S28 Combined Exposures to Multiple Chemicals in Risk Assessment: A Pragmatic Approach
S28-1 Considering combined exposures in risk assessment: Recent developments, opportunities and challenges B. Meek. University of Ottawa, Canada Traditionally, consideration of effects of exposures to multiple chemicals has been based on the toxicity of similar combinations of chemicals or on the toxicity of components. However, the paucity of available data has limited the application of these approaches. As a basis to draw more efficiently on available information including predictive approaches, terminology and more efficient methodology for assessing the impact of combined exposures to multiple chemicals has recently been considered in a project of the World Health Organization (WHO) Programme on Chemical Safety (PCS). This project, which is part of the WHO initiative on Harmonization of Approaches to the Assessment of Risk from Exposure to Chemicals, is being coordinated with additional initiatives of the International Life Sciences Institute (ILSI) and the European Centre for Ecotoxicology and Toxicology of Chemicals. Following an international workshop to review advances in this area, a draft framework has been developed which includes delineation of explicit criteria for initial consideration of the bounds of appropriate grouping for assessment of combined exposures, followed by stepwise consideration of both exposure and hazard in several tiers of increasingly data-informed analyses. These analyses build on recent developments in assessment, incorporating predictive approaches in early tiers and increasingly refined, more data-informed mode of action and probabilistic analyses in later tiers. The extent of assessment and nature of recommendations for generation of additional data are dependent upon the extent of the knowledge base, the magnitude of public health concern and implications of potential risk management decisions. Recommendations regarding terminology and the status of development of the framework, its content, review and application will be described and an example provided. Critical areas for future effort will also be described. doi:10.1016/j.toxlet.2010.03.154
S28-2 Illustration of the WHO combined exposures framework: A tiered and integrative approach to exposure and hazard M. Van Raaij Center of Substances and Integrated Risk Assessment
doi:10.1016/j.toxlet.2010.03.152 Within the WHO/IPCS harmonization project special attention was paid to the issue of combined exposures to chemicals. This reflects single chemical–multiple routes, multiple chemicals–single route, as well as multiple chemicals–multiple routes exposures. A draft framework was established by a WHO working group. The operation of the framework has been illustrated by two case studies and public comments were received and accommodated into the version presented (see contribution SY28-1 for general backgrounds).
relating between the effects of single substances and their mixtures. In experimental rat tests, different biochemical parameters were used as criteria for assessment of mixture toxicity, while in Daphnia magna and Stenocypris werneri we used immobilization following exposure to toxicants. In both cases, interaction was expressed in terms of “additive”, “potentiatian” and “antagonism”; based on the numerical value of the estimated “Interaction Indices”. In mixtures contained pesticides and heavy metals, their interactive toxicity based on alteration in some biochemical parameters in rat’s sera, was accounted to “antagonism” for the majority of the tested combinations. On the other side, the results of joint action estimated by cotoxicity factor (TF) method, in Daphnia and Stenocypris bioassays, showed an agreement with the results of “concentration addition (CA) method” accounting to 93–100% and 79% with “independent action (IA) method”. Therefore, the TF model, as a simple method, was suggested to assess the interactive toxicity of binary mixtures having either similar or dissimilar mode of action, as well as to be used in conjunction with the methods based on CA and IA models. Also, by using a novel Daphnia—bioassay method, it was possible to assess the potential toxicity of mixtures of contaminants (e.g., pesticides and heavy metals) in vegetables, and to categorize toxic hazards in six definite ratings. doi:10.1016/j.toxlet.2010.03.147 HBCD as a Replacement Flame Retardant; Another POP S27-1 Hexabromocyclododecane (HBCD) complex chemistry: Detection and analytical methods A. Covaci 1 , M. Abdallah 2 , L. Roosens 1 , S. Harrad 2 University of Antwerp, Belgium, 2 University of Birmingham, United Kingdom
1
An overview of the analytical methodologies for the determination of HBCD isomers, including sample preparation and instrumental approaches, is presented. HBCD isomers have been measured recently in a variety of matrices including biological tissues, environmental samples, and textiles. Traditionally, HBCD has been determined via GC-ECNI/MS. However, GC techniques have a number of serious limitations, such as interconversion of the HBCD isomers above 160 °C, degradation in dirty injection systems or above 240 °C, and weaker structural identification in ECNI/MS. In contrast, alpha-, beta- and gamma-HBCD isomers can be separated easily using reversed-phase liquid chromatography and determined by mass spectrometry (LC–MS/MS). Furthermore, HBCD stereoisomers can be resolved on an enantioselective LC column. Isomer-specific data are useful to establish profiles and to understand the sources, distribution and fate of individual isomers. Two additional isomers (delta-HBCD and epsilon-HBCD) have been isolated from technical products with delta-HBCD identified recently in fish. However, LC–MS analysis is prone to ion suppression resulting in decreased sensitivity. This problem is avoided by thorough sample clean-up to remove interfering components and by using 13C-labelled and 2Hlabelled HBCDs as internal standards to compensate for potential variations in sensitivity during and between sample runs. The comparability of results obtained using GC–MS or LC–MS has been investigated in a few studies, but results are inconclusive due to substantial variation in the methodologies employed and in the type of samples analysed (fish, dust). Until now, only a limited number of intercalibration studies have been performed for HBCD. Laboratories were able to determine satisfactorily total
S33
HBCD in marine samples (RSD < 35%). However, determination of low HBCD concentrations should be improved. There is also a need for environmentally relevant reference materials certified for HBCDs that can be used for method validation. doi:10.1016/j.toxlet.2010.03.149
S27-2 Hexabromocyclododecane (HBCD) flame retardant in the environment, biota and humans: Sterioisomeric paradox R. Letcher Government of Canada, Canada 1,2,5,6,9,10-Hexabromocyclododecane (HBCD) is a brominated flame retardant (BFR) that is incorporated into a wide range of consumer products to delay the ignition of combustion and thus suppress flammability. A main application is the incorporation of HBCD in polystyrene foam boards used for thermal insulation by the construction industry. HBCD ranks among the largest massproduced BFRs worldwide, with a recent global demand reported to be ∼17,000 metric tons/year. Four technical mixtures of HBCD are currently available for commercial use and are comprised of three major sterioisomers, alpha-HBCD (∼10%), beta-HBCD (∼9%) and gamma-HBCD (∼81%), and minor impurities of other stereoisomers. These structural isomers of HBCD can also possess chirality and thus the number of isomers is even larger. Since HBCD is an additive flame retardant, it is thus pre-disposed to leaching out of commercial products, which leads to contamination and subsequent HBCD accumulation and persistence in the environment. The potential for HBCD to accumulate in organisms and biomagnify within a food web was initially observed in fish and lower aquatic organisms, with subsequent evidence of bioaccumulation in top feeding fish, birds and mammals including humans. Stereoisomer-specific processes have been shown to result in, e.g., the preferential accumulation of the alpha-isomer in biotic samples relative to the dominance of the gamma-isomer in commercial HBCD mixtures. Recent studies have demonstrated that the betaand gamma-HBCD are significantly metabolized in vitro and in vivo assay while the alpha-isomer was not, where isomer-specific (metabolic) biotransformation is the most likely explanation for almost exclusive accumulation of alpha-HBCD in biota. The present study will review and summarize the current state-of-knowledge with respect to stereoisomer-specific presence, bioaccumulation, fate and toxicokinetics of HBCDs in biota and their food webs including in humans, mammals, birds and fish, as well as results from captive studies with HBCD-dosed animals. doi:10.1016/j.toxlet.2010.03.150
S27-3 Different HBCD stereoisomers are metabolized differently H. Hakk Biosciences Research Laboratory, United States The requirement of flame retardancy in most consumer products has resulted in a vast array of chemical compounds that unfortunately have toxic effects and are environmental pollutants. Hexabromocyclododecane (HBCD) is an additive brominated flame retardant applied to extruded and high-impact polystyrene foams (<2.5% by weight) used as thermal insulation in buildings, and HBCD is the only suitable flame retardant for these appli-
S34
Abstracts / Toxicology Letters 196S (2010) S1–S36
cations. HBCD the second highest-volume BFR used in Europe. HBCD was first detected in fish and sediment samples in Sweden in 1998, and have been detected in a variety of environmental samples. Chemically HBCD is a complex mixture of stereoisomers, and the commercial mixture contains a majority of the gammaHBCD (∼81%) with minor amounts of alpha-HBCD (∼10%) and beta-HBCD (∼9%); however, alpha-HBCD is the most abundant isomer reported in biota. Two possible interpretations of these results would be that alpha-HBCD is the most bioavailable stereoisomer, or that metabolic biotransformations favor beta- and gamma-HBCD over alpha-HBCD. This report will present recent laboratory results that lend support to the later hypothesis. Stereoisomer-specific biotransformations have been observed in mice in which gammaHBCD is readily converted to both beta- and alpha-HBCD, but where alpha-HBCD is resistant to such isomerization. The high molecular weight and aliphatic chemical structure of HBCD lend themselves to an intriguing metabolic pathway, and this will be discussed in context of the known metabolic pathways for PCDD/Fs, PBDEs, or PCBs. Possible conjugation of HBCD, urinary volatiles, and involvement of carrier protein systems will be discussed, as well as observed debromination, and oxidative and reductive metabolic events. doi:10.1016/j.toxlet.2010.03.151
S27-4 Toxicity and toxicokinetics of hexabromocylododecane D. Szabo University of North Carolina, United States Hexabromocyclododecane (HBCD) is a brominated flame retardant added to consumer products for over 40 years. Advances in the understanding of HBCD complex chemistry and methods of detection have identified the HBCD commercial mixture to be composed of three main diastereomers; alpha, beta and gamma. We know these stereoisomers to have different chemical and physical properties but differences in biological effects are still yet to be determined. Toxicity studies on HBCD are limited. Furthermore, no stereoisomer toxicity or toxicokinetic study has been reported in any mammalian organism. Toxicity studies performed to date are after exposure to the commercial mixture. Characteristic end points in animal toxicity have focused on hepatotoxicity, thyroid disruption, developmental neurotoxicity and carcinogenisis. Behavioral effects have been observed in mice after administration during a critical period after birth. As the scientific knowledge of today is based on the commercial HBCD mixture, the possible consumer health risk from HBCD appears concerning. However, the presence of many and important data gaps, including those in stereoisomer profiles focusing on carcinogenicity, reproduction, and developmental toxicity, as well as additional routes of exposure, may further heighten this concern as we may currently be underestimating the toxicity effects of HBCD. This report aims to review the current toxicity studies of HBCD and present recent in vivo laboratory results in the kinetics of individual stereoisomers.
S28 Combined Exposures to Multiple Chemicals in Risk Assessment: A Pragmatic Approach
S28-1 Considering combined exposures in risk assessment: Recent developments, opportunities and challenges B. Meek. University of Ottawa, Canada Traditionally, consideration of effects of exposures to multiple chemicals has been based on the toxicity of similar combinations of chemicals or on the toxicity of components. However, the paucity of available data has limited the application of these approaches. As a basis to draw more efficiently on available information including predictive approaches, terminology and more efficient methodology for assessing the impact of combined exposures to multiple chemicals has recently been considered in a project of the World Health Organization (WHO) Programme on Chemical Safety (PCS). This project, which is part of the WHO initiative on Harmonization of Approaches to the Assessment of Risk from Exposure to Chemicals, is being coordinated with additional initiatives of the International Life Sciences Institute (ILSI) and the European Centre for Ecotoxicology and Toxicology of Chemicals. Following an international workshop to review advances in this area, a draft framework has been developed which includes delineation of explicit criteria for initial consideration of the bounds of appropriate grouping for assessment of combined exposures, followed by stepwise consideration of both exposure and hazard in several tiers of increasingly data-informed analyses. These analyses build on recent developments in assessment, incorporating predictive approaches in early tiers and increasingly refined, more data-informed mode of action and probabilistic analyses in later tiers. The extent of assessment and nature of recommendations for generation of additional data are dependent upon the extent of the knowledge base, the magnitude of public health concern and implications of potential risk management decisions. Recommendations regarding terminology and the status of development of the framework, its content, review and application will be described and an example provided. Critical areas for future effort will also be described. doi:10.1016/j.toxlet.2010.03.154
S28-2 Illustration of the WHO combined exposures framework: A tiered and integrative approach to exposure and hazard M. Van Raaij Center of Substances and Integrated Risk Assessment
doi:10.1016/j.toxlet.2010.03.152 Within the WHO/IPCS harmonization project special attention was paid to the issue of combined exposures to chemicals. This reflects single chemical–multiple routes, multiple chemicals–single route, as well as multiple chemicals–multiple routes exposures. A draft framework was established by a WHO working group. The operation of the framework has been illustrated by two case studies and public comments were received and accommodated into the version presented (see contribution SY28-1 for general backgrounds).