Grantee Research Project Results
2003 Progress Report: Pre-natal Exposures of Children to Polybrominated Diphenyl Ethers: The Collection of Animal and Human Data along with the Development and Validation of a PBPK Model
EPA Grant Number: R830756Title: Pre-natal Exposures of Children to Polybrominated Diphenyl Ethers: The Collection of Animal and Human Data along with the Development and Validation of a PBPK Model
Investigators: Raymer, James H. , Hu, Ye A. , Licata, Amy C. , Garner, C Edwin , Mathews, J. M.
Current Investigators: Raymer, James H. , Garner, C Edwin , Emond, C. , Birnbaum, Linda , Studabaker, W.
Institution: Desert Research Institute
Current Institution: RTI International
EPA Project Officer: Aja, Hayley
Project Period: January 1, 2003 through December 31, 2006 (Extended to December 31, 2007)
Project Period Covered by this Report: January 1, 2003 through December 31, 2004
Project Amount: $749,654
RFA: Children's Vulnerability to Toxic Substances in the Environment (2002) RFA Text | Recipients Lists
Research Category: Children's Health , Human Health
Objective:
There are two overall objectives for this research project. The first objective is to develop a physiologically based pharmacokinetic (PBPK) animal model for the polybrominated diphenyl ethers (PBDEs)—2,2',4,4'-tetrabromodiphenyl ether and 2,2',4,4',5'-pentabromodiphenyl ether—that can be used to estimate fetal exposures to PBDEs in humans. The parameters necessary to develop the model for PBDEs will be measured. The second objective is to develop/install and apply analytical methods for PBDEs in human blood and meconium to samples collected during this project to estimate the utility of the model, and to determine if chemical analysis of cord blood and meconium are appropriate media for measurement of cumulative exposures of newborn babies to PBDEs.
The specific hypotheses to be addressed include: (1) a rodent PBPK model for PBDEs can be scaled to be applicable to humans, (2) the PBDE concentrations in cord blood and meconium from newborns are proportional, (3) a mother’s blood concentrations of PBDEs are predictive of the cord blood and/or meconium concentrations in newborn babies, and (4) meconium is a useful medium for assessing cumulative dose of the developing fetus.
Progress Summary:
We obtained pure chemicals to be used in the PBPK model, and we initiated method development and preparations for the PBPK model. Our progress in each of these areas is summarized below.
Synthesis of 2,2',4,4'-Tetrabromodiphenyl Ether and 2,2',4,4',5'-Pentabromodiphenyl Ether
The syntheses of 2,2',4,4'-tetrabromodiphenyl ether and 2,2',4,4',5'-pentabromodiphenyl ether were conducted to provide 4 g of each compound for a reasonable cost. Commercial samples are available for each, but were expensive, as shown for 2,2',4,4'-tetrabromodiphenyl ether ($7,800/100 mg) and 2,2',4,4',5'-pentabromodiphenyl ether ($5,625/50 mg). The synthesis of the two compounds was conducted by following a procedure in the literature (Orn, et al., 1996).
The preparation of 2,2',4,4'-tetrabromodiphenyl ether was accomplished by bromination of phenyl ether catalyzed by iron powder. The crude product (14.41 g) was purified by recrystallization (2 times), followed by column chromatography to give a pure sample (4.65 g) as a white solid. The sample was turned over to J. Beach as Batch 3742-144-2.
The synthesis of 2,2',4,4',5'-pentabromodiphenyl ether was begun by preparing a sample of potassium phenolate from phenol and KOH in ethanol. Evaporation of the solution under vacuum yielded a white solid (19.97 g), which was used without further purification. The potassium phenolate was reacted with 1,3-dibromobenzene and copper bronze at 170°C to give after workup and purification of 3-bromodiphenyl ether as a colorless oil (5.14 g). The sample was characterized by thin-layer chromatography (TLC) and mass spectrometry (MS). The 3-bromodiphenyl ether was brominated with bromine and iron powder in CCl4 to give a mixture of brominated products. The crude mixture was chromatographed (2 times) on silica gel to give a colorless viscous oil (5.73 g). The oil was recrystallized, but no solid was obtained. The crude product was recovered as a viscous sticky oil (6.21 g). This oil was characterized by TLC, 1H nuclear magnetic resonance (NMR), and high performance liquid chromatography (HPLC). By TLC, the sample contained a faster moving impurity. The 1H NMR spectra showed that the main compound was the desired 2,2',4,4',5'-pentabromodiphenyl ether, but there was a significant impurity present in the sample. Reverse phase HPLC analysis showed that the main component was 2,2',4,4',5'-pentabromodiphenyl ether, but four other impurities also were present. The HPLC analysis suggested that the sample might be able to be purified by preparative HPLC. Analysis of the sample by gas chromatography (GC)/MS showed the presence of the desired isomer as the main component; impurities consisted of other pentabromo isomers.
Analytical Method Development
Initial evaluation of an extraction method was started. Standard solutions of the target analytes were purchased and analyzed by GC in conjunction with electron capture detection (ECD). Although it is anticipated that negative ionization/chemical ionization MS will be utilized on actual samples for both specificity and sensitivity, it was decided that initial development work could be performed using the less expensive GC/ECD to evaluate possible gross contamination during sample handling and initial recoveries from tissues. Tissues from rats were collected and placed into a freezer for use when required.
PBPK Modeling
For PBPK modeling, a copy of advanced continuous simulation language software was purchased. This copy was installed on a remote access computer so that several staff members had access to the software, but only one user at a time. This facilitated sharing of the modeling software.
Preliminary work to select the most appropriate model type occurred through literature searches on existing PBPK models for chemicals that have similar chemical and physical properties to PBDEs. Also, literature searches were performed on existing gestational PBPK models. The mathematical modeler/statistician (Amy Licata, Ph.D.) and pharmacokineticist (Ed Garner, Ph.D.) had several discussions about the various PBPK models that could be used as appropriate templates for the PBDE PBPK model. Additional literature searches were performed to obtain recent publications on PBDEs, and to prepare for the U.S. Environmental Protection Agency (EPA)/National Institute of Environmental Health Sciences/Research Triangle Institute meeting that was scheduled to occur in January 2004, at the EPA facility in Research Triangle Park, NC. Relevant literature was stored on a shared electronic folder so that staff members were kept abreast of current findings.
Experimental Details of the Synthetic Approach
2,2',4,4'-Tetrabromodiphenyl Ether. A solution of phenyl ether (5.00 g, 0.029 mole) in CCl4 (50 mL) was stirred and heated to reflux under N2. When at reflux, a solution of bromine (6.5 mL, 0.127 mole) in CCl4 (50 mL) was added dropwise. The addition was stopped when 25 mL remained and iron powder (0.10 g) was added to the reaction mixture. The remaining bromine solution then was added dropwise to the reaction mixture. After refluxing for 20 hours, the reaction mixture was cooled to room temperature and filtered to remove the iron powder. The yellow solution was washed with 5 percent sodium metabisulfite (2 x 200 mL), 0.1 N NaOH (200 mL), and water (200 mL). The CCl4 layer was separated and dried over MgSO4 for 2 hours. The mixture was filtered, and the filtrate was evaporated under vacuum to give a viscous yellow oil (14.41 g, > 100 percent crude).
The oil (14.41 g) was recrystallized from EtOH/CCl4 (2 times) to give a white solid (4.76 g). The solid was purified further by column chromatography on silica gel 60 (300 g, Merck) packed in hexanes. The solid was dissolved in hexanes/CCl4, and fractions were eluted with hexanes. The fractions were followed by TLC on silica gel, and similar fractions were combined and evaporated under vacuum to give 4.65 g (32.6 percent) of the title compound as a white solid: mp 73-74°C (Lit. mp 82-82.5°C); TLC single spot, Rf 0.27, on silica gel using hexanes; 1H NMR (CDCl3) 6.71 (d, H6,6’), 7.38 (dd, H5,5’), 7.78 (d, H3,3’); MS m/z 482 (17 M+), 484 (70), 486 (100), 488 (62), 490 (15), 326 (92 [M+2-2Br]+).
Potassium Phenolate. A mixture of KOH pellets (10.52 g, 0.16 mole; 85 percent KOH) and absolute EtOH (200 mL) was stirred at room temperature to give a cloudy solution. Phenol (15.00 g, 0.16 mole) was added to the reaction mixture, and the mixture was stirred at room temperature for 10 minutes. The pH was basic by pH paper. The cloudy solution was filtered through filter paper to give a clear solution. The solution was evaporated under vacuum, and the solid vacuum dried at room temperature overnight. The solid was suspended in ether (400 mL), and was collected and rinsed with ether. The solid was vacuum dried at room temperature for 60 hours to give 19.97 g (94.8 percent) of the title compound as a powdery white solid. The solid was used directly in the next reaction.
3-Bromodiphenyl Ether. A mixture of potassium phenolate (8.04 g, 0.061 mole), 1,3-dibromobenzene (14.34 g, 0.061 mole) and copper powder (1.5 g) was stirred and immersed in an oil bath at 170°C under N2. After heating for 4 hours, the reaction mixture was cooled to room temperature. The reaction mixture was evaporated under vacuum using a vacuum pump. The resulting residue was triturated with hexanes (300 mL), and the mixture was filtered through filter paper. The filtrate was evaporated under vacuum to give a yellow oil (12.77 g). The oil was chromatographed on silica gel 60 (500 g; Merck) eluting with hexanes. The fractions were followed by TLC on silica gel, and similar fractions were combined and evaporated under vacuum to give 5.14 g (33.9 percent) of the title compound as a colorless oil: TLC single spot, Rf 0.34, on silica gel using hexanes; MS m/z 248 (100 M+), 250 (97), 169 (25 [M-Br]+).
2,2',4,4',5'-Pentabromodiphenyl Ether. A mixture of 3-bromodiphenyl ether (5.00 g, 0.02 mole), iron powder (0.50 g), and CCl4 (50 mL) was stirred and heated to reflux under N2. While refluxing the reaction mixture, a solution of bromine (5 mL, 0.098 mole) in CCl4 (20 mL) was added dropwise during 1 hour. The reaction mixture was refluxed an additional 4 hours, and then was cooled to room temperature. Five percent sodium bisulfite (50 mL) was added, and the mixture was stirred until the bromine color was gone. The layers were separated, and the CCl4 layer was washed with 5 percent sodium bisulfite (2 x 50 mL) and saturated NaCl (50 mL). The CCl4 layer was separated and dried over MgSO4 overnight. The mixture was filtered, and the filtrate was evaporated under vacuum to give a viscous yellow oil (12.10 g, >100 percent crude). The oil was chromatographed on silica gel 60 (500 g; Merck) eluting with hexanes. The fractions were followed by TLC on silica gel, and similar fractions were combined and evaporated under vacuum. The chromatography provided three fractions: fractions (5-9) colorless viscous oil (7.47 g), fraction (10) colorless viscous oil (1.07 g), and fractions (11-16) cloudy viscous oil (2.89 g). TLC analysis showed that none of the fractions were pure. The product was determined to be in fractions (5-9) by 1H NMR analysis.
Fraction (5-9) viscous colorless oil (7.47 g) was dissolved in hexanes/CCl4 and rechromatographed on silica gel 60 (450 g; Merck) eluting with hexanes. The fractions were followed by TLC on silica gel, and similar fractions were combined and evaporated under vacuum. The chromatography provided four fractions: fractions (5-6) colorless oil (0.08 g), fraction (7) colorless viscous oil (1.19 g), fractions (8-14) colorless viscous oil (5.73 g), and fractions (15-17) colorless viscous oil (2.89 g). The largest fraction (5.73 g) was recrystallized from EtOH/hexanes, but no crystals were obtained. The sample was recovered by evaporation under vacuum to give a viscous sticky oil (6.21 g, sample solvated).
References:
Orn U, Eriksson L, Jakobsson E, Bergman A. Synthesis and characterization of polybrominated diphenyl ethers-unlabelled and radiolabelled tetra-, penta- and hexa-bromodiphenylethers. Acta Chemica Scandinavica 1996;50:802-807.
Future Activities:
We will study the acquisition of the 2,2',4,4',4-pentabromodiphenyl ether for the dosing experiments. Method development efforts will continue. Additional PBPK model development will occur in 2004, when PBDE data have been collected. The goal is to develop a rat oral-exposure pregnancy PBPK model and extrapolate that model to describe human pregnancy with oral exposure to PBDEs. During model development and extrapolation, model representation, model parameterization, model simulation, and model validation will occur.
Journal Articles:
No journal articles submitted with this report: View all 8 publications for this projectSupplemental Keywords:
sensitive populations, human health, mathematics, measurement methods, bioavailability, metabolism, vulnerability, infant, animal, modeling, environmental management, health, physical aspects, biochemistry, children's health, environmental microbiology, epidemiology, genetics, health risk assessment, physical processes, risk assessments, susceptibility, sensitive population, genetic susceptibility, toxicology, polybrominated diphenyl ethers, PBDEs, age-related differences, biochemical research, biological markers, biomarkers, children, developmental effects, environmental hazard exposures, exposure, exposure assessment, gene-environment interaction, genetic polymorphisms, health effects, human exposure, human health risk, insecticides, pharmacodynamic model, pharmacokinetic models, risk-based model, toxicity., RFA, Health, Scientific Discipline, PHYSICAL ASPECTS, ENVIRONMENTAL MANAGEMENT, Toxicology, Genetics, Health Risk Assessment, Risk Assessments, Susceptibility/Sensitive Population/Genetic Susceptibility, Environmental Microbiology, Biochemistry, Physical Processes, Children's Health, genetic susceptability, Risk Assessment, health effects, pharmacodynamic model, sensitive populations, biomarkers, age-related differences, PBDE, gene-environment interaction, exposure, developmental effects, children, pharmacokinetic models, toxicity, genetic polymorphisms, insecticides, human exposure, pharmacokinetc model, biological markers, risk based model, exposure assessment, polybrominated diphenyl ethers, biochemical research, environmental hazard exposures, toxicsRelevant Websites:
Progress and Final Reports:
Original AbstractThe perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.