Grantee Research Project Results
Final 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. , Garner, C Edwin , Emond, C. , Birnbaum, Linda , Studabaker, W.
Institution: RTI International
EPA Project Officer: Aja, Hayley
Project Period: January 1, 2003 through December 31, 2006 (Extended to December 31, 2007)
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:
Introduction
Exposures to environmental contaminants in utero and during childhood create the potential for a variety of adverse health effects, including abnormal or disturbed development of systems such as the neurological system and the endocrine system. There is growing concern that the increasing incidence of ailments, such as adult and childhood cancers, reproductive and developmental anomalies, and behavioral deficits, might be linked to exposures early in life. Little is known about in utero exposures to most environmental chemicals, including the polybrominated diphenyl ethers (PBDEs). PBDEs are used commercially as additive flame retardants and have been shown to transfer into environmental compartments where they have the potential to bioaccumulate in wildlife and in people. These PBDEs are known to have neurological effects (Eriksson, et al., 2001; Gill, et al., 2004; Branchi, et al., 2003) and are suspected of having endocrine disruption capability (Darnerud, et al., 2001; Gill, et al., 2004). Of the 209 PBDE congeners, approximately five dominate the profiles measured in humans, particularly 2,2',4,4'-tetrabromodiphenyl ether (BDE-47); this demonstrates a need to understand the toxicokinetic properties associated with this class of chemicals (Staskal, et al., 2005). Only a small subset of the PBDEs have been investigated in toxicokinetic studies; these studies have shown that binding proteins, elimination, adipose tissue mass, and placenta transfer may all play roles in the distribution of PBDEs.
Physiologically based pharmacokinetic (PBPK) models developed in rodents can be extrapolated to humans by incorporating species-specific parameters, such as partition coefficients and physiological parameters. Also, actual human exposure scenarios at ambient concentrations can be incorporated into the model, where high concentrations were given to rodents. The developed model will be evaluated by comparing it to measured exposure data obtained from mothers and newborn babies. PBDE concentrations in mothers’ blood (collected shortly before birth), cord blood, and meconium will be used as potential biomarkers for human exposure. Cord blood and meconium are two easily-obtained biological samples for use in evaluating in utero exposures to toxic compounds. Research into the applicability of meconium to exposure analysis studies for a wide range of chemicals is very active at present.
Objectives
- Develop a PBPK animal model for the PBDE BDE-47 that can be used to estimate fetal exposures to PBDEs in humans. The parameters necessary to develop the model for PBDEs will be measured.
- Analytical methods for PBDEs in human blood and meconium will be developed/installed and applied to samples collected during this project, both 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.
Specific hypotheses include the following: (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) mothers’ 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.
Summary/Accomplishments (Outputs/Outcomes):
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 undertaken to provide 4 g of each compound for a reasonable cost. Commercial samples are available for each but are expensive (at the time they were needed), as shown for 2,2',4,4'-tetrabromodiphenyl ether ($7800/100 mg) and 2,2',4,4',5'-pentabromodiphenyl ether ($5625/50 mg). The synthesis of the two compounds was carried out following the 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 (2x) followed by column chromatography to give a pure sample (4.65 g) as a white solid.
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 Cu bronze at 170°C to give, after workup and purification, 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 (2x) 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, proton nuclear magnetic resonance spectroscopy (1H NMR) and high-performance liquid chromatography (HPLC). By TLC analysis, 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 4 other impurities 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 penta-bromo isomers.
Animal Dosing Studies: Determination of Maternal–Fetal Pharmacokinetics of 2,2',4,4'-Tetrabromodiphenyl Ether
The objectives of these studies were to:
- Administer 2,2',4,4'-tetrabromodiphenyl ether (BDE-47) to pregnant female Sprague Dawley rats via single intravenous (i.v.) or single or repeat oral (p.o.) doses.
- Collect blood and tissue samples for later analysis of parent compound concentrations.
Methods
Animals. Timed pregnant female Sprague Dawley rats (sperm positive on gestation day [GD] 0) with indwelling jugular cannulae were ordered (Charles River Laboratories, Raleigh or equivalent). Animals were approximately 10 weeks of age at the time of breeding at the supplier and were received at RTI’s Animal Research Facility on GD5 (the day the plug is observed is GD0). The animals were quarantined for approximately 3 days prior to use in a given study. Animals that weighed between 250 and 400 g were considered acceptable for use in the study, as long as the animals appeared to be healthy. Extra rats were ordered and dosed to ensure that a sufficient number of pregnant dams was available.
Dose Preparation. Doses were prepared in bulk 1 day prior to administration. Oral formulations were prepared in fresh corn oil (Aldrich) by dissolving the test article in a sufficient volume of acetone, then adding ca. 2 mL corn oil, and evaporating acetone under a nitrogen stream. The concentrated test article/corn oil solution was then brought to volume. Intravenous formulations were prepared with PBDE dissolved in a vehicle consisting of 1:1:8 Emulphor:ethanol:isotonic saline. Formulations were stored in glass bottles with Teflon cap liners at 0–4ºC. Aliquots of dosing material from each formulation were preserved for analysis to confirm dose concentration.
IV Studies. Animals were administered a single i.v. dose of BDE-47 on GD18. Immediately after dose administration, animals were placed in glass metabolism cages designed for the separate collection of urine and feces. Urine and feces were collected at 0–6, 6–12, and 12–24 hours post-dose. Blood was sampled via cannulae at the time points indicated in Table 1. For the final blood collection, the dams were anesthetized by CO2 exposure, and maternal blood was drawn by cardiac puncture. Maternal liver, muscle, kidney, brain, skin (ears), adipose tissue, small intestine, large intestine, cecum, gut contents, placenta, and amniotic fluid, as well as fetal liver, brain, blood, and carcass were collected during necropsy. Samples were frozen at –20°C until extraction and analysis.
PO Studies. Animals were administered single oral doses of test article on GD18 or repeat oral doses on GD10–18. For excreta collection, animals were placed in glass metabolism cages designed for the separate collection of urine and feces. Urine and feces were collected at 0–6, 6–12, and 12–24 hours post-dose during single-dose studies and at 0–6, 6–12, and 12–24 hours following the final dose during multiple-dose studies. Blood was sampled after the doses on GD18 via cannulae, at the timepoints indicated in Table 1. For the terminal blood collection, the dams were anesthetized by CO2 exposure, and maternal blood drawn by cardiac puncture. Maternal liver, muscle, kidney, brain, skin (ears), adipose tissue, small intestine, large intestine, cecum, gut contents, placenta, and amniotic fluid, as well as fetal liver, brain, blood, and carcass were collected during necropsy. Samples were frozen at –20°C until extraction and analysis.
Table 1. Study Design for Determination of Maternal–Fetal Pharmacokinetics of 2,2',4,4'-Tetrabromodiphenyl Ether
Dose (mg/kg/ day)a |
Route |
Animals on Study |
Dose Days |
Blood Collection Timesb |
Excreta Collection Timesb |
1 |
PO |
4 |
1 |
0, 15 and 30 minutes |
0–6, 6–12, 12–24 hr |
50 |
PO |
4 |
1 |
0, 15 and 30 minutes |
0–6, 6–12, 12–24 hr |
1 |
IV |
4 |
1 |
0, 5, 15 and 30 minutes |
0–6, 6–12, 12–24 hr |
50 |
IV |
4 |
1 |
0, 5, 15 and 30 minutes |
0–6, 6–12, 12–24 hr |
1 |
PO |
4 |
10 |
0, 15 and 30 minutes |
0–6, 6–12, 12–24 hr |
20 |
PO |
4 |
10 |
0, 15 and 30 minutes |
0–6, 6–12, 12–24 hr |
20 |
PO |
4 |
1 |
0, 15 and 30 minutes |
0–6, 6–12, 12–24 hr |
20 |
PO |
4 |
1 |
0, 15 and 30 minutes |
0–6, 6–12, 12–24 hr |
20 |
PO |
4 |
1 |
0, 15 and 30 minutes |
0–6, 6–12, 12–24 hr |
20 |
PO |
4 |
10 |
0, 15 and 30 minutes |
0–6, 6–12, 12–24 hr |
20 |
PO |
4 |
10 |
0, 15 and 30 minutes |
0–6, 6–12, 12–24 hr |
20 |
PO |
4 |
10 |
0, 15 and 30 minutes |
0–6, 6–12, 12–24 hr |
a Single p.o. and i.v. doses were delivered in a bolus on GD18. Repeat doses are initiated on GD10 and delivered daily to GD18. |
b Blood and excreta collection initiated on GD18. |
Human Study
A small, focused human study was conducted to provide data that would be useful in testing the ability of the model, which was developed from rodent data, to scale to humans. In order to facilitate this study, a subcontract with Duke University Medical Center, Maternal and Fetal Medicine, was established for the recruitment of, and sample collection from, 25 pregnant women. The Institutional Review Board (IRB) protocol, including questionnaires and consent forms, was prepared and approved by both RTI and Duke University Medical Center. Documentation of approval was supplied to the U.S. Environmental Protection Agency (EPA); EPA also approved the protocol. The sample size was determined based upon simple correlation of two measurements (such as meconium measurement and mother’s blood measurement) without considering other covariates. A sample size of 25 will be able to detect a minimum correlation of 0.4 at a significance level of 0.05.
Recruitment. Upon being admitted to the hospital for delivery, a nurse associated with the study described the study to the expectant women, who were also provided a copy of the informed consent form. After discussion with the nurse, the pregnant women were asked to sign the form and were provided a copy of the form. Consent was also sought prior to admission for delivery during a prenatal visit at the clinic, in order to avoid additional stress on the woman in labor. Duke applied for and was granted by their IRB a “Request for Waiver or Alteration of HIPAA Authorization,” a copy of which was provided to the RTI IRB office.
Sample Collection. After the participants were recruited into the study, the nurse collected a blood sample from the mother within 48 hours before or after delivery. This was conducted as other blood samples were being drawn to avoid an additional “stick.” After delivery, cord blood samples were collected by hospital staff. At that time, a portion (approximately 2 mL) of the blood was collected into a light blue top Vacutainer tube, refrigerated, and later transported on dry ice to RTI. Meconium was collected from diapers by the postpartum staff or the parent at the hospital. If the parent collected the diaper, it was given to the postpartum staff. Patients are normally not released from the hospital until the child passes meconium. Each sample was labeled and stored separately. After receipt of samples at RTI, all samples were stored at –20°C until extraction and analysis. A questionnaire regarding the mother’s dietary habits was administered in person at approximately 4 weeks postpartum. At this time, if the mother was breast-feeding, a breast milk sample was collected. In addition, a dust sample was collected. Other information such as mother’s and baby’s weight, body mass index (BMI), age, etc., were obtained from the hospital from medical records. Details for the biological samples are described as follows.
Mother’s Blood. After a mother was admitted to the hospital for delivery, an extra 2-mL vial of blood was collected as other blood samples were being drawn.The sample was collected in a light blue top Vacutainer containing the following additives: 0.2 mL citrate solution, 4.94 mg sodium citrate, and 0.88 mg citric acid. The whole sample was stored refrigerated and later transported on dry ice to RTI.
Cord Blood. After delivery of the child, the hospital staff collected approximately 2 mL of cord blood into a light blue top Vacutainer, refrigerated, and later transported on dry ice to RTI.
Meconium. A specially marked diaper was put on the infant to indicate that this diaper should be saved to collect the meconium sample. After the infant passed the meconium, the diaper was given to hospital staff by the parent unless it was changed by a hospital staff member. The sample was refrigerated until transport to RTI. The RTI field staff removed the meconium from the diaper. Duke University provided an unused diaper of the same size to use as a pre-weight mass for determining the mass of the meconium collected.
Breast Milk. Four weeks after delivery, 5–10 mL (1–2 teaspoons) of breast milk were collected by the mother into a container using either manual expression or a breast pump. The sample was transported to RTI in a cooler with cold packs and stored frozen until analysis.
Vacuum Dust. A 1-square-foot carpeted area in the participant’s home was marked with a template. This area was vacuumed by RTI field staff. The vacuum dust was collected onto a paper filter placed close to the vacuum inlet. The sample was transported to RTI in a cooler with cold packs and stored frozen until analysis.
Analytical Method
Initial method development was conducted using liver tissue from nondosed rats. A modification of a method used at RTI for organochlorine pesticides and PCBs in brain tissue was evaluated. The modification was intended to adapt the cleanup step to solid-phase extraction (SPE)-based Florisil rather than an open-column approach. Initial work was focused on the validation of the cleanup step.
The procedure for the extraction of tissue is as follows:
- Homogenize tissue with water and hexane; decant the hexane and repeat two additional times;
- Combine the extracts and dry using anhydrous sodium sulfate column;
- Adjust extract volume to obtain 10 mg “tissue” per mL equivalent so that co-extracted tissue components do not vary, add surrogate (PCB-198; 2,2',3,3',4,5,5',6- octachlorobiphenyl) and spike with BDE-47 and BDE-99 to achieve 5 ppb in the extract (approximately 500 ppb in tissue); <
- Apply 1 mL to Florisil SPE cartridge, elute with 30 mL hexane, reduce the volume to 1 mL, and spike with quantitation standard (PCB-119; 2,3',4,4',6 pentachlorobiphenyl); and
- Analyze by GC/electron capture detector (ECD) using a DB5-MS column (30 m x 0.25 mm, 0.25 μm film).
The Florisil SPE cartridges provided recoveries for the target analytes in the range 99 to 101% at the 500 ppb concentration in tissue (5 ppb in extract). Precisions ranged from 1.5 to 11.6% relative standard deviation (RSD). A small contamination for BDE-47 was found and the source is being sought. It is anticipated that the method will be acceptable for the dosing studies but the background contamination issues need to be investigated before application to human samples that are expected to contain much lower concentrations.
Sample analyses were performed using procedures developed and validated for the matrix type. Methods were generally based on existing procedures for the analysis of PCBs in biological matrices. These methods include homogenization (tissues), hexane extraction, Florisil cleanup (tissues), and analysis by GC/ECD. The analyses of human samples (meconium, cord blood, and breast milk) were performed using the blood method for cord blood and milk and the tissue method for meconium, modified as described below. Lipid content was determined using a gravimetric approach following evaporation of 0.5 mL of hexane extract.
Results and Discussion
Spike-Recovery Studies. BDE-47 in methanol was spiked into blood samples immediately prior to extraction. For tissue samples, spikes were made directly into the tissue (~1-g samples) and allowed to permeate for 10 minutes. Tissues evaluated included liver, adipose, abdominal muscle, skin, brain, and fetal. BDE-47 was spiked into tissues at 0, 2.5, 25, and 250 ng/mL. BDE-47 is ubiquitous in the laboratory environment. We observed a consistent background of 0.2–2 ng/mL in laboratory method blanks that we were unable to eliminate despite screening of plastics, baking, and triple hexane rinsing of glassware, and careful cleaning of work areas. BDE-47 recoveries are reported in Table 2. Results for different tissue matrices are pooled; for the most part they were similar except for adipose, which yielded low recoveries (20%–60%). Recoveries of surrogate from both matrices were more variable than for BDE-47 and strongly tissue dependent (blood, 98% ±18%; adipose, 59% ±19%; muscle, 113% ±18%; fetal, 82% ±7%).
Table 2. Recoveries of BDE-47 Spiked into Rat Blood and Rat Tissues.
Matrix |
Spike Level |
n |
Mean Recovery |
% Recovery |
SD |
RSD (%) |
|
0.1 ng/mL |
4 |
0.313 ng/mL |
313 |
0.113 |
36.1 |
Blood |
2.5 |
10 |
3.04 |
122 |
0.160 |
5.3 |
|
25 |
6 |
27.5 |
110 |
1.61 |
5.9 |
|
250 |
8 |
255 |
102 |
23.1 |
9.1 |
|
0 (Reagent) |
13 |
1.04 ng |
n/aa |
0.44 |
42.3 |
Tissue |
0 (Tissue |
24 |
1.97 |
n/a |
0.99 |
50.3 |
(composite |
2.5 ng |
30 |
3.80 |
152 |
1.04 |
27.4 |
results) |
25 |
17 |
25.6 |
102 |
4.06 |
15.9 |
|
250 |
15 |
246 |
98.4 |
32.5 |
13.2 |
a Not applicable |
Discussion. While recovery of BDE-47 from spiked blood samples is essentially quantitative and reproducible, the persistence of a laboratory background confounds the analysis of samples containing low initial concentrations (<10 ng/mL). These observations are consistent with previously reported results for reagent controls. For most of the experimental samples from dosed animals that have been tested so far, actual BDE-47 levels are one or more orders of magnitude higher, and the effect of background may be diluted out of those samples. However, background contamination continues to be a concern for the analysis of human samples. Similarly, recoveries from a variety of spiked tissues are generally quantitative. However, matrix components persist in the extract even following Florisil cleanup, particularly for those derived from tissues high in fat. Comparison of the chromatograms in Figures 1 and 2, obtained for blood and adipose extracts, respectively (spiked at 25 ng/mL and 25 ng, respectively), illustrates the increasing effect of matrix on baseline noise, an effect which frequently persists on dilution. As a result, the range of recoveries is much wider, with imprecision increasing to 15%+ RSD. For both matrices, high concentrations of BDE-47 resulted in masking of the surrogate; dilution pushed it near or below the limits of quantitation.
Figure 1. Representative Chromatogram for Blood Extract
Time (min)
Figure 2. Representative Chromatogram for Adipose Extract
Conclusions for Methods. The methods described yield BDE-47 recoveries of 90–110% from blood in the range 25–250 g/mL and tissues in the range 25 ng/g. Precision in blood samples (<10% RSD) is consistent with previously reported results for reagent controls; precision in tissue samples is impacted by matrix interferences. A persistent low-level contamination will require resolution before analysis of human samples, especially with BDE-47 concentrations less than 10 ng/mL.
Sample Analysis and Results
Samples were extracted and analyzed using the method described above. A summary of the rat data is shown in Table 3. The BDE-47 concentrations in rat samples were not lipid-adjusted. When the concentration data are expressed as a percentage of the total dose, very good mass balance is measured for the i.v. dose. Compartmental volumes—based on the PBPK parameters presented below and the known body masses of the rats—were used. Also, measured mass balance is shown for three percentage adipose values. The data suggest that the adipose content of the rats was between 7 and 10%. Also, a comparison of the recovered dose following single oral doses at 1 and 20 mg/kg shows that a smaller fraction of the higher dose was absorbed. The percentage of dose recoveries for 10-day repeated oral dosing fell between the 1 and 20 mg/kg single oral dose.
Table 3. Summary Data From Intravenous, Single Oral, and Repeated Oral Dosing of Rats With BDE-47 Showing BDE Concentrations (top) and Percentage of Total Dose (bottom)
Some of the BDE-47 was measurable in the fetal rats. Following the 10-day, repeated oral dosing, 0.45% was measured in the fetuses. Of this, 0.03% was measured in the gastrointestinal (GI) tract (surrogate for meconium), 0.05% was measured in the brain, and 0.37% was contained in the remaining fetal tissues.
The concentrations measured in human cord blood, meconium, and milk are shown in Table 4. In this case, data shown are corrected for surrogate recovery and are lipid-adjusted. Lipids were determined gravimetrically following the evaporation of hexane solvent from an aliquot of the original extract. Diapers were lyophilized (freeze-dried) for 48 hours and meconium was removed from diapers by scraping with a spatula. The entire amount removed was extracted with hexane three times (by a combination of sonication and shaking by hand), each time using a volume of ~10 times the mass being extracted. Samples were centrifuged, decanted, and concentrated by Turbovap and the SPE cleanup was then performed as for the rat samples. Method controls were prepared as follows: 25 mL of hexane was added to a centrifuge tube, then 100 μL of surrogate and 5 μL of target analyte spiking solution (BDE-47 at approximately 5 μg/mL) were added to the hexane. Spiked meconium samples were prepared by adding an appropriate volume of hexane (depending on the sample mass extracted) to each centrifuge tube or jar containing the meconium, followed by the addition of 100 μL of surrogate. Field blanks were processed by removing the inside lining of the diaper (avoiding the transfer of the absorbent pad), cutting the lining into small pieces, transferring to a centrifuge tube, and then extracting using the same method as the meconium samples (note: processing of the meconium samples involved extraction of only the meconium, not the lining of the diaper).
Table 4. Concentrations of BDE-47 Measured in Human Samples (Mass per Mass Lipid)
“-“ = Missing Value
The BDE-47 concentrations measured in cord blood are higher than concentrations measured in the blood of pregnant women (2.5–205 ng/g; Bradman et al., 2007), based on total BDE concentrations from pooled U.S. blood (61–80 ng/g; Schecter et al., 2005). Unfortunately, data are not available in this study for maternal blood. Total BDEs in breast milk of 6 to 321 ng/g lipid (She, et al., 2007) and 6.2 to 419 ng/g with a median of 34 ng/g (Schecter, et al., 2003) have been reported and are consistent with the concentrations measured in this study. There are no comparative values in the literature for meconium; to our knowledge this is the first time that BDE-47 has been measured in this medium. It should be noted, however, that the aliquot of extract used to measure lipid content was taken after the SPE cleanup. The extent to which this step perturbs lipid content is not known.
The important message from these data, however, is that BDE-47 can be measured in fetal tissues. This is consistent with data from the rat dosing study, where BDE-47 was measured in the GI tract (a surrogate for meconium in the human) as a small, but consistent, fraction of the maternal dose.
Figures 3–5 were created to investigate potential relationships among the media. Quantitative relationships would be necessary in order to reliably predict the in utero exposure or to provide the basis for prospective studies that include some measure of health outcome, including any developmental abnormalities. Of greatest interest is Figure 3, where meconium might be used to predict the circulating fetal concentration. Meconium, however, accumulates over time and would thus integrate fetal exposure, so the direct comparison of the value with a point measure in blood might not be expected to be meaningful. These plots do not suggest any real associations. However, it should be noted that the measured peak areas were small and that uncertainties associated with concentrations near the method limit of quantitation could obscure associations. These data do suggest that additional studies should be conducted to more conclusively define the meaning of the measured values in the biospecimens to fetal dose. The use of larger samples to provide greater masses of BDEs and the use of a sensitive mass spectral method, such as negative ion chemical ionization in conjunction with GC that also imparts greater specificity in detection, would be good potential analytical chemistry approaches. In addition, the use of an enzymatic approach to lipid determination, as is used by the Centers for Disease Control and Prevention (CDC), would impart greater accuracy and precision, especially for small samples, and would help reduce data variability.
Figure 3. Comparison of BDE-47 Concentrations in Meconium as a Function of Concentration in Cord Blood
Figure 4. Comparison of BDE-47 Concentrations in Breast Milk as a Function of Concentration in Cord Blood
Figure 5. Comparison of BDE-47 Concentrations in Breast Milk as a Function of Concentration in Meconium
These results also support refinement of the PBPK model, described below, to include meconium formation and the associated sequestration of BDEs into the meconium. The model did predict BDE transfer to the fetus in rats, but resources were insufficient to investigate fetal subcompartments, or to even scale the model to the entire human fetus. The study of fetal rat subcompartments and the required scaling to human subcompartments would take substantial effort.
A Physiologically-Based Pharmacokinetic Model for Developmental Exposure to BDE-47 in Rodents
An objective of this project was to describe early PBPK model development based on data generated from the studies described. PBPK modeling efforts are an ongoing collaboration with Dr. Linda Birnbaum of EPA and Dr. Danielle Staskal of Chem Risk (formerly of EPA). Tissue partition coefficients were estimated and the overall model was constructed, based on data generated by Dr. Birnbaum’s laboratory. Work with the mouse data was conducted to help in the generation of a model that was used in the rats when data became available. Model development was begun using Berkley Madonna, version 8.0 by Dr. C.E. Garner at RTI. However, as the complexity of the model was increased, this program became too slow. It was at this point that Dr. Claude Emond of the University of Montreal assumed responsibility for model development on this project using Advanced Computer Simulation Language (ACSL) software (Aegis Technology) version 11.8.4. The remainder of this section is based on Dr. Emond’s work.
a. Model Description, Key Assumptions, Version, Source, and Intended Use. Polybrominated diphenyl ethers (PBDEs) are used commercially as additive flame retardants and have been shown to transfer into environmental compartments where they have the potential to bioaccumulate in wildlife and in people. Of the 209 PBDE congeners, approximately 5 dominate the profiles measured in humans, particularly 2,2',4,4'-tetrabromodiphenyl ether (BDE 47), and indicates the need to understand the toxicokinetic properties associated with this class of chemicals (Staskal, et al., 2005). Only a small subset of the PBDEs have been investigated in toxicokinetic studies; these studies have shown that binding proteins, elimination, adipose tissue mass, and placental transfer may all play roles in the distribution of PBDEs.
The purpose of this modeling work is to develop a PBPK model for developmental description of BDE-47 toxicokinetics in the rat. This description includes the absorption, distribution, metabolism, and elimination (ADME) that reflects the current knowledge of the toxicokinetics of 2,2',4,4'-tetrabromomodiphenyl ether. It also includes a fetal component with the hopes that the model, once scaled, can be used to estimate human fetal exposures to PBDEs.
The model consists of seven compartments for the mother (including blood, liver, brain, fat, kidney, placenta, and the rest of the body). Transfer of BDEs throughout the body relies on the systemic circulation (Figure 6). The fetus was described as a single compartment. During the gestation period, fat, fetal body growth, placenta, and placenta blood are modified and have been described using algebraic equations. Parameters of physiology, such as tissue volumes and blood flows, and physicochemical parameters, such as partition coefficients and extraction ratio, were extracted from literature (Table 5) (Emond, et al., 2004; Krishnan and Andersen, 2001).
Table 5a. Physiological Parameters Used in the PBPK Model for Nonpregnant and Pregnant Rat
Parameters |
Naive |
Pregnant |
Body weight (BW) |
250 |
275 |
Cardiac output (mL/min/kg) |
311.4 |
311.4 |
Tissue volume (fraction of BW) |
|
|
Tissue blood volume (fraction tissue) |
|
|
Table 5b. Physiological Parameters Used in the PBPK Model for Nonpregnant and Pregnant Rat
Parameters |
Naive |
Pregnant |
Tissue blood flow (fraction Qc) |
|
|
Tissue permeability (fraction of Qt) |
|
|
Apparent partition coefficient |
|
|
Three compartments (brain, liver, and fat) were described with a permeability diffusion limitation. Because these parameters are not flow-limited, we used optimization to reach the early experimental distribution. This limited description has an impact on the chemical diffusion into the cellular matrices and that takes place only during the early absorption/distribution phase (approximately within 24 hours). In addition, this influence is negligible at steady-state. For kidney, placenta, and richly perfused tissues, flow limits have been described for the distribution. Different equations were used to describe these two approaches and are presented in section (e) below. The GI tract absorption equation is described as a first-order equation with a Ka (hr-1). Elimination and clearance have been pooled into one expression, such as the extraction coefficient (E) parameter. During gestation, several switches turn on to control maternal and fetal compartment modifications. The initial exchange between placenta and fetus starts a few hours after the start of gestation. This moment is driven by a clearance constant and is controlled by a switch as well.
Figure 6. Conceptual Representation of a PBPK Model for PBDE Developed for Rat
Optimization. Experimental data from Sanders, et al. (2006) were used for optimization and validation in the adult without gestational scenarios. The first dataset used was that following an oral dose of 0.1 mg of BDE-47/kg of body weight to a male rat. Because only a few datasets are available for rats, we made the assumption that no significant differences exist between male and female rats exposed to BDE-47. In fact, Sanders, et al. (2006) report a slight difference with a higher concentration in female rats compared with male rats. The profile of this optimization is presented. Comparison of experimental to simulated data demonstrates that the optimization was performed well (Figure 7).
Figure 7. Comparison of Experimental to Simulated Data for Male Rat
Physiologically change of placenta, fetus and fat tissue took place during gestation. For each compartment equation relate to the measure observed in rat were described (Figures 8 a–d). The gestational module described in this PBPK model was optimized with the RTI International experimental data using a scenario of study A, which consists of a single i.v. injection of 1 mg/kg during gestation at GD18 (Table 6). Simulations were compared to the experimental data in Figure 8. Abbreviations are adipose (CF), hepatic (CLI), the rest of the body (CRE), blood (CB), kidney (CK), and fetal tissue (C FETAL) concentrations of BDE-47 in ng/g or nmol/g tissue. Symbols represent the experimental concentration of BDE-47. This optimization set few parameters in relation to the process (clearance exchange between mother and fetus).
A) | B) |
C) | D) |
Figure 8. Growth Rates for Physiological Changes Occurring During Gestation: (a) Placental Growth (n=10 fetus); (b) Blood Flow Rate in Placenta; (c) Fat Fraction; and (d) Fetal Growth (n=10 fetus)
Figure 9. Comparison of Experimental to Simulated Data for Pregnant Female Rat and Fetus
b. Performance Criteria for the Model Related to the Intended Use. The PBPK model for BDE-47 was intended to be used for i.v. and oral exposure scenarios. We will be able to describe the distribution of PBDE-47 of the parent compounds in different compartments (see Figure 6). This model will predict outcomes for single- and repetitive-exposure scenarios.
Table 6. Experimental Data Generated by RTI for the Simulation and Validation of This Model in the Rat Following i.v. and Oral Exposures
Organs or |
|
Study A 1 mg/kg iv, 24 h at GD 18 |
Study C 1 mg/kg oral, 24 h at GD 18 |
Study E 1 mg/kg oral |
Study P 20 mg/kg oral |
||||
|
Time point |
Mean (% of dose) |
SD (% of dose) |
Mean (% of dose) |
SD (% of dose) |
Mean |
SD (% of dose) |
Mean |
SD (% of dose) |
Blood |
0 |
0.00% |
0.01% |
0.01% |
0.02% |
0.19% |
0.07% |
0.00% |
0.00% |
Blood |
15 |
18.0% |
4.14% |
0.02% |
0.02% |
0.17% |
0.05% |
0.00% |
0.00% |
Blood |
30 |
12.3% |
2.30% |
0.06% |
0.07% |
0.24% |
0.04% |
0.02% |
0.01% |
Blood |
60 |
10.8% |
0.88% |
0.09% |
0.08% |
0.23% |
0.06% |
0.13% |
0.08% |
Blood |
120 |
12.3% |
12.5% |
0.40% |
0.22% |
0.49% |
0.06% |
0.30% |
0.23% |
Blood |
240 |
5.27% |
0.36% |
0.66% |
0.47% |
0.56% |
0.33% |
0.56% |
0.35% |
Blood |
480 |
3.07% |
1.33% |
0.82% |
0.39% |
0.31% |
0.08% |
1.45% |
0.28% |
Blood |
720 |
2.03% |
0.42% |
2.55% |
2.14% |
0.22% |
0.03% |
1.16% |
0.57% |
Blood |
Sacrifice |
2.00% |
0.58% |
1.47% |
0.38% |
0.43% |
0.11% |
0.57% |
0.36% |
Feces |
0 |
0.13% |
0.05% |
0.33% |
0.55% |
8.41% |
4.67% |
- |
- |
Feces |
360 |
0.05% |
0.08% |
0.04% |
0.05% |
0.18% |
0.12% |
0.00% |
0.00% |
Feces |
720 |
2.54% |
3.41% |
- |
- |
1.51% |
0.10% |
0.01% |
0.01% |
Feces |
1400 |
0.35% |
0.39% |
1.05% |
- |
11.3% |
5.33% |
10.8% |
6.95% |
Feces |
Sacrifice |
0.38% |
0.20% |
0.61% |
0.61% |
0.04% |
0.29% |
0.26% |
|
Liver |
Sacrifice |
7.47% |
2.65% |
9.79% |
3.56% |
1.86% |
0.32% |
4.85% |
2.32% |
Kidney |
Sacrifice |
0.31% |
0.14% |
0.13% |
0.06% |
0.05% |
0.03% |
||
Muscle (abdominal + hindleg) |
Sacrifice |
31.0% |
10.4% |
16.6% |
14.9% |
3.66% |
0.46% |
2.79% |
0.78% |
Adipose tissue |
|
|
|
|
|
|
|
|
|
Total recovery: 7wt % |
Sacrifice |
44.0% |
6.28% |
33.4% |
3.85% |
33.3% |
11.9% |
25.2% |
11.1% |
Total recovery: 10wt% |
Sacrifice |
62.9% |
9.0% |
47.8% |
5.49% |
47.6% |
16.9% |
36.1% |
15.9% |
Total recovery: 12wt% |
Sacrifice |
75.5% |
10.8% |
57.3% |
6.59% |
57.2% |
20.3% |
43.3% |
19.1% |
Fetal GI tract |
Sacrifice |
0.03% |
0.02% |
0.04% |
0.01% |
0.03% |
0.04% |
0.01% |
0.01% |
Adipose tissue |
|
|
|
|
|
|
|
|
|
Net fetuses - GI tracts + livers |
Sacrifice |
1.32% |
0.61% |
1.74% |
0.21% |
0.37% |
0.12% |
0.73% |
0.20% |
Fetal blood |
Sacrifice |
0.01% |
0.00% |
0.04% |
0.05% |
0.00% |
0.00% |
0.00% |
0.00% |
Fetal brain |
Sacrifice |
0.20% |
0.07% |
0.22% |
0.02% |
0.05% |
0.01% |
0.11% |
0.08% |
Total recovery: 7% adipose |
|
89.0% |
12.6% |
63.8% |
15.44% |
47.2% |
13.0% |
45.5% |
21.0% |
Total recovery: 10% adipose |
|
107.9% |
13.9% |
78.4% |
17.26% |
61.5% |
17.6% |
56.3% |
25.8% |
Total recovery: 12% adipose |
|
120.5% |
14.9% |
88.1% |
18.49% |
71.0% |
20.8% |
63.5% |
28.9% |
c. Test Results to Demonstrate That Model Performance Criteria Were Met (e.g., Code Verification, Sensitivity Analyses, History Matching With Lab or Field Data, as Appropriate). After the optimization, we used the other dataset generated by Sanders, et al., (2006) for the validation. This group measured only one point at the end of the exposure which is 24 hours post-exposure. Comparison of experimental results to the simulation curve showed an acceptable prediction (Sanders, et al., 2006) (Figure 10 A-D). Even though Sanders, et al. (2006) used radioactivity to measure the tissue concentrations in different compartments, the comparison to the simulated exposure is acceptable.
Figure 10 A–D. Simulation of BDE-47 Exposure at Different Concentrations Corresponding to Single Oral Doses of 1, 10, 100 and 1000 μmol/kg in Naive Adult Rats. Simulations were compared to the experimental data. Abbreviations are adipose (CF), brain tissue (CBR), hepatic (CLI), the rest of the body (CRE), blood (CB), and kidney (CK) concentrations of BDE-47 in nmol/g tissue. Symbols represent the experimental concentration of BDE-47.
Figure 10A.
Figure 10B.
Figure 10C.
Figure 10D.
Our group produced experimental data for rat exposure that were used here for the validation (Table 6). The next three graphs (Figures 11A though 11C) correspond to the experiments C, E, and P, scenarios which described the distribution of BDE-47 single i.v. or oral exposure or repetitive oral exposure scenarios.
Figure 11A. Simulation of BDE-47 Exposed in a Single i.v. Dose of 1 mg of BDE-47 /kg in Gestational Female Rat. Simulations were compared to the experimental data. Abbreviations are adipose (CF), hepatic (CLI), the rest of the body (CRE), blood (CB), kidney (CK), and fetal tissue (C FETAL) concentrations of BDE-47 in ng/g or nmol/g tissue. Symbols represent the experimental concentration of BDE-47.
Figure 11B. Simulation of BDE-47 Exposed at a Concentration Corresponding to Repetitive Oral Exposure Over 10 Days of 1 mg/kg/d During Gestation at GD18. Simulations were compared to the experimental data. Abbreviations are adipose (CF), hepatic (CLI), rest of the body (CRE), blood (CB), kidney (CK), and fetal tissue (C FETAL) concentrations of BDE-47 in ng/g or nmol/g tissue. Symbols represent the experimental concentration of BDE-47.
Figure 11-C. Simulation of BDE-47 Exposed at Different Times and Concentrations Corresponding to a Single 20 mg/kg dose at GD 18. Simulations were compared to the experimental data. Abbreviations are adipose (CF), hepatic (CLI), rest of the body (CRE), blood (CB), kidney (CK) and fetal tissue (C FETAL) concentration of BDE-47 in ng/g or nmol/g tissue. Symbols represent the experimental concentration of BDE-47.
d. Theory Behind the Model, Expressed in Nonmathematical Terms. A literature review suggested that, for the rat, there is metabolic induction, no specific binding, and low biotransformation. In addition, BDE-47 has a high log Kow of around 6 to 7, thus it will extensively spread in the adipose tissue.
e. Mathematics To Be Used, Including Formulas and Calculation Methods. This section will present the mathematical description of organ growth during gestational events (Figure 9).
Many modifications occur during gestation that result in physiological changes. The next section will explain the variability present and the result limit during pregnancy. All descriptions obtained are supported by the literature. Growth rates are for physiological changes occurring during gestation.
Placental Growth During Gestation (Calculated for 10 Placentae). The experimental data came from Sikov (1970). The mathematical expression used was:
Blood Flow Rate in Placental Compartment During Gestation. The experimental data came from Buelke-Sam, et al. (1982a,b). The mathematical expression used is presented below:
Fat Fraction of Body Weight During Gestation. The experimental data came from Fisher, et al. (1989). The mathematical expression used was:
Fetal Growth During Gestation. The experimental data came from Sikov (1970). The mathematical expression used was:
f. Whether or not the Theory and Mathematical Algorithms Were Peer Reviewed, and, if so, Include a Summary of Theoretical Strengths and Weaknesses. All equations used in this model were peer-reviewed and published in 2004 (Emond, et al., 2004).
Equations Used for the Rat PBPK Gestational Model.
Variation of Body Weight with Age
All other tissue growth rates are presented in the legend for Figure 8.
Cardiac Output
A factor of 60 corresponds to the conversion of minutes to hours and 1000 is the conversion of body weight from g to kg.
Blood Compartment
Tissue Compartment (Fat, the Rest of the Body)
i) Tissue blood subcompartment:
ii) Tissue cellular matrices:
Liver Tissue Compartment (Liver)
i) Tissue blood subcompartment:
ii) Tissue cellular matrices:
Placenta Tissue Compartment
i) Tissue blood subcompartment:
ii) Dioxin transfer from placenta to fetuses:
iii) PBDE transfer from fetuses to placenta:
iv) Fetal PBDE concentration (fetuses = 10 per litter):
Gastrointestinal Absorption and Distribution of PBDE to the Portal Lymphatic Circulation
i) Amount of PBDE remaining in lumen cavity:
Lumen: Amount of PBDE remaining in the GI tract (nmole)
Intake: Rate of intake of PBDE during a repetitive exposure (nmol/hour)
ii) Amount of PBDE eliminated in the feces:
iii) Absorption rate of PBDE to the blood via the lymphatic circulation:
iv) Absorption rate of PBDE by the liver via portal circulation:
g. Number and Uncertainty Associated With Parameters (How Data Were Selected/Obtained and Assessed to Assure They Met Requirements, or, Documentation of the Weakness Due to Known Uncertainty and Variability). The model used and the equations have been investigated for variability and uncertainty. The results have been described previously in Emond, et al. (2004).
h. Input Data Requirements and How Data Will be Selected/Obtained and Later Assessed to Assure it Met Requirements, or, Documentation of the Weakness Due to Known Uncertainty and Variability. Few data had been published in the literature, so we tried to select the most accurate data available in recognition of this fact.
i. Hardware Requirements and Documentation (e.g., Users’ Guide, Journal, Publications, Model Code). This model works on a PC computer containing an Intel Pentium processor, 1.86 GHz. The codes were written using a ACSL software Aegis Technology version 11.8.4.
Conclusions:
The rat dosing experiments provided substantial data for PBPK model development. When the concentration data are expressed as a percentage of the total dose, very good mass balance was measured for the i.v. dose; essentially 100% of the i.v. dose was accounted for. A comparison of the recovered dose following single oral doses at 1 and 20 mg/kg shows that a smaller fraction of the higher dose was absorbed (78% and 56%, respectively, for the 1 and 20 mg/kg doses, assuming 10% adipose content). The percentage of the dose recoveries for 10-day repeated oral dosing fell between the 1 and 20 mg/kg single oral dose absorptions at 68%. BDE-47 was measurable in the fetal rats. Following the 10 days, repeated oral dosing at 0.45% was measured in the fetuses. Of this, 0.03% was measured in the GI tract (surrogate for meconium), 0.05% was measured in the brain, and 0.37% was contained in the remaining fetal tissues.
Data obtained from the analysis of the human samples (cord blood, meconium, and breast milk) showed measurable amounts of BDE-47 in each of the media. Ongoing collaborations with CDC for re-analysis of breast milk to confirm or refute the measured concentrations are in process. That BDE-47 was measured in meconium suggests its utility as an integrated measure of in utero exposure. However, the quantitative relationships among media and concentrations in target fetal tissues remain unclear. It is not surprising that cord blood shows little association with meconium since BDE-47 in meconium is integrated over the time of meconium formation while cord blood represents a single point in time. Additional information is needed to understand BDE sequestration.
The PBPK model presented here is the first reported for any PBDE. Good agreement was demonstrated between modeled and measured concentrations in maternal compartments and total content in fetuses. The measured values in rat fetal tissues and human neonate samples also support refinement of the PBPK model to include meconium formation and the associated sequestration of BDEs into the meconium.
References:
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Emond C, Birnbaum LS, DeVito M. Physiologically based pharmacokinetic model for developmental exposures to TCDD in the rat. Toxicological Sciences 2004;80(1):115-133.
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Emond C, Raymer JH, Studabaker WB, Garner CE, Birnbaum LS. A physiologically based pharmacokinetic model for developmental exposure to BDE-47 in rats. Toxicology and Applied Pharmacology 2010;242(3):290-298. |
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Supplemental Keywords:
sensitive populations, human health, mathematics, measurement methods, bioavailability, metabolism, vulnerability, infant,, RFA, Health, Scientific Discipline, PHYSICAL ASPECTS, ENVIRONMENTAL MANAGEMENT, Toxicology, Genetics, Health Risk Assessment, Risk Assessments, Environmental Microbiology, Susceptibility/Sensitive Population/Genetic Susceptibility, 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:
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