Grantee Research Project Results

2004 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: R830756
Title: 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, 2004 through December 31, 2005
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:

The first objective of this research project is to develop a physiologically based pharmacokinetic (PBPK) animal model for the polybrominated diphenyl ethers (PBDEs), 2,2’,4,4’-tetrabromomodiphenyl ether (BDE-47) and 2,2',4,4',5-pentabromodiphenyl ether (BDE-99), which 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 of this research project is to develop analytical methods for PBDEs in human blood and meconium and apply these methods to samples collected during this project to estimate the utility of the model and 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:

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. Mother's blood concentrations of PBDEs are predictive of the cord blood and/or meconium concentrations in newborn babies.
4. Meconium is a useful medium for assessing cumulative dose of the developing fetus.

Progress Summary:

The major efforts during Year 2 of the project were the purification of BDE-47 for dosing studies, execution of the dosing studies using purified BDE-47, development of analytical methods, evaluation of potential for sample contamination arising from environmental PBDEs in dust, and development of the model. Progress in each of these areas is summarized below.

Following discussion with the U.S. Environmental Protection Agency (EPA) Project Officer, a decision was made to drop the studies with BDE-99 in favor of adding a lactational piece for BDE-47. We believed this was justified given the cost of obtaining pure BDE-99 and its similarity (partition, chemical reactivity) to BDE-47. The lactational piece of the model would allow us to determine the excretion after the controlled dosing experiments. In this manner, we can begin to explore the post-natal exposure of rat pups. There is a fair amount of literature that reports measured BDE-47 concentrations in human milk, and the ability to relate breast milk concentrations to known exposures in our research project will add very useful information.

Animal Dosing Studies: Determination of Maternal-Fetal Pharmacokinetics of BDE-47

The objectives of these studies were to:

1. administer BDE-47 to pregnant female Sprague Dawley rats via single IV, single oral, and repeat oral doses;
2. and collect blood and tissue samples for later analysis of parent compound concentrations.

The methods we used to complete this project are described below.

Animals . Timed pregnant female Sprague Dawley rats (sperm positive on gestational day [GD] 0) with indwelling jugular cannulae were ordered. Animals were approximately 10 weeks of age at the time of breeding at the supplier and were received at the Research Triangle Institute (RTI) Animal Research Facility on GD5 (the day 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 were available.

Dose Preparation . Doses were prepared in bulk 1 day prior to administration. Oral formulations were prepared in fresh corn oil by dissolving test article in sufficient volume of acetone, adding approximately 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 intravenous 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, 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 drawn by cardiac puncture. Maternal liver, muscle, kidney, brain, skin (ears), adipose, small intestine, large intestine, cecum, gut contents, placentae, and amniotic fluid and 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, 12-24 hours post-dose during the single dose studies and at 0-6, 6-12, 12-24 hours following the final dose during the 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, small intestine, large intestine, cecum, gut contents, placentae, and amniotic fluid and 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 BDE-47

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, 1, 2, 4, 8, 12 and 24hr	0-6, 6-12, 12-24 hr
50	PO	4	1	0, 15 and 30 minutes, 1, 2, 4, 8, 12 and 24hr	0-6, 6-12, 12-24 hr
1	IV	4	1	0, 5, 15 and 30 minutes, 1, 2, 4, 8, 12 and 24hr	0-6, 6-12, 12-24 hr
50	IV	4	1	0, 5, 15 and 30 minutes, 1, 2, 4, 8, 12 and 24hr	0-6, 6-12, 12-24 hr
1	PO	4	10	0, 15 and 30 minutes, 1, 2, 4, 8, 12 and 24hr	0-6, 6-12, 12-24 hr
50	PO	4	10	0, 15 and 30 minutes, 1, 2, 4, 8, 12 and 24hr	0-6, 6-12, 12-24 hr
20	PO	4	1	0, 15 and 30 minutes, 1, 2, 4, 8, 12 and 24hr	0-6, 6-12, 12-24 hr
20	PO	4	1	0, 15 and 30 minutes, 1, 2, 4, 8, 12 and 24hr	0-6, 6-12, 12-24 hr
20	PO	4	1	0, 15 and 30 minutes, 1, 2, 4, 8, 12 and 24hr	0-6, 6-12, 12-24 hr
20	PO	4	10	0, 15 and 30 minutes, 1, 2, 4, 8, 12 and 24hr	0-6, 6-12, 12-24 hr
20	PO	4	10	0, 15 and 30 minutes, 1, 2, 4, 8, 12 and 24hr	0-6, 6-12, 12-24 hr
20	PO	4	10	0, 15 and 30 minutes, 1, 2, 4, 8, 12 and 24hr	0-6, 6-12, 12-24 hr
a Single PO and IV 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.

Analytical Method Development

Method development was conducted using liver from non-dosed rats. A modification of a method used at RTI for organochlorine pesticides and PCBs in brain was evaluated. The modification sought to adapt the clean-up step to Solid Phase Extraction (SPE)-based Florisil rather than the old, open-column approach. Work conducted during 2004 was focused on the validation of the clean-up step.

The procedure for the extraction of tissue is as follows:

• Homogenize tissue with water and hexane; decant the hexane and repeat twice more;
• Combine the extracts and dry using anhydrous sodium sulfate column;
• Adjust extract volume to obtain 10mg tissue per mL equivalent so that co-extracted tissue components would not vary; add surrogate (PCB-198) 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);
• Analyze by gas chromatography/electron capture detection 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 percent at the 500 ppb concentration in tissue (5 ppb in extract). Precisions ranged from 1.5 to 11.6 percent relative standard deviation. 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.

BDE-47 has been found in household dust and apparently arises, at least in part, from the PBDEs volatilized from electronic equipment that subsequently associate with the dust. Given the widespread occurrence of PBDEs in the environment, it is not surprising that surface wipes of necropsy areas, hoods, and laboratory bench surfaces revealed contamination with BDE-47; BDE-99 was found less frequently. It will be very important as work progresses to generate adequate blanks and controls so that such contamination can be taken into account.

PBPK Modeling

For PBPK Modeling, Mathematical Modeler/Statistician (Amy Licata, Ph.D.) trained a junior statistician (Bing Liu, M.S.; M. CPE.) on the Advanced Continuous Simulation Language (ACSL) software package. With ACSL software, mathematical modeling, statistical analyses, and exploratory data analysis can be conducted and high quality graphs produced. Through literature searches and conversations with colleagues, additional current gestational PBPK models were explored. Also, literature searches and discussions with co-principal investigators were used to evaluate how best to incorporate a lactational component into a gestational PBPK model. Relevant literature was stored on a shared electronic folder so that staff members were kept abreast of current findings.

We presented our work to a local group of scientists in the Research Triangle Park, North Carolina area who are working with brominated flame retardants. The meeting, held in January 2004, was organized by Dr. Linda Birnbaum of EPA.

Future Activities:

We will analyze samples from the timed-pregnancy studies. Partition coefficients will be determined and provided as input to the PBPK model. The PBPK model construction will continue. We will dose rats with BDE-47 to obtain concentrations in breast milk, and these data will be used for the lactational component of the model.

Journal Articles:

No journal articles submitted with this report: View all 8 publications for this project

Supplemental Keywords:

sensitive populations, human health, mathematics, measurement methods, bioavailability, metabolism, vulnerability, infant, children’s health, risk assessment, toxicology, PBDE, developmental effects, environmental hazard exposures, pharmacokinetic models,, 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, toxics

Relevant Websites:

http://www.rti.org Exit
https://cfpub.epa.gov/ncer_abstracts/index.cfm/fuseaction/display.files/fileID/8948 Exit

Progress and Final Reports:

Original Abstract

2003 Progress Report

2005 Progress Report

2006

Final Report