EPA/635/R-07/007F
www.epa.gov/iris
vvEPA
TOXICOLOGICAL REVIEW
OF
2,2',4,4',5,5'-HEXABROMODiPHENYL
ETHER (BDE-153)
(CAS No. 68631-49-2)
In Support of Summary Information on the
Integrated Risk Information System (IRIS)
June 2008
U.S. Environmental Protection Agency
Washington, DC
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DISCLAIMER
This document has been reviewed in accordance with U.S. Environmental Protection
Agency policy and approved for publication. Mention of trade names or commercial products
does not constitute endorsement or recommendation for use.
11
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CONTENTS—TOXICOLOGICAL REVIEW OF
l^M'AS'-HEXABROMODIPHENYL ETHER (CASRN 68631-49-2)
LIST OF TABLES v
LIST OF FIGURES v
LIST OF ACRONYMS vi
FOREWORD vii
AUTHORS, CONTRIBUTORS, AND REVIEWERS viii
1. INTRODUCTION 1
2. CHEMICAL AND PHYSICAL INFORMATION 3
3. TOXICOKINETICS 6
3.1. ABSORPTION 6
3.2. DISTRIBUTION 6
3.2.1. Human Data 6
3.2.1.1. Adipose Tissue 7
3.2.1.2. Liver 8
3.2.1.3. Human Milk 9
3.2.1.4. Blood 10
3.2.1.5. Placental Transport 12
3.2.2. Animal Data 14
3.3. METABOLISM 15
3.4. ELIMINATION 17
3.5. PHYSIOLOGICALLY BASED TOXICOKINETIC MODELS 18
4. HAZARD IDENTIFICATION 19
4.1. STUDIES IN HUMANS—EPIDEMIOLOGY, CASE REPORTS, CLINICAL
CONTROLS 19
4.2. SHORT-TERM, SUBCHRONIC, AND CHRONIC STUDIES AND CANCER
BIOASSAYS IN ANIMALS—ORAL AND INHALATION 19
4.3. REPRODUCTIVE/DEVELOPMENTAL STUDIES 19
4.4. OTHER DURATION-OR ENDPOINT-SPECIFIC STUDIES 22
4.4.1. Receptor Site Interactions 22
4.4.1.1. Aryl Hydrocarbon Receptors 23
4.4.1.2. Other CYP-450 Induction Receptors 25
4.4.1.3. Estrogen Receptors 26
4.4.1.4. Androgen Receptors 26
4.4.1.5. Acetylcholine Receptors 27
4.4.2. Thyroid Effects 28
4.4.3. Genotoxicity 28
4.5. SYNTHESIS OF MAJOR NONCANCER EFFECTS 28
4.5.1. Oral 28
4.5.2. Inhalation 29
4.5.3. Mode-of-Action Information 29
in
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4.6. EVALUATION OF CARCINOGENICITY 31
4.7. SUSCEPTIBLE POPULATIONS AND LIFE STAGES 31
4.7.1. Possible Childhood Susceptibility 31
4.7.2. Possible Gender Differences 31
5. DOSE-RESPONSE ASSESSMENTS 32
5.1. ORAL REFERENCE DOSE (RfD) 32
5.1.1. Choice of Principal Study and Critical Effect—with Rationale and Justification 32
5.1.2. Methods of Analysis 33
5.1.3. RfD Derivation 33
5.1.4. Previous RfD Assessment 34
5.2. INHALATION REFERENCE CONCENTRATION (RfC) 35
5.3. CANCER ASSESSMENT 35
6. MAJOR CONCLUSIONS IN THE CHARACTERIZATION OF HAZARD AND DOSE
RESPONSE 36
6.1. HUMAN HAZARD POTENTIAL 36
6.2. DOSE RESPONSE 36
7. REFERENCES 38
APPENDIX A: SUMMARY OF EXTERNAL PEER REVIEW AND PUBLIC COMMENTS
AND DISPOSITION A-l
IV
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LIST OF TABLES
Table 2-1. IUPAC number and bromine substitution pattern of some hexaBDE congeners 3
Table 2-2. Composition by weight of commercial penta- and octaBDEs 4
Table 2-3. Composition by weight of different congeners in commercial penta- and
octaBDEs 4
Table 2-4. Physical and chemical properties of BDE-153 5
Table 3-1. Median PBDE concentration in human biological media in theU.S 7
Table 3-2. Comparison of median PBDE congener concentrations in maternal and fetal
sera in Sweden and in the U.S 13
Table 4-1. Receptor interaction studies of hexaBDE congeners 23
LIST OF FIGURES
Figure 2-1. Chemical structure of hexabromodiphenyl ether 3
Figure 3-1. Proposed metabolic pathway for BDE-153 inB6C3Fl mice 16
v
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LIST OF ACRONYMS
Ah
AhR
BDE-153
BMD
BMDL
CALUX
CAR
CASRN
cDNA
CYP-450
ER
EROD
FOB
heptaBDE
hexaBDE
IRIS
IUPAC
LOAEL
Iw
mRNA
MUP
NOAEL
octaBDE
PBDE
PCB
PCDD
PCDF
PCR
pentaBDE
PND
PXR
RfC
RfD
T3
T4
TCDD
tetraBDE
triBDE
TTR
UF
aryl hydrocarbon
Ah receptor
2,2',4,4',5,5'-hexabromodiphenyl ether
benchmark dose
95% lower bound on the BMD
Chemical-Activated LUciferase gene expression
constitutive androstane receptor
Chemical Abstracts Service Registry Number
complementary deoxyribonucleic acid
cytochrome P-450
estrogen receptor
ethoxyresorufm O-deethylase
functional observational battery
heptabromodiphenyl ether
hexabromodiphenyl ether
Integrated Risk Information System
International Union of Pure and Applied Chemistry
lowest-observed-adverse-effect level
lipid weight
messenger ribonucleic acid
major urinary protein
no-observed-adverse-effect level
octabromodiphenyl ether
polybrominated diphenyl ether
polychlorinated biphenyl
polychlorinated dibenzo-p-dioxin
polychlorinated dibenzofuran
polymerase chain reaction
pentabromodiphenyl ether
postnatal day
pregnane X receptor
reference concentration
reference dose
triiodothyronine
thyroxine
2,3,7,8-tetrachlorodibenzo-p-dioxin
tetrabromodiphenyl ether
tribromodiphenyl ether
transthyretin
uncertainty factor
VI
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FOREWORD
The purpose of this Toxicological Review is to provide scientific support and rationale
for the hazard and dose-response assessment in IRIS pertaining to chronic exposure to
2,2',4,4',5,5'-hexabromodiphenyl ether (BDE-153). It is not intended to be a comprehensive
treatise on the chemical or toxicological nature of 2,2',4,4',5,5'-hexabromodiphenyl ether.
The majority of the available toxicological information on the hexabromodiphenyl ether
homolog group (CASRN 36483-60-0) relates to the congener 2,2',4,4',5,5'-hexabromodiphenyl
ether or BDE-153 (CASRN 68631-49-2). Toxicological information related to other congeners
in the hexabromodiphenyl ether homolog group is also discussed. However, this health
assessment does not deal with commercial mixtures containing hexabromodiphenyl ether
congeners as one of the ingredients present in the formulation. In addition to BDE-153, IRIS
health assessments have also been prepared for three other polybrominated diphenyl ether
congeners: tetraBDE-47, pentaBDE-99, and decaBDE-209. These four congeners are those for
which toxicological studies suitable for dose-response assessments were available and are the
ones most commonly found in the environment and human biological media.
The intent of section 6, Major Conclusions in the Characterization of Hazard and Dose
Response, is to present the major conclusions reached in the derivation of the reference dose,
reference concentration and cancer assessment, where applicable, and to characterize the overall
confidence in the quantitative and qualitative aspects of hazard and dose response by addressing
the quality of data and related uncertainties. The discussion is intended to convey the limitations
of the assessment and to aid and guide the risk assessor in the ensuing steps of the risk
assessment process.
For other general information about this assessment or other questions relating to IRIS,
the reader is referred to EPA's IRIS Hotline at (202) 566-1676 (phone), (202) 566-1749 (fax), or
hotline.iris@epa.gov (email address).
vn
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AUTHORS, CONTRIBUTORS, AND REVIEWERS
CHEMICAL MANAGER
Joyce Morrissey Donohue, Ph.D.
Office of Water, Office of Science and Technology
Health and Ecological Criteria Division
U.S. Environmental Protection Agency
Washington, DC
AUTHORS
Joyce Morrissey Donohue, Ph.D.
Office of Water, Office of Science and Technology
Health and Ecological Criteria Division
U.S. Environmental Protection Agency
Washington, DC
Hend Galal-Gorchev, Ph.D.
Office of Water, Office of Science and Technology
Health and Ecological Criteria Division
U.S. Environmental Protection Agency
Washington, DC
Mary Manibusan
Office of Research and Development
National Center for Environmental Assessment
U.S. Environmental Protection Agency
Washington, DC
OFFICE OF RESEARCH AND DEVELOPMENT CO-LEAD
Samantha J. Jones, Ph.D.
Office of Research and Development
National Center for Environmental Assessment
U.S. Environmental Protection Agency
Washington, DC
REVIEWERS
This document has been reviewed by EPA scientists, interagency reviewers from other
federal agencies, and the public, and peer reviewed by independent scientists external to EPA. A
summary and EPA's disposition of the comments received from the independent external peer
reviewers and from the public is included in Appendix A of the Toxicological Review of
2,2',4,4',5,5'-hexabromodiphenyl ether (BDE-153).
Vlll
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INTERNAL EPA REVIEWERS
Linda Birnbaum, Ph.D.
Office of Research and Development
Experimental Toxicology Division
National Health and Environmental Effects Research Laboratory
Karen Hogan, M.S.
Office of Research and Development
National Center for Environmental Assessment
Tammy Stoker, Ph.D.
Office of Research and Development
Reproductive Toxicology Division
National Health and Environmental Effects Research Laboratory
EXTERNAL PEER REVIEWERS
Ahmed E. Ahmed, Ph.D.
University of Texas Medical Branch
Richard J. Bull, Ph.D.
MoBull Consulting
Lucio G. Costa, Ph.D.
University of Washington
Ralph Kodell, Ph.D.
University of Arkansas for Medical Sciences
Deborah Rice, Ph.D. (chair)
State of Maine
IX
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1. INTRODUCTION
This document presents background information and justification for the Integrated Risk
Information System (IRIS) Summary of the hazard and dose-response assessment of
2,2',4,4',5,5'-hexabromodiphenyl ether (BDE-153). IRIS Summaries may include oral reference
dose (RfD) and inhalation reference concentration (RfC) values for chronic and other exposure
durations, and a carcinogenicity assessment.
The RfD and RfC, if derived, provide quantitative information for use in risk assessments
for health effects known or assumed to be produced through a nonlinear (presumed threshold)
mode of action. The RfD (expressed in units of mg/kg-day) is defined as an estimate (with
uncertainty spanning perhaps an order of magnitude) of a daily exposure to the human
population (including sensitive subgroups) that is likely to be without an appreciable risk of
deleterious effects during a lifetime. The inhalation RfC (expressed in units of mg/m3) is
analogous to the oral RfD, but provides a continuous inhalation exposure estimate. The
inhalation RfC considers toxic effects for both the respiratory system (portal of entry) and for
effects peripheral to the respiratory system (extrarespiratory or systemic effects). Reference
values are generally derived for chronic exposures (up to a lifetime), but may also be derived for
acute (<24 hours), short-term (>24 hours up to 30 days), and subchronic (>30 days up to 10% of
lifetime) exposure durations, all of which are derived based on an assumption of continuous
exposure throughout the duration specified. Unless specified otherwise, the RfD and RfC are
derived for chronic exposure duration.
The carcinogenicity assessment provides information on the carcinogenic hazard
potential of the substance in question and quantitative estimates of risk from oral and inhalation
exposure may be derived. The information includes a weight-of-evidence judgment of the
likelihood that the agent is a human carcinogen and the conditions under which the carcinogenic
effects may be expressed. Quantitative risk estimates are derived from the application of a low-
dose extrapolation procedure. If derived, the oral slope factor is a plausible upper bound on the
estimate of risk per mg/kg-day of oral exposure. Similarly, an inhalation unit risk is a plausible
upper bound on the estimate of risk per ug/m3 air breathed.
Development of these hazard identification and dose-response assessments for BDE-153
has followed the general guidelines for risk assessment as set forth by the National Research
Council (1983). EPA Guidelines and Risk Assessment Forum Technical Panel Reports that may
have been used in the development of this assessment include the following: Guidelines for the
Health Risk Assessment of'Chemical Mixtures (U.S. EPA, 1986a), Guidelines for Mutagenicity
Risk Assessment (U.S. EPA, 1986b), Recommendations for and Documentation of Biological
Values for Use in Risk Assessment (U.S. EPA, 1988), Guidelines for Developmental Toxicity
Risk Assessment (U.S. EPA, 1991), Interim Policy for Particle Size and Limit Concentration
1
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Issues in Inhalation Toxicity (U.S. EPA, 1994a), Methods for Derivation of Inhalation Reference
Concentrations and Application of Inhalation Dosimetry (U.S. EPA, 1994b), Use of the
Benchmark Dose Approach in Health Risk Assessment (U.S. EPA, 1995), Guidelines for
Reproductive Toxicity Risk Assessment (U.S. EPA, 1996), Guidelines for Neurotoxicity Risk
Assessment (U.S. EPA, 1998), Science Policy Council Handbook: Risk Characterization (U.S.
EPA, 2000a), Benchmark Dose Technical Guidance Document (U.S. EPA, 2000b),
Supplementary Guidance for Conducting Health Risk Assessment of Chemical Mixtures (U.S.
EPA, 2000c), A Review of the Reference Dose and Reference Concentration Processes (U.S.
EPA, 2002), Guidelines for Carcinogen Risk Assessment (U.S. EPA, 2005a), Supplemental
Guidance for Assessing Susceptibility from Early-Life Exposure to Carcinogens (U.S. EPA,
2005b), Science Policy Council Handbook: Peer Review (U.S. EPA, 2006a), and A Framework
for Assessing Health Risks of Environmental Exposures to Children (U.S. EPA, 2006b).
The literature search strategy employed for this compound was based on the Chemical
Abstracts Service Registry Number (CASRN) and at least one common name. Any pertinent
scientific information submitted by the public to the IRIS Submission Desk was also considered
in the development of this document. The relevant literature was reviewed through November
2007.
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2. CHEMICAL AND PHYSICAL INFORMATION
Hexabromodiphenyl ether (hexaBDE) is one of the possible 10 homologs of
polybrominated diphenyl ethers (PBDEs). Figure 2-1 shows the chemical structure of hexaBDE.
The number of possible congeners of hexaBDE is 42, with International Union of Pure and
Applied Chemistry (IUPAC) numbers 128 to 169. The IUPAC numbers and bromine
substitution patterns of some congeners that have been investigated in various studies are given
in Table 2-1.
m + n = 6
Figure 2-1. Chemical structure of hexabromodiphenyl ether.
Table 2-1. IUPAC number and bromine substitution pattern of some
hexaBDE congeners
IUPAC number
BDE-138
BDE-153
BDE-154
BDE-156
BDE-159
BDE-166
Bromine substitution pattern
2,2',3,4,4',5'-HexaBDE
2,2',4,4',5,5'-HexaBDE
2,2',4,4',5,6'-HexaBDE
2,3,3',4,4',5-HexaBDE
2,3,3',4,5,5'-HexaBDE
2,3,4,4',5,6-HexaBDE
HexaBDEs are found in the commercial flame retardants penta- and octabromodiphenyl
ethers (penta- and octaBDEs). The sole U.S. producer of the commercial pentaBDE and
octaBDE mixtures discontinued their production in the U.S. at the end of 2004. Approximate
composition by weight of these flame retardants is given in Table 2-2 (Great Lakes Chemical
Corporation, 2003).
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Table 2-2. Composition by weight of commercial penta- and octaBDEs
Homolog group
Tribromodiphenyl ether
Tetrabromodiphenyl ether
Pentabromodiphenyl ether
Hexabromodiphenyl ether
Heptabromodiphenyl ether
Octabromodiphenyl ether
Nonabromodiphenyl ether
Decabromodiphenyl ether
Commercial pentaBDE
0-1%
24-38%
50-62%
4-12%
-
-
-
-
Commercial octaBDE
-
-
0.5%
12%
45%
33%
10%
0.7%
The relative proportions by weight of different PBDE congeners in the commercial penta
, TM
DE-711M and octa DE-791M are given in Table 2-3 (Great Lakes Chemical Corporation, 2003).
Table 2-3. Composition by weight of different congeners in commercial
penta- and octaBDEs
Congener
BDE-47
BDE-99
BDE-100
BDE-153
BDE-154
BDE-183
DE-71™ pentaBDE
28%
43%
8%
6%
4%
-
DE-79™ octaBDE
<1%
<1%
Not detected
14%
2%
44%
Of the hexaBDE congeners, BDE-153 is present at higher levels than BDE-154 in both
the penta- and octaPBDE commercial products. The predominant hexaBDE congener in
environmental media, biota, and human tissues is BDE-153 (CASRN 68631-49-2), followed by
BDE-154 (CASRN 207122-15-4). Physical and chemical properties of BDE-153 are listed in
Table 2-4.
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Table 2-4. Physical and chemical properties of BDE-153
Parameter
Synonym
CASRN
Chemical formula
Molecular weight
Vapor pressure (Pa) at 25°C
Melting point (°C)
Solubility in water (ug/L)
Henry's law constant (Pa m3 mor1) at 25°C
Log octanol/water partition coefficient
(Kow)at25°C
Log octanol/air partition coefficient (Koa) at
25°C
Value
2,2',4,4',5,5'-Hexabromodiphenyl ether;
benzene; l,r-oxybis-2,4,5-tribromo-;
BDE-153
68631-49-2
C12H4Br6O
643.6
5.8 x 1(T6
183
0.9
0.26
7.90
11.9
Reference
U.S. EPA (2004)
U.S. EPA (2004)
U.S. EPA (2004)
U.S. EPA (2004)
Wong etal. (2001)
Palm et al. (2002)
Tittlemier et al. (2002);
ATSDR3 (2004)
Cetin and Odabasi
(2005)
Braekevelt et al. (2003);
ATSDR (2004)
Chen et al. (2003)
aATSDR = Agency for Toxic Substances and Disease Registry.
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3. TOXICOKINETICS
Data on the toxicokinetics of the hexabrominated diphenyl ethers in humans are limited
to findings on levels in adipose tissues, blood, liver, and maternal milk that demonstrate that they
are absorbed from the environment and distributed to tissues. Studies of BDE-153 in mice and
rats suggest that absorption is about 70% in both species. The highest levels are found in
adipose tissues, followed by muscle, liver, and skin. Metabolism appears to occur in mice to a
greater extent than in rats although results differ among studies. Both species form hydroxylated
metabolites via the activity of cytochrome P-450 (CYP-450) isozymes. There are no data for
metabolites in rats; in mice, monohydroxylated metabolites with six, five, and four bromines
have been identified. Small amounts of the hydroxylated metabolites may become conjugated
with glutathione. Knowledge of the metabolic pathway and tissue distribution of the metabolites
is limited. The parent compound and its metabolites are excreted with bile and feces and to a
lesser extent in urine. Excretion in mice is assisted by a urinary transport protein; a similar
protein has not been identified in rats.
3.1. ABSORPTION
There are no direct studies of BDE-153 absorption in humans. The data that demonstrate
human absorption come from measurements of hexaBDE in human biological media after
anthropogenic exposures but do not permit estimation of route-specific uptake parameters.
About 70% of a 0.6 mg/kg dose (1 umol/kg) was absorbed in groups of five male or
female F344 rats and B6C3F1 mice after gavage dosing with radiolabeled BDE-153 (96% purity;
27.8 mCi/mmol) in corn oil (Sanders et al., 2006). The estimate of absorption was based on the
recovery of the radiolabel from the orally exposed animals as compared with the results from
animals receiving the same dose injected intravenously.
3.2. DISTRIBUTION
The high Kow of hexaBDE suggests a strong potential for accumulation in lipid-rich
tissues. This property of hexaBDE is evident from the data on distribution in humans and
experimental animals described below.
3.2.1. Human Data
The human data come from monitoring of PBDEs in human populations rather than from
measured dosing studies. The data demonstrate that humans are exposed to PBDEs and that
absorption and distribution to some tissues occur. The data do not provide quantitative
information on exposure or the kinetics of absorption, tissue distribution, and retention. The
PBDE congener profiles in human biological media differ from the congener profiles of the
commercial PBDE mixtures. The reasons for this difference in congener distribution are not
6
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known with any certainty. Monitoring data are available for human adipose tissue, liver, milk,
and blood samples and indicate a tendency for PBDEs to distribute to these tissues. However,
distribution studies have not been conducted in humans, and therefore it is not known whether
hexaBDEs distribute to other tissues as well. The number of samples examined in various
studies and countries is small and therefore the data should not be construed as representative at
a national level.
Biomonitoring data with emphasis on levels of PBDE congeners found in the U.S. are
summarized in Tables 3-1 and 3-2.
Table 3-1. Median PBDE concentration in human biological media in the
U.S.
Adipose
tissue
Adipose
tissue
Breast
milk
Breast
milk
Maternal
serum
Fetal
serum
Serum
pools
Serum
pregnant
women
Year3
1996-
1998
2003-
2004
2002
2003
2001
2001
2000-
2002
1999-
2001
Na
23
52
47
40
12
17
7b
24
BDE-
47
BDE-
85
BDE-
99
BDE-
100
BDE-
153
BDE-
154
BDE-
183
£
PBDE
ng/g Iw
18
29
18
28
28
25
34
11
-
<1
0.4
0.6
0.7
0.3
7
10
6
5
6
7
11
2.9
3
12
3
5
4
4
6
1.8
4
<1
2
5
3
4
7
1.5
6
<1
0.2
0.4
0.3
0.7
1
0.3
-
—
0.1
0.2
0
0
-
0.1
41
75
34
50
37
39
61
21
Reference
She et al.
(2002)
Johnson-
Restrepo et
al. (2005)
Schecter et
al. (2003)
She et al.
(2007)
Mazdai et
al. (2003)
Mazdai et
al. (2003)
Sjodin et
al. (2004)
Bradman et
al. (2007)
aYear = year of sampling; N = number of donors.
bSeven serum pools with number of donors in each serum pool ranging from 40-200.
3.2.1.1. Adipose Tissue
Breast adipose samples were collected between 1996 and 1998 from 23 San Francisco
Bay area women as part of a case-control study on organochlorine compounds and breast cancer
(She et al., 2002). Women ranged from 28 to 62 years of age and were predominantly Caucasian
and born in the U.S. Pathology reports indicated that 12 women had malignancies, 8 had benign
tumors, and 3 had ductal carcinomas in situ, a condition considered by some as transitional to
malignancy. Breast adipose samples were collected during biopsy or breast surgery and were
analyzed for tetrabromodiphenyl ether (tetraBDE) (BDE-47), pentaBDEs (BDE-99 and -100),
and hexaBDEs (BDE-153 and -154). Mean and median concentrations of the sum of these
7
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PBDEs were 86 and 41 ng/g lipid weight (Iw), respectively, the highest human levels reported so
far. Median concentrations of individual PBDE congeners are given in Table 3-1. The highest
concentrations found were for tetraBDE, followed by penta- and hexaBDEs, a distribution that
does not follow that of the commercial penta- or octaBDEs used in the U.S. There was an
inverse relationship between the sum of the concentrations of these PBDEs in breast adipose
tissue and age, with women younger than the median age of 48 years having significantly higher
concentrations of PBDEs in adipose tissue than women older than 48. This may imply that
different activities may expose different age groups more than others or that some PBDE
congeners may accumulate differently with age. Five paired samples of breast and abdominal
adipose tissues were also analyzed for tetra- to hexaBDEs. Abdominal and breast concentrations
of PBDEs were highly correlated and of comparable magnitude.
In a study in New York City, adipose tissue samples (n = 52) were collected in 2003-
2004 from patients undergoing liposuction procedures (Johnson-Restrepo et al., 2005). Median
concentrations of individual PBDE congeners are given in Table 3-1. BDE-153 and -154 were
not detected. No significant difference was found in the concentrations of PBDEs between
genders. Concentrations of PBDEs were, on average, similar to those for poly chlorinated
biphenyls (PCBs). PBDE concentrations did not increase with increasing age of the subjects,
whereas concentrations of PCBs increased with increasing age in males but not in females.
These results suggest differences between PBDEs and PCBs in their sources or time course of
exposure and disposition.
Adipose tissues from persons living in the Tokyo area were collected from hospitals in
1970 (n = 10) and 2000 (n = 10). Women in their forties or fifties were selected (Choi et al.,
2003). The samples were analyzed for BDE-28, -47, -99, -100, -153, -154, and -183. Median
concentrations of the sum of these PBDEs were 0.03 and 1.3 ng/g Iw in 1970 and 2000,
respectively. In 2000, median concentrations in ng/g Iw of PBDE congeners were, in decreasing
order, BDE-47 (0.5), BDE-153 (0.4), BDE-100 (0.3), BDE-99 (0.1), BDE-28 (0.08), BDE-154
(0.06), andBDE-183 (0.05).
TetraBDE (BDE-47), pentaBDEs (BDE-85 and -99), and hexaBDEs (BDE-138 and -153)
were analyzed in adipose tissue samples from 3 women and 10 men between the ages of 28 and
83 years and living in Spain for at least 10 years. BDE-85 and -138 were not detected. Mean
concentrations, in decreasing order, were 1.8 ng/g Iw BDE-153, 1.4 ng/g Iw BDE-47, and
0.4 ng/g Iw BDE-99. The predominant congener in both men and women in this study was
BDE-153 (Meneses et al., 1999).
3.2.1.2. Liver
In a Swedish study, paired samples of human liver and adipose tissue obtained at autopsy
from one woman (age 47) and four men (ages 66-83) were analyzed for nine tribromodiphenyl
ether (triBDE) to hexaBDE congeners. PBDEs were found in all samples. TetraBDE-47,
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pentaBDE-99, and hexaBDE-153 were the predominant PBDE congeners in both liver and
adipose tissue. Generally, BDE-47 occurred at similar levels in adipose tissue and liver (mean
approximately 2.7 ng/g Iw). Mean concentrations of BDE-99 were higher in liver (3.8 ng/g Iw)
than in adipose tissue (1.3 ng/g Iw). Average concentrations of BDE-153 were 2.1 and 1.0 ng/g
Iw in liver and adipose tissue, respectively. Lower concentrations were found for BDE-154,
with average concentrations of 0.14 and 0.06 ng/g Iw in liver and adipose tissue, respectively.
Similarly to BDE-99, average concentrations of BDE-153 and -154 were therefore higher in liver
than in adipose tissue, perhaps indicating a trend for selective accumulation in the liver with
increasing degree of bromination (Guvenius et al., 2001).
3.2.1.3. Human Milk
In a study conducted in 2002 of levels of PBDEs in human milk in the U.S., 47 samples
from Caucasian, African-American, and Hispanic nursing mothers 20-41 years of age and living
in Texas were analyzed for 13 PBDE congeners (Schecter et al., 2003). Mean and median total
concentrations of tri- through decaBDEs were 74 and 34 ng/g Iw, respectively. The maximum
and mean concentrations of BDE-153 were 22 and 5 ng/g Iw, respectively. Maximum and mean
levels of the other hexaBDE congeners were 7 and 0.6 ng/g Iw for BDE-138 and 7 and 0.8 ng/g
Iw for BDE-154, respectively, indicating wide variations of congener levels among nursing
women. Median concentrations of individual PBDE congeners and total median concentration
of PBDEs are given in Table 3-1. There was no apparent difference in concentrations between
age groups or ethnic groups.
Milk samples were collected in 2003 from 40 first-time mothers with 2- to 8-week-old
infants and residing in urban areas in the Pacific Northwest of the U.S. (Montana, Oregon, and
Washington State) and Canada (British Columbia) (She et al., 2007). Mean and median total
concentrations of 12 tri- through decaBDE congeners were 96 and 50 ng/g Iw, respectively.
These values are substantially higher than the values reported in the study of Schecter et al.
(2003) and could be due to the fact that the mothers in the later study had been nursing for longer
periods of time. BDE-47 was found at the highest level, with median concentration of 28 ng/g
Iw, followed by BDE-99 (5.4 ng/g Iw), BDE-100 (5.3 ng/g Iw), and hexaBDE-153 (4.8 ng/g Iw).
Except for triBDE-28 with a median concentration of about 2 ng/g Iw, all other concentrations of
PBDE congeners were <1 ng/g Iw. In 7% of the samples, hexaBDE-153 was the dominant
congener. DecaBDE-209 with median concentration of 0.4 ng/g Iw was a minor congener in
breast milk, contributing 1.2% to the total PBDE concentration.
Breast milk was collected from 12 primiparous 24- to 33-year-old nursing women in
Japan, at 1 month after delivery and analyzed for six tri- to hexaBDEs. The most abundant
PBDE congener in human milk was BDE-47, followed by BDE-153, pentaBDEs (BDE-99 and
-100), triBDE (BDE-28), and BDE-154. The sum of the concentrations of six tri- to hexaBDEs
-------
ranged from 0.7 to 2.8 ng/g Iw. There was a strong positive relationship between total PBDE
levels in human milk and the frequency offish consumption (Ohta et al., 2002).
In another study in Japan, 16 tri- to hepta-PBDEs were analyzed in eight pooled human
milk samples collected between 1973 and 2000. PBDEs were not detected in the samples from
1973 at the limit of detection of 0.01 ng/g Iw (Akutsu et al., 2003). In 2000, the sum of the
concentration of these PBDEs was 1.4 ng/g Iw and the predominant congeners were BDE-47
(0.5 ng/g Iw), BDE-153 (0.3 ng/g Iw), and BDE-99 and -100 (approximately 0.2 ng/g Iw each).
Concentrations of the remaining PBDE congeners, including BDE-154, were less than 0.05 ng/g
Iw. The relatively large concentration of BDE-153 in Japanese mothers' milk was explained by
past use of a hexaBDE commercial product in Japan, consisting mostly of BDE-153 (Akutsu et
al., 2003).
The breast milk concentrations of BDE-47, pentaBDEs (BDE-99 and -100), and
hexaBDEs (BDE-153 and -154) were determined in samples from 93 primiparous women
collected from 1996 to 1999 in Uppsala County, Sweden (Lind et al., 2003). The women ranged
in age from 20-35 years. BDE-47 was the major congener (mean value 2.4 ng/g Iw) and
constituted 60% of the mean concentration of PBDEs of 4.0 ng/g Iw, followed by BDE-99 and
BDE-153 (0.6 ng/g Iw each), BDE-100 (0.4 ng/g Iw), and BDE-154 (0.07 ng/g Iw). No
significant relationship was found between breast milk concentrations of PBDEs and dietary
intakes of PBDEs (through fish, meat/poultry, dairy products, and egg consumption), age, body
mass index, alcohol consumption, or computer usage. After adjustments for these factors, a
weak but significant association between PBDE concentrations and smoking was observed.
Time-trend analysis for samples collected between 1996 and 2001 indicated a peak in total
PBDE concentrations around 1998, followed by decreasing levels.
Pooled samples of breast milk collected at eight time periods between 1972 and 1997
from primiparous Swedish women were analyzed for tri- to hexaBDEs. In 1997, BDE-47 was
the most abundant congener (2.3 ng/g Iw), followed by BDE-99 and -153 (approximately
0.5 ng/g Iw each), BDE-100 (0.4 ng/g Iw), BDE-28 (0.2 ng/g Iw), and BDE-154 (0.05 ng/g Iw).
The sum of the concentrations of these PBDE congeners in human milk increased from 0.1 to
4.0 ng/g Iw during the 25-year period studied (Meironyte et al., 1999).
Levels of PBDEs found in breast milk in Japan and Sweden in comparable sampling
years were substantially lower than those found in the U.S. or Canada.
3.2.1.4. Blood
Levels of PBDEs in the blood are representative of either recent exposures or the slow
release of PBDEs from tissue stores. Seven tetra- to decaBDEs were analyzed in serum samples
collected in the U.S. in 1988 from male blood donors (n = 12). The median serum concentration
of the sum of tetra- to decaBDEs was 1.6 ng/g Iw (Sjodin et al., 2001). In 2000-2002, the sum
10
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of the median concentrations of six tetra- to hexaBDEs in serum pools collected in the U.S.
increased to 61 ng/g Iw. Median concentrations of the six individual PBDE congeners analyzed
are given in Table 3-1. BDE-47 followed by BDE-99 and -153 were the most abundant
congeners (Sjodin et al., 2004).
Serum samples from 24 immigrant Mexican pregnant women living in an agricultural
community in California were collected during 1999 and 2001 (Bradman et al., 2007). Tetra-,
penta-, hexa-, and heptaBDE (heptabromodiphenyl ether) congeners were measured in the serum
samples. The median concentration of the sum of tetra-, penta-, hexa-, and heptaBDE congeners
was 21 ng/g Iw with a median concentration for BDE-47, -99, -100, -153, and -154 of 11, 2.9,
1.8, 1.5, and 0.3 ng/g Iw, respectively. There was no clear association among blood levels of
PBDEs and demographic characteristics, including age, lactation, and parity. There was a slight
correlation between number of years living in the U.S. and PBDE blood levels.
Concentrations of tetra-, penta-, hexa-, and decaBDE congeners were measured in serum
samples collected during 2004 from a family residing in Berkeley, California (35- and 36-year-
old father and mother, respectively, 5-year-old daughter, and 18-month-old son) (Fischer et al.,
2006). The 18-month-old was exclusively breast-fed for 6 months and was breast-feeding during
the study period. PBDE levels for the sum of the five lower brominated congeners BDE-47, -99,
-100, -153, and -154 were much higher in the infant (418 ng/g Iw) and child (247 ng/g Iw) than
in their parents (mother 106 ng/g Iw, father 64 ng/g Iw). BDE-47 was the predominant congener
for all ages, followed by hexaBDE-153, BDE-100, -99, and -154. Levels of BDE-209 in the
infant (233 ng/g Iw) and child (143 ng/g Iw) were unusually high compared with those in the
parents (mother 14 ng/g Iw, father 23 ng/g Iw). The authors suspected house dust and breast
milk to contribute appreciably to the child and infant exposures. However, no firm conclusions
can be drawn from this study, given the small number of subjects investigated.
In Norway, pooled serum samples collected in 1998 from eight population groups of
different ages (0 to >60 years) and genders were analyzed for tri- to hexaBDEs. The total
concentration of these PBDEs in men older than 60 years was 5.3 ng/g Iw, with tetraBDE-47
being the most abundant congener (3.4 ng/g Iw), followed by hexaBDE-153 (0.6 ng/g Iw);
pentaBDE-100, pentaBDE-99, and hexaBDE-154 (all at approximately 0.4 ng/g Iw each); and
triBDE-28 (0.1 ng/g Iw). The highest plasma total PBDE concentration was for the 0- to 4-year-
old children (12 ng/g Iw) but was about one-third lower and relatively constant for the different
age groups above 4 years. Except for the 0- to 4-year-olds who seemed to experience elevated
exposure, there was a lack of an age-related trend of PBDE body burden. This may be explained
by the fact that PBDEs are relatively new contaminants in the environment; the time period for
human exposure is therefore relatively short, and different age groups (except the 0- to 4-year-
old group) may thus have experienced a similar exposure period (Thomsen et al., 2002). The
high level of PBDEs in the serum of the 0- to 4-year-olds could be due to higher exposure from
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human milk and/or certain behavioral activity, such as crawling or sucking on flame-retardant
materials.
Concentrations of PBDE congeners BDE-47, hexaBDEs (BDE-153 and -154), heptaBDE
(BDE-183), and decaBDE (BDE-209) were determined in blood serum from groups of
19-20 Swedish male and female subjects in the following occupational groups: hospital workers
(control), clerks working full-time at computer screens, and personnel at an electronic-
dismantling plant (Sjodin et al., 1999). The sums of the median concentrations of these PBDEs
were 3, 4, and 26 ng/g Iw in the hospital cleaners, computer clerks, and electronic dismantlers,
respectively. The median concentration of BDE-153 in serum was 0.6 and 0.9 ng/g Iw in the
controls and computer clerks, respectively, and 4.5 ng/g Iw in the electronic-dismantling
personnel. Serum concentrations of all PBDE congeners decreased in electronic-dismantling
workers after vacation. The median decreases, standardized to 30 days of leave, were 14% for
BDE-47, -153, and -154; 30% for BDE-183; and 66% for BDE-209. These results indicate
shorter half-lives of the more highly brominated diphenyl ethers.
3.2.1.5. Placental Transport
Twelve paired samples of maternal and cord blood collected in 2001 from women
presenting in labor in an Indiana hospital were analyzed for six tetra- to heptaBDE congeners
(Mazdai et al., 2003). None of the mothers had work-related potential for exposure to PBDEs,
and none smoked. Median concentrations of the various PBDEs found in maternal and fetal sera
are given in Table 3-1 and in Table 3-2 for comparison with a Swedish study (Guvenius et al.,
2003) described below. TetraBDE-47 was the most abundant congener, followed by
pentaBDE-99, pentaBDE-100, and hexaBDE-153. PBDE concentrations were highly correlated
between mother and fetal sera, indicating that PBDEs cross the placenta into the fetal circulation.
In addition, the results indicate that all tetra- through hepta-substituted congeners have
approximately the same potential to cross the placenta. There was a decreasing trend in
concentration of PBDE congeners in maternal and fetal sera with increasing degree of
bromination.
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Table 3-2. Comparison of median PBDE congener concentrations in
maternal and fetal sera in Sweden and in the U.S.
PBDE congener
TetraBDE-47
PentaBDE-99
PentaBDE-100
HexaBDE-153
HexaBDE-154
HeptaBDE-183
E PBDEs
Maternal serum
Mazdai et al.
(2003)a
Guvenius et al.
(2003)b
Fetal serum
Mazdai et al.
(2003)a
Guvenius et al.
(2003)b
ng/glw
28
5.7
4.2
2.9
0.3
0
37
0.83
0.19
0.17
0.56
0.04
0.06
2.07
25
7.1
4.1
4.4
0.7
0
39
0.98
0.07
0.07
0.17
0.01
0.01
1.69
aMazdai et al. (2003) (United States): year of sampling 2001; number of donors 12.
bGuvenius et al. (2003) (Sweden): year of sampling 2000-2001; number of donors 15.
Samples of maternal and cord blood plasma were collected during 2000-2001 from
15 Swedish mothers (Guvenius et al., 2003). BDE-47 was the most abundant of all congeners
and comparable median concentrations were found in maternal and cord blood plasma
(Table 3-2). The levels of the higher brominated congeners, BDE-99 to -183, were higher in
maternal blood than in cord blood, suggesting that the higher brominated PBDEs do not pass
through the placenta to the same extent as the lower brominated congener BDE-47. This trend
was not apparent in the Mazdai et al. (2003) study, where comparable levels were found in
maternal and fetal sera for all PBDE congeners studied. There was no relationship between
PBDE concentrations in the samples and frequency offish consumption. The concentrations of
PBDEs found in maternal and fetal blood samples in Indiana women (Mazdai et al., 2003) were
substantially higher than those found in Swedish women (Guvenius et al., 2003).
In summary, median concentrations of PBDE congeners in the U.S. are available for
human adipose tissue (Johnson-Restrepo et al., 2005; She et al., 2002), human milk (She et al.,
2007; Schecter et al., 2003), and serum (Bradman et al., 2007; Sjodin et al., 2004; Mazdai et al.,
2003). In the U.S., the concentration profiles of PBDEs in adipose tissue, serum, and human
milk are similar, although these studies were conducted in different regions of the U.S. (Table
3-1). The predominant congener found in adipose tissue, human milk, and blood samples in the
U.S. is tetraBDE-47, followed by pentaBDE-99, pentaBDE-100, and hexaBDE-153, with current
median concentrations in human biological samples of approximately 20, 7, 4, and 3 ng/g Iw,
respectively. HexaBDE-154 median concentration in human biological samples is usually
<1 ng/g Iw, except in adipose tissue, where BDE-153 and BDE-154 concentrations are
comparable (~5 ng/g Iw). Few measurements have been made of other PBDE congeners, such as
triBDE and heptaBDE to decaBDE. Median concentrations of the sum of PBDEs measured in
13
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human biological media are about 40 ng/g Iw. These levels are substantially higher than the
levels found in human populations in Europe or Japan.
3.2.2. Animal Data
In both F344 rats and B6C3F1 mice receiving a single 0.6 mg/kg dose (1 umol/kg) of
14C-labeled BDE-153 (96% purity; 27.8 mCi/mmol) by gavage, the highest concentrations of
radiolabel were found in adipose tissue, muscle, skin, and liver when measured 24 hours after
dosing (Sanders et al., 2006). The concentrations in adipose and muscle tissues of female mice
were significantly higher than those of female rats, and there was a similar trend in the males,
although the differences between mice and rats were not significant. The levels in the kidney,
lungs, and brain were considerably lower than those in adipose tissue, muscle, skin, and liver.
Concentrations in the lungs and brain of mice were two to three times higher than in rats, and the
differences were statistically significant. The same pattern of tissue distribution was observed in
C57BL/6 mice after intravenous dosing (1 mg/kg) in a study by Staskal et al. (2006). Tissue
levels of BDE-153 were compared with those of BDE-47, BDE-99, and BDE-100 5 days after
the animals received a radiolabeled 1 mg/kg dose. When measured as ng/g wet weight basis, the
levels of BDE-153 in all tissues were higher than the levels of the other tested materials.
In the Sanders et al. (2006) study, when the same radiolabeled dose was given to male
rats for 1, 3, or 10 consecutive days, the tissue concentrations increased with the duration of
exposure. Tissue levels 24 hours after a 10-day exposure to 0.6 mg/kg-day were about twice as
high in adipose tissue and skin as those following a single exposure to 6 mg/kg, demonstrating
the tendency for repeat doses to accumulate in adipose deposits to a greater extent than after a
single dose. On the other hand, the concentrations in the liver and other tissues (except thyroid
and thymus) after the 6 mg/kg single dose were about twice those observed when the same total
dose was distributed across 10 days of dosing (Sanders et al., 2006). The authors hypothesized
that BDE-153 is minimally metabolized, allowing it to accumulate in the liver lipids before its
gradual mobilization to adipose tissues.
Kodavanti et al. (2005) evaluated neuronal uptake by using cultures of cerebellar granule
cells from 7- to 8-day-old Long-Evans rat pups. The cultures were treated with labeled
PBDE-153 (0.05 uCi/mL) combined with unlabeled compound (0-30 uM) for 15 minutes to
1 hour. For each concentration tested, there was a linear increase in percent accumulation over
the 1-hour exposure period, although accumulation for BDE-153 was considerably lower than
that for BDE-99 and -47. When time was held constant and concentration varied, the percent
cellular uptake decreased as the concentration increased from 0.67-30 uM, suggesting a limited
ability to cross the cellular membrane.
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3.3. METABOLISM
No information is available on the metabolism of BDE-153 in humans and little
information is available in experimental animals. In F344 rats, almost all of the radiolabel in
feces appeared to be parent compound (Sanders et al., 2006) in the 24-hour period after
administration of BDE-153 on 1, 3, or 10 consecutive days or an equimolar mixture of BDE-47,
-99, and -153 on 1 or 3 consecutive days. The total radiolabel extracted from the liver after 1 or
3 days of BDE-153 treatment was 85 ± 1% or 94 ± 1%, respectively. Unrecovered radioactivity
(about 5-10%) was considered to be metabolites bound to liver proteins, and recovered material
was considered to be unmetabolized BDE-153 dissolved in liver lipids and not yet transported to
other tissues. These data are consistent with the hypothesis that there is minimal metabolism of
BDE-153 in male rats.
The results from examination of the radiolabel in the feces and urine from mice exposed
to 1 mg/kg-day BDE-153 intravenously by Staskal et al. (2006) are very different from those of
Sanders et al. (2006). In mice, about two-thirds of the label in the feces and four-fifths of that in
the urine were identified as metabolites. Metabolites were extracted, separated using
chromatography, and identified by mass spectroscopy. Three isomers (Figure 3-1) were
identified as monohydroxylated metabolites that retained all six bromines. Two isomers were
identified as monohydroxylated metabolites that had lost one bromine. A single
monohydroxylated isomer that had lost two bromines was identified as well as a trace amount of
a sulfur-containing metabolite. Additional research is needed to determine whether there is a
difference in the ability of rats and mice to metabolize BDE-153 or whether the differences in
the results of Sanders et al. (2006) with rats and Staskal et al. (2006) with mice are
methodological in origin. Much of the fecal BDE-153 from the oral Sanders et al. (2006) study
was unabsorbed parent material, accounting for part of the difference. The BDE-153 in the 4-
hour bile sample was also classified as unmetabolized.
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OH-
Excreted as
parent
compound
Br
Arene epoxide 2 Br'
Br " _ [_ intermediate_J
Br
(2 isomers)
(3 isomers)
OH —
H2O *>
GS Conjugate
Figure 3-1. Proposed metabolic pathway for BDE-153 in B6C3F1 mice.
Source: Derived from Staskal et al. (2006).
Note: GSH = glutathione; ? = uncertainty about pathway.
Br
(1 isomer)
-------
Sanders et al. (2005) examined the effects of BDE-153 on several CYP-450 enzymes in
the liver. The CYP-450s catalyze hepatic oxidation reactions for a variety of xenobiotic
compounds. Accordingly, up-regulation of their expression or gene products can signify the
potential for initial oxidation as part of the metabolic profile. Male F344 rats were treated with
0, 0.6, 6.4, or 64 mg/kg-day BDE-153 for 3 consecutive days and sacrificed 24 hours after the
last dose. Messenger ribonucleic acid (mRNA) was isolated from a portion of the right medial
lobe of the liver and converted to its complementary deoxyribonucleic acid (cDNA), using real-
time polymerase chain reaction (PCR). Target gene amplification was evaluated by using
specific probes for CYP-1A1, CYP-2B, and CYP-3A. These analyses indicated that CYP-1A1
expression was significantly up-regulated (19-fold) only with the 64 mg/kg-day dose of
BDE-153. On the other hand, BDE-153 up-regulated expression of CYP-2B in a dose-related
fashion for the 6.4 and 64 mg/kg-day doses. Expression of the mRNA was increased 20- to
30-fold at the highest dose. The expression of CYP-3 A was up-regulated (sixfold) with the
highest dose in two assays. In one of the two assays, the 6.4 mg/kg dose was associated with a
fivefold increase in mRNA. The results of these assays support the concept that hydroxylated
metabolites are produced by hepatic CYP-450 isozymes. The CYP-2B family isozymes appear
to be favored over the CYP-1A family.
3.4. ELIMINATION
No information is available on the excretion of BDE-153 in feces or urine in humans. In
F344 rats and B6C3F1 mice, about 30% of the radiolabel was found in the feces 24 hours after
administration of a single radiolabeled 0.6 mg/kg dose compared to 4% in animals that received
the same dose intravenously (Sanders et al., 2006). Based on the differences between the oral
and intravenous routes, the authors concluded that most of the fecal material represented
unabsorbed parent BDE-153. Only 0.5% of the dose was found in the bile of rats within 4 hours
of administration, and all of that was present as parent compound. Staskal et al. (2006) found
that cumulative fecal excretion in C57BL/6 mice was 12% 24 hours after a single 1 mg/kg
intravenous dose and 18% 5 days after dosing.
Minimal radiolabel from BDE-153 was excreted in the rat urine (0.1%), and the
percentage did not increase when the dose was increased from 0.6 mg/kg to 6 or 60 mg/kg. The
amount in the urine of mice was higher than in rats, especially in male mice (1%); the level in
female mice was 0.3%. The authors hypothesized that mouse-specific urinary carrier proteins
used for chemosensory signaling may have been responsible for the higher levels of BDE-153
excreted in the urine of male mice. The excreted material appeared to be the parent compound
rather than a metabolite. In the intravenous study of a slightly higher dose (1 mg/kg), Staskal et
al. (2006) found that 55.1% of the urinary material in female C57BL/6 mice was bound to
17
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protein 24 hours after administration. The protein was identified as mouse major urinary protein
(MUP) isoforms MUP-2 and MUP-3.
There is a major difference between the results from the Sanders et al. (2006) and the
Staskal et al. (2006) studies regarding the presence of metabolites in the feces. The oral Sanders
et al. (2006) study in rats did not identify metabolites in the feces, whereas the intravenous
Staskal et al. (2006) study in mice determined that more of the excreted BDE-153 was present as
metabolites than as parent compound. The difference in results can partially be explained by the
differences in the route of exposure (oral versus intravenous). It may also relate to the fact that
Sanders et al. (2006) used 24-hour collections from male rats while Staskal et al. (2006) used
female mice and the samples from a 5-day collection period. Additional research is needed to
clarify the observed differences. Total urinary and fecal excretion of BDE-153 was lower than
that from an equivalent 1 mg/kg dose of BDE-47, BDE-99, and BDE-100 in the study by Staskal
et al. (2006).
3.5. PHYSIOLOGICALLY BASED TOXICOKINETIC MODELS
Little information is available on the absorption, distribution, metabolism, and excretion
of hexaBDEs in humans. A model for human metabolism has not been established. The
database for laboratory animals is more robust, but additional data on the effect of age on
distribution and metabolism are needed. Extrapolation of results from laboratory animals to
humans using physiologically based toxicokinetic models is not possible at this time.
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4. HAZARD IDENTIFICATION
4.1. STUDIES IN HUMANS—EPIDEMIOLOGY, CASE REPORTS, CLINICAL
CONTROLS
Epidemiological studies for BDE-153 were not available.
To assess whether PBDEs may be detrimental to neurodevelopment, Mazdai et al. (2003)
determined concentrations of PBDEs and total and free serum thyroxine (T4) and
triiodothyronine (T3) in human fetal and maternal sera. Twelve paired maternal and cord blood
samples were obtained from women 18-37 years old, presenting in labor at an Indiana hospital.
The PBDE congeners and their concentrations measured in fetal and maternal serum samples are
given in Table 3-1. There was no relationship between infant birth weight and PBDE
concentrations. No birth defects were documented. Thyroid hormones were assayed in 9 of the
12 sample pairs. There was no correlation between total PBDEs and T3 or T4 concentrations
(total or free). The authors cautioned that the sample size may have been too small to detect an
association between serum concentrations of PBDEs and thyroid hormone levels.
In the study of PBDE levels in breast adipose tissue of 23 California women, described in
section 3.2.1 (She et al., 2002), there was no correlation between total concentrations of tetra- to
hexaBDE in breast adipose tissues and disease status (malignancies, benign tumors, or ductal
carcinomas in situ).
In summary, current evidence from these limited human studies does not support an
association between exposure to PBDEs and adverse health outcomes in humans.
4.2. SHORT-TERM, SUBCHRONIC, AND CHRONIC STUDIES AND CANCER
BIOASSAYS IN ANIMALS—ORAL AND INHALATION
Inhalation and oral short-term, subchronic, or chronic toxicity/carcinogenicity studies of
hexaBDE in experimental animals are not available.
4.3. REPRODUCTIVE/DEVELOPMENTAL STUDIES
A study was conducted to determine whether habituation (spontaneous motor behavior in
a novel environment), learning, and memory are affected in adult mice after neonatal exposure to
BDE-153 (Viberg et al., 2003a). Another objective of this study was to investigate whether such
neonatal exposure can affect the development of the cholinergic system by reducing the density
of nicotinic receptors in the hippocampus of the mouse brain at approximately 6 months of age.
Male neonatal NMRI mice were given single doses of BDE-153 (92.5% BDE-153, 7.5%
heptaBDE-183) by gavage on postnatal day (PND) 10 at doses of 0, 0.45, 0.9, or 9.0 mg/kg
dissolved in a 20% fat emulsion of egg lecithin-peanut oil (1:10) and water.
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Motor activity was measured for a 60-minute period, divided into three 20-minute
periods, in male mice at ages 2, 4, and 6 months. The tests were conducted in 10 mice randomly
selected from the three to five litters that comprised each dose group at 2, 4, and 6 months of
age. Motor activity tests measured locomotion (horizontal movement), rearing (vertical
movement), and total activity (all types of vibration within the test cage [i.e., those caused by
mouse movements, shaking/tremors, and grooming]). From the spontaneous motor behavior
test, an habituation ratio was calculated between the performance periods 40-60 minutes and 0-
20 minutes for the three different variables (locomotion, rearing, total activity). This ratio was
used to analyze alteration in habituation among 2-, 4-, and 6-month-old mice. Habituation is
defined as the ability of the animals to adapt to a new environment and is characterized by initial
investigation and exploration of their surroundings followed by a gradual acclimatization and
acceptance of the new area.
There were no clinical signs of toxicity in the BDE-153-treated mice at any given time
during the experimental period, nor was there any significant difference in body-weight gain or
adult weight between controls and mice treated with BDE-153. In control mice, there was a
distinct decrease in locomotion, rearing, and total activity at 2, 4, and 6 months of age, indicating
habituation in response to the diminishing novelty of the test chamber over the 60-minute test
period. Two-month-old mice exposed to 9.0 mg/kg BDE-153 displayed significantly less
activity for all three test variables during the first 20-minute test period compared with controls,
while during the third 20-minute period (40-60 minutes) they were significantly more active (p <
0.01). Mice receiving 0.9 mg/kg BDE-153, compared with the controls, showed significantly
lower activity during the first 20-minute period for the rearing and total activity variables, but,
during the third 20-minute period, they showed a significantly higher activity compared with the
control animals for the two behavioral variables locomotion and total activity. No effects in
activity for any of the three behavioral variables were seen in mice receiving 0.45 mg/kg
BDE-153 compared with controls.
At 4 months of age, mice exposed to 9.0 mg/kg BDE-153 displayed the same pattern of
activity (hypoactive during the first 20 minutes and hyperactive during the last 20 minutes) for
all three behavioral variables: rearing, locomotion, and total activity. Mice receiving 0.9 mg/kg
BDE-153, compared with the controls, showed significantly lower activity during the first
20-minute period for the rearing and total activity variables, but, during the third 20-minute
period, they showed a significantly higher activity compared with the control animals for the
locomotion and total activity variables. No effects in activity for any of the three behavioral
variables were seen in mice receiving 0.45 mg/kg BDE-153 compared with controls.
Six months after neonatal exposure to BDE-153, the only effect seen in mice receiving
the lowest dose of BDE-153 (0.45 mg/kg), compared with controls, was a marginal decrease in
total activity during the first 20-minute period. However, since the total activity returned to
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control levels during the last 20-minute period, this effect was considered of marginal
significance. At 9.0 mg/kg, 6-month-old mice compared with controls were significantly
hypoactive and significantly hyperactive during the first and last 20-minute periods, respectively,
for all three variables. Mice receiving 0.9 mg/kg compared with controls were significantly less
active and more active during the first and last 20-minute period, respectively, for the rearing
variable. They were also significantly more active compared with controls for the locomotion
and total activity variables during the last 20-minute period of the test but were not significantly
hypoactive during the first 20-minute period.
The habituation capability in 2-, 4-, and 6-month-old mice concerning locomotion,
rearing, and total activity decreased significantly with age at 0.9 and 9.0 mg/kg BDE-153
compared with controls. The lowest-observed-adverse-effect level (LOAEL) based on changes
in spontaneous motor behavior, worsening with increasing age, was 0.9 mg/kg, and the no-
observed-adverse-effect level (NOAEL) was 0.45 mg/kg.
Viberg et al. (2003a) also reported observations for the Morris swim maze test performed
at 6 months of age in groups of 19-24 mice from each treatment group. This test was used to
assess spatial learning ability and memory by measuring latencies in locating a submerged
platform during the acquisition period (days 1-4) and during the reversal learning period on the
fifth day. For the acquisition phase of the swim maze test, the mice were first placed on the
submerged platform for 30 seconds to stimulate learning. They were then tested for spatial
memory by being released into the water from a set location and timed to see if they could find
the submerged platform. If the mouse failed to reach the platform in the allotted time, it was
once again placed on the platform to stimulate learning. Five such trials were carried out on
each of the 4 acquisition days, and latencies in finding the platform were measured. On the fifth
day the location of the platform was changed, and the mice were once more presented with the
challenge of finding the submerged platform and given five trials.
In the swim maze test, mice exposed to BDE-153 at 0.9 and 9.0 mg/kg showed
significantly longer latencies in locating the platform for the trials on days 2-4 of the acquisition
period, compared with controls and mice exposed to 0.45 mg/kg. On day 5, after the platform
was relocated in order to measure relearning ability in reversal trials, control mice and the
0.45 mg/kg dose groups displayed significantly (p < 0.001) longer latencies for finding the
location of the platform in its new position. This is a normal behavior during relearning, since
the mouse initially searches close to the previous location of the platform. Mice exposed to 0.9
and 9.0 mg/kg BDE-153 did not show any significant decrease in latency in the first trial on
day 5. However, the latency observed with the fifth trial on day 5 was significantly (p < 0.05)
longer than that of the controls for the 0.9 and 9.0 mg/kg BDE-153 exposed groups.
Changes in the cholinergic receptors have been proposed as affecting learning and
memory. For this reason, 1 week after completion of the behavioral tests, six to nine mice in the
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control, 0.9, and 9.0 mg/kg groups were sacrificed, and measurement of nicotine-binding sites in
the hippocampus was performed by using tritium-labeled a-bungarotoxin, a snake neurotoxin
that specifically binds to nicotinic cholinergic receptors. Density of nicotinic receptors in the
hippocampus of controls and 0.9 mg/kg 6-month-old mice was not affected but was significantly
decreased in mice given 9.0 mg/kg, a dose at which mice showed significant defects in learning
and memory. The authors hypothesized that such changes in the cholinergic system (decrease in
density of nicotinic receptors) may be one mechanism behind the neurodevelopmental effects of
BDE-153. See section 4.4.1.5 for additional details concerning this component of the study.
The NOAEL for BDE-153 (92.5% purity) in this study (Viberg et al., 2003a) was
0.45 mg/kg, and the LOAEL was 0.9 mg/kg for changes in spontaneous motor behavior,
worsening with increasing age, and for effects on learning and memory ability as displayed in
the Morris swim maze test. Data for the three spontaneous behavior variables (horizontal
movement, vertical movement, and total activity) and habituation ratio are only available in
graphic form and could not be used for quantitative assessment.1
4.4. OTHER ENDPOINT-SPECIFIC STUDIES
4.4.1. Receptor Site Interactions
There is considerable evidence from studies of the PCBs, poly chlorinated dibenzo-p-
dioxins (PCDDs), and polychlorinated dibenzofurans (PCDFs) that halogenated aromatic
compounds exert an influence on cells by interacting with membrane receptor sites and
activating cellular transcription factors. Transcription factor complexes then initiate DNA
synthesis, allowing the cell to respond to the extracellular signal by producing a series of
mRNAs that in turn produce a variety of proteins. This process is termed signal transduction.
The structural similarities between PBDEs and the PCBs suggest that PBDEs might activate the
aryl hydrocarbon (Ah) receptor, estrogen receptor (ER), and/or androgen receptor sites. Based
on the data from the well-studied PCBs, PCDDs, and PCDFs, the activation of these receptor
sites is associated with immunosuppression, reproductive effects, and carcinogenesis (Klaassen,
1996; Bock, 1994), endpoints of interest for PBDEs. Table 4-1 provides a summary of the
hexaBDE congeners that have been evaluated in a variety of receptor interaction studies.
Attempts to obtain numerical values and other information on the data from the neurobehavioral studies were not
successful.
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Table 4-1. Receptor interaction studies of hexaBDE congeners
Endpoint evaluated
Estrogen receptor
Androgen receptor
CARb
PXR/SXRb
Congener
138
153
Xa
X
X
X
X
154
156
166
Findings
Effect levels arel(T3 to 1CT6 that of TCDD.b
No estrogenic activity in the ER-CALUXb assay.
No antiandrogenic activity.
Receptor interactions up-regulate expression of
associated CYP-450 isozymes. CAR activation
stronger than PXRb.
Receptor interactions up-regulate expression of
associated CYP-450 isozymes.
aX indicates that the congener was tested for receptor effects; for most congeners several methodologies for
evaluation of receptor interactions were employed.
bTCDD = 2,3,7,8-tetrachlorodibenzo-p-dioxin; ER-CALUX = estrogen receptor-Chemical-Activated LUciferase
gene expression; CAR = constitutive androstane receptor; PXR = pregnane X receptor; SXR = steroid X receptor.
4.4.1.1. Aryl Hydrocarbon Receptors
The transcription of the genes for CYP-450, -1A1, -1A2, and -1B1 is linked to a signal
transduction cascade that is initiated by activation of the Ah receptor (AhR) by an appropriate
ligand. The CYP-1 family of enzymes is highly conserved in mammals and is responsible for
the oxidative metabolism of a variety of planar and near-planar compounds (Lewis et al., 1998).
Enzymes of the CYP-450 family metabolically activate and metabolize poly cyclic aromatic
hydrocarbons and aromatic amines as well as PBDEs. Many substrates for CYP-450 enzymes
are also AhR ligands. Differences in AhR affinity are correlated to variations in CYP-1
inducibility. Receptor-site affinity has been shown to reflect potency and the potential for a
xenobiotic to cause adverse health effects.
Chen et al. (2001) studied the affinity of several PBDE congeners for rat hepatic AhR
through competitive binding assays and determined their ability to induce hepatic CYP-450
enzymes by means of ethoxyresorufin O-deethylase (EROD) assays (a biomarker for CYP-
1 Al/2 induction) in chick and rat hepatocytes, liver cell lines from rainbow trout, and rat and
human tumor cell lines. HexaBDE congeners BDE-153 and -154 had AhR-binding affinities
approximately 2 x 10~5the binding affinity for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD).
Quantitative measures of EROD induction were reported for BDE-153 and -154. BDE-153 was
a very weak inducer in all cells, its relative induction potency being 10~5 to 10~6 that of TCDD.
BDE-154 was not an inducer in any cell line.
Using hepatocyte cultures from Sprague-Dawley rats, Chen and Bunce (2003)
investigated whether nine different PBDE congeners, including hexaBDEs, act as AhR agonists
or antagonists at sequential stages of the AhR signal transduction pathway leading to CYP-1A1
induction. These issues are environmentally relevant because of the strong rank-order
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correlation among strength of AhR binding, CYP-1A induction, and toxicity of many
halogenated aromatic compounds. The hexaBDE congeners evaluated in this study were
BDE-153 and-156.
BDE-153 and -156 were very weak activators of dioxin response element binding,
showing maximum induction at about 10 and 40%, respectively, of that of TCDD but at
concentrations four orders of magnitude higher than TCDD. When tested in combination with
TCDD, BDE-153 and -156 tended to slightly inhibit the activity of a saturating TCDD
concentration. BDE-156 induced responses of CYP-1 Al mRNA protein equivalent to the
maximal response of TCDD in primary Sprague-Dawley rat hepatocytes, although at
concentrations three orders of magnitude greater than TCDD. BDE-153 induced responses of
CYP-1 Al mRNA protein equivalent to about 50% that of TCDD at concentrations four orders of
magnitude greater than TCDD. Induction of CYP-1 Al, measured through EROD assays,
paralleled CYP-1A1 mRNA induction; BDE-153 and -156 gave weak to moderate responses,
showing maximum CYP-1 Al levels 40-60% those of TCDD at concentrations approximately
four orders of magnitude higher than TCDD. The authors concluded that PBDEs, including the
hexaBDE congeners tested, contribute negligibly to dioxin-like toxicity compared with other
environmental contaminants, such as PCBs and TCDD.
The possible dioxin-like effects of BDE-153 and -154 on induction of genes that encode
metabolizing enzymes have also been investigated by Peters et al. (2004). AhR-mediated
induction of CYP-450 enzymes CYP-1A1 and -1B1 was studied in human breast carcinoma
(MCF7), human hepatocellular carcinoma (HepG2), and rat hepatoma (H4IIE) cells, using
EROD activity as a marker for CYP-1A1 and -1B1 activity. BDE-153 and -154 (>98% purity)
did not induce EROD activity when incubated for 72 hours at concentrations that were not
cytotoxic (up to 10 uM) and, therefore, were not found to be AhR agonists in these cells.
Additionally, exposure of the cells to BDE-153 or -154 did not increase mRNA levels of
CYP-1 Al or -1B1, indicating no direct effect of these hexaBDEs on AhR gene expression.
Peters et al. (2006) examined the interaction of BDE-153 and -154 as well as other BDEs
with the AhR in cultured liver cells from four healthy cynomolgus monkeys (three males and one
female), using EROD activation as a biomarker for receptor activation. Both compounds were
weak Ah agonists when coexposures of TCDD and the PBDE were tested, as evidenced by a
decrease in the activation caused by TCDD alone. The impact of the PBDEs was receptor
mediated rather than occurring by inhibition of the enzyme, since no EROD inhibition occurred
if TCDD exposure preceded the PBDE exposure. Environmentally relevant concentrations of
PBDEs (1-10 uM) were evaluated. There was variability in the response of the four monkeys,
likely reflecting individual differences in the animals. Enzyme inhibition for BDE-153 and -154
was between 0 and 30%.
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Villeneuve et al. (2002) examined the ability of BDE-153 to induce AhR-mediated gene
expression in vitro, using H4IIE-luc (luciferase) recombinant rat hepatoma cells. The cells were
grown in culture ViewPlates™ and then exposed to PBDE concentrations ranging from 2 to
500 ng/mL. Luminescence was measured and compared to the maximum response observed
with a 1,500 picomolar TCDD standard (%-TCDD-max). A positive response was defined as
any response that was greater than three standard deviations above the mean value for the
control. BDE-153 failed to induce AhR-mediated gene expression in H4IIE-luc cells. These
results are qualitatively consistent with those of Chen and Bunce (2003).
Sanders et al. (2005) used an in vivo approach to study AhR site activation by BDE-153
as well as several other PBDE congeners. Groups of F344 male rats (three/group),
10-12 weeks old, were dosed by gavage once daily for 3 days with BDE-153 (96% purity) in
corn oil at 0, 1, 10, or 100 umol/kg-day (0, 0.6, 6.4, or 64 mg/kg-day). The animals were
sacrificed 24 hours after receiving the last dose. The liver was removed, and RNA from a
100 mg liver sample was isolated, converted to its cDNA, and amplified by using PCR. The
resultant DNA samples were then analyzed to determine the expression of CYP-1A1, a protein
linked to AhR activation.
BDE-153 had a significant effect on the level of CYP-1A1 expression (19 times that of
vehicle-treated controls) only at 100 umol/kg-day (64 mg/kg-day), making it a weak activator of
the AhR. When the CYP-1A1 expression from BDE-153 was compared with those for
tetraBDE-47 and pentaBDE-99, the impact on the AhR seemed to be correlated to the levels of
polybrominated dibenzofuran contaminants in each congener, which in turn correlated with
increased bromine content of the congeners.
The results from this study confirm in vitro data, suggesting that PBDEs are, at best,
weak activators of the AhR. These results also raise the possibility that brominated dibenzofuran
impurities identified in the congeners studied may, in some cases, have confounded the results
from other studies.
4.4.1.2. Other CYP-450 Induction Receptors
The study of CYP-450 mRNA expression in rat liver by Sanders et al. (2005) (see section
4.4.1.1) found that expression of CYP-2B was up-regulated by BDE-153 in F344 rats to a greater
extent than CYP-1 Al, a biomarker for the activation of the AhR. CYP-3A was slightly up-
regulated but to a lesser extent than the CYP-1 Al and -2B. CYP-2B and -3A are respective
biomarkers for activation of the constitutive androstane receptor (CAR) and pregnane X receptor
(PXR). In the case of BDE-153, the effect on the CAR was greater than that on the PXR. The
CAR and PXR are both involved in the metabolism of xenobiotics and are stimulated by
phenobarbital. The CAR is also involved in steroid metabolism. The impact of BDE-153 on
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these receptors is similar to the impact of noncoplanar PCBs on the same receptors; however,
little is presently known about the physiological effects of stimulation of the CAR and PXR.
4.4.1.3. Estrogen Receptors
Studies have also been conducted to evaluate the interaction between PBDEs and ERs.
Activation of ERs induces cell division in female reproductive organs, mammary glands, and
liver. Receptor-induced mitogenic activity has been linked to tumor formation in the affected
organs (Klaassen, 1996).
The in vitro estrogenic and antiestrogenic potencies of 17 PBDEs, including
hexaBDE-138, -153, and -166, were investigated in a human T47 breast-cancer cell line based
on ER-dependent luciferase reporter gene expression (Meerts et al., 2001). The modified T47D
cells that contained ERa and ERp were trypsinized and seeded in 96 well plates for the ER-
CALUX (Chemical-Activated LUciferase gene expression) assay. After allowing for cell
growth, the wells were exposed to solutions containing the test compounds or estradiol and
incubated. The luciferase activity was measured with a luminometer. The three hexaBDEs
tested did not show any estrogenic activity in the ER-CALUX assay.
Villeneuve et al. (2002) examined the ability of 10 different PBDEs, including BDE-153
(99% purity), to initiate ER-mediated gene expression in vitro. At concentrations up to
500 ng/mL, BDE-153 failed to induce ER-mediated gene expression in MVLN recombinant
human breast carcinoma cells, using a luciferase response element for detection. Overall, the
PBDEs tested were found to be 50,000 times less potent than estradiol for inducing ER-mediated
gene expression.
Villeneuve et al. (2002) also studied the ability of PBDEs to displace steroid hormones
from serum proteins. At concentrations up to 833 ng/mL, BDE-153 as well as the other PBDEs
tested did not show an appreciable capacity for displacing 3H-steroids from carp serum proteins
that had been stripped of hormones before testing. Unlabeled estradiol and testosterone also had
a limited effect on displacing the radiolabeled ligands, suggesting limited sensitivity of the assay
with carp serum.
In summary, the mechanistic studies of the ER indicate that there was no activity of
BDE-138, -153, or -166 at the concentrations tested and the binding affinity of the hexaBDEs
was 50,000 times lower than that of estradiol.
4.4.1.4. Androgen Receptors
DE-71, a commercial pentaPBDE mixture, was found by Stoker et al. (2004) to delay
puberty and suppress the growth of androgen-dependent tissues in male Wistar rats exposed to
doses of 30 or 60 mg/kg, but not to doses of 0 or 3 mg/kg, during the peripubertal period. In
order to examine which components of the mixture might be responsible for the observed effects,
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androgen receptor binding by several of the individual congeners found in DE-71 was examined
in vitro (Stoker et al., 2005). The assays of the individual congeners examined competitive
binding of BDE-153 (99% purity) and BDE-154 (97% purity) in the presence of a tritium-
labeled androgen agonist (R1881) by using ventral prostate cytosolic extracts along with an
assay in an MDA-kb2 cell line containing the human androgen receptor and a transfected
luciferase reporter element.
In the assay with the ventral prostate extract, 0.001, 1.6, 3.3, 16.7, or 33 uM BDE-153
and -154 were incubated in the presence of 1.0 nM R1881 and 10 uM triamcinolone acetonide,
an agent that blocks the progesterone and glucocorticoid receptors. BDE-154 but not BDE-153
showed very slight inhibition of R1881 binding. The maximum inhibition caused by the
BDE-154 was 40% at the highest concentration tested of 33 uM.
In the assay using the MDA-kb2 cell line, BDE-153 and -154 were introduced at
concentrations of 10 pM, 10 nM, 1 uM, or 5 uM in the presence of 0.1 nM of the receptor
agonist dihydrotestosterone. Neither compound demonstrated any antiandrogenic activity in this
assay.
4.4.1.5. Acetylcholine Receptors
As discussed in section 4.3, changes in the cholinergic receptors have been proposed as
affecting movement, learning, and memory. Disturbances in the development of the cholinergic
system have been demonstrated to occur during the brain growth spurt in humans and laboratory
animals (Viberg et al., 2003a). In male mice, sensitivity of the developing cholinergic system to
BDE-153 appears to peak about PND 10 (Viberg et al., 2003a). For this reason, 1 week after
completion of the swim maze test, six to nine mice in the control, 0.9, and 9.0 mg/kg groups
were sacrificed, hippocampal brain tissue was isolated, and measurement of nicotinic-binding
sites was performed using tritium-labeled a-bungarotoxin. Parallel samples were treated with
unlabeled a-bungarotoxin to correct for nonspecific binding. Specific binding was determined
by calculating the difference in the amount of labeled toxin bound in the presence versus absence
of unlabeled a-bungarotoxin. There were no significant differences in the density of nicotinic
receptors in the hippocampus of controls and 0.9 mg/kg 6-month-old mice. However, receptor
density was significantly decreased (20.6%) in mice given 9.0 mg/kg. The authors hypothesized
that the decrease in the density of nicotinic receptors as a result of exposure to BDE-153 may be
one mechanism contributing to the neurodevelopmental effects of BDE-153.
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4.4.2. Thyroid Effects
Because PBDEs have some structural similarity to the thyroid hormone T4, it has been
suggested that they may interfere with thyroid hormone transport by competitively binding with
transthyretin (TTR), one of the thyroid hormone-binding transport proteins in the plasma of
vertebrate species. The possible interference of several hexaBDEs with T4-TTR binding was
investigated in an in vitro competitive binding assay, using human TTR and 125I-labeled T4 as the
displaceable radioligand. The three hexaBDE congeners evaluated (BDE-138, -153, and -166)
did not compete with T4-TTR binding (Meerts et al., 2000).
Meerts et al. (2000) also tested these three hexaBDEs (BDE-138, -153, and -166) before
and after incubation with differently induced hepatic microsomes to examine the ability of their
hydroxylated metabolites to displace T4 from TTR. The hexaBDEs were individually incubated
with liver microsomes prepared in the presence of phenobarbital (a CYP-2B inducer),
p-naphthoflavone (a CYP-1A inducer), or clofibrate (a CYP-4A3 inducer). Incubation of
BDE-166 with CYP-2B-enriched rat liver microsomes resulted in the formation of metabolites
that were able to displace 20-60% of the 125I-T4 from TTR. BDE-138 and -153 did not compete
with T4 for binding. No T4-TTR displacement by hexaBDEs occurred after incubation with liver
microsomes enriched with CYP-1A or -4A3. BDE-166 is therefore able to compete with T4-
TTR binding only after metabolic conversion by induced rat liver microsomes, suggesting an
important role for hydroxylation. The relevance of this observation for humans has yet to be
resolved. T4-binding globulin, rather than TTR, is the major T4-binding protein in humans.
4.4.3. Genotoxicity
Information is not available on the genotoxicity of hexaBDEs.
4.5. SYNTHESIS OF MAJOR NONCANCER EFFECTS
4.5.1. Oral
Alterations of behavioral parameters, namely impaired spontaneous motor behavior
worsening with age, and effects on learning and memory capability have been shown to occur in
adult male mice neonatally exposed to BDE-153 (Viberg et al., 2003a). The Guidelines for
Neurotoxicity Risk Assessment (U.S. EPA, 1998a) consider that an agent that produces detectable
adverse neurotoxic effects in experimental animals will pose a potential hazard to humans.
These adverse neurotoxic effects include behavioral, neurophysiological, neurochemical, and
neuroanatomical effects. Accordingly, the behavioral disturbances seen in adult mice neonatally
exposed to BDE-153 in the Viberg et al. (2003a) study raise concerns about possible
neurobehavioral effects in children and adults. Similar neurodevelopmental effects have been
observed in studies of the tetraBDE-47, pentaBDE-99, and decaBDE-209 congeners.
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BDE-153 has been found in human milk, maternal and cord blood, and adipose tissues.
Concentrations found are high in all human biological samples in the U.S. relative to other
countries. Fetuses and infants are exposed to BDE-153. Whether such exposures constitute a
health risk for neurodevelopmental dysfunction in these population groups is not known at this
time. An association between neonatal exposure to BDE-153 and neurobehavioral effects in
humans has not been established.
4.5.2. Inhalation
No data are available on the toxicity of BDE-153 by the inhalation route of exposure.
4.5.3. Mode-of-Action Information
While evidence exists that demonstrates that BDE-153 (and other PBDEs) interact at the
neurological level, data are inadequate to determine the mode of action for BDE-153.
Researchers from the laboratory of Eriksson/Viberg have hypothesized that the observed effects
on locomotion and habituation are related to impaired development of the cholinergic system
during the postnatal "brain growth spurt" period (Viberg et al., 2005, 2004a, 2003a). In the
study by Viberg et al. (2003a), the density of nicotinic acetylcholine receptors in the
hippocampus of 6-month-old mice was significantly decreased in mice given BDE-153 at 9.0
mg/kg, a dose at which mice showed significant defects in learning and memory, as reflected in
the swim-maze performance test. The authors hypothesized that such a decrease in density of
nicotinic receptors during development may play a role in the behavioral effects of BDE-153.
Similar effects on cholinergic neurons were also observed in the testing of BDE-99 and -209
(Viberg et al., 2007, 2005, 2004a; Ankarberg, 2003).
The Eriksson/Viberg group have further hypothesized that the sensitivity of the
cholinergic system occurs in the vicinity of PND 10 and have tested this hypothesis by varying
the time of dosing and observing differences in the habituation effect for BDE-99 and -209
(Viberg et al., 2007; Eriksson et al., 2002). Impaired development of the cholinergic system
during the postnatal "brain growth spurt" period could change adult responses to endogenous
acetylcholine and other cholinergic agents. The resulting cholinergic receptor deficits persisted
across the duration of testing, apparently causing a hyperactive response to exposure to
cholinergic stimulants in adulthood.
Evidence that BDE-153 can enter the neurological system was reported by Staskal et al.
(2006). BDE-153 was found in the brain tissue of C57BL/6 10-week-old mice when measured
5 days following intravenous administration of a single radiolabeled dose of 1 mg/kg BDE-153.
Interestingly, the concentrations of BDE-153 distributed to the mouse brain tissue were
significantly greater than those for the other measured PBDEs, BDE-47, -99, and -100 (Staskal
et al., 2006). Additional evidence that BDE-153 reaches the brain and potentially interacts with
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the neuronal growth and cognitive function was observed by Kodavanti et al. (2005) in which a
dose-related increase in accumulation of BDE-153 was seen within 1 hour in cultures of
cerebellar neurons and mixed cerebellar neurons and glia from neonatal Long-Evans rats
(Kodavanti et al., 2005). Furthermore, uptake of BDE-153 by the neurons was coincident with
translocation of protein kinase C. Kodavanti et al. (2005) note that concurrent uptake of BDE-
153 and protein kinase C, which is associated with neuronal growth, memory, and learning,
suggests a potential for interaction at the neuronal level; however, data showing this interaction
for BDE-153 are unavailable.
The data on the impact of hexaBDE on the density of nicotinic acetylcholine receptors in
the hippocampal area of the brain in combination with that on neuronal uptake of BDE-153
provide the strongest link to the neurodevelopmental effects observed after BDE-153 exposure.
However, brain development is complex, and many neurochemical changes that occur during the
postnatal growth spurt and that could impact motor activity have not yet been investigated.
While evidence exists that demonstrates that BDE-153 may have an impact on neurochemical
events during early postnatal development, data are inadequate to determine the mode of action
for BDE-153.
Because PBDEs have some structural similarity with the thyroid hormone T4, it has been
suggested that interference with thyroid hormone transport by competitively binding to TTR, one
of the thyroid hormone-binding transport proteins in the plasma of vertebrate species, may
explain the neurobehavioral effects. The possible interference of several hexaBDEs with T4-
TTR binding was investigated in an in vitro competitive binding assay, using human TTR and
125I-labeled T4 as the displaceable radioligand. The three hexaBDE congeners evaluated
(BDE-138, -153, and -166) did not compete with T4-TTR binding (Meerts et al., 2000). Lack of
binding with TTR suggests that diminished T4 transport due to competitive binding of BDE-153
to the transport protein does not provide an explanation for the observed neurodevelopmental
impairment following early life exposures. Thyroid hormone levels and behavioral activity were
not co-measured in the study in mice of Viberg et al. (2003a), and, despite the possibility of
interactions between BDE-153 and thyroid hormone levels, there are no mode-of-action data that
link thyroid hormones to the neurobehavioral observations reported by Viberg et al. (2003a).
HexaBDE-153, -154, and -156 are, at best, weak activators of the AhR (Peters et al.,
2006, 2004; Sanders et al., 2005; Chen and Bunce, 2003; Villeneuve et al., 2002; Chen et al.,
2001). Congeners BDE-138, -153, and -166 did not show any estrogenic activity in tissue
culture studies (Villeneuve et al., 2002; Meerts et al., 2001). Congeners BDE-153 and -154 do
not exhibit antiandrogenic activity in an MDA-kb2 cell line containing the human androgen
receptor and a luciferase reporter (Stoker et al., 2005). Lack of activity in these assays
minimizes concern that hexaBDE is an endocrine disrupter through estrogen or androgen
pathways. However, the implications of these interactions are unknown.
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4.6. EVALUATION OF CARCINOGENICITY
Animal chronic toxicity/carcinogenicity studies have not been conducted for BDE-153.
There is inadequate information to assess the carcinogenic potential of BDE-153 (U.S. EPA,
2005a).
4.7. SUSCEPTIBLE POPULATIONS AND LIFE STAGES
4.7.1. Possible Childhood Susceptibility
A population subgroup is susceptible if exposure occurs during a period of sensitivity as
observed in Viberg et al. (2003a) with adult mice exhibiting neurobehavioral effects following
neonatal exposure to BDE-153. The neonatal stage is a period of rapid development of the
nervous system and is considered a critical window of development. The animal model indicates
a potential concern for early lifetime exposure (i.e., fetal or infant exposure) to the chemical.
The identification of BDE-153 in human maternal and cord blood, milk, and plasma (Bradman et
al., 2007; She et al., 2007; Mazdai et al., 2003; Schecter et al., 2003; Thomsen et al., 2002)
implies humans are exposed to BDE-153 during a period of rapid development of the brain,
indicating a potential for susceptibility. Whether exposure to BDE-153 constitutes a health risk
for adverse neurodevelopmental effects in children is not known at this time because of the
limited toxicological database for BDE-153. An association between prenatal or neonatal
exposures to BDE-153 and neurobehavioral dysfunction in humans has not been established.
4.7.2. Possible Gender Differences
It is unknown whether susceptibility to BDE-153 differs in male and female humans or
experimental animals. Two major urinary proteins promote excretion of BDE-153 in male mice
once the pathways for synthesis become operational. This decreases retention of BDE-153 in
juvenile and mature males compared to females but the pathway is not available during the early
postnatal period of development (Staskal et al., 2006).
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5. DOSE-RESPONSE ASSESSMENTS
5.1. ORAL REFERENCE DOSE (RfD)
5.1.1. Choice of Principal Study and Critical Effect—with Rationale and Justification
There is only one study available for dose-response assessment and derivation of an RfD
for BDE-153. The study of Viberg et al. (2003a) identified a NOAEL and a LOAEL in mice for
effects on spontaneous motor behavior, learning, and memory. In this study, male mice were
administered single oral doses (0, 0.45, 0.9, or 9.0 mg/kg) of BDE-153 (92.5% purity) on PND
10, which is, according to Eriksson et al. (2002), a period of maximum vulnerability of the
developing mouse brain. Adverse effects noted in adult mice at 0.9 and 9.0 mg/kg included
hypoactive spontaneous motor behavior in the beginning of the test period, hyperactive behavior
at the end of the test period (with these disturbances becoming more pronounced with increasing
age), and effects on learning and memory capability.
There are several concerns regarding the design of the Viberg et al. (2003a) study. The
study was conducted in male mice only. The protocol was unique and did not conform to health
effects test guidelines for a neurotoxicity screening battery or developmental neurotoxicity
studies (U.S. EPA, 1998b). The dosing regimen did not include gestation and lactation exposure
(U.S. EPA, 1998a); only single doses were given. In some respects the observation that effects
occurred with such limited dosing argues for the importance of this study. While the study
design appears to identify a developmental window of susceptibility, it is not adequate to
determine the effect of longer dosing. Translating the implications of these data to more
traditional dosing regimens is problematic, particularly with regard to evaluating the
implications of in utero and postnatal exposure. Another concern is that, based on the data
provided in the published report, more than one pup per litter was used for the behavioral testing
(10 mice were randomly selected from three to five different litters in each treatment group).
Increasing the number of samples from each litter may bias the analyses towards false positives,
and the observed neurobehavioral effects may be attributable to differences that are not treatment
related in pups born to a single dam. Another concern regarding the study design is the limited
number of neurobehavioral parameters (related to motor activity and cognitive function) that
were assessed. The absence of a full functional observational battery (FOB) limits the ability to
correlate the reported effects with other FOB parameters. This would be helpful in gauging the
reliability of the parameters that were measured in the study. As indicated in the Guidelines for
Neurotoxicity Risk Assessment (U.S. EPA, 1998a), it is assumed that an agent that produces
detectable adverse neurotoxic effects in experimental animal studies will pose a potential hazard
to humans. For BDE-153, in the absence of human evidence, data from experimental animal
studies are used as the basis for the RfD.
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While study design limitations cloud the utility of this study, several additional
considerations support the use of these data. Acute exposure to a highly lipophilic chemical such
as BDE-153 will result in exposure that lasts longer than the 1 day of dosing. In addition, there
are a wide variety of brain structures that have very limited critical windows during
development. These short critical windows translate to susceptible periods of exposure that can
be very short. The concept that exposure during critical periods can induce functional
neurological effects later in development has been demonstrated with structurally related PBDE
congeners, including
tetra-, penta-, and decaBDEs (Kuriyama et al., 2005; Viberg et al., 2005, 2004a,b, 2003b, 2002;
Ankarberg, 2003; Branch! et al., 2002; Eriksson et al., 2002, 2001). Therefore, the observed
neurobehavioral effects in the Viberg et al. (2003a) study are biologically plausible, and
exposure to BDE-153 may pose a potential hazard to humans (U.S. EPA, 1998a).
Taken together, these considerations support the use of the Viberg et al. (2003a) study for
deriving the RfD for BDE-153.
5.1.2. Methods of Analysis
The study of Viberg et al. (2003a) in mice was not amenable to a benchmark dose (BMD)
approach because the data needed for the use of a BMD approach are not available in the
published study. Experimental data points for locomotion, rearing, and total activity and their
standard deviations are displayed graphically, and such values cannot be determined with any
accuracy from the graphs. Therefore, the NOAEL of 0.45 mg/kg is used as a point of departure
for estimating the RfD for BDE-153.
5.1.3. RfD Derivation
A NOAEL of 0.45 mg/kg for BDE-153 was identified in the Viberg et al. (2003a) study.
To calculate the RfD, a total uncertainty factor (UF) of 3,000 was applied: 10 for extrapolating
animal data to humans (UFA interspecies variability), 10 for susceptible human subpopulation
(UFH interhuman variability), 3 for extrapolating from a single dose to a lifetime exposure
duration (UFS), and 10 to account for a deficient database (UFD). The rationale for application of
the UFs is described below.
A 10-fold UFA was used to account for laboratory animal to human interspecies
differences. Although the toxicokinetics of BDE-153 in animals have been evaluated, no
adequate description of toxicokinetics of BDE-153 in humans exists. The critical effect for
deriving the RfD, altered behavior due to exposure during development, is expected to be
relevant to humans. No quantitative data were identified to compare relative human and rodent
sensitivity to these changes. However, given the longer period of brain development in humans
as compared to rodents and the higher importance of cognitive function, it is appropriate to
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consider that humans may be more sensitive than rodents. Based on these considerations, the
default UFA value of 10 was applied.
A default intraspecies UFH of 10 was applied to account for variations in susceptibility
within the human population (intrahuman variability). This factor accounts for the segment of
the human population that may be more sensitive than the general population to exposure to
BDE-153. A default value is warranted because insufficient information is currently available to
assess human-to-human variability in BDE-153 toxicokinetics or toxicodynamics.
A UFS of 3 was used to account for uncertainties in extrapolating from effects seen in a
single exposure neurodevelopmental study to a lifetime exposure. Exposure on PND 10
occurred during a period of rapid brain development in mice. Brain development does not
continue at an equivalent rate over a mouse's lifespan and is more quiescent during adult life
stages. Many brain structures have a very limited critical window during development in early
life. Following BDE-153 exposure, toxicokinetic data suggest that a mouse urinary protein
becomes functional some time between PNDs 28 and 40, which leads to an increase in BDE-153
urinary excretion, especially in males. This increased excretion reduces the total body burden of
BDE, including the levels of radiolabel reaching the brain 24 hours after dosing in older mice
compared with that in younger mice. These data thus suggest that the risk of
neurodevelopmental effects in neonatal mice may be greater than in older mice because of rapid
postnatal brain growth and coincident increased retention of BDE-153 and/or its metabolites.
Therefore, chronic exposure is not expected to result in more serious effects. However, because
the mice received only a single dose rather than repeated doses over multiple days within the
hypothesized critical window, a threefold UF was applied.
A UFL for LOAEL-to-NOAEL extrapolation was not applied because a NOAEL was
used as the point of departure.
A UFD of 10 was used to account for database uncertainty. The available oral database
for BDE-153 lacks prenatal developmental neurotoxicity studies, reproductive toxicity studies,
and standard chronic or subchronic studies of systemic toxicity. Uncertainties regarding the
effects of exposures during the prenatal period, extended postnatal exposures, and latent
expression of early postnatal changes in the brain are addressed as a component of the database
UF.
Application of a total UF of 3,000 to the NOAEL of 0.45 mg/kg results in a reference
dose for BDE-153 of 1.5 x 10~4 mg/kg-day or 0.2 ug/kg-day.
5.1.4. Previous RfD Assessment
The hexaBDE congener BDE-153 has not been previously assessed in IRIS. However, a
health assessment of the hexaBDE homolog group (CASRN 36483-60-0) was previously entered
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in the IRIS database in 1990 (U.S. EPA, 1990). Information was not available to derive an RfD
or RfC or to assess the carcinogenic potential of the hexaBDE homolog group.
5.2. INHALATION REFERENCE CONCENTRATION (RfC)
No data are available for deriving a chronic RfC for BDE-153.
5.3. CANCER ASSESSMENT
There is inadequate information to assess the carcinogenic potential of BDE-153 (U.S.
EPA, 2005a).
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6. MAJOR CONCLUSIONS IN THE CHARACTERIZATION OF
HAZARD AND DOSE RESPONSE
6.1. HUMAN HAZARD POTENTIAL
BDE-153 (CASRN 68631-49-2) is a component of the commercial penta- and octaBDE
flame retardants. BDE-153 has been found in human milk, adipose tissue, and blood. As a
result, fetuses and infants are exposed to BDE-153.
No data are available regarding the potential toxicity of BDE-153 in humans exposed via
the oral route. However, the available animal data indicate that the nervous system is a sensitive
target organ. Neurobehavioral developmental toxicity has been identified as the critical endpoint
of concern in adult male mice following neonatal oral exposure to BDE-153 (Viberg et al.,
2003a). Since fetuses and infants are exposed to BDE-153 via maternal and cord blood and
human milk, such exposure may constitute a health risk for adverse neurodevelopmental effects
in these population groups.
There are no studies of the potential carcinogenicity of BDE-153 in humans or
experimental animals. Under the Guidelines for Carcinogen Risk Assessment (U.S. EPA,
2005a), there is "inadequate information to assess carcinogenic potential" of BDE-153.
6.2. DOSE RESPONSE
The RfD of 0.2 ug/kg-day (rounded from 1.5 x 10'4 mg/kg-day) for BDE-153 was
derived from the study of Viberg et al. (2003a), which identified a NOAEL of 0.45 mg/kg and a
LOAEL of 0.9 mg/kg for effects on spontaneous motor behavior, learning, and memory
capability in mice. A total UF of 3,000 was applied to the NOAEL: 10 for interspecies
variability, 10 for interindividual variability, 3 for extrapolation from single to lifetime exposure,
and 10 for database deficiencies.
No data are available regarding the potential toxicity of BDE-153 in exposed humans via
the oral route, and no suitable toxicokinetic or toxicodynamic models have been developed to
reduce uncertainty in extrapolating from mice to humans.
The extent of variability in susceptibility to BDE-153 among humans is unknown,
representing another important area of uncertainty in the RfD. However, subpopulations
expected to be more susceptible to BDE-153 toxicity are fetuses and children. Chronic studies
relevant to BDE-153 toxicity have not been performed in experimental animals.
The principal study for the RfD (Viberg et al., 2003a) examined a number of behavioral
parameters in adult male NMRI mice that had been neonatally exposed to BDE-153 (three doses,
administered in a single day, using a limited number of animals). Aside from this study, the oral
database is sparse. No information is available for the testing of BDE-153 in assays of
36
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reproductive toxicity or chronic toxicity. The overall confidence in the RfD assessment for
BDE-153islow.
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Neurotoxicol 28:136-142.
Villeneuve, DL; Kannan, K; Priest, BT; et al. (2002) In vitro assessment of potential mechanism-specific effects of
polybrominated diphenyl ethers. Environ Toxicol Chem 21(11):2431-2433.
Wong, A; Lei, YD; Alaee, M; et al. (2001) Vapor pressures of the polybrominated diphenyl ethers. J ChemEng
Data 46:239-242.
Zhou, T; Taylor, MM; DeVito, MJ; et al. (2002) Developmental exposure to brominated diphenyl ethers results in
thyroid hormone disruption. Toxicol Sci 66(1): 105-116.
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APPENDIX A: SUMMARY OF EXTERNAL PEER REVIEW AND PUBLIC
COMMENTS AND DISPOSITION
The "Toxicological Review" for hexabromodiphenyl Ether (BDE-153) has undergone a
formal external peer review performed by scientists in accordance with EPA guidance on peer
review (U.S. EPA, 2006a, 2000a). The external peer reviewers were tasked with providing
written answers to general questions on the overall assessment and on chemical-specific
questions in areas of scientific controversy or uncertainty. The external peer review for BDE-
153 was conducted in concert with the external peer review of other PBDE congeners (i.e., BDE-
47, BDE-99, and BDE-209), and some external peer review charge questions were specific to
congeners other than BDE-153. External peer reviewer comments on all of the PBDEs and the
Agency response are included below for completeness. A summary of significant comments
made by the external reviewers and EPA's responses to these comments, arranged by charge
question, follow. In many cases the comments of the individual reviewers have been synthesized
and paraphrased in development of Appendix A. Synthesis of comments from individual peer
reviewers resulted in summaries that combine similar statements from peer reviewers that were
mentioned in conjunction with more than one charge question. In such cases, the comment and
its response have been placed under the most relevant charge question. Some peer review
comments were not directly related to charge questions. Those comments are categorized as
miscellaneous and placed after those related to the charge questions. EPA also received
scientific comments from the public. These comments and EPA's responses are included in a
separate section of this appendix.
The peer review of the "Toxicological Review" of BDE-153 was coupled with the review
of the documents for BDE-47, -99, and -209. Accordingly, most of the charge questions address
all four congeners. The responses to the charge questions in this appendix apply primarily to
comments related to BDE-153. The charge to the external peer reviewers and final external peer
review report (February 2007) pertaining to the toxicological reviews of the four PBDE
congeners are available at http://cfpub.epa.gov/ncea/cfm/recordisplay.cfm?deid=161970. The
public comments received can be found at
http://www.regulations.gov/fdmspublic/component/main under the Docket EPA-HQ-ORD-2006-
0838.
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EXTERNAL PEER REVIEWER COMMENTS
The reviewers made several editorial suggestions to clarify specific portions of the text. These
changes were incorporated in the document as appropriate and are not discussed further.
A. General Comments
Charge Question 1. Are you aware of other publishedpeer-reviewed toxicological studies not
included in these toxicological reviews that could be of relevance to the health assessment of
BDE-47, -99, -153, or -209?
Comment 1: Three reviewers stated that they were unaware of any other relevant studies that
would contribute to the BDE-153 IRIS assessment. One reviewer identified the following
potentially relevant, additional literature:
Jones-Ortazo, HA; et al. (2005) Environ. Sci. Technol. 39:5121-5130
Wilford, BH; et al. (2005) Environ. Sci. Technol. 39:7027-7035
Schecter, A; et al. (2005) J. Toxicol. Environ. Health Part A 68:501-513
Kites, RA; et al. (2004) Environ. Sci. Technol. 38:4945-4949
Schecter, A; et al. (2006) Environ. Health Perspect. 114:1515-1520
Fischer, D; et al. (2006) Environ. Health Perspect. 114:1581-1584
Bradman, A; et al. (2007) Environ. Health Perspect. 115:71-74
Kodavanti, PRS; Derr-Yellin, EC. (2002) Toxicol. Sci. 68:451-457
Kodavanti, PRS; et al. (2005) Toxicol. Sci. 88:181-192
Reistad, T; Mariussen, E. (2005) Toxicol. Sci. 87:57-65
Reistad, T; et al. (2006) Arch. Toxicol. 80:785-796
Response: The Agency reviewed and evaluated the studies recommended by the reviewer and
has included the relevant studies for BDE-153. Fischer et al. (2006), Bradman et al. (2007), and
Kodavanti et al. (2005) were found to be relevant and were added to the document. The
remaining studies suggested by the reviewer fell outside the scope of the IRIS assessment (i.e.,
exposure data, commercial mixtures). Additionally, a new literature search was conducted to
ensure that recently published, relevant studies are included in the IRIS assessment. The only
study added to the "Toxicological Review" from the literature search was that by She et al.
(2007).
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B. Oral Reference Dose (RfD) Values
Charge Question 2. Have the rationale and justification for deriving RfD s on the basis of the
neurobehavioral toxicity studies been transparently and objectively described in the draft
toxicological reviews ofBDE-47, -99, -153, and-209? Are there additional studies that should
be considered for deriving the RfDsfor any of the four PBDE congeners?
Comment 1: Three reviewers stated that the rationale for deriving the RfD for BDE-153 based
on the neurobehavioral toxicity study by Viberg et al. (2003a) was clearly and transparently
described. Two reviewers commented that the neurobehavioral study by Viberg et al. (2003a) is
the only study that provides data for deriving the BDE-153 RfD. The peer reviewers
acknowledged the limitations and concerns with the study; however, they felt that its limitations
were transparently discussed in the "Toxicological Review." None of the reviewers suggested
additional studies that should be considered for deriving the RfD.
Response: No response needed.
Charge Question 3. Do you agree or disagree with EPA basing the health assessment ofBDE-
47, -99, -153, and-209 to a large extent on the Eriksson/Viberg neurobehavioral studies?
Comment 1: All peer reviewers supported the use of the Viberg et al. (2003a) neurobehavioral
study as the basis for the derivation of the BDE-153 RfD, given the limited body of toxicological
information available. Two reviewers noted that the neurobehavioral studies for BDE-47, -99,
-153, and -209 are limited by the fact that they originated from the same laboratory. One
reviewer was concerned that the experimental design of the principal study selected more than
one pup per litter, ignoring the "litter" effect. Treating littermates as independent experimental
units could confuse dose effects with litter effects. Another reviewer was concerned with the
specificity of the neurobehavioral data for developmental neurotoxicity and suggested that
independent confirmation of the endpoints is essential. One peer reviewer identified the use of a
single sex (male mice) as a limitation of the critical study that had not been identified in the
"Toxicological Review" discussion of study limitations. One of these reviewers stated that these
limitations do not hinder the derivation of the RfD for BDE-153 but make the confidence low.
Another reviewer noted that the neurobehavioral findings of the Eriksson/Viberg laboratory have
been corroborated in a study examining BDE-99 (Kuriyama et al., 2005). None of the reviewers
stated that the Viberg et al. (2003a) study should not be used as the basis for the derivation of the
RfD for BDE-153.
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Response: The "lexicological Review" contains a detailed summary of the concerns with the
study design and methods utilized in the principal study (see section 5.1.1). A discussion of the
use of only male mice in the study by Viberg et al. (2003a) has been added to the discussion in
section 5.1.1 of the "lexicological Review." Additionally, the neurobehavioral effects reported
in Viberg et al. (2003a) are supported by an expanding body of literature (Rice et al., 2007;
Viberg et al., 2007, 2005, 2004a, b, 2003b, 2002; Kuriyama et al., 2005; Eriksson et al., 2002,
2001; Branch! et al., 2002) that details changes in motor and cognitive activity in rodents
following administration of single or repeated perinatal doses of PBDEs. Some of the concerns
associated with the methodology of the Eriksson/Viberg neurobehavioral studies are alleviated
by other studies (Rice et al., 2007; Kuriyama et al., 2005; Branch! et al., 2002) using more
traditional methodologies that have generated toxic effects similar to the Eriksson/Viberg group.
These studies, although conducted with BDE-99 or -209, support the findings of Viberg et al.
(2003a) that exposure to these PBDE congeners in early developmental stages can result in
lasting changes in the neurobehavioral activity of mice.
Charge Question 4. Are the Eriksson et al. (2001) (BDE-47), Viberg et al. (2004b) (BDE-99),
Viberg et al (2003a) (BDE-153), and Viberg et al (2003b) (BDE-209) studies appropriate for
determining the point of departure? Have the strengths and weaknesses of the Viberg and
Eriksson studies been appropriately characterized and considered?
Comment 1: All four reviewers believed that the Viberg et al. (2003a) study is appropriate for
determining the point of departure for BDE-153. One reviewer felt that the data were
appropriate as long as the document emphasizes that the neurochemical data also show
alterations in normal developmental patterns. Another reviewer noted that Viberg et al. (2003a)
was the only study with dose-response data for determining the point of departure for BDE-153.
None of the reviewers suggested alternative studies for determining the point of departure for
BDE-153. Two reviewers explicitly stated that the strengths and weaknesses were identified and
clearly presented.
Response: The neurochemistry data for BDE-153 are discussed in section 4.4.1.5 of the
"Toxicological Review" because they are studies of the acetylcholine receptor in the brain. At
the suggestion of the reviewers, text has been added to the discussion of the toxicological
endpoints evaluated in the study (section 4.3) referring the reader to section 4.4.1.5 for a more
detailed discussion of the neurochemical data. The neurochemical data for BDE-153 as well as
that from BDE-99 and -209 are also discussed in section 4.5.3 of the "Toxicological Review" on
mode of action.
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Charge Question 5. Have the most appropriate critical effect and point of departure been
selected? And has the rationale for the point of departure been transparently and objectively
described?
Comment 1: All four reviewers agreed with the selection of the neurobehavioral effects of BDE-
153 as the critical effect for identifying a point of departure. One of the reviewers felt that the
neurochemical data also provided critical information and should be presented centrally rather
than as supporting data. One reviewer stated that there was no correlation between PND 10 of
exposure and the concentration of the chemical in the brain. One reviewer added that the
individual motor activity data on decreased habituation might be as appropriate as or more
appropriate than the habituation ratio as an indicator of toxicity, while another believed that the
actual behavioral data, rather than the habituation ratio, should have been presented in the
"lexicological Review." Another reviewer was confused as to why the actual data could not be
recovered from the study authors to allow for dose-response modeling and BMD estimation,
given that the studies were published fairly recently (2003). This reviewer recommended that
the Agency attempt to recover the neurobehavioral toxicity data from the study authors.
Response: Descriptions of the neurochemical data are fully summarized in section 4.4.1.5 and
are referenced in the discussions of the neurodevelopmental data from the principal study
(section 4.3). The evidence of neurochemical interactions and the potential relationship with the
neurobehavioral effects are highlighted in the mode-of-action section of the "Toxicological
Review" (section 4.5.3). The "Toxicological Review" presents the hypothesis proposed by the
Eriksson/Viberg group in which the observed effects on locomotion and habituation are related
to impaired development of the cholinergic system during the postnatal "brain growth spurt."
However, brain development is complex, and many neurochemical changes that occur during the
postnatal growth spurt and that could impact motor activity have not yet been investigated.
Therefore the available data, although supportive of the critical effects, are insufficient to
establish a mode of action. In the case of BDE-153, there are no data on levels in the brains of
mice to illustrate if timing of exposure influenced the amount in the brain.
The actual motor activity components (locomotion, rearing, and total activity) and habituation
ratios for BDE-153 were reported in graphical form only in the published paper and could not be
reasonably estimated as presented. The Agency's attempts to obtain the raw data from the
authors were unsuccessful. The NOAEL for the motor behavior data (activities and ratios)
served as the point of departure for the assessment.
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Comment 2: Two reviewers thought that the rationale for the point of departure had been
transparently and objectively described. Another reviewer felt the document provided clear
rationalization for the selection of the point of departure. None of the reviewers stated that the
rationale for the point of departure was not appropriately described.
Response: No response needed.
Comment 3: One reviewer noted that a 95% lower bound on the BMD (BMDL) would be better
than a NOAEL for deriving an RfD.
Response: The Agency recognizes that a NOAEL can be limiting since it is highly dependent on
the doses selected and the sample size and does not account for the dose-response curve.
Alternatively, a BMDL is more independent of study design, takes into account the dose-
response curve and the statistical significance of the observed effects, and therefore is preferable
to a NOAEL as a point of departure. However, the data from the principal study do not provide
quantitative dose response and can only be used to establish a NOAEL. The graphic reporting of
observational measures of effects in the critical study is insufficient to provide the necessary
information to perform BMD modeling, and the Agency's attempts to obtain the raw data from
the authors were unsuccessful.
Charge Question 6. Have the rationale and justification for each uncertainty factor (UF)
selected in the draft toxicological reviews ofBDE-47, -99, -153, and-209 been transparently
described? If the selected UFs are not appropriate, what alternative UFs would you suggest and
what are the scientific rationales for those suggested? Does the database support the
determinations of the RfDs for BDE-47, -99, -153, and-209?
Comment 1: Two reviewers agreed that the document described the rationale and justification
for each UF and another reviewer noted that the selection of the UFs was described in detail.
Response: No response needed.
Note: The peer reviewers provided fairly extensive comments about the individual components
of the combined UF. For that reason the following reviewer comments and EPA responses have
been grouped by the area of uncertainty to which they apply.
Comment 2: One reviewer suggested decreasing the interspecies UFA, considering the relatively
specific and sensitive nature of the neurobehavioral and neurochemical measures compared with
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conventional endpoints. However, another reviewer felt that the 10-fold UFA was justifiable
based on the lack of data on the mode of action in animals and humans.
Response: The 10-fold UFA for interspecies uncertainty is retained based on the lack of mode of
action, toxicokinetic, and human data that would sufficiently illustrate the similarities and
differences for the effects of BDE-153 in animals and humans. However, additional explanation
for applying the default interspecies UFA was added to section 5.1.3.
Comment 3: Two reviewers suggested lowering the intrahuman UFn. One of these reviewers
felt the relatively specific and sensitive nature of the neurobehavioral and neurochemical
measures compared with conventional endpoints warranted a decrease in the UFH. The other
reviewer recommended decreasing the 10-fold UFn to threefold based on the sensitivity of the
test species population (neonates).
Response: The 10-fold UFH for intraspecies uncertainty is retained based on the lack of
information concerning the toxicokinetics and mode of action of BDE-153 in humans. In the
absence of human data, the effects in potentially susceptible populations exposed to BDE-153
cannot be determined. Additional explanation for applying the default intraspecies UFH was
added to section 5.1.3.
Comment 4: One reviewer disagreed with the treatment of a single-dose experiment as
equivalent to a subchronic exposure when applying a UF to account for differences in exposure
duration. This reviewer stated that the principal study needs to be treated as a single-dose study
and not a subchronic study. The reviewer also felt that the threefold UFS was inappropriate and
suggested raising the UFS from 3 to 10, to consider the extent to which the mother's pre-
pregnancy accumulated body burden would influence the developmental outcome, especially
since these data are unavailable. One reviewer agreed with the application of a threefold UFS,
recognizing that for the observed neurobehavioral effects the timing of exposure is more critical
than the duration of exposure. This reviewer regards the UFs as accounting for uncertainty from
lack of prenatal exposure rather than uncertainty regarding potential effects of chronic exposure.
One reviewer suggested the threefold UFS may not be necessary, considering that exposure
during a window of susceptibility indicates that chronic exposure may not necessarily result in
greater adverse effects.
Response: For BDE-153, the principal study identified endpoints that, for the most part, reflect
an impact during a critical period of postnatal brain development in mice. The hypothesized
window of susceptibility, proposed by the study authors, is based on the observation that the
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developmental neurotoxic effects observed following exposure to BDE-153 on PND 10 will not
occur once the toxicokinetics of intestinal uptake and excretion have matured and the animal
brain is developmentally less active (outside the window of susceptibility). The Eriksson/Viberg
group has suggested that the period of maximum vulnerability for the developing cholinergic
system that coincides with the most pronounced neurodevelopmental effects from BDE-99
exposure is from PNDs 10-14. The UFs was viewed as a dosing duration adjustment rather than
simply a comparison of the effects of a subchronic to a chronic exposure, data that are lacking
for BDE-153. A threefold UFS was applied because the critical study dosed the animals only
once within the hypothesized critical window, not because the chronic exposures would have
exacerbated the impact on habituation.
In response to the comment concerning accumulation of the chemical, the Agency acknowledges
that this is an important uncertainty and that it was considered as part of the database UFo. As
discussed in the response to Charge Question 7, many of the variables needed to determine body
burden for BDE-153 from exposures during pregnancy and lactation are not available.
Accordingly, a UFS as described above was applied. Furthermore, there are no data for BDE-
153 from any conventional studies of chemical toxicity that can be used to determine whether or
not long-term exposures might identify effects that were co-critical with the sensitive
neurodevelomental toxicity identified in mice following a single dose exposure during its critical
window.
Comment 5: One reviewer disagreed with the use of a 10-fold database UFo, stating that "if the
database is so uncertain as to require a UFo of 10, then the database is too limited to allow the
derivation of meaningful RfDs." This reviewer recommended a value of 1 based on the
relatively sensitive nature of the neurobehavioral endpoint, the consistent observation of the
neurobehavioral effects across the four PBDE congeners, and the availability of the dose-
response data for deriving a BMDL. Another reviewer commented on the specificity and
sensitivity of the neurobehavioral and neurochemical measures and stated that it is inappropriate
to apply the database UFo. This reviewer observed that this UFo "addresses questions that go
beyond this endpoint and focus on risks that might occur, but there are no relevant data." The
reviewer felt that this UFo is more appropriately applied at the point of risk management. The
other two reviewers did not comment specifically on the database UFD but noted generally that
the database was poor and the overall confidence in the assessment is low (see next comment).
Response: EPA's practice is to apply a database UFD, generally ranging from 1-10, in the
health assessment to account for the potential for deriving an underprotective RfD as a result of
an incomplete characterization of a chemical's toxicity because of missing studies. In deciding
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to apply this factor to account for deficiencies in the available data set and in identifying its
magnitude, EPA considers both the data lacking and the data available for particular organ
systems as well as life stages. EPA acknowledges that the principal study (involving postnatal
exposure) has identified what appears to be a relatively sensitive effect; however, the database
for BDE-153 lacks a prenatal toxicity study and a two-generation reproduction study, as well as
subchronic and chronic toxicity studies. In light of the inadequate nature of the database, the
Agency retains a 10-fold UFD. The "IRIS Summary" and "lexicological Review" contain
sufficient information on the rationale for the database UFD to allow a risk manager to consider
the impact of this UFD during the risk management process.
Comment 6: Two reviewers believed that the database supports the determination of the RfD but
stated that the overall confidence in the RfD assessment is low. Another reviewer believed the
database is very poor and suggested that the RfDs be acknowledged as temporary while waiting
for additional studies that increase confidence.
Response: The statement that the overall confidence in the RfD is low is included in the
"Toxicological Review" in section 6.2. The Agency does not develop temporary RfDs for IRIS
assessments. However, the availability of new information is one of the factors considered in
selecting a chemical for reassessment.
C. Body Burden Approach
Charge Question 7. Are there adequate data for considering body burden as an alternative
dose metric to administered doses in any of the RfD derivations?
Comment 1: All four reviewers agreed that the data were inadequate to consider body burden as
an alternative dose metric for the derivation of the RfD. Two of the reviewers stated that body
burden is a possible alternative but the data are too limited.
Response: EPA examined the data on BDE-47 and -99 to determine if a body burden approach
could be used for these congeners during the development of the toxicological reviews. It was
determined that existing half-life, exposure, metabolite, and mode-of-action data could not
support a body burden calculation for these congeners. There are no data for BDE-153 that
could support a body burden approach.
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Charge Question 8. Do you agree with the rationale described in the "Toxicological Review "
ofBDE-99 that the data on the window of susceptibility of the cholinergic receptors to BDE-99
tend to minimize body burden concerns?
Comment 1: Three reviewers stated that the question was unclear. One reviewer accepted the
concept as a basis for the experimental design, given the available information. A second
reviewer stated that there was no direct evidence that BDE-99 directly affects cholinergic
receptors and suggested that the mechanism of the interaction must be complex and indirect. A
third reviewer stated that, although there are no definitive data on mode of action, this hypothesis
is plausible. This reviewer acknowledges that the data on the window of susceptibility of the
cholinergic receptors to BDE-99 are suggestive but believes there are too many other
possibilities for mode of action for this rationale to minimize body burden concerns.
Response: Mode-of-action data available that describe the developmental neurotoxicity of
BDE-153 are limited. The Eriksson/Viberg group, the principal study authors, have
hypothesized that the observed effects on locomotion and habituation are related to impaired
development of the cholinergic system during the postnatal "brain growth spurt" period (Viberg
et al., 2005, 2004a, 2003a). They have further hypothesized that the sensitivity of the
cholinergic system occurs in the vicinity of PND 10 and have tested this hypothesis by varying
the time of dosing and observing differences in the habituation effect for BDE-99 and -209
(Viberg et al., 2007; Eriksson et al., 2002). There are neurochemical data for BDE-153, but
there was no testing to determine the impact of varying the time of dosing. The resulting deficit
in cholinergic receptors persisted across the duration of testing and could cause an abnormal
response to exposure to cholinergic stimulants in adulthood. Testing of this hypothesis has not
been conducted in studies of BDE-153. The following statement has been added to the mode-of-
action summary (section 4.5.3): "While evidence exists that demonstrates that BDE-153 may
have an impact on neurochemical events during early postnatal development, data are inadequate
to determine the mode of action for BDE-153."
Miscellaneous Comments
Comment 1: Three reviewers felt that the assessment would benefit from the combination of the
individual documents for the four congeners into one comprehensive document to compare and
cohesively present the similarities and differences among the congeners.
Response: The Agency has recently completed IRIS assessments for four individual PBDE
congeners; tetrabromodiphenyl ether (BDE-47), pentabromodiphenyl ether (BDE-99),
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hexabromodiphenyl ether (BDE-153), and decabromodiphenyl ether (BDE-209) (see Foreword).
These congeners were selected based on frequent detection in human tissues and the
environment, the availability of animal toxicological studies suitable for human health
assessment, and their common occurrence in commercial PBDE mixtures. Although there is
some repetition in the four documents and similarities in the design of many studies, the
outcomes are sufficiently different from one congener to another to support the separation of the
four IRIS assessments. This improves reader comprehension and keeps the information on each
congener separate from that of the others. Keeping the congeners separate improves the ability
of the user to navigate the toxicological reviews and find the appropriate congener-specific data.
However, in response to the comments from the peer reviewers, the Agency has increased the
text that compares the data derived from comparable methodological approaches on BDE-153 to
that for the other congeners evaluated.
Comment 2: One reviewer noted that the document failed to cite the purities of the radioactive
chemicals in most of the studies, position of the label, location of radioactivity in the brain, and
specific activities of the 14C compounds. Another reviewer felt that it was important to be
careful when comparing the data on levels of radiolabel in tissues with data from direct chemical
measurements. The reviewer felt that some of the radiolabel data, especially when the label was
given after a period of dosing with unlabeled compound, might have been misinterpreted.
Response: The requested data were added to the descriptions of the pertinent studies (in section
3) when they were provided by the authors of the paper. Frequently, the position of the
radiolabel was not specified. In a few cases the radiolabel was described as "uniform,"
suggesting that all carbons carried the radiolabel. If the authors of the paper used the term
"uniform," it has been added to the discussion of the study. No change was made if the authors
of the paper did not comment on the position of the radiolabel. In presenting the results of the
studies where the radiolabel was given after a period of dosing with unlabeled compound, the
conclusions were checked against those expressed by the authors of the studies and made
consistent if necessary.
Comment 3: One reviewer was concerned that the doses and concentrations of the compound
and the metabolites in biological tissues were presented in differing units (i.e., umol, ug, and
percent of dose).
Response: Doses and concentrations are reported as given by the authors. If a dose was given in
molar or mole units per unit body weight, the doses have been provided parenthetically as mg/kg
body weight values. Otherwise the units are those provided in the published papers.
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Comment 4: One reviewer suggested including a proposed metabolic pathway that integrates the
available data on the metabolites that have been identified.
Response: A diagram of a proposed metabolic pathway for BDE-153 has been included as
Figure 3-1 in section 3.3. The diagram is based on the metabolites that have been identified in
several studies. The metabolic pathway is described as "proposed," and uncertainties are
indicated. The individual publications that provided data on metabolites are cited in Figure 3-1.
In cases where the data from one study are discussed in more than one section of the
"Toxicological Review," a reference to the location of prior mention of the study is provided.
Comment 5: One reviewer acknowledged that developmental neurotoxicity is consistently
observed following exposure to the PBDEs, despite very different patterns of metabolism,
distribution, and persistence within the body. This reviewer recommended rationalizing the
relative potency of the PBDEs, considering the differences in the extent of metabolism.
Response: Information is currently insufficient to adequately identify differences in the potency
of the four congeners based on the metabolism data. The metabolite data are primarily
qualitative rather than quantitative, and in the case of BDE-153 there is only one study that
provides data on metabolites. That study used an intraveneous rather than oral route of
administration.
Comment 6: One reviewer suggested that the Agency provide conclusions on the extent of
metabolism and the presence of metabolites in excreta for the PBDEs or provide a statement if
conclusions cannot be drawn. One reviewer suggested the addition of a summary at the
beginning of the toxicokinetics section to reduce potential confusion.
Response: An overview has been added to the toxicokinetics section that provides an integration
of the BDE-153 toxicokinetic data for mice and rats and identifies any differences seen between
species. There are no metabolite data from rats and there are inconsistencies between the
intraveneous data on mice from the Staskal et al. (2006) study and the oral Sanders et al. (2006)
study in rats.
Comment 7: One reviewer commented that there appears to be no correlation between PND
exposure, concentration in the brain, and increased adverse responses.
Response: There are no data on the levels of BDE-153 or its metabolites in the brain after oral
dosing during either the postnatal or adult periods.
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Comment 8: One reviewer recommended presenting the receptor site interaction information in
a summary table.
Response: A summary table for the receptor studies has been added to section 4.4 of the
"lexicological Review."
Comment 9: One reviewer recommended the use of a table summarizing the developmental and
reproductive effects of the BDE-99 and suggested adding a similar table to the BDE-153
document.
Response: Considering the lack of other studies to compare to the principal study, a table is not
warranted for only one study.
Comment 10: One reviewer recommended adding data for BDE-209 found in human biological
media to Table 3-1, "Median PBDE concentrations in human biological media in the U.S."
Response: Data for BDE-209 were not included in Table 3-1 for BDE-153 because, except for
the She et al. (2007) study, data on median concentrations of BDE-209 were not provided in the
references used to develop the table.
Comment 11: One reviewer felt that the text that compares levels of congeners in human
biological media for the U.S. to that of other countries and the relative differences in the
congeners identified are important issues and the studies need to be presented to support or
refute the discussions of differences observed.
Response: The Agency has provided information on levels in biological media that reflect
exposure in the U.S. and other countries for comparison purposes. Although the data reflect
exposure, they are not direct measurements of exposure given that there is some metabolism of
PBDEs generating metabolites and, in the case of BDE-209, less highly brominated PBDEs.
Therefore, the data presented are not data on total exposures to any of the congeners. While the
Agency agrees that exposure analysis is a critical component of risk assessment, a
comprehensive presentation and analysis of exposure data is outside the scope of the IRIS health
assessment.
Comment 12: One reviewer stated that the large number of bromine atoms of the PBDEs can
impart electrophilic and lipophilic properties to the aromatic ring of the chemical and also noted
that oily vehicles (e.g., corn oil) were used in most of the in vitro and in vivo animal studies.
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This reviewer was concerned the vehicle could significantly alter the distribution and tissue
uptake of the PBDEs between the oily vehicle and the biological system. These conditions could
lead to decreased absorption and distribution with subsequent alteration in metabolism and
excretion.
Response: The lipophilicity of the BDE-153 is acknowledged in the "Toxicological Review" as
part of section 3, Toxicokinetics. It will be necessary to determine if absorption occurs via the
chylomicrons along with the body lipids or via direct membrane transport before the full impact
of the vehicle on absorption-distribution-metabolism-excretion can be determined. The data are
currently inadequate to determine the impact of the oily vehicle on the distribution and uptake of
BDE-153.
Comment 13: One reviewer noted that considering the antithyroid effects observed with DE-71
(a formulation that might be reasonably linked to neurodevelopmental effects observed with
BDE-47 and -99) in Zhou et al. (2002) occurred at doses that are higher than those that produce
the neurodevelopmental effects of BDE-47 and -99. The reviewer suggested that it could be
concluded that the neurodevelopmental effects cannot be linked to the antithyroid effects of
these compounds. Additionally, the antithyroid effects have not been substantiated.
Response: There are minimal data on the thyroid effects of BDE-153. In human sera there was
no correlation between total PBDEs and TS or T4 concentrations (total or free), but the sample
sizes were small (Mazdai et al., 2003). None of the animal studies of BDE-153 examined the
serum levels of thyroid hormones. Neither BDE-153 nor its hydroxylated metabolites BDE-138
and -153 were found to compete with T4 for binding to TTR (Meerts et al., 2000). In addition,
DE-71 is a commercial mixture and cannot be considered to be fully equivalent to BDE-153.
DE-71 contains only 6% BDE-153.
PUBLIC COMMENTS
The public commenters made several editorial suggestions to clarify specific portions of the text.
These changes were incorporated in the document as appropriate and are not discussed further.
Comment 1: One public commenter suggested that the Agency consider a body burden
approach.
Response: The Agency presented this issue to the peer reviewers in the form of a charge
question. In response to the charge question about use of a body burden approach for dose
evaluation, the peer reviewers agreed that, whereas the body burden approach might be
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appropriate for some of the congeners given their lipophilicity and distribution to adipose tissue,
data to support such an approach are not presently available.
Comment 2: One public commenter questioned the selection of Viberg et al. (2003a) as the
principal study for the derivation of the RfD and questioned the methods utilized by the principal
study authors.
Response: The Agency has included a detailed summary of the concerns with the study design
and methods utilized in the principal study (see section 5.1.1). These issues were raised during
the external peer review of the BDE-153 IRIS assessment. The peer reviewers acknowledged
the limitations and concerns with the study; however, all of the reviewers agreed that this study
was appropriate for derivation of the RfD for BDE-153 and that its limitations were transparently
discussed in the "Toxicological Review." Additionally, the neurobehavioral effects reported in
Viberg et al. (2003a) are supported by an expanding body of literature (Viberg et al., 2007, 2005,
2004a, b, 2003b, 2002; Kuriyama et al., 2005; Eriksson et al., 2002, 2001; Branch! et al., 2002)
that details changes in motor and cognitive activity in rodents following administration of single
or repeated perinatal doses of PBDEs.
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