Draft Risk Evaluation for Cyclic Aliphatic Bromides Cluster (HBCD) Systematic Review Supplemental File: Data Quality Evaluation of Human Health Hazard Studies Animal, In Vitro and Epidemiological Studies


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v>EPA
United States	Office of Chemical Safety and
Environmental Protection Agency	Pollution Prevention
Draft Risk Evaluation for
Cyclic Aliphatic Bromides Cluster
(HBCD)
Systematic Review Supplemental File:
Data Quality Evaluation of
Human Health Hazard Studies - Animal, In Vitro and
Epidemiological Studies
CASRN
NAME
25637-99-4
Hexabromocyclododecane
3194-55-6
1,2,5,6,9,10-Hexabromocyclododecane
3194-57-8
1,2,5,6-Tetrabromocyclooctane
June 2019
1

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TABLE OF CONTENTS
1	Acute Toxicity Studies	5
1, ] Animal toxicity evaluation results of 1990 acute oral study for mortality, body
weight outcomes	5
1.2 Animal toxicity evaluation results of 1990 study for primary skin irritation study on
irritation outcomes	8
13 Animal toxicity evaluation results of Eriksson et al 2006 for oral
neurodevelopmental study (single dose post-natal day 10) study on neurological/behavior,
growth (early life) and development outcomes	11
1.4	Animal toxicity evaluation results of IRDC 1978 for acute toxicity studies (oral,
dermal and ocular) study on gastrointestinal, irritation, and skin and connective tissues
outcomes	15
1.5	Animal toxicity evaluation results of Song et al 2016 for acute and 14-day
inhalation-systemic toxicity study on body weight, hematological and immune, clinical
chemistry/biochemical, hepatic, renal, respiratory, reproductive outcomes	20
1.6	Animal toxicity evaluation results of Szabo et al 2016 for single gavage in mice on
post-natal day 10; metabolomics evaluation only study on gene expression/omics outcomes
23
2	Short-term Toxicity Studies	30
2.1	Animal toxicity evaluation results of Bernhard et al 2016 for 28-day oral exposure in
mice via diet study on hepatic, body weight outcomes	30
2.2	Animal toxicity evaluation results of Genskow et al 2015 for 30 day oral toxicity
study (daily gavage); primarily mechanistic, also contains in vitro data study on
neurological/behavior outcomes	34
2 J Animal toxicity evaluation results of Hachisuka et al 2010 for oral developmental
immunotoxicity study on hematological and immune outcomes	37
2.4	Animal toxicity evaluation results of Maranghi et al 2013 for 28-day dietary study
on hepatic, body weight, thyroid, hematological and immune, reproductive outcomes	40
2.5	Animal toxicity evaluation results of Miller et al 2016 for mechanism of liver and
thyroid toxicity study on hepatic, thyroid outcomes	43
2.6	Animal toxicity evaluation results of Miller-Rhodes et al 2014 for developmental
study; gestation day 1-parturition study on growth (early life) and development,
neurological/behavior outcomes	46
2.7	Animal toxicity evaluation results of van et al 2006 for 280day oral toxicity study
(gavage) study on hepatic, clinical chemistry/biochemical, endocrine,
musculoskeletal/motor function, ADME/PBPK, thyroid, nutrition and metabolic/adult
exposure body weight, hematological and immune, reproductive outcomes	50
2-8 Animal toxicity evaluation results of W. I. L. Research 1997 for 28-day repeated oral
study on mortality, nutrition and metabolic/adult exposure body weight,
neurological/behavior, hematological and immune, clinical chemistry/biochemical, hepatic,
renal, cardiovascular, reproductive, endocrine, gastrointestinal, respiratory outcomes	56
2.9 Animal toxicity evaluation results of Wang et al 2016for 28 day oral gavage
metabolomic study in mice study on nutrition and metabolic/adult exposure body weight,
gene expression/omics outcomes	63
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2.10 Animal toxicity evaluation results of Watanabe et al 2010 for 28 day feeding study
in mice - mechanistic study, animals also infected with rsv study on nutrition and
metabolic/adult exposure body weight, hematological and immune outcomes	66
3	Subchronic Toxicity Studies	68
3.1	Animal toxicity evaluation results of ACC et al 2002 for 90-day gavage-systemic
with sperm evaluations and neurobehavior, same as (2990994) study on reproductive,
hematological, neurological/behavior, renal, hepatic, clinical chemistry/biochemical, body
weight, ocular and sensory, thyroid outcomes	68
3.2	Animal toxicity evaluation results of BASF et al 1990 for 28-day and 90-day dietary
studies study on reproductive, hematological and immune, neurological, renal, hepatic,
endocrine, gastrointestinal, respiratory, thyroid outcomes	73
3 J Animal toxicity evaluation results of van et al 2009 for 1-generation reproduction
study, oral dietary study on endocrine; reproductive; hematological and immune; thyroid;
growth (early life) and development; musculoskeletal/motor function; clinical
chemistry/biochemical; nutrition and metabolic/adult exposure body weight; hepatic
outcomes	75
3.4	Animal toxicity evaluation results of W. I. L. Research 2001 for 90-day gavage
study on reproductive, hematological and immune, neurological/behavior, renal, hepatic,
ocular and sensory, cardiovascular, clinical chemistry/biochemical, endocrine,
gastrointestinal, body weight, respiratory, thyroid outcomes	81
3.5	Animal toxicity evaluation results of Ema et al 2008 study on reproductive, growth
(early life) and development, hepatic, neurological/behavior, thyroid outcomes	84
3.6	Animal toxicity evaluation results of Lilienthal et al 2009 (787693) for 1-generation
reproductive study, dietary exposure study on neurological/behavior outcomes	89
3.7	Animal toxicity evaluation results of Saegusa et al 2009 for 1-generation
developmental toxicity (dietary exposure) study on reproductive, growth (early life) and
development, neurological, hepatic, endocrine, thyroid, nutrition and metabolic/adult
exposure body weight outcomes	92
3.8	Animal toxicity evaluation results of Yanagisawa et al 2014 for 14-week study
(animals dosed by gavage lx per week) study on hepatic, body weight, nutrition and
metabolic/adult exposure body weight outcomes	95
4	In Vitro Studies	99
4.1	In vitro evaluation results of 1990	99
4.2	In vitro evaluation results of Almughamsi et al 2016	101
4.3	In vitro evaluation results of An et al 2016	103
4.4	In vitro evaluation results of Anisuzzaman et al 2016	105
4.5	In vitro evaluation results of Canbaz et al 2016	107
4.6	In vitro evaluation results of Ethyl Corporation 1990	 110
4.7	In vitro evaluation results of Ethyl Corporation 1990	 113
4.8	In vitro evaluation results of Huang et al 2016	115
4.9	In vitro evaluation results of Kim et al 2016	118
4.10	In vitro evaluation results of Koike et al 2016	120
4.1 I In vitro evaluation results of Litton et al 1990	 122

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4.12	In vitro evaluation results of Pharmakologisches et al 1990 	 125
4.13	In vitro evaluation results of S.R.I. International 1990	 127
4.14	In vitro evaluation results of Wang et al 2016	129
4.15	In vitro evaluation results of Wu et al 2016	131
Epidemiological Studies	133
5.1	Epidemiological evaluation results of the Eggesb0 et al 2011 study for thyroid
outcomes for exposure groups in general	133
5.2	Epidemiological evaluation results of the Johnson et al 2013 study for reproductive
outcomes in general	137
5 J Epidemiological evaluation results of the Kicinski et al 2012 study for
neurological/behavior outcomes in general	140
5.4	Epidemiological evaluation results of the Kicinski et al 2012 study for thyroid
outcomes in general	144
5.5	Epidemiological evaluation results of the Kim et al 2014 study for thyroid outcomes
for mothers & infants	148
5.6	Epidemiological evaluation results of the Meijer et al 2012 study for reproductive
outcomes for GIC cohort HBCD sex hormones	152
5.7	Epidemiological evaluation results of the Meijer et al 2012 study for reproductive
outcomes for GIC cohort HBCD male sexual development	156
5.8	Epidemiological evaluation results of the Roze et al 2009 study for
neurological/behavior outcomes in general	160
5.9	Epidemiological evaluation results of the Roze et al 2009 study for
neurological/behavior outcomes for GIC cohort HBCD neuropsychological	164
5.10	Epidemiological evaluation results of the Roze et al 2009 study for
neurological/behavior outcomes for GIC cohort HBCD behavior	168
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1 Acute Toxicity Studies
1.1 Animal toxicity evaluation results of 1990 acute oral study for
mortality, body weight outcomes
Study reference:
(1990). LETTER FROM AMERIBROM INC TO US EPA REGARDING 8D SUBMISSION FOR
HEXABROMOCYCLODODECANE WITH ATTACHMENTS
HERO ID: 1928284
Domain
Metric
Evaluator's Comment
Qualitative
Determination
[i.e., High, Medium,
Low,Unacceptable,
or Not rated]
Metric
Score
Metric
Weighting
Factor
Weighted
Score

1. Test Substance
Identity
The test substance was
identified by
abbreviation.
Medium
2
2
4
Test Substance
2. Test Substance
Source
The source of the test
substance, including
manufacturer, was not
specifically reported. Lot
number was not reported.
Low
3
1
3

3. Test Substance
Purity
Purity and grade were
not reported and there
was no analysis
conducted for
measurement of
impurities, if present.
Low
3
1
3

4. Negative and
Vehicle Controls
Use of a control group
was not reported, but is
not required for studies
of this type and outcome
Low
3
2
6
Test Design
5. Positive Controls

Not Rated (NR)
NR
NR
NR
6. Randomized
Allocation
The study authors did not
report how animals were
allocated to study groups
but there was only one
group.
NR
NR
NR
NR
Exposure
Characterization
7. Preparation and
Storage of Test
Substance
The study authors
reported some details on
test item preparation, but
they were incomplete
(e.g., time of stirring,
temperature, etc.) and the
storage conditions were
not reported,
Low
3
1
3
5

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Study reference:
(1990). LETTER FROM AMERIBROM INC TO US EPA REGARDING 8D SUBMISSION FOR
HEXABROMOCYCLODODECANE WITH ATTACHMENTS
HERO ID: 1928284
Domain
Metric
Evaluator's Comment
Qualitative
Determination
[i.e., High, Medium,
Low,Unacceptable,
or Not rated]
Metric
Score
Metric
Weighting
Factor
Weighted
Score

8. Consistency of
Exposure
Administration
A few details were
reported that indicted
that dosing methods were
equivalent (e.g., similar
dosing volumes at 10
mL/kg), but insufficient
details were reported to
allow determination of
whether exposure
administration was
consistent.
Low
3
1
3

9. Reporting of
Doses/Concentration
s
Administered dose level
was reported.
High
1
2
2

10. Exposure
Frequency and
Duration
The exposure frequency
and duration were
incompletely reported to
allow a determination of
whether they were
suitable. Stated to be an
acute study though, so
suggests one exposure.
Low
3
1
3

11. Number of
Exposure Groups
and Dose Spacing
Only one dose level was
tested, but this is
acceptable for studies of
this type.
High
1
1
1

12. Exposure Route
and Method
The route of exposure
was reported and was
suited to the test
substance.
High
1
1
1

13. Test Animal
Characteristics
The test animal source,
life stage, and starting
body weight were not
reported; species, strain,
and sex were reported.
Medium
2
2
4
Test Organism
14. Adequacy and
Consistency of
Animal Husbandry
Conditions
Husbandry conditions
were not sufficiently
reported to evaluate if
husbandry was adequate
and/or if differences
existed between the
exposed and control
groups. These
deficiencies may have a
substantial impact on the
results.
Low
3
1
3
6

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Study reference:
(1990). LETTER FROM AMERIBROM INC TO US EPA REGARDING 8D SUBMISSION FOR
HEXABROMOCYCLODODECANE WITH ATTACHMENTS
HERO ID: 1928284
Domain
Metric
Evaluator's Comment
Qualitative
Determination
[i.e., High, Medium,
Low,Unacceptable,
or Not rated]
Metric
Score
Metric
Weighting
Factor
Weighted
Score

15. Number per
Group
The number of animals
was appropriate for the
study type and outcome
analysis.
High
1
1
1

16. Outcome
Assessment
Methodology
Details on the outcome
assessment methodology
were incompletely
reported (e.g., the
frequency of
observations during the
post-exposure
observation period). Due
to incomplete reporting,
it's not clear whether
methods were sensitive
for the outcomes of
interest other than non-
lethal outcomes
Low
3
2
6
Outcome
Assessment
17. Consistency of
Outcome
Assessment
Consistency of the
outcome assessments
was not adequately
reported for meaningful
interpretation of results.
These are serious flaws
that make the study
unusable.
Unacceptable
4
1
4

18. Sampling
Adequacy
Details regarding
sampling of outcomes
were not reported and
this deficiency is likely
to have a substantial
impact on results.
Low
3
1
3

19. Blinding of
Assessors

Not Rated
NR
NR
NR

20. Negative Control
Response

Not Rated
NR
NR
NR
Confounding /
Variable Control
21. Confounding
Variables in Test
Design and
Procedures
Lack of reporting of
initial body weights and
whether there were any
differences among the
study groups in this or
other parameters is
considered to have a
substantial impact on the
results.
Low
3
2
6
7

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Study reference:
(1990). LETTER FROM AMERIBROM INC TO US EPA REGARDING 8D SUBMISSION FOR
HEXABROMOCYCLODODECANE WITH ATTACHMENTS
HERO ID: 1928284
Domain
Metric
Evaluator's Comment
Qualitative
Determination
[i.e., High, Medium,
Low,Unacceptable,
or Not rated]
Metric
Score
Metric
Weighting
Factor
Weighted
Score

22. Health Outcomes
Unrelated to
Exposure
Data on attrition and/or
health outcomes
unrelated to exposure for
each study group were
not reported because
only substantial
differences among
groups were noted
Low
3
1
3
Data Presentation
and Analysis
23. Statistical
Methods

Not Rated
NR
NR
NR
24. Reporting of
Data
Data reporting was
minimal and data on
outcomes of exposure
were reported in the text
only.
Low
3
2
6
High: >=1 and <1.7
Medium: >=1.7 and <2.3
Low: >=2.3 and <=3
Sum of scores:

26
65
Overall Score = Sum of Weighted Scores/Sum
of Metric Weighting Factors:
2.5000
Overall Score
(rounded):
2.51
Overall Quality Level:
Unacceptable1
Comment:
The report provides minimal details on methodology and results; however, the results for this acute oral
toxicity study may be useful in a weight of evidence with other similar studies.
Footnote:
1 Consistent with our Application of Systematic Review in TSCA Risk Evaluations document, if a metric for a
data source receives a score of Unacceptable (score = 4), EPA will determine the study to be unacceptable. In
this case, one of the metrics was rated as unacceptable. As such, the study is considered unacceptable and the
score is presented solely to increase transparency.
1.2 Animal toxicity evaluation results of 1990 study for primary
skin irritation study on irritation outcomes
8

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Study reference:
(1990). LETTER FROM AMERIBROM INC TO US EPA REGARDING 8D SUBMISSION FOR
HEXABROMOCYCLODODECANE WITH ATTACHMENTS
HERO ID: 1928284
Domain
Metric
Evaluator's Comment
Qualitative
Determination
[i.e., High, Medium,
Low,Unacceptable,
or Not rated]
Metric
Score
Metric
Weighting
Factor
Weighted
Score
Test Substance
1. Test Substance
Identity
The test substance was
identified by
abbreviation, and a trade
name.
Medium
2
2
4
2. Test Substance
Source
Test substance source
was reported.
High
1
1
1
3. Test Substance
Purity
Purity and grade were
not reported and there
was no analysis
conducted for
measurement of
impurities, if present.
Low
3
1
3
Test Design
4. Negative and
Vehicle Controls
Use of a control group
was not reported, but is
not required for studies
of this type and outcome
Low
3
2
6
5. Positive Controls

Not Rated
NR
NR
NR
6. Randomized
Allocation
The study authors did not
report how animals were
allocated to study groups
but there was only one
group.
Not Rated
NR
NR
NR
Exposure
Characterization
7. Preparation and
Storage of Test
Substance
Test substance
preparation was reported;
however, storage was not
reported.
Medium
2
1
2
8. Consistency of
Exposure
Administration
The study reported
consistent exposure
administration; however,
some details were
lacking, such whether the
exposures occurred at the
same approximate time
for all animals.
Medium
2
1
2
9. Reporting of
Doses/Concentration
s
Administered dose level
was reported.
High
1
2
2
10. Exposure
Frequency and
Duration
Exposure frequency and
duration were reported.
High
1
1
1
11. Number of
Exposure Groups
and Dose Spacing
Only one dose level was
tested, but this is
acceptable for studies of
this type.
High
1
1
1
9

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Study reference:
(1990). LETTER FROM AMERIBROM INC TO US EPA REGARDING 8D SUBMISSION FOR
HEXABROMOCYCLODODECANE WITH ATTACHMENTS
HERO ID: 1928284
Domain
Metric
Evaluator's Comment
Qualitative
Determination
[i.e., High, Medium,
Low,Unacceptable,
or Not rated]
Metric
Score
Metric
Weighting
Factor
Weighted
Score

12. Exposure Route
and Method
The route of exposure
was reported and was
suited to the test
substance.
High
1
1
1

13. Test Animal
Characteristics
Test animal source, life
stage, initial body
weight, species, strain,
and sex were reported;
test animal was from a
laboratory-maintained
colony
High
1
2
2
Test Organism
14. Adequacy and
Consistency of
Animal Husbandry
Conditions
Husbandry conditions
were reported, including
lighting, temperature,
and humidity.
High
1
1
1

15. Number per
Group
The number of animals
per study group (six) and
number of groups (one)
was acceptable for the
study type and outcomes
of interest.
High
1
1
1

16. Outcome
Assessment
Methodology
The outcome assessment
methodology addressed
or reported the intended
outcomes) of interest and
was sensitive for the
outcomes(s) of interest.
High
1
2
2
Outcome
Assessment
17. Consistency of
Outcome
Assessment
Details of the outcome
assessment protocol were
reported for some
outcomes, including time
points for post-exposure
observations, and were
the same across all
groups.
Medium
2
1
2

18. Sampling
Adequacy
Details regarding
sampling for the
outcomes of interest
were partially reported
(e.g., sampling for
general condition was
not indicated, such as
how many animals were
examined.
Medium
2
1
2

19. Blinding of
Assessors

Not Rated
NR
NR
NR
10

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Study reference:
(1990). LETTER FROM AMERIBROM INC TO US EPA REGARDING 8D SUBMISSION FOR
HEXABROMOCYCLODODECANE WITH ATTACHMENTS
HERO ID: 1928284
Domain
Metric
Evaluator's Comment
Qualitative
Determination
[i.e., High, Medium,
Low,Unacceptable,
or Not rated]
Metric
Score
Metric
Weighting
Factor
Weighted
Score

20. Negative Control
Response

Not Rated
NR
NR
NR
Confounding /
Variable Control
21. Confounding
Variables in Test
Design and
Procedures
No initial differences in
body weight were
reported within the
treatment group and
there were no other
reported differences that
could influence the
outcome assessment
Medium
2
2
4
22. Health Outcomes
Unrelated to
Exposure
Data on attrition and/or
health outcomes
unrelated to exposure for
each study group were
not reported because
only substantial
differences among
groups were noted
Low
3
1
3
Data Presentation
and Analysis
23. Statistical
Methods

Not Rated
NR
NR
NR
24. Reporting of
Data
There were some
deficiencies in reporting
of data (e.g., initial body
weights were based on a
range, rather than actual
values.)
Low
3
2
6
High: >=1 and <1.7
Medium: >=1.7 and <2.3
Low: >=2.3 and <=3
Sum of scores:

26
46
Overall Score = Sum of Weighted Scores/Sum
of Metric Weighting Factors:
1.7692
Overall Score:
Nearest *:
1.8
Overall Quality Level:
Medium
13 Animal toxicity evaluation results of Eriksson et al 2006 for
oral neurodevelopmental study (single dose post-natal day 10)
study on neurological/behavior, growth (early life) and
development outcomes
li

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Study reference:
Eriksson, P.,Fischer, C.,Wallin, M.Jakobsson, E.,Fredriksson, A. (2006). Impaired behaviour, learning and
memory, in adult mice neonatally exposed to hexabromocyclododecane (HBCDD) Environmental Toxicology
and Pharmacology, 21(3), 317-322
HERO ID: 787660
Domain
Metric
Evaluator's Comment
Qualitative
Determination
[i.e.,High,Medium,
Low,Unacceptable,
or Not rated]
Metric
Score
Metric
Weighting
Factor
Weighted
Score

1. Test Substance
Identity
Characterized as a
mixture containing three
diastereoisomers alpha-,
beta-, and gamma-
HBCD.
High
1
2
2
Test Substance
2. Test Substance
Source
Prepared from a
commercial mixture, but
the manufacturer and
lot/batch number were
not given. Analytical
verification is not
described.
Low
3
1
3

3. Test Substance
Purity
>98%
High
1
1
1

4. Negative and
Vehicle Controls
Negative vehicle controls
were used.
High
1
2
2
Test Design
5. Positive Controls
Positive controls were
not needed for
neurodevelopmental
studies.
Not Rated
NR
NR
NR

6. Randomized
Allocation
Randomly selected from
3-4 different litters from
each treatment group.
High
1
1
1

7. Preparation and
Storage of Test
Substance
Preparation was well
described and
appropriate. Single dose
study, therefore
prolonged storage is not
a concern.
High
1
1
1
Exposure
Characterization
8. Consistency of
Exposure
Administration
Details of exposure
administration were
reported, and exposures
were administered
consistently across study
groups in a scientifically
sound manner (dose
given via a PVC tube).
High
1
1
1

9. Reporting of
Doses/Concentration
s
Gavage doses were
reported as both mg/kg
and |imol/kg.
High
1
2
2

10. Exposure
Frequency and
Duration
Administered as a single
dose during a critical
period (onPND 10) in
neonatal development of
the mouse brain.
High
1
1
1
12

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Study reference:
Eriksson, P.,Fischer, C.,Wallin, M.Jakohsson, E.,Fredriksson, A. (2006). Impaired behaviour, learning and
memory, in adult mice neonatally exposed to hexabromocyclododecane (HBCDD) Environmental Toxicology
and Pharmacology, 21(3), 317-322
HERO ID: 787660
Domain
Metric
Evaluator's Comment
Qualitative
Determination
[i.e.,High,Medium,
Low,Unacceptable,
or Not rated]
Metric
Score
Metric
Weighting
Factor
Weighted
Score

11. Number of
Exposure Groups
and Dose Spacing
2 doses plus control. A
justification was not
provided for the doses
selected, but the results
suggest they were
appropriate.
Medium
2
1
2

12. Exposure Route
and Method
The route and method of
exposure were reported
and were suited to the
test substance.
High
1
1
1

13. Test Animal
Characteristics
Species, strain and age of
neonatal mice was
specified.
High
1
2
2
Test Organism
14. Adequacy and
Consistency of
Animal Husbandry
Conditions
Most husbandry
conditions were reported
and were adequate and
similar for all groups.
Humidity was not
reported. But this is
unlikely to have a
substantial impact on the
results.
Medium
2
1
2

15. Number per
Group
The number of animals
per study group was
reported, appropriate for
the study type and
outcome analysis, and
consistent with studies of
the same or similar type
(10/group or 12-
17/group)
High
1
1
1

16. Outcome
Assessment
Methodology
Standard tests of
spontaneous behavior
and learning and
memory.
High
1
2
2
Outcome
Assessment
17. Consistency of
Outcome
Assessment
Details of the outcome
assessment protocol were
reported, and outcomes
were assessed
consistently across study
groups (e.g., at the same
time after initial
exposure) using the same
protocol in all study
groups.
High
1
1
1
13

18. Sampling
Adequacy
It is difficult to discern
definitively but based on
the methods description
and a statistical paper
published explaining the
methods used (Eriksson
2005, The Toxicologist)
it appears that the pup
was used as a statistical
unit. While this is less
important because the
mice were not exposed in
utero, it still ignores
known litter effects, as
documented in (Holsen
et al, 2008).
Additionally, Holson et
al 2008 recommends
examining both sexes,
while this study only
examines males.
Low
3
1
3

19. Blinding of
Assessors
Blinding was not
reported; however,
outcomes were objective.
Medium
2
1
2

20. Negative Control
Response
The biological responses
of the negative control
group(s) were adequate.
High
1
1
1
Confounding /
Variable Control
21. Confounding
Variables in Test
Design and
Procedures
There were no significant
deviations in body
weight gain in HBCDD-
treated mice compared
with the vehicle-treated
mice.
High
1
2
2
22. Health Outcomes
Unrelated to
Exposure
Data on attrition and/or
health outcomes
unrelated to exposure
were not reported for
each study group
Low
3
1
3
Data Presentation
and Analysis
23. Statistical
Methods
The specifics of
analyzing pups as
opposed to litters were
not explicitly explained
and failing to account for
litter effects could have a
large statistical impact on
results.
Low
3
1
3
14

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PEER REVIEW DRAFT- DO NOT CITE OR QUOTE
Study reference:
Eriksson, P.,Fischer, C.,Wallin, M.Jakobsson, E.,Fredriksson, A. (2006). Impaired behaviour, learning and
memory, in adult mice neonatally exposed to hexabromocyclododecane (HBCDD) Environmental Toxicology
and Pharmacology, 21(3), 317-322
HERO ID: 787660
Domain
Metric
Evaluator's Comment
Qualitative
Determination
[i.e., High, Medium,
Low,Unacceptable,
or Not rated]
Metric
Score
Metric
Weighting
Factor
Weighted
Score

24. Reporting of
Data
Data for exposure-related
findings were presented
for all outcomes by
exposure group and sex.
High
1
2
2
High: >=1 and <1.7
Medium: >=1.7 and <2.3
Low: >=2.3 and <=3
Sum of scores:

30
41
Overall Score = Sum of Weighted Scores/Sum
of Metric Weighting Factors:
NR
Overall Score:
Nearest *:
NR
Overall Quality Level:
Medium
Study Quality
Comment:
The reviewer downgraded this study. They noted: Downgraded because the statistical methods are
inappropriate based on proper methods for DNT studies according to other publications (e.g. Holman et al,
2008, Neurotoxicology and Teratology) Note: The original calculated score for this study was 1.4. This value is
not presented above because the final rating was changed based on professional judgement.
1.4 Animal toxicity evaluation results of IRDC 1978 for acute
toxicity studies (oral, dermal and ocular) study on
gastrointestinal, irritation, and skin and connective tissues
outcomes
15

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PEER REVIEW DRAFT- DO NOT CITE OR QUOTE
Study reference:
IRDC, (1978). Acute toxicity studies in rabbits and rats with residue of hexabromocyclododecane with
attachments and cover letter dated 030178
HERO ID: 787686
Domain
Metric
Evaluator's Comment
Qualitative
Determination
[i.e.,High,Medium,
Low,Unacceptable,
or Not rated]
Metric
Score
Metric
Weighting
Factor
Weighted
Score
Test Substance
1. Test Substance
Identity
The test substance was
identified as residue of
HBCD (FM 100 residue).
EPA requested additional
information for the
TSCA 8e submitter
(Velsicol Chemical
Corp.) as follows:
"0088-Please provide
information concerning
the composition and
physical/chemical
properties of the "FM
100 Residue" which was
tested. Of particular
interest in this regard is
the amount of
hexabromocyclododecan
e present in the residue.
Available toxicity data
on
hexabromocyclododecan
e would be useful for
correlation purposes."
This information is not
contained in the pdf. The
test substance identity
and form cannot be
determined from the
information provided
Unacceptable
4
2
8

2. Test Substance
Source
The manufacturer was
reported without batch or
lot no.
Medium
2
1
2

3. Test Substance
Purity
Purity was not reported
but is expected to be low
because the 2 samples of
the residue had different
physical descriptions.
Low
3
1
3
Test Design
4. Negative and
Vehicle Controls
No vehicle was used for
irritation studies.
Negative controls are not
used for acute
toxicity/lethality studies.
Not Rated
NR
2
NR

5. Positive Controls
Positive controls are not
required for irritation or
acute toxicity/lethality
studies.
Not Rated
NR
1
NR
16

6. Randomized
Allocation
The study did not report
how animals were
allocated to study groups.
Low
3
1
3

7. Preparation and
Storage of Test
Substance
Information on
preparation and storage
was not reported.
Unacceptable
4
1
4

8. Consistency of
Exposure
Administration
Details of exposure
administration were
reported.
High
1
1
1
Exposure
Characterization
9. Reporting of
Doses/Concentration
s
Doses were reported
mg/kg in oral acute
toxicity studies in
rabbits. But the
concentration of the test
chemical dose (mg)
exposed to rabbits for
eye or skin irritation
study was not specified.
Only volume (mL) was
provided.
Low
3
2
6

10. Exposure
Frequency and
Duration
Adequate follow up time
for examinations for all
experiments.
High
1
1
1

11. Number of
Exposure Groups
and Dose Spacing
5 dose groups dermal
acute; 6 dose groups oral
acute.
High
1
1
1

12. Exposure Route
and Method
The route and method of
exposure were reported
and were suited to the
test substance.
High
1
1
1

13. Test Animal
Characteristics
Species, strain and
starting body weight
were provided
(commercial source, rats
and rabbits).
High
1
2
2
Test Organism
14. Adequacy and
Consistency of
Animal Husbandry
Conditions
Temperature and
humidity controls.
Compliance with animal
care guidance was
indicated.
Medium
2
1
2

15. Number per
Group
4-5/sex for oral acute;
2/sex/group for dermal
acute; adequate numbers
for irritation.
Medium
2
1
2
17

16. Outcome
Assessment
Methodology
EPA requested further
information from the
TSCA 8e submitter
(Velisicol Chemical
Corp.) as follows:
"Please describe any
gross pathological
findings or clinical
observation made on the
test animals."
Medium
2
2
4

17. Consistency of
Outcome
Assessment
Details of the outcome
assessment protocol were
reported.
High
1
1
1
Outcome
Assessment
18. Sampling
Adequacy
Details regarding
sampling for the
outcome(s) of interest
were reported and the
study used adequate
sampling for the
outcome(s) of interest.
High
1
1
1

19. Blinding of
Assessors
Information in the study
report did not report
whether assessors were
blinded to treatment
group for objective
outcomes
Low
3
1
3

20. Negative Control
Response
No negative controls
Not Rated
NR
NR
NR
Confounding /
Variable Control
21. Confounding
Variables in Test
Design and
Procedures
There were no reported
differences among the
study groups in initial
body weight that could
influence the outcome
assessment., Information
on food or water intake,
or respiratory rate was
not reported.
High
1
2
2

22. Health Outcomes
Unrelated to
Exposure
Data on attrition and/or
health outcomes
unrelated to exposure
were not reported for
each study group.
Low
3
1
3
Data Presentation
and Analysis
23. Statistical
Methods
Provided references for
statistical methods.
High
1
1
1
18

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PEER REVIEW DRAFT- DO NOT CITE OR QUOTE
Study reference:
IRDC, (1978). Acute toxicity studies in rabbits and rats with residue of hexabromocyclododecane with
attachments and cover letter dated 030178
HERO ID: 787686
Domain
Metric
Evaluator's Comment
Qualitative
Determination
[i.e., High, Medium,
Low,Unacceptable,
or Not rated]
Metric
Score
Metric
Weighting
Factor
Weighted
Score

24. Reporting of
Data
Data for exposure-related
findings were presented
for all outcomes by
exposure group and sex.
High
1
2
2
High: >=1 and <1.7
Medium: >=1.7 and <2.3
Low: >=2.3 and <=3
Sum of scores:

30
53
Overall Score = Sum of Weighted Scores/Sum
of Metric Weighting Factors:
2.208
Overall Score
(Rounded):
2.21
Overall Quality Level:
Unacceptable1
Comment:
Footnote:
1 Consistent with our Application of Systematic Review in TSCA Risk Evaluations document, if a metric for a
data source receives a score of Unacceptable (score = 4), EPA will determine the study to be unacceptable. In
this case, one of the metrics was rated as unacceptable. As such, the study is considered unacceptable and the
score is presented solely to increase transparency.
19

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PEER REVIEW DRAFT- DO NOT CITE OR QUOTE
1.5 Animal toxicity evaluation results of Song et al 2016 for acute
and 14-day inhalation-systemic toxicity study on body weight,
hematological and immune, clinical chemistry/biochemical,
hepatic, renal, respiratory, reproductive outcomes
Study reference:
Song, N.,Li, L.,Li, H.,Ai, W.,Xic, W.,Yu, W.,Liu, W.,Wang, C.,Shen, G.,Zhou, L.,Wei, C.,Li, D.,Chen, H.
(2016). Single and 14-day repeated dose inhalation toxicity studies of hexabromocyclododecane in rats Food
and Chemical Toxicology, 91, 73-81
HERO ID: 3350482
Domain
Metric
Evaluator's Comment
Qualitative
Determination
[i.e.,High,Medium
,Low,Unacceptable, or
Not rated]
Metric
Score
Metric
Weighting
Factor
Weighted
Score
Test Substance
1. Test Substance
Identity
Test substance was
clearly identified by
name and CASRN.
High
1
2
2
2. Test Substance
Source
The test substance
source/manufacturer was
identified however the
batch/lot number was not
reported
Medium
2
1
2
3. Test Substance
Purity
The test substance purity
was identified
High
1
1
1
Test Design
4. Negative and
Vehicle Controls
Negative control animals
were included in the 14
day. No negative control
required for acute study.
High
1
2
2
5. Positive Controls
Positive controls not
applicable.
Not Rated
NR
NR
NR
6. Randomized
Allocation
Animals were randomly
allocated to each group.
High
1
1
1
Exposure
Characterization
7. Preparation and
Storage of Test
Substance
The method and
equipment used to
generate the dust aerosol
were reported and
appropriate.
High
1
1
1
8. Consistency of
Exposure
Administration
Exposures were
administered
consistently.
High
1
1
1
9. Reporting of
Doses/Concentration
s
Target and measured
concentrations, MMAD,
and GSD were reported
for all groups.
High
1
2
2
10. Exposure
Frequency and
Duration
Frequency and duration
were reported.
High
1
1
1
20

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PEER REVIEW DRAFT- DO NOT CITE OR QUOTE
Study reference:
Song, N.,Li, L.,Li, H.,Ai, W.,Xic, W.,Yu, W.,Liu, W.,Wang, C.,Shen, G.,Zhou, L.,Wei, C.,Li, D.,Chen, H.
(2016). Single and 14-day repeated dose inhalation toxicity studies of hexabromocyclododecane in rats Food
and Chemical Toxicology, 91, 73-81
HERO ID: 3350482
Domain
Metric
Evaluator's Comment
Qualitative
Determination
[i.e.,High,Medium
,Low,Unacceptable, or
Not rated]
Metric
Score
Metric
Weighting
Factor
Weighted
Score

11. Number of
Exposure Groups
and Dose Spacing
The number of groups
and spacing were
reported along with
rationale for
concentration selection.
High
1
1
1

12. Exposure Route
and Method
The route and method
were appropriate.
High
1
1
1

13. Test Animal
Characteristics
The source, health status,
species, strain, age, and
sex were reported. Initial
body weight was not
reported.
Medium
2
2
4
Test Organism
14. Adequacy and
Consistency of
Animal Husbandry
Conditions
All husbandry conditions
were reported and
appropriate.
High
1
1
1

15. Number per
Group
The number of animals
per study group was
appropriate.
High
1
1
1

16. Outcome
Assessment
Methodology
Outcome assessment
methodology was
reported and appropriate.
High
1
2
2
Outcome
Assessment
17. Consistency of
Outcome
Assessment
Outcomes were assessed
consistently.
High
1
1
1
18. Sampling
Adequacy
Sampling size was
adequate.
High
1
1
1

19. Blinding of
Assessors
Blinding not required.
Not Rated
NR
NR
NR

20. Negative Control
Response
Negative control
responses were
appropriate.
High
1
1
1
Confounding /
Variable Control
21. Confounding
Variables in Test
Design and
Procedures
No confounding
variables in test design
were observed.
High
1
2
2
22. Health Outcomes
Unrelated to
Exposure
No health outcomes
unrelated to exposure
were reported.
High
1
1
1
Data Presentation
23. Statistical
Methods
Statistical methods were
reported and appropriate.
High
1
1
1
and Analysis
24. Reporting of
Data
Data were reported.
High
1
2
2
21

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PEER REVIEW DRAFT- DO NOT CITE OR QUOTE
Study reference:
Song, N.,Li, L.,Li, H.,Ai, W.,Xie, W.,Yu, W.,Liu, W.,Wang, C.,Shen, G.,Zhou, L.,Wei, C.,Li, D.,Chen, H.
(2016). Single and 14-day repeated dose inhalation toxicity studies of hexabromocyclododecane in rats Food
and Chemical Toxicology, 91, 73-81
HERO ID: 3350482
Domain
Metric
Evaluator's Comment
Qualitative
Determination
[i.e., High, Medium
,Low,Unacceptable, or
Not rated]
Metric
Score
Metric
Weighting
Factor
Weighted
Score
High: >=1 and <1.7
Medium: >=1.7 and <2.3
Low: >=2.3 and <=3
Sum of scores:

29
32
Overall Score = Sum of Weighted Scores/Sum of
Metric Weighting Factors:
1.1034
Overall Score:
Nearest *:
1.1
Overall Quality Level:
High
22

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PEER REVIEW DRAFT- DO NOT CITE OR QUOTE
1.6 Animal toxicity evaluation results of Szabo et al 2016 for single
gavage in mice on post-natal day 10; metabolomics evaluation
only study on gene expression/omics outcomes
Study reference:
Szabo, D. T.,Pathmasiri, W.,Sumner, S.,Birnbaum, L. S. (2016). Serum Metabolomic Profiles in Neonatal
Mice following Oral Brominated Flame Retardant Exposures to Hexabromocyclododecane (HBCD) Alpha,
Gamma, and Commercial Mixture. Environmental Health Perspectives, 125(4), 651-659
HERO ID: 3546063
Domain
Metric
Evaluator's Comment
Qualitative
Determination
[i.e.,High,Medium,L
ow,Unacceptable, or
Not rated]
Metric
Score
Metric
Weighting
Factor
Weighted
Score
Test Substance
1. Test Substance
Identity
Chemical identity is
clear; CAS #. provided
Test substance is a
commercial mixture of
three stereoisomers.
Percentages of each
isomer are provided.
High
1
2
2
2. Test Substance
Source
Sourced from Sigma-
Aldrich
High
1
1
1
3. Test Substance
Purity
Percentages of isomers in
commercial mixture were
provided.; it is not
indicated whether other
impurities are present,
but the study authors
indicate that chemicals
were purchased at the
highest purity level
available. The authors
did, however, go through
a stereoisomer separation
and thermal conversion
process and it is not clear
how pure the samples
were after this process.
Additionally, dosing
solutions were made
using corn oil and
toluene that was
evaporated under
vacuum. Whether there
was any remaining
toluene is unknown,
although all samples,
including controls were
treated equally.
Medium
2
1
2
Test Design
4. Negative and
Vehicle Controls
Appropriate negative
(vehicle) control was
used.
High
1
2
2
5. Positive Controls
Positive control not
required.
Not Rated
NR
NR
NR
23

6. Randomized
Allocation
Study does not indicate
how dams and
corresponding pups were
allocated into treatment
groups. Given the small
number of total
dams/litters (n = 7), and
the fact that no
statements are made
indicating, for example,
that dams and pup
weights were equivalent,
this introduces
uncertainty that could
impact results.
Low
3
1
3

7. Preparation and
Storage of Test
Substance
Study references
previous publications for
methods used for
stereoisomer separation.
Preparation of dosing
solutions were
appropriate. Since
animals only received a
single dose, storage of
the dosing solutions was
not necessary.
High
1
1
1
Exposure
8. Consistency of
Exposure
Administration
Dosing was equivalent
across treatment groups
(all animals given
lOmg/kg gavage of
appropriate treatment)
High
1
1
1
Characterization
9. Reporting of
Doses/Concentration
s
Doses were clearly stated
High
1
2
2

10. Exposure
Frequency and
Duration
Single exposure via
gavage
High
1
1
1

11. Number of
Exposure Groups
and Dose Spacing
An explanation of chosen
doses was provided
High
1
1
1

12. Exposure Route
and Method
Gavage was appropriate
for pups that were still
lactating, unclear
whether lOml/kg is
appropriate though for
pups that are PND10?
Medium
2
1
2
24

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PEER REVIEW DRAFT- DO NOT CITE OR QUOTE
Study reference:
Szabo, D. T.,Pathmasiri, W.,Sumner, S.,Birnbaum, L. S. (2016). Serum Metabolomic Profiles in Neonatal
Mice following Oral Brominated Flame Retardant Exposures to Hexabromocyclododecane (HBCD) Alpha,
Gamma, and Commercial Mixture. Environmental Health Perspectives, 125(4), 651-659

HERO ID: 3546063




Domain
Metric
Evaluator's Comment
Qualitative
Determination
[i.e.,High,Medium,L
ow,Unacceptable, or
Not rated]
Metric
Score
Metric
Weighting
Factor
Weighted
Score

13. Test Animal
Characteristics
Study clearly explains
reasoning for choosing
mice at this stage of
development
High
1
2
2

14. Adequacy and
Consistency of
Animal Husbandry
Conditions
Animal husbandry
conditions were
appropriate
High
1
1
1
Test Organism
15. Number per
Group
Study indicates that 6
female pups per litter (n
= 7 litters total) were
used for the experiment.
Including the control,
there is a total of 7 dose
groups (control, 3-doses
of alpha-HBCD, 2-doses
of gamma HBCD, and a
single dose of the
commercial mixture). It
is unclear how this would
work, unless one litter
was used exclusively as a
control, and then 1 pup
per litter (out of 6
remaining litters)
received each treatment.?
Overall, the total number
of pups per treatment
group is not explicitly
stated and cannot be
accurately inferred given
the available data.
Low
3
1
3
25

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PEER REVIEW DRAFT- DO NOT CITE OR QUOTE
Study reference:
Szabo, D. T.,Pathmasiri, W.,Sumner, S.,Birnbaum, L. S. (2016). Serum Metabolomic Profiles in Neonatal
Mice following Oral Brominated Flame Retardant Exposures to Hexabromocyclododecane (HBCD) Alpha,
Gamma, and Commercial Mixture. Environmental Health Perspectives, 125(4), 651-659

HERO ID: 3546063




Domain
Metric
Evaluator's Comment
Qualitative
Determination
[i.e.,High,Medium,L
ow,Unacceptable, or
Not rated]
Metric
Score
Metric
Weighting
Factor
Weighted
Score
Outcome
Assessment
16. Outcome
Assessment
Methodology
Metabolomic assessment
of the blood was done
via NMR at a single
time-point (4-days post-
exposure), which
generally could miss key
transitional changes.
However, the study
authors indicate that this
time point was chosen to
coincide with previous
data collected from
various tissues, and
therefore seems
appropriate. -NMRhas
relatively low sensitivity
compared with other
analytical tools for
metabolomics, and no
power analysis was done
to determine an
appropriate sample size.
It is not clear whether
technical replicates were
included in the
methodology.
Medium
2
2
4

17. Consistency of
Outcome
Assessment
Outcome assessment
appeared to be consistent
across groups
High
1
1
1
26

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PEER REVIEW DRAFT- DO NOT CITE OR QUOTE
Study reference:
Szabo, D. T.,Pathmasiri, W.,Sumner, S.,Birnbaum, L. S. (2016). Serum Metabolomic Profiles in Neonatal
Mice following Oral Brominated Flame Retardant Exposures to Hexabromocyclododecane (HBCD) Alpha,
Gamma, and Commercial Mixture. Environmental Health Perspectives, 125(4), 651-659
HERO ID: 3546063
Domain
Metric
Evaluator's Comment
Qualitative
Determination
[i.e.,High,Medium,L
ow,Unacceptable, or
Not rated]
Metric
Score
Metric
Weighting
Factor
Weighted
Score

18. Sampling
Adequacy
Analysis was done on
samples taken from 3 -6
pups/ treatment group.
The number of control
samples were not stated.
It is unclear whether the
differences in sample
numbers across treatment
groups was because
those were the total
number of animals
treated, or whether for
some reason, in some
cases, samples were only
collected from three out
of 6 treated animals.
Three biological
replicates for an omics-
based study is an
absolute minimum and
greatly reduces statistical
power and has increased
noise.
Low
3
1
3
19. Blinding of
Assessors
Blinding was not
indicated, but not
necessarily applicable to
NMR analysis
Not Rated
NR
NR
NR
20. Negative Control
Response
The responses of the
controls are presumed to
be appropriate
High
1
1
1
27

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PEER REVIEW DRAFT- DO NOT CITE OR QUOTE
Study reference:
Szabo, D. T.,Pathmasiri, W.,Sumner, S.,Birnbaum, L. S. (2016). Serum Metabolomic Profiles in Neonatal
Mice following Oral Brominated Flame Retardant Exposures to Hexabromocyclododecane (HBCD) Alpha,
Gamma, and Commercial Mixture. Environmental Health Perspectives, 125(4), 651-659
HERO ID: 3546063
Domain
Metric
Evaluator's Comment
Qualitative
Determination
[i.e.,High,Medium,L
ow,Unacceptable, or
Not rated]
Metric
Score
Metric
Weighting
Factor
Weighted
Score
Confounding /
Variable Control
21. Confounding
Variables in Test
Design and
Procedures
The study authors do not
discuss potential
confounding variables. It
is mentioned that there
were no changes in body
weights between treated
and controls following
treatment, but no
statements were made
indicating that the initial
health and weights of
treated pups were
equivalent across litters
leaving the potential for
unknown confounding
variables. There is also a
potential for litter effects,
however, this was
presumably taken into
account in the study
design by treating across
litters.
Low
3
2
6

22. Health Outcomes
Unrelated to
Exposure
The study does not
include observations
(clinical or otherwise) of
pups during or after
dosing. It is still unclear
why some treatment
groups had three samples
evaluated, and others had
6 samples evaluated, and
whether this could
potentially be due to
problems with some of
the animals, or if only
three animals were
treated.
Low
3
1
3

23. Statistical
Methods
Statistical analysis was
appropriate.
High
1
1
1
Data Presentation
and Analysis
24. Reporting of
Data
Data presentation was
adequate and appropriate
for omics reporting. -
Some data was presented
in supplementary tables
that were not available to
view
High
1
2
2

Sum of scores:

29
45
28

-------
PEER REVIEW DRAFT- DO NOT CITE OR QUOTE
Study reference:
Szabo, D. T.,Pathmasiri, W.,Sumner, S.,Birnbaum, L. S. (2016). Serum Metabolomic Profiles in Neonatal
Mice following Oral Brominated Flame Retardant Exposures to Hexabromocyclododecane (HBCD) Alpha,
Gamma, and Commercial Mixture. Environmental Health Perspectives, 125(4), 651-659
HERO ID: 3546063
Domain
Metric
Evaluator's Comment
Qualitative
Determination
[i.e., High, Medium, L
ow,Unacceptable, or
Not rated]
Metric
Score
Metric
Weighting
Factor
Weighted
Score
High: >=1 and <1.7
Medium: >=1.7 and <2.3
Low: >=2.3 and <=3
Overall Score = Sum of Weighted Scores/Sum
of Metric Weighting Factors:
NR
Overall Score:
Nearest *:
NR
Overall Quality Level:
Medium
Study Quality
Comment:
The reviewer downgraded this study's overall quality rating. They noted: Problems with methods reporting
(specifically the number of animals exposed/treatment group), as well as data indicating animals were of
equivalent health and body weight at study initiation decrease confidence in the study results. Note: The
original calculated score for this study was 1.5. This value is not presented above because the final rating was
changed based on professional judgement.
29

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2
PEER REVIEW DRAFT- DO NOT CITE OR QUOTE
Short-term Toxicity Studies
2.1 Animal toxicity evaluation results of Bernhard et al 2016 for
28-day oral exposure in mice via diet study on hepatic, body
weight outcomes
Study reference:
Bernhard, A.,Berntssen, M. H. G.,Lundebye, A. K.,Ra, Yneberg Alvheim, A.,Secher Myrmel, L.,Fja, Re,
E.,Torstensen, B. E.,Kristiansen, K.,Madsen, L.,Brattelid, T.,Rasinger, J. D. (2016). Marine fatty acids
aggravate hepatotoxicity of HBCD in juvenile female BALB/c mice, 97, 411-423
HERO ID: 3588138
Domain
Metric
Evaluator's Comment
Qualitative
Determination
[i.e., High, Medium,
Low,Unacceptable,
or Not rated]
Metric
Score
Metric
Weighting
Factor
Weighted
Score

1. Test Substance
Identity
Identity and form are
stated, no CAS#
reported.
High
1
2
2

2. Test Substance
Source
alpha-HBCD was
synthesized from from
gamma-HBCD.
Analytical verification of
the product was not
done, however,
concentrations in feed
were analyzed by GC-
MS.
Medium
2
1
2
Test Substance

After production, purity
of the alpha isomer was
described as "pure".

3. Test Substance
Purity
alpha-HBCD was
produced in the
laboratory. Study report
states that "purified
alpha-HBCD" was used
to dose animals but %
purity or details on the
purification methods
were not provided.
Low
3
1
3

4. Negative and
Vehicle Controls
Study used an
appropriate vehicle
negative control diet.
High
1
2
2
Test Design
5. Positive Controls
Positive control not
necessary
Not Rated
NR
NR
NR
6. Randomized
Allocation
It was stated that animals
were randomly assigned,
although the method for
assignment was not
described.
Medium
2
1
2
30

7. Preparation and
Storage of Test
Substance
The frequency of diet
preparation and a
statement about stability
were not provided.
Preparation of diets was
acceptable.
Medium
2
1
2

8. Consistency of
Exposure
Administration
administration was
consistent across groups.
High
1
1
1
Exposure
Characterization
9. Reporting of
Doses/Concentration
s
Both nominal and
measured concentrations
in the diet were provided
with corresponding daily
exposures. However,
these values were
calculated using
estimated (rather than
actual) daily food intake.
It can not be determined
whether there was a
difference in the intake
across treatment groups.
Low
3
2
6
10. Exposure
Frequency and
Duration
Appropriate; study
design was based on
OECD guideline 407 for
short-term repeated dose
toxicity study
High
1
1
1

11. Number of
Exposure Groups
and Dose Spacing
Number of exposure
groups was appropriate.
Authors state that "The
high dose (HD) chosen
was high enough to elicit
molecular aberrations
and the low dose (LD)
was based on the
potentially relevant
Lowest Observed
Adverse Effect Level
(LOAEL) (Table 1;
Yanagisawa et al.,
2014)."
High
1
1
1

12. Exposure Route
and Method
Exposure route
acceptable
High
1
1
1
31

13. Test Animal
Characteristics
Standard animal model
was used. Age was
appropriate for desired
"juvenile" developmental
time point. Only one sex
evaluated. Animals were
obtained from Taconic.
High
1
2
2
Test Organism
14. Adequacy and
Consistency of
Animal Husbandry
Conditions
Animal husbandry
clearly reported and
appropriate.
High
1
1
1

15. Number per
Group
n = 3-8 / group,
depending on the
outcome evaluated.
Sample size is below the
recommended minimum
(n = 10) for OECD 407.
Medium
2
1
2

16. Outcome
Assessment
Methodology
Methodology of outcome
assessments were clearly
described and
appropriate
High
1
2
2

17. Consistency of
Outcome
Assessment
Consistent assessment
across groups.
High
1
1
1
Outcome
Assessment
18. Sampling
Adequacy
Sampling was adequate.
Histology was performed
on a subset of animals
(n=3-4) from each
exposure group,
including controls
High
1
1
1
19. Blinding of
Assessors
Histopathology
evaluations were
subjective. Study report
does not indicate that the
assessor was blinded
during assessment or
whether outcomes were
evaluated independently
by a second pathologist.
Medium
2
1
2

20. Negative Control
Response
No out of the ordinary
control responses were
noted.
High
1
1
1
32

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PEER REVIEW DRAFT- DO NOT CITE OR QUOTE
Study reference:
Bernhard, A.,Berntssen, M. H. G.,Lundebye, A. K.,Ra, Yneberg Alvheim, A.,Secher Myrmel, L.,Fja, Re,
E.,Torstensen, B. E.,Kristiansen, K.,Madsen, L.,Brattelid, T.,Rasinger, J. D. (2016). Marine fatty acids
aggravate hepatotoxicity of HBCD in juvenile female BALB/c mice, 97, 411-423
HERO ID: 3588138
Domain
Metric
Evaluator's Comment
Qualitative
Determination
[i.e., High, Medium,
Low,Unacceptable,
or Not rated]
Metric
Score
Metric
Weighting
Factor
Weighted
Score
Confounding /
Variable Control
21. Confounding
Variables in Test
Design and
Procedures
Initial body weights of
animals were not
reported. It is unclear
whether there were
differences in feed
consumption because a
default value (15% w/w)
was used rather than the
actual dietary intake
Low
3
2
6
22. Health Outcomes
Unrelated to
Exposure
No health outcomes
unrelated to exposure
were reported; animals
were observed daily.
High
1
1
1
Data Presentation
and Analysis
23. Statistical
Methods
Statistical analysis
methodology were
clearly reported and
appropriate.
High
1
1
1
24. Reporting of
Data
Reporting of data was
appropriate for most
outcomes. Confidence
level for histopathology
results is reduced to
Medium because results
are only presented
qualitatively
(representative histology
images from each group
were shown and text
description of the
effects).
High
1
2
2
High: >=1 and <1.7
Medium: >=1.7 and <2.3
Low: >=2.3 and <=3
Sum of scores:

30
45
Overall Score = Sum of Weighted Scores/Sum
of Metric Weighting Factors:
NR
Overall Score:
Nearest *:
NR
Overall Quality Level:
Medium
Study Quality
Comment:
The reviewer downgraded this study's overall quality rating. They noted: I would downgrade this study based
on concerns related to the purity of the chemical and reporting of the doses/concentrations. Note: The original
calculated score for this study was 1.5. This value is not presented above because the final rating was changed
based on professional judgement.
33

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PEER REVIEW DRAFT- DO NOT CITE OR QUOTE
2.2 Animal toxicity evaluation results of Genskow et al 2015 for 30
day oral toxicity study (daily gavage); primarily mechanistic,
also contains in vitro data study on neurological/behavior
outcomes
Study reference:
Genskow, K. R,Bradner, J. M.,Hossain, M. M.,Richardson, J. R,Caudle, W. M. (2015). Selective damage to
dopaminergic transporters following exposure to the brominated flame retardant, HBCDD Neurotoxicology
and Teratology, 52(Pt B), 162-169
HERO ID: 2919804
Domain
Metric
Evaluator's Comment
Qualitative
Determination
[i.e.,High,Medium,
Low,Unacceptable,
or Not rated]
Metric
Score
Metric
Weighting
Factor
Weighted
Score

1. Test Substance
Identity
Test substance name was
provided but CAS# was
not provided
Medium
2
2
4
Test Substance
2. Test Substance
Source
Test substance source
was provided but batch
or lot number was not
reported
Medium
2
1
2

3. Test Substance
Purity
Purity of the test
substance is not reported
Low
3
1
3

4. Negative and
Vehicle Controls
Vehicle control reported
High
1
2
2
Test Design
5. Positive Controls
A positive control was
not necessary, but could
have provided useful
information in this study
that would aid in the
interpretation of the
results
Not Rated
NR
NR
NR

6. Randomized
Allocation
The study does not
indicate whether animals
were randomized, the
endpoints evaluated were
more mechanistic in
nature, and may not have
been impacted greatly by
randomization.
Medium
2
1
2

7. Preparation and
Storage of Test
Substance
Details of preparation,
frequency of preparation,
and storage were lacking
Low
3
1
3

8. Consistency of
Exposure
Administration
Control and treatment
groups were treated
consistently
High
1
1
1
Exposure
Characterization
9. Reporting of
Doses/Concentration
s
Dose concentrations
were clearly reported,
however, no validation of
dose was performed by
the study authors.
Medium
2
2
4

10. Exposure
Frequency and
Duration
Exposure frequency and
duration were clearly
reported
High
1
1
1
34

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PEER REVIEW DRAFT- DO NOT CITE OR QUOTE
Study reference:
Genskow, K. R,Bradner, J. M.,Hossain, M. M.,Richardson, J. R,Caudle, W. M. (2015). Selective damage to
dopaminergic transporters following exposure to the brominated flame retardant, HBCDD Neurotoxicology
and Teratology, 52(Pt B), 162-169
HERO ID: 2919804
Domain
Metric
Evaluator's Comment
Qualitative
Determination
[i.e.,High,Medium,
Low,Unacceptable,
or Not rated]
Metric
Score
Metric
Weighting
Factor
Weighted
Score

11. Number of
Exposure Groups
and Dose Spacing
Single dose exposure that
did not induce effects for
several endpoints
measured. It is unclear
whether HB CD indeed
has no effect, or whether
a dose-limit was not
reached
NK: Single dose
exposure, daily for 30
days. Control had 4 mice
and treatment group had
6 mice.
Medium
2
1
2

12. Exposure Route
and Method
Exposure route and
method were acceptable.
High
1
1
1

13. Test Animal
Characteristics
Animals (C57BL/6 male
mice) were purchased at
8weeks old and the mice
were treated when they
were 3 months old (4
weeks later). Animals are
generally acclimatized
for a week; 4 weeks
seems a bit odd.
Medium
2
2
4
Test Organism
14. Adequacy and
Consistency of
Animal Husbandry
Conditions
Animal husbandry details
were not provided, but
the study authors state
that procedures were
conducted in accordance
with the guide for care
and use of laboratory
animals
Medium
2
1
2

15. Number per
Group
Four control animals and
6 treated animals of a
single sex were used.
OECD guidelines for 28-
day toxicity studies
recommends an n of 10
(5 animals of each sex).
Medium
2
1
2
Outcome
Assessment
16. Outcome
Assessment
Methodology
The outcome assessment
methodology addressed
or reported the intended
outcome(s) of interest
and was sensitive for the
outcome(s) of interest.
High
1
2
2
35

-------
PEER REVIEW DRAFT- DO NOT CITE OR QUOTE
Study reference:
Genskow, K. R,Bradner, J. M.,Hossain, M. M.,Richardson, J. R,Caudle, W. M. (2015). Selective damage to
dopaminergic transporters following exposure to the brominated flame retardant, HBCDD Neurotoxicology
and Teratology, 52(Pt B), 162-169
HERO ID: 2919804
Domain
Metric
Evaluator's Comment
Qualitative
Determination
[i.e., High, Medium,
Low,Unacceptable,
or Not rated]
Metric
Score
Metric
Weighting
Factor
Weighted
Score

17. Consistency of
Outcome
Assessment
Details of the outcome
assessment protocol were
reported, and outcomes
were assessed
consistently across study
groups
High
1
1
1
18. Sampling
Adequacy
The study reported
adequate sampling for
the outcome(s) of interest
High
1
1
1
19. Blinding of
Assessors
Blinding is not required
for this methodology
Not Rated
NR
NR
NR
20. Negative Control
Response
Control responses appear
to be appropriate
High
1
1
1
Confounding /
Variable Control
21. Confounding
Variables in Test
Design and
Procedures
No confounding
variables were noted,
however, data regarding
other potential exposure-
related effects (i.e„
potential effects on body
weight), were not
included in the report.
Medium
2
2
4
22. Health Outcomes
Unrelated to
Exposure
This information was not
included in the study
report or in the study
design.
Medium
2
1
2
Data Presentation
and Analysis
23. Statistical
Methods
Statistical analysis was
acceptable
High
1
1
1
24. Reporting of
Data
Reporting of data (for the
methods used) was
acceptable.
High
1
2
2
High: >=1 and <1.7
Medium: >=1.7 and <2.3
Low: >=2.3 and <=3
Sum of scores:

29
47
Overall Score = Sum of Weighted Scores/Sum
of Metric Weighting Factors:
NR
Overall Score:
Nearest *:
NR
Overall Quality Level:
Medium
Study Quality
Comment:
The reviewer downgraded this study's overall quality rating. They noted: Downgraded the study from 'high' to
'medium' because this is primarily a mechanistic study. The small part of the study that is animal toxicity
study with just one dose and has fewer animals (n=4 for control) and n=6 for treatment group). Note: The
original calculated score for this study was 1.6. This value is not presented above because the final rating was
changed based on professional judgement.
36

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PEER REVIEW DRAFT- DO NOT CITE OR QUOTE
2,3 Animal toxicity evaluation results of Hachisuka et al 2010 for
oral developmental immunotoxicity study on hematological and
immune outcomes
Study reference:
Hachisuka, A.,Nakamura, R,Sato, Y.,Nakamura, R,Shibutani, M.,Teshima, R (2010). [Effects of perinatal
exposure to the brominated flame-retardant hexabromocyclododecane (HBCD) on the developing immune
system in rats] Kokuritsu Iyakuhin Shokuhin Eisei Kenkyusho Hokoku, [2010](128), 58-64
HERO ID: 1403765
Domain
Metric
Evaluator's Comment
Qualitative
Determination
[i.e.,High,Medium,
Low,Unacceptable,
or Not rated]
Metric
Score
Metric
Weighting
Factor
Weighted
Score

1. Test Substance
Identity
Test substance identified
by name.
Medium
2
2
4
Test Substance
2. Test Substance
Source
Source not identified.
Low
3
1
3

3. Test Substance
Purity
Composition and purity
not reported.
Low
3
1
3

4. Negative and
Vehicle Controls
Concurrent negative
control animals are
included.
High
1
2
2
Test Design
5. Positive Controls
Positive controls not
required.
Not Rated
NR
NR
NR

6. Randomized
Allocation
Allocation methods were
not reported.
Low
3
1
3

7. Preparation and
Storage of Test
Substance
Limited details on
preparation (mixed into
the food) and no
information on storage
and stability were
reported.
Low
3
1
3

8. Consistency of
Exposure
Administration
Animals were allowed to
feed freely on the diet,
but no details on the
amount of diet provided
was reported.
Medium
2
1
2
Exposure
Characterization
9. Reporting of
Doses/Concentration
s
Concentrations were
reported.
High
1
2
2

10. Exposure
Frequency and
Duration
Exposure duration was
reported.
High
1
1
1

11. Number of
Exposure Groups
and Dose Spacing
The number of exposure
groups and spacing were
reported, but not
justified.
Medium
2
1
2

12. Exposure Route
and Method
The exposure route and
method were appropriate.
High
1
1
1
37

-------
PEER REVIEW DRAFT- DO NOT CITE OR QUOTE
Study reference:
Hachisuka, A.,Nakamura, R.,Sato, Y.,Nakamura, R.,Shibutani, M.,Teshima, R. (2010). [Effects of perinatal
exposure to the brominated flame-retardant hexabromocyclododecane (HBCD) on the developing immune
system in rats] Kokuritsu Iyakuhin Shokuhin Eisei Kenkyusho Hokoku, [2010](128), 58-64
HERO ID: 1403765
Domain
Metric
Evaluator's Comment
Qualitative
Determination
[i.e.,High,Medium,
Low,Unacceptable,
or Not rated]
Metric
Score
Metric
Weighting
Factor
Weighted
Score
Test Organism
13. Test Animal
Characteristics
The species, strain, and
sex were reported. The
source and starting body
weight of dams were not
reported.
Low
3
2
6
14. Adequacy and
Consistency of
Animal Husbandry
Conditions
Details were not
reported.
Low
3
1
3
15. Number per
Group
The number of animals
per group was
appropriate.
High
1
1
1
Outcome
Assessment
16. Outcome
Assessment
Methodology
Outcome assessment
methodology was
reported for some
outcomes- hematology,
thymus and spleen
weight and pathology,
and immunity. Other
outcomes assessment
methodology, including
body weight and weight
gain, were not reported.
Medium
2
2
4
17. Consistency of
Outcome
Assessment
Outcomes were assessed
consistently.
High
1
1
1
18. Sampling
Adequacy
Sampling for some
outcomes was not
reported or illegible.
Medium
2
1
2
19. Blinding of
Assessors
Blinding not required.
Not Rated
NR
NR
NR
20. Negative Control
Response
Negative control
responses were
appropriate.
High
1
1
1
Confounding /
Variable Control
21. Confounding
Variables in Test
Design and
Procedures
Initial body weight and
food/water intake of
same were not reported
and appear not to have
been measured.
Low
3
2
6
22. Health Outcomes
Unrelated to
Exposure
There were not reported
differences among the
groups in health
outcomes unrelated to
exposures.
High
1
1
1
38

-------
PEER REVIEW DRAFT- DO NOT CITE OR QUOTE
Study reference:
Hachisuka, A.,Nakamura, R.,Sato, Y.,Nakamura, R.,Shibutani, M.,Teshima, R. (2010). [Effects of perinatal
exposure to the brominated flame-retardant hexabromocyclododecane (HBCD) on the developing immune
system in rats] Kokuritsu Iyakuhin Shokuhin Eisei Kenkyusho Hokoku, [2010](128), 58-64
HERO ID: 1403765
Domain
Metric
Evaluator's Comment
Qualitative
Determination
[i.e., High, Medium,
Low,Unacceptable,
or Not rated]
Metric
Score
Metric
Weighting
Factor
Weighted
Score
Data Presentation
and Analysis
23. Statistical
Methods
Statistical methods were
not described but were
conducted, and data were
provided to conduct an
independent analysis.
Medium
2
1
2
24. Reporting of
Data
Data were reported by
groups, however it
appears that not all
outcomes were reported
by sex.
Medium
2
2
4
High: >=1 and <1.7
Medium: >=1.7 and <2.3
Low: >=2.3 and <=3
Sum of scores:

29
57
Overall Score = Sum of Weighted Scores/Sum
of Metric Weighting Factors:
1.9655
Overall Score:
Nearest *:
2
Overall Quality Level:
Medium
39

-------
PEER REVIEW DRAFT- DO NOT CITE OR QUOTE
2.4 Animal toxicity evaluation results of Maranghi et al 2013 for
28-day dietary study on hepatic, body weight, thyroid,
hematological and immune, reproductive outcomes
Study reference:
Maranghi, F.,Tassinari, R.,Moracci, G.,Altieri, I.,Rasinger, J. D.,Carroll, T. S.,Hogstrand, C.,Lundebye, A.
K.,Mantovani, A. (2013). Dietary exposure of juvenile female mice to polyhalogenated seafood contaminants
(HBCD, BDE-47, PCB-153, TCDD): comparative assessment of effects in potential target tissues Food and
Chemical Toxicology, 56, 443-449
HERO ID: 1927558
Domain
Metric
Evaluator's Comment
Qualitative
Determination
[i.e., High, Medium,
Low,Unacceptable,
or Not rated]
Metric
Score
Metric
Weighting
Factor
Weighted
Score
Test Substance
1. Test Substance
Identity
Chemical name
provided, no CAS #, and
no structure provided.
Medium
2
2
4
2. Test Substance
Source
The source was no
reported, no verification
or analytical assessment
Low
3
1
3
3. Test Substance
Purity
Substance purity was not
provided
Low
3
1
3
Test Design
4. Negative and
Vehicle Controls
An appropriate negative
control was used
High
1
2
2
5. Positive Controls
Positive control was not
required
Not Rated
NR
NR
NR
6. Randomized
Allocation
Mice were allocated at
random; method used
was not detailed
High
1
1
1
Exposure
Characterization
7. Preparation and
Storage of Test
Substance
Preparation of exposure
diets were described,
however the frequency of
preparation and details of
storage were not
indicated.
Medium
2
1
2
8. Consistency of
Exposure
Administration
Exposure was consistent
across groups. - Animals
were restricted to 15%
w/w food intake.
High
1
1
1
40

-------
PEER REVIEW DRAFT- DO NOT CITE OR QUOTE
Study reference:
Maranghi, F.,Tassinari, R.,Moracci, G.,Altieri, I.,Rasinger, J. D.,Carroll, T. S.,Hogstrand, C.,Lundebye, A.
K.,Mantovani, A. (2013). Dietary exposure of juvenile female mice to polyhalogenated seafood contaminants
(HBCD, BDE-47, PCB-153, TCDD): comparative assessment of effects in potential target tissues Food and
Chemical Toxicology, 56, 443-449
HERO ID: 1927558
Domain
Metric
Evaluator's Comment
Qualitative
Determination
[i.e.,High,Medium,
Low,Unacceptable,
or Not rated]
Metric
Score
Metric
Weighting
Factor
Weighted
Score

9. Reporting of
Doses/Concentration
s
Do to methodological
limitations, the intended
HBCD concentration in
feed could not be
verified. It was therefore
presumed that the
concentration was
equivalent to the
intended dose. Analysis
of other chemicals
evaluated in the same
study, indicated they
were essentially the same
as the intended inclusion
levels.
Medium
2
2
4

10. Exposure
Frequency and
Duration
Frequency and duration
were clearly reported
High
1
1
1

11. Number of
Exposure Groups
and Dose Spacing
Single dose and a
control. - Justification of
dose was provided.
High
1
1
1

12. Exposure Route
and Method
Exposure route and
method was acceptable
High
1
1
1

13. Test Animal
Characteristics
Appropriate test
organism
High
1
2
2
Test Organism
14. Adequacy and
Consistency of
Animal Husbandry
Conditions
Animal husbandry
acceptable
High
1
1
1

15. Number per
Group
15/control group
10/treatment group
High
1
1
1

16. Outcome
Assessment
Methodology
Methods of outcome
assessment were
appropriate.
High
1
2
2
Outcome
17. Consistency of
Outcome
Assessment
Outcomes were assessed
consistently across
groups
High
1
1
1
Assessment
18. Sampling
Adequacy
Sampling sizes were
adequate
High
1
1
1

19. Blinding of
Assessors
Blinding of assessors
was not reported but is
not required for initial
histology evaluation.
Medium
2
1
2
41

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PEER REVIEW DRAFT- DO NOT CITE OR QUOTE
Study reference:
Maranghi, F.,Tassinari, R.,Moracci, G.,Altieri, I.,Rasinger, J. D.,Carroll, T. S.,Hogstrand, C.,Lundebye, A.
K.,Mantovani, A. (2013). Dietary exposure of juvenile female mice to polyhalogenated seafood contaminants
(HBCD, BDE-47, PCB-153, TCDD): comparative assessment of effects in potential target tissues Food and
Chemical Toxicology, 56, 443-449
HERO ID: 1927558
Domain
Metric
Evaluator's Comment
Qualitative
Determination
[i.e., High, Medium,
Low,Unacceptable,
or Not rated]
Metric
Score
Metric
Weighting
Factor
Weighted
Score

20. Negative Control
Response
No abnormal control
responses were reported
High
1
1
1
Confounding /
Variable Control
21. Confounding
Variables in Test
Design and
Procedures
No confounding
variables were identified.
High
1
2
2
22. Health Outcomes
Unrelated to
Exposure
There were no unrelated
exposure health
outcomes
High
1
1
1
Data Presentation
and Analysis
23. Statistical
Methods
Appropriate statistical
methods were utilized
High
1
1
1
24. Reporting of
Data
Data reporting was
acceptable
High
1
2
2
High: >=1 and <1.7
Medium: >=1.7 and <2.3
Low: >=2.3 and <=3
Sum of scores:

30
40
Overall Score = Sum of Weighted Scores/Sum
of Metric Weighting Factors:
1.3333
Overall Score:
Nearest *:
1.3
Overall Quality Level:
High
42

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PEER REVIEW DRAFT- DO NOT CITE OR QUOTE
2.5 Animal toxicity evaluation results of Miller et al 2016 for
mechanism of liver and thyroid toxicity study on hepatic,
thyroid outcomes
Study reference:
Miller, I.,Serchi, T.,Cambier, S.,Diepenbroek, C.,Renaut, J.,Van der Berg, J. H.,Kwadijk, C.,Gutleb, A.
C.,Rijntjes, E.,Murk, A. J. (2016). Hexabromocyclododecane (HBCD) induced changes in the liver proteome
of eu- and hypothyroid female rats Toxicology Letters, 245, 40-51
HERO ID: 3350495
Domain
Metric
Evaluator's Comment
Qualitative
Determination
[i.e., High, Medium,
Low,Unacceptable,
or Not rated]
Metric
Score
Metric
Weighting
Factor
Weighted
Score
Test Substance
1. Test Substance
Identity
Test substance identified
by name. No CAS # or
other details were
provided
Medium
2
2
4
2. Test Substance
Source
Source or manufacturer
was not identified.
Low
3
1
3

3. Test Substance
Purity
Purity of the substance
was not provided
Low
3
1
3

4. Negative and
Vehicle Controls
Concurrent negative
controls were included.
High
1
2
2
Test Design
5. Positive Controls
Positive controls were
not required.
Not Rated
NR
NR
NR

6. Randomized
Allocation
Allocation methods were
not reported.
Low
3
1
3

7. Preparation and
Storage of Test
Substance
Preparation of the test
substance was reported
but storage prior to
administration was not
reported.
Medium
2
1
2

8. Consistency of
Exposure
Administration
Exposures were
administered
consistently.
High
1
1
1
Exposure
Characterization
9. Reporting of
Doses/Concentration
s
Appropriate doses were
reported
High
1
2
2

10. Exposure
Frequency and
Duration
Frequency and duration
were reported.
High
1
1
1

11. Number of
Exposure Groups
and Dose Spacing
The number of groups
and spacing were
reported
High
1
1
1

12. Exposure Route
and Method
The route and method
were appropriate.
High
1
1
1
43

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PEER REVIEW DRAFT- DO NOT CITE OR QUOTE
Study reference:
Miller, I.,Serchi, T.,Cambier, S.,Diepenbroek, C.,Renaut, J.,Van der Berg, J. H.,Kwadijk, C.,Gutleb, A.
C.,Rijntjes, E.,Murk, A. J. (2016). Hexabromocyclododecane (HBCD) induced changes in the liver proteome
of eu- and hypothyroid female rats Toxicology Letters, 245, 40-51
HERO ID: 3350495
Domain
Metric
Evaluator's Comment
Qualitative
Determination
[i.e., High, Medium,
Low,Unacceptable,
or Not rated]
Metric
Score
Metric
Weighting
Factor
Weighted
Score
Test Organism
13. Test Animal
Characteristics
The source, species,
strain, and age were
reported. Initial body
weight was not reported.
Some animals were
iodine depleted to create
a hypothyroid state
resulting in 2 groups,
normal and hypothyroid.
Medium
2
2
4
14. Adequacy and
Consistency of
Animal Husbandry
Conditions
The temperature,
humidity, lighting, water,
and diet were reported.
No other details were
reported.
Medium
2
1
2

15. Number per
Group
The number of animals
per group was
appropriate.
High
1
1
1

19. Blinding of
Assessors
Blinding was not
required.
Not Rated
NR
NR
NR

20. Negative Control
Response
Negative control
responses were
appropriate.
High
1
1
1
Confounding /
Variable Control
21. Confounding
Variables in Test
Design and
Procedures
Iodine depletion may
have an effect on the
results
Medium
2
2
4
22. Health Outcomes
Unrelated to
Exposure
One group of animals
were exposed in a
hypothyroid state.
Medium
2
1
2
Data Presentation
23. Statistical
Methods
Statistical methods were
reported and appropriate.
High
1
1
1
and Analysis
24. Reporting of
Data
Data were reported.
High
1
2
2

Sum of scores:

29
44
44

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PEER REVIEW DRAFT- DO NOT CITE OR QUOTE
Study reference:
Miller, I.,Serchi, T.,Cambier, S.,Diepenbroek, C.,Renaut, J.,Van der Berg, J. H.,Kwadijk, C.,Gutleb, A.
C.,Rijntjes, E.,Murk, A. J. (2016). Hexabromocyclododecane (HBCD) induced changes in the liver proteome
of eu- and hypothyroid female rats Toxicology Letters, 245, 40-51
HERO ID: 3350495
Domain
Metric
Evaluator's Comment
Qualitative
Determination
[i.e., High, Medium,
Low,Unacceptable,
or Not rated]
Metric
Score
Metric
Weighting
Factor
Weighted
Score
High: >=1 and <1.7
Medium: >=1.7 and <2.3
Low: >=2.3 and <=3
Overall Score = Sum of Weighted Scores/Sum
of Metric Weighting Factors:
NR
Overall Score:
Nearest *:
NR
Overall Quality Level:
Medium
Study Quality
Comment:
The reviewer downgraded this study's overall quality rating. They noted: This seem to be a well conducted
study, however, one major flaw is that the source of HBCD was not reported. Not sure if the chemical was
prepared in the lab or purchased from a manufacturer. Left the rating for metric 2 as low, but could be
changed to unacceptable since information on test material source, manufacturer, purity, other analytical
details of HBCD was not provided. Other parts of the study were appropriately conducted. Note: The original
calculated score for this study was 1.5. This value is not presented above because the final rating was changed
based on professional judgement.
45

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PEER REVIEW DRAFT- DO NOT CITE OR QUOTE
2.6 Animal toxicity evaluation results of Miller-Rhodes et al 2014
for developmental study; gestation day 1-parturition study on
growth (early life) and development, neurological/behavior
outcomes
Study reference:
Miller-Rhodes, P.,Popescu, M.,Goeke, C.,Tirabassi, T.,Johnson, L.,Markowski, V. P. (2014). Prenatal
exposure to the brominated flame retardant hexabromocyclododecane (HBCD) impairs measures of sustained
attention and increases age-related morbidity in the Long-Evans rat Neurotoxicology and Teratology, 45, 34-
43

HERO ID: 2528337

Domain
Metric
Evaluator's Comment
Qualitative
Determination
[i.e., High, Medium,
Low,Unacceptable,
or Not rated]
Metric
Score
Metric
Weighting
Factor
Weighted
Score

1. Test Substance
Identity
Name and product
number provided
High
1
2
2
Test Substance
2. Test Substance
Source
Commercial source
High
1
1
1

3. Test Substance
Purity
Purity >95%
High
1
1
1

4. Negative and
Vehicle Controls
Use of vehicle control
High
1
2
2
Test Design
5. Positive Controls
Positive control not
necessary
Not Rated
NR
NR
NR

6. Randomized
Allocation
Randomized block
design
High
1
1
1

7. Preparation and
Storage of Test
Substance
Prepared fresh daily,
properly mixed.
High
1
1
1

8. Consistency of
Exposure
Administration
Exposure consistent
across groups
High
1
1
1
Exposure
Characterization
9. Reporting of
Doses/Concentration
s
concentrations were
reported
High
1
2
2
10. Exposure
Frequency and
Duration
Daily gavage
High
1
1
1

11. Number of
Exposure Groups
and Dose Spacing
Three dose groups and a
control
High
1
1
1

12. Exposure Route
and Method
Gavage
High
1
1
1

13. Test Animal
Characteristics
Standard animal model
used (Long Evans rats)
High
1
2
2
Test Organism
14. Adequacy and
Consistency of
Animal Husbandry
Conditions
Animal husbandry was
reported and acceptable
High
1
1
1
46

HERO ID: 2528337

Domain
Metric
Evaluator's Comment
Qualitative
Determination
[i.e., High, Medium,
Low,Unacceptable,
or Not rated]
Metric
Score
Metric
Weighting
Factor
Weighted
Score

15. Number per
Group
10-11 pregnant
dams/treatment group,
(litters culled to 8 pups
using randomized
selection procedure)
High
1
1
1

16. Outcome
Assessment
Methodology
Outcome assessment
methods were
appropriate
High
1
2
2

17. Consistency of
Outcome
Assessment
Outcomes were assessed
consistently across
groups
High
1
1
1
Outcome
Assessment
18. Sampling
Adequacy
It is unclear the number
of animals evaluated for
each outcome. The "n"
is consistently stated.
Although it was
mentioned that litters
were culled to 8 pups,
there were a number of
deaths, so it is not clear
how many were left for
further analysis. It is
stated that every pup in
each litter was examined,
for example, for FOB
tests, but it is not known
what differences in n
there is between
exposure groups, or if
there are any. In some
cases, it is mentioned
that one male and one
female from each litter
were used for some
endpoints, but it is not
clear this was always the
case.
Low
3
1
3

19. Blinding of
Assessors
Stated that observers
were blind to the
exposure group
High
1
1
1
47

20. Negative Control
Response
Study authors indicate
that the mean gestation
length of the control
group was shorter than
typically expected for
these rats, which may be
the reason why HBCD
treated rats appeared to
have a longer gestation
period.
Medium
2
1
2
Confounding /
Variable Control
21. Confounding
Variables in Test
Design and
Procedures
Study authors mention
that the ability to detect
an exposure effect for
locomotor activity could
have been confounded by
different body size to
chamber size ratios. It
was also mentioned that
paw sizes were not taken
into account for the grip
strength tests
Medium
2
2
4
22. Health Outcomes
Unrelated to
Exposure
There were a number of
animals that
disproportionately died
unexpectedly or became
ill. The authors indicate
that data from these
animals were not used
for several of the
analyses. Since the
actual numbers of
animals effected were
not reported, it is unclear
how this impacted the
analyses or the actual
number of animals
evaluated for each
endpoint. The timing of
when these animals died
or became ill is also not
reported.
Low
3
1
3
48

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PEER REVIEW DRAFT- DO NOT CITE OR QUOTE
Study reference:
Miller-Rhodes, P.,Popescu, M.,Goeke, C.,Tirabassi, T.,Johnson, L.,Markowski, V. P. (2014). Prenatal
exposure to the brominated flame retardant hexabromocyclododecane (HBCD) impairs measures of sustained
attention and increases age-related morbidity in the Long-Evans rat Neurotoxicology and Teratology, 45, 34-
43
HERO ID: 2528337
Domain
Metric
Evaluator's Comment
Qualitative
Determination
[i.e., High, Medium,
Low,Unacceptable,
or Not rated]
Metric
Score
Metric
Weighting
Factor
Weighted
Score
Data Presentation
and Analysis
23. Statistical
Methods
The described statistical
analysis was appropriate,
and the litter was used as
the unit of analysis for
offspring endpoints,
however, results from
statistical analysis were
not shown in any of the
figures making it
difficult to easily
interpret the data. In
most instances, p-values
were provided within the
text.
Medium
2
1
2
24. Reporting of
Data
No individual offspring
animal data were
reported, therefore the
data cannot be
independently reviewed.
Additionally, most data
are reported in the form
of bar graphs, and text
does not provide the
quantal values. Data
from males and females
were often pooled and
averaged, and therefore
not reported
independently.
Low
3
2
6
High: >=1 and <1.7
Medium: >=1.7 and <2.3
Low: >=2.3 and <=3
Sum of scores:

30
42
Overall Score = Sum of Weighted Scores/Sum
of Metric Weighting Factors:
NR
Overall Score:
Nearest *:
NR
Overall Quality Level:
Medium
Study Quality
Comment:
The reviewer downgraded this study's overall quality rating. They noted: The lack of individual animal data,
and the way the data is presented, make it difficult to interpret the data. Additionally, the lack of clarity
regarding the number of animals evaluated should be considered. There were also a large number of animals
that became ill. Without further transparency or information, it is difficult to know how this could have
impacted the various results with the data provided Note: The original calculated score for this study was 1.4.
This value is not presented above because the final rating was changed based on professional judgement.
49

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2.7 Animal toxicity evaluation results of van et al 2006 for 280day
oral toxicity study (gavage) study on hepatic, clinical
chemistry/biochemical, endocrine, musculoskeletal/motor
function, ADME/PBPK, thyroid, nutrition and metabolic/adult
exposure body weight, hematological and immune,
reproductive outcomes
Study reference:
van der Ven, L. T.,Verhoef, A.,van de Kuil, T.,Slob, W.,Leonards, P. E.,Visser, T. J.,Hamers, T.,Herlin,
M.,Hakansson, H.,01ausson, H.,Piersma, A. H.,Vos, J. G. (2006). A 28-day oral dose toxicity study enhanced
to detect endocrine effects of hexabromocyclododecane in Wistar rats Toxicological Sciences, 94(2), 281-292
HERO ID: 787745
Domain
Metric
Evaluator's Comment
Qualitative
Determination
[i.e.,High,Medium,
Low,Unacceptable,
or Not rated]
Metric
Score
Metric
Weighting
Factor
Weighted
Score

1. Test Substance
Identity
The test substance was
identified definitively
and characterized.
HBCD technical
preparation is a mixture
of three enantiomers,
HBCD-alpha- beta-, and
gamma, and their
respective proportion in
the used batch was 10.28,
8.72, and 81.01%,
respectively.
High
1
2
2
Test Substance
2. Test Substance
Source
The source
(manufacturer) of the test
substance was reported,
but the batch/lot numbers
were omitted; this
omission is unlikely to
have a substantial impact
on results.
Medium
2
1
2

3. Test Substance
Purity
The test substance was
noted to be technical
HBCD as a mixture of
three enantiomers,
HBCD-alpha- beta-, and
gamma, with respective
proportions as 10.28,
8.72, and 81.01%,
respectively. Trace
impurities were
identified as traces of
tetra- and
pentabromocyclododecan
e.
High
1
1
1
Test Design
4. Negative and
Vehicle Controls
An appropriate
concurrent negative
control group was
included.
High
1
2
2
50

5. Positive Controls
The use of a positive
control was reported for
the UDP-
glucuronosyltransferase
assay. This metric was
not rated/applicable for
the other evaluations in
the study.
Medium
2
1
2

6. Randomized
Allocation
"The experimental
protocol followed the
OECD407 28-day sub-
acute toxicity guideline,
which was enhanced for
endocrine and
immunological endpoints
(Andrews et al., 2001).
However, in contrast to
the published protocol,
the animals were
distributed among more
dose groups each with
fewer animals, that is,
five rats per sex per dose
group, for improved
assessment of dose
response relationships
(Kavlock et al., 1996;
Slob, 2002)."
It is unclear if this would
have a substantial impact
on results.
Medium
2
1
2
Exposure
Characterization
7. Preparation and
Storage of Test
Substance
Test substance
preparation was reported,
but with limitations in
reporting. HBCD was
reported to be dissolved
in corn oil. It is not
reported how often the
test solution was
prepared or how it was
stored. This omission is
unlikely to have a
substantial impact on
results.
Medium
2
1
2
51

8. Consistency of
Exposure
Administration
Details of exposure
administration were
reported and
administration was
consistent across study
groups.
High
1
1
1

9. Reporting of
Doses/Concentration
s
Administered doses were
reported without
ambiguity.
High
1
2
2

10. Exposure
Frequency and
Duration
The exposure frequency
and duration of exposure
were reported and
appropriate for this study
type and/or outcome(s)
of interest.
High
1
1
1

11. Number of
Exposure Groups
and Dose Spacing
The number of exposure
groups and spacing was
reported. It was reported
that a larger number of
dose groups was used
(than recommended in
OECD 407) for
improved assessment of
the dose-response
relationship.
High
1
1
1

12. Exposure Route
and Method
The route and method of
exposure were reported
and were suited to the
test substance.
High
1
1
1
Test Organism
13. Test Animal
Characteristics
The test animal species,
strain, sex, and age were
reported. It was noted
that the animals were
inspected daily for
general condition and
clinical abnormalities.
The animals were
obtained from a
commercial breeding
facility.
High
1
2
2
52

14. Adequacy and
Consistency of
Animal Husbandry
Conditions
Most animal husbandry
conditions were reported
and adequate. Humidity
and temperature were not
reported, however, this
limitation in reporting is
unlikely to have a
substantial impact on
results.
Medium
2
1
2

15. Number per
Group
The number of animals
per study group was
reported (5/sex/dose).
OECD 407 requires at
least 10 animals (5/sex)
for each dose level.
Hence, the confidence is
selected as 'medium'.
Medium
2
1
2

16. Outcome
Assessment
Methodology
The outcome assessment
methodology reported
and sensitive to the
intended outcomes of
interest.
High
1
2
2

17. Consistency of
Outcome
Assessment
Details of the outcome
assessment methodology
were reported and
consistent across study
groups for the outcomes
of interest.
High
1
1
1
Outcome
Assessment
18. Sampling
Adequacy
Details regarding the
sampling for the
outcomes of interest
were reported and
adequate for assessment.
High
1
1
1

19. Blinding of
Assessors
This metric is not rated
when outcomes are not
subjective or for initial
histopathology review.
Not Rated
NR
NR
NR

20. Negative Control
Response
The biological response
of the negative control
group was adequate. As
shown in Data tables and
in Supplemental tables
(ID2919527)
High
1
1
1
53

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PEER REVIEW DRAFT- DO NOT CITE OR QUOTE
Study reference:
van der Ven, L. T.,Verhoef, A.,van de Kuil, T.,Slob, W.,Leonards, P. E.,Visser, T. J.,Hamers, T.,Herlin,
M.,Hakansson, H.,01ausson, H.,Picrsma, A. H.,Vos, J. G. (2006). A 28-day oral dose toxicity study enhanced
to detect endocrine effects of hexabromocyclododecane in Wistar rats Toxicological Sciences, 94(2), 281-292
HERO ID: 787745
Domain
Metric
Evaluator's Comment
Qualitative
Determination
[i.e., High, Medium,
Low,Unacceptable,
or Not rated]
Metric
Score
Metric
Weighting
Factor
Weighted
Score
Confounding /
Variable Control
21. Confounding
Variables in Test
Design and
Procedures
There were no reported
differences among the
study groups that could
influence the outcome of
the assessment. Food
consumption was
reported, but initial body
weights were not. The
lack of reporting is not
likely to have a
significant impact on
results.
Medium
2
2
4
22. Health Outcomes
Unrelated to
Exposure
Data on attrition
unrelated to exposure
was reported. No other
health outcomes
unrelated to exposure
were reported. The
incidence of attrition is
unlikely to have a
substantial impact on
results.
Medium
2
1
2
Data Presentation
and Analysis
23. Statistical
Methods
Statistical analysis was
shown for all datasets
included in the published
report and for
supplemental data tables
(ID2919527). BMD
methodology was clearly
described and
appropriate.
High
1
1
1
24. Reporting of
Data
Data for exposure-related
findings were presented
for all outcomes by
exposure group and sex
as evaluated for this
reference and the
supplemental data tables
(ID2919527).
High
1
2
2
High: >=1 and <1.7
Medium: >=1.7 and <2.3
Low: >=2.3 and <=3
Sum of scores:

30
39
Overall Score = Sum of Weighted Scores/Sum
of Metric Weighting Factors:
1.3
Overall Score:
Nearest *:
1.3
Overall Quality Level:
High
54

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PEER REVIEW DRAFT- DO NOT CITE OR QUOTE
55

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PEER REVIEW DRAFT- DO NOT CITE OR QUOTE
2.8 Animal toxicity evaluation results of W. I. L. Research 1997 for
28-day repeated oral study on mortality, nutrition and
metabolic/adult exposure body weight, neurological/behavior,
hematological and immune, clinical chemistry/biochemical,
hepatic, renal, cardiovascular, reproductive, endocrine,
gastrointestinal, respiratory outcomes
Study reference:
W. I. L. Research (1997). Twenty-eight day repeated dose oral toxicity study of HBCD in rats, with cover
letter dated 3/18/1997
HERO ID: 787758
Domain
Metric
Evaluator's Comment
Qualitative
Determination
[i.e.,High,Medium,
Low,Unacceptable,
or Not rated]
Metric
Score
Metric
Weighting
Factor
Weighted
Score
Test Substance
1. Test Substance
Identity
The test substance was
identified definitively.
High
1
2
2
2. Test Substance
Source
The source of the test
substance was reported,
including manufacturer
and lot number.
High
1
1
1
3. Test Substance
Purity
The study authors stated
that the purity was
"considered to be 100%",
but no verification of this
purity was reported.
Medium
2
1
2
Test Design
4. Negative and
Vehicle Controls
The study authors
reported using an
appropriate concurrent
negative control group
(administered the vehicle
via gavage at the same
dose volume).
High
1
2
2
5. Positive Controls
Positive control is not
indicated by study type.
Not Rated
NR
NR
NR
6. Randomized
Allocation
The study reported
methods of allocation of
animals to study groups,
but there were minor
limitations in the
allocation method
(method of distribution
had a non-random
component, including
assignment to minimize
differences in body
weight across groups).
Medium
2
1
2
56

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PEER REVIEW DRAFT- DO NOT CITE OR QUOTE
Study reference:
W. I. L. Research (1997). Twenty-eight day repeated dose oral toxicity study of HBCD in rats, with cover
letter dated 3/18/1997
HERO ID: 787758
Domain
Metric
Evaluator's Comment
Qualitative
Determination
[i.e.,High,Medium,
Low,Unacceptable,
or Not rated]
Metric
Score
Metric
Weighting
Factor
Weighted
Score

7. Preparation and
Storage of Test
Substance
The test substance
preparation and storage
conditions were reported
and appropriate for the
test substance (the test
substance was prepared
daily and stored at room
temperature). Storage of
the bulk test substance
was also reported (sealed
container at room
temperature) and the
bulk test substance was
considered stable under
the storage conditions.
High
1
1
1
Exposure
Characterization
8. Consistency of
Exposure
Administration
Details of the
administration were
reported but minor
limitations in
administration of the
exposures, including
accidental mistakes in
dosing, were identified
that are unlikely to have
a substantial impact on
results. On one particular
day, animals at higher
dose levels were
inadvertently dosed with
lower doses, and a few
lower dose animals were
inadvertently dosed with
higher doses. Lower
doses were corrected so
that the underdosed
animals received the
correct doses.
Medium
2
1
2

9. Reporting of
Doses/Concentration
s
Administered doses were
reported without
ambiguity. Test
concentrations were
evaluated by gravimetric
analysis each day prior to
dosing and homogeneity
was evaluated on three
days during the
administration period (d
0, 13, 27); however, the
results were not reported.
Medium
2
2
4
57

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PEER REVIEW DRAFT- DO NOT CITE OR QUOTE
Study reference:
W. I. L. Research (1997). Twenty-eight day repeated dose oral toxicity study of HBCD in rats, with cover
letter dated 3/18/1997
HERO ID: 787758
Domain
Metric
Evaluator's Comment
Qualitative
Determination
[i.e.,High,Medium,
Low,Unacceptable,
or Not rated]
Metric
Score
Metric
Weighting
Factor
Weighted
Score

10. Exposure
Frequency and
Duration
The exposure frequency
and duration of exposure
(daily exposure for 28
consecutive days) were
reported and appropriate
for the study type and
outcomes of interest.
High
1
1
1

11. Number of
Exposure Groups
and Dose Spacing
The number of exposure
groups and dose spacing
(125, 350, 1000
mg/kg/day) were
considered adequate to
address the purpose of
the study. Although the
basis for selection of the
doses was not reported,
the range of doses was
adequate.
High
1
1
1

12. Exposure Route
and Method
The route and method of
exposure (oral, gavage)
were reported and were
suited to the test
substance.
High
1
1
1

13. Test Animal
Characteristics
The test animal source,
species, strain, sex, age,
and starting body weight
(group means) were
reported; however, health
status was not reported.
Medium
2
2
4
Test Organism
14. Adequacy and
Consistency of
Animal Husbandry
Conditions
All husbandry conditions
(temperature, humidity,
light-dark cycle) were
reported and were
adequate and the same
for control and exposed
populations.
High
1
1
1
58

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PEER REVIEW DRAFT- DO NOT CITE OR QUOTE
Study reference:
W. I. L. Research (1997). Twenty-eight day repeated dose oral toxicity study of HBCD in rats, with cover
letter dated 3/18/1997
HERO ID: 787758
Domain
Metric
Evaluator's Comment
Qualitative
Determination
[i.e.,High,Medium,
Low,Unacceptable,
or Not rated]
Metric
Score
Metric
Weighting
Factor
Weighted
Score

15. Number per
Group
The reported number of
animals was lower than
the typical number used
in studies of the same or
similar type for some
groups; however, the
number was sufficient
for statistical analysis.
The low- and mid-dose
groups had only
6/sex/group, while the
control and high-dose
groups had 12/sex/group
(6/sex/group sacrificed at
the end of the 28-day
administration period and
the remaining
6/sex/group were
maintained for an
additional 14-day
recovery period).
Medium
2
1
2

16. Outcome
Assessment
Methodology
The outcome assessment
methodology addressed
or reported the intended
outcomes of interest and
was sensitive for the
outcomes of interest.
High
1
2
2
Outcome
Assessment
17. Consistency of
Outcome
Assessment
Details of the outcome
assessment protocol were
reported, and outcomes
were assessed
consistently across study
groups.
High
1
1
1

18. Sampling
Adequacy
Details regarding the
sampling for the
outcomes of interest
were reported and the
study used adequate
sampling for the
outcomes of interest.
High
1
1
1
59

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Study reference:
W. I. L. Research (1997). Twenty-eight day repeated dose oral toxicity study of HBCD in rats, with cover
letter dated 3/18/1997
HERO ID: 787758
Domain
Metric
Evaluator's Comment
Qualitative
Determination
[i.e.,High,Medium,
Low,Unacceptable,
or Not rated]
Metric
Score
Metric
Weighting
Factor
Weighted
Score

19. Blinding of
Assessors
The study states that
investigators were
blinded for subjective
outcomes in the
neurological tests (For
FOB parameters "testing
was performed by the
same technicians without
knowledge of the animal
group assignment"). No
other subjective
outcomes were reported
in the study.
High
1
1
1

20. Negative Control
Response
The biological responses
of the negative control
groups were adequate.
High
1
1
1
Confounding /
Variable Control
21. Confounding
Variables in Test
Design and
Procedures
There were no reported
differences among the
study groups related to
confounding variables in
test design or procedures
and no significant
differences in initial
body weights.
High
1
2
2
60

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PEER REVIEW DRAFT- DO NOT CITE OR QUOTE
Study reference:
W. I. L. Research (1997). Twenty-eight day repeated dose oral toxicity study of HBCD in rats, with cover
letter dated 3/18/1997
HERO ID: 787758
Domain
Metric
Evaluator's Comment
Qualitative
Determination
[i.e., High, Medium,
Low,Unacceptable,
or Not rated]
Metric
Score
Metric
Weighting
Factor
Weighted
Score

22. Health Outcomes
Unrelated to
Exposure
Data on attrition and
health outcomes
unrelated to exposure
were reported. The
authors report that
"animal no. 50292 was
replaced by animal
no.50289 on study day -1
as animal no. 50292 died
shortly after being
handled for pretest
clinical observations and
weighing." The authors
also stated that "Several
animals weighed less
than the protocol-
specified minimum
weight (175 g for males,
125 g for females) at the
initiation of dosing. This
deviation had no impact
on the outcome of the
study as all animals were
within the protocol-
specified age range (4-8
weeks) at the initiation of
dosing. "
Medium
2
1
2
Data Presentation
and Analysis
23. Statistical
Methods
Statistical methods were
clearly described and
appropriate for the
datasets.
High
1
1
1
24. Reporting of
Data
Data for exposure-related
findings were presented
for all outcomes by
exposure group and sex
with quantal or
continuous presentation
and negative findings
reported qualitatively or
quantitatively.
High
1
2
2
High: >=1 and <1.7
Medium: >=1.7 and <2.3
Low: >=2.3 and <=3
Sum of scores:

30
39
Overall Score = Sum of Weighted Scores/Sum
of Metric Weighting Factors:
1.3
Overall Score:
Nearest *:
1.3
Overall Quality Level:
High
61

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62

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2.9 Animal toxicity evaluation results of Wang et al 2016for 28 day
oral gavage metabolomic study in mice study on nutrition and
metabolic/adult exposure body weight, gene expression/omics
outcomes
Study reference:
Wang, D.,Zhang, P.,Wang, X.,Wang, Y.,Zhou, Z.,Zhu, W. (2016). NMR- and LC-MS/MS-based urine
metabolomic investigation of the subacute effects of hexabromocyclododecane in mice Environmental Science
and Pollution Research, 23(9), 8500-8507
HERO ID: 3350496
Domain
Metric
Evaluator's Comment
Qualitative
Determination
[i.e.,High,Medium,
Low,Unacceptable,
or Not rated]
Metric
Score
Metric
Weighting
Factor
Weighted
Score

1. Test Substance
Identity
Test substance identified
as technical HBCD with
10% alpha, 10% beta,
and 80% gamma
stereoisomers.
High
1
2
2
Test Substance
2. Test Substance
Source
Test substance obtained
from manufacturer but
without certification or
analytical verification of
identity.
Medium
2
1
2

3. Test Substance
Purity
Test substance purity
reported as 95%
High
1
1
1

4. Negative and
Vehicle Controls
Sham-treated controls
received vehicle
High
1
2
2
Test Design
5. Positive Controls
Positive controls not
typical for study type
Not Rated
NR
NR
NR

6. Randomized
Allocation
Study reports random
allocation to groups
High
1
1
1

7. Preparation and
Storage of Test
Substance
Test substance
preparation was reported
but storage was not
reported
Medium
2
1
2

8. Consistency of
Exposure
Administration
Time of day of gavage
administration was not
reported.
Medium
2
1
2
Exposure
Characterization
9. Reporting of
Doses/Concentration
s
Details of exposure
administration were
reported and exposures
were administered
consistently across study
groups in a scientifically
sound manner
High
1
2
2

10. Exposure
Frequency and
Duration
Doses administered daily
for 28 days
High
1
1
1
63

11. Number of
Exposure Groups
and Dose Spacing
2 nonzero doses were
administered ranging 5-
fold. Doses were
selected based on
reported range of toxic
doses
Medium
2
1
2

12. Exposure Route
and Method
oral gavage exposure
with appropriate vehicle
reported
High
1
1
1
Test Organism
13. Test Animal
Characteristics
Test animal species,
strain, sex, age, and
body weight were
reported. Females were
chosen because they
were reportedly more
sensitive.
High
1
2
2
14. Adequacy and
Consistency of
Animal Husbandry
Conditions
Relative humidity and
diet were not reported.
All other husbandry
conditions were reported
and adequate.
Medium
2
1
2

15. Number per
Group
5 animals/dose tested.
Medium
2
1
2

16. Outcome
Assessment
Methodology
Body weight, organ
weight and both targeted
and untargeted
metabolomics were
evaluated. BW was
measured weekly, but
metabolomics only
performed once on 24
hour urine samples
collected after last dose.
Medium
2
2
4
Outcome
Assessment
17. Consistency of
Outcome
Assessment
No inconsistencies in
outcome assessment
were noted
High
1
1
1

18. Sampling
Adequacy
Body weights and
metabolomics assessed
for individual animals
High
1
1
1

19. Blinding of
Assessors
no subjective outcomes
Not Rated
NR
NR
NR

20. Negative Control
Response
Control responses were
reported and appeared to
be appropriate
High
1
1
1
64

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PEER REVIEW DRAFT- DO NOT CITE OR QUOTE
Study reference:
Wang, D.,Zhang, P.,Wang, X.,Wang, Y.,Zhou, Z.,Zhu, W. (2016). NMR- and LC-MS/MS-based urine
metabolomic investigation of the subacute effects of hexabromocyclododecane in mice Environmental Science
and Pollution Research, 23(9), 8500-8507
HERO ID: 3350496
Domain
Metric
Evaluator's Comment
Qualitative
Determination
[i.e., High, Medium,
Low,Unacceptable,
or Not rated]
Metric
Score
Metric
Weighting
Factor
Weighted
Score
Confounding /
Variable Control
21. Confounding
Variables in Test
Design and
Procedures
Food and water intake
were not reported.
Medium
2
2
4
22. Health Outcomes
Unrelated to
Exposure
One control mouse died
during the study.
Medium
2
1
2
Data Presentation
and Analysis
23. Statistical
Methods
Statistical analysis
methods reported and
appropriate.
High
1
1
1
24. Reporting of
Data
Body weights reported
graphically without
measure of variability in
supplemental material.
Medium
2
2
4
High: >=1 and <1.7
Medium: >=1.7 and <2.3
Low: >=2.3 and <=3
Sum of scores:

29
42
Overall Score = Sum of Weighted Scores/Sum
of Metric Weighting Factors:
NR
Overall Score:
Nearest *:
NR
Overall Quality Level:
Medium
Study Quality
Comment:
The reviewer downgraded this study's overall quality rating. They noted: Although body weight and organ
weights were measured, only average body weight was provided in the supplemental material. The author
reports that organ weight data was not shown but did not have any changes. This study mainly focuses on
metabolomics using urine samples and analyzing amino acids. Even though it is a 28-day study, no useful
information is provided in terms of outcomes for toxicological endpoint. It possibly can be used as a
mechanistic supporting study for understanding the metabolic pathway. Note: The original calculated score for
this study was 1.4. This value is not presented above because the final rating was changed based on
professional judgement.
65

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2.10Animal toxicity evaluation results of Watanabe et al 2010 for 28
day feeding study in mice - mechanistic study, animals also
infected with rsv study on nutrition and metabolic/adult
exposure body weight, hematological and immune outcomes
Study reference:
Watanabe, W.,Shimizu, T.,Sawamura, R.,Hino, A.,Konno, K.,Hirose, A.,Kurokawa, M. (2010). Effects of
tetrabromobisphenol A, a brominated flame retardant, on the immune response to respiratory syncytial virus
infection in mice International Immunopharmacology, 10(4), 393-397
HERO ID: 1927692
Domain
Metric
Evaluator's Comment
Qualitative
Determination
[i.e.,High,Medium,
Low,Unacceptable,
or Not rated]
Metric
Score
Metric
Weighting
Factor
Weighted
Score

1. Test Substance
Identity
Substance reported as
HBCD, no CAS # was
provided
High
1
2
2
Test Substance
2. Test Substance
Source
Purchased from a
commercial source
High
1
1
1

3. Test Substance
Purity
Purity was not reported;
no validation was done to
assess purity
Low
3
1
3
Test Design
4. Negative and
Vehicle Controls
The study indicates there
was a control, it is
presumed that this was
the powdered diet alone.
It does not appear as
though a vehicle was
used?
Medium
2
2
4

5. Positive Controls
Positive control not
necessary
Not Rated
NR
NR
NR

6. Randomized
Allocation
Randomization was not
reported
Low
3
1
3

7. Preparation and
Storage of Test
Substance
Preparation nor storage
was reported. Study
authors only indicate that
HBCD was mixed into a
powder diet.
Low
3
1
3
Exposure
Characterization
8. Consistency of
Exposure
Administration
Control and treated
Animals were fed ad
libitum
High
1
1
1
9. Reporting of
Doses/Concentration
s
Reported as 1% in diet.,
body weights and food
consumption were
provided,
High
1
2
2

10. Exposure
Frequency and
Duration
Daily for 28 days
High
1
1
1
66

11. Number of
Exposure Groups
and Dose Spacing
Single exposure and
control; There was no
explanation or
justification of chosen
dose; not useful for dose-
response analysis, but
single dose may be
appropriate for the
endpoints evaluated.
There were no responses,
so it is unclear whether
the dose used was
appropriate or not.
Medium
2
1
2

12. Exposure Route
and Method
Standard exposure route
and method
High
1
1
1

13. Test Animal
Characteristics
Test animals were
acceptable
High
1
2
2
Test Organism
14. Adequacy and
Consistency of
Animal Husbandry
Conditions
Animal husbandry was
not reported
Low
3
1
3
15. Number per
Group
Study reports use of 6-7
mice/ group; OECD
guidelines for 28-day
repeated dose study
recommends 10
animals/group (5/sex)
Medium
2
1
2

16. Outcome
Assessment
Methodology
CK: The outcome
assessment methodology
addressed the intended
outcomes
High
1
2
2

17. Consistency of
Outcome
Assessment
Methods were acceptable
for what they were
looking at.
High
1
1
1
Outcome
Assessment
18. Sampling
Adequacy
Sampling was done on
all of the mice/group
High
1
1
1

19. Blinding of
Assessors
Histology was not done
on HBCD treated
animals; there were no
other subjective
outcomes
Not Rated
NR
NR
NR

20. Negative Control
Response
Control responses were
as expected
High
1
1
1
67

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Study reference:
Watanabe, W.,Shimizu, T.,Sawamura, R.,Hino, A.,Konno, K.,Hirose, A.,Kurokawa, M. (2010). Effects of
tetrabromobisphenol A, a brominated flame retardant, on the immune response to respiratory syncytial virus
infection in mice International Immunopharmacology, 10(4), 393-397
HERO ID: 1927692
Domain
Metric
Evaluator's Comment
Qualitative
Determination
[i.e., High, Medium,
Low,Unacceptable,
or Not rated]
Metric
Score
Metric
Weighting
Factor
Weighted
Score
Confounding /
Variable Control
21. Confounding
Variables in Test
Design and
Procedures
There were no apparently
confounding factors that
would influence the
outcomes
High
1
2
2
22. Health Outcomes
Unrelated to
Exposure
There were no unrelated
health outcomes
High
1
1
1
Data Presentation
and Analysis
23. Statistical
Methods
Statistical method was
appropriate for outcome
High
1
1
1
24. Reporting of
Data
Reporting of data was
acceptable
High
1
2
2
High: >=1 and <1.7
Medium: >=1.7 and <2.3
Low: >=2.3 and <=3
Sum of scores:

29
41
Overall Score = Sum of Weighted Scores/Sum
of Metric Weighting Factors:
NR
Overall Score:
Nearest *:
NR
Overall Quality Level:
Medium
Study Quality
Comment:
The reviewer downgraded this study's overall quality rating. They noted: Some study details regarding
preparation of diets, and validation of dosing were omitted. Since there was no justification of dose, it is
unknown whether the dose used was appropriate to elicit an effect. The limited endpoints evaluated do not
greatly inform mechanism of the potential effects of HBCD on immunity. Note: The original calculated score
for this study was 1.4. This value is not presented above because the final rating was changed based on
professional judgement.
3 Subchronic Toxicity Studies
3.1 Animal toxicity evaluation results of ACC et al 2002 for 90-day
gavage-systemic with sperm evaluations and neurobehavior,
same as (2990994) study on reproductive, hematological,
neurological/behavior, renal, hepatic, clinical
chemistry/biochemical, body weight, ocular and sensory,
thyroid outcomes
68

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Study reference:
ACC (2002). A 90-Day Oral (Gavage) Toxicity Study of HBCD in Rats
HERO ID: 4269953
Domain
Metric
Evaluator's Comment
Qualitative
Determination
[i.e.,High,Medium,
Low,Unacceptable,
or Not rated]
Metric
Score
Metric
Weighting
Factor
Weighted
Score

1. Test Substance
Identity
Identified by name,
CARSN, structure,
molecular formula, and
isomer distribution (pp.
1235-1236)
High
1
2
2

2. Test Substance
Source
Source and analytical
verification were
included in the study
report.
High
1
1
1
Test Substance
3. Test Substance
Purity
The test substance
composition was such
that any observed effects
were highly likely to be
due to the test substance.
Although the test
chemical was analyzed to
determine the isomer
composition analysis
does not appear to
address the purity of the
chemical.
Medium
2
1
2

4. Negative and
Vehicle Controls
Concurrent vehicle
control groups were
included in the main and
satellite studies.
High
1
2
2
Test Design
5. Positive Controls
This metric not
applicable.
Not Rated
NR
NR
NR
6. Randomized
Allocation
Animals were allocated
by a computerized
randomization procedure
based on body weight
stratification in a block
design.
Medium
2
1
2
Exposure
Characterization
7. Preparation and
Storage of Test
Substance
Preparation and storage
conditions were reported
and appropriate based on
stability and
homogeneity testing (pp.
1242-1268).
High
1
1
1
69

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Study reference:
ACC (2002). A 90-Day Oral (Gavage) Toxicity Study of HBCD in Rats


HERO ID: 4269953




Domain
Metric
Evaluator's Comment
Qualitative
Determination
[i.e.,High,Medium,
Low,Unacceptable,
or Not rated]
Metric
Score
Metric
Weighting
Factor
Weighted
Score

8. Consistency of
Exposure
Administration
Details were reported
and administered
consistently across
groups. Dosing volume
was appropriate. A
dosing error was reported
(pp. 65) but this is
unlikely to have
substantial impact on
results.
Medium
2
1
2

9. Reporting of
Doses/Concentration
s
Doses reported without
ambiguity.
High
1
2
2

10. Exposure
Frequency and
Duration
Duration of study and
frequency of dosing were
reported and appropriate
for this study
High
1
1
1

11. Number of
The selected doses were
not justified by study
authors, but the doses
were adequate to show
results relevant to the
outcomes of interest.





Exposure Groups
and Dose Spacing
Medium
2
1
2

12. Exposure Route
and Method
Exposure route and
method were suitable.
High
1
1
1

13. Test Animal
Characteristics
The test animal species,
strain, sex, health status,
age, and starting body
weight were reported.
Animals obtained from
commercial supplier
(Charles River).
High
1
2
2
Test Organism
14. Adequacy and
Consistency of
Animal Husbandry
Conditions
Temperature, relative
humidity, light/day cycle
were reported.
High
1
1
1

15. Number per
Group
In general, the number of
animals assigned per
group was appropriate
for the study type and
outcome analysis. Group
sizes conformed to
OECD 408.
High
1
1
1
70

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Study reference:
ACC (2002). A 90-Day Oral (Gavage) Toxicity Study of HBCD in Rats
HERO ID: 4269953
Domain
Metric
Evaluator's Comment
Qualitative
Determination
[i.e.,High,Medium,
Low,Unacceptable,
or Not rated]
Metric
Score
Metric
Weighting
Factor
Weighted
Score


In general, outcome
assessment methodology
was described in detail
and sensitive for
outcomes of interest.





16. Outcome
Assessment
Methodology
Serious concerns were
identified for serum
hormone data.
Specifically, the
confidence rating for
TSH data is low because
of a high incidence of
samples in the control
group below the limit of
detection, indicating
insensitivity of the
method. In one instance
data were reported for a
single control animal
(278-281; 916-939)
High
1
2
2
Outcome
Assessment
17. Consistency of
Outcome
Assessment
Details of the protocols
used for outcome
assessment were reported
ad outcomes were
assessed consistently
across study groups.
High
1
1
1

18. Sampling
Adequacy
Sampling details were
well described and
adequate.
High
1
1
1

19. Blinding of
Assessors
Two subjective outcomes
were evaluated:
functional observational
battery and
histopathology.
Functional Observational
Battery : High - the study
report indicates that
assessors were blinded to
treatment group during
observations.
Histopathology: Medium
- Blinding was not
reported in the study and
no indication that tissues
were subjected to a
secondary independent
evaluation.
High
1
1
1
71

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Study reference:
ACC (2002). A 90-Day Oral (Gavage) Toxicity Study of HBCD in Rats
HERO ID: 4269953
Domain
Metric
Evaluator's Comment
Qualitative
Determination
[i.e., High, Medium,
Low,Unacceptable,
or Not rated]
Metric
Score
Metric
Weighting
Factor
Weighted
Score


In general, biological
response of negative
controls was adequate.





20. Negative Control
Response
Serious concerns were
identified for the serum
hormone data.
Specifically, the
confidence rating for
TSH data is low because
of a high variability in
the biological responses
between control
replicates such that, in
some cases, the SD >
mean and there were as
much as two orders of
magnitude difference
across individual controls
(pp. 278-281; 916-939).
High
1
1
1
Confounding /
Variable Control
21. Confounding
Variables in Test
Design and
Procedures
No reported differences
among the groups were
observed.
High
1
2
2
22. Health Outcomes
Unrelated to
Exposure
There were no health
outcomes unrelated to
exposure that would
influence outcome
assessment.
High
1
1
1
Data Presentation
23. Statistical
Methods
Statistical methods were
clearly described and
appropriate.
High
1
1
1
and Analysis
24. Reporting of
Data
Data were reported in
tables and in the text for
all outcomes.
High
1
2
2


Sum of scores:

30
34
High: >=
Medium: >=
1 and <1.7
=1.7 and <2.3
Overall Score = Sum of Weighted Scores/Sum
of Metric Weighting Factors:
1.1333
Overall Score:
Nearest *:
1.1
Low: >=2.5 and <=j







Overall Quality Level:

High

72

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3.2 Animal toxicity evaluation results of BASF et al 1990 for 28-day
and 90-day dietary studies study on reproductive,
hematological and immune, neurological, renal, hepatic,
endocrine, gastrointestinal, respiratory, thyroid outcomes
Study reference:
BASF (1990). Hexabromocyclododecane 28-day feeding trials with rats with test data and cover letter,
900000274, #86-900000274
HERO ID: 787638
Domain
Metric
Evaluator's Comment
Qualitative
Determination
[i.e.,High,Medium,
Low,Unacceptable,
or Not rated]
Metric
Score
Metric
Weighting
Factor
Weighted
Score

1. Test Substance
Identity
Identified by trade name
and isomer designation.
High
1
2
2
Test Substance
2. Test Substance
Source
Source and lot no. were
not reported.
Manufacturer was
assumed to be BASF.
Medium
2
1
2

3. Test Substance
Purity
Purity was not reported.
Low
3
1
3

4. Negative and
Vehicle Controls
A negative dietary
control group was used.
High
1
2
2
Test Design
5. Positive Controls
Positive controls are not
necessary for a 28-day
study.
Not Rated
NR
NR
NR

6. Randomized
Allocation
The study did not report
how animals were
allocated to study groups.
Low
3
1
3

7. Preparation and
Storage of Test
Substance
Analysis showed that
concentrations remained
stable over the week.
High
1
1
1

8. Consistency of
Exposure
Administration
Details of exposure
administration were
reported.
High
1
1
1
Exposure
9. Reporting of
Doses/Concentration
s
Dietary concentrations
were not measured
analytically, but bw and
food consumption were
reported for each group.
Medium
2
2
4
Characterization
10. Exposure
Frequency and
Duration
Diet was administered
over 13 weeks (daily was
assumed).
High
1
1
1

11. Number of
Exposure Groups
and Dose Spacing
4 treatment groups plus
control; dose response
relationships were
apparent.
High
1
1
1

12. Exposure Route
and Method
The route and method of
exposure were reported
and were suited to the
test substance.
High
1
1
1
73

-------
Study reference:
BASF (1990). Hexabromocyclododecane 28-day feeding trials with rats with test data and cover letter,
900000274, #86-900000274
HERO ID: 787638
Domain
Metric
Evaluator's Comment
Qualitative
Determination
[i.e.,High,Medium,
Low,Unacceptable,
or Not rated]
Metric
Score
Metric
Weighting
Factor
Weighted
Score
Test Organism
13. Test Animal
Characteristics
Species, strain and
starting bw were
reported. Not a
commercial source, but a
laboratory maintained
colony.
High
1
2
2
14. Adequacy and
Consistency of
Animal Husbandry
Conditions
Husbandry conditions
were not reported.
Low
3
1
3

15. Number per
Group
10/sex/group
High
1
1
1

16. Outcome
Assessment
Methodology
The outcome assessment
methodology was
reported.
High
1
2
2

17. Consistency of
Outcome
Assessment
See footnote at end of
page.1
High
1
1
1
Outcome
Assessment
18. Sampling
Adequacy
Data tables are difficult
to read, but sampling
appears adequate.
Medium
2
1
2

19. Blinding of
Assessors
Blinding was not
reported; however,
outcomes were objective.
Medium
2
1
2

20. Negative Control
Response
Data tables are difficult
to read; however, several
lesions are noted for
controls.
Low
3
1
3
Confounding /
Variable Control
21. Confounding
Variables in Test
Design and
Procedures
The study reported (in
the text) minor
differences among the
study groups (<20%
difference from control)
with respect to initial
body weight, drinking
water and/or food
consumption. But the
information in the tables
is difficult to read.
Medium
2
2
4
1 Metrics that received a "High" rating met the criteria as discussed in the Applications of Systematic
Review for TSCA Risk Evaluation.
74

-------
Study reference:
BASF (1990). Hexabromocyclododecane 28-day feeding trials with rats with test data and cover letter,
900000274, #86-900000274
HERO ID: 787638
Domain
Metric
Evaluator's Comment
Qualitative
Determination
[i.e., High, Medium,
Low,Unacceptable,
or Not rated]
Metric
Score
Metric
Weighting
Factor
Weighted
Score

22. Health Outcomes
Unrelated to
Exposure
A large proportion of rats
showed signs of
respiratory inflammation
(47% of controls, 26% of
treated rats) which would
not be expected from a
feeding trial.
Unacceptable
4
1
4
Data Presentation
and Analysis
23. Statistical
Methods
Statistical analysis was
not described clearly, and
this deficiency is likely
to have a substantial
impact on results.
Low
3
1
3
24. Reporting of
Data
Data tables are provided
for all outcomes by
exposure group and sex;
however, data are in
German and mostly
illegible.
Low
3
2
6
High: >=1 and <1.7
Medium: >=1.7 and <2.3
Low: >=2.3 and <=3
Sum of scores:

30
54
Overall Score = Sum of Weighted Scores/Sum
of Metric Weighting Factors:
1.8000
Overall Score
(Rounded):
1.81
Overall Quality Level:
Unacceptable1
Comment:
Footnote:
1 Consistent with our Application of Systematic Review in TSCA Risk Evaluations document, if a metric for a
data source receives a score of Unacceptable (score = 4), EPA will determine the study to be unacceptable. In
this case, seven of the metrics were rated as unacceptable. As such, the study is considered unacceptable and
the score is presented solely to increase transparency.
3.3 Animal toxicity evaluation results of van et al 2009 for 1-
generation reproduction study, oral dietary study on
endocrine; reproductive; hematological and immune;
thyroid; growth (early life) and development;
musculoskeletal/motor function; clinical
chemistry/biochemical; nutrition and metabolic/adult
exposure body weight; hepatic outcomes
75

-------
Study reference:
van der Ven, L. T. M.,van de Kuil, T.,Leonards, P. E. G.,Slob, W.,Lilienthal, H.,Litens, S.,Herlin,
M.,Hakansson, H.,Canton, R. F.,van den Berg, M.,Visser, T. J.,van Loveren, H.,Vos, J. G.,Piersma, A. H.
(2009). Endocrine effects of hexabromocyclododecane (HBCD) in a one-generation reproduction study in
Wistar rats Toxicology Letters, 185(1), 51-62
HERO ID: 589273
Domain
Metric
Evaluator's Comment
Qualitative
Determination
[i.e.,High,Medium,
Low,Unacceptable,
or Not rated]
Metric
Score
Metric
Weighting
Factor
Weighted
Score

1. Test Substance
Identity
The test substance was
identified definitively as
HBCD a mixture of three
diastereoisomers, H
alpha-, beta-, and
gamma- HBCD and their
respective proportion in
the used batch was 10.3-
8.7-81.0%.
High
1
2
2
Test Substance
2. Test Substance
Source
The test substance
manufacturer and source
were reported; however,
the batch/lot number was
not specified.
Medium
2
1
2

3. Test Substance
Purity
The test substance was
said to be technical grade
(technical mixture
containing traces of tetra-
and
pentabromocyclododecan
e) it was noted; the test
substance composition is
such that any observed
effects are likely due to
the nominal test
substance.
High
1
1
1
Test Design
4. Negative and
Vehicle Controls
Study authors reported
using an appropriate
concurrent negative
control group. An
additional group was
included to monitor
effects of the carrier oil
contents in the feed.
High
1
2
2

5. Positive Controls
This metric is not
rated/applicable for this
study type
Not Rated
NR
NR
NR
76

-------
Study reference:
van der Ven, L. T. M.,van de Kuil, T.,Leonards, P. E. G.,Slob, W.,Lilienthal, H.,Litens, S.,Herlin,
M.,Hakansson, H.,Canton, R. F.,van den Berg, M.,Visser, T. J.,van Loveren, H.,Vos, J. G.,Picrsma, A. H.
(2009). Endocrine effects of hexabromocyclododecane (HBCD) in a one-generation reproduction study in
Wistar rats Toxicology Letters, 185(1), 51-62
HERO ID: 589273
Domain
Metric
Evaluator's Comment
Qualitative
Determination
[i.e.,High,Medium,
Low,Unacceptable,
or Not rated]
Metric
Score
Metric
Weighting
Factor
Weighted
Score

6. Randomized
Allocation
The study noted that the
protocol was based on
OECD415 (one-
generation reproduction
toxicity study) guideline
and that the animals were
distributed among a
larger number of dose
groups than advised in
guideline. The study did
not explicitly report how
animals were allocated to
study groups. It is
unclear if this would
have a substantial impact
on results.
Low
3
1
3

7. Preparation and
Storage of Test
Substance
Test substance
preparation was reported,
but with limitations in
reporting. HBCD was
reported to be mixed
with corn-based oil and
pelleted for feed. It is not
reported how often feed
was mixed or how it was
stored. This omission is
unlikely to have a
substantial impact on
results.
Medium
2
1
2
Exposure
Characterization
8. Consistency of
Exposure
Administration
Details of exposure
administration were
reported and
administration was
consistent between
across study groups.
High
1
1
1

9. Reporting of
Doses/Concentration
s
The targeted dietary
exposure was reported to
be 0-0.1-0.3-1-3-10-
30-100 mg/kg
bodyweight/day.
High
1
2
2

10. Exposure
Frequency and
Duration
Exposure frequency (ad
libitum) and duration of
exposure were reported
and appropriate.
High
1
1
1
77

11. Number of
Exposure Groups
and Dose Spacing
The number of exposure
groups and spacing was
reported and was
justified based on a
preceding subacute
repeated oral dose study.
High
1
1
1

12. Exposure Route
and Method
The route (oral, dietary)
was reported and suited
to the test substance.
High
1
1
1

13. Test Animal
Characteristics
The test animal species,
strain, sex, and age were
reported. It was noted
that the animals were of
weighed and that animals
were inspected daily for
general condition and
clinical abnormalities.
The animals were
obtained from a
commercial breeding
facility.
High
1
2
2
Test Organism
14. Adequacy and
Consistency of
Animal Husbandry
Conditions
Animal husbandry
conditions were reported
and included
temperature, humidity,
and light-dark cycle.
Husbandry conditions
were adequate and the
same for all animals.
High
1
1
1

15. Number per
Group
The number of animals
per group was reported
and appropriate for the
study type and outcome
analysis.
High
1
1
1
Outcome
Assessment
16. Outcome
Assessment
Methodology
The outcome assessment
methodology reported
and sensitive to the
intended outcomes of
interest.
High
1
2
2
17. Consistency of
Outcome
Assessment
Details of the outcome
assessment methodology
were reported and
consistent across study
groups for the outcomes
of interest.
High
1
1
1
78

-------
Study reference:
van der Ven, L. T. M.,van de Kuil, T.,Leonards, P. E. G.,Slob, W.,Lilienthal, H.,Litens, S.,Herlin,
M.,Hakansson, H.,Canton, R. F.,van den Berg, M.,Visser, T. J.,van Loveren, H.,Vos, J. G.,Picrsma, A. H.
(2009). Endocrine effects of hexabromocyclododecane (HBCD) in a one-generation reproduction study in
Wistar rats Toxicology Letters, 185(1), 51-62
HERO ID: 589273
Domain
Metric
Evaluator's Comment
Qualitative
Determination
[i.e.,High,Medium,
Low,Unacceptable,
or Not rated]
Metric
Score
Metric
Weighting
Factor
Weighted
Score

18. Sampling
Adequacy
Details regarding the
sampling for the
outcomes of interest
were reported and
adequate for assessment.
High
1
1
1

19. Blinding of
Assessors
This metric is not rated
when outcomes are not
subjective or for initial
histopathology review.
Not Rated
NR
NR
NR

20. Negative Control
Response
The biological response
of the negative control
group was adequate. As
shown in Supplemental
tables 1-16 (ID2919529)
High
1
1
1

21. Confounding
Variables in Test
Design and
Procedures
There were no reported
differences among the
study groups that could
influence the outcome
assessment.
Medium
2
2
4
Confounding /
Variable Control
22. Health Outcomes
Unrelated to
Exposure
Data on attrition or
health outcomes not
related to exposure were
not reported. The carrier
oil control group
experienced increased
mortality of F1 pups
during lactation and
several other health
outcomes. While not
related to HBDC
exposure, these effects
were influenced by the
carrier oil in the feed.
Medium
2
1
2
Data Presentation
and Analysis
23. Statistical
Methods
Statistical analysis was
shown for all datasets as
evaluated for
Supplemental tables 1-16
(ID2919529). BMD
methodology was clearly
described and
appropriate.
High
1
1
1
79

-------
Study reference:
van der Ven, L. T. M.,van de Kuil, T.,Leonards, P. E. G.,Slob, W.,Lilienthal, H.,Litens, S.,Herlin,
M.,Hakansson, H.,Canton, R. F.,van den Berg, M.,Visser, T. J.,van Loveren, H.,Vos, J. G.,Picrsma, A. H.
(2009). Endocrine effects of hexabromocyclododecane (HBCD) in a one-generation reproduction study in
Wistar rats Toxicology Letters, 185(1), 51-62
HERO ID: 589273
Domain
Metric
Evaluator's Comment
Qualitative
Determination
[i.e., High, Medium,
Low,Unacceptable,
or Not rated]
Metric
Score
Metric
Weighting
Factor
Weighted
Score

24. Reporting of
Data
Data for exposure-related
findings were presented
for all outcomes by
exposure group and sex -
as evaluated for
Supplemental tables 1-16
(ID2919529).
High
1
2
2
High: >=1 and <1.7
Medium: >=1.7 and <2.3
Low: >=2.3 and <=3
Sum of scores:

29
36
Overall Score = Sum of Weighted Scores/Sum
of Metric Weighting Factors:
1.2414
Overall Score:
Nearest *:
1.2
Overall Quality Level:
High
80

-------
3.4 Animal toxicity evaluation results of W. I. L. Research 2001 for
90-day gavage study on reproductive, hematological and
immune, neurological/behavior, renal, hepatic, ocular and
sensory, cardiovascular, clinical chemistry/biochemical,
endocrine, gastrointestinal, body weight, respiratory, thyroid
outcomes
Study reference:
W. I. L. Research (2001). 90-Day oral (gavage) toxicity study of HBCD in rats

HERO ID: 787787

Domain
Metric
Evaluator's Comment
Qualitative
Determination
[i.e.,High,Medium,
Low,Unacceptable,
or Not rated]
Metric
Score
Metric
Weighting
Factor
Weighted
Score

1. Test Substance
Identity
Identified by name.
High
1
2
2

2. Test Substance
Source
Manufacturer, lot no. and
composite sample nos.
High
1
1
1
Test Substance

Composite made from
commercial HBCD
products.

3. Test Substance
Purity
CK: HBCD, Alpha;
HBCD, Beta;
HBCD, Gamma; CAS
number 3194-55-6. The
standards had reported
purities of 99.4%, 100%
and 98.7%. respectively,
High
1
1
1

4. Negative and
Vehicle Controls
Vehicle (corn oil)
controls were used.
High
1
2
2
Test Design
5. Positive Controls
Positive controls are not
used for 90-day studies.
Not Rated
NR
NR
NR

6. Randomized
Allocation
Computerized
randomization.
High
1
1
1
Exposure
Characterization
7. Preparation and
Storage of Test
Substance
Stirred until uniform and
continuously throughout
used. Dosing
formulations were
prepared weekly.
High
1
1
1

8. Consistency of
Exposure
Administration
See footnote at end of
page.1
High
1
1
1
1 Metrics that received a "High" rating met the criteria as discussed in the Applications of Systematic Review for
TSCA Risk Evaluation.
81

-------

9. Reporting of
Doses/Concentration
s
Doses reported as
mg/kg/day, based on
most recent bw
measurement.
High
1
2
2

10. Exposure
Frequency and
Duration
90 consecutive days.
High
1
1
1

11. Number of
Exposure Groups
and Dose Spacing
3 treatment groups plus
control; not justified by
authors, but did produce
a range of response (i.e.,
thyroid).
High
1
1
1

12. Exposure Route
and Method
CK: Followed OECD
Guidelines
OECD Guideline 408
and OPPTS 870.3 100
High
1
1
1

13. Test Animal
Characteristics
Species, strain, sex, age,
and starting body weight
were reported
(commercial source).
High
1
2
2
Test Organism
14. Adequacy and
Consistency of
Animal Husbandry
Conditions
Husbandry conditions
were reported and
appropriate.
High
1
1
1

15. Number per
Group
15/sex/group
High
1
1
1

16. Outcome
Assessment
Methodology
Thorough outcome
assessments.
High
1
2
2

17. Consistency of
Outcome
Assessment
See footnote at end of
page.1
High
1
1
1

18. Sampling
Adequacy
See footnote at end of
page.1
High
1
1
1
Outcome
Assessment
19. Blinding of
Assessors
FOB testing was
performed without
knowledge of the animal
groups assignment.
Other outcomes were
objective.
CK: Functional
Observational Battery
(FOB) evaluations
High
1
1
1

20. Negative Control
Response
Low incidence of
histopath. lesions.
High
1
1
1
Confounding /
Variable Control
21. Confounding
Variables in Test
Design and
Procedures
See footnote at end of
page.1
High
1
2
2
22. Health Outcomes
See footnote at end of
page.1





Unrelated to
Exposure
High
1
1
1
82

-------
Data Presentation
and Analysis
23. Statistical
Methods
CK: Well described
High
1
1
1
24. Reporting of
Data
Summary and individual
animals tables were
included.
High
1
2
2
High: >=1 and <1.7
Medium: >=1.7 and <2.3
Low: >=2.3 and <=3
Sum of scores:

30
30
Overall Score = Sum of Weighted Scores/Sum
of Metric Weighting Factors:
1
Overall Score:
Nearest *:
1
Overall Quality Level:
High
1 Metrics that received a "High" rating met the criteria as discussec
Systematic Review for TSCA Risk Evaluation.
in the Applications of
83

-------
3.5 Animal toxicity evaluation results of Ema et al 2008 study on
reproductive, growth (early life) and development, hepatic,
neurological/behavior, thyroid outcomes
Study reference:
Ema, M.,Fujii, S.,Hirata-Koizumi, M.,Matsumoto, M. (2008). Two-generation reproductive toxicity study of
the flame retardant hexabromocyclododecane in rats Reproductive Toxicology, 25(3), 335-351
HERO ID: 787657
Domain
Metric
Evaluator's Comment
Qualitative
Determination
[i.e.,High,Medium,
Low,Unacceptable,
or Not rated]
Metric
Score
Metric
Weighting
Factor
Weighted
Score

1. Test Substance
Identity
The CASRN, purity,
mixture components, and
ratios were explicitly
specified.
High
1
2
2
Test Substance
2. Test Substance
Source
The manufacturer was
specified; test substance
number was reported. It
was indicated that the
purity and stability of the
test chemical were
verified using liquid
chromatography.
High
1
1
1

3. Test Substance
Purity
The test substance was
99.7% pure; therefore,
effects in the study were
highly likely to be due to
the test substance itself
(rather than any
unspecified impurities).
High
1
1
1

4. Negative and
Vehicle Controls
An appropriate
concurrent control group
was used (all of the
conditions the same
except exposure).
High
1
2
2
Test Design
5. Positive Controls
Positive control not
indicated by study type.
Not Rated
NR
NR
NR

6. Randomized
Allocation
The study indicates that
rats were randomly
assigned into study
groups.
High
1
1
1
Exposure
Characterization
7. Preparation and
Storage of Test
Substance
It was indicated that the
test substance was stored
in a sealed container
under cool and dark
conditions. The test
substance was well-
mixed in the diet
(homogeneous and stable
for at least 21 days).
High
1
1
1
84

-------
Study reference:
Ema, M.,Fujii, S.,Hirata-Koizumi, M.,Matsumoto, M. (2008). Two-generation reproductive toxicity study of
the flame retardant hexabromocyclododecane in rats Reproductive Toxicology, 25(3), 335-351
HERO ID: 787657
Domain
Metric
Evaluator's Comment
Qualitative
Determination
[i.e.,High,Medium,
Low,Unacceptable,
or Not rated]
Metric
Score
Metric
Weighting
Factor
Weighted
Score

8. Consistency of
Exposure
Administration
Analysis of the diet
indicated that the test
substance was
administered at the
desired feed
concentrations
throughout the study.
Animals were fed ad
libitum.
High
1
1
1

9. Reporting of
Doses/Concentration
s
Food consumption data
were recorded (provided
in the supplemental
data). Mean daily intakes
of the test substance for
various generations and
life stages (i.e. F0 and F1
males and females during
pre-mating, mating,
gestation, lactation, and
for the whole period of
administration) were
reported without
ambiguity.
High
1
2
2

10. Exposure
Frequency and
Duration
The exposure frequency
and duration were
appropriate for the study
type (and consistent with
OECD guidelines).
Mating was 3 weeks
(rather than 2 weeks
outlined by guideline).
High
1
1
1

11. Number of
Exposure Groups
and Dose Spacing
Three dose groups and a
concurrent control group
were used. Dosage levels
were based on the results
of a 90-day repeated-
dose toxicity study.
High
1
1
1

12. Exposure Route
and Method
The test substance was
administered in the diet
(oral route is
recommended by
guideline).
High
1
1
1
85

13. Test Animal
Characteristics
The animal species,
strain, sex, health, age,
and starting body
weights were reported.
Animals were purchased
from a commercial
laboratory. Crl:CD(SD)
rats were used because
they are the most
commonly used in
reproductive and
developmental toxicity
studies; historical control
data are available. The
rat is the preferred
species for testing
(according to guideline).
High
1
2
2
Test Organism
14. Adequacy and
Consistency of
Animal Husbandry
Conditions
Animals were housed
under the same
conditions (at the
temperature and
humidity recommended
by guideline). Animals
were housed individually
except during
acclimation, mating, and
nursing periods.
High
1
1
1

15. Number per
Group
No less than 20 pregnant
females per group is
preferred (but not always
possible). The study
utilized 24
rats/sex/group. Although
the number of pregnant
animals was only 19 for
high-dose F0 females,
the number of pregnant
females was adequate for
meaningful analyses of
the desired outcomes.
High
1
1
1
Outcome
Assessment
16. Outcome
Assessment
Methodology
The outcome assessment
methodology addressed
the intended outcomes
(mirrored guideline
recommendations for a
two-generation
reproductive toxicity
assay).
High
1
2
2
86

17. Consistency of
Outcome
Assessment
The outcomes were
measured consistently
across study groups.
High
1
1
1

18. Sampling
Adequacy
Reporting details were
provided; litter data were
recorded. Sampling was
adequate for the
outcomes of interest.
High
1
1
1

19. Blinding of
Assessors
Although the study does
not indicate that
investigators were
blinded to treatment
group, the study cited
various quality control
methods that were
followed.
High
1
1
1

20. Negative Control
Response
The response of the
negative controls was
reported and were
adequate (e.g. there were
no histological findings
in the thyroid of control
rats).
High
1
1
1

21. Confounding
Variables in Test
Design and
Procedures
There were no
differences in initial
body weights or intake
that could influence the
outcome assessment.
High
1
2
2
Confounding /
Variable Control
22. Health Outcomes
Unrelated to
Exposure
Details regarding animal
outcomes unrelated to
exposure (i.e. accidental
injury in the home cage)
were reported, but these
differences would not
influence the outcome
assessment.
High
1
1
1
Data Presentation
and Analysis
23. Statistical
Methods
Statistical methods were
clearly described.
High
1
1
1
87

-------
Study reference:
Ema, M.,Fujii, S.,Hirata-Koizumi, M.,Matsumoto, M. (2008). Two-generation reproductive toxicity study of
the flame retardant hexabromocyclododecane in rats Reproductive Toxicology, 25(3), 335-351
HERO ID: 787657
Domain
Metric
Evaluator's Comment
Qualitative
Determination
[i.e., High, Medium,
Low,Unacceptable,
or Not rated]
Metric
Score
Metric
Weighting
Factor
Weighted
Score

24. Reporting of
Data
Data were provided for
all exposure-related
findings by dose group.
The cutoff value for
decreased thyroid follicle
size was not reported, but
this is not likely to affect
the outcome of the study.
Additional data are
provided in the
supplemental document
(for example, date for
primordial follicles are
presented graphically in
the primary report;
quantitative data are
available in the
supplemental document).
High
1
2
2
High: >=1 and <1.7
Medium: >=1.7 and <2.3
Low: >=2.3 and <=3
Sum of scores:

30
30
Overall Score = Sum of Weighted Scores/Sum
of Metric Weighting Factors:
1
Overall Score:
Nearest *:
1
Overall Quality Level:
High
88

-------
3.6 Animal toxicity evaluation results of Lilienthal et al 2009
(787693) for 1-generation reproductive study, dietary exposure
study on neurological/behavior outcomes
Study reference:
Lilienthal, H.,van der Ven, L. T.,Piersma, A. H.,Vos, J. G. (2009). Effects of the brominated flame retardant
hexabromocyclododecane (HBCD) on dopamine-dependent behavior and brainstem auditory evoked
potentials in a one-generation reproduction study in Wistar rats Toxicology Letters, 185(1), 63-72
HERO ID: 787693
Domain
Metric
Evaluator's Comment
Qualitative
Determination
[i.e.,High,Medium,
Low,Unacceptable,
or Not rated]
Metric
Score
Metric
Weighting
Factor
Weighted
Score

1. Test Substance
Identity
Isomer composition of
HBCD was reported.
High
1
2
2

2. Test Substance
Source
Supplier was Bromine
Science and
Environmental Forum.
No information on lot or
batch and no analytical
verification was
described.
Medium
2
1
2
Test Substance
3. Test Substance
Purity
HBCD was a technical
mixture of three
diastereoisomers, alpha,
beta, and gamma-HBCD
at respective proportions
of 10.28%, 8.72%, and
81.02% with traces of
tetra- and
pentabromocyclododecan
e.
High
1
1
1

4. Negative and
Vehicle Controls
Untreated and vehicle
controls.
High
1
2
2
Test Design
5. Positive Controls
Positive controls were
not needed for
neurobehavioral studies.
Not Rated
NR
NR
NR

6. Randomized
Allocation
The study did not report
how animals were
allocated to study groups.
Low
3
1
3

7. Preparation and
Storage of Test
Substance
Preparation of test diets
was described; however,
the frequency of
preparation and store was
not indicated.
Medium
2
1
2
Exposure
Characterization
8. Consistency of
Exposure
Administration
Details of exposure
administration were
reported, and exposures
were administered
consistently across study
groups in a scientifically
sound manner.
High
1
1
1
89

-------
Study reference:
Lilienthal, H.,van der Ven, L. T.,Picrsma, A. H.,Vos, J. G. (2009). Effects of the brominated flame retardant
hexabromocyclododecane (HBCD) on dopamine-dependent behavior and brainstem auditory evoked
potentials in a one-generation reproduction study in Wistar rats Toxicology Letters, 185(1), 63-72
HERO ID: 787693
Domain
Metric
Evaluator's Comment
Qualitative
Determination
[i.e.,High,Medium,
Low,Unacceptable,
or Not rated]
Metric
Score
Metric
Weighting
Factor
Weighted
Score

9. Reporting of
Doses/Concentration
s
Dose in mg/kg/day were
calculated by study
authors.
High
1
2
2
10. Exposure
Frequency and
Duration
Continuous paternal and
maternal exposure during
premating, mating,
gestation, lactation and
after weaning in
offspring was reported.
High
1
1
1
11. Number of
Exposure Groups
and Dose Spacing
The number of exposure
groups and
dose/concentration
spacing were justified by
study authors and
considered adequate to
address the purpose of
the study.
High
1
1
1
12. Exposure Route
and Method
The route and method of
exposure were reported
and were suited to the
test substance.
High
1
1
1
Test Organism
13. Test Animal
Characteristics
Species, strain, sex and
starting age were
provided (commercial
source).
High
1
2
2
14. Adequacy and
Consistency of
Animal Husbandry
Conditions
Husbandry conditions
were reported and
appropriate.
Medium
2
1
2
15. Number per
Group
6/sex/group
High
1
1
1
Outcome
Assessment
16. Outcome
Assessment
Methodology
The outcome assessment
methodology addressed
or reported the intended
outcome(s) of interest
and was sensitive for the
outcomes(s) of interest.
High
1
2
2
17. Consistency of
Outcome
Assessment
Details of the outcome
assessment protocol were
reported, and outcomes
were assessed
consistently across study
groups.
High
1
1
1
90

-------
Study reference:
Lilienthal, H.,van der Ven, L. T.,Picrsma, A. H.,Vos, J. G. (2009). Effects of the brominated flame retardant
hexabromocyclododecane (HBCD) on dopamine-dependent behavior and brainstem auditory evoked
potentials in a one-generation reproduction study in Wistar rats Toxicology Letters, 185(1), 63-72
HERO ID: 787693
Domain
Metric
Evaluator's Comment
Qualitative
Determination
[i.e., High, Medium,
Low,Unacceptable,
or Not rated]
Metric
Score
Metric
Weighting
Factor
Weighted
Score

18. Sampling
Adequacy
Details regarding
sampling for the
outcome(s) of interest
were reported.
High
1
1
1
19. Blinding of
Assessors
The authors report that
"personnel conducting
the measurements were
unaware of the exposure
conditions" suggesting
the assessors were
blinded.
High
1
1
1
20. Negative Control
Response
The biological responses
of the negative control
group(s) were adequate.
High
1
1
1
Confounding /
Variable Control
21. Confounding
Variables in Test
Design and
Procedures
Initial body weight and
food/water intake were
not reported.
Low
3
2
6
22. Health Outcomes
Unrelated to
Exposure
Data on attrition and/or
health outcomes
unrelated to exposure
were not reported for
each study group.
Low
3
1
3
Data Presentation
and Analysis
23. Statistical
Methods
Statistics and BMD
modeling was reported.
High
1
1
1
24. Reporting of
Data
Test data and BMD
results were reported.
High
1
2
2
High: >=1 and <1.7
Medium: >=1.7 and <2.3
Low: >=2.3 and <=3
Sum of scores:

30
41
Overall Score = Sum of Weighted Scores/Sum
of Metric Weighting Factors:
1.3667
Overall Score:
Nearest *:
1.4
Overall Quality Level:
High
91

-------
3.7 Animal toxicity evaluation results of Saegusa et al 2009 for 1-
generation developmental toxicity (dietary exposure) study on
reproductive, growth (early life) and development,
neurological, hepatic, endocrine, thyroid, nutrition and
metabolic/adult exposure body weight outcomes
Study reference:
Saegusa, Y.,Fujimoto, H.,Woo, G. H.,Inoue, K.,Takahashi, M.,Mitsumori, K.,Hirose, M.,Nishikawa,
A.,Shibutani, M. (2009). Developmental toxicity of brominated flame retardants, tetrabromobisphenol A and
1,2,5,6,9,10-hexabromocyclododecane, in rat offspring after maternal exposure from mid-gestation through
lactation Reproductive Toxicology, 28(4), 456-467
HERO ID: 787721
Domain
Metric
Evaluator's Comment
Qualitative
Determination
[i.e.,High,Medium,
Low,Unacceptable,
or Not rated]
Metric
Score
Metric
Weighting
Factor
Weighted
Score

1. Test Substance
Identity
Identified by chemical
name and CASRN.
High
1
2
2
Test Substance
2. Test Substance
Source
Manufacturer and lot no.
were reported..
High
1
1
1

3. Test Substance
Purity
>95%
High
1
1
1

4. Negative and
Vehicle Controls
Concurrent negative
control.
High
1
2
2
Test Design
5. Positive Controls
Positive control not
needed developmental
studies.
Not Rated
NR
NR
NR

6. Randomized
Allocation
Randomized allocation.
High
1
1
1

7. Preparation and
Storage of Test
Substance
Test substance
preparation and storage
were not described.
Low
3
1
3

10. Exposure
Frequency and
Duration
Daily exposure during
critical developmental
periods.
High
1
1
1

11. Number of
Range-finding study was
used to set doses: 3
treatment groups plus
controls.

Exposure Groups
and Dose Spacing
High
1
1
1
92

-------
Study reference:
Saegusa, Y.,Fujimoto, H.,Woo, G. H.,Inoue, K.,Takahashi, M.,Mitsumori, K.,Hirose, M.,Nishikawa,
A.,Shibutani, M. (2009). Developmental toxicity of brominated flame retardants, tetrabromobisphenol A and
1,2,5,6,9,10-hexabromocyclododecane, in rat offspring after maternal exposure from mid-gestation through
lactation Reproductive Toxicology, 28(4), 456-467
HERO ID: 787721
Domain
Metric
Evaluator's Comment
Qualitative
Determination
[i.e.,High,Medium,
Low,Unacceptable,
or Not rated]
Metric
Score
Metric
Weighting
Factor
Weighted
Score

12. Exposure Route
and Method
The route and method of
exposure were reported
and were suited to the
test substance.
High
1
1
1

13. Test Animal
Characteristics
Test animals were
obtained from a
commercial source.
Species, strain, and
pregnancy status were
reported.
High
1
2
2
Test Organism
14. Adequacy and
Consistency of
Animal Husbandry
Conditions
Husbandry conditions
were reported and
appropriate.
High
1
1
1

16. Outcome
Assessment
Methodology
Thorough outcome
examinations pubertal
and adult necropsies).
High
1
2
2

17. Consistency of
Outcome
Assessment
Details of the outcome
assessment protocol were
reported and outcomes
were assessed
consistently across study
groups.
High
1
1
1
Outcome
Assessment
18. Sampling
Adequacy
Details regarding
sampling for the
outcome(s) of interest
were reported and the
study used adequate
sampling for the
outcome(s) of interest
(e.g., litter data provided
for developmental
studies; endpoints were
evaluated in an adequate
number of animals in
each group).
High
1
1
1
93

-------
Study reference:
Saegusa, Y.,Fujimoto, H.,Woo, G. H.,Inoue, K.,Takahashi, M.,Mitsumori, K.,Hirose, M.,Nishikawa,
A.,Shibutani, M. (2009). Developmental toxicity of brominated flame retardants, tetrabromobisphenol A and
1,2,5,6,9,10-hexabromocyclododecane, in rat offspring after maternal exposure from mid-gestation through
lactation Reproductive Toxicology, 28(4), 456-467
HERO ID: 787721
Domain
Metric
Evaluator's Comment
Qualitative
Determination
[i.e., High, Medium,
Low,Unacceptable,
or Not rated]
Metric
Score
Metric
Weighting
Factor
Weighted
Score

19. Blinding of
Assessors
Blinding was not
reported, but outcomes
were objective.
Medium
2
1
2
20. Negative Control
Response
No histopathology lesion
in controls.
High
1
1
1
Confounding /
Variable Control
21. Confounding
Variables in Test
Design and
Procedures
No differences among
groups in food
consumption and body
weight.
High
1
2
2
22. Health Outcomes
Unrelated to
Exposure
Data on attrition and/or
health outcomes
unrelated to exposure
were not reported for
each study group
Low
3
1
3
Data Presentation
and Analysis
23. Statistical
Methods
Statistical methods were
clearly described and
appropriate for
dataset(s).
High
1
1
1
24. Reporting of
Data
HBCD caused a dose-
dependent decrease in
Cingulate deep cortex
CNPase (+) cell count,
which was significantly
lower at the highest dose
exposed.
Medium
2
2
4
High: >=1 and <1.7
Medium: >=1.7 and <2.3
Low: >=2.3 and <=3
Sum of scores:

30
37
Overall Score = Sum of Weighted Scores/Sum
of Metric Weighting Factors:
1.2333
Overall Score:
Nearest *:
1.2
Overall Quality Level:
High
94

-------
3.8 Animal toxicity evaluation results of Yanagisawa et al 2014 for
14-week study (animals dosed by gavage lx per week) study on
hepatic, body weight, nutrition and metabolic/adult exposure
body weight outcomes
Study reference:
Yanagisawa, R.,Koike, E.,Win-Shwe, T. T.,Yamamoto, M.,Takano, H. (2014). Impaired lipid and glucose
homeostasis in hexabromocyclododecane-exposed mice fed a high-fat diet Environmental Health Perspectives,
122(3), 277-283

HERO ID: 2343717

Domain
Metric
Evaluator's Comment
Qualitative
Determination
[i.e.,High,Medium,
Low,Unacceptable,
or Not rated]
Metric
Score
Metric
Weighting
Factor
Weighted
Score

1. Test Substance
Identity
Test substance described
as HBCD, study did not
indicate whether the test
substance was composed
of different isomers (as
other studies have).
Medium
2
2
4
Test Substance
2. Test Substance
Source
Sigma Aldrich - no
catalog #
High
1
1
1

3. Test Substance
Purity
Purity was not reported,
however, products
purchased from Sigma
for experimental use are
generally >95% pure.
Medium
2
1
2

4. Negative and
Vehicle Controls
an appropriate vehicle
control was used
High
1
2
2
Test Design
5. Positive Controls
Positive control was not
necessary
Not Rated
NR
NR
NR
6. Randomized
Allocation
Mice were randomly
allocated. There were no
differences in initial
BWs
High
1
1
1

7. Preparation and
Storage of Test
Substance
Preparation of the test
substance was described,
but the frequency of
preparation and storage
were not reported.
Medium
2
1
2
Exposure
Characterization
8. Consistency of
Exposure
Administration
All groups appeared to
be treated consistently
High
1
1
1
9. Reporting of
Doses/Concentration
s
Dosing was clearly
reported, although
reported as mg/kg/week
CK: Dosing was reported
as ng/kg BW/week, not
as mg/kg/week
High
1
2
2
95

-------
Study reference:
Yanagisawa, R.,Koike, E.,Win-Shwe, T. T.,Yamamoto, M.,Takano, H. (2014). Impaired lipid and glucose
homeostasis in hexabromocyclododecane-exposed mice fed a high-fat diet Environmental Health Perspectives,
122(3), 277-283
HERO ID: 2343717
Domain
Metric
Evaluator's Comment
Qualitative
Determination
[i.e.,High,Medium,
Low,Unacceptable,
or Not rated]
Metric
Score
Metric
Weighting
Factor
Weighted
Score

10. Exposure
Frequency and
Duration
Animals were only given
the test substance
lx/week via oral gavage.
This is not a standard
frequency of
administration, and there
is no discussion in the
text indicating reasoning
for the chosen dosing
frequency. It is an
unusual frequency to
evaluate the toxicological
effects of the test
substance on mice fed
different diets.
Unacceptable
4
1
4

11. Number of
Exposure Groups
and Dose Spacing
Three exposure groups
and a control.
Justification for exposure
levels was provided.
High
1
1
1

12. Exposure Route
and Method
Method of gavage is
acceptable, although it is
unclear in this case, why
a spiked dietary
administration wasn't
used instead.
High
1
1
1

13. Test Animal
Characteristics
Animals, and animal
characteristics were all
reported, however, only
males were used, for a
~90-day repeated dose
study; OECD guideline
recommends testing on
both sexes
Medium
2
2
4
Test Organism
14. Adequacy and
Consistency of
Animal Husbandry
Conditions
Animal husbandry
conditions were
appropriate
High
1
1
1

15. Number per
Group
Only 5-6 animals/group;
OECD guideline for 90-
day repeated dose study
recommends a minimum
of 8 animals/group (4
males and 4 females)
Medium
2
1
2
Outcome
Assessment
16. Outcome
Assessment
Methodology
Methods used to assess
outcomes were
appropriate
High
1
2
2
96

-------
Study reference:
Yanagisawa, R.,Koike, E.,Win-Shwe, T. T.,Yamamoto, M.,Takano, H. (2014). Impaired lipid and glucose
homeostasis in hexabromocyclododecane-exposed mice fed a high-fat diet Environmental Health Perspectives,
122(3), 277-283
HERO ID: 2343717
Domain
Metric
Evaluator's Comment
Qualitative
Determination
[i.e., High, Medium,
Low,Unacceptable,
or Not rated]
Metric
Score
Metric
Weighting
Factor
Weighted
Score

17. Consistency of
Outcome
Assessment
There was consistency
across the groups that
were tested
High
1
1
1

18. Sampling
Adequacy
A number of endpoints
were only done using
controls and high-dose
groups, even though
significant changes were
supposedly observed in
the medium-dose group
for other endpoints. This
precludes the ability to
evaluate dose-response
for these endpoints
Medium
2
1
2

19. Blinding of
Assessors
Study indicates histology
was done in a blinded
fashion.
High
1
1
1

20. Negative Control
Response
No unexpected negative
control responses were
reported
High
1
1
1
Confounding /
21. Confounding
Variables in Test
Design and
Procedures
No confounding
variables were identified.
High
1
2
2
Variable Control
22. Health Outcomes
Unrelated to
Exposure
No unusual health
outcomes un-related to
the exposure were
identified
High
1
1
1
Data Presentation
and Analysis
23. Statistical
Methods
Statistical analysis was
clearly described and
appropriate
High
1
1
1
24. Reporting of
Data
Data presentation was
adequate; histological
data was presented as
images only
Medium
2
2
4

Sum of scores:

30
43
High: >=
Medium: >=
1 and <1.7
=1.7 and <2.3
Overall Score = Sum of Weighted Scores/Sum
of Metric Weighting Factors:
1.4333
Overall Score
(Rounded):
1.41
Low: >=2.5 and <=j

Overall Quality Level:

Unacceptable1

-------
Study reference:
Yanagisawa, R.,Koike, E.,Win-Shwe, T. T.,Yamamoto, M.,Takano, H. (2014). Impaired lipid and glucose
homeostasis in hexabromocyclododecane-exposed mice fed a high-fat diet Environmental Health Perspectives,
122(3), 277-283
HERO ID: 2343717
Domain
Metric
Evaluator's Comment
Qualitative
Determination
[i.e.,High,Medium,
Low,Unacceptable,
or Not rated]
Metric
Score
Metric
Weighting
Factor
Weighted
Score
Comment:
Footnote:
1 Consistent with our Application of Systematic Review in TSCA Risk Evaluations document, if a metric for a
data source receives a score of Unacceptable (score = 4), EPA will determine the study to be unacceptable. In
this case, one of the metrics was rated as unacceptable. As such, the study is considered unacceptable and the
score is presented solely to increase transparency.
98

-------
4 In Vitro Studies
4.1 In vitro evaluation results of 1990.
Study reference:
(1990). LETTER FROM AMERIBROM INC TO US EPA REGARDING 8D SUBMISSION FOR
HEXABROMOCYCLODODECANE WITH ATTACHMENTS
HERO ID: 1928284
Domain
Metric
Evaluator's Comment
Qualitative
Determination
[i.e., High, Medium,
Low,Unacceptable,
or Not rated]
Metric
Score
Metric
Weighting
Factor
Weighted
Score
Test Substance
1. Test Substance
Identity
Test substance identified
by name, chemical
formula, and physical
chemical properties.
High
1
2
2
2. Test Substance
Source
Source not identified.
Low
3
1
3

3. Test Substance
Purity
Purity not reported.
Low
3
1
3

4. Negative and
Vehicle Controls
Negative controls were
included.
High
1
2
2
Test Design
5. Positive Controls
Positive controls were
included.
High
1
2
2
6. Assay procedures
Assay procedures were
described.
High
1
1
1

7. Standards for tests
Criteria not required.
Not Rated
NR
NR
NR

8. Preparation and
Storage of Test
Substance
Preparation details were
described.
High
1
1
1

9. Consistency of
Exposure
Administration
Exposures were
administered
consistently.
High
1
1
1
Exposure
Characterization
10. Reporting of
Doses/Concentration
s
Concentrations were
reported.
High
1
2
2
11. Number of
Exposure Groups
and Concentration
Spacing
Duration was reported.
High
1
2
2

12. Exposure Route
and Method
The number of groups
and spacing were
reported with
justification.
High
1
1
1

13. Metabolic
Activation
Activation system and
mix were described.
High
1
1
1
Test Model
14. Test Model
Test models were well
described.
High
1
2
2
99

-------
Study reference:
(1990). LETTER FROM AMERIBROM INC TO US EPA REGARDING 8D SUBMISSION FOR
HEXABROMOCYCLODODECANE WITH ATTACHMENTS
HERO ID: 1928284
Domain
Metric
Evaluator's Comment
Qualitative
Determination
[i.e., High, Medium,
Low,Unacceptable,
or Not rated]
Metric
Score
Metric
Weighting
Factor
Weighted
Score

15. Number per
Group
An overnight culture was
used for experiments, but
exact number of cells not
reported. The number of
replicates was reported.
Medium
2
1
2
Outcome
Assessment
16. Outcome
Assessment
Methodology
Outcome assessment
methodology was
described.
High
1
2
2
17. Consistency of
Outcome
Assessment
Outcomes were assessed
consistently.
High
1
1
1
18. Sampling
Adequacy
Sampling was adequate.
High
1
2
2
19. Blinding of
Assessors
Blinding was not
required.
Not Rated
NR
NR
NR
Confounding /
Variable Control
20. Confounding
Variables in Test
Design and
Procedures
No confounding
variables were reported.
High
1
2
2
21. Confounding
Variables in
Outcomes Unrelated
to Exposure
No outcomes unrelated
to exposure were
reported.
High
1
1
1
Data Presentation
and Analysis
22. Data Analysis
Statistical methods were
described.
High
1
1
1
23. Data
Interpretation
Criteria for positive
finding was described.
High
1
2
2
24. Cytotoxicity
Data
A preliminary
cytotoxicity assay was
conducted.
High
1
1
1
25. Reporting of
Data
Data were reported.
High
1
2
2
High: >=1 and <1.7
Medium: >=1.7 and <2.3
Low: >=2.3 and <=3
Sum of scores:

34
39
Overall Score = Sum of Weighted Scores/Sum
of Metric Weighting Factors:
1.1471
Overall Score:
Nearest *:
1.1
Overall Quality Level:
High
100

-------
4.2 In vitro evaluation results of Almughamsi et al 2016
Study reference:
Almughamsi, H.,Whalen, M. M. (2016). Hexabromocyclododecane and tetrabromobisphenol A alter secretion
of interferon gamma (IFN-?) from human immune cells Archives of Toxicology, 90(7), 1695-1707
HERO ID: 3350524
Domain
Metric
Evaluator's Comment
Qualitative
Determination
[i.e., High, Medium,
Low,Unacceptable,
or Not rated]
Metric
Score
Metric
Weighting
Factor
Weighted
Score

1. Test Substance
Identity
Test substance identified
by name.
Medium
2
2
4
Test Substance
2. Test Substance
Source
Source was identified.
Medium
2
1
2

3. Test Substance
Purity
Purity/grade and/or
composition were not
reported.
Low
3
1
3

4. Negative and
Vehicle Controls
Concurrent controls were
included.
High
1
2
2
Test Design
5. Positive Controls
Positive controls not
required.
Not Rated
NR
NR
NR
6. Assay procedures
Assay procedures were
reported.
High
1
1
1

7. Standards for tests
No standards were
required for the assays.
Not Rated
NR
NR
NR

8. Preparation and
Storage of Test
Substance
Limited preparation
details were provided
and not storage or
stability data were
reported.
Medium
2
1
2

11. Number of
Exposure Groups
and Concentration
Spacing
Durations were reported.
High
1
2
2

12. Exposure Route
and Method
The number of groups
and spacing were
reported but not justified.
Medium
2
1
2

13. Metabolic
Activation
Not required for the
assay.
Not Rated
NR
NR
NR
Test Model
14. Test Model
The test models and
sources were identified
and appropriate.
High
1
2
2

15. Number per
Group
The number of cells
exposure were reported.
High
1
1
1
101

-------
Study reference:
Almughamsi, H.,Whalen, M. M. (2016). Hexabromocyclododecane and tetrabromobisphenol A alter secretion
of interferon gamma (IFN-?) from human immune cells Archives of Toxicology, 90(7), 1695-1707
HERO ID: 3350524
Domain
Metric
Evaluator's Comment
Qualitative
Determination
[i.e., High, Medium,
Low,Unacceptable,
or Not rated]
Metric
Score
Metric
Weighting
Factor
Weighted
Score
Outcome
Assessment
16. Outcome
Assessment
Methodology
Outcome assessment
methodology was
reported.
High
1
2
2
17. Consistency of
Outcome
Assessment
Outcomes were assessed
consistently.
High
1
1
1
18. Sampling
Adequacy
Sampling was adequate.
High
1
2
2
19. Blinding of
Assessors
Blinding not required.
Not Rated
NR
NR
NR
Confounding /
Variable Control
20. Confounding
Variables in Test
Design and
Procedures
No confounding
variables in test design
were reported.
High
1
2
2
21. Confounding
Variables in
Outcomes Unrelated
to Exposure
No confounding
variables in outcomes
unrelated to exposures
were reported.
High
1
1
1
Data Presentation
and Analysis
22. Data Analysis
Statistical methods were
reported and appropriate.
High
1
1
1
23. Data
Interpretation
Metric not required.
Not Rated
NR
NR
NR
24. Cytotoxicity
Data
Cell viability methods
were defined and
described.
High
1
1
1
25. Reporting of
Data
Data were reported.
High
1
2
2
High: >=1 and <1.7
Medium: >=1.7 and <2.3
Low: >=2.3 and <=3
Sum of scores:

29
36
Overall Score = Sum of Weighted Scores/Sum
of Metric Weighting Factors:
1.2414
Overall Score:
Nearest *:
1.2
Overall Quality Level:
High
102

-------
4,3 In vitro evaluation results of An et al 2016
Study reference:
An, J.,Guo, P.,Shang, Y.,Zhong, Y.,Zhang, X.,Yu, Y.,Yu, Z. (2016). The adaptive response; of low
concentrations of HBCD in L02 cells and the underlying molecular mechanisms Chemosphere, 145, 68-76
HERO ID: 3350502
Domain
Metric
Evaluator's Comment
Qualitative
Determination
[i.e., High, Medium,
Low,Unacceptable,
or Not rated]
Metric
Score
Metric
Weighting
Factor
Weighted
Score
Test Substance
1. Test Substance
Identity
Test substance identified
by name.
Medium
2
2
4
2. Test Substance
Source
Source identified.
Medium
2
1
2
3. Test Substance
Purity
Purity/composition was
not reported but was
reported to be analytical
reagents.
Medium
2
1
2
Test Design
4. Negative and
Vehicle Controls
Negative controls were
included.
High
1
2
2
5. Positive Controls
Positive controls not
required.
Not Rated
NR
NR
NR
6. Assay procedures
Assay procedures were
described.
High
1
1
1
7. Standards for tests
No standards were
required.
Not Rated
NR
NR
NR
Exposure
Characterization
8. Preparation and
Storage of Test
Substance
Preparation, storage, and
stability information
were not reported.
Low
3
1
3
9. Consistency of
Exposure
Administration
Exposures were
administered
consistently.
High
1
1
1
10. Reporting of
Doses/Concentration
s
Concentrations were
reported.
High
1
2
2
11. Number of
Exposure Groups
and Concentration
Spacing
Exposure durations were
reported.
High
1
2
2
12. Exposure Route
and Method
The number of exposure
groups and spacing were
reported and justified.
High
1
1
1
13. Metabolic
Activation
Metabolic activation was
not required.
Not Rated
NR
NR
NR
Test Model
14. Test Model
The test model was
described with limited
details, and the source
was not reported.
Medium
2
2
4
103

-------
Study reference:
An, J.,Guo, P.,Shang, Y.,Zhong, Y.,Zhang, X.,Yu, Y.,Yu, Z. (2016). The adaptive response; of low
concentrations of HBCD in L02 cells and the underlying molecular mechanisms Chemosphere, 145, 68-76
HERO ID: 3350502
Domain
Metric
Evaluator's Comment
Qualitative
Determination
[i.e., High, Medium,
Low,Unacceptable,
or Not rated]
Metric
Score
Metric
Weighting
Factor
Weighted
Score

15. Number per
Group
The number of
organisms exposed was
not reported for all
experiments.
Medium
2
1
2
Outcome
Assessment
16. Outcome
Assessment
Methodology
Outcome assessment
methodology was
reported.
High
1
2
2
17. Consistency of
Outcome
Assessment
Outcomes were assessed
consistently.
High
1
1
1
18. Sampling
Adequacy
Sampling was adequate.
High
1
2
2
19. Blinding of
Assessors
Blinding was not
applicable.
Not Rated
NR
NR
NR
Confounding /
Variable Control
20. Confounding
Variables in Test
Design and
Procedures
No confounding
variables in test design
were reported.
High
1
2
2
21. Confounding
Variables in
Outcomes Unrelated
to Exposure
No outcomes unrelated
to exposure were
reported.
High
1
1
1
Data Presentation
and Analysis
22. Data Analysis
Statistical methods were
reported and appropriate.
High
1
1
1
23. Data
Interpretation
Not required for these
assays.
Not Rated
NR
NR
NR
24. Cytotoxicity
Data
Cell viability methods
were described.
High
1
1
1
25. Reporting of
Data
Data were reported.
High
1
2
2
High: >=1 and <1.7
Medium: >=1.7 and <2.3
Low: >=2.3 and <=3
Sum of scores:

29
38
Overall Score = Sum of Weighted Scores/Sum
of Metric Weighting Factors:
1.3103
Overall Score:
Nearest *:
1.3
Overall Quality Level:
High
104

-------
4.4 In vitro evaluation results of Anisuzzaman et al 2016
Study reference:
Anisuzzaman, S.,Whalen, M. M. (2016). Tetrabromobisphenol A and hexabromocyclododecane alter
secretion of IL-1 from human immune cells Journal of Immunotoxicology, 13(3), 403-416
HERO ID: 3350463
Domain
Metric
Evaluator's Comment
Qualitative
Determination
[i.e., High, Medium,
Low,Unacceptable,
or Not rated]
Metric
Score
Metric
Weighting
Factor
Weighted
Score
Test Substance
1. Test Substance
Identity
Test substance identified
by name.
Medium
2
2
4
2. Test Substance
Source
The source was
identified.
Medium
2
1
2
3. Test Substance
Purity
Purity was not reported.
Low
3
1
3
Test Design
4. Negative and
Vehicle Controls
Concurrent negative
controls were included
High
1
2
2
5. Positive Controls
Positive controls not
required.
Not Rated
NR
NR
NR
6. Assay procedures
Assay procedures were
described.
High
1
1
1
7. Standards for tests
No standards required.
Not Rated
NR
NR
NR
Exposure
Characterization
8. Preparation and
Storage of Test
Substance
Limited preparation
details were reported, but
not information about
stability and storage were
reported.
Medium
2
1
2
9. Consistency of
Exposure
Administration
Exposures were
administered
consistently.
Medium
2
1
2
10. Reporting of
Doses/Concentration
s
Concentrations were
reported.
High
1
2
2
11. Number of
Exposure Groups
and Concentration
Spacing
Duration of exposure
was reported.
High
1
2
2
12. Exposure Route
and Method
The number of exposure
groups and concentration
spacing were reported,
and the rationale for
selected was reported.
High
1
1
1
13. Metabolic
Activation
Metabolic activation not
required.
Not Rated
NR
NR
NR
Test Model
14. Test Model
Test model and source
information were
reported.
High
1
2
2
15. Number per
Group
The number of cells was
reported.
High
1
1
1
105

-------
Study reference:
Anisuzzaman, S.,Whalen, M. M. (2016). Tetrabromobisphenol A and hexabromocyclododecane alter
secretion of IL-1 from human immune cells Journal of Immunotoxicology, 13(3), 403-416
HERO ID: 3350463
Domain
Metric
Evaluator's Comment
Qualitative
Determination
[i.e., High, Medium,
Low,Unacceptable,
or Not rated]
Metric
Score
Metric
Weighting
Factor
Weighted
Score

16. Outcome
Outcome assessment was
reported.

Assessment
Methodology
High
1
2
2

17. Consistency of
Outcome
Assessment
Outcomes were assessed
High
1
1
1
Outcome
Assessment
consistently.
18. Sampling
Adequacy
Sampling was adequate
for the outcomes of
interest.
High
1
2
2

19. Blinding of
Assessors
No outcomes required
blinding.
Not Rated
NR
NR
NR
Confounding /
20. Confounding
Variables in Test
Design and
Procedures
There were no reported
differences among study
group parameters.

NR
2
NR
Variable Control
21. Confounding
Variables in
Outcomes Unrelated
to Exposure
No reported outcome
differences among study
groups unrelated to
exposure were reported.
High
1
1
1

22. Data Analysis
Statistical analysis was
reported and appropriate.
High
1
1
1
Data Presentation
and Analysis
23. Data
Interpretation
Scoring and evaluation
criteria not required.
Not Rated
NR
NR
NR
24. Cytotoxicity
Data
Cell viability was
defined and methods
were described.
High
1
1
1

25. Reporting of
Data
All data were reported.
High
1
2
2

Sum of scores:

27
34
High: >=
Medium: >=
1 and <1.7
=1.7 and <2.3
Overall Score = Sum of Weighted Scores/Sum
of Metric Weighting Factors:
1.2593
Overall Score:
Nearest *:
1.3
Low: >=2.5 and <=j

Overall Quality Level:

High

106

-------
4.5 In vitro evaluation results of Canbaz et al 2016
Study reference:
Canbaz, D.,Lebre, M. C.,Logiantara, A.,van Ree, R.,van Rijt, L. S. (2016). Indoor pollutant
hexabromocyclododecane enhances house dust mite-induced activation of human monocyte-derived dendritic
cells Journal of Immunotoxicology, 13(6), 1-7
HERO ID: 3355511
Domain
Metric
Evaluator's Comment
Qualitative
Determination
[i.e., High, Medium,
Low,Unacceptable,
or Not rated]
Metric
Score
Metric
Weighting
Factor
Weighted
Score

1. Test Substance
Identity
Test substance identified
by name.
Medium
2
2
4

2. Test Substance
Source
Source identified.
Medium
2
1
2
Test Substance
3. Test Substance
Purity
Test substance described
as technical mixture, but
purity/grade and/or
composition were not
reported.
Low
3
1
3

4. Negative and
Vehicle Controls
Concurrent negative
controls were used.
High
1
2
2
Test Design
5. Positive Controls
Positive controls not
required.
Not Rated
NR
NR
NR
6. Assay procedures
Assay procedures were
reported.
High
1
1
1

7. Standards for tests
Standards not required
for assays.
Not Rated
NR
NR
NR

8. Preparation and
Storage of Test
Substance
Limited preparation
details were reported, but
stability and storage were
not.
Medium
2
1
2

9. Consistency of
Exposure
Administration
Exposures were
administered
consistently.
High
1
1
1
Exposure
Characterization
10. Reporting of
Doses/Concentration
s
Concentrations were
administered
consistently.
High
1
2
2
11. Number of
Exposure Groups
and Concentration
Spacing
Durations were reported.
High
1
2
2

12. Exposure Route
and Method
The number of groups
and spacing were
reported nut not justified.
Medium
2
1
2

13. Metabolic
Activation
Activation not required.
Not Rated
NR
NR
NR
Test Model
14. Test Model
Test model and donor
information were
provided.
High
1
2
2
107

-------
Study reference:
Canbaz, D.,Lebre, M. C.,Logiantara, A.,van Ree, R.,van Rijt, L. S. (2016). Indoor pollutant
hexabromocyclododecane enhances house dust mite-induced activation of human monocyte-derived dendritic
cells Journal of Immunotoxicology, 13(6), 1-7
HERO ID: 3355511
Domain
Metric
Evaluator's Comment
Qualitative
Determination
[i.e., High, Medium,
Low,Unacceptable,
or Not rated]
Metric
Score
Metric
Weighting
Factor
Weighted
Score

15. Number per
Group
The number of cells per
group in the initial
exposure assay was not
reported, but was
reported for the cytokine
assay.
Medium
2
1
2
Outcome
Assessment
16. Outcome
Assessment
Methodology
Outcome assessment
methodology was
described
High
1
2
2
17. Consistency of
Outcome
Assessment
Outcomes were assessed
consistently.
High
1
1
1
18. Sampling
Adequacy
Sampling was adequate.
High
1
2
2
19. Blinding of
Assessors
Blinding was not
required.
Not Rated
NR
NR
NR
Confounding /
Variable Control
20. Confounding
Variables in Test
Design and
Procedures
No confounding
variables in test design
were observed.
High
1
2
2
21. Confounding
Variables in
Outcomes Unrelated
to Exposure
Two donors did not yield
sufficient cells to
perform all experiments.
Medium
2
1
2
Data Presentation
and Analysis
22. Data Analysis
Statistical methods were
reported and appropriate.
High
1
1
1
23. Data
Interpretation
Data interpretation
criteria not required.
Not Rated
NR
NR
NR
24. Cytotoxicity
Data
Methods were not
reported but the data
were provided.
Low
3
1
3
25. Reporting of
Data
Data were reported.
High
1
2
2
High: >=1 and <1.7
Medium: >=1.7 and <2.3
Low: >=2.3 and <=3
Sum of scores:

29
40
Overall Score = Sum of Weighted Scores/Sum
of Metric Weighting Factors:
1.3793
Overall Score:
Nearest *:
1.4
Overall Quality Level:
High
108

-------
109

-------
4.6 In vitro evaluation results of Ethyl Corporation 1990
Study reference:
Ethyl Corporation (1990). Genetic toxicology salmonella/microsomal assay on hexabromocyclododecane with
cover letter dated 030890
HERO ID: 787661
Domain
Metric
Evaluator's Comment
Qualitative
Determination
[i.e.,High,Medium,
Low,Unacceptable,
or Not rated]
Metric
Score
Metric
Weighting
Factor
Weighted
Score

1. Test Substance
Identity
Test substance identified
as HBCD Bottoms, a
brittle black solid.
Low
3
2
6
Test Substance
2. Test Substance
Source
Source identified as a
person but may have
worked for the Sponsor
of the study.
Low
3
1
3

3. Test Substance
Purity
Purity was not reported.
Low
3
1
3

4. Negative and
Vehicle Controls
Negative and solvent
controls were included.
High
1
2
2

5. Positive Controls
Positive controls were
included but were not
identified.
Medium
2
2
4
Test Design
6. Assay procedures
The methods and
procedures were not well
described and most
details were not reported.
Company SOP numbers
were reported.
Low
3
1
3

7. Standards for tests
Criteria were not
required.
Not Rated
NR
NR
NR

8. Preparation and
Storage of Test
Substance
Limited details on
preparation were
reported and no storage
details were reported.
Low
3
1
3

9. Consistency of
Exposure
Administration

Low
3
1
3
Exposure
Characterization
10. Reporting of
Doses/Concentration
s
Concentrations were
reported.
High
1
2
2
11. Number of
Exposure Groups
and Concentration
Spacing
Duration was not
reported but may be
referenced in the SOP
documents.
Low
3
2
6

12. Exposure Route
and Method
The number of groups
and spacing were
reported, but not
justified.
Medium
2
1
2

13. Metabolic
Activation
Metabolic activation was
reported, but details were
not provided.
Medium
2
1
2
no

-------
Study reference:
Ethyl Corporation (1990). Genetic toxicology salmonella/microsomal assay on hexabromocyclododecane with
cover letter dated 030890
HERO ID: 787661
Domain
Metric
Evaluator's Comment
Qualitative
Determination
[i.e., High, Medium,
Low,Unacceptable,
or Not rated]
Metric
Score
Metric
Weighting
Factor
Weighted
Score
Test Model
14. Test Model
The strains and source
were reported, but
additional details were
not provided
Medium
2
2
4
15. Number per
Group
The number of exposed
cells per group was not
reported but may be
found in the SOP
documents.
Low
3
1
3
Outcome
Assessment
16. Outcome
Assessment
Methodology
The outcome assessment
methodology was not
reported but may be
found in the SOP
documents.
Low
3
2
6
17. Consistency of
Outcome
Assessment
Details were not
reported.
Low
3
1
3
18. Sampling
Adequacy
Details were not
reported.
Low
3
2
6
19. Blinding of
Assessors
Blinding was not
required.
Not Rated
NR
NR
NR
Confounding /
Variable Control
20. Confounding
Variables in Test
Design and
Procedures
Insufficient details were
reported to determine.
Low
3
2
6
21. Confounding
Variables in
Outcomes Unrelated
to Exposure
Insufficient details were
reported to determine.
Low
3
1
3
Data Presentation
and Analysis
22. Data Analysis
Calculations were not
described clearly but
inferences could be
made.
Low
3
1
3
23. Data
Interpretation
Evaluation criteria were
reported.
Medium
2
2
4
24. Cytotoxicity
Data
Cytotoxicity was not
reported to have been
evaluated.
Not Rated
NR
NR
NR
25. Reporting of
Data
Data were reported.
High
1
2
2
High: >-1 and <1.7
Medium: >=1.7 and <2.3
Low: >=2.3 and <=3
Sum of scores:

33
79
Overall Score = Sum of Weighted Scores/Sum
of Metric Weighting Factors:
2.3939
Overall Score:
Nearest *:
2.4
Ill

-------
Study reference:
Ethyl Corporation (1990). Genetic toxicology salmonella/microsomal assay on hexabromocyclododecane with
cover letter dated 030890
HERO ID: 787661
Domain
Metric
Evaluator's Comment
Qualitative
Determination
[i.e.,High,Medium,
Low,Unacceptable,
or Not rated]
Metric
Score
Metric
Weighting
Factor
Weighted
Score

Overall Quality Level:
Low
112

-------
4.7 In vitro evaluation results of Ethyl Corporation 1990
Study reference:
Ethyl Corporation (1990). Genetic toxicology rat hepatocyte primary culture/DNR repair test on
hexabromocyclododecane with cover letter dated 030890
HERO ID: 1928253
Domain
Metric
Evaluator's Comment
Qualitative
Determination
[i.e., High, Medium,
Low,Unacceptable,
or Not rated]
Metric
Score
Metric
Weighting
Factor
Weighted
Score

1. Test Substance
Identity
Test substance identified
as NBCD Bottoms,
brittle black solid.
Medium
2
2
4
Test Substance
2. Test Substance
Source
Source not identified.
Low
3
1
3

3. Test Substance
Purity
Purity not reported.
Low
3
1
3

4. Negative and
Vehicle Controls
Solvent control included.
High
1
2
2

5. Positive Controls
Positive control included.
High
1
2
2
Test Design
6. Assay procedures
Assay methods were
reported for harvesting
cells and data
quantifications, but
limited details regarding
treatment were reported.
Low
3
1
3

7. Standards for tests
Criteria not required.
Not Rated
NR
NR
NR

8. Preparation and
Storage of Test
Substance
Limited preparation
details and no storage
details were reported.
Low
3
1
3

9. Consistency of
Exposure
Administration
Insufficient data were
reported.
Low
3
1
3
Exposure
10. Reporting of
Doses/Concentration
s
Concentrations were
reported.
High
1
2
2
Characterization
11. Number of
Exposure Groups
and Concentration
Spacing
Durations not reported
but may be found in the
reported SOP.
Low
3
2
6

12. Exposure Route
and Method
The number of groups
and spacing were
reported but not justified.
Medium
2
1
2

13. Metabolic
Activation
Metabolic activation was
not required.
Not Rated
NR
NR
NR

14. Test Model
The test model was
described.
High
1
2
2
Test Model
15. Number per
Group
The number of cells was
not reported but may be
found in the reported
SOP.
Low
3
1
3
113

-------
Study reference:
Ethyl Corporation (1990). Genetic toxicology rat hepatocyte primary culture/DNR repair test on
hexabromocyclododecane with cover letter dated 030890
HERO ID: 1928253
Domain
Metric
Evaluator's Comment
Qualitative
Determination
[i.e., High, Medium,
Low,Unacceptable,
or Not rated]
Metric
Score
Metric
Weighting
Factor
Weighted
Score

16. Outcome
Assessment
Methodology
Outcome methodology
assessment was reported.
High
1
2
2
Outcome
Assessment
17. Consistency of
Outcome
Assessment
Outcomes were assessed
consistently.
High
1
1
1

18. Sampling
Adequacy
Sampling was adequate.
High
1
2
2

19. Blinding of
Assessors
Blinding not required.
Not Rated
NR
NR
NR
Confounding /
20. Confounding
Variables in Test
Design and
Procedures
No confounding
variables were reported.
High
1
2
2
Variable Control
21. Confounding
Variables in
Outcomes Unrelated
to Exposure
No outcomes unrelated





to exposure were
reported.
High
1
1
1

22. Data Analysis
Statistical methods were
reported.
High
1
1
1
Data Presentation
and Analysis
23. Data
Interpretation
Criteria for positive or
equivocal findings were
reported.
High
1
2
2
24. Cytotoxicity
Data
Cytotoxicity not
required.
Not Rated
NR
NR
NR

25. Reporting of
Data
Data were reported.
High
1
2
2


Sum of scores:

32
51
High: >=
Medium: >=
1 and <1.7
=1.7 and <2.3
Overall Score = Sum of Weighted Scores/Sum
of Metric Weighting Factors:
1.5938
Overall Score:
Nearest *:
1.6
Low: >=2.5 and <=j







Overall Quality Level:

High

114

-------
4.8 In vitro evaluation results of Huang et al 2016
Study reference:
Huang, X.,Chen, C.,Shang, Y.,Zhong, Y.,Ren, G.,Yu, Z.,An, J. (2016). In vitro study on the biotransformation
and cytotoxicity of three hexabromocyclododecane diastereoisomers in liver cells Chemosphere, 161, 251-258
HERO ID: 3545979
Domain
Metric
Evaluator's Comment
Qualitative
Determination
[i.e.,High,Medium,
Low,Unacceptable,
or Not rated]
Metric
Score
Metric
Weighting
Factor
Weighted
Score

1. Test Substance
Identity
Native-HBCDs and
isomers were named
Medium
2
2
4

2. Test Substance
Source
Source provided but no
other details were
reported
Medium
2
1
2
Test Substance
3. Test Substance
Purity
Uncertainty of purity was
present, however, given
that unlabeled and
labeled products were
used in the studies, any
effects observed are more
likely than not to be due
to the test substance
Medium
2
1
2

4. Negative and
Vehicle Controls
Concurrent negative
controls were used
High
1
2
2
Test Design
5. Positive Controls
Positive controls not
required for these assays
Not Rated
NR
NR
NR
6. Assay procedures
Assays procedures were
described in detail
High
1
1
1

7. Standards for tests
Not applicable for these
assays
Not Rated
NR
NR
NR

8. Preparation and
Storage of Test
Substance
Limited details were
reported regarding stock
solution preparation and
no details were reported
regarding storage.
Medium
2
1
2

9. Consistency of
Exposure
Administration
Consistency of
administration was
reported
High
1
1
1
Exposure
Characterization
10. Reporting of
Doses/Concentration
s
Concentrations were
reported
High
1
2
2

11. Number of
Exposure Groups
and Concentration
Spacing
Durations were reported
for each assay
High
1
2
2

12. Exposure Route
and Method
Number of groups and
concentration spacing
were appropriate
High
1
1
1

13. Metabolic
Activation
Metabolic activation not
required for these assays
Not Rated
NR
NR
NR
115

-------
Study reference:
Huang, X.,Chen, C.,Shang, Y.,Zhong, Y.,Ren, G.,Yu, Z.,An, J. (2016). In vitro study on the biotransformation
and cytotoxicity of three hexabromocyclododecane diastereoisomers in liver cells Chemosphere, 161, 251-258
HERO ID: 3545979
Domain
Metric
Evaluator's Comment
Qualitative
Determination
[i.e., High, Medium,
Low,Unacceptable,
or Not rated]
Metric
Score
Metric
Weighting
Factor
Weighted
Score
Test Model
14. Test Model
The cell types used were
appropriate for the
intended outcomes
High
1
2
2
15. Number per
Group
Limited information on
the number of cells were
reported, number of
replicates were reported
for each assay,
Medium
2
1
2
Outcome
Assessment
16. Outcome
Assessment
Methodology
Outcome assessment
methodologies were
described in detail
High
1
2
2
17. Consistency of
Outcome
Assessment
Outcomes assessments
were conducted
consistently
High
1
1
1
18. Sampling
Adequacy
Details regarding
sampling outcomes were
not fully reported.
Medium
2
2
4
19. Blinding of
Assessors
Blinding was not
applicable for this study
Not Rated
NR
NR
NR
Confounding /
Variable Control
20. Confounding
Variables in Test
Design and
Procedures
No confounding
variables were reported
High
1
2
2
21. Confounding
Variables in
Outcomes Unrelated
to Exposure
No confounding
variables were reported
High
1
1
1
Data Presentation
and Analysis
22. Data Analysis
Statistical analysis was
appropriate
High
1
1
1
23. Data
Interpretation
Not applicable for the
assays
Not Rated
NR
NR
NR
24. Cytotoxicity
Data
Cytotoxicity was defined
and methods were
described sufficiently
High
1
1
1
25. Reporting of
Data
Outcome data were
reported
High
1
2
2
High: >=1 and <1.7
Medium: >=1.7 and <2.3
Low: >=2.3 and <=3
Sum of scores:

29
37
Overall Score = Sum of Weighted Scores/Sum
of Metric Weighting Factors:
1.2759
Overall Score:
Nearest *:
1.3
Overall Quality Level:
High
116

-------
117

-------
4.9 In vitro evaluation results of Kim et al 2016
Study reference:
Kim, S. H.,Nam, K. H.,Hwang, K. A.,Choi, K. C. (2016). Influence of hexabromocyclododecane and 4-
nonylphenol on the regulation of cell growth, apoptosis and migration in prostatic cancer cells Toxicology In
Vitro, 32, 240-247
HERO ID: 3350494
Domain
Metric
Evaluator's Comment
Qualitative
Determination
[i.e., High, Medium,
Low,Unacceptable,
or Not rated]
Metric
Score
Metric
Weighting
Factor
Weighted
Score

1. Test Substance
Identity
Established
nomenclature used
High
1
2
2
Test Substance
2. Test Substance
Source
Source identified, but no
additional information
provided
Medium
2
1
2

3. Test Substance
Purity
Purity such that effects
likely due to test
substance
High
1
1
1

4. Negative and
Vehicle Controls
A concurrent vehicle
control was used
High
1
2
2
Test Design
5. Positive Controls
A concurrent positive
control was used
High
1
2
2
6. Assay procedures
Experimental procedures
were described
High
1
1
1

7. Standards for tests
Not applicable for this
study
Not Rated
NR
NR
NR

8. Preparation and
Storage of Test
Substance
Test substance was
dissolved in solvent but
no other details were
provided
Medium
2
1
2

9. Consistency of
Exposure
Administration
No deficiencies noted
High
1
1
1
Exposure
10. Reporting of
Doses/Concentration
s
Concentrations were
reported
High
1
2
2
Characterization
11. Number of
Exposure Groups
and Concentration
Spacing
Exposure durations are
listed for all experiments
High
1
2
2

12. Exposure Route
and Method
The number of groups
and spacing are
appropriate for these
study types
High
1
1
1

13. Metabolic
Activation
Metabolic activation not
required
Not Rated
NR
NR
NR

14. Test Model
Test model was
appropriate
High
1
2
2
Test Model
15. Number per
Group
The number of cells and
replicates were
appropriate
High
1
1
1
118

-------
Study reference:
Kim, S. H.,Nam, K. H.,Hwang, K. A.,Choi, K. C. (2016). Influence of hexabromocyclododecane and 4-
nonylphenol on the regulation of cell growth, apoptosis and migration in prostatic cancer cells Toxicology In
Vitro, 32, 240-247

HERO ID: 3350494

Domain
Metric
Evaluator's Comment
Qualitative
Determination
[i.e., High, Medium,
Low,Unacceptable,
or Not rated]
Metric
Score
Metric
Weighting
Factor
Weighted
Score

16. Outcome
Assessment
Methodology
Methodology for
outcome assessment was
reported in detail
High
1
2
2
Outcome
Assessment
17. Consistency of
Outcome
Assessment
Outcomes were assessed
consistently across
groups
High
1
1
1

18. Sampling
Adequacy
See footnote at end of
page.4
High
1
2
2

19. Blinding of
Assessors
No subjective outcomes
were assessed
Not Rated
NR
NR
NR
Confounding /
20. Confounding
Variables in Test
Design and
Procedures
No confounding
variables were reported.
High
1
2
2
Variable Control
21. Confounding
Variables in
Outcomes Unrelated
to Exposure
No confounding
variables were reported
High
1
1
1

22. Data Analysis
Appropriate statistical
tests were used
High
1
1
1
Data Presentation
23. Data
Interpretation
This metric is not
applicable for these
studies
Not Rated
NR
NR
NR
and Analysis
24. Cytotoxicity
Data
Methods to determine
cell viability were
described
High
1
1
1

25. Reporting of
Data
Data were reported for
all outcomes
High
1
2
2

Sum of scores:

31
33
High: >=
Medium: >=
1 and <1.7
=1.7 and <2.3
Overall Score = Sum of Weighted Scores/Sum
of Metric Weighting Factors:
1.0645
Overall Score:
Nearest *:
1.1
Low: >=2.5 and <=j

Overall Quality Level:

High

4 Metrics that received a "High" rating met the criteria as discussed in the Applications of Systematic Review for
TSCA Risk Evaluation.
119

-------
4.10 In vitro evaluation results of Koike et al 2016
Study reference:
Koike, E.,Yanagisawa, R.,Takano, H. (2016). Brominated flame retardants, hexabromocyclododecane and
tetrabromobisphenol A, affect proinflammatory protein expression in human bronchial epithelial cells via
disruption of intracellular signaling Toxicology In Vitro, 32, 212-219
HERO ID: 3350501
Domain
Metric
Evaluator's Comment
Qualitative
Determination
[i.e., High, Medium,
Low,Unacceptable,
or Not rated]
Metric
Score
Metric
Weighting
Factor
Weighted
Score

1. Test Substance
Identity
Test substance identified
by name, structure, and
molecular weight.
High
1
2
2
Test Substance
2. Test Substance
Source
The source was
identified.
Medium
2
1
2

3. Test Substance
Purity
The reported purity was
such that observed
effects are likely due to
the test substance.
High
1
1
1

4. Negative and
Vehicle Controls
Concurrent negative
controls were included.
High
1
2
2
Test Design
5. Positive Controls
Positive controls not
required.
Not Rated
NR
NR
NR
6. Assay procedures
Assay procedures were
reported.
High
1
1
1

7. Standards for tests
Metric not required for
the test.
Not Rated
NR
NR
NR

8. Preparation and
Storage of Test
Substance
Preparation details were
reported, but storage and
stability were not
reported.
Medium
2
1
2

9. Consistency of
Exposure
Administration
Exposures were
administered
consistently.
High
1
1
1

10. Reporting of
Doses/Concentration
s
Concentrations were
reported (found in graphs
and/or figure legends).
High
1
2
2
Exposure
Characterization
11. Number of
Exposure Groups
and Concentration
Spacing
Durations were reported,
but ranges were given for
the transcription assay,
and no duration was
given for the ligand-
binding assay.
Medium
2
2
4

12. Exposure Route
and Method
The number of groups
was provided for each
assay, but spacing was
not justified.
Medium
2
1
2

13. Metabolic
Activation
Metabolic activation was
not required.
Not Rated
NR
NR
NR
Test Model
14. Test Model
Test model and
information were
provided.
High
1
2
2
120

-------
Study reference:
Koike, E.,Yanagisawa, R.,Takano, H. (2016). Brominated flame retardants, hexabromocyclododecane and
tetrabromobisphenol A, affect proinflammatory protein expression in human bronchial epithelial cells via
disruption of intracellular signaling Toxicology In Vitro, 32, 212-219
HERO ID: 3350501
Domain
Metric
Evaluator's Comment
Qualitative
Determination
[i.e., High, Medium,
Low,Unacceptable,
or Not rated]
Metric
Score
Metric
Weighting
Factor
Weighted
Score

15. Number per
Group
The number of cells and
replicates used was
reported.
High
1
1
1
Outcome
Assessment
16. Outcome
Assessment
Methodology
Outcome assessment
methodology was
described and
appropriate.
High
1
2
2
17. Consistency of
Outcome
Assessment
Outcomes were assessed
consistently.
High
1
1
1
18. Sampling
Adequacy
Sampling was adequate.
High
1
2
2
19. Blinding of
Assessors
Blinding not required.
Not Rated
NR
NR
NR
Confounding /
Variable Control
20. Confounding
Variables in Test
Design and
Procedures
No confounding variable
in assay design were
reported.
High
1
2
2
21. Confounding
Variables in
Outcomes Unrelated
to Exposure
No confounding
variables in outcomes
were reported.
High
1
1
1
Data Presentation
and Analysis
22. Data Analysis
Statistical methods were
appropriate.
High
1
1
1
23. Data
Interpretation
This metric not
applicable.
Not Rated
NR
NR
NR
24. Cytotoxicity
Data
Cell viability endpoints
were described and
appropriate.
High
1
1
1
25. Reporting of
Data
Data were reported.
High
1
2
2
High: >=1 and <1.7
Medium: >=1.7 and <2.3
Low: >=2.3 and <=3
Sum of scores:

29
34
Overall Score = Sum of Weighted Scores/Sum
of Metric Weighting Factors:
1.1724
Overall Score:
Nearest *:
1.2
Overall Quality Level:
High
121

-------
4.11 In vitro evaluation results of Litton et al 1990
Study reference:
Litton Bionetics (1990). Mutagenicity evaluation of 421-32B (final report) with test data and cover letter
HERO ID: 787698
Domain
Metric
Evaluator's Comment
Qualitative
Determination
[i.e., High, Medium,
Low,Unacceptable, or
Not rated]
Metric
Score
Metric
Weighting
Factor
Weighted
Score
Test Substance
1. Test Substance
Identity
Test substance was
identified by name, and
CASE# was hand-written
on title page.
Medium
2
2
4
2. Test Substance
Source
Source not identified.
Low
3
1
3

3. Test Substance
Purity
Purity was not reported.
Low
3
1
3

4. Negative and
Vehicle Controls
Solvent controls were
included.
High
1
2
2
Test Design
5. Positive Controls
Positive controls were
included and identified.
High
1
2
2
6. Assay procedures
Assay procedures were
reported.
High
1
1
1

7. Standards for tests
Criteria not applicable
Not Rated
NR
NR
NR

8. Preparation and
Storage of Test
Substance
Preparation was reported
with missing details (i.e.,
solvent used) and storage
information was not
reported.
Low
3
1
3

11. Number of
Exposure Groups
and Concentration
Spacing
Exposure durations were
reported.
High
1
2
2

12. Exposure Route
and Method
The number of groups
and spacing were
reported and justified.
High
1
1
1

13. Metabolic
Activation
The system and reaction
mixture were reported.
High
1
1
1
Test Model
14. Test Model
The strains were
reported, but no
additional information
was reported.
Low
3
2
6
122

-------
Study reference:
Litton Bionetics (1990). Mutagenicity evaluation of 421-32B (final report) with test data and cover letter
HERO ID: 787698
Domain
Metric
Evaluator's Comment
Qualitative
Determination
[i.e., High, Medium,
Low,Unacceptable, or
Not rated]
Metric
Score
Metric
Weighting
Factor
Weighted
Score

15. Number per
Group
The number of cells
exposed was reported
and adequate but the
number of replicates was
not reported.
Medium
2
1
2

18. Sampling
Adequacy
Sampling was adequate.
High
1
2
2

19. Blinding of
Assessors
Blinding was not
required.
Not Rated
NR
NR
NR
Confounding /
20. Confounding
Variables in Test
Design and
Procedures
No confounding
variables were reported.
High
1
2
2
Variable Control
21. Confounding
Variables in
Outcomes Unrelated
to Exposure
No outcomes unrelated

to exposure were
reported.
High
1
1
1

22. Data Analysis
Calculation and/or
statistical methods were
not reported, but the data
was present to conduct
analysis.
Low
3
1
3
Data Presentation
and Analysis
23. Data
Interpretation
Evaluation criteria were
not reported.
Low
3
2
6
24. Cytotoxicity
Data
Description of
cytotoxicity wans how it
was determined was not
reported.
Low
3
1
3

25. Reporting of
Data
Data were reported
High
1
2
2

Sum of scores:

34
55
High: >=
Medium: >=
1 and <1.7
=1.7 and <2.3
Overall Score = Sum of Weighted Scores/Sum of
Metric Weighting Factors:
1.6176
Overall Score:
Nearest *:
1.6
Low: >=2.5 and <=j

Overall Quality Level:

High

123

-------
124

-------
4,12 In vitro evaluation results of Pharmakologisches et al 1990
Study reference:
Pharmakologisches Inst (1990). Ames test with hexabromides with cover letter dated 031290
HERO ID: 787701
Domain
Metric
Evaluator's Comment
Qualitative
Determination
[i.e., High, Medium,
Low,Unacceptable, or
Not rated]
Metric
Score
Metric
Weighting
Factor
Weighted
Score

1. Test Substance
Identity
Test substance identified
by chemical name and
structure.
High
1
2
2
Test Substance
2. Test Substance
Source
Source not identified.
Low
3
1
3

3. Test Substance
Purity
Reported purity such that
effects likely due to test
substance.
High
1
1
1

4. Negative and
Vehicle Controls
Negative controls were
included.
High
1
2
2
Test Design
5. Positive Controls
Appropriate positive
controls were included.
High
1
2
2
6. Assay procedures
Assay procedures were
reported.
High
1
1
1

7. Standards for tests
Criteria were not
required.
Not Rated
NR
NR
NR

8. Preparation and
Storage of Test
Substance
Preparation details were
reported, but no
additional details were
provided such as
covering and storage
conditions prior to assay.
Medium
2
1
2

9. Consistency of
Exposure
Administration
Exposures were
administered
consistently.
High
1
1
1
Exposure
Characterization
10. Reporting of
Doses/Concentration
s
Concentrations were
reported.
High
1
2
2
11. Number of
Exposure Groups
and Concentration
Spacing
Exposure duration was
reported.
High
1
2
2

12. Exposure Route
and Method
The number of exposure
groups and spacing were
reported but were not
justified.
Medium
2
1
2

13. Metabolic
Activation
The metabolic activation
system and mix were
reported.
High
1
1
1
Test Model
14. Test Model
Test models were
reported without
additional details.
Medium
2
2
4
125

-------
Study reference:
Pharmakologisches Inst (1990). Ames test with hexabromides with cover letter dated 031290
HERO ID: 787701
Domain
Metric
Evaluator's Comment
Qualitative
Determination
[i.e., High, Medium,
Low,Unacceptable, or
Not rated]
Metric
Score
Metric
Weighting
Factor
Weighted
Score

15. Number per
Group
The number of cells used
was reported, but the
number of replicates was
not.
Medium
2
1
2

16. Outcome
Assessment
Methodology
Outcome assessment
methodology was
reported.
High
1
2
2
Outcome
Assessment
17. Consistency of
Outcome
Assessment
Outcomes were assessed
consistently.
High
1
1
1

18. Sampling
Adequacy
Sampling was adequate.
High
1
2
2

19. Blinding of
Assessors
Counters were blinded.
High
1
1
1
Confounding /
20. Confounding
Variables in Test
Design and
Procedures
No confounding
variables were reported.
High
1
2
2
Variable Control
21. Confounding
Variables in
Outcomes Unrelated
to Exposure
No outcomes unrelated





to exposure were
reported.
High
1
1
1

22. Data Analysis
Data were not analyzed
but were presented so
analysis can be
conducted if needed.
Medium
2
1
2
Data Presentation
and Analysis
23. Data
Interpretation
Criteria were not
reported.
Low
3
2
6
24. Cytotoxicity
Data
Cytotoxicity was not
included in the study.
Not Rated
NR
NR
NR

25. Reporting of
Data
Data were reported.
High
1
2
2


Sum of scores:

34
46
High: >=
Medium: >=
1 and <1.7
=1.7 and <2.3
Overall Score = Sum of Weighted Scores/Sum of
Metric Weighting Factors:
1.3529
Overall Score:
Nearest *:
1.4
Low: >=2.5 and <=j







Overall Quality Level:

High

126

-------
4,13 In vitro evaluation results of S.R.I. International 1990
Study reference:
S. R. I. International (1990). In vitro microbiological mutagenicity studies of four Ciba-Geigy Corporation
compounds (final report) with test data and cover letter
HERO ID: 787716
Domain
Metric
Evaluator's Comment
Qualitative
Determination
[i.e., High, Medium,
Low,Unacceptable,
or Not rated]
Metric
Score
Metric
Weighting
Factor
Weighted
Score

1. Test Substance
Identity
Test substance identified
by name, CAS# on title
page.
Medium
2
2
4
Test Substance
2. Test Substance
Source
Source not identified, but
compound was called
"Ciby-Geigy Corporation
compounds"
Medium
2
1
2

3. Test Substance
Purity
Purity was not reported.
Low
3
1
3

4. Negative and
Vehicle Controls
Negative controls were
included.
High
1
2
2
Test Design
5. Positive Controls
Positive controls were
included.
High
1
2
2
6. Assay procedures
Assay procedures were
partially described.
Medium
2
1
2

7. Standards for tests
Criteria were not
required.
Not Rated
NR
NR
NR

8. Preparation and
Storage of Test
Substance
Preparation was reported;
substances used
immediately after
preparation.
High
1
1
1

9. Consistency of
Exposure
Administration
Exposures were
administered
consistently.
High
1
1
1
Exposure
Characterization
10. Reporting of
Doses/Concentration
s
Concentrations were
reported.
High
1
2
2
11. Number of
Exposure Groups
and Concentration
Spacing
Durations were reported.
High
1
2
2

12. Exposure Route
and Method
The number of groups
and spacing were
reported but not justified.
Medium
2
1
2

13. Metabolic
Activation
The metabolic system
and mix were described.
High
1
1
1
Test Model
14. Test Model
Test model, source, and
descriptive information
was reported.
High
1
2
2
127

-------
Study reference:
S. R. I. International (1990). In vitro microbiological mutagenicity studies of four Ciba-Geigy Corporation
compounds (final report) with test data and cover letter
HERO ID: 787716
Domain
Metric
Evaluator's Comment
Qualitative
Determination
[i.e., High, Medium,
Low,Unacceptable,
or Not rated]
Metric
Score
Metric
Weighting
Factor
Weighted
Score

15. Number per
Group
The number of exposed
cells was not reported,
but cells were shaken for
3-4 hours to ensure
optimal growth.
Low
3
1
3
Outcome
Assessment
16. Outcome
Assessment
Methodology
Outcome methodology
assessment was reported.
High
1
2
2
17. Consistency of
Outcome
Assessment
Outcomes were assessed
consistently.
High
1
1
1
18. Sampling
Adequacy
Sampling was adequate.
High
1
2
2
19. Blinding of
Assessors
Blinding was not
required.
Not Rated
NR
NR
NR
Confounding /
Variable Control
20. Confounding
Variables in Test
Design and
Procedures
No confounding
variables were reported.
High
1
2
2
21. Confounding
Variables in
Outcomes Unrelated
to Exposure
No outcomes unrelated
to exposure were
reported.
High
1
1
1
Data Presentation
and Analysis
22. Data Analysis
Data were provided to
conduct analysis.
Medium
2
1
2
23. Data
Interpretation
Evaluation criteria were
not reported.
Low
3
2
6
24. Cytotoxicity
Data
Cytotoxicity not included
in test.
Not Rated
NR
NR
NR
25. Reporting of
Data
Data were reported.
High
1
2
2
High: >=1 and <1.7
Medium: >=1.7 and <2.3
Low: >=2.3 and <=3
Sum of scores:

33
47
Overall Score = Sum of Weighted Scores/Sum
of Metric Weighting Factors:
1.4242
Overall Score:
Nearest *:
1.4
Overall Quality Level:
High
128

-------
4.14 In vitro evaluation results of Wang et al 2016
Study reference:
Wang, F.,Zhang, H.,Geng, N.,Zhang, B.,Ren, X.,Chen, J. (2016). New Insights into the Cytotoxic Mechanism
of Hexabromocyclododecane from a Metabolomic Approach Environmental Science and Technology, 50(6),
3145-3153

HERO ID: 3350479

Domain
Metric
Evaluator's Comment
Qualitative
Determination
[i.e., High, Medium,
Low,Unacceptable, or
Not rated]
Metric
Score
Metric
Weighting
Factor
Weighted
Score

1. Test Substance
Test substance identified
Medium
9
9
A

Identity
by name.

Test Substance
2. Test Substance
Source
Source identified.
Medium
2
1
2
3. Test Substance
Purity
Reported purity and
grade such that effects
likely due to test
substance.
High
1
1
1

4. Negative and
Vehicle Controls
Negative control groups
were included.
High
1
2
2
Test Design
5. Positive Controls
Positive control groups
were not required.
Not Rated
NR
NR
NR
6. Assay procedures
Assay procedures were
described.
High
1
1
1

7. Standards for tests
Standards not applicable
for the assay.
Not Rated
NR
NR
NR

8. Preparation and
Storage of Test
Substance
Limited preparation
details were provided,
but no storage and
stability information
were provided.
Medium
2
1
2

9. Consistency of
Exposure
Administration
Exposures were
administered
consistently.
High
1
1
1
Exposure
Characterization
10. Reporting of
Doses/Concentration
s
Concentrations were
reported.
High
1
2
2
11. Number of
Exposure Groups
and Concentration
Spacing
Durations were reported.
High
1
2
2

12. Exposure Route
and Method
The number of groups
and spacing were
reported and based on
cell viability testing.
High
1
1
1

13. Metabolic
Activation
Metabolic activation was
not required.
Not Rated
NR
NR
NR
Test Model
14. Test Model
The information was
reported.
High
1
2
2
15. Number per
Group
The number of cells
exposed was reported.
High
1
1
1
129

-------
Study reference:
Wang, F.,Zhang, H.,Geng, N.,Zhang, B.,Ren, X.,Chen, J. (2016). New Insights into the Cytotoxic Mechanism
of Hexabromocyclododecane from a Metabolomic Approach Environmental Science and Technology, 50(6),
3145-3153

HERO ID: 3350479

Domain
Metric
Evaluator's Comment
Qualitative
Determination
[i.e., High, Medium,
Low,Unacceptable, or
Not rated]
Metric
Score
Metric
Weighting
Factor
Weighted
Score

16. Outcome
Outcome assessment was
described.

Assessment
Methodology
High
1
2
2
Outcome
Assessment
17. Consistency of
Outcome
Assessment
Exposures were
administered
consistently.
High
1
1
1

18. Sampling
Adequacy
Sampling was adequate.
High
1
2
2

19. Blinding of
Assessors
Blinding not required.
Not Rated
NR
NR
NR
Confounding /
20. Confounding
Variables in Test
Design and
Procedures
No confounding
variables in test design
and procedures were
reported.
High
1
2
2
Variable Control
21. Confounding
Variables in
Outcomes Unrelated
to Exposure
No confounding
variables in outcomes
unrelated to exposure
were reported.
High
1
1
1

22. Data Analysis
Statistical methods and
data manipulation were
reported and appropriate.
High
1
1
1
Data Presentation
and Analysis
23. Data
Interpretation
Data evaluation not
required.
Not Rated
NR
NR
NR
24. Cytotoxicity
Data
Cytotoxicity studies were
described.
High
1
1
1

25. Reporting of
Data
Data were reported for
outcomes.
High
1
2
2

Sum of scores:

29
33
High: >=
Medium: >=
1 and <1.7
=1.7 and <2.3
Overall Score = Sum of Weighted Scores/Sum of
Metric Weighting Factors:
1.1379
Overall Score:
Nearest *:
1.1
Low: >=z.J ana <=j

Overall Quality Level:

High

130

-------
4.15 In vitro evaluation results of Wu et al 2016
Study reference:
Wu, M.,Wu, D.,Wang, C.,Guo, Z.,Li, B.,Zuo, Z. (2016). Hexabromocyclododecane exposure induces cardiac
hypertrophy and arrhythmia by inhibiting miR-1 expression via up-regulation of the homeobox gene Nkx2.5
Journal of Hazardous Materials, 302, 304-313
HERO ID: 3350515
Domain
Metric
Evaluator's Comment
Qualitative
Determination
[i.e., High, Medium,
Low,Unacceptable,
or Not rated]
Metric
Score
Metric
Weighting
Factor
Weighted
Score
Test Substance
1. Test Substance
Identity
Test substance identified
by structure and name.
High
1
2
2
2. Test Substance
Source
The source was
identified.
Medium
2
1
2
3. Test Substance
Purity
The reported purity was
such that effects likely
due to the test substance.
High
1
1
1
Test Design
4. Negative and
Vehicle Controls
Concurrent controls were
included.
High
1
2
2
5. Positive Controls
Positive controls not
required.
Not Rated
NR
NR
NR
6. Assay procedures
Assay procedures were
described and
appropriate.
High
1
1
1
7. Standards for tests
Metric not applicable to
study type.
Not Rated
NR
NR
NR
Exposure
Characterization
8. Preparation and
Storage of Test
Substance
Limited preparation
details were reported and
no details on storage and
stability were reported.
Medium
2
1
2
9. Consistency of
Exposure
Administration
Exposures were
administered
consistently.
High
1
1
1
10. Reporting of
Doses/Concentration
s
Concentrations were
reported.
High
1
2
2
11. Number of
Exposure Groups
and Concentration
Spacing
Duration was reported.
High
1
2
2
12. Exposure Route
and Method
The number of groups
and spacing were
reported, but justification
was not provided.
Medium
2
1
2
13. Metabolic
Activation
Metabolic activation was
not required.
Not Rated
NR
NR
NR
Test Model
14. Test Model
The test model and
source were reported and
were appropriate.
High
1
2
2
131

-------
Study reference:
Wu, M.,Wu, D.,Wang, C.,Guo, Z.,Li, B.,Zuo, Z. (2016). Hexabromocyclododecane exposure induces cardiac
hypertrophy and arrhythmia by inhibiting miR-1 expression via up-regulation of the homeobox gene Nkx2.5
Journal of Hazardous Materials, 302, 304-313
HERO ID: 3350515
Domain
Metric
Evaluator's Comment
Qualitative
Determination
[i.e., High, Medium,
Low,Unacceptable,
or Not rated]
Metric
Score
Metric
Weighting
Factor
Weighted
Score

15. Number per
Group
n-6 was reported in the
figures, but it is not clear
if that is the number of
replicates. The number
of cells used was not
reported.
Low
3
1
3
Outcome
Assessment
16. Outcome
Assessment
Methodology
Outcome assessment
methodology was
reported and appropriate.
High
1
2
2
17. Consistency of
Outcome
Assessment
Outcomes were assessed
consistently.
High
1
1
1
18. Sampling
Adequacy
Sampling was adequate.
High
1
2
2
19. Blinding of
Assessors
Blinding was not
required.
Not Rated
NR
NR
NR
Confounding /
Variable Control
20. Confounding
Variables in Test
Design and
Procedures
No confounding
variables in test design
were reported.
High
1
2
2
21. Confounding
Variables in
Outcomes Unrelated
to Exposure
No confounding
variables in outcomes
unrelated to exposure
were reported.
High
1
1
1
Data Presentation
and Analysis
22. Data Analysis
Statistical methods were
appropriate.
High
1
1
1
23. Data
Interpretation
This metric not
applicable.
Not Rated
NR
NR
NR
24. Cytotoxicity
Data
This metric not
applicable.
Not Rated
NR
NR
NR
25. Reporting of
Data
Data were reported.
High
1
2
2
High: >=1 and <1.7
Medium: >=1.7 and <2.3
Low: >=2.3 and <=3
Sum of scores:

28
33
Overall Score = Sum of Weighted Scores/Sum
of Metric Weighting Factors:
1.1786
Overall Score:
Nearest *:
1.2
Overall Quality Level:
High
132

-------
5 Epidemiological Studies
5.1 Epidemiological evaluation results of the Eggesbo et al 2011
study for thyroid outcomes for exposure groups in general
Study
reference:
Eggesba, M.,Thomsen, CJurgensen, J. V.,Becher, G.,Odland, J. 0,Longnecker, M. P. (2011). Associations
between brominated flame retardants in human milk and thyroid-stimulating hormone (TSH) in neonates
Environmental Research, 111(6), 737-743

HERO ID: 787656

Domain
Metric
Comments
Qualitative
Determinatio
n
Metric
Score
Metric
Weighting
Factor
Weighted
Score

1. Participant
selection
High rating: key elements of study design were
reported (such as setting, participation rate
described at all steps of the study, inclusion and
exclusion criteria, and methods of participant
selection), and the reported information indicates
selection in or out of the study and participation is
not likely to be biased.
High
1
0.400
0.400
s
_o
"s
.5-
"3
es
&
2. Attrition
Medium rating: 31% of women that agreed to
participate in the study did not provide milk samples
(authors explained this was partly due to lack of
milk); 40% of the 396 babies selected for the study
were excluded from analysis due to inaccessible
TSH values. Attrition was acceptably handled.
Supplemental Fig Al provides a description of
characteristics between participants and non-
participants. No significant differences were
reported between these 2 groups. Missing values
for "age at which TSH was measured" were
replaced by mean values for 80 (33%) participants.
Medium
2
0.400
0.800
¦o
5
Tfl
3. Comparison Group
High rating: differences in baseline characteristics
of groups were considered as potential confounding
or stratification variables and were thereby
controlled by statistical analysis. Covariates
included age at which TSH was
measured(continuously in hours), county of
residence and pre-pregnancy maternal body mass
index. The following potential confounders:
maternal education as a socioeconomic index (<12,
12, 13-16 and >16 years of education), Norwegian
nationality, season, parity, smoking, maternal age at
delivery, sex, pregnancy hypertension and/or
preeclampsia based on maternal reports (yes/no)
and type of delivery (spontaneous, induced, assisted
or cesarean); and continuous variables: gestational
age, HCB, b-HCH,p,pO-DDE,oxychlordane and the
sum of all PCB congeners.
High
1
0.200
0.200
Exposure
Character
ization
4.
Measurem
ent of
Exposure
High rating: exposure was assessed using the same
well-established methods that directly measure
HBCD in breast milk, a frequently used biomarker
of exposure.
High
1
0.400
0.400
133

-------
Study
reference:
Eggesba, M.,Thomsen, CJurgenscn, J. V.,Becher, G.,Odland, J. 0,Longnecker, M. P. (2011). Associations
between brominated flame retardants in human milk and thyroid-stimulating hormone (TSH) in neonates
Environmental Research, 111(6), 737-743
HERO ID: 787656
Domain
Metric
Comments
Qualitative
Determinatio
n
Metric
Score
Metric
Weighting
Factor
Weighted
Score

5. Exposure
levels
Medium rating: range and distribution of exposure
was sufficient to develop an exposure-response
estimate; 3 or more levels of exposure were
reported.
Medium
2
0.200
0.400
6.
Temporalit
y
High rating: temporality is established and the
interval between the exposure and the outcome has
an appropriate consideration of relevant exposure
windows.
High
1
0.400
0.400
Outcome Assessment
7. Outcome
measurement or
characterization
High rating: TSH levels were measured using well-
established methods (i.e., on dried filter paper
bloodspots by an immunoassay) (Auto Delfias
neonatal TSH kits; Perkin Elmer).
High
1
0.670
0.667
8.
Reporting
Bias
High rating: all of the study's measured outcomes
are reported, effect estimates reported with
confidence interval; number of exposed reported for
each analysis.
High
1
0.330
0.333
Potential Confounding/Variable Control
9. Covariate
Adjustment
High rating: appropriate adjustments or explicit
considerations were made for potential confounders
in the final analyses through the use of statistical
models for covariate adjustment. See discussion in
metric 3.
High
1
0.500
0.500
10. Covariate
Characterization
Medium rating: Primary confounders (excluding co-
exposures) were assessed. The paper did not
describe if the survey to gather demographic
characteristics, the amount of breastfeeding/month,
etc. was validated.
Medium
2
0.250
0.500
11. Co-exposure
Confounding
Medium rating: HBCD models were adjusted for
some co-pollutants (PCBs, HCB, DDE, etc);
however, separate models were run for PBDEs and
HBCD, and it difficult to distinguish which
contaminant might have caused an association with
a disease. However, there does not appear to be
direct evidence of an unbalanced provision of
additional co-exposures across the primary study
groups,
Medium
2
0.250
0.500
Analysis
12. Study
Design and
Methods
Medium rating: appropriate design (i.e., prospective
cohort for assessment of TSH levels in relation to
HBCD exposure), and appropriate statistical
methods (i.e., linear and logistic regression
analyses) were employed to analyze data.
Medium
2
0.400
0.800
134

-------
Study
reference:
Eggesba, M.,Thomsen, CJurgensen, J. V.,Becher, G.,Odland, J. 0,Longnecker, M. P. (2011). Associations
between brominated flame retardants in human milk and thyroid-stimulating hormone (TSH) in neonates
Environmental Research, 111(6), 737-743

HERO ID: 787656

Domain
Metric
Comments
Qualitative
Determinatio
n
Metric
Score
Metric
Weighting
Factor
Weighted
Score

13. Statistical
power
Medium rating: the number of participants were
adequate to detect an effect in the exposed
population for HBCD and for most BFRs except
BDE- 209.
Medium
2
0.200
0.400

14.
Reproducibility of
analyses
Medium rating: description of the analyses is
sufficient to understand what has been done and to
be reproducible with access to the data.
Medium
2
0.200
0.400

15. Statistical
models
Medium rating: linear regression models were used
to generate beta coefficients and logistic regression
models were used to generate Odds Ratios.
Rationale for variable selection is stated. Model
assumptions are met.
Medium
2
0.200
0.400

16. Use of
Biomarker of
Exposure
High rating: Evidence exists for a relationship
between HBCD in breast milk and external
exposure.
High
1
0.140
0.143
Other
17. Effect
biomarker
High rating: Effect biomarker measured is an
indicator of a key event in an AOP.
High
1
0.140
0.143

18. Method
Sensitivity
Medium rating: LOD is low enough to detect
HBCD in a sufficient percentage of the samples to
address the research question. Analytical methods
measuring biomarker are adequately reported.
LOD/LOQ (value or %) are reported.
Medium
2
0.140
0.286
U
CJ
19. Biomarker
stability
High rating: samples with a known storage history
(Supplement-03 document)
High
1
0.140
0.140
o
20. Sample
contamination
Low rating: No known sampling contamination
issues are discussed in the paper, but there is no
documentation of the steps taken to provide the
necessary assurance that the study data are reliable.
Low
3
0.140
0.429
135

-------
Study
reference:
Eggesba, M.,Thomsen, CJurgensen, J. V.,Becher, G.,Odland, J. 0,Longnecker, M. P. (2011). Associations
between brominated flame retardants in human milk and thyroid-stimulating hormone (TSH) in neonates
Environmental Research, 111(6), 737-743
HERO ID: 787656
Domain
Metric
Comments
Qualitative
Determinatio
n
Metric
Score
Metric
Weighting
Factor
Weighted
Score
Other
21. Method requirements
High rating: instrumentation that provides
unambiguous identification and quantitation of the
biomarker at the required sensitivity were used.
Specifically, the extracts were analyzed by gas
chromatography
coupled to a mass spectrometer using electron
capture negative ionization (GC- EC/MS) and an
internal standard calibration as described by
Thomsen et al., 2007.
High
1
0.140
0.143
22. Matrix
adjustment
Medium rating: study only provides results using
one method (lipid-adjusted).
Medium
2
0.140
0.286
High: >=1 and <1.7
Medium: >=1.7 and <2.3
Low: >=2.3 and <=3
Sum of scores:

6
8.53
Overall Score = Sum of Weighted Scores/Sum of Metric
Weighting Factors:
1.4217
Overall
Score:
Nearest *:
1.4
Overall Quality Level:
High
136

-------
5.2 Epidemiological evaluation results of the Johnson et al 2013
study for reproductive outcomes in general
Study
reference:
Johnson, P. I.,Stapleton, H. M.,Mukherjee, B.,Hauser, R.,Meeker, J. D. (2013). Associations between brominated
flame retardants in house dust and hormone levels in men Science of the Total Environment, 445-446(Supplement
C), 177-184

HERO ID: 1676758

Domain
Metric
Comments
Qualitative
Determinatio
n
Metric
Score
Metric
Weighting
Factor
Weighted
Score

1.
Participant
selection
No explanation for participation rate of 65%
provided; only male subjects. Information on
participation selection, inclusion and exclusion
criteria are provided in cited publications.
High
1
0.250
0.250
s
_o
"s
.5-
"3
2. Attrition
Attrition is not reported, and n values do not equal
62 in all results presented, (e.g. T3 has n=38 which
is -40% missing samples). No information on how
missing data is handled.
Low
3
0.250
0.750
&
¦a
S
3. Comparison Group
There is no information on a comparison group,
however correlation analysis was performed looking
for trends on a continuum of exposure.
Unacceptable
NR
NR
NR
a
#o
"s
.a
~-
(J
si
~-
si
-fl
U
5
5«
O
4.
Measurem
ent of
Exposure
Dust samples were collected from used vacuum
badge from home. It is unclear if this is an
established method to determine levels of exposure.
HBCD detected in 97% of samples.
Medium
2
0.400
0.800
5. Exposure
levels
The range of exposure is limited but based on the
analysis it does allow limited exploration in the
exposure-response relationship.
Low
3
0.200
0.600
&
UJ
6.
Tempor
ality
Dust samples and serum hormone levels are
sampled in the same year for participants. The
temporality of exposure and outcome is uncertain.
Medium
2
0.400
0.800
c
s
5«
%
5«
5«
s
O
(J
7. Outcome
measurement or
characterization
QA/QC methods described in another paper. The
outcome was assessed using established methods.
High
1
0.670
0.667
5
O
8.
Repo
rting
Bias
Author's discuss results in text for significant results
only
Medium
2
0.330
0.667
Potential
Confounding/
Variable
Control
9. Covariate
Adjustment
Although models were adjusted for age and BMI for
some flame retardants, there is no mention of
covariate consideration for HBCD.
High
1
0.500
0.500
137

-------
Study
reference:
Johnson, P. I.,Stapleton, H. M.,Mukherjee, B.,Hauser, R.,Meeker, J. D. (2013). Associations between brominated
flame retardants in house dust and hormone levels in men Science of the Total Environment, 445-446(Supplement
C), 177-184
HERO ID: 1676758
Domain
Metric
Comments
Qualitative
Determinatio
n
Metric
Score
Metric
Weighting
Factor
Weighted
Score

10. Covariate
Characterization
There is no information to suggest that the
questionnaire used was validated, however there is
no evidence that the method had poor validity.
High
1
0.250
0.250
11. Co-exposure
Confounding
Cannot rule out possibility of that findings are due
to unmeasured co-exposures (e.g. other chemicals in
household dust).
Medium
2
0.250
0.500
Analysis
12. Study
Design and
Methods
The study was exploratory to assess the association
between exposure levels and hormone levels.
However only a correlation analysis between HBCD
and free androgen index was reported.
Unacceptable
NR
NR
NR
13. Statistical
power
The sample size is relatively small and the authors
indicate that the study is exploratory in nature.
Unacceptable
NR
NR
NR
14.
Reproducibility of
analyses
The analysis is sufficiently described.
Medium
2
0.070
0.143
15. Statistical
models
The authors provide an explanation for when data is
combined with previous study data and limitations
of the analysis in detail.
Medium
2
0.070
0.143
Other
16. Use of
Biomarker of
Exposure
No biomarker of exposure measured.
Not Rated
NR
NR
NR
17. Effect
biomarker
Biomarker not specific to a health outcome.
Unacceptable
NR
NR
NR
138

18. Method
Sensitivity
Limit of detection not discussed in study, but no
evidence of insufficient data.
Not Rated
NR
NR
NR
Other
19. Biomarker
stability
samples with known storage history and
documented stability data
High
NR
NR
NR
20. Sample
contamination
No information to indicate sample contamination.
Medium
2
0.140
0.286
Other
21. Method
requirements
Method provides the identification and
quantification of the biomarker.
High
1
0.140
0.143
22. Matrix
adjustment
No matrix adjustment.
Not Rated
NR
NR
NR
High: >=1 and <1.7
Medium: >=1.7 and <2.3
Low: >=2.3 and <=3
Sum of scores:

4.07
6.5
Overall Score = Sum of Weighted Scores/Sum of Metric
Weighting Factors:
2.5273
Overall
Score:
Nearest *:
2.51
Overall Quality Level:
Unacceptable1
Comment:
Footnote:
1 Consistent with our Application of Systematic Review in TSCA Risk Evaluations document, if a metric for a data
source receives a score of Unacceptable (score = 4), EPA will determine the study to be unacceptable. In this case,
four of the metrics were rated as unacceptable. As such, the study is considered unacceptable and the score is
presented solely to increase transparency.
139

-------
5.3 Epidemiological evaluation results of the Kicinski et al 2012
study for neurological/behavior outcomes in general
Study
reference:
Kicinski, M.,Viaene, M. K.,Den Hond, E.,Schoeters, G.,Covaci, A.,Dirtu, A. C.,Nelen, V.,Bruckers, L.,Croes,
K.,Sioen, I.,Baeyens, W.,Van Larebeke, N.,Nawrot, T. S. (2012). Neurobehavioral function and low-level exposure
to brominated flame retardants in adolescents: A cross-sectional study Environmental Health: A Global Access
Science Source, ll(#issue#), 86

HERO ID: 1927571

Domain
Metric
Comments
Qualitative
Determinatio
n
Metric
Score
Metric
Weighting
Factor
Weighted
Score
s
_o
"s
o.
1. Participant selection
Participants were recruited during the same time
frame (2008-2011) from the same two industrial
areas and from the general population of Flemish
adolescents using the same criteria. All adolescents
from Genk and Menen were eligible. Random
sampling of the general population was attained
through a multistage sampling design (which is
described). Details were provided for all aspects of
the selection. The response rates were slightly
higher in Genk, but non-responders were noted to
not be different from the responders except that
there was a higher proportion of girls responding.
High
1
0.400
0.400
si
&
¦o
5
VI
2. Attrition
107 of the 606 subjects included were excluded
because of missing covariates (n=84), missing blood
measurements (n=3), or did not complete neuro-
behavioral tests (n=4). However, results have much
fewer numbers for some results without full
explanation.
Medium
2
0.400
0.800

3. Comparison Group
Although a table of characteristics was provided, it
was not broken down by area or general population.
Differences that were expected to potentially bias
the results were included in the analysis. However,
there is not enough information provided about the
two study areas to determine if there may have been
other differences that varied by exposure.
Medium
2
0.200
0.400
a
#o
N
'C
4. Measurement of
Exposure
HBCD was measured in the serum according to
methods by Covaci and Voorspoels (HERO ID
3113586). However, the method they cite does not
indicate that this is a method for measuring HBCD
nor do they provide recovery rates. Despite that
there is no evidence that there would be poor
validity or misclassification, it may just be more
likely that samples would fall below the LOQ.
Low
3
0.400
1.200
es
~-
cS
-fl
U
£
o
o.
UJ
5. Exposure
levels
For HBCD the effects of the concentrations above
the LOQ compared to the concentrations below the
LOQ were estimated (binary exposure).
Low
3
0.200
0.600

6.
Temporalit
y
The temporality of exposure and outcome is
uncertain. The cross-sectional nature of the study
design makes it difficult to determine if exposure
occurred prior to the outcome.
Low
3
0.400
1.200
140

-------
Study
reference:
Kicinski, M.,Viaene, M. K.,Den Hond, E.,Schoeters, G.,Covaci, A.,Dirtu, A. C.,Nelen, V.,Bruckers, L.,Croes,
K.,Sioen, I.,Baeyens, W.,Van Larebeke, N.,Nawrot, T. S. (2012). Neurobehavioral function and low-level exposure
to brominated flame retardants in adolescents: A cross-sectional study Environmental Health: A Global Access
Science Source, ll(#issue#), 86

HERO ID: 1927571

Domain
Metric
Comments
Qualitative
Determinatio
n
Metric
Score
Metric
Weighting
Factor
Weighted
Score
C
S
5«
5«
5«
5«
«<
s
O
7. Outcome
measurement or
characterization
Neurobehavioral Evaluation System is a
computerized battery of tests developed to study the
neurological effects of an exposure to
environmental agents. This study used four tests
from the NES-3 version of the test battery. Study
authors note these tests are reliable.
High
1
0.670
0.667
(J
5
O
8.
Reporti
ng Bias
Sufficient information is provided. All outcomes
were reported with effect, 95% confidence intervals,
and sample size.
High
1
0.330
0.333
s
c
o
u
9. Covariate
Adjustment
Gender, age, type of education, parental education,
owning the house, smoking, passive smoking, and
blood lipids were included in the assessment. BMI,
physical activity, computer use, alcohol use, fish
consumption, blood lead, serum PCBs were also
included in a stepwise regression procedure with
p=0.15 for entering and p=0.10 for remaining in the
model.
High
1
0.500
0.500
3
2
'~I
es
&
¦3
c
5
£
10. Covariate
Characterization
Information was obtained via questionnaires some
information to be filled out by the adolescent and
some for the parents.
Medium
2
0.250
0.500
C
O
U
.*2
c
4>
O
a.
11. Co-exposure
Confounding
Two of the groups were selected because they lived
near industrial areas. No information was provided
on these industrial areas and what else might be
there. However, they did account for lead and PCBs
(and possibly mercury via fish consumption)
because these may impact the results. Although it is
unclear if there might be other potential co-
exposures, there is no indication that there would be
anything additional that would greatly impact the
results that was not considered.
Medium
2
0.250
0.500
%
*5«
12. Study
Design and
Methods
The cross-sectional study design is appropriate for
evaluating HBCD concentrations in adolescents
with neurobehavioral effects. The study was part of
a biomonitoring program for environmental health
surveillance in Flanders, Belgium.
Medium
2
0.400
0.800
13
c
13. Statistical
power
Sufficient statistical power with 515 included
subjects and outcome results available for 340 to
511 for any specific outcome.
Medium
2
0.200
0.400
141

HERO ID: 1927571

Domain
Metric
Comments
Qualitative
Determinatio
n
Metric
Score
Metric
Weighting
Factor
Weighted
Score

14.
Reproducibility of
analyses
Description is not 100% clear on methods to be
reproducible.
Low
3
0.200
0.600

15. Statistical
models
The use of a linear regression or a negative binomial
model were acceptable for the data with
assumptions met or data transformed.
Medium
2
0.200
0.400

16. Use of
Biomarker of
Exposure
No information is provided to indicate serum HBCD
is the appropriate, but the parent compound was
measured.
Medium
2
0.200
0.400
Other
17. Effect
biomarker
No biomarker of effect was measured.
Not Rated
NR
NR
NR

18. Method
Sensitivity
Frequency of detection was low. Although they did
not provide specific numbers below detection for
HBCD, the P75 was still below the LOQ indicating
that a large percent was below detection.
Low
3
0.200
0.600
U
CJ
19. Biomarker
stability
No information was provided on storage history or
stability of the HBCD in the sample.
Medium
2
0.200
0.400
o
20. Sample
contamination
There is incomplete documentation of the steps
taken to provide the necessary assurance that the
study data are reliable.
Medium
2
0.200
0.400
142

-------
Study
reference:
Kicinski, M.,Viaene, M. K.,Den Hond, E.,Schoeters, G.,Covaci, A.,Dirtu, A. C.,Nelen, V.,Bruckers, L.,Croes,
K.,Sioen, I.,Baeyens, W.,Van Larebeke, N.,Nawrot, T. S. (2012). Neurobehavioral function and low-level exposure
to brominated flame retardants in adolescents: A cross-sectional study Environmental Health: A Global Access
Science Source, ll(#issue#), 86
HERO ID: 1927571
Domain
Metric
Comments
Qualitative
Determinatio
n
Metric
Score
Metric
Weighting
Factor
Weighted
Score
Other
21. Method requirements
Solid phase extraction followed by gas
chromatography mass spectrometry in electron
capture negative ion mode was used. Specifics of
the extraction were not provided but are assumed to
be the same as used in cited reference (HERO ID
311586). Sensitivity of method for HBCD is not
clear as recovery was not reported. The LOQ was
30 ng/L which seems high compared to the other
PBDEs and the majority of the samples fell below
the LOQ.
Medium
2
0.200
0.400
22. Matrix
adjustment
Don't think matrix adjustment would be appropriate
for this biomarker of exposure.
Not Rated
NR
NR
NR
High: >=1 and <1.7
Medium: >=1.7 and <2.3
Low: >=2.3 and <=3
Sum of scores:

6
11.5
Overall Score = Sum of Weighted Scores/Sum of Metric
Weighting Factors:
1.9167
Overall
Score:
Nearest *:
1.9
Overall Quality Level:
Medium
143

-------
5.4 Epidemiological evaluation results of the Kicinski et al 2012
study for thyroid outcomes in general
Study
reference:
Kicinski, M.,Viaene, M. K.,Den Hond, E.,Schoeters, G.,Covaci, A.,Dirtu, A. C.,Nelen, V.,Bruckers, L.,Croes,
K.,Sioen, I.,Baeyens, W.,Van Larebeke, N.,Nawrot, T. S. (2012). Neurobehavioral function and low-level exposure
to brominated flame retardants in adolescents: A cross-sectional study Environmental Health: A Global Access
Science Source, ll(#issue#), 86

HERO ID: 1927571

Domain
Metric
Comments
Qualitative
Determinatio
n
Metric
Score
Metric
Weighting
Factor
Weighted
Score
C
S
5«
5«
5«
5«
«<
s
O
(J
7. Outcome
measurement or
characterization
Thyroid hormones were measured by competitive
immune assays. No other information was provided.
These are assumed to be standard assays.
Medium
2
0.670
1.333
5
O
8.
Repo
rting
Bias
Information is provided, but not enough for
complete extraction (sample size was not specified).
Medium
2
0.330
0.667
s
c
o
u
9. Covariate
Adjustment
Gender, age, type of education, parental education,
owning the house, smoking, passive smoking, and
blood lipids were included in the assessment. BMI,
physical activity, computer use, alcohol use, fish
consumption, blood lead, serum PCBs were also
included in a stepwise regression procedure with
p=0.15 for entering and p=0.10 for remaining in the
model.
High
1
0.500
0.500
3
cS
¦e
es
&
¦3
c
5
£
10. Covariate
Characterization
Information was obtained via questionnaires some
information to be filled out by the adolescent and
some for the parents.
Medium
2
0.250
0.500
C
O
U
.*2
c
4>
O
a.
11. Co-exposure
Confounding
Two of the groups were selected because they lived
near industrial areas. No information was provided
on these industrial areas and what else might be
there. However, they did account for lead and PCBs
(and possibly mercury via fish consumption)
because these may impact the results. Although it is
unclear if there might be other potential co-
exposures, there is no indication that there would be
anything additional that would greatly impact the
results that was not considered.
Medium
2
0.250
0.500
%
*5«
12. Study
Design and
Methods
The cross-sectional study design is appropriate for
evaluating HBCD concentrations in adolescents
with thyroid hormone concentrations. The study was
part of a biomonitoring program for environmental
health surveillance in Flanders, Belgium.
Medium
2
0.400
0.800
13
c
<
13. Statistical
power
Sufficient statistical power with 515 included
subjects.
Medium
2
0.200
0.400
145

14.
Reproducibility of
analyses
Description is not 100% clear on methods to be
reproducible.
Low
3
0.200
0.600
15. Statistical
models
The use of a linear regression or a negative binomial
model were acceptable for the data with
assumptions met or data transformed.
Medium
2
0.200
0.400
Other
16. Use of
Biomarker of
Exposure
No information is provided to indicate serum HBCD
is the appropriate, but the parent compound was
measured.
Medium
2
0.170
0.333
17. Effect
biomarker
Biomarkers of effect shown to have a relationship to
health outcomes, but the method is not well
validated and the mechanism of action is not
understood.
Low
3
0.170
0.500
18. Method
Sensitivity
Frequency of detection of serum HBCD was low.
Although they did not provide specific numbers
below detection for HBCD, the P75 was still below
the LOQ indicating that a large percent was below
detection. Sensitivity was likely okay for the thyroid
hormones.
Low
3
0.170
0.500
Other
19. Biomarker
stability
No information was provided on storage history or
stability of the HBCD or thyroid hormones in the
sample.
Medium
2
0.170
0.333
20. Sample
contamination
There is incomplete documentation of the steps
taken to provide the necessary assurance that the
study data are reliable.
Medium
2
0.170
0.333
146

-------
Study
reference:
Kicinski, M.,Viaene, M. K.,Den Hond, E.,Schoeters, G.,Covaci, A.,Dirtu, A. C.,Nelen, V.,Bruckers, L.,Croes,
K.,Sioen, I.,Baeyens, W.,Van Larebeke, N.,Nawrot, T. S. (2012). Neurobehavioral function and low-level exposure
to brominated flame retardants in adolescents: A cross-sectional study Environmental Health: A Global Access
Science Source, ll(#issue#), 86
HERO ID: 1927571
Domain
Metric
Comments
Qualitative
Determinatio
n
Metric
Score
Metric
Weighting
Factor
Weighted
Score
Other
21. Method requirements
Solid phase extraction followed by gas
chromatography mass spectrometry in electron
capture negative ion mode was used. Specifics of
the extraction were not provided but are assumed to
be the same as used in cited reference (HERO ID
311586). Sensitivity of method for HBCD is not
clear as recovery was not reported. The LOQ was
30 ng/L which seems high compared to the other
PBDEs and the majority of the samples fell below
the LOQ. Few details were provided on the thyroid
hormone tests.
Medium
2
0.170
0.333
22. Matrix
adjustment
Don't think matrix adjustment would be appropriate
for this biomarker of exposure or thyroid hormones.
Not Rated
NR
NR
NR
High: >=1 and <1.7
Medium: >=1.7 and <2.3
Low: >=2.3 and <=3
Sum of scores:

6
12.62
Overall Score = Sum of Weighted Scores/Sum of Metric
Weighting Factors:
2.1033
Overall
Score:
Nearest *:
2.1
Overall Quality Level:
Medium
147

-------
5.5 Epidemiological evaluation results of the Kim et al 2014 study
for thyroid outcomes for mothers & infants
Study
reference:
Kim, U. J.,Oh, J. E. (2014). Tetrabromobisphenol A and hexabromocyclododecane flame retardants in infant-
mother paired serum samples, and their relationships with thyroid hormones and environmental factors
Environmental Pollution, 184(#issue#), 193-200

HERO ID: 2324769

Domain
Metric
Comments
Qualitative
Determinatio
n
Metric
Score
Metric
Weighting
Factor
Weighted
Score

1. Participant selection
Information on participant selection can be found in
a related reference—HERO ID 4182288 (Kim et al.
2012). 38 mother-infant pairs agreed to participate
and had blood collected at a hospital in Seoul
between Nov 2009 and May 2010. Participation
eligibility criteria and participation rate were not
reported. It is unclear whether this sample was
drawn from another previous study (HERO ID
4182289; Kim etal. 2011).
Low
3
0.400
1.200
s
_o
"s
.5-
"3
es
&
2. Attrition
There was no withdrawal of participants from this
sample. Use of imputation methods for missing
exposure data; exposure measurements (BFR)
below the MDL were imputed at 0.5 x MDL to
prevent distortion of the data-set, then the data were
normalized, excluding outliers, to the total BFR.
High
1
0.400
0.400
B
VI
3. Comparison Group
Summary demographic descriptors of the entire
population were reported in a prior study (HERO ID
4182288; Kim et al. 2012). Characteristics were not
reported by case and control group, but there is no
other indication that groups are not similar. It was
reported in this reference that controls did not show
any symptoms of thyroid disease or other metabolic
disorders (including obesity). Therefore, there is
indirect evidence (i.e., stated by the authors without
providing a description of methods) that cases and
controls are similar.
Medium
2
0.200
0.400
a
#o
"s
N
'C
t5
es
~-
es
4. Measurement of
Exposure
HBCD (three diastereomers: alpha-, beta-, gamma-)
concentrations were measured in the serum of
mothers and infants 1 to 3 months after birth.
Quantification methods are provided in Thomsen et
al. 2010 [HERO ID 1927695], HBCDs analyzed by
LC/MS/MS. It should be noted that two infants in
the case group were unable to have blood drawn in
the 1-3 month window. These two infants had
samples collected 18-24 months afterbirth.
High
1
0.400
0.400
U
£
o
s.
H
W
5. Exposure levels
Range is sufficiently large to determine an
exposure-response estimate. Ranges were from
below MDL (0.05 ng/g lipid) to 91 ng/g lipid.
Smallest range was
-------
Study
reference:
Kim, U. J.,Oh, J. E. (2014). Tetrabromobisphenol A and hexabromocyclododecane flame retardants in infant-
mother paired serum samples, and their relationships with thyroid hormones and environmental factors
Environmental Pollution, 184(#issue#), 193-200
HERO ID: 2324769
Domain
Metric
Comments
Qualitative
Determinatio
n
Metric
Score
Metric
Weighting
Factor
Weighted
Score

6. Temporality
Serum samples were taken from mother and infant
within the first three months after birth. This does
not adequately measure prenatal exposure to
HBCDs and serves more as a cross-sectional
measure of HBCD concentrations in cases and
controls. Serum concentrations from the mother or
infant after birth may be related to prenatal exposure
but does not give an accurate indication of prenatal
exposure and its relationship to congenital
hypothyroidism. Thus, the temporality of exposure
and outcome is uncertain.
Low
3
0.400
1.200
Outcome Assessment
7. Outcome
measurement or
characterization
Thyroid hormones were quantified by
radioimmunoassay kits (Diagnostic Products Corp.,
Los Angeles, CA) with a detection limit for T4 and
TSH of 1 ug/dL and 0.02 ug/dL, respectively.
High
1
0.670
0.667
8. Reporting Bias
All of the study's measured outcomes outlined in the
abstract, introduction, and methods were discussed
in the results. Significant results are presented
clearly in tables. However, many non-significant
results were discussed in-text only and this does not
allow for detailed extraction of non-significant
values.
Medium
2
0.330
0.667
Potential Confounding/Variable Control
9. Covariate
Adjustment
There is no indication in this reference or the parent
reference (HERO ID 4182288; Kim et al. 2012) that
potential confounders were considered in the
analysis.
Low
3
0.670
2.000
10. Covariate
Characterization
Covariates were not assessed.
Not Rated
NR
NR
NR
11. Co-exposure
Confounding
Other brominated flame retardants were measured
and reported in this study. There is no indication of
differential exposure between cases and controls.
Medium
2
0.330
0.667
Analysis
12. Study
Design and
Methods
The study design chosen was appropriate for
investigating thyroid hormone levels in relation to
exposure to HBCDs. The study uses an appropriate
statistical method to address the research question.
Medium
2
0.500
1.000
149
-------
Study
reference:
Kim, U. J.,Oh, J. E. (2014). Tetrabromobisphenol A and hexabromocyclododecane flame retardants in infant-
mother paired serum samples, and their relationships with thyroid hormones and environmental factors
Environmental Pollution, 184(#issue#), 193-200

HERO ID: 2324769

Domain
Metric
Comments
Qualitative
Determinatio
n
Metric
Score
Metric
Weighting
Factor
Weighted
Score

13. Statistical power
The sample size of this study is small. There were
38 mother-infant pairs with only 12 mothers and 12
infants with congenital hypothyroidism (diagnosed
in the infant) used in the analysis of correlation
between HBCD concentrations and thyroid
hormones. It is uncertain if the sample size is
adequate to detect an effect in the exposed
population.
Medium
2
0.250
0.500

14.
Reproducibility of
analyses
The analyses (two-sided student's t-test,
normalization of the data set, and outlier exclusions)
are presented clearly in the methods and is sufficient
to understand precisely what has been done and to
be conceptually reproducible with access to the
analytic data.
Medium
2
0.250
0.500

15. Statistical
models
No statistical model used.
Not Rated
NR
NR
NR

16. Use of
Biomarker of
Exposure
Three diastereomers of HBCD were measured in
serum, accurately reflecting exposure to HBCDs.
These biomarkers are in a specified matrix and are
assumed to have an accurate and precise
quantitative relationship with exposure.
High
1
0.140
0.143
Other
17. Effect
biomarker
TSH, T4, and other thyroid hormone levels are
appropriate measures of thyroid conditions.
High
1
0.140
0.143

18. Method
Sensitivity
The lowest rate of detection for HBCDs was 66%
with a MDL of 50 pg/dL. This is low enough to
detect chemicals in a sufficient percentage of the
samples to address the research question. Analytical
methods measuring biomarker are adequately
reported.
Medium
2
0.140
0.286
Other
19. Biomarker
stability
No apparent issues; storage history is documented.
High
NR
NR
NR
150
-------
Study
reference:
Kim, U. J.,Oh, J. E. (2014). Tetrabromobisphenol A and hexabromocyclododecane flame retardants in infant-
mother paired serum samples, and their relationships with thyroid hormones and environmental factors
Environmental Pollution, 184(#issue#), 193-200
HERO ID: 2324769
Domain
Metric
Comments
Qualitative
Determinatio
n
Metric
Score
Metric
Weighting
Factor
Weighted
Score

20. Sample
contamination
Use of blanks and QA/QC documented in detail.
Detailed procedures can be found in the
supplemental material of a parent reference (HERO
ID 4182288; Kim et al. 2012).
High
1
0.140
0.143
Other
21. Method
requirements
HBCDs were analyzed by LC/MS/MS
(Agilentl200/6460QQQMSD, Agilent
Technologies, Santa Clara, CA). Detailed
procedures can be found in the supplemental
material of a parent reference (HERO ID 4182288;
Kimetal. 2012).
High
1
0.140
0.143
22. Matrix
adjustment
HBCDs in serum are presented only as matrix
adjusted (ng/g lipid).
Medium
2
0.140
0.286
High: >=1 and <1.7
Medium: >=1.7 and <2.3
Low: >=2.3 and <=3
Sum of scores:

6
11.15
Overall Score = Sum of Weighted Scores/Sum of Metric
Weighting Factors:
1.8583
Overall
Score:
Nearest *:
1.9
Overall Quality Level:
Medium
151
-------
5.6 Epidemiological evaluation results of the Meijer et al 2012
study for reproductive outcomes for GIC cohort HBCD sex
hormones
Study
reference:
Meijer, L.,Martijn, A.,Melessen, J.,Brouwer, A.,Weiss, J.,de Jong, F. H.,Sauer, P. J. (2012). Influence of prenatal
organohalogen levels on infant male sexual development: sex hormone levels, testes volume and penile length
Human Reproduction, 27(3), 867-872

HERO ID: 1401499

Domain
Metric
Comments
Qualitative
Determinatio
n
Metric
Score
Metric
Weighting
Factor
Weighted
Score
s
1. Participant selection
Subjects were part of the Groningen-infant-compare
cohort (GIC). Cohort consisted of 90 healthy
pregnant women, living in the norther provinces of
the Netherlands, who delivered a single, term,
health infant. This study only focused on the 56
boys born in the cohort; one boy was excluded after
ICSI (intracytoplasmic sperm injection) pregnancy,
which may predispose to aberrations of sexual
development (Wennerholm et al., 2000). How the
initial cohort was selected was not determined nor
do the study authors provide a citation. However,
there is no indication that this sample would not be
representative of the exposure-outcome distribution.
Medium
2
0.400
0.800
Study Participate
2. Attrition
There was minimal subject loss to follow up during
the study. One boy was excluded because he was
born after ICSI pregnancy, which they indicated
could predispose the boy to aberrations of sexual
development. HBCD was only measured in 44 of
the samples, which were randomly selected, due to
financial restraints. Penile length was missing in 8
infants at 18 months due to non-cooperative
behavior or loss to follow-up. There is no indication
how many of these were from the 44 with
measurements for HBCD.
High
1
0.400
0.400

3. Comparison Group
HBCD was evaluated on a continuous basis and
there is no indication that there was anything
different about the exposure in this cohort.
Medium
2
0.200
0.400
Exposure
Characterization
4. Measurement of
Exposure
Maternal serum levels obtained at the 35th week of
pregnancy were measured for HBCD levels at the
Department of Environmental Chemistry,
Stockholm University, Sweden and noted to be
described in Meijer et al., 2008 (HERO ID 787696).
Cited reference provides complete details including
quality control. Therefore, exposure was
consistently assessed using well established
methods of compound in serum.
High
1
0.400
0.400
152
-------
Study
reference:
Meijer, L.,Martijn, A.,Melessen, J.,Brouwer, A.,Weiss, J.,de Jong, F. H.,Sauer, P. J. (2012). Influence of prenatal
organohalogen levels on infant male sexual development: sex hormone levels, testes volume and penile length
Human Reproduction, 27(3), 867-872

HERO ID: 1401499

Domain
Metric
Comments
Qualitative
Determinatio
n
Metric
Score
Metric
Weighting
Factor
Weighted
Score

5. Exposure
levels
Range (not detected to 7.4 ng/g lipid) and
distribution (continuous) of exposure is sufficient to
establish an exposure response estimate.
Medium
2
0.200
0.400

6.
Temporalit
y
Temporality is established, however, it isn't clear if
the levels at 35 weeks of gestation cover the time
window relevant to the outcome of interest (male
sexual development).
Medium
2
0.400
0.800
itcome Assessment
7. Outcome measurement or
characterization
Testes volume was measured by ultrasound.
Measurements were performed by three pediatric
radiologists trained for the examination on the same
Antares ultrasound machine (Siemens, Erlangen,
Germany). Penile length was measured with a
standardized tapeline by the same investigator
throughout the entire study. A detailed description
of how the penile length measurement was made
was included. Thus, these outcomes were
objectively measured with diagnostic methods and
by trained interviewers. There is no reason to
believe that the evaluators would be aware of the
child's exposure status.
High
1
0.670
0.667
O
8. Reporting Bias
All of the study's measured outcomes (primary and
secondary) outlined in the methods, abstract, and/or
introduction (that are relevant for the evaluation)
have not been reported. There are some very
general comments for most of the data relevant to
the assessment and very little of the HBCD data was
actually provided.
Low
3
0.330
1.000
Potential Confounding/Variable
Control
9. Covariate
Adjustment
No consideration was made for any possible
covariates. However, there is no information
provided to indicate that there was a significant
differential distribution that would have affected the
results.
Low
3
0.670
2.000
10. Covariate
Characterization
Covariates were not assessed.
Not Rated
NR
NR
NR
153
-------
Study
reference:
Meijer, L.,Martijn, A.,Melessen, J.,Brouwer, A.,Weiss, J.,de Jong, F. H.,Sauer, P. J. (2012). Influence of prenatal
organohalogen levels on infant male sexual development: sex hormone levels, testes volume and penile length
Human Reproduction, 27(3), 867-872

HERO ID: 1401499

Domain
Metric
Comments
Qualitative
Determinatio
n
Metric
Score
Metric
Weighting
Factor
Weighted
Score

11. Co-exposure
Confounding
The study measured several OHC compounds in the
serum. There is no indication that there is a
correlation between any of these compounds. This is
a general population study with no reason to believe
there would be other differential co-exposures that
would affect the results. However, in this cohort,
compounds, such as phthalates, that also might be
related to sexual development (Hannas et al.,2011)
were not analyzed for.
Medium
2
0.330
0.667

12. Study
Design and
Methods
The study design chosen was appropriate for the
research question. The study used an appropriate
statistical method to address the research question.
Medium
2
0.400
0.800
5«
13. Statistical
power
The number of participants (i.e., 55) seem adequate
to detect an effect in the exposed population.
Medium
2
0.200
0.400
¦a
14.
Reproducibility of
analyses
The description of the analysis is sufficient to
understand precisely what was done and to be
conceptually reproducible with access to the
analytic data.
Medium
2
0.200
0.400

15. Statistical
models
There is a clear description of the analyses.
Medium
2
0.200
0.400
~-
16. Use of
Biomarker of
Exposure
Maternal serum level of HBCD is the biomarker of
exposure and its use is thought to have an accurate
and precise quantitative relationship with external
exposure.
High
1
0.170
0.167
o
17. Effect
biomarker
Sex hormones levels are an acceptable biomarker of
effect and they were determined at the Endocrine
Laboratory, Department of Internal Medicine,
Erasmus Medical Centre, Rotterdam, The
Netherlands as described elsewhere (Laven et al.,
2004).
Medium
2
0.170
0.333
154
-------
Study
reference:
Meijer, L.,Martijn, A.,Melessen, J.,Brouwer, A.,Weiss, J.,de Jong, F. H.,Sauer, P. J. (2012). Influence of prenatal
organohalogen levels on infant male sexual development: sex hormone levels, testes volume and penile length
Human Reproduction, 27(3), 867-872

HERO ID: 1401499

Domain
Metric
Comments
Qualitative
Determinatio
n
Metric
Score
Metric
Weighting
Factor
Weighted
Score

18. Method Sensitivity
Limits of detection are low enough to detect
chemicals in a sufficient percentage of the samples
to address the research question. Analytical methods
measuring biomarker are adequately reported.
LOD/LOQ (value or %) are reported. The limit of
detection (LOD = three times the standard
deviation
of the blank values) was 9 pg/g serum for HBCDD.
Background levels were subtracted from reported
results. HBCDD levels were below LOD in
1/44 samples.
Medium
2
0.170
0.333
0>
19. Biomarker
stability
Although the infant serum was stated to be stored at
-20 degrees C until analysis, there is no information
on how long that was or if there might be any
stability issues. No information was provided on the
storage or stability of the serum samples for HBCD.
Medium
2
0.170
0.333
o
20. Sample
contamination
There is incomplete documentation of the steps
taken to provide the necessary assurance that the
study data are reliable.
Medium
2
0.170
0.333
~-
21. Method
requirements
Instrumentation that provides unambiguous
identification and quantitation of the biomarker at
the required sensitivity (GC-MS) was used.
High
1
0.170
0.167
o
22. Matrix
adjustment
I don't think this is applicable to either matrix
measured.
Not Rated
NR
NR
NR

Sum of scores:

6
11.2
High: >=1 and <1.7
Medium: >=1.7 and <2.3
Overall Score = Sum of Weighted Scores/Sum of Metric
Weighting Factors:
1.8667
Overall
Score:
Nearest *:
1.9
Low: >=2.3 and <=3

Overall Quality Level:

Medium

155
-------
5.7 Epidemiological evaluation results of the Meijer et al 2012
study for reproductive outcomes for GIC cohort HBCD male
sexual development
Study
reference:
Meijer, L.,Martijn, A.,Melessen, J.,Brouwer, A.,Weiss, J.,de Jong, F. H.,Sauer, P. J. (2012). Influence of prenatal
organohalogen levels on infant male sexual development: sex hormone levels, testes volume and penile length
Human Reproduction, 27(3), 867-872

HERO ID: 1401499

Domain
Metric
Comments
Qualitative
Determinatio
n
Metric
Score
Metric
Weighting
Factor
Weighted
Score
s
_o
"s
o.
1. Participant selection
Subjects were part of the Groningen-infant-compare
cohort (GIC). Cohort consisted of 90 healthy
pregnant women, living in the norther provinces of
the Netherlands, who delivered a single, term,
health infant. This study only focused on the 56
boys born in the cohort; one boy was excluded after
ICSI (intracytoplasmic sperm injection) pregnancy,
which may predispose to aberrations of sexual
development (Wennerholm et al., 2000). How the
initial cohort was selected was not determined nor
do the study authors provide a citation. However,
there is no indication that this sample would not be
representative of the exposure-outcome distribution.
Medium
2
0.400
0.800
si
&
¦o
5
Tfl
2. Attrition
There was minimal subject loss to follow up during
the study. One boy was excluded because he was
born after ICSI pregnancy, which they indicated
could predispose the boy to aberrations of sexual
development. HBCD was only measured in 44 of
the samples, which were randomly selected, due to
financial restraints.
High
1
0.400
0.400

3. Comparison Group
HBCD was evaluated on a continuous basis and
there is no indication that there was anything
different about the exposure in this cohort.
Medium
2
0.200
0.400
a
#o
"s
N
'C
t5
si
~-
si
-fl
U
4. Measurement of
Exposure
Maternal serum levels obtained at the 35th week of
pregnancy were measured for HBCD levels at the
Department of Environmental Chemistry,
Stockholm University, Sweden and noted to be
described in Meijer et al., 2008 (HERO ID 787696).
Cited reference provides complete details including
quality control. Therefore, exposure was
consistently assessed using well established
methods of compound in serum.
High
1
0.400
0.400
£
o
o.
UJ
5. Exposure
levels
Range (not detected to 7.4 ng/g lipid) and
distribution (continuous) of exposure is sufficient to
establish an exposure response estimate.
Medium
2
0.200
0.400
156
-------
Study
reference:
Meijer, L.,Martijn, A.,Melessen, J.,Brouwer, A.,Weiss, J.,de Jong, F. H.,Sauer, P. J. (2012). Influence of prenatal
organohalogen levels on infant male sexual development: sex hormone levels, testes volume and penile length
Human Reproduction, 27(3), 867-872

HERO ID: 1401499

Domain
Metric
Comments
Qualitative
Determinatio
n
Metric
Score
Metric
Weighting
Factor
Weighted
Score

6.
Temporalit
y
Temporality is established, however, it isn't clear if
the levels at 35 weeks of gestation cover the time
window relevant to the outcome of interest (male
sexual development).
Medium
2
0.400
0.800
Outcome Assessment
7. Outcome
measurement or
characterization
Sex hormones were measured using acceptable
methods and measured at the Endocrine Laboratory,
Department of Internal Medicine, Erasmus Medical
Centre, Rotterdam, The Netherlands as described
elsewhere (Laven et al., 2004). Sex hormones were
measured in a specific order due to insufficient
amounts of the hormone in some infants.
Medium
2
0.670
1.333
8. Reporting Bias
All of the study's measured outcomes (primary and
secondary) outlined in the methods, abstract, and/or
introduction (that are relevant for the evaluation)
have not been reported. There are some very
general comments for most of the data relevant to
the assessment and very little of the HBCD data was
actually provided.
Low
3
0.330
1.000
"o
~-
c
9. Covariate
Adjustment
No consideration was made for any possible
covariates. However, there is no information
provided to indicate that there was a significant
differential distribution that would have affected the
results.
Low
3
0.670
2.000
U
—
3
es
¦e
1
¦3
c
5
10. Covariate
Characterization
Covariates were not assessed.
Not Rated
NR
NR
NR
£
C
O
U
!§
v*
c
O
a.
11. Co-exposure
Confounding
The study measured several OHC compounds in the
serum. There is no indication that there is a
correlation between any of these compounds. This is
a general population study with no reason to believe
there would be other differential co-exposures that
would affect the results. However, in this cohort,
compounds, such as phthalates, that also might be
related to sexual development (Hannas et al.,2011)
were not analyzed for.
Medium
2
0.330
0.667
Analysis
12. Study
Design and
Methods
The study design chosen was appropriate for the
research question. The study used an appropriate
statistical method to address the research question.
Medium
2
0.400
0.800
157
-------
Study
reference:
Meijer, L.,Martijn, A.,Melessen, J.,Brouwer, A.,Weiss, J.,de Jong, F. H.,Sauer, P. J. (2012). Influence of prenatal
organohalogen levels on infant male sexual development: sex hormone levels, testes volume and penile length
Human Reproduction, 27(3), 867-872

HERO ID: 1401499

Domain
Metric
Comments
Qualitative
Determinatio
n
Metric
Score
Metric
Weighting
Factor
Weighted
Score

13. Statistical
power
The number of participants (i.e., 55) seem adequate
to detect an effect in the exposed population.
Medium
2
0.200
0.400

14.
Reproducibility of
analyses
The description of the analysis is sufficient to
understand precisely what was done and to be
conceptually reproducible with access to the
analytic data.
Medium
2
0.200
0.400

15. Statistical
models
There is a clear description of the analyses.
Medium
2
0.200
0.400

16. Use of
Biomarker of
Exposure
Maternal serum level of HBCD is the biomarker of
exposure and its use is thought to have an accurate
and precise quantitative relationship with external
exposure.
High
1
0.170
0.167
u
CJ
-fl
17. Effect
biomarker
Sex hormones levels are an acceptable biomarker of
effect and they were determined at the Endocrine
Laboratory, Department of Internal Medicine,
Erasmus Medical Centre, Rotterdam, The
Netherlands as described elsewhere (Laven et al.,
2004).
Medium
2
0.170
0.333
o
18. Method Sensitivity
Limits of detection are low enough to detect
chemicals in a sufficient percentage of the samples
to address the research question. Analytical methods
measuring biomarker are adequately reported.
LOD/LOQ (value or %) are reported. The limit of
detection (LOD = three times the standard
deviation
of the blank values) was 9 pg/g serum for HBCDD.
Background levels were subtracted from reported
results. HBCDD levels were below LOD in
1/44 samples.
Medium
2
0.170
0.333
Other
19. Biomarker
stability
Although the infant serum was stated to be stored at
-20 degrees C until analysis, there is no information
on how long that was or if there might be any
stability issues. No information was provided on the
storage or stability of the serum samples for HBCD.
Medium
2
0.170
0.333
158
-------
Study
reference:
Meijer, L.,Martijn, A.,Melessen, J.,Brouwer, A.,Weiss, J.,de Jong, F. H.,Sauer, P. J. (2012). Influence of prenatal
organohalogen levels on infant male sexual development: sex hormone levels, testes volume and penile length
Human Reproduction, 27(3), 867-872
HERO ID: 1401499
Domain
Metric
Comments
Qualitative
Determinatio
n
Metric
Score
Metric
Weighting
Factor
Weighted
Score

20. Sample
contamination
There is incomplete documentation of the steps
taken to provide the necessary assurance that the
study data are reliable.
Medium
2
0.170
0.333
Other
21. Method
requirements
Instrumentation that provides unambiguous
identification and quantitation of the biomarker at
the required sensitivity (GC-MS) was used.
High
1
0.170
0.167
22. Matrix
adjustment
I don't think this is applicable to either matrix
measured.
Not Rated
NR
NR
NR
High: >=1 and <1.7
Medium: >=1.7 and <2.3
Low: >=2.3 and <=3
Sum of scores:

6
11.86
Overall Score = Sum of Weighted Scores/Sum of Metric
Weighting Factors:
1.9767
Overall
Score:
Nearest *:
2
Overall Quality Level:
Medium
159
-------
5.8 Epidemiological evaluation results of the Roze et al 2009 study
for neurological/behavior outcomes in general
Study
reference:
Roze, E.,Meijer, L.,Bakker, A.,Van Braeckel, K. N. J. A.,Sauer, P. J. J.,Bos, A. F. (2009). Prenatal exposure to
organohalogens, including brominated flame retardants, influences motor, cognitive, and behavioral performance
at school age Environmental Health Perspectives, 117(12), 1953-1958

HERO ID: 758049

Domain
Metric
Comments
Qualitative
Determinatio
n
Metric
Score
Metric
Weighting
Factor
Weighted
Score

1. Participant selection
The GIC cohort consisted of 90 white, healthy
pregnant women who were randomly selected from
those who had given birth to a healthy, full-term,
singleton infant. Subjects were selected from the
same general population during the same time frame
using the same methods. Participation rates and
number eligible were not reported. It was noted that
all women who had registered with midwives
between October 2001 and November 2002 were
invited.
Medium
2
0.400
0.800
Study Participation
2. Attrition
HBCD was only measured in 69 of the 90 women
due to financial constraints, but samples were
randomly selected. 62 of these actually participated
in the follow-up programs. The OHC
concentrations of the seven children not followed up
were not different from those who did participate.
Some results were only available in 57 of the
children. Any exclusion of subjects from analyses
was adequately addressed and reasons were
documented when subjects were removed from the
study or excluded from analyses (NTP, 2015a).
High
1
0.400
0.400

3. Comparison Group
There is only indirect evidence (e.g., stated by the
authors without providing a description of methods)
that groups are similar with regard to exposure.
Some differences in baseline characteristics of
groups (such as SES, HOME scores, and sex) were
considered as potential confounding and were
adjusted for in the analyses.
Medium
2
0.200
0.400
a
#o
N
'C
4. Measurement of
Exposure
Maternal serum levels obtained at the 35th week of
pregnancy were measured for HBCD levels. Noted
to be described in Meijer et al., 2008 (HERO ID
787696). Cited reference provides complete details
including quality control. Therefore, exposure was
consistently assessed using well established
methods of compound in the serum.
High
1
0.400
0.400
es
~-
es
-fl
U
£
o
o.
UJ
5. Exposure
levels
Range (0.3-7.5 ng/g lipid) and distribution
(continuous) of exposure is sufficient to establish an
exposure response estimate.
Medium
2
0.200
0.400

6.
Tempor
ality
Temporality is established. However, it isn't clear if
the levels at 35 weeks of gestation cover the time
window relevant to the outcome of interest.
Medium
2
0.400
0.800
160
-------
Study
reference:
Roze, E.,Meijer, L.,Bakker, A.,Van Braeckel, K. N. J. A.,Sauer, P. J. J.,Bos, A. F. (2009). Prenatal exposure to
organohalogens, including brominated flame retardants, influences motor, cognitive, and behavioral performance
at school age Environmental Health Perspectives, 117(12), 1953-1958

HERO ID: 758049

Domain
Metric
Comments
Qualitative
Determinatio
n
Metric
Score
Metric
Weighting
Factor
Weighted
Score
C
S
5«
5«
5«
5«
«<
7. Outcome measurement or
characterization
Children were assessed at 5-6 years of age for motor
performance, cognition, and behavior. Standardized
tests of motor skills for children 4-12 years of age
were used for motor outcome. WPPSI-R was used
for cognitive outcomes, Touwen's age-specific
neurological examination was used to test
coordination, balance, and fine manipulative
abilities. These are standard methods and are
considered to be validated and well-established.
The Dutch version of the Developmental
Coordination Disorder Questionnaire was also filled
out by the parents.
High
1
0.670
0.667
s
O
(J
5
O
8. Reporting Bias
All of the study's measured outcomes (primary and
secondary) outlined in the methods, abstract, and/or
introduction (that are relevant for the evaluation)
have not been reported. Although Table 4 provides
correlation coefficients for a list of outcomes, it
appears that only the significant (less than or equal
to a p value of 0.05) or borderline significant effects
(less than a p value of 0.10) were reported. For
HBCD correlation coefficients were reported for
only 3 outcomes.
Low
3
0.330
1.000
"o
~-
c
o
U
9. Covariate
Adjustment
Results were adjusted for some covariates (such as
SES, HOME, and sex) without providing a
description of methods.
Medium
2
0.500
1.000
3
es
¦e
es
&
¦3
c
5
£
fi
10. Covariate
Characterization
Information was obtained from a questionnaire
during the first year after birth. The validity and
reliability of this questionnaire was not discussed by
the authors.
Medium
2
0.250
0.500
O
U
!§
c
4>
O
a.
11. Co-exposure
Confounding
The study measured several compounds in the
serum. There is no indication that there is a
correlation among any of the compounds. This is a
general population study with no reason to believe
there would be other differential co-exposures that
would influence the results.
Medium
2
0.250
0.500
Analysis
12. Study
Design and
Methods
The prospective cohort study design is appropriate
and uses acceptable statistical method (i.e.,
correlations or Mann-Whitney U test) to address the
research question.
Medium
2
0.400
0.800
161
-------
Study
reference:
Roze, E.,Meijer, L.,Bakker, A.,Van Braeckel, K. N. J. A.,Sauer, P. J. J.,Bos, A. F. (2009). Prenatal exposure to
organohalogens, including brominated flame retardants, influences motor, cognitive, and behavioral performance
at school age Environmental Health Perspectives, 117(12), 1953-1958
HERO ID: 758049
Domain
Metric
Comments
Qualitative
Determinatio
n
Metric
Score
Metric
Weighting
Factor
Weighted
Score

13. Statistical
power
The number of participants (i.e., 62) seem adequate
to detect an effect in the exposed population.
Medium
2
0.200
0.400
14.
Reproducibility of
analyses
The description of the analysis is insufficient to
understand what has been done and to be
reproducible. Table 4 indicates adjustments for SES,
HOME, and sex, but the method description for this
was not complete enough to be reproducible.
Low
3
0.200
0.600
15. Statistical
models
As described, it appears that the method is
appropriate and that assumptions were met (or data
were transformed).
Medium
2
0.200
0.400
Other
16. Use of
Biomarker of
Exposure
Maternal serum levels of HBCD is a biomarker in a
specified matrix that has accurate and precise
relationship with external exposure.
High
1
0.200
0.200
17. Effect
biomarker
No biomarker of effect was measured.
Not Rated
NR
NR
NR
18. Method
Sensitivity
Limits of detection are low enough to detect
chemicals in a sufficient percentage of the samples
to address the research question. Analytical methods
measuring biomarkers are adequately reported.
LOD/LOQ (value or %) are reported.
Medium
2
0.200
0.400
Other
19. Biomarker
stability
No information was provided on storage history or
stability.
Low
3
0.200
0.600
20. Sample
contamination
There is incomplete documentation of the steps
taken to provide necessary assurance that the study
data are reliable.
Medium
2
0.200
0.400
162
-------
Study
reference:
Roze, E.,Meijer, L.,Bakker, A.,Van Braeckel, K. N. J. A.,Sauer, P. J. J.,Bos, A. F. (2009). Prenatal exposure to
organohalogens, including brominated flame retardants, influences motor, cognitive, and behavioral performance
at school age Environmental Health Perspectives, 117(12), 1953-1958
HERO ID: 758049
Domain
Metric
Comments
Qualitative
Determinatio
n
Metric
Score
Metric
Weighting
Factor
Weighted
Score
Other
21. Method
requirements
Instrumentation provides unambiguous
identification and quantification of the biomarker at
the require sensitivity (GC-MS).
Medium
2
0.200
0.400
22. Matrix
adjustment
I don't think any adjustment is needed.
Not Rated
NR
NR
NR
High: >=1 and <1.7
Medium: >=1.7 and <2.3
Low: >=2.3 and <=3
Sum of scores:

6
11.07
Overall Score = Sum of Weighted Scores/Sum of Metric
Weighting Factors:
1.845
Overall
Score:
Nearest *:
1.8
Overall Quality Level:
Medium
163
-------
5.9 Epidemiological evaluation results of the Roze et al 2009 study
for neurological/behavior outcomes for GIC cohort HBCD
neuropsychological
Study
reference:
Roze, E.,Meijer, L.,Bakker, A.,Van Braeckel, K. N. J. A.,Sauer, P. J. J.,Bos, A. F. (2009). Prenatal exposure to
organohalogens, including brominated flame retardants, influences motor, cognitive, and behavioral performance
at school age Environmental Health Perspectives, 117(12), 1953-1958

HERO ID: 758049

Domain
Metric
Comments
Qualitative
Determinatio
n
Metric
Score
Metric
Weighting
Factor
Weighted
Score

3. Comparison Group
There is only indirect evidence (e.g., stated by the
authors without providing a description of methods)
that groups are similar with regard to exposure.
Some differences in baseline characteristics of
groups (such as SES, HOME scores, and sex) were
considered as potential confounding and were
adjusted for in the analyses.
Medium
2
0.200
0.400
a
"s
N
¦e
t5
si
~-
si
4. Measurement of
Exposure
Maternal serum levels obtained at the 35th week of
pregnancy were measured for HBCD levels. Noted
to be described in Meijer et al., 2008 (HERO ID
787696). Cited reference provides complete details
including quality control. Therefore, exposure was
consistently assessed using well established
methods of compound in the serum.
High
1
0.400
0.400
U
5
O
o.
UJ
5. Exposure
levels
Range (0.3-7.5 ng/g lipid) and distribution
(continuous) of exposure is sufficient to establish an
exposure response estimate.
Medium
2
0.200
0.400
164
-------
Study
reference:
Roze, E.,Meijer, L.,Bakker, A.,Van Braeckel, K. N. J. A.,Sauer, P. J. J.,Bos, A. F. (2009). Prenatal exposure to
organohalogens, including brominated flame retardants, influences motor, cognitive, and behavioral performance
at school age Environmental Health Perspectives, 117(12), 1953-1958

HERO ID: 758049

Domain
Metric
Comments
Qualitative
Determinatio
n
Metric
Score
Metric
Weighting
Factor
Weighted
Score

6.
Tempor
ality
Temporality is established. However, it isn't clear if
the levels at 35 weeks of gestation cover the time
window relevant to the outcome of interest.
Medium
2
0.400
0.800
C
S
5«
7. Outcome
measurement or
characterization
Children were assessed at 5-6 years of age for motor
performance, cognition, and behavior. Subtests of
the NEPSY-II were used to assess
neuropsychological function. This is assumed to be
a validated standardized test.
High
1
0.670
0.667
5«
5«
<
s
O
(J
+*
5
O
8. Reporting Bias
All of the study's measured outcomes (primary and
secondary) outlined in the methods, abstract, and/or
introduction (that are relevant for the evaluation)
have not been reported. Although Table 4 provides
correlation coefficients for a list of outcomes, it
appears that only the significant (less than or equal
to a p value of 0.05) or borderline significant effects
(less than a p value of 0.10) were reported. For
HBCD correlation coefficients were reported for
only 3 outcomes.
Low
3
0.330
1.000
"o
~-
c
o
U
9. Covariate
Adjustment
Results were adjusted for some covariates (such as
SES, HOME, and sex) without providing a
description of methods.
Medium
2
0.500
1.000
3
cS
¦e
es
1
C
¦3
c
5
£
fi
10. Covariate
Characterization
Information was obtained from a questionnaire
during the first year after birth. The validity and
reliability of this questionnaire was not discussed by
the authors.
Medium
2
0.250
0.500
O
U
!§
c
o
a.
11. Co-exposure
Confounding
The study measured several compounds in the
serum. There is no indication that there is a
correlation among any of the compounds. This is a
general population study with no reason to believe
there would be other differential co-exposures that
would influence the results.
Medium
2
0.250
0.500
Analysis
12. Study
Design and
Methods
The prospective cohort study design is appropriate
and uses acceptable statistical method (i.e.,
correlations or Mann-Whitney U test) to address the
research question.
Medium
2
0.400
0.800
165
-------
Study
reference:
Roze, E.,Meijer, L.,Bakker, A.,Van Braeckel, K. N. J. A.,Sauer, P. J. J.,Bos, A. F. (2009). Prenatal exposure to
organohalogens, including brominated flame retardants, influences motor, cognitive, and behavioral performance
at school age Environmental Health Perspectives, 117(12), 1953-1958
HERO ID: 758049
Domain
Metric
Comments
Qualitative
Determinatio
n
Metric
Score
Metric
Weighting
Factor
Weighted
Score

13. Statistical
power
The number of participants (i.e., 62) seem adequate
to detect an effect in the exposed population.
Medium
2
0.200
0.400
14.
Reproducibility of
analyses
The description of the analysis is insufficient to
understand what has been done and to be
reproducible. Table 4 indicates adjustments for SES,
HOME, and sex, but the method description for this
was not complete enough to be reproducible.
Low
3
0.200
0.600
15. Statistical
models
As described, it appears that the method is
appropriate and that assumptions were met (or data
were transformed).
Medium
2
0.200
0.400
Other
16. Use of
Biomarker of
Exposure
Maternal serum levels of HBCD is a biomarker in a
specified matrix that has accurate and precise
relationship with external exposure.
High
1
0.200
0.200
17. Effect
biomarker
No biomarker of effect was measured.
Not Rated
NR
NR
NR
18. Method
Sensitivity
Limits of detection are low enough to detect
chemicals in a sufficient percentage of the samples
to address the research question. Analytical methods
measuring biomarkers are adequately reported.
LOD/LOQ (value or %) are reported.
Medium
2
0.200
0.400
Other
19. Biomarker
stability
No information was provided on storage history or
stability.
Low
3
0.200
0.600
20. Sample
contamination
There is incomplete documentation of the steps
taken to provide necessary assurance that the study
data are reliable.
Medium
2
0.200
0.400
166
-------
Study
reference:
Roze, E.,Meijer, L.,Bakker, A.,Van Braeckel, K. N. J. A.,Sauer, P. J. J.,Bos, A. F. (2009). Prenatal exposure to
organohalogens, including brominated flame retardants, influences motor, cognitive, and behavioral performance
at school age Environmental Health Perspectives, 117(12), 1953-1958
HERO ID: 758049
Domain
Metric
Comments
Qualitative
Determinatio
n
Metric
Score
Metric
Weighting
Factor
Weighted
Score
Other
21. Method
requirements
Instrumentation provides unambiguous
identification and quantification of the biomarker at
the require sensitivity (GC-MS).
Medium
2
0.200
0.400
22. Matrix
adjustment
I don't think any adjustment is needed.
Not Rated
NR
NR
NR
High: >=1 and <1.7
Medium: >=1.7 and <2.3
Low: >=2.3 and <=3
Sum of scores:

6
11.07
Overall Score = Sum of Weighted Scores/Sum of Metric
Weighting Factors:
1.845
Overall
Score:
Nearest *:
1.8
Overall Quality Level:
Medium
167
-------
5.10 Epidemiological evaluation results of the Roze et al 2009 study
for neurological/behavior outcomes for GIC cohort HBCD
behavior
Study
reference:
Roze, E.,Meijer, L.,Bakker, A.,Van Braeckel, K. N. J. A.,Sauer, P. J. J.,Bos, A. F. (2009). Prenatal exposure to
organohalogens, including brominated flame retardants, influences motor, cognitive, and behavioral performance
at school age Environmental Health Perspectives, 117(12), 1953-1958

HERO ID: 758049

Domain
Metric
Comments
Qualitative
Determinatio
n
Metric
Score
Metric
Weighting
Factor
Weighted
Score

3. Comparison Group
There is only indirect evidence (e.g., stated by the
authors without providing a description of methods)
that groups are similar with regard to exposure.
Some differences in baseline characteristics of
groups (such as SES, HOME scores, and sex) were
considered as potential confounding and were
adjusted for in the analyses.
Medium
2
0.200
0.400
a
"s
N
¦e
t5
si
~-
si
4. Measurement of
Exposure
Maternal serum levels obtained at the 35th week of
pregnancy were measured for HBCD levels. Noted
to be described in Meijer et al., 2008 (HERO ID
787696). Cited reference provides complete details
including quality control. Therefore, exposure was
consistently assessed using well established
methods of compound in the serum.
High
1
0.400
0.400
U
5
O
o.
UJ
5. Exposure
levels
Range (0.3-7.5 ng/g lipid) and distribution
(continuous) of exposure is sufficient to establish an
exposure response estimate.
Medium
2
0.200
0.400
168
-------
Study
reference:
Roze, E.,Meijer, L.,Bakker, A.,Van Braeckel, K. N. J. A.,Sauer, P. J. J.,Bos, A. F. (2009). Prenatal exposure to
organohalogens, including brominated flame retardants, influences motor, cognitive, and behavioral performance
at school age Environmental Health Perspectives, 117(12), 1953-1958

HERO ID: 758049

Domain
Metric
Comments
Qualitative
Determinatio
n
Metric
Score
Metric
Weighting
Factor
Weighted
Score

6.
Tempor
ality
Temporality is established. However, it isn't clear if
the levels at 35 weeks of gestation cover the time
window relevant to the outcome of interest.
Medium
2
0.400
0.800
C
S
5«
7. Outcome
measurement or
characterization
Children were assessed at 5-6 years of age for motor
performance, cognition, and behavior. To obtain
information on the children's competencies and their
behavioral and emotional problems, the parents
completed the Child behavior checklist and teachers
filled out the Teacher's Report Form. Parents also
filled out an ADHD questionnaire.
Medium
2
0.670
1.333
5«
5«
<
s
O
(J
+*
5
O
8. Reporting Bias
All of the study's measured outcomes (primary and
secondary) outlined in the methods, abstract, and/or
introduction (that are relevant for the evaluation)
have not been reported. Although Table 4 provides
correlation coefficients for a list of outcomes, it
appears that only the significant (less than or equal
to a p value of 0.05) or borderline significant effects
(less than a p value of 0.10) were reported. For
HBCD correlation coefficients were reported for
only 3 outcomes.
Low
3
0.330
1.000
"o
~-
c
o
U
9. Covariate
Adjustment
Results were adjusted for some covariates (such as
SES, HOME, and sex) without providing a
description of methods.
Medium
2
0.500
1.000
3
cS
¦e
es
1
C
¦3
c
5
£
fi
10. Covariate
Characterization
Information was obtained from a questionnaire
during the first year after birth. The validity and
reliability of this questionnaire was not discussed by
the authors.
Medium
2
0.250
0.500
O
U
!§
c
o
a.
11. Co-exposure
Confounding
The study measured several compounds in the
serum. There is no indication that there is a
correlation among any of the compounds. This is a
general population study with no reason to believe
there would be other differential co-exposures that
would influence the results.
Medium
2
0.250
0.500
Analysis
12. Study
Design and
Methods
The prospective cohort study design is appropriate
and uses acceptable statistical method (i.e.,
correlations or Mann-Whitney U test) to address the
research question.
Medium
2
0.400
0.800
169
-------
Study
reference:
Roze, E.,Meijer, L.,Bakker, A.,Van Braeckel, K. N. J. A.,Sauer, P. J. J.,Bos, A. F. (2009). Prenatal exposure to
organohalogens, including brominated flame retardants, influences motor, cognitive, and behavioral performance
at school age Environmental Health Perspectives, 117(12), 1953-1958
HERO ID: 758049
Domain
Metric
Comments
Qualitative
Determinatio
n
Metric
Score
Metric
Weighting
Factor
Weighted
Score

13. Statistical
power
The number of participants (i.e., 62) seem adequate
to detect an effect in the exposed population.
Medium
2
0.200
0.400
14.
Reproducibility of
analyses
The description of the analysis is insufficient to
understand what has been done and to be
reproducible. Table 4 indicates adjustments for SES,
HOME, and sex, but the method description for this
was not complete enough to be reproducible.
Low
3
0.200
0.600
15. Statistical
models
As described, it appears that the method is
appropriate and that assumptions were met (or data
were transformed).
Medium
2
0.200
0.400
Other
16. Use of
Biomarker of
Exposure
Maternal serum levels of HBCD is a biomarker in a
specified matrix that has accurate and precise
relationship with external exposure.
High
1
0.200
0.200
17. Effect
biomarker
No biomarker of effect was measured.
Not Rated
NR
NR
NR
18. Method
Sensitivity
Limits of detection are low enough to detect
chemicals in a sufficient percentage of the samples
to address the research question. Analytical methods
measuring biomarkers are adequately reported.
LOD/LOQ (value or %) are reported.
Medium
2
0.200
0.400
Other
19. Biomarker
stability
No information was provided on storage history or
stability.
Low
3
0.200
0.600
20. Sample
contamination
There is incomplete documentation of the steps
taken to provide necessary assurance that the study
data are reliable.
Medium
2
0.200
0.400
170
-------
Study
reference:
Roze, E.,Meijer, L.,Bakker, A.,Van Braeckel, K. N. J. A.,Sauer, P. J. J.,Bos, A. F. (2009). Prenatal exposure to
organohalogens, including brominated flame retardants, influences motor, cognitive, and behavioral performance
at school age Environmental Health Perspectives, 117(12), 1953-1958
HERO ID: 758049
Domain
Metric
Comments
Qualitative
Determinatio
n
Metric
Score
Metric
Weighting
Factor
Weighted
Score
Other
21. Method
requirements
Instrumentation provides unambiguous
identification and quantification of the biomarker at
the require sensitivity (GC-MS).
Medium
2
0.200
0.400
22. Matrix
adjustment
I don't think any adjustment is needed.
Not Rated
NR
NR
NR
High: >=1 and <1.7
Medium: >=1.7 and <2.3
Low: >=2.3 and <=3
Sum of scores:

6
11.73
Overall Score = Sum of Weighted Scores/Sum of Metric
Weighting Factors:
1.955
Overall
Score:
Nearest *:
2
Overall Quality Level:
Medium
171
-------