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>=,EPA
United States
Environmental Protection Agency
Office of Chemical Safety and
Pollution Prevention
Draft Risk Evaluation for
Cyclic Aliphatic Bromides Cluster
(HBCD)
Supplemental Information on Human Health Hazard
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
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TABLE OF CONTENTS
1 PET ATT,ED HAZARD OVERVIEW 3
1.1 Thyroid Effects 3
1.1.1 Human Evidence 3
1.1.2 Animal Evidence 4
1.1.3 Thyroid Hormones 4
1.1.4 Thyroid Histopathology 5
1.1.5 Thyroid Weight 5
1.1.6 Mechanistic Evidence 17
1.2 Liver Effects 21
1.2.1 Human Evidence 21
1.2.2 Animal Evidence 21
1.2.3 Mechanistic Evidence 31
1.3 Reproductive Effects 32
1.3.1 Female Reproductive Effects 32
1.3.2 Male Reproductive Effects 41
1.4 Developmental Effects 51
1.4.2 Human Evidence 51
1.4.3 Animal Evidence 51
1.4.4 Mechanistic Evidence 58
1.5 Nervous System Effects 59
1.5.2 Human Evidence 59
1.5.3 Animal Evidence 59
1.5.4 Mechanistic Evidence 72
1.6 Immune System Effects 73
1.6.1 Human Evidence 73
1.6.2 Animal Evidence 73
1.6.3 Mechanistic Evidence 87
1.7 Genotoxicity 87
2 DOSE-RESPONSE ANALYSIS 91
2.1 Supplemental Information on Non-Cancer Dose Response Analysis 91
2.1.1 Additional Considerations for Selection of Studies for Dose-Response Analysis.... 91
2.1.2 BMR Selection 94
3 DOSE-RESPONSE MODELING FOR THE DERIVATION OF POINTS OF
DEPARTURE 97
3.1 Noncancer Endpoints for BMD Modeling 98
3.2 Dose-Response Modeling of Non-Cancer Endpoints 101
3.2.1 Evaluation of Model Fit 101
3.2.2 Model Selection 101
3.2.3 Modeling Results 102
4 REFERENCES 189
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Detailed Hazard Overview
1.1 Thyroid Effects
1.1.1 Human Evidence
The association between HBCD exposure and alterations of thyroid hormones was investigated
in populations at different lifestages. Specifically, investigations of the potential effects of
HBCD on the thyroid in humans have been conducted in infants and children participating in
birth cohort studies in the Netherlands (Roze et at.. 2009) and Norway (Eggesbg et at.. 2011).
adolescents participating in a cross-sectional general population study in areas around industrial
sites in Belgium (Kicinski et at.. 2012). and adult men attending an infertility clinic in the United
States (cross-sectional study) (Johnson et at.. 2013). In addition, there is one case-control study
of hypothyroi di sm in Korean mother and infant pairs (Kim and Oh. 2014). Of these five studies,
only two were large scale (>500 participants) (Kicinski et at.. 2012; Eggesbg et at.. 2011). and
only one included an analysis that allowed for the examination of exposure-response patterns
(Eggesbg et at., ). Quantitative methods used by several of the studies resulted in 25-75%
of samples below stated detection limits (Kim and Oh. 1:01 I; Ivi^mski et at.. 2012; Eggesbg et
at.. 2011). While some of the available studies included consideration of other suspected thyroid-
disrupting chemicals, none considered known thyroid antagonists such as perchlorate,
thiocyanate, or nitrate (Steinmaus et at.. 2013; Tonacchera et at.. 2004). Other study limitations
and a summary of overall confidence in the results are noted in Table 1-1. Studies are ordered by
the age at outcome evaluation, and then by overall confidence in the study.
A Norwegian birth cohort did not find a statistically significant association between the levels of
HBCD measured in breast milk and thyroid-stimulating hormone (TSH) levels in newborns
(Eggesbg et at.. ). Elevated, but non-statistically significant, odds ratios (range: 1.3-1.6)
were reported for increased TSH in relation to increasing HBCD levels in breast milk that are
suggestive of a potential association; however, confidence intervals (CIs) around each of the
point estimates were relatively wide (based on approximately 30 individuals per group) and a
clear dose-response was not observed. This analysis controlled for several potential mediators of
normal thyroid hormone variability and several thyroid disruptors (e.g., polychlorinated
biphenyls [PCBs], polybrominated diphenyl ethers [PBDEs], and hexachlorobenzene).
Adjustments for iodine deficiency were not made; however, the study authors noted that this
condition is rare in Norway (Eggesbg et al.J ).
A study in adolescents ages 13-17 years who lived in areas around industrial sites in Belgium (n
= 515) did not find an association between serum concentrations of HBCD and concurrent
measures of TSH, thyroxine (T4), or triiodothyronine (T3) (Kicinski et at.. 2012). Since
approximately 75% of serum concentrations were below the limit of quantitation (LOQ),
analyses were dichotomized to compare effects associated with HBCD concentrations above and
below the LOQ. The three remaining studies (Kim am! iHi 2014; Johnson et at., e et
at., 2009) had reporting deficiencies that limit the ability to interpret results from these studies
(Table 1-2). In studies of infants (Roze et at.. 2009) and adult men (Johnson et at.. 2013). the
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authors did not identify a statistically significant relationship between HBCD and a specific
thyroid hormone; quantitative results pertaining to the magnitude or direction of association
between HBCD and thyroid hormones were not reported. Kim and Olr found no
significant correlations between a-, P-, or y-HBCD and any thyroid hormones in infants with
congenital hypothyroidism; however, reporting limitations of this case-control study (i.e., no
information on participant recruitment) and analysis (i.e., 25% of samples were below the limit
of detection [LOD]) were noted.
The human database for HBCD is inadequate to support conclusions regarding the relationship
between HBCD exposure and thyroid effects. The studies of HBCD exposure in relation to
variation in thyroid hormone levels or thyroid disease (congenital hypothyroidism) do not
provide a basis for assessing a causal association at any lifestage.
1.1.2 Animal Evidence
Several short-term and subchronic rodent studies evaluated the effects of HBCD on the thyroid,
specifically serum thyroid hormone levels, thyroid histopathology, and thyroid weight. Two of
these studies investigated thyroid-related endpoints at time-points approximately 4-8 weeks
following the end of dosing (Saegusa et at.. 2009; 5 search. 2001). The evidence pertaining
to thyroid effects in experimental animals following oral exposure to HBCD is summarized in
Table 1-2 and Figure 1-1. Exposure response array of thyroid effects following oral exposure.
Effect categories with stronger evidence are presented first, with individual studies ordered by
study duration and then species. If not otherwise indicated, endpoint measurements were made in
adults.
1.1.3 Thyroid Hormones
Several studies in rats reported HBCD-related effects on thyroid hormone levels using
radioimmunoassay (van der Yen et al.. 2009; Etna et at.. 2008; van der Yen et al.. 2006) or
electrochemi 1 uminescence immunoassay (Saegusa et al, 2009; WIL Research, 2001).
TSH levels were generally increased in most dosed groups (male and female F0 and F1 CD rats
(Ema et al.. 2008). male and female CD rats (WIL Research. 2001). and male weanling CD rats
(Saegusa et al.. 2009). These increases reached statistical significance in male weanlings
(postnatal day [PND] 20) (Saegusa et al.. 2009) and female adult rats (F0 and F1) (Ema et al..
2008). Additional support for HBCD-mediated increases in TSH are provided by van der Yen et
al. (2006); although serum TSH levels were not directly measured, female rats exposed to 200
mg/kg-day HBCD for 28 days showed a statistically significant increase in pituitary TSH
immunostaining, suggesting elevated synthesis and release of this hormone.
Statistically significant decreases in T4 (up to -38% of control) were observed in F0 rats
exposed to approximately 1,000-1,300 mg/kg-day HBCD (Ema et al., 2008). A dose-related
decrease in T4 was also observed in the F1 generation, with a 28% decrease in T4 in high-dose
females (Ema et al.. 2008). Similarly, male and female rats exposed for 90 days to doses up to
1000 mg/kg-day were observed to have a dose-related decrease in T4 (up to -37% of control)
0 search. 2001). Adult female rats exposed to up to 200 mg/kg-day HBCD for 28 days
also showed a significant dose-dependent decrease in serum T4 (26% decrease at 200 mg/kg-
day) (van der Yen et al., 2006); a dose-related decrease was not observed in male rats in the same
study. The available developmental and one-generation toxicity studies did not detect alterations
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in levels of T4 in offspring at maternal doses ranging from approximately 100 to 1,500 mg/kg-
day (Saeeusa et at.. 2009; van der Yen et at.. 2009). Serum levels of T3 were also investigated in
several studies (Saeeusa et at.. 2009; van der Yen et at.. 2009; Ema et at.. 2008; van der Yen et
at.. 2006; WIL Research. 2001). but only one detected a statistically significant effect. A 15%
decrease in T3 levels relative to controls was observed in male weanling rats treated gestationally
and lactationally at maternal doses of 1,505 mg/kg-day (Saeeusa et at.. 2009).
The pattern of increased TSH and decreased T4 observed in the two-generation reproductive
study (Ema et at.. 2008) is consistent with the multi-loop feedback system of the hypothalamus-
pituitary-thyroid (HPT) axis (Fisher and Nelson. 2012). The same patterns of effect in TSH and
T4 were reported by W.I.L Research (2001); however, confidence in the hormone measurements
from this study is low because approximately 50% of control samples used for TSH
measurements were below the limit of detection and the remaining samples were 1-2 orders of
magnitude lower than controls in other available studies, calling into question the conduct of the
assay.
Two studies also measured thyroid hormone levels 4 weeks (V* search. 2001) or 8 weeks
(Saeeusa et at.. 2009) after the end of dosing. Treatment-related changes in TSH and T3 levels
were still present 8 weeks after the end of dosing in developmentally-exposed rats; however, the
change was statistically significant for T3 only (Saeeusa et at.. 2009). In contrast, T4 and TSH
levels in rats exposed as adults returned to control levels within 4 weeks after cessation of
exposure (WIL R esearch. 2001).
1.1.4 Thyroid Histopathology
Histopathological changes indicative of thyroid activation were observed in some studies in
experimental animals following exposure to HBCD. A 28-day study using doses up to 200
mg/kg-day qualitatively reported a dose-dependent increase in thyroid activation (i.e., follicle
size, epithelial cell height, vacuolization, and nuclear size) in both male and female adult rats
(van der Yen et al.. 2006). A dose-related increase in the incidence of thyroid follicular cell
hypertrophy was reported in adult male and female rats exposed to HBCD for 90 days and in
female rats developmentally exposed to approximately 1,000-1,500 mg/kg-day for 30 days
(Saeeusa et al, 2009; 5 search, 2001). A similar dose-related effect was not observed in a
28-day study at doses up to 1,000 mg/kg-day (WIL Research. 1997) or in a two-generation
reproductive toxicity study at doses up to approximately 1,300 mg/kg-day (Ema et al.. 2008). A
statistically significant increase (46-87%) in the incidence of small thyroid follicles was reported
in both F0 and F1 high-dose animals in a two-generation reproductive toxicity study (Ema et al..
2008). This histological observation is likely indicative of a loss of colloid, which functions as a
reservoir from which T3 and T4 can be released into the bloodstream as needed. With long-term
TSH elevation, endocytosis of colloid occurs faster than synthesis, resulting in the progressive
depletion of colloid and decreased follicle size (Rosot et al.. 2013). Female mice exposed to
approximately 200 mg/kg-day HBCD for 28 days showed a 20 and 26% decrease in follicle and
colloid areas, respectively; however, this change did not reach statistical significance (Maranehi
et al.. 2013).
1.1.5 Thyroid Weight
Several studies in rats reported treatment-related increases in thyroid weight (Saeeusa et al.,
2009; Ema et al.. 2008; van der Yen et al.. 2006; WIL Research. 2001); however, the response
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patterns were not consistently dose-related nor were responses consistent across sexes. In
animals exposed as adults only, several studies reported increased relative thyroid weights in
female rats at doses ranging from approximately 30 to 1,500 mg/kg-day HBCD (Saeeusa et at..
2009; Ema et at.. 2008; van der Yen et at.. 2006; WIL Research. 2001). whereas only one study
reported the same effect in males exposed to approximately 1,000 mg/kg-day (Ema et at.. 2008).
In animals exposed to HBCD during development, statistically significant increases in thyroid
weight were observed in male and female F1 adults exposed to 1,142 and 1,363 mg/kg-day,
respectively (Ema et at.. 2008) and adult males, but not females, 8 weeks after gestational and
lactational exposure to >146 mg/kg-day (Saeeusa et at.. 2009). In a one-generation reproductive
study, no changes in absolute thyroid weight were reported in male or female F1 rats at doses up
to 100 mg/kg-day (van der Yen et al.. 2009); relative thyroid weight was not reported.
Table 1-1. Evidence pertaining to thyroid effects in humans following exposure to
HBCD
Reference and study design
Results
Studies in infants
Eggesba et at. ( (Norway, 2003-2006)
Association between HBCD level in breast milk with
neonatal TSH levels:
Adjusted odds
Adjusted beta ratio for TSH
Exposure category for In TSH >80lh percentile
(ng/g lipid) (N) (95% CI)b (95% CI)C
Population: Birth cohort, recruited within 2 wks of
delivery (able and willing to provide breast milk
sample), 396 randomly selected for analysis; 239 of
these were after February 2004 when the link to the
thyroid screening data became available; 193 with
HBCD data (46% girls)
Exposure measures: Breast milk, collected at a
median of 33 d after delivery (samples pooled over
8 consecutive mornings)
Total HBCD detected in 67.9% of samples
LOQ = 0.2 ng/g lipid
Median 0.54 (range: 0.1-31) ng/g lipid
Effect measures: TSH (whole blood spots)
measured in infants 3 d after delivery (linked data
beginning in February 2004); immunoassay (clinical
lab)
Analysis: Linear regression for In TSH (continuous)
and logistic regression for dichotomized In TSH (at
80th percentile); see results column for consideration
of covariates. Referent category includes all samples
less than the LOQ (n = 62, 32%); remainder of
population divided into four equally-sized
categories.
Data Quality:
High (1.4)
0.10(62) (Referent) (Referent)
0.13-0.52 (31) -0.01 (-0.21,0.20) 1.3 (0.3,4.5)
0.53-0.79 (33) 0.02 (-0.18,0.22) 1.4 (0.3,6.1)
0.80-1.24 (33) 0.12 (-0.08,0.33) 1.6 (0.4,6.1)
1.29-31.2 (34) 0.03 (-0.17,0.23) 1.3 (0.3,5.8)
Per interquartile -0.00 (-0.02, 0.02) 1.0 (0.8, 1.1)
range increase:
Adjusted for age at TSH screening, maternal BMI, county,
p,p-DDE, hexachlorobenzene, delivery type, pregnancy
preeclampsia, and hypertension. Also evaluated but
eliminated were maternal education, age at delivery,
Norwegian nationality, season, parity, smoking, sex,
gestational age, beta-hexachlorocyclohexane, oxychlordane,
and sum of all PCB congeners.
EPA has lower confidence in results per interquartile range
increase than in categorical analysis; this analysis used
HBCD as a continuous variable. The inclusion of non-
detects in this analysis presents considerable uncertainty in
the interpretation of the results.
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Reference and study design
Results
Roze et al. (2009) (the Netherlands, COMPARE
cohort, 2001-2002)
Population: Birth cohort, 90 singleton, term births,
62 of 69 (90%) mother-child pairs randomly selected
from the cohort for HBCD measures in serum
Exposure measures: Prenatal exposure, maternal
serum at 35th week of pregnancy
1,2,5,6,9,10-HBCD (HBCD) detected in all samples
LOD 0.8 pg/g serum
Median 0.8 (range: 0.3-7.5) ng/g lipids
Effect measures: Thyroid hormones (cord blood
samples, n = 51, selected based on amount of sample
available): T4, free T4, reverse T3, T3, TSH,
throxine-binding globulin (assay not described)
Analysis: Pearson correlation (for normally
distributed variables) or Spearman's rank correlation
(for non-normally distributed variables)
Data Quality:a
Medium (1.8)
Results for correlations between HBCD and cord blood
thyroid hormone levels were not shown, but were stated to
be not statistically significant.
Kim and Oh (2( (South Korea, 2009-2010)
Congenital hypothyroidism Healthy controls
Population: 26 infants with congenital
hypothyroidism and their mothers, 12 healthy infant-
mother pairs from the same hospital department also
collected (case-control). Age of infants 1-24 mo;
most 1-3 mo; excluded obese mothers (normal
group only). Sex of infants not reported.
Exposure measures: Serum, a, (3, y-HBCD, most
samples collected 1-3 mo afterbirth, samples from
two congenital hypothyroidism infants collected
18 and 24 mo after birth
LOQ 0.036 ng/g lipid (% less than detection limit
not reported)
Total HBCD: Mean 8.55 ng/g lipid, range from less
than method detection limit to 166 ng/g lipid
Effect measures: Congenital hypothyroidism (not
defined)
Analysis: Two-sided student t-tests; comparisons
between mothers of cases and controls, and between
infant cases and controls. Values below LOQ
replaced by a value of 0.5 times the LOQ;
concentration data normalized, excluding outliers
(not defined), to sum of PBDEs, HBCDs, and
tetrabromobisphenol A.
Data Quality:a
Medium (1.9)
Mothers, mean HBCD level (SD)
a-HBCD 0.494 (1.52) 2.57 (1.48)*
(3-HBCD 0.27 (0.933) 0.461 (1.08)
y-HBCD 2.72 (1.42) 8.86 (2.81)
Infants, mean HBCD level (SD)
a-HBCD 2.42 (3.33) 1.84 (2.5)
(3-HBCD 0.578 (1.71) 0.462 (0.768)
y-HBCD 5.16(2.42) 14.05 (2.87)
Studies in adolescents
Kicinski et al. ( (Belgium, 2008-2011)
Population: 515 adolescents (13-17 yrs old) from
two industrial sites and randomly selected from the
Thyroid hormone results (estimated from Figure 4 of
Kicinski et al. (: :
Beta (95% CI)d
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Reference and study design
Results
general population; participation rates 22-34% in
the three groups, sample size varied by test
Exposure measures: Serum samples, HBCD
>75% were less than the LOQ (LOQ = 30 ng/L);
Median <30 (range: 30 ng/L (LOQ) versus <30 ng/L; 0.0 = no association.
BMI = body mass index; EPA = U.S. Environmental Protection Agency; SD = standard deviation
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Table 1-2. Evidence pertaining to thyroid effects in animals following exposure to
HBCD
Reference and study
design
Results
Scrum thyroid hormones
Doses (mg/kg-d)
Rats, CRL:CD(SD)
Male, F0 0
10
101
1,008
Diet
Two generation
Female, F0 0
Male, Fl 0
14
11
141
115
1,363
1,142
F0: exposure started
Female, Fl 0
14
138
1,363
10 wks prior to mating
TSH (ng/mL)
Fl: dietary exposure post
weaning through
necropsy
Male, F0 (n = 8)
Mean (SD) 16.15(3.78)
16.18(8.61)
19.14(6.02)
23.26 (10.90)
F1/F2 offspring:
% of control3 -
0%
19%
44%
continuous maternal
Female, F0 (n = 8)
exposure throughout
gestation/lactation
Mean (SD) 10.68 (1.35)
% of control3 -
14.83* (2.47)
39%
15.37* (2.17)
44%
21.59* (8.87)
102%
Thyroid hormones were
Male, Fl (n = 8)
measured by
radioimmunoassay in
adults only
Mean (SD) 11.93 (4.62)
% of control3 -
11.50 (2.94)
-4%
15.78 (6.48)
32%
15.54 (5.76)
30%
Female, Fl (n = 8)
Data Quality:d
Mean (SD) 10.35 (2.04)
15.36(4.18)
18.09* (5.23)
17.28* (5.58)
High (1.0)
% of control3 -
48%
75%
67%
T4 (ng/dL)
Male, F0 (n = 8)
Mean (SD) 4.04(1.42)
3.98 (0.89)
2.97 (0.76)
2.49* (0.59)
% of control3 -
-1%
-26%
-38%
Female, F0 (n = 8)
Mean (SD) 2.84(0.61)
3.14(0.48)
3.00 (0.77)
1.96* (0.55)
% of control3 -
11%
6%
-31%
Male, Fl (n = 8)
Mean (SD) 3.54 (0.29)
3.44 (0.86)
3.32 (0.98)
3.18(0.48)
% of control3 -
-3%
-6%
-10%
Female, Fl (n = 8)
Mean (SD) 3.59 (1.08)
3.56 (0.53)
3.39(1.21)
2.58 (0.37)
% of control3 -
-1%
-6%
-28%
T3 (ng/dL)
Male, F0 (n = 8)
Mean (SD) 143.6 (29.0)
138.2 (21.6)
121.6(15.6)
126.9(16.3)
% of control3 -
-4%
-15%
-12%
Female, F0 (n = 8)
Mean (SD) 133.1(15.9)
140.9(16.3)
146.5 (29.5)
134.7 (25.6)
% of control3 -
6%
10%
1%
Male, Fl (n = 8)
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Reference and study
design
Results
Mean (SD) 122.1(9.9)
123 (13.7)
123.6 (22.6)
122.3 (20.4)
% of control3
1%
1%
0%
Female, Fl (n = 8)
Mean (SD) 146.7(17.5)
143.3 (18.1)
132.1 (26.2)
130.4(17.8)
% of control3
-2%
-
10%
-
11%
van der Yen et al.
Doses (mg/kg-d)
(2009)
0
0.1
0.3
1
3
10
30
100
Rats, Wistar
T4 (nmol/L)
Diet
One generation
Male, F0 (n = 5)b
Mean (SD) 62.0
54.2
F0: exposure started one
spermatogenic cycle
(males: 70 d) or two
estrous cycles (females:
14 d) prior to mating
Fl: continuous maternal
exposure throughout
gestation/lactation;
(4.7)
% of control3 -
Female, F0 (n = 5)b
Mean (SD) 44.4
(9.3)
% of control3 -
-
-
-
-
-
-
(13.8)
-13%
38.0
(17.6)
-14%
Male, Fl (n = 3-5)
dietary exposure post
weaning through PNW 11
Mean (SD) 44.8
(4.55)
48.6
(7.6)
46.3
(8.2)
47.2
(3.4)
42.6
(6.6)
45.0
(4.3)
46.6
(5.1)
47.6
(12.4)
Thyroid hormones (total
T3/T4) were measured by
radioimmunoassay in
adults only
% of control3 -
Female, Fl (n = 3-5)
Mean (SD) 50.6
(16.6)
8%
37.8
(13.4)
3%
38.8
(8.2)
5%
49.6
(11.1)
-5%
44.8
(13.5)
0%
59.7
(4.9)
4%
41.4
(12.1)
6%
47.0
(10.8)
Data Quality:d
High (1.2)
% of control3 -
-25%
-23%
-2%
-11%
18%
-18%
-7%
T3 (nmol/L)
Male, F0 (n = 5)b
Mean (SD) 0.9
(0.1)
% of control3 -
Female, F0 (n = 5)b
Mean (SD) 0.8
(0.2)
% of control3 -
-
-
-
-
-
-
0.8
(0.1)
-11%
0.9
(0.3)
12%
Male, Fl (n = 3-5)
Mean (SD) 0.9
1.2
1.0
1.0
1.0
0.9
0.9
1.0
(0.1)
(0.2)
(0.1)
(0.1)
(0.1)
(0.1)
(0.1)
(0.1)
% of control3 -
33%
11%
11%
11%
0%
0%
11%
Female, Fl (n = 3-5)
Mean (SD) 1.1
1.2
1.1
1.1
1.2
1.4
1.0
1.0
(0.3)
(0.2)
(0.2)
(0.1)
(0.2)
(0.1)
(0.1)
(0.1)
% of control3 -
9%
0%
0%
9%
27%
-9%
-9%
Doses (mg/kg-d)
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Reference and study
design
Results
V search (2001)
0
100
300
1,000
Rats, Crl:CD(SD)IGS BR
TSH (ng/mL)
Gavage
90-d exposure starting on
~PNW 7 followed by a
Male (n= 5-10)
Mean (SD) 0.46 (0.42)
3.29 (3.86)
2.65 (2.10)
3.88 (2.98)
28-d recovery period
% of control3
615%
476%
743%
Recovery data not shown
Female (n = 5-10)
Mean (SD) 0.46(0.31)
1.42(1.11)
3.96 (5.15)
2.43 (1.74)
Thyroid hormones (total
% of control3
209%
761%
428%
T3/T4) measured by
T4 (ng/dL)
electro-
chemiluminescence
immunoassay in adults
Male (n= 9-10)
Mean (SD) 7.87(1.22)
6.34* (1.22)
6.28* (1.03)
4.97* (0.76)
only
% of control3
-19%
-20%
-37%
Data Quality:d
High (1.0) - Note: thyroid
hormone metrics were
Female (n =9-10)
Mean (SD) 5.43 (0.86)
% of control3 -
4.96 (0.62)
-9%
4.53* (0.88)
-17%
4.31* (0.76)
-21%
determined to be low
quality due to inadequate
reporting of thyroid
hormone measurement
methods and questionable
control data.
T3 (ng/dL)
Male (n= 9-10)
Mean (SD) 64.36 (9.55)
58.78 (13.01)
58.96 (13.17)
64.23 (9.55)
% of control3
-9%
-8%
0%
Female (n =9-10)
Mean (SD) 73.4 (14.97)
70.78(19.18)
67.02 (17.22)
70.31 (16.78)
% of control3
-4%
-9%
-4%
van der Yen et al.
Doses (mg/kg-d)
(2006)
0
0.3
1 3
10 30
100 200
Rats, Wistar
T4 (nmol/L)
Gavage
28-d exposure starting on
PNW 11
Male (n = 4-5)
Mean (SD) 40.2
40.4
40.6 49.4
43.3 41.9
35.4 41.4
(3.6)
(5.0)
(5.3) (7.2)
(1.3) (4.6)
(4.2) (3.5)
Thyroid hormones (total
% of control3 -
0%
1% 23%
8% 4%
-12% 3%
T3/T4) were measured by
radioimmunoassay
Female (n= 4-5)**
Mean (SD) 41.3
41.9
40.2 37.2
38.6 38
35.8 30.4
(2.6)
(3.1)
(7.3) (4.7)
(1.7) (6.1)
(5.2) (5.9)
% of control3 -
1%
-3% -10%
0s-
00
1
0s-
t"-
1
-13% -26%
T3 (nmol/L)
Data Quality:d
High (1.3)
Male (n = 4-5)
Mean (SD) 0.81
0.84
0.85 0.89
0.97 0.90
0.82 0.89
(0.06)
(0.14)
(0.16) (0.04)
(0.16) (0.13)
(0.06) (0.05)
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% of control3 - 4% 5% 10% 20% 11% 1% 10%
Female (n = 4-5)
Mean (SD) 0.91 0.84 0.88 0.81 0.80 0.74 0.92 0.82
(0.10) (0.15) (0.12) (0.11) (0.09) (0.15) (0.20) (0.13)
% of control3 - -8% -3% -11% -12% -19% 1% -10%
Saegusa et al. (2009)
Rats, Crj:CD(SD)IGS
Diet
Fl: maternal exposure
from GD 10 to PND 20
followed by an 8-wk non-
exposure period through
PNW 11
Thyroid hormones were
measured by
electrochemi-
luminescence
immunoassay in males
only
Data Quality:d
High (1.2)
Doses (mg/kg-d)°
0 15 146 1,505
TSH (ng/mL)
Male, Fl, PND 20 (n = 10)
Mean (SD) 5.40(0.62) 6.66(1.24) 6.07(1.41) 7.00* (1.31)
% of control3 - 23% 12% 30%
Male, Fl, PNW 11 (n= 10)
Mean (SD) 4.74 (0.62) 5.81 (1.72) 5.36 (1.11) 4.96 (0.8)
% of control3 - 23% 13% 5%
T4 (ng/dL)
Male, Fl, PND 20 (n = 10)
Mean (SD) 4.39(0.93) 4.20(0.77) 4.78(0.49) 4.20(0.52)
% of control3 - -4% 9% -4%
Male, Fl, PNW 11 (n= 10)
Mean (SD) 4.77 (0.7) 4.84 (0.59) 5.21 (0.65) 5.20 (0.98)
% of control3 - 1% 9% 9%
T3 (ng/mL)
Male, Fl, PND 20 (n = 10)
Mean (SD) 1.09 (0.11) 1.13 (0.12) 1.06 (0.08) 0.93* (0.10)
% of control3 - 4% -3% -15%
Male, Fl, PNW 11 (n= 10)
Mean (SD) 0.96(0.06) 0.93 (0.07) 0.88* (0.05) 0.89* (0.06)
% of control3 - -3% -8% -7%
'/ hyroid hisiopallio/ogy
Rats, CRL:CD(SD)
Diet
Two generation
F0: exposure started
10 wks prior to mating
Fl: dietary exposure post
weaning until necropsy
F1/F2 offspring:
continuous maternal
Exposure throughout
gestation/lactation
Doses (mg/kg-d)
Male, F0 0 10 101 1,008
Female, F0 0 14 141 1,363
Male, Fl 0 11 115 1,142
Female, Fl 0 14 138 1,363
Decreased thyroid follicle size
Male, F0 (n = 23-24)
Incidence 0/24 0/24 6/24* 20/23*
Female, F0 (n = 23-24)
Incidence 0/24 0/24 5/24* 11/23*
Male, Fl (n = 22-24)
Incidence 0/24 0/24 2/22 11/24*
Female, Fl (n = 24)
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Reference and study
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Results
Incidence 0/24
1/24
5/24*
13/24*
Data Quality:"1
Thyroid follicular cell hypertrophy
High (1.0)
Male, F0 (n = 23-24)
Incidence 0/24
0/24
3/24
1/23
Female, F0 (n = 23-24)
Incidence 0/24
0/24
2/24
0/23
Male, F1 (n = 22-24)
Incidence 0/24
0/24
0/22
0/24
Female, F1 (n = 24)
Incidence 0/24
0/24
0/24
0/24
Thyroid gland histopathology
Treatment-related histopathological thyroid changes were not observed
and F2 animals.
in weanling F1
V search (2001)
Doses (mg/kg-d)
Rats, Crl:CD(SD)IGS BR
0
100
300
1,000
Gavage
90-d exposure starting on
~PNW 7 followed by a
28-d recovery period
Thyroid follicular cell hypertrophy (total incidence, includes all severities)
Male (n= 9-10)
Incidence 1/10
1/10
5/10
8/9
Recovery data not shown
Female (n =9-10)
Incidence 0/10
0/10
4/9
7/10
Data Quality:d
High (1.0)
van der Yen et al.
Doses (mg/kg-d)
(2006)
O
©
3
10 30
100 200
Rats, Wistar
Thyroid activation
Gavage
28-d exposure in adults
starting on PNW 11
Dose-dependent increases in thyroid activation (i.e., follicle size, epithelial cell height,
vacuolization, and nuclear size) were reported qualitatively for both males and females.
Data Quality:d
High (1.3)
WIL Research (1997)
Doses (mg/kg-d)
Rats, Sprague-Dawley
0
125
350
1,000
Gavage
28-d exposure starting on
~PNW 6 followed by a
14-d recovery period
Thyroid follicular cell hypertrophy (total incidence, includes all severities)
Male (n = 6)
Incidence 6/6
Female (n = 6)
6/6
6/6
6/6
Incidence 6/6
5/6
6/6
6/6
Colloid loss (total incidence, includes all severities)
Data Quality:d
High (1.3)
Male (n = 6)
Incidence 5/6
Female (n = 6)
4/6
6/6
6/6
Incidence 4/6
4/6
6/6
6/6
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Reference and study
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Results
Saegusa et al. (2009)
Rats, Crj:CD(SD)IGS
Doses (mg/kg-d)°
0 15
146 1,505
Diet
Thyroid follicular cell hypertrophy
Fl: maternal exposure
from GD 10 to PND 20
Female, F0 (n = 10)
Incidence 3/10 5/10
6/10 9/10*
followed by an 8-wk
recovery period through
PNW 11
Males and females, Fl: no treatment-related histopathological effects.
Data Quality:d
High (1.2)
Maranghi et al.
(2013)
Doses (mg/kg-d)
0
199
Mice, BALB/c
Female (n = 6-8)
Females only
Diet
28-d exposure starting on
PND 26
Colloid area (|inr)
Mean (SD) 1,718 (403)
% of control3 -
1,270 (452)
-26%
Follicle area (|inr)
Data Quality:d
High (1.3)
Mean (SD) 2,402 (500)
% of control3 -
1,927 (610)
-20%
Follicle:colloid ratio
Mean (SD) 1.41(0.07)
% of control3 -
1.53* (0.07)
9%
Thyroid weight
Etna et al. (2008)
Doses (mg/kg-d)
Rats, CRL:CD(SD)
Diet
Two generation
Male, F0 0 10
Female, F0 0 14
Male, Fl 0 11
101 1,008
141 1,363
115 1,142
F0: exposure started
Female, Fl 0 14
138 1,363
10 wks prior to mating
Relative thyroid weight (mg/100 g BW)
Fl: dietary exposure post
weaning through
necropsy
F1/F2 offspring:
continuous maternal
exposure throughout
gestation/lactation
Male, F0 (n = 22-24)
Mean (SD) 4.28(0.71) 4.17(0.77)
% of control3 - -3%
Female, F0 (n = 17-24)
Mean (SD) 6.38 (0.89) 5.99 (1.27)
% of control3 - -6%
4.09 (0.73) 5.17* (1.00)
-4% 21%
6.47 (1.32) 7.20 (1.30)
1% 13%
Thyroid weight measured
in adults only
Data Quality:d
High (1.0)
Male, Fl (n = 22-24)
Mean (SD) 4.03 (0.79) 4.22 (0.63)
% of control3 - 5%
4.15 (0.72) 4.96* (0.87)
3% 23%
Female, Fl (n= 13-22)
Mean (SD) 6.01 (1.01) 6.08(1.05)
% of control3 - 1%
6.54 (1.36) 7.76* (1.36)
9% 29%
Doses (mg/kg-d)
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Reference and study
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Results
van der Yen et al
o
0.1
0.3
1
10
30
100
Absolute thyroid weight (mg)
Rats, Wistar
Diet
One generation
F0: exposure started one
spermatogenic cycle
(males: 70 d) or two
estrous cycles (females:
14 d) prior to mating
Fl: continuous maternal
exposure throughout
gestation/lactation;
dietary exposure post
weaning through PNW 11
Data Quality:d
High (1.2)
Male, Fl (n= 5)
Mean (SD) 26 (3)
% of control3 -
Female, Fl (n = 5)
Mean (SD) 24 (5)
% of control3 -
24 (3)
-8%
30(5)
15%
26 (3)
0%
26 (3)
0%
25 (5) 25 (5)
-4% -4%
26 (1)
0%
21 (3) 19 (4) 20 (5)
-12% -21% -17%
22 (4) 20 (4) 19 (6) 22 (3)
-8% -17% -21% -8%
1 tearch (2001)
Rats, Crl:CD(SD)IGS BR
Gavage
90-d exposure starting on
~PNW 7 followed by a
28-d recovery period
Recovery data not shown
Data Quality:d
High (1.0)
Doses (mg/kg-d)
0
100
300
1,000
Relative thyroid weight (mg/100 mg BW)
Male (n= 9-10)
Mean (SD)
% of control3
Female (n = 10)
Mean (SD)
% of control3
5 (1.2)
6(1.2)
5 (1.6)
0%
7(1.8)
17%
5 (1.6)
0%
6(1.2)
0%
5(1.3)
0%
7 (1.4)
17%
van der Yen et al
Doses (mg/kg-d)
0
0.3
1
10 30 100 200
Rats, Wistar
Gavage
28-d exposure starting on
PNW 11
Data Quality:d
High (1.3)
Relative thyroid weight (g/g BW x 100,000)
Male (n = 3-5)
Response 7.33 4.08 6.13
(1.03) (0.36) (1.68)
% of control3 - -44% -16%
Female (n= 4-5)**
Response 5.98 6.62 8.98
(0.60) (0.68) (1.03)
% of control3 - 11% 50%
6.97
(0.10)
-5%
5.26
(1.35)
-12%
6.02 6.28 5.54 6.46
(2.09) (0.53) (0.39) (1.14)
-18% -14% -24% -12%
7.13 9.52 9.41 9.59
(0.60) (0.59) (2.26) (0.88)
19% 59% 57% 60%
Saeeusa et al
Doses (mg/kg-d)c
Rats, Crj:CD(SD)IGS
Diet
Fl: maternal exposure
from GD 10 to PND 20
followed by an 8-wk non-
0
14.8
146.3
1,505
Relative thyroid weight (mg/100 g BW)
Female, F0 (n = 10)
Mean (SD) 5.73 (0.90) 6.75 (0.99) 6.30 (0.80) 7.47* (1.05)
% of control3 - 18% 10% 30%
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Reference and study
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Results
exposure period through
Male, Fl, PNW 11 (n= 10)
PNW 11
Mean (SD)
4.85 (0.69)
5.66 (0.67)
5.78* (0.82)
6.20* (1.03)
Data Quality:d
High (1.2)
% of control3
-
17%
19%
28%
Female, Fl, PNW 11 (n = 10)
Mean (SD)
8.20 (2.94)
6.84 (0.81)
7.35 (0.87)
7.72 (0.83)
% of control3
-
-17%
-10%
-6%
* Statistically significantly different from the control at p< 0.05 as reported by study authors.
**Significant dose response trend as reported by study authors.
"Percent change compared to control calculated as: (treated value - control value)/control value x 100.
bNot measured; only control and high-dose values reported for endocrine parameters in the F0 animals.
°Time-weighted averages (TWAs) for each exposure group were calculated by multiplying the measured HBCD
intake (mg/kg-day) reported by the study authors for GDs 10-20, PNDs 1-9, and PNDs 9-20 by the number of
inclusive days of exposure for each time.
dBased on OPPT data evaluation criteria
BW = body weight; GD = gestation day; PNW = postnatal week
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4/ follicle size Ema et al., 2008 (rat, Fl F)
4/ follicle size Ema et al., 2008..
nJ/ follicle size Ema et al., 2008 (rat, FO )
t colloid loss WIL, 1997/1998 (rat)
1scolloid ratio Maranghi et al., 2013 (mice)
Saegusa et al., 2009 (rats, FO F)
3 %
= a.
Ema et al., 2008 (rats, FO +F1)
WIL, 1997/1998 (rats)
f WIL, 2001/2002 (rats)
van derVen eta!., 2009 (rats, F0 +F1 )
Van derven et al., 2006 (rats)
Saegusa et al., 2009 (rats, F1 F)
Saegusa etal., 2009 (rats, F1 M)
Saegusa et al., 2009 (rats, FO F)
Ema et al., 2008 (rats, F1 adults F)
Ema et al., 2008 (rats, FO adults F)
Ema et al., 2008 (rats, FO + F1 adults M)
WIL, 2001/2002 (rats, F, wk 17)
WIL, 2001/2002 (rats, M, wk 17)
WIL, 2001/2002 (rats, M+f~wk 13)
Saegusa et al., 2009 (rats, F1M adults)
Saegusa et al,, 2009 (rats, F1 M weanlings)
Ema et al.,2008(rats, F1 F)
Ema et al., 2008 (rats, FO F)
Ema etal.,2008(rats, F0+F1 M)
WIL, 2001/2002 (rats, M + F)
van derVen etal., 2006 (rats, M+F)
Saegusa et al., 2009 (rats, F1M adults)
Saegusa etal., 2009 (rats, F1M weanling)
Ema et al., 2008 (rats, F1 M+F)
Ema et al.,2008 (rats, FO M+F)
van derVen et al., 2009 (rats, F0+ Fl)
WIL, 2001/2002 (rats, F)
WIL, 2001/2002 (rats, M)
van derVen et al., 2006 (rats)
Saegusa et al., 2009 (rats, Fl M adults)
ro Saegusa et al., 2009 (rats, Fl M weanlings)
-> Ema et ai„ 2008 (rats, FO + Fl)
van derVen etal., 2009 (rats, F0+Fl )
WIL, 2001/2002 (rats, M + F)
• significantly changed
O not significantly changed
1 10
Doses (mg/kg-day)
Figure 1-1. Exposure response array of thyroid effects following oral exposure. All studies
scored a High in data quality evaluation.
1.1.6 Mechanistic Evidence
Available mechanistic data suggest that HBCD may interfere with normal thyroid hormone
function. Indirectly, HBCD may decrease circulating thyroid hormone levels by inducing liver
xenobiotic enzymes that are responsible for metabolizing thyroid hormones. Directly, HBCD
may act via the thyroid receptor and regulate thyroid-responsive genes. Evidence to support these
hypothesized modes of action (MOAs) are reviewed below. Other related, but less supported
possible mechanisms, such as competition for thyroid hormone binding proteins and
dysregulation of deiodinases, are also included in this review. The complex interplay of
physiologic processes that regulate thyroid hormone homeostasis and possible sites of disruption
by HBCD are summarized in Figure 1-2 and the text below.
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1.1.6.1 Indirect Pathway: Increased Clearance of Thyroid Hormones
Results from short-term in vivo studies suggest that HBCD induces uridine diphosphate
glucuronyl transferase (UGT), an enzyme that regulates metabolism and irreversible elimination
of T4 (Shelby et at.. 2003; Van sell and Klaassen. 2002; Kelly. 2000). HBCD-mediated activation
of UGT has been observed in both rodent and non-mammalian models (Crump et at.. 2010;
Canton et at.. 2008; Crump et at.. 2008; Palace et at.. 2008; van der Ven et al.. 2006). In rats,
UGT activity showed dose-related increases in both males and females exposed to up to 200
mg/kg-day (van der Ven et al.. 2006) and gene transcription in males exposed to 30 and 100
mg/kg-day HBCD (Canton et al, 2008). Additional support for this mechanism is provided by
data obtained from fish and avian models. Activity of liver UGT increased by approximately
45% in juvenile rainbow trout exposed to a- or P-HBCD isomers in the diet for 56 days (Palace
et al.. 2008). Similarly, the technical mixture or a-HBCD induced hepatic expression of a
UGT 1A1 ortholog in chicken embryos (Crump et al.. 2010; Crump et al.. 2008). These data
suggest that HBCD-mediated induction of UGT could lower serum thyroid hormone levels
through increased thyroid hormone catabolism and excretion (Kato et al, 2008; Klaassen and
Hood. 2001). As shown in Figure 1-2, decreased levels of circulating thyroid hormones trigger
activation of HPT axis feedback mechanisms, which stimulate the release of TSH.
Although the exact mechanism by which HBCD induces UGT is unclear, there is some evidence
to indicate that this effect may be mediated by interaction with the constitutive androstane
receptor (CAR) and/or pregnane X receptor (PXR). Often referred to as xenobiotic sensors, these
nuclear receptors bind to numerous exogenous compounds and regulate metabolizing enzymes
(Chen et al.. 2003; Mackenzie et al.. 2003). HBCD activated CAR in a human breast cancer cell
line (Sakai et al.. 2009). Although Sakai et al. (2009) is the only study that directly investigated
interaction of HBCD with CAR/PXR, these results are supported by studies in HBCD-exposed
animal models showing activation of several other enzymes that are regulated by these nuclear
receptors (Omiecinski et al, 2011; Rosenfeld et al., 2003; Lleda et al, 2002). Upregulation or
increased activity of CYP2B1/2 and CYP3A1/3 was reported in HBCD-exposed rats (Canton et
al.. 2008; Germer et al.. 2006) and chicken embryos (Crump et al.. 2010; Crump et al.. 2008).
Pentoxyresorufin-O-depentylase activity, a biomarker of CYP2B1, was also increased in HBCD-
exposed fish (Zhang et al.. 2008). Additionally, liver weight increases in rats and mice are often
associated with hepatic microsomal induction (Amacher et al.. 1998); thus, the HBCD-induced
liver weight increases (16-108%) observed in rodents (Maranghi et al., 2013; Saegusa et al.,
2009; WIL Research. 2001) are consistent with the findings from these mechanistic studies.
Taken together, these data support the hypothesis that perturbation of thyroid hormones
following HBCD exposure is driven by indirect induction of UGT through interaction with
CAR/PXR.
1.1.6.2 Direct Pathway: Stimulation of Thyroid Hormone Receptor (TR)
Signaling at the Cellular Level
Thyroid hormones bind with the thyroid receptor (TR) to form the thyroid hormone/TR
complex. When formed, this complex translocates into the nucleus to activate transcription via
the thyroid hormone response element (TRE). Xenobiotic chemicals can alter TRE transcription
by interfering with the formation of the thyroid hormone/TR complex or its ability to interact
with the TRE (Kitamura et al.. 2005). Although it is unclear whether HBCD binds to the TR,
there is evidence to support treatment-related TR activation (e.g., proliferation, gene
expression).
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Several in vitro models indicate that HBCD may act as a TR agonist. Two studies evaluated the
effect of HBCD on rat pituitary tumor cells (GH3 cells) that proliferate via TR activation by
T3. Both reported that the technical mixture of HBCD increased GH3 cell proliferation in the
presence of T3 (Hamers et at.. 2006; Schriks et at.. 2006a). In the absence of T3, a-HBCD, but
not other isomers, still induced proliferation; however, the magnitude of the effect was small
(Hamers et at., 2006). Maximal proliferation stimulation by HBCD was observed when T3 was
added simultaneously, which mimics in vivo conditions.
Interaction of HBCD with the TR was also examined in a Xenopus laevis tadpole tail tip
regression model that simulates amphibian metamorphosis. In organ culture, the tail tissue
responds to T3 by undergoing TR-mediated regression (Furlow et at.. 2004; Shaffer.
1963). Schriks et at. (2006b) demonstrated that the T3-induced tadpole tail tip regression was
potentiated by the technical mixture of HBCD. In HeLa cells that constitutively overexpress TRa
and were transfected with TRE luciferase construct, HBCD increased TRE transcription by about
1.8-fold (Yamada-Okabe et at.. 2005). Two studies using green monkey kidney fibroblast (CV-1)
cells transfected with Xenopus TR/TRE luciferase constructs provide inconsistent results
regarding the effects of HBCD on TR activation (Ibhazehiebo et at.. 201 la; Schriks et at..
2007). Notably, this model has less biological relevance in studying TR activation when
compared to those that endogenously express the TR (e.g., "T-screen" assay, X laevis tadpole
tail tip regression, and HeLa cells).
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Indirect Pathway. HBCD induces UGT in the liver, increasing TH elimination, lowering circulating TH levels and
activating the hypothalamic-pituitary-thyroid feedback axis. Direct Pathway: HBCD may interfere with TR
signaling by interfering with binding to the TRE. Other: HBCD may alter thyroid homeostasis through competitive
binding with TTR or dysregulation of deiodinases. CAR/PXR = constitutive antrostane receptor/pregnane X
receptor; Glue = glucuronide; RXR = retinoid X receptor; T4= Thyroxine; T3 = triiodothyronine; TH = thyroid
hormone; TR = thyroid receptor; TRE = thyroid hormone response element; TRH = thyrotropin-releasing hormone;
TSH = thyroid stimulating hormone; TTR = transthretin; UGT = uridine diphosphate glucuronyltransferase;
Figure 1-2. Hypothesized MO As for thyroid effects of HBCD (adapted from Miller et al. (2009))
1.1.6.3 Other Mechanistic Information
Environmental chemicals can alter circulating levels of free T3 and T4 by competitively binding
with the serum transport protein, transthyretin (TTR) (Schussler, 2000; Lans et al.. 1993) or
interacting with deiodinase enzymes (Klammer et al., 2007; Morse et al„ 1993). Two in vitro
studies provide limited evidence of HBCD interaction with TTR. Crump et al. (2008) reported a
>2-fold inhibition of TTR messenger ribonucleic acid (mRNA) transcription in chicken
embryonic hepatocytes following exposure to both the technical mixture and a-HBCD for 24
hours, but this effect diminished after treatment for 36 hours. In a TTR replacement assay, a-
and P-HBCD showed low potency (IC50 >10 |iM), whereas the technical mixture and y-isomer
showed no ability to compete with T4 binding sites (Hamers et al., 2006). Additionally,
dysregulation of deiodinase enzymes that catalyze the deiodination of T4 to T3 can disrupt
thyroid hormone metabolism (Klammer et al.. 2007; Morse et al.. 1993). In the liver, total T4 to
T3 conversion was decreased by approximately 40% in juvenile rainbow trout fed a-, P-, or y-
isomers for 56 days (Palace et al.. 2008); however, the same research group later reported that P-
and y-HBCD increased conversion by approximately 60% in the same species after a 32-day
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dietary exposure (Palace et ai, 2010). Differences in the way enzyme activity was measured in
the two experiments may have contributed to the disparate outcomes. Overall, these data provide
limited evidence for a role of HBCD in dysregulating the conversion of T4 to T3 in the liver.
1.2 Liver Effects
1.2.1 Human Evidence
The potential for HBCD to affect the liver has not been investigated in humans.
1.2.2 Animal Evidence
Several rodent studies have evaluated hepatic effects, including changes in liver weight, liver
chemistry, and histopathology, following oral exposure to HBCD. A summary of liver effects
associated with HBCD exposure is presented in Table 1-3 and Figure 1-3. Effect categories with
stronger evidence are presented first, with individual studies ordered by study duration and then
species. If not otherwise indicated, endpoint measurements were made in adults.
1.2.2.1 Liver Weight
Effects on liver weight were evaluated in eight studies in rats (Saegusa et ai. 2009; van der Yen
et at., 2009; Ema et ai, 2008; van der Yen et ai, 2006; \\ it Kesearch, 2001, I • >• > ' ) and mice
(Yamagisawa et ai. JO I i; Vlaramghi et ai. 2013). With the exception of three studies that
presented only absolute liver weight (Yamagisawa et ai. 2014; van der Yen et ai. 2009; van der
Yen et ai. 2006). study authors reported both absolute and relative liver weights. This discussion
focuses on relative liver weight changes, as this measure has been shown in the general literature
to be more informative in evaluating liver toxicity when there are changes in body weight
(Bailey et ai, 2004); absolute weight data were considered when relative weights were not
available.
Statistically significant increases in relative liver weight were reported in five studies in rats
(Saegusa et ai. 2009; Ema et ai. 2008; V search. 2001. 1997) and mice (Maranghi et ai.
2013) that utilized similar dose ranges (10-1,505 mg/kg-day), generally at concentrations >100
mg/kg-day.
Study authors reported a significant positive trend with dose for absolute liver weight in adult
female, but not male, rats exposed to HBCD for 28 days (van der Yen et ai. 2006). but a later
study by the same research group did not see a similar effect in F1 rats from a one-generation
study (van der Yen et ai. 2009). In a study designed to investigate the influence of HBCD
exposure on metabolic function (Yanagisawa et ai, 2014), absolute liver weight was examined in
male mice dosed once per week for 105 days while being fed either a standard diet or a high-fat
diet (created by mixing lard into the feed) at HBCD dose levels (0.002-0.7 mg/kg-week) several
orders of magnitude lower than other studies. Changes in absolute liver weight were not
observed in mice receiving the standard diet but mice receiving the high-fat diet showed
treatment-related increases. The increased absolute liver weight corresponded with significant
increases in body weight in these animals.
In three rat studies that evaluated animals 2-8 weeks after the end of exposure, liver weight
returned to control levels in all dose groups (Saegusa et ai. 2009; * ssearch. 2001. 1997).
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1.2.2.2 Liver Histopathology
Histopathological changes were investigated following oral exposure to HBCD in six studies in
rats (Saegusa et at., 2009; Ema et at., 2008; ] search, 2001, 1997) and mice (Yanagisawa
et at.. 2014; Maranghi et at.. 2013). Increased hepatocellular vacuolation, which can reflect a
normal physiological process as well as a response to a toxic agent (Henics and Wheats I >»9).
was the most consistently observed histopathological change, with effects seen in male and
female rats and female mice following multiple exposure durations at doses ranging from 100 to
1.505 mg/kg-dav (Maranghi et at.. 2013; Saegusa et at.. 2009;' esearch. 2001. 1997). One
of these studies stained liver sections with lipid- and glycogen-specific stains (Oil Red O and
periodic acid Schiff s reagent, respectively) and characterized the vacuoles as lipid filled (WIL
Research. 2001). With the exception of hypertrophy, which was increased in high-dose females
in the study by ] ^search (2001). no other significant histopathological changes were
reported in the available rat studies; however, some histopathologic changes were observed in
mouse studies. Low HBCD exposures (up to 0.7 mg/kg-week) in male mice showed no
histological changes in mice fed a standard diet; however, increases in microvesicular fatty
changes (steatosis) and hypertrophy (characterized as hepatocyte ballooning) were observed in
the high-dose group given a high-fat diet relative to the high-fat controls. Confidence in these
findings is reduced because other dose groups were not evaluated histologically and data were
presented qualitatively only (Yanagisawa et at.. 2014). In a second mouse study, statistically
significant increases in the incidence of lymphocytic infiltration and tissue congestion, indicators
of inflammation, were observed in female mice administered 199 mg/kg-day (Maranghi et at.,
2013).
In two rat studies that evaluated animals 2-4 weeks after the end of exposure, histopathological
changes returned to control levels in all dose groups (WIL Research. 2001. 1997).
1.2.2.3 Liver Chemistry
Changes in serum liver enzyme levels were investigated as potential indicators of liver damage
following short-term and sub chronic oral exposure to HBCD in five studies in rats (van der Yen
et at., 2009; van der Yen et al., 2006; WIL Research, 2001, 1997) and mice (Yanagisawa et at.,
2014).
Measures of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST),
indicators of hepatocellular injury, showed no biologically or statistically significant increases
with HBCD exposure; indeed, animals in the high-dose groups often showed decreases in these
enzyme levels (Yanagisawa et al., 2014; van der Yen et al., 2009; van der Yen et al., 2006;
Research. 2001. 1997). Although it is generally accepted that increases in serum ALT greater
than 100% of controls is suggestive of hepatocellular damage (Emea. 2008; Boone et al.. 2005).
the biological significance of decreased aminotransferase levels is unclear.
Serum y-glutamyltransferase (GGT) and serum alkaline phosphatase (ALP) activities, markers of
hepatobiliary injury, were also reported in four studies (van der Yen et al., 2009; van der Yen et
al.. 2006; WIL Research. 2001. 1997). GGT was significantly increased in male and female rats
exposed to 1,000 mg/kg-day for 90 days; this effect was not observed following a 4-week
recovery period (WIL Research. 2001) or a shorter (28-day) exposure (WIL Research. 1997). In
general, ALP activity was consistently decreased, sometimes statistically significantly, in male
and female rats (van der Yen et al.. 2009; van der Yen et al.. 2006; WIL Research. 2001. 1997).
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Although decreased ALP levels are not generally associated with liver injury, they can be a
marker of vitamin B«, (pyridoxal phosphate) or zinc deficiency (Hall et at.. 2012; Waner and
Nvska. 19911
Table 1-3. Evidence pertaining to liver effects in animals following exposure to
HBCD
Reference and
study design
Results
Liver weight
Etna et al. (2008)
Doses (mg/kg-d)
Rats, CRL:CD(SD)
Male, F0 0
10
101
1,008
Diet
Two generation
Female, F0 0
F1 offspring3 0
14
17
141
168
1,363
1,570
F0: exposure started
Male, F1 0
11
115
1,142
10 wks prior to mating
Female, F1 0
14
138
1,363
Ft: dietary exposure post
weaning until necropsy
F1/F2 offspring:
continuous maternal
F2 offspring3 0
15
139
1,360
Relative liver weight (g/100 g BW)
Male, F0 (n = 22-24)
exposure throughout
gestation/lactation
Mean (SD) 3.23 (0.26)
% of controlb -
Female, F0 (n = 17-24)
3.33 (0.24)
3%
3.41* (0.31)
6%
4.06* (0.22)
26%
Mean (SD) 4.69 (0.52)
4.76 (0.65)
4.88 (0.48)
6.07* (0.47)
Data Quality:e
High (1.0)
% of controlb -
1%
4%
29%
Male, Fl, PND 26 (n = 17-23)
Mean (SD) 4.60(0.37)
4.60 (0.32)
5.05* (0.32)
6.00* (0.44)
% of controlb -
0%
10%
30%
Female, Fl, PND 26 (n = 14-23)
Mean (SD) 4.57 (0.35)
4.59 (0.28)
5.02* (0.32)
6.07* (0.36)
% of controlb -
0%
10%
33%
Male, Fl, adult (n = 22-24)
Mean (SD) 3.27(0.18)
3.34 (0.26)
3.37 (0.25)
3.86* (0.28)
% of controlb -
2%
3%
18%
Female, Fl, adult (n = 13-22)
Mean (SD) 4.18(0.42)
4.39 (0.44)
4.38 (0.47)
5.05* (0.50)
% of controlb -
5%
5%
21%
Male, F2, PND 26 (n = 13-22)
Mean (SD) 4.72(0.59)
4.74 (0.35)
5.04* (0.4)
6.00* (0.25)
% of controlb -
0%
7%
27%
Female, F2, PND 26 (n = 13-22)
Mean (SD) 4.70 (0.27)
4.70 (0.28)
4.94 (0.32)
5.89* (0.44)
% of controlb -
0%
5%
25%
van der Yen et al.
Doses (mg/kg-d)
(2009)
0 0.1
0.3 1
3 10
30 100
Rats, Wistar
Absolute liver weight (g)
Diet
Male, Fl, PNW 11 (n = 4-5)
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Reference and
study design
Results
One generation
Mean (SD) 11.9
12.3
12.7 14.4
12.2
12.1
14.0
12.0
(1.5)
(0.4)
(0.8) (2.0)
(1.7)
(0.8)
(2.8)
(0.5)
F0: exposure started one
%of
spermatogenic cycle
controlb -
3%
7% 21%
3%
2%
18%
1%
(males: 70 d) or two
estrous cycles (females:
Female, Fl, PNW 11
(n = 4-5)
14 d) prior to mating
Mean (SD) 7.7
7.9
7.8 8.3
7.7
8.3
9.0
8.4
Fl: continuous maternal
(0.9)
(0.8)
(1.4) (0.5)
(0.8)
(0.5)
(1.1)
(0.6)
exposure throughout
%of
3%
1% 8%
0%
8%
17%
9%
gestation/lactation;
controlb
dietary exposure post
weaning through
PNW 11
Data Quality:e
High (1.2)
V search
Doses (mg/kg-d)
0
100
300
1,000
Rats, Crl:CD(SD)IGS
Relative liver weight (g/100
ew)
BR
Male (n = 10)
Gavage
90-d exposure starting on
Mean (SD) 2.71(0.12)
3.18* (0.23)
3.13* (0.27)
3.86* (0.16)
~PNW 7 followed by a
% of control
-
17%
17%
42%
28-d recovery period
Female (n = 10)
Recovery data not shown
Mean (SD) 2.89(0.21)
3.58* (0.27)
3.58* (0.35)
4.31* (0.29)
% of control
—
24%
24%
49%
Data Quality:e
High (1.0)
van der Yen et al.
Doses (mg/kg-d)
(2006)
0
0.3
1 3
10
30
100
200
Rats, Wistar
Absolute liver weight (g)
Gavage
Male (n = 4-5)
28-d exposure starting on
PNW 11
Mean 13.9
17.1
16.2 15.0
17.7
15.7
16.4
16.4
(SD) (0.7)
(3.4)
(3.0) (1.6)
(2.3)
(0.5)
(2.3)
(3.2)
%of
controlb -
23%
17% 8%
27%
13%
18%
18%
Data Quality:e
Female (n= 4-5)**
High (1.3)
Mean 9.7
8.9
8.6 9.5
8.9
11.0
13.0
11.6
(SD) (1.0)
(1.1)
(1.3) (0.4)
(0.6)
(1.0)
(0.5)
(0.6)
%of
controlb -
-8%
-11% -2%
-8%
13%
34%
20%
WIL Research
Doses (mg/kg-d)
0
125
350
1,000
Rats, Sprague-Dawley
Relative liver weight (g/100
e\V)
Gavage
Male (n = 6)
Mean (SD) 3.68(0.16)
4.05 (0.24)
4.29* (0.29)
4.76* (0.44)
% of controlb
-
10%
17%
29%
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Reference and
study design
Results
28-d exposure starting on
~PNW 6 followed by a
14-d recovery period
Female (n = 6)
Mean (SD) 3.84 (0.39)
% of controlb -
4.47* (0.26)
16%
4.69* (0.59)
22%
5.30* (0.25)
38%
Recovery data not shown
Data Quality:e
High (1.3)
Saegusa et al. (2009)
Rats, Crj:CD(SD)IGS
Doses (mg/kg-d)c
0
15
146
1,505
Diet
Relative liver weight (g/100 g
BW)
Fl: maternal exposure
from GD 10 to PND 20
followed by an 8-wk
non-exposure period
through PNW 11
Data Quality:e
High (1.2)
Male, Fl, PND 20 (n = 10)
Mean (SD) 3.68(0.11)
% of controlb -
Female, Fl, PND 20 (n = 10)
Mean (SD) 3.77(0.17)
% of controlb -
Male, Fl, PNW 11 (n= 10)
Mean (SD) 3.45 (0.27)
% of controlb -
Female, Fl, PNW 11 (n = 10)
Mean (SD) 3.35 (0.20)
% of controlb -
3.82 (0.31)
4%
3.83 (0.23)
2%
3.81* (0.23)
10%
3.59(0.19)
7%
3.98 (0.15)
8%
4.01 (0.25)
6%
3.58 (0.24)
4%
3.44 (0.25)
3%
4.66* (0.35)
27%
4.83* (0.26)
28%
3.53 (0.22)
2%
3.30 (0.22)
-1%
Yamagisawa et al.
Doses (mg/kg-wk)
0
0.00175
0.035
0.7
Mice, C57BL/6
Absolute liver weight (mg), standard diet
Males only
Gavage
Animals dosed once
weekly
Male (n = 6)
Mean (SE) 1,261 (54.8)
% of controlb -
1,283 (36.8)
2%
1,159(21.9)
-8%
1,165 (49.4)
-8%
15-week exposure
starting on PNW 6
Dose groups split
between standard and
high-fat diets
Absolute liver weight (mg), high-fat diet
Male (n = 6)
Mean (SE) 1,405 (96.4)
% of controlb -
1,622 (164)
15%
1,662* (87.9)
18%
1,790* (153)
27%
Data Quality:e
Unacceptable (4)*
Maranghi et al.
(2013)
Doses (mg/kg-d)
0
199
Mice, BALB/c
Relative liver weight (%)
Females only
Diet
28-d exposure starting on
PND 26
Female (n= 10-15)
Mean (SD) 4.38 (0.49)
% of controlb -
5.67* (0.4)
29%
Data Quality:e
High (1.3)
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Reference and
study design
Results
Liver hisiopaihology
Ema et al. (2008)
Rats, CRL:CD(SD)
Diet
Two generation
F0: exposure started
10 wks prior to mating
Fl: dietary exposure post
weaning until necropsy
F1/F2: continuous
maternal exposure
throughout gestation/
lactation
Data Quality:e
High (1.3)
Doses (mg/kg-d)
Male, F0
Female, F0
Fl offspring3
Male, Fl
Female, Fl
F2 offspring3
0
10
101
1,008
0
14
141
1,363
0
17
168
1,570
0
11
115
1,142
0
14
138
1,363
0
15
139
1,360
Histopathological findings
Histopathological evaluation did not observe any significant effects with HBCD
exposure.
WIL Research
Doses (mg/kg-d)
(2001)
Rats, Crl:CD(SD)IGS
BR
Gavage
90-d exposure starting on
~PNW 7 followed by a
28-d recovery period
Recovery data not shown
0
100
300
1,000
Hepatocellular hypertrophy
Male (n = 10)
Incidence 0/10
Female (n = 10)
Incidence 0/10
0/10
0/10
0/10
0/10
0/10
5/10
Hepatocellular vacuolation
Male (n= 9-10)
Incidence 2/10
Female (n = 10)
Incidence 3/10
6/10
6/10
5/10
5/10
6/9
9/10
Data Quality:'
High (1.0)
Other histopathological findings
Inflammation was also observed in animals from every treatment group with no pattern
related to dose.
Doses (mg/kg-d)
Rats, Sprague-Dawley
Gavage
28-d exposure starting on
~PNW 6 followed by a
14-d recovery period
Recovery data not shown
Data Quality:e
High (1.3)
0
125
350
1,000
Hepatocellular vacuolation
Male (n = 6)
Incidence 0/6
Female (n = 6)
Incidence 1/6
0/6
4/6
0/6
2/6
0/6
5/6
Other histopathological findings
Inflammation was also observed in animals from every treatment group with no pattern
related to dose.
Saeeusa et al. (2009)
Doses (mg/kg-d)c
Crj:CD(SD)IGS, rat
Diet
0
15
146
1,505
Hepatocellular vacuolar degeneration
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Reference and
study design
Results
Fl: maternal exposure
from GD 10 to PND 20
followed by an 8-wk
non-exposure period
through PNW 11
Data Quality:e
High (1.2)
Male, Fl, PND 20 (n = 10)
Incidence 0/10 0/10
Female, Fl, PND 20 (n = 10)
Incidence 0/10 0/10
0/10
0/10
6/10*
6/10*
Yanaeisawa et al
Doses (mg/kg-wk)
(2014)
Mice, C57BL/6
Males only
Gavage
Animals dosed once
weekly
15-wk exposure starting
on PNW 6
Dose groups split
between standard and
high-fat diets
Data Quality:e
Unacceptable (4)*
0
0.00175
0.035
0.7
Hepatocyte ballooning
The study authors observed development of hepatocyte ballooning following oral high-
dose exposure in male mice fed a high-fat diet.
Microvesicular fatty changes
The study authors observed development of severe microvesicular fatty changes
following oral high-dose exposure in male mice fed a high-fat diet.
Treatment-related effects were not observed in mice fed a standard diet.
Maranghi et al
Doses (mg/kg-d)
(2
BALB/c, mice
Females only
Diet
28-d exposure starting on
PND 26
Data Quality:e
High (1.3)
0
199
Periportal lymphatic filtration
Incidence
0/10
6/8*
Tissue congestion
Incidence
0/10
6/8*
Vacuolation in hepatocytes
Incidence
0/10
5/8*
Liver clicmisirv
Rats, Wistar
Diet
One generation
F0: exposure started one
spermatogenic cycle
(males: 70 d) or two
estrous cycles (females:
14 d) prior to mating
Fl: continuous maternal
exposure throughout
gestation/lactation;
Doses (mg/kg-d)
0
0.1
0.3
10
30
100
ALT (U/L)
Male (n = 4-5)
Mean 37.3
(SD) (1.8)
%of
controlb
Female (n = 5)
Mean 34.7
(SD) (3.3)
%of
controlb
33.6
(4.7)
-10%
37.5
(6.5)
8%
43.6
(7.8)
17%
39.7
(12.6)
14%
43.1
(4.2)
16%
37.3
(4.8)
7%
43.3
(4.4)
16%
33.5
(6.2)
-3%
40.3
(6.8)
8%
30.7
(6.2)
-12%
38.2
(4.7)
2.4%
33.9
(10.4)
-2%
37.2
(2.6)
0%
34.0
(4.6)
-2%
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Reference and
study design
Results
dietary exposure post
ALP (U/L)
weaning through
Male (n = 4-5)
PNW 11
Mean 3.22
4.40
3.28 4.80
3.38 3.20
4.60 3.76
(SD) (2.24) (2.31)
(1.76) (2.79)
(1.90) (0.85)
(2.43) (1.90)
Data Quality:e
%of
37%
2% 49%
5% -1%
43% 17%
High (1.2)
controlb
Female (n= 5)**
Mean 3.78
2.70
3.82 2.64
1.14 3.82
2.66 1.28
(SD) (1.97) (2.37)
(3.23) (0.95)
(0.53) (1.64)
(1.55) (0.59)
%of
-29%
1% -30%
-70% 1%
-30% -66%
controlb
V search
Doses (mg/kg-d)
0
100
300
1,000
Rats, Crl:CD(SD)IGS
ALT (U/L)
BR
Gavage
Male (n= 9-10)
90-d exposure starting on
Mean (SD)
40 (12.8)
31 (4.8)
40 (12)
33 (6)
~PNW 7 followed by a
% of controlb
-
-22%
0%
-18%
28-d recovery period
Female (n = 10)
Recovery data not shown
Mean (SD)
28 (4.9)
30 (5.5)
31 (11.7)
35 (10.2)
% of controlb
-
7%
11%
25%
ALP (U/L)
Male (n = 10)
Mean (SD)
103 (21.5)
87(11.3)
97 (20.1)
87 (17.6)
% of controlb
-
-16%
-6%
-16%
Data Quality:6
Female (n = 10)
High (1.0)
Mean (SD)
58(19.4)
38* (10.7)
39* (10.7)
34* (11.1)
% of controlb
-
-34%
-33%
-41%
AST (U/L)
Male (n= 9-10)
Mean (SD)
89 (21.9)
74 (16.4)
75 (16.9)
67 (10.9)
% of controlb
-
-17%
-16%
-25%
Female (n = 10)
Mean (SD)
83 (17.6)
86 (25.5)
72(19.1)
77 (30.8)
% of controlb
-
4%
-13%
-7%
GGT (U/L)
Male (n= 9-10)
Mean (SD)
0(0)
0 (0.4)
0 (0.7)
1* (1.2)
% of controlb
n/a
n/a
n/a
n/a
Female (n = 10)
Mean (SD)
0(0)
0 (0.4)
0 (0.7)
2* (1.7)
% of controlb
n/a
n/a
n/a
n/a
Doses (mg/kg-d)
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Reference and
study design
Results
van der Yen et al.
0
0.3
1
3
10
30
100
200
(2006)
ALT (U/L)
Rats, Wistar
Male (n = 3-5)
Gavage
28-d exposure starting on
PNW 11
Mean 44.5
(SD) (5.9)
40.9
(4.1)
44.3
(10.3)
38.2
(3.6)
45.0
(14.3)
42.7
(11.0)
40.6
(8.1)
39.2
(10.9)
%of
controlb
Female (n = 3-5)
-8%
0%
-14%
1%
-4%
-9%
-12%
Mean 43.4
44.7
39.8
40.5
34.6
38.2
36.0
42.5
(SD) (4.6)
(6.5)
(4.5)
(6.7)
(6.6)
(5.0)
(5.2)
(7.5)
%of
3%
-8%
-7%
-20%
-12%
-17%
-2%
controlb
Data Quality:6
ALP (U/L)
High (1.3)
Male (n = 3-5)
Mean 7.34
5.30
3.68
7.43
4.88
5.10
2.74
3.48
(SD) (5.59)
(3.66)
(1.82)
(7.43)
(5.75)
(2.54)
(1.61)
(1.95)
%of
-28%
-50%
1%
-34%
-31%
-63%
-53%
controlb
Female (n= 3-5)**
Mean 4.66
3.10
4.74
3.72
2.30
2.36
2.73
2.42
(SD) (2.91)
(2.76)
(2.50)
(2.14)
(1.21)
(0.33)
(1.55)
(2.71)
%of
controlb
-33%
2%
-20%
-51%
-49%
-41%
-48%
V search
Doses (mg/kg-d)
0
125
350
1,000
Rats, Sprague-Dawley
ALT (U/L)
Gavage
28-d exposure starting on
~PNW 6 followed by a
Male (n = 6)
Mean (SD)
31 (4.9)
23
* (5.4)
21* (2.3)
23*
(3.5)
14-d recovery period
% of controlb
-
-26%
-32%
-26%
Recovery data not shown
Female (n = 6)
Mean (SD)
26 (2.1)
24 (3.7)
27 (3.5)
26 (7.9)
% of controlb
-
-8%
4%
0%
ALP (U/L)
Male (n = 6)
Mean (SD) 199 (40.9)
149 (24.7)
165 (34.6)
154 (37.1)
% of controlb
-
-25%
-17%
-23%
Female (n = 6)
Mean (SD) 100 (29.7)
87(11.8)
85 (20.4)
74 (9.7)
% of controlb
-
-13%
-15%
-26%
AST (U/L)
Male (n = 6)
Mean (SD)
30 (18.3)
63
* (5.9)
65 (5.4)
61*
(6.8)
% of controlb
-
-21%
-19%
-24%
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Reference and
study design
Results
Female (n = 6)
Mean (SD)
75 (13.0)
63 (11.5)
61 (9.6)
62 (9.9)
% of controlb
-
-16%
-19%
-17%
Data Quality:6
GGT (U/L)
High (1.3)
Male (n = 6)
Mean (SD)
1 (0.4)
1 (0.5)
1 (0.5)
1 (0.4)
% of controlb
-
0%
0%
0%
Female (n = 6)
Mean (SD)
1 (0.8)
1 (0.8)
1 (0.9)
1 (0.4)
% of controlb
-
0%
0%
0%
Yamagisawa et al.
Doses (ng/kg BW)
0
1.75
35
700
Mice, C57BL/6
ALT (IU/L), standard diet
Males only
Gavage
Animals dosed once
Male (n = 5-6)
Mean (SE)
13.6(1.04)
15.0(1.18)
14.2 (1.59)
10.5 (0.22)
weekly
% of controlb
-
10%
4%
-23%
15-week exposure
starting on PNW 6
ALT (IU/L), high-fat diet
Male (n = 5-6)
Dose groups split
Mean (SE)
34.5 (8.43)
43.0(15.0)
60.0 (12.2)
61.5 (10.2)
between standard and
% of controlb
-
25%
74%
78%
high-fat diets
AST (IU/L), standard diet
Male (n = 5-6)
Data Quality:e
Mean (SE)
73.0 (8.86)
74.2 (7.59)
66.6 (6.57)
46.0* (7.96)
Unacceptable (4)*
% of controlb
-
2%
-9%
-37%
AST (IU/L), high-fat diet
Male (n = 5-6)
Mean (SE)
79.7 (7.44)
78.7 (8.58)
101 (8.39)
85.2 (7.50)
% of controlb
-
-1%
27%
7%
* Statistically significantly different from the control at p< 0.05 as reported by study authors.
**Significant dose response trend as reported by study authors.
aFl and F2 offspring presented as mean maternal gestational and lactational F0 and F1 doses, respectively.
bPercent change compared to control calculated as: (treated value - control value)/control value / 100.
°TWAs for each exposure group were calculated by: (1) multiplying the measured HBCD intake (mg/kg-day)
reported by the study authors for GDs 10-20, PNDs 1-9, and PNDs 9-20 by the number of inclusive days of
exposure for each time period; (2) adding the resulting products together; and (3) dividing the sum by the total
number of inclusive days (33) of HBCD exposure. Example: 100 ppm = (8.1 mg/kg-day x 11 days) +
(14.3 mg/kg-day x 10 days) + (21.3 mg/kg-day x 12 days)/33 days = 14.8 mg/kg-day.
'Based on OPPT data evaluation criteria. *Yanagisawa et at. (20.1.4) was scored unacceptable, so it is assigned a
score of 4. It's calculated score would have been 1.5
"Based on OPPT data evaluation criteria
SE = standard error
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gamma-glutamyl transpeptidase (GGT)WiL, 1997/1998 (rats)
aspartate aminotransferase(AST)WIL, 2001/2002 (rats, F)
aspartate aminotransferase(AST)WIL, 2001/2002 (rats, M)
ALKaiine phosphatase(ALP)WIL 1997/1998 (rats)
alanine arninotransferase(ALT)WIL, 1997/1998 (rats,F)
alanine aminotransferase(ALT)W!L, 1997/1998 (rats,M)
alkaline phosphatase(ALP)van der ven et al., 2006 (rats)
alanine aminotransferase(ALT)van der Ven et al., 2006 (rats)
gamma-glutamyl transpeptidase (GGT)WIL, 2001/2002 (rats)
aspartate aminotransferase(AST)WiL, 2001/2002 (rats)
alkaline phosphatase(ALP)WIL 2001/2002 (rats, F)
ALKaiine phosphatase(ALP)WIL 2001/2002 (rats, M)
alanine aminotransferase(ALT)WIL, 2001/2002 (rats)
alkaline phosphatase(ALP)van derven et al., 2009 (rats)
alanine aminotransferase(AL.T)van derVen et al., 2009 (rats)
(T4-UGT), van der Ven et al., 2006 (rats)
Saegusa et al., 2009 (rats, F, F1 adults)
Saegusa et al., 2009 (rats, M, F1 adults)
Saegusa etal., 2009 (rats, F1 weanlings)
Ema et al., 2008 (rats, F2 weanling F)
Ema etal., 2008 (rats, F2 weanling M)
Ema et al., 2008 (rats, F1 adults)
Ema et al., 2008 (rats, F1 weanling )
Ema et al., 2008 (rats, FO F)
Ema et al., 2008 (rats, FO M)
Maranghi et al., 2013 (mice, F)
WIL, 1997/1998 (rats, M + F)
WIL, 2001/2002 (rats, wk 13)
Yanagisawa et al., 2014 (mice, M, high fat diet
Yanagisawa et al., 2014 (mice, M, normal diet
Van derven et al., 2006 (rats)
(Is vacuolar degeneration) Saegusa et al., 2009 (rats)
(1s vacuolation, tissue congestion, lymphocytic infiltration) Maranghi et al., 2013 (mice)
(1s vacuolation) WIL, 2001/2002 (rats, F)
(t vacuolation) WIL, 2001/2002 (rats, M)
(t hypertrophy) WIL, 2001/2002 (rats)
• significantly changed
O not significantly changed
>__©
o (
o 1
Doses {mg/kg-day)
Figure 1-3. Exposure response array of liver effects following oral exposure. All studies
scored a High in data quality evaluation except for lagisawa et ai. (2014). which scored
Unacceptable. The study is included only for reference (indicated in the chart by X).
1.2.3 Mechanistic Evidence
Studies have reported a generally consistent pattern of increased liver weight related to HBCD
exposure. Increased liver weight is often correlated with induction of hepatic microsomal
enzymes, although the level of induction does not necessarily reflect the magnitude of weight
change, nor it is a requirement for liver weight increases (Amacher et al., 1998). HBCD has been
shown to induce the expression of several hepatic microsomal enzymes (Crump et al.. 2010;
Crump et al.. 2008; Germer et al.. 2006). Specifically, dose-related increases in liver CYP3Al
and CYP2B1 protein levels were observed in rats exposed to HBCD via diet (Germer et al..
2006). In addition, dose-related increases in CYP2H1 and CYP3A37 mRNA levels were
observed in chicken hepatocytes following in ovo (Crump et al.. 2010) and in vitro exposure
(Crump et al., 2008). Furthermore, some data suggest that induction of hepatic microsomal
enzymes responsible for conjugation and elimination of thyroid hormones may contribute to
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HBCD-mediated effects related to thyroid perturbation (Section 1.2.1, Mechanistic Evidence).
Liver weight changes are also associated with increased hepatocellular hypertrophy and
hyperplasia. Hypertrophy was reported in high-dose animals in two studies (\ niiaeisawa et at..
2014; WIL Research. 2001); however, hyperplasia was not noted.
HBCD may also impair lipid homeostasis. Several studies observed increased vacuolation in
hepatocytes (Maranghi et at., 2013; Saeeusa et at., 2009; WIL Research, 2001, 1997). The only
study to evaluate vacuole contents indicated that they predominantly consisted of lipid (WIL
Research. 2001). Chemically-induced impairment of fatty acid metabolism in cells with high
energy demands, such as hepatocytes, has been shown to promote accumulation of triglycerides,
which form nonmembrane bound vacuoles in cells (i.e., fatty change) (W heater and Burkitt.
1996). Various gene expression studies lend supportive evidence for HBCD-mediated disruption
of genes involved in lipid metabolism and transport. A 28-day study in rats reported inhibition of
peroxisome proliferator-activated receptor (PPAR)-mediated genes involved in lipid metabolism,
particularly in females (Canton et at.. 2008). Statistically significant increases in liver
triglyceride levels as well as PPAR-mediated genes involved in lipid metabolism (PPARg) and
transport (FSp27) were also observed in mice exposed to 0.7 mg/kg-week HBCD while being
fed a high-fat diet (Yanaeisawa et at.. 2014).
HBCD-mediated alterations in the regulation of lipid metabolism have also been observed in
avian species and in vitro. HBCD decreased the mRNA expression of liver fatty acid binding
protein in chicken hepatocytes in vitro and following in ovo exposure (Crump et at.. JO 10;
Crump et at.. 2008). The observed effects on lipid homeostasis may be a direct effect or
secondary to perturbation of thyroid function. In humans and animal models, hypothyroidism is
thought to be associated with altered liver metabolism and increased triglycerides and
cholesterol, as well as non-alcoholic fatty liver disease (Eshraehian and Jahromi. 2014; Pucci et
at.. 2000). HBCD studies that evaluated serum lipid profiles did not report any significant
changes in serum cholesterol or triglyceride levels in exposed rats (van der Ven et at.. 2006; WIL
Research. 2001) or mice (Yanaeisawa et at.. 2014) fed a standard diet; however, statistically
significant increases in levels of liver triglycerides were reported in mice exposed concurrently
to HBCD and a high-fat diet (Yanaeisawa et at., 2014).
The lack of increased incidence of necrosis or apoptosis and/or serum enzymatic markers of
hepatocellular damage suggests that HBCD is not highly cytotoxic. However, there is evidence
to suggest the exposure to HBCD can increase the production of reactive oxygen species (ROS).
Dose-related increases in ROS were observed in human hepatocyte and carcinoma cell lines
following in vitro exposures (An et at., 2013; H.u et at., 2009b).
1.3 Reproductive Effects
1.3.1 Female Reproductive Effects
1.3.1.1 Human Evidence
The potential for HBCD to affect the female reproductive system has not been investigated in
humans.
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1.3.1.2 Animal Evidence
Evidence to inform the potential for HBCD to induce female reproductive effects comes from
five studies in rats (Saegusa et at., 2009; van der Yen et at., 2009; Ema et at., 2008; WIL
Research. 2001. 1997) and one study in mice (Maranghi et at.. ) with exposure durations
ranging from 28 days to two generations. Endpoints evaluated in these studies include fertility
and pregnancy outcomes, hormone levels, markers of reproductive differentiation and
development, and reproductive organ weights. Evidence pertaining to female reproductive
effects in experimental animals following oral exposure to HBCD is summarized in Table 1-4
and Figure 1-4. Effect categories with stronger evidence are presented first, with individual
studies ordered by study duration and then species. If not otherwise indicated, endpoint
measurements were made in adults.
Fertility and pregnancy outcomes were evaluated in three rat studies (Saegusa et at.. 2009; van
der Yen et at.. 2009; Ema et at.. 2008). Dose-related decreases in pregnancy incidence in the F0
and F1 dams was reported in the two-generation reproductive toxicity study using doses up to
approximately 1,300 mg/kg-day HBCD (Ema et at.. 2008). In the F1 females, a 36-37%
decrease in the number of primordial follicles was reported at approximately 140 mg/kg-day
HBCD or greater received throughout gestation, lactation, and adulthood (p<0.05) (Ema et at..
2008). This endpoint was only evaluated in the F1 females. The one-generation reproductive
toxicity study, using doses up to 100 mg/kg-day HBCD, reported no significant trend in
successful matings, defined as the rate of matings resulting in offspring (van der Yen et al.,
2009). The results from van der Yen et al. (2009) are not directly comparable to the findings of
Ema et al. (2008) due to the low doses used by investigators (i.e., a dose range lower than doses
associated with effects in Ema et al. (2008)). Incidence of pregnancy was not measured in the
developmental study using doses up to approximately 1,500 mg/kg-day HBCD because the study
began with previously impregnated females (Saegusa et al.. 2009). Other measures of fertility
and pregnancy outcomes (e.g., gestational duration, number of implantation sites, litter size)
reported in these three studies showed no effect with HBCD exposure studies (Saegusa et al..
2009; van der Yen et al.. 2009; Ema et al.. 2008).
HBCD-induced changes in reproductive hormone concentrations were examined in both rats
(Ema et al.. 2008) and mice (Maranghi et al.. 2013). Ema et al. (2008) observed elevated follicle-
stimulating hormone (FSH) concentrations (41%) only in F0 rats exposed to approximately
1,300 mg/kg-day; serum levels of estradiol, testosterone, progesterone, and luteinizing hormone
(LH) were not affected. Statistically significant increases in serum testosterone levels {51%)
were reported in female mice exposed to 199 mg/kg-day for 28 days (Maranghi et al.. 2013).
resulting in a 56% elevation in the testosterone/17P-estradiol ratio.
Effects on reproductive differentiation and development were evaluated in three studies in rats
(Saegusa et al.. 2009; van der Yen et al.. 2009; Ema et al.. 2008). Although van der Yen et al.
(2009) reported a dose-related delay in vaginal opening, a measurement of puberty onset, at
concentrations up to 100 mg/kg-day, no treatment-related effects were observed in the other two
studies that used concentrations up to 1,505 mg/kg-day (Saegusa et al.. 2009; Ema et al.. 2008).
There were no HBCD-mediated effects on anogenital distance (AGD) (Saegusa et al.. 2009; van
der Yen et al.. 2009; Ema et al., 2008).
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Treatment-related effects on female reproductive organ weights were evaluated in six studies
using both rats (Saeeusa et at.. 2009; van der Yen et al.. 2009; Ema et at.. 2008;] ^search.
2001. 1997) and mice (Maranghi et at.. 2013). Absolute uterine weights were decreased by
17-23% in a 90-day oral study in rats (WIL Research. 2001). but the decreases were not dose-
related and returned to control levels after a 4-week recovery period. Absolute, but not relative,
uterine weight showed a statistically significant decrease (22%) in F2 rats (PND 26) in the high-
dose group (approximately 1,300 mg/kg-day) (Ema et al, 2008); no exposure-related effects on
uterine weight were observed in F1 animals. No other clear treatment-related effects were
observed on absolute or relative uterine (Maranghi et at.. 2013; Saeeusa et al.. 2009; van der Yen
et al.. 2009) or ovary weights (Saeeusa et al.. 2009; van der Yen et al.. 2009; Ema et al.. 2008;
search. 2001. 1997).
Table 1-4. Evidence pertaining to female reproductive effects in animals following
exposure to HBCD
Reference and
study design
Results
l-ertility and pregnancy outcomes
Ema et at. (2008)
Doses (mg/kg-d)
Rats, CRL:CD(SD)
Female, F0 0
14
141
1,363
Diet
Two generation
Female, Fl 0
14
138
1,363
Incidence of pregnant females
F0: exposure started
Female, F0 (n = 23-24)
10 wks prior to mating
Fl: dietary exposure post
weaning through
necropsy
F1/F2 offspring:
Incidence 24/24
Female, Fl (n= 21-24)
Incidence 23/24
22/24
23/24
20/24
21/24
19/23
21/24
Primordial follicles (count)
continuous maternal
Female, Fl (n = 10)
exposure throughout
gestation/lactation
Mean (SD) 316.3(119.5)
% of control3 -
294.2 (66.3)
-7%
197.9* (76.9)
-37%
203.4* (79.5)
-36%
Data Quality:d
Other pregnancy outcomes
High (1.0)
No dose-related changes in other outcomes (e.g., number of implantation sites,
gestation duration, litter size) reported in either generation
van der Yen et at.
Doses (mg/kg-d)
(2009)
0 0.1
0.3 1
3 10
30 100
Rats, Wistar
Successful matings
Diet
One generation
Female, F0 (n =8-10)
Incidence 8/10 8/10
4/10 7/10
8/10 6/8
6/10 6/10
F0: exposure started one
spermatogenic cycle
(males: 70 d) or two
estrous cycles (females:
14 d) prior to mating
Fl: continuous maternal
exposure throughout
gestation/lactation;
Other pregnancy outcomes
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Reference and
study design
Results
dietary exposure post
weaning through PNW
11
No significant dose-response trend in other outcomes (e.g., number of implantation
sites, gestation duration, litter size)
Data Quality:d
High (1.0)
Saegusa et al. (2009)
Crj:CD(SD)IGS, rat
Diet
Doses (mg/kg-d)°
0 15 146 1,505
Pregnancy outcomes
Fl: maternal exposure
from GD 10 to PND 20
followed by an 8-wk
non-exposure period
through PNW 11
No dose-related effect on pregnancy outcomes (e.g., number of implantation sites,
gestation duration, litter size)
Data Quality:d
High (1.2)
I1 or man a! measures
Rats, CRL:CD(SD)
Diet
Two generation
Doses (mg/kg-d)
Female, F0 0 14 141 1,363
Female, Fl 0 14 138 1,363
FSH (ng/mL)
F0: exposure started
10 wks prior to mating
Fl: dietary exposure post
weaning through
necropsy
F1/F2 offspring:
continuous maternal
Female, F0 (n = 8)
Mean (SD) 4.17 (0.51) 4.84 (0.63) 4.88 (1.05) 5.86* (1.11)
% of control3 - 16% 17% 41%
Female, Fl (n = 8)
Mean (SD) 5.89 (1.60) 6.07 (0.60) 6.33 (0.82) 6.52 (0.95)
% of control3 - 3% 7% 11%
exposure throughout
gestation/lactation
Data Quality:d
High (1.0)
Other hormone measurements
Exposure-related changes were not found for progesterone, LH, or estradiol in the F0
andFl females.
Maranghi et al.
(2013)
Doses (mg/kg-d)
0 199
Mice, BALB/c
Testosterone (ng/mL)
Females only
Diet
28-d exposure starting on
PND 26
Female (n = 10)
Mean (SD) 0.07(0.02) 0.11* (0.07)
% of control3 - 57%
Testosterone/estradiol
Data Quality:d
High (1.3)
Female (n = 10)
Mean (SD) 8.5(2.1) 13.3* (6.7)
% of control3 - 56%
Other hormone measurements
Exposure-related changes were not found for estradiol.
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Reference and
study design
Results
Reproductive differentiation and development
Etna et al. (2008)
Rats, CRL:CD(SD)
Diet
Two generation
Doses (mg/kg-d)
Fl offspringd 0
F2 offspringd 0
17
15
168
139
1,570
1,360
F0: exposure started
10 wks prior to mating
Fl: dietary exposure post
weaning through
necropsy
Time to vaginal opening (d)
Female Fl (n = 24)
Mean (SD) 30.9 (2.0)
% of control3
30.3 (2.6)
-2%
30.1 (1.8)
-3%
30.8 (2.2)
0%
F1/F2 offspring:
continuous maternal
exposure throughout
gestation/lactation
AGD (mm)
No dose-related changes in the Fl or F2 female pups
Data Quality:d
High (1.0)
van der Yen et al.
(2009)
Doses (mg/kg-d)
0 0.1
0.3 1
3 10
30 100
Rats, Wistar
Time to vaginal opening (days)
Diet
One generation
F0: exposure started one
spermatogenic cycle
Female, Fl (n = 4-5)b **
Mean (SD) 35.4 35.3
(2.3) (2.2)
% of control3 - 0%
36.2 36.8
(2.4) (4.1)
2% 4%
36.8 35.4
(3.3) (2.7)
4% 0%
34.8 39.9
(1.6) (2.6)
-2% 13%
(males: 70 d) or two
estrous cycles (females:
14 d) prior to mating
Fl: continuous maternal
exposure throughout
gestation/lactation;
dietary exposure post
weaning through PNW
11
AGD (mm)
No significant dose-response trend
Data Quality:d
High (1.2)
Saegusa et al. (2009)
Crj:CD(SD)IGS, rat
Doses (mg/kg-d)c
0
15
146
1,505
Diet
Time to vaginal opening (d)
Fl: maternal exposure
from GD 10 to PND 20
followed by an 8-wk
Female Fl (n= 12-14)
Mean (SD) 35.4(1.9)
% of control3 -
35.6(1.8)
1%
34.9 (1.7)
-1%
34.4 (2.1)
-3%
non-exposure period
through PNW 11
Data Quality:d
High (1.3)
AGD (mm)
No dose-related change
Reproductive organ weights
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Reference and
study design
Results
Etna et al. (2008)
Doses (mg/kg-d)
Rats, CRL:CD(SD)
Fl offspring"1 0
17
168
1,570
Diet
Two generation
Female Fl adult 0
F2 offspring"1 0
14
15
138
139
1,363
1,360
F0: exposure started
Absolute ovary weight (mg)
10 wks prior to mating
Female, Fl, PND 26 (n = 14-23)
Fl: dietary exposure post
weaning through
necropsy
F1/F2 offspring:
Mean (SD) 20.8(3.7)
% of control3 -
Female, Fl, adult (n = 13-22)
22.8 (3.6)
10%
21.0 (4.0)
1%
20.9 (3.4)
0%
continuous maternal
exposure throughout
gestation/lactation
Mean (SD) 102.4(12.9)
% of control3 -
Female, F2, PND 26 (n = 13-21)
106.4(13.2)
4%
108.6 (18.0)
6%
104.9 (16.9)
2%
Mean (SD) 20.0(3.9)
22.9* (2.6)
20.9 (3.9)
18.2 (4.0)
Data Quality:d
% of control3 -
14%
4%
-9%
High (1.0)
Relative ovary weight (mg/100 g
BW)
Female, Fl, PND 26 (n = 14-23)
Mean (SD) 26.5(4.5)
27.5 (4.1)
25.0 (3.8)
28.9 (3.7)
% of control3 -
4%
-6%
9%
Female, Fl, adult (n = 13-22)
Mean (SD) 31.8(4.2)
32.6 (3.9)
33.1 (5.3)
34.1 (4.2)
% of control3 -
3%
4%
7%
Female, F2, PND 26 (n = 13-21)
Mean (SD) 26.9(5.1)
30.5* (3.9)
28.8 (4.2)
32.1* (7.5)
% of control3 -
13%
7%
19%
Absolute uterus weight (mg)
Female, Fl, PND 26 (n = 14-23)
Mean (SD) 57.0(10.9)
62.0(14.1)
64.1 (18.6)
51.9(12.4)
% of control3 -
9%
12%
-9%
Female, Fl, adult (n = 13-22)
Mean (SD) 966(216)
913 (188)
955 (204)
949 (156)
% of control3 -
-5%
-1%
-2%
Female, F2, PND 26 (n = 13-21)
Mean (SD) 60.8(16.1)
63.6(15.1)
57.0 (15.7)
47.6* (11.4)
% of control3 -
5%
-6%
-22%
Relative uterus weight (mg/100 g
rBW)
Female, Fl, PND 26 (n = 14-23)
Mean (SD) 73.6(17.5)
74.9 (17.7)
76.0 (18.4)
71.9(16.2)
% of control3 -
2%
3%
-2%
Female, Fl, adult (n = 13-22)
Mean (SD) 299 (64)
282 (65)
291 (64)
313 (69)
% of control3 -
-6%
-3%
5%
Female, F2, PND 26 (n = 13-21)
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Reference and
study design
Results
Mean (SD) 80.9(16.3)
% of control3 -
84.4 (21.0)
4%
78.7 (21.7)
-3%
83.7 (20.3)
3%
van der Yen et al.
(2009)
Doses (mg/kg-d)
0 0.1
0.3 1
3 10
30 100
Rats, Wistar
Absolute ovary weight (left and right) (g)
Diet
One generation
F0: exposure started one
spermatogenic cycle
(males: 70 d) or two
estrous cycles (females:
14 d) prior to mating
Female, Fl, PNW 11 (n = 4-5)
Mean (SD) 0.10 0.13
(0.01) (0.02)
% of control3 - 21%
0.11 0.11
(0.02) (0.003)
11% 9%
0.13 0.11
(0.02) (0.02)
24% 8%
0.12 0.11
(0.02) (0.02)
17% 1%
Fl: continuous maternal
Absolute uterus weight (g)
exposure throughout
gestation/lactation;
dietary exposure post
weaning through PNW
11
Female, Fl, PNW 11 (n = 4-5)
Mean (SD) 0.53 0.60
(0.11) (0.20)
% of control3 - 13%
0.50 0.75
(0.11) (0.38)
-6% 42%
0.71 0.94
(0.39) (0.28)
34% 77%
0.48 0.49
(0.10) (0.22)
-9% -8%
Data Quality:d
High (1.2)
V search
Doses (mg/kg-d)
0
100
300
1,000
Rats, Crl:CD(SD)IGS
Absolute ovary with oviduct weight (g)
BR
Gavage
90 d exposure starting on
~PNW 7 followed by a
Female (n = 10)
Mean (SD) 0.14(0.03)
% of control3 -
0.13 (0.03)
-10%
0.13 (0.03)
-9%
0.15 (0.02)
3%
28-d recovery period
Relative ovary with oviduct weight (g/100 g BW)
Recovery data not shown
Female (n = 10)
Mean (SD) 0.05(0.01)
% of control3 -
0.05 (0.01)
-8%
0.05 (0.01)
-12%
0.05 (0.01)
2%
Data Quality:d
High (1.0)
Absolute uterus with cervix weight (g)
Female (n = 10)
Mean (SD) 0.81(0.25)
% of control3 -
0.64 (0.16)
-21%
0.67 (0.14)
-17%
0.62 (0.17)
-23%
Relative uterus with cervix weight (g/100 g BW)
Female (n = 10)
Mean (SD) 0.29 (0.07)
% of control3 -
0.23 (0.05)
-20%
0.22 (0.04)
-21%
0.22 (0.07)
-23%
WIL Research
Doses (mg/kg-d)
0
125
350
1,000
Rats, Sprague-Dawley
Relative ovary with oviduct weight (g/100 g BW)
Gavage
Female (n = 6)
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Reference and
study design
Results
28-d exposure starting on
~PNW 6 followed by a
14-d recovery period
Recovery data not shown
Data Quality:d
High (1.3)
Mean (SD) 0.06(0.0003) 0.06(0.01) 0.06(0.01) 0.06(0.01)
% of control3 - 0% 0% 0%
Saegusa et al. (2009)
Rats, Crj:CD(SD)IGS
Diet
Fl: maternal exposure
from GD 10 to PND 20
followed by an 8-wk
non-exposure period
through PNW 11
Data Quality:d
High (1.2)
Doses (mg/kg-d)d
0 15 146 1,505
Relative ovary weight (mg/100 g BW)
Female, Fl, PND 20 (n = 10)
Mean (SD) 32.3 (3.9) 30.9 (4.9) 28.1 (6.3) 28.7 (3.4)
% of control3 - -4% -13% -11%
Female, Fl, PNW 11 (n = 10)
Mean (SD) 31.8 (6.1) 32.8 (2.6) 32.2 (5.7) 34.0 (4.8)
% of control3 - 3% 1% 7%
Relative uterus weight (g/100 g BW)
Female, Fl, PND 20 (n = 10)
Mean (SD) 0.08(0.01) 0.08(0.01) 0.08(0.01) 0.07(0.01)
% of control3 - 0% -4% -9%
Female, Fl, PNW 11 (n = 10)
Mean (SD) 0.16(0.04) 0.15 (0.02) 0.16(0.02) 0.17(0.03)
% of control3 - -6% 0% 6%
Maranghi et al.
(2013)
Mice, BALB/c
Females only
Diet
28-d exposure starting on
PND 26
Data Quality:d
High (1.3)
Doses (mg/kg-d)
0 199
Absolute uterus weight (g)
Female (n= 10-15)
Mean (SD) 0.140(0.051) 0.141 (0.041)
% of control3 - 1%
Relative uterus weight (%)
Female (n= 10-15)
Mean (SD) 0.66(0.24) 0.71 (0.21)
% of control3 - 8%
* Statistically significantly different from the control at p< 0.05 as reported by study authors.
**Significant dose response trend as reported by study authors.
"Percent change compared to control calculated as: (treated value - control value)/control value x 100.
bExact number of animals examined per dose group was unclear in the published paper.
°TWAs for each exposure group were calculated by: (1) multiplying the measured HBCD intake (mg/kg-day)
reported by the study authors for GDs 10-20, PNDs 1-9, and PNDs 9-20 by the number of inclusive days of
exposure for each time period; (2) adding the resulting products together; and (3) dividing the sum by the total
number of inclusive days (33) of HBCD exposure. Example: 100 ppm = (8.1 mg/kg-day x 11 days) + (14.3 mg/kg-
day x 10 days) + (21.3 mg/kg-day x 12 days)/33 days = 14.8 mg/kg-day.
dFl and F2 offspring doses presented as maternal F0 and F1 mean gestational and lactational doses, respectively.
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ai .SP
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cancer cell lines (MCF-7) or ovarian cancer cells (Kane et at., 2012; Park et at., 2012; Dorosh et
at.. 2011; Yamada-Okabe et at.. 2005).
In addition to hormone receptor level effects, several studies indicate that HBCD may also
perturb enzymes involved in the synthesis and metabolism of reproductive hormones. In female
rats, HBCD exposure increased mRNA and protein levels as well as activity of the CYP3 A
family of enzymes (Canton et at., 2008; Germer et at., 2006), which play an important role in the
metabolism and excretion of estrogens (Kretschmer and Baldwin. 2005). Studies in rat primary
Leydig and human adrenocortical carcinoma cell lines indicate that HBCD exposure may
interfere with activity and/or cell signaling pathways of several enzymes involved in steroid
synthesis (Scott et at.. 2009; Canton et at.. 2006). including CYP17 (Fa et JO l'<; I -nnandez
Canton et at.. 2005) and CYP19A1 (van den Dun gen et at.. 2015). CYP11A1, and HSD17P (Fa
et at., 2015).
1.3.2 Male Reproductive Effects
1.3.2.1 Human Evidence
Epidemiological studies evaluating HBCD exposure and reproductive endpoints include a birth
cohort (Meiier et at.. 2012) and a cross-sectional study of male infertility patients (Johnson et at..
2013) (Table 1-5). The birth cohort study in the Netherlands examined maternal serum HBCD
levels in relation to male infants' testes volume and penile length at 3 and 18 months (n = 44) as
well as steroidal and gonadotropin hormone levels at 3 months (n = 34) (Meiier et at.. 2012).
Effect estimates for the association with testes volume or penile length were not provided but
were not reported to be statistically significant. A weak to moderate correlation coefficient (r =
-0.31; 0.05
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animals following oral exposure to HBCD is summarized in Table 1-6 and Figure 1-5. Effect
categories with stronger evidence are presented first, with individual studies ordered by study
duration and then species. If not otherwise indicated, endpoint measurements were made in
adults.
The available evidence for an association between HBCD exposure and male reproductive
effects in experimental animals is insufficient for drawing conclusions (Table 1-6). One study
found a significant dose-related increase in AGD, a measure of reproductive differentiation and
development, only on PND 4 (van der Yen et al.. 2009) and the biological significance of
increased AGD is unclear, van der Yen et al. (2009) also reported a significant trend with dose
for epididymal sperm with separate heads in rats continuously exposed to HBCD from gestation
through PNW 11, but not after a 28-day exposure in adults (van der Yen et al.. 2006).
Statistically significant increases (9-12% relative to control) in relative testis weight were
reported for PND 26 F1 rats in all three dose groups (approximately 17-1,500 mg/kg-day) in a
two-generation reproductive study (Ema et al.. 2008). but not in 15-week F1 males or PND 26
F2 males in the same study. Relative testes weights in HBCD-exposed rats were increased (6-
7%) in V search (2001) and decreased (4-7%) in Saeeusa et al. (2009); in both studies,
changes were not statistically significantly different. Two studies reported statistically significant
changes in relative prostate weight in high-dose animals; however, the direction of the effect was
not consistent across studies, with Etna et al. (2008) reporting a decrease and WIL Research
(2001) reporting an increase. Furthermore, this effect was no longer present following a 4-week
recovery period (WIL Research. 2001). No other dose-related effects were observed for other
measures of male reproductive differentiation and development (Saeeusa et al.. 2009; van der
Yen et al.. 2009; Etna et al.. 2008). spermatogenic measures (van der Yen et al.. 2009; Em a et
al.. 2008; van der Yen et al.. 2006; WIL Research, 2001), or male reproductive organ weights
(Saeeusa et al.. 2009; van der Yen et al.. 2009; Etna et al.. 2008; V search. 2001).
Table 1-5. Evidence pertaining to male reproductive toxicity of HBCD in humans
Reference and study design
Results
Meiier et al. (2012) (the Netherlands,
COMPARE cohort, 2001-2002)
Population: Birth cohort, 90 singleton, term births,
55 healthy boys, assessed at 3 mo (n = 55) and
18 mo (n = 52); 44 with HBCD measures, 45 with
hormone measures, 34 with both measures
Exposure measures: Prenatal exposure, maternal
serum at 35th week of pregnancy
1,2,5,6,9,10-HBCD (HBCD) detected in 43 of
44 samples
LOD 0.8 pg/g serum; LOQ = 9 pg/g serum
Median 0.7 (range:
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Reference and study design
Results
• inhibin B
Testes volume, measured by ultrasound (ages 3 and
18 mo); penile length (ages 3 and 18 mo)
Analysis: Spearman correlation
Data quality:3
Medium (1.9)
Johnson et al. € (USA, 2002-2003)
Population: 38 men (18-54 yrs old), from couples
seeking infertility treatment; approximately 65%
participation into general study; participation rate
in the vacuum bag collection phase not reported
Exposure measures: HBCD exposure from
vacuum bag dust; three main stereoisomers of
HBCD presented together; HBCD detected in 97%
of samples; LOD not reported; median 246 ng/g
dust (90th percentile 1,103 ng/g dust)
Effect measures: Non-fasting blood sample
(immunoassay details in immunoassay
details in Meeker et al., 2008)
testosterone
Sex hormone binding globulin (SHBG)
Follicle stimulating hormone (FSH)
Luteinizing hormone (LH)
estradiol
inhibin B
prolactin
Analysis: All variables analyzed as continuous
variables; Spearman's correlation between HBCD
in house dust and serum hormone levels;
multivariable models adjusted for age and BMI, but
results for HBCD model results not reported
Data quality:3
High (1.6)
Spearman r (/j-value)
Free androgen index 0.46 (p = 0.004)
(testosterone/SHBG)
SHBG -0.353 (p = 0.03)
Multivariate models adjusted for age and BMI reportedly
produced similar results to the bivariate results (data not
reported for HBCD).
Results for other hormones not shown.
Note that HBCD was not strongly correlated with other flame
retardants measured (Spearman correlation coefficients
ranging from -0.20 to 0.27, all /^-values > 0.10)
a Based on OPPT data evaluation criteria
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Table 1-6. Evidence pertaining to male reproductive effects in animals following exposure to HBCD
Reference and study
design
Results
Reproductive differentiation and development
Etna et at (2008)
Rats, CRL:CD(SD)
Diet
Two generation
F0: exposure started
10 wks prior to mating
Fl: dietary exposure
post weaning through
necropsy
F1/F2 offspring:
continuous maternal
exposure throughout
gestation/lactation
Data quality:6
High (1.0)
Doses (mg/kg-d)
Fl offspring3
F2 offspring3
17
15
168
139
1,570
1,360
AGD (mm)
Male, Fl, PND 4 (n = 18-24 litters)
Mean 5.37 (0.41) 5.44 (0.36)
(SD)
% changeb - 1%
Male, F2, PND 4 (n = 19-22 litters)
Mean 5.12(0.54) 5.12(0.41)
(SD)
% changeb - 0%
5.38 (0.32)
0%
5.04 (0.42)
-2%
5.20 (0.51)
-3%
4.84 (0.39)
-5%
van der Yen et a I.
(2009)
Rats, Wistar
Diet
One generation
F0: exposure started
one spermatogenic
cycle (males: 70 d) or
two estrous cycles
(females: 14 d) prior to
mating
Fl: continuous maternal
exposure throughout
gestation/lactation;
dietary exposure post
weaning through
PNW 11
Data quality:
High (1.0)
Doses (mg/kg-d)
0
0.1
0.3
10
30
100
AGD (mm)
Male, Fl, PND 4 (n > 14)c **
Mean 4.6 5.1
(SD) (0.8) (1.1)
% changeb - 11%
Male, Fl, PND 7 (n > 14)c
Mean 6.2 6.7
(SD) (1.2) (1.2)
% changeb - 8%
Male, Fl, PND 21 (n> 14)c
Mean 19.0 19.1
(SD) (6.0) (4.1)
% changeb - 1%
4.7
(0.8)
2%
5.5
(1.1)
-11%
14.8
(2.6)
-22%
4.8
(1.0)
4%
6.4
(1.4)
3%
n/a
5.0
(0.8)
9%
6.1
(1.3)
-2%
18.7
(2.9)
-2%
5.0
(0.9)
9%
6.0
(1.3)
-3%
18.3
(5.5)
-4%
4.5
(0.8)
-2%
6.6
(1.0)
6%
18.9
(6.1)
-1%
5.4
(1.0)
17%
6.3
(1.2)
2%
16.0
(2.2)
-16%
Value for male Fl PND 21 rats at 1 mg/kg-d was "n/a" in study report.
Saegusa et a I. (2009)
Rats, Crj:CD(SD)IGS
Diet
Fl: maternal exposure
from GD 10 to PND 20
followed by an 8-wk
non-exposure period
through PNW 11
Data quality:6
Doses (mg/kg-d)d
15
146
1,505
AGD (mm)
Male, Fl, PND 1 (n= 10 litters)
Mean 3.88 (0.23) 3.96 (0.20)
(SD)
% changeb - 2%
4.08 (0.30)
5%
4.01 (0.23)
3%
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Reference and study
design
Results
High (1.2)
Spernialogenic measures
van cler Yen et al.
(2009)
Rats, Wistar
Diet
One generation
F0: exposure started
one spermatogenic
cycle (males: 70 d) or
two estrous cycles
(females: 14 d) prior to
mating
Fl: continuous maternal
exposure throughout
gestation/lactation;
dietary exposure post
weaning through
PNW 11
Data quality:6
High (1.2)
Doses (mg/kg-d)
0 0.1 0.3 1 3 10 30 100
Epididymal sperm with separate heads (% of total)
Male, Fl, PNW 11 (n = 4-5)**
Mean 4.2 3.8 7.5 2.2 4.4 4.1 5.0 0.8
(SD) (1.7) (2.9) (8.1) (1.9) (1.9) (2.1) (1.8) (0.8)
% changeb - -10% 79% -48% 5% -2% 19% -81%
van der Yen et al.
(2006)
Rats, Wistar
Gavage
28-d exposure starting
on PNW 11
Data quality:6
High (1.3)
Doses (mg/kg-d)
0 0.3 1 3 10 30 100 200
Epididymal sperm with separate heads (% of total)
Male (n = 4-5)
Mean 5.3 3.8 7.4 4.7 5.1 6.8 3.5 5.1
(SD) (2.9) (2.2) (3.2) (3.4) (4.0) (4.1) (2.7) (3.6)
% changeb - -28% 40% -11% -4% 28% -34% -4%
Reproductive organ weights
Etna et al. (2008)
Rats, CRL:CD(SD)
Diet
Two generation
F0: exposure started
10 wks prior to mating
Fl: dietary exposure
post weaning through
necropsy
F1/F2 offspring:
continuous maternal
exposure throughout
gestation/lactation
Doses (mg/kg-d)
Fl, offspring3 0 17 168 1,570
Male, Fl, adult 0 11 115 1,142
F2, offspring3 0 15 139 1,360
Relative epididymis weight (left and right) (mg/100 g BW)
Male, Fl, PND 26 (n = 17-23)
Mean 85.9 (9.8) 86.7 (10.3) 89.3 (7.5) 89.9 (15.3)
(SD)
%changeb - 1% 4% 5%
Male, Fl adult (n = 22-24)
Mean 223 (24) 232 (24) 210 (19) 234 (23)
(SD)
% changeb - 4% -6% 5%
Male, F2, PND 26 (n = 13-22)
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Reference and study
design
Results
Data quality:6
High (1.0)
Mean 90.7(14.1)
(SD)
87.2 (10.6)
87.3 (9.6)
96.2 (10.5)
% changeb -
-4%
-4%
6%
Relative testis weight (left and right) (mg/100 g BW)
Male, Fl, PND 26 (n = 17-23)
Mean 0.57 (0.07)
(SD)
0.61* (0.06)
0.62* (0.06)
0.63* (0.07)
% changeb -
9%
9%
12%
Male, Fl adult (n = 22-24)
Mean 0.60 (0.07)
(SD)
0.61 (0.05)
0.58 (0.06)
0.59 (0.07)
% changeb -
2%
-4%
-1%
Male, F2, PND 26 (n = 13-22)
Mean 0.57 (0.01)
(SD)
0.60 (0.06)
0.57 (0.09)
0.59 (0.05)
% changeb -
5%
0%
3%
Relative ventral prostate weight (mg/100 g BW)
Male, Fl, PND 26 (n = 17-23)
Mean 46.4 (10.3)
(SD)
47.1 (8.8)
48.2 (7.3)
44.5 (11.1)
% changeb -
2%
4%
-4%
Male, Fl adult (n = 22-24)
Mean 137 (28)
135 (34)
131 (30)
135 (22)
(SD)
% changeb -
-1%
-4%
-1%
Male, F2, PND 26 (n = 13-22)
Mean 50.2 (9.3)
(SD)
50.2 (10.7)
50.8 (9.6)
47.3 (15.8)
% changeb -
0%
1%
-6%
van cler Yen et al.
Doses (mg/kg-d)
(2009)
Rats, Wistar
Diet
Male, Fl 0 0.1
0.3 1
3 10
30 100
Absolute epididymis weight (left and right) (g)
One generation
Male, Fl, PNW 11 (n = 4-5)
Mean 0.95 0.88
0.95 1.00
0.90 0.85
0.98 0.82
F0: exposure started
one spermatogenic
cycle (males: 70 d) or
two estrous cycles
(SD) (0.13) (0.13)
% changeb - -7%
(0.12) (0.06)
0% 5%
(0.09) (0.13)
-5% -11%
(0.14) (0.06)
3% -14%
Absolute testis weight (left and right) (g)
(females: 14 d) prior to
Male, Fl, PNW 11 (n = 4-5)**
mating
Fl: continuous maternal
exposure throughout
gestation/lactation;
dietary exposure post
Mean 3.01 2.91
(SD) (0.17) (0.08)
3.07 3.18
(0.42) (0.20)
2.88 2.82
(0.28) (0.07)
2.97 2.60
(0.25) (0.06)
% changeb - -3%
2% 6%
0s-
1
0s-
-t
1
-1% -14%
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Reference and study
design
Results
weaning through
PNW 11
Data quality:6
High (1.2)
Absolute prostate weight (g)
Male, Fl, PNW 11 (n = 4-5)**
Mean 0.66 0.73 0.57 0.73 0.57 0.58 0.67 0.42
(SD) (0.18) (0.21) (0.15) (0.21) (0.12) (0.07) (0.09) (0.13)
% changeb - 11% -14% 11% -14% -12% 2% -36%
Absolute seminiferous vesicle weight (g)
Male, Fl, PNW 11 (n = 4-5)
Mean 1.00 1.07 1.32 1.14 1.21 1.07 1.21 1.09
(SD) (0.40) (0.22) (0.23) (0.29) (0.09) (0.29) (0.25) (0.27)
% changeb - 7% 32% 14% 21% 7% 21% 9%
WIL Research (2001)
Rats, Crl:CD(SD)IGS
BR
Gavage
90 d exposure starting
on ~PNW 7 followed
by a 28-d recovery
period
Recovery data not
shown
Data quality:6
High (1.0)
Doses (mg/kg-d)
Male 0 100 300 1,000
Relative prostate weight (g/100 g BW)
Male (n= 9-10)
Mean 0.18(0.03) 0.19(0.03) 0.21 (0.04) 0.26(0.05)
(SD)
% changeb - 3% 17% 42%
Relative testis weight (left) (g/100 g BW)
Male (n= 9-10)
Mean 0.30 (0.08) 0.31 (0.04) 0.31 (0.04) 0.32 (0.04)
(SD)
% changeb - 4% 2% 7%
Relative testis weight (right) (g/100 g BW)
Male (n= 9-10)
Mean 0.31 (0.07) 0.31 (0.04) 0.31 (0.04) 0.32 (0.05)
(SD)
%changeb - 0% 1% 6%
Relative cauda epididymis weight (left) (g/100 g BW)
Male (n= 9-10)
Mean 0.05 (0.01) 0.06(0.01) 0.06(0.01) 0.06(0.01)
(SD)
%changeb - 9% 6% 15%
Relative cauda epididymis weight (right) (g/100 g BW)
Male (n= 9-10)
Mean 0.05 (0.01) 0.06(0.01) 0.06(0.01) 0.06(0.01)
(SD)
%changeb - 6% 4% 17%
Relative epididymis weight (left) (g/100 g BW)
Male (n= 9-10)
Mean 0.12(0.02) 0.13 (0.01) 0.12(0.02) 0.14(0.01)
(SD)
%changeb - 8% 3% 13%
Relative epididymis weight (right) (g/100 g BW)
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Reference and study
design
Results
Male (n= 9-10)
Mean 0.12(0.04) 0.13(0.01)
(SD)
0.13 (0.01)
0.14(0.02)
% changeb - 8%
3%
16%
Saeeusa et al. (2009)
Rats, Crj:CD(SD)IGS
Diet
Doses (mg/kg-d)d
Male, Fl 0 14.8
146.3
1,505
Relative epididymis weight (left and right) (g/100
g BW)
Fl: maternal exposure
Male, Fl, PND 20 (n = 10)
from GD 10 to PND 20
followed by an 8-wk
Mean 0.06 (0.02) 0.07 (0.01)
(SD)
0.07 (0.01)
0.07 (0.01)
non-exposure period
through PNW 11
% changeb - 8%
Male, Fl adult, PNW 11 (n = 10)
13%
8%
Mean 0.23 (0.02) 0.21* (0.01)
(SD)
0.22 (0.02)
0.21 (0.01)
Data quality:6
% changeb - -9%
-4%
-9%
High (1.2)
Relative testis weight (left and right) (g/100 g BW)
Male, Fl, PND 20 (n = 10)
Mean 0.43 (0.04) 0.43 (0.03)
(SD)
0.43 (0.05)
0.40 (0.03)
% changeb - 0%
0%
-7%
Male, Fl adult, PNW 11 (n = 10)
Mean 0.77 (0.07) 0.73 (0.04)
(SD)
0.78 (0.09)
0.74 (0.05)
% changeb - -5%
1%
-4%
Relative dorsolateral prostate weight (mg/100 g B W)
Male, Fl adult, PNW 11 (n = 10)
Mean 0.13(0.03) 0.13(0.01)
(SD)
0.14(0.03)
0.13 (0.02)
% changeb - 0%
8%
0%
Relative ventral prostate weight (mg/100 g BW)
Male, Fl adult, PNW 11 (n = 10)
Mean 0.13 (0.02) 0.13 (0.04)
(SD)
0.12(0.03)
0.12(0.01)
% changeb - 0%
-8%
-8%
Relative seminal vesicle weight (mg/100 g BW)
Male, Fl adult, PNW 11 (n = 10)
Mean 0.27 (0.05) 0.26 (0.03)
(SD)
0.26 (0.05)
0.26 (0.05)
% changeb - -4%
-4%
-4%
* Statistically significantly different from the control at p< 0.05 as reported by study authors.
**Significant dose response trend as reported by study authors.
aFl and F2 offspring doses presented as mean maternal gestational and lactational F0 and F1 doses, respectively.
bPercent change compared to control calculated as: (treated value - control value)/control value / 100.
°Exact number of animals examined per dose group was unclear in the published paper.
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dTWAs for each exposure group were calculated by: (1) multiplying the measured HBCD intake (mg/kg-day)
reported by the study authors for GDs 10-20, PND 1-9, and PND 9-20 by the number of inclusive days of
exposure for each time period; (2) adding the resulting products together; and (3) dividing the sum by the total
number of inclusive days (33) of HBCD exposure. Example: 100 ppm = (8.1 mg/kg-day x 11 days) +
(14.3 mg/kg-day x 10 days) + (21.3 mg/kg-day x 12 days)/33 days = 14.8 mg/kg-day.
"Based on OPPT data evaluation criteria
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§>
6
"O
5:
I
1
Relative seminal vesicle weight Saegusa et al., 2009 ( F1 adults)
Relative prostate weight Saegusa et al., 2009 { F1 adults)
Relative testis weight Saegusa et al., 2009 (F1 weanlings)
Relative testis weight Saegusa et al., 2009 (F1 adults)
Relative epididymis weight Saegusa et al., 2009 (F1 weanlings)
Relative epididymis weight Saegusa et al., 2009 (F1 adults)
Relative epididymis weight WIL, 2001/2002 (rats)
Relative cauda epididymis weight WIL, 2001/2002 (rats)
Testis weight
Ema et al., 2008 (rats, F2 weanlings)
Testis weight
Ema et al., 2008 (rats, F1 weanlings)
Testis weight.
Ema et al., 2008 (rats, FX adults)
Absolute seminiferous vesicle weight van der Ven et al., 2009 (rats)
Absolute Testis weight van der Ven et al., 2009 (rats)
Absolute epididymis weight van der Ven et al., 2009 (rats)
Relative Testis weight WlL, 2001/2002 (rats)
Relative ventral Prostate weight, Ema et al., 2008 (rats, F1 + F2 weanlings)
Prostate weight.
Ema et al., 2008 (rats, FX adults)
1s Prostate weight van de Ven, et al. 2009 (rats)
1* Relative Prostate weight
WIL, 2001/2002 (rats)
ive epididymis weight(left and right) Ema et al., 2008 (F1 adults)
Relative epididymis weightfleft and right) Ema et al., 2008 (F1 weanlings)
van der Ven et al., 2006 (rats)
WIL 2001/2002 (rats)
van der Ven et al., 2009 ( F1 rats)
Ema et a)., 2008 (F0 + F1 rats)
Saegusa et al., 2009 (Fl, rats)
van der Ven et al., 2009 ( Fl rats)
Ema et al., 2008 (rats Fl + F2 weanlings)
• significantly changed
O not significantly changed
o e-
o e-
o e-
(! e-
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1.3.2.3 Mechanistic Evidence
See Section 1.3.1.3 in the Female Reproductive Effects section above (Mechanistic Evidence).
1.4 Developmental Effects
1.4.2 Human Evidence
Epidemiology studies investigating potential thyroid, male reproductive, and nervous system
effects of HBCD following developmental exposure were identified and are discussed in their
respective organ/system-specific hazard sections (Sections 1.1.1, 1.3.2.1, and 1.5.1,
respectively).
1.4.3 Animal Evidence
Evidence to inform organ-system specific effects of HBCD in animals following developmental
exposure are discussed in the individual hazard sections. The current section is limited to
discussion of developmental specific effects, including offspring survival, pup body weight,
developmental markers, and bone measures.
HBCD-induced developmental effects, including offspring survival, body weight, and
developmental markers, were evaluated in five studies in rats (Hachisuka et ai. 2010; Saeeusa et
ai. 2009; van der Yen et ai. 2009; Ema et ai. 2008) and mice (Maranghi et ai. 2013). with
exposure durations ranging from 28 days in juvenile mice to continuous exposure of rats over
two generations. A summary of developmental effects associated with HBCD exposure is
presented in Table 1-7 and Figure 1-6. Effect categories with stronger evidence are presented
first, with individual studies ordered by study duration and then species. For each endpoint, age
at outcome measurement is indicated.
Effects on offspring survival and pup body weight were evaluated in three rat studies (Saeeusa et
ai. 2009; van der Yen et ai. 2009; Ema et ai. 2008) and juvenile body weight was reported in a
single mouse study (Maranghi et ai J ). Two rat studies that utilized similar dose ranges
(approximately 10-1,500 mg/kg-day) reported statistically significant effects in the high-dose
group (Saeeusa et ai. 2009; Ema et ai. 2008). Ema et ai (2008) reported decreases in pup body
weight ranging from 20 to 25% for male and female F2 rat pups on PNDs 7, 14, and 21.
Offspring survival on PNDs 4 and 21 (21 and 42%, respectively) in this dose group was also
decreased (Ema et ai. 2008). Decreases in pup weight in F1 animals were smaller (<10%), did
not show a consistent pattern of effect, and were not associated with decreased viability (Saeeusa
et ai. 2009; Ema et ai. 2008). The remaining studies indicate a potential for HBCD to decrease
body weight (Maranghi et ai. JO I <; van der Yen et ai. 2009) but not viability (van der Yen et
ai. 2009) at lower doses (up to 199 mg/kg-day). van der Yen et ai (2009) reported significant
dose-dependent trends in decreased body weight in male and female rat pups. Similarly,
Maranghi et ai (2013) reported a 14% body weight decrease in juvenile female mice exposed for
28 days, although this effect was not statistically significant. Use of a single-dose study design
did not allow for evaluation of dose-response in this study.
Treatment-related effects on several developmental landmarks were evaluated in F1 and F2
offspring in the two-generation reproductive toxicity study (Ema et ai. 2008). In F1 pups, eye
opening on PND 14 was significantly increased in both sexes in the mid-dose group, but not the
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high-dose group (approximately 170 and 1,500 mg/kg-day, respectively). In contrast, F2
offspring exhibited statistically significant dose-related decreases in eye opening on PND 14 in
both the mid- (females only) and high-dose groups (males and females). Other developmental
landmarks (i.e., pinna unfolding, and incisor eruption) were not affected (Etna et at.. 2008).
Measures of bone development were also evaluated in rats treated continuously from gestation
through adulthood at doses up to 100 mg/kg-day (van der Yen et al., 2009). Trabecular bone
mineral density in females was decreased by 20%. The study authors reported dose-related
decreases in several other tibia related endpoints; however, the magnitude of these effects was
small and inconsistent across dose group and sex, making it difficult to interpret the biological
significance of these findings.
Table 1-7. Evidence pertaining to developmental effects in animals following
exposure to HBCD
Reference and study
design
Results
/¦'clal and early poslnalal survival
Etna et al. (2008)
Doses (mg/kg-d)
Rats, CRL:CD(SD)
Fl 0
17
168
1,570
Diet
Two generation
offspring3
F2 0
15
139
1,360
F0: exposure started
10 wks prior to mating
Fl: dietary exposure post
offspring3
Viability index (%)
Fl, PND 0 (n = 18-24 litters)
weaning through
Mean (SD) 99.6(1.9)
97.5 (8.5)
98.8 (2.8)
99.2 (2.5)
necropsy
F1/F2 offspring:
continuous maternal
exposure throughout
gestation/lactation
% of controlb -
Fl, PND 4 (n = 18-24 litters)
Mean (SD) 95.6 (8.6)
% of controlb -
Fl, PND 21 (n = 18-24 litters)
-2%
98.7 (2.8)
3%
-1%
98.7 (4.4)
3%
0%
95.8(10.3)
0%
Data quality:'
High (1.0)
Mean (SD) 93.2(17.3)
% of controlb -
99.4 (2.7)
7%
98.1 (4.6)
5%
93.8 (23.6)
1%
F2, PND 0 (n = 20-23 litters)
Mean (SD) 98.6(5.3)
97.7 (4.9)
96.0 (9.5)
97.8(5.1)
% of controlb -
-1%
-3%
-1%
F2, PND 4 (pre-culling) (n = 20-23 litters)
Mean (SD) 86.9 (24.8)
87.3 (21.1)
92.1 (12.8)
68.4* (33.5)
% of controlb -
0%
6%
-21%
F2, PND 21 (n = 20-22 litters)
Mean (SD) 85.0(22.0)
89.6(13.9)
71.3 (26.9)
49.7* (41.1)
% of controlb -
5%
-16%
-42%
Saegusa et al. (2009)
Doses (mg/kg-d)c
Rats, Crj:CD(SD)IGS
0
15
146
1,505
Diet
Number of live pups
Female, F0 (n = 10 litters)
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Reference and study
design
Results
Fl: maternal exposure
from GD 10 to PND 20
followed by an 8-wk non-
exposure period through
PNW 11
Data quality:'
High (1.2)
Mean (SD) 13.0(1.8) 13.0(1.6) 11.6(1.6) 12.9(1.4)
% of control13 - 0% -11% -1%
Body weight
Em a et al
Rats, CRL:CD(SD)
Diet
Two generation
FO: exposure started
10 wks prior to mating
Fl: dietary exposure post
weaning through
necropsy
F1/F2 offspring:
continuous maternal
exposure throughout
gestation/lactation
Data quality:'
High (1.0)
Doses (mg/kg-d)
Fl
offspring3
F2
offspring3
17
15
168
139
Pup weight (g)
Male, Fl, PND
Mean (SD)
% of controlb
Male, Fl, PND
Mean (SD)
% of controlb
Male, Fl, PND
Mean (SD)
% of controlb
Male, Fl, PND
Mean (SD)
% of controlb
Male, Fl, PND
Mean (SD)
% of controlb
Female, Fl, PND 0 (n = 18-23 litters)
Mean (SD) 6.3(0.5) 6.6(0.7)
% of control13 - 5%
Female, Fl, PND 4 (n = 18-23 litters)
Mean (SD) 9.6(1.4) 10.3(1.8)
% of control13 - 7%
Female, Fl, PND 7 (n = 17-23 litters)
Mean (SD) 15.4(2.8) 17.0(2.5)
% of control13 - 10%
Female, Fl, PND 14 (n = 17-23 litters)
Mean (SD) 33.5 (5.3) 35.5 (3.6)
% of control13 - 6%
Female, Fl, PND 21 (n = 17-23 litters)
6.8* (0.6)
8%
10.4(1.5)
8%
16.9 (2.3)
10%
35.7 (3.6)
7%
1,570
1,360
0 (n = 18-24 litters)
6.8 (0.5) 6.9 (0.6) 7.2 (0.7) 6.8 (0.6)
1% 6% 0%
4 (n = 18-24 litters)
10.2(1.7) 10.7(1.8) 10.8(1.6) 9.5(1.8)
5% 6% -7%
7 (n = 17-24 litters)
16.4(3.1) 17.5(2.4) 16.9(2.2) 15.6(2.0)
7% 3% -5%
14 (n = 17-23 litters)
36.1 (4.8) 36.3 (3.6) 36.1 (3.9) 33.5 (2.6)
1% 0% -7%
21 (n = 17-23 litters)
61.1 (7.1) 62.3 (6.5) 61.9 (6.5) 55.4* (4.0)
2% 1% -9%
6.5 (0.7)
3%
9.2(1.6)
-4%
15.1 (1.6)
-2%
32.6 (3.0)
-3%
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Reference and study
design
Results
Mean (SD) 56.5 (8.0) 59.9 (6.4)
60.5 (5.9)
53.2 (4.7)
% of controlb - 6%
7%
-6%
Male, F2, PND 0 (n = 20-23 litters)
Mean (SD) 6.8(0.8) 6.7(0.7)
7.1 (0.6)
6.6 (0.6)
%ofcontrolb - -1%
4%
-3%
Male, F2, PND 4 (n = 19-22 litters)
Mean (SD) 9.1(2.3) 9.3(1.3)
9.0(1.8)
8.0(1.3)
% of control13 - 2%
-1%
-12%
Male, F2, PND 7 (n = 17-22 litters)
Mean (SD) 14.7(3.9) 15.4(2.8)
14.3 (3.6)
11.5* (2.9)
% of control13 - 5%
-3%
-22%
Male, F2, PND 14 (n = 14-22 litters)
Mean (SD) 31.4(8.0) 33.8(5.0)
31.0 (7.2)
24.2* (6.6)
% of control13 - 8%
-1%
-23%
Male, F2, PND 21 (n = 13-22 litters)
Mean (SD) 53.0 (12.6) 56.2 (6.7)
54.1 (10.1)
42.6* (8.3)
% of control13 - 6%
2%
-20%
Female, F2, PND 0 (n = 20-23 litters)
Mean (SD) 6.5(0.8) 6.3(0.6)
6.7 (0.6)
6.2 (0.6)
% of control13 - -3%
3%
-5%
Female, F2, PND 4 (n = 20-22 litters)
Mean (SD) 8.9(2.3) 8.5(1.3)
8.8(1.8)
7.3* (1.3)
% of control13 - -5%
-1%
-22%
Female, F2, PND 7 (n = 17-22 litters)
Mean (SD) 14.3(3.5) 14.2(2.8)
13.5 (3.9)
10.7* (2.6)
% of control13 - -1%
-6%
-25%
Female, F2, PND 14 (n = 13-22 litters)
Mean (SD) 31.2(6.5) 31.3(5.1)
29.3 (7.3)
23.9* (5.9)
% of control13 - 0%
-6%
-23%
Female, F2, PND 21 (n = 13-22 litters)
Mean (SD) 52.0(10.0) 52.8(6.6)
51.2 (10.8)
41.6* (8.4)
% of control13 - 2%
-2%
-20%
van der Yen et al.
Doses (mg/kg-d)
(2009)
0 0.1 0.3 1
3 10
30 100
Rats, Wistar
Pup weight (g)
Diet
One generation
Male, Fl, PND 4 (n > 14)d **
Mean (SD) 10.0 10.2 9.8 10.8
10.2 10.8
11.0 9.5(0.9)
F0: exposure started one
(1.3) (0.7) (1.2) (1.9)
(1.7) (1.4)
(1.3)
spermatogenic cycle
% of control13 - 2% -2% 8%
2% 8%
10% -5%
(males: 70 d) or two
Male, F1,PND 7 (n> 14)d
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Reference and study
design
Results
estrous cycles (females:
Mean (SD) 13.4 13.6
12.7
14.7
13.1
13.9
14.6
12.6
14 d) prior to mating
(2.2) (1.6)
(2.0)
(4.1)
(3.0)
(2.7)
(1.7)
(1.0)
Fl: continuous maternal
% of control13 - 1%
-5%
10%
-2%
4%
9%
-6%
exposure throughout
gestation/lactation;
Male, Fl, PND 14 (n > 14)d **
dietary exposure post
Mean (SD) 22.3 24.2
22.0
33.3
24.1
24.6
22.5
20.5
weaning through PNW 11
(6.4) (5.0)
(4.0)
(8.6)
(7.7)
(6.5)
(3.2)
(2.2)
% of controlb - 9%
-1%
49%
8%
10%
1%
-8%
Data quality:'
i_ri(jU /i
Male, Fl, PND 21 (n > 14)d **
Mean (SD) 39.3 41.8
35.1
55.7
39.1
39.5
35.6
32.2
JrLlgn (1.2)
(7.5) (8.9)
(5.2)
(14.4)
(12.0)
(10.0)
(6.2)
(3.0)
% of controlb - 6%
-11%
42%
-1%
1%
-9%
-8%
Female, Fl, PND 4 (n > 14)d **
Mean (SD) 9.5 9.7
9.4
10.6
9.4
10.8
10.7
8.9 (0.9)
(1.5) (0.8)
(1.1)
(2.7)
(1.5)
(1.1)
(1.2)
% of control13 - 2%
-1%
12%
-1%
14%
13%
-6%
Female, Fl, PND 7 (n > 14)d **
Mean (SD) 12.9 12.8
12.4
14.2
12.5
14.4
14.1
11.9
(2.6) (1.4)
(2.1)
(5.1)
(2.7)
(2.2)
(1.7)
(1.3)
% of control13 - -1%
-4%
10%
-3%
12%
9%
-8%
Female, Fl, PND 14 (n> 14)d **
Mean (SD) 23.6 23.1
21.0
31.1
22.4
24.7
22.5
20.0
(5.3) (2.7)
(3.8)
(7.9)
(6.0)
(5.8)
(4.4)
(2.9)
% of control13 - -2%
-11%
32%
-5%
5%
-5%
-15%
Female, Fl, PND 21 (n> 14)d **
Mean (SD) 40.3 40.1
34.1
50.4
37.0
40.0
37.5
32.3
(8.6) (5.9)
(5.4)
(11.9)
(10.3)
(9.5)
(5.9)
(3.9)
% of control13 - 0%
-15%
25%
-8%
-1%
-7%
-20%
Saegusa et al. (2009)
Doses (mg/kg-d)°
Rats, Crj:CD(SD)IGS
0
15
146
1,505
Diet
Pup weight (g)
Fl: maternal exposure
Male, Fl, PND 1 (n = 10 litters)
from GD 10 to PND 20
Mean (SD) 7.11(0.66)
7.22 (0.56)
7.65 (0.95)
7.15 (0.80)
followed by an 8-wk non-
% of control13 -
2%
8%
1%
exposure period through
Male, Fl, PND 20 (n = 10)
PNW 11e
Mean (SD) 54.3(3.5)
51.2 (7.3)
56.7 (4.1)
54.0 (3.3)
% of control13 -
-6%
4%
-1%
Data quality:'
Male, Fl, at puberty onset -PND 40 (n = 12-14)
High (1.2)
Mean (SD) 204.3 (15.7)
198.3 (20.4)
203.2 (15)
195
8(10.1)
% of control13 -
-3%
-1%
-4%
Male, Fl, PNW 11 (n= 10)
Mean (SD) 454.3 (25.4)
456.9 (24.8)
450.
8 (33.4)
435.1 (24.6)
% of control13 -
1%
-1%
-4%
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Reference and study
design
Results
Female, Fl, PND 1 (n = 10 litters)0
Mean (SD) 6.53 (0.59) 6.84 (0.50) 7.28 (0.75) 6.84 (0.81)
%ofcontrolb - 5% 11% 5%
Female, Fl, PND 20 (n = 10)
Mean (SD) 50.3 (3.4) 50.0 (6.0) 53.7 (5.5) 51.3 (2.9)
%ofcontrolb - -1% 7% 2%
Female, Fl, at puberty onset ~PND 35 (n = 12-14)
Mean (SD) 130.8 (11.7) 133.8 (10.8) 129.2 (13.5) 118.6* (11.7)
% of control13 - 2% -1% -9%
Female, Fl, PNW 11 (n = 10)
Mean (SD) 286.2(25.2) 293.4(21.5) 289.2(24.4) 270.7(19.6)
% of control13 - 3% 1% -5%
Maranghi et al.
(2013)
Mice, BALB/c
Females only
Diet
28-d exposure starting on
PND 26
Data quality:'
High (1.2)
Doses (mg/kg-d)
0 199
Body weight gain (g)
Female, PND 54 (n = 10-15)
Mean (SD) 5.80(0.74) 5.00(1.16)
% of control13 - -14%
Developmental markers
.1
Rats, CRL:CD(SD)
Diet
Two generation
F0: exposure started
10 wks prior to mating
Fl: dietary exposure post
weaning through
necropsy
F1/F2 offspring:
continuous maternal
exposure throughout
gestation/lactation
Data quality:'
High (1.0)
Doses (mg/kg-d)
Fl 0 17 168 1,570
offspring3
F2 0 15 139 1,360
offspring3
Eye opening (%)
Male, Fl, PND 14 (n = 17-23 litters)
Mean (SD) 48.2 (41.5) 56.7 (37.9) 77.1* (36.3) 45.8 (34.6)
% of control13 - 18% 60% -5%
Female, Fl, PND 14 (n =17-23 litters)
Mean (SD) 49.3 (37.8) 66.7 (41.3) 82.9* (33.5) 54.9 (41.4)
% of control13 - 35% 68% 11%
Male, F2, PND 14 (n = 14-22 litters)
Mean (SD) 72.7 (40.0) 62.5 (40.6) 47.2 (44.8) 33.9* (34.7)
% of control13 - -14% -35% -53%
Female, F2, PND 14 (n = 13-21 litters)
Mean (SD) 82.9 (26.8) 72.7 (37.7) 53.8* (40.3) 48.1* (42.0)
% of control13 - -12% -35% -42%
No exposure-related changes were found in incisor eruption (PND 11) or pinna
unfolding (PND 3).
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Reference and study
design
Results
Bone measures
van der Yen et al.
Doses (mg/kg-d)
(2009)
0 0.1 0.3
1
3
10
30
100
Rats, Wistar
Trabecular bone mineral density, tibia (mg/cm3)
Diet
One generation
Male, Fl, PNW 11 (n = 4-5)
Mean 145 143 154
167
134
146
156
167
F0: exposure started one
(SD) (25) (20) (23)
(16)
(36)
(25)
(20)
(11)
spermatogenic cycle
% of control13 - -1% 6%
15%
-8%
1%
8%
15%
(males: 70 d) or two
estrous cycles (females:
14 d) prior to mating
Fl: continuous maternal
Female, Fl, PNW 11 (n = 5)**
Mean 294 268 253
231
245
227
200
234
(SD) (19) (27) (30)
(35)
(31)
(28)
(31)
(29)
exposure throughout
% of controlb - -9% -14%
-21%
-17%
-23%
-32%
-20%
gestation/lactation;
dietary exposure post
weaning through PNW 11
Data quality:'
High (1.2)
* Statistically significantly different from the control at p< 0.05 as reported by study authors.
**Significant dose response trend as reported by study authors.
aFl and F2 offspring doses presented as mean maternal gestational and lactational F0 and F1 doses, respectively.
bPercent change compared to control calculated as: (treated value - control value)/control value / 100.
TWA doses for each exposure group were calculated by: (1) multiplying the measured HBCD intake (mg/kg-day)
reported by the study authors for GDs 10-20, PNDs 1-9, and PNDs 9-20 by the number of inclusive days of
exposure for each time period; (2) adding the resulting products together; and (3) dividing the sum by the total
number of inclusive days (33) of HBCD exposure. Example: 100 ppm= (8.1 mg/kg-day x lldays) +
(14.3 mg/kg-day x 10 days) + (21.3 mg/kg-day x 12 days)/33 days = 14.8 mg/kg-day.
dExact number of animals examined per dose group was unclear based on the published paper.
'Saegusa et at. (2009) and Hachisuka et at. (20.1.0) appear to be two publications of the same animal cohort; the
TWA doses calculated for Saegnsa et at. (2009) were applied to Hachisuka et at. (20.1.0).
fBased on OPPT data evaluation criteria
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3 %
*11 f)#r V»« «t it, 2«» ifiti, fll
got* et «L, 200* (rtfts, FS offering f ]
[w ft •!.. 200* {nlj, fi affiprtif )
Em* « »l, TGOIt | littr Vtn et A, St® |rslJ, t I)
t
fW W*«H| Oty «1|lm» et il, 2008 |r«, f2 olfeprti* F}
Pyp Weight 10»y 0! (ira 11 »i. ?00(S Im*, ft etbprinc f|
Pup weight ( 0»r ?-»> EfiBi M *1. 20« Efjfft, H olliutlfif M)
m *•**» IBW MJ to* « 2MB M*.« olliftflitf Ml
!m* a il, JIB i mm. f 1 «¦»(!*)# fj
MP WWf M 00
1000
20000
Owes tmijki-itif I
Figure 1-6. Exposure response array of developmental effects following oral exposure. All
studies scored High in data quality evaluation.
1.4.4 Mechanistic Evidence
Studies directly investigating mechanistic evidence to inform potential developmental effects of
HBCD are limited to a few studies in zebrafish (Wu et al.. 2013; Du et at.. 2012; Deng et at..
2009; I hi et al.. 2009a). which focus on identifying molecular targets that drive HBCD-mediated
perturbation of normal embryonic development. In general, HBCD exposure was associated with
increased ROS generation and induction of apoptotic cell pathways resulting in malformations
and reduced viability in zebrafish (Du et al.. 2012; Deng et al.. 2009; Hu et al.. 2009a). In the
absence of overt teratogenic effects, HBCD exposure was found to affect cardiac function and
development, resulting in increased heart rate, arrhythmia, cardiac hypertrophy, and increased
collagen deposition; these effects were associated with changes in expression of genes associated
with calcium transport and cardiomyocyte conduction (Wu et al.. 2016; Wu et al.. 2013). In rat
cardiomyocytes (H9C2), HBCD treatment altered Ca2+ signaling through changes in expression
of several genes (Ryr2, Serca2a, and Ncxl) involved in Ca2+ regulation (Wu et al.. 2016).
Although no studies were identified that directly investigated the potential for HBCD-driven
thyroid hormone imbalances to induce developmental effects, in vivo studies provide evidence of
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an association between HBCD exposure and disrupted homeostasis of thyroid hormones (see
Section 1.2.1), which are critical regulators of growth and development. In humans, umbilical T4
concentrations are positively correlated with body weight and length at birth (Shields et ai.
2011) and cases of intrauterine growth restriction and small-for-gestational-age fetuses are
associated with reduced thyroid hormone levels in both human populations and experimental
animals (Forhead ami rmoden. 2014; Pererira arn! ^uvianoy. 2003). Thyroidectomy in fetal
sheep reduces total body and organ weights and affects bone development, including delayed
maturation and altered bone strength and mineral density (Forhead and Fowden. JO I I; < jnham
et at.. 2011); these effects were ameliorated by T4 replacement (Forhead and Fowden. 2014).
Furthermore, human congenital hypothyroidism is also associated with neurological and skeletal
abnormalities, even when birth weight is unaffected (Patel et at.. 2011; Shields et at.. 2011).
Based on the broader developmental literature, it is plausible that developmental effects observed
following HBCD exposure could be a consequence of HBCD-induced changes in thyroid
homeostasis; however, HBCD-specific data to support this relationship are not available.
1.5 Nervous System Effects
1.5.2 Human Evidence
Epidemiology studies have been conducted in children participating in birth cohort studies in the
Netherlands (Roze et at.. 2009) and in adolescents in a cross-sectional general population study
in areas around industrial sites in Belgium (Kicihski et at..: ) (Table 1-8). In a study of
children ages 5-6 years (n = 62), maternal HBCD levels measured at week 35 of pregnancy were
associated with increased scores for three neuropsychological domains (coordination, total
intelligence, and verbal intelligence) after adjusting for maternal education, home environment
(Home Observation for Measurement of the Environment [HOME] score), and sex (Roze et at..
2009). The authors stated that no associations were observed between HBCD and the other tested
domains (visual perception, visuomotor integration, inhibitory control, attention, behavior, and
attention deficit/hyperactivity disorder), but did not report effect estimates for these measures.
Kicihski et at. C did not observe associations between HBCD levels and six
neurobehavioral measures assessing attention, visual scanning and information processing,
working memory, and motor function in a study in adolescents (ages 13-17; n = 515); this
analysis was based on HBCD exposure dichotomized at concentrations above and below the
LOQ (30 ng/L) because 75% of values were less than the LOQ. Interpretation of the results of
these studies is limited by poor reporting of results and small sample size in the study by Roze et
at. (2009). and by low HBCD detection rates (<25%) in the study population and measure of
HBCD in adolescents that does not represent a relevant time window of exposure for
neurodevelopmental outcomes in the case of Kicihski et at. (2012). Thus, the available evidence
for an association between HBCD exposure and nervous system effects in humans is insufficient
for drawing conclusions.
1.5.3 Animal Evidence
The potential for HBCD to affect the nervous system has been examined in 10 studies in rats
(Genskow et al.. 2015; Milter-Rhodes et at.. 2014; Lilienthal et at.. 2009; Saeeusa et at.. 2009;
van d-n \ on et at.. 2009; Ema et at.. 2008; Eriksson et al.. 2006; van d-n \ on et al.. 2006; WIL
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Research, 2001, 1997) with exposures ranging from a single gavage dose on PND 10 to
continuous exposure across two generations.
Discussion of nervous system-related effects is organized by the timing of exposure
(i.e., developmental and adult) due to the sensitivity of the developing nervous system to the
effect of chemicals. A summary of the evidence pertaining to nervous system effects in
experimental animals is presented in Table 1-9 and Figure 1-7. Individual studies are ordered by
study duration and then species. If not otherwise indicated measurements were made in adults.
1.5.3.1 Developmental Exposure
Neurodevelopmental Milestones
Neurodevelopmental milestones were evaluated in two rat studies (Miller-Rhodes et at., 2014;
Ema et at.. 2008). Gestational exposure to HBCD heightened tail pinch responses in pooled male
and female rat pups (PNDs 1-21; 3-30 mg/kg-day) and reduced forelimb grip strength in
juvenile male, but not female, rats (PND 26; 10 and 30 mg/kg-day) (Miller-Rhodes et at.. 2014).
Development of sensorimotor reflexes was affected in rats exposed to approximately
1,300 mg/kg-day in a two-generation reproductive toxicity study; however, effects were not
consistent across generations, sex, or the reflex evaluated (Ema et at., 2008) and were not
observed in a separate study (Miller-Rhodes et at.. 2014). Differences in the experimental design
(i.e., multigenerational versus developmental) and outcome recording (i.e., righting latency
versus age at which >85% of pups completed the behavior within 1 minute) may have
contributed to differences in the surface righting reflex responses reported by these research
groups. Furthermore, in the study by Ema et al. (2008). statistically significant effects on righting
reflexes were only observed in exposure groups that also exhibited signs of overt toxicity (e.g.,
decreased body weight gain and pup survival); thus, changes in sensorimotor reflexes may be
due to general toxicity rather than an organ system-specific effect.
Executive Function and Locomotor Activity
The effects of HBCD exposure on executive function (e.g., learning, memory, attention) were
evaluated in three studies in rats (Miller-Rhodes et al.. 201 I; < Ima et al.. 2008) and mice
(Eriksson et al.. 2006). Miller-Rhodes et al. ( evaluated performance on two operant tasks
designed to measure sustained attention, response inhibition, and persistence in adult (11-14
months) and aging rats (19-21 months) that were exposed to HBCD in utero. The go/no-go task
evaluated effects on sustained attention and response inhibition by requiring animals to
discriminate between distinct visual cues that indicate whether a trial is reinforced for pressing
the lever (i.e., go trial) or for abstaining from lever pressing (i.e., no-go trial). Combined
responses from male and female offspring from the low-dose group (3 mg/kg-day) showed a
statistically significant decrease in the number of correct lever presses and an increase in
response latency; however, no effect was observed in the two higher dose groups. No treatment-
related effects were observed in the random ratio task, which evaluated persistence behaviors by
providing animals with intermittent reinforcement (i.e., food pellet reward) for lever pressing.
Although these tests are sensitive indicators of altered cognitive function, the results are difficult
to interpret as data were pooled across age cohorts. Furthermore, some aging animals in the 3
mg/kg-day group developed unexplained loss of hindlimb control that was not observed in
controls or higher dose groups. To minimize the potential effects on these behavioral outcomes,
litters containing animals that developed serious health complications were excluded from
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analysis (Miller-Rhodes et al, 2014); however, it is possible that animals with less severe
muscular degeneration were included.
Two studies evaluated learning ability using swim maze tests. A statistically significant increase
in trial time on a Morris swim maze was observed in young adult (3-month-old) male mice
exposed once to 13.5 mg/kg on PND 10; however, swim speed and visual acuity were not
measured as possible confounders (Eriksson et at., 2006). In contrast, a statistically significant
decrease in trial times on a multiple T-maze was reported on a single day of testing in juvenile
F1 male rats (PNW 6) exposed to approximately 100-1,300 mg/kg-day (Ema et at.. 2008).
Females showed a similar pattern of behavior across multiple testing days, but changes were not
statistically significant and the data showed high standard errors (SEs). Differences in the test
species, exposure, and testing methods may have contributed to the different results of the two
swim maze studies and complicates interpretation of these findings.
Three studies measured effects of early-life exposure on locomotor activity in rats (Miller-
Rhodes et at JO I i; I 'ma et at.. 2008) and mice (Eriksson et at.. 2006). Eriksson et al. (2006)
evaluated effects in young adult (3-month-old) mice that were administered a single dose on
PND 10, which corresponds with a period of rapid growth and maturation for motor and sensory
neural networks in mice. Controls and mice exposed to 0.9 mg/kg showed a normal activity
pattern, characterized by high initial activity that steadily decreased over the course of the
60-minute test period. The 13.5 mg/kg group, however, exhibited a moderate activity level that
remained steady (i.e., significantly lower versus control activity at the beginning and
significantly higher versus controls at the end of the test), suggesting failure to habituate to the
novel environment of the testing arena. Similar testing methods were employed to evaluate
locomotor activity in juvenile (Ema et al., 2008), young adult, and aging rats (Miller-Rhodes et
al.. 2014). Although both of these studies utilized longer exposure durations and higher doses,
they found no effects on spontaneous locomotor activity (Miller-Rhodes et al < s s, < ma et al..
2008).
Other Neurological Effects
Effects on auditory function and dopamine-dependent movement behavior were evaluated in a
single rat study that exposed animals continuously throughout gestation, lactation, and into
adulthood (Lilienthal et al.. 2009). Brainstem evoked auditory potentials (BAEPs) were
measured to evaluate effects on auditory function. Study authors reported that males, but not
females, showed a small dose-related trend towards increased thresholds and signal latency,
suggesting reduced hearing sensitivity. In the same study, dopamine system effects were
evaluated by measuring cataleptic movement latencies, atalepsy is a condition characterized by
muscle rigidity and waxy flexibility (i.e., subject tends to remain in a fixed position, but the
posture/limb position can be altered). A cataleptic state was induced by haloperidol, a drug that
blocks dopamine receptors. Animals were then placed in fixed postures and movement latency
was recorded. Statistically significant dose-dependent decreases in movement latency were
reported in the catalepsy tests for both sexes, although effects were more pronounced in females.
These results suggest that HBCD increases dopamine signaling. It was unclear, however,
whether animals were given a recovery period between certain postures in the catalepsy tests,
which may have stressed the animals and affected the results. In the BAEP test, the average
increase in auditory threshold observed at the highest dose was 9 dB. Although BAEP is a
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sensitive measure of auditory function, the changes observed in this study were below those
generally considered to be biologically significant (10-15 dB).
Three studies evaluated brain weight changes in rats (Saeeusa et at.. 2009; van der Yen et al..
2009; Ema et al.. 2008). Absolute brain weights showed a statistically significant reduction in F1
adults and both F1 and F2 weanlings in the high-dose group (approximately 1,300 mg/kg-day)
(Ema et al, 2008); these animals also exhibited signs of overt toxicity, including decreased
viability and pup weight (Section 1.2.4). van der Yen et al. (2009) also reported a significant
trend for absolute brain weights in male rats at the end of a one-generation exposure, with most
groups showing an increase relative to controls; brain weight changes were not observed in
females. No statistically significant change in relative brain weight was observed in gestationally
and lactationally exposed rats (Saeeusa et al.. 2009); however, relative brain weight changes are
considered to be less informative of nervous system effects. Notably, brain weight changes are
considered to be a relatively insensitive measure of neurotoxicity and, with the exception of the
F2 high dose animals in Ema et al. (2008). the statistically significant effects were below the
level that is considered to be biologically significant.
1.5.3.2 Adult Exposure
The four studies that evaluated neurotoxicity endpoints in adult animals did not provide evidence
that HBCD exposure affects the nervous system at this life stage (Genskow et al.. 2015; van der
Yen et al.. 2006; WIL Research. 2001. 1997). No gross changes in striatal levels of dopamine or
its metabolites were observed in adult male mice exposed to 25 mg/kg-day HBCD for 30 days
(Genskow et al.. 2015). Similarly, no effects on other neurological measures, including a
functional observational battery (FOB), locomotor activity, brain weight, or gross pathology
were observed in adult rats exposed to up to 1,000 mg/kg-day HBCD for 90 (WIL Research.
2001) or 28 days (van der Yen et al.. 2006; \ search. 1997).
Table 1-8. Evidence pertaining to nervous system effects in humans
Reference and study design
Results
Studies in infants and children, neurodevelopment
Roze et al. (2009) (the Netherlands, COMPARE
cohort, 2001-2002 at baseline)
Population: Birth cohort, 90 singleton, term births, 62
of 69 (90%) mother-child pairs randomly selected from
the cohort for HBCD measures in serum; children ages
5-6 years at follow-up
Exposure measures: Prenatal exposure, maternal serum
at 35th week of pregnancy; 1,2,5,6,9,10-HBCD (HBCD)
detected in all samples; LOD 0.8 pg/g serum
Correlations between lipid-adjusted HBCD and outcome
measure adjusted for socioeconomic status (maternal
education), HOME score, and sex
Neuropsychological measure Correlation coefficient
Coordination 0.290 (p < 0.05)
Total intelligence 0.393 (p < 0.05)
Verbal intelligence 0.479 (p < 0.01)
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Median 0.8 (range: 0.3-7.5) ng/g lipids
Effect measures:
Neuropsychological tests (references for procedure
provided)
• Movement ABC test battery for motor performance
(coordination, fine motor skills)
• Developmental Coordination Disorder Questionnaire
for behavior
• Wechsler Preschool and Primary Scale of Intelligence,
Revised for intelligence (total, verbal, performance)
• Neuropsychological Assessment (NEPSY-II) for
visual perception, visuomotor integration, inhibitory
control
• Rey's Auditory Verbal Learning test (verbal memory)
• Test of Everyday Attention for Children (attention)
Behavioral tests (references for procedure provided)
• Child Behavior Checklist and Teacher's Report Form
• Attention Deficit/Hyperactivity Disorder
questionnaire
Analysis: Pearson correlation (for normally distributed
variables) or Spearman's rank correlation (for non-
normally distributed variables)
Data quality:
Medium (1.8)
(Correlations of similar, but somewhat smaller,
magnitude were seen between PCB-153 or 4,4-DDE and
coordination; none of the other nine compounds
examined were associated with either intelligence
measure.)
Results for correlations between HBCD and other
neuropsychological and behavioral outcomes were not
shown, but were stated to be not statistically significant
(p>0.10).
Studies in adolescents, neurodevelopment
ft ski et al. (; (Belgium, 2008-2011)
Beta (95% CI)b
Population: 515 adolescents (13-17 yrs old) residing in
two industrial areas and randomly selected from the
general population; participation rates 22-34% in the
three groups; sample size varied by test (designed as
"biomonitoring program for environmental health
surveillance")
Exposure measures: Serum samples, HBCD
>75% were less than the LOQ (LOQ = 30 ng/L);
Median <30 ng/L (range:
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Effects of levels above the LOQ were estimated.
Models evaluating number of digits in Digital Span test
were also adjusted for the method of test administration.
aBased on OPPT data evaluation criteria
bBeta is for HBCD >30 ng/L (LOQ) versus <30 ng/L; 0.0 = no association.
Table 1-9. Evidence pertaining to neurological effects in animals following
developmental ex
aosure to HBCD
Reference and study
design
Results
Xeurorfevelopnienlal milestones
Etna et al. (2008)
Doses (mg/kg-d)
Rats, CRL:CD(SD
Fl offspring3 0 17
168
1,570
Diet
Two generation
F2 offspring3 0 15
139
1,360
Surface righting reflex response time (s)
F0: exposure started 10 wks
Male, Fl, PND 5 (n = 17-24 litters)
prior to mating
Fl: dietary exposure post
weaning through necropsy
F1/F2 offspring: continuous
Mean (SD) 2.3(1.1) 2(0.6)
%ofcontrolb - -13%
Female, Fl, PND 5 (n = 17-23 litters)
1.8(0.5)
-22%
1.6* (0.3)
-30%
maternal exposure
throughout gestation/
lactation
Mean (SD) 3.1(1.8) 2.4(1.5)
% of controlb - -23%
2.9 (2.6)
-6%
2.6 (2.6)
-16%
Male, F2, PND 5 (n = 19-22 litters)
Data quality:d
High (0)
Mean (SD) 2.1(1.7) 2.0(1.5)
% of control13 - -5%
Female, F2, PND 5 (n = 16-22 litters)
2.8 (2.5)
33%
2.2 (2.3)
5%
Mean (SD) 2.3(0.9) 2.4(1.7)
2.1 (0.9)
3.7 (3.7)
% of controlb - 4%
-9%
61%
Mid-air righting reflex completion rate (%)
Male, Fl, PND 18 (n = 17-23 litters)
Mean 100 100
100
100
% of controlb - 0%
0%
0%
Female, Fl, PND 18 (n = 17-23 litters)
Mean 100 100
100
100
% of controlb - 0%
0%
0%
Male, F2, PND 18 (n = 13-22 litters)
Mean 100 100
94.4
100
% of controlb - 0%
-6%
0%
Female, F2, PND 18 (n = 13-21 litters)
Mean 100 100
90
76.9*
% of controlb - 0%
-10%
-23%
Miller-Rhodes et al.
Doses (mg/kg-d)
0 3
10
30
Rats, Long-Evans
Age at which 85% of pups could perform righting reflex
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Reference and study
design
Results
Gavage
Male, Fl (n = 8-10 litters)
Fl: Continuous maternal
exposure throughout
gestation
PND 5
% of controlb -
Female, Fl (n = 8-10 litters)
5
0%
5
0%
3
-40%
PND 7
5
5
3
% of controlb -
-29%
-29%
-57%
FOB including the righting reflex was conducted every other day from PND 1 to 21.
Every pup in each litter was examined.
Data quality:"1
Animals that did not respond to tail pinch (mean % pups per litter)
Medium (2)*
Males and females, Fl PNDs 1-21 (n = 8-10 litters)
Mean (SE) 39(2)
28* (2)
31* (2)
27* (2)
% of controlb -
-28%
-21%
-31%
Grip strength (Newtons)
Male, Fl, PND 26 (n = 8-10 litters)
Mean (SE) 4.1(0.2)
3.9 (0.2)
2.8* (0.2)
3.3* (0.2)
% of controlb -
-5%
-32%
-20%
Data for tail pinch and grip strength were digitized from figure. No significant
treatment-related effect on grip strength in females.
Executive function and locomotor activity
Etna et al. (2008)
Doses (mg/kg-d)
Rats, CRL:CD(SD)
Diet
Two generation
Male, Fl 0
Female, Fl 0
11
14
115
138
1,142
1,363
Locomotor activity
F0: exposure started 10 wks
Male, Fl, PNW 4 (n = 10)
prior to mating
Fl: dietary exposure post
weaning until necropsy
F1/F2 offspring: continuous
maternal exposure
Mean (SD)
% of controlb
0-10 min 141.9(63.5)
240.9(116.7)
70%
127.4 (79.2)
-10%
162.4 (124.9)
14%
throughout gestation/
lactation
10-20 min 86.1 (59.3)
116.8(86.3)
36%
71.7 (44.4)
-17%
53.3 (53.7)
-38%
Data quality:"1
20-30 min 39.9 (49.4)
58.2 (66.8)
11.8 (11.4)
8.8(13.9)
High (1.0)
-
46%
-70%
-78%
30-40 min 15.6(19.1)
29.5 (45.0)
2.9 (5.9)
7.1 (11.9)
-
89%
-81%
-54%
40-50 min 13.8(21.5)
5.7 (18.0)
0.0 (0.0)
1.0 (2.5)
-
-59%
-100%
-93%
50-60 min 4.8 (15.2)
0.8 (2.5)
0.0 (0.0)
5.7(18.0)
-
-83%
-100%
19%
Female, Fl, PNW 4 (n = 10)
Mean (SD)
% of controlb
0-10 min 196.9 (75.8)
194.1 (112.7)
176.7 (93.8)
172.6(101.9)
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Reference and study
design
Results
-
-1%
-10%
-12%
10-20 min
77.6 (50.0)
70.7 (64.3)
84.7 (66.2)
35.2 (31.8)
-
-9%
9%
-55%
20-30 min
40.4 (44.7)
52.1 (62.3)
39.5 (49.4)
17.7 (31.2)
-
29%
-2%
-56%
30-40 min
13.0(30.9)
15.4 (42.0)
5.6 (12.3)
15.8 (22.0)
-
18%
-57%
22%
40-50 min
5.4(14.2)
2.3 (7.3)
9.9 (31.3)
3.6(11.4)
-
-57%
83%
-33%
50-60 min
0.8(1.9)
1.3 (3.5)
4.9 (12.4)
5.0(11.2)
-
63%
513%
525%
T-maze swim test, trial time (s)
Male, Fl, PNW 6 (n = 10)
Mean (SD)
% of controlb
Day 1
8.3 (2.5)
8.0(1.1)
6.9 (1.3)
8.3 (2.5)
-
-4%
-17%
0%
Day 2
48.7(19.1)
43.5 (18.4)
33.2 (12.0)
40.8 (17.4)
-
-11%
-32%
-16%
Day 3
38.9(14.8)
27.8 (8.8)
32.4* (37.3)
18.4* (4.9)
-
-29%
-17%
-53%
Day 4
27.5 (12.3)
30.4(12.3)
28.0 (24.7)
19.6 (5.2)
-
11%
2%
-29%
Female, Fl, PNW 6 (n = 10)
Mean (SD)
% of controlb
Day 1
12.2 (4.7)
10.8 (4.0)
8.8 (4.4)
10.5 (2.3)
-
-11%
-28%
-14%
Day 2
49.1 (18.2)
43.4(17.1)
40.7 (14.2)
39.2(12.2)
-
-12%
-17%
-20%
Day 3
42.1 (32.6)
35.1 (15.8)
34.5 (23.3)
31.5 (19.4)
-
-17%
-18%
-25%
Day 4
28.3 (8.1)
31.6(19.6)
30.7 (13.0)
25.4(10.1)
-
12%
8%
-10%
Miller-Rhodes et al.
Doses (mg/kg-d)
£
0
3
10
30
Rats, Long-Evans
Go/no-go task (% hits)
Gavage
Males and females, Fl (n = 4)
Fl: Continuous maternal
Mean (SE)
94.8 (0.7)
87.8(1.9)*
94.1 (1.6)
94.8 (0.9)
% of controlb
-
-7%
-1%
0%
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Reference and study
design
Results
exposure throughout
gestation
Go/no-go task: animals
tested on PNM 14 and 21
Random ratio (RR) task (responses per minute)
Males and females, F1 (n = 4)
Mean (SD)
% of controlb
RR1
8.6(1.5)
7.5 (0.1)
7.6(1.2)
8.5 (1.2)
RR task animals tested on
-
-13%
-12%
-1%
PNM 11 and 19
RR2
14.1 (2.6)
12.8(1.8)
12.5 (1.5)
14.9(1.7)
-
-9%
-11%
6%
Data quality:"1
RR5
20.1 (4.0)
20.2 (2.8)
18.9 (2.9)
22.7(1.5)
Medium (2)*
-
1%
-6%
13%
RR10
26.9 (3.7)
26.4 (4.0)
23.0 (3.6)
25.9 (3.2)
-
-2%
-15%
-4%
RR20
24.7 (4.5)
26.5 (3.7)
23.6 (5.3)
30.6 (2.9)
-
7%
-4%
24%
All data were digitized from figure.
Go/no-go task: hit defined as lever press
RR task: Different schedules (e.g., RR1,
of lever presses between reinforcements.
behavior during a "go'
RR2...) correspond to
' trial.
the average number
Eriksson et al. (2006)
Mice, NMRI
Gavage
Fl: single dose onPND 10
Males only
Data quality:d
Medium (2)*
Doses (mg/kg)
0
0.9
13.5
Horizontal locomotion (beam hits)
Male, Fl, PNM 3 (n = 10)
Mean (SD)
% of controlb
0-20 min
20-40 min
40-60 min
499 (81)
209 (62)
12(8)
414* (50)
-17%
256 (50)
22%
12(16)
0%
213* (58)
-57%
232 (39)
11%
256* (47)
2,103%
Rearing (beam hits)
Male, Fl, PNM 3 (n = 10)
Mean (SD)
% of controlb
0-20 min
20-40 min
40-60 min
1,596 (285)
487 (91)
104 (13)
1,206* (260)
-24%
525 (143)
8%
142(13)
37%
322*(78)
-80%
485 (130)
0%
480* (104)
362%
Total activity (beam hits)
Male, Fl, PNM 3 (n = 10)
Mean (SD)
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Reference and study
design
Results
% of controlb
0-20 min 4,741 (606)
4,491 (535)
-5%
2,495* (321)
-47%
20-40 min 2,210(428)
2,424 (606)
10%
2,566 (321)
16%
40-60 min 1,176(214)
998 (214)
-15%
2,709* (570)
130%
Morris water maze (s)
Male, Fl, PNM 3 (n = 12-17)c
Mean
% of controlb
Day 1 27
27
0%
25
-1%
Day 2 20
21
8%
23
18%
Day 3 15
17
13%
19
24%
Day 4 10
14*
20*
-
33%
90%
Day 5 14
20
46%
21*
54%
All data were digitized from figure.
Morris water maze: error data not shown. Day 5, platform relocated.
Other neurological effects
Etna et al. (2008)
Doses (mg/kg-d)
Rats, CRL:CD(SD)
Fl offspring3 0
17
168
1,570
Diet
Two generation
Male, Fl 0
Female, Fl 0
11
14
115
138
1,142
1,363
F0: exposure started 10 wks
F2 offspring3 0
15
139
1,360
prior to mating
Absolute brain weight (mg)
Fl: dietary exposure post
weaning until necropsy
F1/F2 offspring: continuous
Male, Fl PND 26 (n = 17-23)
Mean (SD) 1.64(0.09)
1.66 (0.05) 1.62 (0.07)
1.55* (0.06)
maternal exposure
throughout gestation/
lactation
% of controlb -
Female, Fl PND 26 (n = 14-23)
1%
-1%
-5%
Mean (SD) 1.58 (0.09)
1.61 (0.07) 1.59 (0.08)
1.51* (0.06)
% of controlb -
2%
1%
-4%
Data quality:"1
Male, Fl adult (n = 22-24)
High (1.0)
Mean (SD) 2.18(0.08)
2.22(0.08) 2.18(0.09)
2.11* (0.07)
% of controlb -
2%
0%
-3%
Female, Fl adult (n = 13-22)
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Reference and study
design
Results
Mean (SD) 2.07 (0.09) 2.06 (0.07)
2.06 (0.08)
1.97* (0.06)
% of controlb -
0%
0%
-5%
Male, F2 PND 26 (n = 13-22)
Mean (SD) 1.62 (0.13) 1.65 (0.08)
1.60 (0.10)
1.46* (0.09)
% of controlb -
2%
-1%
-
10%
Female, F2 PND 26 (n = 13-22)
Mean (SD) 1.57 (0.11) 1.58 (0.07)
1.55 (0.12)
1.41
*(0.15)
% of controlb -
1%
-1%
-
10%
Lilienthal et al. (2009)
Doses (mg/kg-d)
Rats, Wistar
0 0.1 0.3
1
3
10
30
100
Diet
BAEPs, click threshold (dB)
F0: exposure started 10 wks
Male, Fl, PNW 20 (n = 4-6)**
(male) or 2 wks (female)
Mean 47 (2) 47 (4) 40 (2)
49 (7)
48 (8)
48 (4)
53 (3)
56 (4)
prior to mating
(SE)
Fl: continuous maternal
%of - 0% -15%
4%
2%
2%
13%
19%
exposure throughout
controlb
gestation/lactation; dietary
Female, Fl, PNW 20 (n = 4-6)
exposure post weaning until
sacrifice (~PNW 20)
Mean 44 (3) 47 (2) 53 (4)
52 (3)
41(3)
54 (2)
49 (2)
48 (2)
(SE)
Data quality:"1
% of - 7% 20%
18%
-7%
23%
11%
9%
High (1.3)
controlb
Data for males were digitized from figure.
Catalepsy, box, foreleg latency (s)
Male, Fl, PNW 15 (n = 5)**
Mean 135 150 105
98
129
140
99
69
(SE) (24) (18) (19)
(26)
(27)
(27)
(33)
(30)
%of - 11% -22%
-27%
-4%
4%
-27%
-49%
controlb
Female, Fl, PNW 15 (n = 5)**
Mean 136 77 128
145
111
65
56
60
(SE) (24) (28) (32)
(34)
(31)
(38)
(25)
(30)
%of - -43% -6%
7%
-18%
-52%
-59%
-56%
controlb
Data for females were digitized from figure.
van der Yen et al.
Doses (mg/kg-d)
(2009)
0 0.1 0.3
1
3
10
30
100
Rats, Wistar
Absolute brain weight (g)
Diet
Male, Fl, PNW 11 (n = 4-5)**
One generation
Mean 1.84 1.87 1.94
1.98
1.91
1.88
1.92
1.78
F0: exposure started one
(SE) (0.12) (0.07) (0.06)
(0.07)
(0.07)
(0.05)
(0.06)
(0.06)
spermatogenic cycle
%of - 2% 5%
8%
4%
2%
4%
-3%
(males: 70 d) or two estrous
controlb
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Reference and study
design
Results
cycles (females: 14 d) prior
to mating
Fl: continuous maternal
exposure throughout
gestation/lactation; dietary
exposure post weaning
through PNW 11
Data quality:"1
High (1.2)
Female, Fl, PNW 11 (n = 4-5)
Mean 1.76 1.71 1.71 1.77 1.62 1.80 1.76 1.66
(SE) (0.14) (0.09) (0.09) (0.08) (0.23) (0.06) (0.08) (0.07)
%of - -3% -3% 1% -8% 2% 0% -6%
controlb
* Statistically significantly different from the control at p< 0.05 as reported by study authors.
**Significant dose response trend as reported by study authors.
aFl and F2 offspring doses presented as mean maternal gestational F0 and F1 doses, respectively.
bPercent change compared to control calculated as: (treated value - control value)/control value / 100.
°Exact number of animals examined per dose group was unclear based on the published paper.
'Based on OPPT data evaluation criteria. *Miller-Rhodes et at. (20.1.4) was downgraded to a Medium. The calculated
score was 1.4. Eriksson et at. (2006) was also downgraded to a Medium. The calculated score was 1.3
PNM = postnatal month
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III
Catalepsy - box, foreleg latency Lilienthal, 2009 (rats)
Absolute brain weight Ema et a!., 2008 (rats, F2 offpsring)
Absolute brain weight Ema et ah, 2008 (rats, F1 adults)
Absolute brain weight Ema et al., 2008 (rats, Fl offspring)
Morris water maze Eriksson et al., 2006 ( mice, F1) Day 5
Morris water maze Eriksson et al., 2006 ( mice, Fl) Days 1-3
Total Activity Eriksson et al., 2006 ( mice, F1)
Rearing Eriksson et al., 2006 ( mice, FX PNM 3 40-60 min)
Rearing Eriksson et al., 2006 ( mice, F1 PNM 3 20-40 min)
Rearing Eriksson et al., 2006 ( mice, F1 PNM 3 0-20 min)
Swim Maze Performance (D3) F1 Mate
Ema et al., 2008 (rats)
Swim Maze Performance F1 Female Ema et al., 2008 (rats)
Locomotor activity Ema et al., 2008 (rats)
Grip strength Miller-Rhodes et al., 2014 (rats, Fl)
Surface Righting Reflex Ema et al., 2008 (rats, F2)
Surface Righting Reflex Ema et al., 2008 (rats, Fl Female)
Surface Righting Reflex Ema et al., 2008 (rats, Fl...
Spontaneous Motor Activity Eriksson et al., 2006 (mice)
Brainstem auditory evoked potentials (BAEPs) - click threshold Lilienthal et al., 2009 (rats, Fl)
• significantly changed
Onot significantly changed
Morris water maze Eriksson et al., 2006 ( mice, Fl) Day 4
Horizontal Locomotion Eriksson et al., 2006 (mice, Fl PNM 3 40-60 min)
Horizontal Locomotion Eriksson et al., 2006 ( mice, Fl PNM 3 20*40 min)
Horizontal Locomotion Eriksson et al., 2006 (mice, Fl PNM 3 0*20 min)
Random ratio task Miller-Rhodes et al., 2014 (rats, Fl)
Go/no-go task Miller-Rhodes et al., 2014 (rats, Fl)
Animals that did not respond to tail pinch Miller-Rhodes eta!., 2014 (rats, Fl)
! Age at which 85% of pups could perform righting reflex Miiler-Rhodes et al., 2014 (rats, Fl)
Mid-air righting reflex completion rate Ema et al., 2008 (rats, F2 offspring F)
Mid-air righting reflex completion rate Ema et al., 2008 (rats, F2 offspring M)
Mid-air righting reflex completion rate Ema et al., 2008 (rats, Fl offspring)
Doses (mg/kg-day)
10000
Figure 1-7. Exposure response array of nervous system effects following oral exposure. Lilienthal et al. (2009) and
Ema et al. (2008) scored a High in data quality evaluation. Miller-Rhodes et al. (2014) and Eriksson et al. (2006) scored a
Medium (indicated with ¦»).
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1.5.4 Mechanistic Evidence
1.5.4.1 Thyroid Perturbation and Neurodifferentiation
Thyroid hormones are known to play a key role in development of the vertebrate central nervous
system, and perinatal exposure to thyroid-disrupting chemicals has been shown to have lasting
effects on cognitive and behavioral outcomes (Gilbert et at.. 2012; Howdeshell. 2002; Koibuchi
and Chin. 2000). The evidence to support mechanisms by which HBCD may affect thyroid
hormones is covered elsewhere (Section 1.2.1, Mechanistic Evidence); therefore, the following
discussion focuses on the available studies that specifically investigated possible associations
between HBCD-mediated thyroid hormone perturbation and neurodevelopmental endpoints
(Fuiimoto et al., 2013; Saegusa et at., 2012; Ibhazehiebo et at., 201 la; Ibhazehiebo et at ).
As discussed in Section 1.2.1, HBCD elicited a decrease in thyroid hormone levels in
developmentally exposed rats (Saegusa et al.. 2009). In two follow-up studies by the same
research group, thyroid perturbation corresponded with several changes in brain morphometry
indicative of altered neuronal migration and neurogenesis in the hippocampus, a region that is
critical for learning and memory (Fuiimoto et at., 2013; Saegusa et al, 2012). Developmental
exposure also elicited a statistically significant increase in the number of astrocytes and
oligodendrocytes in the cingulum, an area of the brain involved in regulating behaviors related to
emotion and cognitive function (Fuiimoto et al.. ). These results mirror those previously
found following developmental exposure to known anti-thyroid drugs, propylthiouracil and
methimazole (Fuiimoto et al.. ). These data are supported by two studies with primary rat
neuronal cell cultures. During normal development, thyroid hormones regulate neurite growth
and arborization of cerebellar granule neurons (CGNs) and Purkinje cells. In the cerebellum,
these cells generate a highly interconnected dendritic network that is critical for motor control
and coordination (Gilbert et al.. 2012; Koibuchi and Chin. 2000). Primary rat Purkinje cell
(Ibhazehiebo et al.. JO I I j) and CGN (Ibhazehiebo et al JO I I h) cultures co-exposed to thyroid
hormone and sub-nanomolar concentrations of a-HBCD showed statistically significant
reductions in thyroid hormone-induced neurite growth and arborization. These effects were seen
at concentrations several orders of magnitude below those that reduced viability by >50% in rat
primary CGNs (Reistad et al.. 2006) and human neuroblastoma cells (Al-Mousa and
Michetamgeti. 2012). indicating that they were not due to cytotoxicity. HBCD-mediated effects
on neurite growth and arborization could be ameliorated by elevated thyroid hormone levels
(Ibhazehiebo et al.. ) or coexposure with brain-derived neurotrophic factor (Ibhazehiebo et
al. 20fib).
1.5.4.2 Calcium Homeostasis
Several studies suggest that HBCD may alter calcium (Ca2+) homeostasis in the brain by
affecting three types of calcium transporters: sarco-endoplasmic reticulum Ca2+-dependent
ATPase (SERCA) pumps (Al-Mousa and Michelangeli. 2014. ), ligand-gated Ca2+
channels (LGCC) (Reistad et al.. 2006). and voltage-gated Ca2+ channels (VGCC) (Din gem an s
et al.. 2009). Within neurons, Ca2+ levels are typically maintained at low concentrations relative
to the extracellular fluid; however, rapid influx can occur through various ion channels. After an
influx event, low cytosolic Ca2+ levels are restored via active transport across the cell membrane
or sequestration into subcellular compartments. Tight regulation of Ca2+ is critical as both
excess and insufficient levels can adversely affect numerous cellular processes.
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SERCA uses ATP to actively transport excess Ca2+ from the cytosol into intracellular
compartments to regulate protein synthesis and neurotransmitter release (Neher and Sakaba.
2008; Rodriguez et at.. 2001). HBCD increased intracellular Ca2+ and cell death in human
neuroblastoma cells (SH-SY5Y) via concentration-dependent SERCA inhibition (Al-Mousa and
Michelangeli. 2014. 2012). HBCD interacts with SERCA in a manner that: (1) reduces ATP
binding affinity and (2) stabilizes the low Ca2+ affinity conformation ("Al-Mousa and
Michelangeli. 2014). Exposure of PC 12 cells to either the technical mixture or individual HBCD
isomers reduced Ca2+ influx through VGCCs, but did not affect resting intracellular Ca2+ levels
(Dingemans et at.. 2009). y-HBCD showed the greatest potency, whereas the a-isomer had a
moderate effect similar to that of the technical mixture. These effects were associated with
decreased catecholamine release, likely due to low cytosolic Ca2+ levels that were insufficient to
trigger synaptic release (Neher and Sakaba, 2008). HBCD may also act as a mild LGCC-agonist.
Co-exposure to MK801, an LGCC antagonist, was found to ameliorate HBCD-induced
cytotoxicity, suggesting a role of this Ca2+ channel in neurotoxicity. Although no significant
changes in intracellular Ca2+ calcium were reported, this was the only study that measured Ca2+
effects as an average across all cells, which may have reduced the sensitivity when compared to
single cell measurements (Al-Mousa and Michelangeli. 2012; Dingemans et at.. 2009).
1.5.4.3 Neurotransmitter Reuptake
Adult male mice exposed to 25 mg/kg-day for 30 days showed decreased striatal levels of
dopamine transporter and vesicular monoamine transporter 2, regulators of dopamine
homeostasis and neurotransmission (Genskow et at.. 2015). Similarly, an in vitro study found a
dose-related reduction in dopamine and gamma-aminobutyric acid uptake in rat synaptosomes
and vesicles exposed to HBCD (Mariussen a num. 2003). Although prolonged deficits in
reuptake mechanisms could result in excessive stimulation of the post synaptic cell or deplete
neurotransmitter stores in the presynaptic cell, Genskow et al. (2015) did not find significant
changes in tissue concentrations of dopamine or its metabolites in adult mice exposed for 30
days.
1.6 Immune System Effects
1.6.1 Human Evidence
The potential for HBCD to affect the immune system has not been investigated in humans.
1.6.2 Animal Evidence
The potential for HBCD to affect the immune system has been examined in eight studies in rats
(Hachisuka et al.. 2010; van der Yen et al.. 2009; Etna et al.. 2008; van der Yen et al.. 2006; WIL
Research, 2001, 1997) and mice (Maranghi et al., 2013; Watanabe et al., ^ ), with exposures
ranging from a 28-day exposure in adults to continuous exposure across two generations.
Discussion of immune-related effects of HBCD is organized first by age of exposure
(i.e., developmental or adult) and second by the type of endpoint evaluated (i.e., functional or
observational). Exposure timing is an important factor that may influence the effect of chemical
exposure on immune function, particularly for early-life exposure studies. In rodents, immune
development occurs in a series of discrete stages until approximately PND 42. The developing
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immune system is susceptible to perturbation resulting from chemical exposure, and exposures
during this period may result in distinct toxicological consequences that would not be observed
in animals exposed only as adults (Burns-Haas et at.. 2008). With regard to the type of endpoint
evaluated, functional immune outcomes, including response to challenge with an infectious agent
or immunization with a foreign antigen, are the most relevant and sensitive for determining
potential immunotoxicity because the primary role of the immune system is to protect host
integrity from foreign challenge and potential insult. Laboratory animals are housed in
environments that limit their exposure to antigenic stimulation or infectious agents, and their
immune systems are typically in a resting state (Who. 2012). In the absence of a foreign
challenge, observational endpoints, including structural alterations or changes in immune cell
populations, can provide information about immune system effects, but are considered less
sensitive and predictive (Luster et at.. 2005).
A summary of the evidence pertaining to functional and observational immune system effects in
experimental animals is presented in Table 1-10, Table 1-11, Table 1-12 and Figure 1-9. Studies
are ordered within effect categories by decreasing exposure duration and then species.
1.6.2.1 Developmental Exposure
Functional immune Effects
Changes in functional immune endpoints (immunoglobulin G [IgG] and immunoglobulin [IgM]
antibody production in response to foreign antigens) following developmental HBCD exposures
were evaluated in two one-generation reproductive toxicity studies in male (van der Yen et al..
2009) or female rats (Hachisuka et al., 2010) (see Table 1-10 and Figure 1-8). Statistically
significant changes in IgG levels were reported in both studies, but with opposite directions of
effect; males exposed to up to 100 mg/kg-day showed a dose-dependent increase in IgG, whereas
females exposed to approximately 1,500 mg/kg-day showed a decrease. Differences in the design
of these two studies, including timing of exposure, immune challenge, and titer measurement
(Figure 1-3), may have contributed to the inconsistent results. IgM activity was unaffected in van
der Yen et al. (2009) and results were not reported by Hachisuka et al. (2010). van der Yen et al.
(2009) also evaluated natural killer (NK) cell activity and found no treatment-related effects.
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HBCD
dosing
begins
Immunization 1
(SRBC)
I
Immunization 2
(SRBC)
I
van der Ven et at, (2009)
i L
Immunization 1
Immunization 2
(KLH)
I Immunisation 3
| {KLH)
I I
Hachisuka et al, (2010)
IgM & IgG
analysis
IgM & IgG
analysis
I I
IgM
analysis
IgG
analysis
KLH = keyhole limpet hemocyanin; SRBC = sheep red blood cell
Horizontal lines represent the experimental timelines, with black indicating the time period when HBCD was
administered (i.e., from 2 weeks prior to mating through IgG analysis in van der Ven et al. (2009). and from GD 10
to PND 21 in Hachisuka et al. (2010).
Figure 1-8. Comparison of study designs used by van der Ven et al. (2009) and Hachisuka
et al. (2010).
Observational Immune Effects
Five studies evaluated effects on observational immune parameters, including organ weights,
hematology, and histopathology, in developmentally-exposed rats (Hachisuka et al., 2010;
Saegusa et al.. 2009; van der Ven et al.. 2009; Ema et al.. 2008) or mice (Maranghi et al.. 2013)
(see Table 1-4 and Figure 1-4).
Thymus weights showed significant dose-response trends in male and female adult rats (PNW
11) continuously exposed to HBCD at doses up to 100 mg/kg-day (van der Ven et al.. 2009) and
in female F2 weanlings exposed to approximately 1,300 mg/kg-day HBCD throughout gestation
and lactation (Ema et al.. 2008). Spleen weight was reduced in both male and female F2
weanlings from the 1,300 mg/kg-day dose group (Ema et al.. 2008). A significant positive trend
was also reported for absolute popliteal lymph node weight in PNW 11 male, but not female, rats
(van der Ven et al.. 2009). No other treatment-related effects were reported for thymus
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(Maranghi et al., 2013; Hachisuka et al., 2010; Saegusa et al., 2009) or spleen weights (Maranghi
et al. 2013; Hachisuka et al.. 2010; Saegusa et al.. 2009; van der Yen et al.. 2009).
Hematological analyses revealed significant treatment-related effects on several blood immune
cell populations, although the pattern of effect was variable across studies, sex, and time point.
Total white blood cell (WBC) count was measured in three studies. Hachisuka et al. (2010)
reported statistically significant increases in WBC count in HBCD-exposed male rats on PNWs 3
and 11 (approximately 8 weeks after the end of the exposure). In contrast, van der Yen et al.
(2009) reported a significant dose-related decrease in continuously exposed PNW 11 male rats,
and Etna et al. (2008) found no effect on total WBCs of F1 males or females. In addition to total
WBCs, several subpopulations were measured, van der Yen et al. (2009) found a significant
dose-related increase and decrease in the fraction of neutrophils and lymphocytes, respectively.
The magnitude of the lymphocyte change was small (<4% change from control) and the
biological significance is unclear. Hachisuka et al. (: also measured subpopulations of
several leukocyte subtypes. On PNW 3, high-dose (1,505 mg/kg-day HBCD) male rats showed a
decrease in activated T-cell and NK cell fractions and an increase in inactive B-cell fractions;
however, cell fractions returned to control levels by PNW 11.
Hachisuka et al. (2010) and van der Yen et al. (2009) reported inconsistent effects on splenic NK
and cytotoxic T-cell populations. Hachisuka et al. (1 reported a statistically significant
decrease in the NK cell fraction (e.g., CD4NKT cells, PNW 3) and an increase in the cytotoxic
T-cell fraction in adult rats (CD8+ cells, PNW 11) that were gestationally and lactationally
exposed to HBCD. In contrast, male rats continuously exposed through PNW 11 showed a dose-
dependent increase in the NK cell fraction and no change in the cytotoxic T-cell fraction. No
other treatment-related effects were observed for other immune cell counts in the spleen (van der
Yen et al.. 2009).
Immune cell counts were also measured in the thymus (Hachisuka et al.J ) and bone marrow
(van der Yen et al.. 2009). Rats showed decreases in the thymus fraction of active and regulatory
T-cells and an increase in NK cells on PNW 3 and PNW 1 1, respectively (Hachisuka et al..
2010). WBC counts in bone marrow showed an increasing dose-related trend in adult males
continuously exposed to HBCD at doses up to 100 mg/kg-day (van der Yen et al.. 2009).
Histological examination of immune-related tissues showed limited changes with no clear
pattern of effect. Thymus tissues showed increased incidence of "starry sky" appearance
(Hachisuka et al.. 2010) and blurring of the corticomedullary demarcation (Maranghi et al..
2013) in rats and mice, respectively. In the spleen, increased incidence of marginal zone
enlargement was also observed in adult (PNW 11) rats continuously exposed to 100 mg/kg-day
HBCD (van der Yen et al.. 2009). No other treatment-related histological changes were observed
(Hachisuka et al.. 2010; van der Yen et al.. 2009; Etna et al.. 2008).
1.6.2.2 Adult Exposure
Functional Immune Effects
Two studies evaluated functional immune endpoints following adult exposure to HBCD for 28
days (Watanabe et al.. 2010; van der Yen et al.. 2006). No statistically significant changes were
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observed in NK cell activity in adult male rats (van der Yen et at., 2006) or host immunity
infection in female mice (Watanabe et at.. 2010).
Observational Immune Effects
Treatment related effects on organ weight, hematology, and histopathology were evaluated in
four rat studies (Etna et at.. 2008; van der Yen et at.. 2006; esearch. 2001. 1997) (see
Table 1-5 and Figure 1-4). Trends identified by the authors as statistically significant were
reported for absolute thymus weight in male rats and for absolute spleen weight in female rats
administered up to 200 mg/kg-day for 28 days (van der Yen et al.. 2006). In both cases, effects
were not consistent across sexes, the magnitude of the effect was small, and the biological
significance of these changes is unclear. Hematological analyses revealed a statistically
significant reduction in the percentage of stabform and segmented neutrophils and increase in the
lymphocyte fraction of F0 females exposed to HBCD for 14 weeks (Ema et al.. 2008); however,
these effects were only seen in the low-dose group (approximately 14 mg/kg-day) in this study
and not in a second study involving adult exposure (van der Yen et al.. 2006). Total splenocyte
number was decreased in adult male rats in the 28-day study by van der Yen et al. (2006). No
other observational immune endpoints were affected (Ema et al.. 2008; WIL Research. 2001.
1997).
Table 1-10. Evidence pertaining to functional immune system effects in animals following
exposure to HBCD during development
Reference and study
design
Results
van der Veil et al. (2009)
Rats, Wistar
Diet
One generation
Fl: continuous maternal
exposure throughout
gestation/lactation; dietary
exposure post weaning
through PNW 11
Data quality:0
High (1.2)
Doses (mg/kg-d)
Male, Fl 0 0.1 0.3 1 3 10 30 100
SRBC antibody titers IgG (extinction)
Male, Fl, PNW 11 (n = 2-4)**
Mean (SD) 0.182 0.362 0.174 0.233 0.152 0.444 0.856 0.469
(0.128) (0.333) (0.143) (0.169) (0.180) (0.143) (0.231) (0.205)
% change3 - 99% -4% 28% -16% 144% 370% 158%
Animals (males only) immunized with SRBCs on PNWs 8 and 10.
Hachisuka et al. (2010)
Rats, SD:IGS
Diet
Fl: maternal exposure from
GD 10 to PND 20 followed
by an 8-wk recovery period
through PNW 11
Data quality:0
Medium (1.9)
Doses (mg/kg-d)b
Female, 0 14.8 146.3 1,505
Fl
Antibody IgG responses to KLH (titer)
Female, Fl, PND 40 (n = 7-8, estimated from graph)
Mean 139,452 63,196 95,592 42,548*
% change3 - -55% -31% -69%
Data were digitized from figure; animals (females only) challenged with KLH on
PNDs 23 and 33. IgM titers (enzyme-linked immunosorbent assay) were measured
on PND 40.
* Statistically significantly different from the control at p< 0.05.
**Significant dose response trend.
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"Percent change compared to control calculated as: (treated value - control value)/control value x 100.
bTWAs for each exposure group were calculated by: (1) multiplying the measured HBCD intake (mg/kg-day)
reported by the study authors for GDs 10-20, PNDs 1-9, and PNDs 9-20 by the number of inclusive days of
exposure for each time period; (2) adding the resulting products together; and (3) dividing the sum by the total
number of inclusive days (33) of HBCD exposure. Example: 100 ppm = (8.1 mg/kg-day x 11 days) +
(14.3 mg/kg-day x 10 days) + (21.3 mg/kg-day x 12 days)/33 days = 14.8 mg/kg-day.
°Based on OPPT data evaluation criteria.
Table 1-11. Evidence pertaining to observational immune system effects in animals
following exposure to HBCD during development
Reference and
study design
Results
Organ w eight
et al. (2008)
Rats, CRL:CD(SD)
Diet
Two generation
Doses (mg/kg-d)
Fl offspring3 0
17
168
1,570
Male, Fl 0
11
115
1,142
Female, Fl 0
14
138
1,363
F0: exposure started
F2 offspring3 0
15
139
1,360
10 wks prior to
mating
Fl: dietary exposure
post weaning until
Absolute spleen weight (mg)
Male, Fl, adult (n = 22-24)
Mean (SD) 885 (168)
840 (147)
878 (163)
851 (113)
necropsy
F1/F2 offspring:
continuous maternal
exposure throughout
gestation/lactation
% changeb -
Male, Fl, PND 26 (n = 17-23)
Mean (SD) 336 (62)
% changeb -
Female, Fl, adult (n = 13-22)
-5%
327 (41)
-3%
-1%
334 (43)
-1%
-4%
309 (69)
-8%
Mean (SD) 632 (124)
595 (68)
624 (93)
578 (70)
Data quality:6
High (1.0)
% changeb -
Female, Fl, PND 26 (n = 14-23)
-6%
-1%
-9%
Mean (SD) 311(53)
306 (44)
304 (59)
280 (40)
% changeb -
-2%
-2%
-10%
Male, F2, PND 26 (n = 13-22)
Mean (SD) 360 (83)
361 (54)
346 (78)
263* (50)
% changeb -
0%
-4%
-27%
Female F2, PND 26 (n = 13-21)
Mean (SD) 325 (59)
302 (42)
299 (62)
225* (45)
% changeb -
-7%
-8%
-31%
Absolute thymus weight (mg)
Male, Fl, adult (n = 22-24)
Mean (SD) 344 (72)
305 (92)
368 (100)
341 (76)
% changeb -
-11%
7%
-1%
Female, Fl, adult (n = 13-22)
Mean (SD) 250 (62)
233 (62)
276 (80)
259 (76)
% changeb -
-7%
10%
4%
Male, Fl, PND 26 (n = 17-23)
Mean (SD) 342 (68)
339 (50)
369 (59)
317(57)
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study design
Results
% changeb -
-1%
8%
7%
Female, Fl, PND 26 (n = 14-23)
Mean (SD) 335 (64)
330 (58)
370 (58)
305 (31)
% changeb -
-1%
10%
9%
Male, F2, PND 26 (n = 13-22)
Mean (SD) 343 (92)
336 (57)
360 (88)
282 (71)
% changeb -
-2%
5%
18%
Female, F2, PND 26 (n = 13-22)
Mean (SD) 338 (85)
324 (50)
331 (69)
260* (80)
% changeb -
-4%
-2%
-23%
van cler Yen et al.
Doses (mg/kg-d)
(2009)
Rats, Wistar
Diet
One generation
0 0.1
0.3
1
3
10
30
100
Absolute popliteal lymph node weight (mg)
Male, Fl(n= 4-5)**
Mean (SD) 9 (2) 10 (3)
9(4)
15(11)
9(3)
8(1)
10(5)
21 (16)
Fl: continuous
maternal exposure
throughout
gestation/lactation;
% changeb - 11%
Female, Fl (n = 4-5)
Mean (SD) 8 (2) 9 (2)
0%
9(2)
67%
8(2)
0%
8(2)
-11%
8(2)
11%
9(1)
133%
7(2)
dietary exposure
% changeb - 12%
12%
0%
0%
0%
12%
-12%
post weaning
through PNW 11
Absolute spleen weight (g)
Male, Fl (n = 4-5)
Mean (SD) 0.49 0.53
0.49
0.58
0.49
0.50
0.58
0.48
(0.12) (0.07)
(0.03)
(0.07)
(0.05)
(0.07)
(0.09)
(0.06)
Data quality:6
High (1.2)
% changeb - 8%
Female, Fl (n = 4-5)
0%
18%
0%
2%
18%
-2%
Mean (SD) 0.40 0.39
0.37
0.56
0.56
0.38
0.40
0.39
(0.04) (0.04)
(0.06)
(0.37)
(0.42)
(0.05)
(0.04)
(0.07)
% changeb - -3%
-8%
40%
40%
-5%
0%
-3%
Absolute thymus weight (g)
Male, Fl(n= 4-5)**
Mean (SD) 0.62 0.54
0.53
0.56
0.50
0.55
0.48
0.45
(0.10) (0.12)
(0.12)
(0.13)
(0.09)
(0.08)
(0.14)
(0.06)
%changeb - -13%
-15%
-10%
-19%
-11%
-23%
-27%
Female, Fl (n = 4-5)**
Mean (SD) 0.49 0.41
0.40
0.42
0.48
0.45
0.44
0.37
(0.07) (0.05)
(0.04)
(0.05)
(0.10)
(0.06)
(0.11)
(0.07)
%changeb - -16%
-18%
-14%
-2%
-8%
-10%
-24%
Hachisuka et al.
Doses (mg/kg-d)c
(2010)
Rats, SD:IGS
Diet
0
15
146
1,505
Absolute spleen weight (g)
Male, Fl, PNW 3 (n = 10)
Fl: maternal
Mean (SD) 0.29 (0.05)
0.25 (0.03)
0.22 (0.04)
0.23 (0.04)
exposure from
% changeb -
14%
-24%
-21%
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Reference and
study design
Results
GD 10 to PND 20
followed by an 8-wk
recovery period
through PNW 11
Only males
evaluated
Data quality:6
Medium (1.9)
Male, Fl, PNW 11
Mean (SD) 0.55 (0.08) 0.55 (0.11) 0.56 (0.08) 0.53 (0.13)
% changeb - 0% 2% -4%
Absolute thymus weight (g)
Male, Fl, PNW 3 (n = 10)
Mean (SD) 0.21 (0.06) 0.24(0.05) 0.21 (0.06) 0.21 (0.03)
%changeb - 14% 0% 0%
Male, Fl, PNW 11 (n= 10)
Mean (SD) 0.79 (0.08) 0.88 (0.17) 0.88 (0.18) 0.81 (0.13)
%changeb - 11% 11% 3%
I h'nialo/ogv
Etna et al (2008)
Rats, CRL:CD(SD)
Diet
Two generation
F0: exposure started
10 wks prior to
mating
Fl: maternal
exposure throughout
gestation/lactation;
dietary exposure
post weaning until
necropsy
Data quality:6
High (1.0)
Doses (mg/kg-d)
Male, Fl 0 11 115 1,142
Female, Fl 0 14 138 1,363
Lymphocyte fraction (%)
Male, Fl (n = 10)
Mean (SD) 88.2 (4.4) 90.9 (2.7) 87.7 (5.9) 87.3 (5.7)
%changeb - 3% -1% -1%
Female, Fl (n = 10)
Mean (SD) 83.6 (9.4) 76.2 (9.6) 83.6 (8.3) 73 (11.6)
%changeb - -9% 0% -13%
van der Yen et al.
(2009)
Rats, Wistar
Diet
One generation
Fl: continuous
maternal exposure
throughout
gestation/lactation;
dietary exposure
post weaning
through PNW 11
Only males
evaluated
Data quality:6
High (1.2)
Doses (mg/kg-d)
0 0.1 0.3 1 3 10 30 100
Basophil cell count in blood (xl09/L)
Male, Fl (n= 3-4)**
Mean (SD) 0.040 0.072 0.063 0.057 0.045 0.048 0.068 0.035
(0.00 (0.016) (0.026) (0.016) (0.016) (0.028) (0.008) (0.030)
4)
% changeb - 80% 57% 43% 12% 20% 70% -12%
Lymphocyte cell fraction in blood (%)
Male, Fl (n= 3-4)**
Mean (SD) 89.64 89.87 89.45 89.72 88.61 89.61 88.65 85.9
(0.29) (0.26) (0.29) (0.18) (0.4) (0.25) (0.15) (0.23)
% changeb - 0% 0% 0% -1% 0% -1% -4%
WBC count in blood (xl09/L)
Male, Fl (n = 3-4)**
Mean (SD) 5.10 7.18 5.72 6.53 4.90 5.92 6.55 4.05
(1.01) (1.44) (1.79) (0.72) (1.71) (2.27) (0.14) (1.50)
% changeb - 41% 12% 28% -4% 16% 28% -21%
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Reference and
study design
Results
Hachisuka et al.
(2010)
Rats, SD:IGS
Diet
Fl: maternal
exposure from
GD 10 to PND 20
followed by an 8-wk
recovery period
through PNW 11
Only males
evaluated
Data quality:6
Medium (1.9)
Doses (mg/kg-d)°
0 14.8 146.3 1,505
Activated T cell fraction in blood (%)
Male, Fl, PNW 3 (n = 10)
Mean (SD) 13.51 (3.47) 14.01 (2.16) 11.81 (1.96) 10.40* (2.02)
% changeb - 4% -13% -23%
Male, Fl, PNW 11 (n= 10)
Mean (SD) 1.45 (0.54) 1.35 (0.6) 1.27 (0.47) 1.32 (0.24)
% changeb - -7% -12% -9%
Lymphocyte fraction in blood (%)
Male, Fl, PNW 3 (n = 9-10)
Mean (SD) 78.88 (4.74) 79.02 (3.18) 81.69 (3.81) 81.41 (4.06)
%changeb - 0% 3% 3%
Male, Fl, PNW 11 (n= 10)
Mean (SD) 84.64 (5.46) 84.27 (4.88) 87.56 (4.33) 86.44 (3.36)
%changeb - 0% 3% 2%
NK cell fraction in blood (%)
Male, Fl, PNW 3 (n = 10)
Mean (SD) 0.12(0.03) 0.1 (0.03) 0.09(0.02) 0.08* (0.04)
% changeb - -17% -25% -33%
Male, Fl, PNW 11 (n= 10)
Mean (SD) 0.27 (0.07) 0.23 (0.08) 0.27 (0.07) 0.25 (0.09)
%changeb - -15% 0% -7%
WBC count in blood (/ 102/|iL)
Male, Fl, PNW 3 (n = 10)
Mean (SD) 35.3 (11.3) 30.9 (10) 47.5* (11.8) 39.6 (7.9)
% changeb - -12% 35% 12%
Male, Fl, PNW 11 (n= 10)
Mean (SD) 82.1 (17.8) 109.8* (30.8) 110* (29.3) 103.4 (34.1)
% changeb - 34% 34% 26%
Ilistopathology
Male,11 1 3 10 30 100
Female, Fl
Rats, Wistar
Diet
One generation
Fl: continuous
maternal exposure
throughout
gestation/lactation;
dietary exposure
post weaning
through PNW 11
WBC count in bone marrow (xl09/L)
Male, Fl (n = 3-4)**
Mean (SD) 9.3 15.0 17.4 13.0 17.9 20.2 16.3 17.6
(3.4) (9.3) (8.5) (3.0) (4.2) (4.1) (5.0) (4.8)
% changeb - 61% 87% 40% 92% 117% 75% 89%
CD161a (NK) subpopulation fraction in spleen (%)
Male, Fl (n = 3-5)**
Mean (SD) 7.9 8.8 8.6 8.9 9.6 8.9 9.0 11.3
(0.4) (0.8) (1.4) (1.3) (0.6) (0.8) (1.5) (1.3)
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Reference and
study design
Results
% change3 - 11%
9% 13%
22% 13%
14% 43%
Data quality:6
High (1.2)
Splenic marginal zone enlargement (incidence)
Male, Fl (n = 8-10)
Incidence 1/8 -d
d d
d d
_d 7/10*
Hachisuka et al.
Doses (mg/kg-d)°
(2010)
Rats, SD:IGS
Diet
Male, Fl
Female, Fl
15
146
1,505
CD4NKT (NK) cell fraction in spleen (%)
Fl: maternal
Male, Fl, PNW 3 (n = 10)
exposure from
GD 10 to PND 20
followed by an 8-wk
recovery period
through PNW 11
Mean (SD) 6.47(0.61)
% changeb -
Male, Fl, PNW 11 (n= 10)
Mean (SD) 12.53 (1.88)
6.28 (0.81)
-4%
12.89 (1.85)
6.4(1.31)
-1%
13.78 (2.66)
5.63* (0.81)
-13%
13.09 (1.72)
% changeb -
3%
10%
4%
Data quality:6
Medium (1.9)
CD8+ CD4- (cytotoxic T-cell) cell fraction in spleen (%)
Male, Fl, PNW 3 (n = 10)
Mean (SD) 6.86 (0.95)
8.12(2.16)
6.99(1.42)
6.43 (1.44)
% changeb -
28%
10%
1%
Male, Fl, PNW 11 (n= 10)
Mean (SD) 14.42 (2.23)
18.54* (4.34)
16.85 (4.31)
18.87* (4.82)
% changeb -
29%
17%
31%
N NKRP1A+CD4- (NK) cell fraction in spleen (%)
Male, Fl, PNW 3 (n = 10)
Mean (SD) 5.75 (0.35)
6.06 (1.09)
5.65 (0.87)
5.09* (0.76)
% changeb -
5%
-2%
-11%
Male, Fl, PNW 11 (n= 10)
Mean (SD) 10.63 (1.63)
9.97 (3.44)
11.38 (2.47)
9.44 (2.39)
% changeb -
-6%
7%
-11%
Activated T-cell fraction in thymus (%)
Male, Fl, PNW 3 (n = 10)
Mean (SD) 2.67 (0.87)
2.46 (0.80)
1.82* (0.55)
1.87(1.15)
% changeb -
-4%
-29%
-27%
Male, Fl, PNW 11 (n= 10)
Mean (SD) 0.92(0.97)
0.74 (0.51)
1.02 (0.84)
1.04 (0.70)
% changeb -
-20%
11%
13%
Increased starry sky appearance in thymus
Male, Fl, PNW 3 (n = 10)
Incidence 0/10
0/10
4/10*
1/10
Male, Fl, PNW 11 (n= 10)
Incidence 0/10
0/10
0/10
0/10
Female, Fl, PNW 3 (n = 10)
Incidence 0/10
0/10
0/10
0/10
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Reference and
study design
Results
Female, Fl, PNW 11 (n = 10)
Incidence 0/10
0/10
3/10
0/10
NK cell fraction in thymus (%)
Male, Fl, PNW 3 (n = 10)
Mean (SD) 0.07 (0.03)
0.07 (0.03)
0.06 (0.02)
0.07 (0.05)
% changeb -
0%
-43%
0%
Male, Fl, PNW 11 (n= 10)
Mean (SD) 0.2 (0.04)
0.2 (0.05)
0.25 (0.09)
0.27* (0.08)
% changeb -
0%
25%
35%
Treg cell fraction in thymus (%)
Male, Fl, PNW 3 (n = 10)
Mean (SD) 7.7(2.57)
5.15* (0.94)
7.69 (1.27)
7.85 (2.85)
% changeb -
-33%
0%
-5%
Male, Fl, PNW 11 (n= 10)
Mean (SD) 4.16(1.09)
3.98 (0.87)
4.41 (0.76)
4.32 (1.22)
% changeb -
-1%
6%
4%
* Statistically significantly different from the control at p< 0.05 as reported by study authors.
**Significant dose response trend as reported by study authors.
aPercent change compared to control calculated as: (treated value - control value)/control value x 100.
bFl and F2 offspring doses presented as mean maternal gestational F0 and F1 doses, respectively.
°TWAs for each exposure group were calculated by: (1) multiplying the measured HBCD intake (mg/kg-day)
reported by the study authors for GDs 10-20, PNDs 1-9, and PNDs 9-20 by the number of inclusive days of
exposure for each time period; (2) adding the resulting products together; and (3) dividing the sum by the total
number of inclusive days (33) of HBCD exposure. Example: 100 ppm = (8.1 mg/kg-day x 11 days) +
(14.3 mg/kg-day x 10 days) + (21.3 mg/kg-day x 12 days)/33 days = 14.8 mg/kg-day.
dNot measured; only control and high-dose values reported.
"Based on OPPT data evaluation criteria.
Table 1-12. Evidence pertaining to observational immune system effects in animals
following exposure to HBCD as adults
Reference and
study design
Results
Organ weight
Ema et al. (2008)
Rats, CRL:CD(SD)
Diet
Two generation
F0: exposure started
10 wks prior to
mating
Fl: dietary exposure
post weaning until
necropsy
Doses (mg/kg-d)
Male, F0 0 10 101 1,008
Female, F0 0 14 141 1,363
Absolute spleen weight (mg)
Male, F0 (n = 22-24)
Mean (SD) 848 (136) 828 (109) 855 (160) 843 (248)
% change3 - -2% 1% -1%
Female, F0 (n = 17-24)
Mean (SD) 588 (75) 577 (83) 570 (89) 584 (72)
% change3 - -2% -3% -1%
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Reference and
study design
Results
F1/F2 offspring:
Absolute thymus weight (mg)
continuous maternal
exposure throughout
gestation/lactation
Male, F0 (n = 22-24)
Mean (SD) 323 (88)
% change3 -
305 (82)
-6%
299 (64)
-7%
315 (71)
-2%
Data quality:b
Female, F0 (n = 17-24)
High (1.0)
Mean (SD) 232 (38)
238 (63)
252 (73)
200 (64)
% change3 -
3%
9%
-14%
van cler Yen et al.
Doses (mg/kg-d)
(2006)
Rats, Wistar
Gavage
28-d exposure
starting on PNW 11
O
©
1 3
10 30
100 200
Absolute spleen weight (g)
Male (n = 4-5)
Mean (SD) 0.51 0.59
0.78 0.52
0.58 0.47
0.49 0.50
(0.09) (0.13)
(0.55) (0.05)
(0.08) (0.03)
(0.05) (0.10)
% change3 - 16%
53% 2%
14% -8%
-4% -2%
Data quality:b
High (1.3)
Female (n= 4-5)**
Mean (SD) 0.41 0.37
(0.04) (0.04)
0.38 0.44
(0.06) (0.01)
0.40 0.49
(0.04) (0.08)
0.53 0.37
(0.04) (0.05)
% change3 - -10%
-7% 7%
-2% 20%
29% -10%
Absolute thymus weight (g)
Male (n=4-5)**
Mean (SD) 0.47 0.45
0.52 0.47
0.50 0.37
0.42 0.38
(0.08) (0.08)
(0.17) (0.07)
(0.09) (0.06)
(0.09) (0.13)
% change3 - -4%
11% 0%
6% -21%
-11% -19%
Female (n = 4-5)
Mean (SD) 0.42 0.28
0.36 0.35
0.44 0.43
0.42 0.37
(0.06) (0.10)
(0.09) (0.07)
(0.07) (0.08)
(0.08) (0.10)
% change3 - -33%
-14% -17%
5% 2%
0% —12%
I lenialo/ogv
Etna et al. (2008)
Doses (mg/kg-d)
Rats, CRL:CD(SD)
Diet
Two generation
Male, F0 0
Female, F0 0
10
14
101
141
1,008
1,363
Lymphocyte fraction (%)
F0: exposure started
Male, F0 (n = 10)
10 wks prior to
mating
Fl: maternal
exposure throughout
Response 88.5 (6.5)
88.8 (2.4)
88.8 (3.9)
87.5 (4.6)
% change3 -
Female, F0 (n = 10)
0%
0%
-1%
gestation/lactation;
Mean (SD) 72.5(8.7)
85* (5)
78.4 (9.5)
70.8 (9)
dietary exposure
post weaning until
necropsy
% change3 -
17%
8%
-2%
Segmented neutrophil fraction (%)
Male, F0 (n = 10)
Data quality:b
High (1.0)
Mean (SD) 8.00(5.24)
% change3 -
Female, F0 (n = 10)
8.24 (1.98)
3%
7.68 (3.26)
-4%
8.68 (4.61)
8%
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Reference and
study design
Results
Mean (SD) 21.68(8.08)
10.56* (4.19)
16.84 (9.19)
23.28 (8.13)
% change3
-51%
-22%
7%
Stab form neutrophil fraction (%)
Male, F0 (n = 10)
Mean (SD) 0.48 (0.73)
0.36(0.3)
0.64 (0.28)
0.56 (0.51)
% change3 -
-25%
33%
17%
Female, F0 (n = 10)
Mean (SD) 1.32 (0.57)
0.60* (0.39)
0.84 (0.55)
1.12(0.7)
% change3 -
-55%
-36%
-15%
van der Yen et al.
Doses (mg/kg-d)
(2006)
Rats, Wistar
Gavage
28-d exposure
Male 0
0.3
1
3
10
30
100
200
Lymphocyte cell fraction in blood (%)
Male (n = 3-5)
starting on PNW 11
Mean (SD) 89.1
89.0
85.4
85.3
86.7
88.9
84.2
88.1
Data quality:b
High (1.3)
(2.5)
% change3 -
(3.7)
0%
(5.9)
-4%
(2.0)
-4%
(3.7)
-3%
(3.8)
0%
(8.1)
-5%
(3.1)
-1%
Ilistopathology
van der Yen et al.
Doses (mg/kg-d)
(2006)
Rats, Wistar
Gavage
28-d exposure
0
0.3
1
3
10
30
100
200
CD4 (Th) cells per spleen (cells
xlO7)
Male (n=l-5)**
starting on PNW 11
Mean (SD) 14.0
15.2
13.3
11.4
10.5
9.0
11.2
10.0
(4.7)
(n/a)
(4.8)
(n/a)
(0.9)
(n/a)
(n/a)
(2.0)
Data quality:b
High (1.3)
% change3 -
9%
-5%
-19%
-25%
-36%
-20%
-29%
Total immune cells per spleen (cells /10 )
Male (n=l-5)**
Mean (SD) 48.7
49.6
47.1
44.4
39.4
29.7
37.0
35.8
(10.5)
(n/a)
(15.4)
(n/a)
(3.8)
(n/a)
(n/a)
(1.1)
% change3 -
2%
-3%
-9%
-19%
-39%
-24%
-26%
* Statistically significantly different from the control at p< 0.05 as reported by study authors.
**Significant dose response trend as reported by study authors.
aPercent change compared to control calculated as: (treated value - control value)/control value x 100
bBased on OPPT data evaluation criteria.
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Treg ceil fraction in thymus Hachisuka et al., 2010 (rats, F1 M PNW 11)
Treg cell fraction in thymus Hachisuka et a!., 2010 (rats, F1 M PNW 3)
NK cell fraction in thymus Hachisuka et al., 2010 (rats, F1 M PNW 11)
NK cell fraction in thymus Hachisuka et al., 2010 (rats, F1 M PNW 3)
Increased starry sky appearance in thymus Hachisuka et a I., 2010 (rats, Fl F)
Increased starry sky appearance in thymus Hachisuka et al., 2010 (rats, F1 M)
Activated T cell fraction in blood Hachisuka et al., 2010 (rats, F1 M)
NK cell fraction in spleen Hachisuka et al,, 2010 ( F1 M PNW 11)
NK cell fraction in spleen Hachisuka et ai., 2010 ( F1 M PNW 3)
CD8+ cell fraction in spleen Hachisuka et al., 2010 ( F1 M PNW 11)
CD8+ cell fraction in spleen Hachisuka et al., 2010 ( F1M PNW 3)
CD4NKTcell fraction in spleen Hachisuka et al., 2010 ( F1 M)
Splenic marginal van der Ven et al., 200 (rats, F1 M)
White blood cell count van der Ven et al., 2009 (rats, IV!)
CD161a (NK) fraction in spleen van der van et al., 2009 (rats, M)
WBC in blood Hachisuka et a I., 2010 ( F1 M PNW 11)
WBC in blood Hachisuka et a!., 2010 ( F1 M PNW 3)
NK cell fraction in blood Hachisuka et al., 2010 ( F1 (VI PNW 11)
NK cell fraction in blood Hachisuka et al., 2010 ( F1IV! PNW 3)
Lymphocyte fraction in blood Hachisuka et a!., 2010 (rats, F1 M )
Activated T cell fraction in blood Hachisuka et al., 2010 (rats, F1 M)
WBC count in blood van der Ven et al., 2009 (rats, M)
Lymphocyte cell fraction in blood van der Ven et al., 2009 (rats, M)
Basophil cell count in blood van der Ven et al., 2009 (rats, M)
Lymphocyte fraction (%) Ema et al., 2008 (rats, Fl)
Absolute thymus weight Hachisuka et al., 2010 (rats, Fl M)
Absolute spleen weight Hachisuka et al., 2010 (rats, Fl M)
Absolute thymus weight van der Ven et al., 2009 ( rats, Fl)
Absolute spleen weight van der Ven et al., 2009 (rats, Fl)
Absolute popliteal lymph node weight van der Ven et al., 2008 (rats)
Absolute thymus weight
Ema et al., 2008 ( rats, F2 F)
Absolute thymus weight
Ema et al., 2008 ( rats, F2 M)
Absolute thymus weight Ema et al., 2008 (rats, Fl)
Absolute spleen weight
Ema et al., 2008 (rats, F2)
Absolute spleen weight Ema et al., 2008 (rats, Fl)
Antibody IgG responses to KLH Hachisuka et al., 2010 (rats, F)
SRBC antibody titers van der Ven et al., 2009 (rats, M)
• significantly changed
O not significantly changed
Doses (mg/kg-day)
Figure 1-9. Exposure response array of immune system following oral exposure. Most data was from Hachisuka et ai.
which scored a Medium in data quality evaluation (indicated with™). AH other studies scored a High.
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1.6.3 Mechanistic Evidence
Mechanistic information to support HBCD-mediated effects on the immune system is limited.
Several recent in vitro studies in human immune cells suggest that HBCD may alter immune
function through activation of MAPK signaling pathways (ERK1/2 and p38) resulting in
increased secretion of IFN y and IL-ip, pro-inflammatory cytokines that regulate immune
function (Almughamsi and Whalen. 2016; Anisuzzaman and Whalen. 2016; Canbaz et at..
2016a). Similarly, pro-inflammatory effects driven by were observed in human brochial
epithelial cells (BEAS-2B); HBCD exposure increased expression of proinflammatory cytokines
(IL-6 and IL-8) and ICAM-1, a cell surface marker often expressed by immune cells, which were
mediated by activation of MAPK signaling pathways (Koike et at.. 2016)). One study using
human monocyte-derived dendritic cells found that co-exposure with HBCD enhanced IL-6 and
IL-8 secretion elicited by environmental allergens (Canbaz et at.. 2016a).
Koike et at. (2012) used bone marrow-derived dendritic cells prepared from atopic-prone
NC/Nga mice to investigate HBCD effects on the immune response in vitro. HBCD (10 (j,g/mL)
increased cell proliferation and expression of a dendritic activation marker, DEC205. Bone
marrow-derived dentritic cells differentiated in the presence of HBCD also showed enhanced
MHC class II, CD80, CD86, and CD1 lc expression. These in vitro data are supported by two
studies using the guinea pig maximization test method that indicated that HBCD may act as a
mild skin allergen (Nakamura et a I; Momma et at.. 1993). Taken together, these studies
suggest that HBCD may stimulate an immune response by increasing the activity of antigen-
presenting cells. In vitro, HBCD altered several aspects of human NK cell function, including
decreased target cell binding, expression of surface binding proteins, lytic function, and ATP
levels (Hinkson and Whalen. 2010. 2009); however, in vivo NK cell activity was unaffected in
rats (van der Yen et at.. 2009; van der Yen et al.. 2006).
1.7 Genotoxicity
A limited number of studies have investigated the genotoxicity of HBCD; these are summarized
in Table 1-13. The majority of these studies were standard Ames tests for detecting mutagenic
potential in Salmonella typhimurium. These tests, which employ different strains of bacteria that
have been developed with pre-existing mutations, including S. typhimurium TA98, TA100,
TA1535, TA1537, and TA1538, are referred to as reversion assays (Maron and Ames. 1983).
Most of these assays conducted with HBCD yielded negative results (Huntingdon Research.
1990; International. 1990; Labs. 1990; Litton. 1990; Pharmakotoeisches. 1990; '^i\er et al..
1987). Among the few assays performed to determine the genotoxicity of HBCD in prokaryotic
systems, one in yeast (Litton, 1990), one detecting chromosomal aberrations in human peripheral
lymphocytes in vitro (Microbiological. 1996). and one in vivo mouse micronucleus test
following intraperitoneal (i.p.) injections of HBCD (Basf. 2000) were negative, even when tested
at cytotoxic concentrations.
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Table 1-13. Summary of genotoxicity studies of HBCD
Test/species/ strain/
route
Test doses
(per plate)3
Resultsb
Notes
Reference
-S9
+S9
Eukaryotic systems, in vitro
S. typhimurium
TA98, TA100,
TA1535, TA1537
50-5,000 jig
(HBCD
bottoms)
in acetone
+
(TA1535
and 100
only)
+
(TA100
only)
No cytotoxicity
observed. Dose-
response observed in
TA1535 (-S9)
>100 jig/plate. TA100
positive at highest dose
only (5,000 jig/plate).
All doses had a black
precipitate thought to be
carbon.
Ettwl (1990b)
S. typhimurium
TA98, TA100,
TA1535, TA1537,
TA1538
50 jig
(421-32B)
(solvent not
reported)
Litton (1990)
S. typhimurium
TA98, TA100,
TA1535, TA1537
2-1,000 jig
(GLS-S6-41A)
in DMSO
Gsri (1978)
S. typhimurium
TA98, TA100,
TA1535, TA1537,
TA1538
100-10,000 Jig
in DMSO
Doses >1,000 jig were
insoluble.
Zeiger et al. (1987)
S. typhimurium
TA98, TA100,
TA1535, TA1537,
TA1538
250 jig
(Firemaster,
FM-100, Lot
53, white
powder)
in DMSO
Doses >250 jig were
insoluble.
Labs (1990)
1,000 Jig
(FM-100, Lot
3322, liquid
residue)
in DMSO
+
(TA1535
only)
Significant in TA1535
at highest dose only.
S. typhimurium
TA98, TA100,
TA1537
3,000 jig
in DMSO
Doses >1,000 jig were
partially insoluble.
Pharmakoloeisches
(1990)
S. typhimurium
TA98, TA100,
TA1535, TA1537,
TA1538
5,000 jig
in DMSO
No cytotoxicity
observed.
International (1990)
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Test/species/strain/
route
Test doses
(per plate)3
Resultsb
Notes
Reference
-S9
+S9
S. typhimurium
TA92, TA94, TA98,
TA100, TA1535,
TA1537
10,000 jig
(Pyroguard
SR-103)
in DMSO
Haiiaftisa (1993)
S. typhimurium
TA98, TA100,
TA1535
10,000 Jig
in DMSO
Insoluble at 10,000 jig.
Huntingdon
Research (1990)
Prokaryotic non-mammalian systems, in vitro
Saccharomyces
cerevisiae D4
50 jig (solvent
not reported)
—
—
Litton (1990)
Mammalian systems, in vivo
Micronucleus test
mouse/NMRI/i .p.
injection
2,000 mg/kg
in DMSO
-(T)
NA
Toxicity evident as a
slight inhibition of
erythropoiesis at
2,000 mg/kg.
Number of
polychromatic
erythrocytes with
micronuclei from
femoral bones evaluated
24 hrs after 2nd
injection.
Basf (2000)
Mammalian systems, in vitro
Chromosomal
aberration test
Human peripheral
blood lymphocytes
750 jig/mL
(-S9)
250 jig/mL
(+S9) in
DMSO
-(T)
-(T)
Doses 750-2,500
jig/mL were partially
insoluble, and fully
insoluble
>2,500 jig/mL.
Repeated test for two
harvest time points: 20-
hr (-S9) or 4-hr (+S9)
incubations, and 20- or
44-hr incubations (-S9
and +S9).
Microbiological
(1996)
Reversion assay
CHO/V79/Sp5 and
SPD8
Intragenic
recombination at
hprt locus in Sp5
(non-HR) and SPD8
(HR) duplication cell
lines
3-20 jig/mL
in DMSO
+
NA
A statistically
significant, dose-
dependent increase in
reversion frequency was
observed in both assays
as determined by linear
regression analysis.
Significant inhibition of
cloning efficiency
Helledav et al.
(1999)
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Test/species/strain/
route
Test doses
(per plate)3
Resultsb
Notes
Reference
-S9
+S9
occurred at doses
>15 (ig/mL in the SPD8
assay and >20 (ig/mL in
the Sp5 assay.
Cytotoxicity (IC50)
measured at
0.02-0.03 mM.
Unscheduled DNA
synthesis
rat/F344
male/primary
hepatocytes
10 (ig/well
in acetone
(HBCD
bottoms)
+
NA
Five highest doses
(from 5 (ig/well)
showed an increased
response with dose over
solvent control, but only
four highest were
statistically significant
(x2). Highest dose
(1,000 (ig/well) was
cytotoxic.
Ettwl (1990a)
aLowest effective dose for positive results; highest dose tested for negative results.
b+ = positive; ± = equivocal or weakly positive; - = negative; T = cytotoxicity; NA = not
applicable.
DMSO = dimethyl sulfoxide
Some positive results have been reported. S. typhimurium strain TA1535 was positive for
reverse mutations at the highest dose only using a liquid residue of HBCD in DMSO (Labs.
1990), and strain TA100 was positive also at the highest dose using an unidentified mixture
characterized only as HBCD bottoms in acetone (Ethyl. 1990b). In this same study, TA1535 was
positive at > 100 |ig/plate without addition of an S9 microsomal fraction (Ettv 3b). The
number of revertants increased with dose. This was the only Ames study to report dissolving the
test article in a solvent other than DMSO (in this case, acetone). DMSO is a free-radical
scavenger and can potentially obscure genetic damage due to oxidative radicals. Both strains
TA1535 and TA100 were designed to be sensitive to detecting reversions by base substitution, a
type of genetic lesion that can result from oxidative DNA damage due to reactive oxygen species
(ROS). However, there is only limited evidence in the literature indicating that HBCD exposure
may induce oxidative stress (An et at.. 2013; H.u et at.. 2009b).
In mammalian systems, a reverse mutation assay with Chinese hamster ovary (CHO) Sp5 and
SPD8 cell lines exposed to HBCD (Hetteday et at., 1999) yielded positive results. These two
clones exhibit a partial duplication of the hprt gene, causing lethality unless a reversion occurs,
either via homologous recombination (SPD8) or non-homologous recombination (Sp5). A
statistically significant, dose-dependent increase in reversion frequency was observed in both
clones, although at higher doses, there was a significant inhibition of cloning efficiency. In
addition, a test of unscheduled DNA synthesis with rat hepatocytes exposed to HBCD bottoms
was positive (Ethyl, 1990a), and also showed an increase in response with dose.
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It is noteworthy that in these three studies (Helleday et al., 1999), the positive results were dose-
dependent, observed at nontoxic doses, and in two assays, specific for detecting mutations.
However, the Ames tests in the same strains that showed positive results (TA1535 and TA100)
were negative in seven other studies, and the results in the reverse mutation assay in CHO cells
(Helleday et al.. 1999) have not been confirmed by another group.
2 DOSE-RESPONSE ANALYSIS
2.1 Supplemental Information on Non-Cancer Dose Response
Analysis
2.1.1 Additional Considerations for Selection of Studies for Dose-Response Analysis
As discussed in Section 1.3.2, studies in humans were not adequate to support conclusions
regarding the relationship between HBCD exposure and effects on the thyroid, male
reproduction, or nervous system, and accordingly do not support dose-response analysis. In the
absence of adequate human data, animal toxicity studies were used for dose-response analysis.
Studies in animals provided evidence of thyroid toxicity, liver toxicity, female reproductive, and
developmental toxicity following oral exposure to hexabromocyclododecane (HBCD). These
hazards have been carried forward for dose-response analysis. While there is also evidence to
support nervous system toxicity following exposure to HBCD during development in animal
studies, these data sets were not carried forward for dose-response analysis. Likewise, data sets
for male reproductive effects, adult neurological effects, immune system effects, genotoxicity,
and cancer were not carried forward for dose-response analysis. For a complete discussion, see
Section 1.3.2.
The effects determined to best represent each of the hazards were identified in Section 1.3.2, and
studies that evaluated these effects are considered in this section for dose-response analysis. In
order to identify the stronger studies for dose-response analysis, several attributes of the studies
were reviewed. Preference was given to studies using designs reasonably expected to detect a
dose-related response. Chronic or subchronic toxicity studies are necessary for estimating risks
related to chronic or subchronic exposures under the conditions of use within the scope of the
TSCA risk evaluation. Studies with a broad exposure range and multiple exposure levels are
preferred to the extent that they can provide information about the shape of the exposure-
response relationship. Additionally, with respect to measurement of the endpoint, studies that can
reliably measure the magnitude and/or degree of severity of the effect are preferred.
Experimental animal studies considered for each hazard and effect were evaluated using general
study quality considerations discussed above and in the Systematic Review Methods section. The
rationales for selecting the strongest studies to represent these hazards are summarized below.
2.1.1.1 Thyroid Effects
Regulation of thyroid hormones is complex and homeostasis is largely maintained via HPT axis
feedback mechanisms. Reductions in serum T3 or T4 triggers release of TSH from the pituitary,
which stimulates the thyroid gland to increase secretion of T3 and T4 stores from the colloid
(Fisher and Nelson. 2012). Decreased T4 is expected to be the primary driver of HBCD-
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mediated thyroid effects that triggers release of TSH. Indeed, this is supported by mechanistic
studies that indicate that that observed decreases in T4 may be largely driven by hepatic
induction of enzymes that metabolize this hormone (See Section 1.1.6, Mechanistic Evidence).
Despite demonstrating a sensitive response to HBCD exposure, follicle size was not selected for
modeling because: (1) quantitative data for follicle size changes were provided only in one study
(Ema, 2008); (2) although this is generally a well conducted study, details of the methods of
analysis (e.g., the criteria used to determine whether an animal showed decreased follicle size)
were not provided; and (3) although changes in thyroid histopathology (e.g., follicle size,
epithelial cell hypertrophy) can be useful indicators of changes in thyroid function/homeostasis,
they are less direct measures of thyroid toxicity and it would be difficult to determine an
appropriate benchmark response (BMR).
Serum thyroxine (T4) was selected for dose-response analysis of thyroid effects (see Section
1.3.2). Three studies in rats reported treatment-related decreases in serum T4 following oral
exposure (Ema et at.. 2008; van der Yen et at.. 2006; WIL Research. 2001). Table 2-1 provides
an overview of the study designs for those studies reporting T4 levels that were evaluated for
dose-response analysis.
Ema et al. (2008) reported a decrease in serum T4 levels in both male and female rats from the
F0 (30 and 31% at the high dose, respectively) and F1 (10 and 28% at the high dose,
respectively) generations, van der Yen et al. (2006) reported similar effects on serum T4 (26%
reduction at the high dose) in adult female rats exposed for 28 days. V search (2001)
reported changes in T4 levels in rats exposed to HBCD for 90 days, but inadequate reporting of
thyroid hormone measurement methods, high proportion (50%) of samples below the limit of
detection, and unusually low control thyroid-stimulating hormone (TSH) levels reduced the
confidence in these results, bringing into question the conduct of the assays.
2.1.1.2 Liver Effects
The most consistently observed liver outcome was liver weight changes. Dose-related increases
were consistently observed across species, sexes, and age from multiple studies of various
designs and exposure durations (Yanagisawa et al.. 2014; Maranghi et al.. 2013; Saeeusa et al..
2009; Ema et al.. 2008; WIL Researcl , ) Limited support for HBCD effects on the
liver are provided by histopathological examination. A subset of the rat studies (Saeeusa et al..
2009; WIL Research, 2001, 1997) and one mouse study (Maranghi et al., ) reported
increased vacuolation (generally of minimal to mild severity) in HBCD-exposed animals, but
these responses were not dose-related. The content of the vacuoles was investigated only by WIL
Research (2001) and characterized as lipid. Other histological findings were less frequently
observed and included some additional evidence of fatty change (steatosis) (Yanagisawa et al..
2014). hypertrophy (Yanagisawa et al.. 2014; WIL Research. 1997). and inflammation
(Maranghi et al., 2013). Statistically or biologically significant elevations in serum liver enzymes
were not associated with HBCD exposure in rats or mice in multiple studies (Yanagisawa et al..
2014; WIL Research. 2001. 1997). however in contrast mechanistic evidence in vitro suggests
that HBCD may in fact induce hepatic microsomal enzymes (Crump et al.. 2010; Crump et al..
2008; Germer et al.. 2006). Microsomal enzyme induction is a proposed key event in initiating
the perturbation of the HPT axis that leads to reduced T4 levels. Given limited evidence of
HBCD-related histopathological changes and no clear evidence of clinical chemistry changes,
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the biological significance of liver weight changes is unclear. While increased liver weight was
not consistently associated with other toxicological evidence of liver toxicity in rodents given a
standard diet, biochemical and histopathological effects indicative of steatosis were observed in
mice fed a high-fat diet (Yanagisawa et at.. 2014). A high-fat diet may therefore represent a
susceptibility factor for TCE toxicity (Bernhard et at.. 2016).
Increased liver weight was selected for dose-response analysis of liver effects (see Section 1.3.2).
This endpoint was reported in six studies in rats (Saegusa et at.. 2009; Ema et at.. 2008; van der
Yen et at.. 2006; WIL Research. 2001. 1997) and mice (Maranghi et a ). The
developmental study by Saegusa et at. (2009) and the 28-day study by v\ ^ 1 mrch (1997)
used similar dose ranges as the longer-duration studies (Ema et at.. 2008; esearch. 2001)
and observed similar findings in pup or adult liver weights. A significant trend in increased liver
weight was reported by van der Yen et at. (2006) following a 28-day adult exposure at lower
doses, but in female rats only. Data from these shorter exposure duration studies were not used
for dose-response analysis because similar effects were observed in the studies with longer
exposure durations (Ema et at.. 2008; WIL Research. 2001) that better reflect effects expected
following subchronic or chronic exposure. Similarly, Maranghi et al. ( was not used for
dose-response analysis because it used a relatively short (28-day) exposure and a single dose
group that is less informative for evaluating a dose-response relationship.
2.1.1.3 Female Reproductive Effects
See the Risk Evaluation document ( ) for details on this endpoint.
2.1.1.4 Developmental Effects
Several studies in animals exposed during gestation and lactation provide some evidence of
developmental effects associated with HBCD, including reduced offspring viability (Ema et at.,
2008). decreased pup body weight (Maranghi et al.. JO I Saegusa et al.. 2009; van der Yen et
al.. 2009; Ema et al.. 2008). altered development of the skeletal system, and delayed eye opening
(Ema et al.. 2008). The strongest evidence of developmental effects is based on findings of
reduced offspring viability and decreased pup body weight. Reduced viability was observed in
the two-generation study by Ema et al. (2008); the decreases in viability were dose-related and
observed on both PND 4 and 21. Effects were seen only in F2 offspring. This is consistent with
decreased viability manifesting after multigenerational exposure, although that hypothesis cannot
be established based on the current developmental literature for HBCD (i.e., a single two-
generation study). Effects on pup body weight were demonstrated in several studies in rats using
different strains and exposure durations (Saegusa et al.. 2009; van der Yen et al.. 2009; Ema et
al.. 2008). Other developmental effects, including changes in bone development and delayed eye
opening, were only reported in a single study and with a less clear dose-response relationship
(van der Yen et al.. 2009; Ema et al.. 2008). Therefore, pup body weight and viability were
selected for dose-response analysis of developmental effects.
Ema et al. (2008) evaluated changes in pup body weight in rats that were continuously exposed
to HBCD across two generations. Treatment-related effects on pup body weight were measured
throughout early postnatal development (PNDs 0, 4, 7, 14, and 21) in three dose groups, covering
a dose range of approximately 2.5 orders of magnitude. This study used an adequate sample size
(n = 13—24) and litter as the statistical unit. Maranghi et al. (2013) was considered less
appropriate to support derivation of an RfD because the study used only one dose group, which
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is less informative for evaluating dose-response relationships, and a relatively short exposure
duration (28 days), van der Yen et al. (2009) used a dose range that was >10-fold lower than
those used in the Ema et al. (2008) and Saegusa et al. (2009) studies and, in general, did not
show a clear pattern of dose-related changes in pup body weight on different days of lactation.
2.1.2 BMR Selection
A set of dose-response models that are consistent with a variety of potentially underlying
biological processes were applied to empirically model the dose-response relationship in the
range of the observed data. The models in EPA's Benchmark Dose Software (BMDS, version
2.6) were applied. Consistent with EPA's Benchmark Dose Technical Guidance Document (U.S.
2012). the benchmark dose (BMD) and 95% lower confidence limit on the BMD (BMDL) were
estimated using a benchmark response (BMR) to represent a minimal, biologically significant
level of change, described here as relative deviation (RD). In the absence of information
regarding the level of change that is considered biologically significant, a BMR of 1 standard
deviation (SD) from the control mean for continuous data or a BMR of 10% extra risk (ER) for
dichotomous data is used to estimate the BMD and BMDL, and to facilitate a consistent basis of
comparison across endpoints, studies, and assessments. Endpoint-specific BMRs are described
further below. Where modeling was feasible, the estimated BMDLs were used as points of
departure (PODs). Further details, including the modeling output and graphical results for the
model selected for each endpoint, can be found in Section 3.2. Where dose-response modeling
was not feasible, NOAELs or LOAELs were identified and are summarized in Table 2-4.
2.1.2.1 Thyroid Effects
Changes in T4 levels described by Ema et al. (2008) were amenable to BMD modeling. In
selecting a BMR (i.e., a change in T4 levels considered biologically significant), pregnant
females and their offspring were addressed separately from adult males. Early life development
is generally recognized as being particularly sensitive to thyroid perturbation. Thyroid hormones
play a critical role in coordinating complex developmental processes, and perturbations of
thyroid hormone levels in a pregnant woman or neonate can have persistent adverse health
effects for the child. During early gestation, the developing fetus relies solely on thyroid
hormones of maternal origin. As the fetus begins to produce thyroid hormones, there is less
reliance on maternal thyroid hormones; however, early development remains a sensitive life
stage for hormone deficits, largely due to minimal reserve capacity when compared to adults
(Gilbert and Zoeller. 2010).
Reductions in maternal T4 during pregnancy or the early postnatal period are strongly associated
with adverse neurological outcomes in offspring. In humans, mild to moderate maternal thyroid
insufficiency is associated with higher risk for persistent cognitive and behavioral deficits in
children. In general, mild to moderate thyroid insufficiency in pregnant women was defined as
serum T4 levels below the 10th percentile for the study population, which is associated with a
15-30%) decrease relative to the corresponding median (Finken et al.. JO IJulvez et al. 2013;
Roman et al.. 2013; Henrichs et al.. 2010; Haddow et al.. 1999). Similar effects have been
described in animal studies, with modest reductions in maternal T4 during gestation resulting in
behavioral alterations, learning deficits, and neuroanatomical changes in offspring (Gilbert et al..
2014; Gilbert et al.. 2013; Gilbert. 2011; Liu et al.. 2010; Auso et al.. 2004). Thyroid inhibition
during gestation and lactation that resulted in drops in mean maternal T4 levels of-10-17%
have been found to elicit neurodevelopmental toxicity in offspring (Gilbert et al.. 2016; Gilbert,
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2011). Although there are some differences in HPT regulation (e.g., serum hormone binding
proteins, hormone turnover rates, and timing of in utero thyroid development), rodents are
generally considered to be a good model for evaluating the potential for thyroid effects of
chemicals in humans (Zoeller et at.. 2007). although a National Academies of Sciences review of
the iodide uptake inhibitor perchlorate (NRC. 2005) concluded that there may be quantitative
differences. Based on the overall data observed in both humans and animals, a BMR of 10% RD
from control mean was determined to be a minimally biologically significant degree of change
when performing BMD modeling using female rat data.
The available thyroid literature does not support identification of a biologically significant
change in T4 levels in adult males as decreases in T4, and more generally thyroid function, have
not been conclusively linked to similarly severe outcomes as in females. Nevertheless, males
with depressed T4 values are part of the subpopulation that experiences thyroid dysfunction.
Selecting a biologically-based BMR is also complicated by the inherent variability of thyroid
hormones. Individuals show relatively narrow variability around a set point; however, set points
can vary considerably between individuals, resulting in a broad population range that is
considered normal (Andersen et at.. 2002). Thus, it is possible for an individual to have thyroid
levels that fall within the normal population range, but are abnormal relative to their homeostatic
set point. Consistent with EPA's Benchmark Dose Technical Guidance Document (U.S. 2012), a
BMR of one control SD change from the control mean was applied in modeling T4 data from
male rats in the absence of a biological basis for selecting a BMR.
Additionally, a BMR of 10% RD from control means, supported by the literature on the effects
of thyroid insufficiency in pregnant females and their offspring, was applied in modeling the
male T4 data. In looking across the available HBCD studies, there does not appear to be a strong
sex-specific effect on T4 responses (see Table 1-3). Differences in dose-response (i.e., similar
responses at the high dose but divergent responses at the lower doses) was observed in the F0
male and female data sets that were modeled (Ema et at.. 2008). These differences likely reflect
the inherent variability of thyroid hormones within a population, especially for a relatively small
sample size as used in Etna et at. (2008). and not a sex-specific difference in response. Under the
assumption that differences in thyroid hormone response in male and female rats exposed to
HBCD are not sex-specific but rather a reflection of hormone variability, using a BMR of 10%
RD was considered reasonable.
2.1.2.2 Liver Effects
See the Risk Evaluation document ( ) for details on this endpoint.
2.1.2.3 Female Reproductive Effects
2.1.2.3.1 Primordial Follicle Count
Decreased primordial follicle count as reported in the two-generation reproductive toxicity study
by Etna et at. (2008) was amenable to BMD modeling. Because primordial follicles are formed
during gestation, the average dose during this critical window was used for BMD modeling. A
BMR of 10%) RD from control levels was applied in modeling this endpoint under the
assumption that it represents a minimal biologically significant effect. There is no consensus in
the scientific community regarding the degree of change that is considered to be adverse. In this
situation, it has been suggested that a detectable decrease in follicle number should be considered
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adverse (Heindel, 1998). Power analyses by Heindel (1998) focused on identifying follicle
counts reduced by >20%, suggesting that a reduction of this magnitude is considered a critical
effect level. Thus, a 10% reduction was selected to represent a minimally important degree of
change.
2.1.2.3.2 Pregnancy Incidence
In the study by Ema et al. (2008). the increased incidence of non-pregnancy in HBCD-exposed
F0 or F1 rats alone was not statistically significant with either pairwise test (as reported by
authors) or Cochran-Armitage trend test (conducted by EPA). Dose-response curves were
shallow and never reached a high response percentage. To increase statistical power and obtain a
more precise estimate of the BMD and BMDL, consideration was given to combining F0 and F1
datasets. Cochran-Mantel-Haenszel statistics on F0 and F1 data stratified by dose groups were
not significant (p = 0.59, a = 0.05), indicating no statistical association between generation and
response after adjusting for dose. Equality of responses in F0 and F1 rats was also not rejected (p
> 0.2, a = 0.05) by the Breslow-Day test for homogeneity of the odds ratios, and their
background response percentages were not detectably different (Fisher's exact, p = 1.00). The
results of these statistical tests suggested that F0 and F1 datasets were compatible for
combining. A statistically significant trend (p = 0.02) was found using the Cochran-Armitage test
applied to the combined data. The Log-logistic model was selected after dropping the highest
dose (see Supplemental Information, Appendix D, Section D.2). F0 and F1 data were also
modeled separately after dropping the highest dose. A Likelihood ratio test (a = 0.05, d.f. = 3)
could not reject equality of the three Log-logistic models from combined dataset and F0, F1
alone. Therefore, the Log-logistic model from the combined dataset was used to derive the BMD
and BMDL for increased incidence of non-pregnancy with increasing dose.
A BMR of 5% ER was applied in modeling this endpoint under the assumption that it represents
a minimal biologically significant degree of change. Selection of a BMR took into consideration
the limited sensitivity of rodent species to effects on fertility and pregnancy outcomes (U.S.
1996). As noted in >96), the limited sensitivity of fertility measures in rodents suggests
that a POD (i.e., NOAEL, LOAEL, or BMD) based on fertility may not reflect completely the
extent of effects on reproduction, such that the BMD may need to be adjusted to reflect that
additional uncertainty. Rather than applying an additional uncertainty factor to the POD based on
reduced fertility in rats, a BMR of 5%, rather than 10%, was selected. A BMR of 5% ER was
also consistent with the functional severity of the endpoint (i.e., reduced fertility).
2.1.2.4 Developmental Effects
2.1.2.4.1 Offspring Loss
Increased offspring loss in the F2 generation from the study was amenable to
BMD nested modeling, using individual animal data obtained from the study authors (personal
communication) (Makris et al.. 2016). Two datasets were modeled: offspring loss from
implantation through PND 4 and offspring loss from PND 4 (post-culling) through PND 21.
Maternal gestational doses (10, 100, and 995 mg/kg-day) were used to model the offspring loss
from the implantation through PND 4 dataset because they are reflective of the majority of the
exposure window being modeled (i.e., 3 weeks of gestation compared to 4 days of lactation) and
early lactational doses are closer to the gestational doses than the average dose during the entire
lactational period. For similar reasons, modeling for the PND 4 post-culling through PND 21
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dataset was performed using the maternal lactational doses (20, 179, and 1,724 mg/kg-day). Use
of maternal lactational doses for modeling the PND 4 to 21 dataset was also consistent with total
litter loss in eight high-dose dams that occurred at time points across the lactational period
(specifically, PNDs 4, 5, 7, 9, 11, 13, and 18).
The use of a 1% ER BMR for offspring loss as reported in Ema et al. (2008) resulted in BMDLoi
values for loss from implantation through PND 4 and for offspring loss from PND 4 post-culling
through PND 21 in F2 rats that fell in the region of the dose-response curve where the response
in dosed animals was similar to the response in the controls (see Figure 2-1).
RaiVR Model, with BMR of 1 % Extra Risk for the BMD and 0.95 Lower Confidence Limit for the BMDL
Nested Logistic Model, with BMR of 1% Extra Risk for the BMD and 0.95 Lower Confidence Limit for the BMDL
200 400 600 800 1000 1200 1400 1600 1800
Figure 2-1. BMD modeling plots of incidence of offspring loss from implantation through
PND 4 in F2 offspring rats (A) and incidence of offspring loss from PND 4 post-culling
through PND 21 in F2 offspring rats (B) from Ema et al. (2008): BMR = 1% ER (see
Appendix D, Figures D-31 and D-33).
A NOAEL was also considered as the POD in addition to the POD derived using a BMD
modeling approach. As shown in Figure 2-1, there is variation around the response at each dose.
Although the responses at the BMDLoi for each data set modeled appear not to be elevated over
the control, the possibility of a small increase in response at these dose levels cannot be
eliminated. Because the BMD approach is generally preferred to the NOAEL/LOAEL approach,
and because the BMDLoi values are similar to the NOAELs (difference of approximately 2-fold),
the BMDLoi values were used to estimate the PODs for offspring loss.
2.1.2.4.2 Pup Body Weight
See the Risk Evaluation document (EPA-HQ-QPPT-2016-0735) for details on this endpoint.
3 DOSE-RESPONSE MODELING FOR THE DERIVATION OF
POINTS OF DEPARTURE
This appendix provides technical detail on dose-response evaluation and determination of points
of departure (PODs) for relevant toxicological endpoints. The endpoints were modeled using the
U.S. Environmental Protection Agency (EPA) Benchmark Dose Software (BMDS, version 2.6).
This appendix describes the common practices used in evaluating the model fit and selecting the
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appropriate model for determining the POD, as outlined in the Benchmark Dose Technical
Guidance Document (U.S. 2012). In some cases, it may be appropriate to use alternative
methods, based on statistical judgment; exceptions are noted as necessary in the summary of the
modeling results.
3.1 Noncancer Knil points for BMD Modeling
The noncancer endpoints that were selected for dose-response modeling are presented in Table
3-1. Noncancer endpoints selected for dose-response modeling for HBCD. For each endpoint,
the doses and response data used for the modeling are presented.
Table 3-1. Noncancer endpoints selected for dose-response modeling for HBCD
Endpoint
Species
(strain)/sex
Dose
(mg/kg-d)a
Incidence [%] or mean ± SD
(number of animals or litters)
BMR(s)
Th> mid
|T4
Etna et al.
(2008)
F0 rats (CRL
Sprague-
Dawley)/male
0
10
101
1,008
TWA of lifetime exposure,
F0
4.04 ± 1.42 (8)
3.98 ±0.89 (8)
2.97 ± 0.76 (8)
2.49 ±0.55 (8)
10% RD, 15%
RD, 20% RD, 1
SD
|T4
Etna et al.
(2008)
F0 rats (CRL
Sprague-
Dawley)/female
0
14
141
1,363
TWA of lifetime exposure,
F0
2.84 ±0.61 (8)
3.14 ±0.48 (8)
3.00 ±0.77 (8)
1.96 ±0.55 (8)
10% RD, 15%
RD,
20% RD, 1 SD
|T4
Etna et al.
(2008)
F1 rats (CRL
Sprague-
Dawley)/female
0
14.3
138
1,363
TWA of lifetime exposure,
F1
3.59 ± 1.08 (8)
3.56 ±0.53 (8)
3.39 ± 1.21 (8)
2.58 ±0.37 (8)
10% RD, 15%
RD,
20% RD, 1 SD
1 .in or
Relative liver
weight
Etna et al.
(2008)
F1 rats (CRL
Sprague-
Dawley)/male
weanlings,
PND 26
0
16.5
168
1,570
TWA of F0 gestational and
lactational doses
4.6 ±0.37 (23)
4.6 ±0.32 (21)
5.05 ± 0.32 (20)
6 ±0.44 (17)
10% RD, 1 SD
Relative liver
weight
Etna et al.
(2008)
F1 rats (CRL
Sprague-
Dawley)/female
weanlings,
PND 26
0
16.5
168
1,570
4.57 ± 0.35 (23)
4.59 ±0.28 (21)
5.02 ± 0.32 (20)
6.07 ±0.36 (14)
10% RD, 1 SD
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Endpoint
Species
(strain)/sex
Dose
(mg/kg-d)a
Incidence [%] or mean ± SD
(number of animals or litters)
BMR(s)
TWA of F0 gestational and
lactational doses
Relative liver
weight
Em a et al.
(2008)
F1 rats (CRL
Sprague-
Dawley)/male
adults
0
11.4
115
1,142
TWA of lifetime exposure,
F1
3.27 ±0.18 (24)
3.34 ±0.26 (24)
3.37 ±0.25 (22)
3.86 ±0.28 (24)
10% RD, 1 SD
Relative liver
weight
Em a et al.
(2008)
F1 rats (CRL
Sprague-
Dawley)/female
adults
0
14.3
138
1,363
TWA of lifetime exposure,
F1
4.18 ±0.42 (22)
4.39 ± 0.44 (22)
4.38 ± 0.47 (20)
5.05 ±0.50 (13)
10% RD, 1 SD
Relative liver
weight
Em a et al.
(2008)
F2 rats (CRL
Sprague-
Dawley)/male
weanlings,
PND 26
0
14.7
139
1,360
TWA of F1 gestational and
lactational doses
4.72 ± 0.59 (22)
4.74 ± 0.35 (22)
5.04 ±0.4 (18)
6.0 ±0.25 (13)
10% RD, 1 SD
Relative liver
weight
Em a et al.
(2008)
F2 rats (CRL
Sprague-
Dawley)/female
weanlings, PND
26
0
14.7
139
1,360
4.70 ±0.27 (21)
4.70 ± 0.28 (22)
4.94 ± 0.32 (20)
5.89 ±0.44 (13)
10% RD, 1 SD
TWA of F1 gestational and
lactational doses
Relative liver
weight and
hepatocellular
vacuolization
\ search
£
Rats (Sprague-
Dawley)/male
0
100
300
1,000
2.709 ±0.1193 (10)
3.175 ± 0.2293 (10)
3.183 ±0.2653 (10)
3.855 ±0.1557 (9)
10% RD, 1 SD
Relative liver
weight and
hepatocellular
vacuolization
\ search
Rats (Sprague-
Dawley)/female
0
100
300
1,000
2.887 ± 0.2062 (10)
3.583 ±0.2734 (10)
3.578 ±0.3454 (10)
4.314 ±0.2869 (10)
10% RD, 1 SD
kcpmducliN e
Primordial follicles
Em a et al.
(2008)
(supplemental)
F1 parental rat
(CRL Sprague-
Dawley)/female
0
9.6
96
941
316.3 ±119.5 (10)
294.2 ±66.3 (10)
197.9 ±76.9 (10)
203.4 ±79.5 (10)
1% ER, 5% ER,
10% ER
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Endpoint
Species
(strain)/sex
Dose
(mg/kg-d)a
Incidence [%] or mean ± SD
(number of animals or litters)
BMR(s)
The F0 adult female
gestational doses
Incidence of non-
pregnancy
Ema et al.
(2008)
F0 and F1
parental rats
combined (CRL
Sprague-
Dawley)/female
0
13.3
132
1,302
TWA F0, F1 female pre-
mating doses
1/48 [2%]
3/48 [6.2%]
7/48 [14.5%]
7/47 [14.9%]
5% ER, 10% ER
1 )e\ elopinenliil
Offspring loss at
PND 4
Ema et al.
(2008)
F2 offspring rats
(CRL Sprague-
Dawley)
0
9.7
100
995
The F1 adult female
gestational doses
28/132 [21%]
26/135 [19.3%]
23/118 [19.5%]
47/120 [39.2%]
1% ER, 5% ER
Offspring loss at
PND 21
Ema et al.
(2008)
F2 offspring rats
(CRL Sprague-
Dawley)
0
19.6
179
1,724
The F1 adult female
lactational doses
11/70 [15.7%]
7/70 [10.0%]
18/64 [28.1%]
32/64 [50.0%]
1% ER, 5% ER
Pup weight during
lactation at PND
21
Ema et al.
(2008)
F2 offspring rats
(CRL Sprague-
Dawley)/male
0
19.6
179
1,724
The F1 adult female
lactational doses
53 ± 12.6 (22)
56.2 ± 6.7 (22)
54.1 ± 10.1 (18)
42.6 ±8.3 (13)
5% RD, 10%
RD,
0.5 SD, 1 SD
Pup weight during
lactation at PND
21
Ema et al.
(2008)
F2 offspring rats
(CRL Sprague-
Dawley)/female
0
19.6
179
1,724
The F1 adult female
lactational doses
52 ± 10(21)
52.8 ± 6.6 (22)
51.2 ± 10.8 (20)
41.6 ±8.4 (13)
5% RD, 10%
RD,
0.5 SD, 1 SD
aDoses were calculated as TWA doses using weekly average doses (in mg/kg-day) as reported in Table 10 of the
Supplemental Materials to Ema et al. (2008).
BMR = benchmark response; ER = extra risk; PND = postnatal day; RD = relative deviation; SD = standard deviation; T4 =
thyroxine; TWA = time-weighted average
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3.2 Dose-Response Modeling of Non-Cancer Endpoints
3.2.1 Evaluation of Model Fit
For each dichotomous endpoint where only summary data (i.e., number affected and total
number exposed per group) were available, BMDS dichotomous models1 were fitted to the data
using the maximum likelihood method. Each model was tested for goodness-of-fit using a
chi-square goodness-of-fit test (%2 p-value < 0.10 indicates lack of fit). Other factors were also
used to assess model fit, such as scaled residuals, visual fit, and adequacy of fit in the low-dose
region and in the vicinity of the benchmark response (BMR).
For each dichotomous endpoint for which incidence data were available for individual animals,
BMDS nested dichotomous models2 were fitted to the data using the maximum likelihood
method. Each nested model was tested for goodness-of-fit using a bootstrap approach. Chi-
square statistics were computed with both bootstrap iterations and original data. The p-value was
the proportion of chi-square values from the iterations that were greater than the original chi-
square value (%2 p-value <0.10 indicates lack of fit). Other factors were also used to assess
model fit, such as scaled residuals, visual fit, and adequacy of fit in the low-dose region and in
the vicinity of the BMR.
For each continuous endpoint, BMDS continuous models3 were fitted to the data using the
maximum likelihood method. Model fit was assessed by a series of tests as follows. For each
model, first the homogeneity of the variances was tested using a likelihood ratio test (BMDS
Test 2). If Test 2 was not rejected (%2 p-value > 0.10), the model was fitted to the data assuming
constant variance. If Test 2 was rejected (%2 p-value < 0.10), the variance was modeled as a
power function of the mean, and the variance model was tested for adequacy of fit using a
likelihood ratio test (BMDS Test 3). For fitting models using either constant variance or modeled
variance, models for the mean response were tested for adequacy of fit using a likelihood ratio
test (BMDS Test 4, with yl p-value <0.10 indicating inadequate fit). Other factors were also
used to assess the model fit, such as scaled residuals, visual fit, and adequacy of fit in the low-
dose region and in the vicinity of the BMR.
3.2.2 Model Selection
To select the appropriate model from which to derive the POD for each endpoint, the BMDL
estimate (95% lower confidence limit on the benchmark dose [BMD], as estimated by the profile
likelihood method) and Akaike's information criterion (AIC) value were used to select the model
from among the models exhibiting adequate fit. If the BMDL estimates were "sufficiently close,"
that is, differed by at most 3-fold, the model selected was the one that yielded the lowest AIC
Unless otherwise specified, all available BMDS dichotomous models besides the alternative and nested
dichotomous models were fitted. The following parameter restrictions were applied: for the LogLogistic model,
restrict slope >1; for the Gamma and Weibull models, restrict power >1.
2Unless otherwise specified, all available BMDS nested dichotomous models were fitted. For the nested Logistic,
NCTR, and Rai and van Ryzin models, power >1 was applied.
3Unless otherwise specified, all available BMDS continuous models were fitted. The following parameter
restrictions were applied: for the polynomial models, restrict the coefficients bl and higher to be nonnegative or
nonpositive if the direction of the adverse effect is upward or downward, respectively; for the Hill, Power, and
Exponential models, restrict power >1.
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value. If the BMDL estimates were not sufficiently close, the lowest BMDL was selected as the
POD.
For nested dichotomous models, there are the options of including a litter-specific covariate and
estimating intralitter correlations, yielding four combinations of option selections, as displayed in
Table 3-2. All the three nested dichotomous models were fitted for every combination in the
table, yielding four sets of models (12 model runs in total).
Table 3-2. The combinations of option selections for the nested dichotomous models
Litter-specific covariates used
Intralitter correlations estimated
Litter-specific covariates used
Intralitter correlations assumed zero
Litter-specific covariates not used
Intralitter correlations estimated
Litter-specific covariates not used
Intralitter correlations assumed zero
The appropriate model was selected from this set of 12 models using the same procedure as for
the non-nested models as described in Section 2.3.9 (page 39) of the Benchmark Dose Technical
Guidance Document ( ). If multiple litter specific covariates were tested, this same set
of 12 modeling options was evaluated for each litter-specific covariate (e.g., litter size,
implantation site, dam body weight) and the appropriate model was selected from the expanded
set of modeling options (12 x number of litter-specific covariates considered) using the same
procedure as for the non-nested models.
3.2.3 Modeling Results
Below are tables summarizing the modeling results for the noncancer endpoints modeled.
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3.2.3.1 Thyroid
Table 3-3. Summary of BMD modeling results for T4 in FO parental male CRL Sprague-
Dawley rats exposed to HBCD by diet for 18 weeks (Etna et ai, 2008); BMR = 15% RD
Goodness of fit
BMDiord
(mg/kg-d)
BMDLiord
(mg/kg-d)
BMD15RD
(mg/kg-d)
BMDL15RD
(mg/kg-d)
Basis for model
Model3
p-value
AIC
selection
Exponential (M2)
Exponential (M3)b
0.0473
33.926
259
177
399
274
Of the models
without saturation
Exponential (M4)
Exponential (M5)c
0.742
29.933
23.9
6.99
39.1
11.5
that provided an
adequate fit and a
valid BMDL
estimate, the
Hill
0.949
29.829
14.4
3.21
25.6
5.66
Power"1
Polynomial 3°e
Polynomial 2of
Linear
0.0418
34.174
303
227
455
341
Exponential 4
model with
modeled variance
was selected
based on lowest
AIC
Goodness of fit
BMD20RD
(mg/kg-d)
BMDL20RD
(mg/kg-d)
BMDisd
(mg/kg-d)
BMDLi sd
(mg/kg-d)
Model3
p-value
AIC
(BMDLs differed
Exponential (M2)
Exponential (M3)b
0.0473
33.926
548
376
866
511
by <3).
Exponential (M4)
Exponential (M5)°
0.742
29.933
57.9
17.2
101
29.5
Hill
0.949
29.829
42.0
9.11
94.9
Errorg
Power1
Polynomial 3°e
Polynomial 2of
Linear
0.0418
34.174
607
454
906
595
aModeled variance case presented (BMDS Test 2 p-value = 0.0756, BMDS Test 3 p-value = 0.553), selected model
in bold; scaled residuals for selected model for doses 0, 10.2, 101, and 1,008 mg/kg-day were -0.1665, 0.166,
0.03642, and -0.03619, respectively.
bFor the Exponential (M3) model, the estimate of d was 1 (boundary). The models in this row reduced to the
Exponential (M2) model.
Tor the Exponential (M5) model, the estimate of d was 1 (boundary). The models in this row reduced to the
Exponential (M4) model.
dFor the Power model, the power parameter estimate was 1. The models in this row reduced to the Linear model.
Tor the Polynomial 3° model, the b3 coefficient estimate was 0 (boundary of parameters space). The models in this
row reduced to the Polynomial 2° model. For the Polynomial 3° model, the b3 and b2 coefficient estimates were 0
(boundary of parameters space). The models in this row reduced to the Linear model.
fFor the Polynomial 2° model, the b2 coefficient estimate was 0 (boundary of parameters space). The models in this
row reduced to the Linear model.
gBMD or BMDL computation failed for this model.
Data from Etna et ai. (2008)
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Exponential 4 Model, with BMR of 0.1 Rel. Dev. for the BMD and 0.95 Lower Confidence Limit for the BMDL
10:52 08/18 2017
BMR = 10% RD from control mean; dose shown in mg/kg-day.
Figure 3-1. Plot of mean response by dose, with fitted curve for Exponential 4 Model, for
T4 in F0 parental CRL Sprague-Dawley male rats exposed to HBCD by diet for 18 weeks
(Ema et al., 2008).
Exponential 4 Model (Version: 1.10; Date: 01/12/2015)
The form of the response function is:
Model 4: Y[dose] = a * [c-(c-l) * exp{-b * dose}]
A modeled variance is fit
Benchmark Dose Computation
BMR = 10% RD
BMD = 23.8946
BMDL at the 95% confidence level = 6.99406
Parameter Estimates
Variable
Estimate
Default initial parameter values
lalpha
-3.94284
-3.54227
rho
2.98463
2.72754
a
4.1075
4.242
b
0.0123219
0.00282274
d
1 (specified)
1 (specified)
Table of Data and Estimated Values of Interest
Dose N Observed mean Estimated mean Observed SD Estimated SD Scaled residuals
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0
8
4.04
4.11
1.42
1.15
-0.167
10.2
8
3.98
3.92
0.89
1.07
0.166
101
8
2.97
2.961
0.76
0.71
0.036
1,008
8
2.49
2.50
0.59
0.56
-0.036
Likelihoods of Interest
Model
Log (likelihood)
Number of parameters
AIC
A1
-12.76333
5
35.52665
A2
-9.319925
8
34.63985
A3
-9.91228
6
31.82456
fitted
-9.966286
5
29.93257
R
-19.64317
2
43.28634
Tests of Interest
Test
-2*log (likelihood ratio)
Test df
p-value
Test 1
20.65
6
0.002123
Test 2
6.887
3
0.07559
Test 3
1.185
2
0.553
Test 6a
0.108
1
0.7424
df = degree(s) of freedom
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Exponential 4 Model, with BMR of 0.15 Rel. Dev. for the BMD and 0.95 Lower Confidence Limit for the BMDL
dose
11:24 08/18 2017
BMR = 15% RD from control mean; dose shown in mg/kg-day.
Figure 3-2. Plot of mean response by dose, with fitted curve for Exponential 4 Model, for
T4 in FO parental CRL Sprague-Dawley male rats exposed to HBCD by diet for 18 weeks
(Ema et al., 2008).
Exponential 4 Model (Version: 1.10; Date: 01/12/2015)
The form of the response function is:
Model 4: Y[dose] = a * [c-(c-l) * exp{-b * dose}]
A modeled variance is fit
Benchmark Dose Computation
BMR = 15% RD
BMD = 39.1317
BMDL at the 95% confidence level = 11.5235
Parameter Estimates
Variable
Estimate
Default initial parameter values
lalpha
-3.94284
-3.54227
rho
2.98463
2.72754
a
4.1075
4.242
b
0.0123219
0.00282274
c
0.607906
0.55903
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1 (specified)
1 (specified)
Table of Data anc
Estimated Values of Interest
Dose
N
Observed mean
Estimated mean
Observed SD
Estimated SD
Scaled residuals
0
8
4.04
4.11
1.42
1.15
-0.167
10.2
8
3.98
3.92
0.89
1.07
0.166
101
8
2.97
2.961
0.76
0.71
0.036
1,008
8
2.49
2.50
0.59
0.55
-0.036
Likelihoods of Interest
Model
Log (likelihood)
Number of parameters
AIC
A1
-12.76333
5
35.52665
A2
-9.319925
8
34.63985
A3
-9.91228
6
31.82456
fitted
-9.966286
5
29.93257
R
-19.64317
2
43.28634
Tests of Interest
Test
-2*log (likelihood ratio)
Test df
p-value
Test 1
20.65
6
0.002123
Test 2
6.887
3
0.07559
Test 3
1.185
2
0.553
Test 6a
0.108
1
0.7424
df = degree(s) of freedom
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Exponential 4 Model, with BMR of 0.2 Rel. Dev. for the BMD and 0.95 Lower Confidence Limit for the BMDL
11:50 08/18 2017
BMR = 20% RD from control mean; dose shown in mg/kg-day.
Figure 3-3. Plot of mean response by dose, with fitted curve for Exponential 4 Model, for
T4 in FO parental CRL Sprague-Dawley male rats exposed to HBCD by diet for 18 weeks
(Ema et al.. 2008).
Exponential 4 Model (Version: 1.10; Date: 01/12/2015)
The form of the response function is:
Model 4: Y[dose] = a * [c-(c-l) * exp{-b * dose}]
A modeled variance is fit
Benchmark Dose Computation
BMR = 20% RD
BMD = 57.9065
BMDL at the 95% confidence level = 17.1892
Parameter Estimates
Variable
Estimate
Default initial parameter values
lalpha
-3.94284
-3.54227
rho
2.98463
2.72754
a
4.1075
4.242
b
0.0123219
0.00282274
c
0.607906
0.55903
d
1 (specified)
1 (specified)
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Table of Data anc
Estimated Values of Interest
Dose
N
Observed mean
Estimated mean
Observed SD
Estimated SD
Scaled residuals
0
8
4.04
4.11
1.42
1.15
-0.167
10.2
8
3.98
3.92
0.89
1.07
0.166
101
8
2.97
2.961
0.76
0.71
0.036
1,008
8
2.49
2.50
0.59
0.55
-0.036
Likelihoods of Interest
Model
Log (likelihood)
Number of parameters
AIC
A1
-12.76333
5
35.52665
A2
-9.319925
8
34.63985
A3
-9.91228
6
31.82456
fitted
-9.966286
5
29.93257
R
-19.64317
2
43.28634
Tests of Interest
Test
-2*log (likelihood ratio)
Test df
p-value
Test 1
20.65
6
0.002123
Test 2
6.887
3
0.07559
Test 3
1.185
2
0.553
Test 6a
0.108
1
0.7424
df = degree(s) of freedom
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Exponential 4 Model, with BMR of 1 Std. Dev. for the BMD and 0.95 Lower Confidence Limit for the BMDL
dose
11:24 08/18 2017
BMR = 1 SD from control mean; dose shown in mg/kg-day.
Figure 3-4. Plot of mean response by dose, with fitted curve for Exponential 4 Model, for
T4 in FO parental CRL Sprague-Dawley male rats exposed to HBCD by diet for 18 weeks
(Ema et al., 2008).
Exponential 4 Model (Version: 1.10; Date: 01/12/2015)
The form of the response function is:
Model 4: Y[dose] = a * [c-(c-l) * exp{-b * dose}]
A modeled variance is fit
Benchmark Dose Computation
BMR = 1 SD
BMD = 101.035
BMDL at the 95% confidence level = 29.4693
Parameter Estimates
Variable
Estimate
Default initial parameter values
lalpha
-3.94284
-3.54227
rho
2.98463
2.72754
a
4.1075
4.242
b
0.0123219
0.00282274
c
0.607906
0.55903
d
1 (specified)
1 (specified)
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Table of Data anc
Estimated Values of Interest
Dose
N
Observed mean
Estimated mean
Observed SD
Estimated SD
Scaled residuals
0
8
4.04
4.11
1.42
1.15
-0.167
10.2
8
3.98
3.92
0.89
1.07
0.166
101
8
2.97
2.961
0.76
0.71
0.036
1,008
8
2.49
2.50
0.59
0.55
-0.036
Likelihoods of Interest
Model
Log (likelihood)
Number of parameters
AIC
A1
-12.76333
5
35.52665
A2
-9.319925
8
34.63985
A3
-9.91228
6
31.82456
fitted
-9.966286
5
29.93257
R
-19.64317
2
43.28634
Tests of Interest
Test
-2*log (likelihood ratio)
Test df
p-value
Test 1
20.65
6
0.002123
Test 2
6.887
3
0.07559
Test 3
1.185
2
0.553
Test 6a
0.108
1
0.7424
df = degree(s) of freedom
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Table 3-4. Summary of BMD modeling results for T4 in FO parental female CRL Sprague-
Dawley rats exposed to HBCD by diet for 18 weeks (Etna et ai. 2008); BMR = 10% RD
from control mean, 15% RD from control mean, 20% RD from control mean, and 1 SD
Goodness of fit
BMDiord
(mg/kg-d)
BMDLiord
(mg/kg-d)
BMD15RD
(mg/kg-d)
BMDL15RD
(mg/kg-d)
Basis for model
selection
Model3
p-value
AIC
Exponential (M2)
0.479
3.7677
334
225
516
348
Of the models
Exponential (M3)
0.298
5.3774
1,065
232
1,150
357
that provided an
adequate fit and a
valid BMDL
estimate, the
Exponential M4
Exponential (M4)
0.479
3.7677
334
93.8
516
154
Exponential (M5)
N/Ab
7.3774
1,086
103
1,158
143
Hill
N/Ab
7.3774
1,067
100
1,138
error0
constant variance
model was
selected based on
Power
0.298
5.3774
1,171
293
1,230
439
Polynomial 3°
0.582
3.3778
902
816
1,032
934
lowest BMDL
(BMDLs differed
by >3).
Polynomial 2°
0.580
3.3836
733
293
897
439
Linear
0.505
3.6625
389
289
584
433
Goodness of fit
BMD20RD
(mg/kg-d)
BMDL20RD
(mg/kg-d)
BMDisd
(mg/kg-d)
BMDLi sd
(mg/kg-d)
Model3
p-value
AIC
Exponential (M2)
0.479
3.7677
708
477
680
433
Exponential (M3)
0.298
5.3774
1,240
491
1,234
446
Exponential (M4)
0.479
3.7677
708
229
680
211
Exponential (M5)
N/Ab
7.3774
1,217
146
1,211
145
Hill
N/Ab
7.3774
1,185
error0
1,178
error0
Power
0.298
5.3774
1,275
586
1,270
532
Polynomial 3°
0.582
3.3778
1,136
1,028
1,126
999
Polynomial 2°
0.580
3.3836
1,036
586
1,021
532
Linear
0.505
3.6625
779
577
751
523
aConstant variance case presented (BMDS Test 2 p-value = 0.579), selected model
selected model for doses 0, 14, 141.3, and 1,363 mg/kg-day were -0.9501, 0.5631,
respectively.
bNo available degrees of freedom to calculate a goodness-of-fit value.
°BMD or BMDL computation failed for this model.
in bold; scaled residuals for
0.4611, and-0.07911,
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Exponential Model 4. with BMR of 0.1 Rel. Dev. for the BMD and 0.95 Lower Confidence Level for BMDL
3.5
1.5
11:19 02/11 2015
BMR = 10% RD from control mean; dose shown in mg/kg-day.
Figure 3-5. Plot of mean response by dose, with fitted curve for Exponential Model 4, for
T4 in FO parental CRL Sprague-Dawley female rats exposed to HBCD by diet for 18 weeks
(Ema et al.. 2008).
Exponential Model (Version: 1.9; Date: 01/29/2013)
The form of the response function is: Y[dose] = a * [c-(c-l) * exp(-b * dose)]
A constant variance model is fit
Benchmark Dose Computation
BMR = 10% RD
BMD = 334.313
BMDL at the 95% confidence level = 93.781
Parameter Estimates
Variable
Estimate
Default initial parameter values
lnalpha
-1.06976
-1.11576
rho(S)
N/A
0
a
3.03677
3.297
b
0.000315155
0.00199958
c
0
0.566171
d
1
1
Table of Data and Estimated Va
ues of Interest
Dose
N
Observed mean
Estimated mean
Observed SD
Estimated SD
Scaled residuals
0
8
2.84
3.037
0.61
0.5857
-0.9501
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14
8
3.14
3.023
0.48
0.5857
0.5631
141.3
8
3
2.905
0.77
0.5857
0.4611
1,363
8
1.96
1.976
0.55
0.5857
-0.07911
Likelihoods of Interest
Model
Log (likelihood)
Number of parameters
AIC
Al
1.852186
5
6.295628
A2
2.83624
8
10.32752
A3
1.852186
5
6.295628
R
-6.115539
2
16.23108
4
1.116152
3
3.767695
Tests of Interest
Test
-2*log (likelihood ratio)
Test df
p-value
Test 1
17.9
6
0.006478
Test 2
1.968
3
0.5791
Test 3
1.968
3
0.5791
Test 6a
1.472
2
0.479
1 1 :21 02/1 1 2015
BMR = 15% RD from control mean; dose shown in mg/kg-day.
Figure 3-6. Plot of mean response by dose, with fitted curve for Exponential Model 4, for
T4 in FO parental female CRL Sprague-Dawley rats exposed to HBCD by diet for 18 weeks
(Ema et al., 2008).
Exponential Model (Version: 1.9; Date: 01/29/2013)
The form of the response function is: Y[dose] = a * [c-(c-l) * exp(-b * dose)]
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A constant variance model is fit
Benchmark Dose Computation
BMR = 15% RD
BMD = 515.679
BMDL at the 95% confidence level = 154.19
Parameter Estimates
Variable
Estimate
Default initial parameter values
lnalpha
-1.06976
-1.11576
rho(S)
N/A
0
a
3.03677
3.297
b
0.000315155
0.00199958
c
0
0.566171
d
1
1
Table of Dal
ta and Estimated Values of Interesi
Dose
N
Observed mean
Estimated mean
Observed SD
Estimated SD
Scaled residuals
0
8
2.84
3.037
0.61
0.5857
-0.9501
14
8
3.14
3.023
0.48
0.5857
0.5631
141.3
8
3
2.905
0.77
0.5857
0.4611
1,363
8
1.96
1.976
0.55
0.5857
-0.07911
Likelihoods of Interest
Model
Log (likelihood)
Number of parameters
AIC
A1
1.852186
5
6.295628
A2
2.83624
8
10.32752
A3
1.852186
5
6.295628
R
-6.115539
2
16.23108
4
1.116152
3
3.767695
Tests of Interest
Test
-2*log (likelihood ratio)
Test df
p-value
Test 1
17.9
6
0.006478
Test 2
1.968
3
0.5791
Test 3
1.968
3
0.5791
Test 6a
1.472
2
0.479
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3.5
3
Cd
= 2.5
2
1.5
O 200 400 600 800 1000 1200 1400
dose
10:06 05/20 2016
BMR = 20% RD from control mean; dose shown in mg/kg-day.
Figure 3-7. Plot of mean response by dose with fitted curve for Exponential (M4) model
with constant variance for T4 in FO parental female CRL Sprague-Dawley rats exposed to
HBCD by diet for 18 weeks (Ema et al.. 2008).
Exponential Model (Version: 1.10; Date: 01/12/2015)
The form of the response function is: Y[dose] = a * [c-(c-l) * exp(-b * dose)]
A constant variance model is fit
Benchmark Dose Computation
BMR = 20% RD
BMD = 708.043
BMDL at the 95% confidence level = 228.829
Parameter Estimates
Variable
Estimate
Default initial parameter values
Lnalpha
-1.06976
-1.11576
Rlio
N/A
0
A
3.03677
3.297
B
0.000315155
0.00199958
C
0
0.566171
D
N/A
1
Table of
Data and Estimated Values of Interest
Dose
N
Observed mean
Estimated mean
Observed SD
Estimated SD
Scaled residuals
0
8
2.84
3.04
0.61
0.59
-0.9501
14
8
3.14
3.02
0.48
0.59
0.5631
Exponential 4 Model, with BMR of 0.2 Rel. Dev. for the BMD and 0.95 Lower Confidence Limit for the BMDL
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141.3
8
3
2.9
0.77
0.59
0.4611
1,363
8
1.96
1.98
0.55
0.59
-0.07911
Likelihoods of Interest
Model
Log (likelihood)
Number of parameters
AIC
Al
1.852186
5
6.295628
A2
2.83624
8
10.32752
A3
1.852186
5
6.295628
R
-6.115539
2
16.23108
4
1.116152
3
3.767695
Tests of Interest
Test
-2*log (likelihood ratio)
Test df
p-value
Test 1
17.9
6
0.006478
Test 2
1.968
3
0.5791
Test 3
1.968
3
0.5791
Test 6a
1.472
2
0.479
Exponential 4 Model, with BMR of 1 Std. Dev. for the BMD and 0.95 Lower Confidence Limit for the BMDL
3.5
3
2
1.5
600
dose
10:13 05/20 2016
BMR = 1 SD change from control mean; dose shown in mg/kg-day.
Figure 3-8. Plot of mean response by dose with fitted curve for Exponential (M4) model
with constant variance for T4 in FO parental female CRL Sprague-Dawley rats exposed to
HBCD by diet for 18 weeks (Ema et al., 2008).
Exponential Model (Version: 1.10; Date: 01/12/2015)
The form of the response function is: Y[dose] = a * [c-(c-l) * exp(-b * dose)]
A constant variance model is fit
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Benchmark Dose Computation
BMR = 1.0000 Estimated SDs from control
BMD = 679.939
BMDL at the 95% confidence level = 210.769
Parameter Estimates
Variable
Estimate
Default initial parameter values
Lnalpha
-1.06976
-1.11576
Rho
N/A
0
A
3.03677
3.297
B
0.000315155
0.00199958
C
0
0.566171
D
N/A
1
Table of Data and Estimated Values of Interest
Dose
N
Observed mean
Estimated mean
Observed SD
Estimated SD
Scaled residuals
0
8
2.84
3.04
0.61
0.59
-0.9501
14
8
3.14
3.02
0.48
0.59
0.5631
141.3
8
3
2.9
0.77
0.59
0.4611
1,363
8
1.96
1.98
0.55
0.59
-0.07911
Likelihoods of Interest
Model
Log (likelihood)
Number of parameters
AIC
A1
1.852186
5
6.295628
A2
2.83624
8
10.32752
A3
1.852186
5
6.295628
R
-6.115539
2
16.23108
4
1.116152
3
3.767695
Tests of Interest
Test
-2*log (likelihood ratio)
Test df
p-value
Test 1
17.9
6
0.006478
Test 2
1.968
3
0.5791
Test 3
1.968
3
0.5791
Test 6a
1.472
2
0.479
Table 3-5. Summary of BMD modeling results for T4 in F1 parental female CRL Sprague-
Dawley rats exposed to HBCD by diet for 18 weeks (Ema et at.. 2008); BMR = 10% RD
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from control mean, 15% RD from control mean, 20% RD from control mean, and 1 SD
change from control mean
Goodness of fit
Model3
p-value
AIC
BMDiord
(mg/kg-d)
BMDLiord
(mg/kg-d)
BMD15RD
(mg/kg-d)
BMDL15RD
(mg/kg-d)
Basis for model
selection
Exponential (M2)
0.305
19.978
448
320
691
493
Of the models that
Exponential (M3)
0.191
21.318
1,184
333
1,254
514
provided an
adequate fit and a
valid BMDL
estimate, the
Exponential M4
Exponential (M4)
0.305
19.978
448
127
691
214
Exponential (M5)
N/Ab
23.318
1,193
153
1,259
144
Hill
N/Ab
23.318
1,131
153
1,204
error0
(modeled variance)
model was selected
based on lowest
Power
0.191
21.318
1,287
389
1,318
583
Polynomial 3°
0.424
19.323
984
898
1,127
1,028
BMDL (BMDLs
differed by >3).
Polynomial 2°
0.414
19.368
835
728
1,023
892
Linear
0.323
19.868
498
379
747
568
Goodness of fit
BMD20RD
(mg/kg-d)
BMDL20RD
(mg/kg-d)
BMDisd
(mg/kg-d)
BMDLi sd
(mg/kg-d)
Model3
p-value
AIC
Exponential (M2)
0.305
19.978
948
677
1,344
828
Exponential (M3)
0.191
21.318
1,305
705
1,362
876
Exponential (M4)
0.305
19.978
948
328
1,344
536
Exponential (M5)
N/Ab
23.318
1,309
148
1,362
152
Hill
N/Ab
23.318
1,269
error0
1,360
error0
Power
0.191
21.318
1,341
777
1,363
932
Polynomial 3°
0.424
19.323
1,240
1,132
1,360
1,193
Polynomial 2°
0.414
19.368
1,181
1,030
1,357
1,115
Linear
0.323
19.868
996
757
1,344
896
aModeled variance case presented (BMDS Test 2 p-value = 0.00445), selected model in bold; scaled residuals for
selected model for doses 0, 14.3, 138.3, and 1,363 mg/kg-day were 0.105, 0.05257, -0.1637, and 0.008804,
respectively.
bNo available degrees of freedom to calculate a goodness-of-fit value.
°BMD or BMDL computation failed for this model.
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Exponential Model 4, with BMR of 0.1 Rel. Dev. for the BMD and 0.95 Lower Confidence Level for BM
1 1 :30 02/11 2015
BMR = 10% RD from control mean; dose shown in mg/kg-day.
Figure 3-9. Plot of mean response by dose, with fitted curve for Exponential Model 4
(modeled variance) for T4 in F1 parental female CRL Sprague-Dawley rats exposed to
HBCD by diet for 18 weeks (Ema et al., 2008).
Exponential Model (Version: 1.9; Date: 01/29/2013)
The form of the response function is: Y[dose] = a * [c-(c-l) * exp(-b * dose)]
A modeled variance is fit
Benchmark Dose Computation
BMR = 10% RD
BMD = 447.782
BMDL at the 95% confidence level = 127.272
Parameter Estimates
Variable
Estimate
Default initial parameter values
lnalpha
-7.9144
-6.73265
rho
6.1823
5.13248
a
3.55422
3.7695
b
0.000235294
0.000283737
c
0
0.000684441
d
1
1
Table oi
'Data and Estimated Values of Interest
Dose
N
Observed mean
Estimated mean
Observed SD
Estimated SD
Scaled residuals
0
8
3.59
3.554
1.08
0.9635
0.105
14.3
8
3.56
3.542
0.53
0.9535
0.05257
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138.3
8
3.39
3.44
1.21
0.8713
-0.1637
1,363
8
2.58
2.579
0.37
0.3574
0.008804
Likelihoods of Interest
Model
Log (likelihood)
Number of parameters
AIC
Al
-9.516133
5
29.03227
A2
-2.971105
8
21.94221
A3
-4.802103
6
21.60421
R
-13.13332
2
30.26663
4
-5.988946
4
19.97789
Tests of Interest
Test
-2*log (likelihood ratio)
Test df
p-value
Test 1
20.32
6
0.002424
Test 2
13.09
3
0.004446
Test 3
3.662
2
0.1603
Test 6a
2.374
2
0.3052
Exponential Model 4, with BMR of 0.15 Rel. Dev. for the BMD and 0.95 Lower Confidence Level for Bl\
15:55 03/11 2015
BMR = 15% RD from control mean; dose shown in mg/kg-day.
Figure 3-10. Plot of mean response by dose, with fitted curve for Exponential Model 4, for
T4 in F1 parental female CRL Sprague-Dawley rats exposed to HBCD by diet for 18 weeks
(Ema et al., 2008).
Exponential Model (Version: 1.9; Date: 01/29/2013)
The form of the response function is: Y[dose] = a * [c-(c-l) * exp(-b * dose)]
A modeled variance is fit
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Benchmark Dose Computation
BMR = 15% RD
BMD = 690.705
BMDL at the 95% confidence level = 213.844
Parameter Estimates
Variable
Estimate
Default initial parameter values
Lnalpha
-7.9144
-6.73265
Rho
6.1823
5.13248
A
3.55422
3.7695
B
0.000235294
0.000283737
C
0
0.000684441
D
1
1
Table of Data and Estimated Values of Interest
Dose
N
Observed mean
Estimated mean
Observed SD
Estimated SD
Scaled residuals
0
8
3.59
3.554
1.08
0.9635
0.105
14.3
8
3.56
3.542
0.53
0.9535
0.05257
138.3
8
3.39
3.44
1.21
0.8713
-0.1637
1,363
8
2.58
2.579
0.37
0.3574
0.008804
Likelihoods of Interest
Model
Log (likelihood)
Number of parameters
AIC
A1
-9.516133
5
29.03227
A2
-2.971105
8
21.94221
A3
-4.802103
6
21.60421
R
-13.13332
2
30.26663
4
-5.988946
4
19.97789
Tests of Interest
Test
-2*log (likelihood ratio)
Test df
p-value
Test 1
20.32
6
0.002424
Test 2
13.09
3
0.004446
Test 3
3.662
2
0.1603
Test 6a
2.374
2
0.3052
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O 200 400 600 800 1000 1200 1400
dose
11:27 05/20 2016
BMR = 20% RD from control mean; dose shown in mg/kg-day.
Figure 3-11. Plot of mean response by dose with fitted curve for Exponential (M4) model
with modeled variance for T4 in F1 parental female CRL Sprague-Dawley rats exposed to
HBCD by diet for 18 weeks (Ema et al., 2008).
Exponential Model (Version: 1.10; Date: 01/12/2015)
The form of the response function is: Y[dose] = a * [c-(c-l) * exp(-b * dose)]
A modeled variance is fit
Benchmark Dose Computation
BMR = 20% RD
BMD = 948.359
BMDL at the 95% confidence level = 328.063
Parameter Estimates
Variable
Estimate
Default initial parameter values
lnalpha
-7.9144
-6.73265
rho
6.1823
5.13248
a
3.55422
3.7695
b
0.000235294
0.000283737
c
0
0.000684441
d
N/A
1
Table of Data and Estimated Values of Interest
Dose
N
Observed mean
Estimated mean
Observed SD
Estimated SD
Scaled residuals
0
8
3.59
3.55
1.08
0.96
0.105
14.3
8
3.56
3.54
0.53
0.95
0.05257
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138.3
8
3.39
3.44
1.21
0.87
-0.1637
1,363
8
2.58
2.58
0.37
0.36
0.008804
Likelihoods of Interest
Model
Log (likelihood)
Number of parameters
AIC
Al
-9.516133
5
29.03227
A2
-2.971105
8
21.94221
A3
-4.802103
6
21.60421
R
-13.13332
2
30.26663
4
-5.988946
4
19.97789
Tests of Interest
Test
-2*log (likelihood ratio)
Test df
p-value
Test 1
20.32
6
0.002424
Test 2
13.09
3
0.004446
Test 3
3.662
2
0.1603
Test 6a
2.374
2
0.3052
11:34 05/20 2016
BMR = 1 SD change from control mean; dose shown in mg/kg-day.
Figure 3-12. Plot of mean response by dose with fitted curve for Exponential (M4) model
with modeled variance for T4 in F1 parental female CRL Sprague-Dawley rats exposed to
HBCD by diet for 18 weeks (Ema et al., 2008).
Exponential Model (Version: 1.10; Date: 01/12/2015)
The form of the response function is: Y[dose] = a * [c-(c-l) * exp(-b * dose)]
A modeled variance is fit
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Benchmark Dose Computation
BMR = 1.0000 Estimated SDs from control
BMD = 1,343.81
BMDL at the 95% confidence level = 536.006
Parameter Estimates
Variable
Estimate
Default initial parameter values
lnalpha
-7.9144
-6.73265
rho
6.1823
5.13248
a
3.55422
3.7695
b
0.000235294
0.000283737
c
0
0.000684441
d
N/A
1
Table of Data anc
Estimated Values of Interest
Dose
N
Observed mean
Estimated mean
Observed SD
Estimated SD
Scaled residuals
0
8
3.59
3.55
1.08
0.96
0.105
14.3
8
3.56
3.54
0.53
0.95
0.05257
138.3
8
3.39
3.44
1.21
0.87
-0.1637
1,363
8
2.58
2.58
0.37
0.36
0.008804
Likelihoods of Interest
Model
Log (likelihood)
Number of parameters
AIC
A1
-9.516133
5
29.03227
A2
-2.971105
8
21.94221
A3
-4.802103
6
21.60421
R
-13.13332
2
30.26663
4
-5.988946
4
19.97789
Tests of Interest
Test
-2*log (likelihood ratio)
Test df
p-value
Test 1
20.32
6
0.002424
Test 2
13.09
3
0.004446
Test 3
3.662
2
0.1603
Test 6a
2.374
2
0.3052
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3.2.3.2 Liver
Table 3-6. Summary of BMD modeling results for relative liver weight (g/100 g BW) in
male F1 CRL rats exposed to HBCD on GD 0-PND 26, dose TWA gestation through
lactation (Etna et ai. 2008); BMR = 10% RD from control mean and 1 SD change from
control mean
Model3
Goodness of fit
BMDiord
(mg/kg-d)
BMDLiord
(mg/kg-d)
BMDisd
(mg/kg-d)
BMDLi sd
(mg/kg-d)
Basis for model
selection
p-value
AIC
Exponential (M2)
Exponential (M3)b
0.00369
-70.405
599
533
488
417
Of the models that
provided an
adequate fit and a
valid BMDL
estimate, the
Exponential M4
constant variance
model was selected
Exponential
(M4)
0.606
-79.345
163
109
120
80.5
Exponential (M5)
N/A°
-77.611
169
111
157
82.0
Hill
N/A°
-77.611
169
104
156
75.4
Powerd
Polynomial 3oe
Polynomial 2of
Linear
0.00590
-71.344
548
480
440
371
based on lowest AIC
and visual fit.
aConstant variance case presented (BMDS Test 2 p-value = 0.462), selected model in bold; scaled residuals for
selected model for doses 0, 16.5, 168, and 1,570 mg/kg-day were 0.3267, -0.3947, 0.05759, and -0.003788,
respectively.
bFor the Exponential (M3) model, the estimate of d was 1 (boundary). The models in this row reduced to the
Exponential (M2) model.
°No available degrees of freedom to calculate a goodness-of-fit value.
dFor the Power model, the power parameter estimate was 1. The models in this row reduced to the Linear model.
Tor the Polynomial 3° model, the b3 and b2 coefficient estimates were 0 (boundary of parameters space). The
models in this row reduced to the Linear model.
fFor the Polynomial 2° model, the b2 coefficient estimate was 0 (boundary of parameters space). The models in this
row reduced to the Linear model.
Data from Etna et al. (2008)
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Exponential 4 Model, with BMR of 0.1 Rel. Dev. for the BMD and 0.95 Lower Confidence Limit for the BMDL
6
5.5
5
4.5
1 2:31 05/20 2016
BMR = 10% RD from control mean; dose shown in mg/kg-day.
Figure 3-13. Plot of mean response by dose with fitted curve for Exponential (M4) model
with constant variance for relative liver weight (g/100 g BW) in F1 weanling male CRL
Sprague-Dawley rats exposed to HBCD on GD 0-PND 26, dose TWA gestation through
lactation (Ema et al., 2008).
Exponential Model (Version: 1.10; Date: 01/12/2015)
The form of the response function is: Y[dose] = a * [c-(c-l) * exp(-b * dose)]
A constant variance model is fit
Benchmark Dose Computation
BMR = 10% RD
BMD = 162.81
BMDL at the 95% confidence level = 108.569
Parameter Estimates
Variable
Estimate
Default initial parameter values
lnalpha
-2.07833
-2.08162
rho
N/A
0
a
4.5759
4.37
b
0.00230233
0.00120199
c
1.3199
1.44165
d
N/A
1
Table of Data ant
Estimated Values of Interest
Dose
N
Observed mean
Estimated mean
Observed SD
Estimated SD
Scaled residuals
0
23
4.6
4.576
0.37
0.3538
0.3267
16.5
21
4.6
4.63
0.32
0.3538
-0.3947
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168
20
5.05
5.045
0.32
0.3538
0.05759
1,570
17
6
6
0.44
0.3538
-0.003788
Likelihoods of Interest
Model
Log (likelihood)
Number of parameters
AIC
Al
43.80548
5
-77.61096
A2
45.09301
8
-74.18602
A3
43.80548
5
-77.61096
R
-5.569318
2
15.13864
4
43.67234
4
-79.34469
Tests of Interest
Test
-2*log (likelihood ratio)
Test df
p-value
Test 1
101.3
6
<0.0001
Test 2
2.575
3
0.4619
Test 3
2.575
3
0.4619
Test 6a
0.2663
1
0.6058
Exponential 4 Model, with BMR of 1 Std. Dev. for the BMD and 0.95 Lower Confidence Limit for the BMDL
5.5
4.5
13:21 05/20 2016
BMR = 1 SD change from control mean; dose shown in mg/kg-day.
Figure 3-14. Plot of mean response by dose with fitted curve for Exponential (M4) model
with constant variance for relative liver weight (g/100 g BW) in F1 weanling male CRL
Sprague-Dawley rats exposed to HBCD on GD O-PND 26, dose TWA gestation through
lactation (Ema et al., 2008).
Exponential Model (Version: 1.10; Date: 01/12/2015)
The form of the response function is: Y[dose] = a * [c-(c-l) * exp(-b * dose)]
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A constant variance model is fit
Benchmark Dose Computation
BMR = 1.0000 Estimated SDs from control
BMD = 120.152
BMDL at the 95% confidence level = 80.5016
Parameter Estimates
Variable
Estimate
Default initial parameter values
lnalpha
-2.07833
-2.08162
rho
N/A
0
a
4.5759
4.37
b
0.00230233
0.00120199
c
1.3199
1.44165
d
N/A
1
Table of Data anc
Estimated Values of Interest
Dose
N
Observed mean
Estimated mean
Observed SD
Estimated SD
Scaled residuals
0
23
4.6
4.576
0.37
0.3538
0.3267
16.5
21
4.6
4.63
0.32
0.3538
-0.3947
168
20
5.05
5.045
0.32
0.3538
0.05759
1,570
17
6
6
0.44
0.3538
-0.003788
Likelihoods of Interest
Model
Log (likelihood)
Number of parameters
AIC
A1
43.80548
5
-77.61096
A2
45.09301
8
-74.18602
A3
43.80548
5
-77.61096
R
-5.569318
2
15.13864
4
43.67234
4
-79.34469
Tests of Interest
Test
-2*log (likelihood ratio)
Test df
p-value
Test 1
101.3
6
<0.0001
Test 2
2.575
3
0.4619
Test 3
2.575
3
0.4619
Test 6a
0.2663
1
0.6058
Table 3-7. Summary of BMD modeling results for relative liver weight (g/100 g BW) in F1
weanling female CRL Sprague-Dawley rats exposed to HBCD on GD 0-PND 26, dose
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TWA of gestation and lactation (Ema et al., 2008); BMR = 10% RD from control mean and
1 SD change from control mean
Model3
Goodness of fit
BMDiord
(mg/kg-d)
BMDLiord
(mg/kg-d)
BMDisd
(mg/kg-d)
BMDLi sd
(mg/kg-d)
Basis for model
selection
p-value
AIC
Exponential (M2)
Exponential (M3)b
0.00217
-82.410
560
503
418
359
Of the models that
provided an adequate
fit and a valid BMDL
estimate, the
Exponential M4
constant variance
model was selected
based on lowest AIC.
Exponential (M4)
0.731
-92.555
165
115
109
75.8
Exponential (M5)
N/A°
-90.673
170
116
126
76.4
Hill
N/A°
-90.673
170
110
124
70.8
Power"1
Polynomial 3°e
Polynomial 2of
Linear6
0.00403
-83.646
507
449
371
315
"¦Constant variance case presented (BMDS Test 2 p-value = 0.711), selected model in bold; scaled residuals for
selected model for doses 0, 16.5, 168, and 1,570 mg/kg-day were 0.2185, -0.263, 0.03719, and -0.002332,
respectively.
bFor the Exponential (M3) model, the estimate of d was 1 (boundary). The models in this row reduced to the
Exponential (M2) model.
°No available degrees of freedom to calculate a goodness-of-fit value.
dThe Power model may appear equivalent to the Linear model; however, differences exist in digits not displayed in
the table.
Tor the Polynomial 3° model, the b3 coefficient estimate was 0 (boundary of parameters space). The models in this
row reduced to the Polynomial 2° model.
fThe Polynomial 2° model may appear equivalent to the Linear model; however, differences exist in digits not
displayed in the table.
gThe Linear model may appear equivalent to the Power model; however, differences exist in digits not displayed in
the table. This also applies to the Polynomial 3° and Polynomial 2° models.
Exponential 4 Model, with BMR of 0.1 Rel. Dev. for the BMD and 0.95 Lower Confidence Limit for the BMDL
5.5
4.5
13:53 05/20 2016
BMR = 10% RD from control mean; dose shown in mg/kg-day.
Figure 3-15. Plot of mean response by dose with fitted curve for Exponential (M4) model
with constant variance for relative liver weight (g/100 g BW) in F1 weanling female CRL
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Sprague-Dawley rats exposed to HBCD GD 0-PND 26, dose TWA of gestation and
lactation (Etna et ai. 2008).
Exponential Model (Version: 1.10; Date: 01/12/2015)
The form of the response function is: Y[dose] = a * [c-(c-l) * exp(-b * dose)]
A constant variance model is fit
Benchmark Dose Computation
BMR = 10% RD
BMD = 165.267
BMDL at the 95% confidence level = 114.71
Parameter Estimates
Variable
Estimate
Default initial parameter values
lnalpha
-2.28916
-2.29068
rho
N/A
0
a
4.5555
4.3415
b
0.00206359
0.00122548
c
1.34605
1.46804
d
N/A
1
Table of Data and Estimated Values of Interest
Dose
N
Observed mean
Estimated mean
Observed SD
Estimated SD
Scaled residuals
0
23
4.57
4.555
0.35
0.3184
0.2185
16.5
21
4.59
4.608
0.28
0.3184
-0.263
168
20
5.02
5.017
0.32
0.3184
0.03719
1,570
14
6.07
6.07
0.36
0.3184
-0.002332
Likelihoods of Interest
Model
Log (likelihood)
Number of parameters
AIC
Al
50.33659
5
-90.67319
A2
51.02517
8
-86.05034
A3
50.33659
5
-90.67319
R
-3.746671
2
11.49334
4
50.2774
4
-92.55481
Tests of Interest
Test
-2*log (likelihood ratio)
Test df
p-value
Test 1
109.5
6
<0.0001
Test 2
1.377
3
0.7109
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Test 3
1.377
3
0.7109
Test 6a
0.1184
1
0.7308
Exponential 4 Model, with BMR of 1 Std. Dev. for the BMD and 0.95 Lower Confidence Limit for the BMDL
5.5
4.5
14:02 05/20 2016
BMR = 1 SD change from control mean; dose shown in mg/kg-day.
Figure 3-16. Plot of mean response by dose with fitted curve for Exponential (M4) model
with constant variance for relative liver weight (g/100 g BW) in F1 weanling female CRL
Sprague-Dawley rats exposed to HBCD on GD O-PND 26, dose TWA of gestation and
lactation (Ema et al.. 2008).
Exponential Model (Version: 1.10; Date: 01/12/2015)
The form of the response function is: Y[dose] = a * [c-(c-l) * exp(-b * dose)]
A constant variance model is fit
Benchmark Dose Computation
BMR = 1.0000 Estimated SDs from control
BMD = 109.314
BMDL at the 95% confidence level = 75.8445
Parameter Estimates
Variable
Estimate
Default initial parameter values
lnalpha
-2.28916
-2.29068
rho
N/A
0
a
4.5555
4.3415
b
0.00206359
0.00122548
c
1.34605
1.46804
d
N/A
1
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Table of
Data and
estimated Values of Interest
Dose
N
Observed mean
Estimated mean
Observed SD
Estimated SD
Scaled residuals
0
23
4.57
4.555
0.35
0.3184
0.2185
16.5
21
4.59
4.608
0.28
0.3184
-0.263
168
20
5.02
5.017
0.32
0.3184
0.03719
1,570
14
6.07
6.07
0.36
0.3184
-0.002332
Likelihoods of Interest
Model
Log (likelihood)
Number of parameters
AIC
Al
50.33659
5
-90.67319
A2
51.02517
8
-86.05034
A3
50.33659
5
-90.67319
R
-3.746671
2
11.49334
4
50.2774
4
-92.55481
Tests of Interest
Test
-2*log (likelihood ratio)
Test df
p-value
Test 1
109.5
6
<0.0001
Test 2
1.377
3
0.7109
Test 3
1.377
3
0.7109
Test 6a
0.1184
1
0.7308
Table 3-8. Summary of BMD modeling results for relative liver weight (g/100 g BW) in F1
adult male CRL Sprague-Dawley rats exposed to HBCD by diet for 15 weeks (Etna et al.
Model3
Goodness of fit
BMDiord
(mg/kg-d)
BMDLiord
(mg/kg-d)
BMDisd
(mg/kg-d)
BMDLi sd
(mg/kg-d)
Basis for model
selection
p-value
AIC
Exponential (M2)
Exponential (M3)b
0.626
-167.34
703
601
519
433
Of the models
that provided an
adequate fit and
a valid BMDL
estimate, the
Linear constant
variance model
was selected
based on lowest
AIC (BMDLs
differed by <3).
Exponential M5
and Hill models
were excluded
because both
were saturated
Exponential (M4)
0.366
-165.46
578
243
402
161
Exponential (M5)
0.366
-165.46
578
121
402
118
Hill
0.367
-165.46
582
error0
404
164
Power"1
Polynomial 3°e
Polynomial 2of
Linear
0.638
-167.38
680
573
496
409
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models in this
case.
aConstant variance case presented (BMDS Test 2 p-value = 0.181), selected model in bold; scaled residuals for
selected model for doses 0, 11.4, 115, and 1,142 mg/kg-day were -0.723, 0.587, 0.165, and -0.0218, respectively.
bFor the Exponential (M3) model, the estimate of d was 1 (boundary). The models in this row reduced to the
Exponential (M2) model.
°BMD or BMDL computation failed for this model.
dFor the Power model, the power parameter estimate was 1. The models in this row reduced to the Linear model.
Tor the Polynomial 3° model, the b3 coefficient estimate was 0 (boundary of parameters space), he models in this
row reduced to the Polynomial 2° model. For the Polynomial 3° model, the b3 and b2 coefficient estimates were 0
(boundary of parameters space). The models in this row reduced to the Linear model.
fFor the Polynomial 2° model, the b2 coefficient estimate was 0 (boundary of parameters space). The models in this
row reduced to the Linear model.
Data from Ema et al. (2008)
3.9
3.8
3.7
3.5
3.4
3.3
3.2
O 200 400 600 800 1000 1200
dose
19:35 12/03 2015
BMR = 10% RD from control mean; dose shown in mg/kg-day.
Figure 3-17. Plot of mean response by dose with fitted curve for Linear model with
constant variance for relative liver weight (g/100 g BW) in F1 adult male CRL Sprague-
Dawley rats exposed to HBCD by diet for 15 weeks (Ema et al., 2008).
Polynomial Model. (Version: 2.20; Date: 10/22/2014)
The form of the response function is: Y[dose] = beta O + beta_l*dose
A constant variance model is fit
Benchmark Dose Computation.
BMR = 10% Relative deviation
BMD = 679.573
BMDL at the 95% confidence level = 572.977
Linear Model, with BMR of 0.1 Rel. Dev. for the BMD and 0.95 Lower Confidence Limit for the BMDL
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Parameter Estimates
Variable
Estimate
Default Initial
Parameter Values
alpha
0.0581671
0.0601744
rho
n/a
0
betaO
3.30558
3.30581
betal
0.00048642
0.000486264
Table of Data and Estimated Values o
'Interest
Dose
N
Obs Mean
Est Mean
Obs Std Dev
Est Std Dev
Scaled Resid
0
24
3.27
3.31
0.18
0.241
-0.723
11.4
24
3.34
3.31
0.26
0.241
0.587
115
22
3.37
3.36
0.25
0.241
0.165
1142
24
3.86
3.86
0.28
0.241
-0.0218
Likelihoods of Interest
Model
Log(likelihood)
# Param's
AIC
Al
87.137654
5
-164.275308
A2
89.578448
8
-163.156897
A3
87.137654
5
-164.275308
fitted
86.688502
3
-167.377004
R
55.373159
2
-106.746318
Tests of Interest
Test
-2*log(Likelihood
Ratio)
Test df
p-value
Test 1
68.4106
6
<0.0001
Test 2
4.88159
3
0.1807
Test 3
4.88159
3
0.1807
Test 4
0.898304
2
0.6382
Table 3-9. Summary of BMD modeling results for relative liver weight (g/lOOg bw) in F1
adult female CRL Sprague-Dawley rats exposed to HBCD by diet for 17 weeks (Ema et ai.
2008); BMR = 10% RD from control mean and 1 SI
> change from contro
mean
Model3
Goodness of fit
BMDiord
(mg/kg-d)
BMDLiord
(mg/kg-d)
BMDisd
(mg/kg-d)
BMDLi sd
(mg/kg-d)
Basis for model
selection
p-value
AIC
Exponential (M2)
Exponential (M3)b
0.311
-40.783
791
615
824
635
Of the models that
provided an adequate
fit and a valid BMDL
estimate, the
Exponential (M4)
Exponential (M5)°
0.139
-38.934
569
184
603
203
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Hill
0.139
-38.937
575
186
610
208
Exponential M4
constant variance
model was selected
based on lowest
BMDL (BMDLs
differed by >3). Hill
model was excluded
because it was a
saturated model in
this case.
Power"1
Polynomial 3oe
Polynomial 2of
Linear
0.316
-40.816
761
578
795
598
"¦Constant variance case presented (BMDS Test 2 p-value = 0.917), selected model in bold; scaled residuals for
selected model for doses 0, 14.3, 138, and 1,363 mg/kg-d were -0.9658, 1.098, -0.1406, and 0.002993, respectively.
bFor the Exponential (M3) model, the estimate of d was 1 (boundary). The models in this row reduced to the
Exponential (M2) model.
The Exponential (M5) model may appear equivalent to the Exponential (M4) model; however, differences exist in
digits not displayed in the table.
dFor the Power model, the power parameter estimate was 1. The models in this row reduced to the Linear model.
Tor the Polynomial 3° model, the b3 and b2 coefficient estimates were 0 (boundary of parameters space). The
models in this row reduced to the Linear model.
fFor the Polynomial 2° model, the b2 coefficient estimate was 0 (boundary of parameters space). The models in this
row reduced to the Linear model.
Data from Ema et al. (2008)
5.4
5.2
§2 4.8
o
C£
§ 4.6
s
4.4
4.2
O 200 400 600 800 1000 1200 1400
dose
19:46 12/03 2015
BMR = 10% RD from control mean; dose shown in mg/kg-day.
Figure 3-18. Plot of mean response by dose with fitted curve for Exponential (M4) model
with constant variance for relative liver weight (g/100 g BW) in F1 adult female CRL
Sprague-Dawley rats exposed to HBCD by diet for 17 weeks (Ema et al.. 2008).
Exponential Model (Version: 1.10; Date: 01/12/2015)
The form of the response function is: Y[dose] = a * [c-(c-l) * exp(-b * dose)]
A constant variance model is fit
Exponential 4 Model, with BMR of 0.1 Rel. Dev. for the BMDand 0.95 Lower Confidence Limit for the BMDL
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Benchmark Dose Computation
BMR = 10% RD
BMD = 568.784
BMDL at the 95% confidence level = 184.198
Parameter Estimates
Variable
Estimate
Default initial parameter values
lnalpha
-1.60953
-1.63795
rho
N/A
0
a
4.27208
3.971
b
0.000792725
0.0012372
c
1.27553
1.33531
d
N/A
1
Table of Data and Estimated Values of Interes
Dose
N
Observed mean
Estimated mean
Observed SD
Estimated SD
Scaled residuals
0
22
4.18
4.272
0.42
0.4472
-0.9658
14.3
22
4.39
4.285
0.44
0.4472
1.098
138
20
4.38
4.394
0.47
0.4472
-0.1406
1,363
13
5.05
5.05
0.5
0.4472
0.002993
Likelihoods of Interest
Model
Log (likelihood)
Number of parameters
AIC
Al
24.56111
5
-39.12222
A2
24.8146
8
-33.6292
A3
24.56111
5
-39.12222
R
10.7627
2
-17.5254
4
23.46704
4
-38.93407
Tests of Interest
Test
-2*log (likelihood ratio)
Test df
p-value
Test 1
28.1
6
<0.0001
Test 2
0.507
3
0.9174
Test 3
0.507
3
0.9174
Test 6a
2.188
1
0.1391
Table 3-10. Summary of BMD modeling results for relative liver weight (g/100 g BW) in
F2 weanling male CRL Sprague-Dawley rats exposed to HBCD on GD 0-PND 26, dose
TWA gestation and lactation (Etna et ai. 2008); BMR = 10% RD from control mean and 1
SD change from control mean
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Model3
Goodness of fit
BMDiord
(mg/kg-d)
BMDLiord
(mg/kg-d)
BMDisd
(mg/kg-d)
BMDLi sd
(mg/kg-d)
Basis for model
selection
p-value
AIC
Exponential (M2)
Exponential (M3)b
0.235
-45.537
563
482
587
488
Of the models that
provided an adequate
fit and a valid BMDL
estimate, the
Exponential M4
constant variance
model was selected
Exponential (M4)
0.882
-46.411
215
116
227
125
Exponential (M5)
N/A°
-44.433
200
116
218
125
Hill
N/A°
-44.433
207
112
223
120
Power"1
Polynomial 3oe
Polynomial 2of
Linear
0.278
-45.874
522
438
540
441
based on lowest
BMDL (BMDLs
differed by >3).
aConstant variance case presented. Both constant variance assumption and modeled variance were not appropriate
in this case: BMDS Tests 2 and 3 with constatnt variance assumption rejected the null hypothesis with p-value =
0.00438; Test 3 of modeled variance also rejected the null hypothesis. A sensitivity analysis (see below) indicated
limited effect of variance on model fitting. Selected model in bold; scaled residuals for selected model for doses 0,
14.7, 139.3, and 1,360 mg/kg-day were 0.09694, -0.1119, 0.01719, and -0.0007502, respectively.
bFor the Exponential (M3) model, the estimate of d was 1 (boundary). The models in this row reduced to the
Exponential (M2) model.
°No available degrees of freedom to calculate a goodness-of-fit value.
dFor the Power model, the power parameter estimate was 1. The models in this row reduced to the Linear model.
Tor the Polynomial 3° model, the b3 and b2 coefficient estimates were 0 (boundary of parameters space). The
models in this row reduced to the Linear model.
fFor the Polynomial 2° model, the b2 coefficient estimate was 0 (boundary of parameters space). The models in this
row reduced to the Linear model.
Data from Ema et al. (2008)
Exponential 4 Model, with BMR of 0.1 Rel. Dev. for the BMD and 0.95 Lower Confidence Limit for the BMDL
BMR = 10% RD from control mean; dose shown in mg/kg-day.
Figure 3-19. Plot of mean response by dose with fitted curve for Exponential (M4) model
with constant variance for relative liver weight (g/100 g BW) in F2 weanling male CRL
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Sprague-Dawley rats exposed to HBCD on GD 0-PND 26, dose TWA gestation and
lactation (Etna et ai. 2008).
Exponential Model (Version: 1.10; Date: 01/12/2015)
The form of the response function is: Y[dose] = a * [c-(c-l) * exp(-b * dose)]
A constant variance model is fit
Benchmark Dose Computation
BMR = 10% RD
BMD = 214.961
BMDL at the 95% confidence level = 115.944
Parameter Estimates
Variable
Estimate
Default initial parameter values
Lnalpha
-1.72548
-1.72578
Rho
N/A
0
A
4.71128
4.484
B
0.00192508
0.00133871
C
1.29509
1.405
D
N/A
1
Table of Dal
ta and
Estimated Values of Interest
Dose
N
Observed mean
Estimated mean
Observed SD
Estimated SD
Scaled residuals
0
22
4.72
4.711
0.59
0.422
0.09694
14.7
22
4.74
4.75
0.35
0.422
-0.1119
139.3
18
5.04
5.038
0.4
0.422
0.01719
1,360
13
6
6
0.25
0.422
-0.0007502
Likelihoods of Interest
Model
Log (likelihood)
Number of parameters
AIC
Al
27.21664
5
-44.43327
A2
33.77721
8
-51.55442
A3
27.21664
5
-44.43327
R
-2.570126
2
9.140253
4
27.20553
4
-46.41105
Tests of Interest
Test
-2*log (likelihood ratio)
Test df
p-value
Test 1
72.69
6
<0.0001
Test 2
13.12
3
0.004382
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Test 3
13.12
3
0.004382
Test 6a
0.02222
1
0.8815
Sensitivity analysis:
The fit to the means was adequate for Exponential M4 with constant variance, and their scaled
residuals were small. However, Tests 2 and 3 rejected the null hypothesis with both constant
variance assumption and modeled variance, indicating lack of fit to variances whether the
variance was constant or modeled as a power of the means. To determine how much
BMDL10%RD (116 mg/kg-day) was affected by the variance used, a sensitivity analysis was
performed with constant variance by setting the standard deviation for all dose groups to the
minimum or maximum observed values (0.25 and 0.59). Because the means were not changed
and the constant-variance option was used, the parameters (including BMD) were unchanged.
BMDLs (low confidence limit of BMD, BMR = 10% RD) were 147 mg/kg-day (with minimum
standard deviation) and 96.7 mg/kg-day (with maximum standard deviation); the BMDLs were
within twofold, suggesting limited effect of variance in this case. Therefore, the M4 model with
constant variance was used to derive the BMD and BMDL for this data set.
Table 3-11. Sensitivity analysis with minimum SD as variance: Summary of BMD
modeling results for relative liver weight (g/100 g BW) in F2 weanling male CRL Sprague-
Dawley rats exposed to HBCD on GD 0-PND 26, dose TWA gestation and lactation (Ema et
Model3
Goodness of fit
BMDiord
(mg/kg-d)
BMDLiord
(mg/kg-d)
Basis for model selection
p-value
AIC
Exponential (M2)
Exponential (M3)b
0.0150
-122.66
563
512
Exponential (M4)
0.796
-128.99
215
147
Exponential (M5)
N/A°
-127.05
200
147
Hill
N/A°
-127.05
207
148
Power"1
Polynomial 3°e
Polynomial 2of
Linear
0.0241
-123.60
522
468
aConstant variance case presented (BMDS Test 2 p-value = 1.000), selected model in bold; scaled residuals for
selected model for doses 0, 14.7, 139.3, and 1,360 mg/kg-day were 0.1681, -0.1941, 0.02981, and -0.001301,
respectively.
bFor the Exponential (M3) model, the estimate of d was 1 (boundary). The models in this row reduced to the
Exponential (M2) model.
°No available degrees of freedom to calculate a goodness-of-fit value.
dFor the Power model, the power parameter estimate was 1. The models in this row reduced to the Linear model.
Tor the Polynomial 3° model, the b3 and b2 coefficient estimates were 0 (boundary of parameters space). The
models in this row reduced to the Linear model.
fFor the Polynomial 2° model, the b2 coefficient estimate was 0 (boundary of parameters space). The models in this
row reduced to the Linear model.
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Data from Ema et al. (2008)
Exponential 4 Model, with BMR of 0.1 Rel. Dev. for the BMD and 0.95 Lower Confidence Limit for the BMDL
BMR = 10% RD from control mean; dose shown in mg/kg-day.
Figure 3-20. Plot of mean response by dose with fitted curve for Exponential (M4) model
with constant variance for relative liver weight (g/100 g BW) in F2 weanling male CRL
Sprague-Dawley rats exposed to HBCD during gestation and lactation on GD 0-PND 26,
dose TWA gestation and lactation (Ema et al.. 2008).
Exponential Model (Version: 1.10; Date: 01/12/2015)
The form of the response function is: Y[dose] = a * [c-(c-l) * exp(-b * dose)]
A constant variance model is fit
Benchmark Dose Computation
BMR = 10% RD
BMD = 214.961
BMDL at the 95% confidence level = 146.85
Parameter Estimates
Variable
Estimate
Default initial parameter values
lnalpha
-2.82651
-2.8274
rho
N/A
0
a
4.71128
4.484
b
0.00192508
0.00133871
c
1.29509
1.405
d
N/A
1
Table of Data and Estimated Values of Interest
Dose
N
Observed mean
Estimated mean
Observed SD
Estimated SD
Scaled residuals
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0
22
4.72
4.711
0.25
0.2434
0.1681
14.7
22
4.74
4.75
0.25
0.2434
-0.1941
139.3
18
5.04
5.038
0.25
0.2434
0.02981
1,360
13
6
6
0.25
0.2434
-0.001301
Likelihoods of Interest
Model
Log (likelihood)
Number of parameters
AIC
Al
68.52739
5
-127.0548
A2
68.53022
8
-121.0604
A3
68.52739
5
-127.0548
R
10.89708
2
-17.79415
4
68.49396
4
-128.9879
Tests of Interest
Test
-2*log (likelihood ratio)
Test df
p-value
Test 1
115.3
6
<0.0001
Test 2
0.00567
3
0.9999
Test 3
0.00567
3
0.9999
Test 6a
0.06685
1
0.796
Table D-3-12. Sensitivity analysis with maximum SD as variance: Summary of BMD
modeling results for relative liver weight (g/10 Og BW) in F2 weanling male CRL Sprague-
Dawley rats exposed to HBCD by gestation and lactation on GD 0-PND 26, dose TWA
gestation and lactation (Etna et al, 2008): BMR = 10% RD from control mean
Model3
Goodness of fit
BMDiord
(mg/kg-d)
BMDLiord
(mg/kg-d)
Basis for model selection
p-value
AIC
Exponential (M2)
Exponential (M3)b
0.454
-0.67698
563
459
Exponential (M4)
0.913
-0.24352
215
96.7
Exponential (M5)
N/A°
1.7445
200
96.9
Hill
N/A°
1.7445
207
90.2
Power"1
Polynomial 3oe
Polynomial 2of
Linear
0.498
-0.86210
522
414
aConstant variance case presented (BMDS Test 2 p-value = 1.000), selected model in bold; scaled residuals for
selected model for doses 0, 14.7, 139.3, and 1,360 mg/kg-day were 0.07126, -0.08227, 0.01264, and -0.0005523,
respectively.
bFor the Exponential (M3) model, the estimate of d was 1 (boundary). The models in this row reduced to the
Exponential (M2) model.
°No available degrees of freedom to calculate a goodness-of-fit value.
dFor the Power model, the power parameter estimate was 1. The models in this row reduced to the Linear model.
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Tor the Polynomial 3° model, the b3 and b2 coefficient estimates were 0 (boundary of parameters space). The
models in this row reduced to the Linear model.
fForthe Polynomial 2° model, the b2 coefficient estimate was 0 (boundary of parameters space). The models in this
row reduced to the Linear model.
Data from Ema et al. (2008)
Exponential 4 Model, with BMR of 0.1 Rel. Dev. for the BMD and 0.95 Lower Confidence Limit for the BMDL
BMR = 10% RD from control mean; dose shown in mg/kg-day.
Figure 3-21. Plot of mean response by dose with fitted curve for Exponential (M4) model
with constant variance for relative liver weight (g/100 g BW) in F2 weanling male CRL
Sprague-Dawley rats exposed to HBCD on GD 0-PND 26, dose TWA gestation and
lactation (Ema et al„ 2008).
Exponential Model (Version: 1.10; Date: 01/12/2015)
The form of the response function is: Y[dose] = a * [c-(c-l) * exp(-b * dose)]
A constant variance model is fit
Benchmark Dose Computation
BMR = 10% RD
BMD = 214.962
BMDL at the 95% confidence level = 96.7112
Parameter Estimates
Variable
Estimate
Default initial parameter values
lnalpha
-1.10991
-1.11007
rho
N/A
0
a
4.71128
4.484
b
0.00192507
0.00133871
c
1.29509
1.405
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d
N/A
1
Table of Data and Estimated Va
ues of Interest
Dose
N
Observed mean
Estimated mean
Observed SD
Estimated SD
Scaled residuals
0
22
4.72
4.711
0.59
0.5741
0.07126
14.7
22
4.74
4.75
0.59
0.5741
-0.08227
139.3
18
5.04
5.038
0.59
0.5741
0.01264
1,360
13
6
6
0.59
0.5741
-0.0005523
Likelihoods of Interest
Model
Log (likelihood)
Number of parameters
AIC
A1
4.127765
5
1.744471
A2
4.130599
8
7.738801
A3
4.127765
5
1.744471
R
-14.77144
2
33.54287
4
4.121761
4
-0.2435229
Tests of Interest
Test
-2*log (likelihood ratio)
Test df
p-value
Test 1
37.8
6
<0.0001
Test 2
0.00567
3
0.9999
Test 3
0.00567
3
0.9999
Test 6a
0.01201
1
0.9127
Exponential 4 Model, with BMR of 1 Std. Dev. for the BMD and 0.95 Lower Confidence Limit for the BMDL
dose
15:08 05/20 2016
BMR = 1 SD change from control mean; dose shown in mg/kg-day.
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Figure 3-22. Plot of mean response by dose with fitted curve for Exponential (M4) model
with constant variance for relative liver weight (g/100 g BW) in F2 weanling male CRL
Sprague-Dawley rats exposed to HBCD on GD 0-PND 26, dose TWA gestation and
lactation (Etna et ai. 2008).
Exponential Model (Version: 1.10; Date: 01/12/2015)
The form of the response function is: Y[dose] = a * [c-(c-l) * exp(-b * dose)]
A constant variance model is fit
Benchmark Dose Computation
BMR = 1.0000 Estimated SDs from control
BMD = 227.183
BMDL at the 95% confidence level = 124.503
Parameter Estimates
Variable
Estimate
Default initial parameter values
lnalpha
-1.72556
-1.72578
rho
N/A
0
a
4.71255
4.484
b
0.00156899
0.00115941
c
1.29864
1.405
d
N/A
1
Table oi
' Data and Estimated Va
ues of Interest
Dose
N
Observed mean
Estimated mean
Observed SD
Estimated SD
Scaled residuals
0
22
4.72
4.713
0.59
0.422
0.08283
16.5
22
4.74
4.749
0.35
0.422
-0.09464
168
18
5.04
5.039
0.4
0.422
0.01356
1,570
13
6
6
0.25
0.422
-0.0006035
Likelihoods of Interest
Model
Log (likelihood)
Number of parameters
AIC
Al
27.21664
5
-44.43327
A2
33.77721
8
-51.55442
A3
27.21664
5
-44.43327
R
-2.570126
2
9.140253
4
27.20864
4
-46.41727
Tests of Interest
Test
-2*log (likelihood ratio)
Test df
p-value
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Test 1
72.69
6
<0.0001
Test 2
13.12
3
0.004382
Test 3
13.12
3
0.004382
Test 6a
0.016
1
0.8993
Table 3-13. Summary of BMD modeling results for relative liver weight (g/100 g BW) in
F2 weanling female CRL Sprague-Dawley rats exposed to HBCD on GD 0-PND 26, dose as
TWA of gestation and lactation (Etna et ai, 2008); BMR = 10% RD from control mean and
Model3
Goodness of fit
BMDiord
(mg/kg-d)
BMDLiord
(mg/kg-d)
BMDisd
(mg/kg-d)
BMDLi sd
(mg/kg-d)
Basis for model
selection
p-value
AIC
Exponential (M2)
Exponential (M3)b
0.265
-92.639
589
520
400
339
Of the models that
provided an adequate
fit and a valid BMDL
estimate, the
Exponential M4
constant variance
model was selected
Exponential (M4)
0.759
-93.205
286
166
177
103
Exponential (M5)
N/A°
-91.299
168
141
149
104
Hill
N/A°
-91.299
153
error"1
144
101
Power6
Polynomial 3of
Polynomial 2og
Linear
0.323
-93.039
549
477
367
307
based on lowest
BMDL (BMDLs
differed by >3).
aConstant variance case presented (BMDS Test 2 p-value = 0.192), selected model in bold; scaled residuals for
selected model for doses 0, 14.7, 139.3, and 1,360 mg/kg-day were 0.2031, -0.2277, 0.03152, and -0.001049,
respectively.
bFor the Exponential (M3) model, the estimate of d was 1 (boundary). The models in this row reduced to the
Exponential (M2) model.
°No available degrees of freedom to calculate a goodness-of-fit value.
dBMD or BMDL computation failed for this model.
Tor the Power model, the power parameter estimate was 1. The models in this row reduced to the Linear model.
fFor the Polynomial 3° model, the b3 and b2 coefficient estimates were 0 (boundary of parameters space) The
models in this row reduced to the Linear model.
gFor the Polynomial 2° model, the b2 coefficient estimate was 0 (boundary of parameters space). The models in this
row reduced to the Linear model.
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Data from Ema et al. (2008)
6.2
6
5.8
5.6
§" 5.4
en
S 5.2
5
4.8
4.6
O 200 400 600 800 1000 1200 1400
dose
16:11 05/20 2016
BMR = 10% RD from control mean; dose shown in mg/kg-day.
Figure 3-23. Plot of mean response by dose with fitted curve for Exponential (M4) model
with constant variance for relative liver weight (g/100 g BW) in F2 weanling female CRL
Sprague-Dawley rats exposed to HBCD on GD O-PND 26, dose as TWA of gestation and
lactation (Ema et al.. 2008).
Exponential Model (Version: 1.10; Date: 01/12/2015)
The form of the response function is: Y[dose] = a * [c-(c-l) * exp(-b * dose)]
A constant variance model is fit
Benchmark Dose Computation
BMR = 10% RD
BMD = 286.259
BMDL at the 95% confidence level = 166.437
Parameter Estimates
Variable
Estimate
Default initial parameter values
lnalpha
-2.33164
-2.33288
rho
N/A
0
a
4.68619
4.465
b
0.00140932
0.00130926
c
1.30123
1.38511
d
N/A
1
Table of Data and Estimated Values of Interest
Dose
N
Observed mean
Estimated mean
Observed SD
Estimated SD
Scaled residuals
Exponential 4 Model, with BMR of 0.1 Rel. Dev. for the BMD and 0.95 Lower Confidence Limit for the BMDL
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0
21
4.7
4.686
0.27
0.3117
0.2031
14.7
22
4.7
4.715
0.28
0.3117
-0.2277
139.3
20
4.94
4.938
0.32
0.3117
0.03152
1,360
13
5.89
5.89
0.44
0.3117
-0.001049
Likelihoods of Interest
Model
Log (likelihood)
Number of parameters
AIC
Al
50.6495
5
-91.299
A2
53.0199
8
-90.03981
A3
50.6495
5
-91.299
R
9.931909
2
-15.86382
4
50.60242
4
-93.20485
Tests of Interest
Test
-2*log (likelihood ratio)
Test df
p-value
Test 1
86.18
6
<0.0001
Test 2
4.741
3
0.1918
Test 3
4.741
3
0.1918
Test 6a
0.09415
1
0.759
Exponential 4 Model, with BMR of 1 Std. Dev. for the BMD and 0.95 Lower Confidence Limit for the BMDL
BMR = 1 SD change from control mean; dose shown in mg/kg-day.
Figure 3-24. Plot of mean response by dose with fitted curve for Exponential (M4) model
with constant variance for relative liver weight (g/100 g BW) in F2 weanling female CRL
Sprague-Dawley rats exposed to HBCD on GD O-PND 26, dose as TWA of gestation and
lactation (Ema et al.. 2008).
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Exponential Model (Version: 1.10; Date: 01/12/2015)
The form of the response function is: Y[dose] = a * [c-(c-l) * exp(-b * dose)]
A constant variance model is fit
Benchmark Dose Computation
BMR = 1.0000 Estimated SDs from control
BMD = 177.017
BMDL at the 95% confidence level = 102.961
Parameter Estimates
Variable
Estimate
Default initial parameter values
lnalpha
-2.33164
-2.33288
rho
N/A
0
a
4.68619
4.465
b
0.00140932
0.00130926
c
1.30123
1.38511
d
N/A
1
Table of Data and Estimated Values of Interest
Dose
N
Observed mean
Estimated mean
Observed SD
Estimated SD
Scaled residuals
0
21
4.7
4.686
0.27
0.3117
0.2031
14.7
22
4.7
4.715
0.28
0.3117
-0.2277
139.3
20
4.94
4.938
0.32
0.3117
0.03152
1,360
13
5.89
5.89
0.44
0.3117
-0.001049
Likelihoods of Interest
Model
Log (likelihood)
Number of parameters
AIC
A1
50.6495
5
-91.299
A2
53.0199
8
-90.03981
A3
50.6495
5
-91.299
R
9.931909
2
-15.86382
4
50.60242
4
-93.20485
Tests of Interest
Test
-2*log (likelihood ratio)
Test df
p-value
Test 1
86.18
6
<0.0001
Test 2
4.741
3
0.1918
Test 3
4.741
3
0.1918
Test 6a
0.09415
1
0.759
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Table 3-14. Summary of BMD modeling results for relative liver weight (g/100 g BW) in
male CRL Sprague-Dawley rats exposed to HBCD by gavage for 13 weeks Q ^search.
2001): BMR =
0% RD from control mean and 1 SD c
lange from control mean
Goodness of fit
BMDiord
(mg/kg-d)
BMDLiord
(mg/kg-d)
BMDisd
(mg/kg-d)
BMDLi sd
(mg/kg-d)
Basis for model
selection
Model3
p-value
AIC
Modeled with constant variance
No model showed
Exponential (M2)
Exponential
(M3)b
3.14 x
10-4
-67.830
328
283
269
219
adequate fit.
Dropping highest
dose is not
expected to help
in this case.
Exponential
(M4)°
3.92 x
10-4
-69.396
164
97.7
128
77.9
Exponential
(M5)d
3.92 x
10-4
-69.396
164
97.7
128
77.9
Hill
4.91 x
10-4
-69.815
145
74.8
113
59.7
Power6
Polynomial 3of
Polynomial 2og
Linear
5.14 x
10-4
-68.817
290
244
234
187
Modeled with modeled variance
Exponential (M2)
Exponential
(M3)b
0.00119
-68.721
337
295
320
245
Exponential
(M4)°
5.50 x
10-4
-68.244
204
103
187
67.5
Exponential
(M5)d
5.50 x
10-4
-68.244
204
103
187
67.5
Hill
5.84 x
10-4
-68.355
192
35.9
173
106
Power6
Polynomial 3of
Polynomial 2og
Linear
0.00161
-69.324
299
256
282
210
aConstant variance (BMDS Test 2 p-value = 0.0644, BMDS Test 3 p-value = 0.0644) and nonconstant variance
cases presented, no model was selected as a best-fitting model.
bFor the Exponential (M3) model, the estimate of d was 1 (boundary). The models in this row reduced to the
Exponential (M2) model.
The Exponential (M4) model may appear equivalent to the Exponential (M5) model; however, differences exist in
digits not displayed in the table.
dThe Exponential (M5) model may appear equivalent to the Exponential (M4) model; however, differences exist in
digits not displayed in the table.
Tor the Power model, the power parameter estimate was 1. The models in this row reduced to the Linear model.
fFor the Polynomial 3° model, the b3 and b2 coefficient estimates were 0 (boundary of parameters space). The
models in this row reduced to the Linear model.
gFor the Polynomial 2° model, the b2 coefficient estimate was 0 (boundary of parameters space). The models in this
row reduced to the Linear model.
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Data from } search (2001)
Table 3-15. Summary of BMD modeling results for relative liver weight (g/100 g BW) in
female CRL Sprague-Dawley rats exposed to HBCD by gavage for 13 weeks (WIL Research.
2001); BMR =
0% RD from con
trol mean and 1 SD c
lange from control mean
Model3
Goodness of fit
BMDLiord
(mg/kg-d)
BMDisd
(mg/kg-d)
BMDLi sd
(mg/kg-d)
Basis for model
selection
p-value
AIC
(mg/kg-d)
Modeled with constant variance
Exponential (M2)
Exponential
(M3)b
<0.0001
-39.545
310
261
332
267
No model showed
adequate fit.
Dropping highest
dose is not
expected to help
in this case
Exponential (M4)
Exponential
(M5)°
2.59 x
10-4
-44.035
101
56.0
106
61.8
Hill
5.71 x
10-4
-45.515
69.3
30.6
73.3
34.6
Power"1
Polynomial 3oe
Polynomial 2of
Linear
<0.0001
-40.679
270
220
287
226
Modeled with modeled variance
Exponential (M2)
Exponential
(M3)b
<0.0001
-38.793
319
269
374
282
Exponential (M4)
Exponential
(M5)°
1.72 x
10-4
-42.217
53.4
28.5
38.3
16.0
Hill
0.00115
-45.763
39.2
20.7
26.0
11.6
Powerd
Polynomial 3oe
Polynomial 2of
Linear
<0.0001
-39.727
278
227
327
237
aConstant variance (BMDS Test 2 p-value = 0.461, BMDS Test 3 p-value = 0.461) and nonconstant variance
presented; no model was selected as a best-fitting model.
bFor the Exponential (M3) model, the estimate of d was 1 (boundary). The models in this row reduced to the
Exponential (M2) model.
Tor the Exponential (M5) model, the estimate of d was 1 (boundary). The models in this row reduced to the
Exponential (M4) model.
dFor the Power model, the power parameter estimate was 1. The models in this row reduced to the Linear model.
Tor the Polynomial 3° model, the b3 and b2 coefficient estimates were 0 (boundary of parameters space). The
models in this row reduced to the Linear model.
fFor the Polynomial 2° model, the b2 coefficient estimate was 0 (boundary of parameters space). The models in this
row reduced to the Linear model.
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3.2.3.3 Reproductive
Table 3-16. Summary of BMD modeling results for primordial follicles in F1 parental
female CRL Sprague-Dawley rats exposed to HBCD by diet for 18 weeks (Etna et ai, 2008);
BMR =1% RD from control mean, 5% RD from control mean, and 10% RD from control
mean
Modela
Goodness of fit
BMDird
(mg/kg-d)
BMDLird
(mg/kg-d)
BMDsrd
(mg/kg-d)
BMDL5RD
(mg/kg-d)
BMDiord
(mg/kg-d)
BMDLiord
(mg/kg-d)
Basis for
model
selection
p-value
AIC
Exponential
(M2)
Exponential
(M3)b
0.0130
408.57
26.8
13.9
137
71.0
281
146
Exponential
M4 constant
variance
selected as
only model
with
adequate fit.
Exponential
(M4)
0.688
402.05
0.883
0.252
4.67
1.33
10.1
2.87
Exponential
(M5)
N/A°
403.91
4.09
0.259
8.23
1.37
11.4
2.95
Hill
N/A°
403.91
8.00
errord
9.28
1.10
9.99
2.50
Power6
Polynomial 2of
Linear
Polynomial 3°g
0.0117
408.78
33.1
19.8
165
99.0
331
198
aConstant variance case presented (BMDS Test 2 p-value = 0.242), selected model in bold; scaled residuals for
selected model for doses 0, 9.6, 96.3, and 940.7 mg/kg-day were -0.129, 0.1915, -0.2611, and 0.1987, respectively.
bFor the Exponential (M3) model, the estimate of d was 1 (boundary). The models in this row reduced to the
Exponential (M2) model.
°No available degrees of freedom to calculate a goodness-of-fit value.
dBMD or BMDL computation failed for this model.
Tor the Power model, the power parameter estimate was 1. The models in this row reduced to the Linear model.
fFor the Polynomial 2° model, the b2 coefficient estimate was 0 (boundary of parameters space). The models in this
row reduced to the Linear model.
gThe Polynomial 3° model may appear equivalent to the Linear model; however, differences exist in digits not
displayed in the table.
Data from Etna et ai. (2008)
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Exponential Model 4, with BMR of 0.1 Rel. Dev. for the BMD and 0.95 Lower Confidence Level for BM
dose
12:48 02/11 2015
BMR = 10% RD from control mean; dose shown in mg/kg-day.
Figure 3-25. Plot of mean response by dose, with fitted curve for Exponential M4, for primordial
follicles in F1 parental female CRL Sprague-Dawley rats exposed to HBCD by diet for 18 weeks
(Ema et al., 2008).
Exponential Model (Version: 1.9; Date: 01/29/2013)
The form of the response function is: Y[dose] = a * [c-(c-l) * exp(-b * dose)]
A constant variance model is fit
Benchmark Dose Computation
BMR = 10% RD
BMD = 10.1143
BMDL at the 95% confidence level = 2.86589
Parameter Estimates
Variable
Estimate
Default initial parameter values
lnalpha
8.85121
8.84717
rho(S)
N/A
0
a
319.71
332.115
b
0.0301725
0.0026785
c
0.619779
0.567503
d
1
1
Table of Data ant
Estimated Values of Interest
Dose
N
Observed mean
Estimated mean
Observed SD
Estimated SD
Scaled residuals
0
10
316.3
319.7
119.5
83.56
-0.129
9.6
10
294.2
289.1
66.3
83.56
0.1915
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96.3
10
197.9
204.8
76.9
83.56
-0.2611
940.7
10
203.4
198.1
79.5
83.56
0.1987
Likelihoods of Interest
Model
Log (likelihood)
Number of parameters
AIC
Al
-196.9435
5
403.8869
A2
-194.8505
8
405.701
A3
-196.9435
5
403.8869
R
-203.7104
2
411.4207
4
-197.0241
4
402.0483
Tests of Interest
Test
-2*log (likelihood ratio)
Test df
p-value
Test 1
17.72
6
0.006972
Test 2
4.186
3
0.2421
Test 3
4.186
3
0.2421
Test 6a
0.1613
1
0.6879
Exponential Model 4, with BMR of 0.01 Rel. Dev. for the BMD and 0.95 Lower Confidence Level for BI\
12:46 02/11 2015
BMR = 1% RD from control mean; dose shown in mg/kg-day.
Figure 3-26. Plot of mean response by dose, with fitted curve for Exponential M4, for
primordial follicles in F1 parental female CRL Sprague-Dawley rats exposed to HBCD by
diet for 18 weeks (Ema et al.. 2008).
Exponential Model (Version: 1.9; Date: 01/29/2013)
The form of the response function is: Y[dose] = a * [c-(c-l) * exp(-b * dose)]
A constant variance model is fit
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Benchmark Dose Computation
BMR = 1% RD
BMD = 0.883338
BMDL at the 95% confidence level = 0.251965
Parameter Estimates
Variable
Estimate
Default initial parameter values
lnalpha
8.85121
8.84717
rho(S)
N/A
0
a
319.71
332.115
b
0.0301725
0.0026785
c
0.619779
0.567503
d
1
1
Table of Data and Estimated Values of Interest
Dose
N
Observed mean
Estimated mean
Observed SD
Estimated SD
Scaled residuals
0
10
316.3
319.7
119.5
83.56
-0.129
9.6
10
294.2
289.1
66.3
83.56
0.1915
96.3
10
197.9
204.8
76.9
83.56
-0.2611
940.7
10
203.4
198.1
79.5
83.56
0.1987
Likelihoods of Interest
Model
Log (likelihood)
Number of parameters
AIC
A1
-196.9435
5
403.8869
A2
-194.8505
8
405.701
A3
-196.9435
5
403.8869
R
-203.7104
2
411.4207
4
-197.0241
4
402.0483
Tests of Interest
Test
-2*log (likelihood ratio)
Test df
p-value
Test 1
17.72
6
0.006972
Test 2
4.186
3
0.2421
Test 3
4.186
3
0.2421
Test 6a
0.1613
1
0.6879
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Exponential Model 4, with BMR of 0.05 Rel. Dev. for the BMD and 0.95 Lower Confidence Level for Bl\
400
350
300
200
150
Br
O 200 400 600 800
dose
12:46 02/11 2015
BMR = 5% RD from control mean; dose shown in mg/kg-day.
Figure 3-27. Plot of mean response by dose, with fitted curve for Exponential Model 4, for
primordial follicles in F1 parental female CRL Sprague-Dawley rats exposed to HBCD by
diet for 18 weeks (Ema et al.. 2008).
Exponential Model (Version: 1.9; Date: 01/29/2013)
The form of the response function is: Y[dose] = a * [c-(c-l) * exp(-b * dose)]
A constant variance model is fit
Benchmark Dose Computation
BMR = 5% RD
BMD = 4.67281
BMDL at the 95% confidence level = 1.32975
Parameter Estimates
Variable
Estimate
Default initial parameter values
lnalpha
8.85121
8.84717
rho(S)
N/A
0
a
319.71
332.115
b
0.0301725
0.0026785
c
0.619779
0.567503
d
1
1
Table of Data and Estimated Values of Interest
Dose
N
Observed mean
Estimated mean
Observed SD
Estimated SD
Scaled residuals
0
10
316.3
319.7
119.5
83.56
-0.129
9.6
10
294.2
289.1
66.3
83.56
0.1915
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96.3
10
197.9
204.8
76.9
83.56
-0.2611
940.7
10
203.4
198.1
79.5
83.56
0.1987
Likelihoods of Interest
Model
Log (likelihood)
Number of parameters
AIC
Al
-196.9435
5
403.8869
A2
-194.8505
8
405.701
A3
-196.9435
5
403.8869
R
-203.7104
2
411.4207
4
-197.0241
4
402.0483
Tests of Interest
Test
-2*log (likelihood ratio)
Test df
p-value
Test 1
17.72
6
0.006972
Test 2
4.186
3
0.2421
Test 3
4.186
3
0.2421
Test 6a
0.1613
1
0.6879
Data from Ema et al. (2008) for incidence of non-pregnancy.
Table 3-17. Summary of BMD modeling results for incidence of non-pregnancy in F0 and
F1 CRL female rats combined exposed to HBCD in diet for 14 weeks, TWA F0 and F1
premating dose (Ema et al., 2008); BMR = 5% ER and 10% ER
Goodness of fit
BMDspct
(mg/kg-d)
BMDL5pct
(mg/kg-d)
BMDiopct
(mg/kg-d)
BMDLiopct
(mg/kg-d)
Basis for model
selection
Model3
p-value
AIC
Gamma
Weibull
Multistage 3°
Multistage 2°
Quantal-Linear
0.0881
120.47
617
263
1,266
541
No models provided
an adequate fit and a
valid BMDL
estimate; therefore no
model was selected.
Dichotomous-
Hill
N/Ab
119.61
15.1
error0
35.8
13.4
Logistic
0.0806
120.75
824
482
1,401
817
LogLogistic
0.0897
120.43
584
230
1,232
486
Probit
0.0815
120.72
797
449
1,392
781
LogProbit
0.396
118.31
6.18
error0
159
error0
aNo model was selected as a best-fitting model.
bNo available degrees of freedom to calculate a goodness-of-fit value.
°BMD or BMDL computation failed for this model.
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Table 3-18. Summary of BMD modeling results for incidence of non-pregnancy in FO and
F1 CRL female rats combined exposed to HBCD in diet for 14 weeks, TWA FO and F1
Model3
Goodness of fit
BMDspct
(mg/kg-d)
BMDLspct
(mg/kg-d)
BMDiopct
(mg/kg-d)
BMDLiopct
(mg/kg-d)
Basis for model
selection
p-value
AIC
Gammab
0.457
76.591
51.1
25.6
105
52.5
Of the models that
provided an adequate
fit and a valid BMDL
estimate, the
LogLogistic model
was selected based
on lowest AIC.
Logistic
0.374
76.860
77.3
53.3
121
85.5
LogLogistic
0.469
76.560
48.5
22.7
102
47.9
Probit
0.382
76.832
73.6
49.3
120
81.1
LogProbit
N/A°
78.045
18.0
errord
74.8
errord
Weibull6
Quantal-Linearf
0.457
76.591
51.1
25.6
105
52.5
Multistage 2°g
0.457
76.591
51.1
25.6
105
52.5
Selected model in bold; scaled residuals for selected model for doses 0, 13.3, and 131.5 mg/kg-day were -0.422,
0.575, and -0.128, respectively.
bThe Gamma model may appear equivalent to the Weibull model; however, differences exist in digits not displayed
in the table. This also applies to the Multistage 2° and Quantal-Linear models.
°No available degrees of freedom to calculate a goodness-of-fit value.
dBMD or BMDL computation failed for this model.
Tor the Weibull model, the power parameter estimate was 1. The models in this row reduced to the Quantal-Linear
model.
fThe Quantal-Linear model may appear equivalent to the Gamma model; however, differences exist in digits not
displayed in the table. This also applies to the Multistage 2° model.
gThe Multistage 2° model may appear equivalent to the Gamma model; however, differences exist in digits not
displayed in the table. This also applies to the Weibull and Quantal-Linear models.
Data from Ema et al. (2008)
Log-Logistic Model, with BMR of 5% Extra Risk for the BMD and 0.95 Lower Confidence Limit for the BMDL
0.3
22:22 05/20 2016
BMR = 5% ER; dose shown in mg/kg-day.
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Figure 3-28. Plot of incidence rate by dose with fitted curve for LogLogistic model for
incidence of non-pregnancy in FO and F1 CRL female rats combined exposed to HBCD in
diet for 14 weeks, TWA FO and F1 premating dose, high dose dropped QEiita et ai. 2008).
Logistic Model (Version: 2.14; Date: 2/28/2013)
The form of the probability function is: P[response] = background+(l-background)/[l+EXP(-
intercept-slope*Log(dose))]
Slope parameter is restricted as slope >= 1
Benchmark Dose Computation
BMR = 5% ER
BMD = 48.4809
BMDL at the 95% confidence level = 22.7093
Parameter Estimates
Variable
Estimate
Default initial parameter values
background
0.0314626
0.0208333
intercept
-6.8256E+00
-6.4682E+00
slope
1
1
Analysis of Deviance Table
Model
Log (likelihood)
Number of parameters
Deviance
Test df
p-value
Full model
-36.0225
3
Fitted model
-36.28
2
0.514904
1
0.473
Reduced model
-38.8598
1
5.6746
2
0.05858
AIC: = 76.56
Goodness-of-Fit Table
Dose
Est. Prob.
Expected
Observed
Size
Scaled residuals
0
0.0315
1.51
1
48
-0.422
13.3
0.0452
2.172
3
48
0.575
131.5
0.1525
7.318
7
48
-0.128
ChiA2 = 0.52, df = 1, p-value = 0.4687
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0.3
0.25
0.2
< 0.15
0.1
0.05
O 20 40 60 80 100 120
dose
22:27 05/20 2016
BMR = 10% ER; dose shown in mg/kg-day.
Figure 3-29. Plot of incidence rate by dose with fitted curve for LogLogistic model for
incidence of non-pregnancy in FO and F1 CRL female rats combined exposed to HBCD in
diet for 14 weeks, TWA FO and F1 premating dose, high dose dropped (Ema et al.. 2008).
Logistic Model (Version: 2.14; Date: 2/28/2013)
The form of the probability function is: P[response] = background+(l-background)/[l+EXP(-
intercept-slope*Log(dose))]
Slope parameter is restricted as slope >= 1
Benchmark Dose Computation
BMR = 10% ER
BMD = 102.349
BMDL at the 95% confidence level = 47.9419
Parameter Estimates
Variable
Estimate
Default initial parameter values
background
0.0314626
0.0208333
intercept
-6.8256E+00
-6.4682E+00
slope
1
1
Analysis of Deviance Table
Model
Log (likelihood)
Number of parameters
Deviance
Test df
p-value
Full model
-36.0225
3
Fitted model
-36.28
2
0.514904
1
0.473
Reduced model
-38.8598
1
5.6746
2
0.05858
AIC: = 76.56
Log-Logistic Model, with BMR of 10% Extra Risk for the BMD and 0.95 Lower Confidence Limit for the BMDL
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Goodness-of-Fit Table
Dose
Est. Prob.
Expected
Observed
Size
Scaled residuals
0
0.0315
1.51
1
48
-0.422
13.3
0.0452
2.172
3
48
0.575
131.5
0.1525
7.318
7
48
-0.128
ChiA2 = 0.52, df = 1, p-value = 0.4687
3.2.3.4 Developmental
Table 3-19. Summary of BMD modeling results for offspring loss from implantation
through PND 4 in F2 offspring CRL Sprague-Dawley rats; gestational doses of F1 dams
(Etna et al. 2008); BMR = 1% ER and 5% ER
Model3
Goodness of Fit
BMDipct
(mg/kg-d)
BMDLipct
(mg/kg-d)
BMDspct
(mg/kg-d)
BMDLspct
(mg/kg-d)
Basis for model
selection
p-value
AIC
Litter-specific covariate = implantation size; intra-litter correlations estimated
Of the models that
provided an adequate
fit, a valid BMDL
estimate and
BMD/BMDL <5, the
NCTR/Rai and Van
Ryzin model (litter-
specific covariate not
used; intra-litter
correlations
estimated) was
selected based on
lowest BMDL
(BMDLs differed by
>3).
Nested Logistic
0.1776
1,236.98
523.682
17.8051
708.771
92.7735
NCTR
0.1770
1,237.29
450.409
225.409
659.055
329.826
Rai and Van Ryzin
0.1984
1,236.26
371.593
185.81
538.091
269.046
Litter-specific covariate = implantation size; intra-litter correlations assumed to be zero
Nested Logistic
0.0000
1,337.62
560.759
26.8162
740.805
139.727
NCTR
0.0000
1,335.98
553.123
460.936
739.356
616.13
Rai and Van Ryzin
0.0000
1,337.63
138.735
86.7096
291.342
291.342
Litter-specific covariate not used; intra-litter correlations estimated
Nested Logistic
0.1377
1,234.32
105.863
17.0526
301.093
88.853
NCTRb
Rai and Van Ryzin
0.1423
1,234.32
108.957
54.4786
315.584
157.792
Litter-specific covariate not used; intra-litter correlations assumed to be zero
Nested Logistic
0.0000
1,336.56
132.255
25.2574
353.37
131.605
NCTRb
Rai and Van Ryzin
0.0000
1,336.56
136.105
68.0523
367.95
183.975
aBecause the individual animal data were available, the BMDS nested models were fitted, with the selected model in
bold. For the selected model, the proportion of litters with scaled residuals above 2 in absolute value for doses 0,9.7,
100, and 995 mg/kg-day were 2/23, 1/23, 1/20, and 1/21, respectively.
bWith the litter-specific covariate not used, the NCTR and Rai and van Ryzin models yielded identical results.
Data from Etna et al. (2008)
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RaiVR Model, with BMR of 1% Extra Risk for the BMD and 0.95 Lower Confidence Limit for the BMDL
dose
15:15 08/09 2016
BMR = 1% ER; dose shown in mg/kg-day.
Figure 3-30. Plot of incidence rate by dose, with fitted curve for the nested Rai and Van
Ryzin model where the litter specific covariate was not used and the intra-litter
correlations were estimated, for incidence of offspring loss from implantation through PND
4 in F2 offspring CRL Sprague-Dawley rats; gestational doses of F1 dams (Ema et al..
2008).
Rai and Van Ryzin Model (Version: 2.12; Date: 04/27/2015)
The form of the probability function is:
Prob. = [l-exp(-Alpha-Beta*DoseARho)]*exp(-(Thl+Th2*Dose)*Rij),
where Rij is the litter specific covariate.
Restrict Power rho >= 1.
Benchmark Dose Computation
To calculate the BMD and BMDL, the litter specific covariate is fixed at the mean litter specific
covariate of all the data: 14.425287
BMR = 1% ER
BMD = 108.957
BMDL at the 95% confidence level = 54.4787
Parameter Estimates
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Variable
Estimate
(Default) Initial Parameter Values
alpha
0.201085
0.201085
beta
7.58104 x 10-6
7.58104 x 10-6
rho
1.53267
1.53267
phil
0.222343
0.222343
phi2
0.0213907
0.0213907
phi3
0.0759418
0.0759418
phi4
0.277171
0.277171
Log-likelihood:-610.162 AIC: 1,234.32
Goodness-of-Fit Table
Lit.-Spec. Litter Scaled
Dose Cov. Est.Prob. Size Expected Observed Residual
0.0000
9.0000
0.182
9
1.639
3
0.7049
0.0000
10.0000
0.182
10
1.822
4
1.0303
0.0000
11.0000
0.182
11
2.004
5
1.3037
0.0000
11.0000
0.182
11
2.004
0
-0.8718
0.0000
12.0000
0.182
12
2.186
1
-0.4778
0.0000
13.0000
0.182
13
2.368
0
-0.8885
0.0000
13.0000
0.182
13
2.368
3
0.2371
0.0000
13.0000
0.182
13
2.368
3
0.2371
0.0000
13.0000
0.182
13
2.368
0
-0.8885
0.0000
14.0000
0.182
14
2.550
1
-0.5442
0.0000
14.0000
0.182
14
2.550
3
0.1579
0.0000
15.0000
0.182
15
2.732
15
4.0466
0.0000
15.0000
0.182
15
2.732
11
2.7271
0.0000
16.0000
0.182
16
2.915
4
0.3377
0.0000
16.0000
0.182
16
2.915
2
-0.2845
0.0000
16.0000
0.182
16
2.915
2
-0.2845
0.0000
16.0000
0.182
16
2.915
1
-0.5956
0.0000
16.0000
0.182
16
2.915
2
-0.2845
0.0000
16.0000
0.182
16
2.915
2
-0.2845
0.0000
17.0000
0.182
17
3.097
3
-0.0285
0.0000
17.0000
0.182
17
3.097
0
-0.9115
0.0000
17.0000
0.182
17
3.097
6
0.8546
0.0000
18.0000
0.182
18
3.279
1
-0.6365
9.7000
2.0000
0.182
2
0.365
2
2.9630
9.7000
12.0000
0.182
12
2.188
5
1.8912
9.7000
13.0000
0.182
13
2.371
3
0.4032
9.7000
13.0000
0.182
13
2.371
0
-1.5189
9.7000
13.0000
0.182
13
2.371
4
1.0439
9.7000
14.0000
0.182
14
2.553
3
0.2736
9.7000
14.0000
0.182
14
2.553
1
-0.9508
9.7000
14.0000
0.182
14
2.553
1
-0.9508
9.7000
14.0000
0.182
14
2.553
0
-1.5630
9.7000
14.0000
0.182
14
2.553
2
-0.3386
9.7000
15.0000
0.182
15
2.735
4
0.7418
9.7000
15.0000
0.182
15
2.735
4
0.7418
9.7000
15.0000
0.182
15
2.735
3
0.1552
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9.7000
15.0000
0.182
15
2.735
2
-0.4314
9.7000
16.0000
0.182
16
2.918
0
-1.6437
9.7000
16.0000
0.182
16
2.918
2
-0.5170
9.7000
16.0000
0.182
16
2.918
1
-1.0803
9.7000
16.0000
0.182
16
2.918
2
-0.5170
9.7000
17.0000
0.182
17
3.100
3
-0.0543
9.7000
17.0000
0.182
17
3.100
1
-1.1386
9.7000
17.0000
0.182
17
3.100
4
0.4879
9.7000
18.0000
0.182
18
3.282
3
-0.1476
9.7000
21.0000
0.182
21
3.830
4
0.0806
100.0000
11.0000
0.189
11
2.083
3
0.5323
100.0000
11.0000
0.189
11
2.083
1
-0.6282
100.0000
12.0000
0.189
12
2.272
0
-1.2357
100.0000
13.0000
0.189
13
2.461
0
-1.2604
100.0000
14.0000
0.189
14
2.651
2
-0.3149
100.0000
14.0000
0.189
14
2.651
3
0.1691
100.0000
14.0000
0.189
14
2.651
5
1.1369
100.0000
14.0000
0.189
14
2.651
2
-0.3149
100.0000
14.0000
0.189
14
2.651
6
1.6208
100.0000
14.0000
0.189
14
2.651
1
-0.7988
100.0000
14.0000
0.189
14
2.651
2
-0.3149
100.0000
15.0000
0.189
15
2.840
1
-0.8442
100.0000
15.0000
0.189
15
2.840
2
-0.3854
100.0000
15.0000
0.189
15
2.840
0
-1.3031
100.0000
15.0000
0.189
15
2.840
3
0.0734
100.0000
16.0000
0.189
16
3.029
4
0.4235
100.0000
16.0000
0.189
16
3.029
2
-0.4491
100.0000
17.0000
0.189
17
3.219
3
-0.0910
100.0000
17.0000
0.189
17
3.219
7
1.5729
100.0000
19.0000
0.189
19
3.597
10
2.4370
995.0000
7.0000
0.393
7
2.751
7
2.0149
995.0000
10.0000
0.393
10
3.930
2
-0.6684
995.0000
11.0000
0.393
11
4.323
3
-0.4205
995.0000
12.0000
0.393
12
4.716
0
-1.3852
995.0000
12.0000
0.393
12
4.716
6
0.3772
995.0000
13.0000
0.393
13
5.109
9
1.0623
995.0000
14.0000
0.393
14
5.502
4
-0.3831
995.0000
14.0000
0.393
14
5.502
0
-1.4032
995.0000
14.0000
0.393
14
5.502
2
-0.8932
995.0000
14.0000
0.393
14
5.502
10
1.1472
995.0000
15.0000
0.393
15
5.895
8
0.5037
995.0000
15.0000
0.393
15
5.895
3
-0.6928
995.0000
15.0000
0.393
15
5.895
9
0.7430
995.0000
15.0000
0.393
15
5.895
11
1.2216
995.0000
16.0000
0.393
16
6.288
15
1.9636
995.0000
16.0000
0.393
16
6.288
4
-0.5157
995.0000
16.0000
0.393
16
6.288
2
-0.9664
995.0000
17.0000
0.393
17
6.681
6
-0.1451
995.0000
17.0000
0.393
17
6.681
1
-1.2101
995.0000
17.0000
0.393
17
6.681
5
-0.3581
995.0000
20.0000
0.393
20
7.860
6
-0.3402
Observed Chi-square = 102.1763 Bootstrap Iterations per run = 10,000
p-value = 0.1423
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RaiVR Model, with BMR of 5% Extra Risk for the BMD and 0.95 Lower Confidence Limit for the BMDL
0.5
0.45
0.4
| 0.35
<1)
<
1 0.3
2
LL
0.25
0.2
0.15
0.1
0 200 400 600 800 1000
dose
15:29 08/09 2016
BMR = 5% ER; dose shown in mg/kg-day.
Figure 3-31. Plot of incidence rate by dose, with fitted curve for the nested Rai and Van
Ryzin model where the litter specific covariate was not used and the intra-litter
correlations were estimated, for incidence of offspring loss from implantation through PND
4 in F2 offspring CRL Sprague-Dawley rats; gestational doses of F1 dams (Ema et al..
2008).
Rai and Van Ryzin Model (Version: 2.12; Date: 04/27/2015)
The form of the probability function is:
Prob. = [l-exp(-Alpha-Beta*DoseARho)]*exp(-(Thl+Th2*Dose)*Rij),
where Rij is the litter specific covariate.
Restrict Power rho >= 1.
Benchmark Dose Computation
To calculate the BMD and BMDL, the litter specific covariate is fixed at the mean litter specific
covariate of all the data: 14.425287
BMR = 5% ER
BMD = 315.585
BMDL at the 95% confidence level = 157.792
Parameter Estimates
Variable
Estimate
(Default) Initial parameter values
alpha
0.201085
0.201085
beta
7.58104 x 10-6
7.58104 x 10-6
rho
1.53267
1.53267
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phil
0.222343
0.222343
phi2
0.0213907
0.0213907
phi3
0.0759418
0.0759418
phi4
0.277171
0.277171
Log-likelihood:-610.162 AIC: 1,234.32
Goodness-of-Fit Table
Lit.-Spec. Litter Scaled
Dose Cov. Est.Prob. Size Expected Observed Residual
0.0000
9.0000
0.182
9
1.639
3
0.7049
0.0000
10.0000
0.182
10
1.822
4
1.0303
0.0000
11.0000
0.182
11
2.004
5
1.3037
0.0000
11.0000
0.182
11
2.004
0
-0.8718
0.0000
12.0000
0.182
12
2.186
1
-0.4778
0.0000
13.0000
0.182
13
2.368
0
-0.8885
0.0000
13.0000
0.182
13
2.368
3
0.2371
0.0000
13.0000
0.182
13
2.368
3
0.2371
0.0000
13.0000
0.182
13
2.368
0
-0.8885
0.0000
14.0000
0.182
14
2.550
1
-0.5442
0.0000
14.0000
0.182
14
2.550
3
0.1579
0.0000
15.0000
0.182
15
2.732
15
4.0466
0.0000
15.0000
0.182
15
2.732
11
2.7271
0.0000
16.0000
0.182
16
2.915
4
0.3377
0.0000
16.0000
0.182
16
2.915
2
-0.2845
0.0000
16.0000
0.182
16
2.915
2
-0.2845
0.0000
16.0000
0.182
16
2.915
1
-0.5956
0.0000
16.0000
0.182
16
2.915
2
-0.2845
0.0000
16.0000
0.182
16
2.915
2
-0.2845
0.0000
17.0000
0.182
17
3.097
3
-0.0285
0.0000
17.0000
0.182
17
3.097
0
-0.9115
0.0000
17.0000
0.182
17
3.097
6
0.8546
0.0000
18.0000
0.182
18
3.279
1
-0.6365
9.7000
2.0000
0.182
2
0.365
2
2.9630
9.7000
12.0000
0.182
12
2.188
5
1.8912
9.7000
13.0000
0.182
13
2.371
3
0.4032
9.7000
13.0000
0.182
13
2.371
0
-1.5189
9.7000
13.0000
0.182
13
2.371
4
1.0439
9.7000
14.0000
0.182
14
2.553
3
0.2736
9.7000
14.0000
0.182
14
2.553
1
-0.9508
9.7000
14.0000
0.182
14
2.553
1
-0.9508
9.7000
14.0000
0.182
14
2.553
0
-1.5630
9.7000
14.0000
0.182
14
2.553
2
-0.3386
9.7000
15.0000
0.182
15
2.735
4
0.7418
9.7000
15.0000
0.182
15
2.735
4
0.7418
9.7000
15.0000
0.182
15
2.735
3
0.1552
9.7000
15.0000
0.182
15
2.735
2
-0.4314
9.7000
16.0000
0.182
16
2.918
0
-1.6437
9.7000
16.0000
0.182
16
2.918
2
-0.5170
9.7000
16.0000
0.182
16
2.918
1
-1.0803
9.7000
16.0000
0.182
16
2.918
2
-0.5170
9.7000
17.0000
0.182
17
3.100
3
-0.0543
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9.7000
17.0000
0.182
17
3.100
1
-1.1386
9.7000
17.0000
0.182
17
3.100
4
0.4879
9.7000
18.0000
0.182
18
3.282
3
-0.1476
9.7000
21.0000
0.182
21
3.830
4
0.0806
100.0000
11.0000
0.189
11
2.083
3
0.5323
100.0000
11.0000
0.189
11
2.083
1
-0.6282
100.0000
12.0000
0.189
12
2.272
0
-1.2357
100.0000
13.0000
0.189
13
2.461
0
-1.2604
100.0000
14.0000
0.189
14
2.651
2
-0.3149
100.0000
14.0000
0.189
14
2.651
3
0.1691
100.0000
14.0000
0.189
14
2.651
5
1.1369
100.0000
14.0000
0.189
14
2.651
2
-0.3149
100.0000
14.0000
0.189
14
2.651
6
1.6208
100.0000
14.0000
0.189
14
2.651
1
-0.7988
100.0000
14.0000
0.189
14
2.651
2
-0.3149
100.0000
15.0000
0.189
15
2.840
1
-0.8442
100.0000
15.0000
0.189
15
2.840
2
-0.3854
100.0000
15.0000
0.189
15
2.840
0
-1.3031
100.0000
15.0000
0.189
15
2.840
3
0.0734
100.0000
16.0000
0.189
16
3.029
4
0.4235
100.0000
16.0000
0.189
16
3.029
2
-0.4491
100.0000
17.0000
0.189
17
3.219
3
-0.0910
100.0000
17.0000
0.189
17
3.219
7
1.5729
100.0000
19.0000
0.189
19
3.597
10
2.4370
995.0000
7.0000
0.393
7
2.751
7
2.0149
995.0000
10.0000
0.393
10
3.930
2
-0.6684
995.0000
11.0000
0.393
11
4.323
3
-0.4205
995.0000
12.0000
0.393
12
4.716
0
-1.3852
995.0000
12.0000
0.393
12
4.716
6
0.3772
995.0000
13.0000
0.393
13
5.109
9
1.0623
995.0000
14.0000
0.393
14
5.502
4
-0.3831
995.0000
14.0000
0.393
14
5.502
0
-1.4032
995.0000
14.0000
0.393
14
5.502
2
-0.8932
995.0000
14.0000
0.393
14
5.502
10
1.1472
995.0000
15.0000
0.393
15
5.895
8
0.5037
995.0000
15.0000
0.393
15
5.895
3
-0.6928
995.0000
15.0000
0.393
15
5.895
9
0.7430
995.0000
15.0000
0.393
15
5.895
11
1.2216
995.0000
16.0000
0.393
16
6.288
15
1.9636
995.0000
16.0000
0.393
16
6.288
4
-0.5157
995.0000
16.0000
0.393
16
6.288
2
-0.9664
995.0000
17.0000
0.393
17
6.681
6
-0.1451
995.0000
17.0000
0.393
17
6.681
1
-1.2101
995.0000
17.0000
0.393
17
6.681
5
-0.3581
995.0000
20.0000
0.393
20
7.860
6
-0.3402
Observed Chi-square = 102.1763 Bootstrap Iterations per run = 10,000
p-value = 0.1416
Table 3-20. Summary of BMD modeling results for offspring loss from PND 4 through
PND 21 in F2 offspring CRL Sprague-Dawley rats; lactational doses of F1 dams (Etna et al.
2008): BMR = 1% ER and 5% ER
Page 167 of 201
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Model3
Goodness of Fit
BMDipct
(mg/kg-d)
BMDLipct
(mg/kg-d)
BMDspct
(mg/kg-d)
BMDLspct
(mg/kg-d)
Basis for model
selection
p-value
AIC
Litter-specific covariate = implantation size; intra-litter correlations estimated
Of the models that
provided an adequate
fit, a valid BMDL
estimate and
BMD/BMDL <5, the
Nested Logistic model
(litter-specific
covariate not used;
intra-litter correlations
Nested Logistic
0.4417
561.04
20.4
10.1841
106.295
53.0644
NCTR
0.4114
561.816
25.079
12.5395
127.994
63.997
Rai and Van Ryzin
0.4056
564.38
25.8561
1.00024
131.96
5.9492
Litter-specific covariate = implantation size; intra-litter correlations assumed to be zero
Nested Logistic
0.0000
643.52
36.1762
22.5296
188.497
117.391
NCTR
0.0000
650.146
33.8744
16.9372
172.883
86.4414
estimated) was
selected based on
lowest AIC (BMDLs
differed by <3).
Rai and Van Ryzin
0.0000
660.111
35.975
17.9875
183.603
91.8017
Litter-specific covariate not used; intra-litter correlations estimated
Nested Logistic
0.3944
559.472
16.9114
9.03491
88.1172
47.0766
NCTRb
Rai and Van Ryzin
0.4051
560.38
25.8566
12.9283
131.963
65.9814
Litter-specific covariate not used; intra-litter correlations assumed to be zero
Nested Logistic
0.0000
654.556
26.3666
18.3313
137.384
95.5159
NCTRb
Rai and Van Ryzin
0.0000
656.111
35.975
17.9875
183.603
91.8017
aBecause the individual animal data were available, the BMDS nested models were fitted, with the selected model in
bold. For the selected model, the proportion of litters with scaled residuals above 2 in absolute value for doses 0,
19.6, 179, and 1,724 mg/kg-d were 2/22, 0/22, 2/20, and 0/20, respectively.
bWith the litter-specific covariate not used, the NCTR and Rai and van Ryzin models yielded identical results.
Data from Etna et at. (2008)
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Nested Logistic Model, with BMR of 1% Extra Riskforthe BMD and 0.95 Lower Confidence Limit for the BMDL
13:22 08/10 2016
BMR = 1% ER; dose shown in mg/kg-day.
Figure 3-32. Plot of incidence rate by dose, with fitted curve for the nested logistic model where
the litter specific covariate was not used and the intra-litter correlations were estimated, for
incidence of offspring loss from PND 4 through PND 21 in F2 offspring CRL Sprague-Dawley
rats; lactational doses of F1 dams (Ema et al., 2008).
Nested Logistic Model (Version: 2.20; Date: 04/27/2015)
The form of the probability function is:
Prob. = alpha + thetal*Rij + [1 - alpha - thetal*Rij]/
[ 1 +exp(-beta-theta2*Rij -rho*log(Dose))],
where Rij is the litter specific covariate.
Restrict Power rho >= 1.
Benchmark Dose Computation
To calculate the BMD and BMDL, the litter specific covariate is fixed at the mean litter specific
covariate of all the data: 14.654762
BMR = 1% ER
BMD = 16.9114
BMDL at the 95% confidence level = 9.03491
Parameter Estimates
Variable
Estimate
(Default) Initial Parameter Values
alpha
0.133513
0.133513
beta
-7.42311
-7.42311
rho
1
1
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phil
0.229222
0.229222
phi2
0.152985
0.152985
phi3
0.247495
0.247495
phi4
0.586386
0.586386
Log-likelihood:-273.736 AIC: 559.472
Goodness-of-Fit Table
Lit.-Spec. Litter Scaled
Dose Cov. Est. Prob. Size Expected Observed Residual
0.0000
9.0000
0.134
6
0.801
0
-0.6563
0.0000
10.0000
0.134
6
0.801
1
0.1630
0.0000
11.0000
0.134
8
1.068
0
-0.6880
0.0000
11.0000
0.134
6
0.801
0
-0.6563
0.0000
12.0000
0.134
8
1.068
1
-0.0439
0.0000
13.0000
0.134
8
1.068
6
3.1766
0.0000
13.0000
0.134
8
1.068
0
-0.6880
0.0000
13.0000
0.134
8
1.068
3
1.2443
0.0000
13.0000
0.134
8
1.068
0
-0.6880
0.0000
14.0000
0.134
8
1.068
1
-0.0439
0.0000
14.0000
0.134
8
1.068
0
-0.6880
0.0000
15.0000
0.134
4
0.534
0
-0.6043
0.0000
16.0000
0.134
8
1.068
1
-0.0439
0.0000
16.0000
0.134
8
1.068
1
-0.0439
0.0000
16.0000
0.134
8
1.068
0
-0.6880
0.0000
16.0000
0.134
8
1.068
2
0.6002
0.0000
16.0000
0.134
8
1.068
1
-0.0439
0.0000
16.0000
0.134
8
1.068
4
1.8884
0.0000
17.0000
0.134
8
1.068
0
-0.6880
0.0000
17.0000
0.134
8
1.068
0
-0.6880
0.0000
17.0000
0.134
8
1.068
5
2.5325
0.0000
18.0000
0.134
8
1.068
0
-0.6880
19.6000
12.0000
0.144
7
1.005
2
0.7747
19.6000
13.0000
0.144
8
1.148
1
-0.1039
19.6000
13.0000
0.144
8
1.148
0
-0.8046
19.6000
13.0000
0.144
8
1.148
3
1.2975
19.6000
14.0000
0.144
8
1.148
2
0.5968
19.6000
14.0000
0.144
8
1.148
0
-0.8046
19.6000
14.0000
0.144
8
1.148
0
-0.8046
19.6000
14.0000
0.144
8
1.148
0
-0.8046
19.6000
14.0000
0.144
8
1.148
0
-0.8046
19.6000
15.0000
0.144
8
1.148
1
-0.1039
19.6000
15.0000
0.144
8
1.148
3
1.2975
19.6000
15.0000
0.144
8
1.148
0
-0.8046
19.6000
15.0000
0.144
8
1.148
1
-0.1039
19.6000
16.0000
0.144
8
1.148
0
-0.8046
19.6000
16.0000
0.144
8
1.148
0
-0.8046
19.6000
16.0000
0.144
8
1.148
0
-0.8046
19.6000
16.0000
0.144
8
1.148
0
-0.8046
19.6000
17.0000
0.144
8
1.148
1
-0.1039
19.6000
17.0000
0.144
8
1.148
0
-0.8046
19.6000
17.0000
0.144
8
1.148
3
1.2975
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19.6000
18.0000
0.144
8
1.148
1
-0.1039
19.6000 :
21.0000
0.144
8
1.148
0
-0.8046
179.0000
11.0000
0.217
8
1.738
4
1.1735
179.0000
11.0000
0.217
8
1.738
2
0.1361
179.0000
12.0000
0.217
8
1.738
2
0.1361
179.0000
13.0000
0.217
8
1.738
0
-0.9013
179.0000
14.0000
0.217
8
1.738
2
0.1361
179.0000
14.0000
0.217
8
1.738
5
1.6922
179.0000
14.0000
0.217
8
1.738
3
0.6548
179.0000
14.0000
0.217
8
1.738
1
-0.3826
179.0000
14.0000
0.217
8
1.738
4
1.1735
179.0000
14.0000
0.217
8
1.738
1
-0.3826
179.0000
14.0000
0.217
8
1.738
6
2.2109
179.0000
15.0000
0.217
8
1.738
0
-0.9013
179.0000
15.0000
0.217
8
1.738
0
-0.9013
179.0000
15.0000
0.217
8
1.738
1
-0.3826
179.0000
15.0000
0.217
8
1.738
6
2.2109
179.0000
16.0000
0.217
8
1.738
0
-0.9013
179.0000
16.0000
0.217
8
1.738
4
1.1735
179.0000
17.0000
0.217
8
1.738
0
-0.9013
179.0000
17.0000
0.217
8
1.738
0
-0.9013
179.0000
19.0000
0.217
8
1.738
5
1.6922
1.724.0000
10.0000
0.573
8
4.585
4
-0.1850
1.724.0000
11.0000
0.573
8
4.585
2
-0.8178
1.724.0000
12.0000
0.573
8
4.585
1
-1.1341
1.724.0000
12.0000
0.573
6
3.439
0
-1.4313
1.724.0000
13.0000
0.573
4
2.292
1
-0.7865
1.724.0000
14.0000
0.573
8
4.585
8
1.0805
1.724.0000
14.0000
0.573
8
4.585
1
-1.1341
1.724.0000
14.0000
0.573
8
4.585
0
-1.4505
1.724.0000
14.0000
0.573
4
2.292
4
1.0392
1.724.0000
15.0000
0.573
7
4.012
3
-0.3637
1.724.0000
15.0000
0.573
8
4.585
0
-1.4505
1.724.0000
15.0000
0.573
6
3.439
6
1.0662
1.724.0000
15.0000
0.573
4
2.292
4
1.0392
1.724.0000
16.0000
0.573
1
0.573
1
0.8631
1.724.0000
16.0000
0.573
8
4.585
5
0.1313
1.724.0000
16.0000
0.573
8
4.585
0
-1.4505
1.724.0000
17.0000
0.573
8
4.585
3
-0.5014
1.724.0000
17.0000
0.573
8
4.585
8
1.0805
1.724.0000
17.0000
0.573
8
4.585
3
-0.5014
1.724.0000
20.0000
0.573
8
4.585
8
1.0805
Observed Chi-square = 86.7400 Bootstrap Iterations per run = 10,000
p-value = 0.3944
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Nested Logistic Model, with BMR of 5% Extra Risk for the BMD and 0.95 Lower Confidence Limit for the BMDL
13:27 08/10 2016
BMR = 5% ER; dose shown in mg/kg-day.
Figure 3-33. Plot of incidence rate by dose, with fitted curve for the nested logistic model
where the litter specific covariate was not used and the intra-litter correlations were
estimated, for incidence of offspring loss from PND 4 through PND 21 in F2 offspring CRL
Sprague-Dawley rats; gestational doses of F1 dams (Ema et al.. 2008).
Nested Logistic Model (Version: 2.20; Date: 04/27/2015)
The form of the probability function is:
Prob. = alpha + thetal*Rij + [1 - alpha - thetal*Rij]/
[ 1 +exp(-beta-theta2*Rij -rho*log(Dose))],
where Rij is the litter specific covariate.
Restrict Power rho >= 1.
Benchmark Dose Computation
To calculate the BMD and BMDL, the litter specific covariate is fixed at the mean litter specific
covariate of all the data: 14.654762
BMR = 5% ER
BMD = 88.1172
BMDL at the 95% confidence level = 47.0766
Parameter Estimates
Variable
Estimate
(Default) Initial Parameter Values
alpha
0.133513
0.133513
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beta
-7.42311
-7.42311
rho
1
1
phil
0.229222
0.229222
phi2
0.152985
0.152985
phi3
0.247495
0.247495
phi4
0.586386
0.586386
Log-likelihood:-273.736 AIC: 559.472
Goodness-of-Fit Table
Lit.-Spec. Litter Scaled
Dose Cov. Est. Prob. Size Expected Observed Residual
0.0000
9.0000
0.134
6
0.801
0
-0.6563
0.0000
10.0000
0.134
6
0.801
1
0.1630
0.0000
11.0000
0.134
8
1.068
0
-0.6880
0.0000
11.0000
0.134
6
0.801
0
-0.6563
0.0000
12.0000
0.134
8
1.068
1
-0.0439
0.0000
13.0000
0.134
8
1.068
6
3.1766
0.0000
13.0000
0.134
8
1.068
0
-0.6880
0.0000
13.0000
0.134
8
1.068
3
1.2443
0.0000
13.0000
0.134
8
1.068
0
-0.6880
0.0000
14.0000
0.134
8
1.068
1
-0.0439
0.0000
14.0000
0.134
8
1.068
0
-0.6880
0.0000
15.0000
0.134
4
0.534
0
-0.6043
0.0000
16.0000
0.134
8
1.068
1
-0.0439
0.0000
16.0000
0.134
8
1.068
1
-0.0439
0.0000
16.0000
0.134
8
1.068
0
-0.6880
0.0000
16.0000
0.134
8
1.068
2
0.6002
0.0000
16.0000
0.134
8
1.068
1
-0.0439
0.0000
16.0000
0.134
8
1.068
4
1.8884
0.0000
17.0000
0.134
8
1.068
0
-0.6880
0.0000
17.0000
0.134
8
1.068
0
-0.6880
0.0000
17.0000
0.134
8
1.068
5
2.5325
0.0000
18.0000
0.134
8
1.068
0
-0.6880
19.6000
12.0000
0.144
7
1.005
2
0.7747
19.6000
13.0000
0.144
8
1.148
1
-0.1039
19.6000
13.0000
0.144
8
1.148
0
-0.8046
19.6000
13.0000
0.144
8
1.148
3
1.2975
19.6000
14.0000
0.144
8
1.148
2
0.5968
19.6000
14.0000
0.144
8
1.148
0
-0.8046
19.6000
14.0000
0.144
8
1.148
0
-0.8046
19.6000
14.0000
0.144
8
1.148
0
-0.8046
19.6000
14.0000
0.144
8
1.148
0
-0.8046
19.6000
15.0000
0.144
8
1.148
1
-0.1039
19.6000
15.0000
0.144
8
1.148
3
1.2975
19.6000
15.0000
0.144
8
1.148
0
-0.8046
19.6000
15.0000
0.144
8
1.148
1
-0.1039
19.6000
16.0000
0.144
8
1.148
0
-0.8046
19.6000
16.0000
0.144
8
1.148
0
-0.8046
19.6000
16.0000
0.144
8
1.148
0
-0.8046
19.6000
16.0000
0.144
8
1.148
0
-0.8046
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19.6000
17.0000
0.144
i 1.148
1
-0.1039
19.6000
17.0000
0.144
I 1.148
0
-0.8046
19.6000
17.0000
0.144
I 1.148
3
1.2975
19.6000
18.0000
0.144
I 1.148
1
-0.1039
19.6000
21.0000
0.144
I 1.148
0
-0.8046
179.0000
11.0000
0.217
8 1.738
4
1.1735
179.0000
11.0000
0.217
8 1.738
2
0.1361
179.0000
12.0000
0.217
8 1.738
2
0.1361
179.0000
13.0000
0.217
8 1.738
0
-0.9013
179.0000
14.0000
0.217
8 1.738
2
0.1361
179.0000
14.0000
0.217
8 1.738
5
1.6922
179.0000
14.0000
0.217
8 1.738
3
0.6548
179.0000
14.0000
0.217
8 1.738
1
-0.3826
179.0000
14.0000
0.217
8 1.738
4
1.1735
179.0000
14.0000
0.217
8 1.738
1
-0.3826
179.0000
14.0000
0.217
8 1.738
6
2.2109
179.0000
15.0000
0.217
8 1.738
0
-0.9013
179.0000
15.0000
0.217
8 1.738
0
-0.9013
179.0000
15.0000
0.217
8 1.738
1
-0.3826
179.0000
15.0000
0.217
8 1.738
6
2.2109
179.0000
16.0000
0.217
8 1.738
0
-0.9013
179.0000
16.0000
0.217
8 1.738
4
1.1735
179.0000
17.0000
0.217
8 1.738
0
-0.9013
179.0000
17.0000
0.217
8 1.738
0
-0.9013
179.0000
19.0000
0.217
8 1.738
5
1.6922
1.724.0000 10.0000 0.573 8 4.585 4 -0.1850
1.724.0000 11.0000 0.573 8 4.585 2 -0.8178
1.724.0000 12.0000 0.573 8 4.585 1 -1.1341
1.724.0000 12.0000 0.573 6 3.439 0 -1.4313
1.724.0000 13.0000 0.573 4 2.292 1 -0.7865
1.724.0000 14.0000 0.573 8 4.585 8 1.0805
1.724.0000 14.0000 0.573 8 4.585 1 -1.1341
1.724.0000 14.0000 0.573 8 4.585 0 -1.4505
1.724.0000 14.0000 0.573 4 2.292 4 1.0392
1.724.0000 15.0000 0.573 7 4.012 3 -0.3637
1.724.0000 15.0000 0.573 8 4.585 0 -1.4505
1.724.0000 15.0000 0.573 6 3.439 6 1.0662
1.724.0000 15.0000 0.573 4 2.292 4 1.0392
1.724.0000 16.0000 0.573 1 0.573 1 0.8631
1.724.0000 16.0000 0.573 8 4.585 5 0.1313
1.724.0000 16.0000 0.573 8 4.585 0 -1.4505
1.724.0000 17.0000 0.573 8 4.585 3 -0.5014
1.724.0000 17.0000 0.573 8 4.585 8 1.0805
1.724.0000 17.0000 0.573 8 4.585 3 -0.5014
1.724.0000 20.0000 0.573 8 4.585 8 1.0805
Observed Chi-square = 86.7400 Bootstrap Iterations per run = 10,000
p-value = 0.4003
Table 3-21. Summary of BMD modeling results for pup weight during lactation in F2 male
offspring CRL Sprague-Dawley rats (PND 21) exposed to HBCD by diet for 3 weeks,
lactational dose(YEma et ai. 2008); BMR = 5% RD from control mean, 10% RD from
control mean, 0.5 SD change from control mean, and 1 SD change from control mean
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Goodness of fit
BMD5RD
(mg/kg-d)
BMDLsri)
(mg/kg-d)
BMDiord
(mg/kg-d)
BMDLiord
(mg/kg-d)
Basis for model
selection
Model3
p-value
AIC
Exponential (M2)
0.486
420.90
354
240
727
494
Of the models that
Exponential (M3)
0.266
422.69
651
244
1016
500
provided an
adequate fit, a
valid BMDL
estimate and
BMD/BMDL <5,
Exponential (M4)
0.486
420.90
354
89.6
727
206
Exponential (M5)
N/Ab
424.68
230
94.0
258
181
Hill
N/Ab
424.68
230
89.2
264
errorc
the Exponential
M4 constant
variance model
Power
0.266
422.69
676
282
1,049
565
Polynomial 3°
Polynomial 2°
0.264
422.70
817
282
1,161
564
was selected based
on lowest BMDL
(BMDLs differed
by >3).
Linear
0.497
420.85
389
280
779
560
Goodness of fit
BMDossd
(mg/kg-d)
BMDL0.5SD
(mg/kg-d)
BMDisd
(mg/kg-d)
BMDLi sd
(mg/kg-d)
Model3
p-value
AIC
Exponential (M2)
0.486
420.90
634
419
1,332
879
Exponential (M3)
0.266
422.69
937
425
1,483
891
Exponential (M4)
0.486
420.90
634
172
1,332
468
Exponential (M5)
N/Ab
424.68
252
176
296
189
Hill
N/Ab
424.68
256
176
324
error0
Power
0.266
422.69
969
482
1,503
965
Polynomial 3°
Polynomial 2°
0.264
422.70
1,091
482
1,549
964
Linear
0.497
420.85
684
478
1,368
956
aConstant variance case presented (BMDS Test 2 p-value = 0.0278), selected model in bold; scaled residuals for
selected model for doses 0, 19.6, 179, and 1,724 mg/kg-day were -0.92, 0.71, 0.27, and -0.06, respectively.
bNo available degrees of freedom to calculate a goodness-of-fit value.
°BMD or BMDL computation failed for this model.
Data from Etna et at. (2008)
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60
55
I 50
a:
M
45
40
O 200 400 600 800 1000 1200 1400 1600 1800
dose
23:10 05/20 2016
BMR = 5% RD from control mean; dose shown in mg/kg-day.
Figure 3-34. Plot of mean response by dose with fitted curve for Exponential (M4) model with
constant variance for pup weight during lactation in F2 male offspring CRL Sprague-Dawley rats
(PND 21) exposed to HBCD by diet for 3 weeks, lactational dose (Ema et al., 2008).
Exponential Model (Version: 1.10; Date: 01/12/2015)
The form of the response function is: Y[dose] = a * [c-(c-l) * exp(-b * dose)]
A constant variance model is fit
Benchmark Dose Computation
BMR = 5% RD
BMD = 353.728
BMDL at the 95% confidence level = 89.5935
Parameter Estimates
Variable
Estimate
Default initial parameter values
lnalpha
4.53195
4.51269
rho
N/A
0
a
54.8883
59.01
b
0.000145008
0.00128594
c
0
0.687535
d
N/A
1
Table of Data and Estimated Values of Interes
Dose
N
Observed mean
Estimated mean
Observed SD
Estimated SD
Scaled residuals
0
22
53
54.89
12.6
9.64
-0.9187
19.6
22
56.2
54.73
6.7
9.64
0.714
Exponential 4 Model, with BMR of 0.05 Rel. Dev. for the BMD and 0.95 Lower Confidence Limit for the BMDL
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179
18
54.1
53.48
10.1
9.64
0.272
1,724
13
42.6
42.75
8.3
9.64
-0.0551
Likelihoods of Interest
Model
Log (likelihood)
Number of parameters
AIC
Al
-206.7258
5
423.4517
A2
-202.1665
8
420.333
A3
-206.7258
5
423.4517
R
-214.7267
2
433.4535
4
-207.4482
3
420.8963
Tests of Interest
Test
-2*log (likelihood ratio)
Test df
p-value
Test 1
25.12
6
0.0003244
Test 2
9.119
3
0.02775
Test 3
9.119
3
0.02775
Test 6a
1.445
2
0.4856
Exponential 4 Model, with BMR of 0.1 Rel. Dev. for the BMD and 0.95 Lower Confidence Limit for the BMDL
60
55
| 50
a:
M
45
40
0 200 400 600 800 1000 1200 1400 1600 1800
dose
23:17 05/20 2016
BMR = 10% RD from control mean; dose shown in mg/kg-day.
Figure 3-35. Plot of mean response by dose with fitted curve for Exponential (M4) model
with constant variance for pup weight during lactation in F2 male offspring CRL Sprague-
Dawley rats (PND 21) exposed to HBCD by diet for 3 weeks, lactational dose (Ema et al..
2008).
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Exponential Model (Version: 1.10; Date: 01/12/2015)
The form of the response function is: Y[dose] = a * [c-(c-l) * exp(-b * dose)]
A constant variance model is fit
Benchmark Dose Computation
BMR = 10% RD
BMD = 726.585
BMDL at the 95% confidence level = 206.377
Parameter Estimates
Variable
Estimate
Default initial parameter values
lnalpha
4.53195
4.51269
rho
N/A
0
a
54.8883
59.01
b
0.000145008
0.00128594
c
0
0.687535
d
N/A
1
Table oi
'Data and Estimated Values of Interest
Dose
N
Observed mean
Estimated mean
Observed SD
Estimated SD
Scaled residuals
0
22
53
54.89
12.6
9.64
-0.9187
19.6
22
56.2
54.73
6.7
9.64
0.714
179
18
54.1
53.48
10.1
9.64
0.272
1,724
13
42.6
42.75
8.3
9.64
-0.0551
Likelihoods of Interest
Model
Log (likelihood)
Number of parameters
AIC
A1
-206.7258
5
423.4517
A2
-202.1665
8
420.333
A3
-206.7258
5
423.4517
R
-214.7267
2
433.4535
4
-207.4482
3
420.8963
Tests of Interest
Test
-2*log (likelihood ratio)
Test df
p-value
Test 1
25.12
6
0.0003244
Test 2
9.119
3
0.02775
Test 3
9.119
3
0.02775
Test 6a
1.445
2
0.4856
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Exponential 4 Model, with BMR of 0.5 Std. Dev. for the BMD and 0.95 Lower Confidence Limit for the BMDL
23:19 05/20 2016
BMR = 0.5 SD change from control mean; dose shown in mg/kg-day.
Figure 3-36. Plot of mean response by dose with fitted curve for Exponential (M4) model
with constant variance for pup weight during lactation in F2 male offspring CRL Sprague-
Dawley rats (PND 21) exposed to HBCD by diet for 3 weeks, lactational dose (Ema et al..
2008).
Exponential Model (Version: 1.10; Date: 01/12/2015)
The form of the response function is: Y[dose] = a * [c-(c-l) * exp(-b * dose)]
A constant variance model is fit
Benchmark Dose Computation
BMR = 50% Estimated SDs from control
BMD = 633.879
BMDL at the 95% confidence level = 171.599
Parameter Estimates
Variable
Estimate
Default initial parameter values
lnalpha
4.53195
4.51269
rho
N/A
0
a
54.8883
59.01
b
0.000145008
0.00128594
c
0
0.687535
d
N/A
1
Table of Data and Estimated Values of Interest
Dose
N
Observed mean
Estimated mean
Observed SD
Estimated SD
Scaled residuals
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0
22
53
54.89
12.6
9.64
-0.9187
19.6
22
56.2
54.73
6.7
9.64
0.714
179
18
54.1
53.48
10.1
9.64
0.272
1,724
13
42.6
42.75
8.3
9.64
-0.0551
Likelihoods of Interest
Model
Log (likelihood)
Number of parameters
AIC
Al
-206.7258
5
423.4517
A2
-202.1665
8
420.333
A3
-206.7258
5
423.4517
R
-214.7267
2
433.4535
4
-207.4482
3
420.8963
Tests of Interest
Test
-2*log (likelihood ratio)
Test df
p-value
Test 1
25.12
6
0.0003244
Test 2
9.119
3
0.02775
Test 3
9.119
3
0.02775
Test 6a
1.445
2
0.4856
Exponential 4 Model, with BMR of 1 Std. Dev. for the BMD and 0.95 Lower Confidence Limit for the BMDL
BMR = 1 SD change from control mean; dose shown in mg/kg-day.
Figure 3-37. Plot of mean response by dose with fitted curve for Exponential (M4) model
with constant variance for pup weight during lactation in F2 male offspring CRL Sprague-
Dawley rats (PND 21) exposed to HBCD by diet for 3 weeks, lactational dose (Ema et al..
2008).
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Exponential Model (Version: 1.10; Date: 01/12/2015)
The form of the response function is: Y[dose] = a * [c-(c-l) * exp(-b * dose)]
A constant variance model is fit
Benchmark Dose Computation
BMR = 1.0000 Estimated SDs from control
BMD = 1331.98
BMDL at the 95% confidence level = 468.431
Parameter Estimates
Variable
Estimate
Default initial parameter values
lnalpha
4.53195
4.51269
rho
N/A
0
a
54.8883
59.01
b
0.000145008
0.00128594
c
0
0.687535
d
N/A
1
Table oi
' Data and Estimated Va
ues of Interest
Dose
N
Observed mean
Estimated mean
Observed SD
Estimated SD
Scaled residuals
0
22
53
54.89
12.6
9.64
-0.9187
19.6
22
56.2
54.73
6.7
9.64
0.714
179
18
54.1
53.48
10.1
9.64
0.272
1,724
13
42.6
42.75
8.3
9.64
-0.0551
Likelihoods of Interest
Model
Log (likelihood)
Number of parameters
AIC
A1
-206.7258
5
423.4517
A2
-202.1665
8
420.333
A3
-206.7258
5
423.4517
R
-214.7267
2
433.4535
4
-207.4482
3
420.8963
Tests of Interest
Test
-2*log (likelihood ratio)
Test df
p-value
Test 1
25.12
6
0.0003244
Test 2
9.119
3
0.02775
Test 3
9.119
3
0.02775
Test 6a
1.445
2
0.4856
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Table 3-22. Summary of BMD modeling results for pup weight during lactation in F2
female offspring CRL Sprague-Dawley rats (PND 21) exposed to HBCD by diet for 3
weeks, lactational dose (Etna et ai. 2008); BMR = 5% RD from control mean, 10% RD
Goodness of fit
BMDsrd
(mg/kg-d)
BMDLsri)
(mg/kg-d)
BMDiord
(mg/kg-d)
BMDLiord
(mg/kg-d)
Basis for model
selection
Model3
p-value
AIC
Exponential
(M2)
0.942
413.8640
381
257
783
528
Of the models that
provided an
Exponential
(M3)
0.732
415.86
411
257
815
529
adequate fit, a
valid BMDL
estimate and
Exponential
(M4)
0.729
415.86
381
257
783
528
BMD/BMDL <5,
the Linear constant
variance model
was selected based
on lowest AIC
Exponential
(M5)
N/Ab
417.83
201
76.5
225
179
Hill
N/Ab
417.83
203
67.7
235
error0
(BMDLs differed
by <3).
Power
0.729
415.86
423
297
840
594
Polynomial 3°°
Polynomial 2od
Linear
0.942
413.8637
417
297
834
594
Goodness of fit
BMDossd
(mg/kg-d)
BMDL0.5SD
(mg/kg-d)
BMDisd
(mg/kg-d)
BMDLi sd
(mg/kg-d)
Modela
p-value
AIC
Exponential
(M2)
0.942
413.864
657
432
1378
903
Exponential
(M3)
0.732
415.86
690
432
1397
903
Exponential
(M4)
0.729
415.86
657
432
1378
903
Exponential
(M5)
N/Ab
417.83
219
140
256
188
Hill
N/Ab
417.83
226
133
291
errorc
Power
0.729
415.86
712
489
1,416
978
Polynomial 3°
Polynomial 2°
Linear
0.942
413.8637
706
489
1,412
978
aConstant variance case presented (BMDS Test 2 p-value = 0.133), selected model in bold; scaled residuals for
selected model for doses 0, 19.6, 179, and 1,724 mg/kg-day were -0.22, 0.26, -0.05, and 0, respectively.
bNo available degrees of freedom to calculate a goodness-of-fit value.
°BMD or BMDL computation failed for this model.
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Linear Model, with BMR of 0.05 Rel. Dev. for the BMD and 0.95 Lower Confidence Limit for the BMDL
55
50
40
35
0 200 400 600 800 1000 1200 1400 1600 1800
dose
00:01 05/21 2016
BMR = 5% RD from control mean; dose shown in mg/kg-day.
Figure 3-38. Plot of mean response by dose with fitted curve for Linear model with
constant variance for pup weight during lactation in F2 female offspring CRL Sprague-
Dawley rats (PND 21) exposed to HBCD by diet for 3 weeks, lactational dose (Ema et al..
2008).
Polynomial Model (Version: 2.20; Date: 10/22/2014)
The form of the response function is: Y[dose] = beta O + beta_l*dose
A constant variance model is fit
Benchmark Dose Computation
BMR = 5% RD
BMD = 417.145
BMDL at the 95% confidence level = 296.948
Parameter Estimates
Variable
Estimate
Default initial parameter values
alpha
78.7776
83.0228
rho
N/A
0
betaO
52.4269
52.4168
betal
-0.00628402
-0.00627654
Table of Data and Estimated Values of Interest
Dose
N
Observed mean
Estimated mean
Observed SD
Estimated SD
Scaled residuals
0
21
52
52.4
10
8.88
-0.22
19.6
22
52.8
52.3
6.6
8.88
0.262
179
20
51.2
51.3
10.8
8.88
-0.0514
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1,724
13
41.6
41.6
8.4
8.88
0.00274
Likelihoods of Interest
Model
Log (likelihood)
Number of parameters
AIC
Al
-203.871816
5
417.743631
A2
-201.070527
8
418.141053
A3
-203.871816
5
417.743631
fitted
-203.931869
3
413.863738
R
-210.813685
2
425.627371
Tests of Interest
Test
-2*log (likelihood ratio)
Test df
p-value
Test 1
19.4863
6
0.003416
Test 2
5.60258
3
0.1326
Test 3
5.60258
3
0.1326
Test 4
0.120106
2
0.9417
Linear Model, with BMR of 0.1 Rel. Dev. for the BMD and 0.95 Lower Confidence Limit for the BMDL
55
50
40
35
0 200 400 600 800 1000 1200 1400 1600 1800
dose
00:07 05/21 2016
BMR = 10% RD from control mean; dose shown in mg/kg-day.
Figure 3-39. Plot of mean response by dose with fitted curve for Linear model with
constant variance for pup weight during lactation in F2 female offspring CRL Sprague-
Dawley rats (PND 21) exposed to HBCD by diet for 3 weeks, lactational dose (Ema et al.,
2008).
Polynomial Model (Version: 2.20; Date: 10/22/2014)
The form of the response function is: Y[dose] = beta O + beta_l*dose
A constant variance model is fit
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Benchmark Dose Computation
BMR = 10% RD
BMD = 834.289
BMDL at the 95% confidence level = 593.896
Parameter Estimates
Variable
Estimate
Default initial parameter values
alpha
78.7776
83.0228
rho
N/A
0
betaO
52.4269
52.4168
betal
-0.00628402
-0.00627654
Table of Data and Estimated Values of Interest
Dose
N
Observed mean
Estimated mean
Observed SD
Estimated SD
Scaled residuals
0
21
52
52.4
10
8.88
-0.22
19.6
22
52.8
52.3
6.6
8.88
0.262
179
20
51.2
51.3
10.8
8.88
-0.0514
1,724
13
41.6
41.6
8.4
8.88
0.00274
Likelihoods of Interest
Model
Log (likelihood)
Number of parameters
AIC
A1
-203.871816
5
417.743631
A2
-201.070527
8
418.141053
A3
-203.871816
5
417.743631
fitted
-203.931869
3
413.863738
R
-210.813685
2
425.627371
Tests of Interest
Test
-2*log (likelihood ratio)
Test df
p-value
Test 1
19.4863
6
0.003416
Test 2
5.60258
3
0.1326
Test 3
5.60258
3
0.1326
Test 4
0.120106
2
0.9417
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Linear Model, with BMR of 0.5 Std. Dev. for the BMD and 0.95 Lower Confidence Limit for the BMDL
55
50
40
35
0 200 400 600 800 1000 1200 1400 1600 1800
dose
00:09 05/21 2016
BMR = 0.5 SD change from control mean; dose shown in mg/kg-day.
Figure 3-40. Plot of mean response by dose with fitted curve for Linear model with
constant variance for pup weight during lactation in F2 female offspring CRL Sprague-
Dawley rats (PND 21) exposed to HBCD by diet for 3 weeks, lactational dose (Ema et al..
2008).
Polynomial Model (Version: 2.20; Date: 10/22/2014)
The form of the response function is: Y[dose] = beta O + beta_l*dose
A constant variance model is fit
Benchmark Dose Computation
BMR = 50% Estimated SDs from the control mean
BMD = 706.21
BMDL at the 95% confidence level = 488.985
Parameter Estimates
Variable
Estimate
Default initial parameter values
alpha
78.7776
83.0228
rho
N/A
0
betaO
52.4269
52.4168
betal
-0.00628402
-0.00627654
Table of Data and Estimated Values of Interest
Dose
N
Observed mean
Estimated mean
Observed SD
Estimated SD
Scaled residuals
0
21
52
52.4
10
8.88
-0.22
19.6
22
52.8
52.3
6.6
8.88
0.262
179
20
51.2
51.3
10.8
8.88
-0.0514
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1,724
13
41.6
41.6
8.4
8.88
0.00274
Likelihoods of Interest
Model
Log (likelihood)
Number of parameters
AIC
Al
-203.871816
5
417.743631
A2
-201.070527
8
418.141053
A3
-203.871816
5
417.743631
fitted
-203.931869
3
413.863738
R
-210.813685
2
425.627371
Tests of Interest
Test
-2*log (likelihood ratio)
Test df
p-value
Test 1
19.4863
6
0.003416
Test 2
5.60258
3
0.1326
Test 3
5.60258
3
0.1326
Test 4
0.120106
2
0.9417
Linear Model, with BMR of 1 Std. Dev. for the BMD and 0.95 Lower Confidence Limit for the BMDL
dose
00:10 05/21 2016
BMR = 1 SD change from control mean; dose shown in mg/kg-day.
Figure 3-41. Plot of mean response by dose with fitted curve for Linear model with
constant variance for pup weight during lactation in F2 female offspring CRL Sprague-
Dawley rats (PND 21) exposed to HBCD by diet for 3 weeks, lactational dose (Ema et al..
2008).
Polynomial Model (Version: 2.20; Date: 10/22/2014)
The form of the response function is: Y[dose] = beta O + beta_l*dose
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A constant variance model is fit
Benchmark Dose Computation
BMR = 1 Estimated SDs from the control mean
BMD = 1412.42
BMDL at the 95% confidence level = 977.97
Parameter Estimates
Variable
Estimate
Default initial parameter values
alpha
78.7776
83.0228
rho
N/A
0
betaO
52.4269
52.4168
betal
-0.00628402
-0.00627654
Table of Data and Estimated Values of Interest
Dose
N
Observed mean
Estimated mean
Observed SD
Estimated SD
Scaled residuals
0
21
52
52.4
10
8.88
-0.22
19.6
22
52.8
52.3
6.6
8.88
0.262
179
20
51.2
51.3
10.8
8.88
-0.0514
1,724
13
41.6
41.6
8.4
8.88
0.00274
Likelihoods of Interest
Model
Log (likelihood)
Number of parameters
AIC
A1
-203.871816
5
417.743631
A2
-201.070527
8
418.141053
A3
-203.871816
5
417.743631
fitted
-203.931869
3
413.863738
R
-210.813685
2
425.627371
Tests of Interest
Test
-2*log (likelihood ratio)
Test df
p-value
Test 1
19.4863
6
0.003416
Test 2
5.60258
3
0.1326
Test 3
5.60258
3
0.1326
Test 4
0.120106
2
0.9417
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4 REFERENCES
Al-Mousa. F; Michelangeli. F. (2012). Some commonly used brominated flame retardants cause Ca2+-ATPase inhibition, beta-
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