Published by Oxford University Press on behalf of the Society of Toxicology 2013.

This work is written by (a) US Government employee(s) and is in the public domain in the US.

The Fate of β-Hexabromocyclododecane in Female C57BL/6 Mice

J. Michael Sanders1, Gabriel A. Knudsen, and Linda S. Birnbaum

Toxicology and Toxicokinetics Group, Basic Research Laboratory, Center for Cancer Research, National Cancer Institute, Research Triangle Park,

North Carolina 27709

1

To whom correspondence should be addressed at NIEHS, P.O. Box 12233, MD C2-02, Research Triangle Park, NC 27709. Fax: 919-541-5136. 

E-mail: sander10@mail.nih.gov.

Received February 27, 2013; accepted May 13, 2013

1,2,5,6,9,10-Hexabromocyclododecane (HBCD) is a high production volume cycloaliphatic used as an additive flame retardant primarily in polystyrene foam building materials. HBCD 

mixtures contain three major stereoisomers, alpha (α), beta (β), 

and gamma (γ), at a typical ratio of 1.2:0.6:8.2. The toxicokinetic 

properties of the α and γ isomers differ. For instance, α-HBCD 

has greater bioavailability and potential for accumulation in mice 

than γ-HBCD. The present study reports comparative kinetics 

data for β-HBCD needed to support toxicological evaluations 

of HBCD mixtures. Results indicated that a single oral dose of 

3mg/kg of [14C]-labeled β-HBCD was absorbed rapidly (≥ 85% 

total dose) in the female C57BL/6 mouse. The Cmax for β-HBCDderived radioactivity in tissues, except adipose, was observed 3h 

following gavage. Approximately 90% of the administered dose 

was excreted in urine and feces within 24h, primarily as β-HBCDderived metabolites. A portion of the dose (circa 9%) was excreted 

in feces as γ-HBCD. Oral administration of 30 or 100mg/kg of 

β-HBCD resulted initially in slower rates of [14C] elimination; 

however, cumulative excretion data were similar across the dosing 

range 4 days postdosing. Residual concentrations of [14C] in tissues were highest in adipose and liver. β-HBCD-derived radioactivity accumulated in most tissues following four consecutive daily 

oral doses of 3mg/kg. The extent of metabolism and excretion of 

β-HBCD in female C57BL/6 mice was similar to that for γ-HBCD. 

The potential for accumulation of β-HBCD-derived material in 

most tissues appeared to be less than for α-HBCD.

Key Words: hexabromocyclododecane; toxicokinetics; mouse; 

brominated flame retardant; disposition; risk assessment; persistent 

organic pollutant.

1,2,5,6,9,10-Hexabromocyclododecane (HBCD) is a high 

production volume cycloaliphatic used as an additive flame 

retardant in polystyrene foam building materials and in 

some textile and electronic products (Marvin et  al., 2011). 

Commercial HBCD mixtures are composed primarily of three 

enantiomer pairs designated as alpha (α)-, beta (β)-, and gamma 

(γ)-HBCD (Heeb et al., 2005; Fig. 1). As a consequence of high 

usage, physiochemical properties, and potential for long-range 

transport, these isomers are detected in the global environment, 

including Arctic regions and have been observed in wildlife and 

humans (Brandsma et al., 2009; Covaci et al., 2006; Darnerud 

et al., 2011; de Wit et al., 2010; Klosterhaus et al., 2012; Toms 

et al., 2012). The chemical is considered to be persistent, bioaccumulative, and toxic and will be reviewed by the Stockholm 

Convention for recommended elimination of production and 

use (Marvin et al., 2011; Stockholm Convention Web site).

Humans may be at greatest exposure risk to HBCD adsorbed 

to indoor dust or present in food products (Schecter et al., 2012; 

Stapleton et al., 2008; Törnkvist et al., 2011). A risk assessment 

conducted by the European Commission (2008) estimated 

consumer exposure to HBCD from these sources to be in the 

low ng/kg/day range and calculated a no observed adverse effect 

level of 22.9mg/kg/day from a repeat-dose study conducted by 

van der Ven et al. (2006) in rats. The HBCD isomeric mixture 

is a developmental neurotoxicant and endocrine disruptor in 

rodents (Ema et al., 2008; Eriksson et al., 2006; van der Ven et al., 

2006, 2009). Disruption of thyroid homeostasis is a purported 

mechanism of toxicity, supported by observations of effects 

on thyroid weight, follicle size, and serum thyroid-stimulating 

hormone in rats, and T4 levels in rats, trout, and captive kestrels 

exposed to HBCD (Ema et al., 2008; Marteinson et al., 2011; 

Palace et al., 2010; van der Ven et al., 2006, 2009). In rats, HBCD 

isomers have been shown to induce CYP2B and CYP3A, a 

phenobarbital-type induction associated with depletion of thyroid 

hormone in rodents (Germer et al., 2006). Exposure to HBCD 

has resulted in cytotoxicity in neuronal and hepatic cells and 

hyperthyroidal, estrogenic, antiandrogenic, and antiprogestagenic 

activity in breast and ovarian cancer cell lines and/or other in vitro

assays (Al-Mousa and Michelangeli, 2012; Dorosh et al., 2010; 

Hamers et al., 2006; Ibhazehiebo et al., 2011; Park et al., 2011; 

Zhang et al., 2008). The toxicity of individual HBCD isomers has 

not been characterized in a mammalian model.

Covaci et  al. (2006) reported that α, β, and γ, comprised 

10–13%, 1–12%, and 75–89%, respectively, of commercial grade 

HBCD mixtures. γ-HBCD is most often found at the greatest 

concentration in sediment, soil, and sludge samples because of its 

toxicological sciences 134(2), 251–257 2013

doi:10.1093/toxsci/kft121

Advance Access publication June 2, 2013
Sanders, Knudsen, and Birnbaum

relative abundance in mixtures (Covaci et al., 2006; Klosterhaus 

et  al., 2012). Conversely, α-HBCD has been detected at a 

higher concentration than γ-HBCD in lipid of aquatic invertebrates, marine mammals, fish, birds, and humans (Abdallah and 

Harrad, 2011; Covaci et al., 2006; Klosterhaus et al., 2012; Xia 

et al., 2011; Zegers et al., 2005). Exceptions have been noted, 

with higher levels of γ-HBCD observed in some human (milk, 

adipose tissue) and fish (muscle) samples (Johnson-Restrepo 

et al., 2008; Toms et al., 2012). Recent evidence indicates that 

the in vivo fates of individual isomers play a substantial role in 

their differential profiles in abiotic and biotic matrices. Studies 

conducted by Szabo et al. (2010, 2011) demonstrated that oral 

doses of [14C]-labeled α-HBCD were less metabolized and eliminated, and retained at much higher concentrations in tissues of 

female C57BL/6 mice over time than similar [14C]-labeled doses 

of γ-HBCD. γ-HBCD has been shown to isomerize thermally 

to α-HBCD (Heeb et  al., 2008), and in vivo isomerization of 

γ-HBCD to α- and β-HBCD was observed by Szabo et al. (2010, 

2011). In contrast, isomerization of α-HBCD was not detected in 

female mice used in those studies.

Exposure to β-HBCD may involve direct contact in the environment and/or in vivo formation from γ-HBCD. β-HBCD was 

detected in brain, liver, adipose, and feces of mice receiving 

oral doses of γ-HBCD (Szabo et al., 2010). Characterization of 

the in vivo fate of HBCD isomers is critical to assessing exposure risk to the mixture. However, disposition data for β-HBCD 

are lacking, and the toxicological implications of exposure are 

unknown. Therefore, the objective of the present study was to 

determine the in vivo fate of β-HBCD in the mouse model used 

in previous toxicokinetic studies of α- and γ-HBCD. These 

data will support toxicological evaluations and risk assessment 

of HBCD isomeric mixtures.

Materials and Methods

Chemicals and dosing solutions. [

14C]-Labeled and nonlabeled β-HBCD 

used in these experiments were prepared by Drs Heldur Hakk and Janice Huwe 

of Agricultural Research Service/United States Department of Agriculture, 

Fargo, ND, as described by Szabo et al. (2010). Briefly, [14C]-labeled β-HBCD 

was obtained by separation of a mixture of [14C] β- and γ-HBCD obtained from 

American Radiochemicals Corporation (St Louis, MO). The specific activity and 

radiochemical purity were 2 mCi/mmol and > 99%, respectively. The identity 

and purity (> 99%) of nonlabeled β-HBCD were determined using liquid chromatography-tandem mass spectrometry, by comparison with an authentic standard of β-HBCD obtained from Wellington (Guelph, Ontario, Canada). Dosing 

solutions were formulated to administer ~10  μCi/kg/mouse and contained 

amounts of nonlabeled β-HBCD for delivery of doses up to 100mg/kg. Doses 

were chosen to match those used in previous toxicokinetic studies of α- and 

γ-HBCD (Szabo et al., 2010, 2011). β-HBCD dissolved in toluene was added 

to a corn oil vehicle for oral administration. The toluene was removed from the 

dosing solution via evaporation under a steady stream of N2

. For iv administration, β-HBCD in toluene was added to one part Cremophor EL (Sigma-Aldrich, 

St Louis, MO). The toluene was evaporated with N2, and one part ethanol and 

three parts water were added to complete the dosing solution. Dose volumes 

were 2 and 5ml/kg for iv-injected and gavaged mice, respectively.

Animals and treatment. All procedures involving animals were approved 

by the Institutional Care and Use Committee of the National Institute of 

Environmental Health Sciences. Female C57BL/6 mice, obtained from Charles 

River (Raleigh, NC), were ~8 weeks old and 20g at time of dosing. Dose response 

was investigated in mice (n = 4/treatment group) receiving single oral administration of 3, 30, or 100mg/kg of β-HBCD. All oral doses were administered using 

a no. 20 ball-tipped stainless steel feeding needle. Following gavage, the mice 

were housed individually in metabolism cages for daily collection of urine and 

feces for 4 days postdosing. Distribution to and clearance of [14C] from tissues 

over time were investigated in treatment groups (n = 4) receiving 3mg/kg following termination at time points of 1, 3, 8, and 24h postdosing. Urine and feces 

were collected from the 8- and 24-h groups. Accumulation of [14C] over time was 

investigated in a group of animals receiving four consecutive daily doses of 3mg/

kg by gavage. These mice were euthanized 24h after the last dose. Mice (n = 4) 

receiving 3mg/kg by tail vein injection were euthanized 24h postdosing to provide comparative data for assessing the extent of absorption in gavaged mice.

Two of the iv-treated mice were placed in cages allowing for the collection 

of [14C] in expired air, either as volatile organic compounds (VOC) or 14CO2, 

using the method described by Sanders et al. (2000). Food (NIH no. 31) and 

water were available for ad libitum consumption by all mice used in this study. 

Euthanasia in all experiments was by CO2

 asphyxiation. Blood was collected 

by cardiac puncture at time of death. Liver, muscle, adipose tissue (from the 

abdominal cavity), brain, lung, skin (ear site), and kidney were collected at 

necropsy. Blood, skin, muscle, and adipose tissue were estimated to be 8, 12, 

35, and 8%, respectively, of body weight as previously reported for female 

C57BL/6 mice of similar age by Szabo et al. (2010). All other tissues were 

weighed gravimetrically. These values were used to calculate % total dose in 

tissues following necropsy.

Analysis of [14C] content in biological samples. The amount of β-HBCDderived radioactivity in urine was determined by pipetting triple aliquots (10 μl) 

of each sample directly into vials containing Ecolume (MP Biomedicals, Solon, 

OH) for analysis in a liquid scintillation counter (LSC) (Beckman Coulter, 

Brea, CA). The [14C] values obtained from triple aliquots (50 μl each) of cage 

rinsates were added to those obtained for urine at each time point. The [14C] 

content in tissue and feces samples was determined by oxidizing triple aliquots 

(≤ 100mg; wet weight, except feces) of these samples in a biological sample 

Fig. 1. Structures of the three major isomers in HBCD commercial flame retardants. Each stereoisomer consists of an enantiomer pair.

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The Fate of β-HBCD in Mice

oxidizer (Perkin Elmer, Waltham, MA) prior to LSC analysis. Feces were airdried, weighed, and ground to a fine powder before combustion.

Analytical high-performance liquid chromatography methods and sample 

preparation. The relative amounts of β-HBCD and its metabolites in urine 

and tissue and feces extracts were determined using high-performance liquid 

chromatography (HPLC). The HPLC analysis was based on a method described 

previously (Yu et al., 2008). The system consisted of a Waters (Milford, MA) 

1525 binary pump coupled to a Waters 2487 dual absorbance detector and a IN/

US β-RAM model 3 radiochemical detector (LabLogic, Brandon, FL). Samples 

were prepared as described below and separated using an Agilent (Santa Clara, 

CA) Eclipse Plus C18 column (3.5 μm, 4.6×150mm) with a Zorbax Reliance 

cartridge guard column. Mobile phases consisted of (A): 0.1% trifluoroacetic 

acid (TFA) in water and (B): methanol/water (80/20, vol/vol). A gradient of 80% 

A, reduced to 10% A in 2min, held at 10% A for 33min, then returned to initial 

conditions over 5min was used. The flow rate throughout the run was 300 μl/

min. For radiochemical analyses, Inflow ES scintillation cocktail (LabLogic) 

was used at a 3:1 ratio based on the manufacturer’s recommendation. Breeze 

(v. 3.30, Waters) and Laura-Lite (v. 3.3.10.49, LabLogic) software packages 

were used for instrument control and data analyses. Prior to HPLC analysis, 

urine samples were filtered by centrifugation using nonsterile Ultrafree MC filter 

units (0.22 μm; Millipore, Billerica, MA) and an Eppendorf 5415 D centrifuge 

(Eppendorf, Hauppauge, NY) at 12,700rpm for 15min. Aliquots (~250mg) of 

air-dried fecal or tissue (wet weight) samples were placed in Ultrafree MC cartridges, rehydrated with 400 μl water (containing 0.1% TFA), and centrifuged 

as described. This process was repeated twice more, with the filtrates transferred 

and pooled. Each retentate was then extracted with 95% ethanol (3×400 μl), 

refiltered, and the filtrates transferred and pooled. Aqueous and organic extracts, 

maintained separately, were concentrated using a Savant SPD1010 SpeedVac 

(ThermoScientific, Waltham, MA) without heating, and aliquots were injected 

onto the HPLC.

Statistical analysis and calculation of half-lives of [14C] in tissues. The 

data were subjected to statistical analysis using one-way ANOVA followed by 

the Tukey-Kramer test for pairwise comparisons (JMP Statistical Software, 

SAS Institute Inc., Cary, NC). Values were considered to be significantly different at p < 0.05. Noncompartmental pharmacokinetic data analysis was performed using the means of the tissue data collected from mice receiving 3mg/

kg by gavage (PK Solutions 2.0, Summit Research, Montrose, CO).

Results

Approximately 90% of the lowest orally administered dose 

(3mg/kg) of β-HBCD was excreted in urine and feces within 

24h of treatment (Fig. 2). The majority of the remaining dose 

was excreted within 48 h, although measurable amounts of 

[

14C] remained in tissues up to 4 days postdosing (Fig. 3 and 

Table 1). Administration of higher doses (30 and 100mg/kg) 

resulted in less excretion of β-HBCD-derived radioactivity in 

urine and feces 24h after gavage (Fig. 2). However, there were 

no statistically significant differences in the cumulative 4-day 

excretion of [14C] over the dose range. No overt signs of toxicity were observed in any mice following dosing. However, loss 

of up to 25% total body weight was noted at the 4-day terminal time point in the mice receiving a single dose of 100mg/

kg. Necropsy results indicated large decreases in the amount of 

adipose tissue in the body cavity relative to mice treated with 

lower doses (data not shown). It was uncertain if these mice 

consumed less food than others because daily food consumption was not measured in this study. Similar weight loss was 

not reported in mice receiving comparable doses of either α- or 

γ-HBCD (Szabo et al., 2010, 2011).

β-HBCD-derived radioactivity was rapidly distributed to all 

assayed tissues following a single oral dose of 3mg/kg, with the 

observed Cmax for most tissues at the 3-h time point (Table 1). The 

highest concentration of [14C] over time was detected in liver. This 

amounted to ~7% of the total dose at the observed Cmax. Adipose 

tissue was also a major target, containing the highest concentration of [14C] at the 8-h time point. The peak concentration of [14C] 

in adipose tissue appeared to be less than that in liver; however, 

[

14C] was more persistent over time in adipose tissue, resulting in 

approximately threefold higher levels of [14C] at 4 days postdosing than in liver. The brain contained the least amount of [14C] of 

the assayed tissues over time. The tissue:blood ratio for [14C] in 

the brain was near unity at the common Cmax, and the concentration of [14C] was at or near background by 24h postdosing. The 

concentration of [14C] in the blood over time was low compared 

with that of liver and adipose tissue and was within twofold of 

background at the 4-day time point.

The amount of [14C] in adipose tissue 24h after a single dose 

of β-HBCD indicated potential for accumulation of β-HBCDderived material in lipid upon repeated exposure (Table  1). 

Further, the elimination half-life of [14C] exceeded 24 h in all 

Fig. 2. Excretion of β-HBCD-derived radioactivity over time following single doses of 3, 30, or 100mg/kg by gavage. Values are the mean ± SD of four mice. 

*The value is significantly higher than that for the 100mg/kg group (p < 0.05).

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Sanders, Knudsen, and Birnbaum

assayed tissues, except brain. The repeat-dose experiment 

demonstrated a higher concentration of [14C] in all assayed tissues, except muscle, 24h following the last of four daily doses 

than following a single dose. The cumulative amount of [14C] 

recovered in urine of repeat-dose mice increased slightly over 

time (29±4% total dose after 1 day vs. 35±5% after 4 days), 

whereas the amount excreted in feces declined from 59±12% 

total dose after 1  day to 51±5% after 4  days. However, the 

differences in the means were not statistically significant. No 

differences were detected in the metabolic profiles of [14C] in 

excreta over time (data not shown).

The % total dose remaining in the assayed tissues 4  days 

following oral administration of single doses of 3, 30, and 

100mg/kg is shown in Figure  3. Less than 1% total dose 

was present in the blood at this time point. The % total dose 

remaining in brain, kidney, and lung was less than observed for 

blood; therefore, those values are not shown. The amount of 

[

14C] in blood, liver, muscle, skin, brain, kidney, and lung was 

generally proportional to dose. However, proportionally more 

[

14C] was present in the adipose tissue of the 30 and 100mg/

kg treatment groups than observed for adipose tissue from the 

low-dose group.

Mice receiving 3 mg/kg of β-HBCD by single iv injection 

excreted significantly less [14C] in feces than mice receiving 

a similar dose by gavage (Fig. 4). Although less [14C] was 

recovered in urine of iv-injected mice, the means were not 

significantly different. Conversely, iv-injected mice retained 

a significantly higher concentration of [14C] in tissues, specifically muscle and skin (not shown), than did gavaged 

mice. Trace amounts of [14C] were excreted in expired air as 

VOC (0.24% total dose) and 14CO2

 (0.87% total dose) from 

iv-treated mice.

β-HBCD was extensively metabolized following oral or 

iv administration to mice. Following oral administration of 

3mg/kg, the [14C] in urine was recovered in two early eluting radiolabeled peaks from the HPLC system (combined 

and listed as hydrophilic metabolites in Table  2). Neither 

of the radiolabeled peaks in urine coeluted with an authentic standard of β-HBCD in the system (Rt

 = 30.9min). The 

retention times of these peaks (7 and 9min) were similar to 

Fig. 3. β-HBCD-derived radioactivity remaining in tissues 4 days following single doses of 3, 30, or 100mg/kg by gavage. Values are the mean ± SD 

for three to four mice. *The value is significantly higher than that for the 3mg/

kg group (p < 0.05).

Table 1

Amount and Half-Lives of β-HBCD-Derived Radioactivity in Tissues of Female Mice Receiving 3mg/kg by Gavage

Single dose Four daily doses Alpha t1/2 Beta t1/2

Tissue 1ha 3 h 8 h 24 h 96 h 24 h Days Days

Blood 148±39b

(0.34±0.1)c

206±29

(0.54±0.08)

125±36

(0.31±0.09)

55±19

(0.13±0.04)

20±12

(0.05±0.04)

120±10

(0.08±0.01)

0.2 2.1

Liver 1632±288

(2.21±0.78)

4462±246

(6.78±0.40)

1638±245

(2.46±0.47)

692±54

(1.07±0.22)

147±37

(0.33±0.18)

1077±46

(0.46±0.03)

0.1 1.3

Muscle 110±93

(1.12±0.98)

443±191

(5.01±2.18)

130±32

(1.39±0.35)

110±79

(1.15±0.75)

74±43

(0.85±0.48)

163±82

(0.44±0.21)

0.04 5.3

Adipose 543±317

(1.26±0.78)

1585±825

(4.08±2.07)

2345±847

(5.07±2.11)

1136±131

(2.79±0.45)

498±148

(1.36±0.38)

3126±1187

(2.14±0.51)

NDd 2.5

Brain 72±33

(0.04±0.02)

228±23

(0.15±0.01)

94±23

(0.06±0.01)

7±3

(0.01±0.00)

8±6

(> 0.01)

41±9

(0.01±0.00)

0.02 0.2

Lung 320±91

(0.07±0.02)

581±103

(0.14±0.01)

374±174

(0.09±0.04)

170±67

(0.03±0.01)

44±28

(0.01±0.01)

431±110

(0.09±0.13)

0.2 1.5

Skin 153±95

(0.53±0.35)

315±178

(1.22±0.68)

249±62

(0.91±0.23)

202±37

(0.74±0.13)

150±42

(0.61±0.16)

464±86

(0.44±0.10)

0.1 7.0

Kidney 646±172

(0.20±0.05)

1222±226

(0.41±0.08)

694±187

(0.21±0.06)

151±47

(0.05±0.02)

55±23

(0.02±0.01)

373±40

(0.03±0.00)

0.2 2.1

Note. a

Terminal time point in hours after receiving a single dose or the last of four daily doses. b

Each value represents the mean and SD of 4 mice/treatment group expressed as ng [14C]-equivalents/gram of tissue. c

Each value in parenthesis represents the data expressed as % of the administered dose.

d

Could not be determined.

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The Fate of β-HBCD in Mice

that of two radiolabeled peaks present in the aqueous extracts 

of feces from β-HBCD-treated mice. The organic extracts of 

feces contained two [14C]-labeled peaks coeluting with β- and 

γ-HBCD standards. The amount of β-HBCD and γ-HBCD 

excreted in feces each represented ~8–9% of the total dose. 

Approximately 30% of the [14C] in the feces was nonextractable and was assumed to represent β-HBCD-derived metabolites. Trace amounts (< 0.3% of the total dose) of α- and 

γ-HBCD were detected in extracts of adipose tissue and liver 

of β-HBCD-treated mice. The [14C] profile in urine and feces 

samples from mice receiving 100mg/kg was similar to that of 

lower dose mice (data not shown).

Discussion

β-HBCD was rapidly absorbed from the gastrointestinal 

tract of female C57BL/6 mice. The extent of absorption was 

estimated to be ≥ 85% of the total dose, based on comparison 

of oral- and iv-derived excretion data and the amount of 

unmetabolized HBCD in extracts of feces from gavaged mice. 

A comparison of the present data with those obtained by Szabo 

et al. (2010, 2011) demonstrated that the extent of absorption, 

specific target tissues, and excretion routes were similar for the 

three major HBCD stereoisomers in C57BL/6 female mice. 

However, differences in the observed Cmax and Tmax of [14C] in 

tissues over time were evident among α-, β-, and γ-HBCDtreated mice receiving a comparable dose (3mg/kg). For 

instance, comparisons of [14C] in major tissues demonstrated 

that liver contained the highest mean concentration following 

oral administration of the three stereoisomers; however, the 

Tmax for the tissue varied from 1 to 3h and the Cmax varied 

up to 21-fold between the isomers. The differences in tissue 

disposition were greatest between α-HBCD-treated mice and 

mice dosed with the other stereoisomers. For example, the 

Cmax for [14C] in adipose, liver, and blood of α-HBCD-treated 

mice was 8-, 11-, and 200-fold higher than mice treated with a 

comparable dose of β-HBCD. On the other hand, the maximum 

concentrations of [14C] in liver, blood, and other assayed tissues 

(except adipose) were within twofold of each other in β- and 

γ- HBCD-treated mice, with the higher amounts (except in 

skin) associated with β-HBCD treatment. Adipose tissue from 

β-HBCD-treated mice contained 24-fold greater amounts at 

the peak concentration than did adipose tissue from γ-HBCDtreated mice. These observations indicate that α-HBCD has 

greater bioavailability than either β- or γ-HBCD and has 

greater potential to accumulate in tissues following repeat 

exposure than the two other major HBCD isomers. However, 

this conclusion does not preclude accumulation of β-HBCDderived material in tissues following chronic exposure, at 

least at a concentration of 3mg/kg in mice, as shown in the 

present study.

In a study conducted by Zegers et al. (2005), rapid metabolism was observed for β- and γ-HBCD, but not α-HBCD, 

when incubated with male Wistar rat liver microsomes. These 

results suggested that observed accumulation of α-HBCD in 

tissues of harbor porpoise and dolphins in European seas was 

driven largely by differences in the extent of metabolism of 

the three stereoisomers. In the present study, the elimination 

Fig. 4. β-HBCD-derived radioactivity remaining in tissues and cumulatively excreted 24 h following oral and iv administration of 3mg/kg. Values are 

for the mean ± SD of four mice. *The value is significantly different from the 

corresponding value (p < 0.05).

Table 2

Profile and Amount of [14C] Contained in Urine or Feces/Tissue Extracts of Mice Receiving 3mg/kg β-HBCD by Gavagea

Sample Hydrophilic metabolites α-HBCD β-HBCD γ-HBCD Nonextractable

Urine (0–8h) 25 (100)b NDc ND ND —

Urine (0–24 h) 39 (100) ND ND ND —

Feces (0–24 h)d 22 (39) ND 8 (14) 9 (16) 17 (30)

Adipose (8h)d < 0.1 (0.3) 0.1 (3) 3 (92) 0.1 (3) < 0.1 (1.4)

Liver (3 h)d 4 (66) < 0.1 (0.2) < 0.1 (0.3) < 0.1 (0.2) 2 (33)

Note. a

Data are derived from HPLC chromatograms of representative samples pooled from all mice in the specified treatment group. b

Values are “% of the administered dose (% of the total [14C] in the assayed sample).” c

Not detected.

d

Values are from combined aqueous and organic extracts.

 255
Sanders, Knudsen, and Birnbaum

half-lives of [14C] in assayed tissues from β-HBCD-treated 

mice were significantly shorter than for α-HBCD, but similar to 

those for γ-HBCD as calculated by Szabo et al. (2010, 2011). 

Furthermore, HPLC analysis demonstrated that the extent of in 

vivo metabolism of β-HBCD was similar to that of γ-HBCD, 

not α-HBCD. The series of kinetic studies of the three major 

HBCD stereoisomers conducted in this laboratory confirmed 

that the extent of metabolism plays a primary role in the pattern 

of accumulation in tissues of mammalian species exposed to 

the HBCD stereoisomers.

In the present study, [14C] β-HBCD was excreted in urine of 

mice solely as metabolic product, as were the other two stereoisomers in the studies reported by Szabo et al. (2010, 2011). 

Metabolites of each of the HBCD stereoisomers were also 

excreted in feces of treated mice. Hakk et  al. (2012) identified α- and γ-HBCD-derived metabolites from the Szabo et al.

(2010, 2011) studies, showing that α-HBCD was monohydroxylated, debrominated to a tri- or tetrabrominated species, 

and conjugated with glutathione. Metabolism of γ-HBCD was 

more complex, with detection of hexa-, tetra-, and tribrominated carboxylic acid moieties, as well as metabolites arising 

from hydroxylation and glutathione conjugation. In the present study, all of the [14C] excreted in urine and most of the 

[

14C] excreted in feces of mice consisted of metabolites of 

β-HBCD. Although these metabolites were not identified, they 

are assumed to arise through similar biotransformation pathways utilized by α- and γ-HBCD. Thus, they probably represent hydroxylation, debromination, and conjugation products 

of parent β-HBCD.

Enrichment of α-HBCD in biota may also involve enzymatic 

isomerization of other HBCD stereoisomers to α-HBCD. 

γ-HBCD has been shown to convert to α- and β-HBCD both 

thermally and in vivo (Heeb et al., 2008; Köppen et al., 2008; 

Szabo et al., 2010). On the other hand, thermal conversion of 

α-HBCD to other isomers is less favored (Heeb et al., 2008; 

Köppen et al., 2008), and there was no evidence for isomerization 

of α-HBCD in mice (Szabo et al., 2011). In the present work, a 

measurable portion of β-HBCD was converted to γ-HBCD and 

excreted in feces. It is uncertain if this conversion occurred as 

the result of microflora activity in the gut or metabolic activity 

in tissues.

In conclusion, the results from the present study demonstrated that β-HBCD was well absorbed, distributed to tissues, 

and rapidly excreted in urine and feces following oral administration to female mice. Administration of higher doses resulted 

in apparent saturation of kinetics as indicated by lower initial 

[

14C] excretion rates, as well as an indication of toxicity (weight 

loss) at the highest dose. β-HBCD was extensively metabolized, with evidence of in vivo isomerization to γ-HBCD. 

Comparison of β-HBCD disposition to that reported for the 

other major isomers (Szabo et al., 2010, 2011) indicated that 

β- and γ-HBCD were more extensively metabolized and had 

less potential to accumulate in tissues of female C57BL/6 mice 

than α-HBCD.

Funding

This research was conducted by employees of the National 

Institutes of Health and was funded by the Intramural Research 

Program of the National Cancer Institute.

Acknowledgments

The authors thank Ms Sherry Coulter and Mr Abdella Sadik 

for excellent technical assistance.

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